# Patent

## Title
Functional and hyperfunctional siRNA directed against Bcl-2

## Abstract

Efficient sequence specific gene silencing is possible through the use of siRNA technology. By selecting particular siRNAs by rationale design, one can maximize the generation of an effective gene silencing reagent, as well as methods for silencing genes.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a divisional application of U.S. patent application Ser. No. 10/714,333, filed Nov. 14, 2003 entitled “Functional and Hyperfunctional siRNA,” which claims the benefit of U.S. Provisional Application Ser. No. 60/502,050, filed Sep. 10, 2003, entitled “Methods for Selecting siRNA,” the entire disclosures of which are hereby incorporated by reference into the present disclosure.


REFERENCE TO TABLES SUBMITTED IN ELECTRONIC FORM
Applicants hereby incorporate by reference the material submitted previously, in duplicate on the compact disks labeled PATENT APPLICATION NO. 60/426,137, DISK 1 of 1, COPY 1 of 2; PATENT APPLICATION NO. 60/426,137, DISK 1 of 1, COPY 2 of 2; which copies are identical, in files entitled Table—12.txt, date of creation Jun. 26, 2003, with a size of 31,045 kb; Table—13.txt, date of creation Nov. 13, 2003, with a size of 78,451 kb; Table—14.txt, date of creation Nov. 13, 2003, with a size of 454 kb; and Table—15.txt date of creation Nov. 13, 2003, with a size of 1,690 kb.
FIELD OF INVENTION
The present invention relates to RNA interference (“RNAi”).
BACKGROUND OF THE INVENTION
Relatively recently, researchers observed that double stranded RNA (“dsRNA”) could be used to inhibit protein expression. This ability to silence a gene has broad potential for treating human diseases, and many researchers and commercial entities are currently investing considerable resources in developing therapies based on this technology.
Double stranded RNA induced gene silencing can occur on at least three different levels: (i) transcription inactivation, which refers to RNA guided DNA or histone methylation; (ii) siRNA induced mRNA degradation; and (iii) mRNA induced transcriptional attenuation.
It is generally considered that the major mechanism of RNA induced silencing (RNA interference, or RNAi) in mammalian cells is mRNA degradation. Initial attempts to use RNAi in mammalian cells focused on the use of long strands of dsRNA. However, these attempts to induce RNAi met with limited success, due in part to the induction of the interferon response, which results in a general, as opposed to a target-specific, inhibition of protein synthesis. Thus, long dsRNA is not a viable option for RNAi in mammalian systems.
More recently it has been shown that when short (18-30 bp) RNA duplexes are introduced into mammalian cells in culture, sequence-specific inhibition of target mRNA can be realized without inducing an interferon response. Certain of these short dsRNAs, referred to as small inhibitory RNAs (“siRNAs”), can act catalytically at sub-molar concentrations to cleave greater than 95% of the target mRNA in the cell. A description of the mechanisms for siRNA activity, as well as some of its applications are described in Provost et al., Ribonuclease Activity and RNA Binding of Recombinant Human Dicer, E.M.B.O. J., 2002 Nov. 1; 21(21): 5864-5874; Tabara et al., The dsRNA Binding Protein RDE-4 Interacts with RDE-1, DCR-1 and a DexH-box Helicase to Direct RNAi in C. elegans, Cell 2002, June 28; 109(7):861-71; Ketting et al., Dicer Functions in RNA Interference and in Synthesis of Small RNA Involved in Developmental Timing in C. elegans; Martinez et al., Single-Stranded Antisense siRNAs Guide Target RNA Cleavage in RNAi, Cell 2002, September. 6; 110(5):563; Hutvagner & Zamore, A microRNA in a multiple-turnover RNAi enzyme complex, Science 2002, 297:2056.
From a mechanistic perspective, introduction of long double stranded RNA into plants and invertebrate cells is broken down into siRNA by a Type III endonuclease known as Dicer. Sharp, RNA interference—2001, Genes Dev. 2001, 15:485. Dicer, a ribonuclease-II-like enzyme, processes the dsRNA into 19-23 base pair short interfering RNAs with characteristic two base 3′ overhangs. Bernstein, Caudy, Hammond, & Hannon, Role for a bidentate ribonuclease in the initiation step of RNA interference, Nature 2001, 409:363. The siRNAs are then incorporated into an RNA-induced silencing complex (RISC) where one or more helicases unwind the siRNA duplex, enabling the complementary antisense strand to guide target recognition. Nykanen, Haley, & Zamore, ATP requirements and small interfering RNA structure in the RNA interference pathway, Cell 2001, 107:309. Upon binding to the appropriate target mRNA, one or more endonucleases within the RISC cleaves the target to induce silencing. Elbashir, Lendeckel, & Tuschl, RNA interference is mediated by 21- and 22-nucleotide RNAs, Genes Dev 2001, 15:188, FIG. 1.
The interference effect can be long lasting and may be detectable after many cell divisions. Moreover, RNAi exhibits sequence specificity. Kisielow, M. et al. (2002) Isoform-specific knockdown and expression of adaptor protein ShcA using small interfering RNA, J. of Biochemistry 363: 1-5. Thus, the RNAi machinery can specifically knock down one type of transcript, while not affecting closely related mRNA. These properties make siRNA a potentially valuable tool for inhibiting gene expression and studying gene function and drug target validation. Moreover, siRNAs are potentially useful as therapeutic agents against: (I) diseases that are caused by over-expression or misexpression of genes; and (2) diseases brought about by expression of genes that contain mutations.
Successful siRNA-dependent gene silencing depends on a number of factors. One of the most contentious issues in RNAi is the question of the necessity of siRNA design, i.e., considering the sequence of the siRNA used. Early work in C. elegans and plants circumvented the issue of design by introducing long dsRNA (see, for instance, Fire, A. et al. (1998) Nature 391:806-811). In this primitive organism, long dsRNA molecules are cleaved into siRNA by Dicer, thus generating a diverse population of duplexes that can potentially cover the entire transcript. While some fraction of these molecules are non-functional (i.e. induce little or no silencing) one or more have the potential to be highly functional, thereby silencing the gene of interest and alleviating the need for siRNA design. Unfortunately, due to the interferon response, this same approach is unavailable for mammalian systems. While this effect can be circumvented by bypassing the Dicer cleavage step and directly introducing siRNA, this tactic carries with it the risk that the chosen siRNA sequence may be non-functional or semi-functional.
A number of researches have expressed the view that siRNA design is not a crucial element of RNAi. On the other hand, others in the field have begun to explore the possibility that RNAi can be made more efficient by paying attention to the design of the siRNA. Unfortunately, none of the reported methods have provided a satisfactory scheme for reliably selecting siRNA with acceptable levels of functionality. Accordingly, there is a need to develop rational criteria by which to select siRNA with an acceptable level of functionality, and to identify siRNA that have this improved level of functionality, as well as to identify siRNAs that are hyperfunctional.
SUMMARY OF THE INVENTION
The present invention is directed to increasing the efficiency of RNAi, particularly in mammalian systems. Accordingly, the present invention provides kits, siRNAs and methods for increasing siRNA efficacy.
According to one embodiment, the present invention provides a kit for gene silencing, wherein said kit is comprised of a pool of at least two siRNA duplexes, each of which is comprised of a sequence that is complementary to a portion of the sequence of one or more target messenger RNA.
According to a second embodiment, the present invention provides a method for optimizing RNA interference by using one or more siRNAs that are optimized according to a formula (or algorithm) selected from:

Relative functionality of siRNA=−(GC/3)+(AU15-19)−(Tm20° C.)*3−(G13)*3−(C19)+(A19)*2+(A3)+(U10)+(A14)−(U5)−(A11)  Formula I

Relative functionality of siRNA=−(GC/3)−(AU15-19)*3−(G13)*3−(C19)+(A19)*2+(A3)  Formula II

Relative functionality of siRNA=−(GC/3)+(AU15-19)−(Tm20° C.)*3  Formula III

Relative functionality of siRNA=−GC/2+(AU15-19)/2−(Tm20° C.)*2−(G13)*3−(C19)+(A9)*2+(A3)+(U10)+(A14)−(U5)−(A11)  Formula IV

Relative functionality of siRNA=−(G13)*3−(C19)+(A19)*2+(A3)+(U10)+(A14)−(U5)−(A11)  Formula V

Relative functionality of siRNA=−(G13)*3−(C19)+(A19)*2+(A3)  Formula VI

Relative functionality of siRNA=−(GC/2)+(AU15-19)/2−(Tm20° C.)*1−(G13)*3−(C19)+(A19)*3+(A3)*3+(U10)/2+(A14)/2−(U5)/2−(A11)/2  Formula VII

wherein in Formulas I-VII:

    
    
        Tm20° C.=1 if the Tm is greater than 20° C.;
        A19=1 if A is the base at position 19 on the sense strand, otherwise its value is 0;
        AU15-19=0-5 depending on the number of A or U bases on the sense strand at positions 15-19;
        G13=1 if G is the base at position 13 on the sense strand, otherwise its value is 0;
        C19=1 if C is the base at position 19 of the sense strand, otherwise its value is 0;
        GC=the number of G and C bases in the entire sense strand;
        A3=1 if A is the base at position 3 on the sense strand, otherwise its value is 0;
        A11=1 if A is the base at position 11 on the sense strand, otherwise its value is 0;
        A14=1 if A is the base at position 14 on the sense strand, otherwise its value is 0;
        U10=1 if U is the base at position 10 on the sense strand, otherwise its value is 0;
        U5=1 if U is the base at position 5 on the sense strand, otherwise its value is 0; or

Relative functionality of siRNA=(−14)*G13−13*A1−12*U7−11*U2−10*A11−10*U4−10*C3−10*C5−10*C6−9*A10−9*U9−9*C18−8*G10−7*U1−7*U16−7*C17−7*C19+7*U17+8*A2+8*A4+8*A5+8*C4+9*G8+10*A7+10*U18+11*A19+11*C9+15*G1+18*A3+19*U10−Tm−3*(GCtotal)−6*(GC15-19)−30*X; and  Formula VIII

Relative functionality of siRNA=(14.1)*A3+(14.9)*A6+(17.6)*A13+(24.7)*A19+(14.2)*U10+(10.5)*C9+(23.9)*G1+(16.3)*G2+(−12.3)*A11+(−19.3)*U1+(−12.1)*U2+(−11)*U3+(−15.2)*U15+(−11.3)*U16+(−11.8)*C3+(−17.4)*C6+(−10.5)*C7+(−13.7)*G13+(−25.9)*G19−Tm−3*(GCtotal)−6*(GC15-19)−30*X  Formula IX

wherein

        A1=1 if A is the base at position 1 of the sense strand, otherwise its value is 0;
        A2=1 if A is the base at position 2 of the sense strand, otherwise its value is 0;
        A3=1 if A is the base at position 3 of the sense strand, otherwise its value is 0;
        A4=1 if A is the base at position 4 of the sense strand, otherwise its value is 0;
        A5=1 if A is the base at position 5 of the sense strand, otherwise its value is 0;
        A6=1 if A is the base at position 6 of the sense strand, otherwise its value is 0;
        A7=1 if A is the base at position 7 of the sense strand, otherwise its value is 0;
        A10=1 if A is the base at position 10 of the sense strand, otherwise its value is 0;
        A11=1 if A is the base at position II of the sense strand, otherwise its value is 0;
        A13=1 if A is the base at position 13 of the sense strand, otherwise its value is 0;
        A19=1 if A is the base at position 19 of the sense strand, otherwise if another base is present or the sense strand is only 18 base pairs in length, its value is 0;
        C3=1 if C is the base at position 3 of the sense strand, otherwise its value is 0;
        C4=1 if C is the base at position 4 of the sense strand, otherwise its value is 0;
        C5=1 if C is the base at position 5 of the sense strand, otherwise its value is 0;
        C6=1 if C is the base at position 6 of the sense strand, otherwise its value is 0;
        C7=1 if C is the base at position 7 of the sense strand, otherwise its value is 0;
        C9=1 if C is the base at position 9 of the sense strand, otherwise its value is 0;
        C17=1 if C is the base at position 17 of the sense strand, otherwise its value is 0;
        C18=1 if C is the base at position 18 of the sense strand, otherwise its value is 0;
        C19=1 if C is the base at position 19 of the sense strand, otherwise if another base is present or the sense strand is only 18 base pairs in length, its value is 0;
        G1=1 if 6 is the base at position 1 on the sense strand, otherwise its value is 0;
        G2=1 if G is the base at position 2 of the sense strand, otherwise its value is 0;
        G8=1 if G is the base at position 8 on the sense strand, otherwise its value is 0;
        G10=1 if G is the base at position 10 on the sense strand, otherwise its value is 0;
        G13=1 if G is the base at position 13 on the sense strand, otherwise its value is 0;
        G19=1 if G is the base at position 19 of the sense strand, otherwise if another base is present or the sense strand is only 18 base pairs in length, its value is 0;
        U1=1 if U is the base at position 1 on the sense strand, otherwise its value is 0;
        U2=1 if U is the base at position 2 on the sense strand, otherwise its value is 0;
        U3=1 if U is the base at position 3 on the sense strand, otherwise its value is 0;
        U4=1 if U is the base at position 4 on the sense strand, otherwise its value is 0;
        U7=1 if U is the base at position 7 on the sense strand, otherwise its value is 0;
        U9=1 if U is the base at position 9 on the sense strand, otherwise its value is 0;
        U10=1 if U is the base at position 10 on the sense strand, otherwise its value is 0;
        U15=1 if U is the base at position 15 on the sense strand, otherwise its value is 0;
        U16=1 if U is the base at position 16 on the sense strand, otherwise its value is 0;
        U17=1 if U is the base at position 17 on the sense strand, otherwise its value is 0;
        U18=1 if U is the base at position 18 on the sense strand, otherwise its value is 0;
        GC15-19=the number of G and C bases within positions 15-19 of the sense strand or within positions 15-18 if the sense strand is only 18 base pairs in length;
        GCtotal=the number of G and C bases in the sense strand;
        Tm=100 if the targeting site contains an inverted repeat longer than 4 base pairs, otherwise its value is 0; and
        X=the number of times that the same nucleotide repeats four or more times in a row.
    
    


According to a third embodiment, the present invention is directed to a kit comprised of at least one siRNA that contains a sequence that is optimized according to one of the formulas above. Preferably the kit contains at least two optimized siRNA, each of which comprises a duplex, wherein one strand of each duplex comprises at least eighteen contiguous bases that are complementary to a region of a target messenger RNA. For mammalian systems, the siRNA preferably comprises between 18 and 30 nucleotide base pairs.
The ability to use the above algorithms, which are not sequence or species specific, allows for the cost-effective selection of optimized siRNAs for specific target sequences. Accordingly, there will be both greater efficiency and reliability in the use of siRNA technologies.
According to a fourth embodiment, the present invention provides a method for developing an siRNA algorithm for selecting functional and hyperfunctional siRNAs for a given sequence. The method comprises:

    
    
        (a) selecting a set of siRNAs;
        (b) measuring the gene silencing ability of each siRNA from said set;
        (c) determining the relative functionality of each siRNA;
        (d) determining the amount of improved functionality by the presence or absence of at least one variable selected from the group consisting of the total GC content, melting temperature of the siRNA, GC content at positions 15-19, the presence or absence of a particular nucleotide at a particular position and the number of times that the same nucleotide repeats within a given sequence; and
        (e) developing an algorithm using the information of step (d).
    
    


According to this embodiment, preferably the set of siRNAs comprises at least 90 siRNAs from at least one gene, more preferably at least 180 siRNAs from at least two different genes, and most preferably at least 270 and 360 siRNAs from at least three and four different genes, respectively. Additionally, in step (d) the determination is made with preferably at least two, more preferably at least three, even more preferably at least four, and most preferably all of the variables. The resulting algorithm is not target sequence specific.
In a fifth embodiment, the present invention provides rationally designed siRNAs identified using the formulas above.
In a sixth embodiment, the present invention is directed to hyperfunctional siRNA.
For a better understanding of the present invention together with other and further advantages and embodiments, reference is made to the following description taken in conjunction with the examples, the scope of which is set forth in the appended claims.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 shows a model for siRNA-RISC interactions. RISC has the ability to interact with either end of the siRNA or miRNA molecule. Following binding, the duplex is unwound, and the relevant target is identified, cleaved, and released.
FIG. 2 is a representation of the functionality of two hundred and seventy siRNA duplexes that were generated to target human cyclophilin, human diazepam-binding inhibitor (DB), and firefly luciferase.
FIG. 3a is a representation of the silencing effect of 30 siRNAs in three different cells lines, HEK293, DU145, and Hela. FIG. 3b shows the frequency of different functional groups (>95% silencing (black), >80% silencing (gray), >50% silencing (dark gray), and <50% silencing (white)) based on GC content. In cases where a given bar is absent from a particular GC percentage, no siRNA were identified for that particular group. FIG. 3c shows the frequency of different functional groups based on melting temperature (Tm). Again, each group has four different divisions: >95% (black), >80% (gray), >50% (dark gray), and <50% (white) silencing.
FIG. 4 is a representation of a statistical analysis that revealed correlations between silencing and five sequence-related properties of siRNA: (A) an A at position 19 of the sense strand, (B) an A at position 3 of the sense strand, (C) a U at position 10 of the sense strand, (D) a base other than G at position 13 of the sense strand, and (E) a base other than C at position 19 of the sense strand. All variables were correlated with siRNA silencing of firefly luciferase and human cyclophilin. SiRNAs satisfying the criterion are grouped on the left (Selected) while those that do not, are grouped on the right (Eliminated). Y-axis is “% Silencing of Control.” Each position on the X-axis represents a unique siRNA.
FIGS. 5 A and 5 B are representations of firefly luciferase and cyclophilin siRNA panels sorted according to functionality and predicted values using Formula VIII. The siRNA found within the circle represent those that have Formula VIII values (SMARTscoresTM) above zero. SiRNA outside the indicated area have calculated Formula VIII values that are below zero. Y-axis is “Expression (% Control).” Each position on the X-axis represents a unique siRNA.
FIG. 6A is a representation of the average internal stability profile (AISP) derived from 270 siRNAs taken from three separate genes (cyclophilin B, DBI and firefly luciferase). Graphs represent AISP values of highly functional, functional, and non-functional siRNA. FIG. 6B is a comparison between the AISP of naturally derived GFP siRNA (filled squares) and the AISP of siRNA from cyclophilin B, DBI, and luciferase having >90% silencing properties (no fill) for the antisense strand. “DG” is the symbol for ΔG, free energy.
FIG. 7 is a histogram showing the differences in duplex functionality upon introduction of basepair mismatches. The X-axis shows the mismatch introduced inot the siRNA and the position it is introduced (e.g., 8C->A reveals that position 8 (which normally has a C) has been changed to an A). The Y-axis is “% Silencing (Normalized to Control).”
FIG. 8a is histogram that shows the effects of 5′sense and antisense strand modification with 2′-O-methylation on functionality. FIG. 8b is an expression profile showing a comparison of sense strand off-target effects for IGF1R-3 and 2′-O-methyl IGF1R-3. Sense strand off-targets (lower white box) are not induced when the 5′ end of the sense strand is modified with 2′-O-methyl groups (top white box).
FIG. 9 shows a graph of SMARTscoresTM versus RNAi silencing values for more than 360 siRNA directed against 30 different genes. SiRNA to the right of the vertical bar represent those siRNA that have desirable SMARTscoresTM.
FIGS. 10A-E compare the RNAi of five different genes (SEAP, DBI, PLK, Firefly Luciferase, and Renila Luciferase) by varying numbers of randomly selected siRNA and four rationally designed (SMART-selected) siRNA chosen using the algorithm described in Formula VIII. In addition, RNAi induced by a pool of the four SMART-selected siRNA is reported at two different concentrations (100 and 400 nM). 10F is a comparison between a pool of randomly selected EGFR siRNA (Pool 1) and a pool of SMART selected EGFR siRNA (Pool 2). Pool 1, S1-S4 and Pool 2 S1-S4 represent the individual members that made up each respective pool. Note that numbers for random siRNAs represent the position of the 5′ end of the sense strand of the duplex. The Y-axis represents the % expression of the control(s). The X-axis is the percent expression of the control.
FIG. 11 shows the Western blot results from cells treated with siRNA directed against twelve different genes involved in the clathrin-dependent endocytosis pathway (CHC, DynII, CALM, CLCa, CLCb, Eps15, Eps15R, Rab5a, Rab5b, Rab5c, β2 subunit of AP-2 and EEA.1). SiRNA were selected using Formula VIII. “Pool” represents a mixture of duplexes 1-4. Total concentration of each siRNA in the pool is 25 nM. Total concentration=4×25=100 nM.
FIG. 12 is a representation of the gene silencing capabilities of rationally-selected siRNA directed against ten different genes (human and mouse cyclophilin, C-myc, human lamin A/C, QB (ubiquinol-cytochrome c reductase core protein I), MEK1 and MEK2, ATE1 (arginyl-tRNA protein transferase), GAPDH, and Eg5). The Y-axis is the percent expression of the control. Numbers 1, 2, 3 and 4 represent individual rationally selected siRNA. “Pool” represents a mixture of the four individual siRNA.
FIG. 13 is the sequence of the top ten Bcl2 siRNAs as determined by Formula VIII. Sequences are listed 5′ to 3′.
FIG. 14 is the knockdown by the top ten Bcl2 siRNAs at 100 nM concentrations. The Y-axis represents the amount of expression relative to the non-specific (ns) and transfection mixture control.
FIG. 15 represents a functional walk where siRNA beginning on every other base pair of a region of the luciferase gene are tested for the ability to silence the luciferase gene. The Y-axis represents the percent expression relative to a control. The X-axis represents the position of each individual siRNA. Reading from left to right across the X-axis, the position designations are 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, and Plasmid.
FIGS. 16A and 16B are histograms demonstrating the inhibition of target gene expression by pools of 2 (16A) and 3 (16B) siRNA duplexes taken from the walk described in FIG. 15. The Y-axis in each represents the percent expression relative to control. The X-axis in each represents the position of the first siRNA in paired pools, or trios of siRNAs. For instance, the first paired pool contains siRNAs 1 and 3. The second paired pool contains siRNAs 3 and 5. Pool 3 (of paired pools) contains siRNAs 5 and 7, and so on. For each of 16A and 16B, the X-axis from left to right reads 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, and Plasmid.
FIGS. 17A and 17B are histograms demonstrating the inhibition of target gene expression by pools of 4 (17A) and 5 (17B) siRNA duplexes. The Y-axis in each represents the percent expression relative to control. The X-axis in each represents the position of the first siRNA in each pool. For each of 17A and 17B, the X-axis from left to right reads 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87 89 and Plasmid.
FIGS. 18A and 18B are histograms demonstrating the inhibition of target gene expression by siRNAs that are ten (18A) and twenty (18B) base pairs apart. The Y-axis represents the percent expression relative to a control. The X-axis represents the position of the first siRNA in each pool. For each of 18A and 18B, the X-axis from left to right reads 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, and Plasmid.
FIG. 19 shows that pools of siRNAs (dark gray bar) work as well (or better) than the best siRNA in the pool (light gray bar). The Y-axis represents the percent expression relative to a control. The X-axis represents the position of the first siRNA in each pool. The X-axis from left to right reads 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, and Plasmid.
FIG. 20 shows that the combination of several semifunctional siRNAs (dark gray) result in a significant improvement of gene expression inhibition over individual (semi-functional; light gray) siRNA. The Y-axis represents the percent expression relative to a control.
FIG. 21A, 21B and 21C show both pools (Library, Lib) and individual siRNAs in inhibition of gene expression of Beta-Galactosidase, Renilla Luciferase and SEAP (alkaline phosphatase). Numbers on the X-axis indicate the position of the 5′-most nucleotide of the sense strand of the duplex. The Y-axis represents the percent expression of each gene relative to a control. Libraries contain siRNAs that begin at the following nucleotides: FIG. 21B: Seap: Lib 1: 206, 766, 812, 923, Lib 2: 1117, 1280, 1300, 1487, Lib 3: 206, 766, 812, 923, 1117, 1280, 1300, 1487, Lib 4: 206, 812, 1117, 1300, Lib 5: 766, 923, 1280, 1487, Lib 6: 206, 1487; FIG. 21A: Bgal: Lib 1 (denoted as I on the figure): 979, 1339, 2029, 2590, Lib 2 (denoted as II on the figure): 1087, 1783, 2399, 3257, Lib 3 (denoted as III on the figure): 979, 1783, 2590, 3257, Lib 4 (denoted as IV on the figure): 979, 1087, 1339, 1783, 2029, 2399, 2590, 3257, Lib 5 (denoted as V on the figure): 979, 1087, 1339, 1783, Lib 6 (denoted as VI on the figure): 2029, 2399, 2590, 3257 ; FIG. 21C: Renilla: Lib 1: 174, 300, 432, 568, Lib 2: 592, 633, 729, 867, Lib 3: 174, 300, 432, 568, 592, 633, 729, 867, Lib 4: 174, 432, 592, 729, Lib 5: 300, 568, 633, 867, Lib 6: 592, 568.
FIG. 22 shows the results of an EGFR and TfnR internalization assay when single gene knockdowns are performed. The Y-axis represents percent internalization relative to control.
FIG. 23 shows the results of an EGFR and TfnR internalization assay when multiple genes are knocked down (e.g. Rab5a, b, c). The Y-axis represents the percent internalization relative to control.
FIG. 24 shows the simultaneous knockdown of four different genes. SiRNAs directed against G6PD, GAPDH, PLK, and UBQ were simultaneously introduced into cells. Twenty-four hours later, cultures were harvested and assayed for mRNA target levels for each of the four genes. A comparison is made between cells transfected with individual siRNAs vs. a pool of siRNAs directed against all four genes.
FIG. 25 shows the functionality of ten siRNAs at 0.3 nM concentrations.



DETAILED DESCRIPTION
Definitions
Unless stated otherwise, the following terms and phrases have the meanings provided below:
siRNA
The term “siRNA” refers to small inhibitory RNA duplexes that induce the RNA interference (RNAi) pathway. These molecules can vary in length (generally between 18-30 basepairS) and contain varying degrees of complementarity to their target mRNA in the antisense strand. Some, but not all, siRNA have unpaired overhanging bases on the 5′ or 3′ end of the sense strand and/or the antisense strand. The term “siRNA” includes duplexes of two separate strands, as well as single strands that can form hairpin structures comprising a duplex region.
SiRNA may be divided into five (5) groups (non-functional, semi-functional, functional, highly functional, and hyper-functional) based on the level or degree of silencing that they induce in cultured cell lines. As used herein, these definitions are based on a set of conditions where the siRNA is transfected into said cell line at a concentration of 100 nM and the level of silencing is tested at a time of roughly 24 hours after transfection, and not exceeding 72 hours after transfection. In this context, “non-functional siRNA” are defined as those siRNA that induce less than 50% (<50%) target silencing. “Semi-functional siRNA” induce 50-79% target silencing. “Functional siRNA” are molecules that induce 80-95% gene silencing. “Highly-functional siRNA” are molecules that induce greater than 95% gene silencing. “Hyperfunctional siRNA” are a special class of molecules. For purposes of this document, hyperfunctional siRNA are defined as those molecules that: (1) induce greater than 95% silencing of a specific target when they are transfected at subnanomolar concentrations (i.e., less than one nanomolar); and/or (2) induce functional (or better) levels of silencing for greater than 96 hours. These relative functionalities (though not intended to be absolutes) may be used to compare siRNAs to a particular target for applications such as functional genomics, target identification and therapeutics.
miRNA
The term “miRNA” refers to microRNA.
Gene Silencing
The phrase “gene silencing” refers to a process by which the expression of a specific gene product is lessened or attenuated. Gene silencing can take place by a variety of pathways. Unless specified otherwise, as used herin, gene silencing refers to decreases in gene product expression that results from RNA interference (RNAi), a defined, though partially characterized pathway whereby small inhibitory RNA (siRNA) act in concert with host proteins (e.g. the RNA induced silencing complex, RISC) to degrade messenger RNA (mRNA) in a sequence-dependent fashion. The level of gene silencing can be measured by a variety of means, including, but not limited to, measurement of transcript levels by Northern Blot Analysis, B-DNA techniques, transcription-sensitive reporter constructs, expression profiling (e.g. DNA chips), and related technologies. Alternatively, the level of silencing can be measured by assessing the level of the protein encoded by a specific gene. This can be accomplished by performing a number of studies including Western Analysis, measuring the levels of expression of a reporter protein that has e.g. fluorescent properties (e.g. GFP) or enzymatic activity (e.g. alkaline phosphatases), or several other procedures.
Transfection
The term “transfection” refers to a process by which agents are introduced into a cell. The list of agents that can be transfected is large and includes, but is not limited to, siRNA, sense and/or anti-sense sequences, DNA encoding one or more genes and organized into an expression plasmid, proteins, protein fragments, and more. There are multiple methods for transfecting agents into a cell including, but not limited to, electroporation, calcium phosphate-based transfections, DEAE-dextran-based transfections, lipid-based transfections, molecular conjugate-based transfections (e.g. polylysine-DNA conjugates), microinjection and others.
Target
The term “target” is used in a variety of different forms throughout this document and is defined by the context in which it is used. “Target mRNA” refers to a messenger RNA to which a given siRNA can be directed against. “Target sequence” and “target site” refer to a sequence within the mRNA to which the sense strand of an siRNA shows varying degrees of homology and the antisense strand exhibits varying degrees of complementarity. The term “siRNA target” can refer to the gene, mRNA, or protein against which an siRNA is directed. Similarly “target silencing” can refer to the state of a gene, or the corresponding mRNA or protein.
Off-Target Silencing and Off-Target Interference
The phrases “off-target silencing” and “off-target interference” are defined as degradation of mRNA other than the intended target mRNA due to overlapping and/or partial homology with secondary mRNA messages.
SMARTscoreTM
The term “SMARTscoreTM” refers to a number determined by applying any of the Formulas I-Formula IX to a given siRNA sequence. The term “SMART-selected” or “rationally selected” or “rational selection” refers to siRNA that have been selected on the basis of their SMARTscoresTM.
Complementary
The term “complementary” refers to the ability of polynucleotides to form base pairs with one another. Base pairs are typically formed by hydrogen bonds between nucleotide units in antiparallel polynucleotide strands. Complementary polynucleotide strands can base pair in the Watson-Crick manner (e.g., A to T, A to U, C to G), or in any other manner that allows for the formation of duplexes. As persons skilled in the art are aware, when using RNA as opposed to DNA, uracil rather than thymine is the base that is considered to be complementary to adenosine. However, when a U is denoted in the context of the present invention, the ability to substitute a T is implied, unless otherwise stated.
Perfect complementarity or 100% complementarity refers to the situation in which each nucleotide unit of one polynucleotide strand can hydrogen bond with a nucleotide unit of a second polynucleotide strand. Less than perfect complementarity refers to the situation in which some, but not all, nucleotide units of two strands can hydrogen bond with each other. For example, for two 20-mers, if only two base pairs on each strand can hydrogen bond with each other, the polynucleotide strands exhibit 10% complementarity. In the same example, if 18 base pairs on each strand can hydrogen bond with each other, the polynucleotide strands exhibit 90% complementarity. “Substantial complementarity” refers to polynucleotide strands exhibiting 79% or greater complementarity, excluding regions of the polynucleotide strands, such as overhangs, that are selected so as to be noncomplementary. (“Substantial similarity” refers to polynucleotide strands exhibiting 79% or greater similarity, excluding regions of the polynucleotide strands, such as overhangs, that are selected so as not to be similar.) Thus, for example, two polynucleotides of 29 nucleotide units each, wherein each comprises a di-dT at the 3′ terminus such that the duplex region spans 27 bases, and wherein 26 of the 27 bases of the duplex region on each strand are complementary, are substantially complementary since they are 96.3% complementary when excluding the di-dT overhangs.
Deoxynucleotide
The term “deoxynucleotide” refers to a nucleotide or polynucleotide lacking a hydroxyl group (OH group) at the 2′ and/or 3′ position of a sugar moiety. Instead, it has a hydrogen bonded to the 2′ and/or 3′ carbon. Within an RNA molecule that comprises one or more deoxynucleotides, “deoxynucleotide” refers to the lack of an OH group at the 2′ position of the sugar moiety, having instead a hydrogen bonded directly to the 2′ carbon.
Deoxyribonucleotide
The terms “deoxyribonucleotide” and “DNA” refer to a nucleotide or polynucleotide comprising at least one sugar moiety that has an H, rather than an OH, at its 2′ and/or 3′position.
Substantially Similar
The phrase “substantially similar” refers to a similarity of at least 90% with respect to the identity of the bases of the sequence.
Duplex Region
The phrase “duplex region” refers to the region in two complementary or substantially complementary polynucleotides that form base pairs with one another, either by Watson-Crick base pairing or any other manner that allows for a stabilized duplex between polynucleotide strands that are complementary or substantially complementary. For example, a polynucleotide strand having 21 nucleotide units can base pair with another polynucleotide of 21 nucleotide units, yet only 19 bases on each strand are complementary or substantially complementary, such that the “duplex region” has 19 base pairs. The remaining bases may, for example, exist as 5′ and 3′ overhangs. Further, within the duplex region, 100% complementarity is not required; substantial complementarity is allowable within a duplex region. Substantial complementarity refers to 79% or greater complementarity. For example, a mismatch in a duplex region consisting of 19 base pairs results in 94.7% complementarity, rendering the duplex region substantially complementary.
Nucleotide
The term “nucleotide” refers to a ribonucleotide or a deoxyribonucleotide or modified form thereof, as well as an analog thereof. Nucleotides include species that comprise purines, e.g., adenine, hypoxanthine, guanine, and their derivatives and analogs, as well as pyrimidines, e.g., cytosine, uracil, thymine, and their derivatives and analogs.
Nucleotide analogs include nucleotides having modifications in the chemical structure of the base, sugar and/or phosphate, including, but not limited to, 5-position pyrimidine modifications, 8-position purine modifications, modifications at cytosine exocyclic amines, and substitution of 5-bromo-uracil; and 2′-position sugar modifications, including but not limited to, sugar-modified ribonucleotides in which the 2′-OH is replaced by a group such as an H, OR, R, halo, SH, SR, NH2, NHR, NR2, or CN, wherein R is an alkyl moiety. Nucleotide analogs are also meant to include nucleotides with bases such as inosine, queuosine, xanthine, sugars such as 2′-methyl ribose, non-natural phosphodiester linkages such as methylphosphonates, phosphorothioates and peptides.
Modified bases refer to nucleotide bases such as, for example, adenine, guanine, cytosine, thymine, uracil, xanthine, inosine, and queuosine that have been modified by the replacement or addition of one or more atoms or groups. Some examples of types of modifications that can comprise nucleotides that are modified with respect to the base moieties include but are not limited to, alkylated, halogenated, thiolated, aminated, amidated, or acetylated bases, individually or in combination. More specific examples include, for example, 5-propynyluridine, 5-propynylcytidine, 6-methyladenine, 6-methylguanine, N,N,-dimethyladenine, 2-propyladenine, 2-propylguanine, 2-aminoadenine, 1-methylinosine, 3-methyluridine, 5-methylcytidine, 5-methyluridine and other nucleotides having a modification at the 5 position, 5-(2-amino)propyl uridine, 5-halocytidine, 5-halouridine, 4-acetylcytidine, 1-methyladenosine, 2-methyladenosine, 3-methylcytidine, 6-methyluridine, 2-methylguanosine, 7-methylguanosine, 2,2-dimethylguanosine, 5-methylaminoethyluridine, 5-methyloxyuridine, deazanucleotides such as 7-deazaadenosine, 6-azouridine, 6-azocytidine, 6-azothymidine, 5-methyl-2-thiouridine, other thio bases such as 2-thiouridine and 4-thiouridine and 2-thiocytidine, dihydrouridine, pseudouridine, queuosine, archaeosine, naphthyl and substituted naphthyl groups, any O- and N-alkylated purines and pyrimidines such as N6-methyladenosine, 5-methylcarbonylmethyluridine, uridine 5-oxyacetic acid, pyridine-4-one, pyridine-2-one, phenyl and modified phenyl groups such as aminophenol or 2,4,6-trimethoxy benzene, modified cytosines that act as G-clamp nucleotides, 8-substituted adenines and guanines, 5-substituted uracils and thymines, azapyrimidines, carboxyhydroxyalkyl nucleotides, carboxyalkylaminoalkyl nucleotides, and alkylcarbonylalkylated nucleotides. Modified nucleotides also include those nucleotides that are modified with respect to the sugar moiety, as well as nucleotides having sugars or analogs thereof that are not ribosyl. For example, the sugar moieties may be, or be based on, mannoses, arabinoses, glucopyranoses, galactopyranoses, 4′-thioribose, and other sugars, heterocycles, or carbocycles.
The term nucleotide is also meant to include what are known in the art as universal bases. By way of example, universal bases include but are not limited to 3-nitropyrrole, 5-nitroindole, or nebularine. The term “nucleotide” is also meant to include the N3′ to P5′ phosphoramidate, resulting from the substitution of a ribosyl 3′ oxygen with an amine group.
Further, the term nucleotide also includes those species that have a detectable label, such as for example a radioactive or fluorescent moiety, or mass label attached to the nucleotide.
Polynucleotide
The term “polynucleotide” refers to polymers of nucleotides, and includes but is not limited to DNA, RNA, DNA/RNA hybrids including polynucleotide chains of regularly and/or irregularly alternating deoxyribosyl moieties and ribosyl moieties (i.e., wherein alternate nucleotide units have an OH, then and —H, then an OH, then an —H, and so on at the 2′ position of a sugar moiety), and modifications of these kinds of polynucleotides, wherein the attachment of various entities or moieties to the nucleotide units at any position are included.
Polyribonucleotide
The term “polyribonucleotide” refers to a polynucleotide comprising two or more modified or unmodified ribonucleotides and/or their analogs. The term “polyribonucleotide” is used interchangeably with the term “oligoribonucleotide.”
Ribonucleotide and Ribonucleic Acid
The term “ribonucleotide” and the phrase “ribonucleic acid” (RNA), refer to a modified or unmodified nucleotide or polynucleotide comprising at least one ribonucleotide unit. A ribonucleotide unit comprises an hydroxyl group attached to the 2′ position of a ribosyl moiety that has a nitrogenous base attached in N-glycosidic linkage at the 1′ position of a ribosyl moiety, and a moiety that either allows for linkage to another nucleotide or precludes linkage.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is directed to improving the efficiency of gene silencing by siRNA. Through the inclusion of multiple siRNA sequences that are targeted to a particular gene and/or selecting an siRNA sequence based on certain defined criteria, improved efficiency may be achieved.
The present invention will now be described in connection with preferred embodiments. These embodiments are presented in order to aid in an understanding of the present invention and are not intended, and should not be construed, to limit the invention in any way. All alternatives, modifications and equivalents that may become apparent to those of ordinary skill upon reading this disclosure are included within the spirit and scope of the present invention.
Furthermore, this disclosure is not a primer on RNA interference. Basic concepts known to persons skilled in the art have not been set forth in detail.
Optimizing siRNA
According to one embodiment, the present invention provides a method for improving the effectiveness of gene silencing for use to silence a particular gene through the selection of an optimal siRNA. An siRNA selected according to this method may be used individually, or in conjunction with the first embodiment, i.e., with one or more other siRNAs, each of which may or may not be selected by this criteria in order to maximize their efficiency.
The degree to which it is possible to select an siRNA for a given mRNA that maximizes these criteria will depend on the sequence of the mRNA itself. However, the selection criteria will be independent of the target sequence. According to this method, an siRNA is selected for a given gene by using a rational design. That said, rational design can be described in a variety of ways. Rational design is, in simplest terms, the application of a proven set of criteria that enhance the probability of identifying a functional or hyperfunctional siRNA. In one method, rationally designed siRNA can be identified by maximizing one or more of the following criteria:

    
    
        1. A low GC content, preferably between about 30-52%.
        2. At least 2, preferably at least 3 A or U bases at positions 15-19 of the siRNA on the sense strand.
        3. An A base at position 19 of the sense strand.
        4. An A base at position 3 of the sense strand.
        5. A U base at position 10 of the sense strand.
        6. An A base at position 14 of the sense strand.
        7. A base other than C at position 19 of the sense strand.
        8. A base other than G at position 13 of the sense strand.
        9. A Tm, which refers to the character of the internal repeat that results in inter- or intramolecular structures for one strand of the duplex, that is preferably not stable at greater than 50° C., more preferably not stable at greater than 37° C., even more preferably not stable at greater than 30° C. and most preferably not stable at greater than 20° C.
        10. A base other than U at position 5 of the sense strand.
        11. A base other than A at position 11 of the sense strand.
    
    


Criteria 5, 6, 10 and 11 are minor criteria, but are nonetheless desirable. Accordingly, preferably an siRNA will satisfy as many of the aforementioned criteria as possible, more preferably at least 1-4 and 7-9, and most preferably all of the criteria
With respect to the criteria, GC content, as well as a high number of AU in positions 15-19, may be important for easement of the unwinding of double stranded siRNA duplex. Duplex unwinding has been shown to be crucial for siRNA functionality in vivo.
With respect to criterion 9, the internal structure is measured in terms of the melting temperature of the single strand of siRNA, which is the temperature at which 50% of the molecules will become denatured. With respect to criteria 2-8 and 10-11, the positions refer to sequence positions on the sense strand, which is the strand that is identical to the mRNA.
In one preferred embodiment, at least criteria 1 and 8 are satisfied. In another preferred embodiment, at least criteria 7 and 8 are satisfied. In still another preferred embodiment, at least criteria 1, 8 and 9 are satisfied.
It should be noted that all of the aforementioned criteria regarding sequence position specifics are with respect to the 5′ end of the sense strand. Reference is made to the sense strand, because most databases contain information that describes the information of the mRNA. Because according to the present invention a chain can be from 18 to 30 bases in length, and the aforementioned criteria assumes a chain 19 base pairs in length, it is important to keep the aforementioned criteria applicable to the correct bases.
When there are only 18 bases, the base pair that is not present is the base pair that is located at the 3′ of the sense strand. When there are twenty to thirty bases present, then additional bases are added at the 5′ end of the sense chain and occupy positions −1 to −11. Accordingly, with respect to SEQ. ID NO. 0001. NNANANNNNUCNAANNNNA and SEQ. ID NO. 0028. GUCNNANANNNNUCNAANNNNA, both would have A at position 3, A at position 5, U at position 10, C at position 11, A and position 13, A and position 14 and A at position 19. However, SEQ. ID NO. 0028 would also have C at position −1, U at position −2 and G at position −3.
For a 19 base pair siRNA, an optimal sequence of one of the strands may be represented below, where N is any base, A, C, G, or U:










SEQ. ID NO. 0001.
NNANANNNNUCNAANNNNA


 



SEQ. ID NO. 0002.
NNANANNNNUGNAANNNNA


 



SEQ. ID NO. 0003.
NNANANNNNUUNAANNNNA


 



SEQ. ID NO. 0004.
NNANANNNNUCNCANNNNA


 



SEQ. ID NO. 0005.
NNANANNNNUGNCANNNNA


 



SEQ. ID NO. 0006.
NNANANNNNUUNCANNNNA


 



SEQ. ID NO. 0007.
NNANANNNNUCNUANNNNA


 



SEQ. ID NO. 0008..
NNANANNNNUGNUANNNNA


 



SEQ. ID NO. 0009.
NNANANNNNUUNUANNNNA


 



SEQ. ID NO. 0010.
NNANCNNNNUCNAANNNNA


 



SEQ. ID NO. 0011.
NNANCNNNNUGNAANNNNA


 



SEQ. ID NO. 0012.
NNANCNNNNUUNAANNNNA


 



SEQ. ID NO. 0013.
NNANCNNNNUCNCANNNNA


 



SEQ. ID NO. 0014.
NNANCNNNNUGNCANNNNA


 



SEQ. ID NO. 0015.
NNANCNNNNUUNCANNNNA


 



SEQ. ID NO. 0016.
NNANCNNNNUCNUANNNNA


 



SEQ. ID NO. 0017.
NNANCNNNNUGNUANNNNA


 



SEQ. ID NO. 0018.
NNANCNNNNUUNUANNNNA


 



SEQ. ID NO. 0019.
NNANGNNNNUCNAANNNNA


 



SEQ. ID NO. 0020.
NNANGNNNNUGNAANNNNA


 



SEQ. ID NO. 0021.
NNANGNNNNUUNAANNNNA


 



SEQ. ID NO. 0022.
NNANGNNNNUCNCANNNNA


 



SEQ. ID NO. 0023.
NNANGNNNNUGNCANNNNA


 



SEQ. ID NO. 0024.
NNANGNNNNUUNCANNNNA


 



SEQ. ID NO. 0025.
NNANGNNNNUCNUANNNNA


 



SEQ. ID NO. 0026.
NNANGNNNNUGNUANNNNA


 



SEQ. ID NO. 0027.
NNANGNNNNNUNUANNNNA






In one embodiment, the sequence used as an siRNA is selected by choosing the siRNA that score highest according to one of the following seven algorithms that are represented by Formulas I-VII:

Relative functionality of siRNA=−(GC/3)+(AU15-19)−(Tm20° C.)*3−(G13)*3−(C19)+(A19)*2+(A3)+(U10)+(A14)−(U5)−(A11)  Formula I

Relative functionality of siRNA=−(GC/3)−(AU15-19)*3−(G13)*3−(C19)+(A19)*2+(A3)  Formula II

Relative functionality of siRNA=−(GC/3)+(AU15-19)−(Tm20° C.)*3  Formula III

Relative functionality of siRNA=−GC/2+(AU15-19)/2−(Tm20° C.)*2−(G13)*3−(C19)+(A9)*2+(A3)+(U10)+(A14)−(U5)−(A11)  Formula IV

Relative functionality of siRNA=−(G13)*3−(C19)+(A19)*2+(A3)+(U10)+(A14)−(U5)−(A11)  Formula V

Relative functionality of siRNA=−(G13)*3−(C19)+(A19)*2+(A3)  Formula VI

Relative functionality of siRNA=−(GC/2)+(AU15-19)/2−(Tm20° C.)*1−(G13)*3−(C19)+(A19)*3+(A3)*3+(U10)/2+(A14)/2−(U5)/2−(A11)/2  Formula VII

In Formulas I-VII:

wherein A19=1 if A is the base at position 19 on the sense strand, otherwise its value is 0,

    
    
        AU15-19=0-5 depending on the number of A or U bases on the sense strand at positions 15-19;
        G13=1 if G is the base at position 13 on the sense strand, otherwise its value is 0;
        C19=1 if C is the base at position 19 of the sense strand, otherwise its value is 0;
        GC=the number of G and C bases in the entire sense strand;
        Tm20° C.=1 if the Tm is greater than 20° C.;
        A3=1 if A is the base at position 3 on the sense strand, otherwise its value is 0;
        U10==1 if U is the base at position 10 on the sense strand, otherwise its value is 0;
        A14=1 if A is the base at position 14 on the sense strand, otherwise its value is 0;
        U5=1 if U is the base at position 5 on the sense strand, otherwise its value is 0; and
        A11=1 if A is the base at position 11 of the sense strand, otherwise its value is 0.
    
    


Formulas I-VII provide relative information regarding functionality. When the values for two sequences are compared for a given formula, the relative functionality is ascertained; a higher positive number indicates a greater functionality. For example, in many applications a value of 5 or greater is beneficial.
Additionally, in many applications, more than one of these formulas would provide useful information as to the relative functionality of potential siRNA sequences. However, it is beneficial to have more than one type of formula, because not every formula will be able to help to differentiate among potential siRNA sequences. For example, in particularly high GC mRNAs, formulas that take that parameter into account would not be useful and application of formulas that lack GC elements (e.g., formulas V and VI) might provide greater insights into duplex functionality. Similarly, formula II might by used in situations where hairpin structures are not observed in duplexes, and formula IV might be applicable for sequences that have higher AU content. Thus, one may consider a particular sequence in light of more than one or even all of these algorithms to obtain the best differentiation among sequences. In some instances, application of a given algorithim may identify an unususally large number of potential siRNA sequences, and in those cases, it may be appropriate to re-analyze that sequence with a second algorithm that is, for instance, more stringent. Alternatively, it is conceivable that analysis of a sequence with a given formula yields no acceptable siRNA sequences (i.e. low SMARTscoresTM). In this instance, it may be appropriate to re-analyze that sequences with a second algorithm that is, for instance, less stringent. In still other instances, analysis of a single sequence with two separate formulas may give rise to conflicting results (i.e. one formula generates a set of siRNA with high SMARTscoresTM while the other formula identifies a set of siRNA with low SMARTscoresTM). In these instances, it may be necessary to determine which weighted factor(s) (e.g. GC content) are contributing to the discrepancy and assessing the sequence to decide whether these factors should or should not be included. Alternatively, the sequence could be analyzed by a third, fourth, or fifth algorithm to identify a set of rationally designed siRNA.
The above-referenced criteria are particularly advantageous when used in combination with pooling techniques as depicted in Table I:










TABLE I









Functional Probability











Oligos
Pools















Criteria
>95%
>80%
<70%
>95%
>80%
<70%



















Current
33.0
50.0
23.0
79.5
97.3
0.3



New
50.0
88.5
8.0
93.8
99.98
0.005



(GC)
28.0
58.9
36.0
72.8
97.1
1.6










The term “current” refers to Tuschl's conventional siRNA parameters (Elbashir, S. M. et al. (2002) “Analysis of gene function in somatic mammalian cells using small interfering RNAs” Methods 26: 199-213). “New” refers to the design parameters described in Formulas I-VII. “GC” refers to criteria that select siRNA solely on the basis of GC content.

As Table I indicates, when more functional siRNA duplexes are chosen, siRNAs that produce <70% silencing drops from 23% to 8% and the number of siRNA duplexes that produce >80% silencing rises from 50% to 88.5%. Further, of the siRNA duplexes with >80% silencing, a larger portion of these siRNAs actually silence >95% of the target expression (the new criteria increases the portion from 33% to 50%). Using this new criteria in pooled siRNAs, shows that, with pooling, the amount of silencing >95% increases from 79.5% to 93.8% and essentially eliminates any siRNA pool from silencing less than 70%.
Table II similarly shows the particularly beneficial results of pooling in combination with the aforementioned criteria. However, Table II, which takes into account each of the aforementioned variables, demonstrates even a greater degree of improvement in functionality.









TABLE II









Functional Probability










Oligos
Pools














Func-

Non-
Func-

Non-



tional
Average
functional
tional
Average
functional

















Random
20
40
50
67
97
3


Criteria 1
52
99
0.1
97
93
0.0040


Criteria 4
89
99
0.1
99
99
0.0000









The terms “functional,” “Average,” and “Non-functional” refer to siRNA that exhibit >80%, >50%, and <50% functionality, respectively. Criteria 1 and 4 refer to specific criteria described above.

The above-described algorithms may be used with or without a computer program that allows for the inputting of the sequence of the mRNA and automatically outputs the optimal siRNA. The computer program may, for example, be accessible from a local terminal or personal computer, over an internal network or over the Internet.
In addition to the formulas above, more detailed algorithms may be used for selecting siRNA. Preferably, at least one RNA duplex of between 18 and 30 base pairs is selected such that it is optimized according a formula selected from:

Relative functionality of siRNA=(−14)*G13−13*A1−12*U7−11*U2−10*A11−10*U4−10*C3−10*C5−10*C6−9*A10−9*U9−9*C18−8*G10−7*U1−7*U16−7*C17−7*C19+7*U17+8*A2+8*A4+8*A5+8*C4+9*G8+10*A7+10*U18+11*A19+11*C9+15*G1+18*A3+19*U10−Tm−3*(GCtotal)−6*(GC15-19)−30*X; and  Formula VIII:

Relative functionality of siRNA=(14.1)*A3+(14.9)*A6+(17.6)*A13+(24.7)*A19+(14.2)*U10+(10.5)*C9+(23.9)*G1+(16.3)*G2+(−12.3)*A11+(−19.3)*U1+(−12.1)*U2+(−11)*U3+(−15.2)*U15+(−11.3)*U16+(−11.8)*C3+(−17.4)*C6+(−10.5)*C7+(−13.7)*G13+(−25.9)*G19−Tm−3*(GCtotal)−6*(GC15-19)−30*X  Formula IX:

wherein

    
    
        A1=1 if A is the base at position 1 of the sense strand, otherwise its value is 0;
        A2=1 if A is the base at position 2 of the sense strand, otherwise its value is 0;
        A3=1 if A is the base at position 3 of the sense strand, otherwise its value is 0;
        A4=1 if A is the base at position 4 of the sense strand, otherwise its value is 0;
        A5=1 if A is the base at position 5 of the sense strand, otherwise its value is 0;
        A6=1 if A is the base at position 6 of the sense strand, otherwise its value is 0;
        A7=1 if A is the base at position 7 of the sense strand, otherwise its value is 0;
        A10=1 if A is the base at position 10 of the sense strand, otherwise its value is 0;
        A11=1 if A is the base at position 11 of the sense strand, otherwise its value is 0;
        A13=1 if A is the base at position 13 of the sense strand, otherwise its value is 0;
        A19=1 if A is the base at position 19 of the sense strand, otherwise if another base is present or the sense strand is only 18 base pairs in length, its value is 0;
        C3=1 if C is the base at position 3 of the sense strand, otherwise its value is 0;
        C4=1 if C is the base at position 4 of the sense strand, otherwise its value is 0;
        C5=1 if C is the base at position 5 of the sense strand, otherwise its value is 0;
        C6=1 if C is the base at position 6 of the sense strand, otherwise its value is 0;
        C7=1 if C is the base at position 7 of the sense strand, otherwise its value is 0;
        C9=1 if C is the base at position 9 of the sense strand, otherwise its value is 0;
        C17=1 if C is the base at position 17 of the sense strand, otherwise its value is 0;
        C18=1 if C is the base at position 18 of the sense strand, otherwise its value is 0;
        C19=1 if C is the base at position 19 of the sense strand, otherwise if another base is present or the sense strand is only 18 base pairs in length, its value is 0;
        G1=1 if G is the base at position 1 on the sense strand, otherwise its value is 0;
        G2=1 if G is the base at position 2 of the sense strand, otherwise its value is 0;
        G8=1 if G is the base at position 8 on the sense strand, otherwise its value is 0;
        G10=1 if G is the base at position 10 on the sense strand, otherwise its value is 0;
        G13=1 if G is the base at position 13 on the sense strand, otherwise its value is 0;
        G19=1 if G is the base at position 19 of the sense strand, otherwise if another base is present or the sense strand is only 18 base pairs in length, its value is 0;
        U1=1 if U is the base at position 1 on the sense strand, otherwise its value is 0;
        U2=1 if U is the base at position 2 on the sense strand, otherwise its value is 0;
        U3=1 if U is the base at position 3 on the sense strand, otherwise its value is 0;
        U4=1 if U is the base at position 4 on the sense strand, otherwise its value is 0;
        U7=1 if U is the base at position 7 on the sense strand, otherwise its value is 0;
        U9=1 if U is the base at position 9 on the sense strand, otherwise its value is 0;
        U10=1 if U is the base at position 10 on the sense strand, otherwise its value is 0;
        U15=1 if U is the base at position 15 on the sense strand, otherwise its value is 0;
        U16=1 if U is the base at position 16 on the sense strand, otherwise its value is 0;
        U17=1 if U is the base at position 17 on the sense strand, otherwise its value is 0;
        U18=1 if U is the base at position 18 on the sense strand, otherwise its value is 0;
        GC15-19=the number of G and C bases within positions 15-19 of the sense strand, or within positions 15-18 if the sense strand is only 18 base pairs in length;
        GCtotal=the number of G and C bases in the sense strand;
        Tm=100 if the siRNA oligo has the internal repeat longer then 4 base pairs, otherwise its value is 0; and
        X=the number of times that the same nucleotide repeats four or more times in a row.
    
    


The above formulas VIII and IX, as well as formulas I-VII, provide methods for selecting siRNA in order to increase the efficiency of gene silencing. A subset of variables of any of the formulas may be used, though when fewer variables are used, the optimization hierarchy becomes less reliable.
With respect to the variables of the above-referenced formulas, a single letter of A or C or G or U followed by a subscript refers to a binary condition. The binary condition is that either the particular base is present at that particular position (wherein the value is “1”) or the base is not present (wherein the value is “0”). Because position 19 is optional, i.e. there might be only 18 base pairs, when there are only 18 base pairs, any base with a subscript of 19 in the formulas above would have a zero value for that parameter. Before or after each variable is a number followed by *, which indicates that the value of the variable is to be multiplied or weighed by that number.
The numbers preceding the variables A, or G, or C, or U in Formulas VIII and IX (or after the variables in Formula I-VII) were determined by comparing the difference in the frequency of individual bases at different positions in functional siRNA and total siRNA. Specifically, the frequency in which a given base was observed at a particular position in functional groups was compared with the frequency that that same base was observed in the total, randomly selected siRNA set. If the absolute value of the difference between the functional and total values was found to be greater than 6%, that parameter was included in the equation. Thus for instance, if the frequency of finding a “G” at position 13 (G13) is found to be 6% in a given functional group, and the frequency of G 3 in the total population of siRNAs is 20%, the difference between the two values is 6%−20%=−14%. As the absolute value is greater than six (6), this factor (−14) is included in the equation. Thus in Formula VIII, in cases where the siRNA under study has a G in position 13, the accrued value is (−14)*(1)=−14. In contrast, when a base other than G is found at position 13, the accrued value is (−14)*(0)=0.
When developing a means to optimize siRNAs, the inventors observed that a bias toward low internal thermodynamic stability of the duplex at the 5′-antisense (AS) end is characteristic of naturally occurring miRNA precursors. The inventors extended this observation to siRNAs for which functionality had been assessed in tissue culture.
With respect to the parameter GC15-19, a value of 0-5 will be ascribed depending on the number of G or C bases at positions 15 to 19. If there are only 18 base pairs, the value is between 0 and 4.
With respect to the criterion GCtotal content, a number from 0-30 will be ascribed, which correlates to the total number of G and C nucleotides on the sense strand, excluding overhangs. Without wishing to be bound by any one theory, it is postulated that the significance of the GC content (as well as AU content at positions 15-19, which is a parameter for formulas III-VII) relates to the easement of the unwinding of a double-stranded siRNA duplex. Duplex unwinding is believed to be crucial for siRNA functionality in vivo and overall low internal stability, especially low internal stability of the first unwound base pair is believed to be important to maintain sufficient processivity of RISC complex-induced duplex unwinding. If the duplex has 19 base pairs, those at positions 15-19 on the sense strand will unwind first if the molecule exhibits a sufficiently low internal stability at that position. As persons skilled in the art are aware, RISC is a complex of approximately twelve proteins; Dicer is one, but not the only, helicase within this complex. Accordingly, although the GC parameters are believed to relate to activity with Dicer, they are also important for activity with other RISC proteins.
The value of the parameter Tm is 0 when there are no internal repeats longer than (or equal to) four base pairs present in the siRNA duplex; otherwise the value is 1. Thus for example, if the sequence ACGUACGU, or any other four nucleotide (or more) palindrome exists within the structure, the value will be one (1). Alternatively if the structure ACGGACG, or any other 3 nucleotide (or less) palindrome exists, the value will be zero (0).
The variable “X” refers to the number of times that the same nucleotide occurs contiguously in a stretch of four or more units. If there are, for example, four contiguous As in one part of the sequence and elsewhere in the sequence four contiguous Cs, X=2. Further, if there are two separate contiguous stretches of four of the same nucleotides or eight or more of the same nucleotides in a row, then X=2. However, X does not increase for five, six or seven contiguous nucleotides.
Again, when applying Formula VIII or Formula IX to a given mRNA, (the “target RNA” or “target molecule”), one may use a computer program to evaluate the criteria for every sequence of 18-30 base pairs or only sequences of a fixed length, e.g., 19 base pairs. Preferably the computer program is designed such that it provides a report ranking of all of the potential siRNAs between 18 and 30 base pairs, ranked according to which sequences generate the highest value. A higher value refers to a more efficient siRNA for a particular target gene. The computer program that may be used, may be developed in any computer language that is known to be useful for scoring nucleotide sequences, or it may be developed with the assistance of commercially available product such as Microsoft's product net. Additionally, rather than run every sequence through one and/or another formula, one may compare a subset of the sequences, which may be desirable if for example only a subset are available. For instance, it may be desirable to first perform a BLAST (Basic Local Alignment Search Tool) search and to identify sequences that have no homology to other targets. Alternatively, it may be desirable to scan the sequence and to identify regions of moderate GC context, then perform relevant calculations using one of the above-described formulas on these regions. These calculations can be done manually or with the aid of a computer.
As with Formulas I-VII, either Formula VIII or Formula IX may be used for a given mRNA target sequence. However, it is possible that according to one or the other formula more than one siRNA will have the same value. Accordingly, it is beneficial to have a second formula by which to differentiate sequences. Formula IX was derived in a similar fashion as Formula VIII, yet used a larger data set and thus yields sequences with higher statistical correlations to highly functional duplexes. The sequence that has the highest value ascribed to it may be referred to as a “first optimized duplex.” The sequence that has the second highest value ascribed to it may be referred to as a “second optimized duplex.” Similarly, the sequences that have the third and fourth highest values ascribed to them may be referred to as a third optimized duplex and a fourth optimized duplex, respectively. When more than one sequence has the same value, each of them may, for example, be referred to as first optimized duplex sequences or co-first optimized duplexes.
SiRNA sequences identified using Formula VIII are contained within the enclosed compact disks. The data included on the enclosed compact disks is described more fully below. The sequences identified by Formula VIII that are disclosed in the compacts disks may be used in gene silencing applications.
It should be noted that for Formulas VIII and IX all of the aforementioned criteria are identified as positions on the sense strand when oriented in the 5′ to 3′ direction as they are identified in connection with Formulas I-VII unless otherwise specified.
Formulas I-IX, may be used to select or to evaluate one, or more than one, siRNA in order to optimize silencing. Preferably, at least two optimized siRNAs that have been selected according to at least one of these formulas are used to silence a gene, more preferably at least three and most preferably at least four. The siRNAs may be used individually or together in a pool or kit. Further, they may be applied to a cell simultaneously or separately. Preferably, the at least two siRNAs are applied simultaneously. Pools are particularly beneficial for many research applications. However, for therapeutics, it may be more desirable to employ a single hyperfunctional siRNA as described elsewhere in this application.
When planning to conduct gene silencing, and it is necessary to choose between two or more siRNAs, one should do so by comparing the relative values when the siRNA are subjected to one of the formulas above. In general a higher scored siRNA should be used.
Useful applications include, but are not limited to, target validation, gene functional analysis, research and drug discovery, gene therapy and therapeutics. Methods for using siRNA in these applications are well known to persons of skill in the art.
Because the ability of siRNA to function is dependent on the sequence of the RNA and not the species into which it is introduced, the present invention is applicable across a broad range of species, including but not limited to all mammalian species, such as humans, dogs, horses, cats, cows, mice, hamsters, chimpanzees and gorillas, as well as other species and organisms such as bacteria, viruses, insects, plants and C. elegans. 
The present invention is also applicable for use for silencing a broad range of genes, including but not limited to the roughly 45,000 genes of a human genome, and has particular relevance in cases where those genes are associated with diseases such as diabetes, Alzheimer's, cancer, as well as all genes in the genomes of the aforementioned organisms.
The siRNA selected according to the aforementioned criteria or one of the aforementioned algorithms are also, for example, useful in the simultaneous screening and functional analysis of multiple genes and gene families using high throughput strategies, as well as in direct gene suppression or silencing.
Development of the Algorithms
To identify siRNA sequence features that promote functionality and to quantify the importance of certain currently accepted conventional factors—such as G/C content and target site accessibility—the inventors synthesized an siRNA panel consisting of 270 siRNAs targeting three genes, Human Cyclophilin, Firefly Luciferase, and Human DBI. In all three cases, siRNAs were directed against specific regions of each gene. For Human Cyclophilin and Firefly Luciferase, ninety siRNAs were directed against a 199 bp segment of each respective mRNA. For DBI, 90 siRNAs were directed against a smaller, 109 base pair region of the mRNA. The sequences to which the siRNAs were directed are provided below.
It should be noted that in certain sequences, “t” is present. This is because many databases contain information in this manner. However, the t denotes a uracil residue in mRNA and siRNA. Any algorithm will, unless otherwise specified, process a t in a sequence as a u.
Human Cyclophilin: 193-390, M60857







SEQ. ID NO. 29:


gttccaaaaacagtggataattttgtggccttagctacaggagagaaagg


 


atttggctacaaaaacagcaaattccatcgtgtaatcaaggacttcatga


 


tccagggcggagacttcaccaggggagatggcacaggaggaaagagcatc


 


tacggtgagcgcttccccgatgagaacttcaaactgaagcactacgggcc


 


tggctggg






Firefly Luciferase: 1434-1631, U47298 (pGL3, Promega)








SEQ. ID NO. 30:


tgaacttcccgccgccgttgttgttttggagcacggaaagacgatgacgg


 


aaaaagagatcgtggattacgtcgccagtcaagtaacaaccgcgaaaaag


 


ttgcgcggaggagttgtgtttgtggacgaagtaccgaaaggtcttaccgg


 


aaaactcgacgcaagaaaaatcagagagatcctcataaaggccaagaagg






DBI, NM—020548 (202-310) (every position)







SEQ. ID NO. 0031:


acgggcaaggccaagtgggatgcctggaatgagctgaaagggacttccaa


 


ggaagatgccatgaaagduacatcaacaaagtagaagagctaaagaaaaa


 


atacggg






A list of the siRNAs appears in Table IV (see Examples Section, Example I)

The set of duplexes was analyzed to identify correlations between siRNA functionality and other biophysical or thermodynamic properties. When the siRNA panel was analyzed in functional and non-functional subgroups, certain nucleotides were much more abundant at certain positions in functional or non-functional groups. More specifically, the frequency of each nucleotide at each position in highly functional siRNA duplexes was compared with that of nonfunctional duplexes in order to assess the preference for or against any given nucleotide at every position. These analyses were used to determine important criteria to be included in the siRNA algorithms (Formulas VIII and IX).
The data set was also analyzed for distinguishing biophysical properties of siRNAs in the functional group, such as optimal percent of GC content, propensity for internal structures and regional thermodynamic stability. Of the presented criteria, several are involved in duplex recognition, RISC activation/duplex unwinding, and target cleavage catalysis.
The original data set that was the source of the statistically derived criteria is shown in FIG. 2. Additionally, this figure shows that random selection yields siRNA duplexes with unpredictable and widely varying silencing potencies as measured in tissue culture using HEK293 cells. In the figure, duplexes are plotted such that each x-axis tick-mark represents an individual siRNA, with each subsequent siRNA differing in target position by two nucleotides for Human Cyclophilin and Firefly Luciferase, and by one nucleotide for Human DBI. Furthermore, the y-axis denotes the level of target expression remaining after transfection of the duplex into cells and subsequent silencing of the target.
SiRNA identified and optimized in this document work equally well in a wide range of cell types. FIG. 3a shows the evaluation of thirty siRNAs targeting the DBI gene in three cell lines derived from different tissues. Each DBI siRNA displays very similar functionality in HEK293 (ATCC, CRL-1573, human embryonic kidney), HeLa (ATCC, CCL-2, cervical epithelial adenocarcinoma) and DU145 (HTB-81, prostate) cells as deterimined by the B-DNA assay. Thus, siRNA functionality is determined by the primary sequence of the siRNA and not by the intracellular environment. Additionally, it should be noted that although the present invention provides for a determination of the functionality of siRNA for a given target, the same siRNA may silence more than one gene. For example, the complementary sequence of the silencing siRNA may be present in more than one gene. Accordingly, in these circumstances, it may be desirable not to use the siRNA with highest SMARTscoreTM. In such circumstances, it may be desirable to use the siRNA with the next highest SMARTscoreTM.
To determine the relevance of G/C content in siRNA function, the G/C content of each duplex in the panel was calculated and the functional classes of siRNAs (<F50, ≧F50, ≧F80, ≧F95 where F refers to the percent gene silencing) were sorted accordingly. The majority of the highly-functional siRNAs (≧F95) fell within the G/C content range of 36%-52% (FIG. 3B). Twice as many non-functional (<F50) duplexes fell within the high 6/C content groups (>57% GC content) compared to the 36%-52% group. The group with extremely low GC content (26% or less) contained a higher proportion of non-functional siRNAs and no highly-functional siRNAs. The G/C content range of 30%-52% was therefore selected as Criterion I for siRNA functionality, consistent with the observation that a G/C range 30%-70% promotes efficient RNAi targeting. Application of this criterion alone provided only a marginal increase in the probability of selecting functional siRNAs from the panel: selection of F50 and F95 siRNAs was improved by 3.6% and 2.2%, respectively. The siRNA panel presented here permitted a more systematic analysis and quantification of the importance of this criterion than that used previously.
A relative measure of local internal stability is the A/U base pair (bp) content; therefore, the frequency of A/U bp was determined for each of the five terminal positions of the duplex (5′ sense (S)/5′ antisense (AS)) of all siRNAs in the panel. Duplexes were then categorized by the number of A/U bp in positions 1-5 and 15-19 of the sense strand. The thermodynamic flexibility of the duplex 5′-end (positions 1-5; S) did not appear to correlate appreciably with silencing potency, while that of the 3′-end (positions 15-19; S) correlated with efficient silencing. No duplexes lacking A/U bp in positions 15-19 were functional. The presence of one A/U bp in this region conferred some degree of functionality, but the presence of three or more A/Us was preferable and therefore defined as Criterion 11. When applied to the test panel, only a marginal increase in the probability of functional siRNA selection was achieved: a 1.8% and 2.3% increase for F50 and F95 duplexes, respectively (Table III).
The complementary strands of siRNAs that contain internal repeats or palindromes may form internal fold-back structures. These hairpin-like structures exist in equilibrium with the duplexed form effectively reducing the concentration of functional duplexes. The propensity to form internal hairpins and their relative stability can be estimated by predicted melting temperatures. High Tm reflects a tendency to form hairpin structures. Lower Tm values indicate a lesser tendency to form hairpins. When the functional classes of siRNAs were sorted by Tm (FIG. 3c), the following trends were identified: duplexes lacking stable internal repeats were the most potent silencers (no F95 duplex with predicted hairpin structure Tm>60° C.). In contrast, about 60% of the duplexes in the groups having internal hairpins with calculated Tm, values less than 20° C. were F80. Thus, the stability of internal repeats is inversely proportional to the silencing effect and defines Criterion III (predicted hairpin structure Tm≦200C).
Sequence-Based Determinants of SiRNA Functionality
When the siRNA panel was sorted into functional and non-functional groups, the frequency of a specific nucleotide at each position in a functional siRNA duplex was compared with that of a nonfunctional duplex in order to assess the preference for or against a certain nucleotide. FIG. 4 shows the results of these queries and the subsequent resorting of the data set (from FIG. 2). The data is separated into two sets: those duplexes that meet the criteria, a specific nucleotide in a certain position—grouped on the left (Selected) and those that do not—grouped on the right (Eliminated). The duplexes are further sorted from most functional to least functional with the y-axis of FIG. 4a-e representing the % expression i.e. the amount of silencing that is elicited by the duplex (Note: each position on the X-axis represents a different duplex). Statistical analysis revealed correlations between silencing and several sequence-related properties of siRNAs. FIG. 4 and Table III show quantitative analysis for the following five sequence-related properties of siRNA: (A) an A at position 19 of the sense strand; (B) an A at position 3 of the sense strand; (C) a U at position 10 of the sense strand; (D) a base other than G at position 13 of the sense strand; and (E) a base other than C at position 19 of the sense strand.
When the siRNAs in the panel were evaluated for the presence of an A at position 19 of the sense strand, the percentage of non-functional duplexes decreased from 20% to 11.8%, and the percentage of F95 duplexes increased from 21.7% to 29.4% (Table III). Thus, the presence of an A in this position defined Criterion IV.
Another sequence-related property correlated with silencing was the presence of an A in position 3 of the sense strand (FIG. 4b). Of the siRNAs with A3, 34.4% were F95, compared with 21.7% randomly selected siRNAs. The presence of a U base in position 10 of the sense strand exhibited an even greater impact (FIG. 4c). Of the duplexes in this group, 41.7% were F95. These properties became criteria V and VI, respectively.
Two negative sequence-related criteria that were identified also appear on FIG. 4. The absence of a G at position 13 of the sense strand, conferred a marginal increase in selecting functional duplexes (FIG. 4d). Similarly, lack of a C at position 19 of the sense strand also correlated with functionality (FIG. 4e). Thus, among functional duplexes, position 19 was most likely occupied by A, and rarely occupied by C. These rules were defined as criteria VII and VIII, respectively.
Application of each criterion individually provided marginal but statistically significant increases in the probability of selecting a potent siRNA. Although the results were informative, the inventors sought to maximize potency and therefore consider multiple criteria or parameters. Optimization is particularly important when developing therapeutics. Interestingly, the probability of selecting a functional siRNA based on each thermodynamic criteria was 2%-4% higher than random, but 4%-8% higher for the sequence-related determinates. Presumably, these sequence-related increases reflect the complexity of the RNAi mechanism and the multitude of protein-RNA interactions that are involved in RNAi-mediated silencing.









TABLE III







Improvement


Criterion
% Functional
over Random



















I.
30%-52% G/C content
<F50
16.4%
−3.6%  




≧F50
83.6%
3.6%




≧F80
60.4%
4.3%




≧F95
23.9%
2.2%


II.
At least 3 A/U bases
<F50
18.2%
−1.8%  



at positions 15-19
≧F50
81.8%
1.8%



of the sense strand
≧F80
59.7%
3.6%




≧F95
24.0%
2.3%


III.
Absence of internal
<F50
16.7%
−3.3%  



repeats, as measured
≧F50
83.3%
3.3%



by Tm of secondary
≧F80
61.1%
5.0%



structure ≦ 20° C.
≧F95
24.6%
2.9%


IV.
An A base at position 19
<F50
11.8%
−8.2%  



of the sense strand
≧F50
88.2%
8.2%




≧F80
75.0%
18.9% 




≧F95
29.4%
7.7%


V.
An A base at position 3
<F50
17.2%
−2.8%  



of the sense strand
≧F50
82.8%
2.8%




≧F80
62.5%
6.4%




≧F95
34.4%
12.7% 


VI.
A U base at position 10
<F50
13.9%
−6.1%  



of the sense strand
≧F50
86.1%
6.1%




≧F80
69.4%
13.3% 




≧F95
41.7%
 20%


VII.
A base other than C at
<F50
18.8%
−1.2%  



position 19 of the
≧F50
81.2%
1.2%



sense strand
≧F80
59.7%
3.6%




≧F95
24.2%
2.5%


VIII.
A base other than G at
<F50
15.2%
−4.8%  



position 13 of the
≧F50
84.8%
4.8%



sense strand
≧F80
61.4%
5.3%




≧F95
26.5%
4.8%









The siRNA Selection Algorithm

In an effort to improve selection further, all identified criteria, including but not limited to those listed in Table III were combined into the algorithms embodied in Formula VIII and Formula IX. Each siRNA was then assigned a score (referred to as a SMARTscoreTM) according to the values derived from the formulas. Duplexes that scored higher than 0 or 20, for Formulas VIII and IX, respectively, effectively selected a set of functional siRNAs and excluded all non-functional siRNAs. Conversely, all duplexes scoring lower than 0 and 20 according to formulas VIII and IX, respectively, contained some functional siRNAs but included all non-functional siRNAs. A graphical representation of this selection is shown in FIG. 5.
The methods for obtaining the seven criteria embodied in Table III are illustrative of the results of the process used to develop the information for Formulas VIII and IX. Thus similar techniques were used to establish the other variables and their multipliers. As described above, basic statistical methods were use to determine the relative values for these multipliers.
To determine the value for “Improvement over Random” the difference in the frequency of a given attribute (e.g. GC content, base preference) at a particular position is determined between individual functional groups (e.g. <F50) and the total siRNA population studied (e.g. 270 siRNA molecules selected randomly). Thus, for instance, in Criterion 1 (30%-52% GC content) members of the <F50 group were observed to have GC contents between 30-52% in 16.4% of the cases. In contrast, the total group of 270 siRNAs had GC contents in this range, 20% of the time. Thus for this particular attribute, there is a small negative correlation between 30%-52% GC content and this functional group (i.e. 16.4%-20%=−3.6%). Similarly, for Criterion VI, (a “U” at position 10 of the sense strand), the >F95 group contained a “U” at this position 41.7% of the time. In contrast, the total group of 270 siRNAs had a “U” at this position 21.7% of the time, thus the improvement over random is calculated to be 20% (or 41.7%-21.7%).
Identifying the Average Internal Stability Profile of Strong siRNA
In order to identify an internal stability profile that is characteristic of strong siRNA, 270 different siRNAs derived from the cyclophilin B, the diazepam binding inhibitor (DBI), and the luciferase gene were individually transfected into HEK293 cells and tested for their ability to induce RNAi of the respective gene. Based on their performance in the in vivo assay, the sequences were then subdivided into three groups, (i) >95% silencing; (ii) 80-95% silencing; and (iii) less than 50% silencing. Sequences exhibiting 51-84% silencing were eliminated from further consideration to reduce the difficulties in identifying relevant thermodynamic patterns.
Following the division of siRNA into three groups, a statistical analysis was performed on each member of each group to determine the average internal stability profile (AISP) of the siRNA. To accomplish this the Oligo 5.0 Primer Analysis Software and other related statistical packages (e.g. Excel) were exploited to determine the internal stability of pentamers using the nearest neighbor method described by Freier et al., (1986) Improved free-energy parameters for predictions of RNA duplex stability, Proc Natl. Acad. Sci. U.S.A. 83(24): 9373-7. Values for each group at each position were then averaged, and the resulting data were graphed on a linear coordinate system with the Y-axis expressing the ΔG (free energy) values in kcal/mole and the X-axis identifying the position of the base relative to the 5′ end.
The results of the analysis identified multiple key regions in siRNA molecules that were critical for successful gene silencing. At the 3′-most end of the sense strand (5′antisense), highly functional siRNA (>95% gene silencing, see FIG. 6a, >F95) have a low internal stability (AISP of position 19= ̃−7.6 kcal/mol). In contrast low-efficiency siRNA (i.e. those exhibiting less than 50% silencing, <F50) display a distinctly different profile, having high ΔG values ( ̃−8.4 kcal/mol) for the same position. Moving in a 5′ (sense strand) direction, the internal stability of highly efficient siRNA rises (position 12= ̃−8.3 kcal/mole) and then drops again (position 7 = ̃−7.7 kcal/mol) before leveling off at a value of approximately −8.1 kcal/mol for the 5′ terminus. SiRNA with poor silencing capabilities show a distinctly different profile. While the AISP value at position 12 is nearly identical with that of strong siRNAs, the values at positions 7 and 8 rise considerably, peaking at a high of  ̃−9.0 kcal/mol. In addition, at the 5′ end of the molecule the AISP profile of strong and weak siRNA differ dramatically. Unlike the relatively strong values exhibited by siRNA in the >95% silencing group, siRNAs that exhibit poor silencing activity have weak AISP values (−7.6, −7.5, and −7.5 kcal/mol for positions 1, 2 and 3 respectively).
Overall the profiles of both strong and weak siRNAs form distinct sinusoidal shapes that are roughly 180° out-of-phase with each other. While these thermodynamic descriptions define the archetypal profile of a strong siRNA, it will likely be the case that neither the ΔG values given for key positions in the profile or the absolute position of the profile along the Y-axis (i.e. the ΔG-axis) are absolutes. Profiles that are shifted upward or downward (i.e. having on an average, higher or lower values at every position) but retain the relative shape and position of the profile along the X-axis can be foreseen as being equally effective as the model profile described here. Moreover, it is likely that siRNA that have strong or even stronger gene-specific silencing effects might have exaggerated ΔG values (either higher or lower) at key positions. Thus, for instance, it is possible that the 5′-most position of the sense strand (position 19) could have ΔG values of 7.4 kcal/mol or lower and still be a strong siRNA if, for instance, a G-C→G-T/U mismatch were substituted at position 19 and altered duplex stability. Similarly, position 12 and position 7 could have values above 8.3 kcal/mol and below 7.7 kcal/mole, respectively, without abating the silencing effectiveness of the molecule. Thus, for instance, at position 12, a stabilizing chemical modification (e.g. a chemical modification of the 2′ position of the sugar backbone) could be added that increases the average internal stability at that position. Similarly, at position 7, mismatches similar to those described previously could be introduced that would lower the ΔG values at that position.
Lastly, it is important to note that while functional and non-functional siRNA were originally defined as those molecules having specific silencing properties, both broader or more limiting parameters can be used to define these molecules. As used herein, unless otherwise specified, “non-functional siRNA” are defined as those siRNA that induce less than 50% (<50%) target silencing, “semi-functional siRNA” induce 50-79% target silencing, “functional siRNA” are molecules that induce 80-95% gene silencing, and “highly-functional siRNA” are molecules that induce great than 95% gene silencing. These definitions are not intended to be rigid and can vary depending upon the design and needs of the application. For instance, it is possible that a researcher attempting to map a gene to a chromosome using a functional assay, may identify an siRNA that reduces gene activity by only 30%. While this level of gene silencing may be “non-functional” for e.g. therapeutic needs, it is sufficient for gene mapping purposes and is, under these uses and conditions, “functional.” For these reasons, functional siRNA can be defined as those molecules having greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% silencing capabilities at 100 nM transfection conditions. Similarly, depending upon the needs of the study and/or application, non-functional and semi-functional siRNA can be defined as having different parameters. For instance, semi-functional siRNA can be defined as being those molecules that induce 20%, 30%, 40%, 50%, 60%, or 70% silencing at 100 nM transfection conditions. Similarly, non-functional siRNA can be defined as being those molecules that silence gene expression by less than 70%, 60%, 50%, 40%, 30%, or less. Nonetheless, unless otherwise stated, the descriptions stated in the “Definitions” section of this text should be applied.
Functional attributes can be assigned to each of the key positions in the AISP of strong siRNA. The low 5′ (sense strand) AISP values of strong siRNAs may be necessary for determining which end of the molecule enters the RISC complex. In contrast, the high and low AISP values observed in the central regions of the molecule may be critical for siRNA-target mRNA interactions and product release, respectively.
If the AISP values described above accurately define the thermodynamic parameters of strong siRNA, it would be expected that similar patterns would be observed in strong siRNA isolated from nature. Natural siRNAs exist in a harsh, RNase-rich environment and it can be hypothesized that only those siRNA that exhibit heightened affinity for RISC (i.e. siRNA that exhibit an average internal stability profile similar to those observed in strong siRNA) would survive in an intracellular environment. This hypothesis was tested using GFP-specific siRNA isolated from N. benthamiana. Llave et al. (2002) Endogenous and Silencing-Associated Small RNAs in Plants, The Plant Cell 14, 1605-1619, introduced long double-stranded GFP-encoding RNA into plants and subsequently re-isolated GFP-specific siRNA from the tissues. The AISP of fifty-nine of these GFP-siRNA were determined, averaged, and subsequently plotted alongside the AISP profile obtained from the cyclophilin B/DBI/luciferase siRNA having >90% silencing properties (FIG. 6b). Comparison of the two groups show that profiles are nearly identical. This finding validates the information provided by the internal stability profiles and demonstrates that: (1) the profile identified by analysis of the cyclophilin B/DBI/luciferase siRNAs are not gene specific; and (2) AISP values can be used to search for strong siRNAs in a variety of species.
Both chemical modifications and base-pair mismatches can be incorporated into siRNA to alter the duplex's AISP and functionality. For instance, introduction of mismatches at positions 1 or 2 of the sense strand destabilized the 5′end of the sense strand and increases the functionality of the molecule (see Luc, FIG. 7). Similarly, addition of 2′-O-methyl groups to positions 1 and 2 of the sense strand can also alter the AISP and (as a result) increase both the functionality of the molecule and eliminate off-target effects that results from sense strand homology with the unrelated targets (FIGS. 8a, 8b).
Rationale for Criteria in a Biological Context
The fate of siRNA in the RNAi pathway may be described in 5 major steps: (1) duplex recognition and pre-RISC complex formation; (2) ATP-dependent duplex unwinding/strand selection and RISC activation; (3) mRNA target identification; (4) mRNA cleavage, and (5) product release (FIG. 1). Given the level of nucleic acid-protein interactions at each step, siRNA functionality is likely influenced by specific biophysical and molecular properties that promote efficient interactions within the context of the multi-component complexes. Indeed, the systematic analysis of the siRNA test set identified multiple factors that correlate well with functionality. When combined into a single algorithm, they proved to be very effective in selecting active siRNAs.
The factors described here may also be predictive of key functional associations important for each step in RNAi. For example, the potential formation of internal hairpin structures correlated negatively with siRNA functionality. Complementary strands with stable internal repeats are more likely to exist as stable hairpins thus decreasing the effective concentration of the functional duplex form. This suggests that the duplex is the preferred conformation for initial pre-RISC association. Indeed, although single complementary strands can induce gene silencing, the effective concentration required is at least two orders of magnitude higher than that of the duplex form.
siRNA-pre-RISC complex formation is followed by an ATP-dependent duplex unwinding step and “activation” of the RISC. The siRNA functionality was shown to correlate with overall low internal stability of the duplex and low internal stability of the 3′ sense end (or differential internal stability of the 3′ sense compare to the 5′ sense strand), which may reflect strand selection and entry into the RISC. Overall duplex stability and low internal stability at the 3′ end of the sense strand were also correlated with siRNA functionality. Interestingly, siRNAs with very high and very low overall stability profiles correlate strongly with non-functional duplexes. One interpretation is that high internal stability prevents efficient unwinding while very low stability reduces siRNA target affinity and subsequent mRNA cleavage by the RISC.
Several criteria describe base preferences at specific positions of the sense strand and are even more intriguing when considering their potential mechanistic roles in target recognition and mRNA cleavage. Base preferences for A at position 19 of the sense strand but not C, are particularly interesting because they reflect the same base preferences observed for naturally occurring miRNA precursors. That is, among the reported miRNA precursor sequences 75% contain a U at position 1 which corresponds to an A in position 19 of the sense strand of siRNAs, while G was under-represented in this same position for miRNA precursors. These observations support the hypothesis that both miRNA precursors and siRNA duplexes are processed by very similar if not identical protein machinery. The functional interpretation of the predominance of a U/A base pair is that it promotes flexibility at the 5′antisense ends of both siRNA duplexes and miRNA precursors and facilitates efficient unwinding and selective strand entrance into an activated RISC.
Among the criteria associated with base preferences that are likely to influence mRNA cleavage or possibly product release, the preference for U at position 10 of the sense strand exhibited the greatest impact, enhancing the probability of selecting an F80 sequence by 13.3%. Activated RISC preferentially cleaves target mRNA between nucleotides 10 and 11 relative to the 5′ end of the complementary targeting strand. Therefore, it may be that U, the preferred base for most endoribonucleases, at this position supports more efficient cleavage. Alternatively, a U/A bp between the targeting siRNA strand and its cognate target mRNA may create an optimal conformation for the RISC-associated “slicing” activity.
Pooling
According to another embodiment, the present invention provides a pool of at least two siRNAs, preferably in the form of a kit or therapeutic reagent, wherein one strand of each of the siRNAs, the sense strand comprises a sequence that is substantially similar to a sequence within a target mRNA. The opposite strand, the antisense strand, will preferably comprise a sequence that is substantially complementary to that of the target mRNA. More preferably, one strand of each siRNA will comprise a sequence that is identical to a sequence that is contained in the target mRNA. Most preferably, each siRNA will be 19 base pairs in length, and one strand of each of the siRNAs will be 100% complementary to a portion of the target mRNA.
By increasing the number of siRNAs directed to a particular target using a pool or kit, one is able both to increase the likelihood that at least one siRNA with satisfactory functionality will be included, as well as to benefit from additive or synergistic effects. Further, when two or more siRNAs directed against a single gene do not have satisfactory levels of functionality alone, if combined, they may satisfactorily promote degradation of the target messenger RNA and successfully inhibit translation. By including multiple siRNAs in the system, not only is the probability of silencing increased, but the economics of operation are also improved when compared to adding different siRNAs sequentially. This effect is contrary to the conventional wisdom that the concurrent use of multiple siRNA will negatively impact gene silencing (e.g. Holen, T. et al. (2003) “Similar behavior of single strand and double strand siRNAs suggests they act through a common RNAi pathway.” NAR 31: 2401-21407).
In fact, when two siRNAs were pooled together, 54% of the pools of two siRNAs induced more than 95% gene silencing. Thus, a 2.5-fold increase in the percentage of functionality was achieved by randomly combining two siRNAs. Further, over 84% of pools containing two siRNAs induced more than 80% gene silencing.
More preferably, the kit is comprised of at least three siRNAs, wherein one strand of each siRNA comprises a sequence that is substantially similar to a sequence of the target mRNA and the other strand comprises a sequence that is substantially complementary to the region of the target mRNA. As with the kit that comprises at least two siRNAs, more preferably one strand will comprise a sequence that is identical to a sequence that is contained in the mRNA and another strand that is 100% complementary to a sequence that is contained in the mRNA. During experiments, when three siRNAs were combined together, 60% of the pools induced more than 95% gene silencing and 92% of the pools induced more than 80% gene silencing.
Further, even more preferably, the kit is comprised of at least four siRNAs, wherein one strand of each siRNA comprises a sequence that is substantially similar to a region of the sequence of the target mRNA, and the other strand comprises a sequence that is substantially complementary to the region of the target mRNA. As with the kit or pool that comprises at least two siRNAs, more preferably one strand of each of the siRNA duplexes will comprise a sequence that is identical to a sequence that is contained in the mRNA, and another strand that is 100% complementary to a sequence that is contained in the mRNA.
Additionally, kits and pools with at least five, at least six, and at least seven siRNAs may also be useful with the present invention. For example, pools of five siRNA induced 95% gene silencing with 77% probability and 80% silencing with 98.8% probability. Thus, pooling of siRNAs together can result in the creation of a target-specific silencing reagent with almost a 99% probability of being functional. The fact that such high levels of success are achievable using such pools of siRNA, enables one to dispense with costly and time-consuming target-specific validation procedures.
For this embodiment, as well as the other aforementioned embodiments, each of the siRNAs within a pool will preferably comprise between 18 and 30 base pairs, more preferably between 18 and 25 base pairs, and most preferably 19 base pairs. Within each siRNA, preferably at least 18 contiguous bases of the antisense strand will be 100% complementary to the target mRNA. More preferably, at least 19 contiguous bases of the antisense strand will be 100% complementary to the target mRNA. Additionally, there may be overhangs on either the sense strand or the antisense strand, and these overhangs may be at either the 5′ end or the 3′ end of either of the strands, for example there may be one or more overhangs of 1-6 bases. When overhangs are present, they are not included in the calculation of the number of base pairs. The two nucleotide 3′ overhangs mimic natural siRNAs and are commonly used but are not essential. Preferably, the overhangs should consist of two nucleotides, most often dTdT or UU at the 3′ end of the sense and antisense strand that are not complementary to the target sequence. The siRNAs may be produced by any method that is now known or that comes to be known for synthesizing double stranded RNA that one skilled in the art would appreciate would be useful in the present invention. Preferably, the siRNAs will be produced by Dharmacon's proprietary ACE® technology. However, other methods for synthesizing siRNAs are well known to persons skilled in the art and include, but are not limited to, any chemical synthesis of RNA oligonucleotides, ligation of shorter oligonucleotides, in vitro transcription of RNA oligonucleotides, the use of vectors for expression within cells, recombinant Dicer products and PCR products.
The siRNA duplexes within the aforementioned pools of siRNAs may correspond to overlapping sequences within a particular mRNA, or non-overlapping sequences of the mRNA. However, preferably they correspond to non-overlapping sequences. Further, each siRNA may be selected randomly, or one or more of the siRNA may be selected according to the criteria discussed above for maximizing the effectiveness of siRNA.
Included in the definition of siRNAs are siRNAs that contain substituted and/or labeled nucleotides that may, for example, be labeled by radioactivity, fluorescence or mass. The most common substitutions are at the 2′ position of the ribose sugar, where moieties such as H (hydrogen) F, NH3, OCH3 and other O-alkyl, alkenyl, alkynyl, and orthoesters, may be substituted, or in the phosphorous backbone, where sulfur, amines or hydrocarbons may be substituted for the bridging of non-bridging atoms in the phosphodiester bond. Examples of modified siRNAs are explained more fully in commonly assigned U.S. patent application Ser. No. 10/613,077, filed Jul. 1, 2003, which is incorporated by reference herein.
Additionally, as noted above, the cell type into which the siRNA is introduced may affect the ability of the siRNA to enter the cell; however, it does not appear to affect the ability of the siRNA to function once it enters the cell. Methods for introducing double-stranded RNA into various cell types are well known to persons skilled in the art.
As persons skilled in the art are aware, in certain species, the presence of proteins such as RdRP, the RNA-dependent RNA polymerase, may catalytically enhance the activity of the siRNA. For example, RdRP propagates the RNAi effect in C. elegans and other non-mammalian organisms. In fact, in organisms that contain these proteins, the siRNA may be inherited. Two other proteins that are well studied and known to be a part of the machinery are members of the Argonaute family and Dicer, as well as their homologues. There is also initial evidence that the RISC complex might be associated with the ribosome so the more efficiently translated mRNAs will be more susceptible to silencing than others.
Another very important factor in the efficacy of siRNA is mRNA localization. In general, only cytoplasmic mRNAs are considered to be accessible to RNAi to any appreciable degree. However, appropriately designed siRNAs, for example, siRNAs modified with internucleotide linkages, may be able to cause silencing by acting in the nucleus. Examples of these types of modifications are described in commonly assigned U.S. patent application Ser. Nos. 10/431,027 and 10/613,077, each of which is incorporated by reference herein.
As described above, even when one selects at least two siRNAs at random, the effectiveness of the two may be greater than one would predict based on the effectiveness of two individual siRNAs. This additive or synergistic effect is particularly noticeable as one increases to at least three siRNAs, and even more noticeable as one moves to at least four siRNAs. Surprisingly, the pooling of the non-functional and semi-functional siRNAs, particularly more than five siRNAs, can lead to a silencing mixture that is as effective if not more effective than any one particular functional siRNA.
Within the kit of the present invention, preferably each siRNA will be present in a concentration of between 0.001 and 200 μM, more preferably between 0.01 and 200 nM, and most preferably between 0.1 and 10 nM.
In addition to preferably comprising at least four or five siRNAs, the kit of the present invention will also preferably comprise a buffer to keep the siRNA duplex stable. Persons skilled in the art are aware of buffers suitable for keeping siRNA stable. For example, the buffer may be comprised of 100 mM KCl, 30 mM HEPES-pH 7.5, and 1 mM MgCl2. Alternatively, kits might contain complementary strands that contain any one of a number of chemical modifications (e.g. a 2′-O-ACE) that protect the agents from degradation by nucleases. In this instance, the user may (or may not) remove the modifying protective group (e.g. deprotect) before annealing the two complementary strands together.
By way of example, the kit may be organized such that pools of siRNA duplexes are provided on an array or microarray of wells or drops for a particular gene set or for unrelated genes. The array may, for example, be in 96 wells, 384 wells or 1284 wells arrayed in a plastic plate or on a glass slide using techniques now known or that come to be known to persons skilled in the art. Within an array, preferably there will be controls such as functional anti-lamin A/C, cyclophilin and two siRNA duplexes that are not specific to the gene of interest.
In order to ensure stability of the siRNA pools prior to usage, they may be retained in lyophilized form at minus twenty degrees (−20° C.) until they are ready for use. Prior to usage, they should be resuspended; however, even once resuspended, for example, in the aforementioned buffer, they should be kept at minus twenty degrees, (−20° C.) until used. The aforementioned buffer, prior to use, may be stored at approximately 4° C. or room temperature. Effective temperatures at which to conduct transfections are well known to persons skilled in the art and include for example, room temperature.
The kit may be applied either in vivo or in vitro. Preferably, the siRNA of the pools or kits is applied to a cell through transfection, employing standard transfection protocols. These methods are well known to persons skilled in the art and include the use of lipid-based carriers, electroporation, cationic carriers, and microinjection. Further, one could apply the present invention by synthesizing equivalent DNA sequences (either as two separate, complementary strands, or as hairpin molecules) instead of siRNA sequences and introducing them into cells through vectors. Once in the cells, the cloned DNA could be transcribed, thereby forcing the cells to generate the siRNA. Examples of vectors suitable for use with the present application include but are not limited to the standard transient expression vectors, adenoviruses, retroviruses, lentivirus-based vectors, as well as other traditional expression vectors. Any vector that has an adequate siRNA expression and procession module may be used. Furthermore, certain chemical modifications to siRNAs, including but not limited to conjugations to other molecules, may be used to facilitate delivery. For certain applications it may be preferable to deliver molecules without transfection by simply formulating in a physiological acceptable solution.
This embodiment may be used in connection with any of the aforementioned embodiments. Accordingly, the sequences within any pool may be selected by rational design.
Multigene Silencing
In addition to developing kits that contain multiple siRNA directed against a single gene, another embodiment includes the use of multiple siRNA targeting multiple genes. Multiple genes may be targeted through the use of high- or hyper-functional siRNA. High- or hyper-functional siRNA that exhibit increased potency, require lower concentrations to induce desired phenotypic (and thus therapeutic) effects. This circumvents RISC saturation. It therefore reasons that if lower concentrations of a single siRNA are needed for knockout or knockdown expression of one gene, then the remaining (uncomplexed) RISC will be free and available to interact with siRNA directed against two, three, four, or more, genes. Thus in this embodiment, the authors describe the use of highly functional or hyper-functional siRNA to knock out three separate genes. More preferably, such reagents could be combined to knockout four distinct genes. Even more preferably, highly functional or hyperfunctional siRNA could be used to knock out five distinct genes. Most preferably, siRNA of this type could be used to knockout or knockdown the expression of six or more genes.
Hyperfunctional siRNA
The term hyperfunctional siRNA (hf-siRNA) describes a subset of the siRNA population that induces RNAi in cells at low- or sub-nanomolar concentrations for extended periods of time. These traits, heightened potency and extended longevity of the RNAi phenotype, are highly attractive from a therapeutic standpoint. Agents having higher potency require lesser amounts of the molecule to achieve the desired physiological response, thus reducing the probability of side effects due to “off-target” interference. In addition to the potential therapeutic benefits associated with hyperfunctional siRNA, hf-siRNA are also desirable from an economic position. Hyperfunctional siRNA may cost less on a per-treatment basis, thus reducing the overall expenditures to both the manufacturer and the consumer.
Identification of hyperfunctional siRNA involves multiple steps that are designed to examine an individual siRNA agent's concentration- and/or longevity-profiles. In one non-limiting example, a population of siRNA directed against a single gene are first analyzed using the previously described algorithm (Formula VIII). Individual siRNA are then introduced into a test cell line and assessed for the ability to degrade the target mRNA. It is important to note that when performing this step it is not necessary to test all of the siRNA. Instead, it is sufficient to test only those siRNA having the highest SMARTscoresTM (i.e. SMARTscoreTM>−10). Subsequently, the gene silencing data is plotted against the SMARTscoresTM (see FIG. 9). SiRNA that (I) induce a high degree of gene silencing (i.e. they induce greater than 80% gene knockdown) and (2) have superior SMARTscoresTM (i.e. a SMARTscoreTM of >−10, suggesting a desirable average internal stability profile) are selected for further investigations designed to better understand the molecule's potency and longevity. In one, non-limiting study dedicated to understanding a molecule's potency, an siRNA is introduced into one (or more) cell types in increasingly diminishing concentrations (e.g. 3.0→0.3 nM). Subsequently, the level of gene silencing induced by each concentration is examined and siRNA that exhibit hyperfunctional potency (i.e. those that induce 80% silencing or greater at e.g. picomolar concentrations) are identified. In a second study, the longevity profiles of siRNA having high (>−10) SMARTscoresTM and greater than 80% silencing are examined. In one non-limiting example of how this is achieved, siRNA are introduced into a test cell line and the levels of RNAi are measured over an extended period of time (e.g. 24-168 hrs). SiRNAs that exhibit strong RNA interference patterns (i.e. >80% interference) for periods of time greater than, e.g., 120 hours are thus identified. Studies similar to those described above can be performed on any and all of the >106 siRNA included in this document to further define the most functional molecule for any given gene. Molecules possessing one or both properties (extended longevity and heightened potency) are labeled “hyperfunctional siRNA,” and earmarked as candidates for future therapeutic studies.
While the example(s) given above describe one means by which hyperfunctional siRNA can be isolated, neither the assays themselves nor the selection parameters used are rigid and can vary with each family of siRNA. Families of siRNA include siRNAs directed against a single gene, or directed against a related family of genes.
The highest quality siRNA achievable for any given gene may vary considerably. Thus, for example, in the case of one gene (gene X), rigorous studies such as those described above may enable the identification of an siRNA that, at picomolar concentrations, induces 99+% silencing for a period of 10 days. Yet identical studies of a second gene (gene Y) may yield an siRNA that at high nanomolar concentrations (e.g. 100 nM) induces only 75% silencing for a period of 2 days. Both molecules represent the very optimum siRNA for their respective gene targets and therefore are designated “hyperfunctional.” Yet due to a variety of factors including but not limited to target concentration, siRNA stability, cell type, off-target interference, and others, equivalent levels of potency and longevity are not achievable. Thus, for these reasons, the parameters described in the before mentioned assays, can vary. While the initial screen selected siRNA that had SMARTscoresTM above −10 and a gene silencing capability of greater than 80%, selections that have stronger (or weaker) parameters can be implemented. Similarly, in the subsequent studies designed to identify molecules with high potency and longevity, the desired cutoff criteria (i.e. the lowest concentration that induces a desirable level of interference, or the longest period of time that interference can be observed) can vary. The experimentation subsequent to application of the rational criteria of this application is significantly reduced where one is trying to obtain a suitable hyperfunctional siRNA for, for example, therapeutic use. When, for example, the additional experimentation of the type described herein is applied by one skilled in the art with this disclosure in hand, a hyperfunctional siRNA is readily identified.
The siRNA may be introduced into a cell by any method that is now known or that comes to be known and that from reading this disclosure, persons skilled in the art would determine would be useful in connection with the present invention in enabling siRNA to cross the cellular membrane. These methods include, but are not limited to, any manner of transfection, such as for example transfection employing DEAE-Dextran, calcium phosphate, cationic lipids/liposomes, micelles, manipulation of pressure, microinjection, electroporation, immunoporation, use of vectors such as viruses, plasmids, cosmids, bacteriophages, cell fusions, and coupling of the polynucleotides to specific conjugates or ligands such as antibodies, antigens, or receptors, passive introduction, adding moieties to the siRNA that facilitate its uptake, and the like.
Having described the invention with a degree of particularity, examples will now be provided. These examples are not intended to and should not be construed to limit the scope of the claims in any way.
EXAMPLES
General Techniques and Nomenclatures
siRNA nomenclature. All siRNA duplexes are referred to by sense strand. The first nucleotide of the 5′-end of the sense strand is position 1, which corresponds to position 19 of the antisense strand for a 19-mer. In most cases, to compare results from different experiments, silencing was determined by measuring specific transcript mRNA levels or enzymatic activity associated with specific transcript levels, 24 hours post-transfection, with siRNA concentrations held constant at 100 nM. For all experiments, unelss otherwise specified transfection efficiency was ensured to be over 95%, and no detectable cellular toxicity was observed. The following system of nomenclature was used to compare and report siRNA-silencing functionality: “F” followed by the degree of minimal knockdown. For example, F50 signifies at least 50% knockdown, F80 means at least 80%, and so forth. For this study, all sub-F50 siRNAs were considered non-functional.
Cell culture and transfection. 96-well plates are coated with 50 ill of 50 mg/ml poly-L-lysine (Sigma) for 1 hr, and then washed 3× with distilled water before being dried for 20 min. HEK293 cells or HEK293Lucs or any other cell type of interest are released from their solid support by trypsinization, diluted to 3.5×105 cells/ml, followed by the addition of 100 μL of cells/well. Plates are then incubated overnight at 37° C., 5% CO2. Transfection procedures can vary widely depending on the cell type and transfection reagents. In one non-limiting example, a transfection mixture consisting of 2 mL Opti-MEM I (Gibco-BRL), 80 μl Lipofectamine 2000 (Invitrogen), 15 μL SUPERNasin at 20 U/μl (Ambion), and 1.5 μl of reporter gene plasmid at 1 μg/μl is prepared in 5-ml polystyrene round bottom tubes. 100 μl of transfection reagent is then combined with 100 μl of siRNAs in polystyrene deep-well titer plates (Beckman) and incubated for 20 to 30 min at room temp. 550 μl of Opti-MEM is then added to each well to bring the final siRNA concentration to 100 nM. Plates are then sealed with parafilm and mixed. Media is removed from HEK293 cells and replaced with 95 μl of transfection mixture. Cells are incubated overnight at 37° C., 5% CO2.
Quantification of gene knockdown. A variety of quantification procedures can be used to measure the level of silencing induced by siRNA or siRNA pools. In one non-limiting example: to measure mRNA levels 24 hrs post-transfection, QuantiGene branched-DNA (bDNA) kits (Bayer) (Wang, et al, Regulation of insulin preRNA splicing by glucose. Proc Natl Acad Sci 1997, 94:4360.) are used according to manufacturer instructions. To measure luciferase activity, media is removed from HEK293 cells 24 hrs post-transfection, and 50 μl of Steady-GLO reagent (Promega) is added. After 5 min, plates are analyzed on a plate reader.
Example I
Sequences Used to Develop the Algorithm
Anti-Firefly and anti-Cyclophilin siRNAs panels (FIG. 5A, B) sorted according to using Formula VIII predicted values. All siRNAs scoring more than 0 (formula VIII) and more then 20 (formula IX) are fully functional. All ninety sequences for each gene (and DBI) appear below in Table IV.










TABLE IV







Cyclo
1
SEQ. ID 0032
GUUCCAAAAACAGUGGAUA


Cyclo
2
SEQ. ID 0033
UCCAAAAACAGUGGAUAAU


Cyclo
3
SEQ. ID 0034
CAAAAACAGUGGAUAAUUU


Cyclo
4
SEQ. ID 0035
AAAACAGUGGAUAAUUUUG


Cyclo
5
SEQ. ID 0036
AACAGUGGAUAAUUUUGUG


Cyclo
6
SEQ. ID 0037
CAGUGGAUAAUUUUGUGGC


Cyclo
7
SEQ. ID 0038
GUGGAUAAUUUUGUGGCCU


Cyclo
8
SEQ. ID 0039
GGAUAAUUUUGUGGCCUUA


Cyclo
9
SEQ. ID 0040
AUAAUUUUGUGGCCUUAGC


Cyclo
10
SEQ. ID 0041
AAUUUUGUGGCCUUAGCUA


Cyclo
11
SEQ. ID 0042
UUUUGUGGCCUUAGCUACA


Cyclo
12
SEQ. ID 0043
UUGUGGCCUUAGCUACAGG


Cyclo
13
SEQ. ID 0044
GUGGCCUUAGCUACAGGAG


Cyclo
14
SEQ. ID 0045
GGCCUUAGCUACAGGAGAG


Cyclo
15
SEQ. ID 0046
CCUUAGCUACAGGAGAGAA


Cyclo
16
SEQ. ID 0047
UUAGCUACAGGAGAGAAAG


Cyclo
17
SEQ. ID 0048
AGCUACAGGAGAGAAAGGA


Cyclo
18
SEQ. ID 0049
CUACAGGAGAGAAAGGAUU


Cyclo
19
SEQ. ID 0050
ACAGGAGAGAAAGGAUUUG


Cyclo
20
SEQ. ID 0051
AGGAGAGAAAGGAUUUGGC


Cyclo
21
SEQ. ID 0052
GAGAGAAAGGAUUUGGCUA


Cyclo
22
SEQ. ID 0053
GAGAAAGGAUUUGGCUACA


Cyclo
23
SEQ. ID 0054
GAAAGGAUUUGGCUACAAA


Cyclo
24
SEQ. ID 0055
AAGGAUUUGGCUACAAAAA


Cyclo
25
SEQ. ID 0056
GGAUUUGGCUACAAAAACA


Cyclo
26
SEQ. ID 0057
AUUUGGCUACAAAAACAGC


Cyclo
27
SEQ. ID 0058
UUGGCUACAAAAACAGCAA


Cyclo
28
SEQ. ID 0059
GGCUACAAAAACAGCAAAU


Cyclo
29
SEQ. ID 0060
CUACAAAAACAGCAAAUUC


Cyclo
30
SEQ. ID 0061
ACAAAAACAGCAAAUUCCA


Cyclo
31
SEQ. ID 0062
AAAAACAGCAAAUUCCAUC


Cyclo
32
SEQ. ID 0063
AAACAGCAAAUUCCAUCGU


Cyclo
33
SEQ. ID 0064
ACAGCAAAUUCCAUCGUGU


Cyclo
34
SEQ. ID 0065
AGCAAAUUCCAUCGUGUAA


Cyclo
35
SEQ. ID 0066
CAAAUUCCAUCGUGUAAUC


Cyclo
36
SEQ. ID 0067
AAUUCCAUCGUGUAAUCAA


Cyclo
37
SEQ. ID 0068
UUCCAUCGUGUAAUCAAGG


Cyclo
38
SEQ. ID 0069
CCAUCGUGUAAUCAAGGAC


Cyclo
39
SEQ. ID 0070
AUCGUGUAAUCAAGGACUU


Cyclo
40
SEQ. ID 0071
CGUGUAAUCAAGGACUUCA


Cyclo
41
SEQ. ID 0072
UGUAAUCAAGGACUUCAUG


Cyclo
42
SEQ. ID 0073
UAAUCAAGGACUUCAUGAU


Cyclo
43
SEQ. ID 0074
AUCAAGGACUUCAUGAUCC


Cyclo
44
SEQ. ID 0075
CAAGGACUUCAUGAUCCAG


Cyclo
45
SEQ. ID 0076
AGGACUUCAUGAUCCAGGG


Cyclo
46
SEQ. ID 0077
GACUUCAUGAUCCAGGGCG


Cyclo
47
SEQ. ID 0078
CUUCAUGAUCCAGGGCGGA


Cyclo
48
SEQ. ID 0079
UCAUGAUCCAGGGCGGAGA


Cyclo
49
SEQ. ID 0080
AUGAUCCAGGGCGGAGACU


Cyclo
50
SEQ. ID 0081
GAUCCAGGGCGGAGACUUC


Cyclo
51
SEQ. ID 0082
UCCAGGGCGGAGACUUCAC


Cyclo
52
SEQ. ID 0083
CAGGGCGGAGACUUCACCA


Cyclo
53
SEQ. ID 0084
GGGCGGAGACUUCACCAGG


Cyclo
54
SEQ. ID 0085
GCGGAGACUUCACCAGGGG


Cyclo
55
SEQ. ID 0086
GGAGACUUCACCAGGGGAG


Cyclo
56
SEQ. ID 0087
AGACUUCACCAGGGGAGAU


Cyclo
57
SEQ. ID 0088
ACUUCACCAGGGGAGAUGG


Cyclo
58
SEQ. ID 0089
UUCACCAGGGGAGAUGGCA


Cyclo
59
SEQ. ID 0090
CACCAGGGGAGAUGGCACA


Cyclo
60
SEQ. ID 0091
CCAGGGGAGAUGGCACAGG


Cyclo
61
SEQ. ID 0092
AGGGGAGAUGGCACAGGAG


Cyclo
62
SEQ. ID 0093
GGGAGAUGGCACAGGAGGA


Cyclo
63
SEQ. ID 0431
GAGAUGGCACAGGAGGAAA


Cyclo
64
SEQ. ID 0095
GAUGGCACAGGAGGAAAGA


Cyclo
65
SEQ. ID 0094
UGGCACAGGAGGAAAGAGC


Cyclo
66
SEQ. ID 0096
GCACAGGAGGAAAGAGCAU


Cyclo
67
SEQ. ID 0097
ACAGGAGGAAAGAGCAUCU


Cyclo
68
SEQ. ID 0098
AGGAGGAAAGAGCAUCUAC


Cyclo
69
SEQ. ID 0099
GAGGAAAGAGCAUCUACGG


Cyclo
70
SEQ. ID 0100
GGAAAGAGCAUCUACGGUG


Cyclo
71
SEQ. ID 0101
AAAGAGCAUCUACGGUGAG


Cyclo
72
SEQ. ID 0102
AGAGCAUCUACGGUGAGCG


Cyclo
73
SEQ. ID 0103
AGCAUCUACGGUGAGCGCU


Cyclo
74
SEQ. ID 0104
CAUCUACGGUGAGCGCUUC


Cyclo
75
SEQ. ID 0105
UCUACGGUGAGCGCUUCCC


Cyclo
76
SEQ. ID 0106
UACGGUGAGCGCUUCCCCG


Cyclo
77
SEQ. ID 0107
CGGUGAGCGCUUCCCCGAU


Cyclo
78
SEQ. ID 0108
GUGAGCGCUUCCCCGAUGA


Cyclo
79
SEQ. ID 0109
GAGCGCUUCCCCGAUGAGA


Cyclo
80
SEQ. ID 0110
GCGCUUCCCCGAUGAGAAC


Cyclo
81
SEQ. ID 0111
GCUUCCCCGAUGAGAACUU


Cyclo
82
SEQ. ID 0112
UUCCCCGAUGAGAACUUCA


Cyclo
83
SEQ. ID 0113
CCCCGAUGAGAACUUCAAA


Cyclo
84
SEQ. ID 0114
CCGAUGAGAACUUCAAACU


Cyclo
85
SEQ. ID 0115
GAUGAGAACUUCAAACUGA


Cyclo
86
SEQ. ID 0116
UGAGAACUUCAAACUGAAG


Cyclo
87
SEQ. ID 0117
AGAACUUCAAACUGAAGCA


Cyclo
88
SEQ. ID 0118
AACUUCAAACUGAAGCACU


Cyclo
89
SEQ. ID 0119
CUUCAAACUGAAGCACUAC


Cyclo
90
SEQ. ID 0120
UCAAACUGAAGCACUACGG


DB
1
SEQ. ID 0121
ACGGGCAAGGCCAAGUGGG


DB
2
SEQ. ID 0122
CGGGCAAGGCCAAGUGGGA


DB
3
SEQ. ID 0123
GGGCAAGGCCAAGUGGGAU


DB
4
SEQ. ID 0124
GGCAAGGCCAAGUGGGAUG


DB
5
SEQ. ID 0125
GCAAGGCCAAGUGGGAUGC


DB
6
SEQ. ID 0126
CAAGGCCAAGUGGGAUGCC


DB
7
SEQ. ID 0127
AAGGCCAAGUGGGAUGCCU


DB
8
SEQ. ID 0128
AGGCCAAGUGGGAUGCCUG


DB
9
SEQ. ID 0129
GGCCAAGUGGGAUGCCUGG


DB
10
SEQ. ID 0130
GCCAAGUGGGAUGCCUGGA


DB
11
SEQ. ID 0131
CGAAGUGGGAUGCCUGGAA


DB
12
SEQ. ID 0132
CAAGUGGGAUGCCUGGAAU


DB
13
SEQ. ID 0133
AAGUGGGAUGCCUGGAAUG


DB
14
SEQ. ID 0134
AGUGGGAUGCCUGGAAUGA


DB
15
SEQ. ID 0135
GUGGGAUGCCUGGAAUGAG


DB
16
SEQ. ID 0136
UGGGAUGCCUGGAAUGAGC


DB
17
SEQ. ID 0137
GGGAUGCCUGGAAUGAGCU


DB
18
SEQ. ID 0138
GGAUGCCUGGAAUGAGCUG


DB
19
SEQ. ID 0139
GAUGCCUGGAAUGAGCUGA


DB
20
SEQ. ID 0140
AUGCCUGGAAUGAGCUGAA


DB
21
SEQ. ID 0141
UGCCUGGAAUGAGCUGAAA


DB
22
SEQ. ID 0142
GCCUGGAAUGAGCUGAAAG


DB
23
SEQ. ID 0143
CCUGGAAUGAGCUGAAAGG


DB
24
SEQ. ID 0144
CUGGAAUGAGCUGAAAGGG


DB
25
SEQ. ID 0145
UGGAAUGAGCUGAAAGGGA


DB
26
SEQ. ID 0146
GGAAUGAGCUGAAAGGGAC


DB
27
SEQ. ID 0147
GAAUGAGCUGAAAGGGACU


DB
28
SEQ. ID 0148
AAUGAGCUGAAAGGGACUU


DB
29
SEQ. ID 0149
AUGAGCUGAAAGGGACUUC


DB
30
SEQ. ID 0150
UGAGCUGAAAGGGACUUCC


DB
31
SEQ. ID 0151
GAGCUGAAAGGGACUUCCA


DB
32
SEQ. ID 0152
AGCUGAAAGGGACUUCCAA


DB
33
SEQ. ID 0153
GCUGAAAGGGACUUCCAAG


DB
34
SEQ. ID 0154
CUGAAAGGGACUUCCAAGG


DB
35
SEQ. ID 0155
UGAAAGGGACUUCCAAGGA


DB
36
SEQ. ID 0156
GAAAGGGACUUCCAAGGAA


DB
37
SEQ. ID 0157
AAAGGGACUUCCAAGGAAG


DB
38
SEQ. ID 0158
AAGGGACUUCCAAGGAAGA


DB
39
SEQ. ID 0159
AGGGACUUCCAAGGAAGAU


DB
40
SEQ. ID 0160
GGGACUUCCAAGGAAGAUG


DB
41
SEQ. ID 0161
GGACUUCCAAGGAAGAUGC


DB
42
SEQ. ID 0162
GACUUCCAAGGAAGAUGCC


DB
43
SEQ. ID 0163
ACUUCCAAGGAAGAUGCCA


DB
44
SEQ. ID 0164
CUUCCAAGGAAGAUGCCAU


DB
45
SEQ. ID 0165
UUCCAAGGAAGAUGCCAUG


DB
46
SEQ. ID 0166
UCCAAGGAAGAUGCCAUGA


DB
47
SEQ. ID 0167
CCAAGGAAGAUGCCAUGAA


DB
48
SEQ. ID 0168
CAAGGAAGAUGCCAUGAAA


DB
49
SEQ. ID 0169
AAGGAAGAUGCCAUGAAAG


DB
50
SEQ. ID 0170
AGGAAGAUGCCAUGAAAGC


DB
51
SEQ. ID 0171
GGAAGAUGCCAUGAAAGCU


DB
52
SEQ. ID 0172
GAAGAUGCCAUGAAAGCUU


DB
53
SEQ. ID 0173
AAGAUGCCAUGAAAGCUUA


DB
54
SEQ. ID 0174
AGAUGCCAUGAAAGCUUAC


DB
55
SEQ. ID 0175
GAUGCCAUGAAAGCUUACA


DB
56
SEQ. ID 0176
AUGCCAUGAAAGCUUACAU


DB
57
SEQ. ID 0177
UGCCAUGAAAGCUUACAUC


DB
58
SEQ. ID 0178
GCCAUGAAAGCUUACAUCA


DB
59
SEQ. ID 0179
CCAUGAAAGCUUACAUCAA


DB
60
SEQ. ID 0180
CAUGAAAGCUUACAUCAAC


DB
61
SEQ. ID 0181
AUGAAAGCUUACAUCAACA


DB
62
SEQ. ID 0182
UGAAAGCUUACAUCAACAA


DB
63
SEQ. ID 0183
GAAAGCUUACAUCAACAAA


DB
64
SEQ. ID 0184
AAAGCUUACAUCAACAAAG


DB
65
SEQ. ID 0185
AAGCUUACAUCAACAAAGU


DB
66
SEQ. ID 0186
AGCUUACAUCAACAAAGUA


DB
67
SEQ. ID 0187
GCUUACAUCAACAAAGUAG


DB
68
SEQ. ID 0188
CUUACAUCAACAAAGUAGA


DB
69
SEQ. ID 0189
UUACAUCAACAAAGUAGAA


DB
70
SEQ. ID 0190
UACAUCAACAAAGUAGAAG


DB
71
SEQ. ID 0191
ACAUCAACAAAGUAGAAGA


DB
72
SEQ. ID 0192
CAUCAACAAAGUAGAAGAG


DB
73
SEQ. ID 0193
AUCAACAAAGUAGAAGAGC


DB
74
SEQ. ID 0194
UCAACAAAGUAGAAGAGCU


DB
75
SEQ. ID 0195
CAACAAAGUAGAAGAGCUA


DB
76
SEQ. ID 0196
AACAAAGUAGAAGAGCUAA


DB
77
SEQ. ID 0197
ACAAAGUAGAAGAGCUAAA


DB
78
SEQ. ID 0198
CAAAGUAGAAGAGCUAAAG


DB
79
SEQ. ID 0199
AAAGUAGAAGAGCUAAAGA


DB
80
SEQ. ID 0200
AAGUAGAAGAGCUAAAGAA


DB
81
SEQ. ID 0201
AGUAGAAGAGCUAAAGAAA


DB
82
SEQ. ID 0202
GUAGAAGAGCUAAAGAAAA


DB
83
SEQ. ID 0203
UAGAAGAGCUAAAGAAAAA


DB
84
SEQ. ID 0204
AGAAGAGCUAAAGAAAAAA


DB
85
SEQ. ID 0205
GAAGAGCUAAAGAAAAAAU


DB
86
SEQ. ID 0206
AAGAGCUAAAGAAAAAAUA


DB
87
SEQ. ID 0207
AGAGCUAAAGAAAAAAUAC


DB
88
SEQ. ID 0208
GAGCUAAAGAAAAAAUACG


DB
89
SEQ. ID 0209
AGCUAAAGAAAAAAUACGG


DB
90
SEQ. ID 0210
GCUAAAGAAAAAAUACGGG


Luc
1
SEQ. ID 0211
AUCCUCAUAAAGGCCAAGA


Luc
2
SEQ. ID 0212
AGAUCCUCAUAAAGGCCAA


Luc
3
SEQ. ID 0213
AGAGAUCCUCAUAAAGGCC


Luc
4
SEQ. ID 0214
AGAGAGAUGCUCAUAAAGG


Luc
5
SEQ. ID 0215
UGAGAGAGAUCCUCAUAAA


Luc
6
SEQ. ID 0216
AAUCAGAGAGAUCCUCAUA


Luc
7
SEQ. ID 0217
AAAAUCAGAGAGAUCCUCA


Luc
8
SEQ. ID 0218
GAAAAAUCAGAGAGAUCCU


Luc
9
SEQ. ID 0219
AAGAAAAAUCAGAGAGAUC


Luc
10
SEQ. ID 0220
GCAAGAAAAAUCAGAGAGA


Luc
11
SEQ. ID 0221
ACGCAAGAAAAAUCAGAGA


Luc
12
SEQ. ID 0222
CGACGCAAGAAAAAUCAGA


Luc
13
SEQ. ID 0223
CUCGACCCAAGAAAAAUCA


Luc
14
SEQ. ID 0224
AACUCGACGCAAGAAAAAU


Luc
15
SEQ. ID 0225
AAAACUCGACGCAAGAAAA


Luc
16
SEQ. ID 0226
GGAAAACUCGACGCAAGAA


Luc
17
SEQ. ID 0227
CCGGAAAACUCGACGCAAG


Luc
18
SEQ. ID 0228
UACCGGAAAACUCGACGCA


Luc
19
SEQ. ID 0229
GUUACCGGAAAACUCGACG


Luc
20
SEQ. ID 0230
GUCUUACCGGAAAACUCGA


Luc
21
SEQ. ID 0231
AGGUCUUACCGGAAAACUC


Luc
22
SEQ. ID 0232
AAAGGUGUUACCGGAAAAC


Luc
23
SEQ. ID 0233
CGAAAGGUCUUACCGGAAA


Luc
24
SEQ. ID 0234
ACCGAAAGGUGUUACCGGA


Luc
25
SEQ. ID 0235
GUACCGAAAGGUCUUACCG


Luc
26
SEQ. ID 0236
AAGUACCGAAAGGUCUUAC


Luc
27
SEQ. ID 0237
CGAAGUACCGAAAGGUCUU


Luc
28
SEQ. ID 0238
GACGAAGUACCGAAAGGUC


Luc
29
SEQ. ID 0239
UGGACGAAGUACCGAAAGG


Luc
30
SEQ. ID 0240
UGUGGACGAAGUACCGAAA


Luc
31
SEQ. ID 0241
UUUGUGGACGAAGUACCGA


Luc
32
SEQ. ID 0242
UGUUUGUGGACGAAGUACC


Luc
33
SEQ. ID 0243
UGUGUUUGUGGACGAAGUA


Luc
34
SEQ. ID 0244
GUUGUGUUUGUGGACGAAG


Luc
35
SEQ. ID 0245
GAGUUGUGUUUGUGGACGA


Luc
36
SEQ. ID 0246
AGGAGUUGUGUUUGUGGAC


Luc
37
SEQ. ID 0247
GGAGGAGUUGUGUUUGUGG


Luc
38
SEQ. ID 0248
GCGGAGGAGUUGUGUUUGU


Luc
39
SEQ. ID 0249
GCGCGGAGGAGUUGUGUUU


Luc
40
SEQ. ID 0250
UUGCGCGGAGGAGUUGUGU


Luc
41
SEQ. ID 0251
AGUUGCGCGGAGGAGUUGU


Luc
42
SEQ. ID 0252
AAAGUUGCGCGGAGGAGUU


Luc
43
SEQ. ID 0253
AAAAAGUUGCGCGGAGGAG


Luc
44
SEQ. ID 0254
CGAAAAAGUUGCGCGGAGG


Luc
45
SEQ. ID 0255
CGCGAAAAAGUUGCGCGGA


Luc
46
SEQ. ID 0256
ACCGCGAAAAAGUUGCGCG


Luc
47
SEQ. ID 0257
CAACCGCGAAAAAGUUGCG


Luc
48
SEQ. ID 0258
AACAACCGCGAAAAAGUUG


Luc
49
SEQ. ID 0259
GUAACAACCGCGAAAAAGU


Luc
50
SEQ. ID 0260
AAGUAACAACCGCGAAAAA


Luc
51
SEQ. ID 0261
UCAAGUAACAACCGCGAAA


Luc
52
SEQ. ID 0262
AGUCAAGUAACAACCGCGA


Luc
53
SEQ. ID 0263
CCAGUCAAGUAACAACCGC


Luc
54
SEQ. ID 0264
CGCCAGUCAAGUAACAACC


Luc
55
SEQ. ID 0265
GUCGCCAGUCAAGUAACAA


Luc
56
SEQ. ID 0266
ACGUCGCCAGUCAAGUAAC


Luc
57
SEQ. ID 0267
UUACGUCGCCAGUCAAGUA


Luc
58
SEQ. ID 0268
GAUUACGUCGCCAGUCAAG


Luc
59
SEQ. ID 0269
UGGAUUACGUCGCCAGUCA


Luc
60
SEQ. ID 0270
CGUGGAUUACGUCGCCAGU


Luc
61
SEQ. ID 0271
AUCGUGGAUUACGUCGCCA


Luc
62
SEQ. ID 0272
AGAUCGUGGAUUACGUCGC


Luc
63
SEQ. ID 0273
AGAGAUCGUGGAUUACGUC


Luc
64
SEQ. ID 0274
AAAGAGAUCGUGGAUUACG


Luc
65
SEQ. ID 0275
AAAAAGAGAUCGUGGAUUA


Luc
66
SEQ. ID 0276
GGAAAAAGAGAUCGUGGAU


Luc
67
SEQ. ID 0277
ACGGAAAAAGAGAUCGUGG


Luc
68
SEQ. ID 0278
UGACGGAAAAAGAGAUCGU


Luc
69
SEQ. ID 0279
GAUGACGGAAAAAGAGAUC


Luc
70
SEQ. ID 0280
ACGAUGACGGAAAAAGAGA


Luc
71
SEQ. ID 0281
AGACGAUGACGGAAAAAGA


Luc
72
SEQ. ID 0282
AAAGACGAUGACGGAAAAA


Luc
73
SEQ. ID 0283
GGAAAGACGAUGACGGAAA


Luc
74
SEQ. ID 0284
ACGGAAAGACGAUGACGGA


Luc
75
SEQ. ID 0285
GCACGGAAAGACGAUGACG


Luc
76
SEQ. ID 0286
GAGCACGGAAAGACGAUGA


Luc
77
SEQ. ID 0287
UGGAGCACGGAAAGACGAU


Luc
78
SEQ. ID 0288
UUUGGAGCACGGAAAGACG


Luc
79
SEQ. ID 0289
GUUUUGGAGCACGGAAAGA


Luc
80
SEQ. ID 0290
UUGUUUUGGAGGACGGAAA


Luc
81
SEQ. ID 0291
UGUUGUUUUGGAGCACGGA


Luc
82
SEQ. ID 0292
GUUGUUGUUUUGGAGCACG


Luc
83
SEQ. ID 0293
CCGUUGUUGUUUUGGAGCA


Luc
84
SEQ. ID 0294
CGCCGUUGUUGUUUUGGAG


Luc
85
SEQ. ID 0295
GCCGCCGUUGUUGUUUUGG


Luc
86
SEQ. ID 0296
CCGCCGCCGUUGUUGUUUU


Luc
87
SEQ. ID 0297
UCCCGCCGCCGUUGUUGUU


Luc
88
SEQ. ID 0298
CUUCCCGCCGCCGUUGUUG


Luc
89
SEQ. ID 0299
AACUUCCCGCCGCCGUUGU


Luc
90
SEQ. ID 0300
UGAACUUCCCGCCGCCGUU









Example II
Validation of the Algorithm using DBI, Luciferase, PLK, EGFR, and SEAP
The algorithm (Formula VIII) identified siRNAs for five genes, human DBI, firefly luciferase (fLuc), renilla luciferase (rLuc), human PLK, and human secreted alkaline phosphatase (SEAP). Four individual siRNAs were selected on the basis of their SMARTscoresTM derived by analysis of their sequence using Formula VIII (all of the siRNAs would be selected with Formula IX as well) and analyzed for their ability to silence their targets' expression. In addition to the scoring, a BLAST search was conducted for each siRNA. To minimize the potential for off-target silencing effects, only those target sequences with more than three mismatches against un-related sequences were selected. Semizarov, et al, Specificity of short interfering RNA determined through gene expression signatures. Proc. Natl. Acad. Sci. U.S.A. 2003, 100:6347. These duplexes were analyzed individually and in pools of 4 and compared with several siRNAs that were randomly selected. The functionality was measured a percentage of targeted gene knockdown as compared to controls. All siRNAs were transfected as described by the methods above at 100 nM concentration into HEK293 using Lipofectamine 2000. The level of the targeted gene expression was evaluated by B-DNA as described above and normalized to the non-specific control. FIG. 10 shows that the siRNAs selected by the algorithm disclosed herein were significantly more potent than randomly selected siRNAs. The algorithm increased the chances of identifying an F50 siRNA from 48% to 91%, and an F80 siRNA from 13% to 57%. In addition, pools of SMART siRNA silence the selected target better than randomly selected pools (see FIG. 10F).
Example III
Validation of the Algorithm Using Genes Involved in Clathrin-Dependent Endocytosis
Components of clathrin-mediated endocytosis pathway are key to modulating intracellular signaling and play important roles in disease. Chromosomal rearrangements that result in fusion transcripts between the Mixed-Lineage Leukemia gene (MLL) and CALM (Clathrin assembly lymphoid myeloid leukemia gene) are believed to play a role in leukemogenesis. Similarly, disruptions in Rab7 and Rab9, as well as HIP1 (Huntingtin-interacting protein), genes that are believed to be involved in endocytosis, are potentially responsible for ailments resulting in lipid storage, and neuronal diseases, respectively. For these reasons, siRNA directed against clathrin and other genes involved in the clathrin-mediated endocytotic pathway are potentially important research and therapeutic tools.
siRNAs directed against genes involved in the clathrin-mediated endocytosis pathways were selected using Formula VIII. The targeted genes were clathrin heavy chain (CHC, accession # NM—004859), clathrin light chain A (CLCa, NM—001833), clathrin light chain B (CLCb, NM—001834), CALM (U45976), β2 subunit of AP-2 (β2, NM—001282), Eps15 (NM—001981), Eps15R (NM—021235), dynamin II (DYNII, NM—004945), Rab5a (BC001267), Rab5b (NM—002868), Rab5c (AF141304), and EEA.1 (XM—018197).
For each gene, four siRNAs duplexes with the highest scores were selected and a BLAST search was conducted for each of them using the Human EST database. In order to minimize the potential for off-target silencing effects, only those sequences with more than three mismatches against un-related sequences were used. All duplexes were synthesized at Dharmacon, Inc. as 21-mers with 3′-UU overhangs using a modified method of 2′-ACE chemistry Scaringe, Advanced 5′-silyl-2′-orthoester approach to RNA oligonucleotide synthesis, Methods Enzymol 2000, 317:3 and the antisense strand was chemically phosphorylated to insure maximized activity.
HeLa cells were grown in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum, antibiotics and glutamine. siRNA duplexes were resuspended in 1× siRNA Universal buffer (Dharmacon, Inc.) to 20μM prior to transfection. HeLa cells in 12-well plates were transfected twice with 4 μl of 20 μM siRNA duplex in 3 μl Lipofectamine 2000 reagent (Invitrogen, Carlsbad, Calif., USA) at 24-hour intervals. For the transfections in which 2 or 3 siRNA duplexes were included, the amount of each duplex was decreased, so that the total amount was the same as in transfections with single siRNAs. Cells were plated into normal culture medium 12 hours prior to experiments, and protein levels were measured 2 or 4 days after the first transfection.
Equal amounts of lysates were resolved by electrophoresis, blotted, and stained with the antibody specific to targeted protein, as well as antibodies specific to unrelated proteins, PPI phosphatase and Tsg101 (not shown). The cells were lysed in Triton X— 100/glycerol solubilization buffer as described previously. Tebar, Bohlander, & Sorkin, Clathrin Assembly Lymphoid Myeloid Leukemia (CALM) Protein: Localization in Endocytic-coated Pits, Interactions with Clathrin, and the Impact of Overexpression on Clathrin-mediated Traffic, Mol. Biol. Cell August 1999, 10:2687. Cell lysates were electrophoresed, transferred to nitrocellulose membranes, and Western blotting was performed with several antibodies followed by detection using enhanced chemiluminescence system (Pierce, Inc). Several x-ray films were analyzed to determine the linear range of the chemiluminescence signals, and the quantifications were performed using densitometry and Alphalmager v5.5 software (Alpha Innotech Corporation). In experiments with Eps15R-targeted siRNAs, cell lysates were subjected to immunoprecipitation with Ab860, and Eps15R was detected in immunoprecipitates by Western blotting as described above.
The antibodies to assess the levels of each protein by Western blot were obtained from the following sources: monoclonal antibody to clathrin heavy chain (TD.1) was obtained from American Type Culture Collection (Rockville, Md., USA); polyclonal antibody to dynamin II was obtained from Affinity Bioreagents, Inc. (Golden, Colo., USA); monoclonal antibodies to EEA.1 and Rab5a were purchased from BD Transduction Laboratories (Los Angeles, Calif., USA); the monoclonal antibody to Tsg101 was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, Calif., USA); the monoclonal antibody to GFP was from ZYMED Laboratories Inc. (South San Francisco, Calif., USA); the rabbit polyclonal antibodies Ab32 specific to α-adaptins and Ab20 to CALM were described previously Sorkin, et al, Stoichiometric Interaction of the Epidermal Growth Factor Receptor with the Clathrin-associated Protein Complex AP-2, J. Biol. Chem. January 1995, 270:619, the polyclonal antibodies to clathrin light chains A and B were kindly provided by Dr. F. Brodsky (UCSF); monoclonal antibodies to PP1 (BD Transduction Laboratories) and α-Actinin (Chemicon) were kindly provided by Dr. M. Dell'Acqua (University of Colorado); Eps15 Ab577 and Eps15R Ab860 were kindly provided by Dr. P. P. Di Fiore (European Cancer Institute).
FIG. 11 demonstrates the in vivo functionality of 48 individual siRNAs, selected using Formula VIII (most of them will meet the criteria incorporated by Formula IX as well) targeting 12 genes. Various cell lines were transfected with siRNA duplexes (Dup1-4) or pools of siRNA duplexes (Pool), and the cells were lysed 3 days after transfection with the exception of CALM (2 days) and β2 (4 days).
Note a β1-adaptin band (part of AP-1 Golgi adaptor complex) that runs slightly slower than P2 adaptin. CALM has two splice variants, 66 and 72 kD. The full-length Eps15R (a doublet of  ̃130 kD) and several truncated spliced forms of  ̃100 kD and  ̃70 kD were detected in Eps15R immunoprecipitates (shown by arrows). The cells were lysed 3 days after transfection. Equal amounts of lysates were resolved by electrophoresis and blotted with the antibody specific to a targeted protein (GFP antibody for YFP fusion proteins) and the antibody specific to unrelated proteins PP1 phosphatase or α-actinin, and TSG101. The amount of protein in each specific band was normalized to the amount of non-specific proteins in each lane of the gel. Nearly all of them appear to be functional, which establishes that Formula VIII and IX can be used to predict siRNAs' functionality in general in a genome wide manner.
To generate the fusion of yellow fluorescent protein (YFP) with Rab5b or Rab5c (YFP-Rab5b or YFP-Rab5c), a DNA fragment encoding the full-length human Rab5b or Rab5c was obtained by PCR using Pfu polymerase (Stratagene) with a SacI restriction site introduced into the 5′ end and a KpnI site into the 3′ end and cloned into pEYFP-C1 vector (CLONTECH, Palo Alto, Calif., USA). GFP-CALM and YFP-Rab5a were described previously Tebar, Bohlander, & Sorkin, Clathrin Assembly Lymphoid Myeloid Leukemia (CALM) Protein: Localization in Endocytic-coated Pits, Interactions with Clathrin, and the Impact of Overexpression on Clathrin-mediated Traffic, Mol. Biol. Cell August 1999, 10:2687.
Example IV
Validation of the Algorithm Using Eg5, GADPH, ATEI, MEK2, MEK1, QB, LaminA/C, c-myc, Human Cyclophilin, and Mouse Cyclophilin
A number of genes have been identified as playing potentially important roles in disease etiology. Expression profiles of normal and diseased kidneys has implicated Edg5 in immunoglobulin A neuropathy, a common renal glomerular disease. Myc1, MEK1/2 and other related kinases have been associated with one or more cancers, while lamins have been implicated in muscular dystrophy and other diseases. For these reasons, siRNA directed against the genes encoding these classes of molecules would be important research and therapeutic tools.
FIG. 12 illustrates four siRNAs targeting 10 different genes (Table V for sequence and accession number information) that were selected according to the Formula VIII and assayed as individuals and pools in HEK293 cells. The level of siRNA induced silencing was measured using the B-DNA assay. These studies demonstrated that thirty-six out of the forty individual SMART-selected siRNA tested are functional (90%) and all 10 pools are fully functional.
Example V
Validation of the Algorithm Using Bcl2
Bcl-2 is a  ̃25 kD, 205-239 amino acid, anti-apoptotic protein that contains considerable homology with other members of the BCL family including BCLX, MCL1, BAX, BAD, and BIK. The protein exists in at least two forms (Bcl2a, which has a hydrophobic tail for membrane anchorage, and Bcl2b, which lacks the hydrophobic tail) and is predominantly localized to the mitochondrial membrane. While Bcl2 expression is widely distributed, particular interest has focused on the expression of this molecule in B and T cells. Bcl2 expression is down-regulated in normal germinal center B cells yet in a high percentage of follicular lymphomas, Bcl2 expression has been observed to be elevated. Cytological studies have identified a common translocation ((14;18)(q32;q32)) amongst a high percentage (>70%) of these lymphomas. This genetic lesion places the Bcl2 gene in juxtaposition to immunoglobulin heavy chain gene (IgH) encoding sequences and is believed to enforce inappropriate levels of gene expression, and resistance to programmed cell death in the follicle center B cells. In other cases, hypomethylation of the Bcl2 promoter leads to enhanced expression and again, inhibition of apoptosis. In addition to cancer, dysregulated expression of Bcl-2 has been correlated with multiple sclerosis and various neurological diseases.
The correlation between Bcl-2 translocation and cancer makes this gene an attractive target for RNAi. Identification of siRNA directed against the bcl2 transcript (or Bcl2-IgH fusions) would further our understanding Bcl2 gene function and possibly provide a future therapeutic agent to battle diseases that result from altered expression or function of this gene.
In Silico Identification of Functional siRNA
To identify functional and hyperfunctional siRNA against the Bcl2 gene, the sequence for Bcl-2 was downloaded from the NCBI Unigene database and analyzed using the Formula VIII algorithm. As a result of these procedures, both the sequence and SMARTscoresTM of the Bcl2 siRNA were obtained and ranked according to their functionality. Subsequently, these sequences were BLAST'ed (database) to insure that the selected sequences were specific and contained minimal overlap with unrealated genes. The SMARTscoresTM for 6 of the top 10 Bcl-2 siRNA (SEQ ID NOs 301-310) are identified in Table V under SEQ. ID NOs. 0409-0414.
In Vivo Testing of Bcl-2 SiRNA
Bcl-2 siRNAs having the top ten SMARTscoresTM were selected and tested in a functional assay to determine silencing efficiency. To accomplish this, each of the ten duplexes were synthesized using 2′-O-ACE chemistry and transfected at 100 nM concentrations into cells. Twenty-four hours later assays were performed on cell extracts to assess the degree of target silencing. Controls used in these experiments included mock transfected cells, and cells that were transfected with a non-specific siRNA duplex.
The results of these experiments are presented below (and in FIG. 14) and show that all ten of the selected siRNA induce 80% or better silencing of the Bcl2 message at 100 nM concentrations. These data verify that the algorithm successfully identified functional Bcl2 siRNA and provide a set of functional agents that can be used in experimental and therapeutic environments.










siRNA
1
GGGAGAUAGUGAUGAAGUA
SEQ. ID NO. 301


 


siRNA
2
GAAGUACAUCCAUUAUAAG
SEQ. ID NO. 302


 


siRNA
3
GUACGACAACCGGGAGAUA
SEQ. ID NO. 303


 


siRNA
4
AGAUAGUGAUGAAGUACAU
SEQ. ID NO. 304


 


siRNA
5
UGAAGACUCUGCUCAGUUU
SEQ. ID NO. 305


 


siRNA
6
GCAUGCGGCCUCUGUUUGA
SEQ. ID NO. 306


 


siRNA
7
UGCGGCCUCUGUUUGAUUU
SEQ. ID NO. 307


 


siRNA
8
GAGAUAGUGAUGAAGUACA
SEQ. ID NO. 308


 


siRNA
9
GGAGAUAGUGAUGAAGUAC
SEQ. ID NO. 309


 


siRNA
10
GAAGACUCUGCUCAGUUUG
SEQ. ID NO. 310






Bcl2 siRNA: Sense Strand, 5′→3′
Example VI
Sequences Selected by the Algorithm
Sequences of the siRNAs selected using Formulas (Algorithms) VIII and IX with their corresponding ranking, which have been evaluated for the silencing activity in vivo in the present study (Formula VIII and IX, respectively).












TABLE V





Gene
Accession


Formula
Formula


Name
Number
SEQ. ID NO.
FTllSeqTence
VIII
IX




















CLTC
NM_004859
SEQ. ID NO. 2400
GAAAGAATCTGTAGAGAAA
76
94.2


 


CLTC
NM_004859
SEQ. ID NO. 2401
GCAATGAGCTGTTTGAAGA
65
39.9


 


CLTC
NM_004859
SEQ. ID NO. 2402
TGACAAAGGTGGATAAATT
57
38.2


 


CLTC
NM_004859
SEQ. ID NO. 2403
GGAAATGGATCTCTTTGAA
54
49.4


 


CLTA
NM_001833
SEQ. ID NO. 2404
GGAAAGTAATGGTCCAACA
22
55.5


 


CLTA
NM_001833
SEQ. ID NO. 2405
AGACAGTTATGCAGCTATT
4
22.9


 


CLTA
NM_001833
SEQ. ID NO. 2406
CCAATTCTCGGAAGCAAGA
1
17


 


CLTA
NM_001833
SEQ. ID NO. 2407
GAAAGTAATGGTCCAACAG
−1
−13


 


CLTB
NM_001834
SEQ. ID NO. 2408
GCGCCAGAGTGAACAAGTA
17
57.5


 


CLTB
NM_001834
SEQ. ID NO. 2409
GAAGGTGGCCCAGCTATGT
15
−8.6


 


CLTB
NM_001834
SEQ. ID NO. 0311
GGAACCAGCGCCAGAGTGA
13
40.5


 


CLTB
NM_001834
SEQ. ID NO. 0312
GAGCGAGATTGCAGGCATA
20
61.7


 


CALM
U45976
SEQ. ID NO. 0313
GTTAGTATCTGATGACTTG
36
−34.6


 


CALM
U45976
SEQ. ID NO. 0314
GAAATGGAACCACTAAGAA
33
46.1


 


CALM
U45976
SEQ. ID NO. 0315
GGAAATGGAACCACTAAGA
30
61.2


 


CALM
U45976
SEQ. ID NO. 0316
CAACTACACTTTCCAATGC
28
6.8


 


EPS15
NM_001981
SEQ. ID NO. 0317
CCACCAAGATTTCATGATA
48
25.2


 


EPS15
NM_001981
SEQ. ID NO. 0318
GATCGGAACTCCAACAAGA
43
49.3


 


EPS15
NM_001981
SEQ. ID NO. 0319
AAACGGAGCTACAGATTAT
39
11.5


 


EPS15
NM_001981
SEQ. ID NO. 0320
CCACAGAGCATTCTTGTAA
33
−23.6


 


EPS15R
NM_021235
SEQ. ID NO. 0321
GAAGTTACCTTGAGCAATC
48
33


 


EPS15R
NM_021235
SEQ. ID NO. 0322
GGACTTGGCCGATCCAGAA
27
33


 


EPS15R
NM_021235
SEQ. ID NO. 0323
GCACTTGGATCGAGATGAG
20
1.3


 


EPS15R
NM_021235
SEQ. ID NO. 0324
CAAAGACCAATTCGCGTTA
17
27.7


 


DNM2
NM_004945
SEQ. ID NO. 0325
CCGAATCAATCGCATCTTC
6
−29.6


 


DNM2
NM_004945
SEQ. ID NO. 0326
GACATGATCCTGCAGTTCA
5
−14


 


DNM2
NM_004945
SEQ. ID NO. 0327
GAGCGAATCGTCACCACTT
5
24


 


DNM2
NM_004945
SEQ. ID NO. 0328
CCTCCGAGCTGGCGTCTAC
−4
−63.6


 


ARF6
AF93885
SEQ. ID NO. 0329
TCACATGGTTAACCTCTAA
27
−21.1


 


ARF6
AF93885
SEQ. ID NO. 0330
GATGAGGGACGGCATAATC
7
−38.4


 


ARF6
AF93885
SEQ. ID NO. 0331
CCTCTAACTACAAATCTTA
4
16.9


 


ARF6
AF93885
SEQ. ID NO. 0332
GGAAGGTGCTATCCAAAAT
4
11.5


 


RAB5A
BC001267
SEQ. ID NO. 0333
GCAAGCAAGTCCTAACATT
40
25.1


 


RAB5A
BC001267
SEQ. ID NO. 0334
GGAAGAGGAGTAGACCTTA
17
50.1


 


RAB5A
BC001267
SEQ. ID NO. 0335
AGGAATCAGTGTTGTAGTA
16
11.5


 


RAB5A
BC001267
SEQ. ID NO. 0336
GAAGAGGAGTAGACCTTAC
12
7


 


RAB5B
NM_002868
SEQ. ID NO. 0337
GAAAGTCAAGCCTGGTATT
14
18.1


 


RAB5B
NM_002868
SEQ. ID NO. 0338
AAAGTCAAGCCTGGTATTA
6
−17.8


 


RAB5B
NM_002868
SEQ. ID NO. 0339
GCTATGAACGTGAATGATC
3
−21.1


 


RAB5B
NM_002868
SEQ. ID NO. 0340
CAAGCCTGGTATTACGTTT
−7
−37.5


 


RAB5C
AF141304
SEQ. ID NO. 0341
GGAAGAAGATCTGTCAATT
38
51.9


 


RAB5C
AF141304
SEQ. ID NO. 0342
GCAATGAACGTGAACGAAA
29
43.7


 


RAB5C
AF141304
SEQ. ID NO. 0343
CAATGAACGTGAACGAAAT
18
43.3


 


RAB5C
AF141304
SEQ. ID NO. 0344
GGACAGGAGGGGTATCACA
6
18.2


 


EEA1
XM_018197
SEQ. ID NO. 0345
AGACAGAGCTTGAGAATAA
67
64.1


 


EEA1
XM_018197
SEQ. ID NO. 0346
GAGAAGATCTTTATGCAAA
60
48.7


 


EEA1
XM_018197
SEQ. ID NO. 0347
GAAGAGAAATGAGCAGATA
58
45.7


 


EEA1
XM_018197
SEQ. ID NO. 0348
GGAAGTAACTCAACTAACA
56
72.3


 


AP2B1
NM_001282
SEQ. ID NO. 0349
GAGCTAATCTGCCACATTG
49
−12.4


 


AP2B1
NM_001282
SEQ. ID NO. 0350
GCAGATGAGTTACTAGAAA
44
48.9


 


AP2B1
NM_001282
SEQ. ID NO. 0351
CAACTTAATTGTCCAGAAA
41
28.2


 


AP2B1
NM_001282
SEQ. ID NO. 0352
CAACACAGGATTCTGATAA
33
−5.8


 


PLK
NM_005030
SEQ. ID NO. 0353
AGATTGTGCCTAAGTCTCT
−35
−3.4


 


PLK
NM_005030
SEQ. ID NO. 0354
ATGAAGATCTGGAGGTGAA
0
−4.3


 


PLK
NM_005030
SEQ. ID NO. 0355
TTTGAGACTTCTTGCCTAA
−5
−27.7


 


PLK
NM_005030
SEQ. ID NO. 0356
AGATCACCCTCCTTAAATA
15
72.3


 


GAPDH
NM_002046
SEQ. ID NO. 0357
CAACGGATTTGGTCGTATT
27
−2.8


 


GAPDH
NM_002046
SEQ. ID NO. 0358
GAAATCCCATCACCATCTT
24
3.9


 


GAPDH
NM_002046
SEQ. ID NO. 0359
GACCTCAACTACATGGTTT
22
−22.9


 


GAPDH
NM_002046
SEQ. ID NO. 0360
TGGTTTACATGTTCCAATA
9
9.8


 


c-Myc

SEQ. ID NO. 0361
GAAGAAATCGATGTTGTTT
31
−11.7


 


c-Myc

SEQ. ID NO. 0362
ACACAAACTTGAACAGCTA
22
51.3


 


c-Myc

SEQ. ID NO. 0363
GGAAGAAATCGATGTTGTT
18
26


 


c-Myc

SEQ. ID NO. 0364
GAAACGACGAGAACAGTTG
18
−8.9


 


MAP2K1
NM_002755
SEQ. ID NO. 0365
GCACATGGATGGAGGTTCT
26
16


 


MAP2K1
NM_002755
SEQ. ID NO. 0366
GCAGAGAGAGCAGATTTGA
16
0.4


 


MAP2K1
NM_002755
SEQ. ID NO. 0367
GAGGTTCTCTGGATCAAGT
14
15.5


 


MAP2K1
NM_002755
SEQ. ID NO. 0368
GAGCAGATTTGAAGCAACT
14
18.5


 


MAP2K2
NM_030662
SEQ. ID NO. 0369
CAAAGACGATGACTTCGAA
37
26.4


 


MAP2K2
NM_030662
SEQ. ID NO. 0370
GATCAGCATTTGCATGGAA
24
−0.7


 


MAP2K2
NM_030662
SEQ. ID NO. 0371
TCCAGGAGTTTGTCAATAA
17
−4.5


 


MAP2K2
NM_030662
SEQ. ID NO. 0372
GGAAGCTGATCCACCTTGA
16
59.2


 


KNSL1(EGS)
NM_004523
SEQ. ID NO. 0373
GCAGAAATCTAAGGATATA
53
35.8


 


KNSL1(EGS)
NM_004523
SEQ. ID NO. 0374
CAACAAGGATGAAGTCTAT
50
18.3


 


KNSL1(EGS)
NM_004523
SEQ. ID NO. 0375
CAGCAGAAATCTAAGGATA
41
32.7


 


KNSL1(EGS)
NM_004523
SEQ. ID NO. 0376
CTAGATGGCTTTCTCAGTA
39
3.9


 


CyclophilinA_
NM_021130
SEQ. ID NO. 0377
AGACAAGGTCCCAAAGACA
−16
58.1


 


CyclophilinA_
NM_021130
SEQ. ID NO. 0378
GGAATGGCAAGACCAGCAA
−6
36


 


CyclophilinA_
NM_021130
SEQ. ID NO. 0379
AGAATTATTCCAGGGTTTA
−3
16.1


 


CyclophilinA_
NM_021130
SEQ. ID NO. 0380
GCAGACAAGGTCCCAAAGA
8
8.9


 


LAMIN A/C
NM_170707
SEQ. ID NO. 0381
AGAAGCAGCTTGAGGATGA
31
38.8


 


LAMIN A/C
NM_170707
SEQ. ID NO. 0382
GAGCTTGACTTCCAGAAGA
33
22.4


 


LAMIN A/C
NM_170707
SEQ. ID NO. 0383
CCACCGAAGTTCACCCTAA
21
27.5


 


LAMIN A/C
NM_170707
SEQ. ID NO. 0384
GAGAAGAGCTCCTCCATCA
55
30.1


 


CyclophilinB
M60857
SEQ. ID NO. 0385
GAAAGAGCATCTACGGTGA
41
83.9


 


CyclophilinB
M60857
SEQ. ID NO. 0386
GAAAGGATTTGGCTACAAA
53
59.1


 


CyclophilinB
M60857
SEQ. ID NO. 0387
ACAGCAAATTCCATCGTGT
−20
28.8


 


CyclophilinB
M60857
SEQ. ID NO. 0388
GGAAAGACTGTTCCAAAAA
2
27


 


DB11
NM_020548
SEQ. ID NO. 0389
CAACACGCCTCATCCTCTA
27
−7.6


 


DB12
NM_020548
SEQ. ID NO. 0390
CATGAAAGCTTACATCAAC
25
−30.8


 


DB13
NM_020548
SEQ. ID NO. 0391
AAGATGCCATGAAAGCTTA
17
22


 


DB14
NM_020548
SEQ. ID NO. 0392
GCACATACCGCCTGAGTCT
15
3.9


 


rLUC1

SEQ. ID NO. 0393
GATCAAATCTGAAGAAGGA
57
49.2


 


rLUC2

SEQ. ID NO. 0394
GCCAAGAAGTTTCCTAATA
50
13.7


 


rLUC3

SEQ. ID NO. 0395
CAGCATATCTTGAACCATT
41
−2.2


 


rLUC4

SEQ. ID NO. 0396
GAACAAAGGAAACGGATGA
39
29.2


 


SeAP1
NM_031313
SEQ. ID NO. 0397
CGGAAACGGTCCAGGCTAT
6
26.9


 


SeAP2
NM_031313
SEQ. ID NO. 0398
GCTTCGAGCAGACATGATA
4
−11.2


 


SeAP3
NM_031313
SEQ. ID NO. 0399
CCTACAGGGTCCTCCTATA
4
4.9


 


SeAP4
NM_031313
SEQ. ID NO. 0400
GCCAAGAACCTCATCATCT
1
−9.9


 


fLUC1

SEQ. ID NO. 0401
GATATGGGCTGAATACAAA
54
40.4


 


fLUC2

SEQ. ID NO. 0402
GCACTCTGATTGACAAATA
47
54.7


 


fLUC3

SEQ. ID NO. 0403
TGAAGTCTCTGATTAAGTA
46
34.5


 


fLUC4

SEQ. ID NO. 0404
TCAGAGAGATCCTCATAAA
40
11.4


 


mCyclo1
NM_008907
SEQ. ID NO. 0405
GCAAGAAGATCACCATTTC
52
46.4


 


mCyclo2
NM_008907
SEQ. ID NO. 0406
GAGAGAAATTTGAGGATGA
36
70.7


 


mCyclo3
NM_008907
SEQ. ID NO. 0407
GAAAGGATTTGGCTATAAG
35
−1.5


 


mCyclo4
NM_008907
SEQ. ID NO. 0408
GAAAGAAGGCATGAACATT
27
10.3


 


BCL2_1
NM_000633
SEQ. ID NO. 0409
GGGAGATAGTGATGAAGTA
21
72


 


BCL2_2
NM_000633
SEQ. ID NO. 0410
GAAGTACATCCATTATAAG
1
3.3


 


BCL2_3
NM_000633
SEQ. ID NO. 0411
GTACGACAACCGGGAGATA
1
35.9


 


BCL2_4
NM_000633
SEQ. ID NO. 0412
AGATAGTGATGAAGTACAT
−12
22.1


 


BCL2_5
NM_000633
SEQ. ID NO. 0413
TGAAGACTCTGCTCAGTTT
36
19.1


 


BCL2_6
NM_000633
SEQ. ID NO. 0414
GCATGCGGCCTCTGTTTGA
5
−9.7


 


QB1
NM_003365.1
SEQ. ID NO. 0415
GCACACAGCUUACUACAUC
52
−4.8


 


QB2
NM_003365.1
SEQ. ID NO. 0416
GAAAUGCCCUGGUAUCUCA
49
22.1


 


QB3
NM_003365.1
SEQ. ID NO. 0417
GAAGGAACGUGAUGUGAUC
34
22.9


 


QB4
NM_003365.1
SEQ. ID NO. 0418
GCACUACUCCUGUGUGUGA
28
20.4


 


ATE1-1
NM_007041
SEQ. ID NO. 0419
GAACCCAGCUGGAGAACUU
45
15.5


 


ATE1-2
NM_007041
SEQ. ID NO. 0420
GAUAUACAGUGUGAUCUUA
40
12.2


 


ATE1-3
NM_007041
SEQ. ID NO. 0421
GUACUACGAUCCUGAUUAU
37
32.9


 


ATE1-4
NM_007041
SEQ. ID NO. 0422
GUGCCGACCUUUACAAUUU
35
18.2


 


EGFR-1
NM_005228
SEQ. ID NO. 0423
GAAGGAAACTGAATTCAAA
68
79.4


 


EGFR-1
NM_005228
SEQ. ID NO. 0424
GGAAATATGTACTACGAAA
49
49.5


 


EGFR-1
NM_005228
SEQ. ID NO. 0425
CCACAAAGCAGTGAATTTA
41
7.6


 


EGFR-1
NM_005228
SEQ. ID NO. 0426
GTAACAAGCTCACGCAGTT
40
25.9









Example VII
Genome-Wide Application of the Algorithm
The examples described above demonstrate that the algorithm(s) can successfully identify functional siRNA and that these duplexes can be used to induce the desirable phenotype of transcriptional knockdown or knockout. Each gene or family of genes in each organism plays an important role in maintaining physiological homeostasis and the algorithm can be used to develop functional, highly functional, or hyperfunctional siRNA to each gene. To accomplish this for the human genome, the entire online ncbi refseq database was accessed through Entrez (efetch). The database was processed through Formula VIII. For each gene the top 80-100 scores for siRNAs were obtained and BLAST'ed to insure that the selected sequences are specific in targeting the gene of choice. These sequences are provided on the enclosed CDs in electronic form.
With respect to the disks, there are four tables on each disk copy in text format: Tables XII-XV. Table XII, which is located in a file entitled Table—12.txt, provides a list of the 80-100 sequences for each target, identified by Formula VIII as having the highest relative SMARTscoresTM for the target analyzed. Table XIII, which is located in a file entitled Table—13.txt, provides the SMARTscoresTM, and for each gene, a pool pick of up to four sequences is denoted. (The denotation of “1” in Table XIII means that it is a pool pick.) These pool pick sequences represent the most functional siRNAs for the corresponding target. Any 1, 2, 3, or 4 of the pool pick sequences could be used for gene silencing. Further, sequences that are not denoted as pool pick sequences, but that are included on the compact disks may also be used for gene silencing either alone or in combination with other sequences. However, their individual relative functionality would be less than that of a pool pick sequence. Table XIV, which is located in a file entitled Table 14.txt, provides an identification of genes by accession number, and Table XV, which is located in a file entitled Table—15.txt, provides a short name for the genes identified on the disk. The information contained on the disks is part of this patent application and are incorporated into the specification by reference. One may use these tables in order to identify functional siRNAs for the gene provided therein, by simply looking for the gene of interest and an siRNA that is listed as functional. Preferably, one would select one or more of the siRNA that most optimized for the target of interest and is denoted as a pool pick.
Table XII: siRNA selected by Formula VIII
See data submitted herewith on a CD-ROM in accordance with PCT Administrative Instructions Section 801(a)
Table XIII: SMARTscoresTM
See data submitted herewith on a CD-ROM in accordance with PCT Administrative Instructions Section 801(a)
Table XIV: Identification of Targets
See data submitted herewith on a CD-ROM in accordance with PCT Administrative Instructions Section 801(a)
Table XV: Description of Targts
See data submitted herewith on a CD-ROM in accordance with PCT Administrative Instructions Section 801(a)
Many of the genes to which the described siRNA are directed play critical roles in disease etiology. For this reason, the siRNA listed in the accompanying compact disk may potentially act as therapeutic agents. A number of prophetic examples follow and should be understood in view of the siRNA that are identified on the accompanying CD. To isolate these siRNA, the appropriate message sequence for each gene is analyzed using one of the before mentioned formulas (preferably formula VIII) to identify potential siRNA targets. Subsequently these targets are BLAST'ed to eliminate homology with potentially off-targets.
The list of potential disease targets is extensive. For instance, over-expression of Bcl10 has been implicated in the development of MALT lymphoma (mucosa associated lymphoid tissue lymphoma) and thus, functional, highly functional, or hyperfunctional siRNA directed against that gene (e.g. SEQ. ID NO. 0427: GGAAACCUCUCAUUGCUAA; SEQ. ID NO. 0428: GAAAGAACCUUGCCGAUCA; SEQ. ID NO. 0429: GGAAAUACAUCAGAGCUUA, or SEQ. ID NO. 0430: GAAAGUAUGUGUCUUAAGU) may contribute to treatment of this disorder.
In another example, studies have shown that molecules that inhibit glutamine:fructose-6-phosphate aminotransferase (GFA) may act to limit the symptoms suffered by Type II diabetics. Thus, functional, highly functional, or hyperfunctional siRNA directed against GFA (also known as GFPT1: siRNA=SEQ. ID NO. 0433 UGAAACGGCUGCCUGAUUU; SEQ. ID NO. 0434 GAAGWUACCUCUUACAUUU; SEQ. ID NO. 0435 GUACGAAACUGUAUGAWUA; SEQ. ID NO. 0436 GGACGAGGCUAUCAUUAUG) may contribute to treatment of this disorder.
In another example, the von Hippel-Lindau (VHL) tumor suppressor has been observed to be inactivated at a high frequency in sporadic clear cell renal cell carcinoma (RCC) and RCCs associated with VHL disease. The VHL tumor suppressor targets hypoxia-inducible factor-1 alpha (HIF-1 alpha), a transcription factor that can induce vascular endothelial growth factor (VEGF) expression, for ubiquitination and degradation. Inactivation of VHL can lead to increased levels of HIF-1 alpha, and subsequent VEGF over expression. Such over expression of VEGF has been used to explain the increased (and possibly necessary) vascularity observed in RCC. Thus, functional, highly functional, or hyperfunctional siRNAs directed against either HIF-1 alpha (SEQ. ID NO. 0437 GAAGGAACCUGAUGCUUUA; SEQ ID NO. 0438 GCAUAUAUCUAGAAGGUAU; SEQ. ID NO. 0439 GAACAAAUACAUGGGAUUA; SEQ. ID NO. 0440 GGACACAGAUUUAGACUUG) or VEGF (SEQ. ID NO. 0441 GAACGUACUUGCAGAUGUG; SEQ. ID NO. 0442 GAGAAAGCAUUUGUUUGUA; SEQ. ID NO. 0443 GGAGAAAGCAUUUGUUUGU; SEQ. ID NO. 0444 CGAGGCAGCUUGAGUUAAA) may be useful in the treatment of renal cell carcinoma.
In another example, gene expression of platelet derived growth factor A and B (PDGF-A and PDGF-B) has been observed to be increased 22- and 6-fold, respectively, in renal tissues taken from patients with diabetic nephropathy as compared with controls. These findings suggest that over expression of PDGF A and B may play a role in the development of the progressive fibrosis that characterizes human diabetic kidney disease. Thus, functional, highly functional, or hyperfunctional siRNAs directed against either PDGF A (SEQ. ID NO. 0445: GGUAAGAUAWUGUGCUUUA; SEQ. ID NO. 0446: CCGCAAAUAUGCAGAAUUA; SEQ. ID NO. 0447: GGAUGUACAUGGCGUGUUA; SEQ. ID NO. 0448: GGUGAAGUUUGUAUGWUUUA) or PDGF B (SEQ. ID NO. 0449: CCGAGGAGCWUUAUGAGAU; SEQ. ID NO. 0450: GCUCCGCGCUUUCCGAUUU; SEQ. ID NO. 0451 GAGCAGGAAUGGUGAGAUG; SEQ. ID NO. 0452: GAACUUGGGAUAAGAGUGU; SEQ. ID NO. 0453 CCGAGGAGCUUUAUGAGAU; SEQ. ID NO. 0454 UUUAUGAGAUGCUGAGUGA) may be useful in the treatment of this form of kidney disorder.
In another example, a strong correlation exists between the over-expression of glucose transporters (e.g. GLUT12) and cancer cells. It is predicted that cells undergoing uncontrolled cell growth up-regulate GLUT molecules so that they can cope with the heightened energy needs associated with increased rates of proliferation and metastasis. Thus, siRNA-based therapies that target the molecules such as GLUT1 (also known as SLC2A1: siRNA=SEQ. ID NO.: 0455 GCAAUGAUGUCCAGAAGAA; SEQ. ID NO.: 0456 GAAGAAUAUUCAGGACUUA; SEQ. ID NO.: 0457 GAAGAGAGUCGGCAGAUGA; SEQ. ID NO.: 0458 CCAAGAGUGUGCUAAAGAA) GLUT12 (also known as SLCA12: siRNA SEQ. ID NO. 0459: GAGACACUCUGAAAUGAUA; SEQ. ID NO. 0460: GAAAUGAUGUGGAUAAGAG; SEQ. ID NO. 0461: GAUCAAAUCCUCCCUGAAA; SEQ. ID NO. 0462: UGAAUGAGCUGAUGAUUGU) and other related transporters, may be of value in treating a multitude of malignancies.
The siRNA sequences listed above are presented in a 5′ 3′ sense strand direction. In addition, siRNA directed against the targets listed above as well as those directed against other targets and listed in the accompanying compact disk may be useful as therapeutic agents.
Example VIII
Evidence for the Benefits of Pooling
Evidence for the benefits of pooling have been demonstrated using the Reporter gene, luciferase. Ninety siRNA duplexes were synthesized using Dharmacon proprietary ACE® chemistry against one of the standard reporter genes: firefly luciferase. The duplexes were designed to start two base pairs apart and to cover approximately 180 base pairs of the luciferase gene (see sequences in Table IV). Subsequently, the siRNA duplexes were co-transfected with a luciferase expression reporter plasmid into HEK293 cells using standard transfection protocols and luciferase activity was assayed at 24 and 48 hours.
Transfection of individual siRNAs showed standard distribution of inhibitory effect. Some duplexes were active, while others were not. FIG. 15 represents a typical screen of ninety siRNA duplexes (SEQ. ID NO. 0032-0120) positioned two base pairs apart. As the figure suggests, the functionality of the siRNA duplex is determined more by a particular sequence of the oligonucleotide than by the relative oligonucleotide position within a gene or excessively sensitive part of the mRNA, which is important for traditional anti-sense technology.
When two continuous oligonucleotides were pooled together, a significant increase in gene silencing activity was observed (see FIGS. 16A and 16B). A gradual increase in efficacy and the frequency of pools functionality was observed when the number of siRNAs increased to 3 and 4 (see FIGS. 16A, 16B, 17A, and 17B). Further, the relative positioning of the oligonucleotides within a pool did not determine whether a particular pool was functional (see FIGS. 18A and 18B, in which 100% of pools of oligonucleotides distanced by 2, 10 and 20 base pairs were functional).
However, relative positioning may nonetheless have an impact. An increased functionality may exist when the siRNA are positioned continuously head to toe (5′ end of one directly adjacent to the 3′ end of the others).
Additionally, siRNA pools that were tested performed at least as well as the best oligonucleotide in the pool, under the experimental conditions whose results are depicted in FIG. 19. Moreover, when previously identified non-functional and marginally (semi) functional siRNA duplexes were pooled together in groups of five at a time, a significant functional cooperative action was observed. (See FIG. 20) In fact, pools of semi-active oligonucleotides were 5 to 25 times more functional than the most potent oligonucleotide in the pool. Therefore, pooling several siRNA duplexes together does not interfere with the functionality of the most potent siRNAs within a pool, and pooling provides an unexpected significant increase in overall functionality
Example IX
Pooling Across Species
Experiments were performed on the following genes: β-galactosidase, Renilla luciferase, and Secreted alkaline phosphatase, which demonstrates the benefits of pooling. (see FIGS. 21A, 21B and 21C) Approximately 50% of individual siRNAs designed to silence the above-specified genes were functional, while 100% of the pools that contain the same siRNA duplexes were functional.
Example X
Highly Functional siRNA
Pools of five siRNAs in which each two siRNAs overlap to 10-90% resulted in 98% functional entities (>80% silencing). Pools of siRNAs distributed throughout the mRNA that were evenly spaced, covering an approximate 20-2000 base pair range, were also functional. When the pools of siRNA were positioned continuously head to tail relative to mRNA sequences and mimicked the natural products of Dicer cleaved long double stranded RNA, 98% of the pools evidenced highly functional activity (>95% silencing).
Example XI
Human Cyclophyline
Table IV above lists the siRNA sequences for the human cyclophyline protein. A particularly functional siRNA may be selected by applying these sequences to any of Formula I to VII above.
Alternatively, one could pool 2, 3, 4, 5 or more of these sequences to create a kit for silencing a gene. Preferably, within the kit there would be at least one sequence that has a relatively high predicted functionality when any of Formulas 1-VII is applied.
Example XII
Sample Pools of siRNAs and their Application to Human Disease
The genetic basis behind human disease is well documented and siRNA may be used as both research or diagnostic tools and therapeutic agents, either individually or in pools. Genes involved in signal transduction, the immune response, apoptosis, DNA repair, cell cycle control, and a variety of other physiological functions have clinical relevance and therapeutic agents that can modulate expression of these genes may alleviate some or all of the associated symptoms. In some instances, these genes can be described as a member of a family or class of genes and siRNA (randomly, conventionally, or rationally designed) can be directed against one or multiple members of the family to induce a desired result.
To identify rationally designed siRNA to each gene, the sequence was analyzed using Formula VIII to identify a SMARTpool containing the functional sequences. To confirm the activity of these sequences, the siRNA are introduced into a cell type of choice (e.g HeLa cells, HEK293 cells) and the levels of the appropriate message are analyzed using one of several art proven techniques. SiRNA having heightened levels of potency can be identified by testing each of the before mentioned duplexes at increasingly limiting concentrations. Similarly, siRNA having increased levels of longevity can be identified by introducing each duplex into cells and testing functionality at 24, 48, 72, 96, 120, 144, 168, and 192 hours after transfection. Agents that induce >95% silencing at sub-nanomolar concentrations and/or induce functional levels of silencing for >96 hours are considered hyperfunctional.
The following are non-limiting examples of families of proteins to which siRNA described in this document are targeted against:
Transporters, Pumps, and Channels
Transporters, pumps, and channels represent one class of genes that are attractive targets for siRNAs. One major class of transporter molecules are the ATP-binding cassette (ABC) transporters. To date, nearly 50 human ABC-transporter genes have been characterized and have been shown to be involved in a variety of physiological functions including transport of bile salts, nucleosides, chloride ions, cholesterol, toxins, and more. Predominant among this group are MDR1 (which encodes the P-glycoprotein, NP—000918), the MDR-related proteins (MRP1-7), and the breast cancer resistance protein (BCRP). In general, these transporters share a common structure, with each protein containing a pair of ATP-binding domains (also known as nucleotide binding folds, NBF) and two sets of transmembrane (TM) domains, each of which typically contains six membrane-spanning α-helices. The genes encoding this class of transporter are organized as either full transporters (i.e. containing two TM and two NBF domains) or as half transporters that assemble as either homodimers or heterodimers to create functional transporters. As a whole, members of the family are widely dispersed throughout the genome and show a high degree of amino acid sequence identify among eukaryotes.
ABC-transporters have been implicated in several human diseases. For instance, molecular efflux pumps of this type play a major role in the development of drug resistance exhibited by a variety of cancers and pathogenic microorganisms. In the case of human cancers, increased expression of the MDR1 gene and related pumps have been observed to generate drug resistance to a broad collection of commonly used chemotherapeutics including doxorubicin, daunorubicin, vinblastine, vincristine, colchicines. In addition to the contribution these transporters make to the development of multi-drug resistance, there are currently 13 human genetic diseases associated with defects in 14 different transporters. The most common of these conditions include cystic fibrosis, Stargardt disease, age-related macular degeneration, adrenoleukodystrophy, Tangier disease, Dubin-Johnson syndrome and progressive familial intrahepatic cholestasis. For this reason, siRNAs directed against members of this, and related, families are potentially valuable research and therapeutic tools.
With respect to channels, analysis of Drosophila mutants has enabled the initial molecular isolation and characterization of several distinct channels including (but not limited to) potassium (K+) channels. This list includes shaker (Sh), which encodes a voltage activated K+ channel, slowpoke (Slo), a Ca2+ activated K+ channel, and ether-a-go-go (Eag). The Eag family is further divided into three subfamilies: Eag, Elk (eag-like K channels), and Erg (Eag related genes).
The Erg subfamily contains three separate family members (Erg 1-3) that are distantly related to the sh family of voltage activated K+ channels. Like sh, erg polypetides contain the classic six membrane spanning architecture of K+ channels (S1-S6) but differ in that each includes a segment associated with the C-terminal cytoplasmic region that is homologous to cyclic nucleotide binding domains (cNBD). Like many isolated ion channel mutants, erg mutants are temperature-sensitive paralytics, a phenotype caused by spontaneous repetitive firing (hyperactivity) in neurons and enhanced transmitter release at the neuromuscular junction.
Initial studies on the tissue distribution of all three members of the erg subfamily show two general patterns of expression. Erg1 and erg3 are broadly expressed throughout the nervous system and are observed in the heart, the superior mesenteric ganglia, the celiac ganglia, the retina, and the brain. In contrast, erg2 shows a much more restricted pattern of expression and is only observed in celiac ganglia and superior mesenteric ganglia. Similarly, the kinetic properties of the three erg potassium channels are not homogeneous. Erg1 and erg2 channels are relatively slow activating delayed rectifiers whereas the erg3 current activates rapidly and then exhibits a predominantly transient component that decays to a sustained plateau. The current properties of all three channels are sensitive to methanesulfonanilides, suggesting a high degree of conservation in the pore structure of all three proteins.
Recently, the erg family of K+ channels has been implicated in human disease. Consistent with the observation that erg1 is expressed in the heart, single strand conformation polymorphism and DNA sequence analyses have identified HERG (human erg1) mutations in six long-QT-syndrome (LQT) families, an inherited disorder that results in sudden death from a ventricular tachyarrythmia. Thus siRNA directed against this group of molecules (e.g. KCNH1-8) will be of extreme therapeutic value.
Another group of channels that are potential targets of siRNAs are the CLCA family that mediate a Ca2+-activated Cl− conductance in a variety of tissues. To date, two bovine (bCLC1; bCLCA2 (Lu-ECAM-1)), three mouse (mCLCA1; mCLCA2; mCLCA3) and four human (hCLCA1; hCLCA2; hCLCA3; hCLCA4) CLCA family members have been isolated and patch-clamp studies with transfected human embryonic kidney (HEK-293) cells have shown that bCLCA1, mCLCA1, and hCLCA1 mediate a Ca2+-activated Cl− conductance that can be inhibited by the anion channel blocker DIDS and the reducing agent dithiothreitol (DTT).
The protein size, structure, and processing seem to be similar among different CLCA family members and has been studied in greatest detail for Lu-ECAM-1. The Lu-ECAM-1 open reading frame encodes a precursor glycoprotein of 130 kDa that is processed to a 90-kDa amino-terminal cleavage product and a group of 30- to 40-kDa glycoproteins that are glycosylation variants of a single polypeptide derived from its carboxy terminus. Both subunits are associated with the outer cell surface, but only the 90-kDa subunit is thought to be anchored to the cell membrane via four transmembrane domains.
Although the protein processing and function appear to be conserved among CLCA homologs, significant differences exist in their tissue expression patterns. For example, bovine Lu-ECAM-1 is expressed primarily in vascular endothelia, bCLCA1 is exclusively detected in the trachea, and hCLCA1 is selectively expressed in a subset of human intestinal epithelial cells. Thus the emerging picture is that of a multigene family with members that are highly tissue specific, similar to the ClC family of voltage-gated Cl− channels. The human channel, hCLCA2, is particular interesting from a medical and pharmacological standpoint. CLCA2 is expressed on the luminal surface of lung vascular endothelia and serves as an adhesion molecule for lung metastatic cancer cells, thus mediating vascular arrest and lung colonization. Expression of this molecule in normal mammary epithelium is consistently lost in human breast cancer and in nearly all tumorigenic breast cancer cell lines. Moreover, re-expression of hCLCA2 in human breast cancer cells abrogates tumorigenicity in nude mice, implying that hCLCA2 acts as a tumour suppressor in breast cancer. For these reasons, siRNA directed against CLCA family members and related channels may prove to be valuable in research and therapeutic venues.
Transporters Involved in Synaptic Transmission.
Synaptic transmission involves the release of a neurotransmitter into the synaptic cleft, interaction of that transmitter with a postsynaptic receptor, and subsequent removal of the transmitter from the cleft. In most synapses the signal is terminated by a rapid reaccumulation of the neurotransmitter into presynaptic terminals. This process is catalyzed by specific neurotransmitter transporters that are often energized by the electrochemical gradient of sodium across the plasma membrane of the presynaptic cells.
Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the central nervous system. The inhibitory action of GABA, mediated through GABAA/GABAB receptors, and is regulated by GABA transporters (GATs), integral membrane proteins located perisynaptically on neurons and glia. So far four different carriers (GAT1-(GAT4) have been cloned and their cellular distribution has been partly worked out. Comparative sequence analysis has revealed that GABA transporters are related to several other proteins involved in neurotransmitter uptake including gamma-aminobutyric acid transporters, monoamine transporters, amino acid transporters, certain “orphan” transporters, and the recently discovered bacterial transporters. Each of these proteins has a similar 12 transmembrane helices topology and relies upon the Na+/Cl− gradient for transport function. Transport rates are dependent on substrate concentrations, with half-maximal effective concentrations for transport frequently occurring in the submicromolar to low micromolar range. In addition, transporter function is bidirectional, and non-vesicular efflux of transmitter may contribute to ambient extracellular transmitter levels.
Recent evidence suggests that GABA transporters, and neurotransmitter transporters in general, are not passive players in regulating neuronal signaling; rather, transporter function can be altered by a variety of initiating factors and signal transduction cascades. In general, this functional regulation occurs in two ways, either by changing the rate of transmitter flux through the transporter or by changing the number of functional transporters on the plasma membrane. A recurring theme in transporter regulation is the rapid redistribution of the transporter protein between intracellular locations and the cell surface. In general, this functional modulation occurs in part through activation of second messengers such as kinases, phosphatases, arachidonic acid, and pH. However, the mechanisms underlying transporter phosphorylation and transporter redistribution have yet to be fully elucidated.
GABA transporters play a pathophysiological role in a number of human diseases including temporal lobe epilepsy and are the targets of pharmacological interventions. Studies in seizure sensitive animals show some (but not all) of the GAT transporters have altered levels of expression at times prior to and post seizure, suggesting this class of transporter may affect epileptogenesis, and that alterations following seizure may be compensatory responses to modulate seizure activity. For these reasons, siRNAs directed against members of this family of genes (including but not limited to SLCG6A1-12) may prove to be valuable research and therapeutic tools.
Organic Ion Transporters.
The human body is continuously exposed to a great variety of xenobiotics, via food, drugs, occupation, and environment. Excretory organs such as kidney, liver, and intestine defend the body against the potentially harmful effects of these compounds by transforming them into less active metabolites that are subsequently secreted from the system.
Carrier-mediated transport of xenobiotics and their metabolites exist for the active secretion of organic anions and cations. Both systems are characterized by a high clearance capacity and tremendous diversity of substances accepted, properties that result from the existance of multiple transporters with overlapping substrate specificities. The class of organic anion transporters plays a critical role in the elimination of a large number of drugs (e.g., antibiotics, chemotherapeutics, diuretics, nonsteroidal anti-inflammatory drugs, radiocontrast agents, cytostatics); drug metabolites (especially conjugation products with glutathione, glucuronide, glycine, sulfate, acetate); and toxicants and their metabolites (e.g., mycotoxins, herbicides, plasticizers, glutathione S-conjugates of polyhaloalkanes, polyhaloalkenes, hydroquinones, aminophenols), many of which are specifically harmful to the kidney.
Over the past couple of years the number of identified anion transporting molecules has grown tremendously. Uptake of organic anions (OA−) across the basolateral membrane is mediated by the classic sodium-dependent organic anion transport system, which includes α-ketoglutarate (α-KG2−)/OA− exchange via the organic anion transporter (OAT1) and sodium-ketoglutarate cotransport via the Na+/dicarboxylate cotransporter (SDCT2). The organic anion transporting polypetide, Oatp1, and the kidney-specific OAT-K1 and OAT-K2 are seen as potential molecules that mediate facilitated OA− efflux but could also be involved in reabsorption via an exchange mechanism. Lastly the PEPT1 and PEPT2 mediate luminal uptake of peptide drugs, whereas CNT1 and CNT2 are involved in reabsorption of nucleosides
The organic anion-transporting polypeptide 1 (Oatp1) is a Na+- and ATP-independent transporter originally cloned from rat liver. The tissue distribution and transport properties of the Oatp1 gene product are complex. Oatp1 is localized to the basolateral membrane of hepatocytes, and is found on the apical membrane of S3 proximal tubules. Studies with transiently transfected cells (e.g. HeLa cells) have indicated that Oatp1 mediates transport of a variety of molecules including taurocholate, estrone-3-sulfate, aldosterone, cortisol, and others. The observed uptake of taurocholate by Oatp1 expressed in X. laevis oocytes is accompanied by efflux of GSH, suggesting that transport by this molecule may be glutathione dependent.
Computer modeling suggests that members of the Oatp family are highly conserved, hydrophobic, and have 12 transmembrane domains. Decreases in expression of Oatp family members have been associated with cholestatic liver diseases and human hepatoblastomas, making this family of proteins of key interest to researchers and the medical community. For these reasons, siRNAs directed against OAT family members (including but not limited to SLC21A2, 3, 6, 8, 9, 11, 12, 14, 15, and related transporters) are potentially useful as research and therapeutic tools.
Nucleoside Transporters.
Nucleoside transporters play key roles in physiology and pharmacology. Uptake of exogenous nucleosides is a critical first step of nucleotide synthesis in tissues such as bone marrow and intestinal epithelium and certain parasitic organisms that lack de novo pathways for purine biosynthesis. Nucleoside transporters also control the extracellular concentration of adenosine in the vicinity of its cell surface receptors and regulate processes such as neurotransmission and cardiovascular activity. Adenosine itself is used clinically to treat cardiac arrhythmias, and nucleoside transport inhibitors such as dipyridamole, dilazep, and draflazine function as coronary vasodilators.
In mammals, plasma membrane transport of nucleosides is brought about by members of the concentrative, Na+-dependent (CNT) and equilibrative, Na+-independent (ENT) nucleoside transporter families. CNTs are expressed in a tissue-specific fashion; ENTs are present in most, possibly all, cell types and are responsible for the movement of hydrophilic nucleosides and nucleoside analogs down their concentration gradients. In addition, structure/function studies of ENT family members have predicted these molecules to contain eleven transmembrane helical segments with an amino terminus that is intracellular and a carboxyl terminus that is extracellular. The proteins have a large glycosylated loop between TMs 1 and 2 and a large cytoplasmic loop between TMs 6 and 7. Recent investigations have implicated the TM 3-6 region as playing a central role in solute recognition. The medical importance of the ENT family of proteins is broad. In humans adenosine exerts a range of cardioprotective effects and inhibitors of ENTs are seen as being valuable in alleviating a variety of cardio/cardiovascular ailments. In addition, responses to nucleoside analog drugs has been observed to vary considerably amongst e.g. cancer patients. While some forms of drug resistance have been shown to be tied to the up-regulation of ABC-transporters (e.g. MDR1), resistance may also be the result of reduced drug uptake (i.e. reduced ENT expression). Thus, a clearer understanding of ENT transporters may aid in optimizing drug treatments for patients suffering a wide range of malignancies. For these reasons, siRNAs directed against this class of molecules (including SLC28A1-3, SLC29A1-4, and related molecules) may be useful as therapeutic and research tools. Sulfate Transporters.
All cells require inorganic sulfate for normal function. Sulfate is the fourth most abundant anion in human plasma and is the major source of sulfur in many organisms. Sulfation of extracellular matrix proteins is critical for maintaining normal cartilage metabolism and sulfate is an important constituent of myelin membranes found in the brain
Because sulfate is a hydrophilic anion that cannot passively cross the lipid bilayer of cell membranes, all cells require a mechanism for sulfate influx and efflux to ensure an optimal supply. To date, a variety of sulfate transporters have been identified in tissues from many origins. These include the renal sulfate transporters (NaSi-1 and Sat-1), the ubiquitously expressed diastrophic dysplasia sulfate transporter (DTDST), the intestinal sulfate transporter (DRA), and the erythrocyte anion exchanger (AE1). Most, if not all, of these molecules contain the classic 12 transmembrane spanning domain architecture commonly found amongst members of the anion transporter superfamily.
Recently three different sulfate transporters have been associated with specific human genetic diseases. Family members SLC26A2, SLC26A3, and SLC26A4 have been recognized as the disease genes mutated in diastrophic dysplasia, congenital chloride diarrhea (CLD), and Pendred syndrome (PDS), respectively. DTDST is a particularly complex disorder. The gene encoding this molecule maps to chromosome 5q, and encodes two distinct transcripts due to alternative exon usage. In contrast to other sulfate transporters (e.g. Sat-1) anion movement by the DTDST protein is markedly inhibited by either extracellular chloride or bicarbonate. Impaired function of the DTDST gene product leads to undersulfation of proteoglycans and a complex family of recessively inherited osteochondrodysplasias (achondrogenesis type I B, atelosteogenesis type II, and diastrophic dysplasia) with clinical features including but not limited to, dwarfism, spinal deformation, and specific joint abnormalities. Interestingly, while epidemiological studies have shown that the disease occurs in most populations, it is particularly prevalent in Finland owing to an apparent founder effect. For these reasons, siRNAs directed against this class of genes (including but not limited to SLC26A1-9, and related molecules) may be potentially helpful in both therapeutic and research venues.
Ion Exchangers
Intracellular pH regulatory mechanisms are critical for the maintenance of countless cellular processes. For instance, in muscle cells, contractile processes and metabolic reactions are influenced by pH. During periods of increased energy demands and ischemia, muscle cells produce large amounts of lactic acid that, without quick and efficient disposal, would lead to acidification of the sarcoplasm.
Several different transport mechanisms have evolved to maintain a relatively constant intracellular pH. The relative contribution of each of these processes varies with cell type, the metabolic requirements of the cell, and the local environmental conditions. Intracellular pH regulatory processes that have been characterized functionally include but are not limited to the Na+/H+ exchange, the Na(HCO3)n cotransport, and the Na+-dependent and -independent Cl−/base exchangers. As bicarbonate and CO2 comprise the major pH buffer of biological fluids, sodium biocarbonate cotransporters (NBCs) are critical. Studies have shown that these molecules exist in numerous tissues including the kidney, brain, liver, cornea, heart, and lung, suggesting that NBCs play an important role in mediating HCO3− transport in both epithelial as well as nonepithelial cells.
Recent molecular cloning experiments have identified the existence of four NBC isoforms (NBC1, 2, 3 and 4) and two NBC-related proteins, AE4 and NCBE (Anion Exchanger 4 and Na-dependent Chloride-Bicarbonate Exchanger). The secondary structure analyses and hydropathy profile of this family predict them to be intrinsic membrane proteins with 12 putative transmembrane domains and several family members exhibit N-linked glycosylation sites, protein kinases A and C, casein kinase II, and ATP/GTP-binding consensus phosphorylation sites, as well as potential sites for myristylation and amidation. AE4 is a relatively recent addition to this family of proteins and shows between 30-48% homology with the other family members. When expressed in COS-7 cells and Xenopus oocytes AE4 exhibits sodium-independent and DIDS-insensitive anion exchanger activity. Exchangers have been shown to be responsible for a variety of human diseases For instance, mutations in three genes of the anion transporter family (SLC) are believed to cause known hereditary diseases, including chondrodysplasia (SLC26A2, DTD), diarrhea (A3, down-regulated in adenoma/chloride-losing diarrhea protein: DRA/CLD), and goiter/deafness syndrome (A4, pendrin). Moreover, mutations in Na+/HCO3 co-transporters have also been associated with various human maladies. For these reasons, siRNAs directed against these sorts of genes (e.g. SLC4A4-10, and related genes) may be useful for therapeutic and research purposes.
Receptors Involved in Synaptic Transmission
In all vertebrates, fast inhibitory synaptic transmission is the result of the interaction between the neurotransmitters glycine (Gly) and γ-aminobutyric acid (GABA) and their respective receptors. The strychnine-sensitive glycine receptor is especially important in that it acts in the mammalian spinal cord and brain stem and has a well-established role in the regulation of locomotor behavior.
Glycine receptors display significant sequence homology to several other receptors including the nicotinic acetylcholine receptor, the aminobutyric acid receptor type A (GABAAR), and the serotonin receptor type 3 (5-HT3R) subunits. As members of the superfamily of ligand-gated ion channels, these polypeptides share common topological features. The glycine receptor is composed of two types of glycosylated integral membrane proteins (α1-α4 and β) arranged in a pentamenc suprastructure. The alpha subunit encodes a large extracellular, N-terminal domain that carries the structural determinants essential for agonist and antagonist binding, followed by four transmembrane spanning regions (TM1-TM4), with TM2 playing the critical role of forming the inner wall of the chloride channel.
The density, location, and subunit composition of glycine neurotransmitter receptors changes over the course of development. It has been observed that the amount of GlyR gene translation (assessed by the injection of developing rat cerebral cortex mRNA into Xenopus oocytes) decreases with age, whereas that of GABARs increases. In addition, the type and location of mRNAs coding for GlyR changes over the course of development. For instance in a study of the expression of alpha 1 and alpha 2 subunits in the rat, it was observed that (in embryonic periods E11-18) the mantle zone was scarce in the alpha 1 mRNA, but the germinal zone (matrix layer) at E11-14 expressed higher levels of the message. At postnatal day 0 (P0), the alpha 1 signals became manifested throughout the gray matter of the spinal cord. By contrast, the spinal tissues at P0 exhibited the highest levels of alpha 2 mRNA, which decreased with the postnatal development.
In both, man and mouse mutant lines, mutations of GlyR subunit genes result in hereditary motor disorders characterized by exaggerated startle responses and increased muscle tone. Pathological alleles of the Glra1 gene are associated with the murine phenotypes oscillator (spdot) and spasmodic (spd). Similarly, a mutant allele of Glrb has been found to underly the molecular pathology of the spastic mouse (spa). Resembling the situation in the mouse, a variety of GLRA1 mutant alleles have been shown to be associated with the human neurological disorder hyperekplexia or startle disease. For these reasons, siRNA directed against glycine receptors (GLRA1-3, GLRB, and related molecules), glutamate receptors, GABA receptors, ATP receptors, and related neurotransmitter receptor molecules may be valuable therapeutic and research reagents.
Proteases
Kallikreins
One important class of proteases are the kallikreins, serine endopeptidases that split peptide substrates preferentially on the C-terminal side of internal arginyl and lysyl residues Kallikreins are generally divided into two distinct groups, plasma kallikreins and tissue kallikreins. Tissue kallikreins represent a large group of enzymes that have substantial similarities at both the gene and protein level. The genes encoding this group are frequently found on a single chromosome, are organized in clusters, and are expressed in a broad range of tissues (e.g. pancreas, ovaries, breast). In contrast, the plasma form of the enzyme is encoded by a single gene (e.g KLK3) that has been localized to chromosome 4q34-35 in humans. The gene encoding plasma kallikrein is expressed solely in the liver, contains 15 exons, and encodes a glycoprotein that is translated as a preprotein called prekallikrein Kallikreins are believed to play an important role in a host of physiological events For instance, the immediate consequence of plasma prekallikrein activation is the cleavage of high molecular weight kininogen (HK) and the subsequent liberation of bradykinin, a nine amino acid vasoactive peptide that is an important mediator of inflammatory responses. Similarly, plasma kallikrein promotes single-chain urokinase activation and subsequent plasminogen activation, events that are critical to blood coaggulation and wound healing.
Disruptions in the function of kallikreins have been implicated in a variety of pathological processes including imbalances in renal function and inflammatory processes. For these reasons, siRNAs directed against this class of genes (e.g. KLK1-15) may prove valuable in both research and therapeutic settings.
ADAM Proteins
The process of fertilization takes place in a series of discrete steps whereby the sperm interacts with, i) the cumulus cells and the hyaluronic acid extracellular matrix (ECM) in which they are embedded, ii) the egg's own ECM, called the zona pellucida (ZP), and iii) the egg plasma membrane. During the course of these interactions, the “acrosome reaction,” the exocytosis of the acrosome vesicle on the head of the sperm, is induced, allowing the sperm to penetrate the ZP and gain access to the perivitelline space. This process exposes new portions of the sperm membrane, including the inner acrosomal membrane and the equatorial segment, regions of the sperm head that can participate in initial gamete membrane binding.
The interactions of the gamete plasma membranes appear to involve multiple ligands and receptors and are frequently compared to leukocyte-endothelial interactions. These interactions lead to a series of signal transduction events in the egg, known as collectively as egg activation and include the initiation of oscillations in intracellular calcium concentration, the exit from meiosis, the entry into the first embryonic mitosis, and the formation of a block to polyspermy via the release of ZP-modifying enzymes from the egg's cortical granules. Ultimately, sperm and egg not only adhere to each other but also go on to undergo membrane fusion, making one cell (the zygote) from two.
Studies on the process of sperm-egg interactions have identified a number of proteins that are crucial for fertilization. One class of proteins, called the ADAM family (A Disintegrin And Metalloprotease), has been found to be important in spermatogenesis and fertilization, as well as various developmental systems including myogenesis and neurogenesis. Members of the family contain a disintegrin and metalloprotease domain (and therefore have (potentially) both cell adhesion and protease activities), as well as cysteine-rich regions, epidermal growth factor (EGF)-like domains, a transmembrane region, and a cytoplasmic tail. Currently, the ADAM gene family has 29 members and constituents are widely distributed in many tissues including the brain, testis, epididymis, ovary, breast, placenta, liver, heart, lung, bone, and muscle.
One of the best-studied members of the ADAM family is fertilin, a heterodimeric protein comprised of at least two subunits, fertilin alpha and fertilin beta. The fertilin beta gene (ADAM2) has been disrupted with a targeting gene construct corresponding to the exon encoding the fertilin beta disintegrin domain. Sperm from males homozygous for disruptions in this region exhibit defects in multiple facets of sperm function including reduced levels of sperm transit from the uterus to the oviduct, reduced sperm-ZP binding, and reduced sperm-egg binding, all of which contribute to male infertility.
Recently, four new ADAM family members (ADAM 24-27) have been isolated. The deduced amino acid sequences show that all four contain the complete domain organization common to ADAM family members and Northern Blot analysis has shown all four to be specific to the testes. SiRNAs directed against this class of genes (e.g. ADAM2 and related proteins) may be useful as research tools and therapeutics directed toward fertility and birth control.
Aminopeptidases
Aminopeptidases are proteases that play critical roles in processes such as protein maturation, protein digestion in its terminal stage, regulation of hormone levels, selective or homeostatic protein turnover, and plasmid stabilization. These enzymes generally have broad substrate specificity, occur in several forms and play a major role in physiological homeostasis. For instance, the effects of bradykinin, angiotensin converting enzyme (ACE), and other vasoactive molecules are muted by one of several peptidases that cleave the molecule at an internal position and eliminate its ability to bind its cognate receptor (e.g. for bradykinin, the B2-receptor).
Among the enzymes that can cleave bradykinin is the membrane bound aminopeptidase P, also referred to as aminoacylproline aminopeptidase, proline aminopeptidase; X-Pro aminopeptidase (eukaryote) and XPNPEP2. Aminopeptidase P is an aminoacylproline aminopeptidase specific for NH2-terminal Xaa-proline bonds. The enzyme i) is a mono-zinc-containing molecule that lacks any of the typical metal binding motifs found in other zinc metalloproteases, ii) has an active-site configuration similar to that of other members of the MG peptidase family, and iii) is present in a variety of tissues including but not limited to the lung, kidney, brain, and intestine.
Aminopeptidases play an important role in a diverse set of human diseases. Low plasma concentrations of aminopeptidase P are a potential predisposing factor for development of angio-oedema in patients treated with ACE inhibitors, and inhibitors of aminopeptidase P may act as cardioprotectors against other forms of illness including, but not limited to myocardial infarction. For these reasons, siRNAs directed against this family of proteins (including but not limited to XPNPEP1 and related proteins) may be useful as research and therapeutic tools.
Serine Proteases
One important class of proteases are the serine proteases. Serine proteases share a common catalytic triad of three amino acids in their active site (serine (nucleophile), aspartate (electrophile), and histidine (base)) and can hydrolyze either esters or peptide bonds utilizing mechanisms of covalent catalysis and preferential binding of the transition state. Based on the position of their introns serine proteases have been classified into a minimum of four groups including those in which 1) the gene has no introns interrupting the exon coding for the catalytic triad (e.g. the haptoglobin gene,); 2) each gene contains an intron just downstream from the codon for the histidine residue at the active site, a second intron downstream from the exon containing the aspartic acid residue of the active site and a third intron just upstream from the exon containing the serine of the active site (e.g. trypsinogen, chymotrypsinogen, kallikrein and proelastase); 3) the genes contain seven introns interrupting the exons coding the catalytic region (e.g. complement factor B gene); and 4) the genes contain two introns resulting in a large exon that contains both the active site aspartatic acid and serine residues (e.g. factor X, factor 1× and protein C genes).
Cytotoxic lymphocytes (e.g. CD8(+) cytotoxic T cells and natural killer cells) form the major defense of higher organisms against virus-infected and transformed cells. A key function of these cells is to detect and eliminate potentially harmful cells by inducing them to undergo apoptosis. This is achieved through two principal pathways, both of which require direct but transient contact between the killer cell and its target. The first pathway involves ligation of TNF receptor-like molecules such as Fas/CD95 to their cognate ligands, and results in mobilization of conventional, programmed cell-death pathways centered on activation of pro-apoptotic caspases. The second mechanism consists of a pathway whereby the toxic contents of a specialized class of secretory vesicles are introduced into the target cell. Studies over the last two decades have identified the toxic components as Granzymes, a family of serine proteases that are expressed exclusively by cytotoxic T lymphocytes and natural killer (NK) cells. These agents are stored in specialized lytic granules and enter the target cell via endocytosis. Like caspases, cysteine proteases that play an important role in apoptosis, granzymes can cleave proteins after acidic residues, especially aspartic acid, and induce apoptosis in the recipient cell.
Granzymes have been grouped into three subfamilies according to substrate specificity. Members of the granzyme family that have enzymatic activity similar to the serine protease chymotrypsin are encoded by a gene cluster termed the ‘chymase locus’. Similarly, granzymes with trypsin-like specificities are encoded by the ‘tryptase locus’, and a third subfamily cleaves after unbranched hydrophobic residues, especially methionine, and are encoded by the ‘Met-ase locus’. All granzymes are synthesized as zymogens and, after clipping of the leader peptide, obtain maximal enzymatic activity subsequent to the removal of an amino-terminal dipeptide.
Granzymes have been found to be important in a number of important biological functions including defense against intracellular pathogens, graft versus host reactions, the susceptibility to transplantable and spontaneous malignancies, lymphoid homeostasis, and the tendency toward auto-immune diseases. For these reasons, siRNAs directed against granszymes (e.g. GZMA, GZMB, GZMH, GZHK, GZMM) and related serine proteases may be useful research and therapeutic reagents.
Kinases
Protein Kinases (PKs) have been implicated in a number of biological processes. Kinase molecules play a central role in modulating cellular physiology and developmental decisions, and have been implicated in a large list of human maladies including cancer, diabetes, and others.
During the course of the last three decades, over a hundred distinct protein kinases have been identified, all with presumed specific cellular functions. A few of these enzymes have been isolated to sufficient purity to perform in vitro studies, but most remain intractable due to the low abundance of these molecules in the cell. To counter this technical difficulty, a number of protein kinases have been isolated by molecular cloning strategies that utilize the conserved sequences of the catalytic domain to isolate closely related homologs. Alternatively, some kinases have been purified (and subsequently studied) based on their interactions with other molecules.
p58 is a member of the p34cdc2-related supergene family and contains a large domain that is highly homologous to the cell division control kinase, cdc2. This new cell division control-related protein kinase was originally identified as a component of semipurified galactosyltransferase; thus, it has been denoted galactosyltransferase-associated protein kinase (GTA-kinase). GTA-kinase has been found to be expressed in both adult and embryonic tissues and is known to phosphorylate a number of substrates, including histone H1, and casein. Interestingly enough, over expression of this molecule in CHO cells has shown that elevated levels of p58 result in a prolonged late telophase and an early G1 phase, thus hinting of an important role for GTA-kinase in cell cycle regulation.
Cyclin Dependent Kinases
The cyclin-dependent kinases (Cdks) are a family of highly conserved serine/threonine kinases that mediate many of the cell cycle transitions that occur during duplication. Each of these Cdk catalytic subunits associates with a specific subset of regulatory subunits, termed cyclins, to produce a distinct Cdk-cyclin kinase complex that, in general, functions to execute a unique cell cycle event.
Activation of the Cdk-cyclin kinases during cellular transitions is controlled by a variety of regulatory mechanisms. For the Cdc2cyclin B complex, inhibition of kinase activity during S phase and G2 is accomplished by phosphorylation of two Cdc2 residues, Thr14 and Tyr15, which are positioned within the ATP-binding cleft. Phosphorylation of Thr14 and/or Tyr15 suppresses the catalytic activity of the molecule by disrupting the orientation of the ATP present within this cleft. In contrast, the abrupt dephosphorylation of these residues by the Cdc25 phosphatase results in the rapid activation of Cdc2cyclin B kinase activity and subsequent downstream mitotic events. While the exact details of this pathway have yet to be elucidated, it has been proposed that Thr14/Tyr15 phosphorylation functions to permit a cell to attain a critical concentration of inactive Cdkcyclin complexes, which, upon activation, induces a rapid and complete cell cycle transition Furthermore, there is evidence in mammalian cells that Thr14/Tyr15 phosphorylation also functions to delay Cdk activation after DNA damage.
The Schizosaccharomyces pombe wee1 gene product was the first kinase identified that is capable of phosphorylating Tyr15 in Cdc2. Homologs of the Wee1 kinase have been subsequently identified and biochemically characterized from a wide range of species including human, mouse, frog, Saccharomyces cerevisiae, and Drosophila. In vertebrate systems, where Thr14 in Cdc2 is also phosphorylated, the Wee1 kinase was capable of phosphorylating Cdc2 on Tyr15, but not Thr14, indicating that another kinase was responsible for Thr14 phosphorylation. This gene, Myt1 kinase, was recently isolated from the membrane fractions of Xenopus egg extracts and has been shown to be capable of phosphorylating Thr14 and, to a lessor extent, Tyr15 in Cdc2. A human Myt1 homolog displaying similar properties has been isolated, as well as a non-membrane-associated molecule with Thr14 kinase activity.
In the past decade it has been shown that cancer can originate from overexpression of positive regulators, such as cyclins, or from underexpression of negative regulators (e.g. p16 (INK4a), p15 (INK4b), p21 (Cip1)). Inhibitors such as Myt1 are the focus of much cancer research because they are capable of controlling cell cycle proliferation, now considered the Holy Grail for cancer treatment. For these reasons, siRNA directed against kinases and kinase inhibitors including but not limited to ABL1, ABL2, ACK1, ALK, AXL, BLK, BMX, BTK, C20orf64, CSF1R, SCK, DDR1, DDR2, DKFZp761P1010, EGFR, EPHA1, EPHA2, EPHA3, EPHA4, EPHA7, EPHA8, EPHB1, EPHB2, EPHB3, EPHB4. EPHB6, ERBB2, ERBB3, ERBB4, FER, FES, FGFR1, FGFR2, FGFR3, FGFR4, FGR, FLT1, FLT3, FLT4, FRK, FYN, HCK, IGF1R, INSR, ITK, JAK1, JAK2, JAK3, KDR, KIAA1079, KIT, LCK, LTK, LYN, MATK, MERTK, MET, MSTIR, MUSK, NTRK1, NTRK2, NTRK3, PDGFRA, PDGFRB, PTK2, PTK2B, PTK6, PTK7, PTK9, PTK9L, RET, ROR1, ROR2, ROS1, RYK, SRC, SYK, TEC, TEK, TIE, TNK1, TXK, TYK2, TYRO3, YES1, and related proteins, may be useful for research and therapeutic purposes.
G Protein Coupled Receptors
One important class of genes to which siRNAs can be directed are G-protein coupled receptors (GPCRs). GPCRs constitute a superfamily of seven transmembrane spanning proteins that respond to a diverse array of sensory and chemical stimuli, such as light, odor, taste, pheromones, hormones and neurotransmitters. GPCRs play a central role in cell proliferation, differentiation, and have been implicated in the etiology of disease.
The mechanism by which 6 protein-coupled receptors translate extracellular signals into cellular changes was initially envisioned as a simple linear model: activation of the receptor by agonist binding leads to dissociation of the heterotrimeric GTP-binding G protein (Gs, Gi, or Gq) into its alpha and beta/gamma subunits, both of which can activate or inhibit various downstream effector molecules. More specifically, activation of the GPCR induces a conformational change in the Goa subunit, causing GDP to be released and GTP to be bound in its place. The Ga and Gβγ subunits then dissociate from the receptor and interact with a variety of effector molecules. For instance in the case of the Gs family, the primary function is to stimulate the intracellular messenger adenylate cyclase (AC), which catalyzes the conversion of cytoplasmic ATP into the secondary messenger cyclic AMP (cAMP). In contrast, the Gi family inhibits this pathway and the Gq family activates phospholipases C (PLC), which cleaves phosphatidylinositol 4,5, bisphosphate (PIP2) to generate inositol-1,4,5-phosphate (IP3) and diacylglycerol (DAG).
More recently, studies have shown that the functions of GPCRs are not limited to their actions on G-proteins and that considerable cross-talk exists between this diverse group of receptor molecules and a second class of membrane bound proteins, the receptor tyrosine kinases (RTKs). A number of GPCRs such as endothelin-1, thrombin, bombesin, and dopamine receptors can activate MAPKs, a downstream effector of the RTK/Ras pathway. Interestingly, the interaction between these two families is not unidirectional and RTKs can also modulate the activity of signaling pathways traditionally thought to be controlled exclusively by ligands that couple to GPCRs. For instance, EGF, which normally activates the MAPK cascade via the EGF receptor can stimulate adenylate cyclase activity by activating Gαs.
There are dozens of members of the G Protein-Coupled Receptor family that have emerged as prominent drug targets in the last decade. One non-limiting list of potential GPCR-siRNA targets is as follows:
CMKLR1
CML1/CMKLR1 (Accession No. Q99788) is a member of the chemokine receptor family of GPCRs that may play a role in a number of diseases including those involved in inflammation and immunological responses (e.g asthma, arthritis). For this reason, siRNA directed against this protein may prove to be important therapeutic reagents.
Studies of juvenile-onset neuronal ceroid lipofuscinosis (JNCL, Batten disease), the most common form of childhood encephalopathy that is characterized by progressive neural degeneration, show that it is brought on by mutations in a novel lysosomal membrane protein (CLN3). In addition to being implicated in JNCL, CLN3 (GPCR-like protein, Accession No. A57219) expression studies have shown that the CLN3 mRNA and protein are highly over-expressed in a number of cancers (e.g. glioblastomas, neuroblastomas, as well as cancers of the prostate, ovaries, breast, and colon) suggesting a possible contribution of this gene to tumor growth. For this reason, siRNA directed against this protein may prove to be important therapeutic reagents.
CLACR
The calcitonin receptor (CTR/CALCR, Accession No. NM—001742) belongs to “family B” of GPCRs which typically recognized regulatory peptides such as parathyroid hormone, secretin, glucagons and vasoactive intestinal polypeptide. Although the CT receptor typically binds to calcitonin (CT), a 32 amino acid peptide hormone produced primarily by the thyroid, association of the receptor with RAMP (Receptor Activity Modulating Protein) enables it to readily bind other members of the calcitonin peptide family including amylin (AMY) and other CT gene-related peptides (e.g α(CGRP and βCGRP). While the primary function of the calcitonin receptor pertains to regulating osteoclast mediated bone resorption and enhanced Ca+2 excretion by the kidney, recent studies have shown that CT and CTRs may play an important role in a variety of processes as wide ranging as embryonic/foetal development and sperm function/physiology. In addition, studies have shown that patients with particular CTR genotypes may be at higher risk to lose bone mass and that this GPCR may contribute to the formation of calcium oxalate urinary stones. For this reason, siRNA directed against CTR may be useful as therapeutic reagents.
OXTR
The human oxytocin receptor (OTR, OXTR) is a 389 amino acid polypeptide that exhibits the seven transmembrane domain structure and belongs to the Class-I (rhodopsin-type) family of G-protein coupled receptors. OTR is expressed in a wide variety of tissues throughout development and mediates physiological changes through G(q) proteins and phospholipase C-beta. Studies on the functions of oxytocin and the oxytocin receptor have revealed a broad list of duties. OT and OTR play a role in a host of sexual, maternal and social behaviors that include egg-laying, birth, milk-letdown, feeding, grooming, memory and learning. In addition, it has been hypothesized that abnormalities in the functionality of oxytocin-OTR receptor-ligand system can lead to a host of irregularities including compulsive behavior, eating disorders (such as anorexia), depression, and various forms of neurodegenerative diseases. For these reasons, siRNA directed against this gene (NM—000916) may play an important role in combating OTR-associated illnesses.
EDG GPCRs
Lysophosphatidic acid and other lipid-based hormones/growth factors induce their effects by activating signaling pathways through the G-protein coupled receptors (GPCRs) and have been observed to play important roles in a number of human diseases including cancer, asthma, and vascular pathologies. For instance, during studies of immunoglobulin A nephropathy (IgAN), researchers have observed an enhanced expression of EDG5 (NP—004221) suggesting a contribution of this gene product in the development of IgAN. For that reasons, siRNA directed against Edg5 (NM—004230), Edg4 (NM—004720), Edg7 (Nm—012152) and related genes may play an important role in combating human disease.
Genes Involved in Cholesterol Signaling and Biosynthesis
Studies on model genetic organisms such as Drosophila and C. elegans have led to the identification of a plethora of genes that are essential for early development. Mutational analysis and ectopic expression studies have allowed many of these genes to be grouped into discreet signal transduction pathways and have shown that these elements play critical roles in pattern formation and cell differentiation. Disruption of one or more of these genes during early stages of development frequently leads to birth defects whereas as alteration of gene function at later stages in life can result in tumorigenesis.
One critical set of interactions known to exist in both invertebrates and vertebrates is the Sonic Hedgehog-Patched-Gli pathway. Originally documented as a Drosophila segmentation mutant, several labs have recently identified human and mouse orthologs of many of the pathways members and have successfully related disruptions in these genes to known diseases. Pathway activation is initiated with the secretion of Sonic hedgehog. There are three closely related members of the Shh family (Sonic hedgehog, Desert, and Indian) with Shh being the most widely expressed form of the group. The Shh gene product is secreted as a small pro-signal molecule. To successfully initiate its developmental role, Shh is first cleaved, whereupon the N-terminal truncated fragment is covalently modified with cholesterol. The addition of the sterol moiety promotes the interaction between Shh and its cognate membrane bound receptor, Patched (Ptch). There are at least two isoforms of the Patched gene, Ptch1 and Ptch2. Both isoforms contain a sterol-sensing domain (SSD); a roughly 180 amino acid cluster that is found in at least seven different classes of molecules including those involved in cholesterol biosynthesis, vesicular traffic, signal transduction, cholesterol transport, and sterol homeostasis. In the absence of Shh, the Patched protein is a negative regulator of the pathway. In contrast, binding of Shh-cholesterol to the Patched receptor releases the negative inhibition which that molecule enforces on a G-protein coupled receptor known as Smoothened. Subsequent activation of Smoothened (directly or indirectly) leads to the triggering of a trio of transcription factors that belong to the Gli family. All three factors are relatively large, contain a characteristic C2-H2 zinc-finger pentamer, and recognize one of two consensus sequences (SEQ. ID NO 0463 GACCACCCA or SEQ. ID NO. 0464 GAACCACCCA). In the absence of Shh, Gli proteins are cleaved by the proteosome and the C-terminally truncated fragment translocates to the nucleus and acts as a dominant transcription repressor. In the presence of Shh-cholesterol, Gli repressor formation is inhibited and full-length Gli functions as a transcriptional activator.
Shh and other members of the Shh-PTCH-Gli pathway are expressed in a broad range of tissues (e.g. the notochord, the floorplate of the neural tube, the brain, and the gut) at early stages in development. Not surprisingly, mutations that lead to altered protein expression or function have been shown to induce developmental abnormalities. Defects in the human Shh gene have been shown to cause holoprosencephaly, a midline defect that manifests itself as cleft lip or palate, CNS septation, and a wide range of other phenotypes. Interestingly, defects in cholesterol biosynthesis generate similar Shh-like disorders (e.g. Smith-Lemli-Opitz syndrome) suggesting that cholesterol modification of the Shh gene product is crucial for pathway function. Both the Patched and Smoothened genes have also been shown to be clinically relevant with Smoothened now being recognized as an oncogene that, like PTCH-1 and PTCH-2, is believed to be the causative agent of several forms of adult tumors. For these reasons, siRNA directed against Smoothened (SMO, NM—005631), Patched (PTCH, nm—000264), and additional genes that participate in cholesterol signaling, biosynthesis, and degradation, have potentially useful research and therapeutic applications.
Targeted Pathways.
In addition to targeting siRNA against one or more members of a family of proteins, siRNA can be directed against members of a pathway. Thus, for instance, siRNA can be directed against members of a signal transduction pathway (e.g. the insulin pathway, including AKT1-3, CBL, CBLB, EIF4EBP1, FOXO1A, FOXO3A, FRAP1, GSK3A, GSK3B, IGF1, IGF1R, INPP5D, INSR, IRS1, MLLT7, PDPK1, PIK3CA, PIK3CB, PIK3R1, PIK3R2, PPP2R2B, PTEN, RPS6, RPS6KA1, RPX6KA3, SGK, TSC1, TSC2, AND XPO1), an apoptotic pathway (CASP3,6,7,8,9, DSH1/2, P110, P85, PDK1/2, CATENIN, HSP90, CDC37, P23, BAD, BCLXL, BCL2, SMAC, and others), pathways, involved in DNA damage, cell cycle, and other physiological (p53, MDM2, CHK1/2, BRCA1/2, ATM, ATR, P15INK4, P27, P21, SKP2, CDC25C/A, 14-3-3, PLK, RB, CDK4, GLUT4, Inos, Mtor, FKBP, PPAR, RXR, ER). Similarly, genes involved in immune system function including TNFR1, IL-IR, IRAK1/2, TRAF2, TRAF6, TRADD, FADD, IKKε, IKKγ, IKKβ, IKKα, IkBα, IkBβ, p50, p65, Rac, RhoA, Cdc42, ROCK, Pak1/2/3/4/5/6, cIAP, HDAC1/2, CBP, β-TrCP, Rip2/4, and others are also important targets for the siRNAs described in this document and may be useful in treating immune system disorders. Genes involved in apoptosis, such as Dsh1/2, PTEN, P110 (pan), P85, PDK1/2, Akt1, Akt2, Akt (pan), p70S6K, GSK3β, PP2A (cat), β-catenin, HSP90, Cdc37/p50, P23, Bad, BclxL, Bcl2, Smac/Diablo, and Ask1 are potentially useful in the treatment of diseases that involve defects in programmed cell death (e.g. cancer), while siRNA agents directed against p53, MDM2, Chk1/2, BRCA1/2, ATM, ATR, p15INK4, P27, P21, Skp2, Cdc25C/A, 14-3-3σ/ε, PLK, Rb, Cdk4, Glut4, iNOS, mTOR, FKBP, PPARγ, RXRcα, ERα and related genes may play a critical role in combating diseases associated with disruptions in DNA repair, and cell cycle abnormalities.
Tables VI-Table X below provide examples of useful pools for inhibiting different genes in the human insulin pathway and tyrosine kinase pathways, proteins involved in the cell cycle, the production of nuclear receptors, and other genes. These particular pools are particularly useful in humans, but would be useful in any species that generates an appropriately homologous mRNA. Further, within each of the listed pools any one sequence maybe used independently but preferably at least two of the listed sequences, more preferably at least three, and most preferably all of the listed sequences for a given gene is present.













TABLE VI





Gene





SEQ.


Name
Acc#
GI
L.L.
Duplex #
Sequence
ID NO.







AKT1
NM_005163
4885060
207
D-003000-05
GACAAGGACGGGCACATTA
465


 


AKT1
NM_005163
4885060
207
D-003000-06
GGACAAGGACGGGCACATT
466


 


AKT1
NM_005163
4885060
207
D-003000-07
GCTACTTCCTCCTCAAGAA
467


 


AKT1
NM_005163
4885060
207
D-003000-08
GACCGCCTCTGCTTTGTCA
468


 


AKT2








AKT2
NM_001626
6715585
208
D-003001-05
GTACTTCGATGATGAATTT
469


 


AKT2
NM_001626
6715585
208
D-003001-06
GCAAAGAGGGCATCAGTGA
470


 


AKT2
NM_001626
6715585
208
D-003001-07
GGGCTAAAGTGACCATGAA
471


 


AKT2
NM_001626
6715585
208
D-003001-08
GCAGAATGCCAGCTGATGA
472


 


AKT3








AKT3
NM_005465
32307164
10000
D-003002-05
GGAGTAAACTGGCAAGATG
473


 


AKT3
NM_005465
32307164
10000
D-003002-06
GACATTAAATTTCCTCGAA
474


 


AKT3
NM_005465
32307164
10000
D-003002-07
GACCAAAGCCAAACACATT
475


 


AKT3
NM_005465
32307164
10000
D-003002-08
GAGGAGAGAATGAATTGTA
476


 


CBL








CBL
NM_005188
4885116
867
D-003003-05
GGAGACACATTTCGGATTA
477


 


CBL
NM_005188
4885116
867
D-003003-06
GATCTGACCTGCAATGATT
478


 


CBL
NM_005188
4885116
867
D-003003-07
GACAATCCCTCACAATAAA
479


 


CBL
NM_005188
4885116
867
D-003003-08
CCAGAAAGCTTTGGTCATT
480


 


CBLB








CBLB
NM_170662
29366807
868
D-003004-05
GACCATACCTCATAACAAG
481


 


CBLB
NM_170662
29366807
868
D-003004-06
TGAAAGACCTCCACCAATC
482


 


CBLB
NM_170662
29366807
868
D-003004-07
GATGAAGGCTCCAGGTGTT
483


 


CBLB
NM_170662
29366807
868
D-003004-08
TATCAGCATTTACGACTTA
484


 


EIF4EBP1








EIF4EBP1
NM_004095
20070179
1978
D-003005-05
GCAATAGCCCAGAAGATAA
485


 


EIF4EBP1
NM_004095
20070179
1978
D-003005-06
CGCAATAGCCCAGAAGATA
486


 


EIF4EBP1
NM_004095
20070179
1978
D-003005-07
GAGATGGACATTTAAAGCA
487


 


EIF4EBP1
NM_004095
20070179
1978
D-003005-08
CAATAGCCCAGAAGATAAG
488


 


FOXO1A








FOXO1A
NM_002015
9257221
2308
D-003006-05
CCAGGCATCTGATAACAAA
489


 


FOXO1A
NM_002015
9257221
2308
D-003006-06
CCAGATGCCTATAGAAACA
490


 


FOXO1A
NM_002015
9257221
2308
D-003006-07
GGAGGTATGAGTCAGTATA
491


 


FOXO1A
NM_002015
9257221
2308
D-003006-08
GAGGTATGAGTCAGTATAA
492


 


FOXO3A








FOXO3A
NM_001455
4503738
2309
D-003007-01
CAATAGCAACAAGTATACC
493


 


FOXO3A
NM_001455
4503738
2309
D-003007-02
TGAAGTCCAGGACGATGAT
494


 


FOXO3A
NM_001455
4503738
2309
D-003007-03
TGTCACACTATGGTAACCA
495


 


FOXO3A
NM_001455
4503738
2309
D-003007-04
TGTTCAATGGGAGCTTGGA
496


 


FRAP1








FRAP1
NM_004958
19924298
2475
D-003008-05
GAGAAGAAATGGAAGAAAT
497


 


FRAP1
NM_004958
19924298
2475
D-003008-06
CGAAAGTGCTGCAGTACTA
498


 


FRAP1
NM_004958
19924298
2475
D-003008-07
GAGCATGCCGTCAATAATA
499


 


FRAP1
NM_004958
19924298
2475
D-003008-08
GGTCTGAACTGAATGAAGA
500


 


GSK3A








GSK3A
NM_019884
11995473
2931
D-003009-05
GGACAAAGGTGTTGAAATC
501


 


GSK3A
NM_019884
11995473
2931
D-003009-06
GAACCCAGCTGCCTAACAA
502


 


GSK3A
NM_019884
11995473
2931
D-003009-07
GCGGAGAGGTTCTTTGATG
503


 


GSK3A
NM_019884
11995473
2931
D-003009-08
GCTCTAGCCTGCTGGAGTA
504


 


GSK3B








GSK3B
NM_002093
21361339
2932
D-003010-05
GAAGAAAGATGAGGTCTAT
505


 


GSK3B
NM_002093
21361339
2932
D-003010-06
GGACCCAAATGTCAAACTA
506


 


GSK3B
NM_002093
21361339
2932
D-003010-07
GAAATGAAGCCAAACTACA
507


 


GSK3B
NM_002093
21361339
2932
D-003010-08
GATGAGGTCTATCTTAATC
508


 


IGF1








IGF1
NM_000618


D-003011-05
GGAAGTACATTTGAAGAAC
509


 


IGF1
NM_000618


D-003011-06
AGAAGGAAGTACATTTGAA
510


 


IGF1
NM_000618


D-003011-07
CCTCAAGCGTGCCAAGTCA
511


 


IGF1
NM_000618


D-003011-08
GGTGGATGCTGTTCAGTTC
512


 


IGF1R








IGF1R
NM_000875
11068002
3480
D-003012-05
CAACGAAGCTTCTGTGATG
513


 


IGF1R
NM_000875
11068002
3480
D-003012-06
GGCCAGAAATGGAGAATAA
514


 


IGF1R
NM_000875
11068002
3480
D-003012-07
GAAGCACCCTTTAAGAATG
515


 


IGF1R
NM_000875
11068002
3480
D-003012-08
GCAGACACCTACAACATCA
516


 


INPP5D








INPP5D
NM_005541
5031798
3635
D-003013-05
GGAATTGCGTTTACACTTA
517


 


INPP5D
NM_005541
5031798
3635
D-003013-06
GGAAACTGATCATTAAGAA
518


 


INPP5D
NM_005541
5031798
3635
D-003013-07
CGACAGGGATGAAGTACAA
519


 


INPP5D
NM_005541
5031798
3635
D-003013-08
AAACGCAGCTGCGCATCTA
520


 


INSR








INSR
NM_000208
4557883
3643
D-003014-05
GGAAGACGTTTGAGGATTA
521


 


INSR
NM_000208
4557883
3643
D-003014-06
GAACAAGGCTCCCGAGAGT
522


 


INSR
NM_000208
4557883
3643
D-003014-07
GGAGAGACCTTGGAAATTG
523


 


INSR
NM_000208
4557883
3643
D-003014-08
GGACGGAACCCACCTATTT
524


 


IRS1








IRS1
NM_005544
5031804
3667
D-003015-05
AAAGAGGTCTGGCAAGTGA
525


 


IRS1
NM_005544
5031804
3667
D-003015-06
GAACCTGATTGGTATCTAC
526


 


IRS1
NM_005544
5031804
3667
D-003015-07
CCAGGGCGATCTAGTGCTT
527


 


IRS1
NM_005544
5031804
3667
D-003015-08
GTCAGTCTGTCGTCCAGTA
528


 


MLLT7








MLLT7
NM_005938
5174578
4303
D-003016-05
GGACTGGACTTCAACTTTG
529


 


MLLT7
NM_005938
5174578
4303
D-003016-06
CCAGGAAGCAGTTCAAATG
530


 


MLLT7
NM_005938
5174578
4303
D-003016-07
GAGAAGCGACTGACACTTG
531


 


MLLT7
NM_005938
5174578
4303
D-003016-08
GACCAGAGATCGCTAACCA
532


 


PDPK1








PDPK1
NM_002613
4505694
5170
D-003017-05
CAAGAGACCTCGTGGAGAA
533


 


PDPK1
NM_002613
4505694
5170
D-003017-06
GACCAGAGGCCAAGAATTT
534


 


PDPK1
NM_002613
4505694
5170
D-003017-07
GGAAACGAGTATCTTATAT
535


 


PDPK1
NM_002613
4505694
5170
D-003017-08
GAGAAGCGAGATATCATAA
536


 


PIK3CA








PIK3CA
NM_006218
5453891
5290
D-003018-05
GCTATCATCTGAACAATTA
537


 


PIK3CA
NM_006218
5453891
5290
D-003018-06
GGATAGAGGCCAAATAATA
538


 


PIK3CA
NM_006218
5453891
5290
D-003018-07
GGACAACTGTTTCATATAG
539


 


PIK3CA
NM_006218
5453891
5290
D-003018-08
GCCAGTACCTCATGGATTA
540


 


PIK3CB








PIK3CB
NM_006219
5453893
5291
D-003019-05
CGACAAGACTGCCGAGAGA
541


 


PIK3CB
NM_006219
5453893
5291
D-003019-06
TCAAGTGTCTCCTAATATG
542


 


PIK3CB
NM_006219
5453893
5291
D-003019-07
GGATTCAGTTGGAGTGATT
543


 


PIK3CB
NM_006219
5453893
5291
D-003019-08
TTTCAAGTGTCTCCTAATA
544


 


PIK3R1








PIK3R1
NM_181504
32455251
5295
D-003020-05
GGAAATATGGGTTCTCTGA
545


 


PIK3R1
NM_181504
32455251
5295
D-003020-06
GAAAGACGAGAGACCAATA
546


 


PIK3R1
NM_181504
32455251
5295
D-003020-07
GTAAAGCATTGTGTCATAA
547


 


PIK3R1
NM_181504
32455251
5295
D-003020-08
GGATCAAGTTGTCAAAGAA
548


 


PIK3R2








PIK3R2
NM_005027
4826907
5296
D-003021-05
GGAAAGGCGGGAACAATAA
549


 


PIK3R2
NM_005027
4826907
5296
D-003021-06
GATGAAGCGTACTGCAATT
550


 


PIK3R2
NM_005027
4826907
5296
D-003021-07
GGACAGCGAATCTCACTAC
551


 


PIK3R2
NM_005027
4826907
5296
D-003021-08
GCAAGATCCGAGACCAGTA
552


 


PPP2R2B








PPP2R2B
NM_004576
4758953
5521
D-003022-05
GAATGCAGCTTACTTTCTT
553


 


PPP2R2B
NM_004576
4758953
5521
D-003022-06
GACCGAAGCTGACATTATC
554


 


PPP2R2B
NM_004576
4758953
5521
D-003022-07
TCGATTACCTGAAGAGTTT
555


 


PPP2R2B
NM_004576
4758953
5521
D-003022-08
CCTGAAGAGTTTAGAAATA
556


 


PTEN








PTEN
NM_000314
4506248
5728
D-003023-05
GTGAAGATCTTGACCAATG
557


 


PTEN
NM_000314
4506248
5728
D-003023-06
GATCAGCATACACAAATTA
558


 


PTEN
NM_000314
4506248
5728
D-003023-07
GGCGCTATGTGTATTATTA
559


 


PTEN
NM_000314
4506248
5728
D-003023-08
GTATAGAGCGTGCAGATAA
560


 


RPS6








RPS6
NM_001010
17158043
6194
D-003024-05
GCCAGAAACTCATTGAAGT
561


 


RPS6
NM_001010
17158043
6194
D-003024-06
GGATATTCCTGGACTGACT
562


 


RPS6
NM_001010
17158043
6194
D-003024-07
CCAAGGAGAACTGGAGAAA
563


 


RPS6
NM_001010
17158043
6194
D-003024-08
GCGTATGGGCACAGAAGTT
564


 


RPS6KA1








RPS6KA1
NM_002953
20149546
6195
D-003025-05
GATGACACCTTCTACTTTG
565


 


RPS6KA1
NM_002953
20149546
6195
D-003025-06
GAGAATGGGCTCCTCATGA
566


 


RPS6KA1
NM_002953
20149546
6195
D-003025-07
CAAGCGGGATCCTTCAGAA
567


 


RPS6KA1
NM_002953
20149546
6195
D-003025-08
CCACCGGCCTGATGGAAGA
568


 


RPS6KA3








RPS6KA3
NM_004586
4759049
6197
D-003026-05
GAAGGGAAGTTGTATCTTA
569


 


RPS6KA3
NM_004586
4759049
6197
D-003026-06
GAAAGTATGTGTATGTAGT
570


 


RPS6KA3
NM_004586
4759049
6197
D-003026-07
GGACAGCATCCAAACATTA
571


 


RPS6KA3
NM_004586
4759049
6197
D-003026-08
GGAGGTGAATTGCTGGATA
572


 


SGK








SGK
NM_005627
5032090
6446
D-003027-01
TTAATGGTGGAGAGTTGTT
573


 


SGK
NM_005627
5032090
6446
D-003027-04
ATTAACTGGGATGATCTCA
574


 


SGK
NM_005627
25168262
6446
D-003027-05
GAAGAAAGCAATCCTGAAA
575


 


SGK
NM_005627
25168262
6446
D-003027-06
AAACACAGCTGAAATGTAC
576


 


TSC1








TSC1
NM_000368
24475626
7248
D-003028-05
GAAGATGGCTATTCTGTGT
577


 


TSC1
NM_000368
24475626
7248
D-003028-06
TATGAAGGCTCGAGAGTTA
578


 


TSC1
NM_000368
24475626
7248
D-003028-07
CGACACGGCTGATAACTGA
579


 


TSC1
NM_000368
24475626
7248
D-003028-08
CGGCTGATGTTGTTAAATA
580


 


TSC2








TSC2
NM_000548
10938006
7249
D-003029-05
GCATTAATCTCTTACCATA
581


 


TSC2
NM_000548
10938006
7249
D-003029-06
CCAATGTCCTCTTGTCTTT
582


 


TSC2
NM_000548
10938006
7249
D-003029-07
GGAGACACATCACCTACTT
583


 


TSC2
NM_000548
10938006
7249
D-003029-08
TCACCAGGCTCATCAAGAA
584


 


XPO1








XPO1
NM_003400
8051634
7514
D-003030-05
GAAAGTCTCTGTCAAAATA
585


 


XPO1
NM_003400
8051634
7514
D-003030-06
GCAATAGGCTCCATTAGTG
586


 


XPO1
NM_003400
8051634
7514
D-003030-07
GGAACATGATCAACTTATA
587


 


XPO1
NM_003400
8051634
7514
D-003030-08
GGATACAGATTCCATAAAT
588






















TABLE VII





Gene





SEQ.


Name
Acc#
GI
L.L
Duplex #
Sequence
ID NO.







ABL1








ABL1
NM_007313
6382057
25
D-003100-05
GGAAATCAGTGACATAGTG
589


 


ABL1
NM_007313
6382057
25
D-003100-06
GGTCCACACTGCAATGTTT
590


 


ABL1
NM_007313
6382057
25
D-003100-07
GAAGGAAATCAGTGACATA
591


 


ABL1
NM_007313
6382057
25
D-003100-08
TCACTGAGTTCATGACCTA
592


 


ABL2








ABL2
NM_007314
6382061
27
D-003101-05
GAAATGGAGCGAACAGATA
593


 


ABL2
NM_007314
6382061
27
D-003101-06
GAGCCAAATTTCCTATTAA
594


 


ABL2
NM_007314
6382061
27
D-003101-07
GTAATAAGCCTACAGTCTA
595


 


ABL2
NM_007314
6382061
27
D-003101-08
GGAGTGAAGTTCGCTCTAA
596


 


ACK1








ACK1
NM_005781
8922074
10188
D-003102-05
AAACGCAAGTCGTGGATGA
597


 


ACK1
NM_005781
8922074
10188
D-003102-06
GCAAGTCGTGGATGAGTAA
598


 


ACK1
NM_005781
8922074
10188
D-003102-07
GAGCACTACCTCAGAATGA
599


 


ACK1
NM_005781
8922074
10188
D-003102-08
TCAGCAGCACCCACTATTA
600


 


ALK








ALK
NM_004304
29029631
238
D-003103-05
GACAAGATCCTGCAGAATA
601


 


ALK
NM_004304
29029631
238
D-003103-06
GGAAGAGTCTGGGAGTTGA
602


 


ALK
NM_004304
29029631
238
D-003103-07
GCACGTGGCTCGGGACATT
603


 


ALK
NM_004304
29029631
238
D-003103-08
GAACTGCAGTGAAGGAACA
604


 


AXL








AXL
NM_021913
21536465
558
D-003104-05
GGTCAGAGCTGGAGGATTT
605


 


AXL
NM_021913
21536465
558
D-003104-06
GAAAGAAGGAGAGCCGTTA
606


 


AXL
NM_021913
21536465
558
D-003104-07
CCAAGAAGATCTACAATGG
607


 


AXL
NM_021913
21536465
558
D-003104-08
GGAACTGCATGCTGAATGA
608


 


BLK








BLK
NM_001715
4502412
640
D-003105-05
GAGGATGCCTGCTGGATTT
609


 


BLK
NM_001715
4502412
640
D-003105-06
ACATGAAGGTGGCCATTAA
610


 


BLK
NM_001715
4502412
640
D-003105-07
GGTGAGCGGCCAAGAGAAG
611


 


BLK
NM_001715
4502412
640
D-003105-08
GAAACTCGGGTCTGGACAA
612


 


BMX








BMX
NM_001721
21359831
660
D-003106-05
AAACAAACCTTTCCTACTA
613


 


BMX
NM_001721
21359831
660
D-003106-06
GAAGGAGCATTTATGGTTA
614


 


BMX
NM_001721
21359831
660
D-003106-07
GAGAAGAGATTACCTTGTT
615


 


BMX
NM_001721
21359831
660
D-003106-08
GTAAGGCTGTGAATGATAA
616


 


BTK








BTK
NM_000061
4557376
695
D-003107-05
GAACAGGAATGGAAGCTTA
617


 


BTK
NM_000061
4557376
695
D-003107-06
GCTATGGGCTGGGAAATTT
618


 


BTK
NM_000061
4557376
695
D-003107-07
GAAAGCAACTTACCATGGT
619


 


BTK
NM_000061
4557376
695
D-003107-08
GGTAAACGATCAAGGAGTT
620


 


C20orf64








C20orf64
NM_033550
19923655
11285
D-003108-05
CAACTTAGCCAAGACAATT
621


 


C20orf64
NM_033550
19923655
11285
D-003108-06
GAAATTGAAGGCTCAGTGA
622


 


C20orf64
NM_033550
19923655
11285
D-003108-07
TGGAACAGCTGAACATTGT
623


 


C20orf64
NM_033550
19923655
11285
D-003108-08
GCTTCCAACTGCTTATATA
624


 


CSF1R








CSF1R
NM_005211
27262658
1436
D-003109-05
GGAGAGCTCTGACGTTTGA
625


 


CSF1R
NM_005211
27262658
1436
D-003109-06
CAACAACGCTACCTTCCAA
626


 


CSF1R
NM_005211
27262658
1436
D-003109-07
CCACGCAGCTGCCTTACAA
627


 


CSF1R
NM_005211
27262658
1436
D-003109-08
GGAACAACCTGCAGTTTGG
628


 


CSK








CSK
NM_004383
4758077
1445
D-003110-05
CAGAATGTATTGCCAAGTA
629


 


CSK
NM_004383
4758077
1445
D-003110-06
GAACAAAGTCGCCGTCAAG
630


 


CSK
NM_004383
4758077
1445
D-003110-07
GCGAGTGCCTTATCCAAGA
631


 


CSK
NM_004383
4758077
1445
D-003110-08
GGAGAAGGGCTACAAGATG
632


 


DDR1








DDR1
NM_013994
7669484
780
D-003111-05
GGAGATGGAGTTTGAGTTT
633


 


DDR1
NM_013994
7669484
780
D-003111-06
CAGAGGCCCTGTCATCTTT
634


 


DDR1
NM_013994
7669484
780
D-003111-07
GCTGGTAGCTGTCAAGATC
635


 


DDR1
NM_013994
7669484
780
D-003111-08
TGAAAGAGGTGAAGATCAT
636


 


DDR2








DDR2
NM_006182
5453813
4921
D-003112-05
GGTAAGAACTACACAATCA
637


 


DDR2
NM_006182
5453813
4921
D-003112-06
GAACGAGAGTGCCACCAAT
638


 


DDR2
NM_006182
5453813
4921
D-003112-07
ACACCAATCTGAAGTTTAT
639


 


DDR2
NM_006182
5453813
4921
D-003112-08
CAACAAGAATGCCAGGAAT
640


 


DKFZp761








P1010








 


DKFZp761
NM_018423
8922178
55359
D-003113-05
CCTAGAAGCTGCCATTAAA
641


P1010








 


DKFZp761
NM_018423
8922178
55359
D-003113-06
GATTAGGCCTGGCTTATGA
642


P1010








 


DKFZp761
NM_018423
8922178
55359
D-003113-07
CCCAGTAGCTGCACACATA
643


P1010








 


DKFZp761
NM_018423
8922178
55359
D-003113-08
GGTGGTACCTGAACTGTAT
644


P1010








 


EGFR








EGFR
NM_005228
4885198
1956
D-003114-05
GAAGGAAACTGAATTCAAA
645


 


EGFR
NM_005228
4885198
1956
D-003114-06
GGAAATATGTACTACGAAA
646


 


EGFR
NM_005228
4885198
1956
D-003114-07
CCACAAAGCAGTGAATTTA
647


 


EGFR
NM_005228
4885198
1956
D-003114-08
GTAACAAGCTCACGCAGTT
648


 


EPHA1








EPHA1
NM_005232
4885208
2041
D-003115-05
GACCAGAGCTTCACCATTC
649


 


EPHA1
NM_005232
4885208
2041
D-003115-06
GCAAGACTGTGGCGATTAA
650


 


EPHA1
NM_005232
4885208
2041
D-003115-07
GGGCGAACCTGACCTATGA
651


 


EPHA1
NM_005232
4885208
2041
D-003115-08
GATTGTAGCGGTCATCTTT
652


 


EPHA2








EPHA2
NM_004431
4758277
1969
D-003116-05
GGAGGGATCTGGCAACTTG
653


 


EPHA2
NM_004431
4758277
1969
D-003116-06
GCAGCAAGGTGCACGAATT
654


 


EPHA2
NM_004431
4758277
1969
D-003116-07
GGAGAAGGATGGCGAGTTC
655


 


EPHA2
NM_004431
4758277
1969
D-003116-08
GAAGTTCACTACCGAGATC
656


 


EPHA3








EPHA3
NM_005233
21361240
2042
D-003117-05
GATCGGACCTCCAGAAATA
657


 


EPHA3
NM_005233
21361240
2042
D-003117-06
GAACTCAGCTCAGAAGATT
658


 


EPHA3
NM_005233
21361240
2042
D-003117-07
GCAAGAGGCACAAATGTTA
659


 


EPHA3
NM_005233
21361240
2042
D-003117-08
GAGCATCAGTTTACAAAGA
660


 


EPHA4








EPHA4
NM_004438
4758279
2043
D-003118-05
GGTCTGGGATGAAGTATTT
661


 


EPHA4
NM_004438
4758279
2043
D-003118-06
GAATGAAGTTACCTTATTG
662


 


EPHA4
NM_004438
4758279
2043
D-003118-07
GAACTTGGGTGGATAGCAA
663


 


EPHA4
NM_004438
4758279
2043
D-003118-08
GAGATTAAATTCACCTTGA
664


 


EPHA7








EPHA7
NM_004440
4758281
2045
D-003119-05
GAAAAGAGATGTTGCAGTA
665


 


EPHA7
NM_004440
4758281
2045
D-003119-06
CTAGATGCCTCCTGTATTA
666


 


EPHA7
NM_004440
4758281
2045
D-003119-07
AGAAGAAGGTTATCGTTTA
667


 


EPHA7
NM_004440
4758281
2045
D-003119-08
TAGCAAAGCTGACCAAGAA
668


 


EPHA8








EPHA8
NM_020526
18201903
2046
D-003120-05
GAAGATGCACTATCAGAAT
669


 


EPHA8
NM_020526
18201903
2046
D-003120-06
GAGAAGATGCACTATCAGA
670


 


EPHA8
NM_020526
18201903
2046
D-003120-07
AACCTGATCTCCAGTGTGA
671


 


EPHA8
NM_020526
18201903
2046
D-003120-08
TCTGAGACGTGGGCTATGT
672


 


EPHB1








EPHB1
NM_004441
21396502
2047
D-003121-05
GCGATAAGCTCCAGCATTA
673


 


EPHB1
NM_004441
21396502
2047
D-003121-06
GAAACGGGCTTATAGCAAA
674


 


EPHB1
NM_004441
21396502
2047
D-003121-07
GGATGAAGATCTACATTGA
675


 


EPHB1
NM_004441
21396502
2047
D-003121-08
GCACGTCTCTGTCAACATG
676


 


EPHB2








EPHB2
NM_017449
17975764
2048
D-003122-05
ACTATGAGCTGCAGTACTA
677


 


EPHB2
NM_017449
17975764
2048
D-003122-06
GTACAACGCCACAGCCATA
678


 


EPHB2
NM_017449
17975764
2048
D-003122-07
GGAAAGCAATGACTGTTCT
679


 


EPHB2
NM_017449
17975764
2048
D-003122-08
CGGACAAGCTGCAACACTA
680


 


EPHB3








EPHB3
NM_004443
17975767
2049
D-003123-05
GGTGTGATCTCCAATGTGA
681


 


EPHB3
NM_004443
17975767
2049
D-003123-06
GGGATGACCTCCTGTACAA
682


 


EPHB3
NM_004443
17975767
2049
D-003123-07
CAGAAGACCTGCTCCGTAT
683


 


EPHB3
NM_004443
17975767
2049
D-003123-08
GAGATGAAGTACTTTGAGA
684


 


EPHB4








EPHB4
NM_004444
17975769
2050
D-003124-05
GGACAAACACGGACAGTAT
685


 


EPHB4
NM_004444
17975769
2050
D-003124-06
GTACTAAGGTCTACATCGA
686


 


EPHB4
NM_004444
17975769
2050
D-003124-07
GGAGAGAAGCAGAATATTC
687


 


EPHB4
NM_004444
17975769
2050
D-003124-08
GCCAATAGCCACTCTAACA
688


 


EPHB6








EPHB6
NM_004445
4758291
2051
D-003125-05
GGAAGTCGATCCTGCTTAT
689


 


EPHB6
NM_004445
4758291
2051
D-003125-06
GGACCAAGGTGGACACAAT
690


 


EPHB6
NM_004445
4758291
2051
D-003125-07
TGTGGGAAGTGATGAGTTA
691


 


EPHB6
NM_004445
4758291
2051
D-003125-08
CGGGAGACCTTCACGCTTT
692


 


ERBB2








ERBB2
NM_004448
4758297
2064
D-003126-05
GGAGGAATTCTGCACAATG
693


 


ERBB2
NM_004448
4758297
2064
D-003126-06
GACGAATTCTGCAGAATGG
694


 


ERBB2
NM_004448
4758297
2064
D-003126-07
CTACAACACAGACACGTTT
695


 


ERBB2
NM_004448
4758297
2064
D-003126-08
AGACGAAGCATACGTGATG
696


 


ERBB3








ERBB3
NM_001982
4503596
2065
D-003127-05
AAGAGGATGTCAACGGTTA
697


 


ERBB3
NM_001982
4503596
2065
D-003127-06
GAAGACTGCCAGACATTGA
698


 


ERBB3
NM_001982
4503596
2065
D-003127-07
GACAAACACTGGTGCTGAT
699


 


ERBB3
NM_001982
4503596
2065
D-003127-08
GCAGTGGATTCGAGAAGTG
700


 


ERBB4








ERBB4
NM_005235
4885214
2066
D-003128-05
GAGGAAAGATGCCAATTAA
701


 


ERBB4
NM_005235
4885214
2066
D-003128-06
GCAGGAAACATCTATATTA
702


 


ERBB4
NM_005235
4885214
2066
D-003128-07
GATCACAACTGCTGCTTAA
703


 


ERBB4
NM_005235
4885214
2066
D-003128-08
CCTCAAAGATACCTAGTTA
704


 


FER








FER
NM_005246
4885230
2241
D-003129-05
GGAGTGACCTGAAGAATTC
705


 


FER
NM_005246
4885230
2241
D-003129-06
TAAAGCAGATTCCCATTAA
706


 


FER
NM_005246
4885230
2241
D-003129-07
GGAAAGTACTGTCCAAATG
707


 


FER
NM_005246
4885230
2241
D-003129-08
GAACAACGGCTGCTAAAGA
708


 


FES








FES
NM_002005
13376997
2242
D-003130-05
CGAGGATGCTGAAGCAGTA
709


 


FES
NM_002005
13376997
2242
D-003130-06
AGGAATACCTGGAGATTAG
710


 


FES
NM_002005
13376997
2242
D-003130-07
CAACAGGAGCTCCGGAATG
711


 


FES
NM_002005
13376997
2242
D-003130-08
GGTGTTGGGTGAGCAGATT
712


 


FGFR1








FGFR1
NM_000604
13186232
2260
D-003131-05
TAAGAAATGTCTCCTTTGA
713


 


FGFR1
NM_000604
13186232
2260
D-003131-06
GAAGACTGCTGGAGTTAAT
714


 


FGFR1
NM_000604
13186232
2260
D-003131-07
GATGGTCCCTTGTATGTCA
715


 


FGFR1
NM_000604
13186232
2260
D-003131-08
CTTAAGAAATGTGTCCTTT
716


 


FGFR2








FGFR2
NM_000141
13186239
2263
D-003132-05
CCAAATGTCTCAACCAGAA
717


 


FGFR2
NM_000141
13186239
2263
D-003132-06
GAACAGTATTCACCTAGTT
718


 


FGFR2
NM_000141
13186239
2263
D-003132-07
GGCCAACACTGTCAAGTTT
719


 


FGFR2
NM_000141
13186239
2263
D-003132-08
GTGAAGATGTTGAAAGATG
720


 


FGFR3








FGFR3
NM_000142
13112046
2261
D-003133-05
TGTCGGACCTGGTGTCTGA
721


 


FGFR3
NM_000142
13112046
2261
D-003133-06
GCATCAAGCTGCGGCATCA
722


 


FGFR3
NM_000142
13112046
2261
D-003133-07
GGACGGCACACCCTACGTT
723


 


FGFR3
NM_000142
13112046
2261
D-003133-08
TGCACAACCTCGACTACTA
724


 


FGFR4








FGFR4
NM_002011
13112051
2264
D-003134-05
GCACTGGAGTCTCGTGATG
725


 


FGFR4
NM_002011
13112051
2264
D-003134-06
GATAGGGACCTCTCGAATA
726


 


FGFR4
NM_002011
13112051
2264
D-003134-07
ATACGGACATCATCCTGTA
727


 


FGFR4
NM_002011
13112051
2264
D-003134-08
ATAGGGACCTCTCGAATAG
728


 


FGR








FGR
NM_005248
4885234
2268
D-003135-05
GCGATCATGTGAAGCATTA
729


 


FGR
NM_005248
4885234
2268
D-003135-06
TGACTGAGCTCATCACCAA
730


 


FGR
NM_005248
4885234
2268
D-003135-07
GAAGAGTGGTACTTTGGAA
731


 


FGR
NM_005248
4885234
2268
D-003135-08
CCCAGAAGGTGCCCTCTTT
732


 


FLT1








FLT1
NM_002019
4503748
2321
D-003136-05
GAGCAAACGTGACTTATTT
733


 


FLT1
NM_002019
4503748
2321
D-003136-06
CCAAATGGGTTTCATGTTA
734


 


FLT1
NM_002019
4503748
2321
D-003136-07
CAACAAGGATGCAGCACTA
735


 


FLT1
NM_002019
4503748
2321
D-003136-08
GGACGTAACTGAAGAGGAT
736


 


FLT3








FLT3
NM_004119
4758395
2322
D-003137-05
GAAGGGATCTACACCATTA
737


 


FLT3
NM_004119
4758395
2322
D-003137-06
GAAGGAGTCTGGAATAGAA
738


 


FLT3
NM_004119
4758395
2322
D-003137-07
GAATTTAAGTCGTGTGTTC
739


 


FLT3
NM_004119
4758395
2322
D-003137-08
GGAATTCATTTCACTCTGA
740


 


FLT4








FLT4
NM_002020
4503752
2324
D-003138-05
GCAAGAACGTGGATCTGTT
741


 


FLT4
NM_002020
4503752
2324
D-003138-06
GCGAATACCTGTCCTACGA
742


 


FLT4
NM_002020
4503752
2324
D-003138-07
GAAGACATTTGAGGAATTC
743


 


FLT4
NM_002020
4503752
2324
D-003138-08
GAGCAGCCATTCATCAACA
744


 


FRK








FRK
NM_002031
4503786
2444
D-003139-05
GAAACAGACTCTTCATATT
745


 


FRK
NM_002031
4503786
2444
D-003139-06
GAACAATACCACTCCAGTA
746


 


FRK
NM_002031
4503786
2444
D-003139-07
CAAGACCGGTTCCTTTCTA
747


 


FRK
NM_002031
4503786
2444
D-003139-08
GCAAGAATATCTCCAAAAT
748


 


FYN








FYN
NM_002037
23510344
2534
D-003140-05
GGAATGGACTCATATGCAA
749


 


FYN
NM_002037
23510344
2534
D-003140-06
GCAGAAGAGTGGTACTTTG
750


 


FYN
NM_002037
23510344
2534
D-003140-07
CAAAGGAAGTTTACTGGAT
751


 


FYN
NM_002037
23510344
2534
D-003140-08
GAAGAGTGGTAGTTTGGAA
752


 


HCK








HCK
NM_002110
4504356
3055
D-003141-05
GAGATACCGTGAAACATTA
753


 


HCK
NM_002110
4504356
3055
D-003141-06
GCAGGGAGATACCGTGAAA
754


 


HCK
NM_002110
4504356
3055
D-003141-07
CATGGTGGTTGCCCTGTAT
755


 


HCK
NM_002110
4504356
3055
D-003141-08
TGTGTAAGATTGGTGACTT
756


 


ITK








ITK
NM_005546
21614549
3702
D-003144-05
CAAATAATCTGGAAACCTA
757


 


ITK
NM_005546
21614549
3702
D-003144-06
GAAGAAACGAGGAATAATA
758


 


ITK
NM_005546
21614549
3702
D-003144-07
GAAACTCTCTCATCCCAAA
759


 


ITK
NM_005546
21614549
3702
D-003144-08
GGAATGGGCATGAAGGATA
760


 


JAK1








JAK1
NM_002227
4504802
3716
D-003145-05
CCACATAGCTGATCTGAAA
761


 


JAK1
NM_002227
4504802
3716
D-003145-06
TGAAATCACTCACATTGTA
762


 


JAK1
NM_002227
4504802
3716
D-003145-07
TAAGGAACCTCTATCATGA
763


 


JAK1
NM_002227
4504802
3716
D-003145-08
GCAGGTGGCTGTTAAATCT
764


 


JAK2








JAK2
NM_004972
13325062
3717
D-003146-05
GCAAATAGATCCAGTTCTT
765


 


JAK2
NM_004972
13325062
3717
D-003146-06
GAGGAAAGATCCAAGACTA
766


 


JAK2
NM_004972
13325062
3717
D-003146-07
GCCAGAAACTTGAAACTTA
767


 


JAK2
NM_004972
13325062
3717
D-003146-08
GTACAGATTTCGCAGATTT
768


 


JAK3








JAK3
NM_000215
4557680
3718
D-003147-05
GCGCCTATCTTTCTCCTTT
769


 


JAK3
NM_000215
4557680
3718
D-003147-06
CCAGAAATCGTAGACATTA
770


 


JAK3
NM_000215
4557680
3718
D-003147-07
CCTCATCTCTTCAGACTAT
771


 


JAK3
NM_000215
4557680
3718
D-003147-08
TGTACGAGCTCTTCACCTA
772


 


KDR








KDR
NM_002253
11321596
3791
D-003148-05
GGAAATCTCTTGCAAGCTA
773


 


KDR
NM_002253
11321596
3791
D-003148-06
GATTACAGATCTCCATTTA
774


 


KDR
NM_002253
11321596
3791
D-003148-07
GCAGACAGATCTACGTTTG
775


 


KDR
NM_002253
11321596
3791
D-003148-08
GCGATGGCCTCTTCTGTAA
776


 


KIAA1079








KIAA1079
NM_014916
7662475
22853
D-003149-05
GAAATTCTCTCAACTGATG
777


 


KIAA1079
NM_014916
7662475
22853
D-003149-06
GCAGAGGTCTTCACACTTT
778


 


KIAA1079
NM_014916
7662475
22853
D-003149-07
TAAATGATCTTCAGACAGA
779


 


KIAA1079
NM_014916
7662475
22853
D-003149-08
GAGCAGCCCTACTCTGATA
780


 


KIT








KIT
NM_000222
4557694
3815
D-003150-05
AAACACGGCTTAAGCAATT
781


 


KIT
NM_000222
4557694
3815
D-003150-06
GAACAGAACCTTCACTGAT
782


 


KIT
NM_000222
4557694
3815
D-003150-07
GGGAAGCCCTCATGTCTGA
783


 


KIT
NM_000222
4557694
3815
D-003150-08
GCAATTCCATTTATGTGTT
784


 


LCK








LCK
NM_005356
20428651
3932
D-003151-05
GAACTGCCATTATCCCATA
785


 


LCK
NM_005356
20428651
3932
D-003151-06
GAGAGGTGGTGAAACATTA
786


 


LCK
NM_005356
20428651
3932
D-003151-07
GGGCCAAGTTTCCCATTAA
787


 


LCK
NM_005356
20428651
3932
D-003151-08
GCACGCTGCTCATCCGAAA
788


 


LTK








LTK
NM_002344
4505044
4058
D-003152-05
TGAATTCACTCCTGCCAAT
789


 


LTK
NM_002344
4505044
4058
D-003152-06
GTGGCAACCTGAACACTGA
790


 


LTK
NM_002344
4505044
4058
D-003152-07
GGAGCTAGCTGTGGATAAC
791


 


LTK
NM_002344
4505044
4058
D-003152-08
GCAAGTTTCGCCATCAGAA
792


 


LYN








LYN
NM_002350
4505054
4067
D-003153-05
GCAGATGGCTTGTGCAGAA
793


 


LYN
NM_002350
4505054
4067
D-003153-06
GGAGAAGGCTTGTATTAGT
794


 


LYN
NM_002350
4505054
4067
D-003153-07
GATGAGCTCTATGACATTA
795


 


LYN
NM_002350
4505054
4067
D-003153-08
GGTGCTAAGTTCCCTATTA
796


 


MATK








MATK
NM_002378
21450841
4145
D-003154-05
TGAAGAATATGAAGTGTGA
797


 


MATK
NM_002378
21450841
4145
D-003154-06
CCGCTCAGCTCCTGCAGTT
798


 


MATK
NM_002378
21450841
4145
D-003154-07
TACTGAAGCTGCAGCATTT
799


 


MATK
NM_002378
21450841
4145
D-003154-08
TGGGAGGTCTTCTCATATG
800


 


MERTK








MERTK
NM_006343
5453737
10461
D-003155-05
GAACTTACCTTACATAGCT
801


 


MERTK
NM_006343
5453737
10461
D-003155-06
GGACCTGCATACTTACTTA
802


 


MERTK
NM_006343
5453737
10461
D-003155-07
TGACAGGAATCTTCTAATT
803


 


MERTK
NM_006343
5453737
10461
D-003155-08
GGTAATGGCTCAGTCATGA
804


 


MET








MET
NM_000245
4557746
4233
D-003156-05
GAAAGAACCTCTCAACATT
805


 


MET
NM_000245
4557746
4233
D-003156-06
GGACAAGGCTGACCATATG
806


 


MET
NM_000245
4557746
4233
D-003156-07
CCAATGACCTGCTGAAATT
807


 


MET
NM_000245
4557746
4233
D-003156-08
GAGCATACATTAAACCAAA
808


 


MST1R








MST1R
NM_002447
4505264
4486
D-003157-05
GGATGGAGCTGCTGGCTTT
809


 


MST1R
NM_002447
4505264
4486
D-003157-06
CTGCAGACCTATAGATTTA
810


 


MST1R
NM_002447
4505264
4486
D-003157-07
GCACCTGTCTCACTCTTGA
811


 


MST1R
NM_002447
4505264
4486
D-003157-08
GAAAGAGTCCATCCAGCTA
812


 


MUSK








MUSK
NM_005592
5031926
4593
D-003158-05
GAAGAAGCCTCGGCAGATA
813


 


MUSK
NM_005592
5031926
4593
D-003158-06
GTAATAATCTCCATCATGT
814


 


MUSK
NM_005592
5031926
4593
D-003158-07
GGAATGAACTGAAAGTAGT
815


 


MUSK
NM_005592
5031926
4593
D-003158-08
GAGATTTCCTGGACTAGAA
816


 


NTRK1








NTRK1
NM_002529
4585711
4914
D-003159-05
GGACAACCCTTTCGAGTTC
817


 


NTRK1
NM_002529
4585711
4914
D-003159-06
CCAGTGACCTCAACAGGAA
818


 


NTRK1
NM_002529
4585711
4914
D-003159-07
CCACAATACTTCAGTGATG
819


 


NTRK1
NM_002529
4585711
4914
D-003159-08
GAAGAGTGGTCTCCGTTTC
820


 


NTRK2








NTRK2
NM_006180
21361305
4915
D-003160-05
GAACAGAAGTAATGAAATC
821


 


NTRK2
NM_006180
21361305
4915
D-003160-06
GTAATGCTGTTTCTGCTTA
822


 


NTRK2
NM_006180
21361305
4915
D-003160-07
GCAAGACACTGCAAGTTTG
823


 


NTRK2
NM_006180
21361305
4915
D-003160-08
GAAAGTCTATCACATTATC
824


 


NTRK3








NTRK3
NM_002530
4505474
4916
D-003161-05
GAGCGAATCTGCTAGTGAA
825


 


NTRK3
NM_002530
4505474
4916
D-003161-06
GAAGTTCACTACAGAGAGT
826


 


NTRK3
NM_002530
4505474
4916
D-003161-07
GGTCGAGGGTCCAAATTTG
827


 


NTRK3
NM_002530
4505474
4916
D-003161-08
GAATATGACTTCCATACAC
828


 


PDGFRA








PDGFRA
NM_006206
15451787
5156
D-003162-05
GAAACTTCCTGGACTATTT
829


 


PDGFRA
NM_006206
15451787
5156
D-003162-06
GAGATTTGGTCAACTATTT
830


 


PDGFRA
NM_006206
15451787
5156
D-003162-07
GCACGCCGCTTCCTGATAT
831


 


PDGFRA
NM_006206
15451787
5156
D-003162-08
CATCAGAGCTGGATCTAGA
832


 


PDGFRB








PDGFRB
NM_002609
15451788
5159
D-003163-05
GAAAGGAGACGTCAAATAT
833


 


PDGFRB
NM_002609
15451788
5159
D-003163-06
GGAATGAGGTGGTCAACTT
834


 


PDGFRB
NM_002609
15451788
5159
D-003163-07
CAACGAGTCTCCAGTGCTA
835


 


PDGFRB
NM_002609
15451788
5159
D-003163-08
GAGAGGACCTGCCGAGCAA
836


 


PTK2








PTK2
NM_005607
27886592
5747
D-003164-05
GAAGTTGGGTTGTCTAGAA
837


 


PTK2
NM_005607
27886592
5747
D-003164-06
GAAGAACAATGATGTAATC
838


 


PTK2
NM_005607
27886592
5747
D-003164-07
GGAAATTGCTTTGAAGTTG
839


 


PTK2
NM_005607
27886592
5747
D-003164-08
GGTTCAAGCTGGATTATTT
840


 


PTK2B








PTK2B
NM_004103
27886583
2185
D-003165-05
GAACATGGCTGACCTCATA
841


 


PTK2B
NM_004103
27886583
2185
D-003165-06
GGACCACGGTGCTCTATTT
842


 


PTK2B
NM_004103
27886583
2185
D-003165-07
GGACGAGGACTATTACAAA
843


 


PTK2B
NM_004103
27886583
2185
D-003165-08
TGGCAGAGCTCATCAACAA
844


 


PTK6








PTK6
NM_005975
27886594
5753
D-003166-05
GAGAAAGTCCTGCCCGTTT
845


 


PTK6
NM_005975
27886594
5753
D-003166-06
TGAAGAAGCTGCGGCACAA
846


 


PTK6
NM_005975
27886594
5753
D-003166-07
CCGCGACTCTGATGAGAAA
847


 


PTK6
NM_005975
27886594
5753
D-003166-08
TGCGCGAGCTTGTGAACTA
848


 


PTK7








PTK7
NM_002821
27886610
5754
D-003167-05
GAGAGAAGCGCACTATTAA
849


 


PTK7
NM_002821
27886610
5754
D-003167-06
CGAGAGAAGCCCACTATTA
850


 


PTK7
NM_002821
27886610
5754
D-003167-07
GGAGGGAGTTGGAGATGTT
851


 


PTK7
NM_002821
27886610
5754
D-003167-08
GAAGACATGCCGCTATTTG
852


 


PTK9








PTK9
NM_002822
4506274
5756
D-003168-05
GAAGAAGTACGAGAGATTA
853


 


PTK9
NM_002822
4506274
5756
D-003168-09
GAAGGAGACTATTTAGAGT
854


 


PTK9
NM_002822
4506274
5756
D-003168-10
GAGCGGATGCTGTATTCTA
855


 


PTK9
NM_002822
4506274
5756
D-003168-11
CTGCAGACTTCCTTTATGA
856


 


PTK9L








PTK9L
NM_007284
31543446
11344
D-003169-05
AGAGAGAGCTCCAGCAGAT
857


 


PTK9L
NM_007284
31543446
11344
D-003169-06
TTAACGAGGTGAAGACAGA
858


 


PTK9L
NM_007284
31543446
11344
D-003169-07
ACACAGAGCCCACGGATGT
859


 


PTK9L
NM_007284
31543446
11344
D-003169-08
GCTGGGATCAGGACTATGA
860


 


RET








RET
NM_000323
21536316
5979
D-003170-05
GCAAAGACCTGGAGAAGAT
861


 


RET
NM_000323
21536316
5979
D-003170-06
GCACACGGCTGCATGAGAA
862


 


RET
NM_000323
21536316
5979
D-003170-07
GAACTGGCCTGGAGAGAGT
863


 


RET
NM_000323
21536316
5979
D-003170-08
TTAAATGGATGGCAATTGA
864


 


ROR1








ROR1
NM_005012
4826867
4919
D-003171-05
GCAAGCATCTTTACTAGGA
865


 


ROR1
NM_005012
4826867
4919
D-003171-06
GAGCAAGGCTAAAGAGCTA
866


 


ROR1
NM_005012
4826867
4919
D-003171-07
GAGAGCAACTTCATGTAAA
867


 


ROR1
NM_005012
4826867
4919
D-003171-08
GAGAATGTCCTGTGTCAAA
868


 


ROR2








ROR2
NM_004560
19743897
4920
D-003172-05
GGAACTCGCTGCTGCCTAT
869


 


ROR2
NM_004560
19743897
4920
D-003172-06
GCAGGTGCCTCCTCAGATG
870


 


ROR2
NM_004560
19743897
4920
D-003172-07
GCAATGTGCTAGTGTACGA
871


 


ROR2
NM_004560
19743897
4920
D-003172-08
GAAGACAGAATATGGTTCA
872


 


ROS1








ROS1
NM_002944
19924164
6098
D-003173-05
GAGGAGACCTTCTTACTTA
873


 


ROS1
NM_002944
19924164
6098
D-003173-06
TTACAGAGGTTCAGGATTA
874


 


ROS1
NM_002944
19924164
6098
D-003173-07
GAACAAACCTAAGCATGAA
875


 


ROS1
NM_002944
19924164
6098
D-003173-08
GAAAGAGCACTTCAAATAA
876


 


RYK








RYK
NM_002958
11863158
6259
D-003174-05
GAAAGATGGTTACCGAATA
877


 


RYK
NM_002958
11863158
6259
D-003174-06
CAAAGTAGATTCTGAAGTT
878


 


RYK
NM_002958
11863158
6259
D-003174-07
TCACTACGCTCTATCCTTT
879


 


RYK
NM_002958
11863158
6259
D-003174-08
GGTGAAGGATATAGCAATA
880


 


SRC








SRC
NM_005417
21361210
6714
D-003175-05
GAGAACCTGGTGTGCAAAG
881


 


SRC
NM_005417
21361210
6714
D-003175-09
GAGAGAAGCTGGTGTGCAA
882


 


SRC
NM_005417
21361210
6714
D-003175-10
GGAGTTTGCTGGACTTTCT
883


 


SRC
NM_005417
21361210
6714
D-003175-11
GAAAGTGAGACCACGAAAG
884


 


SYK








SYK
NM_003177
21361552
6850
D-003176-05
GGAATAATCTCAAGAATCA
885


 


SYK
NM_003177
21361552
6850
D-003176-06
GAACTGGGCTCTGGTAATT
886


 


SYK
NM_003177
21361552
6850
D-003176-07
GGAAGAATCTGAGCAAATT
887


 


SYK
NM_003177
21361552
6850
D-003176-08
GAACAGACATGTCAAGGAT
888


 


TEC








TEC
NM_003215
4507428
7006
D-003177-05
GAAATTGTCTAGTAAGTGA
889


 


TEC
NM_003215
4507428
7006
D-003177-06
CACCTGAAGTGTTTAATTA
890


 


TEC
NM_003215
4507428
7006
D-003177-07
GTACAAAGTCGGAATCAAA
891


 


TEC
NM_003215
4507428
7006
D-003177-08
TGGAGGAGATTCTTATTAA
892


 


TEK








TEK
NM_000459
4557868
7010
D-003178-05
GAAAGAATATGCCTCCAAA
893


 


TEK
NM_000459
4557868
7010
D-003178-06
GGAATGACATCAAATTTCA
894


 


TEK
NM_000459
4557868
7010
D-003178-07
TGAAGTACCTGATATTCTA
895


 


TEK
NM_000459
4557868
7010
D-003178-08
CGAAAGACCTACGTGAATA
896


 


TIE








TIE
NM_005424
4885630
7075
D-003179-05
GAGAGGAGGTTTATGTGAA
897


 


TIE
NM_005424
4885630
7075
D-003179-06
GGGACAGCCTCTACCCTTA
898


 


TIE
NM_005424
4885630
7075
D-003179-07
GAAGTTCTGTGCAAATTGG
899


 


TIE
NM_005424
4885630
7075
D-003179-08
CAACATGGCCTCAGAACTG
900


 


TNK1








TNK1
NM_003985
4507610
8711
D-003180-05
GTTCTGGGCCTAAGTCTAA
901


 


TNK1
NM_003985
4507610
8711
D-003180-06
GAACTGGGTCTACAAGATC
902


 


TNK1
NM_003985
4507610
8711
D-003180-07
CGAGAGGTATCGGTCATGA
903


 


TNK1
NM_003985
4507610
8711
D-003180-08
GGCGCATCCTGGAGCATTA
904


 


TXK








TXK
NM_003328
4507742
7294
D-003181-05
GAACATCTATTGAGACAAG
905


 


TXK
NM_003328
4507742
7294
D-003181-06
TCAAGGCACTTTATGATTT
906


 


TXK
NM_003328
4507742
7294
D-003181-07
GGAGAGGAATGGCTATATT
907


 


TXK
NM_003328
4507742
7294
D-003181-08
GGATATATGTGAAGGAATG
908


 


TYK2








TYK2
NM_003331
4507748
7297
D-003182-05
GAGGAGATCGACCACTTTA
909


 


TYK2
NM_003331
4507748
7297
D-003182-06
GCATCCACATTGCACATAA
910


 


TYK2
NM_003331
4507748
7297
D-003182-07
TCAAATACCTAGCCACACT
911


 


TYK2
NM_003331
4507748
7297
D-003182-08
CAATGTTGCTGACGTCTTG
912


 


TYRO3








TYRO3
NM_006293
27597077
7301
D-003183-05
GGTAGAAGGTGTGCCATTT
913


 


TYRO3
NM_006293
27597077
7301
D-003183-06
ACGCTGAGATTTACAACTA
914


 


TYRO3
NM_006293
27597077
7301
D-003183-07
GGATGGCTCCTTTGTGAAA
915


 


TYRO3
NM_006293
27597077
7301
D-003183-08
GAGAGGAACTACGAAGATC
916


 


YES1








YES1
NM_005433
21071041
7525
D-003184-05
GAAGGACCCTGATGAAAGA
917


 


YES1
NM_005433
21071041
7525
D-003184-06
TAAGAAGGGTGAAAGATTT
918


 


YES1
NM_005433
21071041
7525
D-003184-07
TCAAGAAGCTCAGATAATG
919


 


YES1
NM_005433
21071041
7525
D-003184-08
CAGAATCCCTCCATGAATT
920






















TABLE VIII





Gene


Locus


SEQ. ID


Name
Acc#
GI
Link
Duplex #
Full Sequence
NO.





















APC2








APC2
NM_013366
7549800
29882
D-003200-05
GCAAGGACCTCTTCATCAA
921


 


APC2
NM_013366
7549800
29882
D-003200-06
GAGAAGAAGTCCACACTAT
922


 


APC2
NM_013366
7549800
29882
D-003200-07
GGAATGCCATCTCCCAATG
923


 


APC2
NM_013366
7549800
29882
D-003200-09
CAACACGTGTGACATCATC
924


 


ATM








ATM
NM_000051
20336202
472
D-003201-05
GCAAGCAGCTGAAACAAAT
925


 


ATM
NM_000051
20336202
472
D-003201-06
GAATGTTGCTTTCTGAATT
926


 


ATM
NM_000051
20336202
472
D-003201-07
GACCTGAAGTCTTATTTAA
927


 


ATM
NM_000051
20336202
472
D-003201-08
AGACAGAATTCCCAAATAA
928


 


ATR








ATR
NM_001184
20143978
545
D-003202-05
GAACAACACTGCTGGTTTG
929


 


ATR
NM_001184
20143978
545
D-003202-06
GAAGTCATCTGTTCATTAT
930


 


ATR
NM_001184
20143978
545
D-003202-07
GAAATAAGGTAGACTCAAT
931


 


ATR
NM_001184
20143978
545
D-003202-08
CAACATAAATCCAAGAAGA
932


 


BTAK








BTAK
NM_003600
3213196
6790
D-003545-04
CAAAGAATCAGCTAGCAAA
933


 


BTAK
NM_003600
3213196
6790
D-003203-05
GAAGAGAGTTATTCATAGA
934


 


BTAK
NM_003600
3213196
6790
D-003203-07
CAAATGCCCTGTCTTACTG
935


 


STK6
NM_003600
3213196
6790
D-003203-09
TCTCGTGACTCAGCAAATT
936


 


CCNA1








CCNA1
NM_003914
16306528
8900
D-003204-05
GAACCTGGCTAAGTACGTA
937


 


CCNA1
NM_003914
16306528
8900
D-003204-06
GCAGATCCATTCTTGAAAT
938


 


CCNA1
NM_003914
16306528
8900
D-003204-07
TCACAAGAATCAGGTGTTA
939


 


CCNA1
NM_003914
16306528
8900
D-003204-08
CATAAAGCGTACCTTGATA
940


 


CCNA2








CCNA2
NM_001237
16950653
890
D-003205-05
GCTGTGAACTACATTGATA
941


 


CCNA2
NM_001237
16950653
890
D-003205-06
GATGATACCTACACCAAGA
942


 


CCNA2
NM_001237
16950653
890
D-003205-07
GCTGTTAGCCTCAAAGTTT
943


 


CCNA2
NM_001237
16950653
890
D-003205-08
AAGCTGGCCTGAATCATTA
944


 


CCNB1








CCNB1
NM_031966
14327895
891
D-003206-05
CAACATTACCTGTCATATA
945


 


CCNB1
NM_031966
14327895
891
D-003206-06
CCAAATACCTGATGGAACT
946


 


CCNB1
NM_031966
14327895
891
D-003206-07
GAAATGTACCCTCCAGAAA
947


 


CCNB1
NM_031966
14327895
891
D-003206-08
GCACCTGGCTAAGAATGTA
948


 


CCNB2








CCNB2
NM_004701
10938017
9133
D-003207-05
CAACAAATGTCAACAAAGA
949


 


CCNB2
NM_004701
10938017
9133
D-003207-06
GCAGCAAACTCCTGAAGAT
950


 


CCNB2
NM_004701
10938017
9133
D-003207-07
CCAGTGATTTGGAGAATAT
951


 


CCNB2
NM_004701
10938017
9133
D-003207-08
GTGACTACGTTAAGGATAT
952


 


CCNB3








CCNB3
NM_033031
14719419
85417
D-003208-05
TGAACAAACTGCTGACTTT
953


 


CCNB3
NM_033031
14719419
85417
D-003208-06
GCTAGCTGCTGGCTCCTTA
954


 


CCNB3
NM_033031
14719419
85417
D-003208-07
CAACTCAGCTCGTGTGGAT
955


 


CCNB3
NM_033031
14719419
85417
D-003208-08
GTGGATCTCTACCTAATGA
956


 


CCNC








CCNC
NM_005190
7382485
892
D-003209-05
GCAGAGCTCCCACTATTTG
957


 


CCNC
NM_005190
7382485
892
D-003209-06
GGAGTAGTTTCAAATACAA
958


 


CCNC
NM_005190
7382485
892
D-003209-07
GACGTTTGCTCCAGTATGT
959


 


CCNC
NM_005190
7382485
892
D-003209-08
GAGATTGTATGCCAGGTAT
960


 


CCND1








CCND1
NM_053056
16950654
595
D-003210-05
TGAACAAGCTCAAGTGGAA
961


 


CCND1
NM_053056
16950654
595
D-003210-06
CCAGAGTGATCAAGTGTGA
962


 


CCND1
NM_053056
16950654
595
D-003210-07
GTTCGTGGCCTCTAAGATG
963


 


CCND1
NM_053056
16950654
595
D-003210-08
CCGAGAAGCTGTGCATCTA
964


 


CCND2








CCND2
NM_001759
16950656
894
D-003211-06
TGAATTACCTGGACCGTTT
965


 


CCND2
NM_001759
16950656
894
D-003211-07
CGGAGAAGCTGTGCATTTA
966


 


CCND2
NM_001759
16950656
894
D-003211-08
CTACAGACGTGCGGGATAT
967


 


CCND2
NM_001759
16950656
894
D-003211-09
CAACACAGACGTGGATTGT
968


 


CCND3








CCND3
NM_001760
16950657
896
D-003212-05
GGACCTGGCTGCTGTGATT
969


 


CCND3
NM_001760
16950657
896
D-003212-06
GATTATACCTTTGCCATGT
970


 


CCND3
NM_001760
16950657
896
D-003212-07
GACCAGCACTGGTACAGAT
971


 


CCND3
NM_001760
16950657
896
D-003212-08
TGCGGAAGATGCTGGCTTA
972


 


CCNE1








CCNE1
NM_001238
17318558
898
D-003213-05
GTACTGAGCTGGGCAAATA
973


 


CCNE1
NM_001238
17318558
898
D-003213-06
GGAAATCTATCCTCCAAAG
974


 


CCNE1
NM_001238
17318558
898
D-003213-07
GGAGGTGTGTGAAGTCTAT
975


 


CCNE1
NM_001238
17318558
898
D-003213-08
CTAAATGACTTACATGAAG
976


 


CCNE2








CCNE2
NM_057749
17318564
9134
D-003214-05
GGATGGAACTCATTATATT
977


 


CCNE2
NM_057749
17318564
9134
D-003214-06
GCAGATATGTTCATGACAA
978


 


CCNE2
NM_057749
17318564
9134
D-003214-07
CATAATATCCAGACACATA
979


 


CCNE2
NM_057749
17318564
9134
D-003214-08
TAAGAAAGCCTCAGGTTTG
980


 


CCNF








CCNF
NM_001761
4502620
899
D-003215-05
TCACAAAGCATCCATATTG
981


 


CCNF
NM_001761
4502620
899
D-003215-06
GAAGTCATGTTTACAGTGT
982


 


CCNF
NM_001761
4502620
899
D-003215-07
TAGCCTACCTCTACAATGA
983


 


CCNF
NM_001761
4502620
899
D-003215-08
GCACCCGGTTTATCAGTAA
984


 


CCNG1








CCNG1
NM_004060
8670528
900
D-003216-05
GATAATGGCCTCAGAATGA
985


 


CCNG1
NM_004060
8670528
900
D-003216-06
GCACGGCAATTGAAGCATA
986


 


CCNG1
NM_004060
8670528
900
D-003216-07
GGAATAGAATGTCTTCAGA
987


 


CCNG1
NM_004060
8670528
900
D-003216-08
TAACTCACCTTCCAACAAT
988


 


CCNG2








CCNG2
NM_004354
4757935
901
D-003217-05
GGAGAGAGTTGGTTTCTAA
989


 


CCNG2
NM_004354
4757935
901
D-003217-06
GGTGAAACCTAAACATTTG
990


 


CCNG2
NM_004354
4757935
901
D-003217-07
GAAATACTGAGCCTTGATA
991


 


CCNG2
NM_004354
4757935
901
D-003217-08
TGCCAAAGTTGAAGATTTA
992


 


CCNH








CCNH
NM_001239
17738313
902
D-003218-05
GCTGATGACTTTCTTAATA
993


 


CCNH
NM_001239
17738313
902
D-003218-06
CAACTTAATTTCCACCTTA
994


 


CCNH
NM_001239
17738313
902
D-003218-07
ATACACACCTTCCCAAATT
995


 


CCNH
NM_001239
17738313
902
D-003218-08
GCTATGAAGATGATGATTA
996


 


CCNI








CCNI
NM_006835
17738314
10983
D-003219-05
GCAAGCAGACCTGTACTAA
997


 


CCNI
NM_006835
17738314
10983
D-003219-07
TGAGAGAATTCCAGTACTA
998


 


CCNI
NM_006835
17738314
10983
D-003219-08
GGAATCAAACGGCTCTATA
999


 


CCNI
NM_006835
17738314
10983
D-003219-09
GAATTGGGATCTTCACACA
1000


 


CCNT1








CCNT1
NM_001240
17978465
904
D-003220-05
TATCAACACTGCTATAGTA
1001


 


CCNT1
NM_001240
17978465
904
D-003220-06
GAACAAACGTCCTGGTGAT
1002


 


CCNT1
NM_001240
17978465
904
D-003220-07
GCACAAGACTCACCCATCT
1003


 


CCNT1
NM_001240
17978465
904
D-003220-08
GCACAGACTTCTTACTTCA
1004


 


CCNT2A








CCNT2A
NM_001241
17978467
905
D-003221-05
GCACAGACATCCTATTTCA
1005


 


CCNT2A
NM_001241
17978467
905
D-003221-06
GCAGGGACCTTCTATATCA
1006


 


CCNT2A
NM_001241
17978467
905
D-003221-07
GAACAGCTATATTCACAGA
1007


 


CCNT2A
NM_001241
17978467
905
D-003221-09
TTATATAGCTGCCCAGGTA
1008


 


CCNT2B








CCNT2B
NM_058241
17978468
905
D-003222-05
GCACAGACATGCTATTTCA
1009


 


CCNT2B
NM_058241
17978468
905
D-003222-06
GCAGGGACCTTCTATATCA
1010


 


CCNT2B
NM_058241
17978468
905
D-003222-07
GAACAGCTATATTCACAGA
1011


 


CCNT2B
NM_058241
17978468
905
D-003222-08
GGTGAAATGTACCCAGTTA
1012


 


CDC16








CDC16
NM_003903
14110370
8881
D-003223-05
GTAGATGGCTTGCAAGAGA
1013


 


CDC16
NM_003903
14110370
8881
D-003223-06
TAAAGTAGCTTCACTCTCT
1014


 


CDC16
NM_003903
14110370
8881
D-003223-07
GGTACAAGCTTACTTCTGT
1015


 


CDC16
NM_003903
14110370
8881
D-003223-08
TGGAAGAGCCCATCAATAA
1016


 


CDC2








CDC2
NM_033379
27886643
983
D-003552-01
GTACAGATCTCCAGAAGTA
1017


 


CDC2
NM_033379
27886643
983
D-003552-02
GATCAACTCTTCAGGATTT
1018


 


CDC2
NM_033379
27886643
983
D-003552-03
GGTTATATCTCATCTTTGA
1019


 


CDC2
NM_033379
27886643
983
D-003552-04
GAACTTCGTCATCCAAATA
1020


 


CDC20








CDC20
NM_001255
4557436
991
D-003225-05
GGGAATATATATCCTCTGT
1021


 


CDC20
NM_001255
4557436
991
D-003225-06
GAAACGGCTTCGAAATATG
1022


 


CDC20
NM_001255
4557436
991
D-003225-07
GAAGACCTGCCGTTACATT
1023


 


CDC20
NM_001255
4557436
991
D-003225-08
CACCAGTGATCGACACATT
1024


 


CDC25A








CDC25A
NM_001789
4502704
993
D-003226-05
GAAATTATGGCATCTGTTT
1025


 


CDC25A
NM_001789
4502704
993
D-003226-06
TACAAGGAGTTCTTTATGA
1026


 


CDC25A
NM_001789
4502704
993
D-003226-07
CCACGAGGACTTTAAAGAA
1027


 


CDC25A
NM_001789
4502704
993
D-003226-08
TGGGAAACATCAGGATTTA
1028


 


CDC25B








CDC25B
NM_004358
11641416
994
D-003227-05
GCAGATACCCCTATGAATA
1029


 


CDC25B
NM_004358
11641416
994
D-003227-06
CTAGGTGGCTTCTCTCTGA
1030


 


CDC25B
NM_004358
11641416
994
D-003227-07
GAGAGCTGATTGGAGATTA
1031


 


CDC25B
NM_004358
11641416
994
D-003227-08
AAAAGGACCTCGTCATGTA
1032


 


CDC25C








CDC25C
NM_001790
12408659
995
D-003228-05
GAGCAGAAGTGGCCTATAT
1033


 


CDC25C
NM_001790
12408659
995
D-003228-06
CAGAAGAGATTTCAGATGA
1034


 


CDC25C
NM_001790
12408659
995
D-003228-07
CCAGGGAGCCTTAAACTTA
1035


 


CDC25C
NM_001790
12408659
995
D-003228-08
GAAACTTGGTGGACAGTGA
1036


 


CDC27








CDC27
NM_001256
16554576
996
D-003229-06
CATGCAAGCTGAAAGAATA
1037


 


CDC27
NM_001256
16554576
996
D-003229-07
CAACACAAGTACCTAATCA
1038


 


CDC27
NM_001256
16554576
996
D-003229-08
GGAGATGGATCCTATTTAC
1039


 


CDC27
NM_001256
16554576
996
D-003229-09
GAAAAGCCATGATGATATT
1040


 


CDC34








CDC34
NM_004359
16357476
997
D-003230-05
GCTCAGACCTCTTCTACGA
1041


 


CDC34
NM_004359
16357476
997
D-003230-06
GGACGAGGGCGATCTATAC
1042


 


CDC34
NM_004359
16357476
997
D-003230-07
GATCGGGAGTACACAGACA
1043


 


CDC34
NM_004359
16357476
997
D-003230-08
TGAACGAGCCCAACACCTT
1044


 


CDC37








CDC37
NM_007065
16357478
11140
D-003231-05
GCGAGGAGAGAGCCAATTA
1045


 


CDC37
NM_007065
16357478
11140
D-003231-06
CACAAGACCTTCGTGGAAA
1046


 


CDC37
NM_007065
16357478
11140
D-003231-07
ACAATCGTCATGCAATTTA
1047


 


CDC37
NM_007065
16357478
11140
D-003231-08
GAGGAGAAATGTGCACTCA
1048


 


CDC45L








CDC45L
NM_003504
34335230
8318
D-003232-05
GCACACGGATCTCCTTTGA
1049


 


CDC45L
NM_003504
34335230
8318
D-003232-06
GCAAACACCTGCTCAAGTC
1050


 


CDC45L
NM_003504
34335230
8318
D-003232-07
TGAAGAGTCTGCAAATAAA
1051


 


CDC45L
NM_003504
34335230
8318
D-003232-08
GGACGTGGATGCTCTGTGT
1052


 


CDC6








CDC6
NM_001254
16357469
990
D-003233-05
GAACACAGCTGTCCCAGAT
1053


 


CDC6
NM_001254
16357469
990
D-003233-06
GAGCAGAGATGTCCACTGA
1054


 


CDC6
NM_001254
16357469
990
D-003233-07
GGAAATATCTTAGCTACTG
1055


 


CDC6
NM_001254
16357469
990
D-003233-08
GGACGAAGATTGGTATTTG
1056


 


CDC7








CDC7
NM_003503
11038647
8317
D-003234-05
GGAATGAGGTACCTGATGA
1057


 


CDC7
NM_003503
11038647
8317
D-003234-06
CAGGAAAGGTGTTCACAAA
1058


 


CDC7
NM_003503
11038647
8317
D-003234-07
CTACACAAATGCAGAAATT
1059


 


CDC7
NM_003503
11038647
8317
D-003234-08
GTACGGGAATATATGCTTA
1060


 


CDK10








CDK10
NM_003674
32528262
8558
D-003235-05
GAACTGCTGTTGGGAACCA
1061


 


CDK10
NM_003674
32528262
8558
D-003235-06
GGAAGCAGCCCTACAACAA
1062


 


CDK10
NM_003674
32528262
8558
D-003235-07
GCACGCCCAGTGAGAACAT
1063


 


CDK10
NM_003674
32528262
8558
D-003235-08
GGAAGCAGCCCTAGAACAA
1064


 


CDK2








CDK2
NM_001798
16936527
1017
D-003236-05
GAGCTTAACCATCCTAATA
1065


 


CDK2
NM_001798
16936527
1017
D-003236-06
GAGGTTAACCATCCTAATA
1066


 


CDK2
NM_001798
16936527
1017
D-003236-07
GTACCGAGCTCCTGAAATC
1067


 


CDK2
NM_001798
16936527
1017
D-003236-08
GAGAGGTGGTGGCGCTTAA
1068


 


CDK3








CDK3
NM_001258
4557438
1018
D-003237-05
GAGCATTGGTTGCATCTTT
1069


 


CDK3
NM_001258
4557438
1018
D-003237-06
GATCGGAGAGGGCACCTAT
1070


 


CDK3
NM_001258
4557438
1018
D-003237-07
GAAGCTCTATCTGGTGTTT
1071


 


CDK3
NM_001258
4557438
1018
D-003237-08
GCAGAGATGGTGACTCGAA
1072


 


CDK4








CDK4
NM_000075
456426
1019
D-003238-05
GCAGCACTCTTATCTACAT
1073


 


CDK4
NM_000075
456426
1019
D-003238-06
GGAGGAGGGCTTCCCATCA
1074


 


CDK4
NM_000075
456426
1019
D-003238-07
TCGAAAGCCTCTCTTCTGT
1075


 


CDK4
NM_000075
456426
1019
D-003238-08
GTACCGAGCTCCCGAAGTT
1076


 


CDK5








CDK5
NM_004935
4826674
1020
D-003239-05
TGACCAAGCTGCCAGACTA
1077


 


CDK5
NM_004935
4826674
1020
D-003239-06
GAGCTGAAATTGGCTGATT
1078


 


CDK5
NM_004935
4826674
1020
D-003239-07
CAACATCCCTGGTGAACGT
1079


 


CDK5
NM_004935
4826674
1020
D-003239-08
GGATTCCCGTCCGCTGTTA
1080


 


CDK6








CDK6
NM_001259
16950658
1021
D-003240-05
GCAAAGACCTACTTCTGAA
1081


 


CDK6
NM_001259
16950658
1021
D-003240-06
GAAGAAGACTGGCCTAGAG
1082


 


CDK6
NM_001259
16950658
1021
D-003240-07
GGTGTGGACTTTCTTCATT
1083


 


CDK6
NM_001259
16950658
1021
D-003240-08
TAACAGATATCGATGAACT
1084


 


CDK7








CDK7
NM_001799
16950659
1022
D-003241-05
GGACATAGATCAGAAGCTA
1085


 


CDK7
NM_001799
16950659
1022
D-003241-06
CAATAGAGCTTATACACAT
1086


 


CDK7
NM_001799
16950659
1022
D-003241-07
GATACAAGGCTTATTCTTA
1087


 


CDK7
NM_001799
16950659
1022
D-003241-08
GGAGACGACTTACTAGATC
1088


 


CDK8








CDK8
NM_001260
4502744
1024
D-003242-05
CCACAGTACTCACATCAGA
1089


 


CDK8
NM_001260
4502744
1024
D-003242-06
GCAATAACCACACTAATGG
1090


 


CDK8
NM_001260
4502744
1024
D-003242-07
GAAGAAAGTGAGAGTTGTT
1091


 


CDK8
NM_001260
4502744
1024
D-003242-08
GAACATGACCTCTGGCATA
1092


 


CDK9








CDK9
NM_001261
17017983
1025
D-003243-05
GGCCAAACGTGGACAACTA
1093


 


CDK9
NM_001261
17017983
1025
D-003243-06
TGACGTCCATGTTCGAGTA
1094


 


CDK9
NM_001261
17017983
1025
D-003243-07
CCAACCAGACGGAGTTTGA
1095


 


CDK9
NM_001261
17017983
1025
D-003243-08
GAAGGTGGCTCTGAAGAAG
1096


 


CDKN1C








CDKN1C
NM_000076
4557440
1028
D-003244-05
GACCAGAACCGCTGGGATT
1097


 


CDKN1C
NM_000076
4557440
1028
D-003244-06
GGACCGAAGTGGACAGCGA
1098


 


CDKN1C
NM_000076
4557440
1028
D-003244-08
GCAAGAGATCAGCGCCTGA
1099


 


CDKN1C
NM_000076
4557440
1028
D-003244-09
CCGCTGGGATTACGAGTTC
1100


 


CDKN2B








CDKN2B
NM_004936
17981693
1030
D-003245-05
GCGAGGAGAACAAGGGCAT
1101


 


CDKN2B
NM_004936
17981693
1030
D-003245-06
CCAACGGAGTCAACCGTTT
1102


 


CDKN2B
NM_004936
17981693
1030
D-003245-07
CGATCCAGGTCATGATGAT
1103


 


CDKN2B
NM_004936
17981693
1030
D-003245-08
CCTGGAAGCCGGCGCGGAT
1104


 


CDKN2C








CDKN2C
NM_001262
17981697
1031
D-003246-05
GGACACCGCCTGTGATTTG
1105


 


CDKN2C
NM_001262
17981697
1031
D-003246-06
GCCAGGAGACTGCTACTTA
1106


 


CDKN2C
NM_001262
17981697
1031
D-003246-07
TGAAAGACCGAACTGGTTT
1107


 


CDKN2C
NM_001262
17981697
1031
D-003246-08
GAACCTGCCCTTGCACTTG
1108


 


CDKN2D








CDKN2D
NM_001800
17981700
1032
D-003247-05
TGGCAGTTGAAGAGGGTCA
1109


 


CDKN2D
NM_001800
17981700
1032
D-003247-06
CTCAGGACCTCGTGGACAT
1110


 


CDKN2D
NM_001800
17981700
1032
D-003247-07
TGAAGGTGCTAGTGGAGCA
1111


 


CDKN2D
NM_001800
17981700
1032
D-003247-08
AGAGGGCGCTGCAGGTCAT
1112


 


CDT1








CDT1
NM_030928
19923847
81620
D-003248-05
CCAAGGAGGCACAGAAGCA
1113


 


CDT1
NM_030928
19923847
81620
D-003248-06
GCTTCAACGTGGATGAAGT
1114


 


CDT1
NM_030928
19923847
81620
D-003248-07
TCTCCGGGCCAGAAGATAA
1115


 


CDT1
NM_030928
19923847
81620
D-003248-08
GCGCAATGTTGGCCAGATC
1116


 


CENPA








CENPA
NM_001809
4585861
1058
D-003249-05
GCACACACCTCTTGATAAG
1117


 


CENPA
NM_001809
4585861
1058
D-003249-06
GCAAGAGAAATATGTGTTA
1118


 


CENPA
NM_001809
4585861
1058
D-003249-07
TTACATGCAGGCCGAGTTA
1119


 


CENPA
NM_001809
4585861
1058
D-003249-08
GAGACAAGGTTGGCTAAAG
1120


 


CENPB








CENPB
NM_001810
26105977
1059
D-003250-05
GGACATAGCCGCCTGCTTT
1121


 


CENPB
NM_001810
26105977
1059
D-003250-06
GCACGATCCTGAAGAACAA
1122


 


CENPB
NM_001810
26105977
1059
D-003250-07
GGAGGAGGGTGATGTTGAT
1123


 


CENPB
NM_001810
26105977
1059
D-003250-08
CCGAATGGCTGCAGAGTCT
1124


 


CENPC1








CENPC1
NM_001812
4502778
1060
D-003251-05
GCGAATAGATTATCAAGGA
1125


 


CENPC1
NM_001812
4502778
1060
D-003251-06
GAACAGAATCCATCACAAA
1126


 


CENPC1
NM_001812
4502778
1060
D-003251-07
CCATAAACCTCACCCAGTA
1127


 


CENPC1
NM_001812
4502778
1060
D-003251-08
CAAGAGAACACGTTTGAAA
1128


 


CENPE








CENPE
NM_001813
4502780
1062
D-003252-05
GAAGACAGCTCAAATAATA
1129


 


CENPE
NM_001813
4502780
1062
D-003252-06
CAACAAAGCTACTAAATCA
1130


 


CENPE
NM_001813
4502780
1062
D-003252-07
GGAAAGAAGTGCTACCATA
1131


 


CENPE
NM_001813
4502780
1062
D-003252-08
GGAAAGAAATGACACAGTT
1132


 


CENPF








CENPF
NM_016343
14670380
1063
D-003253-05
GCGAATATCTGAATTAGAA
1133


 


CENPF
NM_016343
14670380
1063
D-003253-06
GGAAATTAATGCATCCTTA
1134


 


CENPF
NM_016343
14670380
1063
D-003253-07
GAGCGAGGCTGGTGGTTTA
1135


 


CENPF
NM_016343
14670380
1063
D-003253-08
CAAGTCATCTTTCATCTAA
1136


 


CENPH








CENPH
NM_022909
21264590
64946
D-003254-05
GAAAGAAGAGATTGCAATT
1137


 


CENPH
NM_022909
21264590
64946
D-003254-06
CAGAACAAATTATGCAAGA
1138


 


CENPH
NM_022909
21264590
64946
D-003254-07
CTAGTGTGCTCATGGATAA
1139


 


CENPH
NM_022909
21264590
64946
D-003254-08
GAAACACCTATTAGAGCTA
1140


 


CHEK1








CHEK1
NM_001274
20127419
1111
D-003255-05
CAAATTGGATGCAGACAAA
1141


 


CHEK1
NM_001274
20127419
1111
D-003255-06
GCAACAGTATTTCGGTATA
1142


 


CHEK1
NM_001274
20127419
1111
D-003255-07
GGACTTCTCTCCAGTAAAC
1143


 


CHEK1
NM_001274
20127419
1111
D-003255-08
AAAGATAGATGGTACAACA
1144


 


CHEK2








CHEK2
NM_007194
22209010
11200
D-003256-02
CTCTTACATTGCATACATA
1145


 


CHEK2
NM_007194
22209010
11200
D-003256-03
TAAACGCCTGAAAGAAGCT
1146


 


CHEK2
NM_007194
22209010
11200
D-003256-04
GCATAGGACTCAAGTGTCA
1147


 


CHEK2
NM_007194
22209010
11200
D-003256-05
GAAATTGCACTGTCACTAA
1148


 


CNK








CNK
NM_004073
4758015
1263
D-003257-05
GCGAGAAGATCCTAAATGA
1149


 


CNK
NM_004073
4758015
1263
D-003257-07
GCAAGTGGGTTGACTACTC
1150


 


CNK
NM_004073
4758015
1263
D-003257-08
GCACATCCGTTGGCCATCA
1151


 


CNK
NM_004073
4758015
1263
D-003257-09
GACCTCAAGTTGGGAAATT
1152


 


CR11








CR11
NM_014335
7656937
23741
D-003258-05
GTGATGAGATTATTGATAG
1153


 


CR11
NM_014335
7656937
23741
D-003258-06
GGACGAGGGCGAGGAATTT
1154


 


CR11
NM_014335
7656937
23741
D-003258-07
GGAAACGGAGCCTTGCTAA
1155


 


CR11
NM_014335
7656937
23741
D-003258-08
TCAATCGTCTGACCGAAGA
1156


 


E2F1








E2F1
NM_005225
12669910
1869
D-003259-05
GAACAGGGCCACTGACTCT
1157


 


E2F1
NM_005225
12669910
1869
D-003259-06
TGGACCACCTGATGAATAT
1158


 


E2F1
NM_005225
12669910
1869
D-003259-07
CCCAGGAGGTCACTTCTGA
1159


 


E2F1
NM_005225
12669910
1869
D-003259-08
GGCTGGACCTGGAAACTGA
1160


 


E2F2








E2F2
NM_004091
34485718
1870
D-003260-05
GGGAGAAGACTCGGTATGA
1161


 


E2F2
NM_004091
34485718
1870
D-003260-06
GAGGACAACCTGCAGATAT
1162


 


E2F2
NM_004091
34485718
1870
D-003260-07
TGAAGGAGCTGATGAACAC
1163


 


E2F2
NM_004091
34485718
1870
D-003260-08
CCAAGAAGTTCATTTACCT
1164


 


E2F3








E2F3
NM_001949
12669913
1871
D-003261-05
GAAATTAGATGAACTGATC
1165


 


E2F3
NM_001949
12669913
1871
D-003261-06
TGAAGTGCCTGACTCAATA
1166


 


E2F3
NM_001949
12669913
1871
D-003261-07
GAACAAGGCAGCAGAAGTG
1167


 


E2F3
NM_001949
12669913
1871
D-003261-08
GAAACACACAGTCCAATGA
1168


 


E2F4








E2F4
NM_001950
12669914
1874
D-003262-05
GGAGATTGCTGACAAACTG
1169


 


E2F4
NM_001950
12669914
1874
D-003262-06
GAAGGTATCGGGCTAATCG
1170


 


E2F4
NM_001950
12669914
1874
D-003262-07
GTGCAGAAGTCCAGGGAAT
1171


 


E2F4
NM_001950
12669914
1874
D-003262-08
GGACAGTGGTGAGCTGAGT
1172


 


E2F5








E2F5
NM_001951
12669916
1875
D-003263-05
GCAGATGACTACAACTTTA
1173


 


E2F5
NM_001951
12669916
1875
D-003263-06
GACATCAGCTACAGATATA
1174


 


E2F5
NM_001951
12669916
1875
D-003263-07
CAACATGTGTCTGAAAGAA
1175


 


E2F5
NM_001951
12669916
1875
D-003263-08
GAAGACATCTGTAATTGCT
1176


 


E2F6








E2F6
NM_001952
12669917
1876
D-003264-05
TAAACAAGGTTGCAACGAA
1177


 


E2F6
NM_001952
12669917
1876
D-003264-06
TAGCATATGTGACCTATCA
1178


 


E2F6
NM_001952
12669917
1876
D-003264-07
GAAACCAGATTGGATGTTC
1179


 


E2F6
NM_001952
12669917
1876
D-003264-09
GGAACTTTCTGACTTATCA
1180


 


FOS








FOS
NM_005252
6552332
2353
D-003265-05
GGGATAGCCTCTCTTACTA
1181


 


FOS
NM_005252
6552332
2353
D-003265-06
GAACAGTTATCTCCAGAAG
1182


 


FOS
NM_005252
6552332
2353
D-003265-07
GGAGACAGACCAACTAGAA
1183


 


FOS
NM_005252
6552332
2353
D-003265-08
AGACCGAGCCCTTTGATGA
1184


 


HIPK2








HIPK2
NM_022740
13430859
28996
D-003266-06
GAGAATCACTCCAATCGAA
1185


 


HIPK2
NM_022740
13430859
28996
D-003266-07
AGACAGGGATTAAGTCAAA
1186


 


HIPK2
NM_022740
13430859
28996
D-003266-08
GGACAAAGACAACTAGGTT
1187


 


HIPK2
NM_022740
13430859
28996
D-003266-09
GCACACACGTCAAATGATG
1188


 


HUS1








HUS1
NM_004507
31077213
3364
D-003267-05
ACAAAGGCCTTATGCAATA
1189


 


HUS1
NM_004507
31077213
3364
D-003267-06
GAAGTGGACATAGATATTA
1190


 


HUS1
NM_004507
31077213
3364
D-003267-07
AAGCTTAACTTCATCCTTT
1191


 


HUS1
NM_004507
31077213
3364
D-003267-08
GAACTTCTTCAACGAATTT
1192


 


JUN








JUN
NM_002228
7710122
3725
D-003268-05
TGGAAACGACCTTCTATGA
1193


 


JUN
NM_002228
7710122
3725
D-003268-06
GAACTGCACAGCCAGAACA
1194


 


JUN
NM_002228
7710122
3725
D-003268-07
GAGCTGGAGCGCCTGATAA
1195


 


JUN
NM_002228
7710122
3725
D-003268-08
TAACGCAGCAGTTGCAAAC
1196


 


JUNB








JUNB
NM_002229
4504808
3726
D-003269-05
GCATCAAAGTGGAGCGCAA
1197


 


JUNB
NM_002229
4504808
3726
D-003269-06
TGGAAGACCAAGAGCGCAT
1198


 


JUNB
NM_002229
4504808
3726
D-003269-07
CATACACAGCTACGGGATA
1199


 


JUNB
NM_002229
4504808
3726
D-003269-08
CCATCAACATGGAAGACCA
1200


 


LOC510








53








LOC510
NM_015895
20127542
51053
D-003270-05
GGAGAAAGGCGCTGTATGA
1201


53








 


LOC510
NM_015895
20127542
51053
D-003270-06
GAATAGTTCTGTCCCAAGA
1202


53








 


LOC510
NM_015895
20127542
51053
D-003270-07
GAACATGTACAGTATATGG
1203


53








 


LOC510
NM_015895
20127542
51053
D-003270-08
GCAGAAACAAGAAGAAATC
1204


53








 


MAD2L1








MAD2L1
NM_002358
6466452
4085
D-003271-05
GAAAGATGGGAGTTTGATA
1205


 


MAD2L1
NM_002358
6466452
4085
D-003271-06
TAAATAATGTGGTGGAACA
1206


 


MAD2L1
NM_002358
6466452
4085
D-003271-07
GAAATCCGTTCAGTGATCA
1207


 


MAD2L1
NM_002358
6466452
4085
D-003271-08
TTACTCGAGTGCAGAAATA
1208


 


MAD2L2








MAD2L2
NM_006341
6006019
10459
D-003272-05
GGAAGAGCGCGCTCATAAA
1209


 


MAD2L2
NM_006341
6006019
10459
D-003272-06
TGGAAGAGCGCGCTCATAA
1210


 


MAD2L2
NM_006341
6006019
10459
D-003272-07
AGCCACTCCTGGAGAAGAA
1211


 


MAD2L2
NM_006341
6006019
10459
D-003272-08
TGGAGAAATTCGTCTTTGA
1212


 


MCM2








MCM2
NM_004526
33356546
4171
D-003273-05
GAAGATCTTTGCCAGGATT
1213


 


MCM2
NM_004526
33356546
4171
D-003273-06
GGATAAGGCTGGTCAGATC
1214


 


MCM2
NM_004526
33356546
4171
D-003273-07
CAGAGCAGGTGACATATCA
1215


 


MCM2
NM_004526
33356546
4171
D-003273-08
GCCGTGGGCTCCTGTATGA
1216


 


MCM3








MCM3
NM_002388
33356548
4172
D-003274-05
GGACATCAATATTCTTCTA
1217


 


MCM3
NM_002388
33356548
4172
D-003274-06
GCCAGGACATCTCCAGTTA
1218


 


MCM3
NM_002388
33356548
4172
D-003274-07
GCAGGTATGACCAGTATAA
1219


 


MCM3
NM_002388
33356548
4172
D-003274-08
GGAAATGCCTCAAGTACAC
1220


 


MCM4








MCM4
XM_030274
22047061
4173
D-003275-05
GGACATATCTATTCTTACT
1221


 


MCM4
XM_030274
22047061
4173
D-003275-06
GATGTTAGTTCACCACTGA
1222


 


MCM4
XM_030274
22047061
4173
D-003275-07
CCAGCTGCCTCATACTTTA
1223


 


MCM4
XM_030274
22047061
4173
D-003275-08
GAAAGTACAAGATCGGTAT
1224


 


MCM5








MCM5
NM_006739
23510447
4174
D-003276-05
GAAGATCCCTGGCATCATC
1225


 


MCM5
NM_006739
23510447
4174
D-003276-06
GAACAGGGTTACCATCATG
1226


 


MCM5
NM_006739
23510447
4174
D-003276-07
GGACAACATTGACTTCATG
1227


 


MCM5
NM_006739
23510447
4174
D-003276-08
CCAAGGAGGTAGCTGATGA
1228


 


MCM6








MCM6
NM_005915
33469920
4175
D-003277-05
GGAAAGAGCTCAGAGATGA
1229


 


MCM6
NM_005915
33469920
4175
D-003277-06
GAGCAGCGATGGAGAAATT
1230


 


MCM6
NM_005915
33469920
4175
D-003277-07
GGAAACACGTGATGTCAAT
1231


 


MCM6
NM_005915
33469920
4175
D-003277-08
CCAAACATCTGCCGAAATC
1232


 


MCM7








MCM7
NM_005916
33469967
4176
D-003278-05
GGAAATATCCCTCGTAGTA
1233


 


MCM7
NM_005916
33469967
4176
D-003278-06
GGAAGAAGCAGTTCAAGTA
1234


 


MCM7
NM_005916
33469967
4176
D-003278-07
CAACAAGCCTCGTGTGATC
1235


 


MCM7
NM_005916
33469967
4176
D-003278-08
GGAGAGAACACAAGGATTG
1236


 


MDM2








MDM2
NM_002392
4505136
4193
D-003279-05
GGAGATATGTTGTGAAAGA
1237


 


MDM2
NM_002392
4505136
4193
D-003279-06
CCAGAAATCTGATAGTATT
1238


 


MDM2
NM_002392
4505136
4193
D-003279-07
GATGAGGTATATCAAGTTA
1239


 


MDM2
NM_002392
4505136
4193
D-003279-08
GGAAGAAACCCAAGACAAA
1240


 


MK167








MK167
NM_002417
19923216
4288
D-003280-05
GCACAAAGCTTGGTTATAA
1241


 


MK167
NM_002417
19923216
4288
D-003280-06
CCTAAGAGCTGAACTATTT
1242


 


MK167
NM_002417
19923216
4288
D-003280-07
CAAAGAGGAACACAAATTA
1243


 


MK167
NM_002417
19923216
4288
D-003280-08
GTAAATGGGTCTGTTATTG
1244


 


MNAT1








MNAT1
NM_002431
4505224
4331
D-003281-05
GGAAGAAGCTTTAGAAGTG
1245


 


MNAT1
NM_002431
4505224
4331
D-003281-06
TAGATGAGCTGGAGAGTTC
1246


 


MNAT1
NM_002431
4505224
4331
D-003281-07
GGACCTTGCTGGAGGCTAT
1247


 


MNAT1
NM_002431
4505224
4331
D-003281-08
GCAGATAGAGACATATGGA
1248


 


MYC








MYC
NM_002467
31543215
4609
D-003282-05
CAGAGAAGCTGGCCTCCTA
1249


 


MYC
NM_002467
31543215
4609
D-003282-06
GAAACGACGAGAACAGTTG
1250


 


MYC
NM_002467
31543215
4609
D-003282-07
CGACGAGACCTTCATCAAA
1251


 


MYC
NM_002467
31543215
4609
D-003282-08
CCACACATCAGCACAACTA
1252


 


ORC1L








ORC1L
NM_004153
31795543
4998
D-003283-05
GAACAGGAATTCCAAGACA
1253


 


ORC1L
NM_004153
31795543
4998
D-003283-06
TAAGAAACGTGCTCGAGTA
1254


 


ORC1L
NM_004153
31795543
4998
D-003283-07
GAGATCACCTCACCTTCTA
1255


 


ORC1L
NM_004153
31795543
4998
D-003283-08
GCAGAGAGCCCTTCTTGGA
1256


 


ORC2L








ORC2L
NM_006190
32454751
4999
D-003284-05
GAAGAAACCTCCTATGAGA
1257


 


ORC2L
NM_006190
32454751
4999
D-003284-06
GAAGGGAACTGATGGAGTA
1258


 


ORC2L
NM_006190
32454751
4999
D-003284-07
GAAGAATGATCCTGAGATT
1259


 


ORC2L
NM_006190
32454751
4999
D-003284-08
GAAGAGATGTTCAAGAATC
1260


 


ORC3L








ORC3L
NM_012381
32483366
23595
D-003285-05
GGACTGCTGTGTAGATATA
1261


 


ORC3L
NM_012381
32483366
23595
D-003285-06
GAACTGATGACCATACTTG
1262


 


ORC3L
NM_012381
32483366
23595
D-003285-07
AAAGATCTCTCTGCCAATA
1263


 


ORC3L
NM_012381
32483366
23595
D-003285-08
CAGCACAGCTAAGAGAATA
1264


 


ORC4L








ORC4L
NM_002552
32454749
5000
D-003286-06
GAAAGCACATTCCGTTTAT
1265


 


ORC4L
NM_002552
32454749
5000
D-003286-07
TGAAAGAACTCATGGAAAT
1266


 


ORC4L
NM_002552
32454749
5000
D-003286-08
GCTGAGAAGTGGAATGAAA
1267


 


ORC4L
NM_002552
32454749
5000
D-003286-09
CCAGTGATCTTCATATTAG
1268


 


ORC5L








ORC5L
NM_002553
32454752
5001
D-003287-05
GAAATAACCTGTGAAACAT
1269


 


ORC5L
NM_002553
32454752
5001
D-003287-06
CAGATTACCTCTCTAGTGA
1270


 


ORC5L
NM_002553
32454752
5001
D-003287-07
GAACTTCCATATTACTGTA
1271


 


ORC5L
NM_002553
32454752
5001
D-003287-08
GTATTCAGCTGATTTGTAT
1272


 


ORC6L








ORC6L
NM_014321
32454755
23594
D-003288-05
GAACATGGCTTCAAAGATA
1273


 


ORC6L
NM_014321
32454755
23594
D-003288-06
GGAGAGGGCTTATTTAATT
1274


 


ORC6L
NM_014321
32454755
23594
D-003288-07
GAAAGAAGATAGTGGTTGA
1275


 


ORC6L
NM_014321
32454755
23594
D-003288-08
TATCAGAGCTGTCTTAAAT
1276


 


PCNA








PCNA
NM_002592
33239449
5111
D-003289-05
GATCGAGGATGAAGAAGGA
1277


 


PCNA
NM_002592
33239449
5111
D-003289-07
GCCGAGATCTCAGCCATAT
1278


 


PCNA
NM_002592
33239449
5111
D-003289-09
GAGGCCTGCTGGGATATTA
1279


 


PCNA
NM_002592
33239449
5111
D-003289-10
GTGGAGAACTTGGAAATGG
1280


 


PLK








PLK
NM_005030
21359872
5347
D-003290-05
CAACCAAAGTGGAATATGA
1281


 


PLK
NM_005030
21359872
5347
D-003290-06
CAAGAAGAATGAATACAGT
1282


 


PLK
NM_005030
21359872
5347
D-003290-07
GAAGATGTCCATGGAAATA
1283


 


PLK
NM_005030
21359872
5347
D-003290-08
CAACACGCCTCATCCTCTA
1284


 


PIN1








PIN1
NM_006221
5453897
5300
D-003291-05
GGACGAAGGAGGAGGCCCT
1285


 


PIN1
NM_006221
5453897
5300
D-003291-06
GGTCCTGGCGGCAGGAGAA
1286


 


PIN1
NM_006221
5453897
5300
D-003291-07
CGGGAGAGGAGGACTTTGA
1287


 


PIN1
NM_006221
5453897
5300
D-003291-08
AGTCGGGAGAGGAGGACTT
1288


 


PIN1L








PIN1L
NM_006222
5453899
5301
D-003292-06
CGACCTGGGGGCAGGAAAT
1289


 


PIN1L
NM_006222
5453899
5301
D-003292-07
AGGCAGGAGAGAAGGACTT
1290


 


PIN1L
NM_006222
5453899
5301
D-003292-08
GCTACATCCAGAAGATCAA
1291


 


PIN1L
NM_006222
5453899
5301
D-003292-09
GGACAGTGTTCACGGATTC
1292


 


RAD1








RAD1
NM_002853
19718797
5810
D-003293-05
GAAGATGGACAAATATGTT
1293


 


RAD1
NM_002853
19718797
5810
D-003293-06
GGAAGAGTCTGTTACTTTT
1294


 


RAD1
NM_002853
19718797
5810
D-003293-07
GATAACAGAGGCTTCCTTT
1295


 


RAD1
NM_002853
19718797
5810
D-003293-08
GCATTAGTCCTATCTTGTA
1296


 


RAD17








RAD17
NM_133338
19718783
5884
D-003294-05
GAATCAAGCTTCCATATGT
1297


 


RAD17
NM_133338
19718783
5884
D-003294-06
CAACAAAGCCCGAGGATAT
1298


 


RAD17
NM_133338
19718783
5884
D-003294-07
ACACATGCCTGGAGACTTA
1299


 


RAD17
NM_133338
19718783
5884
D-003294-08
CTACATAGATTTCTTCATG
1300


 


RAD9A








RAD9A
NM_004584
19924112
5883
D-003295-05
TCAGCAAACTTGAATCTTA
1301


 


RAD9A
NM_004584
19924112
5883
D-003295-06
GACATTGACTCTTACATGA
1302


 


RAD9A
NM_004584
19924112
5883
D-003295-08
GGAAACCACTATAGGCAAT
1303


 


RAD9A
NM 004584
19924112
5883
D-003295-09
CGGACGACTTTGCCAATGA
1304


 


RB1








RB1
NM_000321
19924112
5925
D-003296-05
GAAAGGACATGTGAACTTA
1305


 


RB1
NM_000321
19924112
5925
D-003296-06
GAAGAAGTATGATGTATTG
1306


 


RB1
NM_000321
4506434
5925
D-003296-07
GAAATGACTTCTACTCGAA
1307


 


RB1
NM_000321
4506434
5925
D-003296-08
GGAGGGAACATCTATATTT
1308


 


RBBP2








RBBP2
NM_005056
4826967
5927
D-003297-05
CAAAGAAGCTGAATAAACT
1309


 


RBBP2
NM_005056
4826967
5927
D-003297-06
CAACACATATGGCGGATTT
1310


 


RBBP2
NM_005056
4826967
5927
D-003297-07
GGACAAACCTAGAAAGAAG
1311


 


RBBP2
NM_005056
4826967
5927
D-003297-08
GAAAGGCACTCTCTCTGTT
1312


 


RBL1








RBL1
NM_002895
34577078
5933
D-003298-05
CAAGAGAAGTTGTGGCATA
1313


 


RBL1
NM_002895
34577078
5933
D-003298-06
CAGCAGCACTCCATTTATA
1314


 


RBL1
NM_002895
34577078
5933
D-003298-07
ACAGAAAGGTCTATCATTT
1315


 


RBL1
NM_002895
34577078
5933
D-003298-08
GGACATAAAGTTACAATTC
1316


 


RBL2








RBL2
NM_005611
21361291
5934
D-003299-05
GAGCAGAGCTTAATCGAAT
1317


 


RBL2
NM_005611
21361291
5934
D-003299-06
GAGAATAGCCCTTGTGTGA
1318


 


RBL2
NM_005611
21361291
5934
D-003299-07
GGACTTAGTTTATGGAAAT
1319


 


RBL2
NM_005611
21361291
5934
D-003299-08
GAATTTAGATGAGCGGATA
1320


 


RBP1








RBP1
NM_002899
8400726
5947
D-003300-05
GAGACAAGCTCCAGTGTGT
1321


 


RBP1
NM_002899
8400726
5947
D-003300-06
GCAAGGAAGTATTCAAGAA
1322


 


RBP1
NM_002899
8400726
5947
D-003300-07
GCAGGACGGTGACCATATG
1323


 


RBP1
NM_002899
8400726
5947
D-003300-08
GCAAGTGCATGACAACAGT
1324


 


RPA3








RPA3
NM_002947
19923751
6119
D-003322-05
GGAAGTGGTTGGAAGAGTA
1325


 


RPA3
NM_002947
19923751
6119
D-003322-06
GAAGATAGCCATCCTTTTG
1326


 


RPA3
NM_002947
19923751
6119
D-003322-07
CATGCTAGCTCAATTCATC
1327


 


RPA3
NM_002947
19923751
6119
D-003322-08
GATCTTGGACTTTACAATG
1328


 


SKP1A








SKP1A
NM_006930
25777710
6500
D-003323-05
GGAGAGATATTTGAAGTTG
1329


 


SKP1A
NM_006930
25777710
6500
D-003323-06
GGGAATGGATGATGAAGGA
1330


 


SKP1A
NM_006930
25777710
6500
D-003323-07
CAAACAATCTGTGACTATT
1331


 


SKP1A
NM_006930
25777710
6500
D-003323-08
TCAATTAAGTTGCAGAGTT
1332


 


SKP2








SKP2
NM_005983
16306594
6502
D-003324-05
CATCTAGACTTAAGTGATA
1333


 


SKP2
NM_005983
16306594
6502
D-003324-06
GAAATCAGATCTGTCTAGT
1334


 


SKP2
NM_005983
16306594
6502
D-003324-07
CTAAAGGTCTCTGGTGTTT
1335


 


SKP2
NM_005983
16306594
6502
D-003324-08
GATGGTACCCTTCAACTGT
1336


 


SNK








SNK
NM_006622
5730054
10769
D-003325-05
GAAGACATCTACAAGCTTA
1337


 


SNK
NM_006622
5730054
10769
D-003325-06
GAAATACCTTCATGAACAA
1338


 


SNK
NM_006622
5730054
10769
D-003325-07
GAAGGTCAATGGCTGATAT
1339


 


SNK
NM_006622
5730054
10769
D-003325-08
CCGGAGATCTCGCGGATTA
1340


 


STK12








STK12
NM_004217
4759177
9212
D-003326-07
GAGAAGAGCTGCACATTTG
1341


 


STK12
NM_004217
4759177
9212
D-003326-08
CCAAACTGCTCAGGCATAA
1342


 


STK12
NM_004217
4759177
9212
D-003326-09
ACGCGGGACTTCACAATTG
1343


 


STK12
NM_004217
4759177
9212
D-003326-10
TGGGACACCCGACATCTTA
1344


 


TFDP1








TFDP1
NM_007111
34147667
7027
D-003327-05
GGAAGCAGCTCTTGCCAAA
1345


 


TFDP1
NM_007111
34147667
7027
D-003327-06
GAGGAGACTTGAAAGAATA
1346


 


TFDP1
NM_007111
34147667
7027
D-003327-07
GAACTTAGAGGTGGAAAGA
1347


 


TFDP1
NM_007111
34147667
7027
D-003327-08
GCGAGAAGGTGCAGAGGAA
1348


 


TFDP2








TFDP2
NM_006286
5454111
7029
D-003328-05
GAAAGTGTGTGAGAAAGTT
1349


 


TFDP2
NM_006286
5454111
7029
D-003328-06
CACAGGACCTTCTTGGTTA
1350


 


TFDP2
NM_006286
5454111
7029
D-003328-07
CGAAATCCCTGGTGCCAAA
1351


 


TFDP2
NM_006286
5454111
7029
D-003328-08
TGAGATCCATGATGACATA
1352


 


TP53








TP53
NM_000546
8400737
7157
D-003329-05
GAGGTTGGCTCTGACTGTA
1353


 


TP53
NM_000546
8400737
7157
D-003329-06
CAGTCTACCTCCCGCCATA
1354


 


TP53
NM_000546
8400737
7157
D-003329-07
GCACAGAGGAAGAGAATCT
1355


 


TP53
NM_000546
8400737
7157
D-003329-08
GAAGAAACCACTGGATGGA
1356


 


TP63








TP63
NM_003722
31543817
8626
D-003330-05
CATCATGTCTGGACTATTT
1357


 


TP63
NM_003722
31543817
8626
D-003330-06
CAAACAAGATTGAGATTAG
1358


 


TP63
NM_003722
31543817
8626
D-003330-07
GCACACAGACAAATGAATT
1359


 


TP63
NM_003722
31543817
8626
D-003330-08
CGACAGTCTTGTACAATTT
1360


 


TP73








TP73
NM_005427
4885644
7161
D-003331-05
GCAAGCAGCCCATCAAGGA
1361


 


TP73
NM_005427
4885644
7161
D-003331-06
GAGACGAGGACACGTACTA
1362


 


TP73
NM_005427
4885644
7161
D-003331-07
CTGCAGAACCTGACCATTG
1363


 


TP73
NM_005427
4885644
7161
D-003331-08
GGCCATGCCTGTTTACAAG
1364


 


YWHAZ








YWHAZ
NM_003406
21735623
7534
D-003332-05
GCAAGGAGCTGAATTATCC
1365


 


YWHAZ
NM_003406
21735623
7534
D-003332-06
TAAGAGATATCTGCAATGA
1366


 


YWHAZ
NM_003406
21735623
7534
D-003332-07
GAGGGAAGGTGCTGAGAAA
1367


 


YWHAZ
NM_003406
21735623
7534
D-003332-08
AGAGCAAAGTCTTCTATTT
1368





















TABLE IX





Gene




SEQ. ID


Name
Accession #
GI#
Duplex #
Sequence
NO.







AR
NM_000044
21322251
D-003400-01
GGAACTCGATCGTATCATT
1369


 


AR
NM_000044
21322251
D-003400-02
CAAGGGAGGTTACACCAAA
1370


 


AR
NM_000044
21322251
D-003400-03
TCAAGGAACTCGATCGTAT
1371


 


AR
NM_000044
21322251
D-003400-04
GAAATGATTGCACTATTGA
1372


 


ESR1
NM_000125
4503602
D-003401-01
GAATGTGCCTGGCTAGAGA
1373


 


ESR1
NM_000125
4503602
D-003401-02
CATGAGAGCTGCCAACCTT
1374


 


ESR1
NM_000125
4503602
D-003401-03
AGAGAAAGATTGGCCAGTA
1375


 


ESR1
NM_000125
4503602
D-003401-04
CAAGGAGACTCGCTACTGT
1376


 


ESR2
NM_001437
10835012
D-003402-01
GAACATCTGGTCAACATGA
1377


 


ESR2
NM_001437
10835012
D-003402-02
GCACGGCTCCATATACATA
1378


 


ESR2
NM_001437
10835012
D-003402-03
CAAGAAGATTCCCGGCTTT
1379


 


ESR2
NM_001437
10835012
D-003402-04
GGAAATGCGTAGAAGGAAT
1380


 


ESRRA
NM_004451
18860919
D-003403-01
GGCCTTCGCTGAGGACTTA
1381


 


ESRRA
NM_004451
18860919
D-003403-02
TGAATGCACTGGTGTCTCA
1382


 


ESRRA
NM_004451
18860919
D-003403-03
GCATTGAGCCTCTCTACAT
1383


 


ESRRA
NM_004451
18860919
D-003403-04
CCAGACAGCGGGCAAAGTG
1384


 


ESRRB
NM_004452
22035686
D-003404-01
TACCTGAGCTTACAAATTT
1385


 


ESRRB
NM_004452
22035686
D-003404-02
GCACTTCTATAGCGTCAAA
1386


 


ESRRB
NM_004452
22035686
D-003404-03
CAACTCCGATTCCATGTAC
1387


 


ESRRB
NM_004452
22035686
D-003404-04
GGACTCGCCACCCATGTTT
1388


 


ESRRG
NM_001438
4503604
D-003405-01
AAACAAAGATCGACACATT
1389


 


ESRRG
NM_001438
4503604
D-003405-02
TCAGGAAACTGTATGATGA
1390


 


ESRRG
NM_001438
4503604
D-003405-03
GAAGACCAGTCCAAATTAG
1391


 


ESRRG
NM_001438
4503604
D-003405-04
ATGAAGCGCTGCAGGATTA
1392


 


HNF4A
NM_000457
21361184
D-003406-01
CGACATCACTGGAGCATAT
1393


 


HNF4A
NM_000457
21361184
D-003406-02
GAAGGAAGCCGTCCAGAAT
1394


 


HNF4A
NM_000457
21361184
D-003406-03
CCAAGTACATCCCAGCTTT
1395


 


HNF4A
NM_000457
21361184
D-003406-04
GGACATGGCCGACTACAGT
1396


 


HNF4G
NM_004133
6631087
D-003407-01
GGACTGACATAAACGTTAA
1397


 


HNF4G
NM_004133
6631087
D-003407-02
ACAAAGAGATCCATGATGT
1398


 


HNF4G
NM_004133
6631087
D-003407-03
AGAGATCCATGATGTATAA
1399


 


HNF4G
NM_004133
6631087
D-003407-04
AAATGAACGTGACAGAATA
1400


 


HSAJ2425
NM_017532
8923776
D-003408-01
GAATGAATCTACACCTTTG
1401


 


HSAJ2425
NM_017532
8923776
D-003408-02
GGAAATACGTGGAGACACT
1402


 


HSAJ2425
NM_017532
8923776
D-003408-03
CCAGATAACTACGGCGATA
1403


 


HSAJ2425
NM_017532
8923776
D-003408-04
TGGCGTACCTTCTCATTGA
1404


 


NR0B1
NM_000475
5016089
D-003409-01
CAGCATGGATGATATGATG
1405


 


NR0B1
NM_000475
5016089
D-003409-02
CTGCTGAGATTCATCAATG
1406


 


NR0B1
NM_000475
5016089
D-003409-03
ACAGATTCATCGAACTTAA
1407


 


NR0B1
NM_000475
5016089
D-003409-04
GAACGTGGCGCTCCTGTAC
1408


 


NR0B2
NM_021969
13259502
D-003410-01
GAATATGCCTGCCTGAAAG
1409


 


NR0B2
NM_021969
13259502
D-003410-02
GGAATATGCCTGCCTGAAA
1410


 


NR0B2
NM_021969
13259502
D-003410-03
CGTAGCCGCTGCCTATGTA
1411


 


NR0B2
NM_021969
13259502
D-003410-04
GCCATTCTCTACGCACTTG
1412


 


NR1D1
NM_021724
13430847
D-003411-01
CAACACAGGTGGCGTCATC
1413


 


NR1D1
NM_021724
13430847
D-003411-02
GGCATGGTGTTACTGTGTA
1414


 


NR1D1
NM_021724
13430847
D-003411-03
GAACATGCATTCCGAGAAG
1415


 


NR1D1
NM_021724
13430847
D-003411-04
GCGCTTTGGTTCGTTGTTC
1416


 


NR1H2
NM_007121
11321629
D-003412-01
GAACAGATCCGGAAGAAGA
1417


 


NR1H2
NM_007121
11321629
D-003412-02
GAAGAACAGATCCGGAAGA
1418


 


NR1H2
NM_007121
11321629
D-003412-03
CTAAGCAAGTGCCTGGTTT
1419


 


NR1H2
NM_007121
11321629
D-003412-04
GCTAACAGCGGCTCAAGAA
1420


 


NR1H3
NM_005693
5031892
D-003413-01
GAACAGATCCGCCTGAAGA
1421


 


NR1H3
NM_005693
5031892
D-003413-02
GGAGATAGTTGACTTTGGT
1422


 


NR1H3
NM_005693
5031892
D-003413-03
GAGTTTGCCTTGCTCATTG
1423


 


NR1H3
NM_005693
5031892
D-003413-04
TGACTTTGCTAAACAGCTA
1424


 


NR1H4
NM_005123
4826979
D-003414-01
CAAGTGACCTCGACAACAA
1425


 


NR1H4
NM_005123
4826979
D-003414-02
GAAAGAATTCGAAATAGTG
1426


 


NR1H4
NM_005123
4826979
D-003414-03
CAACAGACTCTTCTACA1T
1427


 


NR1H4
NM_005123
4826979
D-003414-04
GAACCATACTCGCAATACA
1428


 


NR1I2
NM_003889
11863133
D-003415-01
GAACCATGCTGACTTTGTA
1429


 


NR1I2
NM_003889
11863133
D-003415-02
GATGGACGCTCAGATGAAA
1430


 


NR1I2
NM_003889
11863133
D-003415-03
CAACCTACATGTTCAAAGG
1431


 


NR1I2
NM_003889
11863133
D-003415-04
CAGGAGCAATTCGCCATTA
1432


 


NR1I3
NM_005122
4826660
D-003416-01
GGAAATCTGTCACATCGTA
1433


 


NR1I3
NM_005122
4826660
D-003416-02
TCGCAGACATCAACACTTT
1434


 


NR1I3
NM_005122
4826660
D-003416-03
CCTCTTCGCTACAGAATTG
1435


 


NR1I3
NM_005122
4826660
D-003416-04
GAACAGTTTGTGCAGTTTA
1436


 


NR2C1
NM_003297
4507672
D-003417-01
TGACAGCACTTGATGATAA
1437


 


NR2C1
NM_003297
4507672
D-003417-02
GGAAGGAAGTGTACAGCTA
1438


 


NR2C1
NM_003297
4507672
D-003417-03
GAGCACATGTTGAAACTAC
1439


 


NR2C1
NM_003297
4507672
D-003417-04
GAAGAAATTGCACATCAAA
1440


 


NR2C2
NM_003298
4507674
D-003418-01
GAACAACGGTGAGACTTGA
1441


 


NR2C2
NM_003298
4507674
D-003418-02
CTGATGAGCTCCAACATAA
1442


 


NR2C2
NM_003298
4507674
D-003418-03
CAACCTAAGTGAATCTTTG
1443


 


NR2C2
NM_003298
4507674
D-003418-04
GAAGACACCTACCGATTGG
1444


 


NR2E1
NM_003269
21361108
D-003419-01
GATCATATCTGAAATACAG
1445


 


NR2E1
NM_003269
21361108
D-003419-02
CAAGACTGCTTTCAGATAT
1446


 


NR2E1
NM_003269
21361108
D-003419-03
GTTAGATGCTACTGAATTT
1447


 


NR2E1
NM_003269
21361108
D-003419-04
CAATGTATCTCTATGAAGT
1448


 


NR2E3
NM_014249
7657394
D-003420-01
GAGAAGCTCCTTTGTGATA
1449


 


NR2E3
NM_014249
7657394
D-003420-02
GAAGCACTATGGCATCTAT
1450


 


NR2E3
NM_014249
7657394
D-003420-03
GAAGGATCCTGAGCACGTA
1451


 


NR2E3
NM_014249
7657394
D-003420-04
GAAGCTCCTTTGTGATATG
1452


 


NR2F1
NM_005654
20127484
D-003421-01
GAAACTCTCATCCGCGATA
1453


 


NR2F1
NM_005654
20127484
D-003421-02
TCTCATCCGCGATATGTTA
1454


 


NR2F1
NM_005654
20127484
D-003421-03
CAAGAAGTGCCTCAAAGTG
1455


 


NR2F1
NM_005654
20127484
D-003421-04
GGAACTTAACTTACACATG
1456


 


NR2F2
NM_021005
14149745
D-003422-01
GTACCTGTCCGGATATATT
1457


 


NR2F2
NM_021005
14149745
D-003422-02
CCAACCAGCCGACGAGATT
1458


 


NR2F2
NM_021005
14149745
D-003422-03
ACTCGTACCTGTCCGGATA
1459


 


NR2F2
NM_021005
14149745
D-003422-04
GGCCGTATATGGCAATTCA
1460


 


NR2F6
NM_005234
20070198
D-003423-01
CGACGCCTGTGGCCTCTCA
1461


 


NR2F6
NM_005234
20070198
D-003423-02
GAGCCGGTGTCCGAACTGA
1462


 


NR2F6
NM_005234
20070198
D-003423-03
CAACCGTGACTGCCAGATC
1463


 


NR2F6
NM_005234
20070198
D-003423-04
GTACTGCCGTCTCAAGAAG
1464


 


NR3C1
NM_000176
4504132
D-003424-01
GAGGACAGATGTACCACTA
1465


 


NR3C1
NM_000176
4504132
D-003424-02
GATAAGACCATGAGTATTG
1466


 


NR3C1
NM_000176
4504132
D-003424-03
GAAGACGATTCATTCCTTT
1467


 


NR3C1
NM_000176
4504132
D-003424-04
GGACAGATGTACCACTATG
1468


 


NR3C2
NM_000901
4505198
D-003425-01
GCAAACAGATGATCCAAGT
1469


 


NR3C2
NM_000901
4505198
D-003425-02
CAGCTAAGATTTATCAGAA
1470


 


NR3C2
NM_000901
4505198
D-003425-03
GCACGAAAGTCAAAGAAGT
1471


 


NR3C2
NM_000901
4505198
D-003425-04
GGTATCCGGTCTTAGAATA
1472


 


NR4A1
NM_002135
21361341
D-003426-01
GAAGGAAGTTGTCCGAACA
1473


 


NR4A1
NM_002135
21361341
D-003426-02
CAGGAGAGTTTGACACCTT
1474


 


NR4A1
NM_002135
21361341
D-003426-03
CAGTGGCTCTGACTACTAT
1475


 


NR4A1
NM_002135
21361341
D-003426-04
GAAGGCCGCTGTGCTGTGT
1476


 


NR4A2
NM_006186
5453821
D-003427-01
GCAATGCGTTCGTGGCTTT
1477


 


NR4A2
NM_006186
5453821
D-003427-02
CGGCTACACAGGAGAGTTT
1478


 


NR4A2
NM_006186
5453821
D-003427-03
CCACGTGACTTTCAAGAAT
1479


 


NR4A2
NM_006186
5453821
D-003427-04
GAATACAGCTCCGATTTCT
1480


 


NR4A3
NM_006981
11276070
D-003428-01
CAAAGAAGATCAGACATTA
1481


 


NR4A3
NM_006981
11276070
D-003428-02
GATCAGACATTACTTATTG
1482


 


NR4A3
NM_006981
11276070
D-003428-03
CCAGAGATCTTGATTATTC
1483


 


NR4A3
NM_006981
11276070
D-003428-04
GAAGTTGTCCGTACAGATA
1484


 


NR5A1
NM_004959
20070192
D-003429-01
GATTTGAAGTTCCTGAATA
1485


 


NR5A1
NM_004959
20070192
D-003429-02
GGAGCGAGCTGCTGGTGTT
1486


 


NR5A1
NM_004959
20070192
D-003429-03
GGAGGTGGCCGACCAGATG
1487


 


NR5A1
NM_004959
20070192
D-003429-04
CAACGTGCCTGAGCTCATC
1488


 


NR5A2
NM_003822
20070161
D-003430-01
CCAAACATATGGCCACTTT
1489


 


NR5A2
NM_003822
20070161
D-003430-02
TCAGAGAACTTAAGGTTGA
1490


 


NR5A2
NM_003822
20070161
D-003430-03
GGATCCATCTTCCTGGTTA
1491


 


NR5A2
NM_003822
20070161
D-003430-04
AAGAATACCTCTACTACAA
1492


 


NR6A1
NM_033334
15451847
D-003431-01
CAACGAACCTGTCTCATTT
1493


 


NR6A1
NM_033334
15451847
D-003431-02
GAAGAACTACACAGATTTA
1494


 


NR6A1
NM_033334
15451847
D-003431-03
GAAGATGGATACGCTGTGA
1495


 


NR6A1
NM_033334
15451847
D-003431-04
AAACGATACTGGTACATTT
1496


 


null
D16815
2116671
D-003432-01
GAAGAATGATCGAATAGAT
1497


 


null
D16815
2116671
D-003432-02
GAACATGGAGCAATATAAT
1498


 


null
D16815
2116671
D-003432-03
GAGGAGCTCTTGGCCTTTA
1499


 


null
D16815
2116671
D-003432-04
TAAACAACATGCACTCTGA
1500


 


PGR
NM_000926
4505766
D-003433-01
GAGATGAGGTCAAGCTACA
1501


 


PGR
NM_000926
4505766
D-003433-02
CAGCGTTTCTATCAACTTA
1502


 


PGR
NM_000926
4505766
D-003433-03
AGATAACTCTCATTCAGTA
1503


 


PGR
NM_000926
4505766
D-003433-04
GTAGTCAAGTGGTGTAAAT
1504


 


PPARA
NM_005036
7549810
D-003434-01
TGACGGAGCTCACGGAATT
1505


 


PPARA
NM_005036
7549810
D-003434-02
GAACATGACATAGAAGATT
1506


 


PPARA
NM_005036
7549810
D-003434-03
GGATAGTTCTGGAAGCTTT
1507


 


PPARA
NM_005036
7549810
D-003434-04
GACTCAAGCTGGTGTATGA
1508


 


PPARD
NM_006238
5453939
D-003435-01
GAGCGCAGCTGCAAGATTC
1509


 


PPARD
NM_006238
5453939
D-003435-02
GCATGAAGCTGGAGTACGA
1510


 


PPARD
NM_006238
5453939
D-003435-03
GGAAGCAGTTGGTGAATGG
1511


 


PPARD
NM_006238
5453939
D-003435-04
GCTGCAAGATTCAGAAGAA
1512


 


PPARG
NM_138712
20336234
D-003436-01
AGACTCAGCTCTACAATAA
1513


 


PPARG
NM_138712
20336234
D-003436-02
GATTGAAGCTTATCTATGA
1514


 


PPARG
NM_138712
20336234
D-003436-03
AAGTAACTCTCCTCAAATA
1515


 


PPARG
NM_138712
20336234
D-003436-04
GCATTTCTACTCCACATTA
1516


 


RARA
NM_000964
4506418
D-003437-01
GACAAGAACTGCATCATCA
1517


 


RARA
NM_000964
4506418
D-003437-02
GCAAATACACTACGAACAA
1518


 


RARA
NM_000964
4506418
D-003437-03
GAAGAACAGCTCAGAACAA
1519


 


RARA
NM_000964
4506418
D-003437-04
GAGCAGCAGTTCTGAAGAG
1520


 


RARB
NM_000965
14916493
D-003438-01
GCACACTGCTCAATCAATT
1521


 


RARB
NM_000965
14916493
D-003438-02
GCAGAAGTATTCAGAAGAA
1522


 


RARB
NM_000965
14916493
D-003438-03
GGAATGACAGGAACAAGAA
1523


 


RARB
NM_000965
14916493
D-003438-04
GCACAGTCCTAGCATCTCA
1524


 


RARG
NM_000966
21359851
D-003439-01
GAAATGACCGGAACAAGAA
1525


 


RARG
NM_000966
21359851
D-003439-02
TAGAAGAGCTCATCACCAA
1526


 


RARG
NM_000966
21359851
D-003439-03
CAAGGAAGCTGTGCGAAAT
1527


 


RARG
NM_000966
21359851
D-003439-04
TCAGTGAGCTGGCTACCAA
1528


 


RORA
NM_134261
19743902
D-003440-01
GGAAAGAGTTTATGTTCTA
1529


 


RORA
NM_134261
19743902
D-003440-02
CAAGATCTGTGGAGACAAA
1530


 


RORA
NM_134261
19743902
D-003440-03
GCACCTGACTGAAGATGAA
1531


 


RORA
NM_134261
19743902
D-003440-04
CCGAGAAGATGGAATACTA
1532


 


RORB
NM_006914
19743906
D-003441-01
GCACAGAACATCATTAAGT
1533


 


RORB
NM_006914
19743906
D-003441-02
CCACACCTATGAAGAAATT
1534


 


RORB
NM_006914
19743906
D-003441-03
GATCAAATTCTACTTCTGA
1535


 


RORB
NM_006914
19743906
D-003441-04
TCAAACAGATAAAGCAAGA
1536


 


RORC
NM_005060
19743908
D-003442-01
TAGAACAGCTGCAGTACAA
1537


 


RORC
NM_005060
19743908
D-003442-02
TCACCGAGGCCATTCAGTA
1538


 


RORC
NM_005060
19743908
D-003442-03
GAACAGCTGCAGTACAATC
1539


 


RORC
NM_005060
19743908
D-003442-04
CCTCATGCCACCTTGAATA
1540


 


RXRA
NM_002957
21536318
D-003443-01
TGACGGAGCTTGTGTCCAA
1541


 


RXRA
NM_002957
21536318
D-003443-02
CAACAAGGACTGCCTGATT
1542


 


RXRA
NM_002957
21536318
D-003443-03
GCAAGGACCTGACCTACAC
1543


 


RXRA
NM_002957
21536318
D-003443-04
GCAAGGACCGGAACGAGAA
1544


 


RXRB
NM_021976
21687229
D-003444-01
GCAAAGACCTTAGATACTC
1545


 


RXRB
NM_021976
21687229
D-003444-02
GCAATCATTCTGTTTAATC
1546


 


RXRB
NM_021976
21687229
D-003444-03
TCACACCGATCCATTGATG
1547


 


RXRB
NM_021976
21687229
D-003444-04
GCAAACGGCTATGTGCAAT
1548


 


RXRG
NM_006917
21361386
D-003445-01
GGAAGGACCTCATCTACAC
1549


 


RXRG
NM_006917
21361386
D-003445-02
CCGGATCTCTGGTTAAACA
1550


 


RXRG
NM_006917
21361386
D-003445-03
GCGAGCCATTGTACTCTTT
1551


 


RXRG
NM_006917
21361386
D-003445-04
GAGCCATTGTACTCTTTAA
1552


 


THRA
NM_003250
20127451
D-003446-01
GGACAAAGACGAGCAGTGT
1553


 


THRA
NM_003250
20127451
D-003446-02
GGAAACAGAGGCGGAAATT
1554


 


THRA
NM_003250
20127451
D-003446-03
GTAAGCTGATTGAGCAGAA
1555


 


THRA
NM_003250
20127451
D-003446-04
GAACCTCCATCCCACCTAT
1556


 


THRB
NM_000461
10835122
D-003447-01
GAATGTCGCTTTAAGAAAT
1557


 


THRB
NM_000461
10835122
D-003447-02
GAACAGTCGTCGCCACATC
1558


 


THRB
NM_000461
10835122
D-003447-03
GGACAAGCACCAATAGTGA
1559


 


THRB
NM_000461
10835122
D-003447-04
GTGGAAAGGTTGACTTGGA
1560


 


VDR
NM_000376
4507882
D-003448-01
TGAAGAAGCTGAACTTGCA
1561


 


VDR
NM_000376
4507882
D-003448-02
GCAACCAAGACTACAAGTA
1562


 


VDR
NM_000376
4507882
D-003448-03
TCAATGCTATGACCTGTGA
1563


 


VDR
NM_000376
4507882
D-003448-04
CCATTGAGGTCATCATGTT
1564


















TABLE X





Gene




Symbol
Sense
SEQ ID NO







ABCB1
GACCAUAAAUGUAAGGUUU
1565



UAGAAGAUCUGAUGUCAAA
1566



GAAAUGUUCACUUCAGUUA
1567



GAAGAUCGCUACUGAAGCA
1568


ABCC1
GGAAGCAAGUGCAGAGACA
1569



GAUGACACCUCUCAACAAA
1570



UAAAGUUGCUCAUCAAGUU
1571



CAACGAGUCUGCCGAAGGA
1572


ABCG2
GCAGAUGCCUUCUUCGUUA
1573



AGGCAAAUCUUCGUUAUUA
1574



GGGAAGAAAUCUGGUCUAA
1575



UGACUCAUCCCAACAUUUA
1576


KCNH2
CCGACGUGCUGCCUGAGUA
1577



GAGAAGAGCAGCGACACUU
1578



GAUCAUAGCACCUAAGAUA
1579



GCUAUUUACUGCUCUUAUU
1580



UCACUGGGCUCCUUUAAUU
1581



GUGCGAGCCUUCUGAAUAU
1582



GCUAAGCUAUACUACUGUA
1583



UGAGGGCGCUCUACUUCAC
1584


KCNH1
GAGAUGAAUUCCUUUGAAA
1585



GAAGAACGCAUGAAACGAA
1586



GAUAAAGACACGAUUGAAA
1587



GCUGAGAGGUCUAUUUAAA
1588


CLCA1
GAACAACAAUGGCUAUGAA
1589



GUACAUACCUGGCUGGAUU
1590



GAACAGCUCACAAGUAUAU
1591



GGAAACGUGUGUCUAUAUU
1592


SLC6A1
GGAGGUGGGAGGACAGUUA
1593



UCACAGCCCUGGUGGAUGA
1594



GAAGCUGGCUCCUAUGUUC
1595



GGUCAACACUACCAACAUG
1596


SLC6A2
GAACACAAGGUCAACAUUG
1597



AGAAGGAGCUGGCCUAGUG
1598



CGGAAACUCUUCACAUUUG
1599



CAACAAAUUUGACAACAAC
1600


SLC21A2
GUACAUCUCCAUCUUAUUU
1601



GGAAGUGGCUGAGUUAUUA
1602



GAAGGGAGGCUCAAUGUAA
1603



GAAGGAAGUGGCUGAGUUA
1604


SLC21A3
GUAGAAACAGGAGCUAUUA
1605



CAAGAUUACUGUCAAACAA
1606



GCACAAGAGUAUUUGGUAA
1607



GCAAAUGUCCCUUCUGUAU
1608



GCAUGACUCCUAUAUAAUA
1609



AAACAGCAAUUUCCCUUAA
1610



GGACUGCAGUUUGUACUUG
1611


SLC28A1
GUUCAUCGCUCUCCUCUUU
1612



GGAUCAAGCUGUUUCUGAA
1613



GGACUGCAGUUUGUACUUG
1614



GAGUGAAACUGACCUAUGG
1615


SLC29A1
GAACGCUGCUCCCGUGGAA
1616



GAAAGCCACUCUAUCAAAG
1617



GAAACCAGGUGCCUUCAGA
1618



CCUCACAGCUGUAUUCAUG
1619


SLC26A1
CCACGGAGCUGCUGGUCAU
1620



GGGUUGACAUCUUAUUUGA
1621



GCACGAGGGUCUCUGUGUU
1622



GGCCAUCGCCUACUCAUUG
1623



CAACACCCAUGGCAAUUAA
1624



GAGGAAAGAUCUUGCUGAU
1625



GAGCAAGCGUCCUCCAAAU
1626



GCAACACCCAUGGCAAUUA
1627


SLC26A2
CCAAAGAACUCAAUGAACA
1628



ACAAGAACCUUCAGACUAA
1629



GAAGGUAGAUAGAAGAAUG
1630



GUAUUGAACUGUACUGUAA
1631


SLC4A4
GCAAUUCUCUUCAUUUAUC
1632



GGAAAGAUGUCCACUGAAA
1633



GGACAAAGCCUUCUUCAAU
1634



GGAAUGGGAUCCAGCAAUU
1635


GLRA1
UGAAAGCCAUUGACAUUUG
1636



CAGACACGCUGGAGUUUAA
1637



CAAUAGCGCUUUCUGGUUU
1638



GCAGGUAGCAGAUGGACUA
1639


KLK1
UCAGAGUGCUGUCUUAUGU
1640



CAACUUGUUUGACGACGAA
1641



UGACAGAGCCUGCUGAUAC
1642



AGGCGGCUCUGUACCAUUU
1643


ADAM2
GAAACAUGCUGUGAUAUUG
1644



GCAGAUGUUUCCUUAUAUA
1645



CAACAGAGAUGCCAUGAUA
1646



GAAAGGCGCUACAUUGAGA
1647


XPNPEP1
GACCUGAGCUUCCCAACAA
1648



GCGACUGGCUCAACAAUUA
1649



GAGAUUGCGUGGCUAUUUA
1650



GACAGCAACUGGACACUUA
1651


GZMA
GGAAGAGACUCGUGCAAUG
1652



GGAACCAUGUGCCAAGUUG
1653



GAAGUAACUCCUCAUUCAA
1654



GAACUCCUAUAGAUUUCUG
1655


CMKLR1
CAUAGAAGCUUUACCAAGA
1656



GAAUGGAGGAUGAAGAUUA
1657



GGUCAAUGCUCUAAGUGAA
1658



GGUCAAUGGUCUAAGUGAA
1659



GAGAGGACUUCUAUGAAUG
1659


CLN3
CAUCAUGCCUUCUGAAUAA
1660



CAACAGCUCAUCACGAUUU
1661



GCAACAACUUCUCUUAUGU
1662



GGUCUUCGCUAGCAUCUCA
1663


CALCR
GGACCUAGCUGUUGUAAAG
1664



GAAAGACCAUGCAUUUAAA
1665



GCAGGAAGAUGUAUGCUUU
1666



GAAUAAACCAGUAUCGUUA
1667


OXTR
GGACCCAGAUAUCCAAAUA
1668



GCAAUACUAUCCUAACUGA
1669



GAAUAUAGAUUAGCGUUUG
1670



GAUGAGGCAUGACUACUAA
1671


EDG4
GCGAGUCUGUCCACUAUAC
1672



GAGAACGGCCACCCACUGA
1673



GAACGGCCACCCACUGAUG
1674



GGUCAAUGCUGCUGUGUAC
1675


EDG5
UCCAGGAACACUAUAAUUA
1676



GUGACCAUCUUCUCCAUCA
1677



CAUCCUCUGUUGCGCCAUU
1678



CCAACAAGGUCCAGGAACA
1679


EDG7
ACACUGAUACUGUCGAUGA
1680



AAUAGGAGCAACACUGAUA
1681



CAGCAGGAGUUACCUUGUU
1682



GGACACCCAUGAAGCUAAU
1683


PTCH
GCACAGAACUCCACUCAAA
1684



GGACAGCAGUUCAUUGUUA
1685



GAGAAGAGGCUAUGUUUAA
1686



GGACAAACUUGGACCCUUU
1687


SMO
UCGCUACCCUGCUGUUAUU
1688



GCUACAAGAACUACCGAUA
1689



CAAGAAAGCUUCCUUCAAC
1690



GAGAAGAAAUACAGUCAAU
1691


CASP3
CAAUAUAUCUGAAGAGCUA
1692



GAACUGGACUGUGGCAUUG
1693



GUGAGAAGAUGGUAUAUUU
1694



GAGGGUACUUUAAGACAUA
1695


CASP6
CAUGAGGUGUCAACUGUUA
1696



GAAGUGAAAUGCUUUAAUG
1697



AAAUAUGGCUCCUCCUUAG
1698



GCAAUCACAUUUAUGCAUA
1699



CAACAUAACUGAGGUGGAU
1700



CAUGGUACAUUCAAGAUUU
1701


CASP7
GAACUCUACUUCAGUCAAU
1704



GGGCAAAUGCAUCAUAAUA
1703



CAACAGAGGGAGUUUAAUA
1704



GAACAAAGCCACUGACUGA
1705


CASP8
GAAGUGAACUAUGAAGUAA
1706



CAACAAGGAUGAGAAGAAA
1707



GGACAAAGUUUACCAAAUG
1708



GAGGGUCGAUCAUCUAUUA
1709



GAAUAUAGAGGGCUUAUGA
1710



CAACGACUAUGAAGAAUUC
1711



GAAGUGAGCAGAUCAGAAU
1712



GAGGAAAUCUCCAAAUGGA
1713


CASP9
CCAGGCAGCUGAUCAUAGA
1714



GAACAGCUGUAAUCUAUGA
1715



GAACAGCUGUAAUCUAUGA
1716



CCACUGGUCUGUAGGGAUU
1717


DVL1
UCGUAAAGCUGUUGAUAUC
1718



GAGGAGAUCUUUGAUGACA
1719



GUAAAGCUGUUGAUAUCGA
1720



GAUCGUAAAGCUGUUGAUA
1721


DVL2
AGACGAAGGUGAUUUACCA
1722



UGUGAGAGGUACCUAGUCA
1723



GAAGAAAUUUCAGAUGACA
1724



UAAUAGGCAUUUCCUCUUU
1725


PTEN
GUGAAGAUCUUGACCAAUG
1726



GAUCAGCAUACACAAAUUA
1727



GAAUGAACCUUCUGCAACA
1728



GGCGCUAUGUGUAUUAUUA
1729


PDK1
GUACAAAGCUGGUAUAUCC
1730



GAAAGACUCCCAGUGUAUA
1731



GGAAGUCCAUCUCAUCGAA
1732



CCAAAGACAUGACGACGUU
1733


PDK2
GUAAAGAGGAGACUGAAUG
1734



GGUCUGUGAUGGUCCCUAA
1735



CAAAGAUGCCUACGACAUG
1736



GGGCGAUGCCUGAGGGUUA
1737


PPP2CA
UCACACAAGUUUAUGGUUU
1738



CAACAGCCGUGACCACUUU
1739



UAACCAAGCUGCAAUCAUG
1740



GAACUUGACGAUACUCUAA
1741


CTNNA1
GAAGAGAGGUCGUUCUAAG
1742



AAGCAGAUGUGCAUGAUUA
1743



UCUAAUAACUGCAGUGUUU
1744



GUAAAGGGCCCUCUAAUAA
1745


CTNNA2
GAAAGAAUAUGCCCAAGUU
1746



GAAGAAGAAUGCCACAAUG
1747



GCAGGAAGAUUAUGAUGUG
1748



AAAGAAAGCCCAUGUACUA
1749


HSPCA
GCUUAGAACUCUUUACUGA
1751



UAUAAGAGCUUGACCAAUG
1752



GGAGAUAUCUCUAUGAUUG
1753


DCTN2
CAACUCAUGUCCAAUACUG
1754



GGAAUGAGCCAGAUGUUUA
1755



GGAGACAGCUGUACGUUGU
1756



UCCAAGAGCUGACAACUGA
1757


CD2
GUAAGGAGAAGCAAUAUAA
1758



AAGAUGAGCUUUCCAUGUA
1759



GGACAUCUAUCUCAUCAUU
1760



GACAAGAGCCCACAGAGUA
1761


BAD
GUACUUCCCUCAGGCCUAU
1762



GCUGUGCCUUGACUACGUA
1763



GUACUUCGCUCAGGCCUAU
1764



GGUCAGGUGCCUCGAGAUC
1765


SMAC
CAGCGUAACUUCAUUCUUC
1766



UAACUUCAUUCUUCAGGUA
1767



CAGCUGCUCUUACCCAUUU
1768



GAUUGAAGCUAUUACUGAA
1769



UAGAAGAGCUCCGUCAGAA
1770



CCACAUAUGCGUUGAUUGA
1771



GCGCAGGGCUCUCUACCUA
1772


MAP3K5
GAACAGCCUUCAAAUCAAA
1773



GAUGUUCUCUACUAUGUUA
1774



GGAAAUAGUGGAAGGAUUA
1775



CAGGAAAGCUCGUAAUUUA
1776



GAUCGGGAUUUAUUUCUAU
1780


ERBB2
UGUGGGAGCUGAUGACUUU
1781



UCACAGAGAUCUUGAAAGG
1782



UGGAAGAGAUCACAGGUUA
1783



GCUCAUCGCUCACAACCAA
1784


SOS1
GAGCACCACUUCUAUGAUU
1785



CAAAGAAGCUGUUCAAUAU
1786



UGAAAGCCCUCCCUUAUUA
1787



GAAAUAGCAUGGAGAAGGA
1788


BRCA1
CCAUACAGCUUCAUAAAUA
1789



GAAGAGAACUUAUCUAGUG
1790



GAAGUGGGCUCCAGUAUUA
1791



GCAAGAUGCUGAUUCAUUA
1792



GAAGUGGGCUCCAGUAUUA
1793



GAACGGACACUGAAAUAUU
1794



GCAGAUAGUUCUACCAGUA
1795


CDKN1A
GAACAAGGAGUCAGACAUU
1796



AAACUAGGCGGUUGAAUGA
1797



GAUGGAACUUCGACUUUGU
1798



GUAAACAGAUGGGACUUUG
1799


CDKN1B
GGAAUGGACAUCCUGUAUA
1800



GGAGAAAGAUGUCAAACGU
1801



GAAUGGACAUCCUGUAUAA
1802



GUAAACAGCUCGAAUUAAG
1803


SLC2A4
CAGAUAGGCUCCGAAGAUG
1804



AGACUCAGCUCCAGAAUAC
1805



GAUCGGUUGUUUCAUCUUC
1806



CAGGAUCGGUUCUUUCAUC
1807


NOS2A
CCAGAUAAGUGACAUAAGU
1808



UAAGUGACCUGCUUUGUAA
1809



GAAGAGAGAUUCCAUUGAA
1810



UGAAAGAGCUCAACAACAA
1811


FRAP1
GAGCAUGCCGUCAAUAAUA
1812



CAAGAGAACUCAUCAUAAG
1813



CCAAAGUGCUGCAGUACUA
1814



UAAGAAAGCUAUCCAGAUU
1815


FKBP1A
GAAACAAGCCCUUUAAGUU
1816



GAAUUACUCUGCAAGUUGA
1817



CAGCACAAGUGGUAGGUUA
1818



GUUGAGGACUGAAUUACUC
1819



GAUGGCAGCUGUUUAAAUG
1820



GAGUAUCCUUUCAGUGUUA
1821


TNFRSF1A
CAAAGGAACCUACUUGUAC
1822



GGAACCUACUUGUACAAUG
1823



GAACCUACUUGUACAAUGA
1824



GAGUGUGUCUCCUGUAGUA
1825


IL1R1
GGACAAGAAUCAAUGGAUA
1826



GAACAAGCCUCCAGGAUUC
1827



GGACUUGUGUGCCCUUAUA
1828



GAACACAAAGGCACUAUAA
1829


IRAK1
CGAAGAAAGUGAUGAAUUU
1830



GCUCUUUGCCCAUCUCUUU
1831



UGAAAGACCUGGUGGAAGA
1832



GCAAUUCAGUUUCUACAUC
1833


TRAF2
GAAGACAGAGUUAUUAAAC
1834



UCACGAAGACAGAGUUAUU
1835



AGACAGAGUUAUUAAACCA
1836



CACGAAGACAGAGUUAUUA
1837



GCUGAAGCCUGUCUGAUGU
1838


TRAF6
CAAAUGAUCUGAGGCAGUU
1839



GUUCAUAGUUUGAGCGUUA
1840



GGAGAAACCUGUUGUGAUU
1841



GGACAAAGUUGCUGAAAUC
1842



CAAAUGAUCUGAGGCAGUU
1843



GGAGAAACCUGUUGUGAUU
1844



GGACAAAGUUGCUGAAAUC
1845



GUUCAUAGUUUGAGCGUUA
1846


TRADD
UGAAGCACCUUGAUCUUUG
1847



GGGGAGCGCAUACCUGUUU
1848



GAGGAGCGCUGUUUGAGUU
1849



GGAGGAGGAGCGCUGUUUG
1850



GAGGAGCGGUGUUUGAGUU
1851



GGAUGUCUCUCUCCUCUUU
1852



GCUCACUCCUUUCUACUAA
1853



UGAAGCACCUUGAUCUUUG
1854


FADD
GCACAGAUAUUUCCAUUUC
1855



GCAGUCCUCUUAUUCCUAA
1856



GAACUCAAGCUGCGUUUAU
1857



GGACGAAUUGAGAUAAUAU
1858


IKBKE
UAAGAACACUGCUCAUGAA
1859



GAGGCAUCCUGAAGCAUUA
1860



GAAGGCGGCUGCAGAACUG
1861



GGAACAAGGAGAUCAUGUA
1862


IKBKG
CUAUCGAGGUCGUUAAAUU
1863



GAAUGCAGCUGGAAGAUCU
1864



GCGGCGAGCUGGACUGUUU
1865



CCAGACCGAUGUGUAUUUA
1866


TNFRSF5
GGUCUCACCUCGCUAUGGU
1867



GAAAGCGAAUUCCUAGACA
1868



GCACAAACAAGACUGAUGU
1869



GAAGGGCACCUCAGAAACA
1870



UCUCCCAACUUGUAUUAAA
1871


RELA
UCAAGUGUCUUCCAUCAUG
1872



UCAAGUGCCUUAAUAGUAG
1873



GGAGUACCCUGAGGCUAUA
1874



GAUGAGAUCUUCCUACUGU
1875


ARHA
GAGCUGGGCUAAGUAAAUA
1876



GACCAAAGAUGGAGUGAGA
1877



GGAAGAAACUGGUGAUUGU
1878



GGCUGUAACUACUUUAUAA
1879


CDC42
GGACAUUUGUUUGCCAUUU
1880



GGAGAACCAUAUACUCUUG
1881



GAACCAAUGCUUUCUCAUG
1882



GAAGACCUGUUAUGUAGAG
1883



GAUCAAGAAUUGCAAUAUC
1884



GAAAAGGGGUGACCUAGUA
1885



UGACAAACCUUAUGGAAAA
1886


ROCK1
GGAAUGAGCUUCAGAUGCA
1887



GGACACAGCUGUAAGAUUG
1888



GACAAGAGAUUACAGAUAA
1889



GAAGAAACAUUCCCUAUUC
1890


PAK1
GAGGGUGGUUUAUGAUUAA
1891



CAACAAAGAACAAUCACUA
1892



GAAGAAAUAUACACGGUUU
1893



UACAUGAGCUUUACAGAUA
1894


PAK2
GGUAGGAGAUGAAUUGUUU
1895



AGAAGGAACUGAUCAUUAA
1896



CUACAGACCUCCAAUAUCA
1897



GAAACUGGCCAAACCGUUA
1898


PAK3
GAUUAUCGCUGCAAAGGAA
1899



GAGAGUGCCUGCAAGCUUU
1900



GACAAGAGGUGGCCAUAAA
1901



UUAAAUCGGUGUCUUGAGA
1902


PAK4
ACUAAGAGGUGAACAUGUA
1903



GAUCAUGAAUGUCCGAAGA
1904



GAUGAGACCCUACUACUGA
1905



CAGCAAAGGUGCCAAAGAU
1906


PAK6
UAAAGGCAGUUGUCCACUA
1907



GAAGGGACCUGCUUUCUUG
1908



GCAAAGACGUGCCUAAGAG
1909



CCAAUGGGCUGGCUGCAAA
1910


PAK7
GAGCACGGCUUUAAUAAGU
1911



CAAACUCCGUUAUGAUAUA
1912



GGAUAAAGUUGUCUGAUUU
1913



GGAAAUGCCUGCAUAAAUA
1914


HDAC1
GGACAUCGCUGUGAAUUGG
1915



AGAAAGAAGUCACCGAAGA
1916



GGACAAGGCCACCCAAUGA
1917



CCACAGCGAUGACUAGAUU
1918


HDAC2
GCUGUUAAAUUAUGGCUUA
1919



GCAAAGAAAGCUAGAAUUG
1920



CAUCAGAGAGUCUUAUAUA
1921



CCAAUGAGUUGCCAUAUAA
1922


CREBBP
GGCCAUAGCUUAAUUAAUC
1923



GCACAGCCGUUUACCAUGA
1924



GGACAGCCCUUUAGUCAAG
1925



GAACUGAUUCCUGAAAUAA
1926


BTRC
CACAUAAACUCGUAUCUUA
1927



GAGAAGGCACUCAAGUUUA
1928



AGACAUAGUUUACAGAGAA
1929



GCAGAGAGAUUUCAUAACU
1930


RIPK2
GAACAUACCUGUAAAUCAU
1931



GGACAUCGACCUGUUAUUA
1932



UAAAUGAACUCCUACAUAG
1933



GGAAUUAUCUCUGAACAUA
1934


VAV1
GCAGAAAUACAUCUACUAA
1935



GCUAUGAGCUGUUCUUCAA
1936



GGACAAAGCUCUAGUCAUC
1937



GCUCAACGGUGGAGACAUU
1938


VAV2
GGACAAGACUCGCAGAUUU
1939



GCUGAGCGCUUUGCAAUAA
1940



CAAGAAGUCUCACGGGAAA
1941



UCAGAGAGGCCAAGAAAUU
1942


GRB2
UGGAAGGCAUCGCCAAAUA
 432



CAUCAGUGCAUGACGUUUA
1943



UGAAUGAGCUGGUGGAUUA
1944



UGCCAAAACUUACCUAUAA
1945


PLCG1
GAGCUGCACUCCAAUGAGA
1946



GAAACCAAGCCAUUAAUGA
1947



CCAAGGAGCUACUGACAUU
1948



AGAGAAACAUGGCCCAAUA
1949


ITGB1
CCAGAGACAUUUACAUUAA
1950



GAAGGGAGUUUGCUAAAUU
1951



GAACAGAUCUGAUGAAUGA
1952



GAAGAGAGGUGAAGAGUAU
1953


ITGA4
GCAUAUAUAUUCAGCAUUG
1954



CAACUUGACUGCAGUAUUG
1955



GAACUUAACUUUCCAUGUU
1956



GACAAGACCUGUAGUAAUU
1957


STAT1
AGAAAGAGCUUGACAGUAA
1958



GGAAGUAGUUCACAAAAUA
1959



UGAAGUAUCUGUAUCCAAA
1960



GAGCUUCACUCCCUUAGUU
1961


KRAS2
UAAGGACUCUGAAGAUGUA
1962



GACAAAGUGUGUAAUUAUG
1963



GCUGAGGACUUAGCAAGAA
1964



GAAACUGAAUACCUAAGAU
1965



GAAACUGAAUACCUAAGAU
1966



UAAGGACUCUGAAGAUGUA
1967



GACAAAGUGUGUAAUUAUG
1968



GCUCAGGACUUAGCAAGAA
1969


HRAS
CCAUCCAGCUGAUCCAGAA
1970



GAACCCUCCUGAUGAGAGU
1971



GAGGACAUCCACCAGUACA
1972


BRAF
GAUUAGAGACCAAGGAUUU
2410



CCACUGAUGUGUGUUAAUU
1973



CAAUAGAACCUGUCAAUAU
1974



GAAGACAGGAAUCGAAUGA
1975


ELK1
GAUGUGAGUAGAAGAGUUA
2411



GGAAGAAUUUGUACCAUUU
1976



GAACGACCUUUCUUUCUUU
1977



GGAGUCAUCUCUUCCUAUA
1978


RALGDS
GGAGAAGCCUCACCUCUUG
1979



GCAGAAAGGACUCAAGAUU
1980



GAGAACAACUACUCAUUGA
1981



GAACUUCUCGUCACUGUAU
1982


PRKCA
GGAUUGUUCUUUCUUCAUA
1983



GAAGGGUUCUCGUAUGUCA
1984



GAAGAAGGAUGUGGUGAUU
1985



GGACUGGGAUCGAACAAGA
1986


MAP2K4
GGACAGAAGUGGAAAUAUU
1987



UCAAAGAGGUGAACAUUAA
1988



GACCAAAUCUCAGUUGUUU
1989



GGAGAAUGGUGCUGUUUAA
1990


MAP2K7
GAAGAGACCAAAGUAUAAU
1991



GAAGACCGGCGACGUCAUU
1992



GGAAGAGACCAAAGUAUAA
1993



GCAUUGAGAUUGACCAGAA
1994



UGAGAGAACGAGAAAGUUG
1995



GUGAAACCCUGUCUGCAUU
1996



GGAUCUCUCUCAACAACUA
1997



ACAACUAGGUGAACACAUA
1998


MAPK8
UCACAGUCCUGAAACGAUA
1999



GAUUGGAGAUUCUACAUUC
2000



GCUCAUGGAUGCAAAUCUU
2001



GAAGCUAAGCCGACCAUUU
2002


MAPK9
AAAGAGAGCUUAUCGUGAA
2003



GAUGAUAGGUUAGAAAUAG
2004



ACAAAGAAGUCAUGGAUUG
2005



GGAGCUGGAUCAUGAAAGA
2006


AIF1
GAAAAGGGAUGAUGGGAUU
2007



CCUAGACGAUCCCAAAUAU
2008



GAGCCAAACCAGGGAUUUA
2009



UGAAACGAAUGCUGGAGAA
2010



UCACUCACCCAGAGAAAUA
2011



CCAAGAAAGCUAUCUCUGA
2012



AGACUCACCUAGAGCUAAA
2013


BBC3
CCUGGAGGGUCCUGUACAA
2014



GAGCAAAUGAGCCAAACGU
2015



GGAGGGUCCUGUACAAUCU
2016



GACUUUCUCUGCACCAUGU
2017


BCL2L1
CCAGGGAGCUUGAAAGUUU
2018



AAAGUGCAGUUCAGUAAUA
2019



GAGAAUCACUAACCAGAGA
2020



GAGCCCAUCCCUAUUAUAA
2021


BCL2L11
GAGAGGAGUUUAACGCUUA
2022



AAAGCAACCUUCUGAUGUA
2023



CCGAGAAGGUAGACAAUUG
2024



GCAAAGCAACCUUCUGAUG
2025



AGACAGAGCCACAAGGUAA
2026



GCAAGGAGGUUAGAGAAAU
2027



CAAGGAGGUUAGAGAAAUA
2028



UCUUACGACUGUUACGUUA
2029


BID
GAAGACAUCAUCCGGAAUA
2030



CAACAGCGUUCCUAGAGAA
2031



GAAAUGGGAUGGACUGAAC
2032



AGGAUGAGCUGCAGACUGA
2033


BIRC2
GAAAGAAGCCUGCAUAUAA
2034



GAAAUUGACUCUACAUUGU
2035



ACAAAUAGCACUUAGGUUA
2036



GAAUACACCUGUGGUUAAA
2037


BIRC3
GGAGAUGCCUGCCAUUAAA
2038



UCAAUGAUCUUGUGUUAGA
2039



GAAAGAACAUGUAAAGUGU
2040



GAAGAAAGAACAUGUAAAG
2041


BIRC4
GUAGAUAGAUGGCAAUAUG
2042



GAGGAGGGCUAACUGAUUG
2043



GAGGAACCCUGCCAUGUAU
2044



GCACGGAUCUUUACUUUUG
2045


BIRC5
GGCGUAAGAUGAUGGAUUU
2046



GCAAAGGAAACCAACAAUA
2047



GCACAAAGCCAUUCUAAGU
2048



CAAAGGAAACCAACAAUAA
2049


BRCA1
CCAUACAGCUUCAUAAAUA
2050



GAAGAGAACUUAUCUAGUG
2051



GAAGUGGGCUCCAGUAUUA
2052



GCAAGAUGCUGAUUCAUUA
2053



CCAUACAGCUUCAUAAAUA
2054


CARD4
GAAAGUUAAUGUCAAGGAA
2055



GAGCAAGACUGGCAUAAGA
2056



UAACAGAGAUUUGCCUAAA
2057



GCGAAGAGCUGACCAAAUA
2058


CASP10
CAAAGGGUUUCUCUGUUUA
2059



GAAAUGACCUCCCUAAGUU
2060



GAAGGCAGCUGGUAUAUUC
2061



GACAUGAUCUUCCUUCUGA
2062



GCACUCUUCUGUUCCCUUA
2063


CASP2
GUAUUAAACUCUCCUUUGA
2064



GCAAGGAGAUGUCUGAAUA
2065



CAACUUCCCUGAUCUUUAA
2066



GCUCAAAGAUGUAAUGUAG
2067


CDKN1A
GAACAAGGAGUCAGACAUU
2068



AAACUAGGCGGUUGAAUGA
2069



GAUGGAACUUCGACUUUGU
2070



GUAAACAGAUGGCACUUUG
2071


CFLAR
GAUGUGUCCUCAUUAAUUU
2072



GAAGAGAGAUACAAGAUGA
2073



GAGCAUACCUGAAGAGAGA
2074



GCUAUGAAGUCCAGAAAUU
2075


CLK2
GUGAAUAUGUGAAAUAGUG
2076



AAAGCAUGCUAGAGUAUGA
2077



UUAAGAAUGUGGAGAAGUA
2078



GAUAACAAGCUGACACAUA
2079


CLSPN
GGACGUAAUUGAUGAAGUA
2080



GCAGAUGGGUUCUUAAAUG
2081



CAAAUGAGGUUGAGGAAAU
2082



GGAAAUACCUGGAGGAUGA
2083


CSNK2A1
UGAAUUAGAUCCACGUUUC
2084



GCAUUUAGGUGGAGACUUC
2085



GAUGUACGAUUAUAGUUUG
2086



UGAAUUAGAUCCACGUUUC
2087


CTNNB1
GCACAAGAAUGGAUCACAA
2088



GCUGAAACAUGCAGUUGUA
2089



GUACGUACCAUGCAGAAUA
2090



GAACUUGCAUUGUGAUUGG
2091


CXCR4
GAAGCAUGACGGACAAGUA
2092



GAACAUUCCAGAGCGUGUA
2093



GUUCUUAGUUGCUGUAUGU
2094



CAUCAUGGUUGGCCUUAUC
2095


CXCR6
GGAACAAACUGGCAAAGCA
2096



GAUCAGAGCAGCAGUGAAA
2097



GGGCAAAACUGAAUUAUAA
2098



GAUCUCAGGUUCUCCUUGA
2099


DAXX
CUACAGAUCUCCAAUGAAA
2100



GCUACAAGCUGGAGAAUGA
2101



GGAAACAGCUAUGUGGAAA
2102



GGAGUUGGAUCUCUCAGAA
2103


GAS41
GUAGUAAGCUAAACUGAAA
2104



GACAAUAUGUUCAAGAGAA
2105



GACAACAUCUCGUCAGCUA
2106



UAUAUGAUGUGUCCAGUAA
2107


GTSE1
CAAAGAAGCUCACUUACUG
2108



GAACAGCCCUAAAGUGGUU
2109



GAACAUGGAUGACCCUAAG
2110



GGGCAAAGCUAAAUCAAGU
2111


HDAC3
GGAAAGCGAUGUGGAGAUU
2112



CCAAGACCGUGGCCUAUUU
2113



AAAGCGAUGUGGAGAUUUA
2114



GUGAGGAGCUUCCCUAUAG
2115


HDAC5
GAAUUCCUCUUGUCGAAGU
2116



GUUAUUAGCACCUUUAAGA
2117



GGAGGGAGGCCAUGACUUG
2118



CAGGAGAGCUCAAGAAUGG
2119



GGAUAUGGAUUUCAGUUAA
2120



GGAAGUCGGUGCCUUGGUU
2121



GGAAGGAGAGGACUGGUUU
2122


HEC
GCAGAUACUUGCACGGUUU
2123



GAGUAGAACUAGAAUGUGA
2124



GCGAAUAAAUCAUGAAAGA
2125



GAAGAUGGAAUUAUGCAUA
2126


HIST1H2AA
GGCAAUGCGUCUCGCGAUA
2127



GAUCCGCAAUGAUGAGGAA
2128



GCAAUGCGUCUCGCGAUAA
2129



GAGGAACUCAAUAAGCUUU
2130


LMNB1
AAUAGAAGCUGUGCAAUUA
2131



CAACUGACCUCAUCUGGAA
2132



GAAGGAAUCUGAUCUUAAU
2133



GGGAAGGGUUUCUCUAUUA
2134


LMNB2
GGAGGUUCAUUGAGAAUUG
2134



GGCAAUAGCUCACCGUUUA
2135



CAAAUACGCUUAGCUGUGU
2136



GGAGAUCGCCUACAAGUUC
2137


MYB
GCAGAAACACUCCAAUUUA
2138



GUAAAUACGUGAAUGCAUU
2139



GCACUGAACUUUUGAGAUA
2140



GAAGAACAGUCAUUUGAUG
2141


MYT1
GAGGUGAGCUGUUAAAUCA
2142



GCAGGGUGAUUUCCUAAUA
2143



GGGAGAAGAUAUUUAAUUG
2144



CAACUUCUCUCCUGAACUU
2145


NFKBIB
GGACACGGCACUGCACUUG
2146



GCACUUGGCUGUGAUUCAU
2148



GAGACGAGGGCGAUGAAUA
2149



CAUGAACCCUUCCUGGAUU
2150


NFKBIA
GAACAUGGACUUGUAUAUU
2151



GAUGUGGGGUGAAAAGUUA
2152



GGACGAGAAAGAUCAUUGA
2153



AGGACGAGCUGCCCUAUGA
2154


NFKBIE
GAAGGGAAGUUUCAGUAAC
2155



GGAAGGGAAGUUUCAGUAA
2156



GGAAACUGCUGCUGUGUAC
2157



GAACCAACCACUCAUGGAA
2158


NUMA1
GGGAACAGUUUGAAUAUAA
2159



GCAGUAGCCUGAAGCAGAA
2160



CGAGAAGGAUGCACAGAUA
2161



GCAAGAGGCUGAGAGGAAA
2162


NUP153
GAAGACAAAUGAAAGCUAA
2163



GAUAAAGACUGCUGUUAGA
2164



GAGGAGAGCUCUAAUAUUA
2165



GAGGAAGCCUGAUUAAAGA
2166


OPA1
GAAAGAGCAUGAUGACAUA
2167



GAGGAGAGCUCUAUUAUGU
2168



GAAACUGAAUGGAAGAAUA
2169



AAAGAAGGCUGUACCGUUA
2170


PARVA
CUACAUGUCUUUGCUCUUA
2171



GCUAAGUCCUGUAAGAAUA
2172



CAAAGGCAAUGUACUGUUU
2173



GAACAAUGGUGGAUCCAAA
2174


PIK3CG
AAGUUCAGCUUCUCUAUUA
2175



GAAGAAAUCUCUGAUGGAU
2176



GAACACCUUUACUCUAUAA
2177



GCAUGGAGCUGGAGAACUA
2178


PRKDC
GAUGAAAGCUCUAAAGAUG
2179



GAAAGGAGGUUCUAAACUA
2180



GGAAGAAGCUCAUUUGAUU
2181



GCAAAGAGGUGGCAGUUAA
2182


RASA1
GGAAGAAGAUCCACAUGAA
2183



GAACAUACUUUCAGAGCUU
2184



GAACAAUCUUUGCUGUAUA
2185



UAACAGAACUGCUUCAACA
2186


SLC9A1
GAAGAGAUCCACACACAGU
2187



UCAAUGAGCUGCUGCACAU
2188



GAAGAUAGGUUUCCAUGUG
2189



GAAUUACCCUUCCUCAUCU
2190


TEGT
CUACAGAGCUUCAGUGUGA
2191



GAACAUAUUUGAUCGAAAG
2192



GAGCAAACCUAGAUAAGGA
2193



GCAUUGAUCUCUUCUUAGA
2194


TERT
GGAAGACAGUGGUGAACUU
2195



GCAAAGCAUUGGAAUCAGA
2196



GAGCUGACGUGGAAGAUGA
2197



GAACGGGCCUGGAACCAUA
2198


TNFRSF6
GAUACUAACUGCUCUCAGA
2199



GAAAGAAUGGUGUCAAUGA
2200



UCAAUAAUGUCCCAUGUAA
2201



UCAUGAAUCUCCAACCUUA
2202



GAUGUUGACUUGAGUAAAU
2203


TOP1
GAAAGGAAAUGACUAAUGA
2204



GAAGAAGGCUGUUCAGAGA
2205



GGAAGUAGCUACGUUCUUU
2206



GGACAUAAGUGGAAAGAAG
2207


TOP2A
GAAAGAGUCCAUCAGAUUU
2208



CAAACUACAUUGGCAUUUA
2209



AAACAGACAUGGAUGGAUA
2210



CGAAAGGAAUGGUUAACUA
2211


TOP3A
CCAGAAAUCUUCCACAGAA
2212



GAAACUAUCUGGAUGUGUA
2213



CCACAAAGAUGGUAUCGUA
2214



GGAAAUGGCUGUGGUAACA
2215


TOP3B
GAGACAAGAUGAAGACUGU
2216



GCACAUGGGCUGCGUCUUU
2217



CCAGUGGGCUUCAAGAUGA
2218



GAACAUCUGCUUUGAGGUU
2219


WEE1
GGUAUUGCCUUGUGAAUUU
2220



GCAGAACAAUUACGAAUAG
2221



GUACAUAGCUGUUUGAAAU
2222



GCUGUAAACUUGUAGCAUU
2223









In addition, to identifying functional siRNA against gene families or pathways, it is possible to design duplexes against genes known to be involved in specific diseases. For example when dealing with human disorders associated with allergies, it will be beneficial to develop siRNA against a number of genes including but not limited to:









the interleukin 4 receptor gene










(SEQ. ID NO. 2224:
UAGAGGUGCUCAUUCAUUU,


 



SEQ. ID NO. 2225:
GGUAUAAGCCUUUCCAAGA,


 



SEQ. ID NO. 2412:
ACACACAGCUGGAAGAAAU,


 



SEQ. ID NO. 2226:
UAACAGAGCUUCCUUAGGU),


 









the Beta-arrestin-2










(SEQ. ID NO. 2227:
GGAUGAAGGAUGACGACUA,


 



SEQ. ID NO. 2228:
ACACCAACCUCAUUGAAUU,


 



SEQ. ID NO. 2229:
CGAACAAGAUGACCAGGUA,


 



SEQ. ID NO. 2230:
GAUGAAGGAUGACGACUAU,),


 









the interferon-gamma receptor 1 gene










(SEQ. ID NO. 2231:
CAGCAUGGCUCUCCUCUUU,


 



SEQ. ID NO. 2232:
GUAAAGAACUAUGGUGUUA,


 



SEQ. ID NO. 2233:
GAAACUACCUGUUACAUUA,


 



SEQ. ID NO. 2234:
GAAGUGAGAUCCAGUAUAA),


 









the matrix metalloproteinase MMP-9










(SEQ. ID NO. 2235:
GGAACCAGCUGUAUUUGUU,


 



SEQ. ID NO. 2236:
GUUGGAGUGUUUCUAAUAA,


 



SEQ. ID NO. 2237:
GCGCUGGGCUUAGAUCAUU,


 



SEQ. ID NO. 2238:
GGAGCCAGUUUGCCGGAUA),


 









the Slcllal (Nramp1) gene










(SEQ. ID NO. 2239:
CCAAUGGCCUGCUGAACAA,


 



SEQ. ID NO. 2240:
GGGCCUGGCUUCCUCAUGA,


 



SEQ. ID NO. 2241:
GGGCAGAGCUCCACCAUGA,


 



SEQ. ID NO. 2242:
GCACGGCCAUUGCAUUCAA),


 



SPINK5




(SEQ. ID NO. 2243:
CCAACUGCCUGUUCAAUAA,


 



SEQ. ID NO. 2244:
GGAUACAUGUGAUGAGUUU,


 



SEQ. ID NO. 2245:
GGACGAAUGUGCUGAGUAU,


 



SEQ. ID NO. 2246:
GAGCUUGUCUUAUUUGCUA,),


 



the CYP1A2 gene




(SEQ. ID NO. 2247:
GAAAUGCUGUGUCUUCGUA,


 



SEQ. ID NO. 2248:
GGACAGCACUUCCCUGAGA,


 



SEQ. ID NO. 2249:
GAAGACACCACCAUUCUGA,


 



SEQ. ID NO. 2250:
GGCCAGAGCUUGACCUUCA),


 



thymosin-beta4Y




(SEQ. ID NO. 2251:
GGACAGGCCUGCGUUGUUU,


 



SEQ. ID NO. 2252:
GGAAAGAGGAAGCUCAUGA,


 



SEQ. ID NO. 2253:
GCAAACACGUUGGAUGAGU,


 



SEQ. ID NO. 2254:
GGACUAUGCUGCCCUUUUG,


 









activin A receptor IB










(SEQ. ID NO. 2255:
ACAAGACGCUCCAGGAUCU,


 



SEQ. ID NO. 2413:
GCAACAGGAUCGACUUGAG,


 



SEQ. ID NO. 2414:
GAAGCUGCGUCCCAACAUC,


 



SEQ. ID NO. 2256:
GCAUAGGCCUGUAAUCGUA,


 



SEQ. ID NO. 2257:
UCAGAGAGUUCGAGACAAA,


 



SEQ. ID NO. 2258:
UGCGAAAGGUUGUAUGUGA,


 



SEQ. ID NO. 2259:
GGAACAGGAUCGACUUGAG,


 



SEQ. ID NO. 2260:
GAAUAGCGUUGUGUGUUAU,


 



SEQ. ID NO. 2261:
UGAAUAGCGUUGUGUGUUA,


 



SEQ. ID NO. 2262:
GGGAUCAGUUUGUUGAAUA,


 



SEQ. ID NO. 2263:
GAGCCUGAAUCAUCGUUUA,),


 



ADAM33




(SEQ. ID NO. 2264:
GGAAGUACCUGGAACUGUA,


 



SEQ. ID NO. 2265:
GGACAGAGGGAACCAUUUA,


 



SEQ. ID NO. 2266:
GGUGAGAGGUAGCUCCUAA,


 



SEQ. ID NO. 2267:
AAAGACAGGUGGCCACUGA),


 



the TAP1 gene




(SEQ. ID NO. 2268:
GAAAGAUGAUCAGCUAUUU,


 



SEQ. ID NO. 2269:
CAACAGAACCAGACAGGUA,


 



SEQ. ID NO. 2270:
UGAGAAAUGUUCAGAAUGU,


 



SEQ. ID NO. 2271:
UACCUUCACUCGAAACUUA,


 



COX-2




(SEQ. ID NO. 2272:
GAACGAAAGUAAAGAUGUU,


 



SEQ. ID NO. 2273:
GGACUUAUGGGUAAUGUUA,


 



SEQ. ID NO. 2274:
UGAAAGGACUUAUGGGUAA,


 



SEQ. ID NO. 2275:
GAUCAGAGUUCACUUUCUU),


 



ADPRT




(SEQ. ID NO. 2276:
GGAAAGAUGUUAAGCAUUU,


 



SEQ. ID NO. 2277:
CAUGGGAGCUCUUGAAAUA,


 



SEQ. ID NO. 2278:
GAACAAGGAUGAAGUGAAG,


 



SEQ. ID NO. 2279:
UGAAGAAGCUCACAGUAAA,),


 



HDC




(SEQ. ID NO. 2280:
CAGCAGACCUUCAGUGUGA,


 



SEQ. ID NO. 2281:
GGAGAGAGAUGGUGGAUUA,


 



SEQ. ID NO. 2282:
GUACAGAGCUGGAGAUGAA,


 



SEQ. ID NO. 2283:
GAACGUCCCUUCAGUCUGU),


 



HnmT




(SEQ. ID NO. 2284:
CAAAUUCUCUCCAAAGUUC,


 



SEQ. ID NO. 2285:
GGAUAUAUCUGACUGCUUU,


 



SEQ. ID NO. 2286:
GAGCAGAGCUUGGGAAAGA,


 



SEQ. ID NO. 2287:
GAUAUGAGAUGUAGCAAAU),


 



GATA-3




(SEQ. ID NO. 2288:
GAACUGCUUUCUUUCGUUU,


 



SEQ. ID NO. 2289:
GCAGUAUCAUGAAGCCUAA,


 



SEQ. ID NO. 2290:
GAAACUAGGUCUGAUAUUC,


 



SEQ. ID NO. 2291:
GUACAGCUCCGGACUCUUC),


 



Gab2




(SEQ. ID NO. 2292:
GCACAACCAUUCUGAAGUU,


 



SEQ. ID NO. 2293:
GGACUUAGAUGCCCAGAUG,


 



SEQ. ID NO. 2294:
GAAGGUGGAUUCUAGGAAA,


 



SEQ. ID NO. 2295:
GGACUAGCCCUGCUGUUUA),



and



 



STAT6




(SEQ. ID NO. 2296:
GAUAGAAACUCCUGCUAAU,


 



SEQ. ID NO. 2297:
GGACAUUUAUUCCCAGCUA,


 



SEQ. ID NO. 2298:
GGACAGAGCUACAGACCUA,


 



SEQ. ID NO. 2299:
GGAUGGCUCUCCACAGAUA).






In addition, rationally designed siRNA or siRNA pools can be directed against genes involved in anemia, hemophila or hypercholesterolermia. Such genes would include, but are not be limited to:










APOA5




(SEQ. ID NO. 2300:
GAAAGACAGCCUUGAGCAA,


 



SEQ. ID NO. 2301:
GGACAGGGAGGCCACCAAA,


 



SEQ. ID NO. 2302:
GGACGAGGCUUGGGCUUUG,


 



SEQ. ID NO. 2303:
AGCAAGACCUCAAGAAUAU),


 



HMG-CoA reductase




(SEQ. ID NO. 2304:
GAAUGAAGCUUUGCCCUUU,


 



SEQ. ID NO. 2305:
GAACACAGUUUAGUGCUUU,


 



SEQ. ID NO. 2306:
UAUCAGAGGUCUUAAUGUU,


 



SEQ. ID NO. 2307:
UGAAGAAUGUCUACAGAUA),


 



NOS3




(SEQ. ID NO. 2308:
UGAAGCACCUGGAGAAUGA,


 



SEQ. ID NO. 2309:
CGGAACAGCACAAGAGUUA,


 



SEQ. ID NO. 2310:
GGAAGAAGACCUUUAAAGA,


 



SEQ. ID NO. 2415:
GCACAAGAGUUAUAAGAUC),


 



ARH




(SEQ. ID NO. 2416:
CGAUACAGCUUGGCACUUU,


 



SEQ. ID NO. 2311:
GAGAAGCGCUGCCCUGUGA,


 



SEQ. ID NO. 2312:
GAAUCAUGGUGUUCUCUUU,


 



SEQ. ID NO. 2313:
GGAGUAACCGGACAGCUUA),


 



CYP7A1




(SEQ. ID NO. 2314:
UAAGGUGACUCGAGUGUUU,


 



SEQ. ID NO. 2315:
AAACGACACUUUCAUCAAA,


 



SEQ. ID NO. 2316:
GGACUCAAGUUAAAGUAUU,


 



SEQ. ID NO. 2317:
GUAAUGGACUCAAGUUAAA),


 



FANCA




(SEQ. ID NO. 2318:
GGACAUCACUGCCCACUUC,


 



SEQ. ID NO. 2319:
AGAGGAAGAUGUUCACUUA,


 



SEQ. ID NO. 2320:
GAUCGUGGCUCUUCAGGAA,


 



SEQ. ID NO. 2321:
GGACAGAGGCAGAUAAGAA),


 



FANCG




(SEQ. ID NO. 2322:
GCACUAAGCAGCCUUCAUG,


 



SEQ. ID NO. 2323:
GCAAGCAGGUGCCUACAGA,


 



SEQ. ID NO. 2324:
GGAAUUAGAUGCUCCAUUG,


 



SEQ. ID NO. 2325:
GGACAUCUCUGCCAAAGUC),


 



ALAS




(SEQ. ID NO. 2326:
CAAUAUGCCUGGAAACUAU,


 



SEQ. ID NO. 2327:
GGUUAAGACUCACCAGUUC,


 



SEQ. ID NO. 2328:
CAACAGGACUUUAGGUUCA,


 



SEQ. ID NO. 2329:
GCAUAAGAUUGACAUCAUC),


 



PIGA




(SEQ. ID NO. 2330:
GAAAGAGGGCAUAAGGUUA,


 



SEQ. ID NO. 2331:
GGACUGAUCUUUAAACUAU,


 



SEQ. ID NO. 2332:
UCAAAUGGCUUACUUCAUC,


 



SEQ. ID NO. 2333:
UCUAAGAACUGAUGUCUAA),



and



 



factor VIII




(SEQ. ID NO. 2334:
GCAAAUAGAUCUCCAUUAC,


 



SEQ. ID NO. 2335:
CCAGAUAUGUCGUUCUUUA,


 



SEQ. ID NO. 2336:
GAAAGGCUGUGCUCUCAAA,


 



SEQ. ID NO. 2337:
GGAGAAACCUGCAUGAAAG,


 



SEQ. ID NO. 2338:
CUUGAAGCCUCCUGAAUUA,


 



SEQ. ID NO. 2339:
GAGGAAGCAUCCAAAGAUU,


 



SEQ. ID NO. 2340:
GAUAGGAGAUACAAACUUU).






Furthermore, rationally designed siRNA or siRNA pools can be directed against genes involved in disorders of the brain and nervous system. Such genes would include, but are not be limited to:










APBB1




(SEQ. ID NO. 2341:
CUACGUAGCUCGUGAUAAG,


 



SEQ. ID NO. 2342:
GCAGAGAUGUCCACACGUU,


 



SEQ. ID NO. 2343:
CAUGAGAUCUGCUCUAAGA,


 



SEQ. ID NO. 2344:
GGGCACCUCUGCUGUAUUG),


 



BACE1




(SEQ. ID NO. 2345:
CCACAGAGCAAGUGAUUUA,


 



SEQ. ID NO. 2346:
GCAGAAAGGAGAUCAUUUA,


 



SEQ. ID NO. 2347:
GUAGCAAGAUCUUUACAUA,


 



SEQ. ID NO. 2348:
UGUCAGAGCUUGAUUAGAA),


 



PSEN1




(SEQ. ID NO. 2349:
GAGCUGACAUUGAAAUAUG,


 



SEQ. ID NO. 2350:
GUACAGCUAUUUCUCAUCA,


 



SEQ. ID NO. 2351:
GAGGUUAGGUGAAGUGGUU,


 



SEQ. ID NO. 2352:
GAAAGGGAGUCACAAGACA,


 



SEQ. ID NO. 2353:
GAACUGGAGUGGAGUAGGA,


 



SEQ. ID NO. 2354:
CAGCAGGCAUAUCUCAUUA,


 



SEQ. ID NO. 2355:
UCAAGUACCUCCCUGAAUG),


 



PSEN2




(SEQ. ID NO. 2356:
GCUGGGAAGUGGCUUAAUA,


 



SEQ. ID NO. 2357:
CAUAUUCCCUGCCCUGAUA,


 



SEQ. ID NO. 2358:
GGGAAGUGCUCAAGACCUA,


 



SEQ. ID NO. 2359:
CAUAGAAAGUGACGUGUUA),


 



MASS1




(SEQ. ID NO. 2360:
GGAAGGAGCUGUUAUGAGA,


 



SEQ. ID NO. 2361:
GAAAGGAGAAGCUAAAUUA,


 



SEQ. ID NO. 2362:
GGAGGAAGGUCAAGAUUUA,


 



SEQ. ID NO. 2363:
GGAAAUAGCUGAGAUAAUG,),


 



ARX




(SEQ. ID NO. 2364:
CCAGACGCCUGAUAUUGAA,


 



SEQ. ID NO. 2365:
CAGCACCACUCAAGACCAA,


 



SEQ. ID NO. 2366:
CGCCUGAUAUUGAAGUAAA,


 



SEQ. ID NO. 2367:
CAACAUCCACUCUCUCUUG)



and



 



NNMT




(SEQ. ID NO. 2368:
GGGCAGUGCUCCAGUGGUA,


 



SEQ. ID NO. 2369:
GAAAGAGGCUGGCUACACA,


 



SEQ. ID NO. 2370:
GUACAGAAGUGAGACAUAA,


 



SEQ. ID NO. 2371:
GAGGUGAUCUCGCAAAGUU).






In addition, rationally designed siRNA or siRNA pools can be directed against genes involved in hypertension and related disorders. Such genes would include, but are not be limited to:









angiotensin II type 1 receptor










(SEQ. ID NO. 2372:
CAAGAAGCCUGGACCAUGU,


 



SEQ. ID NO. 2373:
GCACUUCACUACCAAAUGA,


 



SEQ. ID NO. 2374:
GCACUGGUCCCAAGUAGUA,


 



SEQ. ID NO. 2375:
CCAAAGGGCAGUAAAGUUU,


 



SEQ. ID NO. 2376:
GCUCAGAGGAGGUGUAUUU,


 



SEQ. ID NO. 2377:
GCACUUCACUACCAAAUGA,


 



SEQ. ID NO. 2378:
AAAGGGCAGUAAAGUUU),


 



AGTR2




(SEQ. ID NO. 2379:
GAACAUCUCUGGCAACAAU,


 



SEQ. ID NO. 2380:
GGUGAUAUAUCUCAAAUUG,


 



SEQ. ID NO. 2381:
GCAAGCAUCUUAUAUAGUU,


 



SEQ. ID NO. 2382:
GAACCAGUCUUUCAACUCA),






and other related targets.

Example XIII
Validation of Multigene Knockout using Rab5 and Eps
Two or more genes having similar, overlapping functions often leads to genetic redundancy. Mutations that knockout only one of, e.g. a pair of such genes (also referred to as homologs) results in little or no phenotype due to the fact that the remaining intact gene is capable of fulfilling the role of the disrupted counterpart. To fully understand the function of such genes in cellular physiology, it is often necessary to knockout or knockdown both homologs simultaneously. Unfortunately, concomitant knockdown of two or more genes is frequently difficult to achieve in higher organisms (e.g. mice) thus it is necessary to introduce new technologies dissect gene function. One such approach to knocking down multiple genes simultaneously is by using siRNA. For example, FIG. 11 showed that rationally designed siRNA directed against a number of genes involved in the clathrin-mediated endocytosis pathway resulted in significant levels of protein reduction (e.g. >80%). To determine the effects of gene knockdown on clathrin-related endocytosis, internalization assays were performed using epidermal growth factor and transferrin. Specifically, mouse receptor-grade EGF (Collaborative Research Inc.) and iron-saturated human transferrin (Sigma) were iodinated as described previously (Jiang, X., Huang, F., Marusyk, A. & Sorkin, A. (2003) Mol Biol Cell 14, 858-70) HeLa cells grown in 12-well dishes were incubated with 125I-EGF (1 ng/ml) or 125I-transferrin (1 μg/ml) in binding medium (DMEM, 0.1% bovine serum albumin) at 37° C., and the ratio of internalized and surface radioactivity was determined during 5-min time course to calculate specific internalization rate constant ke as described previously (Jiang, X et al.). The measurements of the uptakes of radiolabeled transferrin and EGF were performed using short time-course assays to avoid influence of the recycling on the uptake kinetics, and using low ligand concentration to avoid saturation of the clathrin-dependent pathway (for EGF Lund, K. A., Opresko, L. K., Strarbuck, C., Walsh, B. J. & Wiley, H. S. (1990) J. Biol. Chem. 265, 15713-13723).
The effects of knocking down Rab5a, 5b, 5c, Eps, or Eps 15R (individually) are shown in FIG. 22 and demonstrate that disruption of single genes has little or no effect on EGF or Tfn internalization. In contrast, simultaneous knock down of Rab5a, 5b, and 5c, or Eps and Eps 15R, leads to a distinct phenotype (note: total concentration of siRNA in these experiments remained constant with that in experiments in which a single siRNA was introduced, see FIG. 23). These experiments demonstrate the effectiveness of using rationally designed siRNA to knockdown multiple genes and validates the utility of these reagents to override genetic redundancy.
Example XIV
Validation of Multigene Targeting Using G6PD, GAPDH, PLK, and UQC
Further demonstration of the ability to knock down expression of multiple genes using rationally designed siRNA was performed using pools of siRNA directed against four separate genes. To achieve this, siRNA were transfected into cells (total siRNA concentration of 100 nM) and assayed twenty-four hours later by B-DNA. Results shown in FIG. 24 show that pools of rationally designed molecules are capable of simultaneously silencing four different genes.
Example XV
Validation of Multigene Knockouts as Demonstrated by Gene Expression Profiling, a Prophetic Example
To further demonstrate the ability to concomitantly knockdown the expression of multiple gene targets, single siRNA or siRNA pools directed against a collection of genes (e.g. 4, 8, 16, or 23 different targets) are simultaneously transfected into cells and cultured for twenty-four hours. Subsequently, mRNA is harvested from treated (and untreated) cells and labeled with one of two fluorescent probes dyes (e.g. a red fluorescent probe for the treated cells, a green fluorescent probe for the control cells.). Equivalent amounts of labeled RNA from each sample is then mixed together and hybridized to sequences that have been linked to a solid support (e.g. a slide, “DNA CHIP”). Following hybridization, the slides are washed and analyzed to assess changes in the levels of target genes induced by siRNA
Example XVI
Identifying Hyperfunctional siRNA
Identification of Hyperfunctional Bcl-2 siRNA
The ten rationally designed Bcl2 siRNA (identified in FIG. 14) and as SEQ ID Nos. 301-310) were tested to identify hyperpotent reagents. To accomplish this, each of the ten Bcl-2 siRNA were individually transfected into cells at 300 pM (0.3 nM) concentrations. Twenty-four hours later, transcript levels were assessed by B-DNA assays and compared with relevant controls. As shown in FIG. 25, while the majority of Bcl-2 siRNA failed to induce functional levels of silencing at this concentration, siRNA 1 and 8 induced>80% silencing, and siRNA 6 exhibited greater than 90% silencing at this subnanomolar concentration.
By way of prophetic examples, similar assays could be performed with any of the groups of rationally designed genes described in Example VII or Example VIII. Thus for instance, rationally designed siRNA sequences directed against PDGFA










(SEQ. ID NO. 2383:
GGUAAGAUAUUGUGCUUUA,


 



SEQ. ID NO. 2384:
CCGCAAAUAUGCAGAAUUA,


 



SEQ. ID NO. 2385:
GGAUGUACAUGGCGUGUUA,


 



SEQ. ID NO. 2386:
GGUGAAGUUUGUAUGUUUA),



or



 



PDGFB




(SEQ. ID NO. 2387:
GCUCCGCGCUUUCCGAUUU,


 



SEQ. ID NO. 2388:
GAGCAGGAAUGGUGAGAUG,


 



SEQ. ID NO. 2389:
GAACUUGGGAUAAGAGUGU,


 



SEQ. ID NO. 2390:
CCGAGGAGCUUUAUGAGAU,


 



SEQ. ID NO. 2391:
UUUAUGAGAUGCUGAGUGA)






could be introduced into cells at increasingly limiting concentrations to determine whether any of the duplexes are hyperfunctional. Similarly, rationally designed sequences directed against











HIF1 alpha




(SEQ. ID NO. 2392:
GAAGGAACCUGAUGCUUUA,


 



SEQ. ID NO. 2393:
GCAUAUAUCUAGAAGGUAU,


 



SEQ. ID NO. 2394:
GAACAAAUACAUGGGAUUA,


 



SEQ. ID NO. 2395:
GGACACAGAUUUAGACUUG),



or



 



VEGF




(SEQ. ID NO. 2396:
GAACGUACUUGCAGAUGUG,


 



SEQ. ID NO. 2397:
GAGAAAGCAUUUGUUUGUA,


 



SEQ. ID NO. 2398:
GGAGAAAGCAUUUGUUUGU,


 



SEQ. ID NO. 2399:
CGAGGCAGCUUGAGUUAAA)






could be introduced into cells at increasingly limiting concentrations and screened for hyperfunctional duplexes.

Example XVII
Gene Silencing: Prophetic Example
Below is an example of how one might transfect a cell.

    
    
        a. Select a cell line. The selection of a cell line is usually determined by the desired application. The most important feature to RNAi is the level of expression of the gene of interest. It is highly recommended to use cell lines for which siRNA transfection conditions have been specified and validated.
        b. Plate the cells. Approximately 24 hours prior to transfection, plate the cells at the appropriate density so that they will be approximately 70-90% confluent, or approximately 1×105 cells/ml at the time of transfection. Cell densities that are too low may lead to toxicity due to excess exposure and uptake of transfection reagent-siRNA complexes. Cell densities that are too high may lead to low transfection efficiencies and little or no silencing. Incubate the cells overnight. Standard incubation conditions for mammalian cells are 37° C. in 5% CO2. Other cell types, such as insect cells, require different temperatures and CO2 concentrations that are readily ascertainable by persons skilled in the art. Use conditions appropriate for the cell type of interest.
        c. SiRNA re-suspension. Add 20 μl siRNA universal buffer to each siRNA to generate a final concentration of 50 μM.
        d. SiRNA-lipid complex formation. Use RNase-free solutions and tubes. Using the following table, Table XI:
        e.
    
    












TABLE XI







96-well
24-well















Mixture 1 (TransIT-TKO-Plasmid dilution mixture)













Opti-MEM
9.3
μl
46.5
μl



TransIT-TKO (1 μg/μl)
0.5
μl
2.5
μl



Mixture 1 Final Volume
10.0
μl
50.0
μl







Mixture 2 (siRNA dilution mixture)













Opti-MEM
9.0
μl
45.0
μl



siRNA (1 μM)
1.0
μl
5.0
μl



Mixture 2 Final Volume
10.0
μl
50.0
μl







Mixture 3 (siRNA-Transfection reagent mixture)













Mixture 1
10
μl
50
μl



Mixture 2
10
μl
50
μl



Mixture 3 Final Volume
20
μl
100
μl









Incubate 20 minutes at room temperature.







Mixture 4 (Media-siRNA/Transfection reagent mixture)













Mixture 3
20
μl
100
μl



Complete media
80
μl
400
μl



Mixture 4 Final Volume
100
μl
500
μl









Incubate 48 hours at 37° C.









Transfection. Create a Mixture 1 by combining the specified amounts of OPTI-MEM serum free media and transfection reagent in a sterile polystyrene tube. Create a Mixture 2 by combining specified amounts of each siRNA with OPTI-MEM media in sterile 1 ml tubes. Create a Mixture 3 by combining specified amounts of Mixture 1 and Mixture 2. Mix gently (do not vortex) and incubate at room temperature for 20 minutes. Create a Mixture 4 by combining specified amounts of Mixture 3 to complete media. Add appropriate volume to each cell culture well. Incubate cells with transfection reagent mixture for 24-72 hours at 37° C. This incubation time is flexible. The ratio of silencing will remain consistent at any point in the time period. Assay for gene silencing using an appropriate detection method such as RT-PCR, Western blot analysis, immunohistochemistry, phenotypic analysis, mass spectrometry, fluorescence, radioactive decay, or any other method that is now known or that comes to be known to persons skilled in the art and that from reading this disclosure would useful with the present invention. The optimal window for observing a knockdown phenotype is related to the mRNA turnover of the gene of interest, although 24-72 hours is standard. Final Volume reflects amount needed in each well for the desired cell culture format. When adjusting volumes for a Stock Mix, an additional 10% should be used to accommodate variability in pipetting, etc. Duplicate or triplicate assays should be carried out when possible.
While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departure from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth and as follows in the scope of the appended claims.

## Claims

1. An siRNA molecule effective at silencing Bcl-2 expression, wherein said siRNA molecule consists of: (a) a duplex region; and (b) either no overhang regions or at least one overhang region, wherein each overhang region comprises six or fewer nucleotides, wherein said duplex region consists of a sense region and an antisense region, wherein said sense region and said antisense region together form said duplex region and said duplex region is 19 base pairs in length and said antisense region comprises a sequence that is the complement of SEQ ID NO: 301.

2. The siRNA molecule of claim 1, wherein said siRNA molecule has at least one overhang region.

3. The siRNA molecule of claim 1, wherein said siRNA molecule has no overhang regions.

4. A chemically synthesized double-stranded siRNA molecule, wherein: (a) each strand of said double-stranded siRNA molecule is 19 nucleotides in length; and (b) one strand of said double-stranded siRNA molecule comprises a sequence that is the complement of SEQ ID NO: 301.

5. An siRNA molecule consisting of:
(a) a duplex region; and
(b) either no overhang regions or at least one overhang region, wherein each overhang region comprises six or fewer nucleotides,
wherein the duplex region is nineteen base pairs in length and consists of a sense region that has a sequence that is the same as SEQ ID NO: 301 and an antisense sequence that is the complement of SEQ ID NO: 301.

6. The siRNA molecule of claim 5, wherein said siRNA molecule has no overhang regions.

7. The siRNA molecule of claim 5, wherein said siRNA molecule has at least one overhang region.

8. The siRNA molecule of claim 7, wherein said siRNA molecule has one overhang region.

9. The siRNA molecule of claim 7, wherein said siRNA molecule has two overhang regions.