PATENT ABSTRACT
The present invention concerns methods and reagents useful in modulating HIV gene expression in a variety of applications, including use in therapeutic, diagnostic, target validation, and genomic discovery applications. Specifically, the invention relates to small interfering RNA (siRNA) molecules capable of mediating RNA interference (RNAi) against HIV polypeptide and polynucleotide targets.

PATENT DESCRIPTION
PRIORITY  
       [0001]    This application claims the benefit of U.S. Application serial No. 60/294,140, filed May 29, 2001 and U.S. Application No. 60/398,036 filed Jul. 23, 2002. This application claims priority to U.S. Application Ser. No. 10/157,580 filed May 29, 2002. 
     
    
     
       BACKGROUND OF THE INVENTION  
         [0002]    The present invention concerns methods and reagents useful in modulating HIV gene expression in a variety of applications, including use in therapeutic, diagnostic, target validation, and genomic discovery applications. Specifically, the invention relates to short interfering nucleic acid molecules capable of mediating RNA interference (RNAi) against HIV expression.  
           [0003]    The following is a discussion of relevant art pertaining to RNAi. The discussion is provided only for understanding of the invention that follows. The summary is not an admission that any of the work described below is prior art to the claimed invention.  
           [0004]    RNA interference refers to the process of sequence-specific post transcriptional gene silencing in animals mediated by short interfering RNAs (siRNA) (Fire et al., 1998,  Nature,  391, 806). The corresponding process in plants is commonly referred to as post transcriptional gene silencing or RNA silencing and is also referred to as quelling in fungi. The process of post transcriptional gene silencing is thought to be an evolutionarily conserved cellular defense mechanism used to prevent the expression of foreign genes which is commonly shared by diverse flora and phyla (Fire et al., 1999,  Trends Genet.,  15, 358). Such protection from foreign gene expression may have evolved in response to the production of double stranded RNAs (dsRNA) derived from viral infection or the random integration of transposon elements into a host genome via a cellular response that specifically destroys homologous single stranded RNA or viral genomic RNA. The presence of dsRNA in cells triggers the RNAi response though a mechanism that has yet to be fully characterized. This mechanism appears to be different from the interferon response that results from dsRNA mediated activation of protein kinase PKR and 2′,5′-oligoadenylate synthetase resulting in non-specific cleavage of mRNA by ribonuclease L.  
           [0005]    The presence of long dsRNAs in cells stimulates the activity of a ribonuclease III enzyme referred to as dicer. Dicer is involved in the processing of the dsRNA into short pieces of dsRNA known as short interfering RNAs (siRNA) (Berstein et al., 2001,  Nature,  409, 363). Short interfering RNAs derived from dicer activity are typically about 21-23 nucleotides in length and comprise about 19 base pair duplexes. Dicer has also been implicated in the excision of 21 and 22 nucleotide small temporal RNAs (stRNA) from precursor RNA of conserved structure that are implicated in translational control (Hutvagner et al., 2001,  Science,  293, 834). The RNAi response also features an endonuclease complex containing a siRNA, commonly referred to as an RNA-induced silencing complex (RISC), which mediates cleavage of single stranded RNA having sequence complimentary to the antisense strand of the siRNA duplex. Cleavage of the target RNA takes place in the middle of the region complementary to the antisense strand of the siRNA duplex (Elbashir et al., 2001,  Genes Dev.,  15, 188).  
           [0006]    Short interfering RNA mediated RNAi has been studied in a variety of systems. Fire et al., 1998,  Nature,  391, 806, were the first to observe RNAi in  C. elegans.  Wianny and Goetz, 1999,  Nature Cell Biol.,  2, 70, describe RNAi mediated by dsRNA in mouse embryos. Hammond et al., 2000,  Nature,  404, 293, describe RNAi in Drosophila cells transfected with dsRNA. Elbashir et al., 2001,  Nature,  411, 494, describe RNAi induced by introduction of duplexes of synthetic 21-nucleotide RNAs in cultured mammalian cells including human embryonic kidney and HeLa cells. Recent work in Drosophila embryonic lysates (Elbashir et al., 2001,  EMBO J.,  20, 6877) has revealed certain requirements for siRNA length, structure, chemical composition, and sequence that are essential to mediate efficient RNAi activity. These studies have shown that 21 nucleotide siRNA duplexes are most active when containing two nucleotide 3′-overhangs. Furthermore, complete substitution of one or both siRNA strands with 2′-deoxy (2′-H) or 2′-O-methyl nucleotides abolishes RNAi activity, whereas substitution of the 3 40  -terminal siRNA overhang nucleotides with deoxy nucleotides (2′-H) was shown to be tolerated. Single mismatch sequences in the center of the siRNA duplex were also shown to abolish RNAi activity. In addition, these studies also indicate that the position of the cleavage site in the target RNA is defined by the 5′-end of the siRNA guide sequence rather than the 3′-end (Elbashir et al., 2001,  EMBO J.,  20, 6877). Other studies have indicated that a 5′-phosphate on the target-complementary strand of a siRNA duplex is required for siRNA activity and that ATP is utilized to maintain the 5′-phosphate moiety on the siRNA (Nykanen et al., 2001,  Cell,  107, 309).  
           [0007]    Studies have shown that replacing the 3′-overhanging segments of a 21-mer siRNA duplex having 2 nucleotide 3′ overhangs with deoxyribonucleotides does not have an adverse effect on RNAi activity. Replacing up to 4 nucleotides on each end of the siRNA with deoxyribonucleotides has been reported to be well tolerated whereas complete substitution with deoxyribonucleotides results in no RNAi activity (Elbashir et al., 2001,  EMBO J.,  20, 6877). In addition, Elbashir et al., supra, also report that substitution of siRNA with 2′-O-methyl nucleotides completely abolishes RNAi activity. Li et al., International PCT Publication No. WO 00/44914, and Beach et al., International PCT Publication No. WO 01/68836 both suggest that siRNA “may include modifications to either the phosphate-sugar back bone or the nucleoside to include at least one of a nitrogen or sulfur heteroatom”, however neither application teaches to what extent these modifications are tolerated in siRNA molecules nor provide any examples of such modified siRNA. Kreutzer and Limmer, Canadian Patent Application No. 2,359,180, also describe certain chemical modifications for use in dsRNA constructs in order to counteract activation of double stranded-RNA-dependent protein kinase PKR, specifically 2′-amino or 2′-O-methyl nucleotides, and nucleotides containing a 2′-O or 4′-C methylene bridge. However, Kreutzer and Limmer similarly fail to show to what extent these modifications are tolerated in siRNA molecules nor do they provide any examples of such modified siRNA.  
           [0008]    Parrish et al., 2000,  Molecular Cell,  6, 1977-1087, tested certain chemical modifications targeting the unc-22 gene in  C. elegans  using long (&gt;25 nt) siRNA transcripts. The authors describe the introduction of thiophosphate residues into these siRNA transcripts by incorporating thiophosphate nucleotide analogs with T7 and T3 RNA polymerase and observed that “RNAs with two [phosphorothioate] modified bases also had substantial decreases in effectiveness as RNAi triggers (data not shown); [phosphorothioate] modification of more than two residues greatly destabilized the RNAs in vitro and we were not able to assay interference activities.” Id. at 1081. The authors also tested certain modifications at the 2′-position of the nucleotide sugar in the long siRNA transcripts and observed that substituting deoxynucleotides for ribonucleotides “produced a substantial decrease in interference activity”, especially in the case of Uridine to Thymidine and/or Cytidine to deoxy-Cytidine substitutions. Id. In addition, the authors tested certain base modifications, including substituting 4-thiouracil, 5-bromouracil, 5-iodouracil, 3-(aminoallyl)uracil for uracil, and inosine for guanosine in sense and antisense strands of the siRNA, and found that whereas 4-thiouracil and 5-bromouracil were all well tolerated, inosine “produced a substantial decrease in interference activity” when incorporated in either strand. Incorporation of 5-iodouracil and 3-(aminoallyl)uracil in the antisense strand resulted in substantial decrease in RNAi activity as well.  
           [0009]    Beach et al., International PCT Publication No. WO 01/68836, describes specific methods for attenuating gene expression using endogenously derived dsRNA. Tuschl et al., International PCT Publication No. WO 01/75164, describes a Drosophila in vitro RNAi system and the use of specific siRNA molecules for certain functional genomic and certain therapeutic applications; although Tuschl, 2001,  Chem. Biochem.,  2, 239-245, doubts that RNAi can be used to cure genetic diseases or viral infection due “to the danger of activating interferon response”. Li et al., International PCT Publication No. WO 00/44914, describes the use of specific dsRNAs for use in attenuating the expression of certain target genes. Zernicka-Goetz et al., International PCT Publication No. WO 01/36646, describes certain methods for inhibiting the expression of particular genes in mammalian cells using certain dsRNA molecules. Fire et al., International PCT Publication No. WO 99/32619, describes particular methods for introducing certain dsRNA molecules into cells for use in inhibiting gene expression. Plaetinck et al., International PCT Publication No. WO 00/01846, describes certain methods for identifying specific genes responsible for conferring a particular phenotype in a cell using specific dsRNA molecules. Mello et al., International PCT Publication No. WO 01/29058, describes the identification of specific genes involved in dsRNA mediated RNAi. Deschamps Depaillette et al., International PCT Publication No. WO 99/07409, describes specific compositions consisting of particular dsRNA molecules combined with certain anti-viral agents. Driscoll et al., International PCT Publication No. WO 01/49844, describes specific DNA constructs for use in facilitating gene silencing in targeted organisms. Parrish et al., 2000,  Molecular Cell,  6, 1977-1087, describes specific chemically modified siRNA constructs targeting the unc-22 gene of  C. elegans.  Tuschl et al., International PCT Publication No. WO 02/44321, describe certain synthetic siRNA constructs.  
           [0010]    Acquired immunodeficiency syndrome (AIDS) is thought to be caused by infection with the human immunodeficiency virus, for example HIV-1. Draper et al., U.S. Pat. Nos. 6,159,692, 5,972,704, 5,693,535, and International PCT Publication Nos. WO 93/23569 and WO 95/04818, describes enzymatic nucleic acid molecules targeting HIV. Novina et al., 2002,  Nature Medicine,  advance online publication, doi:10.1039/nm725, 1-6, describes certain siRNA constructs targeting HIV-1 infection. Lee et al., 2002,  Nature Biotechnology,  19, 500-505, describes certain siRNA targeted against HIV-1 rev.  
         SUMMARY OF THE INVENTION  
         [0011]    This invention relates to compounds, compositions, and methods useful for modulating human immunodeficiency virus (HIV) function and/or gene expression in a cell by RNA interference (RNAi) using short interfering RNA (siRNA). In particular, the instant invention features siRNA molecules and methods to modulate the expression of HIV RNA. The siRNA of the invention can be unmodified or chemically modified. The siRNA of the instant invention can be chemically synthesized, expressed from a vector or enzymatically synthesized. The instant invention also features various chemically modified synthetic short interfering RNA (siRNA) molecules capable of modulating HIV gene expression/activity in cells by RNA inference (RNAi). The use of chemically modified siRNA is expected to improve various properties of native siRNA molecules through increased resistance to nuclease degradation in vivo and/or improved cellular uptake. The siRNA molecules of the instant invention provide useful reagents and methods for a variety of therapeutic, diagnostic, agricultural, target validation, genomic discovery, genetic engineering and pharmacogenomic applications.  
           [0012]    In one embodiment, the invention features one or more siRNA molecules and methods that independently or in combination modulate the expression of gene(s) encoding HIV and/or HIV polypeptides. Specifically, the present invention features siRNA molecules that modulate the expression of HIV, for example HIV-1, HIV-2, and related viruses such as FIV-1 and SIV-1; or a HIV gene, for example LTR, nef, vif, tat, or rev. In particular embodiments, the invention features nucleic acid-based molecules and methods that modulate the expression of HIV-1 encoded genes, for example (Genbank Accession No. AJ302647); HIV-2 gene, for example (Genbank Accession No. NC — 001722), FIV-1, for example (Genbank Accession No. NC — 001482), SIV-1, for example (Genbank Accession No. M66437), LTR, for example included in (Genbank Accession No. AJ302647), nef, for example included in (Genbank Accession No. AJ302647), vif, for example included in (Genbank Accession No. AJ302647), tat, for example included in (Genbank Accession No. AJ302647), and rev, for example included in (Genbank Accession No. AJ302647).  
           [0013]    In another embodiment, the invention features one or more siRNA molecules and methods that independently or in combination modulate the expression of gene(s) encoding the HIV-1 envelope glycoprotein (env, for example Genbank accession number NC — 001802), such as to inhibit CD4 receptor mediated fusion of HIV-1. In particular, the present invention describes the selection and function of siRNA molecules capable of modulating HIV-1 envelope glycoprotein expression, for example expression of the gp120 and gp41 subunits of HIV-1 envelope glycoprotein. These siRNA molecules can be used to treat diseases and disorders associated with HIV infection, or as a prophylactic measure to prevent HIV-1 infection.  
           [0014]    In one embodiment, the invention features one or more siRNA molecules and methods that independently or in combination modulate the expression of genes representing cellular targets for HIV infection, such as cellular receptors, cell surface molecules, cellular enzymes, cellular transcription factors, and/or cytokines, second messengers, and cellular accessory molecules.  
           [0015]    Non-limiting examples of such cellular receptors involved in HIV infection contemplated by the instant invention include CD4 receptors, CXCR4 (also known as Fusin; LESTR; NPY3R, such as Genbank Accession No. NM — 003467),CCR5 (also known as CKR-5; CMKRB5 such as Genbank Accession No. NM — 000579), CCR3 (also known as CC-CKR-3; CKR-3; CMKBR3, such as Genbank Accession No. NM — 001837), CCR2 (also known as CCR2b; CMKBR2, such as Genbank Accession Nos. NM — 000647 and NM — 000648), CCR1 (also known as CKR1; CMKBR1, such as Genbank Accession No. NM — 001295), CCR4 (also known as CKR-4, such as Genbank Accession No. NM — 005508), CCR8 (also known as ChemR1; TER1; CMKBR8, such as Genbank Accession No. NM — 005201), CCR9 (also known as D6, such as Genbank Accession Nos. NM — 006641 and NM — 031200), CXCR2 (also known as IL-8RB, such as Genbank Accession No. NM — 001557), STRL33 (also known as Bonzo; TYMSTR, such as Genbank Accession No. NM — 006564), US28, V28 (also known as CMKBRL1; CX3CR1; GPR13, such as Genbank Accession No. NM — 001337), gpr1 (also known as GPR1, such as Genbank Accession No. NM — 005279), gpr15 (also known as BOB; GPR15, such as Genbank Accession No. NM — 005290), Apj (also known as angiotensin-receptor-like; AGTRL1, such as Genbank Accession No. NM — 005161), and ChemR23 receptors (such as Genbank Accession No. NM — 004072).  
           [0016]    Non-limiting examples of cell surface molecules involved in HIV infection contemplated by the instant invention include Heparan Sulfate Proteoglycans, HSPG2 (such as Genbank Accession No. NM — 005529), SDC2 (such as Genbank Accession Nos. AK025488, J04621, J04621), SDC4 (such as Genbank Accession No. NM — 002999), GPC1 (such as Genbank Accession No. NM — 002081), SDC3 (such as Genbank Accession No. NM — 014654), SDC1 (such as Genbank Accession No. NM — 002997), Galactoceramides, (such as Genbank Accession Nos. NM — 000153, NM — 003360, NM — 001478.2, NM — 004775, and NM — 004861) and Erythrocyte-expressed Glycolipids (such as Genbank Accession Nos. NM — 003778, NM — 003779, NM — 003780, NM — 030587, and NM — 001497).  
           [0017]    Non-limiting examples of cellular enzymes involved in HIV infection contemplated by the invention include N-myristoyltransferase (NMT1, such as Genbank Accession No. NM — 021079, and NMT2, such as Genbank Accession No. NM — 004808), Glycosylation Enzymes (such as Genbank Accession Nos. NM — 000303, NM — 013339, NM — 003358, NM — 005787, NM — 002408, NM — 002676, NM — 002435), NM — 002409, NM — 006122, NM — 002372, NM — 006699), NM — 005907, NM — 004479, NM — 000150, NM — 005216 and NM — 005668), gp-160 Processing Enzymes (such as PCSK5, Genbank Accession No. NM — 006200), Ribonucleotide Reductase (such as Genbank Accession Nos. NM — 001034, NM — 001033, AB036063, AB036063, AB036532, AK001965, AK001965, AK023605, AL137348, and AL137348), and Polyamine Biosynthesis enzymes (such as Genbank Accession Nos. NM — 002539, NM — 003132 and NM — 001634).  
           [0018]    Non-limiting examples of cellular transcription factors involved in HIV infection contemplated by the invention include SP-1 and NF-kappa B (such as NFKB2, Genbank Accession No. NM — 002502, RELA, Genbank Accession No. NM — 021975, and NFKB1 Genbank Accession No. NM — 003998). Non-limiting examples of cytokines and second messengers involved in HIV infection contemplated by the invention include Tumor Necrosis Factor-a (TNF-a, such as Genbank Accession No. NM — 000594), Interleukin 1a (IL-1a, such as Genbank Accession No. NM — 000575), Interleukin 6 (IL-6, such as Genbank Accession No. NM — 000600), Phospholipase C (such as Genbank Accession No. NM — 000933) and Protein Kinase C (such as Genbank Accession No. NM — 006255). Non-limiting examples of cellular accessory molecules involved in HIV infection contemplated by the invention include, Cyclophilins, (such as PPID, Genbank Accession No. NM — 005038, PPIA, Genbank Accession No. NM — 021130, PPIE, Genbank Accession No. NM — 006112, PPIB, Genbank Accession No. NM — 000942, PPIF Genbank Accession No. NM — 005729, PPIG Genbank Accession No. NM — 004792, and PPIC, Genbank Accession No. NM — 000943), MAP-Kinase (Mitogen Activated Protein Kinase, such as MAPK1 Genbank Accession Nos. NM — 002745 and NM — 138957), and ERK-Kinase (Extracellular Signal-Regulated Kinase).  
           [0019]    The description below of the various aspects and embodiments is provided with reference to the exemplary HIV-1 gene, referred to herein as HIV. However, the various aspects and embodiments are also directed to other genes which encode HIV polypeptides and/or similar viruses to HIV, as well as cellular targets as described herein. Those additional genes can be analyzed for target sites using the methods described for HIV. Thus, the inhibition and the effects of such inhibition of the other genes can be performed as described herein.  
           [0020]    Due to the high sequence variability of the HIV genome, selection of nucleic acid molecules for broad therapeutic applications would likely involve the conserved regions of the HIV genome. Specifically, the present invention describes nucleic acid molecules that cleave the conserved regions of the HIV genome. Therefore, one nucleic acid molecule can be designed to cleave all the different isolates of HIV. Nucleic acid molecules designed against conserved regions of various HIV isolates can enable efficient inhibition of HIV replication in diverse subject populations and can ensure the effectiveness of the nucleic acid molecules against HIV quasi species which evolve due to mutations in the non-conserved regions of the HIV genome.  
           [0021]    In one embodiment, the invention features a siRNA molecule that down regulates expression of a HIV gene by RNA interference, for example, wherein the HIV gene comprises HIV encoding sequence.  
           [0022]    A siRNA molecule can be adapted for use to treat HIV infection or acquired immunodeficiency syndrome (AIDS). A siRNA molecule can comprise a sense region and an antisense region and wherein said antisense region comprises sequence complementary to a HIV RNA sequence and the sense region comprises sequence complementary to the antisense region. A siRNA molecule can be assembled from two nucleic acid fragments wherein one fragment comprises the sense region and the second fragment comprises the antisense region of said siRNA molecule. The sense region and antisense region can be covalently connected via a linker molecule. The linker molecule can be a polynucleotide linker or a non-nucleotide linker.  
           [0023]    In one embodiment, the invention features a siRNA molecule having RNAi activity against HIV-1 RNA, wherein the siRNA molecule comprises a sequence complimentary to any RNA having HIV-1 encoding sequence, for example Genbank Accession No. AJ302647. In another embodiment, the invention features a siRNA molecule having RNAi activity against HIV-2 RNA, wherein the siRNA molecule comprises a sequence complimentary to any RNA having HIV-2 encoding sequence, for example Genbank Accession No. NC — 001722. In another embodiment, the invention features a siRNA molecule having RNAi activity against FIV-1 RNA, wherein the siRNA molecule comprises a sequence complimentary to any RNA having FIV-1 encoding sequence, for example Genbank Accession No. NC — 001482. In another embodiment, the invention features a siRNA molecule having RNAi activity against SIV-1 RNA, wherein the siRNA molecule comprises a sequence complimentary to any RNA having SIV-1 encoding sequence, for example Genbank Accession No. M66437.  
           [0024]    In another embodiment, the invention features a siRNA molecule comprising sequences selected from the group consisting of SEQ ID NOs: 1-1476. A siRNA molecule can comprise and antisense region that comprises sequence complementary to sequence having any of SEQ ID NOs. 1-738. The antisense region can comprises sequence having any of SEQ ID NOs. 739-1476. The sense region can comprise sequence having any of SEQ ID NOs. 1-738. The sequences shown in SEQ ID NO:1-1476 are not limiting. A siRNA molecule of the invention can comprise any contiguous HIV sequences (e.g., about 19 contiguous HIV nucleotides).  
           [0025]    In yet another embodiment, the invention features a siRNA molecule comprising a sequence complementary to a sequence comprising Genbank Accession Nos. AJ302647 (HIV-1), NC — 001722 (HIV-2), NC — 001482 (FIV-1) and/or M66437 (SIV-1).  
           [0026]    In one embodiment, a siRNA molecule of the invention has RNAi activity that modulates expression of RNA encoded by a HIV gene.  
           [0027]    A sense region of a siRNA molecule of the invention can comprise a 3′-terminal overhang and the antisense region can comprises a 3′-terminal overhang. The 3′-terminal overhangs each can comprise about 2 nucleotides. The antisense region 3′-terminal nucleotide overhang can be complementary to a HIV RNA.  
           [0028]    In one embodiment, nucleic acid molecules of the invention that act as mediators of the RNA interference gene silencing response are double stranded RNA molecules. In another embodiment, the siRNA molecules of the invention consist of duplexes containing about 19 base pairs between oligonucleotides comprising about 19 to about 25 nucleotides, for example, about 19, 20, 21, 22, 23, 24 or 25 nucleotides. In yet another embodiment, siRNA molecules of the invention comprise duplexes with overhanging ends of 1-3 (i.e., 1, 2 or 3) nucleotides, for example 21 nucleotide duplexes with 19 base pairs and 2 nucleotide 3′-overhangs. These nucleotide overhangs in the antisense strand are optionally complimentary to the target sequence.  
           [0029]    In one embodiment, the invention features one or more chemically modified siRNA constructs having specificity for HIV expressing nucleic acid molecules. Non-limiting examples of such chemical modifications include without limitation phosphorothioate internucleotide linkages, 2′-O-methyl ribonucleotides, 2′-O-methyl modified pyrimidine nucleotides, 2′-deoxy-2′-fluoro ribonucleotides, 2′-deoxy-2′-fluoro modified pyrimidine nucleotides, “universal base” nucleotides, 5-C-methyl nucleotides, and inverted deoxyabasic residue incorporation. These chemical modifications, when used in various siRNA constructs, are shown to preserve RNAi activity in cells while at the same time, dramatically increasing the serum stability of these compounds. Furthermore, contrary to the data published by Parrish et al., supra, applicant demonstrates that multiple (greater than one) phosphorothioate substitutions are well tolerated and confer substantial increases in serum stability for modified siRNA constructs. Chemical modifications of the siRNA constructs can also be used to improve the stability of the interaction with target RNA sequence and to improve nuclease resistance.  
           [0030]    In one embodiment of the invention a siRNA molecule has an antisense region comprising a phosphorothioate internucleotide linkage at the 3′ end of said antisense region. An antisense region can comprise between about one and about five phosphorothioate internucleotide linkages at the 5′ end of said antisense region. The 3′-terminal nucleotide overhangs can comprise ribonucleotides or deoxyribonucleotides that are chemically modified at a nucleic acid sugar, base, or backbone. The 3′-terminal nucleotide overhangs can comprise one or more universal base ribonucleotides. The 3′-terminal nucleotide overhangs can comprise one or more acyclic nucleotides.  
           [0031]    In another embodiment of the invention, an expression vector comprising a nucleic acid sequence encoding at least one siRNA molecule of the invention in a manner that allows expression of the nucleic acid molecule. Another embodiment of the invention comprises a mammalian cell comprising an expression vector comprising a nucleic acid sequence encoding at least one siRNA molecule of the invention in a manner that allows expression of the nucleic acid molecule. The mammalian cell can be a human cell. The expression vector can comprise a siRNA molecule that comprises a sense region and an antisense region and wherein said antisense region comprises sequence complementary to a HIV RNA sequence and the sense region comprises sequence complementary to the antisense region. The expression vector can comprise a siRNA molecule that comprises two distinct strands having complementarity sense and antisense regions. The expression vector can comprise a siRNA molecule that comprises a single strand having complementary sense and antisense regions. In a non-limiting example, the introduction of chemically modified nucleotides into nucleic acid molecules will provide a powerful tool in overcoming potential limitations of in vivo stability and bioavailability inherent to native RNA molecules that are delivered exogenously. For example, the use of chemically modified nucleic acid molecules can enable a lower dose of a particular nucleic acid molecule for a given therapeutic effect since chemically modified nucleic acid molecules tend to have a longer half-life in serum. Furthermore, certain chemical modifications can improve the bioavailability of nucleic acid molecules by targeting particular cells or tissues and/or improving cellular uptake of the nucleic acid molecule. Therefore, even if the activity of a chemically modified nucleic acid molecule is reduced as compared to a native nucleic acid molecule, for example when compared to an all RNA nucleic acid molecule, the overall activity of the modified nucleic acid molecule can be greater than the native molecule due to improved stability and/or delivery of the molecule. Unlike native unmodified siRNA, chemically modified siRNA can also minimize the possibility of activating interferon activity in humans.  
           [0032]    In one embodiment, the invention features a chemically modified short interfering RNA (siRNA) molecule capable of mediating RNA interference (RNAi) against HIV inside a cell, wherein the chemical modification comprises one or more nucleotides comprising a backbone modified internucleotide linkage having Formula I:  
                         
 
           [0033]    wherein each R1 and R2 is independently any nucleotide, non-nucleotide, or polynucleotide which can be naturally occurring or chemically modified, each X and Y is independently O, S, N, alkyl, or substituted alkyl, each Z and W is independently O, S, N, alkyl, substituted alkyl, O-alkyl, S-alkyl, alkaryl, or aralkyl, and wherein W, X, Y and Z are not all O.  
           [0034]    The chemically modified internucleotide linkages having Formula I, for example wherein any Z, W, X, and/or Y independently comprises a sulphur atom, can be present in one or both oligonucleotide strands of the siRNA duplex, for example in the sense strand, antisense strand, or both strands. The siRNA molecules of the invention can comprise one or more chemically modified internucleotide linkages having Formula I at the 3′-end, 5′-end, or both 3′ and 5′-ends of the sense strand, antisense strand, or both strands. For example, an exemplary siRNA molecule of the invention can comprise between about 1 and about 5 or more, for example, about 1, 2, 3, 4, 5 or more chemically modified internucleotide linkages having Formula I at the 5′-end of the sense strand, antisense strand, or both strands. In another non-limiting example, an exemplary siRNA molecule of the invention can comprise one or more pyrimidine nucleotides with chemically modified internucleotide linkages having Formula I in the sense strand, antisense strand, or both strands. In yet another non-limiting example, an exemplary siRNA molecule of the invention can comprise one or more purine nucleotides with chemically modified internucleotide linkages having Formula I in the sense strand, antisense strand, or both strands. In another embodiment, a siRNA molecule of the invention having internucleotide linkage(s) of Formula I also comprises a chemically modified nucleotide or non-nucleotide having any of Formulae II, III, V, or VI.  
           [0035]    In one embodiment, the invention features a chemically modified short interfering RNA (siRNA) molecule capable of mediating RNA interference (RNAi) against HIV inside a cell, wherein the chemical modification comprises one or more nucleotides or non-nucleotides having Formula II:  
                         
 
           [0036]    wherein each R3, R4, R5, R6, R7, R8, R10, R11 and R12 is independently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF3, OCF3, OCN, 0-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, SO-alkyl, alkyl-OSH, alkyl-OH, O-alkyl-OH, O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl, ONO2, NO2, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl, O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalklylamino, substituted silyl, or group having Formula I; R9 is O, S, CH2, S═O, CHF, or CF2, and B is a nucleosidic base such as adenine, guanine, uracil, cytosine, thymine, 2-aminoadenosine, 5-methylcytosine, 2,6-diaminopurine, or any other non-naturally occurring base that can be employed to form a stable duplex with RNA or a non-nucleosidic base such as phenyl, naphthyl, 3-nitropyrrole, 5-nitroindole, nebularine, pyridone, pyridinone, or any other non-naturally occurring universal base that can be employed to form a stable duplex with RNA.  
           [0037]    The chemically modified nucleotide or non-nucleotide of Formula II can be present in one or both oligonucleotide strands of the siRNA duplex, for example in the sense strand, antisense strand, or both strands. The siRNA molecules of the invention can comprise one or more chemically modified nucleotide or non-nucleotide of Formula II at the 3′-end, 5′-end, or both 3′ and 5′-ends of the sense strand, antisense strand, or both strands. For example, an exemplary siRNA molecule of the invention can comprise between about 1 and about 5 or more, for example, about 1, 2, 3, 4, 5 or more chemically modified nucleotide or non-nucleotide of Formula II at the 5′-end of the sense strand, antisense strand, or both strands. In anther non-limiting example, an exemplary siRNA molecule of the invention can comprise between about 1 and about 5 or more, for example, 1, 2, 3, 4, 5 or more chemically modified nucleotide or non-nucleotide of Formula II at the 3′-end of the sense strand, antisense strand, or both strands.  
           [0038]    In one embodiment, the invention features a chemically modified short interfering RNA (siRNA) molecule capable of mediating RNA interference (RNAi) against HIV inside a cell, wherein the chemical modification comprises one or more nucleotides or non-nucleotides having Formula III:  
                         
 
           [0039]    wherein each R3, R4, R5, R6, R7, R8, R10, R11 and R12 is independently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, SO-alkyl, alkyl-OSH, alkyl-OH, O-alkyl-OH, O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl, ONO2, NO2, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl, O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalklylamino, substituted silyl, or group having Formula I; R9 is O, S, CH2, S═O, CHF, or CF2, and B is a nucleosidic base such as adenine, guanine, uracil, cytosine, thymine, 2-aminoadenosine, 5-methylcytosine, 2,6-diaminopurine, or any other non-naturally occurring base that can be employed to form a stable duplex with RNA or a non-nucleosidic base such as phenyl, naphthyl, 3-nitropyrrole, 5-nitroindole, nebularine, pyridone, pyridinone, or any other non-naturally occurring universal base that can be employed to form a stable duplex with RNA.  
           [0040]    The chemically modified nucleotide or non-nucleotide of Formula III can be present in one or both oligonucleotide strands of the siRNA duplex, for example in the sense strand, antisense strand, or both strands. The siRNA molecules of the invention can comprise one or more chemically modified nucleotide or non-nucleotide of Formula III at the 3′-end, 5′-end, or both 3′ and 5′-ends of the sense strand, antisense strand, or both strands. For example, an exemplary siRNA molecule of the invention can comprise between about 1 and about 5 or more, for example, about 1, 2, 3, 4, 5 or more chemically modified nucleotide or non-nucleotide of Formula III at the 5′-end of the sense strand, antisense strand, or both strands. In anther non-limiting example, an exemplary siRNA molecule of the invention can comprise between about 1 and about 5 or more, for example, about 1, 2, 3, 4, 5 or more chemically modified nucleotide or non-nucleotide of Formula III at the 3′-end of the sense strand, antisense strand, or both strands.  
           [0041]    In another embodiment, a siRNA molecule of the invention comprises a nucleotide having Formula II or III, wherein the nucleotide having Formula II or III is in an inverted configuration. For example, the nucleotide having Formula II or III is connected to the siRNA construct in a 3′,3′, 3′−2′, 2′−3′, or 5′,5′ configuration, such as at the 3′-end, 5′-end, or both 3′ and 5′ ends of one or both siRNA strands.  
           [0042]    In one embodiment, the invention features a chemically modified short interfering RNA (siRNA) molecule capable of mediating RNA interference (RNAi) against HIV inside a cell, wherein the chemical modification comprises a 5′-terminal phosphate group having Formula IV:  
                         
 
           [0043]    wherein each X and Y is independently O, S, N, alkyl, substituted alkyl, or alkylhalo; each Z and W is independently O, S, N, alkyl, substituted alkyl, O-alkyl, S-alkyl, alkaryl, aralkyl, or alkylhalo; and wherein W, X, Y and Z are not all O.  
           [0044]    In one embodiment, the invention features a siRNA molecule having a 5′-terminal phosphate group having Formula IV on the target-complimentary strand, for example a strand complimentary to HIV RNA, wherein the siRNA molecule comprises an all RNA siRNA molecule. In another embodiment, the invention features a siRNA molecule having a 5′-terminal phosphate group having Formula IV on the target-complimentary strand wherein the siRNA molecule also comprises 1-3 (i.e., 1, 2 or 3) nucleotide 3′-overhangs having between about 1 and about 4, for example, about 1, 2, 3 or 4 deoxyribonucleotides on the 3′-end of one or both strands. In another embodiment, a 5′-terminal phosphate group having Formula IV is present on the target-complimentary strand of a siRNA molecule of the invention, for example a siRNA molecule having chemical modifications having Formula I, Formula II and/or Formula III.  
           [0045]    In one embodiment, the invention features a chemically modified short interfering RNA (siRNA) molecule capable of mediating RNA interference (RNAi) against HIV inside a cell, wherein the chemical modification comprises one or more phosphorothioate internucleotide linkages. For example, in a non-limiting example, the invention features a chemically modified short interfering RNA (siRNA) having about 1, 2, 3, 4, 5, 6, 7, 8 or more phosphorothioate internucleotide linkages in one siRNA strand. In yet another embodiment, the invention features a chemically modified short interfering RNA (siRNA) individually having about 1, 2, 3, 4, 5, 6, 7, 8 or more phosphorothioate internucleotide linkages in both siRNA strands. The phosphorothioate internucleotide linkages can be present in one or both oligonucleotide strands of the siRNA duplex, for example in the sense strand, antisense strand, or both strands. The siRNA molecules of the invention can comprise one or more phosphorothioate internucleotide linkages at the 3′-end, 5′-end, or both 3′ and 5′-ends of the sense strand, antisense strand, or both strands. For example, an exemplary siRNA molecule of the invention can comprise between about 1 and about 5 or more, for example, about 1, 2, 3, 4, 5 or more phosphorothioate internucleotide linkages at the 5′-end of the sense strand, antisense strand, or both strands. In another non-limiting example, an exemplary siRNA molecule of the invention can comprise one or more pyrimidine phosphorothioate internucleotide linkages in the sense strand, antisense strand, or both strands. In yet another non-limiting example, an exemplary siRNA molecule of the invention can comprise one or more purine phosphorothioate internucleotide linkages in the sense strand, antisense strand, or both strands.  
           [0046]    In one embodiment, the invention features a siRNA molecule, wherein the sense strand comprises one or more, for example about 1, 2, 3, 4, 5, 6, 7, 8 , 9, or 10 phosphorothioate internucleotide linkages, and/or one or more, for example, about 1, 2, 3, 4, 5 or more 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or one or more, for example, about 1, 2, 3, 4, 5 or more universal base modified nucleotides, and optionally a terminal cap molecule at the 3′, 5′, or both 3′ and 5′-ends of the sense strand; and wherein the antisense strand comprises any of between 1 and 10, specifically about 1, 2, 3, 4, 5, 6, 7, 8 , 9, or 10 phosphorothioate internucleotide linkages, and/or one or more 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or one or more universal base modified nucleotides, and optionally a terminal cap molecule at the 3′, 5′, or both 3′ and 5′-ends of the antisense strand. In another embodiment, one or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 pyrimidine nucleotides of the sense and/or antisense siRNA stand are chemically modified with 2′-deoxy, 2′-O-methyl and/or 2′-deoxy-2′-fluoro nucleotides, with or without one or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 phosphorothioate internucleotide linkages and/or a terminal cap molecule at the 3′, 5′, or both 3′ and 5′-ends, being present in the same or different strand.  
           [0047]    In another embodiment, the invention features a siRNA molecule, wherein the sense strand comprises between 1 and 5, specifically about 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages, and/or one or more, for example, about 1, 2, 3, 4, 5 or more 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or one or more, for example, about 1, 2, 3, 4, 5 or more universal base modified nucleotides, and optionally a terminal cap molecule at the 3′, 5′, or both 3′ and 5′-ends of the sense strand; and wherein the antisense strand comprises any of between 1 and 5, specifically about 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages, and/or one or more, for example, about 1, 2, 3, 4, 5 or more 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or one or more, for example, about 1, 2, 3, 4, 5 or more universal base modified nucleotides, and optionally a terminal cap molecule at the 3′, 5′, or both 3′ and 5′-ends of the antisense strand. In another embodiment, one or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 pyrimidine nucleotides of the sense and/or antisense siRNA stand are chemically modified with 2′-deoxy, 2′-O-methyl and/or 2′-deoxy-2′-fluoro nucleotides, with or without between 1 and 5, for example about 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages and/or a terminal cap molecule at the 3′, 5′, or both 3′ and 5′-ends, being present in the same or different strand.  
           [0048]    In one embodiment, the invention features a siRNA molecule, wherein the antisense strand comprises one or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 phosphorothioate internucleotide linkages, and/or one or more, for example, about 1, 2, 3, 4, 5 or more 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or one or more, for example, 1, 2, 3, 4, 5 or more universal base modified nucleotides, and optionally a terminal cap molecule at the 3′, 5′, or both 3′ and 5′-ends of the sense strand; and wherein the antisense strand comprises any of between 1 and 10, specifically about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 phosphorothioate internucleotide linkages, and/or one or more, for example, about 1, 2, 3, 4, 5 or more 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or one or more universal base modified nucleotides, and optionally a terminal cap molecule at the 3′, 5′, or both 3′ and 5′-ends of the antisense strand. In another embodiment, one or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 pyrimidine nucleotides of the sense and/or antisense siRNA stand are chemically modified with 2′-deoxy, 2′-O-methyl and/or 2′-deoxy-2′-fluoro nucleotides, with or without one or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 phosphorothioate internucleotide linkages and/or a terminal cap molecule at the 3′, 5′, or both 3′ and 5′-ends, being present in the same or different strand.  
           [0049]    In another embodiment, the invention features a siRNA molecule, wherein the antisense strand comprises between 1 and 5, specifically about 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages, and/or one or more, for example, about 1, 2, 3, 4, 5 or more 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or one or more universal base modified nucleotides, and optionally a terminal cap molecule at the 3′, 5′, or both 3′ and 5′-ends of the sense strand; and wherein the antisense strand comprises any of between 1 and 5, specifically about 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages, and/or one or more, for example, about 1, 2, 3, 4, 5 or more 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or one or more, for example, about 1, 2, 3, 4, 5 or more universal base modified nucleotides, and optionally a terminal cap molecule at the 3′, 5′, or both 3′ and 5′-ends of the antisense strand. In another embodiment, one or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 pyrimidine nucleotides of the sense and/or antisense siRNA stand are chemically modified with 2′-deoxy, 2′-O-methyl and/or 2′-deoxy-2′-fluoro nucleotides, with or without between 1 and 5, for example about 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages and/or a terminal cap molecule at the 3′, 5′, or both 3′ and 5′-ends, being present in the same or different strand.  
           [0050]    In one embodiment, the invention features a chemically modified short interfering RNA (siRNA) molecule having between about 1 and 5, specifically about 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages in each strand of the siRNA molecule.  
           [0051]    In another embodiment, the invention features a siRNA molecule comprising 2′-5′ internucleotide linkages. The 2′-5′ internucleotide linkage(s) can be at the 5′-end, 3′-end, or both 5′ and 3′ ends of one or both siRNA sequence strands. In addition, the 2′-5′ internucleotide linkage(s) can be present at various other positions within one or both siRNA sequence strands, for example, every internucleotide linkage of a pyrimidine nucleotide in one or both strands of the siRNA molecule can comprise a 2′-5′ internucleotide linkage, or every internucleotide linkage of a purine nucleotide in one or both strands of the siRNA molecule can comprise a 2′-5′ internucleotide linkage.  
           [0052]    In another embodiment, a chemically modified siRNA molecule of the invention comprises a duplex having two strands, one or both of which can be chemically modified, wherein each strand is between about 18 and about 27, for example, about 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27, nucleotides in length, wherein the duplex has between about 18 and about 23, for example, about 18, 19, 20, 21, 22, 23, base pairs, and wherein the chemical modification comprises a structure having Formula I, Formula II, Formula III and/or Formula IV. For example, an exemplary chemically modified siRNA molecule of the invention comprises a duplex having two strands, one or both of which can be chemically modified with a chemical modification having Formula I, Formula II, Formula III, and/or Formula IV, wherein each strand consists of 21 nucleotides, each having 2 nucleotide 3′-overhangs, and wherein the duplex has 19 base pairs.  
           [0053]    In another embodiment, a siRNA molecule of the invention comprises a single stranded hairpin structure, wherein the siRNA is between about 36 and about 70, for example, about 36, 40, 45, 50, 55, 60, 65, or 70, nucleotides in length having between about 18 and about 23, for example, about 18, 19, 20, 21, 22, or 23 base pairs, and wherein the siRNA can include a chemical modification comprising a structure having Formula I, Formula II, Formula III and/or Formula IV. For example, an exemplary chemically modified siRNA molecule of the invention comprises a linear oligonucleotide having between about 42 and about 50, for example, 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides that is chemically modified with a chemical modification having Formula I, Formula II, Formula III, and/or Formula IV, wherein the linear oligonucleotide forms a hairpin structure having 19 base pairs and a 2 nucleotide 3′-overhang.  
           [0054]    In another embodiment, a linear hairpin siRNA molecule of the invention contains a stem loop motif, wherein the loop portion of the siRNA molecule is biodegradable. For example, a linear hairpin siRNA molecule of the invention is designed such that degradation of the loop portion of the siRNA molecule in vivo can generate a double stranded siRNA molecule with 3′-overhangs, such as 3′-overhangs comprising about 2 nucleotides.  
           [0055]    In another embodiment, a siRNA molecule of the invention comprises a circular nucleic acid molecule, wherein the siRNA is between about 38 and about 70, for example, about 38, 40, 45, 50, 55, 60, 65 or 70 nucleotides in length having between about 18 and about 23, for example, about 18, 19, 20, 21, 22 or 23 base pairs, and wherein the siRNA can include a chemical modification, which comprises a structure having Formula I, Formula II, Formula III and/or Formula IV. For example, an exemplary chemically modified siRNA molecule of the invention comprises a circular oligonucleotide having between about 42 and about 50, for example, 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides that is chemically modified with a chemical modification having Formula I, Formula II, Formula III, and/or Formula IV, wherein the circular oligonucleotide forms a dumbbell shaped structure having 19 base pairs and 2 loops.  
           [0056]    In another embodiment, a circular siRNA molecule of the invention contains two loop motifs, wherein one or both loop portions of the siRNA molecule is biodegradable. For example, a circular siRNA molecule of the invention is designed such that degradation of the loop portions of the siRNA molecule in vivo can generate a double stranded siRNA molecule with 3′-overhangs, such as 3′-overhangs comprising about 2 nucleotides.  
           [0057]    In one embodiment, a siRNA molecule of the invention comprises one or more abasic residues, for example a compound having Formula V:  
                         
 
           [0058]    wherein each R3, R4, R5, R6, R7, R8, R10, R11, R12, and R13 is independently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF3, OCF3, OCN, 0-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, SO-alkyl, alkyl-OSH, alkyl-OH, O-alkyl-OH, O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl, ONO2, NO2, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl, O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalklylamino, substituted silyl, or group having Formula I; R9 is O, S, CH2, S═O, CHF, or CF2.  
           [0059]    In one embodiment, a siRNA molecule of the invention comprises one or more inverted abasic residues, for example a compound having Formula VI:  
                         
 
           [0060]    wherein each R3, R4, R5, R6, R7, R8, R10, R11, R12, and R13 is independently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, SO-alkyl, alkyl-OSH, alkyl-OH, O-alkyl-OH, O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl, ONO2, NO2, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl, O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalklylamino, substituted silyl, or group having Formula I; R9 is O, S, CH2, S═O, CHF, or CF2, and either R2, R3, R8 or R13 serve as points of attachment to the siRNA molecule of the invention.  
           [0061]    In another embodiment, a siRNA molecule of the invention comprises an abasic residue having Formula II or III, wherein the abasic residue having Formula II or III is connected to the siRNA construct in a 3′,3′, 3′−2′, 2′−3′, or 5′, 5′ configuration, such as that the 3′-end, 5′-end, or both 3′ and 5′ ends of one or both siRNA strands.  
           [0062]    In one embodiment, a siRNA molecule of the invention comprises one or more, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more locked nucleic acid (LNA) nucleotides, for example at the 5′-end, 3′-end, 5′ and 3′-end, or any combination thereof, of the siRNA molecule.  
           [0063]    In one embodiment, the invention features a chemically modified short interfering RNA (siRNA) molecule capable of mediating RNA interference (RNAi) against HIV inside a cell, wherein the chemical modification comprises a conjugate covalently attached to the siRNA molecule. In another embodiment, the conjugate is covalently attached to the siRNA molecule via a biodegradable linker. In one embodiment, the conjugate molecule is attached at the 3′-end of either the sense strand, antisense strand, or both strands of the siRNA. In another embodiment, the conjugate molecule is attached at the 5′-end of either the sense strand, antisense strand, or both strands of the siRNA. In yet another embodiment, the conjugate molecule is attached both the 3′-end and 5′-end of either the sense strand, antisense strand, or both strands of the siRNA, or any combination thereof. In one embodiment, a conjugate molecule of the invention comprises a molecule that facilitates delivery of a siRNA molecule into a biological system such as a cell. In another embodiment, the conjugate molecule attached to the siRNA is a poly ethylene glycol, human serum albumin, or a ligand for a cellular receptor that can mediate cellular uptake. Examples of specific conjugate molecules contemplated by the instant invention that can be attached to siRNA molecules are described in Vargeese et al., U.S. Serial No. 60/311,865, incorporated by reference herein.  
           [0064]    In one embodiment, the invention features a siRNA molecule capable of mediating RNA interference (RNAi) against HIV inside a cell, wherein one or both strands of the siRNA comprise ribonucleotides at positions withing the siRNA that are critical for siRNA mediated RNAi in a cell. All other positions within the siRNA can include chemically modified nucleotides and/or non-nucleotides such as nucleotides and or non-nucleotides having Formula I, II, III, IV, V, or VI, or any combination thereof to the extent that the ability of the siRNA molecule to support RNAi activity in a cell is maintained.  
           [0065]    In one embodiment, the invention features a method for modulating the expression of a HIV gene within a cell, comprising: (a) synthesizing a siRNA molecule of the invention, which can be chemically modified, wherein one of the siRNA strands includes a sequence complimentary to RNA of the HIV gene; and (b) introducing the siRNA molecule into a cell under conditions suitable to modulate the expression of the HIV gene in the cell.  
           [0066]    In one embodiment, the invention features a method for modulating the expression of a HIV gene within a cell, comprising: (a) synthesizing a siRNA molecule of the invention, which can be chemically modified, wherein one of the siRNA strands includes a sequence complimentary to RNA of the HIV gene and wherein the sense strand sequence of the siRNA is identical to the complimentary sequence of the HIV RNA; and (b) introducing the siRNA molecule into a cell under conditions suitable to modulate the expression of the HIV gene in the cell.  
           [0067]    In another embodiment, the invention features a method for modulating the expression of more than one HIV gene within a cell, comprising: (a) synthesizing siRNA molecules of the invention, which can be chemically modified, wherein one of the siRNA strands includes a sequence complimentary to RNA of the HIV genes; and (b) introducing the siRNA molecules into a cell under conditions suitable to modulate the expression of the HIV genes in the cell.  
           [0068]    In another embodiment, the invention features a method for modulating the expression of more than one HIV gene within a cell, comprising: (a) synthesizing a siRNA molecule of the invention, which can be chemically modified, wherein one of the siRNA strands includes a sequence complimentary to RNA of the HIV gene and wherein the sense strand sequence of the siRNA is identical to the complimentary sequence of the HIV RNA; and (b) introducing the siRNA molecules into a cell under conditions suitable to modulate the expression of the HIV genes in the cell.  
           [0069]    In one embodiment, the invention features a method of modulating the expression of a HIV gene in a tissue explant, comprising: (a) synthesizing a siRNA molecule of the invention, which can be chemically modified, wherein one of the siRNA strands includes a sequence complimentary to RNA of the HIV gene; (b) introducing the siRNA molecule into a cell of the tissue explant derived from a particular organism under conditions suitable to modulate the expression of the HIV gene in the tissue explant, and (c) optionally introducing the tissue explant back into the organism the tissue was derived from or into another organism under conditions suitable to modulate the expression of the HIV gene in that organism.  
           [0070]    In one embodiment, the invention features a method of modulating the expression of a HIV gene in a tissue explant, comprising: (a) synthesizing a siRNA molecule of the invention, which can be chemically modified, wherein one of the siRNA strands includes a sequence complimentary to RNA of the HIV gene and wherein the sense strand sequence of the siRNA is identical to the complimentary sequence of the HIV RNA; (b) introducing the siRNA molecule into a cell of the tissue explant derived from a particular organism under conditions suitable to modulate the expression of the HIV gene in the tissue explant, and (c) optionally introducing the tissue explant back into the organism the tissue was derived from or into another organism under conditions suitable to modulate the expression of the HIV gene in that organism.  
           [0071]    In another embodiment, the invention features a method of modulating the expression of more than one HIV gene in a tissue explant, comprising: (a) synthesizing siRNA molecules of the invention, which can be chemically modified, wherein one of the siRNA strands includes a sequence complimentary to RNA of the HIV genes; (b) introducing the siRNA molecules into a cell of the tissue explant derived from a particular organism under conditions suitable to modulate the expression of the HIV genes in the tissue explant, and (c) optionally introducing the tissue explant back into the organism the tissue was derived from or into another organism under conditions suitable to modulate the expression of the HIV genes in that organism.  
           [0072]    In one embodiment, the invention features a method of modulating the expression of a HIV gene in an organism, comprising: (a) synthesizing a siRNA molecule of the invention, which can be chemically modified, wherein one of the siRNA strands includes a sequence complimentary to RNA of the HIV gene; and (b) introducing the siRNA molecule into the organism under conditions suitable to modulate the expression of the HIV gene in the organism.  
           [0073]    In another embodiment, the invention features a method of modulating the expression of more than one HIV gene in an organism, comprising: (a) synthesizing siRNA molecules of the invention, which can be chemically modified, wherein one of the siRNA strands includes a sequence complimentary to RNA of the HIV genes; and (b) introducing the siRNA molecules into the organism under conditions suitable to modulate the expression of the HIV genes in the organism.  
           [0074]    The siRNA molecules of the invention can be designed to inhibit HIV gene expression through RNAi targeting of a variety of RNA molecules. In one embodiment, the siRNA molecules of the invention are used to target various RNAs corresponding to a target gene. Non-limiting examples of such RNAs include messenger RNA (mRNA), alternate RNA splice variants of target gene(s), post-transcriptionally modified RNA of target gene(s), pre-mRNA of target gene(s), and/or RNA templates used for HIV activity. If alternate splicing produces a family of transcipts that are distinguished by usage of appropriate exons, the instant invention can be used to inhibit gene expression through the appropriate exons to specifically inhibit or to distinguish among the functions of gene family members. For example, a protein that contains an alternatively spliced transmembrane domain can be expressed in both membrane bound and secreted forms. Use of the invention to target the exon containing the transmembrane domain can be used to determine the functional consequences of pharmaceutical targeting of membrane bound as opposed to the secreted form of the protein. Non-limiting examples of applications of the invention relating to targeting these RNA molecules include therapeutic pharmaceutical applications, pharmaceutical discovery applications, molecular diagnostic and gene function applications, and gene mapping, for example using single nucleotide polymorphism mapping with siRNA molecules of the invention. Such applications can be implemented using known gene sequences or from partial sequences available from an expressed sequence tag (EST).  
           [0075]    In another embodiment, the siRNA molecules of the invention are used to target conserved sequences corresponding to a gene family or gene families such as HIV genes. As such, siRNA molecules targeting multiple HIV targets can provide increased therapeutic effect. In addition, siRNA can be used to characterize pathways of gene function in a variety of applications. For example, the present invention can be used to inhibit the activity of target gene(s) in a pathway to determine the function of uncharacterized gene(s) in gene function analysis, mRNA function analysis, or translational analysis. The invention can be used to determine potential target gene pathways involved in various diseases and conditions toward pharmaceutical development. The invention can be used to understand pathways of gene expression involved in development, such as prenatal development, postnatal development and/or aging.  
           [0076]    In one embodiment, siRNA molecule(s) and/or methods of the invention are used to inhibit the expression of gene(s) that encode RNA referred to by Genbank Accession number, for example HIV genes such as Genbank Accession Nos. AJ302647 (HIV-1), NC — 001722 (HIV-2), NC — 001482 (FIV-1) and/or M66437 (SIV-1). Such sequences are readily obtained using these Genbank Accession numbers.  
           [0077]    In one embodiment, the invention features a method comprising: (a) analyzing the sequence of a RNA target encoded by a HIV gene; (b) synthesizing one or more sets of siRNA molecules having sequence complimentary to one or more regions of the RNA of (a); and (c) assaying the siRNA molecules of (b) under conditions suitable to determine RNAi targets within the target RNA sequence. In another embodiment, the siRNA molecules of (b) have strands of a fixed length, for example 23 nucleotides in length. In yet another embodiment, the siRNA molecules of (b) are of differing length, for example having strands of about 19 to about 25, for example, about 19, 20, 21, 22, 23, 24 or 25 nucleotides in length.  
           [0078]    In one embodiment, the invention features a composition comprising a siRNA molecule of the invention, which can be chemically modified, in a pharmaceutically acceptable carrier or diluent. In another embodiment, the invention features a pharmaceutical composition comprising siRNA molecules of the invention, which can be chemically modified, targeting one or more genes in a pharmaceutically acceptable carrier or diluent. In another embodiment, the invention features a method for treating or preventing a disease or condition in a subject, comprising administering to the subject a composition of the invention under conditions suitable for the treatment or prevention of the disease or condition in the subject, alone or in conjunction with one or more other therapeutic compounds. In yet another embodiment, the invention features a method for reducing or preventing tissue rejection in a subject comprising administering to the subject a composition of the invention under conditions suitable for the reduction or prevention of tissue rejection in the subject.  
           [0079]    In another embodiment, the invention features a method for validating a HIV gene target, comprising: (a) synthesizing a siRNA molecule of the invention, which can be chemically modified, wherein one of the siRNA strands includes a sequence complimentary to RNA of a HIV target gene; (b) introducing the siRNA molecule into a cell, tissue, or organism under conditions suitable for modulating expression of the HIV target gene in the cell, tissue, or organism; and (c) determining the function of the gene by assaying for any phenotypic change in the cell, tissue, or organism.  
           [0080]    In one embodiment, the invention features a kit containing a siRNA molecule of the invention, which can be chemically modified, that can be used to modulate the expression of a HIV target gene in a cell, tissue, or organism. In another embodiment, the invention features a kit containing more than one siRNA molecule of the invention, which can be chemically modified, that can be used to modulate the expression of more than one HIV target gene in a cell, tissue, or organism.  
           [0081]    In one embodiment, the invention features a cell containing one or more siRNA molecules of the invention, which can be chemically modified. In another embodiment, the cell containing a siRNA molecule of the invention is a mammalian cell. In yet another embodiment, the cell containing a siRNA molecule of the invention is a human cell.  
           [0082]    In one embodiment, the synthesis of a siRNA molecule of the invention, which can be chemically modified, comprises: (a) synthesis of two complimentary strands of the siRNA molecule; (b) annealing the two complimentary strands together under conditions suitable to obtain a double stranded siRNA molecule. In another embodiment, synthesis of the two complimentary strands of the siRNA molecule is by solid phase oligonucleotide synthesis. In yet another embodiment, synthesis of the two complimentary strands of the siRNA molecule is by solid phase tandem oligonucleotide synthesis.  
           [0083]    In one embodiment, the invention features a method for synthesizing a siRNA duplex molecule comprising: (a) synthesizing a first oligonucleotide sequence strand of the siRNA molecule, wherein the first oligonucleotide sequence strand comprises a cleavable linker molecule that can be used as a scaffold for the synthesis of the second oligonucleotide sequence strand of the siRNA; (b) synthesizing the second oligonucleotide sequence strand of siRNA on the scaffold of the first oligonucleotide sequence strand, wherein the second oligonucleotide sequence strand further comprises a chemical moiety than can be used to purify the siRNA duplex; (c) cleaving the linker molecule of (a) under conditions suitable for the two siRNA oligonucleotide strands to hybridize and form a stable duplex; and (d) purifying the siRNA duplex utilizing the chemical moiety of the second oligonucleotide sequence strand. In another embodiment, cleavage of the linker molecule in (c) above takes place during deprotection of the oligonucleotide, for example under hydrolysis conditions using an alkylamine base such as methylamine. In another embodiment, the method of synthesis comprises solid phase synthesis on a solid support such as controlled pore glass (CPG) or polystyrene, wherein the first sequence of (a) is synthesized on a cleavable linker, such as a succinyl linker, using the solid support as a scaffold. The cleavable linker in (a) used as a scaffold for synthesizing the second strand can comprise similar reactivity as the solid support derivatized linker, such that cleavage of the solid support derivatized linker and the cleavable linker of (a) takes place concomitantly. In another embodiment, the chemical moiety of (b) that can used to isolate the attached oligonucleotide sequence comprises a trityl group, for example a dimethoxytrityl group, which can be employed in a trityl-on synthesis strategy as described herein. In yet another embodiment, the chemical moiety, such as a dimethoxytrityl group, is removed during purification, for example using acidic conditions.  
           [0084]    In a further embodiment, the method for siRNA synthesis is a solution phase synthesis or hybrid phase synthesis wherein both strands of the siRNA duplex are synthesized in tandem using a cleavable linker attached to the first sequence which acts a scaffold for synthesis of the second sequence. Cleavage of the linker under conditions suitable for hybridization of the separate siRNA sequence strands results in formation of the double stranded siRNA molecule.  
           [0085]    In another embodiment, the invention features a method for synthesizing a siRNA duplex molecule comprising: (a) synthesizing one oligonucleotide sequence strand of the siRNA molecule, wherein the sequence comprises a cleavable linker molecule that can be used as a scaffold for the synthesis of another oligonucleotide sequence; (b) synthesizing a second oligonucleotide sequence having complementarity to the first sequence strand on the scaffold of (a), wherein the second sequence comprises the other strand of the double stranded siRNA molecule and wherein the second sequence further comprises a chemical moiety than can be used to isolate the attached oligonucleotide sequence; (c) purifying the product of (b) utilizing the chemical moiety of the second oligonucleotide sequence strand under conditions suitable for isolating the full length sequence comprising both siRNA oligonucleotide strands connected by the cleavable linker; and (d) under conditions suitable for the two siRNA oligonucleotide strands to hybridize and form a stable duplex. In another embodiment, cleavage of the linker molecule in (c) above takes place during deprotection of the oligonucleotide, for example under hydrolysis conditions. In another embodiment, cleavage of the linker molecule in (c) above takes place after deprotection of the oligonucleotide. In another embodiment, the method of synthesis comprises solid phase synthesis on a solid support such as controlled pore glass (CPG) or polystyrene, wherein the first sequence of (a) is synthesized on a cleavable linker, such as a succinyl linker, using the solid support as a scaffold. The cleavable linker in (a) used as a scaffold for synthesizing the second strand can comprise similar reactivity or differing reactivity as the solid support derivatized linker, such that cleavage of the solid support derivatized linker and the cleavable linker of (a) takes place either concomitantly or sequentially. In another embodiment, the chemical moiety of (b) that can used to isolate the attached oligonucleotide sequence comprises a trityl group, for example a dimethoxytrityl group.  
           [0086]    In another embodiment, the invention features a method for making a double stranded siRNA molecule in a single synthetic process, comprising: (a) synthesizing an oligonucleotide having a first and a second sequence, wherein the first sequence is complimentary to the second sequence, and the first oligonucleotide sequence is linked to the second sequence via a cleavable linker, and wherein a terminal 5′-protecting group, for example a 5′-O-dimethoxytrityl group (5′-O-DMT) remains on the oligonucleotide having the second sequence; (b) deprotecting the oligonucleotide whereby the deprotection results in the cleavage of the linker joining the two oligonucleotide sequences; and (c) purifying the product of (b) under conditions suitable for isolating the double stranded siRNA molecule, for example using a trityl-on synthesis strategy as described herein.  
           [0087]    In one embodiment, the invention features siRNA constructs that mediate RNAi against HIV, wherein the siRNA construct comprises one or more chemical modifications, for example one or more chemical modifications having Formula I, II, III, IV, or V, that increases the nuclease resistance of the siRNA construct.  
           [0088]    In another embodiment, the invention features a method for generating siRNA molecules with increased nuclease resistance comprising (a) introducing nucleotides having any of Formula I-VI into a siRNA molecule, and (b) assaying the siRNA molecule of step (a) under conditions suitable for isolating siRNA molecules having increased nuclease resistance.  
           [0089]    In one embodiment, the invention features siRNA constructs that mediate RNAi against HIV, wherein the siRNA construct comprises one or more chemical modifications described herein that modulates the binding affinity between the sense and antisense strands of the siRNA construct.  
           [0090]    In another embodiment, the invention features a method for generating siRNA molecules with increased binding affinity between the sense and antisense strands of the siRNA molecule comprising (a) introducing nucleotides having any of Formula I-VI into a siRNA molecule, and (b) assaying the siRNA molecule of step (a) under conditions suitable for isolating siRNA molecules having increased binding affinity between the sense and antisense strands of the siRNA molecule.  
           [0091]    In one embodiment, the invention features siRNA constructs that mediate RNAi against HIV, wherein the siRNA construct comprises one or more chemical modifications described herein that modulates the binding affinity between the antisense strand of the siRNA construct and a complimentary target RNA sequence within a cell.  
           [0092]    In another embodiment, the invention features a method for generating siRNA molecules with increased binding affinity between the antisense strand of the siRNA molecule and a complimentary target RNA sequence, comprising (a) introducing nucleotides having any of Formula I-VI into a siRNA molecule, and (b) assaying the siRNA molecule of step (a) under conditions suitable for isolating siRNA molecules having increased binding affinity between the antisense strand of the siRNA molecule and a complimentary target RNA sequence.  
           [0093]    In one embodiment, the invention features siRNA constructs that mediate RNAi against HIV, wherein the siRNA construct comprises one or more chemical modifications described herein that modulate the polymerase activity of a cellular polymerase capable of generating additional endogenous siRNA molecules having sequence homology to the chemically modified siRNA construct.  
           [0094]    In another embodiment, the invention features a method for generating siRNA molecules capable of mediating increased polymerase activity of a cellular polymerase capable of generating additional endogenous siRNA molecules having sequence homology to the chemically modified siRNA molecule comprising (a) introducing nucleotides having any of Formula I-VI into a siRNA molecule, and (b) assaying the siRNA molecule of step (a) under conditions suitable for isolating siRNA molecules capable of mediating increased polymerase activity of a cellular polymerase capable of generating additional endogenous siRNA molecules having sequence homology to the chemically modified siRNA molecule.  
           [0095]    In one embodiment, the invention features chemically modified siRNA constructs that mediate RNAi against HIV in a cell, wherein the chemical modifications do not significantly effect the interaction of siRNA with a target RNA molecule and/or proteins or other factors that are essential for RNAi in a manner that would decrease the efficacy of RNAi mediated by such siRNA constructs.  
           [0096]    In another embodiment, the invention features a method for generating siRNA molecules with improved RNAi activity against HIV, comprising (a) introducing nucleotides having any of Formula I-VI into a siRNA molecule, and (b) assaying the siRNA molecule of step (a) under conditions suitable for isolating siRNA molecules having improved RNAi activity.  
           [0097]    In yet another embodiment, the invention features a method for generating siRNA molecules with improved RNAi activity against a HIV target RNA, comprising (a) introducing nucleotides having any of Formula I-VI into a siRNA molecule, and (b) assaying the siRNA molecule of step (a) under conditions suitable for isolating siRNA molecules having improved RNAi activity against the target RNA.  
           [0098]    In one embodiment, the invention features siRNA constructs that mediate RNAi against HIV, wherein the siRNA construct comprises one or more chemical modifications described herein that modulates the cellular uptake of the siRNA construct.  
           [0099]    In another embodiment, the invention features a method for generating siRNA molecules against HIV with improved cellular uptake, comprising (a) introducing nucleotides having any of Formula I-VI into a siRNA molecule, and (b) assaying the siRNA molecule of step (a) under conditions suitable for isolating siRNA molecules having improved cellular uptake.  
           [0100]    In one embodiment, the invention features siRNA constructs that mediate RNAi against HIV, wherein the siRNA construct comprises one or more chemical modifications described herein that increases the bioavailability of the siRNA construct, for example by attaching polymeric conjugates such as polyethyleneglycol or equivalent conjugates that improve the pharmacokinetics of the siRNA construct, or by attaching conjugates that target specific tissue types or cell types in vivo. Non-limiting examples of such conjugates are described in Vargeese et al., U.S. Serial No. 60/311,865 incorporated by reference herein.  
           [0101]    In one embodiment, the invention features a method for generating siRNA molecules of the invention with improved bioavailability, comprising (a) introducing a conjugate into the structure of a siRNA molecule, and (b) assaying the siRNA molecule of step (a) under conditions suitable for isolating siRNA molecules having improved bioavailability. Such conjugates can include ligands for cellular receptors such as peptides derived from naturally occurring protein ligands, protein localization sequences including cellular ZIP code sequences, antibodies, nucleic acid aptamers, vitamins and other co-factors such as folate and N-acetylgalactosamine, polymers such as polyethyleneglycol (PEG), phospholipids, polyamines such as spermine or spermidine, and others.  
           [0102]    In another embodiment, the invention features a method for generating siRNA molecules of the invention with improved bioavailability, comprising (a) introducing an excipient formulation to a siRNA molecule, and (b) assaying the siRNA molecule of step (a) under conditions suitable for isolating siRNA molecules having improved bioavailability. Such excipients include polymers such as cyclodextrins, lipids, cationic lipids, polyamines, phospholipids, and others.  
           [0103]    In another embodiment, the invention features a method for generating siRNA molecules of the invention with improved bioavailability, comprising (a) introducing nucleotides having any of Formula I-VI into a siRNA molecule, and (b) assaying the siRNA molecule of step (a) under conditions suitable for isolating siRNA molecules having improved bioavailability.  
           [0104]    In another embodiment, polyethylene glycol (PEG) can be covalently attached to siRNA compounds of the present invention. The attached PEG can be any molecular weight, preferably from about 2,000 to about 50,000 daltons (Da).  
           [0105]    The present invention can be used alone or as a component of a kit having at least one of the reagents necessary to carry out the in vitro or in vivo introduction of RNA to test samples and/or subjects. For example, preferred components of the kit include the siRNA and a vehicle that promotes introduction of the siRNA. Such a kit can also include instructions to allow a user of the kit to practice the invention.  
           [0106]    The term “short interfering RNA” or “siRNA” as used herein refers to any nucleic acid molecule capable of mediating RNA interference “RNAi” or gene silencing; see for example Bass, 2001,  Nature,  411, 428-429; Elbashir et al., 2001,  Nature,  411, 494-498; and Kreutzer et al., International PCT Publication No. WO 00/44895; Zernicka-Goetz et al., International PCT Publication No. WO 01/36646; Fire, International PCT Publication No. WO 99/32619; Plaetinck et al., International PCT Publication No. WO 00/01846; Mello and Fire, International PCT Publication No. WO 01/29058; Deschamps-Depaillette, International PCT Publication No. WO 99/07409; and Li et al., International PCT Publication No. WO 00/44914. Non limiting examples of siRNA molecules of the invention are shown in FIG. 6. For example the siRNA can be a double stranded polynucleotide molecule comprising self complementary sense and antisense regions, wherein the antisense region comprises complementarity to a target nucleic acid molecule. The siRNA can be a single stranded hairpin polynucleotide having self complementary sense and antisense regions, wherein the antisense region comprises complementarity to a target nucleic acid molecule. The siRNA can be a circular single stranded polynucleotide having two or more loop structures and a stem comprising self complementary sense and antisense regions, wherein the antisense region comprises complementarity to a target nucleic acid molecule, and wherein the circular polynucleotide can be processed either in vivo or in vitro to generate an active siRNA capable of mediating RNAi. As used herein, siRNA molecules need not be limited to those molecules containing only RNA, but further encompasses chemically modified nucleotides and non-nucleotides..  
           [0107]    By “modulate” is meant that the expression of the gene, or level of RNA molecule or equivalent RNA molecules encoding one or more proteins or protein subunits, or activity of one or more proteins or protein subunits is up regulated or down regulated, such that expression, level, or activity is greater than or less than that observed in the absence of the modulator. For example, the term “modulate” can mean “inhibit,” but the use of the word “modulate” is not limited to this definition.  
           [0108]    By “inhibit” it is meant that the activity of a gene expression product or level of RNAs or equivalent RNAs encoding one or more gene products is reduced below that observed in the absence of the nucleic acid molecule of the invention. In one embodiment, inhibition with a siRNA molecule preferably is below that level observed in the presence of an inactive or attenuated molecule that is unable to mediate an RNAi response. In another embodiment, inhibition of gene expression with the siRNA molecule of the instant invention is greater in the presence of the siRNA molecule than in its absence.  
           [0109]    By “gene” or “target gene” is meant, a nucleic acid that encodes an RNA, for example, nucleic acid sequences including, but not limited to, structural genes encoding a polypeptide. The target gene can be a gene derived from a cell, an endogenous gene, a transgene, or exogenous genes such as genes of a pathogen, for example a virus, which is present in the cell after infection thereof. The cell containing the target gene can be derived from or contained in any organism, for example a plant, animal, protozoan, virus, bacterium, or fungus. Non-limiting examples of plants include monocots, dicots, or gymnosperms. Non-limiting examples of animals include vertebrates or invertebrates. Non-limiting examples of fungi include molds or yeasts.  
           [0110]    By “HIV” as used herein is meant, any virus, protein, peptide, polypeptide, and/or polynucleotide expressed from a HIV gene, for example entire viruses such as HIV-1, HIV-2, FIV-1, SIV-1 or viral components such as nef, vif, tat, or rev viral gene products.  
           [0111]    By “highly conserved sequence region” is meant, a nucleotide sequence of one or more regions in a target gene does not vary significantly from one generation to the other or from one biological system to the other.  
           [0112]    By “complementarity” or “complementary” is meant that a nucleic acid can form hydrogen bond(s) with another nucleic acid sequence by either traditional Watson-Crick or other non-traditional types of interaction. In reference to the nucleic molecules of the present invention, the binding free energy for a nucleic acid molecule with its complementary sequence is sufficient to allow the relevant function of the nucleic acid to proceed, e.g., RNAi activity. For example, the degree of complementarity between the sense and antisense strand of the siRNA construct can be the same or different from the degree of complementarity between the antisense strand of the siRNA and the target RNA sequence. Complementarity to the target sequence of less than 100% in the antisense strand of the siRNA duplex, including point mutations, is reported not to be tolerated when these changes are located between the 3′-end and the middle of the antisense siRNA (completely abolishes siRNA activity), whereas mutations near the 5 ′-end of the antisense siRNA strand can exhibit a small degree of RNAi activity (Elbashir et al., 2001,  The EMBO Journal,  20, 6877-6888). Determination of binding free energies for nucleic acid molecules is well known in the art (see, e.g., Turner et al., 1987,  CSH Symp. Quant. Biol.  LII pp.123-133; Frier et al., 1986,  Proc. Nat. Acad. Sci.  USA 83:9373-9377; Turner et al., 1987,  J Am. Chem. Soc.  109:3783-3785). A percent complementarity indicates the percentage of contiguous residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementary). “Perfectly complementary” means that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence.  
           [0113]    The siRNA molecules of the invention represent a novel therapeutic approach to treat a variety of pathologic indications or other conditions, such as HIV infection or acquired immunodeficiency syndrome (AIDS) and any other diseases or conditions that are related to the levels of HIV in a cell or tissue, alone or in combination with other therapies. The reduction of HIV expression (specifically HIV RNA levels) and thus reduction in the level of the respective protein(s) relieves, to some extent, the symptoms of the disease or condition.  
           [0114]    In one embodiment of the present invention, each sequence of a siRNA molecule of the invention is independently about 18 to about 24 nucleotides in length, in specific embodiments about 18, 19, 20, 21, 22, 23, or 24 nucleotides in length. In another embodiment, the siRNA duplexes of the invention independently comprise between about 17 and about 23, for example, about 17, 18, 19, 20, 21, 22, or 23 base pairs. In yet another embodiment, siRNA molecules of the invention comprising hairpin or circular structures are about 35 to about 55, for example, about 35, 40, 45, 50 or 55 nucleotides in length, or about 38 to about 44, for example, about 38, 39, 40, 41, 42, 43 or 44 nucleotides in length and comprising about 16 to about 22, for example, about 16, 17, 18, 19, 20, 21 or 22 base pairs. Exemplary siRNA molecules of the invention are shown in Table I and/or FIGS. 4 and 5.  
           [0115]    As used herein “cell” is used in its usual biological sense, and does not refer to an entire multicellular organism, e.g., specifically does not refer to a human. The cell can be present in an organism, e.g mammals such as humans, cows, sheep, apes, monkeys, swine, dogs, and cats. The cell can be eukaryotic (e.g., a mammalian cell). The cell can be of somatic or germ line origin, totipotent or pluripotent, dividing or non-dividing. The cell can also be derived from or can comprise a gamete or embryo, a stem cell, or a fully differentiated cell.  
           [0116]    The siRNA molecules of the invention are added directly, or can be complexed with cationic lipids, packaged within liposomes, or otherwise delivered to target cells or tissues. The nucleic acid or nucleic acid complexes can be locally administered to relevant tissues ex vivo, or in vivo through injection, infusion pump or stent, with or without their incorporation in biopolymers. In particular embodiments, the nucleic acid molecules of the invention comprise sequences shown in Table I and/or FIGS. 4 and 5. Examples of such nucleic acid molecules consist essentially of sequences defined in this table.  
           [0117]    In another aspect, the invention provides mammalian cells containing one or more siRNA molecules of this invention. The one or more siRNA molecules can independently be targeted to the same or different sites.  
           [0118]    By “RNA” is meant a molecule comprising at least one ribonucleotide residue. By “ribonucleotide” is meant a nucleotide with a hydroxyl group at the 2′ position of a β-D-ribo-furanose moiety. The terms include double stranded RNA, single stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as altered RNA that differs from naturally occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides. Such alterations can include addition of non-nucleotide material, such as to the end(s) of the siRNA or internally, for example at one or more nucleotides of the RNA. Nucleotides in the RNA molecules of the instant invention can also comprise non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs can be referred to as analogs or analogs of naturally-occurring RNA.  
           [0119]    By “subject” is meant an organism, which is a donor or recipient of explanted cells or the cells themselves. “Subject” also refers to an organism to which the nucleic acid molecules of the invention can be administered. In one embodiment, a subject is a mammal or mammalian cells. In another embodiment, a subject is a human or human cells.  
           [0120]    The term “phosphorothioate” as used herein refers to an internucleotide linkage having Formula I, wherein Z and/or W comprise a sulfur atom. Hence, the term phosphorothioate refers to both phosphorothioate and phosphorodithioate internucleotide linkages.  
           [0121]    The term “universal base” as used herein refers to nucleotide base analogs that form base pairs with each of the natural DNA/RNA bases with little discrimination between them. Non-limiting examples of universal bases include C-phenyl, C-naphthyl and other aromatic derivatives, inosine, azole carboxamides, and nitroazole derivatives such as 3-nitropyrrole, 4-nitroindole, 5-nitroindole, and 6-nitroindole as known in the art (see for example Loakes, 2001,  Nucleic Acids Research,  29, 2437-2447).  
           [0122]    The term “acyclic nucleotide” as used herein refers to any nucleotide having an acyclic ribose sugar, for example where any of the ribose carbons (C1, C2, C3, C4, or C5), are independently or in combination absent from the nucleotide.  
           [0123]    The nucleic acid molecules of the instant invention, individually, or in combination or in conjunction with other drugs, can be used to treat diseases or conditions discussed herein. For example, to treat a particular disease or condition, the siRNA molecules can be administered to a subject or can be administered to other appropriate cells evident to those skilled in the art, individually or in combination with one or more drugs under conditions suitable for the treatment.  
           [0124]    In a further embodiment, the siRNA molecules can be used in combination with other known treatments to treat conditions or diseases discussed above. For example, the described molecules could be used in combination with one or more known therapeutic agents to treat a disease or condition. Non-limiting examples of other therapeutic agents that can be readily combined with a siRNA molecule of the invention are enzymatic nucleic acid molecules, allosteric nucleic acid molecules, antisense, decoy, or aptamer nucleic acid molecules, antibodies such as monoclonal antibodies, small molecules, and other organic and/or inorganic compounds including metals, salts and ions.  
           [0125]    In one embodiment, the invention features an expression vector comprising a nucleic acid sequence encoding at least one siRNA molecule of the invention, in a manner which allows expression of the siRNA molecule. For example, the vector can contain sequence(s) encoding both strands of a siRNA molecule comprising a duplex. The vector can also contain sequence(s) encoding a single nucleic acid molecule that is self complimentary and thus forms a siRNA molecule. Non-limiting examples of such expression vectors are described in Paul et al., 2002,  Nature Biotechnology,  19, 505; Miyagishi and Taira, 2002,  Nature Biotechnology,  19, 497; Lee et al., 2002,  Nature Biotechnology,  19, 500; and Novina et al., 2002,  Nature Medicine,  advance online publication doi:10.1038/nm725.  
           [0126]    In another embodiment, the invention features a mammalian cell, for example, a human cell, including an expression vector of the invention.  
           [0127]    In yet another embodiment, the expression vector of the invention comprises a sequence for a siRNA molecule having complementarity to a RNA molecule referred to by a Genbank Accession numbers, for example HIV genes such as Genbank Accession Nos. AJ302647 (HIV-1), NC — 001722 (HIV-2), NC — 001482 (FIV-1) and/or M66437 (SIV-1).  
           [0128]    In one embodiment, an expression vector of the invention comprises a nucleic acid sequence encoding two or more siRNA molecules, which can be the same or different.  
           [0129]    In another aspect of the invention, siRNA molecules that interact with target RNA molecules and down-regulate gene encoding target RNA molecules (for example target RNA molecules referred to by Genbank Accession numbers herein) are expressed from transcription units inserted into DNA or RNA vectors. The recombinant vectors can be DNA plasmids or viral vectors. siRNA expressing viral vectors can be constructed based on, but not limited to, adeno-associated virus, retrovirus, adenovirus, or alphavirus. The recombinant vectors capable of expressing the siRNA molecules can be delivered as described herein, and persist in target cells. Alternatively, viral vectors can be used that provide for transient expression of siRNA molecules. Such vectors can be repeatedly administered as necessary. Once expressed, the siRNA molecules bind and down-regulate gene function or expression via RNA interference (RNAi). Delivery of siRNA expressing vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from a subject followed by reintroduction into the subject, or by any other means that would allow for introduction into the desired target cell.  
           [0130]    By “vectors” is meant any nucleic acid- and/or viral-based technique used to deliver a desired nucleic acid.  
           [0131]    By “comprising” is meant including, but not limited to, whatever follows the word “comprising”. Thus, use of the term “comprising” indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present. By “consisting of” is meant including, and limited to, whatever follows the phrase “consisting of”. Thus, the phrase “consisting of” indicates that the listed elements are required or mandatory, and that no other elements may be present. By “consisting essentially of” is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase “consisting essentially of” indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they affect the activity or action of the listed elements.  
           [0132]    Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims. 
       
    
    
     DESCRIPTION OF THE PREFERRED EMBODIMENTS  
       [0133]    First the drawings will be described briefly.  
         [0134]    Drawings  
         [0135]    [0135]FIG. 1 shows a non-limiting example of a scheme for the synthesis of siRNA molecules. The complimentary siRNA sequence strands, strand 1 and strand 2, are synthesized in tandem and are connected by a cleavable linkage, such as a nucleotide succinate or abasic succinate, which can be the same or different from the cleavable linker used for solid phase synthesis on a solid support. The synthesis can be either solid phase or solution phase, in the example shown, the synthesis is a solid phase synthesis. The synthesis is performed such that a protecting group, such as a dimethoxytrityl group, remains intact on the terminal nucleotide of the tandem oligonucleotide. Upon cleavage and deprotection of the oligonucleotide, the two siRNA strands spontaneously hybridize to form a siRNA duplex, which allows the purification of the duplex by utilizing the properties of the terminal protecting group, for example by applying a trityl on purification method wherein only duplexes/oligonucleotides with the terminal protecting group are isolated.  
         [0136]    [0136]FIG. 2 shows a MALDI-TOV mass spectrum of a purified siRNA duplex synthesized by a method of the invention. The two peaks shown correspond to the predicted mass of the separate siRNA sequence strands. This result demonstrates that the siRNA duplex generated from tandem synthesis can be purified as a single entity using a simple trityl-on purification methodology.  
         [0137]    [0137]FIG. 3 shows a non-limiting proposed mechanistic representation of target RNA degradation involved in RNAi. Double stranded RNA (dsRNA), which is generated by RNA dependent RNA polymerase (RdRP) from foreign single stranded RNA, for example viral, transposon, or other exogenous RNA, activates the DICER enzyme which in turn generates siRNA duplexes having terminal phosphate groups (P). An active siRNA complex forms which recognizes a target RNA, resulting in degradation of the target RNA by the RISC endonuclease complex or in the synthesis of additional RNA by RNA dependent RNA polymerase (RdRP), which can activate DICER and result in additional siRNA molecules, thereby amplifying the RNAi response.  
         [0138]    [0138]FIG. 4 shows non-limiting examples of chemically modified siRNA constructs of the present invention. In the figure, N stands for any nucleotide (adenosine, guanosine, cytosine, uridine, or optionally thymidine, for example thymidine can be substituted in the overhanging regions designated by parenthesis (N N). Various modifications are shown for the sense and antisense strands of the siRNA constructs. A The sense strand comprises 21 nucleotides having four phosphorothioate 5′ and 3′-terminal internucleotide linkages, wherein the two terminal 3′-nucleotides are optionally base paired and wherein all pyrimidine nucleotides that may be present are 2′-O-methyl modified nucleotides except for (N N) nucleotides, which can comprise naturally occurring ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. The antisense strand comprises 21 nucleotides, wherein the two terminal 3′-nucleotides are optionally complimentary to the target RNA sequence, and having one 3′-terminal phosphorothioate internucleotide linkage and four 5′-terminal phosphorothioate internucleotide linkages and wherein all pyrimidine nucleotides that may be present are 2′-deoxy-2′-fluoro modified nucleotides except for (N N) nucleotides, which can comprise naturally occurring ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. B The sense strand comprises 21 nucleotides wherein the two terminal 3′-nucleotides are optionally base paired and wherein all pyrimidine nucleotides that may be present are 2′-O-methyl modified nucleotides except for (N N) nucleotides, which can comprise naturally occurring ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. The antisense strand comprises 21 nucleotides, wherein the two terminal 3′-nucleotides are optionally complimentary to the target RNA sequence, and wherein all pyrimidine nucleotides that may be present are 2′-deoxy-2′-fluoro modified nucleotides except for (N N) nucleotides, which can comprise naturally occurring ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. C The sense strand comprises 21 nucleotides having 5′- and 3′-terminal cap moieties wherein the two terminal 3′-nucleotides are optionally base paired and wherein all pyrimidine nucleotides that may be present are 2′-O-methyl modified nucleotides except for (N N) nucleotides, which can comprise naturally occurring ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. The antisense strand comprises 21 nucleotides, wherein the two terminal 3′-nucleotides are optionally complimentary to the target RNA sequence, and wherein all pyrimidine nucleotides that may be present are 2′-deoxy-2′-fluoro modified nucleotides except for (N N) nucleotides, which can comprise naturally occurring ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. D The sense strand comprises 21 nucleotides having five phosphorothioate 5′ and 3′-terminal internucleotide linkages, wherein the two terminal 3′-nucleotides are optionally base paired and wherein all nucleotides are ribonucleotides except for (N N) nucleotides, which can comprise naturally occurring ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. The antisense strand comprises 21 nucleotides, wherein the two terminal 3′-nucleotides are optionally complimentary to the target RNA sequence, and having one 3′-terminal phosphorothioate internucleotide linkage and five 5′-terminal phosphorothioate internucleotide linkages and wherein all nucleotides are ribonucleotides except for (N N) nucleotides, which can comprise naturally occurring ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. E The sense strand comprises 21 nucleotides wherein the two terminal 3′-nucleotides are optionally base paired and wherein all pyrimidine nucleotides that may be present are 2′-O-methyl nucleotides except for (N N) nucleotides, which can comprise naturally occurring ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. The antisense strand comprises 21 nucleotides all having phosphorothioate internucleotide linkages, wherein the two terminal 3′-nucleotides are optionally complimentary to the target RNA sequence, and wherein all nucleotides are ribonucleotides except for (N N) nucleotides, which can comprise naturally occurring ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. F The sense strand comprises 21 nucleotides having 5′- and 3′-terminal cap moieties, wherein the two terminal 3′-nucleotides are optionally base paired and wherein all pyrimidine nucleotides that may be present are 2′-O-methyl nucleotides except for (N N) nucleotides, which can comprise naturally occurring ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. The antisense strand comprises 21 nucleotides, wherein the two terminal 3′-nucleotides are optionally complimentary to the target RNA sequence, and having one 3′-terminal phosphorothioate internucleotide linkage and wherein all pyrimidine nucleotides that may be present are 2′-deoxy-2′-fluoro nucleotides except for (N N) nucleotides, which can comprise naturally occurring ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. The antisense strand of constructs A-F comprise sequence complimentary to target RNA sequence of the invention.  
         [0139]    [0139]FIG. 5 shows non-limiting examples of specific chemically modified siRNA sequences of the invention. A-F applies the chemical modifications described in FIGS.  4 A-F to a HIV siRNA sequence.  
         [0140]    [0140]FIG. 6 shows non-limiting examples of different siRNA constructs of the invention. The examples shown (constructs 1, 2, and 3) have 19 representative base pairs, however, different embodiments of the invention include any number of base pairs described herein. Bracketed regions represent nucleotide overhangs, for example comprising between about 1, 2, 3, or 4 nucleotides in length, preferably about 2 nucleotides. Constructs 1 and 2 can be used independently for RNAi activity. Construct 2 can comprise a polynucleotide or non-nucleotide linker, which can optionally be designed as a biodegradable linker. In one embodiment, the loop structure shown in construct 2 can comprise a biodegradable linker that results in the formation of construct 1 in vivo and/or in vitro. In another example, construct 3 can be used to generate construct 2 under the same principle wherein a linker is used to generate the active siRNA construct 2 in vivo and/or in vitro, which can optionally utilize another biodegradable linker to generate the active siRNA construct 1 in vivo and/or in vitro. As such, the stability and/or activity of the siRNA constructs can be modulated based on the design of the siRNA construct for use in vivo or in vitro and/or in vitro.  
         [0141]    [0141]FIG. 7 is a diagrammatic representation of a scheme utilized in generating an expression cassette to generate siRNA hairpin constructs. (A) A DNA oligomer is synthesized with a 5′-restriction site (R1) sequence followed by a region having sequence identical (sense region of siRNA) to a predetermined HIV target sequence, wherein the sense region comprises, for example, about 19, 20, 21, or 22 nucleotides (N) in length, which is followed by a loop sequence of defined sequence (X), comprising, for example, between about 3 and 10 nucleotides. (B) The synthetic construct is then extended by DNA polymerase to generate a hairpin structure having self complementary sequence that will result in a siRNA transcript having specificity for an HIV target sequence and having self complementary sense and antisense regions. (C) The construct is heated (for example to about 95° C.) to linearize the sequence, thus allowing extension of a complementary second DNA strand using a primer to the 3′-restriction sequence of the first strand. The double stranded DNA is then inserted into an appropriate vector for expression in cells. The construct can be designed such that a 3′-overhang results from the transcription, for example by engineering restriction sites and/or utilizing a poly-U termination region as described in Paul et al., 2002,  Nature Biotechnology,  29, 505-508.  
         [0142]    [0142]FIG. 8 is a diagrammatic representation of a scheme utilized in generating an expression cassette to generate double stranded siRNA constructs. (A) A DNA oligomer is synthesized with a 5′-restriction (R1) site sequence followed by a region having sequence identical (sense region of siRNA) to a predetermined HIV target sequence, wherein the sense region comprises, for example, about 19, 20, 21, or 22 nucleotides (N) in length, and which is followed by a 3′-restriction site (R2) which is adjacent to a loop sequence of defined sequence (X). (B) The synthetic construct is then extended by DNA polymerase to generate a hairpin structure having self complementary sequence. (C) The construct is processed by restriction enzymes specific to R1 and R2 to generate a double stranded DNA which is then inserted into an appropriate vector for expression in cells. The transcription cassette is designed such that a U6 promoter region flanks each side of the dsDNA which generates the separate sense and antisense strands of the siRNA. Poly T termination sequences can be added to the constructs to generate U overhangs in the resulting transcript.  
         [0143]    [0143]FIG. 9 is a diagrammatic representation of a method used to determine target sites for siRNA mediated RNAi within a particular target nucleic acid sequence, such as messenger RNA. (A) A pool of siRNA oligonucleotides are synthesized wherein the antisense region of the siRNA constructs has complementarity to target sites across the target nucleic acid sequence, and wherein the sense region comprises sequence complementary to the antisense region of the siRNA. (B) The sequences are pooled and are inserted into vectors such that (C) transfection of a vector into cells results in the expression of the siRNA. (D) Cells are sorted based on phenotypic change that is associated with modulation of the target nucleic acid sequence. (E) The siRNA is isolated from the sorted cells and is sequenced to identify efficacious target sites within the target nucleic acid sequence. 
     
    
       [0144]    Mechanism of Action of Nucleic Acid Molecules of the Invention  
         [0145]    RNA interference refers to the process of sequence specific post transcriptional gene silencing in animals mediated by short interfering RNAs (siRNA) (Fire et al., 1998,  Nature,  391, 806). The corresponding process in plants is commonly referred to as post transcriptional gene silencing or RNA silencing and is also referred to as quelling in fungi. The process of post transcriptional gene silencing is thought to be an evolutionarily conserved cellular defense mechanism used to prevent the expression of foreign genes which is commonly shared by diverse flora and phyla (Fire et al., 1999,  Trends Genet.,  15, 358). Such protection from foreign gene expression may have evolved in response to the production of double stranded RNAs (dsRNA) derived from viral infection or the random integration of transposon elements into a host genome via a cellular response that specifically destroys homologous single stranded RNA or viral genomic RNA. The presence of dsRNA in cells triggers the RNAi response though a mechanism that has yet to be fully characterized. This mechanism appears to be different from the interferon response that results from dsRNA mediated activation of protein kinase PKR and 2′,5′-oligoadenylate synthetase resulting in non-specific cleavage of mRNA by ribonuclease L.  
         [0146]    The presence of long dsRNAs in cells stimulates the activity of a ribonuclease III enzyme referred to as dicer. Dicer is involved in the processing of the dsRNA into short pieces of dsRNA known as short interfering RNAs (siRNA) (Berstein et al., 2001,  Nature,  409, 363). Short interfering RNAs derived from dicer activity are typically about 21 to about 23 (i.e., about 21, 22 or 23) nucleotides in length and comprise about 19 base pair duplexes. Dicer has also been implicated in the excision of 21 and 22 nucleotide small temporal RNAs (stRNA) from precursor RNA of conserved structure that are implicated in translational control (Hutvagner et al., 2001,  Science,  293, 834). The RNAi response also features an endonuclease complex containing a siRNA, commonly referred to as an RNA-induced silencing complex (RISC), which mediates cleavage of single stranded RNA having sequence homologous to the siRNA. Cleavage of the target RNA takes place in the middle of the region complementary to the guide sequence of the siRNA duplex (Elbashir et al., 2001,  Genes Dev.,  15, 188).  
         [0147]    Short interfering RNA mediated RNAi has been studied in a variety of systems. Fire et al., 1998,  Nature,  391, 806, were the first to observe RNAi in  C. Elegans.  Wianny and Goetz, 1999,  Nature Cell Biol.,  2, 70, describes RNAi mediated by dsRNA in mouse embryos. Hammond et al., 2000,  Nature,  404, 293, describe RNAi in Drosophila cells transfected with dsRNA. Elbashir et al., 2001,  Nature,  411, 494, describe RNAi induced by introduction of duplexes of synthetic 21-nucleotide RNAs in cultured mammalian cells including human embryonic kidney and HeLa cells. Recent work in Drosophila embryonic lysates has revealed certain requirements for siRNA length, structure, chemical composition, and sequence that are essential to mediate efficient RNAi activity. These studies have shown that 21 nucleotide siRNA duplexes are most active when containing two nucleotide 3′-overhangs. Furthermore, substitution of one or both siRNA strands with 2′-deoxy or 2′-O-methyl nucleotides abolishes RNAi activity, whereas substitution of 3′-terminal siRNA nucleotides with deoxy nucleotides was shown to be tolerated. Mismatch sequences in the center of the siRNA duplex were also shown to abolish RNAi activity. In addition, these studies also indicate that the position of the cleavage site in the target RNA is defined by the 5′-end of the siRNA guide sequence rather than the 3′-end (Elbashir et al., 2001,  EMBO J.,  20, 6877). Other studies have indicated that a 5′-phosphate on the target-complementary strand of a siRNA duplex is required for siRNA activity and that ATP is utilized to maintain the 5′-phosphate moiety on the siRNA (Nykanen et al., 2001,  Cell,  107, 309), however siRNA molecules lacking a 5′-phosphate are active when introduced exogenously, suggesting that 5′-phosphorylation of siRNA constructs may occur in vivo.  
         [0148]    Synthesis of Nucleic Acid Molecules  
         [0149]    Synthesis of nucleic acids greater than 100 nucleotides in length is difficult using automated methods, and the therapeutic cost of such molecules is prohibitive. In this invention, small nucleic acid motifs (“small” refers to nucleic acid motifs no more than 100 nucleotides in length, preferably no more than 80 nucleotides in length, and most preferably no more than 50 nucleotides in length; e.g., individual siRNA oligonucleotide sequences or siRNA sequences synthesized in tandem) are preferably used for exogenous delivery. The simple structure of these molecules increases the ability of the nucleic acid to invade targeted regions of protein and/or RNA structure. Exemplary molecules of the instant invention are chemically synthesized, and others can similarly be synthesized.  
         [0150]    Oligonucleotides (e.g., certain modified oligonucleotides or portions of oligonucleotides lacking ribonucleotides) are synthesized using protocols known in the art, for example as described in Caruthers et al., 1992,  Methods in Enzymology  211, 3-19, Thompson et al., International PCT Publication No. WO 99/54459, Wincott et al., 1995,  Nucleic Acids Res.  23, 2677-2684, Wincott et al., 1997,  Methods Mol. Bio.,  74, 59, Brennan et al., 1998,  Biotechnol Bioeng.,  61, 33-45, and Brennan, U.S. Pat. No. 6,001,311. All of these references are incorporated herein by reference. The synthesis of oligonucleotides makes use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5′-end, and phosphoramidites at the 3′-end. In a non-limiting example, small scale syntheses are conducted on a 394 Applied Biosystems, Inc. synthesizer using a 0.2 μmol scale protocol with a 2.5 min coupling step for 2′-O-methylated nucleotides and a 45 sec coupling step for 2′-deoxy nucleotides or 2′-deoxy-2′-fluoro nucleotides. Table II outlines the amounts and the contact times of the reagents used in the synthesis cycle. Alternatively, syntheses at the 0.2 μmol scale can be performed on a 96-well plate synthesizer, such as the instrument produced by Protogene (Palo Alto, Calif.) with minimal modification to the cycle. A 33-fold excess (60 μL of 0.11 M=6.6 μmol) of 2′-O-methyl phosphoramidite and a 105-fold excess of S-ethyl tetrazole (60 μL of 0.25 M=15 μmol) can be used in each coupling cycle of 2′-O-methyl residues relative to polymer-bound 5′-hydroxyl. A 22-fold excess (40 μL of 0.11 M=4.4 μmol) of deoxy phosphoramidite and a 70-fold excess of S-ethyl tetrazole (40 μL of 0.25 M=10 μmol) can be used in each coupling cycle of deoxy residues relative to polymer-bound 5′-hydroxyl. Average coupling yields on the 394 Applied Biosystems, Inc. synthesizer, determined by colorimetric quantitation of the trityl fractions, are typically 97.5-99%. Other oligonucleotide synthesis reagents for the 394 Applied Biosystems, Inc. synthesizer include the following: detritylation solution is 3% TCA in methylene chloride (ABI); capping is performed with 16% N-methyl imidazole in THF (ABI) and 10% acetic anhydride/10% 2,6-lutidine in THF (ABI); and oxidation solution is 16.9 mM I 2 , 49 mM pyridine, 9% water in THF (PERSEPTIVE™). Burdick &amp; Jackson Synthesis Grade acetonitrile is used directly from the reagent bottle. S-Ethyltetrazole solution (0.25 M in acetonitrile) is made up from the solid obtained from American International Chemical, Inc. Alternately, for the introduction of phosphorothioate linkages, Beaucage reagent (3H-1,2-Benzodithiol-3-one 1,1-dioxide, 0.05 M in acetonitrile) is used.  
         [0151]    Deprotection of the DNA-based oligonucleotides is performed as follows: the polymer-bound trityl-on oligoribonucleotide is transferred to a 4 mL glass screw top vial and suspended in a solution of 40% aq. methylamine (1 mL) at 65° C. for 10 min. After cooling to −20° C., the supernatant is removed from the polymer support. The support is washed three times with 1.0 mL of EtOH:MeCN:H2O/3:1:1, vortexed and the supernatant is then added to the first supernatant. The combined supernatants, containing the oligoribonucleotide, are dried to a white powder.  
         [0152]    The method of synthesis used for RNA including certain siRNA molecules of the invention follows the procedure as described in Usman et al., 1987,  J. Am. Chem. Soc.,  109, 7845; Scaringe et al., 1990,  Nucleic Acids Res.,  18, 5433; and Wincott et al., 1995,  Nucleic Acids Res.  23, 2677-2684 Wincott et al., 1997,  Methods Mol. Bio.,  74, 59, and makes use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5′-end, and phosphoramidites at the 3′-end. In a non-limiting example, small scale syntheses are conducted on a 394 Applied Biosystems, Inc. synthesizer using a 0.2 μmol scale protocol with a 7.5 min coupling step for alkylsilyl protected nucleotides and a 2.5 min coupling step for 2′-O-methylated nucleotides. Table II outlines the amounts and the contact times of the reagents used in the synthesis cycle. Alternatively, syntheses at the 0.2 μmol scale can be done on a 96-well plate synthesizer, such as the instrument produced by Protogene (Palo Alto, Calif.) with minimal modification to the cycle. A 33-fold excess (60 μL of 0.11 M=6.6 μmol) of 2′-O-methyl phosphoramidite and a 75-fold excess of S-ethyl tetrazole (60 μL of 0.25 M=15 μmol) can be used in each coupling cycle of 2′-O-methyl residues relative to polymer-bound 5′-hydroxyl. A 66-fold excess (120 μL of 0.11 M=13.2 μmol) of alkylsilyl (ribo) protected phosphoramidite and a 150-fold excess of S-ethyl tetrazole (120 μL of 0.25 M=30 μmol) can be used in each coupling cycle of ribo residues relative to polymer-bound 5′-hydroxyl. Average coupling yields on the 394 Applied Biosystems, Inc. synthesizer, determined by colorimetric quantitation of the trityl fractions, are typically 97.5-99%. Other oligonucleotide synthesis reagents for the 394 Applied Biosystems, Inc. synthesizer include the following: detritylation solution is 3% TCA in methylene chloride (ABI); capping is performed with 16% N-methyl imidazole in THF (ABI) and 10% acetic anhydride/10% 2,6-lutidine in THF (ABI); oxidation solution is 16.9 mM I 2 , 49 mM pyridine, 9% water in THF (PERSEPTIVE™). Burdick &amp; Jackson Synthesis Grade acetonitrile is used directly from the reagent bottle. S-Ethyltetrazole solution (0.25 M in acetonitrile) is made up from the solid obtained from American International Chemical, Inc. Alternately, for the introduction of phosphorothioate linkages, Beaucage reagent (3H-1,2-Benzodithiol-3-one 1,1-dioxide 0.05 M in acetonitrile) is used.  
         [0153]    Deprotection of the RNA is performed using either a two-pot or one-pot protocol. For the two-pot protocol, the polymer-bound trityl-on oligoribonucleotide is transferred to a 4 mL glass screw top vial and suspended in a solution of 40% aq. methylamine (1 mL) at 65° C. for 10 min. After cooling to −20° C., the supernatant is removed from the polymer support. The support is washed three times with 1.0 mL of EtOH:MeCN:H2O/3:1:1, vortexed and the supernatant is then added to the first supernatant. The combined supernatants, containing the oligoribonucleotide, are dried to a white powder. The base deprotected oligoribonucleotide is resuspended in anhydrous TEA/HF/NMP solution (300 μL of a solution of 1.5 mL N-methylpyrrolidinone, 750 μL TEA and 1 mL TEA.3HF to provide a 1.4 M HF concentration) and heated to 65° C. After 1.5 h, the oligomer is quenched with 1.5 M NH 4 HCO 3 .  
         [0154]    Alternatively, for the one-pot protocol, the polymer-bound trityl-on oligoribonucleotide is transferred to a 4 mL glass screw top vial and suspended in a solution of 33% ethanolic methylamine/DMSO: 1/1 (0.8 mL) at 65° C. for 15 min. The vial is brought to r.t. TEA.3HF (0.1 mL) is added and the vial is heated at 65° C. for 15 min. The sample is cooled at −20° C. and then quenched with 1.5 M NH 4 HCO 3 .  
         [0155]    For purification of the trityl-on oligomers, the quenched NH 4 HCO 3  solution is loaded onto a C-18 containing cartridge that had been prewashed with acetonitrile followed by 50 mM TEAA. After washing the loaded cartridge with water, the RNA is detritylated with 0.5% TFA for 13 min. The cartridge is then washed again with water, salt exchanged with 1 M NaCl and washed with water again. The oligonucleotide is then eluted with 30% acetonitrile.  
         [0156]    The average stepwise coupling yields are typically &gt;98% (Wincott et al., 1995  Nucleic Acids Res.  23, 2677-2684). Those of ordinary skill in the art will recognize that the scale of synthesis can be adapted to be larger or smaller than the example described above including but not limited to 96-well format, all that is important is the ratio of chemicals used in the reaction.  
         [0157]    Alternatively, the nucleic acid molecules of the present invention can be synthesized separately and joined together post-synthetically, for example, by ligation (Moore et al., 1992,  Science  256, 9923; Draper et al., International PCT publication No. WO 93/23569; Shabarova et al., 1991,  Nucleic Acids Research  19, 4247; Bellon et al., 1997,  Nucleosides  &amp;  Nucleotides,  16, 951; Bellon et al., 1997,  Bioconjugate Chem.  8, 204), or by hybridization following synthesis and/or deprotection.  
         [0158]    The siRNA molecules of the invention can also be synthesized via a tandem synthesis methodology as described in Example 1 herein, wherein both siRNA strands are synthesized as a contiguous oligonucleotide sequence separated by a cleavable linker which is subsequently cleaved to provide separate siRNA sequences that hybridize and permit purification of the siRNA duplex. The tandem synthesis of siRNA as described herein can be readily adapted to both multiwell/multiplate synthesis platforms such as 96 well or similarly larger multi-well platforms. The tandem synthesis of siRNA as described herein can also be readily adapted to large scale synthesis platforms employing batch reactors, synthesis columns and the like.  
         [0159]    The nucleic acid molecules of the present invention can be modified extensively to enhance stability by modification with nuclease resistant groups, for example, 2′-amino, 2′-C-allyl, 2′-flouro, 2′-O-methyl, 2′-H (for a review see Usman and Cedergren, 1992,  TIBS  17, 34; Usman et al., 1994,  Nucleic Acids Symp. Ser.  31, 163). siRNA constructs can be purified by gel electrophoresis using general methods or can be purified by high pressure liquid chromatography (HPLC; see Wincott et al., supra, the totality of which is hereby incorporated herein by reference) and re-suspended in water.  
         [0160]    In another aspect of the invention, siRNA molecules of the invention are expressed from transcription units inserted into DNA or RNA vectors. The recombinant vectors can be DNA plasmids or viral vectors. siRNA expressing viral vectors can be constructed based on, but not limited to, adeno-associated virus, retrovirus, adenovirus, or alphavirus. The recombinant vectors capable of expressing the siRNA molecules can be delivered as described herein, and persist in target cells. Alternatively, viral vectors can be used that provide for transient expression of siRNA molecules.  
         [0161]    Optimizing Activity of the Nucleic Acid Molecule of the Invention.  
         [0162]    Chemically synthesizing nucleic acid molecules with modifications (base, sugar and/or phosphate) can prevent their degradation by serum ribonucleases, which can increase their potency (see e.g., Eckstein et al., International Publication No. WO 92/07065; Perrault et al., 1990  Nature  344, 565; Pieken et al., 1991,  Science  253, 314; Usman and Cedergren, 1992,  Trends in Biochem. Sci.  17, 334; Usman et al., International Publication No. WO 93/15187; and Rossi et al., International Publication No. WO 91/03162; Sproat, U.S. Pat. No. 5,334,711; Gold et al., U.S. Pat. No. 6,300,074; and Burgin et al., supra; all of which are incorporated by reference herein). All of the above references describe various chemical modifications that can be made to the base, phosphate and/or sugar moieties of the nucleic acid molecules described herein. Modifications that enhance their efficacy in cells, and removal of bases from nucleic acid molecules to shorten oligonucleotide synthesis times and reduce chemical requirements are desired.  
         [0163]    There are several examples in the art describing sugar, base and phosphate modifications that can be introduced into nucleic acid molecules with significant enhancement in their nuclease stability and efficacy. For example, oligonucleotides are modified to enhance stability and/or enhance biological activity by modification with nuclease resistant groups, for example, 2′-amino, 2′-C-allyl, 2′-flouro, 2′-O-methyl, 2′-O-allyl, 2′-H, nucleotide base modifications (for a review see Usman and Cedergren, 1992,  TIBS.  17, 34; Usman et al., 1994,  Nucleic Acids Symp. Ser.  31, 163; Burgin et al., 1996,  Biochemistry,  35, 14090). Sugar modification of nucleic acid molecules have been extensively described in the art (see Eckstein et al.,  International Publication  PCT No. WO 92/07065; Perrault et al.  Nature,  1990, 344, 565-568; Pieken et al.  Science,  1991, 253, 314-317; Usman and Cedergren,  Trends in Biochem. Sci.,  1992, 17, 334-339; Usman et al.  International Publication  PCT No. WO 93/15187; Sproat, U.S. Pat. No. 5,334,711 and Beigelman et al., 1995,  J. Biol. Chem.,  270, 25702; Beigelman et al., International PCT publication No. WO 97/26270; Beigelman et al., U.S. Pat. No. 5,716,824; Usman et al., U.S. Pat. No. 5,627,053; Woolf et al., International PCT Publication No. WO 98/13526; Thompson et al., U.S. Ser. No. 60/082,404 which was filed on Apr. 20, 1998; Karpeisky et al., 1998,  Tetrahedron Lett.,  39, 1131; Earnshaw and Gait, 1998,  Biopolymers  ( Nucleic Acid Sciences ), 48, 39-55; Verma and Eckstein, 1998,  Annu. Rev. Biochem.,  67, 99-134; and Burlina et al., 1997,  Bioorg. Med. Chem.,  5, 1999-2010; all of the references are hereby incorporated in their totality by reference herein). Such publications describe general methods and strategies to determine the location of incorporation of sugar, base and/or phosphate modifications and the like into nucleic acid molecules without modulating catalysis, and are incorporated by reference herein. In view of such teachings, similar modifications can be used as described herein to modify the siRNA nucleic acid molecules of the instant invention so long as the ability of siRNA to promote RNAi is cells is not significantly inhibited.  
         [0164]    While chemical modification of oligonucleotide internucleotide linkages with phosphorothioate, phosphorothioate, and/or 5′-methylphosphonate linkages improves stability, excessive modifications can cause some toxicity or decreased activity. Therefore, when designing nucleic acid molecules, the amount of these internucleotide linkages should be minimized. The reduction in the concentration of these linkages should lower toxicity, resulting in increased efficacy and higher specificity of these molecules.  
         [0165]    Small interfering RNA (siRNA) molecules having chemical modifications that maintain or enhance activity are provided. Such a nucleic acid is also generally more resistant to nucleases than an unmodified nucleic acid. Accordingly, the in vitro and/or in vivo activity should not be significantly lowered. In cases in which modulation is the goal, therapeutic nucleic acid molecules delivered exogenously should optimally be stable within cells until translation of the target RNA has been modulated long enough to reduce the levels of the undesirable protein. This period of time varies between hours to days depending upon the disease state. Improvements in the chemical synthesis of RNA and DNA (Wincott et al., 1995  Nucleic Acids Res.  23, 2677; Caruthers et al., 1992,  Methods in Enzymology  211,3-19 (incorporated by reference herein)) have expanded the ability to modify nucleic acid molecules by introducing nucleotide modifications to enhance their nuclease stability, as described above.  
         [0166]    In one embodiment, nucleic acid molecules of the invention include one or more, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more G-clamp nucleotides. A G-clamp nucleotide is a modified cytosine analog wherein the modifications confer the ability to hydrogen bond both Watson-Crick and Hoogsteen faces of a complementary guanine within a duplex, see for example Lin and Matteucci, 1998,  J. Am. Chem. Soc.,  120, 8531-8532. A single G-clamp analog substitution within an oligonucleotide can result in substantially enhanced helical thermal stability and mismatch discrimination when hybridized to complementary oligonucleotides. The inclusion of such nucleotides in nucleic acid molecules of the invention results in both enhanced affinity and specificity to nucleic acid targets, complimentary sequences, or template strands. In another embodiment, nucleic acid molecules of the invention include one or more, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more LNA “locked nucleic acid” nucleotides such as a 2′, 4′-C mythylene bicyclo nucleotide (see for example Wengel et al., International PCT Publication No. WO 00/66604 and WO 99/14226).  
         [0167]    In another embodiment, the invention features conjugates and/or complexes of siRNA molecules of the invention. Such conjugates and/or complexes can be used to facilitate delivery of siRNA molecules into a biological system, such as a cell. The conjugates and complexes provided by the instant invention can impart therapeutic activity by transferring therapeutic compounds across cellular membranes, altering the pharmacokinetics, and/or modulating the localization of nucleic acid molecules of the invention. The present invention encompasses the design and synthesis of novel conjugates and complexes for the delivery of molecules, including, but not limited to, small molecules, lipids, phospholipids, nucleosides, nucleotides, nucleic acids, antibodies, toxins, negatively charged polymers and other polymers, for example proteins, peptides, hormones, carbohydrates, polyethylene glycols, or polyamines, across cellular membranes. In general, the transporters described are designed to be used either individually or as part of a multi-component system, with or without degradable linkers. These compounds are expected to improve delivery and/or localization of nucleic acid molecules of the invention into a number of cell types originating from different tissues, in the presence or absence of serum (see Sullenger and Cech, U.S. Pat. No. 5,854,038). Conjugates of the molecules described herein can be attached to biologically active molecules via linkers that are biodegradable, such as biodegradable nucleic acid linker molecules.  
         [0168]    The term “biodegradable nucleic acid linker molecule” as used herein, refers to a nucleic acid molecule that is designed as a biodegradable linker to connect one molecule to another molecule, for example, a biologically active molecule. The stability of the biodegradable nucleic acid linker molecule can be modulated by using various combinations of ribonucleotides, deoxyribonucleotides, and chemically modified nucleotides, for example, 2′-O-methyl, 2′-fluoro, 2′-amino, 2′-O-amino, 2′-C-allyl, 2′-O-allyl, and other 2′-modified or base modified nucleotides. The biodegradable nucleic acid linker molecule can be a dimer, trimer, tetramer or longer nucleic acid molecule, for example, an oligonucleotide of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length, or can comprise a single nucleotide with a phosphorus-based linkage, for example, a phosphoramidate or phosphodiester linkage. The biodegradable nucleic acid linker molecule can also comprise nucleic acid backbone, nucleic acid sugar, or nucleic acid base modifications.  
         [0169]    The term “biodegradable” as used herein, refers to degradation in a biological system, for example enzymatic degradation or chemical degradation.  
         [0170]    The term “biologically active molecule” as used herein, refers to compounds or molecules that are capable of eliciting or modifying a biological response in a system. Non-limiting examples of biologically active siRNA molecules either alone or in combination with othe molecules contemplated by the instant invention include therapeutically active molecules such as antibodies, hormones, antivirals, peptides, proteins, chemotherapeutics, small molecules, vitamins, co-factors, nucleosides, nucleotides, oligonucleotides, enzymatic nucleic acids, antisense nucleic acids, triplex forming oligonucleotides, 2,5-A chimeras, siRNA, dsRNA, allozymes, aptamers, decoys and analogs thereof. Biologically active molecules of the invention also include molecules capable of modulating the pharmacokinetics and/or pharmacodynamics of other biologically active molecules, for example, lipids and polymers such as polyamines, polyamides, polyethylene glycol and other polyethers.  
         [0171]    The term “phospholipid” as used herein, refers to a hydrophobic molecule comprising at least one phosphorus group. For example, a phospholipid can comprise a phosphorus-containing group and saturated or unsaturated alkyl group, optionally substituted with OH, COOH, oxo, amine, or substituted or unsubstituted aryl groups.  
         [0172]    Therapeutic nucleic acid molecules (e.g., siRNA molecules) delivered exogenously optimally are stable within cells until reverse trascription of the RNA has been modulated long enough to reduce the levels of the RNA transcript. The nucleic acid molecules are resistant to nucleases in order to function as effective intracellular therapeutic agents. Improvements in the chemical synthesis of nucleic acid molecules described in the instant invention and in the art have expanded the ability to modify nucleic acid molecules by introducing nucleotide modifications to enhance their nuclease stability as described above.  
         [0173]    In yet another embodiment, siRNA molecules having chemical modifications that maintain or enhance enzymatic activity of proteins involved in RNAi are provided. Such nucleic acids are also generally more resistant to nucleases than unmodified nucleic acids. Thus, in vitro and/or in vivo the activity should not be significantly lowered.  
         [0174]    Use of the nucleic acid-based molecules of the invention will lead to better treatment of the disease progression by affording the possibility of combination therapies (e.g., multiple siRNA molecules targeted to different genes; nucleic acid molecules coupled with known small molecule modulators; or intermittent treatment with combinations of molecules, including different motifs and/or other chemical or biological molecules). The treatment of subjects with siRNA molecules can also include combinations of different types of nucleic acid molecules, such as enzymatic nucleic acid molecules (ribozymes), allozymes, antisense, 2,5-A oligoadenylate, decoys, aptamers etc.  
         [0175]    In another aspect a siRNA molecule of the invention comprises one or more 5′ and/or a 3′-cap structure, for example on only the sense siRNA strand, antisense siRNA strand, or both siRNA strands.  
         [0176]    By “cap structure” is meant chemical modifications, which have been incorporated at either terminus of the oligonucleotide (see, for example, Adamic et al., U.S. Pat. No. 5,998,203, incorporated by reference herein). These terminal modifications protect the nucleic acid molecule from exonuclease degradation, and can help in delivery and/or localization within a cell. The cap can be present at the 5′-terminus (5′-cap) or at the 3′-terminal (3′-cap) or can be present on both termini. In non-limiting examples: the 5′-cap is selected from the group comprising inverted abasic residue (moiety); 4′,5′-methylene nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide, 4′-thio nucleotide; carbocyclic nucleotide; 1,5-anhydrohexitol nucleotide; L-nucleotides; alpha-nucleotides; modified base nucleotide; phosphorodithioate linkage; threo-pentofuranosyl nucleotide; acyclic 3′,4′-seco nucleotide; acyclic 3,4-dihydroxybutyl nucleotide; acyclic 3,5-dihydroxypentyl nucleotide, 3′-3′-inverted nucleotide moiety; 3′-3′-inverted abasic moiety; 3′-2′-inverted nucleotide moiety; 3′-2′-inverted abasic moiety; 1,4-butanediol phosphate; 3′-phosphoramidate; hexylphosphate; aminohexyl phosphate; 3′-phosphate; 3′-phosphorothioate; phosphorodithioate; or bridging or non-bridging methylphosphonate moiety.  
         [0177]    In yet another preferred embodiment, the 3′-cap is selected from a group comprising, 4′,5′-methylene nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide; 4′-thio nucleotide, carbocyclic nucleotide; 5′-amino-alkyl phosphate; 1,3-diamino-2-propyl phosphate; 3-aminopropyl phosphate; 6-aminohexyl phosphate; 1,2-aminododecyl phosphate; hydroxypropyl phosphate; 1,5-anhydrohexitol nucleotide; L-nucleotide; alpha-nucleotide; modified base nucleotide; phosphorodithioate; threo-pentofuranosyl nucleotide; acyclic 3′,4′-seco nucleotide; 3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentyl nucleotide, 5′-5′-inverted nucleotide moiety; 5′-5′-inverted abasic moiety; 5′-phosphoramidate; 5′-phosphorothioate; 1,4-butanediol phosphate; 5′-amino; bridging and/or non-bridging 5′-phosphoramidate, phosphorothioate and/or phosphorodithioate, bridging or non bridging methylphosphonate and 5′-mercapto moieties (for more details see Beaucage and Iyer, 1993,  Tetrahedron  49, 1925; incorporated by reference herein).  
         [0178]    By the term “non-nucleotide” is meant any group or compound which can be incorporated into a nucleic acid chain in the place of one or more nucleotide units, including either sugar and/or phosphate substitutions, and allows the remaining bases to exhibit their enzymatic activity. The group or compound is abasic in that it does not contain a commonly recognized nucleotide base, such as adenosine, guanine, cytosine, uracil or thymine and therefore lacks a base at the 1′-position.  
         [0179]    An “alkyl” group refers to a saturated aliphatic hydrocarbon, including straight-chain, branched-chain, and cyclic alkyl groups. Preferably, the alkyl group has 1 to 12 carbons. More preferably, it is a lower alkyl of from 1 to 7 carbons, more preferably 1 to 4 carbons. The alkyl group can be substituted or unsubstituted. When substituted the substituted group(s) is preferably, hydroxyl, cyano, alkoxy, ═O, ═S, NO 2  or N(CH 3 ) 2 , amino, or SH. The term also includes alkenyl groups that are unsaturated hydrocarbon groups containing at least one carbon-carbon double bond, including straight-chain, branched-chain, and cyclic groups. Preferably, the alkenyl group has 1 to 12 carbons. More preferably, it is a lower alkenyl of from 1 to 7 carbons, more preferably 1 to 4 carbons. The alkenyl group may be substituted or unsubstituted. When substituted the substituted group(s) is preferably, hydroxyl, cyano, alkoxy, ═O, ═S, NO 2 , halogen, N(CH 3 ) 2 , amino, or SH. The term “alkyl” also includes alkynyl groups that have an unsaturated hydrocarbon group containing at least one carbon-carbon triple bond, including straight-chain, branched-chain, and cyclic groups. Preferably, the alkynyl group has 1 to 12 carbons. More preferably, it is a lower alkynyl of from 1 to 7 carbons, more preferably 1 to 4 carbons. The alkynyl group may be substituted or unsubstituted. When substituted the substituted group(s) is preferably, hydroxyl, cyano, alkoxy, ═O, ═S, NO 2  or N(CH 3 ) 2 , amino or SH.  
         [0180]    Such alkyl groups may also include aryl, alkylaryl, carbocyclic aryl, heterocyclic aryl, amide and ester groups. An “aryl” group refers to an aromatic group that has at least one ring having a conjugated pi electron system and includes carbocyclic aryl, heterocyclic aryl and biaryl groups, all of which may be optionally substituted. The preferred substituent(s) of aryl groups are halogen, trihalomethyl, hydroxyl, SH, OH, cyano, alkoxy, alkyl, alkenyl, alkynyl, and amino groups. An “alkylaryl” group refers to an alkyl group (as described above) covalently joined to an aryl group (as described above). Carbocyclic aryl groups are groups wherein the ring atoms on the aromatic ring are all carbon atoms. The carbon atoms are optionally substituted. Heterocyclic aryl groups are groups having from 1 to 3 heteroatoms as ring atoms in the aromatic ring and the remainder of the ring atoms are carbon atoms. Suitable heteroatoms include oxygen, sulfur, and nitrogen, and include furanyl, thienyl, pyridyl, pyrrolyl, N-lower alkyl pyrrolo, pyrimidyl, pyrazinyl, imidazolyl and the like, all optionally substituted. An “amide” refers to an —C(O)—NH—R, where R is either alkyl, aryl, alkylaryl or hydrogen. An “ester” refers to an —C(O)—OR′, where R is either alkyl, aryl, alkylaryl or hydrogen.  
         [0181]    By “nucleotide” as used herein is as recognized in the art to include natural bases (standard), and modified bases well known in the art. Such bases are generally located at the 1′ position of a nucleotide sugar moiety. Nucleotides generally comprise a base, sugar and a phosphate group. The nucleotides can be unmodified or modified at the sugar, phosphate and/or base moiety, (also referred to interchangeably as nucleotide analogs, modified nucleotides, non-natural nucleotides, non-standard nucleotides and other; see, for example, Usman and McSwiggen, supra; Eckstein et al., International PCT Publication No. WO 92/07065; Usman et al., International PCT Publication No. WO 93/15187; Uhlman &amp; Peyman, supra, all are hereby incorporated by reference herein). There are several examples of modified nucleic acid bases known in the art as summarized by Limbach et al., 1994,  Nucleic Acids Res.  22, 2183. Some of the non-limiting examples of base modifications that can be introduced into nucleic acid molecules include, inosine, purine, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2, 4, 6-trimethoxy benzene, 3-methyl uracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidines (e.g., 5-methylcytidine), 5-alkyluridines (e.g., ribothymidine), 5-halouridine (e.g., 5-bromouridine) or 6-azapyrimidines or 6-alkylpyrimidines (e.g. 6-methyluridine), propyne, and others (Burgin et al., 1996,  Biochemistry,  35, 14090; Uhlman &amp; Peyman, supra). By “modified bases” in this aspect is meant nucleotide bases other than adenine, guanine, cytosine and uracil at 1′ position or their equivalents.  
         [0182]    In one embodiment, the invention features modified siRNA molecules, with phosphate backbone modifications comprising one or more phosphorothioate, phosphorodithioate, methylphosphonate, phosphotriester, morpholino, amidate carbamate, carboxymethyl, acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, formacetal, thioformacetal, and/or alkylsilyl, substitutions. For a review of oligonucleotide backbone modifications, see Hunziker and Leumann, 1995,  Nucleic Acid Analogues: Synthesis and Properties,  in  Modern Synthetic Methods,  VCH, 331-417, and Mesmaeker et al., 1994,  Novel Backbone Replacements for Oligonucleotides, in Carbohydrate Modifications in Antisense Research,  ACS, 24-39.  
         [0183]    By “abasic” is meant sugar moieties lacking a base or having other chemical groups in place of a base at the 1′ position, see for example Adamic et al., U.S. Pat. No. 5,998,203.  
         [0184]    By “unmodified nucleoside” is meant one of the bases adenine, cytosine, guanine, thymine, uracil joined to the 1′ carbon of β-D-ribo-furanose.  
         [0185]    By “modified nucleoside” is meant any nucleotide base which contains a modification in the chemical structure of an unmodified nucleotide base, sugar and/or phosphate.  
         [0186]    In connection with 2′-modified nucleotides as described for the present invention, by “amino” is meant 2′-NH 2  or 2′-O—NH 2 , which may be modified or unmodified. Such modified groups are described, for example, in Eckstein et al., U.S. Pat. No. 5,672,695 and Matulic-Adamic et al., U.S. Pat. No. 6,248,878, which are both incorporated by reference in their entireties.  
         [0187]    Various modifications to nucleic acid siRNA structure can be made to enhance the utility of these molecules. Such modifications will enhance shelf-life, half-life in vitro, stability, and ease of introduction of such oligonucleotides to the target site, e.g., to enhance penetration of cellular membranes, and confer the ability to recognize and bind to targeted cells.  
         [0188]    Administration of Nucleic Acid Molecules  
         [0189]    A siRNA molecule of the invention can be adapted for use to treat, for example conditions related to HIV infection and/or AIDS, alone or in combination with other therapies. For example, a siRNA molecule can comprise a delivery vehicle, including liposomes, for administration to a subject, carriers and diluents and their salts, and/or can be present in pharmaceutically acceptable formulations. Methods for the delivery of nucleic acid molecules are described in Akhtar et al., 1992,  Trends Cell Bio.,  2, 139;  Delivery Strategies for Antisense Oligonucleotide Therapeutics,  ed. Akhtar, 1995, Maurer et al., 1999,  Mol. Membr. Biol.,  16, 129-140; Hofland and Huang, 1999,  Handb. Exp. Pharmacol.,  137, 165-192; and Lee et al., 2000,  ACS Symp. Ser.,  752, 184-192, all of which are incorporated herein by reference. Beigelman et al., U.S. Pat. No. 6,395,713 and Sullivan et al., PCT WO 94/02595, further describes the general methods for delivery of nucleic acid molecules. Delivery of nucleic acid molecules of the invention to hematopoietic cells, such as T-cells, can be accomplished as is known in the art, see for example Draper, U.S. Pat. No. 6,622,854; Phillips et al., 1996,  Nature Medicine,  2(10), 1154-1156; Smith et al., 1996,  Antiviral Research,  32(2), 99-115; and Rudoll et al., 1996,  Gene Therapy,  3(8), 695-705.  
         [0190]    These protocols can be utilized for the delivery of virtually any nucleic acid molecule. Nucleic acid molecules can be administered to cells by a variety of methods known to those of skill in the art, including, but not restricted to, encapsulation in liposomes, by iontophoresis, or by incorporation into other vehicles, such as hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres, or by proteinaceous vectors (O&#39;Hare and Normand, International PCT Publication No. WO 00/53722). Alternatively, the nucleic acid/vehicle combination is locally delivered by direct injection or by use of an infusion pump. Direct injection of the nucleic acid molecules of the invention, whether subcutaneous, intramuscular, or intradermal, can take place using standard needle and syringe methodologies, or by needle-free technologies such as those described in Conry et al., 1999,  Clin. Cancer Res.,  5, 2330-2337 and Barry et al., International PCT Publication No. WO 99/31262. The molecules of the instant invention can be used as pharmaceutical agents. Pharmaceutical agents prevent, modulate the occurrence, or treat (alleviate a symptom to some extent, preferably all of the symptoms) of a disease state in a subject.  
         [0191]    Thus, the invention features a pharmaceutical composition comprising one or more nucleic acid(s) of the invention in an acceptable carrier, such as a stabilizer, buffer, and the like. The polynucleotides of the invention can be administered (e.g., RNA, DNA or protein) and introduced into a subject by any standard means, with or without stabilizers, buffers, and the like, to form a pharmaceutical composition. When it is desired to use a liposome delivery mechanism, standard protocols for formation of liposomes can be followed. The compositions of the present invention can also be formulated and used as tablets, capsules or elixirs for oral administration, suppositories for rectal administration, sterile solutions, suspensions for injectable administration, and the other compositions known in the art.  
         [0192]    The present invention also includes pharmaceutically acceptable formulations of the compounds described. These formulations include salts of the above compounds, e.g., acid addition salts, for example, salts of hydrochloric, hydrobromic, acetic acid, and benzene sulfonic acid.  
         [0193]    A pharmacological composition or formulation refers to a composition or formulation in a form suitable for administration, e.g., systemic administration, into a cell or subject, including for example a human. Suitable forms, in part, depend upon the use or the route of entry, for example oral, transdermal, or by injection. Such forms should not prevent the composition or formulation from reaching a target cell (i.e., a cell to which the negatively charged nucleic acid is desirable for delivery). For example, pharmacological compositions injected into the blood stream should be soluble. Other factors are known in the art, and include considerations such as toxicity and forms that prevent the composition or formulation from exerting its effect.  
         [0194]    By “systemic administration” is meant in vivo systemic absorption or accumulation of drugs in the blood stream followed by distribution throughout the entire body. Administration routes which lead to systemic absorption include, without limitation: intravenous, subcutaneous, intraperitoneal, inhalation, oral, intrapulmonary and intramuscular. Each of these administration routes expose the siRNA molecules of the invention to an accessible diseased tissue. The rate of entry of a drug into the circulation has been shown to be a function of molecular weight or size. The use of a liposome or other drug carrier comprising the compounds of the instant invention can potentially localize the drug, for example, in certain tissue types, such as the tissues of the reticular endothelial system (RES). A liposome formulation that can facilitate the association of drug with the surface of cells, such as, lymphocytes and macrophages is also useful. This approach can provide enhanced delivery of the drug to target cells by taking advantage of the specificity of macrophage and lymphocyte immune recognition of abnormal cells, such as cancer cells.  
         [0195]    By “pharmaceutically acceptable formulation” is meant, a composition or formulation that allows for the effective distribution of the nucleic acid molecules of the instant invention in the physical location most suitable for their desired activity. Non-limiting examples of agents suitable for formulation with the nucleic acid molecules of the instant invention include: P-glycoprotein inhibitors (such as Pluronic P85), which can enhance entry of drugs into the CNS (Jolliet-Riant and Tillement, 1999,  Fundam. Clin. Pharmacol.,  13, 16-26); biodegradable polymers, such as poly (DL-lactide-coglycolide) microspheres for sustained release delivery after intracerebral implantation (Emerich, DF et al, 1999,  Cell Transplant,  8, 47-58) (Alkermes, Inc. Cambridge, Mass.); and loaded nanoparticles, such as those made of polybutylcyanoacrylate, which can deliver drugs across the blood brain barrier and can alter neuronal uptake mechanisms ( Prog Neuropsychopharmacol Biol Psychiatry,  23, 941-949, 1999). Other non-limiting examples of delivery strategies for the nucleic acid molecules of the instant invention include material described in Boado et al., 1998,  J. Pharm. Sci.,  87, 1308-1315; Tyler et al., 1999,  FEBS Lett.,  421, 280-284; Pardridge et al., 1995,  PNAS USA.,  92, 5592-5596; Boado, 1995,  Adv. Drug Delivery Rev.,  15, 73-107; Aldrian-Herrada et al., 1998,  Nucleic Acids Res.,  26, 4910-4916; and Tyler et al., 1999,  PNAS USA.,  96, 7053-7058.  
         [0196]    The invention also features the use of the composition comprising surface-modified liposomes containing poly (ethylene glycol) lipids (PEG-modified, or long-circulating liposomes or stealth liposomes). These formulations offer a method for increasing the accumulation of drugs in target tissues. This class of drug carriers resists opsonization and elimination by the mononuclear phagocytic system (MPS or RES), thereby enabling longer blood circulation times and enhanced tissue exposure for the encapsulated drug (Lasic et al.  Chem. Rev.  1995, 95, 2601-2627; Ishiwata et al.,  Chem. Pharm. Bull.  1995, 43, 1005-1011). Such liposomes have been shown to accumulate selectively in tumors, presumably by extravasation and capture in the neovascularized target tissues (Lasic et al.,  Science  1995, 267, 1275-1276; Oku et al., 1995,  Biochim. Biophys. Acta,  1238, 86-90). The long-circulating liposomes enhance the pharmacokinetics and pharmacodynamics of DNA and RNA, particularly compared to conventional cationic liposomes which are known to accumulate in tissues of the MPS (Liu et al.,  J. Biol. Chem.  1995, 42, 24864-24870; Choi et al., International PCT Publication No. WO 96/10391; Ansell et al., International PCT Publication No. WO 96/10390; Holland et al., International PCT Publication No. WO 96/10392). Long-circulating liposomes are also likely to protect drugs from nuclease degradation to a greater extent compared to cationic liposomes, based on their ability to avoid accumulation in metabolically aggressive MPS tissues such as the liver and spleen.  
         [0197]    The present invention also includes compositions prepared for storage or administration, which include a pharmaceutically effective amount of the desired compounds in a pharmaceutically acceptable carrier or diluent. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in  Remington&#39;s Pharmaceutical Sciences,  Mack Publishing Co. (A. R. Gennaro edit. 1985) hereby incorporated by reference herein. For example, preservatives, stabilizers, dyes and flavoring agents can be provided. These include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. In addition, antioxidants and suspending agents can be used.  
         [0198]    The present invention also includes compositions prepared for storage or administration that include a pharmaceutically effective amount of the desired compounds in a pharmaceutically acceptable carrier or diluent. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in  Remington&#39;s Pharmaceutical Sciences,  Mack Publishing Co. (A. R. Gennaro edit. 1985), hereby incorporated by reference herein. For example, preservatives, stabilizers, dyes and flavoring agents can be provided. These include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. In addition, antioxidants and suspending agents can be used.  
         [0199]    A pharmaceutically effective dose is that dose required to prevent, inhibit the occurrence, or treat (alleviate a symptom to some extent, preferably all of the symptoms) of a disease state. The pharmaceutically effective dose depends on the type of disease, the composition used, the route of administration, the type of mammal being treated, the physical characteristics of the specific mammal under consideration, concurrent medication, and other factors that those skilled in the medical arts will recognize. Generally, an amount between 0.1 mg/kg and 100 mg/kg body weight/day of active ingredients is administered dependent upon potency of the negatively charged polymer.  
         [0200]    The nucleic acid molecules of the invention and formulations thereof can be administered orally, topically, parenterally, by inhalation or spray, or rectally in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and/or vehicles. The term parenteral as used herein includes percutaneous, subcutaneous, intravascular (e.g., intravenous), intramuscular, or intrathecal injection or infusion techniques and the like. In addition, there is provided a pharmaceutical formulation comprising a nucleic acid molecule of the invention and a pharmaceutically acceptable carrier. One or more nucleic acid molecules of the invention can be present in association with one or more non-toxic pharmaceutically acceptable carriers and/or diluents and/or adjuvants, and if desired other active ingredients. The pharmaceutical compositions containing nucleic acid molecules of the invention can be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsion, hard or soft capsules, or syrups or elixirs.  
         [0201]    Compositions intended for oral use can be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions can contain one or more such sweetening agents, flavoring agents, coloring agents or preservative agents in order to provide pharmaceutically elegant and palatable preparations. Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients that are suitable for the manufacture of tablets. These excipients can be, for example, inert diluents; such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch, or alginic acid; binding agents, for example starch, gelatin or acacia; and lubricating agents, for example magnesium stearate, stearic acid or talc. The tablets can be uncoated or they can be coated by known techniques. In some cases such coatings can be prepared by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monosterate or glyceryl distearate can be employed.  
         [0202]    Formulations for oral use can also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin or olive oil.  
         [0203]    Aqueous suspensions contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydropropyl-methylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents can be a naturally-occurring phosphatide, for example, lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. The aqueous suspensions can also contain one or more preservatives, for example ethyl, or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin.  
         [0204]    Oily suspensions can be formulated by suspending the active ingredients in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspensions can contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents and flavoring agents can be added to provide palatable oral preparations. These compositions can be preserved by the addition of an anti-oxidant such as ascorbic acid.  
         [0205]    Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents or suspending agents are exemplified by those already mentioned above. Additional excipients, for example sweetening, flavoring and coloring agents, can also be present.  
         [0206]    Pharmaceutical compositions of the invention can also be in the form of oil-in-water emulsions. The oily phase can be a vegetable oil or a mineral oil or mixtures of these. Suitable emulsifying agents can be naturally-occurring gums, for example gum acacia or gum tragacanth, naturally-occurring phosphatides, for example soy bean, lecithin, and esters or partial esters derived from fatty acids and hexitol, anhydrides, for example sorbitan monooleate, and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate. The emulsions can also contain sweetening and flavoring agents.  
         [0207]    Syrups and elixirs can be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol, glucose or sucrose. Such formulations can also contain a demulcent, a preservative and flavoring and coloring agents. The pharmaceutical compositions can be in the form of a sterile injectable aqueous or oleaginous suspension. This suspension can be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents that have been mentioned above. The sterele injectable preparation can also be a sterile injectable solution or suspension in a non-toxic parentally acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that can be employed are water, Ringer&#39;s solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.  
         [0208]    The nucleic acid molecules of the invention can also be administered in the form of suppositories, e.g., for rectal administration of the drug. These compositions can be prepared by mixing the drug with a suitable non-irritating excipient that is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Such materials include cocoa butter and polyethylene glycols.  
         [0209]    Nucleic acid molecules of the invention can be administered parenterally in a sterile medium. The drug, depending on the vehicle and concentration used, can either be suspended or dissolved in the vehicle. Advantageously, adjuvants such as local anesthetics, preservatives and buffering agents can be dissolved in the vehicle.  
         [0210]    Dosage levels of the order of from about 0.1 mg to about 140 mg per kilogram of body weight per day are useful in the treatment of the above-indicated conditions (about 0.5 mg to about 7 g per subject per day). The amount of active ingredient that can be combined with the carrier materials to produce a single dosage form varies depending upon the host treated and the particular mode of administration. Dosage unit forms generally contain between from about 1 mg to about 500 mg of an active ingredient.  
         [0211]    It is understood that the specific dose level for any particular subject depends upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, and rate of excretion, drug combination and the severity of the particular disease undergoing therapy.  
         [0212]    For administration to non-human animals, the composition can also be added to the animal feed or drinking water. It can be convenient to formulate the animal feed and drinking water compositions so that the animal takes in a therapeutically appropriate quantity of the composition along with its diet. It can also be convenient to present the composition as a premix for addition to the feed or drinking water.  
         [0213]    The nucleic acid molecules of the present invention can also be administered to a subject in combination with other therapeutic compounds to increase the overall therapeutic effect. The use of multiple compounds to treat an indication can increase the beneficial effects while reducing the presence of side effects.  
         [0214]    In one embodiment, the invention compositions suitable for administering nucleic acid molecules of the invention to specific cell types, such as hepatocytes. For example, the asialoglycoprotein receptor (ASGPr) (Wu and Wu, 1987,  J. Biol. Chem.  262, 4429-4432) is unique to hepatocytes and binds branched galactose-terminal glycoproteins, such as asialoorosomucoid (ASOR). Binding of such glycoproteins or synthetic glycoconjugates to the receptor takes place with an affinity that strongly depends on the degree of branching of the oligosaccharide chain, for example, triatennary structures are bound with greater affinity than biatenarry or monoatennary chains (Baenziger and Fiete, 1980,  Cell,  22, 611-620; Connolly et al., 1982,  J. Biol. Chem.,  257, 939-945). Lee and Lee, 1987,  Glycoconjugate J.,  4, 317-328, obtained this high specificity through the use of N-acetyl-D-galactosamine as the carbohydrate moiety, which has higher affinity for the receptor, compared to galactose. This “clustering effect” has also been described for the binding and uptake of mannosyl-terminating glycoproteins or glycoconjugates (Ponpipom et al., 1981,  J. Med. Chem.,  24, 1388-1395). The use of galactose and galactosamine based conjugates to transport exogenous compounds across cell membranes can provide a targeted delivery approach to the treatment of liver disease such as HBV infection or hepatocellular carcinoma. The use of bioconjugates can also provide a reduction in the required dose of therapeutic compounds required for treatment. Furthermore, therapeutic bioavialability, pharmacodynamics, and pharmacokinetic parameters can be modulated through the use of nucleic acid bioconjugates of the invention.  
         [0215]    Alternatively, certain siRNA molecules of the instant invention can be expressed within cells from eukaryotic promoters (e.g., Izant and Weintraub, 1985,  Science,  229, 345; McGarry and Lindquist, 1986,  Proc. Natl. Acad. Sci.,  USA 83, 399; Scanlon et al., 1991,  Proc. Natl. Acad. Sci. USA,  88, 10591-5; Kashani-Sabet et al., 1992,  Antisense Res. Dev.,  2, 3-15; Dropulic et al., 1992,  J. Virol.,  66, 1432-41; Weerasinghe et al., 1991,  J. Virol.,  65, 5531-4; Ojwang et al., 1992,  Proc. Natl. Acad. Sci. USA,  89, 10802-6; Chen et al., 1992,  Nucleic Acids Res.,  20, 4581-9; Sarver et al., 1990  Science,  247, 1222-1225; Thompson et al., 1995,  Nucleic Acids Res.,  23, 2259; Good et al., 1997,  Gene Therapy,  4, 45. Those skilled in the art realize that any nucleic acid can be expressed in eukaryotic cells from the appropriate DNA/RNA vector. The activity of such nucleic acids can be augmented by their release from the primary transcript by a enzymatic nucleic acid (Draper et al., PCT WO 93/23569, and Sullivan et al., PCT WO 94/02595; Ohkawa et al., 1992,  Nucleic Acids Symp. Ser.,  27, 15-6; Taira et al., 1991,  Nucleic Acids Res.,  19, 5125-30; Ventura et al., 1993,  Nucleic Acids Res.,  21, 3249-55; Chowrira et al., 1994,  J. Biol. Chem.,  269, 25856.  
         [0216]    In another aspect of the invention, RNA molecules of the present invention can be expressed from transcription units (see for example Couture et al., 1996,  TIG.,  12, 510) inserted into DNA or RNA vectors. The recombinant vectors can be DNA plasmids or viral vectors. siRNA expressing viral vectors can be constructed based on, but not limited to, adeno-associated virus, retrovirus, adenovirus, or alphavirus. In another embodiment, pol III based constructs are used to express nucleic acid molecules of the invention (see for example Thompson, U.S. Pat. Nos. 5,902,880 and 6,146,886). The recombinant vectors capable of expressing the siRNA molecules can be delivered as described above, and persist in target cells. Alternatively, viral vectors can be used that provide for transient expression of nucleic acid molecules. Such vectors can be repeatedly administered as necessary. Once expressed, the siRNA molecule interacts with the target mRNA and generates an RNAi response. Delivery of siRNA molecule expressing vectors can be systemic, such as by intravenous or intra-muscular administration, by administration to target cells ex-planted from a subject followed by reintroduction into the subject, or by any other means that would allow for introduction into the desired target cell (for a review see Couture et al., 1996,  TIG.,  12, 510).  
         [0217]    In one aspect the invention features an expression vector comprising a nucleic acid sequence encoding at least one siRNA molecule of the instant invention. The expression vector can encode one or both strands of a siRNA duplex, or a single self complimentary strand that self hybridizes into a siRNA duplex. The nucleic acid sequences encoding the siRNA molecules of the instant invention can be operably linked in a manner that allows expression of the siRNA molecule (see for example Paul et al., 2002,  Nature Biotechnology,  19, 505; Miyagishi and Taira, 2002,  Nature Biotechnology,  19, 497; Lee et al., 2002,  Nature Biotechnology,  19, 500; and Novina et al., 2002,  Nature Medicine,  advance online publication doi:10.1038/nm725).  
         [0218]    In another aspect, the invention features an expression vector comprising: a) a transcription initiation region (e.g., eukaryotic pol I, II or III initiation region); b) a transcription termination region (e.g., eukaryotic pol I, II or III termination region); and c) a nucleic acid sequence encoding at least one of the siRNA molecules of the instant invention; wherein said sequence is operably linked to said initiation region and said termination region, in a manner that allows expression and/or delivery of the siRNA molecule. The vector can optionally include an open reading frame (ORF) for a protein operably linked on the 5′ side or the 3′-side of the sequence encoding the siRNA of the invention; and/or an intron (intervening sequences).  
         [0219]    Transcription of the siRNA molecule sequences can be driven from a promoter for eukaryotic RNA polymerase I (pol I), RNA polymerase II (pol II), or RNA polymerase III (pol III). Transcripts from pol II or pol III promoters are expressed at high levels in all cells; the levels of a given pol II promoter in a given cell type depends on the nature of the gene regulatory sequences (enhancers, silencers, etc.) present nearby. Prokaryotic RNA polymerase promoters are also used, providing that the prokaryotic RNA polymerase enzyme is expressed in the appropriate cells (Elroy-Stein and Moss, 1990,  Proc. Natl. Acad. Sci. U S A,  87, 6743-7; Gao and Huang 1993,  Nucleic Acids Res.,  21, 2867-72; Lieber et al., 1993,  Methods Enzymol.,  217, 47-66; Zhou et al., 1990,  Mol. Cell. Biol.,  10, 4529-37). Several investigators have demonstrated that nucleic acid molecules expressed from such promoters can function in mammalian cells (e.g. Kashani-Sabet et al., 1992,  Antisense Res. Dev.,  2, 3-15; Ojwang et al., 1992,  Proc. Natl. Acad. Sci. USA,  89, 10802-6; Chen et al., 1992,  Nucleic Acids Res.,  20, 4581-9; Yu et al., 1993,  Proc. Natl. Acad. Sci. U S A,  90, 6340-4; L&#39;Huillier et al., 1992,  EMBO J.,  11, 4411-8; Lisziewicz et al., 1993,  Proc. Natl. Acad. Sci. U. S. A,  90, 8000-4; Thompson et al., 1995,  Nucleic Acids Res.,  23, 2259; Sullenger &amp; Cech, 1993,  Science,  262, 1566). More specifically, transcription units such as the ones derived from genes encoding U6 small nuclear (snRNA), transfer RNA (tRNA) and adenovirus VA RNA are useful in generating high concentrations of desired RNA molecules such as siRNA in cells (Thompson et al., supra; Couture and Stinchcomb, 1996, supra; Noonberg et al., 1994,  Nucleic Acid Res.,  22, 2830; Noonberg et al., U.S. Pat. No. 5,624,803; Good et al., 1997,  Gene Ther.,  4, 45; Beigelman et al., International PCT Publication No. WO 96/18736. The above siRNA transcription units can be incorporated into a variety of vectors for introduction into mammalian cells, including but not restricted to, plasmid DNA vectors, viral DNA vectors (such as adenovirus or adeno-associated virus vectors), or viral RNA vectors (such as retroviral or alphavirus vectors) (for a review see Couture and Stinchcomb, 1996, supra).  
         [0220]    In another aspect the invention features an expression vector comprising a nucleic acid sequence encoding at least one of the siRNA molecules of the invention, in a manner that allows expression of that siRNA molecule. The expression vector comprises in one embodiment; a) a transcription initiation region; b) a transcription termination region; and c) a nucleic acid sequence encoding at least one strand of the siRNA molecule; wherein the sequence is operably linked to the initiation region and the termination region, in a manner that allows expression and/or delivery of the siRNA molecule.  
         [0221]    In another embodiment the expression vector comprises: a) a transcription initiation region; b) a transcription termination region; c) an open reading frame; and d) a nucleic acid sequence encoding at least one strand of a siRNA molecule, wherein the sequence is operably linked to the 3′-end of the open reading frame; and wherein the sequence is operably linked to the initiation region, the open reading frame and the termination region, in a manner that allows expression and/or delivery of the siRNA molecule. In yet another embodiment the expression vector comprises: a) a transcription initiation region; b) a transcription termination region; c) an intron; and d) a nucleic acid sequence encoding at least one siRNA molecule; wherein the sequence is operably linked to the initiation region, the intron and the termination region, in a manner which allows expression and/or delivery of the nucleic acid molecule.  
         [0222]    In another embodiment, the expression vector comprises: a) a transcription initiation region; b) a transcription termination region; c) an intron; d) an open reading frame; and e) a nucleic acid sequence encoding at least one strand of a siRNA molecule, wherein the sequence is operably linked to the 3′-end of the open reading frame; and wherein the sequence is operably linked to the initiation region, the intron, the open reading frame and the termination region, in a manner which allows expression and/or delivery of the siRNA molecule.  
       EXAMPLES  
       [0223]    The following are non-limiting examples showing the selection, isolation, synthesis and activity of nucleic acids of the instant invention.  
       Example 1  
     Tandem Synthesis of siRNA Constructs  
       [0224]    Exemplary siRNA molecules of the invention are synthesized in tandem using a cleavable linker, for example a succinyl-based linker. Tandem synthesis as described herein is followed by a one step purification process that provides RNAi molecules in high yield. This approach is highly amenable to siRNA synthesis in support of high throughput RNAi screening, and can be readily adapted to multi-column or multi-well synthesis platforms.  
         [0225]    After completing a tandem synthesis of an siRNA oligo and its compliment in which the 5′-terminal dimethoxytrityl (5′-O-DMT) group remains intact (trityl on synthesis), the oligonucleotides are deprotected as described above. Following deprotection, the siRNA sequence strands are allowed to spontaneously hybridize. This hybridization yields a duplex in which one strand has retained the 5′-O-DMT group while the complimentary strand comprises a terminal 5′-hydroxyl. The newly formed duplex to behaves as a single molecule during routine solid-phase extraction purification (Trityl-On purification) even though only one molecule has a dimethoxytrityl group. Because the strands form a stable duplex, this dimethoxytrityl group (or an equivalent group, such as other trityl groups or other hydrophobic moieties) is all that is required to purify the pair of oligos, for example by using a C18 cartridge.  
         [0226]    Standard phosphoramidite synthesis chemistry is used up to point of introducing a tandem linker, such as an inverted deoxyabasic succinate linker (see FIG. 1) or an equivalent cleavable linker. A non-limiting example of linker coupling conditions that can be used includes a hindered base such as diisopropylethylamine (DIPA) and/or DMAP in the presence of an activator reagent such as Bromotripyrrolidinophosphoniumhexaflurorophosphate (PyBrOP). After the linker is coupled, standard synthesis chemistry is utilized to complete synthesis of the second sequence leaving the terminal the 5′-O-DMT intact. Following synthesis, the resulting oligonucleotide is deprotected according to the procedures described herein and quenched with a suitable buffer, for example with 50 mM NaOAc or 1.5M NH 4 H 2 CO 3 .  
         [0227]    Purification of the siRNA duplex can be readily accomplished using solid phase extraction, for example using a Waters C18 SepPak 1 g cartridge conditioned with 1 column volume (CV) of acetonitrile, 2 CV H20, and 2 CV 50 mM NaOAc. The sample is loaded and then washed with 1 CV H20 or 50 mM NaOAc. Failure sequences are eluted with 1 CV 14% ACN (Aqueous with 50 mM NaOAc and 50 mM NaCl). The column is then washed, for example with 1 CV H20 followed by on-column detritylation, for example by passing 1 CV of 1% aqueous trifluoroacetic acid (TFA) over the column, then adding a second CV of 1% aqueous TFA to the column and allowing to stand for approx. 10 minutes. The remaining TFA solution is removed and the column washed with H20 followed by 1 CV 1M NaCl and additional H20. The siRNA duplex product is then eluted, for example using 1 CV 20% aqueous CAN.  
         [0228]    [0228]FIG. 2 provides an example of MALDI-TOV mass spectrometry analysis of a purified siRNA construct in which each peak corresponds to the calculated mass of an individual siRNA strand of the siRNA duplex. The same purified siRNA provides three peaks when analyzed by capillary gel electrophoresis (CGE), one peak presumably corresponding to the duplex siRNA, and two peaks presumably corresponding to the separate siRNA sequence strands. Ion exchange HPLC analysis of the same siRNA contract only shows a single peak.  
       Example 2  
     Identification of Potential siRNA Target Sites in any RNA Sequence  
       [0229]    The sequence of an RNA target of interest, such as a HIV-1, is screened for target sites, for example by using a computer folding algorithm. In a non-limiting example, the sequence of gene or RNA gene transcripts derived from a database, such as Genbank Accession numbers shown in Table III, is used to generate siRNA targets having complimentarity to the target. Such sequences can be obtained from a database, or can be determined experimentally as known in the art. Target sites that are known, for example, those target sites determined to be effective target sites based on studies with other nucleic acid molecules, for example ribozymes or antisense, or those targets known to be associated with a disease or condition such as those sites containing mutations or deletions, can be used to design siRNA molecules targeting those sites as well. Various parameters can be used to determine which sites are the most suitable target sites within the target RNA sequence. These parameters include but are not limited to secondary or tertiary RNA structure, the nucleotide base composition of the target sequence, the degree of homology between various regions of the target sequence, or the relative position of the target sequence within the RNA transcript. Based on these determinations, any number of target sites within the RNA transcript can be chosen to screen siRNA molecules for efficacy, for example by using in vitro RNA cleavage assays, cell culture, or animal models. In a non-limiting example, anywhere from 1 to 1000 target sites are chosen within the transcript based on the size of the siRNA contruct to be used. High throughput screening assays can be developed for screening siRNA molecules using methods known in the art, such as with multi-well or multi-plate assays to determine efficient reduction in target gene expression.  
       Example 3  
     Selection of siRNA Molecule Target Sites in a RNA  
       [0230]    The following non-limiting steps can be used to carry out the selection of siRNAs targeting a given gene sequence or transcript, eg HIV-1.  
         [0231]    1. The target sequence is parsed in silico into a list of all fragments or subsequences of a particular length, for example 23 nucleotide fragments, contained within the target sequence. This step is typically carried out using a custom Perl script, but commercial sequence analysis programs such as Oligo, MacVector, or the GCG Wisconsin Package can be employed as well.  
         [0232]    2. In some instances the siRNAs correspond to more than one target sequence; such would be the case for example in targeting many different strains of a viral sequence, for targeting different transcipts of the same gene, targeting different transcipts of more than one gene, or for targeting both the human gene and an animal homolog. In this case, a subsequence list of a particular length is generated for each of the targets, and then the lists are compared to find matching sequences in each list. The subsequences are then ranked according to the number of target sequences that contain the given subsequence; the goal is to find subsequences that are present in most or all of the target sequences. Alternately, the ranking can indentify subsequences that are unique to a target sequence, such as a mutant target sequence. Such an approach would enable the use of siRNA to target specifically the mutant sequence and not effect the expression of the normal sequence.  
         [0233]    3. In some instances the siRNA subsequences are absent in one or more sequences while present in the desired target sequence; such would be the case if the siRNA targets a gene with a paralogous family member that is to remain untargeted. As in case 2 above, a subsequence list of a particular length is generated for each of the targets, and then the lists are compared to find sequences that are present in the target gene but are absent in the untargeted paralog.  
         [0234]    4. The ranked siRNA subsequences can be further analyzed and ranked according to GC content. A preference can be given to sites containing 30-70% GC, with a further preference to sites containing 40-60% GC.  
         [0235]    5. The ranked siRNA subsequences can be further analyzed and ranked according to self-folding and internal hairpins. Weaker internal folds are preferred; strong hairpin structures are to be avoided.  
         [0236]    6. The ranked siRNA subsequences can be further analyzed and ranked according to whether they have runs of GGG or CCC in the sequence. GGG (or even more Gs) in either strand can make oligonucleotide synthesis problematic, so it is avoided whenever better sequences are available. CCC is searched in the target strand because that will place GGG in the antisense strand.  
         [0237]    7. The ranked siRNA subsequences can be further analyzed and ranked according to whether they have the dinucleotide UU (uridine dinucleotide) on the 3′ end of the sequence, and/or AA on the 5′ end of the sequence (to yield 3′ UU on the antisense sequence). These sequences allow one to design siRNA molecules with terminal TT thymidine dinucleotides.  
         [0238]    8. Four or five target sites are chosen from the ranked list of subsequences as described above. For example, in subsequences having 23 nucleotides, the right 21 nucleotides of each chosen 23-mer subsequence are then designed and synthesized for the upper (sense) strand of the siRNA duplex, while the reverse complement of the left 21 nucleotides of each chosen 23-mer subsequence are then designed and synthesized for the lower (antisense) strand of the siRNA duplex. If terminal TT residues are desired for the sequence (as described in paragraph 7), then the two 3′ terminal nucleotides of both the sense and antisense strands are replaced by TT prior to synthesizing the oligos.  
         [0239]    9. The siRNA molecules are screened in an in vitro, cell culture or animal model system to identify the most active siRNA molecule or the most preferred target site within the target RNA sequence.  
         [0240]    In an alternate approach, a pool of siRNA constructs specific to a HIV target sequence is used to screen for target sites in cells expressing HIV RNA. The general strategy used in this approach is shown in FIG. 9. A non-limiting example of such as pool is a pool comprising sequences having sense sequences comprising SEQ ID NOs. 1-738 and antisense sequences comprising SEQ ID NOs. 739-1476 respectively. Cells expressing HIV are transfected with the pool of siRNA constructs and cells that demonstrate a phenotype associated with HIV inhibition are sorted. The pool of siRNA constructs can be expressed from transcription cassettes inserted into appropriate vectors (see for example FIG. 7 and FIG. 8). Cells in which HIV expression is decreased due to siRNA treatment demonstrate a phenotypic change, for example decreased production of HIV RNA or HIV protein(s) compared to untreated cells or cells treated with a control siRNA. The siRNA from cells demonstrating a positive phenotypic change (e.g., decreased HIV RNA or protein), are sequenced to determine the most suitable target site(s) within the target HIV RNA sequence.  
       Example 4  
     HIV Targeted siRNA Design  
       [0241]    siRNA target sites were chosen by analyzing sequences of the HIV-1 RNA target (for example Genbank Accession Nos. shown in Table III) and optionally prioritizing the target sites on the basis of folding (structure of any given sequence analyzed to determine siRNA accessibility to the target). The sequence alignments of all known A and B strains of HIV were screened for homology and siRNA molecules were designed to target conserved sequences across these strains since the A and B strains are currently the most prevalent strains. Alternately, all known strains or other subclasses of HIV can be similarly screened for homology (see Table IV) and homologous sequences used as targets. A cutoff for % homology between the different strains can be used to increase or decrease the number of targets considered, for example 70%, 75%, 80%, 85%, 90% or 95% homology. The sequences shown in Table I represent 80% homology between the HIV strains shown in Table III. siRNA molecules were designed that could bind each target sequence and are optionally individually analyzed by computer folding to assess whether the siRNA molecule can interact with the target sequence. Varying the length of the siRNA molecules can be chosen to optimize activity. The siRNA sense (upper sequence) and antisense (lower sequence) sequences shown in Table I comprise 19 nucleotides in length, with the sense strand comprising the same sequence as the target sequence and the antisense strand comprising a complimentary sequence to the sense/target sequence. The sense and antisense strands can further comprise nucleotide 3′-overhangs as described herein, preferably the overhangs comprise about 2 nucleotides which can optionally be complimentary to the target sequence in the antisense siRNA strand, and/or optionally analogous to the adjacent nucleotides in the target sequence when present in the sense siRNA strand. Generally, a sufficient number of complimentary nucleotide bases are chosen to bind to, or otherwise interact with, the target RNA, but the degree of complementarity can be modulated to accommodate siRNA duplexes or varying length or base composition. By using such methodologies, siRNA molecules can be designed to target sites within any known RNA sequence, for example those RNA sequences corresponding to the any gene transcript.  
       Example 5  
     Chemical Synthesis and Purification of siRNA  
       [0242]    siRNA molecules can be designed to interact with various sites in the RNA message, for example target sequences within the RNA sequences described herein. The sequence of one strand of the siRNA molecule(s) are complementary to the target site sequences described above. The siRNA molecules can be chemically synthesized using methods described herein. Inactive siRNA molecules that are used as control sequences can be synthesized by scrambling the sequence of the siRNA molecules such that it is not complimentary to the target sequence.  
       Example 6  
     RNAi in vitro Assay to Assess siRNA Activity  
       [0243]    An in vitro assay that recapitulates RNAi in a cell free system is used to evaluate siRNA constructs targeting HIV RNA targets. The assay comprises the system described by Tuschl et al., 1999,  Genes and Development,  13, 3191-3197 and Zamore et al., 2000,  Cell,  101, 25-33 adapted for use with HIV target RNA. A Drosophila extract derived from syncytial blastoderm is used to reconstitute RNAi activity in vitro. Target RNA is generated via in vitro transcription from an appropriate HIV expressing plasmid using T7 RNA polymerase. The target RNA can also be synthesized chemically as described herein. Sense and antisense siRNA strands (for example 20 uM each) are annealed by incubation in buffer (such as 100 mM potassium acetate, 30 mM HEPES-KOH, pH 7.4, 2 mM magnesium acetate) for 1 min. at 90° C. followed by 1 hour at 37° C., then diluted in lysis buffer (for example 100 mM potassium acetate, 30 mM HEPES-KOH at pH 7.4, 2 mM magnesium acetate). Annealing can be monitored by gel electrophoresis on an agarose gel in TBE buffer and stained with ethidium bromide. The Drosophila lysate is prepared using zero to two hour old embryos from Oregon R flies collected on yeasted molasses agar that are dechorionated and lysed. The lysate is centrifuged and the supernatant isolated. The assay comprises a reaction mixture containing 50% lysate [vol/vol], RNA (10-50 pM final concentration), and 10% [vol/vol] lysis buffer containing siRNA (10 nM final concentration). The reaction mixture also contains 10 mM creatine phosphate, 10 ug.ml creatine phosphokinase, 100 um GTP, 100 uM UTP, 100 uM CTP, 500 uM ATP, 5 mM DTT, 0.1 U/uL RNasin (Promega), and 100 uM of each amino acid. The final concentration of potassium acetate is adjusted to 100 mM. The reactions are pre-assembled on ice and preincubated at 25° C. for 10 minutes before adding RNA, then incubated at 25° C. for an additional 60 minutes. Reactions are quenched with 4 volumes of 1.25×Passive Lysis Buffer (Promega). Target RNA cleavage is assayed by RT-PCR analysis or other methods known in the art and are compared to control reactions in which siRNA is omitted from the reaction.  
         [0244]    Alternately, internally-labeled target RNA for the assay is prepared by in vitro transcription in the presence of [a- 32 P] CTP, passed over a G 50 Sephadex column by spin chromatography and used as target RNA without further purification. Optionally, target RNA is 5′- 32 P-end labeled using T4 polynucleotide kinase enzyme. Assays are performed as described above and target RNA and the specific RNA cleavage products generated by RNAi are visualized on an autoradiograph of a gel. The percentage of cleavage is determined by Phosphor Imager® quantitation of bands representing intact control RNA or RNA from control reactions without siRNA and the cleavage products generated by the assay.  
       Example 7  
     Cell Culture  
       [0245]    The siRNA constructs of the invention can be used in various cell culture systems as are commonly known in the art to screen for compounds having anti-HIV activity. B cell, T cell, macrophage and endothelial cell culture systems are non-limiting examples of cell culture systems that can be readily adapted for screening siRNA molecules of the invention. In a non-limiting example, siRNA molecules of the invention are co-transfected with HIV-1 pNL4-3 proviral DNA into 293/EcR cells as described by Lee et al., 2002,  Nature Biotechnology,  19, 500-505, using a U6 snRNA promoter driven expression system.  
         [0246]    In a non-limiting example, the siRNA expression vectors are prepared using the pTZ U6+1 vector described in Lee et al. supra. as follows. One cassette harbors the 21-nucleotide sense sequences and the other a 21-nucleotide antisense sequence (Table I). These sequences are designed to target HIV-1 RNA targets described herein. As a control to verify a siRNA mechanism, irrelevant sense and antisense (S/AS) sequences lacking complementarity to HIV-1 (S/AS (IR)) are subcloned in pTZ U6+1. RNA samples are prepared from 293/EcR cells transiently co-transfected with siRNA or control constructs, and subjected to Ponasterone A induction. RNAs are also prepared from 293 cells co-transfected with HIV-1 pNL4-3 proviral DNA and siRNA or control constructs. For determination of anti-HIV-1 activity of the siRNAs, transient assays are done by co-transfection of siRNA constructs and infectious HIV-1 proviral DNA, pNL4-3 into 293 cells as described above, followed by Northern analysis as known in the art. The p24 values are calculated with the aid of, for example, a Dynatech MR5000 ELISA plate reader (Dynatech Labs Inc., Chantilly, Va.). Cell viability can also be assessed using a Trypan Blue dye exclusion count at four days after transfection.  
         [0247]    Other cell culture model systems are generally known in the art, see for example Duzgunes et al., 2001, Nucleosides,  Nucleotides &amp; Nucleic Acids,  20(4-7), 515-523; Cagnun et al., 2000,  Antisense Nucleic Acid Drug Dev.,  10, 251; Ho et al., 1995,  Stem Cells,  13 supp 3, 100; and Baur et al., 1997,  Blood,  89, 2259. These cell culture systems can be readily adapted for use with the compositions of the instant invention.  
         [0248]    Animal Models  
         [0249]    The siRNA constructs of the invention can be evaluated in a variety of animal models, including for example a hollow fiber HIV model (see for example Gruenberg, U.S. Pat. No. 5,627,070), mouse models for AIDS using transgenic mice expressing HIV-1 genes from CD4 promoters and enhancers (see for example Jolicoeur, International PCT Publication No. WO 98/50535) and/or the HIV/SIV/SHIV non-human primate models (see for example Narayan, U.S. Pat. No. 5,849,994). The siRNA compounds and virus can be administered by a variety of methods and routes as described herein and as known in the art. Quantitation of results in these models can be performed by a variety of methods, including quantitative PCR, quantitative and bulk co-cultivation assays, plasma co-cultivation assays, antigen and antibody detection assays, lymphocyte proliferation, intracellular cytokines, flow cytometry, as well as hematology and CBC evaluation. Additional animal models are generally known in the art, see for example Bai et al., 2000,  Mol. Ther.,  1, 244.  
         [0250]    Indications  
         [0251]    Particular degenerative and disease states that can be associated with HIV expression modulation include but are not limited to acquired immunodeficiency disease (AIDS) and related diseases and conditions, including but not limited to Kaposi&#39;s sarcoma, lymphoma, cervical cancer, squamous cell carcinoma, cardiac myopathy, rheumatic diseases, and opportunistic infection, for example  Pneumocystis carinii,  Cytomegalovirus, Herpes simplex, Mycobacteria, Cryptococcus, Toxoplasma, Progressive multifocal leuco-encephalopathy (Papovavirus), Mycobacteria, Aspergillus, Cryptococcus, Candida, Cryptosporidium,  Isospora belli,  Microsporidia and any other diseases or conditions that are related to or will respond to the levels of HIV in a cell or tissue, alone or in combination with other therapies  
         [0252]    The present body of knowledge in HIV research indicates the need for methods to assay HIV activity and for compounds that can regulate HIV expression for research, diagnostic, and therapeutic use.  
         [0253]    The use of antiviral compounds, monoclonal antibodies, chemotherapy, radiation therapy, analgesics, and/or anti-inflammatory compounds, are all non-limiting examples of a methods that can be combined with or used in conjunction with the nucleic acid molecules (e.g. ribozymes and antisense molecules) of the instant invention. Examples of antiviral compounds that can be used in conjunction with the nucleic acid molecules of the invention include but are not limited to AZT (also known as zidovudine or ZDV), ddC (zalcitabine), ddI (dideoxyinosine), d4T (stavudine), and 3TC (lamivudine) Ribavirin, delvaridine (Rescriptor), nevirapine (Viramune), efravirenz (Sustiva), ritonavir (Norvir), saquinivir (Invirase), indinavir (Crixivan), amprenivir (Agenerase), nelfinavir (Viracept), and/or lopinavir (Kaletra). Common chemotherapies that can be combined with nucleic acid molecules of the instant invention include various combinations of cytotoxic drugs to kill cancer cells. These drugs include but are not limited to paclitaxel (Taxol), docetaxel, cisplatin, methotrexate, cyclophosphamide, doxorubin, fluorouracil carboplatin, edatrexate, gemcitabine, vinorelbine etc. Those skilled in the art will recognize that other drug compounds and therapies can be similarly be readily combined with the nucleic acid molecules of the instant invention (e.g. ribozymes, siRNA and antisense molecules) are hence within the scope of the instant invention.  
         [0254]    Diagnostic Uses  
         [0255]    The siRNA molecules of the invention can be used in a variety of diagnostic applications, such as in identifying molecular targets such as RNA in a variety of applications, for example, in clinical, industrial, environmental, agricultural and/or research settings. Such diagnostic use of siRNA molecules involves utilizing reconstituted RNAi systems, for example using cellular lysates or partially purified cellular lysates. siRNA molecules of this invention can be used as diagnostic tools to examine genetic drift and mutations within diseased cells or to detect the presence of endogenous or exogenous, for example viral, RNA in a cell. The close relationship between siRNA activity and the structure of the target RNA allows the detection of mutations in any region of the molecule, which alters the base-pairing and three-dimensional structure of the target RNA. By using multiple siRNA molecules described in this invention, one can map nucleotide changes, which are important to RNA structure and function in vitro, as well as in cells and tissues. Cleavage of target RNAs with siRNA molecules can be used to inhibit gene expression and define the role (essentially) of specified gene products in the progression of disease or infection. In this manner, other genetic targets can be defined as important mediators of the disease. These experiments will lead to better treatment of the disease progression by affording the possibility of combination therapies (e.g., multiple siRNA molecules targeted to different genes, siRNA molecules coupled with known small molecule inhibitors, or intermittent treatment with combinations siRNA molecules and/or other chemical or biological molecules). Other in vitro uses of siRNA molecules of this invention are well known in the art, and include detection of the presence of mRNAs associated with a disease, infection, or related condition. Such RNA is detected by determining the presence of a cleavage product after treatment with a siRNA using standard methodologies, for example fluorescence resonance emission transfer (FRET).  
         [0256]    In a specific example, siRNA molecules that can cleave only wild-type or mutant forms of the target RNA are used for the assay. The first siRNA molecules is used to identify wild-type RNA present in the sample and the second siRNA molecules will be used to identify mutant RNA in the sample. As reaction controls, synthetic substrates of both wild-type and mutant RNA will be cleaved by both siRNA molecules to demonstrate the relative siRNA efficiencies in the reactions and the absence of cleavage of the “non-targeted” RNA species. The cleavage products from the synthetic substrates will also serve to generate size markers for the analysis of wild-type and mutant RNAs in the sample population. Thus each analysis will require two siRNA molecules, two substrates and one unknown sample which will be combined into six reactions. The presence of cleavage products will be determined using an RNase protection assay so that full-length and cleavage fragments of each RNA can be analyzed in one lane of a polyacrylamide gel. It is not absolutely required to quantify the results to gain insight into the expression of mutant RNAs and putative risk of the desired phenotypic changes in target cells. The expression of mRNA whose protein product is implicated in the development of the phenotype (i.e., disease related or infection related) is adequate to establish risk. If probes of comparable specific activity are used for both transcripts, then a qualitative comparison of RNA levels will be adequate and will decrease the cost of the initial diagnosis. Higher mutant form to wild-type ratios will be correlated with higher risk whether RNA levels are compared qualitatively or quantitatively.  
         [0257]    All patents and publications mentioned in the specification are indicative of the levels of skill of those skilled in the art to which the invention pertains. All references cited in this disclosure are incorporated by reference to the same extent as if each reference had been incorporated by reference in its entirety individually.  
         [0258]    One skilled in the art would readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The methods and compositions described herein as presently representative of preferred embodiments are exemplary and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art, which are encompassed within the spirit of the invention, are defined by the scope of the claims.  
         [0259]    It will be readily apparent to one skilled in the art that varying substitutions and modifications can be made to the invention disclosed herein without departing from the scope and spirit of the invention. Thus, such additional embodiments are within the scope of the present invention and the following claims.  
         [0260]    The invention illustratively described herein suitably can be practiced in the absence of any element or elements, limitation or limitations that are not specifically disclosed herein. Thus, for example, in each instance herein any of the terms “comprising”, “consisting essentially of” and “consisting of” may be replaced with either of the other two terms. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments, optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the description and the appended claims.  
         [0261]    In addition, where features or aspects of the invention are described in terms of Markush groups or other grouping of alternatives, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group or other group.  
                                                                               TABLE I                           HIV target and siRNA sequences                    Seq       Seq       Seq           Sequence   ID   Upper seq   ID   Lower seq   ID                    UUUGGAAAGGACCAGCAAA   1   UUUGGAAAGGACCAGCAAA   1   UUUGCUGGUCCUUUCCAAA   739                   CAGGAGCAGAUGAUACAGU   2   CAGGAGCAGAUGAUACAGU   2   ACUGUAUCAUCUGCUCCUG   740               AGAAAAGGGGGGAUUGGGG   3   AGAAAAGGGGGGAUUGGGG   3   CCCCAAUCCCCCCUUUUCU   741               GUAGACAGGAUGAGGAUUA   4   GUAGACAGGAUGAGGAUUA   4   UAAUCCUCAUCCUGUCUAC   742               ACAGGAGCAGAUGAUACAG   5   ACAGGAGCAGAUGAUACAG   5   CUGUAUCAUCUGCUCCUGU   743               GAAAAGGGGGGAUUGGGGG   6   GAAAAGGGGGGAUUGGGGG   6   CCCCCAAUCCCCCCUUUUC   744               UUAGAUACAGGAGCAGAUG   7   UUAGAUACAGGAGCAGAUG   7   CAUCUGCUCCUGUAUCUAA   745               UAGAUACAGGAGCAGAUGA   8   UAGAUACAGGAGCAGAUGA   8   UCAUCUGCUCCUGUAUCUA   746               AGCAGAAGACAGUGGCAAU   9   AGCAGAAGACAGUGGCAAU   9   AUUGCCACUGUCUUCUGCU   747               AUUAGAUACAGGAGCAGAU   10   AUUAGAUACAGGAGCAGAU   10   AUCUGCUCCUGUAUCUAAU   748               AUACAGGAGCAGAUGAUAC   11   AUACAGGAGCAGAUGAUAC   11   GUAUCAUCUGCUCCUGUAU   749               GAGCAGAAGACAGUGGCAA   12   GAGCAGAAGACAGUGGCAA   12   UUGCCACUGUCUUCUGCUC   750               AGAGCAGAAGACAGUGGCA   13   AGAGCAGAAGACAGUGGCA   13   UGCCACUGUCUUCUGCUCU   751               GCAGAAGACAGUGGCAAUG   14   GCAGAAGACAGUGGCAAUG   14   CAUUGCCACUGUCUUCUGC   752               AGAUACAGGAGCAGAUGAU   15   AGAUACAGGAGCAGAUGAU   15   AUCAUCUGCUCCUGUAUCU   753               UACAGGAGCAGAUGAUACA   16   UACAGGAGCAGAUGAUACA   16   UGUAUCAUCUGCUCCUGUA   754               UAUUAGAUACAGGAGCAGA   17   UAUUAGAUACAGGAGCAGA   17   UCUGCUCCUGUAUCUAAUA   755               GAUACAGGAGCAGAUGAUA   18   GAUACAGGAGCAGAUGAUA   18   UAUCAUCUGCUCCUGUAUC   756               AUGGAAAACAGAUGGCAGG   19   AUGGAAAACAGAUGGCAGG   19   CCUGCCAUCUGUUUUCCAU   757               GUCAACAUAAUUGGAAGAA   20   GUCAACAUAAUUGGAAGAA   20   UUCUUCCAAUUAUGUUGAC   758               UAUGGAAAACAGAUGGCAG   21   UAUGGAAAACAGAUGGCAG   21   CUGCCAUCUGUUUUCCAUA   759               AUGAUAGGGGGAAUUGGAG   22   AUGAUAGGGGGAAUUGGAG   22   CUCCAAUUCCCCCUAUCAU   760               CAGAAGACAGUGGCAAUGA   23   CAGAAGACAGUGGCAAUGA   23   UCAUUGCCACUGUCUUCUG   761               CAAUGGCCAUUGACAGAAG   24   CAAUGGCCAUUGACAGAAG   24   CUUCUGUCAAUGGCCAUUG   762               UCAACAUAAUUGGAAGAAA   25   UCAACAUAAUUGGAAGAAA   25   UUUCUUCCAAUUAUGUUGA   763               AAUGGCCAUUGACAGAAGA   26   AAUGGCCAUUGACAGAAGA   26   UCUUCUGUCAAUGGCCAUU   764               UGAUAGGGGGAAUUGGAGG   27   UGAUAGGGGGAAUUGGAGG   27   CCUCCAAUUCCCCCUAUCA   765               GACAGGCUAAUUUUUUAGG   28   GACAGGCUAAUUUUUUAGG   28   CCUAAAAAAUUAGCCUGUC   766               AUUUUCGGGUUUAUUACAG   29   AUUUUCGGGUUUAUUACAG   29   CUGUAAUAAACCCGAAAAU   767               CUAUUAGAUACAGGAGCAG   30   CUAUUAGAUACAGGAGCAG   30   CUGCUCCUGUAUCUAAUAG   768               AGACAGGCUAAUUUUUUAG   31   AGACAGGCUAAUUUUUUAG   31   CUAAAAAAUUAGCCUGUCU   769               AAAUGAUAGGGGGAAUUGG   32   AAAUGAUAGGGGGAAUUGG   32   CCAAUUCCCCCUAUCAUUU   770               UAUGGGCAAGCAGGGAGCU   33   UAUGGGCAAGCAGGGAGCU   33   AGCUCCCUGCUUGCCCAUA   771               UAGUAUGGGCAAGCAGGGA   34   UAGUAUGGGCAAGCAGGGA   34   UCCCUGCUUGCCCAUACUA   772               GAAAACAGAUGGCAGGUGA   35   GAAAACAGAUGGCAGGUGA   35   UCACCUGCCAUCUGUUUUC   773               ACCAUCAAUGAGGAAGCUG   36   ACCAUCAAUGAGGAAGCUG   36   CAGCUUCCUCAUUGAUGGU   774               AAUGAUAGGGGGAAUUGGA   37   AAUGAUAGGGGGAAUUGGA   37   UCCAAUUCCCCCUAUCAUU   775               UGGAAAACAGAUGGCAGGU   38   UGGAAAACAGAUGGCAGGU   38   ACCUGCCAUCUGUUUUCCA   776               GGAAAACAGAUGGCAGGUG   39   GGAAAACAGAUGGCAGGUG   39   CACCUGCCAUCUGUUUUCC   777               GAUUAUGGAAAACAGAUGG   40   GAUUAUGGAAAACAGAUGG   40   CCAUCUGUUUUCCAUAAUC   778               AAAAUGAUAGGGGGAAUUG   41   AAAAUGAUAGGGGGAAUUG   41   CAAUUCCCCCUAUCAUUUU   779               UGGAAAGGUGAAGGGGCAG   42   UGGAAAGGUGAAGGGGCAG   42   CUGCCCCUUCACCUUUCCA   780               AUCAAUGAGGAAGCUGCAG   43   AUCAAUGAGGAAGCUGCAG   43   CUGCAGCUUCCUCAUUGAU   781               UGGAAACCAAAAAUGAUAG   44   UGGAAACCAAAAAUGAUAG   44   CUAUCAUUUUUGGUUUCCA   782               CCAUCAAUGAGGAAGCUGC   45   CCAUCAAUGAGGAAGCUGC   45   GCAGCUUCCUCAUUGAUGG   783               AGGGAUUAUGGAAAACAGA   46   AGGGAUUAUGGAAAACAGA   46   UCUGUUUUCCAUAAUCCCU   784               GGAAACCAAAAAUGAUAGG   47   GGAAACCAAAAAUGAUAGG   47   CCUAUCAUUUUUGGUUUCC   785               UAGGGGGAAUUGGAGGUUU   48   UAGGGGGAAUUGGAGGUUU   48   AAACCUCCAAUUCCCCCUA   786               UACAGUGCAGGGGAAAGAA   49   UACAGUGCAGGGGAAAGAA   49   UUCUUUCCCCUGCACUGUA   787               CUCUAUUAGAUACAGGAGC   50   CUCUAUUAGAUACAGGAGC   50   GCUCCUGUAUCUAAUAGAG   788               GGAUUAUGGAAAACAGAUG   51   GGAUUAUGGAAAACAGAUG   51   CAUCUGUUUUCCAUAAUCC   789               CCAAAAAUGAUAGGGGGAA   52   CCAAAAAUGAUAGGGGGAA   52   UUCCCCCUAUCAUUUUUGG   790               AUGGAAACCAAAAAUGAUA   53   AUGGAAACCAAAAAUGAUA   53   UAUCAUUUUUGGUUUCCAU   791               CAGUGCAGGGGAAAGAAUA   54   CAGUGCAGGGGAAAGAAUA   54   UAUUCUUUCCCCUGCACUG   792               ACAAUGGCCAUUGACAGAA   55   ACAAUGGCCAUUGACAGAA   55   UUCUGUCAAUGGCCAUUGU   793               CCAUGCAUGGACAAGUAGA   56   CCAUGCAUGGACAAGUAGA   56   UCUACUUGUCCAUGCAUGG   794               AUUAUGGAAAACAGAUGGC   57   AUUAUGGAAAACAGAUGGC   57   GCCAUCUGUUUUCCAUAAU   795               AACAAUGGCCAUUGACAGA   58   AACAAUGGCCAUUGACAGA   58   UCUGUCAAUGGCCAUUGUU   796               AAAAAUGAUAGGGGGAAUU   59   AAAAAUGAUAGGGGGAAUU   59   AAUUCCCCCUAUCAUUUUU   797               GCCAUGCAUGGACAAGUAG   60   GCCAUGCAUGGACAAGUAG   60   CUACUUGUCCAUGCAUGGC   798               UAGCAGGAAGAUGGCCAGU   61   UAGCAGGAAGAUGGCCAGU   61   ACUGGCCAUCUUCCUGCUA   799               CAAAAAUGAUAGGGGGAAU   62   CAAAAAUGAUAGGGGGAAU   62   AUUCCCCCUAUCAUUUUUG   800               AAGAAAUGAUGACAGCAUG   63   AAGAAAUGAUGACAGCAUG   63   CAUGCUGUCAUCAUUUCUU   801               UCUAUUAGAUACAGGAGCA   64   UCUAUUAGAUACAGGAGCA   64   UGCUCCUGUAUCUAAUAGA   802               GCUCUAUUAGAUACAGGAG   65   GCUCUAUUAGAUACAGGAG   65   CUCCUGUAUCUAAUAGAGC   803               CAGGCUAAUUUUUUAGGGA   66   CAGGCUAAUUUUUUAGGGA   66   UCCCUAAAAAAUUAGCCUG   804               AGGAGCAGAUGAUACAGUA   67   AGGAGCAGAUGAUACAGUA   67   UACUGUAUCAUCUGCUCCU   805               AAACAAUGGCCAUUGACAG   68   AAACAAUGGCCAUUGACAG   68   CUGUCAAUGGCCAUUGUUU   806               CGGGUUUAUUACAGGGACA   69   CGGGUUUAUUACAGGGACA   69   UGUCCCUGUAAUAAACCCG   807               CAACAUAAUUGGAAGAAAU   70   CAACAUAAUUGGAAGAAAU   70   AUUUCUUCCAAUUAUGUUG   808               UCAAUGAGGAAGCUGCAGA   71   UCAAUGAGGAAGCUGCAGA   71   UCUGCAGCUUCCUCAUUGA   809               GGAAAGGUGAAGGGGCAGU   72   GGAAAGGUGAAGGGGCAGU   72   ACUGCCCCUUCACCUUUCC   810               UUUCGGGUUUAUUACAGGG   73   UUUCGGGUUUAUUACAGGG   73   CCCUGUAAUAAACCCGAAA   811               UCGGGUUUAUUACAGGGAC   74   UCGGGUUUAUUACAGGGAC   74   GUCCCUGUAAUAAACCCGA   812               ACAGUGCAGGGGAAAGAAU   75   ACAGUGCAGGGGAAAGAAU   75   AUUCUUUCCCCUGCACUGU   813               AUGCAUGGACAAGUAGACU   76   AUGCAUGGACAAGUAGACU   76   AGUCUACUUGUCCAUGCAU   814               AAGCCAUGCAUGGACAAGU   77   AAGCCAUGCAUGGACAAGU   77   ACUUGUCCAUGCAUGGCUU   815               AGCCAUGCAUGGACAAGUA   78   AGCCAUGCAUGGACAAGUA   78   UACUUGUCCAUGCAUGGCU   816               GCAUUAUCAGAAGGAGCCA   79   GCAUUAUCAGAAGGAGCCA   79   UGGCUCCUUCUGAUAAUGC   817               AAUUGGAGAAGUGAAUUAU   80   AAUUGGAGAAGUGAAUUAU   80   AUAAUUCACUUCUCCAAUU   818               AGAAAAAAUCAGUAACAGU   81   AGAAAAAAUCAGUAACAGU   81   ACUGUUACUGAUUUUUUCU   819               GAAGCCAUGCAUGGACAAG   82   GAAGCCAUGCAUGGACAAG   82   CUUGUCCAUGCAUGGCUUC   820               ACAGGCUAAUUUUUUAGGG   83   ACAGGCUAAUUUUUUAGGG   83   CCCUAAAAAAUUAGCCUGU   821               GAAGAAAUGAUGACAGCAU   84   GAAGAAAUGAUGACAGCAU   84   AUGCUGUCAUCAUUUCUUC   822               UUUUCGGGUUUAUUACAGG   85   UUUUCGGGUUUAUUACAGG   85   CCUGUAAUAAACCCGAAAA   823               ACCAAAAAUGAUAGGGGGA   86   ACCAAAAAUGAUAGGGGGA   86   UCCCCCUAUCAUUUUUGGU   824               GAAGUGACAUAGCAGGAAC   87   GAAGUGACAUAGCAGGAAC   87   GUUCCUGCUAUGUCACUUC   825               UUCGGGUUUAUUACAGGGA   88   UUCGGGUUUAUUACAGGGA   88   UCCCUGUAAUAAACCCGAA   826               AUAGGGGGAAUUGGAGGUU   89   AUAGGGGGAAUUGGAGGUU   89   AACCUCCAAUUCCCCCUAU   827               AGAAGAAAUGAUGACAGCA   90   AGAAGAAAUGAUGACAGCA   90   UGCUGUCAUCAUUUCUUCU   828               AUUGGAGAAGUGAAUUAUA   91   AUUGGAGAAGUGAAUUAUA   91   UAUAAUUCACUUCUCCAAU   829               GGAAGUGACAUAGCAGGAA   92   GGAAGUGACAUAGCAGGAA   92   UUCCUGCUAUGUCACUUCC   830               AGGCUAAUUUUUUAGGGAA   93   AGGCUAAUUUUUUAGGGAA   93   UUCCCUAAAAAAUUAGCCU   831               UUAUGGAAAACAGAUGGCA   94   UUAUGGAAAACAGAUGGCA   94   UGCCAUCUGUUUUCCAUAA   832               GGGAUUAUGGAAAACAGAU   95   GGGAUUAUGGAAAACAGAU   95   AUCUGUUUUCCAUAAUCCC   833               UAGAAGAAAUGAUGACAGC   96   UAGAAGAAAUGAUGACAGC   96   GCUGUCAUCAUUUCUUCUA   834               AGCUCUAUUAGAUACAGGA   97   AGCUCUAUUAGAUACAGGA   97   UCCUGUAUCUAAUAGAGCU   835               GUAUGGGCAAGCAGGGAGC   98   GUAUGGGCAAGCAGGGAGC   98   GCUCCCUGCUUGCCCAUAC   836               CUUAGGCAUCUCCUAUGGC   99   CUUAGGCAUCUCCUAUGGC   99   GCCAUAGGAGAUGCCUAAG   837               GCAGGAACUACUAGUACCC   100   GCAGGAACUACUAGUACCC   100   GGGUACUAGUAGUUCCUGC   838               GGGGAAGUGACAUAGCAGG   101   GGGGAAGUGACAUAGCAGG   101   CCUGCUAUGUCACUUCCCC   839               UACAAUCCCCAAAGUCAAG   102   UACAAUCCCCAAAGUCAAG   102   CUUGACUUUGGGGAUUGUA   840               UUCCCUACAAUCCCCAAAG   103   UUCCCUACAAUCCCCAAAG   103   CUUUGGGGAUUGUAGGGAA   841               AAGCUCUAUUAGAUACAGG   104   AAGCUCUAUUAGAUACAGG   104   CCUGUAUCUAAUAGAGCUU   842               CCUAUGGCAGGAAGAAGCG   105   CCUAUGGCAGGAAGAAGCG   105   CGCUUCUUCCUGCCAUAGG   843               AGGGGAAGUGACAUAGCAG   106   AGGGGAAGUGACAUAGCAG   106   CUGCUAUGUCACUUCCCCU   844               UCCUAUGGCAGGAAGAAGC   107   UCCUAUGGCAGGAAGAAGC   107   GCUUCUUCCUGCCAUAGGA   845               CAGCAUUAUCAGAAGGAGC   108   CAGCAUUAUCAGAAGGAGC   108   GCUCCUUCUGAUAAUGCUG   846               AUCUCCUAUGGCAGGAAGA   109   AUCUCCUAUGGCAGGAAGA   109   UCUUCCUGCCAUAGGAGAU   847               AGCAGGAACUACUAGUACC   110   AGCAGGAACUACUAGUACC   110   GGUACUAGUAGUUCCUGCU   848               GAAACCAAAAAUGAUAGGG   111   GAAACCAAAAAUGAUAGGG   111   CCCUAUCAUUUUUGGUUUC   849               AAACCAAAAAUGAUAGGGG   112   AAACCAAAAAUGAUAGGGG   112   CCCCUAUCAUUUUUGGUUU   850               CAGAAGGAGCCACCCCACA   113   CAGAAGGAGCCACCCCACA   113   UGUGGGGUGGCUCCUUCUG   851               UAGCAGGAACUACUAGUAC   114   UAGCAGGAACUACUAGUAC   114   GUACUAGUAGUUCCUGCUA   852               UGCAUGGACAAGUAGACUG   115   UGCAUGGACAAGUAGACUG   115   CAGUCUACUUGUCCAUGCA   853               UUAGGCAUCUCCUAUGGCA   116   UUAGGCAUCUCCUAUGGCA   116   UGCCAUAGGAGAUGCCUAA   854               UAUGGCAGGAAGAAGCGGA   117   UAUGGCAGGAAGAAGCGGA   117   UCCGCUUCUUCCUGCCAUA   855               AUAGCAGGAACUACUAGUA   118   AUAGCAGGAACUACUAGUA   118   UACUAGUAGUUCCUGCUAU   856               UAGACAUAAUAGCAACAGA   119   UAGACAUAAUAGCAACAGA   119   UCUGUUGCUAUUAUGUCUA   857               CAUUAUCAGAAGGAGCCAC   120   CAUUAUCAGAAGGAGCCAC   120   GUGGCUCCUUCUGAUAAUG   858               CUAUGGCAGGAAGAAGCGG   121   CUAUGGCAGGAAGAAGCGG   121   CCGCUUCUUCCUGCCAUAG   859               GAUAGGGGGAAUUGGAGGU   122   GAUAGGGGGAAUUGGAGGU   122   ACCUCCAAUUCCCCCUAUC   860               ACAAUCCCCAAAGUCAAGG   123   ACAAUCCCCAAAGUCAAGG   123   CCUUGACUUUGGGGAUUGU   861               AUUCCCUACAAUCCCCAAA   124   AUUCCCUACAAUCCCCAAA   124   UUUGGGGAUUGUAGGGAAU   862               AACCAAAAAUGAUAGGGGG   125   AACCAAAAAUGAUAGGGGG   125   CCCCCUAUCAUUUUUGGUU   863               UCUCCUAUGGCAGGAAGAA   126   UCUCCUAUGGCAGGAAGAA   126   UUCUUCCUGCCAUAGGAGA   864               CAUGCAUGGACAAGUAGAC   127   CAUGCAUGGACAAGUAGAC   127   GUCUACUUGUCCAUGCAUG   865               CCUGUGUACCCACAGACCC   128   CCUGUGUACCCACAGACCC   128   GGGUCUGUGGGUACACAGG   866               CAUCAAUGAGGAAGCUGCA   129   CAUCAAUGAGGAAGCUGCA   129   UGCAGCUUCCUCAUUGAUG   867               GACAUAGCAGGAACUACUA   130   GACAUAGCAGGAACUACUA   130   UAGUAGUUCCUGCUAUGUC   868               GAAAGGUGAAGGGGCAGUA   131   GAAAGGUGAAGGGGCAGUA   131   UACUGCCCCUUCACCUUUC   869               AGUGACAUAGCAGGAACUA   132   AGUGACAUAGCAGGAACUA   132   UAGUUCCUGCUAUGUCACU   870               GCAGAUGAUACAGUAUUAG   133   GCAGAUGAUACAGUAUUAG   133   CUAAUACUGUAUCAUCUGC   871               GGAGCAGAUGAUACAGUAU   134   GGAGCAGAUGAUACAGUAU   134   AUACUGUAUCAUCUGCUCC   872               CCAAGGGGAAGUGACAUAG   135   CCAAGGGGAAGUGACAUAG   135   CUAUGUCACUUCCCCUUGG   873               GAAGCUCUAUUAGAUACAG   136   GAAGCUCUAUUAGAUACAG   136   CUGUAUCUAAUAGAGCUUC   874               GGGAAGUGACAUAGCAGGA   137   GGGAAGUGACAUAGCAGGA   137   UCCUGCUAUGUCACUUCCC   875               CAUGCCUGUGUACCCACAG   138   CAUGCCUGUGUACCCACAG   138   CUGUGGGUACACAGGCAUG   876               GAAAGAGCAGAAGACAGUG   139   GAAAGAGCAGAAGACAGUG   139   CACUGUCUUCUGCUCUUUC   877               ACAUAGCAGGAACUACUAG   140   ACAUAGCAGGAACUACUAG   140   CUAGUAGUUCCUGCUAUGU   878               CAUCUCCUAUGGCAGGAAG   141   CAUCUCCUAUGGCAGGAAG   141   CUUCCUGCCAUAGGAGAUG   879               GAGCAGAUGAUACAGUAUU   142   GAGCAGAUGAUACAGUAUU   142   AAUACUGUAUCAUCUGCUC   880               AGCAUUAUCAGAAGGAGCC   143   AGCAUUAUCAGAAGGAGCC   143   GGCUCCUUCUGAUAAUGCU   881               CACCAGGCCAGAUGAGAGA   144   CACCAGGCCAGAUGAGAGA   144   UCUCUCAUCUGGCCUGGUG   882               GUGACAUAGCAGGAACUAC   145   GUGACAUAGCAGGAACUAC   145   GUAGUUCCUGCUAUGUCAC   883               AGCAGGAAGAUGGCCAGUA   146   AGCAGGAAGAUGGCCAGUA   146   UACUGGCCAUCUUCCUGCU   884               GAGAACCAAGGGGAAGUGA   147   GAGAACCAAGGGGAAGUGA   147   UCACUUCCCCUUGGUUCUC   885               AGUAUGGGCAAGCAGGGAG   148   AGUAUGGGCAAGCAGGGAG   148   CUCCCUGCUUGCCCAUACU   886               CCUACAAUCCCCAAAGUCA   149   CCUACAAUCCCCAAAGUCA   149   UGACUUUGGGGAUUGUAGG   887               CUACAAUCCCCAAAGUCAA   150   CUACAAUCCCCAAAGUCAA   150   UUGACUUUGGGGAUUGUAG   888               GCCUGUGUACCCACAGACC   151   GCCUGUGUACCCACAGACC   151   GGUCUGUGGGUACACAGGC   889               AGCAGAUGAUACAGUAUUA   152   AGCAGAUGAUACAGUAUUA   152   UAAUACUGUAUCAUCUGCU   890               AGAGAACCAAGGGGAAGUG   153   AGAGAACCAAGGGGAAGUG   153   CACUUCCCCUUGGUUCUCU   891               CCCUACAAUCCCCAAAGUC   154   CCCUACAAUCCCCAAAGUC   154   GACUUUGGGGAUUGUAGGG   892               UGACAUAGCAGGAACUACU   155   UGACAUAGCAGGAACUACU   155   AGUAGUUCCUGCUAUGUCA   893               UUAUCAGAAGGAGCCACCC   156   UUAUCAGAAGGAGCCACCC   156   GGGUGGCUCCUUCUGAUAA   894               AAGUGACAUAGCAGGAACU   157   AAGUGACAUAGCAGGAACU   157   AGUUCCUGCUAUGUCACUU   895               GCAGGAAGAUGGCCAGUAA   158   GCAGGAAGAUGGCCAGUAA   158   UUACUGGCCAUCUUCCUGC   896               UAGGCAUCUCCUAUGGCAG   159   UAGGCAUCUCCUAUGGCAG   159   CUGCCAUAGGAGAUGCCUA   897               CAAGGGGAAGUGACAUAGC   160   CAAGGGGAAGUGACAUAGC   160   GCUAUGUCACUUCCCCUUG   898               AAAGAGCAGAAGACAGUGG   161   AAAGAGCAGAAGACAGUGG   161   CCACUGUCUUCUGCUCUUU   899               CUCCUAUGGCAGGAAGAAG   162   CUCCUAUGGCAGGAAGAAG   162   CUUCUUCCUGCCAUAGGAG   900               UAUCAGAAGGAGCCACCCC   163   UAUCAGAAGGAGCCACCCC   163   GGGGUGGCUCCUUCUGAUA   901               AUUAUCAGAAGGAGCCACC   164   AUUAUCAGAAGGAGCCACC   164   GGUGGCUCCUUCUGAUAAU   902               AUGCCUGUGUACCCACAGA   165   AUGCCUGUGUACCCACAGA   165   UCUGUGGGUACACAGGCAU   903               AAAUUAGUAGAUUUCAGAG   166   AAAUUAGUAGAUUUCAGAG   166   CUCUGAAAUCUACUAAUUU   904               UGCAUAUAAGCAGCUGCUU   167   UGCAUAUAAGCAGCUGCUU   167   AAGCAGCUGCUUAUAUGCA   905               AAUUAGUAGAUUUCAGAGA   168   AAUUAGUAGAUUUCAGAGA   168   UCUCUGAAAUCUACUAAUU   906               GCAUCUCCUAUGGCAGGAA   169   GCAUCUCCUAUGGCAGGAA   169   UUCCUGCCAUAGGAGAUGC   907               AGAACCAAGGGGAAGUGAC   170   AGAACCAAGGGGAAGUGAC   170   GUCACUUCCCCUUGGUUCU   908               UCAAAAUUUUCGGGUUUAU   171   UCAAAAUUUUCGGGUUUAU   171   AUAAACCCGAAAAUUUUGA   909               CAGGGAUGGAAAGGAUCAC   172   CAGGGAUGGAAAGGAUCAC   172   GUGAUCCUUUCCAUCCCUG   910               GAAGGAGCCACCCCACAAG   173   GAAGGAGCCACCCCACAAG   173   CUUGUGGGGUGGCUCCUUC   911               AAUUUUCGGGUUUAUUACA   174   AAUUUUCGGGUUUAUUACA   174   UGUAAUAAACCCGAPAAUU   912               AGCAGGAAGCACUAUGGGC   175   AGCAGGAAGCACUAUGGGC   175   GCCCAUAGUGCUUCCUGCU   913               AUCAGAAGGAGCCACCCCA   176   AUCAGAAGGAGCCACCCCA   176   UGGGGUGGCUCCUUCUGAU   914               UGAGAGAACCAAGGGGAAG   177   UGAGAGAACCAAGGGGAAG   177   CUUCCCCUUGGUUCUCUCA   915               AAGGUGAAGGGGCAGUAGU   178   AAGGUGAAGGGGCAGUAGU   178   ACUACUGCCCCUUCACCUU   916               GAAAAAAUCAGUAACAGUA   179   GAAAAAAUCAGUAACAGUA   179   UACUGUUACUGAUUUUUUC   917               CAAUGAGGAAGCUGCAGAA   180   CAAUGAGGAAGCUGCAGAA   180   UUCUGCAGCUUCCUCAUUG   918               AGAUGAUACAGUAUUAGAA   181   AGAUGAUACAGUAUUAGAA   181   UUCUAAUACUGUAUCAUCU   919               UGAGGAAGCUGCAGAAUGG   182   UGAGGAAGCUGCAGAAUGG   182   CCAUUCUGCAGCUUCCUCA   920               UAUUAUGACCCAUCAAAAG   183   UAUUAUGACCCAUCAAAAG   183   CUUUUGAUGGGUCAUAAUA   921               UCACUCUUUGGCAACGACC   184   UCACUCUUUGGCAACGACC   184   GGUCGUUGCCAAAGAGUGA   922               UGGAGAAAAUUAGUAGAUU   185   UGGAGAAAAUUAGUAGAUU   185   AAUCUACUAAUUUUCUCCA   923               AGACAGGAUGAGGAUUAGA   186   AGACAGGAUGAGGAUUAGA   186   UCUAAUCCUCAUCCUGUCU   924               AAAGGUGAAGGGGCAGUAG   187   AAAGGUGAAGGGGCAGUAG   187   CUACUGCCCCUUCACCUUU   925               GGCAUCUCCUAUGGCAGGA   188   GGCAUCUCCUAUGGCAGGA   188   UCCUGCCAUAGGAGAUGCC   926               AAGGAGCCACCCCACAAGA   189   AAGGAGCCACCCCACAAGA   189   UCUUGUGGGGUGGCUCCUU   927               UAAAGCCAGGAAUGGAUGG   190   UAAAGCCAGGAAUGGAUGG   190   CCAUCCAUUCCUGGCUUUA   928               GGAGAAAAUUAGUAGAUUU   191   GGAGAAAAUUAGUAGAUUU   191   AAAUCUACUAAUUUUCUCC   929               AAGAGCAGAAGACAGUGGC   192   AAGAGCAGAAGACAGUGGC   192   GCCACUGUCUUCUGCUCUU   930               UCAGAAGGAGCCACCCCAC   193   UCAGAAGGAGCCACCCCAC   193   GUGGGGUGGCUCCUUCUGA   931               AGGCAUCUCCUAUGGCAGG   194   AGGCAUCUCCUAUGGCAGG   194   CCUGCCAUAGGAGAUGCCU   932               AGGGAUGGAAAGGAUCACC   195   AGGGAUGGAAAGGAUCACC   195   GGUGAUCCUUUCCAUCCCU   933               AGGAAGCUGCAGAAUGGGA   196   AGGAAGCUGCAGAAUGGGA   196   UCCCAUUCUGCAGCUUCCU   934               CUGCAUAUAAGCAGCUGCU   197   CUGCAUAUAAGCAGCUGCU   197   AGCAGCUGCUUAUAUGCAG   935               AAGGGGCAGUAGUAAUACA   198   AAGGGGCAGUAGUAAUACA   198   UGUAUUACUACUGCCCCUU   936               UUGACUAGCGGAGGCUAGA   199   UUGACUAGCGGAGGCUAGA   199   UCUAGCCUCCGCUAGUCAA   937               UAAAAGACACCAAGGAAGC   200   UAAAAGACACCAAGGAAGC   200   GCUUCCUUGGUGUCUUUUA   938               GAGGAAGCUGCAGAAUGGG   201   GAGGAAGCUGCAGAAUGGG   201   CCCAUUCUGCAGCUUCCUC   939               CAGCAGGAAGCACUAUGGG   202   CAGCAGGAAGCACUAUGGG   202   CCCAUAGUGCUUCCUGCUG   940               GGAGCCACCCCACAAGAUU   203   GGAGCCACCCCACAAGAUU   203   AAUCUUGUGGGGUGGCUCC   941               AUUAUGACCCAUCAAAAGA   204   AUUAUGACCCAUCAAAAGA   204   UCUUUUGAUGGGUCAUAAU   942               CAGAUGAUACAGUAUUAGA   205   CAGAUGAUACAGUAUUAGA   205   UCUAAUACUGUAUCAUCUG   943               AUGAGAGAACCAAGGGGAA   206   AUGAGAGAACCAAGGGGAA   206   UUCCCCUUGGUUCUCUCAU   944               AUGAGGAAGCUGCAGAAUG   207   AUGAGGAAGCUGCAGAAUG   207   CAUUCUGCAGCUUCCUCAU   945               UGCCUGUGUACCCACAGAC   208   UGCCUGUGUACCCACAGAC   208   GUCUGUGGGUACACAGGCA   946               GAAGGGGCAGUAGUAAUAC   209   GAAGGGGCAGUAGUAAUAC   209   GUAUUACUACUGCCCCUUC   947               UCAGCAUUAUCAGAAGGAG   210   UCAGCAUUAUCAGAAGGAG   210   CUCCUUCUGAUAAUGCUGA   948               UUCAAAAUUUUCGGGUUUA   211   UUCAAAAUUUUCGGGUUUA   211   UAAACCCGAAAAUUUUGAA   949               UCUGGAAAGGUGAAGGGGC   212   UCUGGAAAGGUGAAGGGGC   212   GCCCCUUCACCUUUCCAGA   950               UUAGCAGGAAGAUGGCCAG   213   UUAGCAGGAAGAUGGCCAG   213   CUGGCCAUCUUCCUGCUAA   951               GAACCAAGGGGAAGUGACA   214   GAACCAAGGGGAAGUGACA   214   UGUCACUUCCCCUUGGUUC   952               AGAAGGAGCCACCCCACAA   215   AGAAGGAGCCACCCCACAA   215   UUGUGGGGUGGCUCCUUCU   953               AAUGAGGAAGCUGCAGAAU   216   AAUGAGGAAGCUGCAGAAU   216   AUUCUGCAGCUUCCUCAUU   954               AAGAAAAAAUCAGUAACAG   217   AAGAAAAAAUCAGUAACAG   217   CUGUUACUGAUUUUUUCUU   955               GGAAUUGGAGGUUUUAUCA   218   GGAAUUGGAGGUUUUAUCA   218   UGAUAAAACCUCCAAUUCC   956               UACAGUAUUAGUAGGACCU   219   UACAGUAUUAGUAGGACCU   219   AGGUCCUACUAAUACUGUA   957               CCAGGAAUGGAUGGCCCAA   220   CCAGGAAUGGAUGGCCCAA   220   UUGGGCCAUCCAUUCCUGG   958               UUCUAUGUAGAUGGGGCAG   221   UUCUAUGUAGAUGGGGCAG   221   CUGCCCCAUCUACAUAGAA   959               CAAAAUUUUCGGGUUUAUU   222   CAAAAUUUUCGGGUUUAUU   222   AAUAAACCCGAAAAUUUUG   960               UAGACAGGAUGAGGAUUAG   223   UAGACAGGAUGAGGAUUAG   223   CUAAUCCUCAUCCUGUCUA   961               UGACAGAAGAAAAAAUAAA   224   UGACAGAAGAAAAAAUAAA   224   UUUAUUUUUUCUUCUGUCA   962               UUUAUUACAGGGACAGCAG   225   UUUAUUACAGGGACAGCAG   225   CUGCUGUCCCUGUAAUAAA   963               GGGUUUAUUACAGGGACAG   226   GGGUUUAUUACAGGGACAG   226   CUGUCCCUGUAAUAAACCC   964               AGAUGGAACAAGCCCCAGA   227   AGAUGGAACAAGCCCCAGA   227   UCUGGGGCUUGUUCCAUCU   965               CUAGCGGAGGCUAGAAGGA   228   CUAGCGGAGGCUAGAAGGA   228   UCCUUCUAGCCUCCGCUAG   966               UGACUAGCGGAGGCUAGAA   229   UGACUAGCGGAGGCUAGAA   229   UUCUAGCCUCCGCUAGUCA   967               GACAUAAUAGCAACAGACA   230   GACAUAAUAGCAACAGACA   230   UGUCUGUUGCUAUUAUGUC   968               GGUUUAUUACAGGGACAGC   231   GGUUUAUUACAGGGACAGC   231   GCUGUCCCUGUAAUAAACC   969               GCAGGUGAUGAUUGUGUGG   232   GCAGGUGAUGAUUGUGUGG   232   CCACACAAUCAUCACCUGC   970               AUGGCAGGAAGAAGCGGAG   233   AUGGCAGGAAGAAGCGGAG   233   CUCCGCUUCUUCCUGCCAU   971               AGGUGAUGAUUGUGUGGCA   234   AGGUGAUGAUUGUGUGGCA   234   UGCCACACAAUCAUCACCU   972               CCACCCCACAAGAUUUAAA   235   CCACCCCACAAGAUUUAAA   235   UUUAAAUCUUGUGGGGUGG   973               GUAAAAAAUUGGAUGACAG   236   GUAAAAAAUUGGAUGACAG   236   CUGUCAUCCAAUUUUUUAC   974               AUAAUAGCAACAGACAUAC   237   AUAAUAGCAACAGACAUAC   237   GUAUGUCUGUUGCUAUUAU   975               GCAUAUAAGCAGCUGCUUU   238   GCAUAUAAGCAGCUGCUUU   238   AAAGCAGCUGCUUAUAUGC   976               GGCAGGUGAUGAUUGUGUG   239   GGCAGGUGAUGAUUGUGUG   239   CACACAAUCAUCACCUGCC   977               AUGAUACAGUAUUAGAAGA   240   AUGAUACAGUAUUAGAAGA   240   UCUUCUAAUACUGUAUCAU   978               GAUGGCAGGUGAUGAUUGU   241   GAUGGCAGGUGAUGAUUGU   241   ACAAUCAUCACCUGCCAUC   979               CAUAAUAGCAACAGACAUA   242   CAUAAUAGCAACAGACAUA   242   UAUGUCUGUUGCUAUUAUG   980               AAAAUUUUCGGGUUUAUUA   243   AAAAUUUUCGGGUUUAUUA   243   UAAUAAACCCGAAAAUUUU   981               ACAUAAUAGCAACAGACAU   244   ACAUAAUAGCAACAGACAU   244   AUGUCUGUUGCUAUUAUGU   982               AUUUCAAAAAUUGGGCCUG   245   AUUUCAAAAAUUGGGCCUG   245   CAGGCCCAAUUUUUGAAAU   983               CUGGAAAGGUGAAGGGGCA   246   CUGGAAAGGUGAAGGGGCA   246   UGCCCCUUCACCUUUCCAG   984               AAAACAGAUGGCAGGUGAU   247   AAAACAGAUGGCAGGUGAU   247   AUCACCUGCCAUCUGUUUU   985               UUUCAAAAAUUGGGCCUGA   248   UUUCAAAAAUUGGGCCUGA   248   UCAGGCCCAAUUUUUGAAA   986               GAGAGAACCAAGGGGAAGU   249   GAGAGAACCAAGGGGAAGU   249   ACUUCCCCUUGGUUCUCUC   987               CUCUGGAAAGGUGAAGGGG   250   CUCUGGAAAGGUGAAGGGG   250   CCCCUUCACCUUUCCAGAG   988               AUUAGCAGGAAGAUGGCCA   251   AUUAGCAGGAAGAUGGCCA   251   UGGCCAUCUUCCUGCUAAU   989               GAGCCACCCCACAAGAUUU   252   GAGCCACCCCACAAGAUUU   252   AAAUCUUGUGGGGUGGCUC   990               CAUAGCAGGAACUACUAGU   253   CAUAGCAGGAACUACUAGU   253   ACUAGUAGUUCCUGCUAUG   991               UUUUAAAAGAAAAGGGGGG   254   UUUUAAAAGAAAAGGGGGG   254   CCCCCCUUUUCUUUUAAAA   992               GCGGAGGCUAGAAGGAGAG   255   GCGGAGGCUAGAAGGAGAG   255   CUCUCCUUCUAGCCUCCGC   993               CAGUAUUAGUAGGACCUAC   256   CAGUAUUAGUAGGACCUAC   256   GUAGGUCCUACUAAUACUG   994               AGGGGGAAUUGGAGGUUUU   257   AGGGGGAAUUGGAGGUUUU   257   AAAACCUCCAAUUCCCCCU   995               ACAGUAUUAGUAGGACCUA   258   ACAGUAUUAGUAGGACCUA   258   UAGGUCCUACUAAUACUGU   996               GACUAGCGGAGGCUAGAAG   259   GACUAGCGGAGGCUAGAAG   259   CUUCUAGCCUCCGCUAGUC   997               GUUUAUUACAGGGACAGCA   260   GUUUAUUACAGGGACAGCA   260   UGCUGUCCCUGUAAUAAAC   998               CAGGUGAUGAUUGUGUGGC   261   CAGGUGAUGAUUGUGUGGC   261   GCCACACAAUCAUCACCUG   999               AGCGGAGGCUAGAAGGAGA   262   AGCGGAGGCUAGAAGGAGA   262   UCUCCUUCUAGCCUCCGCU   1000               UCUAUGUAGAUGGGGCAGC   263   UCUAUGUAGAUGGGGCAGC   263   GCUGCCCCAUCUACAUAGA   1001               UAAAAAAUUGGAUGACAGA   264   UAAAAAAUUGGAUGACAGA   264   UCUGUCAUCCAAUUUUUUA   1002               GCAGCAGGAAGCACUAUGG   265   GCAGCAGGAAGCACUAUGG   265   CCAUAGUGCUUCCUGCUGC   1003               UUAUUACAGGGACAGCAGA   266   UUAUUACAGGGACAGCAGA   266   UCUGCUGUCCCUGUAAUAA   1004               AAACAGAUGGCAGGUGAUG   267   AAACAGAUGGCAGGUGAUG   267   CAUCACCUGCCAUCUGUUU   1005               AUUCAAAAUUUUCGGGUUU   268   AUUCAAAAUUUUCGGGUUU   268   AAACCCGAAAAUUUUGAAU   1006               GGGGAAUUGGAGGUUUUAU   269   GGGGAAUUGGAGGUUUUAU   269   AUAAAACCUCCAAUUCCCC   1007               GCCACCCCACAAGAUUUAA   270   GCCACCCCACAAGAUUUAA   270   UUAAAUCUUGUGGGGUGGC   1008               GAUGAUACAGUAUUAGAAG   271   GAUGAUACAGUAUUAGAAG   271   CUUCUAAUACUGUAUCAUC   1009               UAAUAGCAACAGACAUACA   272   UAAUAGCAACAGACAUACA   272   UGUAUGUCUGUUGCUAUUA   1010               GAGGCUAGAAGGAGAGAGA   273   GAGGCUAGAAGGAGAGAGA   273   UCUCUCUCCUUCUAGCCUC   1011               GUACAGUAUUAGUAGGACC   274   GUACAGUAUUAGUAGGACC   274   GGUCCUACUAAUACUGUAC   1012               UAGCGGAGGCUAGAAGGAG   275   UAGCGGAGGCUAGAAGGAG   275   CUCCUUCUAGCCUCCGCUA   1013               CGGAGGCUAGAAGGAGAGA   276   CGGAGGCUAGAAGGAGAGA   276   UCUCUCCUUCUAGCCUCCG   1014               GGUACAGUAUUAGUAGGAC   277   GGUACAGUAUUAGUAGGAC   277   GUCCUACUAAUACUGUACC   1015               AAAUUUUCGGGUUUAUUAC   278   AAAUUUUCGGGUUUAUUAC   278   GUAAUAAACCCGAAAAUUU   1016               AGCAGCAGGAAGCACUAUG   279   AGCAGCAGGAAGCACUAUG   279   CAUAGUGCUUCCUGCUGCU   1017               AGCCACCCCACAAGAUUUA   280   AGCCACCCCACAAGAUUUA   280   UAAAUCUUGUGGGGUGGCU   1018               AACCAAGGGGPAGUGACAU   281   AACCAAGGGGAAGUGACAU   281   AUGUCACUUCCCCUUGGUU   1019               AAGGGGAAGUGACAUAGCA   282   AAGGGGAAGUGACAUAGCA   282   UGCUAUGUCACUUCCCCUU   1020               UUAAAGCCAGGAAUGGAUG   283   UUAAAGCCAGGAAUGGAUG   283   CAUCCAUUCCUGGCUUUAA   1021               ACUAGCGGAGGCUAGAAGG   284   ACUAGCGGAGGCUAGAAGG   284   CCUUCUAGCCUCCGCUAGU   1022               UAGGUACAGUAUUAGUAGG   285   UAGGUACAGUAUUAGUAGG   285   CCUACUAAUACUGUACCUA   1023               GGGGGAAUUGGAGGUUUUA   286   GGGGGAAUUGGAGGUUUUA   286   UAAAACCUCCAAUUCCCCC   1024               AGAUGGCAGGUGAUGAUUG   287   AGAUGGCAGGUGAUGAUUG   287   CAAUCAUCACCUGCCAUCU   1025               UUAAACAAUGGCCAUUGAC   288   UUAAACAAUGGCCAUUGAC   288   GUCAAUGGCCAUUGUUUAA   1026               UGGCAGGUGAUGAUUGUGU   289   UGGCAGGUGAUGAUUGUGU   289   ACACAAUCAUCACCUGCCA   1027               UAAAAUUAGCAGGAAGAUG   290   UAAAAUUAGCAGGAAGAUG   290   CAUCUUCCUGCUAAUUUUA   1028               AGGAGCCACCCCACAAGAU   291   AGGAGCCACCCCACAAGAU   291   AUCUUGUGGGGUGGCUCCU   1029               GUAUUAGUAGGACCUACAC   292   GUAUUAGUAGGACCUACAC   292   GUGUAGGUCCUACUAAUAC   1030               AAUCCCCAAAGUCAAGGAG   293   AAUCCCCAAAGUCAAGGAG   293   CUCCUUGACUUUGGGGAUU   1031               CCAGGCCAGAUGAGAGAAC   294   CCAGGCCAGAUGAGAGAAC   294   GUUCUCUCAUCUGGCCUGG   1032               CCAUUGACAGAAGAAAAAA   295   CCAUUGACAGAAGAAAAAA   295   UUUUUUCUUCUGUCAAUGG   1033               CAGAUGGCAGGUGAUGAUU   296   CAGAUGGCAGGUGAUGAUU   296   AAUCAUCACCUGCCAUCUG   1034               CAGAUGAGAGAACCAAGGG   297   CAGAUGAGAGAACCAAGGG   297   CCCUUGGUUCUCUCAUCUG   1035               GCCAUUGACAGAAGAAAAA   298   GCCAUUGACAGAAGAAAAA   298   UUUUUCUUCUGUCAAUGGC   1036               UAUUAGUAGGACCUACACC   299   UAUUAGUAGGACCUACACC   299   GGUGUAGGUCCUACUAAUA   1037               UCUCGACGCAGGACUCGGC   300   UCUCGACGCAGGACUCGGC   300   GCCGAGUCCUGCGUCGAGA   1038               AGAUGAGAGAACCAAGGGG   301   AGAUGAGAGAACCAAGGGG   301   CCCCUUGGUUCUCUCAUCU   1039               AUCCCCAAAGUCAAGGAGU   302   AUCCCCAAAGUCAAGGAGU   302   ACUCCUUGACUUUGGGGAU   1040               AAUUAGCAGGAAGAUGGCC   303   AAUUAGCAGGAAGAUGGCC   303   GGCCAUCUUCCUGCUAAUU   1041               GGGAAUUGGAGGUUUUAUC   304   GGGAAUUGGAGGUUUUAUC   304   GAUAAAACCUCCAAUUCCC   1042               CUCGACGCAGGACUCGGCU   305   CUCGACGCAGGACUCGGCU   305   AGCCGAGUCCUGCGUCGAG   1043               AUGGCCAUUGACAGAAGAA   306   AUGGCCAUUGACAGAAGAA   306   UUCUUCUGUCAAUGGCCAU   1044               AAAAUUAGCAGGAAGAUGG   307   AAAAUUAGCAGGAAGAUGG   307   CCAUCUUCCUGCUAAUUUU   1045               ACGCAGGACUCGGCUUGCU   308   ACGCAGGACUCGGCUUGCU   308   AGCAAGCCGAGUCCUGCGU   1046               UAAACAAUGGCCAUUGACA   309   UAAACAAUGGCCAUUGACA   309   UGUCAAUGGCCAUUGUUUA   1047               GAUGGAACAAGCCCCAGAA   310   GAUGGAACAAGCCCCAGAA   310   UUCUGGGGCUUGUUCCAUC   1048               AAUGAACAAGUAGAUAAAU   311   AAUGAACAAGUAGAUAAAU   311   AUUUAUCUACUUGUUCAUU   1049               AUUGGAGGUUUUAUCAAAG   312   AUUGGAGGUUUUAUCAAAG   312   CUUUGAUAAAACCUCCAAU   1050               AGGCUAGAAGGAGAGAGAU   313   AGGCUAGAAGGAGAGAGAU   313   AUCUCUCUCCUUCUAGCCU   1051               AGAUGGGUGCGAGAGCGUC   314   AGAUGGGUGCGAGAGCGUC   314   GACGCUCUCGCACCCAUCU   1052               AGGUACAGUAUUAGUAGGA   315   AGGUACAGUAUUAGUAGGA   315   UCCUACUAAUACUGUACCU   1053               GGAGGCUAGAAGGAGAGAG   316   GGAGGCUAGAAGGAGAGAG   316   CUCUCUCCUUCUAGCCUCC   1054               CAGGACAUAACAAGGUAGG   317   CAGGACAUAACAAGGUAGG   317   CCUACCUUGUUAUGUCCUG   1055               AGUAUUAGUAGGACCUACA   318   AGUAUUAGUAGGACCUACA   318   UGUAGGUCCUACUAAUACU   1056               UUGACAGAAGAAAAAAUAA   319   UUGACAGAAGAAAAAAUAA   319   UUAUUUUUUCUUCUGUCAA   1057               UGGAGAAGUGAAUUAUAUA   320   UGGAGAAGUGAAUUAUAUA   320   UAUAUAAUUCACUUCUCCA   1058               CUCUCGACGCAGGACUCGG   321   CUCUCGACGCAGGACUCGG   321   CCGAGUCCUGCGUCGAGAG   1059               AUGAACAAGUAGAUAAAUU   322   AUGAACAAGUAGAUAAAUU   322   AAUUUAUCUACUUGUUCAU   1060               UGGCCAUUGACAGAAGAAA   323   UGGCCAUUGACAGAAGAAA   323   UUUCUUCUGUCAAUGGCCA   1061               AUACCCAUGUUUUCAGCAU   324   AUACCCAUGUUUUCAGCAU   324   AUGCUGAAAACAUGGGUAU   1062               UUUAAAAGAAAAGGGGGGA   325   UUUAAAAGAAAAGGGGGGA   325   UCCCCCCUUUUCUUUUAAA   1063               CGACGCAGGACUCGGCUUG   326   CGACGCAGGACUCGGCUUG   326   CAAGCCGAGUCCUGCGUCG   1064               AUUGACAGAAGAAAAAAUA   327   AUUGACAGAAGAAAAAAUA   327   UAUUUUUUCUUCUGUCAAU   1065               CUAGAAGGAGAGAGAUGGG   328   CUAGAAGGAGAGAGAUGGG   328   CCCAUCUCUCUCCUUCUAG   1066               UGGCAGGAAGAAGCGGAGA   329   UGGCAGGAAGAAGCGGAGA   329   UCUCCGCUUCUUCCUGCCA   1067               CAAUCCCCAAAGUCAAGGA   330   CAAUCCCCAAAGUCAAGGA   330   UCCUUGACUUUGGGGAUUG   1068               AAAUUCAAAAUUUUCGGGU   331   AAAUUCAAAAUUUUCGGGU   331   ACCCGAAAAUUUUGAAUUU   1069               GAAUUGGAGGUUUUAUCAA   332   GAAUUGGAGGUUUUAUCAA   332   UUGAUAAAACCUCCAAUUC   1070               GACGCAGGACUCGGCUUGC   333   GACGCAGGACUCGGCUUGC   333   GCAAGCCGAGUCCUGCGUC   1071               UUUGACUAGCGGAGGCUAG   334   UUUGACUAGCGGAGGCUAG   334   CUAGCCUCCGCUAGUCAAA   1072               AUAGGUACAGUAUUAGUAG   335   AUAGGUACAGUAUUAGUAG   335   CUACUAAUACUGUACCUAU   1073               GGCUAGAAGGAGAGAGAUG   336   GGCUAGAAGGAGAGAGAUG   336   CAUCUCUCUCCUUCUAGCC   1074               ACCAGGCCAGAUGAGAGAA   337   ACCAGGCCAGAUGAGAGAA   337   UUCUCUCAUCUGGCCUGGU   1075               GAUGAGAGAACCAAGGGGA   338   GAUGAGAGAACCAAGGGGA   338   UCCCCUUGGUUCUCUCAUC   1076               GGAGCAGCAGGAAGCACUA   339   GGAGCAGCAGGAAGCACUA   339   UAGUGCUUCCUGCUGCUCC   1077               UCUCUCGACGCAGGACUCG   340   UCUCUCGACGCAGGACUCG   340   CGAGUCCUGCGUCGAGAGA   1078               UCCCUACAAUCCCCAAAGU   341   UCCCUACAAUCCCCAAAGU   341   ACUUUGGGGAUUGUAGGGA   1079               UUGGAGGUUUUAUCAAAGU   342   UUGGAGGUUUUAUCAAAGU   342   ACUUUGAUAAAACCUCCAA   1080               ACUGUACCAGUAAAAUUAA   343   ACUGUACCAGUAAAAUUAA   343   UUAAUUUUACUGGUACAGU   1081               AUGGCAGGUGAUGAUUGUG   344   AUGGCAGGUGAUGAUUGUG   344   CACAAUCAUCACCUGCCAU   1082               GAGGAAAUGAACAAGUAGA   345   GAGGAAAUGAACAAGUAGA   345   UCUACUUGUUCAUUUCCUC   1083               AGACAUAAUAGCAACAGAC   346   AGACAUAAUAGCAACAGAC   346   GUCUGUUGCUAUUAUGUCU   1084               AAAUUAGCAGGAAGAUGGC   347   AAAUUAGCAGGAAGAUGGC   347   GCCAUCUUCCUGCUAAUUU   1085               UUGGAGAAGUGAAUUAUAU   348   UUGGAGAAGUGAAUUAUAU   348   AUAUAAUUCACUUCUCCAA   1086               UCGACGCAGGACUCGGCUU   349   UCGACGCAGGACUCGGCUU   349   AAGCCGAGUCCUGCGUCGA   1087               AAAAUUCAAAAUUUUCGGG   350   AAAAUUCAAAAUUUUCGGG   350   CCCGAAAAUUUUGAAUUUU   1088               CAGGCCAGAUGAGAGAACC   351   CAGGCCAGAUGAGAGAACC   351   GGUUCUCUCAUCUGGCCUG   1089               UACCCAUGUUUUCAGCAUU   352   UACCCAUGUUUUCAGCAUU   352   AAUGCUGAAAACAUGGGUA   1090               ACACAUGCCUGUGUACCCA   353   ACACAUGCCUGUGUACCCA   353   UGGGUACACAGGCAUGUGU   1091               GGCCAUUGACAGAAGAAAA   354   GGCCAUUGACAGAAGAAAA   354   UUUUCUUCUGUCAAUGGCC   1092               GAGCAGCAGGAAGCACUAU   355   GAGCAGCAGGAAGCACUAU   355   AUAGUGCUUCCUGCUGCUC   1093               CUGUACCAGUAAAAUUAAA   356   CUGUACCAGUAAAAUUAAA   356   UUUAAUUUUACUGGUACAG   1094               GAAAUGAUGACAGCAUGUC   357   GAAAUGAUGACAGCAUGUC   357   GACAUGCUGUCAUCAUUUC   1095               CAUUGACAGAAGAAAAAAU   358   CAUUGACAGAAGAAAAAAU   358   AUUUUUUCUUCUGUCAAUG   1096               AAAUGAUGACAGCAUGUCA   359   AAAUGAUGACAGCAUGUCA   359   UGACAUGCUGUCAUCAUUU   1097               GCUAGAAGGAGAGAGAUGG   360   GCUAGAAGGAGAGAGAUGG   360   CCAUCUCUCUCCUUCUAGC   1098               UAGGGAUUAUGGAAAACAG   361   UAGGGAUUAUGGAAAACAG   361   CUGUUUUCCAUAAUCCCUA   1099               GAAAAUUAGUAGAUUUCAG   362   GAAAAUUAGUAGAUUUCAG   362   CUGAAAUCUACUAAUUUUC   1100               CUACACCUGUCAACAUAAU   363   CUACACCUGUCAACAUAAU   363   AUUAUGUUGACAGGUGUAG   1101               ACAGAUGGCAGGUGAUGAU   364   ACAGAUGGCAGGUGAUGAU   364   AUCAUCACCUGCCAUCUGU   1102               CCACAGGGAUGGAAAGGAU   365   CCACAGGGAUGGAAAGGAU   365   AUCCUUUCCAUCCCUGUGG   1103               UUAGGGAUUAUGGAAAACA   366   UUAGGGAUUAUGGAAAACA   366   UGUUUUCCAUAAUCCCUAA   1104               AGAUGCUGCAUAUAAGCAG   367   AGAUGCUGCAUAUAAGCAG   367   CUGCUUAUAUGCAGCAUCU   1105               AAUAGCAACAGACAUACAA   368   AAUAGCAACAGACAUACAA   368   UUGUAUGUCUGUUGCUAUU   1106               AAUUCAAAAUUUUCGGGUU   369   AAUUCAAAAUUUUCGGGUU   369   AACCCGAAAAUUUUGAAUU   1107               CAGACUCACAAUAUGCAUU   370   CAGACUCACAAUAUGCAUU   370   AAUGCAUAUUGUGAGUCUG   1108               UAUGCAUUAGGAAUCAUUC   371   UAUGCAUUAGGAAUCAUUC   371   GAAUGAUUCCUAAUGCAUA   1109               UACACCUGUCAACAUAAUU   372   UACACCUGUCAACAUAAUU   372   AAUUAUGUUGACAGGUGUA   1110               UGGAGGAAAUGAACAAGUA   373   UGGAGGAAAUGAACAAGUA   373   UACUUGUUCAUUUCCUCCA   1111               ACCAAGGGGAAGUGACAUA   374   ACCAAGGGGAAGUGACAUA   374   UAUGUCACUUCCCCUUGGU   1112               GAGAUGGGUGCGAGAGCGU   375   GAGAUGGGUGCGAGAGCGU   375   ACGCUCUCGCACCCAUCUC   1113               UAUAGGUACAGUAUUAGUA   376   UAUAGGUACAGUAUUAGUA   376   UACUAAUACUGUACCUAUA   1114               AUUAGGGAUUAUGGAAAAC   377   AUUAGGGAUUAUGGAAAAC   377   GUUUUCCAUAAUCCCUAAU   1115               UGGCUGUGGAAAGAUACCU   378   UGGCUGUGGAAAGAUACCU   378   AGGUAUCUUUCCACAGCCA   1116               GAGAGAUGGGUGCGAGAGC   379   GAGAGAUGGGUGCGAGAGC   379   GCUCUCGCACCCAUCUCUC   1117               CCUACACCUGUCAACAUAA   380   CCUACACCUGUCAACAUAA   380   UUAUGUUGACAGGUGUAGG   1118               CAGCAGUACAAAUGGCAGU   381   CAGCAGUACAAAUGGCAGU   381   ACUGCCAUUUGUACUGCUG   1119               GGCUGUGGAAAGAUACCUA   382   GGCUGUGGAAAGAUACCUA   382   UAGGUAUCUUUCCACAGCC   1120               AGAAAAUUAGUAGAUUUCA   383   AGAAAAUUAGUAGAUUUCA   383   UGAAAUCUACUAAUUUUCU   1121               GCCACCUUUGCCUAGUGUU   384   GCCACCUUUGCCUAGUGUU   384   AACACUAGGCAAAGGUGGC   1122               GAUGCUGCAUAUAAGCAGC   385   GAUGCUGCAUAUAAGCAGC   385   GCUGCUUAUAUGCAGCAUC   1123               GCUAUAGGUACAGUAUUAG   386   GCUAUAGGUACAGUAUUAG   386   CUAAUACUGUACCUAUAGC   1124               AACAGAUGGCAGGUGAUGA   387   AACAGAUGGCAGGUGAUGA   387   UCAUCACCUGCCAUCUGUU   1125               AUCACUCUUUGGCPACGAC   388   AUCACUCUUUGGCAACGAC   388   GUCGUUGCCAAAGAGUGAU   1126               ACAUGCCUGUGUACCCACA   389   ACAUGCCUGUGUACCCACA   389   UGUGGGUACACAGGCAUGU   1127               ACAGCAGUACAAAUGGCAG   390   ACAGCAGUACAAAUGGCAG   390   CUGCCAUUUGUACUGCUGU   1128               AUGCAUUAGGAAUCAUUCA   391   AUGCAUUAGGAAUCAUUCA   391   UGAAUGAUUCCUAAUGCAU   1129               AAUUGGAGGUUUUAUCAAA   392   AAUUGGAGGUUUUAUCAAA   392   UUUGAUAAAACCUCCAAUU   1130               UUGGAGGAAAUGAACAAGU   393   UUGGAGGAAAUGAACAAGU   393   ACUUGUUCAUUUCCUCCAA   1131               AUUGGAGGAAAUGAACAAG   394   AUUGGAGGAAAUGAACAAG   394   CUUGUUCAUUUCCUCCAAU   1132               AAAAAUUCAAAAUUUUCGG   395   AAAAAUUCAAAAUUUUCGG   395   CCGAAAAUUUUGAAUUUUU   1133               AGGUGAAGGGGCAGUAGUA   396   AGGUGAAGGGGCAGUAGUA   396   UACUACUGCCCCUUCACCU   1134               CUAUAGGUACAGUAUUAGU   397   CUAUAGGUACAGUAUUAGU   397   ACUAAUACUGUACCUAUAG   1135               AUUAAAGCCAGGAAUGGAU   398   AUUAAAGCCAGGAAUGGAU   398   AUCCAUUCCUGGCUUUAAU   1136               GGAGGAAAUGAACAAGUAG   399   GGAGGAAAUGAACAAGUAG   399   CUACUUGUUCAUUUCCUCC   1137               AGCAGUACAAAUGGCAGUA   400   AGCAGUACAAAUGGCAGUA   400   UACUGCCAUUUGUACUGCU   1138               AUCAGUACAAUGUGCUUCC   401   AUCAGUACAAUGUGCUUCC   401   GGAAGCACAUUGUACUGAU   1139               UAUGGGGUACCUGUGUGGA   402   UAUGGGGUACCUGUGUGGA   402   UCCACACAGGUACCCCAUA   1140               AGAGAUGGGUGCGAGAGCG   403   AGAGAUGGGUGCGAGAGCG   403   CGCUCUCGCACCCAUCUCU   1141               GGUGAAGGGGCAGUAGUAA   404   GGUGAAGGGGCAGUAGUAA   404   UUACUACUGCCCCUUCACC   1142               GUGAAGGGGCAGUAGUAAU   405   GUGAAGGGGCAGUAGUAAU   405   AUUACUACUGCCCCUUCAC   1143               CGCAGGACUCGGCUUGCUG   406   CGCAGGACUCGGCUUGCUG   406   CAGCAAGCCGAGUCCUGCG   1144               CACAUGCCUGUGUACCCAC   407   CACAUGCCUGUGUACCCAC   407   GUGGGUACACAGGCAUGUG   1145               GAGAGAGAUGGGUGCGAGA   408   GAGAGAGAUGGGUGCGAGA   408   UCUCGCACCCAUCUCUCUC   1146               UAGAAGGAGAGAGAUGGGU   409   UAGAAGGAGAGAGAUGGGU   409   ACCCAUCUCUCUCCUUCUA   1147               CACAGGGAUGGAAAGGAUC   410   CACAGGGAUGGAAAGGAUC   410   GAUCCUUUCCAUCCCUGUG   1148               GGCAGGAAGAAGCGGAGAC   411   GGCAGGAAGAAGCGGAGAC   411   GUCUCCGCUUCUUCCUGCC   1149               UCCCCAAAGUCAAGGAGUA   412   UCCCCAAAGUCAAGGAGUA   412   UACUCCUUGACUUUGGGGA   1150               CCUGUCAACAUAAUUGGAA   413   CCUGUCAACAUAAUUGGAA   413   UUCCAAUUAUGUUGACAGG   1151               UAUCAGUACAAUGUGCUUC   414   UAUCAGUACAAUGUGCUUC   414   GAAGCACAUUGUACUGAUA   1152               UGAAGGGGCAGUAGUAAUA   415   UGAAGGGGCAGUAGUAAUA   415   UAUUACUACUGCCCCUUCA   1153               CUCAGAUGCUGCAUAUAAG   416   CUCAGAUGCUGCAUAUAAG   416   CUUAUAUGCAGCAUCUGAG   1154               ACAGGGAUGGAAAGGAUCA   417   ACAGGGAUGGAAAGGAUCA   417   UGAUCCUUUCCAUCCCUGU   1155               AAGAAAAGGGGGGAUUGGG   418   AAGAAAAGGGGGGAUUGGG   418   CCCAAUCCCCCCUUUUCUU   1156               UCAUUAGGGAUUAUGGAAA   419   UCAUUAGGGAUUAUGGAAA   419   UUUCCAUAAUCCCUAAUGA   1157               GAAGGAGAGAGAUGGGUGC   420   GAAGGAGAGAGAUGGGUGC   420   GCACCCAUCUCUCUCCUUC   1158               GUUAAACAAUGGCCAUUGA   421   GUUAAACAAUGGCCAUUGA   421   UCAAUGGCCAUUGUUUAAC   1159               AUGGACAAGUAGACUGUAG   422   AUGGACAAGUAGACUGUAG   422   CUACAGUCUACUUGUCCAU   1160               UAGUAGAUUUCAGAGAACU   423   UAGUAGAUUUCAGAGAACU   423   AGUUCUCUGAAAUCUACUA   1161               CUGUCAACAUAAUUGGAAG   424   CUGUCAACAUAAUUGGAAG   424   CUUCCAAUUAUGUUGACAG   1162               GGGGCAGUAGUAAUACAAG   425   GGGGCAGUAGUAAUACAAG   425   CUUGUAUUACUACUGCCCC   1163               CAUUAGGGAUUAUGGAAAA   426   CAUUAGGGAUUAUGGAAAA   426   UUUUCCAUAAUCCCUAAUG   1164               GAACUACUAGUACCCUUCA   427   GAACUACUAGUACCCUUCA   427   UGAAGGGUACUAGUAGUUC   1165               GCAGGAAGCACUAUGGGCG   428   GCAGGAAGCACUAUGGGCG   428   CGCCCAUAGUGCUUCCUGC   1166               AAGGAGAGAGAUGGGUGCG   429   AAGGAGAGAGAUGGGUGCG   429   CGCACCCAUCUCUCUCCUU   1167               CAGGAAUGGAUGGCCCAAA   430   CAGGAAUGGAUGGCCCAAA   430   UUUGGGCCAUCCAUUCCUG   1168               GGAAAUGAACAAGUAGAUA   431   GGAAAUGAACAAGUAGAUA   431   UAUCUACUUGUUCAUUUCC   1169               AAAAGACACCAAGGAAGCU   432   AAAAGACACCAAGGAAGCU   432   AGCUUCCUUGGUGUCUUUU   1170               AUCAUUCAAGCACAACCAG   433   AUCAUUCAAGCACAACCAG   433   CUGGUUGUGCUUGAAUGAU   1171               AACAAGUAGAUAAAUUAGU   434   AACAAGUAGAUAAAUUAGU   434   ACUAAUUUAUCUACUUGUU   1172               AGGAAAUGAACAAGUAGAU   435   AGGAAAUGAACAAGUAGAU   435   AUCUACUUGUUCAUUUCCU   1173               GCAGGACUCGGCUUGCUGA   436   GCAGGACUCGGCUUGCUGA   436   UCAGCAAGCCGAGUCCUGC   1174               GAAUCAUUCAAGCACAACC   437   GAAUCAUUCAAGCACAACC   437   GGUUGUGCUUGAAUGAUUC   1175               CCUCAGAUGCUGCAUAUAA   438   CCUCAGAUGCUGCAUAUAA   438   UUAUAUGCAGCAUCUGAGG   1176               GAUGGAAAGGAUCACCAGC   439   GAUGGAAAGGAUCACCAGC   439   GCUGGUGAUCCUUUCCAUC   1177               AGGAGAGAGAUGGGUGCGA   440   AGGAGAGAGAUGGGUGCGA   440   UCGCACCCAUCUCUCUCCU   1178               CAUGGACAAGUAGACUGUA   441   CAUGGACAAGUAGACUGUA   441   UACAGUCUACUUGUCCAUG   1179               UCAGAUGCUGCAUAUAAGC   442   UCAGAUGCUGCAUAUAAGC   442   GCUUAUAUGCAGCAUCUGA   1180               AUGGAGAAAAUUAGUAGAU   443   AUGGAGAAAAUUAGUAGAU   443   AUCUACUAAUUUUCUCCAU   1181               GAGAAAAUUAGUAGAUUUC   444   GAGAAAAUUAGUAGAUUUC   444   GAAAUCUACUAAUUUUCUC   1182               AUGACAGCAUGUCAGGGAG   445   AUGACAGCAUGUCAGGGAG   445   CUCCCUGACAUGCUGUCAU   1183               AGGCCAGAUGAGAGAACCA   446   AGGCCAGAUGAGAGAACCA   446   UGGUUCUCUCAUCUGGCCU   1184               AGAGAGAUGGGUGCGAGAG   447   AGAGAGAUGGGUGCGAGAG   447   CUCUCGCACCCAUCUCUCU   1185               ACCCAUGUUUUCAGCAUUA   448   ACCCAUGUUUUCAGCAUUA   448   UAAUGCUGAAAACAUGGGU   1186               GAUGACAGCAUGUCAGGGA   449   GAUGACAGCAUGUCAGGGA   449   UCCCUGACAUGGUGUCAUC   1187               AGCCAGGAAUGGAUGGCCC   450   AGCCAGGAAUGGAUGGCCC   450   GGGCCAUCCAUUCCUGGCU   1188               UGAUGACAGCAUGUCAGGG   451   UGAUGACAGCAUGUCAGGG   451   CCCUGACAUGCUGUCAUCA   1189               CAGGAAGCACUAUGGGCGC   452   CAGGAAGCACUAUGGGCGC   452   GCGCCCAUAGUGCUUCCUG   1190               ACAGACUCACAAUAUGCAU   453   ACAGACUCACAAUAUGCAU   453   AUGCAUAUUGUGAGUCUGU   1191               UGGAGGUUUUAUCAAAGUA   454   UGGAGGUUUUAUCAAAGUA   454   UACUUUGAUAAAACCUCCA   1192               AAGCCAGGAAUGGAUGGCC   455   AAGCCAGGAAUGGAUGGCC   455   GGCCAUCCAUUCCUGGCUU   1193               UUUUGACUAGCGGAGGCUA   456   UUUUGACUAGCGGAGGCUA   456   UAGCCUCCGCUAGUCAAAA   1194               CAGAUGCUGCAUAUAAGCA   457   CAGAUGCUGCAUAUAAGCA   457   UGCUUAUAUGCAGCAUCUG   1195               UUGGGCCUGAAAAUCCAUA   458   UUGGGCCUGAAAAUCCAUA   458   UAUGGAUUUUCAGGCCCAA   1196               GCAUGGACAAGUAGACUGU   459   GCAUGGACAAGUAGACUGU   459   ACAGUCUACUUGUCCAUGC   1197               ACCUGUCAACAUAAUUGGA   460   ACCUGUCAACAUAAUUGGA   460   UCCAAUUAUGUUGACAGGU   1198               CAGGAACUACUAGUACCCU   461   CAGGAACUACUAGUACCCU   461   AGGGUACUAGUAGUUCCUG   1199               AUAGCAACAGACAUACAAA   462   AUAGCAACAGACAUACAAA   462   UUUGUAUGUCUGUUGCUAU   1200               GGAGAGAGAUGGGUGCGAG   463   GGAGAGAGAUGGGUGCGAG   463   CUCGCACCCAUCUCUCUCC   1201               ACACCUGUCAACAUAAUUG   464   ACACCUGUCAACAUAAUUG   464   CAAUUAUGUUGACAGGUGU   1202               AGAAAUGAUGACAGCAUGU   465   AGAAAUGAUGACAGCAUGU   465   ACAUGCUGUCAUCAUUUCU   1203               AGAAGGAGAGAGAUGGGUG   466   AGAAGGAGAGAGAUGGGUG   466   CACCCAUCUCUCUCCUUCU   1204               AAUCAUUCAAGCACAACCA   467   AAUCAUUCAAGCACAACCA   467   UGGUUGUGCUUGAAUGAUU   1205               CAAAAAUUGGGCCUGAAAA   468   CAAAAAUUGGGCCUGAAAA   468   UUUUCAGGCCCAAUUUUUG   1206               GCAGUACAAAUGGCAGUAU   469   GCAGUACAAAUGGCAGUAU   469   AUACUGCCAUUUGUACUGC   1207               GGGCAGUAGUAAUACAAGA   470   GGGCAGUAGUAAUACAAGA   470   UCUUGUAUUACUACUGCCC   1208               UCAUUCAAGCACAACCAGA   471   UCAUUCAAGCACAACCAGA   471   UCUGGUUGUGCUUGAAUGA   1209               AUGAUGACAGCAUGUCAGG   472   AUGAUGACAGCAUGUCAGG   472   CCUGACAUGCUGUCAUCAU   1210               GAACAAGUAGAUAAAUUAG   473   GAACAAGUAGAUAAAUUAG   473   CUAAUUUAUCUACUUGUUC   1211               UGACAGCAUGUCAGGGAGU   474   UGACAGCAUGUCAGGGAGU   474   ACUCCCUGACAUGCUGUCA   1212               GGAACUACUAGUACCCUUC   475   GGAACUACUAGUACCCUUC   475   GAAGGGUACUAGUAGUUCC   1213               CACCUGUCAACAUAAUUGG   476   CACCUGUCAACAUAAUUGG   476   CCAAUUAUGUUGACAGGUG   1214               GGCCAGAUGAGAGAACCAA   477   GGCCAGAUGAGAGAACCAA   477   UUGGUUCUCUCAUCUGGCC   1215               UGUGUACCCACAGACCCCA   478   UGUGUACCCACAGACCCCA   478   UGGGGUCUGUGGGUACACA   1216               GGAAUCAUUCAAGCACAAC   479   GGAAUCAUUCAAGCACAAC   479   GUUGUGCUUGAAUGAUUCC   1217               CAGUACAAAUGGCAGUAUU   480   CAGUACAAAUGGCAGUAUU   480   AAUACUGCCAUUUGUACUG   1218               GCAGGAAGAAGCGGAGACA   481   GCAGGAAGAAGCGGAGACA   481   UGUCUCCGCUUCUUCCUGC   1219               AAAGCCAGGAAUGGAUGGC   482   AAAGCCAGGAAUGGAUGGC   482   GCCAUCCAUUCCUGGCUUU   1220               UGAACAAGUAGAUAAAUUA   483   UGAACAAGUAGAUAAAUUA   483   UAAUUUAUCUACUUGUUCA   1221               CAAAAAUUCAAAAUUUUCG   484   CAAAAAUUCAAAAUUUUCG   484   CGAAAAUUUUGAAUUUUUG   1222               UAGGACCUACACCUGUCAA   485   UAGGACCUACACCUGUCAA   485   UUGACAGGUGUAGGUCCUA   1223               GCCAGAUGAGAGAACCAAG   486   GCCAGAUGAGAGAACCAAG   486   CUUGGUUCUCUCAUCUGGC   1224               GACAGCUGGACUGUCAAUG   487   GACAGCUGGACUGUCAAUG   487   CAUUGACAGUCCAGCUGUC   1225               AAAGCCACCUUUGCCUAGU   488   AAAGCCACCUUUGCCUAGU   488   ACUAGGCAAAGGUGGCUUU   1226               GAAAUGAACAAGUAGAUAA   489   GAAAUGAACAAGUAGAUAA   489   UUAUCUACUUGUUCAUUUC   1227               ACAAUUUUAAAAGAAAAGG   490   ACAAUUUUAAAAGAAAAGG   490   CCUUUUCUUUUAAAAUUGU   1228               GCUGUGGAAAGAUACCUAA   491   GCUGUGGAAAGAUACCUAA   491   UUAGGUAUCUUUCCACAGC   1229               UGUCAACAUAAUUGGAAGA   492   UGUCAACAUAAUUGGAAGA   492   UCUUCCAAUUAUGUUGACA   1230               UAAAAGAAAAGGGGGGAUU   493   UAAAAGAAAAGGGGGGAUU   493   AAUCCCCCCUUUUCUUUUA   1231               CAAUUUUAAAAGAAAAGGG   494   CAAUUUUAAAAGAAAAGGG   494   CCCUUUUCUUUUAAAAUUG   1232               UUAGUAGAUUUCAGAGAAC   495   UUAGUAGAUUUCAGAGAAC   495   GUUCUCUGAAAUCUACUAA   1233               AAUUUUAAAAGAAAAGGGG   496   AAUUUUAAAAGAAAAGGGG   496   CCCCUUUUCUUUUAAAAUU   1234               UAGCAACAGACAUACAAAC   497   UAGCAACAGACAUACAAAC   497   GUUUGUAUGUCUGUUGCUA   1235               UGGAACAAGCCCCAGAAGA   498   UGGAACAAGCCCCAGAAGA   498   UCUUCUGGGGCUUGUUCCA   1236               AGGAUGAGGAUUAGAACAU   499   AGGAUGAGGAUUAGAACAU   499   AUGUUCUAAUCCUCAUCCU   1237               GACAAUUGGAGAAGUGAAU   500   GACAAUUGGAGAAGUGAAU   500   AUUCACUUCUCCAAUUGUC   1238               ACAGACCCCAACCCACAAG   501   ACAGACCCCAACCCACAAG   501   CUUGUGGGUUGGGGUCUGU   1239               CACCUAGAACUUUAAAUGC   502   CACCUAGAACUUUAAAUGC   502   GCAUUUAAAGUUCUAGGUG   1240               GAGCCAACAGCCCCACCAG   503   GAGCCAACAGCCCCACCAG   503   CUGGUGGGGCUGUUGGCUC   1241               AGGACCUACACCUGUCAAC   504   AGGACCUACACCUGUCAAC   504   GUUGACAGGUGUAGGUCCU   1242               UUACAAAAAUUCAAAAUUU   505   UUACAAAAAUUCAAAAUUU   505   AAAUUUUGAAUUUUUGUAA   1243               GGAGGUUUUAUCAAAGUAA   506   GGAGGUUUUAUCAAAGUAA   506   UUACUUUGAUAAAACCUCC   1244               CUGGCUGUGGAAAGAUACC   507   CUGGCUGUGGAAAGAUACC   507   GGUAUCUUUCCACAGCCAG   1245               GGAGAAGUGAAUUAUAUAA   508   GGAGAAGUGAAUUAUAUAA   508   UUAUAUAAUUCACUUCUCC   1246               AAUGAUGACAGCAUGUCAG   509   AAUGAUGACAGCAUGUCAG   509   CUGACAUGCUGUCAUCAUU   1247               AUCAUUAGGGAUUAUGGAA   510   AUCAUUAGGGAUUAUGGAA   510   UUCCAUAAUCCCUAAUGAU   1248               UCAAAAAUUGGGCCUGAAA   511   UCAAAAAUUGGGCCUGAAA   511   UUUCAGGCCCAAUUUUUGA   1249               ACCUACACCUGUCAACAUA   512   ACCUACACCUGUCAACAUA   512   UAUGUUGACAGGUGUAGGU   1250               GAUGAGGAUUAGAACAUGG   513   GAUGAGGAUUAGAACAUGG   513   CCAUGUUCUAAUCCUCAUC   1251               ACAGCUGGACUGUCAAUGA   514   ACAGCUGGACUGUCAAUGA   514   UCAUUGACAGUCCAGCUGU   1252               CCCUCAGAUGCUGCAUAUA   515   CCCUCAGAUGCUGCAUAUA   515   UAUAUGCAGCAUCUGAGGG   1253               AUUAGUAGAUUUCAGAGAA   516   AUUAGUAGAUUUCAGAGAA   516   UUCUCUGAAAUCUACUAAU   1254               AGAAAGAGCAGAAGACAGU   517   AGAAAGAGCAGAAGACAGU   517   ACUGUCUUCUGCUCUUUCU   1255               GACCUACACCUGUCAACAU   518   GACCUACACCUGUCAACAU   518   AUGUUGACAGGUGUAGGUC   1256               CACUCUUUGGCAACGACCC   519   CACUCUUUGGCAACGACCC   519   GGGUCGUUGCCAAAGAGUG   1257               AUGAGGAUUAGAACAUGGA   520   AUGAGGAUUAGAACAUGGA   520   UCCAUGUUCUAAUCCUCAU   1258               AUUUUAAAAGAAAAGGGGG   521   AUUUUAAAAGAAAAGGGGG   521   CCCCCUUUUCUUUUAAAAU   1259               AGAACUUUAAAUGCAUGGG   522   AGAACUUUAAAUGCAUGGG   522   CCCAUGCAUUUAAAGUUCU   1260               AUCUAUCAAUACAUGGAUG   523   AUCUAUCAAUACAUGGAUG   523   CAUCCAUGUAUUGAUAGAU   1261               AUGGAACAAGCCCCAGAAG   524   AUGGAACAAGCCCCAGAAG   524   CUUCUGGGGCUUGUUCCAU   1262               UUAUGACCCAUCAAAAGAC   525   UUAUGACCCAUCAAAAGAC   525   GUCUUUUGAUGGGUCAUAA   1263               CACAAUUUUAAAAGAAAAG   526   CACAAUUUUAAAAGAAAAG   526   CUUUUCUUUUAAAAUUGUG   1264               GAACUUUAAAUGCAUGGGU   527   GAACUUUAAAUGCAUGGGU   527   ACCCAUGCAUUUAAAGUUC   1265               AAAAGAAAAGGGGGGAUUG   528   AAAAGAAAAGGGGGGAUUG   528   CAAUCCCCCCUUUUCUUUU   1266               GGAUGGAAAGGAUCACCAG   529   GGAUGGAAAGGAUCACCAG   529   CUGGUGAUCCUUUCCAUCC   1267               AGGGGCAGUAGUAAUACAA   530   AGGGGCAGUAGUAAUACAA   530   UUGUAUUACUACUGCCCCU   1268               AAAGGGGGGAUUGGGGGGU   531   AAAGGGGGGAUUGGGGGGU   531   ACCCCCCAAUCCCCCCUUU   1269               AAGGGGGGAUUGGGGGGUA   532   AAGGGGGGAUUGGGGGGUA   532   UACCCCCCAAUCCCCCCUU   1270               CAGGAUGAGGAUUAGAACA   533   CAGGAUGAGGAUUAGAACA   533   UGUUCUAAUCCUCAUCCUG   1271               AAAAUUAGUAGAUUUCAGA   534   AAAAUUAGUAGAUUUCAGA   534   UCUGAAAUCUACUAAUUUU   1272               GAAUUGGAGGAAAUGAACA   535   GAAUUGGAGGAAAUGAACA   535   UGUUCAUUUCCUCCAAUUC   1273               UACAAAAAUUCAAAAUUUU   536   UACAAAAAUUCAAAAUUUU   536   AAAAUUUUGAAUUUUUGUA   1274               AGGAACUACUAGUACCCUU   537   AGGAACUACUAGUACCCUU   537   AAGGGUACUAGUAGUUCCU   1275               AAAGAAAAGGGGGGAUUGG   538   AAAGAAAAGGGGGGAUUGG   538   CCAAUCCCCCCUUUUCUUU   1276               AAAAAUUGGAUGACAGAAA   539   AAAAAUUGGAUGACAGAAA   539   UUUCUGUCAUCCAAUUUUU   1277               ACAGGAUGAGGAUUAGAAC   540   ACAGGAUGAGGAUUAGAAC   540   GUUCUAAUCCUCAUCCUGU   1278               ACAAUUGGAGAAGUGAAUU   541   ACAAUUGGAGAAGUGAAUU   541   AAUUCACUUCUCCAAUUGU   1279               GGAUGAGGAUUAGAACAUG   542   GGAUGAGGAUUAGAACAUG   542   CAUGUUCUAAUCCUCAUCC   1280               UCACCUAGAACUUUAAAUG   543   UCACCUAGAACUUUAAAUG   543   CAUUUAAAGUUCUAGGUGA   1281               AUUGGGCCUGAAAAUCCAU   544   AUUGGGCCUGAAAAUCCAU   544   AUGGAUUUUCAGGCCCAAU   1282               AAUUGGGCCUGAAAAUCCA   545   AAUUGGGCCUGAAAAUCCA   545   UGGAUUUUCAGGCCCAAUU   1283               GGACCUACACCUGUCAACA   546   GGACCUACACCUGUCAACA   546   UGUUGACAGGUGUAGGUCC   1284               GACAGGAUGAGGAUUAGAA   547   GACAGGAUGAGGAUUAGAA   547   UUCUAAUCCUCAUCCUGUC   1285               UCUAUCAAUACAUGGAUGA   548   UCUAUCAAUACAUGGAUGA   548   UCAUCCAUGUAUUGAUAGA   1286               GGAAUUGGAGGAAAUGAAC   549   GGAAUUGGAGGAAAUGAAC   549   GUUCAUUUCCUCCAAUUCC   1287               AAAAGGGGGGAUUGGGGGG   550   AAAAGGGGGGAUUGGGGGG   550   CCCCCCAAUCCCCCCUUUU   1288               AAAAUUGGAUGACAGAAAC   551   AAAAUUGGAUGACAGAAAC   551   GUUUCUGUCAUCCAAUUUU   1289               CAAUUGGAGAAGUGAAUUA   552   CAAUUGGAGAAGUGAAUUA   552   UAAUUCACUUCUCCAAUUG   1290               AUGACCCAUCAAAAGACUU   553   AUGACCCAUCAAAAGACUU   553   AAGUCUUUUGAUGGGUCAU   1291               CUUAAGCCUCAAUAAAGCU   554   CUUAAGCCUCAAUAAAGCU   554   AGCUUUAUUGAGGCUUAAG   1292               AGUACAAUGUGCUUCCACA   555   AGUACAAUGUGCUUCCACA   555   UGUGGAAGCACAUUGUACU   1293               UUUCCGCUGGGGACUUUCC   556   UUUCCGCUGGGGACUUUCC   556   GGAAAGUCCCCAGCGGAAA   1294               CAGACAUACAAACUAAAGA   557   CAGACAUACAAACUAAAGA   557   UCUUUAGUUUGUAUGUCUG   1295               UUAAGCCUCAAUAAAGCUU   558   UUAAGCCUCAAUAAAGCUU   558   AAGCUUUAUUGAGGCUUAA   1296               GGACAAUUGGAGAAGUGAA   559   GGACAAUUGGAGAAGUGAA   559   UUCACUUCUCCAAUUGUCC   1297               GGAUUGGGGGGUACAGUGC   560   GGAUUGGGGGGUACAGUGC   560   GCACUGUACCCCCCAAUCC   1298               AAAUUGGGCCUGAAAAUCC   561   AAAUUGGGCCUGAAAAUCC   561   GGAUUUUCAGGCCCAAUUU   1299               GGGGGAUUGGGGGGUACAG   562   GGGGGAUUGGGGGGUACAG   562   CUGUACCCCCCAAUCCCCC   1300               GUGGGGGGACAUCAAGCAG   563   GUGGGGGGACAUCAAGCAG   563   CUGCUUGAUGUCCCCCCAC   1301               UCCUGGCUGUGGAAAGAUA   564   UCCUGGCUGUGGAAAGAUA   564   UAUCUUUCCACAGCCAGGA   1302               ACAAAAAUUCAAAAUUUUC   565   ACAAAAAUUCAAAAUUUUC   565   GAAAAUUUUGAAUUUUUGU   1303               GGGGAUUGGGGGGUACAGU   566   GGGGAUUGGGGGGUACAGU   566   ACUGUACCCCCCAAUCCCC   1304               UAAACACAGUGGGGGGACA   567   UAAACACAGUGGGGGGACA   567   UGUCCCCCCACUGUGUUUA   1305               CAGACCCCAACCCACAAGA   568   CAGACCCCAACCCACAAGA   568   UCUUGUGGGUUGGGGUCUG   1306               AGGGGCAAAUGGUACAUCA   569   AGGGGCAAAUGGUACAUCA   569   UGAUGUACCAUUUGCCCCU   1307               AAUUGGAGGAAAUGAACAA   570   AAUUGGAGGAAAUGAACAA   570   UUGUUCAUUUCCUCCAAUU   1308               AAGCCACCUUUGCCUAGUG   571   AAGCCACCUUUGCCUAGUG   571   CACUAGGCAAAGGUGGCUU   1309               CCAUGUUUUCAGCAUUAUC   572   CCAUGUUUUCAGCAUUAUC   572   GAUAAUGCUGAAAACAUGG   1310               AAAGAAAAAAUCAGUAACA   573   AAAGAAAAAAUCAGUAACA   573   UGUUACUGAUUUUUUCUUU   1311               AAAAAAUUGGAUGACAGAA   574   AAAAAAUUGGAUGACAGAA   574   UUCUGUCAUCCAAUUUUUU   1312               CAGUACAAUGUGCUUCCAC   575   CAGUACAAUGUGCUUCCAC   575   GUGGAAGCACAUUGUACUG   1313               CUUUCCGCUGGGGACUUUC   576   CUUUCCGCUGGGGACUUUC   576   GAAAGUCCCCAGCGGAAAG   1314               GCAACAGACAUACAAACUA   577   GCAACAGACAUACAAACUA   577   UAGUUUGUAUGUCUGUUGC   1315               UAUCACCUAGAACUUUAAA   578   UAUCACCUAGAACUUUAAA   578   UUUAAAGUUCUAGGUGAUA   1316               ACCCACAGACCCCAACCCA   579   ACCCACAGACCCCAACCCA   579   UGGGUUGGGGUCUGUGGGU   1317               GAUAGAUGGAACAAGCCCC   580   GAUAGAUGGAACAAGCCCC   580   GGGGCUUGUUCCAUCUAUC   1318               GCUUAAGCCUCAAUAAAGC   581   GCUUAAGCCUCAAUAAAGC   581   GCUUUAUUGAGGCUUAAGC   1319               AUUGGGGGGUACAGUGCAG   582   AUUGGGGGGUACAGUGCAG   582   CUGCACUGUACCCCCCAAU   1320               CCCACAGACCCCAACCCAC   583   CCCACAGACCCCAACCCAC   583   GUGGGUUGGGGUCUGUGGG   1321               AAAAUUGGGCCUGAAAAUC   584   AAAAUUGGGCCUGAAAAUC   584   GAUUUUCAGGCCCAAUUUU   1322               CAUUCAAGCACAACCAGAU   585   CAUUCAAGCACAACCAGAU   585   AUCUGGUUGUGCUUGAAUG   1323               ACUUUAAAUGCAUGGGUAA   586   ACUUUAAAUGCAUGGGUAA   586   UUACCCAUGCAUUUAAAGU   1324               UAGAACUUUAAAUGCAUGG   587   UAGAACUUUAAAUGCAUGG   587   CCAUGCAUUUAAAGUUCUA   1325               CUUUAAAUGCAUGGGUAAA   588   CUUUAAAUGCAUGGGUAAA   588   UUUACCCAUGCAUUUAAAG   1326               GGGAUUGGGGGGUACAGUG   589   GGGAUUGGGGGGUACAGUG   589   CACUGUACCCCCCAAUCCC   1327               UAUGACCCAUCAAAAGACU   590   UAUGACCCAUCAAAAGACU   590   AGUCUUUUGAUGGGUCAUA   1328               GAAGAAGCGGAGACAGCGA   591   GAAGAAGCGGAGACAGCGA   591   UCGCUGUCUCCGCUUCUUC   1329               CCCAUGUUUUCAGCAUUAU   592   CCCAUGUUUUCAGCAUUAU   592   AUAAUGCUGAAAACAUGGG   1330               AGGAAUUGGAGGAAAUGAA   593   AGGAAUUGGAGGAAAUGAA   593   UUCAUUUCCUCCAAUUCCU   1331               AGAGACAGGCUAAUUUUUU   594   AGAGACAGGCUAAUUUUUU   594   AAAAAAUUAGCCUGUCUCU   1332               AAGUAGAUAAAUUAGUCAG   595   AAGUAGAUAAAUUAGUCAG   595   CUGACUAAUUUAUCUACUU   1333               AUGUUUUCAGCAUUAUCAG   596   AUGUUUUCAGCAUUAUCAG   596   CUGAUAAUGCUGAAAACAU   1334               UUAUUGUCUGGUAUAGUGC   597   UUAUUGUCUGGUAUAGUGC   597   GCACUAUACCAGACAAUAA   1335               AUUACAAAAAUUCAAAAUU   598   AUUACAAAAAUUCAAAAUU   598   AAUUUUGAAUUUUUGUAAU   1336               GCCAGGAAUGGAUGGCCCA   599   GCCAGGAAUGGAUGGCCCA   599   UGGGCCAUCCAUUCCUGGC   1337               CCUGGCUGUGGAAAGAUAC   600   CCUGGCUGUGGAAAGAUAC   600   GUAUCUUUCCACAGCCAGG   1338               UGUUUUCAGCAUUAUCAGA   601   UGUUUUCAGCAUUAUCAGA   601   UCUGAUAAUGCUGAAAACA   1339               ACCUAGAACUUUAAAUGCA   602   ACCUAGAACUUUAAAUGCA   602   UGCAUUUAAAGUUCUAGGU   1340               GGGAUGGAAAGGAUCACCA   603   GGGAUGGAAAGGAUCACCA   603   UGGUGAUCCUUUCCAUCCC   1341               AAUUAAAGCCAGGAAUGGA   604   AAUUAAAGCCAGGAAUGGA   604   UCCAUUCCUGGCUUUAAUU   1342               AAAGGAAUUGGAGGAAAUG   605   AAAGGAAUUGGAGGAAAUG   605   CAUUUCCUCCAAUUCCUUU   1343               ACUUUCCGCUGGGGACUUU   606   ACUUUCCGCUGGGGACUUU   606   AAAGUCCCCAGCGGAAAGU   1344               ACAGAAGAAAAAAUAAAAG   607   ACAGAAGAAAAAAUAAAAG   607   CUUUUAUUUUUUCUUCUGU   1345               AGCAACAGACAUACAAACU   608   AGCAACAGACAUACAAACU   608   AGUUUGUAUGUCUGUUGCU   1346               UAUUGUCUGGUAUAGUGCA   609   UAUUGUCUGGUAUAGUGCA   609   UGCACUAUACCAGACAAUA   1347               UUAAAAGAAAAGGGGGGAU   610   UUAAAAGAAAAGGGGGGAU   610   AUCCCCCCUUUUCUUUUAA   1348               UGCUUAAGCCUCAAUAAAG   611   UGCUUAAGCCUCAAUAAAG   611   CUUUAUUGAGGCUUAAGCA   1349               CAGGAAGAUGGCCAGUAAA   612   CAGGAAGAUGGCCAGUAAA   612   UUUACUGGCCAUCUUCCUG   1350               CCAGAUGAGAGAACCAAGG   613   CCAGAUGAGAGAACCAAGG   613   CCUUGGUUCUCUCAUCUGG   1351               GAUUGGGGGGUACAGUGCA   614   GAUUGGGGGGUACAGUGCA   614   UGCACUGUACCCCCCAAUC   1352               AAAUGAACAAGUAGAUAAA   615   AAAUGAACAAGUAGAUAAA   615   UUUAUCUACUUGUUCAUUU   1353               AGCCACCUUUGCCUAGUGU   616   AGCCACCUUUGCCUAGUGU   616   ACACUAGGCAAAGGUGGCU   1354               GACUUUCCGCUGGGGACUU   617   GACUUUCCGCUGGGGACUU   617   AAGUCCCCAGCGGAAAGUC   1355               CCAGUAAAAUUAAAGCCAG   618   CCAGUAAAAUUAAAGCCAG   618   CUGGCUUUAAUUUUACUGG   1356               GCAAUGUAUGCCCCUCCCA   619   GCAAUGUAUGCCCCUCCCA   619   UGGGAGGGGCAUACAUUGC   1357               AACUUUAAAUGCAUGGGUA   620   AACUUUAAAUGCAUGGGUA   620   UACCCAUGCAUUUAAAGUU   1358               UUGGGGGGUACAGUGCAGG   621   UUGGGGGGUACAGUGCAGG   621   CCUGCACUGUACCCCCCAA   1359               GGACUUUCCGCUGGGGACU   622   GGACUUUCCGCUGGGGACU   622   AGUCCCCAGCGGAAAGUCC   1360               CUAGAACUUUAAAUGCAUG   623   CUAGAACUUUAAAUGCAUG   623   CAUGCAUUUAAAGUUCUAG   1361               UCAGUACAAUGUGCUUCCA   624   UCAGUACAAUGUGCUUCCA   624   UGGAAGCACAUUGUACUGA   1362               AAGGAAUUGGAGGAAAUGA   625   AAGGAAUUGGAGGAAAUGA   625   UCAUUUCCUCCAAUUCCUU   1363               UACCCACAGACCCCAACCC   626   UACCCACAGACCCCAACCC   626   GGGUUGGGGUCUGUGGGUA   1364               GAGACAGGCUAAUUUUUUA   627   GAGACAGGCUAAUUUUUUA   627   UAAAAAAUUAGCCUGUCUC   1365               CUGCUUAAGCCUCAAUAAA   628   CUGCUUAAGCCUCAAUAAA   628   UUUAUUGAGGCUUAAGCAG   1366               AGGAAGAUGGCCAGUAAAA   629   AGGAAGAUGGCCAGUAAAA   629   UUUUACUGGCCAUCUUCCU   1367               AGACAUACAAACUAAAGAA   630   AGACAUACAAACUAAAGAA   630   UUCUUUAGUUUGUAUGUCU   1368               CAUGUUUUCAGCAUUAUCA   631   CAUGUUUUCAGCAUUAUCA   631   UGAUAAUGCUGAAAACAUG   1369               UUGGAAAGGACCAGCAAAG   632   UUGGAAAGGACCAGCAAAG   632   CUUUGCUGGUCCUUUCCAA   1370               GGCUGUUGGAAAUGUGGAA   633   GGCUGUUGGAAAUGUGGAA   633   UUCCACAUUUCCAACAGCC   1371               UAAAUGGAGAAAAUUAGUA   634   UAAAUGGAGAAAAUUAGUA   634   UACUAAUUUUCUCCAUUUA   1372               AGGAAGAAGCGGAGACAGC   635   AGGAAGAAGCGGAGACAGC   635   GCUGUCUCCGCUUCUUCCU   1373               AAAAAAGAAAAAAUCAGUA   636   AAAAAAGAAAAAAUCAGUA   636   UACUGAUUUUUUCUUUUUU   1374               AUCAGAAAGAACCUCCAUU   637   AUCAGAAAGAACCUCCAUU   637   AAUGGAGGUUCUUUCUGAU   1375               AGACCCCAACCCACAAGAA   638   AGACCCCAACCCACAAGAA   638   UUCUUGUGGGUUGGGGUCU   1376               CAAGUAGAUAAAUUAGUCA   639   CAAGUAGAUAAAUUAGUCA   639   UGACUAAUUUAUCUACUUG   1377               AAAGCUAUAGGUACAGUAU   640   AAAGCUAUAGGUACAGUAU   640   AUACUGUACCUAUAGCUUU   1378               UGCUGCAUAUAAGCAGCUG   641   UGCUGCAUAUAAGCAGCUG   641   CAGCUGCUUAUAUGCAGCA   1379               UUUAAAUGCAUGGGUAAAA   642   UUUAAAUGCAUGGGUAAAA   642   UUUUACCCAUGCAUUUAAA   1380               UUUUCAGCAUUAUCAGAAG   643   UUUUCAGCAUUAUCAGAAG   643   CUUCUGAUAAUGCUGAAAA   1381               ACUGCUUAAGCCUCAAUAA   644   ACUGCUUAAGCCUCAAUAA   644   UUAUUGAGGCUUAAGCAGU   1382               GGAAAGGACCAGCAAAGCU   645   GGAAAGGACCAGCAAAGCU   645   AGCUUUGCUGGUCCUUUCC   1383               UGUACCAGUAAAAUUAAAG   646   UGUACCAGUAAAAUUAAAG   646   CUUUAAUUUUACUGGUACA   1384               GAAGAAAAAAUAAAAGCAU   647   GAAGAAAAAAUAAAAGCAU   647   AUGCUUUUAUUUUUUCUUC   1385               GUGUACCCACAGACCCCAA   648   GUGUACCCACAGACCCCAA   648   UUGGGGUCUGUGGGUACAC   1386               GGGGGGAUUGGGGGGUACA   649   GGGGGGAUUGGGGGGUACA   649   UGUACCCCCCAAUCCCCCC   1387               GGAAGAAGCGGAGACAGCG   650   GGAAGAAGCGGAGACAGCG   650   CGCUGUCUCCGCUUCUUCC   1388               GAAGCGGAGACAGCGACGA   651   GAAGCGGAGACAGCGACGA   651   UCGUCGCUGUCUCCGCUUC   1389               UUAAAUGCAUGGGUAAAAG   652   UUAAAUGCAUGGGUAAAAG   652   CUUUUACCCAUGCAUUUAA   1390               AACCCACUGCUUAAGCCUC   653   AACCCACUGCUUAAGCCUC   653   GAGGCUUAAGCAGUGGGUU   1391               GUUUUCAGCAUUAUCAGAA   654   GUUUUCAGCAUUAUCAGAA   654   UUCUGAUAAUGCUGAAAAC   1392               GGAUUAAAUAAAAUAGUAA   655   GGAUUAAAUAAAAUAGUAA   655   UUACUAUUUUAUUUAAUCC   1393               GUACCCACAGACCCCAACC   656   GUACCCACAGACCCCAACC   656   GGUUGGGGUCUGUGGGUAC   1394               GAUUAAAUAAAAUAGUAAG   657   GAUUAAAUAAAAUAGUAAG   657   CUUACUAUUUUAUUUAAUC   1395               AAGCCUCAAUAAAGCUUGC   658   AAGCCUCAAUAAAGCUUGC   658   GCAAGCUUUAUUGAGGCUU   1396               GCAGGACAUAACAAGGUAG   659   GCAGGACAUAACAAGGUAG   659   CUACCUUGUUAUGUCCUGC   1397               CCCACUGCUUAAGCCUCAA   660   CCCACUGCUUAAGCCUCAA   660   UUGAGGCUUAAGCAGUGGG   1398               GGGACUUUCCGCUGGGGAC   661   GGGACUUUCCGCUGGGGAC   661   GUCCCCAGCGGAAAGUCCC   1399               AUCACCUAGAACUUUAAAU   662   AUCACCUAGAACUUUAAAU   662   AUUUAAAGUUCUAGGUGAU   1400               UAGAGCCCUGGAAGCAUCC   663   UAGAGCCCUGGAAGCAUCC   663   GGAUGCUUCCAGGGCUCUA   1401               GGGCUGUUGGAAAUGUGGA   664   GGGCUGUUGGAAAUGUGGA   664   UCCACAUUUCCAACAGCCC   1402               UUUCAGCAUUAUCAGAAGG   665   UUUCAGCAUUAUCAGAAGG   665   CCUUCUGAUAAUGCUGAAA   1403               UGACCCAUCAAAAGACUUA   666   UGACCCAUCAAAAGACUUA   666   UAAGUCUUUUGAUGGGUCA   1404               AGAAAAAAUAAAAGCAUUA   667   AGAAAAAAUAAAAGCAUUA   667   UAAUGCUUUUAUUUUUUCU   1405               AGAAGCGGAGACAGCGACG   668   AGAAGCGGAGACAGCGACG   668   CGUCGCUGUCUCCGCUUCU   1406               AAGAAAAAAUAAAAGCAUU   669   AAGAAAAAAUAAAAGCAUU   669   AAUGCUUUUAUUUUUUCUU   1407               AAUGGAGAAAAUUAGUAGA   670   AAUGGAGAAAAUUAGUAGA   670   UCUACUAAUUUUCUCCAUU   1408               GCUGAACAUCUUAAGACAG   671   GCUGAACAUCUUAAGACAG   671   CUGUCUUAAGAUGUUCAGC   1409               AAAAAGAAAAAAUCAGUAA   672   AAAAAGAAAAAAUCAGUAA   672   UUACUGAUUUUUUCUUUUU   1410               GAACAAGCCCCAGAAGACC   673   GAACAAGCCCCAGAAGACC   673   GGUCUUCUGGGGCUUGUUC   1411               GUGAUAAAUGUCAGCUAAA   674   GUGAUAAAUGUCAGCUAAA   674   UUUAGCUGACAUUUAUCAC   1412               GAGCCCUGGAAGCAUCCAG   675   GAGCCCUGGAAGCAUCCAG   675   CUGGAUGCUUCCAGGGCUC   1413               AGUGGGGGGACAUCAAGCA   676   AGUGGGGGGACAUCAAGCA   676   UGCUUGAUGUCCCCCCACU   1414               GCCUGGGAGCUCUCUGGCU   677   GCCUGGGAGCUCUCUGGCU   677   AGCCAGAGAGCUCCCAGGC   1415               UGGAAAGGACCAGCAAAGC   678   UGGAAAGGACCAGCAAAGC   678   GCUUUGCUGGUCCUUUCCA   1416               AGCAGGACAUAACAAGGUA   679   AGCAGGACAUAACAAGGUA   679   UACCUUGUUAUGUCCUGCU   1417               CCUAGAACUUUAAAUGCAU   680   CCUAGAACUUUAAAUGCAU   680   AUGCAUUUAAAGUUCUAGG   1418               AGUAGAUAAAUUAGUCAGU   681   AGUAGAUAAAUUAGUCAGU   681   ACUGACUAAUUUAUCUACU   1419               AAAUUAAAGCCAGGAAUGG   682   AAAUUAAAGCCAGGAAUGG   682   CCAUUCCUGGCUUUAAUUU   1420               AGUAAAAUUAAAGCCAGGA   683   AGUAAAAUUAAAGCCAGGA   683   UCCUGGCUUUAAUUUUACU   1421               UGUGAUAAAUGUCAGCUAA   684   UGUGAUAAAUGUCAGCUAA   684   UUAGCUGACAUUUAUCACA   1422               AGCCCUGGAAGCAUCCAGG   685   AGCCCUGGAAGCAUCCAGG   685   CCUGGAUGCUUCCAGGGCU   1423               CACUGCUUAAGCCUCAAUA   686   CACUGCUUAAGCCUCAAUA   686   UAUUGAGGCUUAAGCAGUG   1424               AAAAAAUCAGUAACAGUAC   687   AAAAAAUCAGUAACAGUAC   687   GUACUGUUACUGAUUUUUU   1425               GAGCCUGGGAGCUCUCUGG   688   GAGCCUGGGAGCUCUCUGG   688   CCAGAGAGCUCCCAGGCUC   1426               UUCCGCUGGGGACUUUCCA   689   UUCCGCUGGGGACUUUCCA   689   UGGAAAGUCCCCAGCGGAA   1427               GAGAGACAGGCUAAUUUUU   690   GAGAGACAGGCUAAUUUUU   690   AAAAAUUAGCCUGUCUCUC   1428               GCUGUGAUAAAUGUCAGCU   691   GCUGUGAUAAAUGUCAGCU   691   AGCUGACAUUUAUCACAGC   1429               CCACAGACCCCAACCCACA   692   CCACAGACCCCAACCCACA   692   UGUGGGUUGGGGUCUGUGG   1430               CAGGAAGAAGCGGAGACAG   693   CAGGAAGAAGCGGAGACAG   693   CUGUCUCCGCUUCUUCCUG   1431               UAAGCCUCAAUAAAGCUUG   694   UAAGCCUCAAUAAAGCUUG   694   CAAGCUUUAUUGAGGCUUA   1432               UAAAAAAGAAAAAAUCAGU   695   UAAAAAAGAAAAAAUCAGU   695   ACUGAUUUUUUCUUUUUUA   1433               GACAGAAGAAAAAAUAAAA   696   GACAGAAGAAAAAAUAAAA   696   UUUUAUUUUUUCUUCUGUC   1434               GUACCAGUAAAAUUAAAGC   697   GUACCAGUAAAAUUAAAGC   697   GCUUUAAUUUUACUGGUAC   1435               AAAAGAAAAAAUCAGUAAC   698   AAAAGAAAAAAUCAGUAAC   698   GUUACUGAUUUUUUCUUUU   1436               AAAAAUCAGUAACAGUACU   699   AAAAAUCAGUAACAGUACU   699   AGUACUGUUACUGAUUUUU   1437               AGAGCCCUGGAAGCAUCCA   700   AGAGCCCUGGAAGCAUCCA   700   UGGAUGCUUCCAGGGCUCU   1438               CAGGGGCAAAUGGUACAUC   701   CAGGGGCAAAUGGUACAUC   701   GAUGUACCAUUUGCCCCUG   1439               CUGCAUUUACCAUACCUAG   702   CUGCAUUUACCAUACCUAG   702   CUAGGUAUGGUAAAUGCAG   1440               UAAAUGCAUGGGUAAAAGU   703   UAAAUGCAUGGGUAAAAGU   703   ACUUUUACCCAUGCAUUUA   1441               AAGUAAACAUAGUAACAGA   704   AAGUAAACAUAGUAACAGA   704   UCUGUUACUAUGUUUACUU   1442               CCACACAUGCCUGUGUACC   705   CCACACAUGCCUGUGUACC   705   GGUACACAGGCAUGUGUGG   1443               AGUAGAUUUCAGAGAACUU   706   AGUAGAUUUCAGAGAACUU   706   AAGUUCUCUGAAAUCUACU   1444               CAUCAGAAAGAACCUCCAU   707   CAUCAGAAAGAACCUCCAU   707   AUGGAGGUUCUUUCUGAUG   1445               ACCAGUAAAAUUAAAGCCA   708   ACCAGUAAAAUUAAAGCCA   708   UGGCUUUAAUUUUACUGGU   1446               CACAGACCCCAACCCACAA   709   CACAGACCCCAACCCACAA   709   UUGUGGGUUGGGGUCUGUG   1447               AGGGGGGAUUGGGGGGUAC   710   AGGGGGGAUUGGGGGGUAC   710   GUACCCCCCAAUCCCCCCU   1448               UGCAUUUACCAUACCUAGU   711   UGCAUUUACCAUACCUAGU   711   ACUAGGUAUGGUAAAUGCA   1449               CAAUGGACAUAUCAAAUUU   712   CAAUGGACAUAUCAAAUUU   712   AAAUUUGAUAUGUCCAUUG   1450               CUGAACAUCUUAAGACAGC   713   CUGAACAUCUUAAGACAGC   713   GCUGUCUUAAGAUGUUCAG   1451               GCCUCAAUAAAGCUUGCCU   714   GCCUCAAUAAAGCUUGCCU   714   AGGCAAGCUUUAUUGAGGC   1452               UGUACCCACAGACCCCAAC   715   UGUACCCACAGACCCCAAC   715   GUUGGGGUCUGUGGGUACA   1453               GAAGUAAACAUAGUAACAG   716   GAAGUAAACAUAGUAACAG   716   CUGUUACUAUGUUUACUUC   1454               GUAGGACCUACACCUGUCA   717   GUAGGACCUACACCUGUCA   717   UGACAGGUGUAGGUCCUAC   1455               CAGUGGGGGGACAUCAAGC   718   CAGUGGGGGGACAUCAAGC   718   GCUUGAUGUCCCCCCACUG   1456               ACCCACUGCUUAAGCCUCA   719   ACCCACUGCUUAAGCCUCA   719   UGAGGCUUAAGCAGUGGGU   1457               AAAAAUUGGGCCUGAAAAU   720   AAAAAUUGGGCCUGAAAAU   720   AUUUUCAGGCCCAAUUUUU   1458               UGGGGGGACAUCAAGCAGC   721   UGGGGGGACAUCAAGCAGC   721   GCUGCUUGAUGUCCCCCCA   1459               GUACAAAUGGCAGUAUUCA   722   GUACAAAUGGCAGUAUUCA   722   UGAAUACUGCCAUUUGUAC   1460               AAGCUAUAGGUACAGUAUU   723   AAGCUAUAGGUACAGUAUU   723   AAUACUGUACCUAUAGCUU   1461               CAGAAGAAAAAAUAAAAGC   724   CAGAAGAAAAAAUAAAAGC   724   GCUUUUAUUUUUUCUUCUG   1462               AAAUGCAUGGGUAAAAGUA   725   AAAUGCAUGGGUAAAAGUA   725   UACUUUUACCCAUGCAUUU   1463               AGCCUCAAUAAAGCUUGCC   726   AGCCUCAAUAAAGCUUGCC   726   GGCAAGCUUUAUUGAGGCU   1464               CCACUGCUUAAGCCUCAAU   727   CCACUGCUUAAGCCUCAAU   727   AUUGAGGCUUAAGCAGUGG   1465               AAGAAGCGGAGACAGCGAC   728   AAGAAGCGGAGACAGCGAC   728   GUCGCUGUCUCCGCUUCUU   1466               AAAUGGAGAAAAUUAGUAG   729   AAAUGGAGAAAAUUAGUAG   729   CUACUAAUUUUCUCCAUUU   1467               AGCCUGGGAGCUCUCUGGC   730   AGCCUGGGAGCUCUCUGGC   730   GCCAGAGAGCUCCCAGGCU   1468               AACAAGCCCCAGAAGACCA   731   AACAAGCCCCAGAAGACCA   731   UGGUCUUCUGGGGCUUGUU   1469               UACCAGUAAAAUUAAAGCC   732   UACCAGUAAAAUUAAAGCC   732   GGCUUUAAUUUUACUGGUA   1470               UUCAAAAAUUGGGCCUGAA   733   UUCAAAAAUUGGGCCUGAA   733   UUCAGGCCCAAUUUUUGAA   1471               AGAAGAAAAAAUAAAAGCA   734   AGAAGAAAAAAUAAAAGCA   734   UGCUUUUAUUUUUUCUUCU   1472               CUGUGUACCCACAGACCCC   735   CUGUGUACCCACAGACCCC   735   GGGGUCUGUGGGUACACAG   1473               GCCUGUACUGGGUCUCUCU   736   GCCUGUACUGGGUCUCUCU   736   AGAGAGACCCAGUACAGGC   1474               CAGUAAAAUUAAAGCCAGG   737   CAGUAAAAUUAAAGCCAGG   737   CCUGGCUUUAAUUUUACUG   1475               UACAAAUGGCAGUAUUCAU   738   UACAAAUGGCAGUAUUCAU   738   AUGAAUACUGCCAUUUGUA   1476                                  
 
         [0262]    [0262]                                                                                                                                                                                                                                                                                                                                 TABLE II                           A. 2.5 μmol Synthesis Cycle ABI 394 Instrument            Reagent   Equivalents   Amount   Wait Time* DNA   Wait Time* 2′-O-methyl   Wait Time* RNA                    Phosphoramidites   6.5   163   μL   45   sec   2.5   min   7.5   min       S-Ethyl Tetrazole   23.8   238   μL   45   sec   2.5   min   7.5   min       Acetic Anhydride   100   233   μL   5   sec   5   sec   5   sec       N-Methyl   186   233   μL   5   sec   5   sec   5   sec       Imidazole       TCA   176   2.3   mL   21   sec   21   sec   21   sec       Iodine   11.2   1.7   mL   45   sec   45   sec   45   sec       Beaucage   12.9   645   μL   100   sec   300   sec   300   sec            Acetonitrile   NA   6.67   mL   NA   NA   NA                    B. 0.2 μmol Synthesis Cycle ABI 394 Instrument            Reagent   Equivalents   Amount   Wait Time* DNA   Wait Time* 2′-O-methyl   Wait Time* RNA                    Phosphoramidites   15   31   μL   45   sec   233   sec   465   sec       S-Ethyl Tetrazole   38.7   31   μL   45   sec   233   mm   465   sec       Acetic Anhydride   655   124   μL   5   sec   5   sec   5   sec       N-Methyl   1245   124   μL   5   sec   5   sec   5   sec       Imidazole       TCA   700   732   μL   10   sec   10   sec   10   sec       Iodine   20.6   244   μL   15   sec   15   sec   15   sec       Beaucage   7.7   232   μL   100   sec   300   sec   300   sec            Acetonitrile   NA   2.64   mL   NA   NA   NA                    C. 0.2 μmol Synthesis Cycle 96 well Instrument                Equivalents: DNA/   Amount: DNA/2′-O-       Wait Time* 2′-O-           Reagent   2′-O-methyl/Ribo   methyl/Ribo   Wait Time* DNA   methyl   Wait Time* Ribo                    Phosphoramidites   22/33/66   40/60/120   μL   60   sec   180   sec   36O   sec       S-Ethyl Tetrazole   70/105/210   40/60/120   μL   60   sec   180   min   360   sec       Acetic Anhydride   265/265/265   50/50/50   μL   10   sec   10   sec   10   sec       N-Methyl   502/502/502   50/50/50   μL   10   sec   10   sec   10   sec       Imidazole       TCA   238/475/475   250/500/500   μL   15   sec   15   sec   15   sec       Iodine   6.8/6.8/6.8   80/80/80   μL   30   sec   30   sec   30   sec       Beaucage   34/51/51   80/120/120       100   sec   200   sec   200   sec            Acetonitrile   NA   1150/1150/1150   μL   NA   NA   NA                                    
         [0263]    [0263]                             TABLE III                           HUMAN HIV-1 SEQUENCES            Accession   Name   Subtype               AF069669   SE8538   A       AF069671   SE7535   A       AF069673   SE8891   A       AF107771   UGSE8131   A       AF193275   97BL006 AF193275   A       AF361872   97TZ02 AF361872   A       AF361873   97TZ03 AF361873   A       AF413987   98UA0116 AF413987   A       AF004885   Q23-17   A1       AF069670   SE7253   A1       M62320   U455 U455A   A1       U51190   92UG037   A1       AF286237   94CY017.41   A2       AF286238   97CDKTB48   A2       A04321   IIIB LAI   B       AB078005   ARES2 AB078005   B       AF003887   WC001   B       AF003888   NL43WC001   B       AF004394   AD87 ADA   B       AF033819   HXB2-copy LAI   B       AF042100   MBC200   B       AF042101   MBC925   B       AF042102   MBC18 MBCC18   B       AF042103   MBCC54   B       AF042104   MBCC98   B       AF042105   MBCD36   B       AF042106   MBCC08R01 C18R01   B       AF049494   499JC16   B       AF049495   NC7   B       AF069140   DH12-3   B       AF070521   NL43E9 LAI IIIB/NY5   B       AF075719   MNTQ MNclone TQ   B       AF086817   TWCYS LM49   B       AF146728   VH   B       AF224507   WK   B       AF256204   S61I1 AF256204   B       AF256205   S61D15 AF256205   B       AF256206   S61G1 AF256206   B       AF256207   S61G7 AF256207   B       AF256208   S61I15 AF256208   B       AF256209   S61K1 AF256209   B       AF256210   S61K15 AF256210   B       AF256211   S61Dl1   B       AF286365   WR27   B       AJ006287   89SP061 89ES061   B       AJ271445   GB8 GB8-46R HIM271445   B       AX078307   BH10   B       AY037268   ARCH054   B       AY037269   ARMS008   B       AY037270   BOL 122   B       AY037274   ARMA173   B       AY037282   ARMA132   B       D10112   CAM1   B       D86068   MCK1   B       D86069   PM213   B       K02007   SF2 LAV2 ARV2   B       K02013   LAI BRU   B       K02083   PV22   B       K03455   HXB2 HXB2CG HXB2R LAI   B       L02317   BC BCSG3   B       L31963   TH475A LAI   B       M15654   BH102 BH10   B       M17449   MNCG MN   B       M17451   RF HAT3   B       M19921   NL43 pNL43 NL4-3   B       M26727   OYI, 397   B       M38429   JRCSF JR-CSF   B       M38431   NY5CG   B       M93258   YU2 YU2X   B       M93259   YU10   B       NC_001802   HXB2R   B       U12055   LW123   B       U21135   WEAU160 GHOSH   B       U23487   contaminant MANC   B       U26546   WR27   B       U26942   NL4-3 LAI/NY5 pNL43 NL43   B       U34603   H0320-2A12 ACH3202A12   B       U34604   3202A21 ACH3202A21   B       U37270   C18MBC   B       U39362   P896 89.6   B       U43096   D31   B       U43141   HAN   B       U63632   JRFL JR-FL   B       U69584   85WCIPR54   B       U69585   WCIPR854   B       U69586   WCIPR8546   B       U69587   WCIPR8552   B       U69588   WCIPR855   B       U69589   WCIPR9011   B       U69590   WCIPR9012   B       U69591   WCIPR9018   B       U69592   WCIPR9031   B       U69593   WCIPR9032   B       U71182   RL42   B       X01762   REHTLV3 LAI IIIB   B       Z11530   F12CG   B                    
         [0264]    [0264]                             TABLE IV                           HUMAN HIV-1 SEQUENCES            Accession   Name   Subtype               AB032740   95TNIH022   01_AE       AB032741   95TNIH047   01_AE       AB052995   93JPNH1   01_AE       AB070352   NH25 93JPNH25T 93JP-NH2.5T   01_AE       AB070353   NH2 93JPNH2ENV   01_AE       AF164485   93TH9021   01_AE       AF197338   93TH057   01_AE       AF197339   93TH065   01_AE       AF197340   90CF11697 AF197340   01_AE       AF197341   90CF4071 AF197341   01_AE       AF259954   CM235-2   01_AE       AF259955   CM235-4   01_AE       AY008714   97CNGX2F 97CNGX-2F   01_AE       AY008718   97CNGX11F   01_AE       U51188   90CF402 90CR402 CAR-E 4002   01_AE       U51189   93TH253   01_AE       U54771   CM240   01_AE       AF362994   NP1623   01B       AY082968   TH1326 AY082968   01B       AJ404325   97DCKTB49 97CDKTB49 HIM404325   01GHJKU       AB049811   97GHAG1 AB049811   02_AG       AB052867   AB052867   02_AG       AF063223   DJ263   02_AG       AF063224   DJ264   02_AG       AF107770   SE7812   02_AG       AF184155   G829   02_AG       AF377954   CM52885 AF377954   02_AG       AF377955   CM53658 AF377955   02_AG       AJ251056   MP1211 98SE-MP1211   02_AG       AJ251057   MP1213 98SEMP1213 HIM251057   02_AG       AJ286133   97CM-MP807   02_AG       L39106   IBNG   02_AG       AF193276   KAL153-2   03_AB       AF193277   RU98001 98RU001   03_AB       AF414006   98BY10443 AF414006   03-AB       AF049337   94CY032-3 CY032.3   04_cpx       AF119819   97PVMY GR84   04_cpx       AF119820   97PVCH GR11   04_cpx       AF076998   VI961   05_DF       AF193253   VI1310 AF193253   05_DF       AF064699   BFP90   06_cpx       AJ245481   95ML84   06_cpx       AJ288981   97SE1078   06_cpx       AJ288982   95ML127   06_cpx       AF286226   97CN001 054   07_BC       AF286230   98CN009   07_BC       AX149647   C54A C54   07_BC       AX149672   C54D AX149672   07_BC       AX149771   CN54b   07_BC       AX149898   C54C   07_BC       AF286229   98CN006   08_BC       AY008715   97CNGX6F   08_BC       AY008716   97CNGX7F   08_BC       AY008717   97CNGX9F   08_BC       AF289548   96TZBF061   10_CD       AF289549   96TZBF071   10_CD       AF289550   96TZBF110   10_CD       AF179368   GR17   11_cpx       AJ291718   MP818   11_cpx       AJ291719   MP1298   11_cpx       AJ291720   MP1307   11_cpx       AF385934   URTR23   12_BF       AF385935   URTR35   12_BF       AF385936   ARMA159   12_BF       AF408629   A32879 AF408629   12_BF       AF408630   A32989 AF408630   12_BF       AY037279   ARMA185   12_BF       AF423756   X397 AF423756   14_BG       AF423757   X421 AF423757   14_BG       AF423758   X475 AF423758   14_BG       AF423759   X477 AF423759   14_BG       AF450096   X605 AF450096   14_BG       AF450097   X623 AF450097   14_BG       AF069669   SE8538   A       AF069671   SE7535   A       AF069673   SE8891   A       AF107771   UGSE8131   A       AF193275   97BL006 AF193275   A       AF361872   97TZ02 AF361872   A       AF361873   97TZ03 AF361873   A       AF413987   98UA0116 AF413987   A       AF004885   Q23-17   A1       AF069670   SE7253   A1       M62320   U455 U455A   A1       U51190   92UG037   A1       AF286237   94CY017.41   A2       AF286238   97CDKTB48   A2       U86780   ZAM184   A2C       AF286239   97KR004   A2D       AF316544   97CDKP58   A2G       AF067156   95IN21301   AC       AF071474   SE9488   AC       AF361871   97TZ01 AF361871   AC       AF361876   97TZ06 AF361876   AC       AF361878   97TZ08 AF361878   AC       AF361879   97TZ09 AF361879   AC       U88823   92RW009   AC       AF075702   SE8603   ACD       AJ276595   VI1035   ACG       AF071473   SE7108   AD       AF075701   SE6954   AD       AJ237565   97NOGIL3   ADHK       X04415   MAL MALCG   ADK       AF377959   CM53379 AF377959   AFGHJU       AF377957   CM53392 AF377957   AG       AJ276596   VI1197   AG       U88825   92NG003   AG       AF076474   VI354   AGHU       AF192135   BW2117   AGJ       AJ293865   B76 HIM293865   AGJ       AF069672   SE6594   AU       A04321   IIIB LAI   B       AB078005   ARES2 AB078005   B       AF003887   WC001   B       AF003888   NL43WC001   B       AF004394   AD87 ADA   B       AF033819   HXB2-copy LAI   B       AF042100   MBC200   B       AF042101   MBC925   B       AF042102   MBC18 MBCC18   B       AF042103   MBCC54   B       AF042104   MBCC98   B       AF042105   MBCD36   B       AF042106   MBCC18R01 C18R01   B       AF049494   499JC16   B       AF049495   NC7   B       AF069140   DH12-3   B       AF070521   NL43E9 LAI IIIB/NY5   B       AF075719   MNTQ MNcloneTQ   B       AF086817   TWCYS LM49   B       AF146728   VH   B       AF224507   WK   B       AF256204   S61I1 AF256204   B       AF256205   S61D15 AF256205   B       AF256206   S61G1 AF256206   B       AF256207   S61G7 AF256207   B       AF256208   S61I15 AF256208   B       AF256209   S61K1 AF256209   B       AF256210   S61K15 AF256210   B       AF256211   S61D1   B       AF286365   WR27   B       AJ006287   89SP061 89ES061   B       AJ271445   GB8 GB8-46R HIM271445   B       AX078307   BH10   B       AY037268   ARCH054   B       AY037269   ARMS008   B       AY037270   BOL122   B       AY037274   ARMA173   B       AY037282   ARMA132   B       D10112   CAM1   B       D86068   MCK1   B       D86069   PM213   B       K02007   SF2 LAV2 ARV2   B       K02013   LAI BRU   B       K02083   PV22   B       K03455   HXB2 HXB2CG HXB2R LAI   B       L02317   BC BCSG3   B       L31963   TH475A LAI   B       M15654   BH102 BH10   B       M17449   MNCG MN   B       M17451   RF HAT3   B       M19921   NL43 pNL43 NL4-3   B       M26727   OYI, 397   B       M38429   JRCSF JR-CSF   B       M38431   NY5CG   B       M93258   YU2 YU2X   B       M93259   YU10   B       NC_001802   HXB2R   B       U12055   LW123   B       U21135   WEAU160 GHOSH   B       U23487   contaminant MANC   B       U26546   WR27   B       U26942   NL4-3 LAI/NY5 pNL43 NL43   B       U34603   H0320-2A12 ACH3202A12   B       U34604   3202A21 ACH3202A21   B       U37270   C18MBC   B       U39362   P896 89.6   B       U43096   D31   B       U43141   HAN   B       U63632   JRFL JR-FL   B       U69584   85WCIPR54   B       U69585   WCIPR854   B       U69586   WCIPR8546   B       U69587   WCIPR8552   B       U69588   WCIPR855   B       U69589   WCIPR9011   B       U69590   WCIPR9012   B       U69591   WCIPR9018   B       U69592   WCIPR9031   B       U69593   WCIPR9032   B       U71182   RL42   B       X01762   REHTLV3 LAI IIIB   B       Z11530   F12CG   B       AF332867   A027 AF332867   BF       AF408626   A025 AF408626   BF       AF408627   A047 AF408627   BF       AF408628   A063 AF408628   BF       AF408631   A050 AF408631   BF       AE408632   A32878 AF408632   BF       AY037266   ARCH014   BF       AY037267   ARCH003   BF       AY037271   BOL137   BF       AY037272   URTR17   BF       AY037273   ARMA062   BF       AY037275   ARMA036   BF       AY037276   ARMA070   BF       AY037277   ARMA037   BF       AY037278   ARMA006   BF       AY037280   ARMA097   BF       AY037281   ARMA038   BF       AY037283   ARMA029   BF       AF005495   93BR029.4   BF1       AF423755   X254 AF423755   BG       AB023804   93IN101   C       AF067154   93IN999 301999   C       AF067155   95IN21068   C       AF067157   93IN904 301904   C       AF067158   93IN905 301905   C       AF067159   94IN11246   C       AF110959   96BW01B03 96BW01B03   C       AF110960   96BW01B21   C       AF110961   96BW01B22   C       AF110962   96BW0402   C       AF110963   96BW0407   C       AF110964   96BW0408   C       AF110965   96BW0409   C       AF110966   96BW0410   C       AF110967   96BW0502   C       AF110968   96BW0504   C       AF110969   96BW1104   C       AF110970   96BW1106   C       AF110971   96BW11B01   C       AF110972   96BW1210   C       AF110973   96BW15B03   C       AF110974   96BW15C02   C       AF110975   96BW15C05   C       AF110976   96BW16B01   C       AF110977   96BW16D14   C       AF110978   96BW1626   C       AF110979   96BW17A09   C       AF110980   96BW17B03   C       AF110981   96BW17B05   C       AF286223   94IN476   C       AF286224   96ZM651   C       AF286225   96ZM751   C       AF286227   97ZA012   C       AF286228   98BR004   C       AF286231   98IN012   C       AF286232   98IN022   C       AF286233   98IS002   C       AF286234   98TZ013   C       AF286235   98TZ017   C       AF290027   96BW06H51 96BW06-H51   C       AF290028   96BW06J4   C       AF290029   96BW06J7 AF290029   C       AF290030   96BW06K18 AF290030   C       AF321523   MJ4   C       AF361874   97TZ04 AF361874   C       AF361875   97TZ05 AF361875   C       AF443074   96BWMO15   C       AF443075   96BWM032 AF443075   C       AF443076   98BWMC122 AF443076   C       AF443077   98BWMC134 AF443077   C       AF443078   98BWMC14A3 AF443078   C       AF443079   988WMO1410 AF443079   C       AF443080   98BWMO18D5 AF443080   C       AF443081   98BWMO36A5 AF443081   C       AF443082   98BWMO37D5 AF443082   C       AF443083   99BW393212 AF443083   C       AF443084   99BW46424 AF443084   C       AF443085   99BW47458 AF443085   C       AF443086   99BW47547 AF443086   C       AF443087   99BWMC168 AF443087   C       AF443088   00BW07621 AF443088   C       AF443089   00BW076820 AF443089   C       AF443090   00BW087421 AF443090   C       AF443091   00BW147127 AF443091   C       AF443092   00BW16162 AF443092   C       AF443093   00BW1686. 00BW16868 AF443093   C       AF443094   00BW17593 AF443094   C       AF443095   00BW17732 AF443095   C       AF443096   00BW17835 AF443096   C       AF443097   00BW17956 AF443097   C       AF443098   00BW18113 AF443098   C       AF443099   00BW18595 AF443099   C       AF443100   00BW18802 AF443100   C       AF443101   00BW192113 AF443101   C       AF443102   00BW20361 AF443102   C       AF443103   00BW20636 AF443103   C       AF443104   00BW20872 AF443104   C       AF443105   00BW2127214 AF443105   C       AF443106   00BW21283 AF443106   C       AF443107   00BW22767 AF443107   C       AF443108   00BW38193 AF443108   C       AF443109   00BW38428 AF443109   C       AF443110   00BW38713 AF443110   C       AF443111   00BW38769   C       AF443112   00BW38868   C       AF443113   00BW38916   C       AF443114   00BW39702   C       AF443115   00BW50311   C       AY043173   DU151 AY043173   C       AY043174   DU179 AY043174   C       AY043175   DU422 AY043175   C       AY043176   CTSC2 AY043176   C       U46016   ETH2220 02220   C       U52953   92BR025   C       AF361877   97TZ07 AF361877   CD       AY074891   00BWMO351 AY074891   CD       AF133821   MB2059   D       AJ320484   HIM320484   D       K03454   ELI   D       M22639   Z2Z6 Z2 CDC-Z34   D       M27323   NDK   D       U88822   84ZR085   D       U88824   94UG1141   D       AF005494   93BR020.1   F1       AF075703   FIN9363   F1       AF077336   VI850   F1       AJ249238   MP411 96FRMP411   F1       AF377956   CM53657 AF377956   F2       AJ249236   MP255 95CMMP255   F2       AJ249237   MP257 95CM-MP257C   F2       AF076475   VI1126   F2KU       AF061640   HH8793-1.1   G       AF061641   HH8793-12.1   G       AF061642   SE6165 G6165   G       AF084936   DRCBL   G       AF423760   X558 AF423760   G       AF450098   X138 AF450098   G       U88826   92NG083 JV10832   G       AF005496   90CF056 90CR056   H       AF190127   VI991   H       AF190128   VI997   H       AF082394   SE7887 SE92809   J       AF082395   SE7022 SE9173   J       AJ249235   EQTB11C 97ZR-EQTB11C   K       AJ249239   MP535 96CM-MP535C   K       AJ239083   97CA-MP645M/O   MO       AJ006022   YBF30   N       AJ271370   YBF106   N       AF407418   VAU AF407418   O       AF407419   VAU AF407419   O       AJ302646   SEMP1299 HIM302646   O       AJ302647   SEMP1300 HIM302647   O       L20571   MVP5180   O       L20587   ANT70   O       NC_002787   SEMP1299 NC_002787   O       AF286236   83CD003 Z3 AF286236   U       AF457101   90CD121E12 AF457101   U       AY046058   GR303 99GR303 AY046058   U