# Patent

## Title
Trans-acting RNA switches

## Abstract

Disclosed are RNA constructs which function to activate or inactivate a biological process, e.g., may be designed for attachment to a polypeptide coding region. Such RNA constructs modulate translation of a polypeptide from the coding region in response to the presence of a target polynucleotide in an expression environment. Such RNA constructs include a weakened stem-loop structure which, when bound to the target polynucleotide, assumes stem-loop secondary structure and associates with an RNA binding protein. Association with the RNA binding protein modulates translation of the polypeptide coding region. Such RNA constructs also have three-way junction joining regions 3′ and 5′ of the stem-loop structure.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a divisional of U.S. application Ser. No. 13/321,816, filed Jun. 12, 2012, issued as U.S. Pat. No. 8,841,438 on Sep. 23, 2014, which is a national stage of International (PCT) Patent Application Serial No. PCT/US2010/035870, filed May 21, 2010, and published under PCT Article 21(2) in English, which claims priority to and the benefit of U.S. provisional patent application Ser. No. 61/180,670, filed May 22, 2009, the disclosure of each of which is incorporated herein by reference in its entirety.


STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
This invention was made with government support under grant no. HG003679 awarded by the National Institutes of Health. The government has certain rights in the invention.


FIELD OF THE INVENTION
The invention relates generally to recombinant or synthetic nucleic acid constructs that are capable of interacting with target polynucleotides such that the interaction modulates the structure of the nucleic acid construct, leading to a targeted biochemical change such as an altered expression of a polypeptide from an associated nucleic acid. More specifically, the invention relates to RNA constructs that either express or cease to express a polypeptide depending on the presence of a target polynucleotide.
BACKGROUND
In nature, RNA not only functions as a component of the information transfer processes, but also performs tasks that are typically accomplished by proteins, including molecular recognition and catalysis. For example, naturally-occurring RNA molecules can interact to induce conformational changes, which in turn initiate biochemical reactions and other cellular processes. A significant advantage could be gained if these interactions could be harnessed to induce specific cellular processes under particular conditions.
SUMMARY OF THE INVENTION
This invention relates, in part, to a class of new chemical structures that can modulate a biochemical activity within a selected environment. For example, these structures can modulate biochemical activity, through expression of polypeptides, in cells and tissue infected by a pathogen, neoplastic cells and tissues, cells and tissues deficient in one or more factors, and scientific and diagnostic assays. Selectivity is achieved by targeting a polynucleotide that is unique to, or in abundance in, the cells or tissue relative to other cells or tissue. Nucleic acid constructs within this class of chemical structures include RNA constructs which are an RNA that hybridizes with all or a portion of the target polynucleotide and which are selected such that the hybridization induces a new conformation of the RNA construct. The conformational change can take many forms, but typically acts as a trigger to activate, suppress, or modulate a cascade of biochemical events, leading to some prophylactic or therapeutic effect or providing a means of diagnosing the presence of a given target polynucleotide. For example, the new molecular conformation may allow for a polypeptide from an attached polypeptide coding region to be expressed in all cells containing the target polynucleotide. The polypeptide coding region may code for an apoptotic gene, for example. Thus, an RNA construct could target, for example, a viral RNA with the result that all virus-infected cells die and/or all viruses in virally-infected cells die and the infection is arrested. Alternatively, when placed in an expression environment containing a target polynucleotide, the RNA construct could allow for the expression of a encoded polypeptide which can be detected, thus providing a diagnostic tool.
Thus, one aspect of the invention involves an RNA construct that includes a non-naturally occurring, continuous sequence of ribonucleotide bases. These bases define a stem-loop. The RNA construct has simulated, three way junction joining regions 3′ and 5′ of the stem-loop structure. Each joining region can include no more than about 10 nucleotides. In addition, the RNA construct has a first region 5′ of the 5′ joining region, including bases complementary to a 3′ region of the target polynucleotide, and a second region 3′ of the 3′ joining region, including bases complementary to a 5′ region of the target polynucleotide. The base sequence of the first and second regions are selected to hybridize with complementary bases on the target polynucleotide and are spaced apart by an intermediate region on the target polynucleotide defining another three way junction joining region. In the presence of the target polynucleotide, the stem-loop conformation of the construct is stabilized compared to when the target polynucleotide is absent.
In one embodiment, at least one base in the stem of the structure is mismatched with its potential binding partner so as to reduce the stability of the stem-loop structure as compared with a wild-type form of the stem-loop structure. Alternatively, the stem-loop, in the context of the RNA construct, has increased minimum free energy (MFE) compared to the stem-loop in a wild-type context, such that the stem-loop of the RNA construct will be less stable than will the stem-loop in a wild-type context.
In another embodiment, the RNA construct also defines an RNA polypeptide coding region. In the absence of the target polynucleotide, the construct assumes a conformation that inhibiting association with an RNA binding protein and suppressing translation of the coding region. On the other hand, in the presence of the target polynucleotide, the construct assumes a stem-loop conformation promoting association with the RNA binding protein and promoting translation of the coding region.
In other embodiments, the construct is expressed is a cell and the polypeptide coding region encodes a polypeptide needed for survival of the cell, whereby only in the presence of a the target polynucleotide can the cell survive. In another embodiment, the construct is expressed in a cell and the polypeptide coding region encodes a polypeptide lethal to the cell. The cell can be an infected cell or a neoplastic cell, and the target polynucleotide can be expressed by the infecting organism or by the neoplastic cell. In another embodiment, the polypeptide coding region encodes a detectable marker polypeptide whose detection in the expression environment indicates the presence of an analyte comprising the target polynucleotide. In yet another embodiment, the construct has more than one stem-loop structure.
Another aspect of the invention involves an RNA construct for attachment to a polypeptide coding region of an RNA. The RNA construct modulates translation of a polypeptide from the coding region in response to the presence of a target single-stranded polynucleotide in an expression environment. The RNA construct comprises a non-naturally occurring, continuous sequence of ribonucleotide bases. These bases define a stem-loop structure which associates with an RNA binding protein present in the expression environment to suppress translation of the polypeptide coding region when the RNA construct is in a stem-loop conformation. The RNA construct has simulated, three way junction joining regions 3′ and 5′ of the stem-loop structure. Each joining region can include no more than about 10 nucleotides. In addition, the RNA construct has a first region 5′ of the 5′ joining region, including bases complementary to a 3′ region of the target polynucleotide, and a second region 3′ of the 3′ joining region, including bases complementary to a 5′ region of the target polynucleotide. The base sequence of the first and second regions are selected to hybridize with complementary bases on the target polynucleotide and are spaced apart by an intermediate region on the target polynucleotide defining another three way junction joining region. In the absence of the target polynucleotide, the construct assumes a conformation inhibiting association with the RNA binding protein and promoting translation of the polypeptide coding region. In the presence of the target polynucleotide, the construct assumes a stem-loop conformation promoting association with the RNA binding protein and inhibiting translation of the polypeptide coding region.
Any of the above aspects can include any of the following features. At least one base in the stem of the structure can be mismatched with its potential binding partner so as to reduce the stability of the stem-loop structure as compared with a wild-type form of the stem-loop structure. Alternatively, the stem-loop, in the context of the RNA construct, can have increased minimum free energy (MFE) compared to the stem-loop in a wild-type context, such that the stem-loop of the RNA construct will be less stable than will the stem-loop in a wild-type context. The continuous sequence of ribonucleotide bases can also include the polypeptide coding region of an RNA disposed 5′ of the first region. Alternatively, the continuous sequence of ribonucleotide bases can include the polypeptide coding region of an RNA disposed 3′ of the second region. The target polynucleotide can be characteristic of a pathogen, for example, a virus or a single-celled microorganism. The target polynucleotide can be expressed preferentially in a cell type of a multicellular organism, for example, a neoplastic cell. The target polynucleotide can include a synthetic target polynucleotide or single stranded DNA for transfection into a cell. The target polynucleotide can be double-stranded.
Another embodiment of any of the above aspects involves a DNA vector that includes a transcription unit encoding the RNA construct. Still another embodiment involves a cell comprising the RNA construct. The expression environment can be an infected cell or a neoplastic cell that expresses the target polynucleotide. The construct can have more than one stem-loop structure.
Another aspect of the invention involves a method for modulating translation of a polypeptide coding region, which involves providing a translatable RNA construct that includes a polypeptide coding region ligated to a non-naturally occurring RNA and introducing the translatable RNA construct into an expression system. The RNA includes a continuous sequence of ribonucleotide bases which define a stem-loop structure which associates with an RNA binding protein present in the expression environment to modulate translation of the polypeptide coding region when the RNA construct is in a stem-loop conformation. The RNA construct has simulated, three way junction joining regions 3′ and 5′ of the stem-loop structure. Each joining region can include no more than about 10 nucleotides. In addition, the RNA construct has a first region 5′ of the 5′ joining region, including bases complementary to a 3′ region of the target polynucleotide, and a second region 3′ of the 3′ joining region, including bases complementary to a 5′ region of the target polynucleotide. The base sequence of the first and second regions are selected to hybridize with complementary bases on the target polynucleotide and are spaced apart by an intermediate region on the target polynucleotide defining another three way junction joining region. The construct assumes a conformation inhibiting association with the RNA binding protein, and in the presence of the target polynucleotide, the construct assumes a stem-loop conformation promoting association with the RNA binding protein.
In one embodiment of this aspect of the invention, at least one base in the stem of the structure can be mismatched with its potential binding partner so as to reduce the stability of the stem duplex as compared with a wild-type form of the stem-loop structure. Alternatively, the stem-loop, in the context of the RNA construct, can have increased minimum free energy (MFE) compared to the stem-loop in a wild-type context, such that the stem-loop of the RNA construct will be less stable than will the stem-loop in a wild-type context. In another embodiment, the RNA construct is introduced into the expression environment by transfecting a DNA encoding the translatable RNA construct. The target polynucleotide can be characteristic of a pathogen, or can be expressed preferentially in a cell type of a multicellular organism. The cell type can be, for example, a neoplastic cell. The method can also include the additional step of introducing the target polynucleotide into the expression system. In another embodiment, the expression system is a cell and the polypeptide coding region encodes a polypeptide lethal to the cell. The cell can be an infected cell or a neoplastic cell, and the target polynucleotide, for example, an enzyme or pore-forming protein, can be expressed by the neoplastic cell or by the infecting organism. The construct can have more than one stem-loop structure.
In another embodiment of this aspect of the invention, the polypeptide coding region can encode a detectable marker, for example, a fluorescent marker. In yet another embodiment, translation of the coding region can be suppressed when the RNA binding protein binds to the stem-loop structure. Translation of the coding region can alternatively be promoted when the RNA binding protein binds to the stem-loop structure.
Another aspect of the invention can involve a method of killing preferentially, in a heterogeneous population of animal cells, target cells that harbor a single-stranded target polynucleotide having a known sequence. The method involves introducing into the heterogeneous cell population an RNA construct attached to a polypeptide coding region which encodes a polypeptide lethal to the target cells. The RNA construct is capable of modulating translation of the polypeptide coding region in response to the presence of the target single-stranded polynucleotide in the target cells. The RNA construct includes a non-naturally occurring, continuous sequence of ribonucleotide bases which define a stem-loop structure which associates with an RNA binding protein present in the target cells to promote translation of the polypeptide coding region when the construct is in a stem-loop conformation. The RNA construct has simulated, three way junction joining regions 3′ and 5′ of the stem-loop structure. Each joining region can include no more than about 10 nucleotides. In addition, the RNA construct has a first region 5′ of the 5′ joining region, including bases complementary to a 3′ region of the target polynucleotide, and a second region 3′ of the 3′ joining region, including bases complementary to a 5′ region of the target polynucleotide. The base sequence of the first and second regions are selected to hybridize with complementary bases on the target polynucleotide and are spaced apart by an intermediate region on the target polynucleotide defining another three way junction joining region. In cells where the target polynucleotide is absent, the construct assumes a conformation inhibiting association with the RNA binding protein and suppressing translation of the coding region. In cells where the target polynucleotide is present, the construct assumes a stem-loop conformation promoting association with the RNA binding protein and promoting translation of the coding region thereby to kill the cells.
In certain embodiments of this aspect of the invention, at least one base in the stem of the structure can be mismatched with its potential binding partner so as to reduce the stability of the stem duplex as compared with a wild-type form of the stem-loop structure. Alternatively, the stem-loop, in the context of the RNA construct, can have increased minimum free energy (MFE) compared to the stem-loop in a wild-type context, such that the stem-loop of the RNA construct will be less stable than will the stem-loop in a wild-type context. In other embodiments, the target polynucleotide is characteristic of a pathogen, such as a virus or a single-celled microorganism. In other embodiments, the target polynucleotide is expressed preferentially in a target cell type, such as a neoplastic cell, of a multicellular organism. The target cell can be an infected cell or a neoplastic cell, and the target polynucleotide can be expressed by the infecting organism or by the neoplastic cell. The polypeptide coding region can include a sequence encoding an enzyme or pore-forming protein. The construct can have more than one stem-loop structure.
Another aspect of the invention involves an RNA construct for attachment to a polypeptide coding region of an RNA and for modulating translation of a polypeptide from the coding region in response to the presence of a target single-stranded polynucleotide in an expression environment. The RNA construct includes a non-naturally occurring, continuous sequence of ribonucleotide bases defining a stem-loop structure, which promotes translation of the polypeptide coding region when the construct is in a stem-loop conformation; simulated, three way junction joining regions 3′ and 5′ of the stem-loop structure; a first region 5′ of the 5′ joining region including bases complementary to a 3′ region of the target polynucleotide; and a second region 3′ of the 3′ joining region including bases complementary to a 5′ region of the target polynucleotide. The base sequence of the first and second regions are selected to hybridize with complementary bases on the target polynucleotide spaced apart by an intermediate region on the target polynucleotide defining another three way junction joining region. In the absence of the target polynucleotide, the construct assumes a conformation suppressing translation of the polypeptide coding region, and in the presence of the target polynucleotide, the construct assumes a conformation promoting translation of the polypeptide coding region. This aspect of the invention can have any of the features described herein (above or below).



BRIEF DESCRIPTION OF THE FIGURES
The foregoing and other objects, features, and advantages of the present invention, as well as the invention itself, may be more fully understood from the following description of preferred embodiments, when read together with the accompanying drawings.
FIG. 1A shows an RNA stem-loop motif interacting with an RNA binding protein (RBP). FIG. 1B shows an RNA construct of the present invention with a weakened stem that prevents formation of the stem-loop structure, therefore preventing RBP binding. FIG. 1C shows an RNA construct, stabilized by a target polynucleotide and which forms a stem-loop structure capable of being bound by an RBP.
FIGS. 2A-D show examples of RNA construct-target polynucleotide interactions. It should be understood that these examples are illustrative of the general concept of a nucleic acid target binding to and changing the conformation of an RNA construct. FIG. 2A shows a target polynucleotide stabilizing a stem-loop motif by binding at least a portion of the 5′ and 3′ flank regions. This represents a stabilizing interaction. FIG. 2B depicts a target polynucleotide binding an RNA construct and preventing the formation of the native secondary structure motif. This represents a destabilizing interaction. FIG. 2C is an example of a target polynucleotide binding to an RNA construct to form a double-stranded site that could be bound by an RBP. FIG. 2D shows a target polynucleotide ablating one RBP binding-site motif to allow formation of a second motif in a manner suggestive of a bacterial operon attenuation mechanism.
FIG. 3A shows an example of a strategy that uses an RNA construct-target polynucleotide interaction to affect the structure of an RNA construct, which modulates the activity of a secondary item such as an attached polypeptide coding region. FIG. 3B shows an example of a target polynucleotide binding to an stem-loop motif, thereby disrupting the stem-loop structure such that an RBP can no longer bind.
FIGS. 4A-C show the components and nomenclature of a three-way junction. FIG. 4D shows the range of relative sizes of joining regions that give rise to the different three-way junction family members shown in FIG. 5. J1 corresponds to the junction region that connects the two helices most closely approaching a coaxial conformation. (See, e.g., Lescoute et al. (2006) RNA 12:83-93.)
FIG. 5 shows examples of three-way junction family types. An RNA construct can form three basic conformations when bound to a target polynucleotide, corresponding to the three family types (A, B and C) of three-way junctions. Three-way junction conformation can affect the ability of RBPs to bind to a stem-loop.
FIGS. 6A-D show a mock example of an RNA construct using a stem-loop element. FIG. 6A shows a consensus structure (5′ NNNGGGGANNCNUCCCCNN-3′ (SEQ ID NO:1)) of an RBP binding site, and the gray area denotes a region where point mutations may be made to weaken stem-loop structure (asterisks show bases that are required for protein binding). The shaded boxes of FIG. 6B show point mutations made to the primary structure (sequence) at spots calculated to increase Minimum Free Energy (MFE) and reduce spontaneous formation of required secondary structure for RBP binding. The sequence of this RNA construct is (5′ NNNUGGGANNCNUCUUUNN-3′ (SEQ ID NO:2)). FIGS. 6C and D show an simulated three way junction (depicted generally in FIG. 6C), that will form when a target polynucleotide hybridizes to the RNA construct (SEQ ID NO:2) to stabilize the formation of the RBP binding site.
FIGS. 7A-C show an example of the RNA construct (SEQ ID NO: 2) of FIGS. 6A-D (FIG. 7A) designed to hybridize to a HCMV miRNA-US4 target polynucleotide (5′-CGACAUGGACGUGCAGGGGGAU-3′ (SEQ ID NO:3) (FIG. 7B). FIG. 7C shows the RNA construct with flank sequences (5′-AUCCCCUGCNNNUGGGANNCNUCUUUNNGUCCGUGUCG-3′) (SEQ ID NO:4), hybridized to the target polynucleotide to form a three-way junction.
FIGS. 8A-C show that the interaction between flank regions of an RNA construct and target polynucleotide (hsa-miR-373, mirBase No. MIMAT0000726; 5′-GAAGUGCUUCGAUUUUGGGGUGU-3′ (SEQ ID NO:5)) can produce J3 regions of varying sizes. FIG. 8A shows a J3 region of 0 nucleotides produced by the interaction of the target polynucleotide with the 5′ flank region ACACCCCAAAA (SEQ ID NO:6) and the 3′ flank region UCGAAGCACUUC (SEQ ID NO:7); FIG. 8B shows a J3 region of 1 nucleotide produced by the interaction of the target polynucleotide with the 5′ flank region ACACCCCAAAA (SEQ ID NO:6) and the 3′ flank region CGAAGCACUUC (SEQ ID NO:8); and FIG. 8C shows a J3 region of 2 nucleotides produced by the interaction of the target polynucleotide with the 5′ flank region ACACCCCAAA (SEQ ID NO:9) and the 3′ flank region CGAAGCACUUC (SEQ ID NO:8).
FIG. 9 shows an example of a SELEX approach to designing an RNA construct. A library of sequences is constructed in which regions containing nucleotides critical for RBP binding (5′-AAAGGCUCUUUUCA-3′ (SEQ ID NO:10), in this example) and regions designed to hybridize to a target polynucleotide are held constant, while remaining regions are varied at random.
FIG. 10 shows a model of the proposed biological mechanism of action of the HSL motif (and an RNA construct based on a HSL motif). The stem-loop binding protein (SLBP) bound to an RNA construct may act through one or more complexes to promote translation of an attached polypeptide coding region.
FIGS. 11A-NN provide GenBank accession numbers for exemplary HSL sequences. “Start” and “end” refer to nucleotide positions at which the HSL starts and ends in the larger corresponding GenBank sequence indicated in the last column.
FIG. 12 shows an example of an RNA construct based on histone stem-loop Consensus Sequence I. Two GU pairings and one UG pairing are designed at the lower portion of the stem to weaken it, such that formation of the stem-loop structure is dependent on the association with a target polynucleotide (hsa-miR-373 (SEQ ID NO:11) in this example). This RNA construct has the nucleotide sequence 5′-ACACCCCAAAAAAAGGUUCUUUUCAGAGUUACUCGAAGCACUUC-3′ (SEQ ID NO:12)).
FIG. 13A shows an RNA construct incorporating a weakened Iron Response Element (IRE) motif that does not effectively form in the absence of a target polynucleotide. FIG. 13B shows the reconstituted IRE motif hybridized to a target polynucleotide. FIG. 13C shows examples of IRE1 (5′-NNCNNNNNCAGWGHNNNNNNN-3′ (SEQ ID NO:13)), IRE2 (5′-NNNNCNNNNNCAGWGHNNNNNNNN-3′ (SEQ ID NO:14)), and IRE3 (5′-NNCNNNNNCAGWGHNNNUNNNN-3′ (SEQ ID NO:15)) consensus sequences (left to right, respectively).
FIGS. 14A-S provide GenBank accession numbers for exemplary IRE1 sequences. “Start” and “end” refer to the positions in the larger corresponding GenBank sequences at which the IRE1 sequence can be found.
FIGS. 15A-N provide GenBank accession numbers for exemplary IRE2 sequences. “Start” and “end” refer to the positions in the larger corresponding GenBank sequences at which the IRE2 sequence can be found.
FIG. 16 provides GenBank accession numbers for exemplary IRE3 sequences. “Start” and “end” refer to the positions in the larger corresponding GenBank sequences at which the IRE3 sequence can be found.
FIG. 17A shows an RNA construct incorporating a weakened Selenocysteine insertion sequence (SECIS) Element that does not effectively form in the absence of a target polynucleotide. FIG. 17B shows the reconstituted SECIS motif hybridized to a target polynucleotide. FIG. 17C shows an example of a SECIS consensus sequence (5′-NNNNNNNAUGANRRNNNNNNNAARNNNNNNNNNNNYYBGANNNNNNNN-3′) (SEQ ID NO:16).
FIGS. 18A-G provide GenBank accession numbers for exemplary SECIS1 sequences. “Start” and “end” refer to the positions in the larger corresponding GenBank sequences at which the SECIS1 sequence can be found.
FIGS. 19A-O provide GenBank accession numbers for exemplary SECIS2 sequences. “Start” and “end” refer to the positions in the larger corresponding GenBank sequences at which the SECIS2 sequence can be found.
FIGS. 20A-B show a motif from the internal ribosome entry site (IRES) element which can be used as the RBP target in an RNA construct. FIG. 20A shows the general structure of an IRES element with domains II-IV depicted. FIG. 20B shows an IRES element from the Hepatitis C virus (SEQ ID NO:17). The internal start codon (AUG), located in domain IV, is shaded.
FIGS. 21A-B show an exemplary portion of an IRES element that can be used as the basis of an RNA construct. This portion is the region of the internal ribosome entry site (IRES) element that corresponds to domain II in the Hepatitis C virus. FIG. 21A shows the sequence of domain II (SEQ ID NO:18) from the IRES of Hepatitis C virus. FIG. 21B shows the base stem region (“N” nucleotides enclosed in a box) of domain II of the Hepatitis C virus that can be weakened by introduction of non-canonical base pairings (SEQ ID NO:19).
FIG. 22 shows sequences encoding naturally-occurring IRESes, with domain II sequences in bold uppercase. The first sequence (From Hepatitis C virus) corresponds to the sequence shown within the dotted line of FIG. 20B, with the bolded uppercase portion corresponding to the sequence shown in FIG. 21A.
FIG. 23 shows domain II sequences from the naturally-occurring IRESes of FIG. 22, with 5′ stem, internal, and 3′ stem regions indicated. Rows 1 to 5 correspond to SEQ ID NOS: 25 to 29.
FIGS. 24A-B show target polynucleotide stabilization of an RNA construct incorporating an IRES motif. FIG. 24A shows an RNA construct incorporating a weakened IRES motif that does not effectively form in the absence of a target polynucleotide. FIG. 24B shows the reconstituted IRES motif hybridized to a target polynucleotide.
FIGS. 25A-C show the results of a mirBase search for viral microRNAs (miRNAs). These miRNAs can be used as target polynucleotides. “Parent structure” refers to the RNA construct to which the miRNA binds. Rows 1-62 correspond to SEQ ID NOS: 30 to 91.
FIGS. 26A-C show the results from Rfam search on “virus” (www.sanger.ac.uk/cgi-bin/Rfam/queryrfam.pl?db=rfam&db=prosite&db=swiss&size=100&terms=virus).
FIGS. 27A-B show examples of polypeptides capable of causing cell death that can be encoded by a polypeptide coding region.
FIG. 28 shows a naturally-occurring three-way junction region from the 16S ribosomal subunit of Thermus thermophilus, (Protein Data Bank entry 1J5E, Chain A). Nucleotides labeled with numbers (561, 585, 621, 646, 727, and 748) indicate the corresponding nucleotide positions in the sequence of the 16S ribosomal subunit. (SEQ ID NO:192). FIG. 28 discloses nucleotides 561-585, 621-646 and 727-748 of SEQ ID NO: 192.
FIG. 29 shows an RNA construct (5′-GGGAUACGCUCAGGCUAGACGGUGGGGUGAAAUCCACCCGUGACGCUGAGGCGC GGAGUAACGAACCUAUCCGUAACAUAAUU-3′) (SEQ ID NO:93)) and target polynucleotide (5′-GCGUGUAGGCGGCCUGGGGCGUCCC-3′) (SEQ ID NO:94)) designed from the naturally-occurring three-way junction from the 16S ribosomal subunit of Thermus thermophilus, shown in FIG. 28. The loop sequence was taken from a naturally-occurring stem-loop (positions 675-680) in the same naturally-occurring 16S ribosomal subunit RNA (SEQ ID NO:192).
FIG. 30 shows that, due to the size of the junction joining regions in the RNA construct of FIG. 29, the RNA construct is expected to produce a Family A type junction, in which P3 is located about midway between P1 and P2. This configuration will allow an RBP to access the stem-loop of the RNA construct.
FIG. 31 shows the fold prediction for the Minimum Free Energy (MFE) structure of the RNA construct (SEQ ID NO:96) of FIG. 29. The reduction in free energy predicted to occur as a result of the folding of the RNA construct alone is −25.00 kcal/mol.
FIG. 32 shows the fold prediction for the Minimum Free Energy (MFE) structure of the target polynucleotide (SEQ ID NO:97) of FIG. 29. The reduction in free energy predicted to occur as a result of the folding of the target polynucleotide alone is −4.60 kcal/mol.
FIG. 33 shows the fold prediction for the Minimum Free Energy (MFE) structure of the RNA construct (SEQ ID NO:96) bound to the target polynucleotide (SEQ ID NO:97) as shown in FIG. 29. The reduction in free energy predicted to occur as a result of the folding of the RNA construct alone is −45.00 kcal/mol, greater than the sum of the reduction in free energy (−29.60 kcal/mol) for the RNA construct alone and the target polynucleotide alone, indicating that the combined structure is more stable and therefore more likely to form.
FIG. 34 shows an RNA construct (5′-GGGAUACGCUCAGGCAAAGGCUCUUUUCAGAGCCAGCUGAGGCGCGGAGUAACG AACCUAUCCGUAACAUAAUU-3′) (SEQ ID NO:98)), which can be designed using a histone stem-loop consensus sequence rather than the stem-loop designed based on a Thermus thermophilus three-way junction shown in FIG. 29. The target polynucleotide (SEQ ID NO:97) in this example is the same as that shown in FIG. 29.
FIG. 35 shows that the RNA construct of FIG. 34 is predicted to form a Family B type three-way junction when bound to a target polynucleotide.
FIG. 36A depicts a stem-loop structure (SEQ ID NO:99) to which the MS2 capsid protein binds. FIG. 36B depicts an MS2 binding site consensus sequence (SEQ ID NO:100).
FIG. 37 shows RNA constructs MS2-B1 (SEQ ID NO:101) and MS2-B2 (SEQ ID NO:102) complexed with target polynucleotide hcmv-mir-us25-2-3p (T-1 (SEQ ID NO:103)).
FIG. 38 shows the predicted folding pattern and minimum free energy (MFE) of the target hcmv-mir-us25-2-3p (T-1 (SEQ ID NO:103)) alone.
FIG. 39 shows the predicted folding pattern and minimum free energy (MFE) of the RNA construct MS2-B1 (SEQ ID NO:101) alone.
FIG. 40 shows the predicted folding pattern and minimum free energy (MFE) of the RNA construct MS2-B2 (SEQ ID NO:102) alone.
FIG. 41 shows the predicted folding pattern and minimum free energy (MFE) of the RNA construct MS2-B1 (SEQ ID NO:101) bound to its target polynucleotide, hcmv-mir-us25-2-3p (SEQ ID NO:103).
FIG. 42 shows the predicted folding pattern and minimum free energy (MFE) of the RNA construct MS2-B2 (SEQ ID NO:102) bound to its target polynucleotide, hcmv-mir-us25-2-3p (SEQ ID NO:103).
FIG. 43 shows a native PAGE in which a structure with a higher molecular weight than either RNA construct or target alone forms (see box), indicating that RNA constructs MS2-B1 and MS2-B2 are capable of stably binding target polynucleotide.
FIG. 44 shows a schematic representation of the immunoprecipitation of MS2 protein bound to an RNA construct-target complex.
FIG. 45A provides a TBE-urea denatured gel showing that RNA construct-target complex (MS2-B1+T-1, lane 3 and MS2-B2+T-1, lane 4) is more efficiently co-immunoprecipitated with MS2 protein than is RNA construct alone (MS2-B1, lane 1 and MS2-B2, lane 2), indicating that the target polynucleotide stabilizes the structure of each RNA construct in a manner that is conducive to MS2 protein binding. FIG. 45B shows quantitation of band intensities for the lanes indicated, which was performed using Quantity One® software (BioRad, Hercules, Calif.).
FIG. 46 depicts the sequence and structure of the MS2-B3 RNA construct (SEQ ID NO:104) bound to target polynucleotide T-2 (SEQ ID NO: 105).
FIG. 47 depicts the sequence and structure of the MS2-B4 (SEQ ID NO:106) and MS2-B5 (SEQ ID NO:107) RNA constructs bound to target polynucleotides T-2 (SEQ ID NO: 105) and T-3 (SEQ ID NO: 108), respectively.
FIG. 48A provides a TBE-urea denatured gel showing that RNA construct-target complex (MS2-B3+T-2 or MS2-B5+T-3) is more efficiently co-immunoprecipitated with MS2 protein than is RNA construct alone (MS2-B3 and MS2-B5), indicating that the target polynucleotide stabilizes the structure of each RNA construct in a manner that is conducive to MS2 protein binding. FIG. 48B shows quantitation of band intensities for the lanes indicated, which was performed using Quantity One® software (BioRad, Hercules, Calif.).
FIG. 49 shows an HSL consensus sequence (SEQ ID NO:109) acquired from the: UTRSITE Signal Manager (www2.ba.itb.cnr.it/UTRSite/index.php/UTRSite%20signal/Signal/frmID/U0035), which was used as the starting point to design HSL-based RNA constructs.
FIG. 50 depicts the sequence and structure of the HSL-B1 (SEQ ID NO:110) and HSL-B2 (SEQ ID NO:111) RNA constructs bound to target polynucleotide T4 (SEQ ID NO:112).
FIG. 51 depicts the sequence and structure of the HSL-B3 (nucleotides 23-57 of SEQ ID NO:113) and HSL-B4 (SEQ ID NO:114) RNA constructs bound to target polynucleotides T-5 (SEQ ID NO:115) and T-6 (SEQ ID NO:116), respectively.
FIG. 52 provides a TBE-urea denatured gel showing that RNA construct-target complex (HSL-B1+T-4, HSL-B2+T-4, HSL-B3+T-5, and HSL-B4+T6) is more efficiently co-immunoprecipitated with Stem-loop Binding Protein (SLBP) than is RNA construct alone (HSL-B1, HSL-B2, HSL-B3, and HSL-B4), indicating that the target polynucleotide stabilizes the structure of each RNA construct in a manner that is conducive to SLBP binding. FIG. 52 shows quantitation of band intensities, which was performed using Quantity One® software (BioRad, Hercules, Calif.).
FIG. 53 shows Histone Stem-Loop (HSL) sequence information, with STLAB_C01_01 to STLAB_C01_25 corresponding to SEQ ID NOS: 117 to 141. FIG. 53 discloses “MMMMMGGYYYUHHUHARRRCCMM” as SEQ ID NO: 109.
FIG. 54 shows miRNAs that are aligned with the HSLs of FIG. 36 in a manner shown for FIG. 38. “Parent structure” refers to the structure to which the miRNA primarily binds. Rows 1-28 correspond to SEQ ID NOS: 142 to 169.
FIG. 55 shows a specific example of a histone stem-loop sequence (5′-CAAAAGGCUCUUUUCAGAGCCAC-3′ (SEQ ID NO:170)) and its potential targeting by a microRNA. The upper sequences of the figure show the alignment of the stem-loop sequence to hsa-mir-518c* sequence (5′-UCUCUGGAGGGAAGCACUUUCUG-3′ (SEQ ID NO:171)). This interaction is predicted to have a energy of −20.81 kcal/mol.
FIGS. 56A-B show a sequence for an RNA construct attached to a green fluorescent protein (GFP), a reporter construct (Pd4EGFP-N1 with stem-loop sequence) (SEQ ID NO:172). Double underlining shows the GFP sequence. Dashed underlining shows mouse ornithine decarboxylase (3′ end of CDS) sequence. The histone sequence is shown in italics. Bold underlining shows the stem-loop sequence.
FIG. 57 shows sequences used for the design of shRNAs. shRNAs were expressed using standard retroviral delivery methods. The shRNA is initially expressed as a double-stranded hairpin precursor molecule that is processed by cellular machinery into the eventual expressed single-stranded miRNA, which will function as the target polynucleotide. The sequence for the eventual processed target polynucleotide is depicted in the “antisense sequence” column, but listed in the DNA form in which “T's” are listed in place of “U's.” Rows 1-4 correspond to SEQ ID NOS: 173 to 175[[6]].
FIGS. 58A-B show direct (FIG. 58A) and indirect (FIG. 58B) evidence of the ON/OFF activity of the GFP-HSL trans-acting structural RNA switch. In FIG. 58A, RT-PCR was used to detect the presence or absence of GFP coding sequence (cds) RNA associated with the stem-loop binding protein (SLBP). This was determined with and without the co-expression of the SV40 target polynucleotide. A significant decrease can be observed in the presence of co-expressed SV40 microRNA as compared to the amount in the total RNA. FIG. 58B, GFP fluorescence was used determine the expression of the GFP fused HSL-RNA construct in the presence or absence of SV40 or mir518 competing miRNAs (target polynucleotide). An approximately 20% decrease in GFP expression is observable when SV40 miRNA is co-expressed with the GFP-HSL-RNA construct.



DETAILED DESCRIPTION
I. Introduction
A. Concept
The present invention relates to the use of interactions between nucleic acid construct, such as an RNA construct, and a target polynucleotide, such as an RNA, as a functional switch. As disclosed herein, a very large number of RNA constructs of the type illustrated in FIG. 1 can be designed, each of which are stabilized in the presence of a target polynucleotide. In this scenario, an RNA construct is able to form a structure in the presence of a target polynucleotide, which results in some type of activity. For example, the formation of a stem-loop structure allows an RNA binding protein (RBP) to associate with the construct, thus promoting or inhibiting translation of a polypeptide coding region attached to the RNA construct. FIG. 1A illustrates an example of a stem-loop structure, which can act as an association-site for a specific protein involved in gene expression. In the presence of this structure, the protein associates with the RNA construct and promotes expression of the attached polypeptide coding region, often increasing protein production by many orders of magnitude.
In the design of RNA constructs according to the invention, the stem-loop structure of 1A is weakened so that the weakened structure will no longer efficiently associate with the protein (FIG. 1B). In the absence of a target polynucleotide, the RNA construct is less capable or incapable of forming a structure with which the RBP will associate. Thus, in the absence of a target polynucleotide, the attached polypeptide coding region will not be effectively expressed.
In order to be able to “turn on” this weakened RNA construct, a portion of the RNA construct is designed to base-pair with a target polynucleotide, often an RNA, such as an RNA from a pathogen or one present in a neoplastic cell. The target polynucleotide acts as a “splint” to reform the original stem-loop structure. FIG. 1C shows an example of an RNA construct-target polynucleotide complex with re-formed binding site potential for the RBP. This re-formed structure has the capacity to interact with the RBP in order to stimulate protein production of whatever polypeptide coding region is attached to the RNA construct. This approach may be used to express a polypeptide that restores function to an impaired cell, stimulates the immune system or kills a diseased or infected cell (or a pathogen within an infected cell).
More generally, at least four categories of RNA construct interactions with target polynucleotides can be used to influence RNA construct structure and, therefore, RBP binding site availability (FIGS. 2A-D). It should be understood that FIGS. 2A-D are intended to illustrate the principles of the invention and that although RNA is shown as the RNA construct in FIGS. 2A-D, the RNA construct may be any nucleic acid, such as RNA or DNA, which occurs naturally or is rationally designed. FIGS. 2A and 3A illustrate a first type of interaction, described above with reference to FIGS. 1A-C, relating to stabilizing a protein binding site. FIGS. 2B and 3B illustrate a second type of interaction in which RNA construct-target polynucleotide interactions are designed so that the target polynucleotide ablates the RNA construct structure and/or an RNA-binding protein binding site. These RNA construct-target polynucleotide interactions can facilitate or disrupt the association of an RNA-binding protein with its targeted regulatory element (FIGS. 2A and 2B, respectively). This type of interaction can exist in either a positive or negative fashion (i.e. “on” or “off”) with respect to the stabilization of the RNA construct and can function upstream or downstream of the structure. Example 1 is representative of the interaction shown in FIG. 2A which may be used to turn on the expression of a polypeptide coding region. Examples 2 and 3 are representative of the interaction shown in FIG. 2B which be used to turn off the expression of a polypeptide coding region.
Another category of interaction is one in which the target polynucleotide-RNA construct interaction is itself the target of an RNA-binding protein (FIG. 2C) and therefore forms the switch. A double-stranded RNA-binding protein, or possibly a moonlighting DNA-binding protein, can target this type of structure. The final category of interaction combines one or more of the already described interactions into a complex switch (FIG. 2D). In this group, multiple RNA regulatory elements are simultaneously influenced by one or more target polynucleotide-RNA construct interactions. Specifically, the binding of one or more target polynucleotides to the RNA construct results in conformational changes in the structure of the message, such that a second regulatory element is revealed or masked. This class of target polynucleotide-RNA construct interaction utilizes a series of secondary structures, such as stem-loops, that are simultaneously influenced in a mutually exclusive switch-like manner dictating which of several possible regulatory elements will be accessible for use.
B. Uses
1. Expression of a Polypeptide to Kill a Cell or Pathogen
Polypeptide coding regions can be attached to RNA constructs that bind to any target polynucleotide that is a marker for infection or disease. This strategy can be used to target any pathogen, including viruses, bacteria, fungi or protozoa. Thus, the strategy can kill the cell harboring a pathogen and/or the pathogen itself. Beyond pathogens, this strategy can be used to eliminate diseased cells, such as neoplastic cells (e.g., cancer cells). In certain embodiments, an RNA construct connected to polypeptide coding region encoding a polypeptide able to kill a cell (e.g., a neoplastic cell or pathogenically infected cell) or pathogen is designed to bind to a target polynucleotide specifically expressed by, for example, a neoplastic cell or a pathogen. Polypeptide coding regions encoding death polypeptides include, for example, genes that induce apoptosis (e.g., caspases), pore-forming genes, or any other gene that would be toxic to a cell or pathogen. Multiple stem loop structures may be designed as part of the same RNA molecule to increase specificity. For example, two stem loop structures within the construct may be designed to bind different target polynucleotides, such that both target polynucleotides must be present to stabilize the RNA construct and promote translation. In another embodiment, two different types of stem-loop motifs (e.g., HSL and IRE) may be used as part of the same RNA molecule, so that the expression of the attached polypeptide coding region is more robust in a particular cell type (e.g., a cancer cell), in which SLBP and IRE RBPs are likely to be active. While selectivity is particularly important when expressing genes that are toxic to the cell, a construct with two or more stem-loop structures may be used in any of the embodiments of the invention described herein.
2. Diagnostic Testing and Imaging
In another embodiment, an RNA construct can be used to diagnose the presence of a particular target polynucleotide in a cell. In this strategy, a marker gene encoding any protein that can be visualized or otherwise detected (e.g., a fluorescent or luminescent protein) is attached to an RNA construct. This strategy can be used, for example, for diagnostic testing or to highlight specific cells or tissues during imaging. In one example, an RNA construct is designed to bind a viral target polynucleotide and is attached to Green Fluorescent Protein (GFP). When the construct is delivered to cells, only cells infected with the virus will fluoresce.
3. Directing Protein Expression in Certain Cells
Rather than simply killing a diseased cell, an RNA construct can be used to direct expression of an attached polypeptide coding region in a specific cell or tissue having a protein deficiency. For example, an RNA construct can be designed to bind to a target polynucleotide indicative of the protein deficiency. When the construct is delivered to cells, it will only direct expression of the protein in cells lacking the protein because the cells contain the target polynucleotide. In one alternative, if all cells of an organism (or cell culture) were lacking a given protein, an RNA construct could be designed to bind to a ubiquitously-expressed target polynucleotide, such that the attached gene would be expressed in all cells. In another alternative, an RNA construct could be designed to bind to a ubiquitously-expressed target polynucleotide, but the RNA construct could be delivered only to the cells or tissues lacking a given polypeptide. Using this strategy, the construct will only direct expression of the protein in cells lacking the protein because it will be delivered to only these cells. Combining administration of an RNA construct attached to a polypeptide coding region and RNAi is also contemplated. For example, for cells that not only lack a certain protein, but also make a defective version of that protein which contributes to the disease, an RNA construct can be administered together with RNAi which specifically targets the defective version of the protein. This strategy will not only eliminate the defective protein, but will also supply the wild-type protein. For example, RNAi targeting a defective Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) and an RNA construct comprising a polypeptide coding region encoding wild-type CFTR can be coadministered to a cell containing a defective CFTR. Table 1 provides examples of diseases that could be ameliorated by expression of the corresponding polypeptide using this type of a system.








TABLE 1





Disease or Condition
Polypeptide(s) to be Expressed by Construct







Cystic Fibrosis
Cystic Fibrosis Transmembrane Conductance



Regulator (CFTR)


Lysosomal storage 
Sulfatases, N-acetylglucosamine-1-phosphate


disorders
transferase, Cathepsin A, GM2-AP,



sphingolipid activator proteins, NPC1 and



NPC2, or sialin


Mucopolysaccharidosis 
a-L-iduronidase, iduronate sulfatase, heparan


(subset type of lysosomal 
sulfamidase, N-acetylglucosaminidase, N-


storage disease)
acetylglucosamine 6-sulfatase, galactose 6-



sulfatase, N-acetylgalactosamine 4-sulfatase,



β-glucuronidase, or hyaluronidase


Fabry's disease
Enzyme alpha galactosidase A


Addiction Disorders
Dopamine receptor


Depression
Serotonin receptor, norepinephrine receptor,



or dopamine receptor


Gaucher Disease
Acid β-glucosidase


Diabetes
Insulin









Additionally, other polypeptides that may be useful include interleukins, reproductive hormones, plasminogen activators, human growth hormones, colony-stimulating factors, blood-clotting factors, erythropoietins, and interferons.

4. Selecting Cells Containing a Target Polynucleotide

In another embodiment of the invention, a DNA construct encoding the RNA construct of the invention can be transformed into a cell. The polypeptide coding region attached to the RNA construct encodes a polypeptide necessary for the continued survival of the cell. In the presence of a target polynucleotide, an RBP binds to the stabilized RNA construct and promotes translation of the polypeptide. In this scenario, only cells that contain the target polynucleotide will survive. In a variation of this embodiment, a population of cells is transformed with a DNA construct encoding the RNA construct in which the polypeptide coding region attached to the RNA construct encodes a polypeptide, for example a drug- or antibiotic-resistance gene (e.g., dihydrofolate reductase (DHFR), thymidine kinase, gene conferring resistance to neomycin, a neomycin analog such as G418, hydromycin, or puromycin) necessary for survival of the cell in a given environment. When transformed cells are placed into this environment (e.g., an environment containing a drug or antibiotic), only cells that (1) have been successfully transformed and (2) contain the target polynucleotide will survive.
5. Suppressing Translation of an Encoded Polypeptide in the Presence of a Target Polynucleotide
In another embodiment, an RNA construct that binds to an RBP that suppresses, rather than enhances, expression may be used to express an attached polypeptide coding region in a cell that lacks a target polynucleotide. Then, if the target polynucleotide enters or becomes expressed in the cell, binding of the target polynucleotide to the RNA construct stabilizes the secondary structure, allowing the RBP to bind, which suppresses translation of the encoded polypeptide. This strategy can be used as a type of feedback loop, wherein, for example, a therapeutic protein is expressed until the cell achieves a certain level of health, at which time the target polypeptide is expressed, and the production of the therapeutic protein ceases. Should the cell return to an unhealthy state wherein the target polypeptide is no longer present, production of the therapeutic protein resumes.
6. Other Applications of an RNA Construct
Many other applications of this invention are contemplated. Essentially, the expression of any polypeptide coding region encoding a polypeptide can be activated using this approach as long as the environment in which the RNA construct is to be delivered contains the activating target polynucleotide. RNA-constructs can also be designed to activate proteins that would enhance processes already occurring within a cell. Genes that could be targeted include any cellular gene, such as those involved in immune cell activation (IL-2), cell growth (cyclin B2, hTert), or vascular development (VEGF), as well as any gene product from any pathogen such as viruses, bacteria, fungi or protozoa.
II. RNA Construct
A. General
Returning to FIG. 2A, the stabilizing interaction depicted is comprised of two nucleic acid components, the RNA construct that is engineered and ectopically supplied and a target polynucleotide, such as an RNA produced by a pathogen. The RNA construct is delivered as a weakened form of a stem-loop with an attached polypeptide coding region. The RNA construct comprises additional structural elements that aid in the binding of the target polynucleotide to the RNA construct and in the assumption of the proper three-dimensional structure. Specifically, the RNA construct typically contains nucleotides that will lack binding partners at joining regions formed when bound to a target polynucleotide, providing flexibility which enhances the RNA construct's ability to assume the conformation necessary for the RBP to associate and activate translation of an attached polypeptide coding region.
B. Construct Design
The following section describes how to design an RNA construct of the type illustrated in FIGS. 1A-C. Typically, the RNA construct will include a stem-loop structure that is designed to be unstable in the absence of a target polynucleotide, although other motifs are contemplated. In the presence of the target polynucleotide, the structure of the RNA construct is stabilized, allowing an RBP to associate with the construct and promote translation of the attached polypeptide coding region of interest. While some details of design will depend upon the stem-loop structure, and its corresponding mode of action, typically design of RNA constructs will involve the following steps (which need not be performed in the order listed).
1. Design of Three-Way Junctions
Three-way junctions are found in nature. (See, e.g., Lescoute et al. (2006) RNA 12:83-93.) The present invention simulates these natural structures and utilizes these simulated three-way junctions as a pattern upon which to build an RNA construct. FIGS. 4A-C provide the nomenclature of the RNA constructs patterned on three-way junctions. This nomenclature will be used throughout the present patent application. FIG. 4A shows a three-way junction having three base-paired stems (P1, P2, and P3) of variable length and three corresponding joining regions (J1, J2, and J3). The joining regions each independently can be from 0 to about 10 unbound nucleotides, for example from about 2 to about 6 nucleotides. FIG. 4B shows the three-way junction of FIG. 4A, indicating the positions of the three RNA molecules of the junction, S1 through S3. S1 is the strand that connects P1 to P2 through J1, S2 is the strand that connects P2 to P3 through J2, and S3 is the strand that connects P3 to P1 through J3. FIG. 4C further divides the strands S1-S3 into 5′ to 3′ segments labeled “a” and “b.” For example, 51 is divided into two segments, S1a being the segment of P1 leading into the J1 joining region and S1b being the segment of P2 leading out of the J1 joining region. S2 and S3 are each similarly divided into “a” and “b” segments.
When developing an RNA construct, the joining regions of the construct as well as the target polynucleotide are chosen according to FIG. 4D. Depending upon how the joining regions are chosen, one can design a construct from one of three families, Families A, B, or C (FIG. 4D). If an RNA construct patterned on Family A is desired, J3 is selected to include any number of nucleotides from 0 to about 10 and J2 is selected to include any number of nucleotides, from 0 to about 10, such that the number of nucleotides selected for J3 is less that the number selected for J2. J1 includes any number of nucleotides from 0 to about 10. In certain embodiments, the number of nucleotides selected for J3 is between 1 and 3, the number of unpaired nucleotides in region J2 is between 3 and 9, and the number of unpaired nucleotides in region J1 is between 0 and 4. If an RNA construct patterned on the Family B conformation is desired, J3 is selected to include any number of nucleotides from 0 to about 10 and should be approximately equal (e.g., equal to 0 to about 10) to the number selected for J2. J1 includes any number of nucleotides from 0 to about 10. In certain embodiments, the number of unpaired nucleotides in each region (J1, J2 and J3) is between 2 and 6. If an RNA construct patterned on Family C is desired, J3 is selected to include any number of nucleotides from 0 to about 10 and J2 is selected to include any number of nucleotides, from 0 to about 10, such that the number of nucleotides in J3 is greater than the number selected for J2. J1 includes any number of nucleotides from 0 to about 10. In certain embodiments, the number of nucleotides selected for J3 is between 3 and 9, the number of unpaired nucleotides in region J2 is between 1 and 5, and the number of unpaired nucleotides in region J1 is between 0 and 5.
The choice of three-way junction family will be governed by which of these conformations will not obstruct the activity (e.g., RBP binding) of the reinforced stem-loop. FIG. 5 depicts a generalized example of the family selection process, using a construct in which stem P3 forms the stem-loop, a polypeptide coding region is attached to strand S2a, and strand S1 is the target polynucleotide. In this example, the Family A structure was chosen because it allows the RBP of interest to associate with the stem-loop. Families B and C are not used in this example because P2 and P1, respectively, obstruct the RBP from binding to the stem-loop. In other examples, Family B or Family C might provide a better platform, depending upon the steric interactions of the RBP with the stem-loop. In the exemplary RNA construct and attached polynucleotide coding region, strand S2a is attached to the polynucleotide region which optionally can be flanked by 3′ and 5′ untranslated regions (UTRs). Also, although this exemplary RNA construct has the stem-loop structure 3′ to the polynucleotide coding region, in some instances it might be located 5′ to the polynucleotide coding region.
Tertiary interactions of bases in the joining regions with bases in the stem regions, as identified by crystallography in natural forms of three way junctions, may be engineered. On this basis, one can change the conformation to improve a given activity or increase stability, so long as the changes do not interfere with the RBP associating with the stem-loop.
2. Design of Weakened Stem-Loop
To design a weakened stem-loop, point mutations are engineered in the wild-type (including a consensus motif sequence) by introducing non-canonical base pairs (i.e., mismatches) so that structure formation, without a trans-acting support, is no longer energetically favorable. The resulting weakened structure typically should not form bonds strong enough to spontaneously establish an active form of the structure, and when structure is re-formed via trans-acting stabilization, the bond distances typically should not interfere with function. The positions and identity of nucleotides important for activity typically are identified so that these bases are not changed. While vital nucleotides typically are not altered, the nucleotide with which a given vital nucleotide pairs may be able to be altered to weaken the stem without sacrificing binding to the reconstituted structure. Stem nucleotides that only provide structural integrity can be altered to reduce thermodynamic favorability of stem formation. In this case, both bases of a pair can be altered if desired. Canonical base pairs are defined as A-U, U-A, G-C, C-G. There are a number of alternative pairings such as G-U and U-U with varying energy levels and bond distance. The choice as to which bases should be substituted is made with the awareness that if relative bond distance and/or conformation of certain bases in the stem is important to activity, appropriate non-canonical pairings should be chosen (See, for example, Leontis, et al. (2002) NUCLEIC ACIDS RESEARCH 30(16):3497-3531.) For example, to weaken a canonical cis Watson-Crick/Watson-Crick G-C base pair while preserving C1′-C1′ bond length distance (10.3 angstroms), a G-U base pair (10.2 angstroms) should be substituted rather than, for example, a U-U base pair (8.1 angstroms). Table 2 provides bond lengths of canonical and non-canonical cis Watson-Crick/Watson-Crick base pairings and allows for selection of appropriate pairings.









TABLE 2







C1′-C1′



Pairing
distance








U-U
 8.1 Å



C-C
 8.5 Å



U-G
10.2 Å



G-U
10.2 Å



C-G
10.3 Å



A-U
10.3 Å



U-A
10.3 Å



G-C
10.3 Å



C-A
10.4 Å



A-C
10.4 Å



U-C
11.8 Å



C-U
11.8 Å



A-A
12.3 Å



G-A
12.5 Å



A-G
12.5 Å









In addition to bond distance, bond angles and their effects on helix twist and stem “kink” may be considered.

In the case of an RBP binding site, a consensus structure, which can be generated by binding assays, typically is the starting point, but any wild-type sequence (including any consensus sequence) can be used. Although the discussion of RNA construct design principles herein typically refers to an RNA sequence as the starting point, it should be understood that not only RNA sequences but also equivalent DNA sequences can be used. As the skilled artisan understands, an RNA sequence is equivalent to the sequence of a coding strand of genomic DNA or cDNA, because RNA that is transcribed from genomic DNA or cDNA has an identical sequence to the coding strand sequence of the genomic DNA or cDNA (with “T” nucleotides in the DNA sequence and “U” nucleotides in the RNA sequence). Thus, the design principles described herein are applicable when using RNA as a starting point or DNA as a starting point.
In order to demonstrate the concepts of the present invention, a generalized, mock stem-loop consensus sequence is depicted in FIG. 6A, with “N” denoting any nucleotide. Nucleotides in the paired stem portion of the structure, indicated by a shaded box, are mutated so that the structure can no longer spontaneously form. Nucleotides indicated by an asterisk are vital to RBP binding and therefore are not altered. FIG. 6B shows that a G to U mutation was made at the fourth nucleotide from the 5′ end, and C to U mutations were made for the three nucleotides at positions 3, 4 and 5, counting from the 3′ end. Each mutation depicted in FIG. 6B is indicated by a shaded box. Point mutations were made at spots calculated to increase minimum free energy (MFE) and reduce spontaneous formation of required secondary structure for RBP binding. FIG. 6D shows the structure formed by the binding of the weakened stem-loop structure to its target polynucleotide which follows the pattern of the three-way junction structure depicted in FIG. 6C. In this instance, J3 is 2, J2 is 3, and J1 is 2. Once the weakened stem-loop has been reinforced by the binding of the target polynucleotide, the relevant RBP can associate with the RNA construct and alter transcription of an attached polynucleotide coding region (not shown).
3. Design of Flank Regions
Flank regions are the regions of nucleotides adjacent to the stem on the 5′ and 3′ sides of the stem-loop structure that form two of the three joining regions and that bind to the target polynucleotide. FIGS. 7A-C illustrate the design of the 3′ and 5′ regions that flank the stem-loop of the mock RNA construct of FIGS. 6A-D. FIG. 7A depicts the hypothetical stem-loop previously shown in FIG. 6D. FIG. 7B shows an example target polynucleotide, a naturally-occurring microRNA from human cytomegalovirus, hcmv-miR-US4. In this example, the “N” nucleotides directly adjacent to the stem portion of the RNA construct form the unpaired nucleotides of the joining region. The three unpaired Ns correspond to the J2 region and the two unpaired Ns correspond to the J3 region of the three-way junction. Because the J2 region contains more unpaired nucleotides than does the J3 region, a Family A structure will form. These unpaired nucleotides can be any nucleotide, provided that they do not hybridize to the unpaired U and G nucleotides that make up the J1 joining region formed by the target polynucleotide or otherwise disrupt the structure of the junction. The nucleotides that make up the flank regions of the RNA construct distal to the joining regions are selected to hybridize to the chosen target polynucleotide, as shown in FIG. 7C. Flank sequences should either have no specific nucleotide requirements, so that perfect pairing can be achieved, or those bases that must retain specific identity should fall in an simulated joining region, be a canonical match for the appropriate bases in the target polynucleotide or provide a beneficial mismatch to avoid a non-reversible hybridization with the target polynucleotide. Regardless of whether specific requirements for flank nucleotides exist, the strength of binding of the RNA construct to the target polynucleotide can be affected by introducing non-canonical base pairings (i.e., mismatches) (e.g., “lower energy G-U pairing,” FIG. 7C) or by leaving out a base in the flank such that a nucleotide in the target polynucleotide is left unpaired, forming a bulge (“bulge,” FIG. 7C).
As shown in FIGS. 8A-C, the nucleotides chosen for the flank regions will dictate the size of the joining region defined by the target polynucleotide sequence. Again, these figures are intended to illustrate the principles of the invention. In FIG. 8A, the flank sequences are designed to pair with each nucleotide of the target polynucleotide sequence, forming a junction size of zero. In FIG. 8B, the flank sequences are designed such that one nucleotide of the target polynucleotide sequence is left without a binding partner, forming a junction size of one. FIG. 8C shows an example of flank sequences designed to leave two nucleotides of the target polynucleotide sequence without binding partners, forming a junction size of two.
Design of an RNA construct can be aided by use of any number of existing informatic RNA folding programs such as mfold, Sfold and the Vienna software. In addition, RNAFold can be used to examine optimal folds of a single RNA molecule; RNACofold can be used to examine optimal folds of two interacting RNA molecules; MiRanda or RNAHybrid can be used as part of high throughput screens to find naturally occurring instances of RNA-target polynucleotide interactions; and JMol can be used for visualizing existing crystal structures. These tools predict the probable structure of RNA and can be used in the design of complementary RNA constructs with predictable structural influences upon contact with the target polynucleotide. Depending on the target polynucleotide sequence, structural aspects may be exploited in the design of the RNA construct, especially with respect to areas of uniqueness in the target polynucleotide. Examples of this strategy include hairpin, stem-loops, cloverleafs, kissing complexes and other conformational structures known to result from traditional RNA-RNA interactions.
4. SELEX Approach
Generally, SELEX (Systematic Evolution of Ligands by Exponential Enrichment) is a method for generating a ligand of interest by taking a population of randomly-generated ligands (e.g., small molecules, polynucleotides or polypeptides) and systematically selecting and amplifying ligands that meet chosen criteria. To select for a ligand of interest, a very large polynucleotide library of random sequences is synthesized. Next, the sequences are exposed to a target binding partner (e.g., a protein or small molecule). Unbound sequences are removed, and the bound sequences are eluted and amplified. The amplified population is again exposed to the target binding partner, and the process is repeated. The stringency of binding and elution conditions can be increased to preferentially enrich only the tightest binding sequences. After a number of cycles, the remaining sequences are identified.
An RNA construct can be produced using a combination of the design approach outlined above and a SELEX-like approach. Use of a SELEX-like approach allows efficient screening of a large range of all possible RNA construct sequences for the best functional candidates. Designing an RNA construct with a SELEX-like approach has several steps in common with the above-described design approach, including choosing a functional motif for the RNA construct (e.g., histone stem-loop); identifying nucleotides important for desired activity (e.g., binding to an RBP); choosing a target polynucleotide sequence, such as an miRNA (e.g., HCMV-miR-US4 or miR-373), which is specific to a desired environment (e.g., HCMV-infected cells or breast cancer cells); and designing the flank sequences of the RNA construct by choosing the region of the target polynucleotide sequence to which the RNA construct will bind.
A SELEX-like approach may be taken by keeping the flank sequences and nucleotides critical to the functioning of the motif constant and randomly generating sequences for the remaining positions. Specifically, a library of nucleic acids is generated having sequences in which the constant positions are the same in each sequence, and random nucleotides are placed into the variable positions. FIG. 9 depicts an example of this approach using the histone stem-loop motif. In FIG. 9, three regions of nucleotides are kept constant during the SELEX procedure: a region that includes nucleotides required for the stem-loop binding protein (SLBP) to bind, as well as the two flank regions that correspond to the target polynucleotide sequence that has been chosen. FIG. 9 also depicts the two regions that are varied with randomly generated sequences: the 5′-most joining region (bold line flanked by “+” signs) and the right side of the stem to the 3′-most joining region (bold line flanked by “+” signs). The 5′ most joining region can be 0-7 nucleotides long, which will produce a joining region of 3-10 bases including the three adenines directly 3′ to the stem. The 3′ side of the stem and 3′-most joining region can be 5-15 nucleotides long. This region should include at least 5 nucleotides, so that each base of the 3′ side of the stem will be paired, and can include up to 10 nucleotides to make up the 3′ most joining region.
A starting library of potential RNA constructs having sequences in which constant positions are kept constant, and variable regions containing random sequences, is initially screened. In the initial screen, nucleic acids having sequences that bind to a protein of interest (e.g., an RBP) in the absence of the target polynucleotide are removed. Then, remaining nucleic acids are exposed to the protein of interest in the presence of the target polynucleotide, and unbound nucleic acids are removed. Bound nucleic acids are eluted, amplified, and the process is repeated under desired stringency conditions. Nucleic acids that remain after multiple rounds of selection are identified and further tested for desired activity. For example, identified nucleic acids may be attached to a polypeptide coding region that includes one or more reporter genes, placed in an expression environment in the presence or absence of the target polynucleotide and assayed to determine whether the reporter gene is expressed. Desired RNA constructs will be those that express the reporter gene only in the presence of the target polynucleotide.
C. Histone Stem-Loop (HSL)
1. HSL Biology and Sequences
The principles of RNA construct design can be applied to a histone stem-loop starting point. For example, a wild-type histone stem-loop (HSL) motif (including a consensus sequence of a HSL motif) can be used as the starting point for further design of an RNA construct in accordance with the principles described above.
Metazoan cell cycle-regulated histone mRNAs are the only known cellular mRNAs that do not terminate in a poly(A) tail. Instead, these messages terminate in a conserved HSL motif, which functions to increase translation. The HSL motif is recognized and bound by “Stem-loop Binding Protein” (SLBP), an RBP, which is believed to upregulate the translation of one or more adjacent polypeptide coding regions through the action of a protein complex, shown in FIG. 10 (see, Gorgoni et al. (2005) RNA 11:1030-1042). As illustrated in FIG. 10, the SLBP binds the HSL in the 3′ UTR of the encoded polypeptide. Without wishing to be bound by the theory, the SLBP may interact with eukaryotic initiation factor 3 (eIF3) and other factors, represented by protein “X,” that interact with eIF4E (“4E” in FIG. 10) and eIF4G (“4G” in FIG. 10), which in turn binds to the 5′ cap (black circle). The formation of this complex may aid in the recruitment of the small ribosomal subunit (40s). In addition, the SLBP may directly bind Paip1, a protein thought to act in a manner similar to eIF4E, which binds eIF4A (“4A” in FIG. 10) to mediate translation. Alternatively, Paip1 simply may act to stabilize the complex.
According to the methods of the present invention, an RNA construct can be designed with a HSL weakened sufficiently to prevent formation of the stem-loop, thereby preventing upregulation of an adjacent polypeptide coding region. The HSL of the RNA construct can be reactivated by binding to a target polynucleotide that stabilizes the stem-loop structure, thereby enabling activation of translation. The advantages of using the HSL motif are that it is well-studied and described, minimal translation occurs when it is inactive, and it achieves orders of magnitude of increased translation when active.
The HSL motif has a conserved size and structure. The visual depiction of this is shown in the following HSL consensus sequence.











TABLE 3







shows the FASTA nucleic acid codes 


that are used throughout the description.








FASTA code
Nucleotide





A
Adenosine


C
Cytidine


G
Guanine


T
Thymidine


U
Uridine


R
G A (purine)


Y
T U C (pyrimidine)


K
G T U (keto)


M
A C (amino)


S
G C (strong)


W
A T U (weak)


B
G T U C


D
G A T U


H
A C T U


V
G C A


N
A G C T U (any)


—
Gap of indeterminate length









In the HSL, one or more of the guanines at the base of the 5′ side stem, the adenine on the 3′ side stem, and the first and third uridines in the loop are typically needed for binding to a SLBP. The remaining nucleotides are more variable. However, in some instances the bases that are typically needed for binding to a SLBP may be altered even if some amount of activity is lost. Examples of naturally occurring HSL sequences are identified in FIG. 11 by GenBank number, and can be accessed at www.ncbi.nlm.nih.gov/. “Start” and “end” indicate the location of the HSL sequence within the entire nucleotide sequence. Accordingly, any HSL consensus sequence or sequence from FIG. 11 can by used as the starting point to construct an RNA construct according to the invention.
While the HSL consensus sequence can be described visually, it can also be described using the logic search pattern (“PatSearch description”) described in Grillo, et al. (2003) NUCLEIC ACIDS RESEARCH 31 (13)3608-3612.
The PatSearch description for the HSL described visually above is:









r1 = {AU, UA, GC, CG, GU, UG}



 



MMM p1 = GGYYY U HHUH A r1~p1 MM






“r1” indicates the rules for pairing to be applied to the consensus sequence. For example, the HSL stem can include “AU” base pairings, “UA” base pairings, “GC” base pairings, “CG” base pairings, “GU” base pairings, and “UG” base pairings. The next line shows the consensus sequence, beginning at the 5′ end with three “M” nucleotides. Next, “p1” indicates the first pattern of the consensus sequence, “GGYYY,” which represents the 5′ side of the stem. The next nucleotides in the sequence are UHHUHA, which represent the loop. Next, “r1 ̃p1” indicates that the pairing rules “r1” must be applied to the “p1” sequence to furnish the 3′ side of the stem. Finally, the 3′ end of the consensus sequence has two amino nucleotides, “MM.”
Other examples of HSL consensus sequences are shown below.




It should be understood that any wild-type HSL sequence (including a consensus sequence) can be used as the starting point to make any number of RNA constructs. From this wild-type HSL, at least one stem-weakening mutation is incorporated into the construct to decrease the formation of the stem-loop structure in the absence of a target polynucleotide. Consensus sequences that include non-canonical base pairings (i.e., mismatches) are considered to be weakened when such a pairing (e.g., a GU or a UG pairing) is chosen, even if the consensus sequence is broad enough to include non-canonical pairings at those same positions (e.g., because some wild-type sequences falling within the consensus sequence have canonical base pairing, rather than non-canonical base pairing, at the location). Alternatively, an RNA construct can contain a wild-type stem-loop if the stem-loop, in the context of the RNA construct, has an increased Minimum Free Energy (MFE) compared to the stem-loop in a wild-type context, such that the stem-loop of the RNA construct will be less stable than will the stem-loop in a wild-type context.
2. Design of an Example RNA Construct when Starting from an HSL Motif
This section illustrates the design of an RNA construct, shown in FIG. 12, starting from Consensus Sequence I.
a. Joining Regions
When designing an RNA construct based on an HSL motif, the size of the J2 joining region can be chosen so that the RNA construct-target polynucleotide structure forms a Family A, Family B or Family C structure. In the example depicted in FIG. 12, Family A is chosen. Family A likely allows the most favorable nucleic acid structure for binding to the SLBP. This is typically true of RNA constructs based upon the HSL structure.
HSL Consensus Sequence I begins with three “M” nucleotides. In FIG. 12, all three “M” nucleotides are chosen to be unpaired “A” nucleotides, thereby forming a J2 region of 3 nucleotides. However, the size of the joining region can be decreased by choosing all or some of the “M” nucleotides to be nucleotides that pair with the target polynucleotide. The size of the J2 region also can be increased by adding nucleotides 5′ to the “MMM” region that do not pair with the target polynucleotide.
The HSL consensus sequence ends with two “M” nucleotides. In FIG. 12, the two “M” nucleotides are chosen to be unpaired “AC,” thereby forming the J3 joining region. As with the J2 joining region, the size of the J3 joining region can be decreased by choosing all or some of the “M” nucleotides to be nucleotides that pair with the target polynucleotide, or it can be increased by adding nucleotides 5′ to the “MM” region that do not pair with the target polynucleotide. The J1 joining region in this example contains no nucleotides. Thus, this example has a Family A structure due to the choice of joining regions.
b. Weakening the Stem
The HSL consensus sequence has the sequence “GGYYY” on the 5′ side of the stem, corresponding to “GGUUC” in FIG. 12. Although the “r1” pairing rules are broad enough to include the non-canonical base pairings GU and UG in the stem, use of non-canonical base pairings (i.e., mismatches) at these positions, rather than canonical base pairs, can produce a weakened stem suitable for use with the present invention. Therefore, the choice of three GU base pairings in the stem, depicted in FIG. 12, weakens the stem to prevent formation of the stem-loop in the absence of the target polynucleotide.
c. Flank Regions
Flank regions can be designed according to the principles discussed above. The flank regions of the RNA construct include two of the three joining regions as well as sequence that is complementary to the target polynucleotide. In the example shown in FIG. 12, the flank regions include the three-way joining regions J2 (“AAA”) and J3 (“AC”), corresponding to “MMM and “MM,” respectively, in the consensus sequence. The nucleotides distal to the joining regions contain nucleotides that hybridize to the target sequence, which in this example is the human microRNA hsa-miR-373.
As described above, the selected HSL sequence is then further modified to attach the polypeptide coding region using standard molecular biological protocols.
D. Iron Response Element (IRE)
Alternatively, a wild type Iron Response Element (IRE), including a consensus sequence of an IRE motif, can be used as the starting point for functional design of an RNA construct in accordance with the principles described above. The IRE is an stem-loop motif with a binding site for RBPs such as iron-responsive binding proteins, for example, aconitase 1 (ACO1, also known as iron-responsive element binding protein 1 or IRP1) and iron-responsive element binding protein 2 (IREB-2, also know as IRP2). IREs occur naturally in the 3′ and 5′UTRs of mRNAs that encode iron metabolism proteins. Translation may be up or down regulated depending upon whether the IRE is located in the 3′ or 5′ UTR and on the concentration of iron. Typically, IREs located in the 5′ UTR of an mRNA inhibit translation, while IREs located in the 3′ UTR stabilize mRNA, leading to increased translation. Additionally, iron-responsive binding proteins actively bind IREs when cellular iron concentration is low. For example, the transferrin receptor mRNA contains an IRE in its 3′ UTR, and therefore more transferrin is produced and more iron will be transported into the cell. Conversely, the ferritin H and L subunit mRNAs contain IREs in their 5′ UTRs and are suppressed when iron concentration is low (i.e., do not make proteins that incorporate iron if it is not available). As cellular iron concentration increases, IRP1 degrades and IRP2 deactivates, thus decreasing production of transferrin and increasing production of H and L ferritin subunits. (See also Matthias W. Hentze and Lukas C. Kuhn (1996) PROC. NATL. ACAD. SCI. USA, Vol. 93, pp. 8175-8182).
In one strategy, depicted in FIG. 13A, an RNA construct is engineered from the wild-type sequence of an IRE (including a consensus sequence). The RNA construct includes one or more weakened IREs and is located in the 3′ UTR adjacent to the polypeptide coding region. As shown in FIG. 13B, the presence of a target polynucleotide stabilizes the IRE. The stabilized IRE associates with an RBP, which prevents degradation of the RNA construct, leading to increased translation. This strategy can be modified to have an RNA construct including at least two weakened IREs located in the 3′ UTR adjacent to the polypeptide coding region. These two IREs can be designed to bind to two different target polynucleotides, such that the presence of both target polynucleotides allows for the desired increase in translation.
In another strategy, an RNA construct is engineered from the wild-type sequence of an IRE (including a consensus sequence). The RNA construct includes one or more weakened IREs and is located in the 5′ UTR adjacent to the polypeptide coding region. The presence of a target polynucleotide stabilizes the IRE. The stabilized IRE associates with an RBP, leading to translation inhibition.
In another strategy, RNA constructs are weakened IREs and are located both in the 5′ and 3′ UTRs adjacent to the polypeptide coding region. In this strategy, the flank regions of the weakened IREs in the 5′ and 3′ UTRs are designed to bind to different target polynucleotides. For example, the flank regions of the 3′ UTR RNA construct are designed to bind a target polynucleotide (“target 1”) found in target cells, and the flank regions of the 5′ UTR RNA construct are designed to bind a target polynucleotide (“target 2”) not found in the target cells. When the two RNA constructs adjacent the polypeptide coding region are introduced into target cells, The 3′ RNA construct is stabilized by the binding of target 1, and translation of the encoded polypeptide will increase. At a desired time point, target 2 can be introduced in the target cells to inhibit translation.
The structure of the IRE motif is conserved but the exact composition of nucleotides can be variable, providing a useful platform for design of an RNA construct. Three subtypes of IRE consensus sequences can be used as a starting point from which to design an RNA construct. FIG. 13C shows an example of each type of IRE consensus sequence. No specific requirements exist for the nucleotide composition of the paired bases of the stem (except that they must follow the “r1” pairing rules described below) or for the composition of the flank sequences. The lack of specific base requirements gives a large degree of flexibility for designing an RNA construct based on the consensus IRE sequences or specific naturally-occurring IRE sequences in accordance with the strategies outlined above. The boxed portions of the consensus sequences indicate the regions where the stem may be weakened by introducing non-canonical base pairings (i.e., mismatches). This lower stem region can be 2 to 6 nucleotides long in certain embodiments of the invention.
The PatSearch pattern for IRE subtype 1 is as follows:









1.) IRE1



r1 = {AU, UA, GC, CG, GU, UG}



 



p1 = 2 . . . 8 C p2 = 5 . . . 5 CAGWGH r1~p2



 



r1~p1






“r1” indicates the rules for pairing to be applied to the consensus sequence. For example, the IRE1 consensus sequence can include “AU” base pairings, “UA” base pairings, “GC” base pairings, “CG” base pairings, “GU” base pairings, and “UG” base pairings. The IRE1 consensus sequence begins at the 5′ end with the first pattern, “p1,” which forms the first part of the 5′ side of the stem. “p1” defines a string of 2 to 8 nucleotides of any composition (A, G, C, or U). The next part of the 5′ side of the stem is an unpaired “C” nucleotide, followed by the second pattern “p2,” the last part of the 5′ stem. “p2” defines a string of exactly 5 nucleotides of any composition (A, G, C, or U). Following “p2” is the loop, which consists of the sequence “CAGWGH.” Following the loop is the first part of the 3′ side of the stem, which can be any string of nucleotides that pairs to the “p2” pattern, provided that the nucleotides follow the “r1” rules for pairing (as indicated by the PatSearch description “r1 ̃p2”). The final part of the 3′ stem can be any string of nucleotides that pairs to the “p1” pattern, provided that the nucleotides follow the “r1” rules for pairing (as indicated by the PatSearch description “r1 ̃p1”). Examples of specific naturally-occurring IRE1 sequences are identified in FIG. 14 by GenBank number, and can be accessed at www.ncbi.nlm.nih.gov/. “Start” and “end” indicate the location of the IRE1 sequence within the entire nucleotide sequence.
The PatSearch pattern for IRE subtype 2 is as follows:







2.) IRE2


r1 = {AU, UA, GC, CG, GU, UG}


 


p3 = 2 . . . 8 NNC p4 = 5 . . . 5 CAGWGH r1~p4 N


 


r1~p3






“r1” indicates the rules for pairing to be applied to the consensus sequence. For example, the IRE2 consensus sequence can include “AU” base pairings, “UA” base pairings, “GC” base pairings, “CG” base pairings, “GU” base pairings, and “UG” base pairings. The IRE2 consensus sequence begins at the 5′ end with the first pattern, “p3,” which forms the first part of the 5′ side of the stem. “p3” defines a string of 2 to 8 nucleotides of any composition (A, G, C, or U). The next part of the 5′ side of the stem is the sequence “NNC,” which will be unpaired. Next, the second pattern, “p4,” forms the last part of the 5′ stem. “p4” defines a string of exactly 5 nucleotides of any composition (A, G, C, or U). Following “p4” is the loop, which consists of the sequence “CAGWGH.” Following the loop is the first part of the 3′ side of the stem, which can be any string of nucleotides that pairs to the “p4” pattern, provided that the nucleotides follow the “r1” rules for pairing (as indicated by the PatSearch description “r1 ̃p4”). The second part of the 3′ stem can be any single unpaired nucleotide “N.” The final part of the 3′ stem can be any string of nucleotides that pairs to the “p3” pattern, provided that the nucleotides follow the “r1” rules for pairing (as indicated by the PatSearch description “r1 ̃p3”). Examples of specific naturally-occurring IRE2 sequences are identified in FIG. 15 by GenBank number, and can be accessed at www.ncbi.nlm.nih.gov/. “Start” and “end” indicate the location of the IRE2 sequence within the entire nucleotide sequence.
The PatSearch pattern for IRE subtype 3 is as follows:









3.) IRE3



r1 = {AU, UA, GC, CG, GU, UG}



 



p5 = 6 . . . 8 C p6 = 2 . . . 2 p7 = 3 . . . 3



 



CAGWGH r1~p7 U r1~p6 r1~p5






“r1” indicates the rules for pairing to be applied to the consensus sequence. For example, the IRE3 consensus sequence can include “AU” base pairings, “UA” base pairings, “GC” base pairings, “CG” base pairings, “GU” base pairings, and “UG” base pairings. The IRE3 consensus sequence begins at the 5′ end with the first pattern, “p5,” which forms the first part of the 5′ side of the stem. “p5” defines a string of 6 to 8 nucleotides of any composition (A, G, C, or U). The next part of the 5′ side of the stem is an unpaired “C” nucleotide, followed by the second pattern “p6,” which makes up the third part of the 5′ stem. “p6” defines a string of exactly 2 nucleotides of any composition (A, G, C, or U). The final part of the 5′ stem is the pattern “p7,” which consists of exactly 3 nucleotides. Following “p7” is the loop, which consists of the sequence “CAGWGH.” Following the loop is the first part of the 3′ side of the stem, which can be any string of nucleotides that pairs to the “p7” pattern, provided that the nucleotides follow the “r1” rules for pairing (as indicated by the PatSearch description “r1 ̃p7”). The second part of the 3′ stem is the unpaired nucleotide “U.” The next part of the 3′ stem can be any string of nucleotides that pairs to the “p6” pattern, provided that the nucleotides follow the “r1” rules for pairing (as indicated by the PatSearch description “r1 ̃p6”). The final part of the 3′ stem can be any string of nucleotides that pairs to the “p5” pattern, provided that the nucleotides follow the “r1” rules for pairing (as indicated by the PatSearch description “r1 ̃p5”). Examples of specific naturally-occurring IRE3 sequences are identified in FIG. 16 by GenBank number, and can be accessed at www.ncbi.nlm.nih.gov/. “Start” and “end” indicate the location of the IRE3 sequence within the entire nucleotide sequence.
E. SECIS
Alternatively, a wild type Selenocysteine Insertion Element (SECIS), including a consensus sequence of a SECIS motif, can be used as the starting point for functional design of an RNA construct in accordance with the principles described above. Two varieties of SECIS elements exist, type 1 and type 2 (SECIS1, SECIS2). An active SECIS element present in the 3′UTR of an mRNA causes the translation machinery to reinterpret a UGA stop codon as a selenocysteine residue. Selenocysteine incorporation at UGA codons also uses specialized trans-acting factors. They include a selenocysteine-specific tRNA, an elongation factor specific for this tRNA and a SECIS-binding protein, (e.g., SBP2, which is an RBP), which recruits the elongation factor to the selenoprotein mRNA. Ribosomal Protein L30 also binds the SECIS element. It is believed that a complex of SELB (the specialized elongation factor), Sec-tRNAsec (selenocysteine tRNA), GTP (guanosine triphosphate), SBP2 and SECIS form and then associate with the ribosome via L30. At this point, a conformational change in the SECIS element triggers the release of Sec-tRNASec and GTP hydrolysis, which allows incorporation of the selenocysteine residue into the polypeptide. (See, e.g., Chavatte et al. (2005) NATURE STRUCTURAL AND MOLECULAR BIOLOGY 12:408-418.)
As shown in FIG. 17A, an RNA construct is designed and placed adjacent to a region encoding an innocuous, small polypeptide ending in a UGA stop codon, which is followed by the desired polypeptide coding region. The RNA construct is a weakened SECIS element located in the 3′ UTR adjacent the polypeptide coding region. In the absence of a stabilizing target polynucleotide, only the small, innocuous peptide is translated. As shown in FIG. 17B, the presence of a stabilizing target polynucleotide causes the SECIS element (the RNA construct) to become active, which in turn causes the translation machinery to read the UGA stop codon as a selenocysteine, thereby producing a polypeptide that includes the innocuous peptide, the selenocysteine residue, and the desired polypeptide from the polypeptide coding region. In addition, when the SECIS element of the RNA construct is inactive (for example, when no target polynucleotide is present) the polypeptide encoded by the polypeptide coding region will not be produced. FIG. 17C shows an example of a SECIS consensus sequence for SECIS1.
The PatSearch description for the SECIS type 1 consensus sequence is as follows:









A.) SECIS1



r1 = {AU, UA, GC, CG, GU, UG}



 



p1 = 4 . . . 19



 



p2 = 2 . . . 9 R



 



UGAN



 



p3 = 8 . . . 12 p4 = 0 . . . 3 p5 = aav



 



p7 = 7 . . . 10 r1~p3[1, 0, 0]



 



p3: ((({circumflex over ( )}RR|{circumflex over ( )}MC)|{circumflex over ( )}SU)6 . . . 10)



 



NGAN



 



p8 = 2 . . . 9



 



r1~p1






“r1” indicates the rules for pairing to be applied to the consensus sequence. For example, the SECIS1 consensus sequence can include “AU” base pairings, “UA” base pairings, “GC” base pairings, “CG” base pairings, “GU” base pairings, and “UG” base pairings. The SECIS1 consensus sequence begins at the 5′ end with the first pattern, “p1,” which forms the first part of the 5′ side of the stem. “p1” defines a string of 4 to 19 nucleotides of any composition (A, G, C, or U). The next part of the 5′ side of the stem is the second pattern “p2,” which defines a string of 2 to 9 unpaired nucleotides of any composition (A, G, C, or U), followed by an “R.” The next part of the 5′ side of the stem is the string of nucleotides “UGAN.” The next part of the 5′ side of the stem is defined by the pattern “p3,” which consists of 8-12 nucleotides. “p3” is further defined by the PatSearch description, “p3:(((^RR|^MC)|^SU) 6 . . . 10),” which indicates that “p3 must start with either “RR,” “MC,” or “SU” followed by 6-10 nucleotides of any composition (making up 8-12 nucleotides total). Pattern “p4” defines the 5′ end of the loop, which can be 0 to 3 nucleotides of any composition (A, G, C, or U). The loop continues with pattern “p5,” defined by the string of nucleotides “AAV.” The loop continues with pattern “p7,” which consists of 7 to 10 nucleotides of any composition (A, G, C, or U). Following the loop is the first part of the 3′ side of the stem, which can be any string of nucleotides that pairs to the “p3” pattern, provided that the nucleotides follow the “r1” rules for pairing. The PatSearch description, “r1 ̃p3[1,0,0]” indicates that the nucleotides in this section can, but are not required to, incorporate 1 mismatch, 0 insertions, and 0 deletions. The second part of the 3′ stem is the string of nucleotides “NGAN.” The next part of the 3′ stem consists of 2 to 9 unpaired nucleotides of any composition (A, G, C, or U). The final part of the 3′ stem can be any string of nucleotides that pairs to the “p1” pattern, provided that the nucleotides follow the “r1” rules for pairing. Examples of specific naturally-occurring SECIS type 1 sequences are identified in FIG. 18 by GenBank number, and can be accessed at www.ncbi.nlm.nih.gov/. “Start” and “end” indicate the location of the SECIS1 sequence within the entire nucleotide sequence.

The PatSearch description for the SECIS type 2 consensus sequence is as follows:









B.) SECIS2



r1 = {AU, UA, GC, CG, GU, UG}



 



p1 = 4 . . . 19



 



p2 = 2 . . . 9 A



 



UGAN



 



p3 = 8 . . . 12 p3: ((({circumflex over ( )}RR|{circumflex over ( )}MC)|{circumflex over ( )}SU)6 . . . 10)



 



p4 = 0 . . . 3 p5 = AAV p6 = 11 . . . 14 p6:



 



(0 . . . 1 p7 = 2 . . . 7 3 . . . 6 r1~p7



 



0 . . . 3$)



 



(((r1~p3[1, 0, 0]|r1~p3[1, 1, 0])|r1~p3



 



[1, 0, 1])|r1~p3[0, 1, 1])



 



NGAN



 



pl0 = 2 . . . 9



 



r1~p1






“r1” indicates the rules for pairing to be applied to the consensus sequence. For example, the SECIS2 consensus sequence can include “AU” base pairings, “UA” base pairings, “GC” base pairings, “CG” base pairings, “GU” base pairings, and “UG” base pairings. The SECIS2 consensus sequence begins at the 5′ end with the first pattern, “p1,” which forms the first part of the 5′ side of the stem. “p1” defines a string of 4 to 19 nucleotides of any composition (A, G, C, or U). The next part of the 5′ side of the stem is the second pattern “p2,” which defines a string of 2 to 9 unpaired nucleotides of any composition (A, G, C, or U), followed by an “A.” The next part of the 5′ side of the stem is the string of nucleotides “UGAN.” The next part of the 5′ side of the stem is defined by the pattern “p3,” which consists of 8-12 nucleotides. “p3” is further defined by the PatSearch description, “p3:(((^rr|^mc)|^su) 6 . . . 10),” which indicates that “p3 must start with either “RR,” “MC,” or “SU” followed by 6-10 nucleotides of any composition (making up 8-12 nucleotides total). Pattern “p4” defines the 5′ end of the loop, and can be 0 to 3 nucleotides of any composition (A, G, C, or U). The loop continues with pattern “p5,” defined by the string of nucleotides “AAV.” The loop continues with pattern “p6,” which consists of 11 to 14 nucleotides which begins with 0 to 1 nucleotide of any compositions (A, G, C, or U), followed by the pattern p7, which can be 2 to 7 nucleotides of any composition (A, G, C, or U) followed by 3 to 6 nucleotides, provided that the last 3 nucleotides, which will form the first part of the 3′ side of the stem, follow the “r1” rules for pairing. The next part of the 3′ side of the stem can be any string of nucleotides that pairs to the “p3” pattern, provided that the nucleotides follow the “r1” rules for pairing. The PatSearch description, “(((r1 ̃p3[1,0,0]|r1 ̃p3[1,1,0])|r1 ̃p3[1,0,1])|r1 ̃p3[0,1,1])” indicates that the nucleotides in this section can (but are not required to) incorporate 1 mismatch, 0 insertions, and 0 deletions; 1 mismatch, 1 insertion, and 0 deletions; 1 mismatch, 0 insertions, and 1 deletion; or 0 mismatches, 1 insertion, and 1 deletion. The second part of the 3′ stem is the string of nucleotides “NGAN.” Pattern 10 defines the next part of the 3′ stem, which consists of 2 to 9 unpaired nucleotides of any composition (A, G, C, or U). The final part of the 3′ stem can be any string of nucleotides that pairs to the “p1” pattern, provided that the nucleotides follow the “r1” rules for pairing. Examples of specific naturally-occurring SECIS type 2 sequences are identified in FIG. 19 by GenBank number, and can be accessed at www.ncbi.nlm.nih.gov/. “Start” and “end” indicate the location of the SECIS2 sequence within the entire nucleotide sequence.
F. Internal Ribosome Entry Site (IRES)
Alternatively, a wild type Internal Ribosomal Entry Site (IRES), including a consensus sequence of a IRES motif, can be used as the starting point for functional design of an RNA construct in accordance with the principles described above. The IRES is located in the 5′ UTR of mRNA and allows for 5′ cap-independent initiation of translation. Multiple forms have been identified and some interact with different factors to begin translation. For example, some IRESes bind eIF4G to initiate translation, while some act by directly binding to the 40S subunit. However, IRESes are not known to bind to an RBP. Instead, proper formation of the IRES facilitates binding of proteins that make up the translation machinery itself (e.g., eIF4G or the 40S subunit).
IRESes typically are large and more complex than a simple stem-loop. A few flaviviruses have an IRES with similar structure, shown generally in FIG. 20A. One example of an IRES of this type is the Hepatitis C virus IRES shown in FIG. 20B. One part of that structure, referred to as “domain II” in the Hepatitis C virus, can be used as a starting point for making an RNA construct (see FIG. 21A). FIG. 21A shows domain II (labeled IIa and IIb) from the Hepatitis C virus, and FIG. 21B depicts the region of nucleotides, labeled “N,” that can be modified to design an RNA construct. For example, nucleotides can be modified to weaken the stem, and flank nucleotides can be designed to match a target polynucleotide sequence. Examples of flavivirus IRESes with similar structure, including a domain II region in bold, are shown in FIG. 22. The sequences for other specific naturally-occurring IRES motifs from which an RNA construct can be designed are identified in FIG. 23 by GenBank number, and can be accessed at www.ncbi.nlm.nih.gov/. “Start” and “end” indicate the location of the IRES sequence within the entire nucleotide sequence. Any of the sequences can by modified to produce an RNA construct.
As shown in FIG. 24A, an RNA construct can be designed to encode a weakened domain II-like structure, which is followed by the remaining IRES structure, including a start codon in the stem-loop corresponding to domain IV of the Hepatitis C IRES, which is in turn followed by the desired polypeptide coding region. The 5′ cap can be modified with an alternate base such that the cap is able to prevent exonuclease degradation of the RNA construct, but translation cannot be initiated from the cap. More particularly, the syntheses of mRNA cap analogs modified with boranophosphate moiety at either the alpha or beta-position of the 5′,5′-triphosphate bridge (m(7)Gppp(BH3)G, m(7)Gpp(BH3)pG and m(7)Gpp(BH3)pm(7)G) have been described. The preliminary biological characterization of these compounds revealed that they have high affinity for translational factor eIF4E and high potency to inhibit cap-dependent translation in a cell free system. The analogs modified at the beta-position were also found to be resistant to DcpS decapping pyrophosphatase. Thus, in the absence of a stabilizing target polynucleotide, translation cannot be initiated from the cap or from the IRES, so nothing is translated. As shown in FIG. 24B, the presence of a stabilizing target polynucleotide causes the weakened IRES element of the RNA construct to become active, which in turn causes the translation machinery to initiate translation at the internal start codon, thereby translating the polypeptide coding region. Unlike RNA constructs based on HSL, IRE, or SECIS motifs, RNA constructs based on IRES motifs do not bind to an RBP. Rather, proper formation of the IRES element is required for binding of one or more proteins that make up the translation machinery itself. Thus, although the invention typically is described herein with regard to a change in structure of the RNA construct that leads to a change in the ability of an RBP to bind to the RNA construct and, thus modulates expression of a polypeptide coding region, it should be understood that the description of how to make and use RNA constructs applies equally to embodiments, such as RNA constructs based on the IRES, where a change in RNA structure leads to modulation of polypeptide coding region expression without involvement of an RBP. Use of the IRES motif is advantageous because it is independent of the cell cycle, and multiple variations of the IRES exist that can be used to produce a variety of RNA constructs suitable for use in many different expression environments.
III. Target Polynucleotides
Target polynucleotides can be any endogenous polynucleotide (e.g., a DNA or an RNA, such as a coding RNA, non-coding RNA, regulatory RNA, or microRNA) that is present in, or characterizes, a particular cell type or tissue in which a polypeptide coding region attached to an RNA construct of the present invention is to be expressed. Alternatively, a target polynucleotide can be one that has been exogenously supplied to an expression environment. An expression environment is any environment in which a polypeptide coding region can be expressed (e.g., a naturally-occurring cell, such as a neoplastic cell or infected cell; a non-naturally occurring cell; or a cell-free expression system (e.g., available from Life Technologies, Foster City, Calif.), such as rabbit reticulocyte lysate, wheat germ extract or E. coli cell-free system). Target polynucleotides bind to the flank regions of an RNA construct, typically stabilizing the secondary structure of the RNA construct, for example, the stem-loop. (It should be understood that, while the text focuses on a target polynucleotide that stabilizes an RNA construct, it can have other effects, such as those described for FIGS. 2A-D.) Stabilization of the secondary structure allows for the modulation of translation of a polypeptide coding region, allowing for specific expression of, or the inhibition of expression of, a desired polypeptide in an expression environment containing a target polynucleotide. A target polynucleotide can be, but is not limited to, a viral polynucleotide, a bacterial polynucleotide, an polynucleotide expressed in a neoplastic cell, or a polynucleotide characteristic of a protein deficiency. Target polynucleotides can be found in, for example, but not limited to, a virally- or bacterially-infected cell, a neoplastic cell, a diseased cell, a tissue, a cell culture, or a sample containing polynucleotides (e.g., a blood sample).
Target polynucleotides can be any non-coding or coding polynucleotide. Typically, though, they are non-coding RNAs, such as microRNAs. Factors for choosing a target polynucleotide having appropriate activity at the RNA construct are that the target polynucleotide be found in the expression environment into which the RNA construct is placed and, preferably, in an abundant manner. In general, the target polynucleotide can function regardless of specific size, sequence, or structure, and a complementary flanking region of an RNA construct can be designed to be specific for any target polynucleotide once it has been identified and sequenced. The RNA construct so designed should change its structure in the presence of the target polynucleotide, thus creating a “switch” with one structural state forming in the absence of the target polynucleotide and another structural state being created in the presence of a target polynucleotide. The specific structure and/or sequence of the target polynucleotide is chosen for its ability to confer a change in the RNA construct structure and thus its activity state.
Selection of a target polynucleotide is based on multiple criteria including, but not limited to, uniqueness, prevalence, and energetics. For example, a target polynucleotide sequence should be unique enough so that the structure of the RNA construct is reinforced and becomes active only in the targeted environment. For instance, a viral microRNA (miRNA) that occurs in infected cells can be used as a target polynucleotide if it is sufficiently different from host sequences in non-infected cells to prevent activation of the RNA construct and translation of the polypeptide coding region where not intended. In addition, the target polynucleotide should be present in sufficient quantity to increase the likelihood that it will interact with the RNA construct. The sequence of the target polynucleotide also should allow for the RNA construct to base pair with a favorable energy minimization to promote structure formation. For example, the calculated MFE (minimum free energy) structure should be such that the target polynucleotide is predicted to hybridize the RNA construct's flank regions as desired (rather than the RNA construct folding with itself and the target polynucleotide folding with itself or the RNA construct and the target polynucleotide hybridizing in a manner other than what is desired).
1. Uniqueness
One factor in selecting a target polynucleotide, such as a viral RNA, is the degree to which the target polynucleotide can selectively (and specifically) activate the RNA construct. Ideally, this occurs uniquely in the desired environment such as the targeted cell or tissue. The desired selectivity may result from several factors including the target polynucleotide sequence and/or quantity.
The most direct manner to achieve specificity of the target polynucleotide will result as a consequence of the uniqueness of its primary sequence. For example, there are many examples of viral specific expressed RNAs that are different from any known human sequence. Example 2 details just one of these situations, where SV40 microRNA differs from its cellular microRNA counterpart by several nucleotides. These sequence differences can be used to design the RNA construct such that they are optimized. Logically, it is expected that the longer the sequence is, the more likely it will have sufficient uniqueness to serve as the basis for designing a complementary specific RNA construct.
2. Prevalence
An additional approach to the specificity issue is to take advantage of target polynucleotides that are uniquely expressed in a desired tissue or cell-type. For instance, one could still use a viral target polynucleotide that had an exact cellular counterpart, if the cellular RNA was only expressed at an irrelevant time point (such as during embryogenesis) or in an unrelated cell type that would not be exposed to the RNA construct. Despite the lack of sequence or structural uniqueness, distinctively expressed (or distributed) target polynucleotides could still be used to activate the RNA construct.
Likewise, if there is a substantial difference in the quantity of the target polynucleotide being expressed in a desired environment relative to an environment in which expression of the RNA construct is not desired, then selective activation of the RNA construct can still be achieved. For example, in the case of a viral target polynucleotide that has an exact cellular (host) counterpart, the cellular version could be expressed at very low background levels but during the course of viral infection, a 10.000-fold increase could be produced in infected cells. This quantitative difference could serve to produce the desired selectivity.
3. Identifying Target Polynucleotides
Target polynucleotides according to the present invention can be identified from a number of sources. For example, to target virally-infected cells, the flanking regions of an RNA construct can be designed to be complementary to microRNAs expressed by viruses. First, using the mirBase web resource (www.microrna.sanger.ac.uk/sequences/) for microRNAs, a search using the keywords “virus” and “viral” was performed. microRNA sequences from viruses known to infect humans were compiled (FIG. 25). These sequences can be used as target polynucleotides, and particularly as target polynucleotides for anti-viral compounds effective against the organisms listed in FIG. 25 as well as related organisms. Second, the rFam web resource (www.sanger.ac.uk/Software/Rfam/) provides a collection of known and putative non-coding RNA “families.” A keyword search of this set using “virus” returned a list of 69 “families” shown in FIG. 26. Many sequences belonging to viruses that infect humans are contained in this set. Again, these sequences can be used as targets. Third and more broadly, using the NCBI (National Center for Biotechnology Information) viral genome resource (www.ncbi.nlm.nih.gov/genomes/VIRUSES/viruses.html), a list of viruses known to infect human hosts also was developed. Complete genome sequences with annotation are downloaded for these viruses and are parsed to extract sequence portions not designated as protein coding regions. This will include well-defined 3′ and 5′ UTR segments from known messages. However, it also will consist of sequence regions whose purpose has not been identified, but that may be expressed as part of the post transcriptional regulatory process. Again, these sequences can be used as targets. All of these resources can be checked periodically for new data.
The same approach can be taken for any pathogen. For example, the rFam database, which includes many non coding RNA families, can be used and the mirBase databases, which includes many microRNAs coded by viruses and animals, can be used. Additionally, NCBI genome entries can be reviewed individually for particular organisms of interest. Moreover, the same approach can be taken for any diseased cell, such as a neoplastic cell. For example, miRNAs from the mirBase database (microrna.sanger.ac.uk/sequences/) can be examined. Additionally, other disease-specific target polynucleotides, such as RNAs, known in the art can be used. Examples of cancer-specific RNAs include, but are not limited to, the human breast cancer-specific microRNA hsa-miR-373 and the testicular cancer-specific microRNA hsa-miR-520c. miR-373 and miR-520c have been shown to be involved in metastatic breast cancer and testicular cancer, respectively, and not present in normal adult tissue. They may play a role in other cancer types as well. The other isoforms of hsa-miR-520 (see Table 4) are also contemplated to be useful.









TABLE 4






mirBase



mirBaseID
Accession
Mature miRNA Sequence







hsa-miR-520a-5p
MIMAT0002833
CUCCAGAGGGAAGUACUUUCU (SEQ ID NO: 180)


 


hsa-miR-520a-3p
MIMAT0002834
AAAGUGCUUCCCUUUGGACUGU (SEQ ID NO: 181)


 


hsa-miR-520b
MIMAT0002843
AAAGUGCUUCCUUUUAGAGGG (SEQ ID NO: 182)


 


hsa-miR-520c-5p
MIMAT0005455
CUCUAGAGGGAAGCACUUUCUG (SEQ ID NO: 183)


 


hsa-miR-520c-3p
MIMAT0002846
AAAGUGCUUCCUUUUAGAGGGU (SEQ ID NO: 184)


 


hsa-miR-520d-
MIMAT0002855
CUACAAAGGGAAGCCCUUUC (SEQ ID NO: 185)


5p




 


hsa-miR-520d-
MIMAT0002856
AAAGUGCUUCUCUUUGGUGGGU (SEQ ID NO: 186)


3p




 


hsa-miR-520e
MIMAT0002825
AAAGUGCUUCCUUUUUGAGGG (SEQ ID NO: 187)


 


hsa-miR-520f
MIMAT0002830
AAGUGCUUCCUUUUAGAGGGUU (SEQ ID NO: 188)


 


hsa-miR-520g
MIMAT0002858
ACAAAGUGCUUCCCUUUAGAGUGU (SEQ ID NO: 189)


 


hsa-miR-520h
MIMAT0002867
ACAAAGUGCUUCCCUUUAGAGU (SEQ ID NO: 190)









4. Experimental Validation of Target Polynucleotide Expression
One direct way to empirically identify viral target polynucleotides is to infect cell cultures with virus and sequence the expressed polynucleotides using a viral specific custom tiling-array strategy or second generation sequencing technologies. The resulting polynucleotide that is identified is compared to polynucleotides from non-infected control cell cultures. Unique viral polynucleotides found to be expressed in abundance are good candidates for use as a viral specific target polynucleotides. These could include microRNAs, non-coding RNAs, regulatory RNAs and even genic/coding RNAs.
A similar procedure can be used to identify target polynucleotides for other pathogens. Moreover, target polynucleotides can be identified for diseased cells, such as neoplastic cells, by comparing the RNA being expressed between a diseased cell and a normal cell. Unique polynucleotides expressed in abundance in a diseased cell are good candidates for use as a diseased-cell specific target polynucleotide.
IV. Polypeptide Coding Region
A polypeptide coding region may be attached 5′ or 3′ of an RNA construct of the present invention. In the presence of a target polynucleotide, the RNA construct will undergo a conformational change which can either promote or inhibit association with an RBP. The RBP will, in turn, promote or inhibit translation of the polypeptide coding region. In one embodiment of the invention, the presence of a target polynucleotide will promote translation of an attached polypeptide coding region, allowing for the directed expression of a desired polypeptide in a target expression environment, for example, a cell. For example, a death gene may be chosen to kill an undesirable cell (or pathogen in a cell), or a reporter gene may be chosen to detect a target polynucleotide. Generally, it is contemplated that the polypeptide coding region will be chosen to suit any of the uses described herein.
A polypeptide coding region can be attached to an RNA construct in any number of ways known in molecular biology. If an RNA construct and polypeptide coding region are to be produced from a DNA vector, a DNA vector incorporating both the RNA construct and polypeptide coding region can be produced. DNA fragments encoding all or a portion of the polypeptide coding region can be obtained from any source, for example, from a cDNA library or amplified from genomic DNA using polymerase chain reaction (PCR). DNA encoding the polypeptide coding region, or portions thereof, together with the remaining portions of the RNA construct can be inserted into an appropriate expression or amplification vector using standard ligation techniques. Alternatively, an RNA construct can be synthesized using any of a number of methods known in the art, and chemically linked to a polypeptide coding region. Vectors suitable for use with the present invention and methods of construction are discussed in more detail below.
1. Death Genes
It is contemplated that the stabilization of the RNA construct in the presence of a target polynucleotide can either turn on or off expression of a death gene (a polypeptide coding region), as described previously. Examples of turning on a death gene would be for the RNA construct, when activated, to allow the expression of killer genes, such as one of the caspases. Alternatively, when the RNA construct is activated, the expression of a gene is turned off which in turn turns “ON” the production of a death gene. Examples of death genes include, but are not limited to, caspase family genes such as CASP1, CASP2, CASP3, CASP4, CASP5, CASP6, CASP7, CASP8, CASP9, CASP10, CASP14 and death domain family genes such as CRADD, DAPK1, DAPK2, FADD, RIPK1, TNFRSF10A, TNFRSF10B, TNFRSF11B, TNFRSF1A, TNFRSF21, TNFRSF25, TNFRSF6, TRADD. Other death genes that may be used are Bcl-2, Bax, Bcl-x, p53, ICE, and Cytochrome C. Additional death polypeptides encoded by “death genes” are listed in FIG. 27 (Parts 1 and 2).
2. Reporter Genes
It is further contemplated that the stabilization of the RNA construct in the presence of a target polynucleotide can either turn on or off expression of a reporter gene (a polypeptide coding region), as described previously. A reporter gene is a gene that encodes a detectable marker. For example, the RNA construct, when activated by the presence of a target polynucleotide, can allow for the expression of any number of reporter genes suitable for use with the present invention. Examples of target polynucleotides that can activate an RNA construct include but are not limited to a viral target polynucleotide, a bacterial target polynucleotide, a target polynucleotide characteristic of a neoplastic cell, or a target polynucleotide characteristic of any disease or condition listed in Table 1. Examples of reporter genes suitable for use with the present invention include LacZ, luciferase (e.g., firefly luciferase (FL) from Photinus pyralis and Renilla luciferase (RL) from Renilla reniformis), green fluorescent protein (GFP) and its derivatives (e.g., eGFP, eCFP, eYFP), ferritin, tyrosine hydroxylase, myoglobin, hemoglobin, protein amide H, transferrin receptor, truncated progesterone receptor, β-galactosidase, and non human receptor proteins whose ligands can incorporate detectable tags (e.g., radiolabels, gadolinium). Any protein that can be detected (i.e. a detectable marker) can function as a reporter gene for use with the present invention. Methods of detection are described in detail below.
V. Production, Administration and Testing of an RNA Construct
1. Production of an RNA Construct
DNA encoding the RNA construct (and, optionally, a polypeptide coding region) of the present invention can be prepared in any number of ways known in the art. DNA fragments encoding portions of the RNA construct can be obtained from any source, for example, from a cDNA library or amplified from genomic DNA using polymerase chain reaction (PCR). Methods for isolating nucleic acids, synthesizing nucleic acids, hybridizing nucleic acids, making and screening cDNA libraries, and performing PCR are well known in the art (see, e.g., Gubler et al. (1983), GENE 25:263-269; Sambrook et al. (2nd ed. 1989) MOLECULAR CLONING, A LABORATORY MANUAL; Ausubel et al., eds. (1994) CURRENT PROTOCOLS IN MOLECULAR BIOLOGY), as are PCR methods (see U.S. Pat. Nos. 4,683,195 and 4,683,202; Innis et al., eds (1990) PCR PROTOCOLS: A GUIDE TO METHODS AND APPLICATIONS). Expression libraries are also well known to those of skill in the art. Additional basic texts disclosing the general methods of use in this invention include Kriegler (1990) GENE TRANSFER AND EXPRESSION: A LABORATORY MANUAL. 
In an exemplary embodiment, at least a portion of a contemplated RNA constructs are chemically synthesized. The single stranded molecules that comprise RNA constructs may be synthesized using any of a variety of techniques known in the art, such as those described in Usman et al. (1987) J. AM. CHEM. SOC. 109:7845; Scaringe et al. (1990) NUCLEIC ACIDS RES. 18:5433; Wincott et al. (1995) NUCLEIC ACIDS RES. 23:2677-2684; and Wincott et al. (1997) METHODS MOL. BIO. 74:59. 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. As a non-limiting example, small scale syntheses may be conducted on an Applied Biosystems synthesizer using a 0.2 μmol scale protocol with a 2.5 min. coupling step for 2′-O-methylated nucleotides. Alternatively, syntheses at the 0.2 μmol scale may be performed on a 96-well plate synthesizer from Protogene (Palo Alto, Calif.). However, a larger or smaller scale of synthesis is also within the scope of the present invention. Suitable reagents for oligonucleotide synthesis, methods for RNA deprotection, and methods for RNA purification are known to those of skill in the art.
Alternative methods of chemical synthesis well known to the skilled artisan (see, for example, Engels et al. (1989) ANGEW. CHEM. INTL. ED. 28:716-734) include, for example, phosphotriester, phosphoramidite, and H-phosphonate methods for nucleic acid synthesis. Another method for such chemical synthesis is polymer-supported synthesis using standard phosphoramidite chemistry. Nucleic acids larger than about 100 nucleotides can be synthesized as several fragments using these methods. The fragments then can be ligated together to form the full length encoded RNA construct.
DNA encoding the RNA construct, or portions thereof, (and, optionally, a polypeptide coding region) can be inserted into an appropriate expression or amplification vector using standard ligation techniques. The vector is typically selected to be functional in the particular expression environment employed (i.e., the vector is compatible with the host cell machinery such that amplification of the RNA construct and/or expression of the RNA construct can occur). DNA encoding the RNA construct may be amplified/expressed in prokaryotic, yeast, insect (baculovirus systems) and/or eukaryotic host cells. These transgenes can be introduced as a linear construct, a circular plasmid, or a viral vector, which can be incorporated and inherited as a transgene integrated into the host genome. The transgene can also be constructed to permit it to be inherited as an extrachromosomal plasmid (Gassmann et al. (1995) PROC. NATL. ACAD. SCI. USA 92:1292).
RNA constructs and, optionally, polypeptide coding regions can be expressed and purified using common molecular biology and biochemistry techniques. For example, recombinant expression vectors can be used which can be engineered to carry an encoded RNA construct and, optionally, polypeptide coding region into a host cell to provide for expression of the RNA construct and attached polypeptide coding region. Such vectors, for example, can be introduced into a host cell by transfection means including, but not limited to, heat shock, calcium phosphate, DEAE-dextran, electroporation or liposome-mediated transfer. Recombinant expression vectors include, but are not limited to, Escherichia coli based expression vectors such as BL21 (DE3) pLysS, COS cell-based expression vectors such as CDM8 or pDC201, or CHO cell-based expression vectors such as pED vectors. An RNA construct and, optionally, polypeptide coding region can be linked to one of any number of promoters in an expression vector that can be activated in the chosen cell line. In an embodiment, a cassette (RNA construct and promoter) is carried by a vector that contains a selectable marker such that cells receiving the vector can be identified.
For example, promoters to express the RNA construct within a cell line can be drawn from those that are functionally active within the host cell. Such promoters can include, but are not limited to, a T7 promoter, a CMV promoter, a SV40 early promoter, a herpes TK promoter, and others known in recombinant DNA technology. Inducible promoters can be used, and include promoters such as metallothionine promoter (MT), mouse mammary tumor virus promoter (MMTV), and others known to those skilled in the art. Exemplary selectable markers and their attendant selection agents can be drawn, for example, from the group including, but not limited to, ampicillin, kanamycin, aminoglycoside phosphotransferase/G418, hygromycin-B phosphotransferase/hygromycin-B, and amplifiable selection markers such as dihydrofolate reductase/methotrexate and others known to skilled practitioners.
Additional elements for directing the replication and transcription of an RNA construct and, optionally, polypeptide coding region) can be included in a vector to express RNA constructs in a variety of cell types, including but not limited to, eukaryotic, prokaryotic, insect, plant and yeast. For example, microorganisms such as bacteria can be transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing the RNA construct coding sequences; yeast can be transformed with recombinant yeast expression vectors containing the RNA construct coding sequences; insect cell systems can be infected with recombinant virus expression vectors (e.g., baculovirus) containing the RNA construct coding sequences; plant cell systems can be infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing the RNA construct coding sequences.
Typically, the vectors used in any of the host cells will contain at least a 5′ flanking sequence (also referred to as a promoter) and, optionally, other regulatory elements, such as an enhancer(s), an origin of replication element, a transcriptional termination element, a complete intron sequence containing a donor and acceptor splice site, a signal peptide sequence, a ribosome binding site element, a polyadenylation sequence, a polylinker region for inserting the DNA encoding the polypeptide coding region, and a selectable marker element. Suitable modifications may be made to the 5′ cap in order to direct translation of the encoded polypeptide using cap-independent mechanisms. Such modifications are well-known in the art (Kowalska, J. et al. (2008) NUCLEIC ACIDS SYMP. SER. 52:289-90).
The recombinant RNA construct expression vectors can be DNA plasmids or viral vectors. RNA construct-expressing viral vectors can be constructed based on, but not limited to, adeno-associated virus (for a review, see Muzyczka et al. (1992) CURR. TOPICS IN MICRO. AND IMMUNOL. 158:97-129), adenovirus (see, for example, Berkner et al. (1988) BIOTECHNIQUES 6:616; Rosenfeld et al. (1991) SCIENCE 252:431-434; and Rosenfeld et al. (1992) CELL 68:143-155), or alphavirus as well as others known in the art. Retroviruses have been used to introduce a variety of genes into many different cell types, including epithelial cells, in vitro and/or in vivo (see for example Eglitis, et al. (1985) SCIENCE 230:1395-1398; Danos and Mulligan (1988) PROC. NATL. ACAD. SCI. USA 85:6460-6464; Wilson et al. (1988) PROC. NATL. ACAD. SCI. USA 85:3014-3018; Armentano et al., (1990) PROC. NATL. ACAD. SCI. USA 87:61416145; Huber et al. (1991) PROC. NATL. ACAD. SCI. USA 88:8039-8043; Ferry et al. (1991), PROC. NATL. ACAD. SCI. USA 88:8377-8381; Chowdhury et al. (1991), SCIENCE 254:1802-1805; van Beusechem. et al. (1992) PROC. NATL. ACAD. SCI. USA 89:7640-19; Kay et al. (1992) Human Gene Therapy 3:641-647; Dai et al. (1992) PROC. NATL. ACAD. SCI. USA 89:10892-10895; Hwu et al. (1993) J. Immunol. 150:4104-4115; U.S. Pat. No. 4,868,116; U.S. Pat. No. 4,980,286; PCT Application WO 89/07136; PCT Application WO 89/02468; PCT Application WO 89/05345; and PCT Application WO 92/07573). Recombinant retroviral vectors capable of transcribing the RNA constructs of the present invention can be produced by transfecting the recombinant retroviral genome into suitable packaging cell lines such as PA317 and Psi-CRIP (Comette et al. (1991) HUMAN GENE THERAPY 2:5-10; Cone et al., 1984, PROC. NATL. ACAD. SCI. USA 81:6349). Recombinant adenoviral vectors can be used to infect a wide variety of cells and tissues in susceptible hosts (e.g., rat, hamster, dog, and chimpanzee) (Hsu et al. (1992) J. INFECTIOUS DISEASE, 166:769), and also have the advantage of not requiring mitotically active cells for infection.
2. Testing an RNA Construct
A DNA vector encoding an RNA construct, and optionally a polypeptide coding region can be transfected into desired cell culture lines or cell-free expression systems, with or without target polynucleotide, and tested to determine whether stem formation, recruitment of an RBP, and/or translation of an encoded polypeptide can occur in the presence of a target polynucleotide.
The ability of an RNA construct to form a stem-loop and bind an RBP in the presence of a target sequence can be tested by incorporating a label, for example, a radiolabel, fluorescent label, or luminescent label, into the RNA construct. The RNA construct, target polynucleotide and any additional desired reagents, such as buffers, may be added to the appropriate RBP. If a target polynucleotide is present, the RNA construct assumes a structure to which the RBP can bind. The RBP is then isolated and the presence of label is assayed. Detection of the label indicates that the RNA construct has attained the structure required for binding to the RBP, which further indicates that the target nucleotide is present. Alternatively, assays useful for detecting the binding of an RNA construct to an RBP, but that do not require labeled RNA construct, include, but are not limited to, absorbance assays, immunoassays, assays for enzymatic activity of the RBP and Western blots.
Examples of suitable fluorescent labels include fluorescein isothiocyanate (FITC), 5,6-carboxymethyl fluorescein, Texas red, nitrobenz-2-oxa-1,3-diazol-4-yl (NBD), coumarin, dansyl chloride, rhodamine, amino-methyl coumarin (AMCA), Eosin, Erythrosin, BODIPY®, Cascade Blue®, Oregon Green®, pyrene, lissamine, xanthenes, acridines, oxazines, phycoerythrin, macrocyclic chelates of lanthanide ions such as quantum Dye®, fluorescent energy transfer dyes, such as thiazole orange-ethidium heterodimer, and the cyanine dyes Cy3, Cy3.5, Cy5, Cy5.5 and Cy7. Examples of other specific fluorescent labels include 3-Hydroxypyrene 5,8,10-Tri Sulfonic acid, 5-Hydroxy Tryptamine (5-HT), Acid Fuchsin, Alizarin Complexon, Alizarin Red, Allophycocyanin, Aminocoumarin, Anthroyl Stearate, Astrazon Brilliant Red 4G, Astrazon Orange R, Astrazon Red 6B, Astrazon Yellow 7 GLL, Atabrine, Auramine, Aurophosphine, Aurophosphine G, BAO 9 (Bisaminophenyloxadiazole), BCECF, Berberine Sulphate, Bisbenzamide, Blancophor FFG Solution, Blancophor SV, Bodipy F1, Brilliant Sulphoflavin FF, Calcien Blue, Calcium Green, Calcofluor RW Solution, Calcofluor White, Calcophor White ABT Solution, Calcophor White Standard Solution, Carbostyryl, Cascade Yellow, Catecholamine, Chinacrine, Coriphosphine O, Coumarin-Phalloidin, CY3.1 8, CY5.1 8, CY7, Dans (1-Dimethyl Amino Naphaline 5 Sulphonic Acid), Dansa (Diamino Naphtyl Sulphonic Acid), Dansyl NH—CH3, Diamino Phenyl Oxydiazole (DAO), Dimethylamino-5-Sulphonic acid, Dipyrrometheneboron Difluoride, Diphenyl Brilliant Flavine 7GFF, Dopamine, Erythrosin ITC, Euchrysin, FIF (Formaldehyde Induced Fluorescence), Flazo Orange, Fluo 3, Fluorescamine, Fura-2, Genacryl Brilliant Red B, Genacryl Brilliant Yellow 10GF, Genacryl Pink 3G, Genacryl Yellow 5GF, Gloxalic Acid, Granular Blue, Haematoporphyrin, Indo-1, Intrawhite Cf Liquid, Leucophor PAF, Leucophor SF, Leucophor WS, Lissamine Rhodamine B200 (RD200), Lucifer Yellow CH, Lucifer Yellow VS, Magdala Red, Marina Blue, Maxilon Brilliant Flavin 10 GFF, Maxilon Brilliant Flavin 8 GFF, MPS (Methyl Green Pyronine Stilbene), Mithramycin, NBD Amine, Nitrobenzoxadidole, Noradrenaline, Nuclear Fast Red, Nuclear Yellow, Nylosan Brilliant Flavin EBG, Oxadiazole, Pacific Blue, Pararosaniline (Feulgen), Phorwite AR Solution, Phorwite BKL, Phorwite Rev, Phorwite RPA, Phosphine 3R, Phthalocyanine, Phycoerythrin R, Polyazaindacene Pontochrome Blue Black, Porphyrin, Primuline, Procion Yellow, Pyronine, Pyronine B, Pyrozal Brilliant Flavin 7GF, Quinacrine Mustard, Rhodamine 123, Rhodamine 5 GLD, Rhodamine 6G, Rhodamine B, Rhodamine B 200, Rhodamine B Extra, Rhodamine BB, Rhodamine BG, Rhodamine WT, Serotonin, Sevron Brilliant Red 2B, Sevron Brilliant Red 4G, Sevron Brilliant Red B, Sevron Orange, Sevron Yellow L, SITS (Primuline), SITS (Stilbene Isothiosulphonic acid), Stilbene, Snarf 1, sulpho Rhodamine B Can C, Sulpho Rhodamine G Extra, Tetracycline, Thiazine Red R, Thioflavin S, Thioflavin TCN, Thioflavin 5, Thiolyte, Thiozol Orange, Tinopol CBS, True Blue, Ultralite, Uranine B, Uvitex SFC, Xylene Orange, and XRITC.
Other useful fluorescent labels include fluorescein (5-carboxyfluorescein-N-hydroxysuccinimide ester), rhodamine (5,6-tetramethyl rhodamine), and the cyanine dyes Cy3, Cy3.5, Cy5, Cy5.5 and Cy7. The absorption and emission maxima, respectively, for these fluors are: FITC (490 nm; 520 nm), Cy3 (554 nm; 568 nm), Cy3.5 (581 nm; 588 nm), Cy5 (652 nm: 672 nm), Cy5.5 (682 nm; 703 nm) and Cy7 (755 nm; 778 nm), thus allowing their simultaneous detection. Other examples of fluorescein dyes include 6-carboxyfluorescein (6-FAM), 2′,4′,1,4,-tetrachlorofluorescein (TET), 2′,4′,5′,7′,1,4-hexachlorofluorescein (HEX), 2′,7′-dimethoxy-4′,5′-dichloro-6-carboxyrhodamine (JOE), 2′-chloro-5′-fluoro-7′,8′-fused phenyl-1,4-dichloro-6-carboxyfluorescein (NED), and 2′-chloro-7′-phenyl-1,4-dichloro-6-carboxyfluorescein (VIC). Fluorescent labels can be obtained from a variety of commercial sources, including Amersham Pharmacia Biotech, Piscataway, N.J.; Molecular Probes, Eugene, Oreg.; and Research Organics, Cleveland, Ohio.
It is further contemplated that labels that detect a structural change in a polynucleotide can be incorporated into an RNA construct or directly into a nucleotide of the RNA construct to detect the conformational change that results upon binding to a target polynucleotide. Many such labels are known to those of skill in the art. Examples of labels suitable for use in the disclosed method are radioactive isotopes, fluorescent molecules, phosphorescent molecules, enzymes, antibodies, and ligands. More specifically, molecular beacons, Amplifluors®, FRET probes, cleavable FRET probes, TagMan® probes, scorpion primers, fluorescent triplex oligonucleotides including but not limited to triplex molecular beacons or triplex FRET probes, fluorescent water-soluble conjugated polymers, PNA probes or QPNA probes, for example, can be used to activate or quench a given label based on stem formation.
Stem activated labels are labels or pairs of labels where fluorescence is increased or altered by formation of a stem structure. Stem activated labels can include an acceptor fluorescent label and a donor moiety such that, when the acceptor and donor are in proximity (when the nucleic acid strands containing the labels form a stem structure), fluorescence resonance energy transfer from the donor to the acceptor causes the acceptor to fluoresce. Stem activated labels are typically pairs of labels positioned on nucleic acid molecules, such as the sides of the stem-loop, such that the acceptor and donor are brought into proximity when a stem structure is formed in the nucleic acid molecule. If the donor moiety of a stem activated label is itself a fluorescent label, it can release energy as fluorescence (typically at a different wavelength than the fluorescence of the acceptor) when not in proximity to an acceptor (that is, when a stem structure is not formed). When the stem structure forms, the overall effect would then be a reduction of donor fluorescence and an increase in acceptor fluorescence. FRET probes are an example of the use of stem activated labels, the operational principles of which can be adapted for use with RNA constructs.
Stem quenched labels are fluorescent labels positioned on a nucleic acid such that when a stem structure forms a quenching moiety is brought into proximity such that fluorescence from the label is quenched. When the stem is disrupted, the quenching moiety is no longer in proximity to the fluorescent label and fluorescence increases. Examples of this effect can be found in molecular beacons, fluorescent triplex oligonucleotides, triplex molecular beacons, triplex FRET probes, and QPNA probes, the operational principles of which can be adapted for use with RNA constructs.
Examples of labels that can be incorporated into nucleic acids include nucleotide analogs such as BrdUrd (5-bromodeoxyuridine, Hoy et al., (1993) MUTATION RESEARCH 290:217-230), aminoallyldeoxyuridine (Henegariu et al., (2000) NATURE BIOTECHNOLOGY 18:345-348), 5-methylcytosine (Sano et al., (1988) BIOCHIM. BIOPHYS. ACTA 951:157-165), bromouridine (Wansick et al., (1993) J. CELL BIOLOGY 122:283-293) and nucleotides modified with biotin (Langer et al., (1981) PROC. NATL. ACAD. SCI. USA 78:6633) or with suitable haptens such as digoxygenin (Kerkhof (1992) ANAL. BIOCHEM. 205:359-364). Suitable fluorescence-labeled nucleotides are Fluorescein-isothiocyanate-dUTP, Cyanine-3-dUTP and Cyanine-5-dUTP (Yu et al. (1994) NUCLEIC ACIDS RES. 22:3226-3232). An exemplary nucleotide analog label for DNA is BrdUrd (bromodeoxyuridine, BrdUrd, BrdU, BUdR, Sigma-Aldrich Co.). Other useful nucleotide analogs for incorporation of label into DNA are AA-dUTP (aminoallyl-deoxyuridine triphosphate, Sigma-Aldrich Co.), and 5-methyl-dCTP (Roche Molecular Biochemicals). A useful nucleotide analog for incorporation of label into RNA is biotin-16-UTP (biotin-16-uridine-5′-triphosphate, Roche Molecular Biochemicals). Fluorescein, Cy3, and Cy5 can be linked to dUTP for direct labelling. Cy3.5 and Cy7 are available as avidin or anti-digoxygenin conjugates for secondary detection of biotin- or digoxygenin-labelled probes.
Labels that are incorporated into nucleic acid, such as biotin, can be subsequently detected using sensitive methods well-known in the art. For example, biotin can be detected using streptavidin-alkaline phosphatase conjugate (Tropix, Inc.), which is bound to the biotin and subsequently detected by chemiluminescence of suitable substrates (for example, chemiluminescent substrate CSPD: disodium, 3-(4-methoxyspiro-[1,2,-dioxetane-3-2′-(5′-chloro)tricyclo[3.3.1.13,7]decane]-4-yl)phenyl phosphate; Tropix, Inc.). Labels can also be enzymes, such as alkaline phosphatase, soybean peroxidase, horseradish peroxidase and polymerases, that can be detected, for example, with chemical signal amplification or by using a substrate to the enzyme which produces light (for example, a chemiluminescent 1,2-dioxetane substrate) or fluorescent signal.
Methods for detecting and measuring signals generated by labels are also known to those of skill in the art. For example, radioactive isotopes can be detected by scintillation counting or direct visualization; fluorescent molecules can be detected with fluorescent spectrophotometers; phosphorescent molecules can be detected with a spectrophotometer or directly visualized with a camera; enzymes can be detected by detection or visualization of the product of a reaction catalyzed by the enzyme; antibodies can be detected by detecting a secondary label coupled to the antibody.
3. Administration of an RNA Construct
Any of the delivery methods known in the art suitable for use with RNA-based drugs are contemplated to work for RNA constructs according to the invention. For example, recombinant vectors capable of expressing RNA constructs are delivered as described above, and persist in target cells. Alternatively, viral vectors can be used that provide for transient expression of RNA constructs. Such vectors can be repeatedly administered as necessary. Once expressed, the RNA constructs bind to target polynucleotides and undergo a conformational change, for example, strengthening of a stem-loop structure. Delivery of RNA construct-expressing vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from a multicellular organism followed by reintroduction into the multicellular organism, or by any other means that allows for introduction into a desired target cell.
DNA plasmids carrying RNA constructs can be transfected into target cells as a complex with cationic lipid carriers (e.g. Oligofectamine) or non-cationic lipid-based carriers (e.g. Transit-TKOTM). Multiple lipid transfections for RNA constructs targeting different regions of a single target polynucleotide or multiple target polynucleotides over a period of a week or more are also contemplated by the present invention. Successful introduction of the vectors of the invention into host cells can be monitored using various known methods. For example, transient transfection can be signaled with a reporter, such as a fluorescent marker, such as Green Fluorescent Protein (GFP). Stable transfection of ex vivo cells can be ensured using markers that provide the transfected cell with resistance to specific environmental factors (e.g., antibiotics and drugs), such as hygromycin B resistance.
Alternatively, RNA constructs can be produced in cell culture and isolated, or can be chemically synthesized, and administered. Absorption or uptake of an RNA construct can occur through unaided diffusive or active cellular processes, or by auxiliary agents or devices. An RNA construct may be introduced into a cell, either in vitro or where the cell is part of a living organism. If the cell is part of an organism, introduction into the cell will include delivery to the organism. For example, for in vivo delivery, an RNA construct can be injected into a tissue site or administered systemically. In vitro introduction into a cell includes methods known in the art such as electroporation and lipofection.
An RNA construct can be chemically modified to enhance stability. The nucleic acids of the invention may be synthesized and/or modified by methods well established in the art, such as those described in CURRENT PROTOCOLS IN NUCLEIC ACID CHEMISTRY, Beaucage, S. L. et al. (Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA, which is hereby incorporated herein by reference. Chemical modifications may include, but are not limited to 2′ modifications, introduction of non-natural bases, covalent attachment to a ligand, and replacement of phosphate linkages with thiophosphate linkages.
In yet another embodiment, nucleotides may be modified to prevent or inhibit the activation of cellular enzymes, such as, for example, without limitation, certain nucleases. Techniques for inhibiting the activation of cellular enzymes are known in the art including, but not limited to, 2′-amino modifications, 2′-amino sugar modifications, 2′-F sugar modifications, 2′-F modifications, 2′-alkyl sugar modifications, uncharged backbone modifications, morpholino modifications, 2′-O-methyl modifications, and phosphoramidate (see, e.g., Wagner (1995) NAT. MED. 1:1116-8). Thus, at least one 2′-hydroxyl group of the nucleotides on an RNA construct is replaced by a chemical group, for example, by a 2′-amino or a 2′-methyl group.
Conjugating a ligand to an RNA construct can enhance its cellular absorption. In certain instances, a hydrophobic ligand is conjugated to the RNA construct to facilitate direct permeation of the cellular membrane. Alternatively, the ligand conjugated to the RNA construct is a substrate for receptor-mediated endocytosis. These approaches have been used to facilitate cell permeation of antisense oligonucleotides. For example, cholesterol has been conjugated to various antisense oligonucleotides resulting in compounds that are substantially more active compared to their non-conjugated analogs. See Manoharan et al., (2002) ANTISENSE & NUCLEIC ACID DRUG DEVELOPMENT 12(2):103-28. Other lipophilic compounds that have been conjugated to oligonucleotides include 1-pyrene butyric acid, 1,3-bis-O -(hexadecyl)glycerol, and menthol. One example of a ligand for receptor-mediated endocytosis is folic acid. Folic acid enters the cell by folate-receptor-mediated endocytosis. RNA constructs bearing folic acid would be efficiently transported into the cell via the folate-receptor-mediated endocytosis. Li and coworkers report that attachment of folic acid to the 3′-terminus of an oligonucleotide resulted in an 8-fold increase in cellular uptake of the oligonucleotide. Li, et al. (1998) PHARM. RES. 15:1540-5. Other ligands that have been conjugated to oligonucleotides include polyethylene glycols, carbohydrate clusters, cross-linking agents, porphyrin conjugates, and delivery peptides.
In certain instances, conjugation of a cationic ligand to oligonucleotides results in improved resistance to nucleases. Representative examples of cationic ligands are propylammonium and dimethylpropylammonium.
A composition that includes an RNA construct (it should be understood that the RNA construct compositions and administration methods referenced below include both RNA constructs and DNA vectors encoding an RNA construct) can be delivered to a subject by a variety of routes. Exemplary routes include intrathecal, parenchymal, intravenous, nasal, oral, parenteral and ocular delivery. An RNA construct can be incorporated into pharmaceutical compositions suitable for administration. For example, compositions can include one or more species of an RNA construct and a pharmaceutically acceptable carrier. As used herein the language “pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic, intranasal, transdermal), oral or parenteral. Parenteral administration includes intravenous drip, subcutaneous, intraperitoneal or intramuscular injection, site-specific injection (e.g. delivery directly to a tumor or other site of disease), or intrathecal or intraventricular administration.
In general, an RNA construct can be administered by any suitable method. As used herein, topical delivery can refer to the direct application of an RNA construct to any surface of the body, including the eye, a mucous membrane, surfaces of a body cavity, or to any internal surface. Formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, sprays, and liquids. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Topical administration can also be used as a means to selectively deliver the RNA construct to the epidermis or dermis of a subject, or to specific strata thereof, or to an underlying tissue.
Compositions for intrathecal or intraventricular administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives.
Formulations for parenteral administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives. Intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir. For intravenous use, the total concentration of solutes should be controlled to render the preparation isotonic.
An RNA construct can be administered to a subject by pulmonary delivery. Pulmonary delivery compositions can be delivered by inhalation by the patient of a dispersion so that the RNA construct within the dispersion can reach the lung where it can be readily absorbed through the alveolar region directly into blood circulation. Pulmonary delivery can be effective both for systemic delivery and for localized delivery to treat diseases of the lungs.
Pulmonary delivery can be achieved by different approaches, including the use of nebulized, aerosolized, micellular and dry powder-based formulations. Delivery can be achieved with liquid nebulizers, aerosol-based inhalers, metered-dose devices, and dry powder dispersion devices. One of the benefits of using an atomizer or inhaler is that the potential for contamination is minimized because the devices are self contained. Dry powder dispersion devices, for example, deliver drugs that may be readily formulated as dry powders. An RNA construct may be stably stored as lyophilized or spray-dried powders by itself or in combination with suitable powder carriers. The delivery of a composition for inhalation can be mediated by a dosing timing element which can include a timer, a dose counter, time measuring device, or a time indicator which when incorporated into the device enables dose tracking, compliance monitoring, and/or dose triggering to a patient during administration of the aerosol medicament.
An RNA construct can be modified such that it is capable of traversing the blood brain barrier. For example, the RNA construct can be conjugated to a molecule that enables the agent to traverse the barrier. Such modified RNA construct can be administered by any desired method, such as by intraventricular or intramuscular injection, or by pulmonary delivery, for example.
An RNA construct can be administered ocularly, such as to treat retinal disorder, e.g., a retinopathy. For example, the pharmaceutical compositions can be applied to the surface of the eye or nearby tissue, e.g., the inside of the eyelid. They can be applied topically, e.g., by spraying, in drops, as an eyewash, or an ointment. Ointments or droppable liquids may be delivered by ocular delivery systems known in the art such as applicators or eye droppers. Such compositions can include mucomimetics such as hyaluronic acid, chondroitin sulfate, hydroxypropyl methylcellulose or poly(vinyl alcohol), preservatives such as sorbic acid, EDTA or benzylchronium chloride, and the usual quantities of diluents and/or carriers. The pharmaceutical composition can also be administered to the interior of the eye, and can be introduced by a needle or other delivery device which can introduce it to a selected area or structure. The composition containing the RNA construct can also be applied via an ocular patch.
An RNA construct can be administered by an oral or nasal delivery. For example, drugs administered through these membranes have a rapid onset of action, provide therapeutic plasma levels, avoid first pass effect of hepatic metabolism, and avoid exposure of the drug to the hostile gastrointestinal (GI) environment. Additional advantages include easy access to the membrane sites so that the drug can be applied, localized and removed easily.
The types of pharmaceutical excipients that are useful as carrier include stabilizers such as human serum albumin (HSA), bulking agents such as carbohydrates, amino acids and polypeptides; pH adjusters or buffers; salts such as sodium chloride; and the like. These carriers may be in a crystalline or amorphous form or may be a mixture of the two.
VI. Detection Assays
In one aspect of the invention, the RNA construct is used to detect the presence of a target polynucleotide. The target polynucleotide may exist in any expression environment, including, but not limited to, a cell, such as an infected or neoplastic cell; a sample comprising components of a cell; a mixture of polynucleotides; or a cell culture. The RNA construct is designed such that, in the presence of a target polynucleotide, a conformational change occurs, which leads to detection of the RNA construct.
The RNA construct of the invention, when operably linked with a reporter gene, allows one to determine whether a target polynucleotide is present or absent in a variety of expression environments and for a variety of purposes. For example, methods utilizing the RNA construct can either be scientific or commercial diagnostic assays. The target polynucleotide is either itself the molecule of interest to be detected or is a marker whose detection allows identification of another molecule, an organism of interest, or a health state of a patient. For example, the target polynucleotide could be unique to a particular pathogen, and the presence of the target polynucleotide would indicate presence of the pathogen. Alternatively, the target polynucleotide could be a marker for whether a patient has, or is likely to develop, a disease and/or whether a patient is likely to respond to, or is responding to, a particular therapeutic intervention. Samples to be assayed can be a cell culture, a disrupted cell culture, media from a cell culture, or a patient sample. Patient samples can include, but are not limited to, a tissue or body fluid sample. The tissue sample can be any tissue of the body, such as breast tissue, colon tissue, prostate tissue, pancreas tissue, liver tissue, bone tissue, cardiac tissue, lung tissue, skin, endothelium, and hair. The body fluid sample can be selected from any body fluid, such as blood, serum, plasma, stool, urine, sputum, nipple aspirate, pus, semen, saliva, and cerebrospinal fluid.
In practice, a sample to be assayed is obtained. To the extent the sample contains the cellular components needed to express the reporter gene when the RNA construct associates with the target polynucleotide (if present), the RNA construct linked with the reported gene can be directly added to the sample and incubated to allow expression of the reporter gene. The expressed reporter gene is then detected. If the sample does not have the cellular components needed to express the reporter gene, these can be added along with the RNA construct linked to the reporter gene. The reporter gene can be a detectable marker. For example, a color-forming, luminescent or fluorescent molecule such as LacZ, luciferase (e.g., firefly luciferase (FL) from Photinus pyralis and Renilla luciferase (RL) from Renilla reniformis), green fluorescent protein (GFP) and its derivatives (e.g., eGFP, eCFP, eYFP), can be used. In the presence of a target polynucleotide, the RNA construct assumes a conformation to which an RBP can bind, promoting translation of the encoded detectable molecule. Detection of the expressed reporter gene (directly or indirectly) indicates the presence of the target polynucleotide.
Alternatively, any polypeptide that can be detected by any number of methods known in the art, such as a Western blot, can be encoded by the polynucleotide coding region and used in accordance with the methods of the present invention to detect the presence of a target polynucleotide. Polypeptides that can be detected in living organisms using magnetic resonance or a PET scan, for example, can also be used. For example, the polypeptide coding region can encode ferritin, tyrosine hydroxylase, myoglobin, hemoglobin, protein amide H, transferrin receptor, truncated progesterone receptor, β-galactosidase, or non human receptor proteins whose ligands can incorporate detectable tags (e.g., radiolabels, gadolinium).
EXAMPLES
Example 1
Design of a Stem-Loop RNA Construct Starting from a Naturally-Occurring Thermus thermophilus Three-Way Junction
To validate the design principles of the present invention, an RNA construct based on a naturally occurring, Family A three-way junction in the 16S ribosomal subunit of Thermus thermophilus (Protein Data Bank entry 1J5E, Chain A), shown in FIG. 28, is examined. The structure of the ribosomal subunit has been determined by X-ray crystallography. While this example does not begin with a wild type sequence and build the RNA construct as described above, it does validate the principles of construction of an RNA construct insofar as this structure follows the rules and principles of the invention. The sequence of the 16S ribosomal subunit is as follows:







(SEQ ID NO: 192)


UUUGUUGGAGAGUUUGAUCCUGGCUCAGGGUGAACGCUGGCGGCGUGCCU


 


AAGACAUGCAAGUCGUGCGGGCCGCGGGGUUUUACUCCGUGGUCAGCGGC


 


GGACGGGUGAGUAACGCGUGGGUGACCUACCCGGAAGAGGGGGACAACCC


 


GGGGAAACUCGGGCUAAUCCCCCAUGUGGACCCGCCCCUUGGGGUGUGUC


 


CAAAGGGCUUUGCCCGCUUCCGGAUGGGCCCGCGUCCCAUCAGCUAGUUG


 


GUGGGGUAAUGGCCCACCAAGGCGACGACGGGUAGCCGGUCUGAGAGGAU


 


GGCCGGCCACAGGGGCACUGAGACACGGGCCCCACUCCUACGGGAGGCAG


 


CAGUUAGGAAUCUUCCGCAAUGGGCGCAAGCCUGACGGAGCGACGCCGCU


 


UGGAGGAAGAAGCCCUUCGGGGUGUAAACUCCUGAACCCGGGACGAAACC


 


CCCGACGAGGGGACUGACGGUACCGGGGUAAUAGCGCCGGCCAACUCCGU


 


GCCAGCAGCCGCGGUAAUACGGAGGGCGCGAGCGUUACCCGGAUUCACUG


 


GGCGUAAAGGGCGUGUAGGCGGCCUGGGGCGUCCCAUGUGAAAGACCACG


 


GCUCAACCGUGGGGGAGCGUGGGAUACGCUCAGGCUAGACGGUGGGAGAG


 


GGUGGUGGAAUUCCCGGAGUAGCGGUGAAAUGCGCAGAUACCGGGAGGAA


 


CGCCGAUGGCGAAGGCAGCCACCUGGUCCACCCGUGACGCUGAGGCGCGA


 


AAGCGUGGGGAGCAAACCGGAUUAGAUACCCGGGUAGUCCACGCCCUAAA


 


CGAUGCGCGCUAGGUCUCUGGGUCUCCUGGGGGCCGAAGCUAACGCGUUA


 


AGCGCGCCGCCUGGGGAGUACGGCCGCAAGGCUGAAACUCAAAGGAAUUG


 


ACGGGGGCCCGCACAAGCGGUGGAGCAUGUGGUUUAAUUCGAAGCAACGC


 


GAAGAACCUUACCAGGCCUUGACAUGCUAGGGAACCCGGGUGAAAGCCUG


 


GGGUGCCCCGCGAGGGGAGCCCUAGCACAGGUGCUGCAUGGCCGUCGUCA


 


GCUCGUGCCGUGAGGUGUUGGGUUAAGUCCCGCAACGAGCGCAACCCCCG


 


CCGUUAGUUGCCAGCGGUUCGGCCGGGCACUCUAACGGGACUGCCCGCGA


 


AAGCGGGAGGAAGGAGGGGACGACGUCUGGUCAGCAUGGCCCUUACGGCC


 


UGGGCGACACACGUGCUACAAUGCCCACUACAAAGCGAUGCCACCCGGCA


 


ACGGGGAGCUAAUCGCAAAAAGGUGGGCCCAGUUCGGAUUGGGGUCUGCA


 


ACCCGACCCCAUGAAGCCGGAAUCGCUAGUAAUCGCGGAUCAGCCAUGCC


 


GCGGUGAAUACGUUCCGGGCCUUGUACACACCGCCCGUCACGCCAUGGGA


 


GCGGGCUCUACCCGAAGUCGCCGGGAGCCUACGGGCAGGCGCCGAGGGUA


 


GGGCCCGUGACUGGGGCGAAGUCGUAACAAGGUAGCUGUACCGGAAGGUG


 


CGGCUGGAUCACCUCCUUUCU






Part I: Design of an RNA Construct and Target Sequence
The sequences of the three-way junction of Thermus thermophilus are separated into their constituent segments and two of these segments are joined using the naturally-occurring loop found within the same Thermus thermophilus 16S ribosomal subunit sequence (positions 675-680 of Protein Data Bank entry 1J5E). The predicted three way junction structure of this RNA construct (in this instance, two sides of the three-way junction of Thermus thermophilus along with the naturally-occurring loop found within the same Thermus thermophilus 16S ribosomal subunit sequence), when bound to a target polynucleotide (in this instance, one side of the three-way junction of Thermus thermophilus), is shown in FIG. 29. The underlined portions of SEQ ID NO: 192 correspond to the sequences used in the RNA construct (the second underlined portion corresponds to the 5′ side of RNA construct, the third underlined portion corresponds to the loop, and the fourth underlined portion corresponds to the 3′ side of the RNA construct) and target polynucleotide (the first underlined portion). The region notated as “Twort Primer” in FIG. 29 is used for experimental validation of secondary structure formation as explained below. Additionally, as shown in FIG. 29, J1 is three bases, J2 is three bases and J3 is one base. According to FIG. 4D, this combination of joining regions forms a Family A structure. FIG. 30 shows the Family A structure that this RNA construct is predicted to form, and the loop of the RNA construct is highlighted. Thus, as shown in FIGS. 29 and 30, the experimental construct follows the joining region guidelines of the invention for a stem-loop RNA construct.
Prior to performing an in vivo experiment, the potential secondary structures formed by the RNA construct and target polynucleotide separately and together were analyzed with secondary structure predicting tools (RNAFold for each individual sequence and RNACofold for two sequences acting on each other). The intent of this analysis was to predict the structure and the associated reduction in free energy for the sequences folded alone versus together. If the overall reduction in free energy is greater for the sequences folded together (into the desired secondary structure) versus the sum of their individual folds (i.e., the secondary structure assumed), they are more likely to interact as desired. FIG. 31 shows the fold prediction for the Minimum Free Energy (MFE) structure of the RNA construct. FIGS. 32 and 33 show the same for the target polynucleotide and the combined RNA construct-target polynucleotide, respectively.
The reduction in free energy predicted to occur as a result of the folding of the RNA construct alone is −25.00 kcal/mol. The reduction in free energy predicted to occur as a result of the folding of the target polynucleotide alone is −4.60 kcal/mol. Thus, the sum reduction in free energy for the individual sequence predictions is −29.60 kcal/mol (−25.00 kcal/mol+−4.60 kcal/mol). The MFE value of the two sequences folding together into the desired structure is −45.00 kcal/mol. This interaction shows a net free energy reduction of more than 15 kcal/mol beyond the two sequences folding independently, indicating that it is energetically favorable for the RNA construct and target polynucleotide to fold together rather than separately. Therefore, the RNA construct and target polynucleotide are more likely to interact to form the predicted structure than not.
Part II: Expressing and Testing the RNA Construct In Vitro
The next steps of the experiment are performed as follows.
DNA oligonucleotide templates are designed using the T7 promoter to express both the RNA construct and the target polynucleotide. This RNA construct and target polynucleotide is transcribed and used for in vitro experiments.
To test whether the RNA construct and target polynucleotide form the desired structure in vivo, the RNA construct and target polynucleotide are exposed to chemical probes. The more reactive bases (e.g., unpaired adenines and cytosines for DMS probing) are altered in such a way that they stop transcription during a primer extension. By comparing relative counts of varying length transcripts, the reactivity (hence the likelihood of being base paired) of each base in the sequence is determined. That information is used in conjunction with a partition function based algorithm (i.e., RNAFold) to make a determination as to what structure is present. The Twort primer that is incorporated into the RNA construct is present specifically to allow this analysis, as explained below.
In order to experimentally validate a change in conformation of RNA structure, the high-throughput chemical mapping approach “CAFA” (Capillary Automated Footprinting Analysis, Mitra et al. (2008) NUCLEIC ACIDS RES. 36, e63) is used. Chemical mapping (specifically dimethyl sulfoxide (DMS) and hydroxyl radical (.OH) footprinting) in conjunction with the CAFA high-throughput approach is used to experimentally validate the predicted conformational rearrangements in the three-way junction (Mitra et al. (2008) NUCLEIC ACIDS RES. 36, e63).
Chemical mapping techniques leverage the unique chemical reactivity of probes (either chemical or enzymatic) to detect aspects of nucleic acid structure (Donis-Keller et al. (1977) NUCLEIC ACIDS RES. 4, 2527-2538; Goldrick and Kessler (2003) CURRENT PROTOCOLS IN NEUROSCIENCE/EDITORIAL BOARD, Jacqueline N Crawley et al., Chapter 5, Unit 5 l; Vicens et al. (2007) RNA 13, 536-548). Certain chemicals react differently depending on the conformation of the nucleic acid. One such probe is dimethyl sulfoxide (DMS), which reacts selectively with unpaired adenines and cytosines (Tijerina et al. (2007) NATURE PROTOCOLS 2, 2608-2623). DMS is often used to determine the secondary structure of an RNA molecule (Lempereur et al. (1985) NUCLEIC ACIDS RES. 13, 8339-8357). Another probe is the hydroxyl radical (.OH), which selectively cleaves surface exposed nucleotides defining the inside and outside of an RNA molecule (Latham and Cech (1989) SCIENCE 245, 276-282). Common to all forms of chemical mapping is a selective modification or cleavage of the nucleic acid. Determining the degree of cleavage at each nucleotide provides structural information on the RNA. In the case of DMS mapping, adenines and cytosines that exhibit a high degree of modification after DMS treatment are unpaired, while low degree of modification indicates base-paired nucleotides (Lempereur et al. (1985) NUCLEIC ACIDS RES. 13, 8339-8357; Tijerina et al. (2007) NATURE PROTOCOLS 2, 2608-2623).
Detecting chemical modification or cleavage in a nucleic acid is straight forward thanks to reverse transcription coupled with primer extension (Tijerina et al. (2007) NATURE PROTOCOLS 2, 2608-2623). Chemical modifications (e.g. methylation) stop the reverse transcription reaction selectively, allowing for the degree of modification to be probed at each site (Lempereur et al., 1985, supra). The higher the extent of methylation at a particular site, the more cDNA of that length will be produced. Determining the relative populations of different cDNA products (of different length) therefore yields the degree of chemical modification at the corresponding site on the RNA.
Recent advances in capillary electrophoresis sequencing technology (Araya et al. (2007) METHODS 42, 141-149; Quigley and Dovichi (2004) ANALYTICAL CHEMISTRY 76, 4645-4658) now allow for such analyses to be carried out in highly optimized, multiplex systems like the Beckman CEQ 8000 instrument. CAFA allows chemical mapping reactions to be performed and analyzed in 96-well plates, completely eliminating the need for gel electrophoresis. CAFA is also a software package with custom algorithms that allow accurate determination of the peak intensities, even in overlapping regions of the spectrum.
DMS Chemical Probing:
DMS chemical probing measures the degree of base-pairing by selectively methylating unpaired nucleotides. The degree of methylation at each nucleotide is evaluated by reverse-transcription, as the reverse-transcriptase will stop if it encounters a methylated nucleotide. The results of a partition function calculation can be directly compared to this type of probing. The DMS results are used to confirm the computational predictions and assess the predictive ability of the partition function calculation.
.OH Radical Chemical Probing (Footprinting):
.OH footprinting measures the solvent-accessibility of nucleotides by selectively cleaving the backbone of exposed nucleotides (Latham and Cech (1989) SCIENCE 245, 276-282). The .OH data complements DMS data as it reports on any global rearrangements in the RNA. Collecting this data allows examination of actual differences in nucleotide accessibility to solvent (and thus exogenous molecule binding) that result from the RNA structure reconstitution. Together, these two data sets will provide both the 2D and 3D consequences of structure/conformation changes of the 3-way junctions of the experimental RNA construct.
Structural characterizations will be performed under three experimental conditions; CE buffer, 100 mM KCl and 10 mM MgCl2. Salt conditions are well known to affect RNA folding (Laederach et al. (2007) PROC. NATL. ACAD. SCI. USA 104, 7045-7050), and characterizing the effect of mutations on RNA in different electrostatic environments will provide further insight into the degree of structural rearrangements that are caused by the mutation. The experimental data collected will provide comprehensive insight into the structure/conformation changes of the 3-way junctions in the RNA construct.
Alternatively, in vitro RNA binding assays will be done to demonstrate that the target polynucleotide and RNA construct associate, reinforcing the stem structure. Association also can be determined experimentally by separation on a native polyacrylamide electrophoretic gel.
Part III: Use of a Reporter Gene to Test the RNA Construct In Vivo
Once it has been confirmed that the simulated three way junction can form as a result of the RNA construct and target polynucleotide interacting, additional experiments described below can be performed to confirm that the structure of the RNA construct, when bound to the target polynucleotide, will allow it to interact with an RBP and promote translation of an attached polypeptide coding region. Instead of a Twort primer, a polypeptide coding region that encodes a fluorescent reporter will be included in the RNA construct. Following transfection of the RNA construct into an in vivo system, translation of the reporter gene in the presence or absence of target polynucleotide can be measured. It is anticipated that some translation of the reporter gene will take place even in the absence of the target polynucleotide, because the stem will not have been weakened. However, removing the three-way junction from the surrounding context of the entire 16S ribosomal subunit may weaken stem formation, in which case it is anticipated that translation of the reporter gene will only occur in the presence of the target polynucleotide.
Mutations that will modify the strength of the stem, or the binding of the flanking regions to the target polynucleotide, can be introduced as desired to prevent stem formation in the absence of the target polynucleotide. The RNA construct then can be introduced into cells and fluorescence can be measured in the absence of, and in the presence of, a target polynucleotide. If fluorescence occurs in the absence of the target polynucleotide, additional stem-weakening mutations can be introduced. If no fluorescence occurs in the presence of the target polynucleotide, nucleotides can be altered to increase either the likelihood of association of the RNA construct with the target polynucleotide, or to increase the strength of the stem.
Part IV: Variations
To demonstrate that RNA constructs can be made from different stem-loop motifs, a variation of the RNA construct that incorporates a Histone Stem-loop (HSL) motif can be designed (FIG. 34). Based on the free nucleotide proportions in the joining region, this RNA construct is likely to adopt a Family B conformation, such that the motif-bearing helix P2 will be in closer proximity to helix P3 (see FIG. 35). Insofar as J1 is three bases, J2 is one base and J3 is one base, this conformation is also predicted by FIG. 4D. Steps similar to those illustrated above can be used to test the ability of the RNA construct to promote translation of a polypeptide coding region, and to modify the construct if necessary.
It is anticipated that the RNA construct described in FIG. 29 will interact with the target polynucleotide as shown and that the interaction will adhere to the principles outlined above. Moreover, it is anticipated that the HSL variation, shown in FIG. 34, will interact with the target polynucleotide as shown and will promote translation of the polypeptide coding region.
Example 2
MS2 Protein-Binding RNA Constructs
The following demonstrates design of an RNA construct capable of binding an RBP only in the presence of a target.
Part I: Background
The bacteriophage MS2 capsid protein acts as a translational repressor by binding to a specific RNA stem-loop structure that contains the viral replicase translation initiation region (Peabody (1990) J. BIOL. CHEM. 265(10): 5684-5689). The structure of this stem-loop (FIG. 36) has been well studied, and the effects of various base mutations in the wild type have been characterized (Table 5, Schneider, et al. (1992) J. MOL. BIOL. 228(3):862-869). Additionally, high affinity variations have been found using the SELEX technique (Schneider, et al., supra).









TABLE 5







Relative


Wild-type
Nucleotide
Dissociation


Position(s)
Variation
Constant

















A−17
deletion
>3000



G
1.0


A−16
G
1.0


U−12-A+1
A-U
1.1


G−11-C−1
C-G
4.2



U-A
3.9



G-G or C-C
>3000


A−10
deletion
>3000



C
>3000



U
>3000



G
1.7


G−9-C−2
C-G
3.4



U-A
9.5


G−8-C−3
C-G
1.0


A−7
C
10



U
>3000


U−6
A
3.0



C
1.0



G
3.0


U−5
A
10



C
0.02-0.2



G
15


A−4
C
>3000



U
>3000



G
>3000


A+1 to U+4
deletion
330


U+2 to U+4
deletion
1.7


G+3 to U+4
deletion
1.0


U+4
deletion
1.0









Although MS2, in its natural context, functions as a translational repressor, the opposite effect can be attained by incorporating it as part of a Xenopus Poly-A Binding Protein (PABP) fusion protein (e.g., Coller et al., (1998) GENES AND DEVELOPMENT 12:3226-3235). Additionally, a mutant variation (LeCuyer, et al., (1995) BIOCHEM. 34(33): 10600-10606) of MS2 has been described that retains RNA binding capability but significantly reduces its tendency to form capsid particles. Previously, that mutant was incorporated as part of the fusion protein and used to show translational enhancement in messages where an MS2 recognition motif replaced the usual mRNA poly-A tail (Coller, supra). Here, a variant was designed using human PABP (MS2-PABP[human], Table 6) fused to the C-terminal end of MS2 protein and a hexa-his-containing peptide (“hexa-his” disclosed as SEQ ID NO: 92) preceding the N terminal region of the MS2.







TABLE 6







(Proteins)









Label
Full Name
Sequence





MS2-PABP
MS2-
MGSSHHHHHHSSGLVPRGSHMGPRASNFTQFVLVDNGGTGDVTVAPSN



PABP[human]
FANGVAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKVATQTV



fusion protein
GGEELPVAGWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPS




AIAANSGIYASPSAPSYPMASLYVGDLHPDVTEAMLYEKFSPAGPILS




IRVCRDMITRRSLGYAYVNFQQPADAERALDTMNFDVIKGKPVRIMWS




QRDPSLRKSGVGNIFIKNLDKSIDNKALYDTFSAFGNILSCKVVCDEN




GSKGYGFVHFETQEAAERAIEKMNGMLLNDRKVFVGRFKSRKEREAEL




GARAKEFTNVYIKNFGEDMDDERLKDLFGKFGPALSVKVMTDESGKSK




GFGFVSFERHEDAQKAVDEMNGKELNGKQIYVGRAQKKVERQTELKRK




FEQMKQDRITRYQGVNLYVKNLDDGIDDERLRKEFSPFGTITSAKVMM




EGGRSKGFGFVCFSSPEEATKAVTEMNGRIVATKPLYVALAQRKEERQ




AHLTNQYMQRMASVRAVPNPVINPYQPAPPSGYFMAAIPQTQNRAAYY




PPSQIAQLRPSPRWTAQGARPHPFQNMPGAIRPAAPRPPFSTMRPASS




QVPRVMSTQRVANTSTQTMGPRPAAAAAAATPAVRTVPQYKYAAGVRN




PQQHLNAQPQVTMQQPAVHVQGQEPLTASMLASAPPQEQKQMLGERLF




PLIQAMHPTLAGKITGMLLEIDNSELLHMLESPESLRSKVDEAVAVLQ




AHQAKEAAQKAVNSATGVPTV (SEQ ID NO: 95)


 


SLBP
Stem-loop
MACRPRSPPRHQSRCDGDASPPSPARWSLGRKRRADGRRWRPEDAEEA



Binding Protein
EHRGAERRPESFTTPEGPKPRSRCSDWASAVEEDEMRTRVNKEMARYK



[human
RKLLINDFGRERKSSSGSSDSKESMSTVPADFETDESVLMRRQKQINY



recombinant]
GKNTIAYDRYIKEVPRHLRQPGIHPKTPNKFKKYSRRSWDQQIKLWKV




ALHFWDPPAEEGCDLQEIHPVDLESAESSSEPQTSSQDDFDVYSGTPT




KVRHMDSQVEDEFDLEACLTEPLRDFSAMS (SEQ ID NO: 196)









As shown in FIG. 37, two versions of an RNA construct, designated MS2-B1 and MS2-B2 (Table 7), were designed to bind to the target hcmv-mir-us25-2-3p (from human cytomegalovirus, Table 8). This target is an example of a naturally occurring non-coding RNA in a human pathogen. The RNA constructs were designated. Minor variations in stem strength and junctions region size exist between the two constructs.







TABLE 7







(RNA Constructs)









Label
Description
Sequence





MS2-
Wild Type MS2 Motif
AAACAUGAGGAUUACCCAUGU (SEQ ID NO: 99)


WT




 


MS2-P1
Positive Control
AGAGAAACAUGAGGAUCACCCAUGUGAGAGAGAGAGAG




AG (SEQ ID NO: 191)


 


MS2-B1
MS2_engineered_sxRNA2.0
CCGCGGGAGCUCGUUUUUGGGCGCCAUCAGGGUUUUUU




GCCAAGUGGAU (SEQ ID NO: 101)


 


MS2-B2
MS2_engineered_sxRNA2.1
CCGCGGGAGCUCGUUUUUGGGGCGCCAUCAGGGUUUUU




UGCCAAGUGGAU (SEQ ID NO: 102)


 


MS2-B3
MS2_engineered_sxRNA3.1
AACUAUACAACCGUUUUUGGGGCGCCAUUAGGGUUUUU




UGCCUACCUCA (SEQ ID NO: 104)


 


MS2-B5
MS2_engineered_sxRNA4.0
AAAGUGUCAGAGUUUUUGGGGCGCCAUUAGGGUUUUUU




GACGGUGUGG (SEQ ID NO: 107)


 


HSL-C1
Histone Stem-loop Consensus
MMMMGGYYYUHHUHARRRCCMM (SEQ ID NO: 109)


 


HSL-P1
NM_001005464.2(HIST2H3A): position
AGGUUCCAUCGUAUCCAAAAGGCUCUUUUCAGAGCCAC



467-507 positive control histone
CCA (SEQ ID NO: 193)



stem-loop



 


HSL-Bl
NM_001005464.2_minor_mutant
AGGUUCCAUCGUAUCCAAAAGGCUCUUUUCAGAGCUAC



(HIST2H3A): 467-507(U sub at 502)
CCA (SEQ ID NO: 110)


 


HSL-B2
NM_001005464.2_major_mutant
AGGUUCCAUCGUAUCCAAAAGGUUCUUUUCAGGGCUAC



(HIST2H3A): 467-507(U sub at 489,
CCA (SEQ ID NO: 111)



G sub at 499,U sub at 502)



 


HSL-B3
NG_011683.1(SEPT 9): 215412-
GGGGCCGUGGGGGUAACACCCUGUGGGCCACAGGUUUU



215468
CUUUAAAAUCCCAGCUCCG (SEQ ID NO: 113)


 


HSL-B4
HSL_engineered_sxRNA1.0
GAAGAGAGCCGCCCAAAACAGAAAGAAAGGCUCUUUUC




AGAGCUACACUUU (SEQ ID NO: 114)
















TABLE 8







(Targets)









Label
Description
Sequence





T-1
hcmv-miR-us25-2-3p
AUCCACUUGGAGAGCUCCCGCGG




(SEQ ID NO: 103)


 


T-2
hsa-let-7e
UGAGGUAGGAGGUUGUAUAGUU




(SEQ ID NO: 105)


 


T-3
hsa-miR-220a
CCACACCGUAUCUGACACUUU




(SEQ ID NO: 108)


 


T-4
hsa-miR-363*
CGGGUGGAUCACGAUGCAAUUU




(SEQ ID NO: 112)


 


T-5
hsa-miR-1250
ACGGUGCUGGAUGUGGCCUUU




(SEQ ID NO: 115)


 


T-6
xla-miR-427
AAAGUGCUUUCUGUUUUGGGCG




(SEQ ID NO: 116)









Part II: Design Considerations

    1) To generally weaken the wild type structure in the MS2-B1 construct, the stem's first base pairing was changed to U-U, and pairs 2-4 were changed to G-U. In MS2-B2, the first four pairs were changed to G-U which makes the pairing slightly stronger.
    2) The A bulge at position −10 was changed to a G to prevent base pairing with the U's introduced to the lower stem.
    3) The top 3 base pairings in the stem were changed from G-C to C-G to
    
        a.) prevent the upper C's from pairing with the G's introduced in the lower opposite side of the stem,
        b.) prevent the upper G's pairing with the U's introduced into the lower opposite side of the stem
        c.) promote the G bulge.
    
    
    4) The U at position −5 was changed to a C to increase affinity for the protein when stem-loop structure forms and partially compensate for some of the other base changes made to weaken the structure.
    5) The desired three-way junction conformation was “family A” as defined in FIG. 4 (and as described by Lescoute et al, supra) with the stem bearing helix at P3.
    6) Unpaired nucleotides in the 5′ portion of the stem-loop were therefore designated as part of the intended junction J2.
    7) To avoid pairing interactions with the engineered stem's bases or with J3 bases, both J2 and J3 were composed primarily of U's, the exception being a single G at the 5′ terminus of J2 and at the 3′ terminus of J3 (explained below). While there is some propensity for U-U pairings, the torsion forces induced at the junction regions should prevent this, particularly in the lower part of the stem.
    8) To obtain a family A-type three-way junction, the length of J2 was designed to be larger than J3. Because there may be some propensity for U-U pairings, particularly further away from the junction and which may be reinforced by the canonical and G-U-based helix, U's were included at position −15 and +4 as part of the stem and not the junction. If pairing does not occur, these bases contribute one nucleotide to both J2 and J3, maintaining the J2>J3 relationship. Therefore, the number of nucleotides comprising the J2 and J3 junction regions for MS2-B1 and MS2-B2 are 5 and 3 and 6 and 3, respectively.
    9) A common characteristic of “family A” constructs is the presence of an A-G trans Hoogsteen-Sugar-Edge pairing at the interface side of P2. In order to promote this pairing, the flank region of the RNA construct was designed to leave an ‘A’ base near the center of the target sequence unpaired, and a ‘G’ base was placed at the 5′ terminus of the J2 segment. To prevent an undesired interaction with the 3′ terminus of the J3 region, a ‘G’ base was placed there, because a ‘U’ might have formed a canonical pairing with the target ‘A’, and a ‘C’ may have interacted with the ‘G’ introduced to J2 or with the ‘G’ upstream of the target ‘A’. The potential for the trans Hoogsteen-Sugar-Edge interaction exists at the ‘G’ in the J2 interface due to the particular strand conformation and presentation of that base in relation to the ‘A’ in the target. Therefore, the ‘G’ in the J3 segment is less likely to interact with the ‘A’ in the target.
    10) Finally, the flanking 5′ and 3′ sequences were designed to be perfectly complementary to the target, leaving a J1 of size 0 if the predicted trans Hoogsteen-Sugar-Edge pair forms, or size 1 if it does not form. In either case, J1 conforms to the “family A” junction region size requirement.


Secondary structure and minimum free energy predictions for the target alone, the RNA construct alone and the RNA construct and target together were determined using RNAFold and RNACoFold and are shown in FIGS. 38-42. The MS2-B1 RNA construct and target showed a −24.9 kcal/mol net change versus the RNA construct and target individually, and the MS2-B2 RNA construct and target showed a −26.6 kcal/mol net change. These predictions indicated that the RNA construct and target would likely hybridize as intended.
Part III: The RNA Construct Selectively Binds the MS2 Fusion Protein in the Presence of the Target Polynucleotide
a. Native Gel Binding Experiment
To determine whether the RNA construct bound the target, a binding experiment under native conditions was performed. All RNA used for the experiment was dissolved in a buffer containing 10 mM Tris-HCl (pH 8.0) and 100 mM NaCl. The MS2-B1 or MS2-B2 RNA construct and target were mixed in a 1:2 ratio and annealed at 85° C. for 3 minutes and gradually cooled to room temperature for about an hour. The resulting mixture was run on a native PAGE (15% TBE-PAGE) gel. FIG. 43, shows an upper band (identified by a box) with a lower electrophoretic mobility than either the RNA construct or target alone. This higher molecular weight species was most likely the RNA construct complexed with the target, indicating that the RNA construct was capable of binding the target.
b. RNA-Protein Interaction Studies Using Immunoprecipitation (IP)
Immunoprecipitation commonly used to precipitate a target protein from a solution using an antibody specific to the target protein. If the protein is also bound to other proteins or to RNA, forming a complex, the complex can be pulled down as an intact unit by co-precipitation. FIG. 44 is a schematic representation of a co-immunoprecipitation experiment for precipitating MS2 with an RNA construct and target bound thereto.
A slurry of magnetic beads (Dynabeads®, Invitrogen, Carlsbad, Calif.) coated in Protein A and G were mixed by vortexing, and 75 μl were transferred to a new tube and washed twice with 0.5 ml of NT-2. The beads were resuspended in 100 μl of NT-2 (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM MgCl2, 0.05% NP-40), and 5 μg of anti-His tag antibody were added. Anti-his antibody was chosen because the MS2-PABP fusion protein has a His tag at the N-terminal end. The tube was mixed at room temperature overnight at 4° C., and then washed five times with 1 ml of NT-2. In parallel, RNA construct, target or RNA construct and target were added to respective tubes, heated to 85° C. for 5 minutes and gradually cooled down to room temperature to allow binding of RNA construct to target to occur. MS2-PABP was added to the tubes containing RNA construct, target or RNA construct and target and incubated at 37° C. for 1 hour. The beads were resuspended in 900 μl of NET-2 (NT2, with 20 mM EDTA (pH 8.0), 10 μL 1 mM DTT, 200 Units Rnase OUTTM (Invitrogen, Carlsbad, Calif.)). RNA-protein complexes were added to the tubes containing the beads to which the anti-His antibody was bound and were mixed overnight at 4° C. The beads were washed six times with 1.0 ml of ice cold NT-2. Before the beads were spun down for the sixth wash, 100 μl of the bead slurry (out of 1 ml total volume) were removed for confirming protein pull down by Western. To remove protein, the beads were resuspended in 150 μl Proteinase K buffer and mixed at 55° C. for 30 min using a thermomixer. To precipitate RNA, 150 μl phenol-chloroform were added, mixed by vortexing, and centrifuged at 12,000 rpm for 5 min. The upper aqueous layer was removed and 150 μl chloroform added. The tubes were then mixed by vortexing and centrifuged. 50 μl 5M ammonium acetate, 5 μl of glycogen and 1.0 ml of cold 100% ethanol were added to the supernatant. The tubes were placed at −80° C. for half an hour. The tubes were then centrifuged at 14,000 rpm for 30 min, washed with 80% ethanol, and centrifuged again for 30 min. The resulting pellets were dried in a Speed-vac and resuspended in 10 μl of buffer (10 mm Tris (pH8.0), 100 mM NaCl).
Magnetic beads (Dynabeads®, Invitrogen, Carlsbad, Calif.) coated with protein A/G were used for the IPs. Since the MS2 fusion protein has a His tag at the N-terminal end, anti-His was chosen as the antibody. Anti-His was incubated with the beads overnight at 4° C. MS2 protein was incubated with the RNA construct and target, the RNA construct alone, or the target alone at 37° C. for 1 hour. The whole mixture was added to the anti-His bound beads and incubated overnight at 4° C. To verify RNA pull down, the beads were treated with proteinase k for protein digestion followed by phenol-chloroform extraction and ethanol precipitation.
The precipitated samples were tested on a denaturing PAGE (10% TBE-Urea) gel. The bands of interest are marked on the gel (FIG. 45A). These bands represent the RNA construct-target complex which was pulled down along with the MS2 as a result of the protein-RNA interaction. An increase in binding of the RNA construct (MS2-B1 or MS2-B2) occurred in the presence of target, which was not seen in the absence of the target. A positive control was used which naturally forms a stem-loop which preferentially binds to MS2. A band that may represent target-target binding was also seen. Self-hybridization of the target might have occurred, which according to RNA cofold prediction software, forms a stem-loop similar to the MS2 RNA. Band intensities were quantified using Quantity One® software (FIG. 45B, BioRad, Hercules, Calif.).
Additional MS2-RNA constructs were designed and designated MS2-B3-MS2-B5 (FIGS. 46-47, Table 7). Target polynucleotide sequences are shown in Table 8. Co-immunoprecipitation experiments were performed in the same manner for MS2-B3 and MS2-B5. Results are shown in FIG. 48.
Part IV: RNA Constructs Based on a Histone Stem-Loop (HSL) Motif
A HSL consensus sequence shown in FIG. 49 (acquired from the: UTRSITE Signal Manager (www2.ba.itb.cnr.it/UTRSite/index.php/UTRSite%20signal/Signal/formID/U0035) was used as the starting point to design HSL-based RNA constructs (HSL-B1-4, see Table 8, and FIGS. 50-51), which were tested as described above for their ability to bind SLBP in the presence of a target sequence (Table 7). Recombinant human SLBP was obtained by expression in an insect/baculovirus system by Genscript (Piscataway, N.J.). Results are shown in FIG. 52.
Example 3
Increasing Expression of a Polypeptide Coding Region 5′ of an RNA Construct
To demonstrate that an RNA construct placed downstream of a polypeptide coding region is capable of increasing expression of the polypeptide coding region in the presence of a target polynucleotide, MS2-based RNA constructs are engineered and inserted downstream of a luciferase reporter gene. In the presence of a target sequence capable of binding to the RNA construct, the RNA construct is stabilized, allowing it to bind an MS2-polyA binding protein (PABP) fusion protein, which increases translation of the luciferase reported gene.
The luciferase gene from the Luciferase T7 control DNA vector (#L4821, Promega, Madison, Wis.) vector is used to construct a DNA vector for transcribing the RNA construct and subsequently translating luciferase. Restriction sites downstream of the T7-expressed luciferase gene are used to clone in DNA sequences encoding MS2-engineered RNA construct sequences, which allow for binding by either wild-type or MS2-PABP fusion protein. Control MS2-engineered RNA constructs, termed Positive control MS2 and Wild-Type MS2 motif, are constructed by annealing designed oligonucleotides to each other in overlapping region, filling in bases with DNA Polymerase I, Large (Klenow) fragment (#M0210, New England BioLabs, Ipswitch, Mass.), amplifying by PCR, and digesting the resulting DNA with SacI and BtgI (#R0156 and #R0608, respectively, New England BioLabs, Ipswitch, Mass.). Once the vector and MS2-RNA construct sequence insert are digested with their respective enzymes, T4 DNA ligase is used (#M0202, New England BioLabs, Ipswitch, Mass.) to attach the two sequences together and the resulting clones are transformed into Escherichia coli DHSα bacterial cells. Once the series of MS2-engineered RNA constructs are cloned downstream of the luciferase gene, purified DNA is linearized for in vitro transcription followed by in vitro translation in with and without the addition of the MS2-PABP fusion protein.
Part I: In Vitro Transcription of the Templates
After linearization of the plasmid with SacI, the DNA is purified with DNA clean and ConcentratorTM (#D4003, Zymo Research, Orange, Calif.). The DNA is then transcribed into uncapped RNA using Ambion® MEGAscript® T7 Kit (#AM1333, Applied Biosystems, Carlsbad, Calif.). 1 μg of linear template DNA is added to a 20 μL transcription reaction containing 8 μL ribonucleotides solution, 2 μL of 10× reaction buffer, 2 μL enzyme mix, and nuclease-free water. Also, capped RNA is manufactured using Ambion® mMESSAGE mMACHINE® T7 Kit (#AM1344, Applied Biosystems, Carlsbad, Calif.), with 1 μg of linear template DNA added to a 20 μL transcription reaction containing 10 μL 2×NTP/CAP solution, 2 μL of 10× reaction buffer, 2 μL enzyme mix, and nuclease-free water. The transcription reactions are incubated in an undisturbed environment at 37° C. overnight. After incubation, 1 μL of Ambion® TURBOTM DNase (2 U/μL, Applied Biosystems, Carlsbad, Calif.) is added and incubated at 37° C. for another 15 minutes to remove the template DNA. The RNA is recovered by lithium chloride precipitation as the transcription reactions are mixed thoroughly with 30 μL of nuclease-free water and 30 μL of 7.5 M LiCl. The mixture is chilled at −20° C. for 30 minutes, and centrifuged at 4° C. for 15 minutes at 14,000 rpm to pellet the RNA. The supernatants are carefully removed and the pellets are washed with 1 mL of 70% ethanol to maximize removal of unincorporated nucleotides. The 70% ethanol is carefully removed after centrifuging again at 4° C. for 15 minutes at 14,000 rpm. The pellets are placed in a Speed vac for 5 minutes for drying, and the RNA is resuspended in nuclease-free water. The concentration of the RNA is determined by Nanodrop analysis (Nanodrop® 2000, Thermo Scientific, Waltham, Mass.), and the transcription products are analyzed by 1% agarose gel electrophoresis.
Part II: In Vitro Translation and Western Blot Analysis
To demonstrate that an RNA construct attached to a polypeptide coding region is capable of increasing expression of the polypeptide coding region in the presence of a target polynucleotide, the RNA construct attached to luciferase RNA and target RNA are placed into an in vitro translation system (Pierce Human in vitro protein expression kit (#88857, Thermo Scientific, Waltham, Mass.)). A 25 μL translation reaction is performed with 2 μg of RNA combined with 12.5 μL of lysate mixture, 2.5 μL accessory proteins, 1 μL salt solution B, 0.5 μL of amino acid minus methionine, 0.5 μL of amino acid minus leucine, 1 μL RNAse inhibitor (40 U/mL), 1.5 μL energy mix, and nuclease-free water. The reaction is assembled in a 0.5 mL centrifuge tube on ice, and the lysate is preincubated with accessory proteins for 5 minutes before adding the subsequent components to enable better translation. Nuclease-free water is substituted with varying quantities of MS2-PABP fusion protein. The reaction is mixed thoroughly by stirring with the pipette tip and gently flicking the tube before incubating at 30° C. overnight. The reaction is stopped by placing the centrifuge tube on ice. Translation products are then analyzed using Western blotting. SDS-PAGE electrophoresis is performed using 4-20% linear gradient polyacrylamide precast gels (#161-1105, BioRad, Hercules, Calif.) before transferring to a nitrocellulose membrane. The nitrocellulose is developed by ONE-HOUR WESTERNTM (#L00228, Genscript, Piscataway, N.J.) using 10 ng of goat monoclonal anti-Luciferase antibody (Millipore, Billerica, Mass.). The intensity of the expression is analyzed by the BioRad Versa DocTM 4000 imaging system (BioRad, Hercules, Calif.). An increase in intensity of luciferase expression is expected to be seen in tubes containing both RNA construct and target, compared to tubes containing RNA construct or target alone. The target stabilizes the RNA construct, which allows the RNA construct to be stably bound by the MS2-PABP fusion RBP, leading to an increase in expression of the attached luciferase polypeptide coding region.
Example 4
Naturally-Occurring RNA Switch
This example relates to a naturally-occurring RNA switch. The principles described in this example further validate RNA constructs of the invention.
MicroRNAs Target mRNA-Binding Protein Sites
Several recent studies suggested that the biological targets of microRNAs reside in the 3′UTRs of many mRNAs. The present invention contemplated that the cis-regulatory code targeted by trans-acting microRNAs was, at least in part, the same as that read by trans-acting RNA binding proteins. This model provides a new mechanism for gene regulation that provides a biochemical means to explain how the expression patterns of genes are selected for the required needs of a cell. It also provides a novel strategy for turning ON or OFF a polynucleotide coding region.
Confirmation that microRNAs Target mRNA-Binding Protein Sites
To confirm this model, it was determined whether any of the presently known microRNAs targeted a well-characterized mRNA regulatory element, such as the stem-loop structure, which is present in the 3′-untranslated region (UTR) of many higher eukaryotic histone mRNAs. Unlike most mRNAs, histone messages are not poly-adenylated. Instead, this family of mRNAs utilizes a 26 base stem-loop structure in its 3′-UTR called the histone stem-loop (HSL), which is regulated by the histone stem-loop binding protein (SLBP) and facilitates efficient translation.
Twenty-five human histone mRNA sequences containing the HSL (FIG. 53) were compared with a dataset containing the presently characterized microRNAs. This comparison was done in parallel with several control datasets matched for the histone mRNA dataset. The box labeled “Structure” in FIG. 53 is a consensus sequence for the twenty-file HSL sequence in FIG. 53. This sequence is the same as Consensus Sequence III, above.
Found microRNA and RBP Target Overlapping Code
A group of microRNAs were identified that are contemplated to specifically interact with the HSL sequence with binding activity centered on the HSL regulatory element. These included the mir-518 and mir-319 microRNA families (FIG. 54). Strikingly, the microRNA-mRNA interaction is predicted to bind the HSL in a manner that melts the stem-loop portion, making the microRNA-mRNA complex and the SLBP binding site (or HSL regulatory element) mutually exclusive, in a scenario similar to that which is depicted in FIG. 55. More specifically, this model presents a mechanism that can be exploited whereby trans-acting non-coding RNAs are used to modulate an RNA construct in a manner that masks or reveals the regulatory targets of RBPs.
This first informatic search focused on human microRNAs. It was then expanded to include all presently characterized microRNAs. This expanded search additionally identified a microRNA encoded by SV40 (first example listed in FIG. 54) that has predicted binding affinity for the HSL sequence that is even higher than that predicted for the earlier identified cellular microRNAs (FIG. 54). It should be noted that all of the miRNAs of FIG. 54 are predicted to bind and melt all of the HSL mRNA elements.
It is likely that this SV40 microRNA represents a viral counterpart to what was a scavenged cellular microRNA, both performing a similar function, but in one case to perform a cellular need and the other case to satisfy a viral function. The cellular microRNA mir-518 family as well as the SV40 microRNA are likely all competing with the SLBP for binding to the HSL. When the SLBP binds, the histone mRNA that it is attached to is actively translated and the histone protein readily produced. When the microRNA binds the HSL, the histone protein is not expressed and translation is repressed.
The cell likely uses the mir-518 to help regulate SLBP binding, and thus the expression of histones, in an elaborate post-transcriptional regulatory mechanism that affords tight control that is directly linked to the cell cycle. SV40 may have developed to use this pathway for its own purposes so that it can better control the cellular production of histone protein, around which it wraps its genomic DNA as part of its replication process. Therefore, the mir-518/SLBP system appears to represent a naturally occurring RNA-based structural switch that has been scavenged by the SV40 virus.
Example 5
An RNA Construct that Turns Off a Reporter Gene
The inventive concept has been validated using an SV40-specific non-coding RNA (a microRNA), a target polynucleotide, that melts a stem-loop structured element based upon those known to exist in the 3′-untranslated region of many human histone-mRNAs (the RNA construct). This structure, called the histone stem-loop (HSL), is the natural target of a regulatory RNA-binding-protein called the stem-loop binding protein (SLBP). When SLBP binds to the HSL present in the histone mRNA, it activates translation, resulting in the increased production of histone protein. The SV40 non-coding RNA can act as a competitor to this process because it base pairs with the HSL in a manner that melts the stem-loop structure and thus destroys the natural binding site for SLBP (an example of the schema depicted in FIG. 2B and FIG. 3B). Thus, the SV40 non-coding RNA and SLBP interact with the HSL RNA construct in a mutually exclusive manner.
The naturally occurring histone mRNA has been rationally designed into a RNA construct with improved base-pairing kinetics for the SV40 non-coding RNA, and the histone mRNA coding region has been replaced with that of a green fluorescent protein (GFP) reporter gene. This trans-acting RNA switch strategy can be used as a molecular tool for biological research as a post-transcriptional reporter assay by inserting a gene of interest to then be turned ON or OFF, post-transcriptionally when the RNA switch is activated. An anti-viral strategy is specifically contemplated that utilizes virally-encoded RNA as the target to turn ON or OFF the RNA construct, resulting in cell death or inhibition of viral replication using one or more of the following additional strategies. The SV40/HSL construct, more fully described below, is able to be used as an antiviral compound when the reporter gene is replaced with, for example, a death gene.
The SV40/HSL/GFP construct was engineered such that the expression of the SV40 microRNA (the first entry in FIG. 54) will be in direct competition with SLBP for the HSL-RNA construct. Binding of the SLBP to the HSL is required for efficient GFP translation and, therefore, protein expression. In the presence of sufficient SV40 microRNA expression, the amount of GFP expression is decreased. Thus, this experiment involved modifying the naturally occurring HSL-containing, histone-mRNA/microRNA structural-RNA switch system (which is an example of a negative switch) to effect the expression of a reporter gene. Any fluorescent protein, for example luciferase, may be used in place of GFP.
The following approach was used to create a trans-acting RNA-structural switch.

    
    
        (1) A hybrid gene (construct) was made containing the reporter gene, GFP, coding sequence (CDS) fused to a histone stem-loop (HSL) regulatory element (conforming to Consensus Sequence I), which serves as the RNA construct. This hybrid gene is shown in FIG. 56 (and the bold, underlined, italic sequence codes for the stem-loop sequence included in the RNA construct). These fused gene constructs were then transfected into mammalian cell culture lines, and the amount of reporter protein expression was determined.
        (2) After optimizing the amount of reporter protein being expressed, the human mir518 or viral SV40 microRNAs were simultaneously expressed in the RNA construct-containing cell lines using a standard retroviral based shRNA delivery approach. The subsequent expressed microRNAs represent the target polynucleotides. The sequences used to produce viral SV40 microRNA or human mir518 microRNA are listed in the first two rows of the table in FIG. 57 (and one further microRNAs to be tested is listed in the final row of the table). The portion of the sequences in the column “antisense sequence” represents the sequence of the microRNA targets of interest (i.e., the sequence of microRNA target is as listed in the “antisense sequence” column, but as DNA, and thus the U's are listed as T's in the shRNA sequences).
        (3) The effect of presence or absence of target RNA on the expression of GFP reporter protein was then assessed as a degree of fluorescence.
    
    


The HSL is the natural target of the stem-loop binding protein (SLBP), and this protein needs to bind in order for the upstream gene to be efficiently translated into protein (in this case it is the reporter GFP). In the presence of the SV40 miRNA, which is the target polynucleotide for this RNA construct, it base-pairs with the melted HSL, preventing SLBP binding and reducing GFP expression.
The On-Off nature of the RNA-structural switch can be directly measured by assessing the amount of GFP-HSL-RNA construct that can be immunoprecipitated with the SLBP in the presence or absence of the SV40 miRNA. This is what is depicted in FIG. 58A in which RT-PCR is used to determine the amount of immunoprecipitated GFP-HSL bound to SLBP, with and without the SV40 miRNA. As can be seen, there is a substantial reduction in the amount of GFP-HSL-RNA construct that can be immunoprecipitated with SLBP when the SV40 miRNA is co-expressed (because the target polynucleotide is preventing binding of SLBP to the RNA construct).
The activity of the switch can also be indirectly assessed by determining the amount of reporter gene being expressed. As can be seen in FIG. 58B, there is about a 20% reduction in GFP expression when the GFP-HSL RNA construct is expressed in the presence of the SV40 miRNA. Interestingly, the human mir518 RNA did not have the same effect on the expression of GFP. There are several nucleotide differences between the SV40 miRNA and the mir518 so this was not that surprising because the HSL sequence that was used for RNA construct was specifically designed to interact preferentially with the SV40 miRNA. While there is overlap with the random fluctuation of the reporter, the experiment serves as an initial example validating this technology. The assay will be optimized to enhance the effect on reporter gene expression. Optimizing and titrating the amount of reporter gene basal expression as well as the amount of microRNA-target expression are accomplishing this.
Additional Experimental Validation
Further experimental work is contemplated to confirm and develop an RNA construct which can bind a viral RNA.

    
    
        (1) The existing technology as described above will be optimized to show a greater difference in reporter gene expression in cells that contain the mir-518 or SV40 microRNA versus cells that do not. This will be accomplished by adjusting the amount of basal reporter gene expression as well as the amount of mir-518 or SV40 microRNA co-expressed in the cells.
        (2) Once a change (reduction) in reporter gene expression by approximately 50% is achieved, use of mir-518 or SV40 microRNA co-expression will be replaced with actual SV40 virus infection in cell culture. This system will be used to determine the effect of SV40 virus on the expression of the reporter gene. It is contemplated that those cells infected with SV40 virus will show a decrease in reporter gene expression.
        (3) The present version of the trans-acting RNA structural switch used in the experiment described above acts as a repressor and decreases the translation (protein production) of the gene fused to it. To produce an effective anti-viral biological (or other anti-pathogen or anti-neoplastic biological), it is desired to activate the expression of a death gene. To accomplish this, the approach of a repressor and an operator will be used. The RNA construct will be fused downstream of a gene coding for a repressor protein (such as the lac-repressor) instead of a reporter gene. This repressor protein will be constitutively expressed because the switch is in the “ON” state in the absence of target polynucleotide (e.g., virus microRNA) and therefore, translationally active. Additionally, the expression of a “Death-gene” will be driven by an inducible promoter, which is the target of the repressor gene being expressed from the RNA construct. This will keep the death-gene expression in the basal “OFF” state because the repressor protein binds it. In the presence of SV40, or the expression of the mir-518 or SV40 microRNA, the repressor gene expression will be reduced or shut OFF, which will activate the expression of the death-gene, thus killing the infected cell.
        (4) Specificity could then be determined by measuring the degree to which virally infected cells or those expressing mir-518 or SV40 microRNA are selectively killed and the degree to which the anti-viral drug is inert in non-infected cells.
    
    


Although the anti-viral described above reverses a negative-type switch (i.e. is an indirect mechanism), it is contemplated that activating a positive-type regulating switch also will be a useful anti-viral mechanism. A rationally designed positive-activating RNA structural switch can be used to directly turn on a death gene when it is activated by a viral target polynucleotide, such as the mechanism depicted in FIG. 1A. Upon completion of the anti-viral (either negative or positive acting), the biological drug would be further optimized and delivery systems would be implemented. Next, animal testing would confirm that the drug does not affect some other functioning of the cells throughout the animals, and subsequent clinical testing will show efficacy within the human population.
EQUIVALENTS
The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting on the invention described herein. Scope of the invention is thus indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are intended to be embraced therein.
INCORPORATION BY REFERENCE
The entire disclosure of each of the patent documents, scientific publications, and other database references disclosed hereinabove is expressly incorporated herein by reference for all purposes.

## Claims

1. A method for modulating translation of a polypeptide coding region comprising the step of:
(A) providing a translatable RNA construct comprising a polypeptide coding region ligated to a non-naturally occurring RNA comprising a continuous sequence of ribonucleotide bases, the continuous sequence of ribonucleotide bases defining:
a stem-loop structure which associates with an RNA binding protein present in an expression environment to modulate translation of the polypeptide coding region when the RNA construct is in a stem-loop conformation;

simulated, three way junction joining regions 3′ and 5′ of the stem-loop structure;
a first region 5′ of the 5′ joining region comprising bases complementary to a 3′ region of a target polynucleotide;
a second region 3′ of the 3′ joining region comprising bases complementary to a 5′ region of the target polynucleotide;
the base sequence of the first and second regions being selected to hybridize with complementary bases on the target polynucleotide spaced apart by an intermediate region on the target polynucleotide defining another three way junction joining region; and

(B) introducing the translatable RNA construct into an expression system, whereby,
in the absence of the target polynucleotide, the construct assumes a conformation inhibiting association with the RNA binding protein, and
in the presence of the target polynucleotide, the construct assumes a stem-loop conformation promoting association with the RNA binding protein.

2. The method of claim 1, wherein at least one base in the stem of the stem-loop structure is mismatched with its potential binding partner so as to reduce the stability of the stem-loop structure.

3. The method of claim 1, wherein a joining region comprises no more than about 10 nucleotides.

4. The method of claim 1, wherein step (B) is effected by transfecting a DNA encoding the translatable RNA construct.

5. The method of claim 1, wherein the target polynucleotide is characteristic of a pathogen.

6. The method of claim 1, wherein the target polynucleotide is expressed preferentially in a cell type of a multicellular organism.

7. The method of claim 6, wherein the cell type is a neoplastic cell.

8. The method of claim 1, comprising the additional step of introducing the target polynucleotide into the expression environment.

9. The method of claim 1, wherein the expression environment is a cell and the polypeptide coding region encodes a polypeptide lethal to the cell.

10. The method of claim 9, wherein the cell is an infected cell or a neoplastic cell, and the target polynucleotide is expressed by the neoplastic cell or by an organism infecting the cell.

11. The method of claim 1, wherein the polypeptide coding region comprises a sequence encoding an enzyme or pore-forming protein.

12. The method of claim 1, wherein the polypeptide coding region encodes a detectable marker.

13. The method of claim 12, wherein the detectable marker is fluorescent.

14. The method of claim 1, wherein translation of the coding region is suppressed when the RNA binding protein binds to the stem-loop structure.

15. The method of claim 1, wherein translation of the coding region is promoted when the RNA binding protein binds to the stem-loop structure.

16. The method of claim 1, wherein the construct comprises more than one stem-loop structure.