AU2013200531B2 - Anti-microRNA oligonucleotide molecules - Google Patents

Abstract

C:NRPonblDCC\LLUJ4754 I L.DOC-211120I The invention relates to isolated anti-microRNA molecules. In another embodiment, the invention relates to an isolated microRNA molecule. In yet another embodiment, the invention 5 provides a method for inhibiting microRNP activity in a cell.

Description

Anti-MicroRNA Oligonucleotide Molecules This application is a divisional application derived from Australian Patent Application No. 2011239266 which is a divisional of Australian Patent Application No. 2005214904, the entire contents of which, as originally filed, are incorporated herein by reference. The invention claimedherein was made with the help of grant number I Rol GM068476 01 from NIH/NIGMS. The U.S. government has certain rights in the invention. BACKGROUND OF THE INVENTION RNA silencing is a fundamental mechanism of gene regulation that uses double-stranded RNA (dsRNA) derived 21- to 28-nucleotide (nt) small RNAs to guide mRNA degradation, control miRNA translation or chromatin modification. Recently, several hundred novel genes were identified in plants and animals that encode transcripts that contain short dsRNA hairpins. Defined 22-nt RNAs, referred to as microRNAs (miRNAs), are reported to be excised by dsRNA specific endonucleases from the hairpin precursors. The miRNAs are incorporated into ribonucleoprotein particles (miRNPs). Plant miRNAs target mRNAs containing sequence segments with high.complementarity for degradation or suppress translation of partially complementary mRNAs. Animal minRNAs appear to act predominantly as translational repressors. However, animal miRNAs have also been reported to guide RNA degradation. This indicates that animal miRNPs act like small interfering RNA (siRNA)-induced silencing complexes (RISCs), Understanding the biological function of miRNAs requires knowledge of their mRNA .targets. Bioinformatic approaches have been used to predict uRNA targets, among which transcription factors and proapoptotic genes were prominent candidates. Processes such as Notch signaling, cell proliferation, morphogenesis and axon guidance appear to be controlled by miRNA genes. Therefore, there is a need for materials and methods that can help elucidate the function of known and future microRNAs. Due to the ability of microRNAs to induce RNA degradation WO 2005/079397 PCT/US2005/004714 or repress translation of mRNA which encode important proteins, there is also a need for novel compositions for inhibiting microRNA-indcued cleavage or repression of mRNAs. SUMMARY THE INVENTION In one embodiment, the invention provides an isolated single stranded anti-microRNA molecule comprising a minimum of ten moieties and a maximum of fifty moieties on a molecular backbone, the molecular backbone comprising backbone units, each moiety comprising a base bonded to a backbone unit, each base forming a Watson-Crick base pair with a complementary base wherein at least ten contiguous bases have the same sequence as a sequence of bases in any one of the anti-iicroRNA molecules shown in Tables 1-4, except that up to thirty percent of the bases pairs may be wobble base pairs, and up to 10% of the contiguous bases may be additions, deletions, mismatches, or combinations thereof; no more than fifty percent of the contiguous moieties contain deoxyribonuleotide backbone units; the moiety in the molecule at the position corresponding to position 11 of the microR NA is non-complementary; and the molecule is capable Of inhibiting microRNP activity. In another embodiment, the invention provides a method for inhibiting microRNP activity in a cell, the microRNP comprising a microRNA molecule, the microRNA molecule comprising a sequences of bases complementary of the sequence of bases in a single stranded anti-microRNA molecule, the method comprising introducing into the cell the single-stranded anti-microRNA molecule comprising a sequence of a minimum of ten moieties and a maximum of fifty moieties on a molecular backbone, the molecular backbone comprising backbone units, each moiety comprising a base bonded to a backbone unit, each base forming a Watson-Crick base pair with a complementary base, wherein at least ten contiguous bases of the anti microRNA molecule are complementary to the microRNA, except that up to thirty percent of the bases may be substituted by wobble base pairs, and up to ten percent of the at least ten moieties may be additions, deletions, mismatches, or combinations thereof; no more than fifty percent of the contiguous moieties contain deoxyribonuleotide backbone units; and the moiety in the molecule at the position corresponding to position 11 of the microRNA is non-complementary. In another embodiment, the invention provides an isolated nicroRNA molecule comprising a minimum of ten moieties and a maximum of fifty moieties on a molecular 2 WO 2005/079397 PCT/US2005/004714 backbone, the molecular backbone comprising backbone units, each moiety comprising a base bonded to a backbone unit, wherein at least ten contiguous bases have the same sequence as a sequence of bases in any one of the microRNA molecules shown in Table 2, except that up to thirty percent of the bases pairs may be wobble base pairs, and up to 10% of the contiguous bases may be additions, deletions, mismatches, or combinations thereof; and no more than fifty percent of the contiguous moieties contain deoxyribonuleotide backbone units. In another embodiment, the invention provides an isolated microRNA molecule comprising a minimum of ten moieties and a maximum of fifty moieties on a molecular backbone, the molecular backbone comprising backbone units, each moiety comprising a base bonded to a backbone unit, wherein at least ten contiguous bases have any one of the microRNA sequences shown in Tables 1, 3 and 4, except that up to thirty percent of the bases pairs may be wobble base pairs, and up to 10% of the contiguous bases may be additions, deletions, mismatches, or combinations thereof; no more than fifty percent of the contiguous moieties contain deoxyribonuleotide backbone units; and is modified for increased nuclease resistance. In yet another embodiment, the invention provides an isolated single stranded anti microRNA molecule comprising a minimum of ten moieties and a maximum of fifty moieties on a molecular backbone, the molecular backbone comprising backbone units, each moiety comprising a base bonded to a backbone unit, each base forming a Watson-Crick base pair with a complementary base wherein at least ten contiguous bases have the same sequence as a sequence of bases in any one of the anti-microRNA molecules shown in Tables 1-4, except that up to thirty percent of the bases pairs may be wobble base pairs, and up to 10% of the contiguous bases may be additions, deletions, mismatches, or combinations thereof; no more than fifty percent of the contiguous moieties contain deoxyribonuleotide backbone units; and the molecule is capable of inhibiting microRNP activity. In yet a further embodiment, the invention provides a method for inhibiting microRNP activity in a cell, the microRNP comprising a microRNA molecule, the microRNA molecule comprising a sequences of bases complementary of the sequence of bases in a single stranded anti-microRNA molecule, the method comprising introducing into the cell the single-stranded anti-microRNA molecule comprising a sequence of a minimum of ten moieties and a maximum 3 WO 2005/079397 PCT/US2005/004714 of fifty moieties on a molecular backbone, the molecular backbone comprising backbone units, each moiety comprising a base bonded to a backbone unit, each base forming a Watson-Crick base pair with a complementary base, wherein at least ten contiguous bases of the anti microRNA molecule are complementary to the microRNA, except that up to thirty percent of the bases may be substituted by wobble base pairs, and up to ten percent of the at least ten moieties may be additions, deletions, mismatches, or combinations thereof; and no more than fifty percent of the contiguous moieties contain deoxyribonuleotide backbone units. DESCRIPTION OF THE FIGURES Figure I shows the modified nucleotide units discussed in the specification. B denotes any one of the following nucleic acid bases: adenosine, cytidine, guanosine, thymine, or uridine. Figure 2. Antisense 2-C-methyl oligoribonucleotide specifically inhibit miR-21 guided cleavage activity in HeLa cell S100 cytoplasmic extracts. The black bar to the left of the RNase T1 ladder represents the region of the target RNA complementary to miR-21. Oligonucleotides complementary to miR-21 were pre-incubated in S100 extracts prior to the addition of 3 2 P-cap labelled cleavage substrate. Cleavage bands and Ti hydrolysis bands appear as doublets after a 1-nt slipping of the T7 RNA polymerase near the middle of the transcript indicated by the asterisk, Figure 3. Antisense 2'-O-methyl oligoribonucleotides interfere with endogenous miR-21 RNP cleavage in HeLa cells. HeLa cells were transfected with pHcRed and pEGFP or its derivatives, with or without inhibitory or control oligonucleotides. EGFP and HcRed protein fluorescence were excited and recorded individually by fluorescence microscopy 24 h after transfection. Co-expression of co-transfected reporter plasmids.was documented by superimposing of the fluorescence images in the right panel. DETAILED DESCRIPTION OF THE INVENTION The invention relates to an isolated single stranded anti-microRNA molecule. The molecule comprises a minimum number of ten moieties, preferably a minimum of thirteen, more preferably a minimum of fifteen, even more preferably a minimum of 18, and most preferably a minimum of 21 moieties. 4 WO 2005/079397 PCT/US2005/004714 The anti-microRNA molecule comprises a maximum number of fifty moieties, preferably a maximum of forty, more preferably a maximum of thirty, even more preferably a maximum of twenty-five, and most preferably a maximum of twenty-three moieties. A suitable range of minimum and maximum number of moieties may be obtained by combining any of the above minima with any of the above maxima. Each moiety comprises a base bonded to a backbone unit. In this specification, a base refers to any one of the nucleic acid bases present in DNA or RNA. The base can be a purine or pyrimidine. Examples of purine bases include adenine (A) and guanine (G). Examples of pyrimidine bases include thymine (T), cytosine (C) and uracil (U). Each base of the moiety forms a Watson-Crick base pair with a complementary base. Watson-Crick base pairs as used herein refers to the hydrogen bonding interaction between, for example, the following bases: adenine and thymine (A = T); adenine and uracil (A = U); and cytosine and guanine (C = G) The -adenine can be replaced with 2,6-dianinopurine without compromising base-pairing. The backbone unit may be any molecular unit that is able stably to bind to a base and to form an oligomeric chain. Suitable backbone units are well known to those in the art. For example, suitable backbone units include sugar-phosphate groups, such as the sugar phosphate groups present in ribonucleotides, d6oxyribonucleotides, phosphorothioate deoxyribose groups, N'3-N'5 phosphoroanidate deoxyribose groups, 2'O-alkyl-ribose phosphate groups, 2'-O-alkyl-alkoxy ribose phosphate groups, ribose phosphate group containing a methylene bridge, 2'-Fluororibose phosphate groups, morpholino phosphoroamidate groups, cyclohexene groups, tricyclo phosphate groups, and amino acid molecules. In one embodiment, the anti-microRNA molecule comprises at least one moiety which is a ribonucleotide moiety or a deoxyribonucleotide moiety. In another embodiment, the anti-microRNA molecule comprises at least one moiety which confers increased nuclease resistance. The nuclease can be an exonuclease, an endonuclease, or both. The exonuclease can be a 3'->5' exonuclease or a 5'-+3' exonuclease. Examples of 3'-+5' human exonuclease include PNPT1, Werner syndrome helicase, RRP40, 5 WO 2005/079397 PCT/US2005/004714 RRP41, RRP42, RRP45, and RRP46. Examples of 5'-+3' exonuclease include XRN2, and FEN1. Examples of endonucleases include Dicer, Drosha, RNase4, Ribonuclease P, Ribonuclease H1, DHP1, ERCC-1 and OGG1. Examples of nucleases which function as both an exonuclease and an endonuclease include APEl and EXO 1. An anti-microRNA molecule comprising at least one moiety which confers increased nuclease resistance means a sequence of moieties wherein at least one moiety is not recognized by a nuclease. Therefore, the nuclease resistance of the molecule is increased compared to a sequence containing only unmodified ribonucleotide, unmodified deoxyribonucleotide or both. Such modified moieties are well known in the art, and were reviewed, for example, by Kurreck, Eur. J. Biochem. 270, 1628-1644 (2003). A modified moiety can occur at any position in the anti-microRNA molecule. For example, to protect the anti-microRNA molecule against 3'->5' exonucleases, the molecule can have at least one modified moiety at the 3' end of the molecule and preferably at least two modified moieties at the 3' end. If it is desirable to protect the molecule against 5'-3' exonuclease, the anti-microRNA molecule can have at least one modified moiety and preferably at least two modified moieties at the 5' end of the molecule. The anti-microRNA molecule can also have at least one and preferably at least two modified moieties between the 5' and 3' end of the molecule to increase resistance of the molecule to endonucleases. In one embodiment, all of the moieties are nuclease resistant. In another embodiment, the anti-microRNA molecule comprises at least one modified deoxyribonucleotide moiety. Suitable modified deoxyribonucleotide moieties ard known in the art. A suitable example of a modified deoxyribonucleotide moiety is a phosphorothioate deoxyribonucleotide moiety. See structure 1 in figure 1. An anti-microRNA molecule comprising more than one phosphorothioate deoxyribonucleotide moiety is referred to as phosphorothioate (PS) DNA, See, for example, Eckstein, Antisense Nucleic Acids Drug Dev. 10, 117-121 (2000). 6 WO 2005/079397 PCT/US2005/004714 Another suitable example of a modified deoxyribonucleotide moiety is an N'3-N'5 phosphoroamidate deoxyribonucleotide moiety. See structure 2 in figure 1. An oligonucleotide molecule comprising more than one phosphoroamidate deoxyribonucleotide moiety is referred to as phosphoroamidate (NP) DNA. See, for example, Gryaznov et al, J. Am. Chem. Soc. 116, 3143-3144 (1994). In another embodiment, the molecule comprises at least one modified ribonucleotide moiety. Suitable modified ribonucleotide moieties are known in the art. A suitable example of a modified ribonucleotide moiety is a ribonucleotide moiety that is substituted at the 2' position. The substituents at the 2' position may, for example, be a C 1 to C 4 alkyl group. The C, to C 4 alkyl group may be saturated or unsaturated, and unbranched or branched. Some examples of C 1 to C 4 alkyl groups include ethyl, isopropyl, and allyl. The preferred C 1 to C 4 alkyl group is methyl. See structure 3 in figure 1. An oligoribonucleotide molecule comprising more than one ribonucleotide moeity that is substituted at the 2' position with a C1 to C 4 alkyl group is referred to as a 2'-0 -(CI-C 4 alkyl) RNA, e.g.,2'-O-methyl RNA (OMe RNA). Another suitable example of a substituent at the 2' position of a modified ribonucleotide moiety is a C 1 to C 4 alkoxy - CI to C 4 alkyl group. The CI to C 4 alkoxy (alkyloxy) and C 1 to C 4 alkyl group may comprise any of the alkyl groups described above. The preferred C 1 to C 4 alkoxy - C 1 to C 4 alkyl group is methoxyethyl. See structure 4 in figure 1. An oligonucleotide molecule comprising more than one ribonucleotide moiety that is substituted at the 2' position with a Cl to C 4 alkoxy-C 1 to C 4 alkyl group is referred to as a 2'-O-(C1 to C 4 alkoxy - C 3 to C 4 alkyl) RNA, e.g., 2.-O-methoxyethyl RNA (MOE RNA). Another suitable example of a modified ribonucleotide moiety is a ribonucleotide that has a methylene bridge between the 2'-oxygen atom and the 4'-carbon atom. See structure 5 in figure 1. An oligoribonucleotide molecule comprising more than one ribonucleotide moiety that has a methylene bridge between the 2'-oxygen atom and the 4'-carbon atom is referred to as locked nucleic acid (LNA). See, for example, Kurreck et al., Nucleic Acids Res. 30, 1911-1918 (2002); Elayadi et al, Curr. Opinion Invest. Drugs 2, 558-561 (2001); Orum et al., Curr. Opinion Mol. There. 3, 239-243 (2001); Koshkin etaL., Tetrahedron .54, 3607-3630 (1998); Obika et aL, 7 WO 2005/079397 PCT/US2005/004714 Tetrahedron Lett.39, 5401-5404 (1998). Locked nucleic acids are commercially available from Proligo (Paris, France and Boulder, Colorado, USA). Another suitable example of a modified ribonucleotide moiety is a ribonucleotide that is substituted at the 2' position with fluoro group. A modified ribonucleotide moiety having a fluoro group at the 2' position is a 2'-fluororibonucleotide moiety. Such moieties are known in the art. Molecules comprising more than one 2'-fluororibonucleotide moiety are referred to herein as 2'-.fluororibo nucleic acids (FANA). See structure 7 in figure 1. Damha et al., J. Am. Chem. Soc. 120, 12976-12977 (1998). In another embodiment, the anti-microRNA molecule comprises at least one base bonded to an amino acid residue. Moieties that have at least one. base bonded to an amino acid residue will be referred to herein as peptide nucleic acid (PNA) moieties. Such moieties are nuclease resistance, and are known in the art. Molecules having more than one PNA moiety are referred to as peptide nucleic acids. See structure 6 in figure 1. Nielson, Methods Enzymol. 313, 156 164 (1999); Elayadi, et al, id.; Braasch et al., Biochemistry 41, 4503-4509 (2002), Nielsen et al., Science 254, 1497-1500 (1991). The amino acids can be any amino acid, including natural or non-natural amino acids. Naturally occurring amino acids include, for example, the twenty most common amino acids normally found in proteins, i.e., alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine (Cys), glutamine (Glu), glutamic acid (Glu), glycine (Gly), histidine (His), isoleucine (Ileu), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan, (Trp), tyrosine (Tyr), and valine (Val). The non-natural amino acids may, for example, comprise alkyl, aryl, or alkylaxyl groups. Some examples of alkyl amino acids include a-aminobutyric acid, P-aminobutyric acid, y aninobutyric acid, 5-aminovaleric acid, and e-aminocaproic acid. Some examples of aryl amino acids include ortho-, meta, and para-aminobenzoic acid. Some examples of alkylary amino acids include ortho-, meta-, and para-aminophenylacetic acid, and y-phenyl-p-aminobutyric acid. 8 WO 2005/079397 PCT/US2005/004714 Non-naturally occurring amino acids also include derivatives of naturally occurring anino acids. The derivative of a naturally occurring amino acid may, for example, include the addition or one or more chemical groups to the naturally occurring amino acid. For example, one or more chemical groups can be added to one or more of the 2', 3', 4' 5', or 6' position of the aromatic ring of a phenylalanine or tyrosine residue, or the 4', 5', 6', or 7' position of the benzo ring of a tryptophan residue. The group can be any chemical group that can be added to an aromatic ring. Some examples of such groups include hydroxyl, C-C 4 alkoxy, amino, methylamino, dimethylamino, nitro, halo (i.e., fluoro, chloro, bromo, or iodo), or branched or unbranched CrC4 alkyl, such as methyl, ethyl, n-propyl, isopropyl, butyl, isobutyl, or t-butyl. Furthermore, other examples of non-naturally occurring amino acids which are derivatives of naturally occurring amino acids include norvaline (Nva), norleucine (Nie), and hydroxyproline (Hyp). The amino acids can be identical or different from one another. Bases are attached to the amino acid unit by molecular linkages. Examples of linkages are methylene carbonyl, ethylene carbonyl and ethyl linkages. (Nielsen et al., Peptide Nucleic Acids-Protocols and Applications, Horizon Scientific Press, pages 1-19; Nielsen et al., Science 254: 1497-1500.) One example of a PNA moiety is N-(2-aminoethyl)-glycine. Further examples of PNA moieties include cyclohexyl PNA, retro-inverso, phosphone, propionyl and aminoproline PNA. PNA can be chemically synthesized by methods known in the art, e.g. by modified Fmoc or tBoc peptide synthesis protocols. The PNA has many desirable properties, including high melting temperatures (Tm), high base-pairing specificity with nucleic acid and an uncharged molecular backbone. Additionally, the PNA does not confer RNase H sensitivity on the target RNA, and generally has good metabolic stability. Peptide nucleic acids are also commercially available from Applied Biosystems (Foster City, California, USA). 9 WO 2005/079397 PCT/US2005/004714 In another embodiment, the anti-niicroRNA molecule comprises at least one morpholino phosphoroamidate nucleotide moiety. A morpholino phosphoroamidate nucleotide moiety is a modified moiety which is nuclease resistant. Such moieties are known in the art. Molecules comprising more than one morpholino phosphoroamidate nucleotide moiety are referred to as morpholino (MF) nucleic acids. See structure 8 in figure 1. Heasman, Dev. Biol. 243, 209-214 (2002), Morpholono oligonucleotides are commercially available from Gene Tools LLC (Corvallis, Oregon, USA). In another embodiment, the anti-microRNA molecule comprises at least one cyclohexene nucleotide moiety. A cyclohexene nucleotide moiety is a modified moiety which is nuclease resistant. Such moieties are known in the art. Molecules comprising more than one cyclohexene nucleotide moiety are referred to as cyclohexene nucleic acids (CeNA). See structure 10 in figure 1. Wang et al, J. Am. Chem, Soc. 122, 8595-8602 (2000), Verbeure et al., Nucleic Acids Res. 29, 4941-4947 (2001). In another embodiment, the anti-microRNA molecule comprises at least one tricyclo nucleotide moiety. A tricyclo nucleotide moiety is a modified moiety which is nuclease resistant. Such moieties are known in the art, Steffens et al, J. Am. Chem. Soc. 119, 11548 11549 (1997), Renneberg et al., J. Am. Chem. Soc. 124, 5993-6002 (2002). Molecules comprising more than one tricyclo nucleotide moiety are referred to as tricyclo nucleic acids (tcDNA). See structure 9 in figure 1. In another embodiment, to increase nuclease resistance of the anti-microRNA molecules of the present invention to exonucleases, inverted nucleotide caps can be attached to the 5' end, the 3' end, or both ends of the molecule. An inverted nucleotide cap refers to a 3-5' sequence of nucleic acids attached to the anti-microRNA molecule at the 5' and/or the 3' end. There is no limit to the maximum number of nucleotides in the inverted cap just as long as it does not interfere with binding of the anti-microRNA molecule to its target microRNA, Any nucleotide can be used in the inverted nucleotide cap. Typically, the inverted nucleotide cap is one nucleotide in length, The nucleotide for the inverted cap is generally thymine, but can be any nucleotide such as adenine, guanine, uracil, or cytosine. 10 WO 2005/079397 PCT/US2005/004714 Alternatively, an ethylene glycol compound and/or amino linkers can be attached to the either or both ends of the anti-microRNA molecule. Amino linkers can also be used to increase nuclease resistance of the anti-microRNA molecules to endonucleases. The table below lists some examples of amino linkers. The below listed amino linker are commercially available from TriLink Biotechnologies, San Diego, CA. 2 1 -Deoxycytidine-5-C6 Amino Linker (3' Terminus) 2'-Deoxycytidine-5-C6 Amino Linker (5' or Internal) ' C3 Amino Linker 3' C6 Amino Linker 3' C7 Amino Linker 5' C12 Amino Linker 5' C3 Amino Linker 5' C6 Amino Linker C7 Internal Amino Linker Thyindine-5-C2 Amino Linker (5' or Internal) Thymidine-5-C6 Amino Linker (31 Terminus) Thymidine-5-C6 Amino Linker (Internal) Chimeric anti-microRNA molecules containing a mixture of any of the moieties mentioned above are also known, and may be made by methods known, in the art. See, for example, references cited above, and Wang et al, Proc. Natl. Acad. Sci. USA 96,13989-13994 (1999), Liang et al., Eur. J. Biochem. 269, 5753-5758 (2002), Lok et al., Biochemistry 4], 3457 3467 (2002), and Damha et al., J. Am. Chern. Soc. 120, 12976-12977 (2002). The molecules of the invention comprise at least ten contiguous, preferably at least thirteen contiguous, more preferably at least fifteen contiguous, and even more preferably at least twenty contiguous bases that have the same sequence as a sequence of bases in any one of the anti-microRNA, molecules shown in Tables 1-4. The anti-microRNA molecules optimally 11 WO 2005/079397 PCT/US2005/004714 comprise the entire sequence of any one of the anti-microRNA molecule sequences shown in Tables 1-4. For the contiguous bases mentioned above, up to thirty percent of the base pairs may be substituted by wobble base pairs. As used herein, wobble base pairs refers to either: i) substitution of a cytosine with a uracil, or 2) the substitution of a adenine with a guanine, in the sequence of the anti-microRNA molecule. These wobble base pairs are generally referred to as UG or GU wobbles. Below is a table showing the number of contiguous bases and the maximum number of wobble base pairs in the anti-microRNA molecule: Table for Number of Wobble Bases No. of 10 11 12 13 14 15 16 17 18 Contiguous Bases Max. No. of 3 3 3 4 4 5 Wobble Base -Fairs o. of Contiguous 19 20 21 22 23 BasesI Max. No. of 5 6 6 6 6 Wobble Base t air Further, up to ten percent, and preferably up to five percent of the contiguous bases can be additions, deletions, mismatches or combinations thereof, Additions refer to the insertion in the contiguous sequence of any moiety described above comprising any one of the bases described above, Deletions refer to the removal of any moiety present in the contiguous sequence. Mismatches refer to the substitution of one of the moieties comprising a base in the contiguous sequence with any of the above described moieties comprising a different base. The additions, deletions or mismatches can occur anywhere in the contiguous sequence, for example, at either end of the contiguous sequence or within the contiguous sequence of the anti-microRNA molecule. If the contiguous sequence is relatively short, such as from about ten 12 WO 2005/079397 PCT/US2005/004714 to about 15 moieties in length, preferably the additions, deletions or mismatches occur at the end of the contiguous sequence. If the contiguous sequence is relatively long, such as a minimum of sixteen contiguous sequences, then the additions, deletions, or mismatches can occur anywhere in the contiguous sequence. Below is a table showing the number of contiguous bases and the maximum number of additions, deletions, mismatches or combinations thereof: Table for Up to 10% o. of 10 11 12 13 14 15 16 17 18 Contiguous Bases Mlax.No. of 1 1 1 1 1 1 1 1 1 Additions, Deletions and/or Mismatches No. of 19 20 21 22 23 Contiguous Base Max. No. of 1 2 2 2 2 Additions, Deletions and/or Mismatches 13 WO 2005/079397 PCT/US2005/004714 Table for Up to 5% No. of 10 11 12 13 14 15 16 17 18 Contiguous Bases Max. No. of o 0 0 0 0 0 0 0 0 Additions, Deletions and/or Mismatches Sof .19 20 21 22 23 Contiguous Bases jNax.No. of 0 1 1 1 dditions, deletions and/or ismatches Furthermore, no more than fifty percent, and preferably no more than thirty percent, of the contiguous moieties contain deoxyribonucleotide backbone units. Below is a table showing the number of contiguous bases and the maximum number of deoxyribonucleotide backbone units: Table for Fifty Percent Deoxyribonucleotide Backbone Units No, of 10 11 12 13 14 15 16 1 18 Contiguons Bases Max. No. of 5 5 6 6 7 7 8 8 9 Deoxyribonucleotide _BackboneU1nits..... No. of 19 20 21 22 23 Contiguous Bases Max. No.of 9 10 10 11 11 Doxyribonucleotide ackbone Units 14 WO 2005/079397 PCT/US2005/004714 Table for Thirty Percent Deoxyribonucleotide Backbone Units No of 10 11 12 13 14 15 16 17 18 Contiguous Bases Max. No. of 3 3 3 3 5 eoxyribonucleotide ackbone Units INO. f 19 20 21 '22 23 contiguous Bases Max. No. of 5 6 6 6 6 Deoxyribonucleotide Backbone Units The moiety in the anti-RNA molecule at the position corresponding to position 11 of the microRNA is optionally non-complementary to a microRNA. The moiety in the anti-microRNA molecule corresponding to position 11 of the microRNA can be rendered non-complementary by an addition, deletion or mismatch as described above. In another embodiment, if the anti-microRNA molecule comprises only unmodified moieties, then the anti-microRNA molecules comprises at least one base, in the at least ten contiguous bases, which is non-complementary to the microRNA and/or comprises an inverted nucleotide cap, ethylene glycol compound or an amino linker, In yet another embodiment, if the at least ten contiguous bases in an anti-microRNA molecule is perfectly (i.e., 100%) complementary to ten contiguous bases in a microRNA, then the anti-microRNA molecule contains at least one modified moiety in the at least ten contiguous bases and/or comprises an inverted nucleotide cap, ethylene glycol compound or an amino linked. As stated above, the maximum length of the anti-microRNA molecule is 50 moieties. Any number of moieties having any base sequence can be added to the contiguous base sequence. The additional moieties can be added to the 5' end, the 3' end, or to both ends of the contiguous sequence. 15 WO 20051079397 PCT/US2005/004'714 MicroRNA molecules are derived from genomnic loci and are produced from specific microRNA genes. Mature microRNA molecules are processed from precursor transcripts that form local hairpin structures. The hairpin structures are typically cleaved by an enzyme known as Dicer, which generates one microRNA duplex. See Bartel, Cell 116, 281-297 (2004) for a review on microRNA molecules. The article by Bartel is hereby incorporated by reference. Each strand of a microRNA is packaged in a microRNA ribonucleoprotein complex (microRNP). A microRNP in, for example, humans, also includes the proteins eIF2C2, the helicase Gemin3, and Gemin 4. The sequence of bases in the anti-microRNA molecules of the present invention can be derived from a microRNA from any species e.g. such as a fly (e.g., Drosophila melanogaster), a worm (e.g., C. elegans). Preferably the sequence of bases is found in mammals, especially humans (H sapiens), mice (e.g., M musculus), and rats (R. norvegicus). The anti-microRNA molecule is preferably isolated, which means that it is essentially free of other nucleic acids. Essentially free from other nucleic acids means that it is at least 90%, preferably at least 95% and, more preferably, at least 98% free of other nucleic acids. Preferably, the molecule is essentially pure, which means that the molecules is free not only of other nucleic acids, but also of other materials used in the synthesis of the molecule, such as, for example, enzymes used in the synthesis of the molecule. The molecule is at least 90% free, preferably at least 95% free and, more preferably, at least 98% free of such materials. The anti-microRNA molecules of the present invention are capable of inhibiting microRNP activity, preferable in a cell. hiIbiting microRNP activity refers to the inhibition of cleavage of the microRNA's target sequence or the repression of translation of the microRNA's target sequence. The method comprises introducing into the cell a single-stranded microRNA molecule. Any anti-microRNA molecule can be used in the methods of the present invention, as long as the anti-microRNA is complementary, subject to the restrictions described above, to the microRNA present in the microRNP. Such anti-microRNAs include, for example, the anti 16 WO 2005/079397 PCT/US2005/004714 microRNA molecules mentioned above (see Table 1-4), and the anti-nicroRNAs molecules described in international PCT application number WO 03/029459 A2, the sequences of which are incorporated herein by reference. The invention also includes any one of the microRNA molecules having the sequences as shown in Table 2. The novel microRNA molecules in Table 2 may optionally be modified as described above for anti-microRNA molecules. The other microRNA molecules in Tables 1, 3 and 4 are modified for increased nuclease resistance as described above for anti-microRNA molecules. Utility The anti-microRNA molecules and the microRNA molecules of the present invention have numerous in vivo, in vitro, and ex vivo applications. For example, the anti-microRNA-molecules and microRNA of the present invention may be used as a modulator of the expression of genes which are at least partially complementary to the anti-microRNA molecules and microRNA. For example, if a particular microRNA is beneficial for the survival of a cell, an appropriate isolated microRNA of the present invention may be introduced into the cell to promote survival. Alternatively, if a particular microRNA is harmful (e.g., induces apoptosis, induces cancer, etc.), an appropriate anti-microRNA molecule can be introduced into the cell in order to inhibit the activity of the microRNA and reduce the harm. In addition, anti-microRNA molecules and/or microRNAs of the present invention can be introduced into a cell to study the function of the microRNA. Any of the anti-microRNA molecules and/or microRNAs listed above can be introduced into a cell for studying their function. For example, a microRNA in a cell can be inhibited with a suitable anti-microRNA molecule. The function of the microRNA can be inferred by observing changes associated with inhibition of the microRNA in the cell in order to inhibit the activity of the microRNA and reduce the harm. 17 WO 2005/079397 PCT/US2005/004714 The cell can be any cell which expresses rmicroRNA molecules, including the microRNA molecules listed herein. Alternatively, the cell can be any cell transfected with an expression vector containing the nucleotide sequence of a microRNA. Examples of cells include, but are not limited to, endothelial cells, epithelial cells, leukocytes (e.g., T cells, B cells, neutrophils, macrophages, eosinophils, basophils, dendritic cells, natural killer cells and monocytes), stem cells, hemopoietic cells, embryonic cells, cancer cells, The anti-microRNA molecules or microRNAs can be introduced into a cell by any method known to those skilled in the art. Useful delivery systems, include for example, liposomes and charged lipids. Liposomes typically encapsulate oligonucleotide molecules within their aqueous center. Charged lipids generally form lipid- oligonucleotide molecule complexes as a result of opposing charges. These liposomes-oligonucleotide molecule complexes or lipid- oligonucleotide molecule complexes are usually internalized by endocytosis. The liposomes or charged lipids generally comprise helper lipids which disrupt the endosomal membrane and release the oligonucleotide molecules. Other methods for introducing an anti-microRNA molecule or a microRNA into a cell include use of delivery vehicles, such as dendrimers, biodegradable polymers, polymers of amino acids, polymers of sugars, and oligonucleotide-binding nanoparticles. In addition, pluoronic gel as a depot reservoir can be used to deliver the anti-microRNA oligonucleotide molecules over a prolonged period. The above methods are described in, for example, Hughes et al., Drug Discovery Today 6, 303-315 (2001); Liang et al, Eur. J. Biochem. 269 5753-5758 (2002); and Becker et al., In Antisense Technology in the Central Nervous System (Leslie, R.A., Hunter, A.J. & Robertson, H.A., eds), pp.147-157, Oxford University Press. Targeting of an anti-microRNA molecule or a microRNA to a particular cell can be performed by any method known to those skilled in the art. For example, the anti-microRNA molecule or microRNA can be conjugated to an antibody or ligand specifically recognized by receptors on the cell. 18 WO 20051079397 PCT/US2005/004714 The sequences of microRNA and anti-microRNA molecules are shown in Tables 1-4 below. Human sequences are indicated with the prefix "hsa." Mouse sequences are indicated with the prefix "mmu." Rat sequences are indicated with the prefix "mo." C. elegan sequences are indicated with the prefix "cel." Drosophila sequences are indicated with the prefix "dme." Table 1: Human, Mouse and Rat microRNA and anti-microRNA sequences. microRNA name microRNA sequence Anti-microRNA molecule

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5 ' to 3') sequence (51 to 3') hsa-miR-100 AACCCGUAGAUCCGAACUUGUG CACAAGUUCGGAUCUACGGGUU hsa-miR-103 AGCAGCAUUGUACAGGGCUAUG CAUAGCCCUGUACAAUGCUGCU hsa-miR-103-Sp UCAAAUGCUCAGACUCCUGUGG CCACAGGAGUCUGAGCAUUUGA hsa-miR-106a AAAAGUGCUUACAGUGCAGGUA UACCUGCACUGUAAGCACUUUJ hsa-miR-106b UAAAGUGCUGACAGUGCAGAUA UAUCUGCACUGUCAGCACUUUA hsa-miR-107 AGCAGCAUUGUACAGGGCUAUC GAUAGCCCUGUACAAUGCUGCU hsa-miR-10b UACCCUGUAGAACCGAAUUUGU ACAAAUUCGGUUCUACAGGGUA hsa-miR-128b UCACAGUGAACCGGUCUCUUUC GAAAGAGACCGGUUCACUGUGA hsa-miR-130b CAGUGCAAUGAUGAAAGGGCAU AUGCCCUUUCAUCAUUGCACUG hsa-miR-140- 3p UACCACAGGGUAGAACCACGGA UCCGUGGUUCUACCCUGUGGUA hsa-miR-142-5p CCCATAAAGUAGAAAGCACUAC GUAGUGCUUUCUACJAUGGG hsa-miR-151 -Sp UCGAGGAGCUCACAGUCUAGUA UACUAGACUGUGAGCUCCUCGA hsa-miR-15S UUAAUGCUAAUCGUGAUAGGGG CCCCUAUCACGAUUAGCAUUAA hsa-miR-181a AACAUUCAACGCUGTCGGUGAG CUCACCGACAGCGUUGAAUGUU hsa-miR-181b AACATTUCAUUGCUGUCGGUGGG CCCACCGACAGCAAUGAAUGUU hsa-miR-181c AACAUUCAACCUGUCGGUGAGU ACUCACCGACAGGUUGAAUGUU hsa-miR-182 UUUGGCAAUGGUAGAACUCACA UGUGAGUUCUACCAUUGCCAAA hsa-miR-183 UAUGGCACUGGUAGAAUUCACU AGUGAAUUCUACCAGUGCCAUA hsa-miR-184 UGGACGGAGAACUGAUAAGGGU ACCCUUAUCAGUUCUCCGUCCA hsa-miR-185 UGGAGAGAAAGGCAGUTUCCUGA UCAGGAACUGCCUUUCUCUCCA ha-miR-186 CAAAGAAUJUCUCCUUUUGGGCU AGCCCAAAAGGAGAAUJCUUUG hsa-miR-187 UCGUGUCUUGUGUUGCAGCCGG CCGGCUGCAACACAAGACACGA hsa-miR-188-3p CUCCCACAUGCAGGGUUUGCAG CUGCAAACCCUGCAUGUGGGAG hsa-miR-188-Sp CAUCCCUUGCAUGGUGGAGGGU ACCCUCCACCAUGCAAGGGAUG hsa-miR-189 GUGCCUACUGAGCUGAUAUCAG CUGAUAUCAGCUCAGUAGGCAC hsa-miR-190 UGAUATJGUUUGAUAUAUUAGGU ACCUAAUAUAUCAAACAUAUCA hsa-miR-191 CAACGGAAUCCCAAAAGCAGCU AGCUGCUUTUGGGAUUCCGUUG hsa-miR-192 CUGACCUAUGAAUUGACAGCCA UGGCUGUCAATJUCAUAGGUCAG hsa-miR-193 - 3p AACUGGCCUACAAAGUCCCAGU ACUGGGACUUUGUAGGCCAGUU hsa-miR-193-Sp UGGGUCUUGCGGGCAAGAUGA UCAUCUUGCCCGCAAAGACCCA hsa-miR--194 UGUAACAGCAACUCCAUGUGGA UCCACAUGGAGUUGCUGUUACA hsa-miR-195 UAGCAGCACAGAAAUAUUGGCA UGCCAAUAUUUCUGUGCUGCUA hsa-mfiR-196 UAGGUAGUUUCAUGUUGTUGGG CCCAACAACAUGAAACUACCUA hsa-miR-197 UUCACCACCUUCUCCACCCAGC GCUGGGUGGAGAAGGUGGUGAA hsa-miR-198 GGUCCAGAGGGGAGAUAGGUTUC GAACCUAUCUCCCCUCUGGACC hsa-miR-199a-3p ACAGUAGUCUGCACAUUGGUUA UAACCAAUGUGCAGACUACUGU hsa-miR-199a-5p CCCAGUGUUCAGACUACCUGUU AACAGGUAGUCUGAACACUGGG 19 WO 2005/079397 PCT1US20051004'714 MicroRNA name rnicrcRNA sequence Ati-nicrRNA molecule (51 to 3') sequence- (5' to 3') hsa-miR- 19 9b CCCAGUGtJTAGACUAUCUGJU AACAGAUAGUCUAAACACUGGG hsa -miR- 20 0a UAACAcUGUCUGGTTAAcQAUGU ACAUCGUTJACCAGACAGUGUUA hsa -miP.-20 Ob cUCUAAUACUCCUGGUAAUGA UCAUUACCAGGCAGJAUTJAGAG hsa-miR- 20 Oc AAUACUGCCGGGCTAAUGAUGGA UCCAUCAtTUACCCGGCAGUATJU fsa-miR- 203 GUGAAAUGUULIAGGACCACUAG CUAGUGGUCCUAAACATJUCAC hsa-miR- 204 IUCCCUUUGUCXUCCUAUGCCU AGGCAUAGGAUGACAAAGGGAA haa-mik- 205 UCCUUCATTtCCACCGGAGUCUG CAGACUCCGGI3GGAAUGAAGGA hsa-mriR- 206 UGGAAUGUAI&GGAAGUGUGUGG CCACACACUJCUACAUZTCCA hsa -miR -208 AUAAGACGAGCA2.AAGCUUGU ACAAGCUTJUUUGCUCGUCUJAU hsa-rniR- 210 CUGUGCGUG{YGACAGCGGCUGA UCAGCCGCI3GICACACGCACAG hsa-miR- 211 UUCCCUUTJGUCAtYCCUUCGCCU AGGCGAAGGAUGACAAAGGGAA hsa-miR- 212 UAACAGUCUCCAGUJCACGGCCA UGGCCGUGACTJGGAGACUGUJA hsa -miR -213 ACCAUCGACCGUTGAU3GUACC. GGUACAAUCAACGGtYCGAUGGU hsa-miR- 214 ACAGCAGGCACA5ACAGGCAGU ACUGCCUGUCUGUGCCJGCUGY hsa-rniR- 215 AUGACCUAUGAAUUGACAGACA UGUCUGUCAAUQCAUAGGUCAU hsa -miR-2 16 UAAUCUCAGCUGGCAACUGUGA UCACAGUTJGCCAGCUGAGAUUA hsa-rniR- 217 UACTJGCAUCAGGAACUGAThYGG CCAAUCAGUUCCUGAUGCAGUA ha a -mik -218 UUGUCrJUGAUCUAAC CATJGUG CACAUGGIJTAGAUCAAGCACAA baa- miR- 219 UGAU-UGUCCAAAkCGCAAUJCU AAGAAUUGCGTJUUGGACAAUCA haa-miR- 220 CCACACCGYAUCtYGACACUUJG CAAAGUGtJCAGAUACGGUGUGG hsa-rniR-221 AGCUACAUJGUCJGCUGGGJTUt AAACCCAGCAGACAAIJGUAGCU haa -riR- 222 AGCUACAUCUGGCUACUGGGUC GACCCAGUAGCCAGAUGUAGCU hsa-miR- 223 UGUJCAGUTJGTJCAAAI3ACCCCA UGGGGUAXYUGACAAACLTGACA haa-miR-224 CAAGUCACJAGJGGUJCCGIYU AA.ACGGAACCACUAGUGACUJC haa-miR- 28-5 AAGGAGCUJCACAGUCUAUUGAG CUCAAUAGACUGUGAGCUCCUJ haa-miR- 290 CUCAAACUGJGGGGCACTTC GAAAGUGCCCCCACAGTJUGAG haa-rniR- 296 AGGGCCCCCCCUC.AAUCCJGUU AACAGGAUTJGAGCGGGGGCCCU hsa-miR-299 UGGUOTACCGUC CCACA3ACAJ AUGUAUGUGGGACGGU.AAAC CA haa-n±R- 301 CAGUGCAAJAGUACJGUCAAAG CUUEJGACAAU-ACUAtYUGCACUG hsa-m±R- 302 UAAGUGCIUhCCAUGtXQCUGGUG CACCAAAACAUGGAAGCACJUA hs a -mik -Je UGUAAACAUCCUJGACUGGAAG CUUCCAGUCAAGGAUGtYACA haa-nik- 320 AAAAGCUGGGUTIGAGAGGGCGA UCGCCCUCUCAACCCAGCUJU haa-miR- 321' UAAGCCAGGGAUJGUGGGUTCG- CGAACCCACAAUCCCUGGCUUA haa-miR- 322 AAACAUGAAJEGCUGCUGUAUC GAUACAGCAGCAAUJCAUGUTU haa-miR- 323 GCACAUUACACGGUJCGACCUCU AGAGGUCGACCGUGUAAUGUGC haa-rniR- 324 -Jp CCACUGCCCCAGGUGCUGCUGG CCAGCAGCACCUGGGGCAGUGG hsa-rniR- 324 -5p CGCAUCCCCUAGGGCAUTJGGUG CACCA7J3GCCCtAGGGGAUGCG hsa-rniR-326 CCUCUGGGCCCUCCUCCAGCC GGCUGGAGG7AACGGCCCAGAGG hsa-rniR-32 S CUGGCCCUCUCUGCCCUJCCGU ACGGAAGGGCAGAGAGGGCCAG hsa-miR- 329 AACACACCCAGCUAACCUUUUJ AAAAAGGUTJAGCUGGGUGUGUU hsa-miR-3S4a UGGCAGUGYCUIJAGCUGGUJGU ACAACCAGCUAAGACACtJGCCA hsa -miR- 34b AGGCAGUGUCAUCJUAGCUGAU-UG CAAUJCAGCUAAUGACACUGCCU hsa -miR- 34 c AGGCAQUGUAGITCJAGCUGAUJG CAAUCAGCU7AACUACACUGCCU hsa-rn±R-92 UAUtJGCACUUJGUCCCGGCCUGU ACAGGCCGGGACAAGUGCAAUA hsa-miR- 93 AAAGUGCUGUQCGU-GCAGGUAG CUACCTJGCACGA3ACAGCACTUU hs a -miR -95 UUCAACGGGUATTAUUGAGCA UGCUCAAU-AAAtJACCCGDJGAA hsa-mriR- 95 UTIUGGCACtJAGCACAUUTJGC GCAAAAAtJGUGCUAGUGCCAAA hsa-miR- 98 UGAGGUAG-AAGUTUGUAUJGtX AA.CAAUACAAOUIACUACCUCA rnmu-riR-1O Sa CAAAGUGCUAACAGUGCAGGUA UACCUGCACUGUJAGCACJJG mrnu-miR-1 Ob CCCUGUAGAACCGAAUUUGUGU ACACAAAUUCGGUUCUACAGGG rnru -miR -135 b UAUGGCtUUTCAOUCCUAUGUG CACAUAGGAAUGAAAAGCCAUA 20 WO 20051079397 PCT/US20051004 714 microRNA name m-icroRNA sequence A-nti-m-ICZoRNA molecule (5r to 31) sequence (5' to 31) ttu - miR -148 b UCAGUGCAUCACAGAACUUUGU ACAAAGUTJUCUGUGAUGCACUGA mrnu-miR- 151- 3p CEAGACUGAG3CUCCJTGAGGA UCCUCAAGGAGCCUCAGUCUAG rnru -rniR-155 UUAAUGCUAATJUGUGAUAGGGC CCCCUAUCACAAUUAGCAUJAA mmu -mi R 19 9b CCCAGUGUTJTAGACLACCUGUU AACAGGUAGUCUAAACACUGGG mmu -miR- 20 Ob UAAUACUGCCEJGGUJAAUGAUGA UCAUCAUEYACCAGGCAGUAUU-A mmu -miR -203 UGAAAUGJTJTAGGAC CACUA3A UCUAGUGGUCCUAAACATYCA mmu-miR-211 UUCCCUUJGUCAUCCtUIGCCU AGGCAAAGGAUGACAAAGGGAA mrnu-miR- 217 UACUGCAUCAGGAACUGACUGG CCAGUCAGUTJCCUGAUGCAGUA tntu -miR- 224 UAASUCACAGUGGTJTCCGUTT AAACGGAkCCACUAGUGACUUA mmu-mik-28 -3p CACUAGAUUGUGAGCTJGCUGGA UCCA3CAGCUCACAAUC{YAGUG tnmu -miR -290 CUCAAACUAUGGGGGCACUUJ AAAAGUGCCCCCAUAGUTJGAG mmu-miR- 291-3p AAAGUGCUUCCACUDTJGUGU3C GCACACAAAGUGGAAGCACTU mmu-miR-2 91- 5p CAUCAAAQUGGAGGCCCUCUCU AGAGAGGGCCUCCACDU=JGAUG mmu-rniR- 292- 3p AAGU3CCGCCAGGUJ1JUGAGUG CACUCAAAACCUGGCGGCACUJ mmu-miR- 292" 5p ACUCAAACUGGCGGCTJCUUUUG CAAAAGAGCCCCCAGUUYGAGU mmu-miR- 293 AGUGCCGCAGAGUUCGUAGUGU ACACUACAAACUCUGCGGCACU mmu -miR- 294 AAAGUGCUJCCCTUTGUGUGU ACACACAAAAGGGAAGCACUTJ rnru-rniR- 295 AAAGUGCUACTJACUtTUUGAGUC GACUCAAAAGUAGUAGCACUJT mmu -mi R-297 AUG:UAUGUGUGCAtJGTYGCAUGU ACAIJGCACAUGCACACAUACAU mmu-m±R- 298 GGCAGAGGAGGG3CUG-TJUCUUCC GGAAGAACAGCCCUCCUCUGCC mmu-miR-3 00 UAUGCAAGGGCAAGC1TCUCUUC GAAGAGA3CUUGCCCUfJG CAUA tnmu -miR- 31: AGGCAAGAUGCUGGCAUAGCUG CAGCtJAUGCCAGCATUT=GCCU mmu -miR -322 AAACAUGAAGCGCUGCAACACC 3GUGUUGCAGCGCUUCAUGJTU mmu -miR -325 CCUAGUAGGUGCUCAGUJAAGUG CACUIJUACUGAGCACCUACUAGG mmu-miR- 326 CCUCUGGGCCCUUCCLTCCAGUC GACUGGAGGAAGGGCCCAGAGG mmu -miR- 330 GCAAAGCACA3GGCCUGCAGAG CUCUGCAGGCCCJGUGCTTC mmu -mih- 331 GCCCCUGGGCCUAUCCJAGAAC GUUCUAGGAUAGGCCC7XGGGGC mu-miR- 337 UTCAGCUCCUAUAUGAUGCCUJ AAGGCAUCAUAUAGGAGCUJGAA tnmu -miR-338 UCCAkGCAUCAGUGATUUUGUJG CAACAAAA.UCACUGAUGCUJGGA mtnu- miR- 339 UCCCTGUCCUCCAGGAGCUCAC GUGAGCUCCUGGAGGACAGGGA mmu-miR- 340 UCCGUCUCAGUTJUACUTYTUAUAGC GCUAUAAAGUAACUGAGACGGA mmu-miR- 341 UCGAUC3GUCGUTCGGUCAGUC GACUGACCGACCGACCGAUCGA mu -miR- 342 UCICACACAGAAAUCGCACCCG C3GGUGCGAJTJVCUUGUGAGA rmu-miR- 344 UCAUCUAGCCAAAGCCUGACUG CAGUCAGGCTUTGGCUAGAUCA rnzu -miR- 345 UGCUGACCCCUAGUCCA3UGCU AGCACU3GACUAGGGGUCAGCA mmu-miR- 346 UGUCUGCCCGAGUGCCUGCCUC GAGGCAGGCACUCGGGCAGACA mu -miR- 34b UAGGCAGUGUAAUUAGCUGAUQ. AAUCAGCUAAUJACACJGC QUA mmu -miR -350 UTJCACAAAGCCCAUACACUTJC GAAAGUGUAUGGGCUTJGUGAA mmu-miR-3 $1 UCCCUGAGGAGCCCTYUUGAGCC GGCUCAAAGGGCUCCUCAGGGA mmu - ml - 7b UGGAAGACUJGUGAUTY&GUTJG CAACA-AAAUCACAAGUCUTJCCA mmu-miR-92 UAUUOCACLUGUCCCGGCCUGA UCAGGCCGGGACAAGUGCAAUA mmu -miR -93 CAAAGUGCUGTJCGUflCAGGUA UACCUGCACGAACAGCACTTG ma -miR- 327 CCUUGAG3GGCAUJGAGGGUA3U- ACUACCCUCAUGCCCCUCAAGG rno-miR- 333 GUGGUGU3CUAGUUACYUtGG CCAAAAGIUhACUAGCACACCAC ra -miiR -335 UCAAGAGCAAUAACGAAA UtG CAUUUUCGUUATJUGCUCUTJGA rno-miR- 33 6 UCACCCUTJCCAUAUCUAGUCUC GAGACUAGAUAUGGAASGGUGA rue-miR- 343 UCUCCCUCCGUGUGCCCAGUAU AUACUGGCCACACGGAG3GAGA rno-miR- 347 UGUCCCUCUGGGUCGCCCAGCU AGCUGGGCGACCCAGAOACA rno-rniR-349 CAGCCCUGCTGUCTTUAACCJCU AGAGGUUAAGACAGQAGGGCUG ra -mlR- 352 AGAGUAGUAGGXJUGCAUAGUAC GUACUAUGCAACCUACUACUCU 21 WO 2005/079397 PCT/US2005/004714 Table 2: Novel Human microRNA and anti-microRNA sequences. microRNA name microRNA sequence Anti-microRNA molecule (5' to 3') sequence (5' to 3') hsa-miR- 361 UUAUCAGAAUCUCCAGGGGUAC GUACCCCUGGAGAUUCUGAUAA hsa-miR-362 AAUCCUTJGGAACCUAGGUGUGA UCACACCUAGGUUCCAAGGAUU hsa-miR-363 AUUGCACGGUAUCCAUCUGUAA UUACAGAUGGAUACCGUGCAAU hsa-miR-364. CGGCGGGGACGGCGAUUGGUCC GGACCAAUCGCCGUCCCCGCCG hsa-miR-365 UAAUGCCCCUAAAAAUCCUUAU AUAAGGAUUUUUAGGGGCAUUA hs a -miR -366 UAACUGGUUGAACAACUGAACC GGUUCAGUUGUUCAACCAGUUA Table 3: C. elegans microRNA and anti-microRNA sequences. microRNA name microRNA sequence Anti-microRNA molecule (5' to 3') sequence (5' to 3') Cel - let -7 UGAGGUAGUAGGUUGUAUAGUTU AACUAUACAACCUACUACCUCA Cel-lin-4 UCCCUGAGACCUCAAGUGUGAG CUCACACUUGAGGUCUCAGGGA Cel-miR-1 UGGAAUGUAAAGAAGUAUGUAG CUACAUACUUCUUUACAUUCCA Cel-miR-2 UAUCACAGCCAGCUUUGAUGUG CACAUCAAAGCUGGCUGUGAUA Cel-miR-34 AGGCAGUGUGGTUAGCUGGUUG CAACCAGCUAACCACACUGCCU Cel-miR-35 UCACCGGGUGGAAACUAGCAGU ACUGCUAGTUUCCACCCGGUGA Cel-miR-36 UCACCGGGUGAAAAUUCGCAUG CAUGCGAAUUUCACCCGGUGA Cel-miR-37 UCACCGGGUGAACACUUGCAGU ACUGCAAGUGUTUCACCCGGUGA Cel-miR-3 8 UCACCGGGAGAAAAACUGGAGU ACUCCAGUUUUCUCCCGGUGA Cel-miR-39 UCACCGGGUGUAAAUCAGCUUG CAAGCUGAUUUACACCCGGUGA Cel-miR-40 UCACCGGGUGUACAUCAGCUAA UUAGCUGAUGUACACCCGGUGA Cel-miR-41 UCACCGGGUGAAAAAUCACCUA UAGGUGAUUUUUCACCOGGUGA Cel-miR-42 CACCGGGUUAACAUCUACAGAG CUCUGUAGAUGUUAACCCGGUG Cel-miR-43 UAUCACAGUUUACUUGCUGUCG CGACAGCAAGUAAACUGUGAUA Cel-miR-44 UGACUAGAGACACAUUCAGCUTU AAGCUGAAUGUGUCUCUAGUCA Cel-miR-45 UGACUAGAGACACAUUCAGCUU AAGCUGAAUGUGUCUCUAGUCA Cel-miR-46 UGUCAUGGAGUCGCUCUCUCA UGAAGAGAGCGACUCCAUGACA Cel-miR-47 UGUCAUGGAGGCGCUCUCUTJUCA UGAAGAGAGCGCCUCCAUGACA Cel-miR-48 UGAGGUAGGCUCAGUAGAUGCG CGCAUCUACUGAGCCUACCUCA Cel-miR-49 AAGCACCACGAGAAGCUGCAGA UCUGCAGCUUCUCGUGGUGCUU Cel -miR-50 UGAUAUGUCUGGUAUUCUUGGG CCCAAGAAUACCAGACAUAUCA Cel-miR-51 UACCCGUAGCUCCUAUCCAUGU ACAUGGAUAGGAGCUACGGGUA Cel-miR-52 CACCCGUACAUAUGUUUCCGUG CACGGAAACAUAUGUACGGGUG Cel-miR-53 CACCCGUACAUUUGUUUCCGUG CACGGAAACAAAUGUACGGGUG Ce1-miR-54 UACCCGUAAUCUUCAUAAUCCG CGGAUUAUGAAGAUACGGGUA Cel-miR-55 UACCCGUAUAAGUTJUCUGCUGA UCAGCAGAAACUUAUACGGGUA Cel-miR-56 UACCCGUAAUGUUUCCGCUGAG CUCAGCGGAAACAUTUACGGGUA Cel-miR-57 UACCCUGUAGAUCGAGCUGUGU ACACAGCUCGAUCUACAGGGUA Cel-miR-58 UGAGAUCGUUCAGUACGGCAAU AUUGCCGUACUGAACGAUCUCA Cel -miR- 59 UCGAAUCGUUUAUCAGGAUGAU AUCAUCCUGAUAAACGAUUCGA Cel-miR-60 UAUUAUGCACAUUTJCUAGUUC GAACUAGAAAAUGUGCAUAAUA Cel-miR-61 UGACUAGAACCGUUACUCAUCU AGAUGAGUAACGGUUCUAGUCA Cel-miR-62 UGAUAUGUAAUCUAGCUUACAG CUGUAAGCUAGAUTJUACAUAUCA Cel-miR-63 AUGACACUGAAGCGAGUUGGAA UUCCAACUCGCUUCAGUGUCAU 22 WO 20051079397 PCT/US2005/004714 micreRNA name microRNA sequence Anti-microRNA molecule (51 to 3,) sequence (5' to 3'1) Gel -miR- 64 UAUGACACUGAAGGUTACCGA UCGGUAACGCTJUCAGUGUGAUA Gel -miR -65 UAUGACACUGAAGCGUAACCGA UCGGJEACGCTJECAGUGUCAUA Gel -miR -6 ESAUGACACTJGAUEJAGGGAUGUG CAGATJCCCTUhAUCAGUGUAJG Gel -miR- 67 UGACAACCUCGUAGAAAGAGUA UACUCJTJTCUAGGAGGUUGUGA Gel -t-iR- 68 UCCAAGACUGAAAAGJGUAGAC GUCUACACUUTJGAGUCUUCGA Gel -miR -69 UCGAAAAUT}AAAAAGUGUAGAA UEJCUACACUIIJJTAAUUtJTIGA eel -miR- 70 UAAUACGUCGU-UGGUGUUGCCA UGGAAACAGCAACGACGUAUUA eel -miR- 71 UGAAAGACAUGGGUAGUGAACG CGUUCACUACCCAUGUCUJTCA Gel -miR- 72 AGGCAAGAUGUYGGCAUAGCUG CAGCUAUGCGAACAUCUQGCCU eel -miR- 73 (GGCAAGAUGUAGGCAGUUCAG CUGAACUGCCUACAUCQUGCCA Gel -miR-74 UGGCAAGAAAUGGCAGJCACA UGUAGACTJGCCAUUUTJUGCGA Cel -miR- 75 UYCAAAGCUACCAACCGGCUJGA UGAAGCCGGJUGGUAGCUtJTAA Cel -miR- 76 UUCGUUGUJGAUGAAGCCUYGA UCA-AGGCUUCAUQAACAACGAA Gel -miR- 77 UTCAUCAGGCCAUAGCUGUGCA UGGACAGCUAUGGCUTGAUGAA Cel -miR- 78 UGGAGGGGUGGUJGUUGUGCU ACCACAAACAACCAGGCCUCGA Gel -miR- 79 AUAAAGCUAGGU-UACCJAACGU AGCUJTGGUAAGCUAGCUTJtYAU Cel -miR- 227 AGCUTJTCGACAUGAUEJCUGAAC GUTICAGAAECAUGUCGAAAQCU Gel -miR- 80 UGAGAUCAUJAGIJUGAAAGCCG CGGCU1JUCAZCUAAUGAUCUCA Gel -miR- 81 UCAGAUCATJCGUGAAAGGUAGU AGUAGCUIUCACGAUGAJCUGA Cel -miR- 82 UcAQAUCAUCGUGAAAGCCAGU AGUGGGOTJTCACGACJGAUGUCA Gel -riR -83 UAGCAC CATAUAAAUUJCAGUAA UEJACTJGAA = UtYAUGGTJGGUA Cel -miR- 84 UGAGGUAGTJAUGUAAUAUUGUA UACAAUAT-TUACAUACUACCUCA Cel -miR- 85 UACA.AAGUAUUTJGAAAACuCGU AGGACUTUUCAAAUACUUJTGUA Cel1-miR- 86 UAAGUGAMJGCUTJUGCGAGAGU ACUGUGOCAAAGGAUTUCAGUrA Gel -miR- 87 GUGAGCAAAGUUCAGGUGUGG GCACACCUGAAACUJTGCUGAC Gel -miR- 90 UGAUAUGUUGTTEGAAUGCGCG GGGGCAUtJCAAACAACAUAUCA Cel -miR- 124 UAAGGCACGCGGUGAAUGCCAC GUGGCAUUCACCCCGUGGGUUA eel miR- 228 AAUGGCACUGCAUGAAUJCAGG CGUGAATJUCAUGCAGUGCCAUT Gel -miR -229 AAUGACACUGGUJAUGUUUUCC GGAMAGAUAACCAUGUCAID eel -miR- 230 GUAUJUAGTTUGUGCGACCAGGAG CUCCUGGUCGCACI4ACUAAUAC Cel -miR- 231 UAAGCUGGUGAUGAACAGGCAG CUGCCUGUCYGAUCACGAGGUJA Cel -miR-232 UAAAUGCAUCUUAAGUGCGGUG CACCGGAGUJUAAGAUGCAUJUA Cel -miR- 233 UUGAGGAAUGCGGAUGUGCGGG CGCGCAGAUGCGAUTJGCUCAA Cel -miR- 234 UUAUUGCUCGAGAXUACCCUUU AAAGQGUAUUCUCGAGCAAUAA CeJ.-miR- 235 UAIUGCCACUCUCGCGGCCUGA UCAGGCCGGGGAGAGUGCAAUA Cel -miR- 236 UAALUACUGUCAGGUAAUGAGGC GCGUCAUUACCUGACAGUAUJA Ce 2.-miR- 237 UCCCUCAG4AUUTCUCC3AACACG GCUGUTJCGAGAATYUCUCAG3GA Cel-miR-238 B UUTJGUACUCCGAUGCCAtTUCAG CUGAAUGGCAUCGCAGuACAAA eel -miR -239 a UtTUGUACUACACAUA3GUACUC CAGUACCUAUGUGUAGIYACAAA Ce1- miR- 23 9b TJTUGUACUACACAAAAGU-ACUG CAGUACUOJQGUGUAGUJACAAA Cel -miR- 240 UAGUGGCCCCCAAAUCUOCOCU AGCGAAGAUUtJGGGG~cCAGUA Cel -miR- 241 UGAGGUAGGUGCGAGAAAUGAC GUCAULUGUCGCACCUACCUCA Cel-miR-242 UUGCGUAGGCCUUUGCUUCCAG CUCGAAGCAAAGGGCUACGCAA Cel -miR- 243 GGGUACGAUCGCGGCGGGAUAU AUAIJCCCGCCGCGAUCGUACG Cel1-miR- 244 UCUXYQGCUUGUACAAAGUGGUA UACCACtUXGUAQAAccAA-AGA Cel -miR- 245 AU{TGGUCCCCUCCAAGUAGCUC GAGCUACUEJGGAGGGGACCAAU Cel -miR- 246 UUACAUGTUTJGGGGUAGGAGCU AGGUCCUACGCGAAACAUGUAA Ce 1-miR -247 UGACUAGAGCCUAT-TJCUCUUCU AGAAGAGAZXUAGGGUCUAGUCA Cel -miR -248 UAGACGUGCACGGAUAACGCUG GAGCGTUtAUCCGUGCACGUGUA Ce 1-mi R-249 UCACACGACUUGAGCGtWGC GCAACGCUCAAAAGUccUGUGA .Cel-miR -250 UCACAGTJCAACUGJTJUGGGAUGG CCAUGCCAACAGU-UGACuGUGA 23 WO 2005/0179397 PCT/US200S/004 714 micrRNAnam rncro~lA equnceAntf"-microRNA molecule (5' to 3') sequence (5' to) 3' ) C e 1 -mil- 251 UUAAGUAGUGGtUGGGGCUUJA UAAGAGGGGAGGAGAUAA Gel -uiR -252 UZAGUAGUAGUGCGGCAGGUAA UTACGUGGGGCAGUACUACUUA Cel1-miR- 253 CACACCUGACUAACACUGACCA UGGUGAGUGtYUAGUGAGGUGUG Gel -miR- 254 UGCAAAUCtUCGCGACUGUAG GUACAGUGGCGAAAGAUJUGGA Gel -miR -256 tGGAAUGGATAGAAGACUGUAG GUACAGUGUCGUAUGCAUUCGA Gel -miR- 257 GAGUAUCAGGAGUACCCAG}GA UCACUGGGUAGUGGU-GAUAGUG Gel -miR -258 GGUIUQTGAGAGGAAUCCUUJTA UAAAAGGATYUCCUCU-CAAAAGC Gel -miR -259 AGUAAAUCUCAUGCTJAAUCUGG C CAGA ,UTAGGAUGAGAUUJAGU Gel -miR- 260 GUGAUGUGGAACTGUUGUAGGA UGGUACAAGAGOTYGGAGAUGAG, Gel -miR- 261 UAGGUUTUUUAGUTJUOCAGGGUG GACG GUGAAAAGTJAAAAAGGUA Gel -miR- 262 GUUUGAUGUUUUUGAUAG GUAUGAGAAAAGAUGGAAAG Gel -miR- 264 GGGGGGUGGUUGUJGJTAUGGG GGGAUAAGAAGAAGGAGGGGGG Gel -miR- 265 UGAGGGAGGAAGGGUJGGUAJ AAAUAGGAGGCUtJTJGGCGUGA Gel -miR -266 AGGGAAGACUUUGCAAAGGUU AAGG{YUGGGAAAGtYCUUGGGU Gel -miR -267 GGGGUGAAGUGTUGUGGUGGAAU AUQGGCAGCAGAGAGYJUGAGGGG Gel -miR- 268 GGCAAGAALUAGAAGAGUTJG GAAAGUGGOTJGUAAUTJGUUGGG Gel -miR- 269 GGGAAGAGUCUGGGAAA.ACUUG GAAGU=JJGGGAGAGTJGUJGGG Gel -miR -270 GGGAUGAUGUAGGAGt}GGAGAU AUCUCGAGUGGUAGAUGAJGGG Gel -miR- 271 UGGGGGUtGGGAAAGGAUUGG GGAATJGGUUTJGGGACGGGGGA Gel -miR- 272 UGUAGGCAUGGGUGUUTGGAAG GUTGGAAAGAGGGATTGGCUAGA Gel -miR- 273 UGGGGGUACGUTGUCGGGUGU AGGAGGGGAGAGAGUJAGGGGGA 24 WO 2005/079397 PCT/US2005/004 714 Table 4. Drosophila microRNA and anbi-microRlfl sequences. tn1CQRNAname micoRNAsequnceAnti-microRNA molecule (51 to 31) sequence ( 5 t to 3') Dine-miR-2 63 a GUTUAATYGGCACUGGAAGAAUJUC GAAUTIUCUUCCAGUGCCAUJAAC Dine -iR- 184 UGGACCGAGAACUGAUAAGGGC GCCCUUAIYCAGLTUCUCCGUCCA Dine-miR- 274 UTJIGUGACCCACACUAACGGG CCCGUJUAGUGUCGGUCACAAAA Dine-miR-275 TCAGGUACCUGAAGUAGCGCGC GCGCGCUACUJUCAGGTJAiCCUGA Dine -miR -92 a CAUTJGCACUTJGUCCCGGCC{JAU AUAGGCCGGGACAAGUGCAAYG Dine - iR -219 TJGAUUGUCCAAACGCAAUVJCUU AAGAAJTGCGUOhJUGACAAUCA Ine -miiR -276 a UAGGAACUJCAUACCCUGCUCY AGAGCACGGUAUGAAGUUCCUA ine -miR- 277 UAAAUCCACQAUCUGGUACGAC GUCGUACCAGAUACUGCATFA Dxe -miR- 278 UCGGUGGGACUJTCGUCCGUUJ AAACGGACGAAAGTCCCACCGA Dine -iiR- 133 JUGGUCCCCUTUCAACCAGCUGU ACAGCUGGUUGAAGGGGACCAA ine -miiR -279 UGACT3AGAUCCACACUCATUA7A UEAAtGAGUGUGGAU(2UAGUCA Dine -riR- 33 AGGUGCAU-UGUAGUCGCAUUGU ACAAUGCGACUACA.UGCACCJ Dyne -miR- 280 UGtIAUUUTACGUUGCAUAUGAAA TUUCAUAUGCAACGUAAAUACA Dine -miR -281 UGUCAUGGAAUTJGCUCUCUUUG CAAAGAGAGCAATUC CAUCACA Dine -iiR-282 AAUCUAGCCIYCUACUAGGCKTU AAAGCCUAG3UAGAGGCUAGAUU Dine -iiR-283 UAAAUAUCAGCTYCGUAAUUCUG CACAAJUACCAGCUGAUAUUA Dne -iS-284 UGAAGUCACCAACUGAUJCCA UGGAAUCAAGUTGCUGACtYUCA Dine -i-34 IGGCAGQGUGUTUAGCUGGUJG CAACCAGCUAACCACACYGCCA Dine -miR -124 UAAGGCACGCGGUGAAUGCCAA TUGGCAtJUCACCGCGDUGCCU-UA Dine -miR-79 UAAAGCTJAGATJACCAAAGCAU AtJGCUIJTGGUAAUCUAGCUJ(A Dine -iiR- 27Gb UAGGAACUTUAAIYACCGUGCUCU AGAGCACGGUAU.AAGTCCCUA Dnxe -miR- 210 UUGIJGCGTJGZGACAGCGGCUATJ AUA.GCCGCUGUCACACGCACAA Dine -minS-285 UAGCACCAUUCGAAAYCAGUGC G3CACT3GAtXUCGA-AUGGUGC3A Dine -mAY-TOO AACCCGUAAAUCCGAACUUGUG CACAAGUGCGGAUUUACGGG-U Dine-MiR- 92b AAUTJGCACUAGIYCCCGGCCUGC GCAGCCCGGGACUAGUGCAAUU Dine -iS-286 TGACUAGACCGAACACUCGUGC GCACGAGUGUVCGGUCUAGUCA Dine -iiR -287 UGUGU.UGAAAAUCGUIJGCACG CGUGCAAACGAUIUUCAACACA Dine - mlR-87 IJUGAGCAAAAJUTJCAGGUGUGY ACACACCYGAAAUTJTTJGCUCAA Dine -miR -263 b CTJTJGGCACUGGGAGAAUUCACA UGUGAAUUCUCCCAOGTGCCbAG Dine-miR- 288 UUTCAUGUCGAUUCAUUTUCAU AUGPAAAUGAAAUCGACAUGAAA Dine-MiR- 289 TYAAATAtUtJAAGUGGAGCCUGC GCAGGCUCCACUUAAAUAYUUA Dine -bantam UGAGAQCAU3EJTGAAAGCUGAUJ AUCAGCUUTJCAAAAUJGAUCUCA, Dine -iiR -303 UUXAGGUUUCACAGGAAACUGG CCAGIUI(JCCUGUGAAACCUAAA Dine-iniR-3 lb UGGCAAGAIGTCGGAAUAGCJG CAGCTYA{YtCCGACATYCUTJGCCA Dine-iniR.-304 UAAUCUCAA&ULTGUAAAUGUGA UCACAUUUACAAAJIGAGAUUA Dine -iiR- 305 AUTJGUACEUCATCAGGUGCUCU AGAGCACCUGAUGAAGtIACAAU Dine-iniR- Dc UCUUUGGUJAUUCIAGCUGYAGA ICACAGCUAGAPMACCAAAGA Dme-miR-3 06 UCAGGUACUJAGTYGACUCUCAA TUUGAGAGUCACUAAGUACCUGA Dme-miR- Sb UCUI=GGUGAUUJUUAGCUGUAU AUACAGCUAAAAUCACCAAAGA Dine-iniR- 125 UCCCUGAGACCCUAACUUGUGA UCACA-AGTAGGGUCUCAGGGA Dme-miR-3 07 UCACAACCUCCT-UAGUGAGCG CGCUCACUCAACGAGGUTJGUGA Dine -iiR- 308 AAUCACAGGAUJTAUACUGTJGAG CUCACAGTYAUAAUCCUGUGALU dine -iiR- 3 a UGGCAAGAUJGUCGGCAUAGCUG CAGCT3AUGCCGACATUGCCA dine-rniR- 309 GCACtIGGGIAAAGUUUGUCCUA UAGGACAAACUJTACCCAGUGC dine -miR- 210 UAUXJGCACACUIJCCCGGCCUU3 AAAGGCCGGGAAGUGUGCAAUA dine-iniR- 311 UAU(GCACA-UVCACCGGCCUGA UCAGGCCGGUGAAUGUGCAAUA dine -miR- 312 UAUTJGCACU{GACACGGCCUGA UCAGGCCGUCUCAAGUGCAAUA dine- miR -313 -UAtUtGCACUUUUICACAGCCCGA TCGGGCUCUGAAAAGuGCAAUA dine -iiR -314 UAEJUCGAGCCAAUAAGUUCGG CCGAACUAUTJGGCUCGAAUA 25 WO 2005/079397 PCT/US2005/004714 muicroRNA name microRNA sequence Anti-microRNA molecule (5' to 3') sequence (5' to 3') dme-miR-315 UUUGAUUGUUGCUCAGAAAGC GCUUUCUGAGCAACAAUCAAAA dme-miR- 316 UGUCUUUUUCCGCUUACUGGCG CGCCAGUAAGCGGAAAAAGACA dme - miR -317 UGAACACAGCUGGUGGUAUCCA UGGAUACCACCAGCUGUGJUCA dme-miR-318 UCACUGGGCUUUGUUUAUCUCA UGAGAUAAACAAAGCCCAGUGA dme-miR-2c UAUCACAGCCAGCUUUGAUGGG CCCAUCAAAGCUGGCUGUGAUA Dme-miR-iab45p ACGUAUACUGAAUGUAUCCUGA UCAGGAUACAUUCAGUAUACGU Dme-miR-iab43p CGGUAUACCUUCAGUAUACGUA UACGUAUACUGAAGGUAUACCG EXAMPLES Example 1: Materials and Methods Oligonucleotide synthesis MiR-21 were synthesized using 5-silyl, 2'-ACE phosphoramidites (Dhannacon, Lafayette, CO, USA) on 0.2 pmol synthesis columns using a modified ABI 394 synthesizer (Foster City, CA, USA) (Scaringe, Methods Enzymol. 317, 3-18 (2001) and Scaringe, Methods 23, 206-217 (2001)). The phosphate methyl group was removed by flushing the column with 2 ml of 0.2 M 2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydIrate in DMF/water (98:2 v/v) for 30 min at room temperature. The reagent was removed and the column rinsed with 10 ml water followed by 10 ml acetonitrile. The oligonucleotide was cleaved and eluted from the solid support by flushing with 1.6 ml of 40% aqueous methylamine over 2 min, collected in a screwcap vial and incubated for 10 min at 55 *C. Subsequently, the base-treated oligonucleotide was dried down in an Eppendorf concentrator to remove methylamine and water. The residue was dissolved in sterile 2T-deprotection buffer (400 pL of 100 mM acetate-TEMED, pH 3.8, for a 0.2 pmol scale synthesis) and incubated for 30 minutes at 60 *C to remove the 2' ACE group. The oligoribonucleotide was precipitated from the acetate-TEMED solution by adding 24 1 5 M NaCl and 1.2 ml of absolute ethanol. 2-O-Methyl oligoribonucleotides were synthesized using 5'-DMT, 2'-0methyl phosphoramidites (Proligo, Hamburg, Germany) on I grmol synthesis columns loaded with 3' aminomodifier (TFA) C7 Icaa control pore glass support (Cherngenes, MA, USA). The aminolinker was added in order to also use the oligonucleotides for conjugation to amino group 26 WO 2005/079397 PCT/US2005/004714 reactive reagents, such as biotin succinimidyl esters. The synthesis products were deprotected for 16 h at 55 IC in 30% aqueous ammonia and then precipitated by the addition of 12 mril absolute 1-butanol. The full-length product was then gel-purified using a denaturing 20% polyacrylamide gel. 2-Deoxyoligonucleotides were prepared using 0.2 pmol scale synthesis and standard DNA synthesis reagents (Proligo, Hamburg, Germany). The sequences of the 2'-0-methyl oligoribonucleotides were 5' GUCAACAUCAGUCUGAUAAGCUAL (L, 3' aminolinker) for 2'-OMe miR-21, and 5' AAGGCAAGCUGACCCUGAAGUL for EGFP 2'-OMe antisense, 5' UGAAGUCCCAGUCGAACGGAAL for EGFP 2'-OMe reverse; the sequence of chimeric 2' OMe/DNA oligonucleotides was 5'-GTCAACATCAGTCTGATAAGCTAGCGL for 2'-deoxy miR-21 (underlined, 2'-OMe residues), and 5'-AAGGCAAGCTGACCCTGAAGTGCGL for EGFP 2'-deoxy antisense. The miR-21 cleavage substrate was prepared by PCR-based extension of the partially complementary synthetic DNA oligonucleotides 5' GAACAATTGCTTTTACAGATGCACATATCGAGGTGAACATCACGTACGTCAACATCA GTCTGATAAGCTATCGGTTGGCAGAAGCTAT and. 5' GGCATAAAGAATTGAAGAGAGTTTTCACTGCATACGACGATTCTGTGATTTGTATTC AGCCCATATCGTTTCATAGCTTCTGCCAACCGA. The extended dsDNA was then used as template for a new PCR with primers 5' TAATACGACTCACTATAGAACAATTGCTTTTACAG and 5' ATTTAGGTGACACTATAGGCATAAAGAATTGAAGA to introduce the T7 and SP6 promoter sequences for in vitro transcription. The PCR product was ligated into pCR2.1 -TOPO (Invitrogen). Plasmids isolated from sequence-verified clones were used as templates for PCR to produce sufficient template for run-off in vitro transcription reactions using phage RNA polymerases (Elbashir et al., EMBO 20, 6877-6888 (2001)). 32 P-Cap-labelling was performed as reported (Martinez et al., Cell 110, 563-574 (2002)). Plasmids Plasmids pEGFP-S-21 and pEGFP-A-21 were generated by T4 DNA ligation of preannealed oligodeoxynucleotides 5'-GGCCTCAACATCAGTCTGATAAGCTAGGTACCT 27 WO 2005/079397 PCT/US2005/004714 and 5'-GGCCAGGTACCTAGCTTATCAGACTGATGTTGA into Noti digested pEGFP-N-1 (Clontech). The plasmid pHcRed-C1 was from Clontech. HeLa extracts and miR-21 quantification HeLa cell extracts were prepared as described (Dignam et al., Nucleic Acid Res. 11 1475-1489 (1983)). 5x10 9 cells from HeLa suspension cultures were collected by centrifugation and washed with PBS (pH7.

4 ). The cell pellet (approx. 15 ml) was re-suspended in two times of its volume with 10mM KCI/1.5 mM MgC12/0.5 mM dithiothreitol/1OmM HEPES-KOH (pH 7.9) and homogenized by douncing. The nuclei were then removed by centrifugation of the cell lysate at 1000 g for 10 min. The supernatant was spun in an ultracentrifuge for 1 h at 10,5000 g to obtain the cytoplasmic S100 extract. The concentration of KCl of the S 100 extract was subsequently raised to 100 mM by the addition of 1 M KCL. The extract was then supplemented with 10% glycerol and frozen in liquid nitrogen, 280 pg of total RNA was isolated from 1 ml of S 100 extract using the acidic guanidinium thiocyanate-phenol-chloroform extraction method (Chomozynski et al., AnaL. Biochem. 162, 156-159 (1987)). A calibration curve for miR-21 Northern signals was produced by loading increasing amounts (10 to 30000 pg) of synthetically made miR-21 (Lim et al. et al., Genes & Devel. 17, 991-1008 (2003)). Northern blot analysis was performed as described using 30 pg of total RNA per well (Lagos-Quintana et al., Science 294, 853-858 (2001)). In vitro mi RNA cleavage and inhibition assay 2'-O-Methyl oligoribonucleotides or 2-deoxyoligonucleotides were pre-incubated with HeLa S100 at 30 'C for 20 min prior to the addition of the cap-labeled miR-21 target RNA. The concentration of the reaction components were 5 nM target RNA, I mM ATP, 0.2 mM GTP, 10 U/ml RNasin (Promega) and 50% HeLa S100 extract in a final reaction volume of 25 pil. The reaction time was 1.5 h at 30 'C. The reaction was stopped by addition of 200 plI of 300 mM NaC1/25 mM EDTA/20% w/v SDS/200 mM Tris HCI (pH7.5). Subsequently, proteinase K was added to a final concentration of 0.6 mg/ml and the sample was incubated for 15 min at 65 C. After phenol/chloroform extraction, the RNA was ethanol-precipitated and separated on a 6% denaturing polyacrylamide gel. Radioactivity was detected by phosphorimaging, 28 WO 2005/079397 PCT/US2005/004714 Cell culture and transfection HeLa S3 and HeLa S3/GFP were grown in 5% C02 at 37 'C in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 unit/nal penicillin, and 100 ag/mi streptomycin. One day before transfection, 105 cells were plated in 500 p1 DMEM containing 10% FBS per well of a 24-well plate. Plasinid and plasmid/oligonucleotide transfection was carried out with Lipofectamine2000 (Invitrogen). 0.2 tg pEGFP or its derivatives were cotransfected with 0.3 pg pHeRed with or without 10 pmol of 2'-O-methyl oligoribonucleotide or 10 pmol of 2'-deoxyoligonucleotide per well. Fluorescent cell images were recorded on a Zeiss Axiovert 200 inverted fluorescence microscope (Plan Apochromat lOx/0.45) equipped with Chroma Technology Corp. filter sets 41001 (EGFP) and 41002c (HcRed) and AxioVision 3.1 software. Example 2: MicroRNA-21 Cleavage of Target RNA In order to assess the ability of modified oligonucleotides to specifically interfere with miRNA function, we used our previously described mammalian biochemical system developed for assaying RISC activity (Martinez et al., Cell 100, 563-574 (2002)). Zamore and colleagues (Hutvigner et al., Science 297, 2056-2050 (2002)) showed that crude cytoplasmic cell lysates and eIF2C2 immunoprecipitates prepared from these lysates contain let-7 RNPs that specifically cleave let-7-complementary target RNAs. We previously reported that in HeLa cells, numerous miRNAs are expressed including several let-7 miRNA variants (Lagos-Quintana et al., Science 294, 853-858 (2001)). To assess if other HeLa cell milRNAs are also engaged in RISC like miRNPs we examined the cleavage of a 32P-cap-labelled substrate RNA with a complementary site to the highly expressed miR-21 (Lagos-Quintana et al., Science 294, 853-858 (2001); Mourelatos et al., Genes & Dev. 16, 720-728 (2002)). Sequence-specific target RNA degradation was readily observed and appeared to be approximately 2- to 5-fold more effective than cleavage of a similar let-7 target RNA (Figure 2A, lane 1, and data not shown). We therefore decided to interfere with miR-21 guided target RNA cleavage. 29 WO 2005/079397 PCT/US2005/004714 Example 3: Anti MicroRNA-21 2-O-methyl Oligoribonucleotide Inhibited MicroRNA-21 Induced Cleavage of Target RNA A 24-nucleotide 2'O-methyl oligoribonucleotide that contained a 3t C7 aminolinker and was complementary to the longest form of the miR-21 was synthesized. The aninolinker was introduced in order to enable post-synthetic conjugation of non-nucleotidic residues such as biotin. Increasing concentrations of anti tmiR-21 2-0-methyl oligoribonucleotide and a control 2'-0-methyl oligoribonucleotide cognate to an EGFP sequence were added to the S100 extract 20 min prior to the addition of 32P-cap-labelled substrate. We determined the concentration of miR-21 in the S100 extract by quantitative Northern blotting to be 50 pM (Lim et al., renes & Devel. 17, 991-1008 (2003)). The control EGFP oligonucleotide did not interfere with miR-21 cleavage even at the highest applied concentration (Figure 2A, lanes 2-3). In Contrast, the activity of miR-21 was completely blocked at a concentration of only 3 nM (Figure 2A, lane 5), and a concentration of 0.3 nM showed a substantial 60%-70% reduction of cleavage activity (Figure 2, lane 6). At a concentration of 0,03 nM, the cleavage activity of miR-21 was not affected when compared to the lysate alone (Figure 2, lane 1, 7). Antisense 2'-deoxyoligonucleotides (approximately 90% DNA molecules) at concentrations identical to those of 2'-0-methyl oligoribonucleotides, we could not detect blockage of miR-21 induced cleavage (Figure 2A, lanes 8-10). The 2-deoxynucleotides used in this study were protected against 3'-exonucleases by the addition of three 2'O-methyl ribonucleotide residues. Example 4: Anti MicroRNA-21 2-O-methyl Oligoribonucleotide Inhibited MicroRNA-21 Induced Cleavage of Target RNA In Vitro In order to monitor the activity of miR-21 in HeLa cells, we constructed reporter plasmids that express EGFP mRNA that contains in its 3' UTR a 22-nt sequence complimentary to miR-21 (pEGFP-S-21) or in sense orientation to miR-21 (p-EGFP-A-21). Endogenous miRNAs have previously been shown to act like siRNAs by cleaving reporter mRNAs carrying 30 C:NRPonb\DCCiLLU94754 LDOC-2//In 2011 sequences perfectly complementary to miRNA. To monitor transfection efficiency and specific interference with the EGFP indicator plasmids, the far-red fluorescent protein encoding plasmid pHcRed-Cl was cotransfected. Expression of EGFP was observed in HeLa cells transfected with pEGFP and pEGFP-A-21 (Figure 3, rows 1 and 2), but not from those transfected with pEGFP-S-21 (Figure 3, row 3). However, expression of EGFP from pEGFP-S-21 was restored upon cotransfection with anti miR 21 2WO-methyl oligoribonucleotide (Figure 3, row 4). Consistent with our above observation, the 2'-deoxy anti miR-21 oligonucleotide showed no effect (Figure 3, row 5). Similarly, cotransfection of the EGFP 2'-0-methyl oligoribonucleotide in sense orientation with respect to the EGFP mRNA (or antisense to EGFP guide siRNA) had no effect (Figure 3, row 6). We have demonstrated that miRNP complexes can be effectively and sequence-specifically inhibited with 2-0-methyl oligoribonucleotides antisense to the guide strand positioned in the RNA silencing complex. The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates. Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. 31

Claims (21)

1. An isolated single stranded anti-microRNA molecule comprising the sequence of SEQ ID NO: 446 and having a maximum of fifty moieties.

2. A molecule according to claim 1, wherein at least one of the moieties is a deoxyribonucleotide, a ribonucleotide moiety, a peptide nucleic acid moiety, a 2' fluororibonucleotide moiety, a morpholino phosphoroamidate nucleotide moiety, a tricyclo nucleotide moiety, a cyclohexene nucleotide moiety, or a moiety modified for increased nuclease resistance.

3. A molecule according to claim 2, wherein the deoxyribonucleotide is a modified deoxyribonucleotide moiety.

4. A molecule according to claim 3, wherein the modified deoxyribonucleotide is a phosphorothioate deoxyribonucleotide moiety, or a N'3-N'5 phosphoroamidate deoxyribonucleotide moiety.

5. A molecule according to claim 2, wherein at least one of the moieties is a modified ribonucleotide moiety.

6. A molecule according to claim 5, wherein the modified ribonucleotide is substituted at the 2' position.

7. A molecule according to claim 6, wherein the substituent at the 2' position is a C to C 4 alkyl group.

8. A molecule according to claim 7, wherein the alkyl group is methyl or allyl.

9. A molecule according to claim 6, wherein the substituent at the 2' position is a Ci to C 4 alkoxy - C, to C 4 alkyl group.

10. A molecule according to claim 9, wherein the C 1 to C 4 alkoxy - C to C 4 alkyl group is methoxyethyl. 32 CANRPrbhlDCC\SCGC490)633_ ,DOC-I/22013

11. A molecule according to claim 5, wherein the modified ribonucleotide has a methylene bridge between the 2'-oxygen atom and the 4'-carbon atom.

12. A molecule according to claim 2, wherein the nuclease is an exonuclease.

13. A molecule according to claim 12, wherein the molecule comprises: (a) at least one modified moiety at the 5' end; or (b) at least two modified moieties at the 5' end; or (c) at least one modified moiety at the 3' end; or (d) at least two modified moieties at the 3' end; or (e) at least one modified moiety at the 5' end and at least one modified moiety at the 3'end; or (f) at least two modified moieties at the 5' end and at least two modified moieties at the 3'end; or (g) a nucleotide cap at the 5' end, the 3' end or both ends; or (h) an ethylene glycol compound and/or amino linkers at the 5' end, the 3' end, or both ends.

14. A molecule according to claim 2, wherein the nuclease is an endonuclease.

15. A molecule according to claim 14, wherein the molecule comprises at least one modified moiety between the 5' end and 3' end and/or an ethylene glycol compound and/or amino linker between the 5' end and 3' end.

16. A molecule according to claim 1, wherein all of the moieties are nuclease resistant.

17. A method for inhibiting microRNP activity in a cell, the microRNP comprising a microRNA molecule, the microRNA molecule comprising a sequence of bases complementary to the sequence of bases in a single stranded anti-microRNA molecule, the method comprising introducing into the cell the single-stranded anti-microRNA molecule of claim 1.

18. An isolated single stranded anti-m icroRNA molecule for use in inhibiting microRNP activity in a cell, the microRNP comprising a microRNA molecule, the microRNA molecule comprising a sequence of bases complementary to the sequence of bases in the single stranded anti 33 C:\WRPotbLDCC\SCCM'901633 LDOC-M0020iL3 microRNA molecule of claim 1.

19. An isolated molecule comprising the sequence of SEQ ID NO: 140 and having a maximum of fifty moieties.

20. A molecule according to claim 19, wherein the molecule is modified for increased nuclease resistance.

21. An isolated single stranded anti-microRNA molecule according to any one of claim I to 16 or 18, or a method according to claim 17, or a molecule according to claim 19 or claim 20 substantially as hereinbefore defined. 34