# Patent

## Title
Anti-MicroRNA oligonucleotide molecules

## Abstract

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 provides a method for inhibiting microRNP activity in a cell.

## Background
This application is a divisional of U.S. application Ser. No. 12/794,085 filed on Jun. 4, 2010, which is a divisional of U.S. application Ser. No. 10/589,449 filed on Aug. 11, 2006, issued on Aug. 10, 2010 as U.S. Pat. No. 7,772,389, which is a U.S. National Phase Application of International Application No. PCT/US05/04714 filed on Feb. 11, 2005 and asserts priority to U.S. application Ser. No. 10/845,057 filed on May 13, 2004, which is a continuing application of U.S. application Ser. Number 10/778,908 filed on Feb. 13, 2004; all of which are hereby incorporated by reference in their entirety.


The invention claimed herein was made with the help of grant number 1 R01 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 mRNA 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 miRNAs 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 mRNA 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 or repress translation of mRNA which encode important proteins, there is also a need for novel compositions for inhibiting microRNA-induced 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-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; the moiety in the molecule at the position corresponding to position 11 of the microRNA 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 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 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 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
FIG. 1 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.
FIG. 2. Antisense 2′-O-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 32P-cap-labelled cleavage substrate. Cleavage bands and T1 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.
FIG. 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.
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-diaminopurine 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, deoxyribonucleotides, phosphorothioate deoxyribose groups, N′3-N′5 phosphoroamidate 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, 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 APE1 and EXO1.
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 are known in the art.
A suitable example of a modified deoxyribonucleotide moiety is a phosphorothioate deoxyribonucleotide moiety. See structure 1 in FIG. 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).
Another suitable example of a modified deoxyribonucleotide moiety is an N′3-N′5 phosphoroamidate deoxyribonucleotide moiety. See structure 2 in FIG. 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 C1 to C4 alkyl group. The C1 to C4 alkyl group may be saturated or unsaturated, and unbranched or branched. Some examples of C1 to C4 alkyl groups include ethyl, isopropyl, and allyl. The preferred C1 to C4 alkyl group is methyl. See structure 3 in FIG. 1. An oligoribonucleotide molecule comprising more than one ribonucleotide moiety that is substituted at the 2′ position with a C1 to C4 alkyl group is referred to as a 2′-O—(C1-C4 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 C1 to C4 alkoxy-C1 to C4 alkyl group. The C1 to C4 alkoxy(alkyloxy) and C1 to C4 alkyl group may comprise any of the alkyl groups described above. The preferred C1 to C4 alkoxy-C1 to C4 alkyl group is methoxyethyl. See structure 4 in FIG. 1. An oligonucleotide molecule comprising more than one ribonucleotide moiety that is substituted at the 2′ position with a C1 to C4 alkoxy-C1 to C4 alkyl group is referred to as a 2′-O—(C1 to C4 alkoxy-C1 to C4 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 FIG. 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); Ørum et al., Curr. Opinion Mol. Ther. 3, 239-243 (2001); Koshkin et al., Tetrahedron 54, 3607-3630 (1998); Obika et al., Tetrahedron Lett. 39, 5401-5404 (1998). Locked nucleic acids are commercially available from Proligo (Paris, France and Boulder, Colo., 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 FIG. 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 FIG. 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 alkylaryl groups. Some examples of alkyl amino acids include α-aminobutyric acid, β-aminobutyric acid, γ-aminobutyric acid, δ-aminovaleric acid, and ε-aminocaproic acid. Some examples of aryl amino acids include ortho-, meta, and para-aminobenzoic acid. Some examples of alkylaryl amino acids include ortho-, meta-, and para-aminophenylacetic acid, and γ-phenyl-β-aminobutyric acid.
Non-naturally occurring amino acids also include derivatives of naturally occurring amino 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, C1-C4 alkoxy, amino, methylamino, dimethylamino, nitro, halo (i.e., fluoro, chloro, bromo, or iodo), or branched or unbranched C1-C4 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 (Nle), 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, Calif., USA).
In another embodiment, the anti-microRNA 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 FIG. 1. Heasman, Dev. Biol. 243, 209-214 (2002). Morpholono oligonucleotides are commercially available from Gene Tools LLC (Corvallis, Oreg., 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 FIG. 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 FIG. 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.
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, Calif.















2′-Deoxycytidine-5-C6 Amino Linker (3′ Terminus)



2′-Deoxycytidine-5-C6 Amino Linker (5′ or Internal)



3′ 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



Thymidine-5-C2 Amino Linker (5′ or Internal)



Thymidine-5-C6 Amino Linker (3′ 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 41, 3457-3467 (2002), and Damha et al., J. Am. Chem. 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 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 Contiguous Bases
10
11
12
13
14
15
16





Max. No. of Wobble Base
3
3
3
3
4
4
4


Pairs





No. of Contiguous Bases
17
18
19
20
21
22
23





Max. No. of Wobble Base
5
5
5
6
6
6
6


Pairs









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 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%






















No. of Contiguous Bases
10
11
12
13
14
15
16





Max. No. of Additions,
1
1
1
1
1
1
1


Deletions and/or


Mismatches





No. of Contiguous Bases
17
18
19
20
21
22
23





Max. No. of Additions,
1
1
1
2
2
2
2


Deletions and/or


Mismatches



















Table for Up to 5%






















No. of Contiguous Bases
10
11
12
13
14
15
16





Max. No. of Additions,
0
0
0
0
0
0
0


Deletions and/or


Mismatches





No. of Contiguous Bases
17
18
19
20
21
22
23





Max. No. of Additions,
0
0
0
1
1
1
1


Deletions and/or


Mismatches









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 Deoxyribonucleotide Units






















No. of Contiguous Bases
10
11
12
13
14
15
16





Max. No. of
5
5
6
6
7
7
8


Deoxyribonucleotide


Backbone Units





No. of Contiguous Bases
17
18
19
20
21
22
23





Max. No. of
8
9
9
10
10
11
11


Deoxyribonucleotide


Backbone Units



















Table for Thirty Percent Deoxyribonucleotide Backbone Units






















No. of Contiguous Bases
10
11
12
13
14
15
16





Max. No. of
3
3
3
3
4
4
4


Deoxyribonucleotide


Backbone Units





No. of Contiguous Bases
17
18
19
20
21
22
23





Max. No. of
5
5
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 linker.
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.
MicroRNA molecules are derived from genomic 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. Inhibiting 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-microRNA molecules mentioned above (see Table 1-4), and the anti-microRNAs 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.
The cell can be any cell which expresses microRNA 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.
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 (5′ to 3′)
Anti-microRNA molecule sequence (5′ to 3′)














hsa-miR-100
SEQ ID NO. 1
AACCCGUAGAUCCGAACUUGUG
SEQ ID NO. 307
CACAAGUUCGGAUCUACGGGUU


 


hsa-miR-103
SEQ ID NO. 2
AGCAGCAUUGUACAGGGCUAUG
SEQ ID NO. 308
CAUAGCCCUGUACAAUGCUGCU


 


hsa-miR-105-5p
SEQ ID NO. 3
UCAAAUGCUCAGACUCCUGUGG
SEQ ID NO. 309
CCACAGGAGUCUGAGCAUUUGA


 


hsa-miR-106a
SEQ ID NO. 4
AAAAGUGCUUACAGUGCAGGUA
SEQ ID NO. 310
UACCUGCACUGUAAGCACUUUU


 


hsa-miR-106b
SEQ ID NO. 5
UAAAGUGCUGACAGUGCAGAUA
SEQ ID NO. 311
UAUCUGCACUGUCAGCACUUUA


 


hsa-miR-107
SEQ ID NO. 6
AGCAGCAUUGUACAGGGCUAUC
SEQ ID NO. 312
GAUAGCCCUGUACAAUGCUGCU


 


hsa-miR-10b
SEQ ID NO. 7
UACCCUGUAGAACCGAAUUUGU
SEQ ID NO. 313
ACAAAUUCGGUUCUACAGGGUA


 


hsa-miR-128b
SEQ ID NO. 8
UCACAGUGAACCGGUCUCUUUC
SEQ ID NO. 314
GAAAGAGACCGGUUCACUGUGA


 


hsa-miR-130b
SEQ ID NO. 9
CAGUGCAAUGAUGAAAGGGCAU
SEQ ID NO. 315
AUGCCCUUUCAUCAUUGCACUG


 


hsa-miR-140-3p
SEQ ID NO. 10
UACCACAGGGUAGAACCACGGA
SEQ ID NO. 316
UCCGUGGUUCUACCCUGUGGUA


 


hsa-miR-142-5p
SEQ ID NO. 11
CCCAUAAAGUAGAAAGCACUAC
SEQ ID NO. 317
GUAGUGCUUUCUACUUUAUGGG


 


hsa-miR-151-5p
SEQ ID NO. 12
UCGAGGAGCUCACAGUCUAGUA
SEQ ID NO. 318
UACUAGACUGUGAGCUCCUCGA


 


hsa-miR-155
SEQ ID NO. 13
UUAAUGCUAAUCGUGAUAGGGG
SEQ ID NO. 319
CCCCUAUCACGAUUAGCAUUAA


 


hsa-miR-181a
SEQ ID NO. 14
AACAUUCAACGCUGUCGGUGAG
SEQ ID NO. 320
CUCACCGACAGCGUUGAAUGUU


 


hsa-miR-181b
SEQ ID NO. 15
AACAUUCAUUGCUGUCGGUGGG
SEQ ID NO. 321
CCCACCGACAGCAAUGAAUGUU


 


hsa-miR-181c
SEQ ID NO. 16
AACAUUCAACCUGUCGGUGAGU
SEQ ID NO. 322
ACUCACCGACAGGUUGAAUGUU


 


hsa-miR-182
SEQ ID NO. 17
UUUGGCAAUGGUAGAACUCACA
SEQ ID NO. 323
UGUGAGUUCUACCAUUGCCAAA


 


hsa-miR-183
SEQ ID NO. 18
UAUGGCACUGGUAGAAUUCACU
SEQ ID NO. 324
AGUGAAUUCUACCAGUGCCAUA


 


hsa-miR-184
SEQ ID NO. 19
UGGACGGAGAACUGAUAAGGGU
SEQ ID NO. 325
ACCCUUAUCAGUUCUCCGUCCA


 


hsa-miR-185
SEQ ID NO. 20
UGGAGAGAAAGGCAGUUCCUGA
SEQ ID NO. 326
UCAGGAACUGCCUUUCUCUCCA


 


hsa-miR-186
SEQ ID NO. 21
CAAAGAAUUCUCCUUUUGGGCU
SEQ ID NO. 327
AGCCCAAAAGGAGAAUUCUUUG


 


hsa-miR-187
SEQ ID NO. 22
UCGUGUCUUGUGUUGCAGCCGG
SEQ ID NO. 328
CCGGCUGCAACACAAGACACGA


 


hsa-miR-188-3p
SEQ ID NO. 23
CUCCCACAUGCAGGGUUUGCAG
SEQ ID NO. 329
CUGCAAACCCUGCAUGUGGGAG


 


hsa-miR-188-5p
SEQ ID NO. 24
CAUCCCUUGCAUGGUGGAGGGU
SEQ ID NO. 330
ACCCUCCACCAUGCAAGGGAUG


 


hsa-miR-189
SEQ ID NO. 25
GUGCCUACUGAGCUGAUAUCAG
SEQ ID NO. 331
CUGAUAUCAGCUCAGUAGGCAC


 


hsa-miR-190
SEQ ID NO. 26
UGAUAUGUUUGAUAUAUUAGGU
SEQ ID NO. 332
ACCUAAUAUAUCAAACAUAUCA


 


hsa-miR-191
SEQ ID NO. 27
CAACGGAAUCCCAAAAGCAGCU
SEQ ID NO. 333
AGCUGCUUUUGGGAUUCCGUUG


 


hsa-miR-192
SEQ ID NO. 28
CUGACCUAUGAAUUGACAGCCA
SEQ ID NO. 334
UGGCUGUCAAUUCAUAGGUCAG


 


hsa-miR-193-3p
SEQ ID NO. 29
AACUGGCCUACAAAGUCCCAGU
SEQ ID NO. 335
ACUGGGACUUUGUAGGCCAGUU


 


hsa-miR-193-5p
SEQ ID NO. 30
UGGGUCUUUGCGGGCAAGAUGA
SEQ ID NO. 336
UCAUCUUGCCCGCAAAGACCCA


 


hsa-miR-194
SEQ ID NO. 31
UGUAACAGCAACUCCAUGUGGA
SEQ ID NO. 337
UCCACAUGGAGUUGCUGUUACA


 


hsa-miR-195
SEQ ID NO. 32
UAGCAGCACAGAAAUAUUGGCA
SEQ ID NO. 338
UGCCAAUAUUUCUGUGCUGCUA


 


hsa-miR-196
SEQ ID NO. 33
UAGGUAGUUUCAUGUUGUUGGG
SEQ ID NO. 339
CCCAACAACAUGAAACUACCUA


 


hsa-miR-197
SEQ ID NO. 34
UUCACCACCUUCUCCACCCAGC
SEQ ID NO. 340
GCUGGGUGGAGAAGGUGGUGAA


 


hsa-miR-198
SEQ ID NO. 35
GGUCCAGAGGGGAGAUAGGUUC
SEQ ID NO. 341
GAACCUAUCUCCCCUCUGGACC


 


hsa-miR-199a-3p
SEQ ID NO. 36
ACAGUAGUCUGCACAUUGGUUA
SEQ ID NO. 342
UAACCAAUGUGCAGACUACUGU


 


hsa-miR-199a-5p
SEQ ID NO. 37
CCCAGUGUUCAGACUACCUGUU
SEQ ID NO. 343
AACAGGUAGUCUGAACACUGGG


 


hsa-miR-199b
SEQ ID NO. 38
CCCAGUGUUUAGACUAUCUGUU
SEQ ID NO. 344
AACAGAUAGUCUAAACACUGGG


 


hsa-miR-200a
SEQ ID NO. 39
UAACACUGUCUGGUAACGAUGU
SEQ ID NO. 345
ACAUCGUUACCAGACAGUGUUA


 


hsa-miR-200b
SEQ ID NO. 40
CUCUAAUACUGCCUGGUAAUGA
SEQ ID NO. 346
UCAUUACCAGGCAGUAUUAGAG


 


hsa-miR-200c
SEQ ID NO. 41
AAUACUGCCGGGUAAUGAUGGA
SEQ ID NO. 347
UCCAUCAUUACCCGGCAGUAUU


 


hsa-miR-203
SEQ ID NO. 42
GUGAAAUGUUUAGGACCACUAG
SEQ ID NO. 348
CUAGUGGUCCUAAACAUUUCAC


 


hsa-miR-204
SEQ ID NO. 43
UUCCCUUUGUCAUCCUAUGCCU
SEQ ID NO. 349
AGGCAUAGGAUGACAAAGGGAA


 


hsa-miR-205
SEQ ID NO. 44
UCCUUCAUUCCACCGGAGUCUG
SEQ ID NO. 350
CAGACUCCGGUGGAAUGAAGGA


 


hsa-miR-206
SEQ ID NO. 45
UGGAAUGUAAGGAAGUGUGUGG
SEQ ID NO. 351
CCACACACUUCCUUACAUUCCA


 


hsa-miR-208
SEQ ID NO. 46
AUAAGACGAGCAAAAAGCUUGU
SEQ ID NO. 352
ACAAGCUUUUUGCUCGUCUUAU


 


hsa-miR-210
SEQ ID NO. 47
CUGUGCGUGUGACAGCGGCUGA
SEQ ID NO. 353
UCAGCCGCUGUCACACGCACAG


 


hsa-miR-211
SEQ ID NO. 48
UUCCCUUUGUCAUCCUUCGCCU
SEQ ID NO. 354
AGGCGAAGGAUGACAAAGGGAA


 


hsa-miR-212
SEQ ID NO. 49
UAACAGUCUCCAGUCACGGCCA
SEQ ID NO. 355
UGGCCGUGACUGGAGACUGUUA


 


hsa-miR-213
SEQ ID NO. 50
ACCAUCGACCGUUGAUUGUACC
SEQ ID NO. 356
GGUACAAUCAACGGUCGAUGGU


 


hsa-miR-214
SEQ ID NO. 51
ACAGCAGGCACAGACAGGCAGU
SEQ ID NO. 357
ACUGCCUGUCUGUGCCUGCUGU


 


hsa-miR-215
SEQ ID NO. 52
AUGACCUAUGAAUUGACAGACA
SEQ ID NO. 358
UGUCUGUCAAUUCAUAGGUCAU


 


hsa-miR-216
SEQ ID NO. 53
UAAUCUCAGCUGGCAACUGUGA
SEQ ID NO. 359
UCACAGUUGCCAGCUGAGAUUA


 


hsa-miR-217
SEQ ID NO. 54
UACUGCAUCAGGAACUGAUUGG
SEQ ID NO. 360
CCAAUCAGUUCCUGAUGCAGUA


 


hsa-miR-218
SEQ ID NO. 55
UUGUGCUUGAUCUAACCAUGUG
SEQ ID NO. 361
CACAUGGUUAGAUCAAGCACAA


 


hsa-miR-219
SEQ ID NO. 56
UGAUUGUCCAAACGCAAUUCUU
SEQ ID NO. 362
AAGAAUUGCGUUUGGACAAUCA


 


hsa-miR-220
SEQ ID NO. 57
CCACACCGUAUCUGACACUUUG
SEQ ID NO. 363
CAAAGUGUCAGAUACGGUGUGG


 


hsa-miR-221
SEQ ID NO. 58
AGCUACAUUGUCUGCUGGGUUU
SEQ ID NO. 364
AAACCCAGCAGACAAUGUAGCU


 


hsa-miR-222
SEQ ID NO. 59
AGCUACAUCUGGCUACUGGGUC
SEQ ID NO. 365
GACCCAGUAGCCAGAUGUAGCU


 


hsa-miR-223
SEQ ID NO. 60
UGUCAGUUUGUCAAAUACCCCA
SEQ ID NO. 366
UGGGGUAUUUGACAAACUGACA


 


hsa-miR-224
SEQ ID NO. 61
CAAGUCACUAGUGGUUCCGUUU
SEQ ID NO. 367
AAACGGAACCACUAGUGACUUG


 


hsa-miR-28-5p
SEQ ID NO. 62
AAGGAGCUCACAGUCUAUUGAG
SEQ ID NO. 368
CUCAAUAGACUGUGAGCUCCUU


 


hsa-miR-290
SEQ ID NO. 63
CUCAAACUGUGGGGGCACUUUC
SEQ ID NO. 369
GAAAGUGCCCCCACAGUUUGAG


 


hsa-miR-296
SEQ ID NO. 64
AGGGCCCCCCCUCAAUCCUGUU
SEQ ID NO. 370
AACAGGAUUGAGGGGGGGCCCU


 


hsa-miR-299
SEQ ID NO. 65
UGGUUUACCGUCCCACAUACAU
SEQ ID NO. 371
AUGUAUGUGGGACGGUAAACCA


 


hsa-miR-301
SEQ ID NO. 66
CAGUGCAAUAGUAUUGUCAAAG
SEQ ID NO. 372
CUUUGACAAUACUAUUGCACUG


 


hsa-miR-302
SEQ ID NO. 67
UAAGUGCUUCCAUGULUUGGUG
SEQ ID NO. 373
CACCAAAACAUGGAAGCACUUA


 


hsa-miR-30e
SEQ ID NO. 68
UGUAAACAUCCUUGACUGGAAG
SEQ ID NO. 374
CUUCCAGUCAAGGAUGUUUACA


 


hsa-miR-320
SEQ ID NO. 69
AAAAGCUGGGUUGAGAGGGCGA
SEQ ID NO. 375
UCGCCCUCUCAACCCAGCUUUU


 


hsa-miR-321
SEQ ID NO. 70
UAAGCCAGGGAUUGUGGGUUCG
SEQ ID NO. 376
CGAACCCACAAUCCCUGGCUUA


 


hsa-miR-322
SEQ ID NO. 71
AAACAUGAAUUGCUGCUGUAUC
SEQ ID NO. 377
GAUACAGCAGCAAUUCAUGUUU


 


hsa-miR-323
SEQ ID NO. 72
GCACAUUACACGGUCGACCUCU
SEQ ID NO. 378
AGAGGUCGACCGUGUAAUGUGC


 


hsa-miR-324-3p
SEQ ID NO. 73
CCACUGCCCCAGGUGCUGCUGG
SEQ ID NO. 379
CCAGCAGCACCUGGGGCAGUGG


 


hsa-miR-324-5p
SEQ ID NO. 74
CGCAUCCCCUAGGGCAUUGGUG
SEQ ID NO. 380
CACCAAUGCCCUAGGGGAUGCG


 


hsa-miR-326
SEQ ID NO. 75
CCUCUGGGCCCUUCCUCCAGCC
SEQ ID NO. 381
GGCUGGAGGAAGGGCCCAGAGG


 


hsa-miR-328
SEQ ID NO. 76
CUGGCCCUCUCUGCCCUUCCGU
SEQ ID NO. 382
ACGGAAGGGCAGAGAGGGCCAG


 


hsa-miR-329
SEQ ID NO. 77
AACACACCCAGCUAACCUUUUU
SEQ ID NO. 383
AAAAAGGUUAGCUGGGUGUGUU


 


hsa-miR-34a
SEQ ID NO. 78
UGGCAGUGUCUUAGCUGGUUGU
SEQ ID NO. 384
ACAACCAGCUAAGACACUGCCA


 


hsa-miR-34b
SEQ ID NO. 79
AGGCAGUGUCAUUAGCUGAUUG
SEQ ID NO. 385
CAAUCAGCUAAUGACACUGCCU


 


hsa-miR-34c
SEQ ID NO. 80
AGGCAGUGUAGUUAGCUGAUUG
SEQ ID NO. 386
CAAUCAGCUAACUACACUGCCU


 


hsa-miR-92
SEQ ID NO. 81
UAUUGCACUUGUCCCGGCCUGU
SEQ ID NO. 387
ACAGGCCGGGACAAGUGCAAUA


 


hsa-miR-93
SEQ ID NO. 82
AAAGUGCUGUUCGUGCAGGUAG
SEQ ID NO. 388
CUACCUGCACGAACAGCACUUU


 


hsa-miR-95
SEQ ID NO. 83
UUCAACGGGUAUUUAUUGAGCA
SEQ ID NO. 389
UGCUCAAUAAAUACCCGUUGAA


 


hsa-miR-96
SEQ ID NO. 84
UUUGGCACUAGCACAUUUUUGC
SEQ ID NO. 390
GCAAAAAUGUGCUAGUGCCAAA


 


hsa-miR-98
SEQ ID NO. 85
UGAGGUAGUAAGUUGUAUUGUU
SEQ ID NO. 391
AACAAUACAACUUACUACCUCA


 


mmu-miR-106a
SEQ ID NO. 86
CAAAGUGCUAACAGUGCAGGUA
SEQ ID NO. 392
UACCUGCACUGUUAGCACUUUG


 


mmu-miR-10b
SEQ ID NO. 87
CCCUGUAGAACCGAAUUUGUGU
SEQ ID NO. 393
ACACAAAUUCGGUUCUACAGGG


 


mmu-miR-135b
SEQ ID NO. 88
UAUGGCUUUUCAUUCCUAUGUG
SEQ ID NO. 394
CACAUAGGAAUGAAAAGCCAUA


 


mmu-miR-148b
SEQ ID NO. 89
UCAGUGCAUCACAGAACUUUGU
SEQ ID NO. 395
ACAAAGUUCUGUGAUGCACUGA


 


mmu-miR-151-3p
SEQ ID NO. 90
CUAGACUGAGGCUCCUUGAGGA
SEQ ID NO. 396
UCCUCAAGGAGCCUCAGUCUAG


 


mmu-miR-155
SEQ ID NO. 91
UUAAUGCUAAUUGUGAUAGGGG
SEQ ID NO. 397
CCCCUAUCACAAUUAGCAUUAA


 


mmu-miR-199b
SEQ ID NO. 92
CCCAGUGUUUAGACUACCUGUU
SEQ ID NO. 398
AACAGGUAGUCUAAACACUGGG


 


mmu-miR-200b
SEQ ID NO. 93
UAAUACUGCCUGGUAAUGAUGA
SEQ ID NO. 399
UCAUCAUUACCAGGCAGUAUUA


 


mmu-miR-203
SEQ ID NO. 94
UGAAAUGUUUAGGACCACUAGA
SEQ ID NO. 400
UCUAGUGGUCCUAAACAUUUCA


 


mmu-miR-211
SEQ ID NO. 95
UUCCCUUUGUCAUCCUUUGCCU
SEQ ID NO. 401
AGGCAAAGGAUGACAAAGGGAA


 


mmu-miR-217
SEQ ID NO. 96
UACUGCAUCAGGAACUGACUGG
SEQ ID NO. 402
CCAGUCAGUUCCUGAUGCAGUA


 


mmu-miR-224
SEQ ID NO. 97
UAAGUCACUAGUGGUUCCGUUU
SEQ ID NO. 403
AAACGGAACCACUAGUGACUUA


 


mmu-miR-28-3p
SEQ ID NO. 98
CACUAGAUUGUGAGCUGCUGGA
SEQ ID NO. 404
UCCAGCAGCUCACAAUCUAGUG


 


mmu-miR-290
SEQ ID NO. 99
CUCAAACUAUGGGGGCACUUUU
SEQ ID NO. 405
AAAAGUGCCCCCAUAGUUUGAG


 


mmu-miR-291-3p
SEQ ID NO. 100
AAAGUGCUUCCACUUUGUGUGC
SEQ ID NO. 406
GCACACAAAGUGGAAGCACUUU


 


mmu-miR-291-5p
SEQ ID NO. 101
CAUCAAAGUGGAGGCCCUCUCU
SEQ ID NO. 407
AGAGAGGGCCUCCACUUUGAUG


 


mmu-miR-292-3p
SEQ ID NO. 102
AAGUGCCGCCAGGUUUUGAGUG
SEQ ID NO. 408
CACUCAAAACCUGGCGGCACUU


 


mmu-miR-292-5p
SEQ ID NO. 103
ACUCAAACUGGGGGCUCUUUUG
SEQ ID NO. 409
CAAAAGAGCCCCCAGUUUGAGU


 


mmu-miR-293
SEQ ID NO. 104
AGUGCCGCAGAGUUUGUAGUGU
SEQ ID NO. 410
ACACUACAAACUCUGCGGCACU


 


mmu-miR-294
SEQ ID NO. 105
AAAGUGCUUCCCUUUUGUGUGU
SEQ ID NO. 411
ACACACAAAAGGGAAGCACUUU


 


mmu-miR-295
SEQ ID NO. 106
AAAGUGCUACUACUUUUGAGUC
SEQ ID NO. 412
GACUCAAAAGUAGUAGCACUUU


 


mmu-miR-297
SEQ ID NO. 107
AUGUAUGUGUGCAUGUGCAUGU
SEQ ID NO. 413
ACAUGCACAUGCACACAUACAU


 


mmu-miR-298
SEQ ID NO. 108
GGCAGAGGAGGGCUGUUCUUCC
SEQ ID NO. 414
GGAAGAACAGCCCUCCUCUGCC


 


mmu-miR-300
SEQ ID NO. 109
UAUGCAAGGGCAAGCUCUCUUC
SEQ ID NO. 415
GAAGAGAGCUUGCCCUUGCAUA


 


mmu-miR-31
SEQ ID NO. 110
AGGCAAGAUGCUGGCAUAGCUG
SEQ ID NO. 416
CAGCUAUGCCAGCAUCUUGCCU


 


mmu-miR-322
SEQ ID NO. 111
AAACAUGAAGCGCUGCAACACC
SEQ ID NO. 417
GGUGUUGCAGCGCUUCAUGUUU


 


mmu-miR-325
SEQ ID NO. 112
CCUAGUAGGUGCUCAGUAAGUG
SEQ ID NO. 418
CACUUACUGAGCACCUACUAGG


 


mmu-miR-326
SEQ ID NO. 113
CCUCUGGGCCCUUCCUCCAGUC
SEQ ID NO. 419
GACUGGAGGAAGGGCCCAGAGG


 


mmu-miR-330
SEQ ID NO. 114
GCAAAGCACAGGGCCUGCAGAG
SEQ ID NO. 420
CUCUGCAGGCCCUGUGCUUUGC


 


mmu-miR-331
SEQ ID NO. 115
GCCCCUGGGCCUAUCCUAGAAC
SEQ ID NO. 421
GUUCUAGGAUAGGCCCAGGGGC


 


mmu-miR-337
SEQ ID NO. 116
UUCAGCUCCUAUAUGAUGCCUU
SEQ ID NO. 422
AAGGCAUCAUAUAGGAGCUGAA


 


mmu-miR-338
SEQ ID NO. 117
UCCAGCAUCAGUGAUUUUGUUG
SEQ ID NO. 423
CAACAAAAUCACUGAUGCUGGA


 


mmu-miR-339
SEQ ID NO. 118
UCCCUGUCCUCCAGGAGCUCAC
SEQ ID NO. 424
GUGAGCUCCUGGAGGACAGGGA


 


mmu-miR-340
SEQ ID NO. 119
UCCGUCUCAGUUACUUUAUAGC
SEQ ID NO. 425
GCUAUAAAGUAACUGAGACGGA


 


mmu-miR-341
SEQ ID NO. 120
UCGAUCGGUCGGUCGGUCAGUC
SEQ ID NO. 426
GACUGACCGACCGACCGAUCGA


 


mmu-miR-342
SEQ ID NO. 121
UCUCACACAGAAAUCGCACCCG
SEQ ID NO. 427
CGGGUGCGAUUUCUGUGUGAGA


 


mmu-miR-344
SEQ ID NO. 122
UGAUCUAGCCAAAGCCUGACUG
SEQ ID NO. 428
CAGUCAGGCUUUGGCUAGAUCA


 


mmu-miR-345
SEQ ID NO. 123
UGCUGACCCCUAGUCCAGUGCU
SEQ ID NO. 429
AGCACUGGACUAGGGGUCAGCA


 


mmu-miR-346
SEQ ID NO. 124
UGUCUGCCCGAGUGCCUGCCUC
SEQ ID NO. 430
GAGGCAGGCACUCGGGCAGACA


 


mmu-miR-34b
SEQ ID NO. 125
UAGGCAGUGUAAUUAGCUGAUU
SEQ ID NO. 431
AAUCAGCUAAUUACACUGCCUA


 


mmu-miR-350
SEQ ID NO. 126
UUCACAAAGCCCAUACACUUUC
SEQ ID NO. 432
GAAAGUGUAUGGGCUUUGUGAA


 


mmu-miR-351
SEQ ID NO. 127
UCCCUGAGGAGCCCUUUGAGCC
SEQ ID NO. 433
GGCUCAAAGGGCUCCUCAGGGA


 


mmu-miR-7b
SEQ ID NO. 128
UGGAAGACUUGUGAUUUUGUUG
SEQ ID NO. 434
CAACAAAAUCACAAGUCUUCCA


 


mmu-miR-92
SEQ ID NO. 129
UAUUGCACUUGUCCCGGCCUGA
SEQ ID NO. 435
UCAGGCCGGGACAAGUGCAAUA


 


mmu-miR-93
SEQ ID NO. 130
CAAAGUGCUGUUCGUGCAGGUA
SEQ ID NO. 436
UACCUGCACGAACAGCACUUUG


 


rno-miR-327
SEQ ID NO. 131
CCUUGAGGGGCAUGAGGGUAGU
SEQ ID NO. 437
ACUACCCUCAUGCCCCUCAAGG


 


rno-miR-333
SEQ ID NO. 132
GUGGUGUGCUAGUUACUUUUGG
SEQ ID NO. 438
CCAAAAGUAACUAGCACACCAC


 


rno-miR-335
SEQ ID NO. 133
UCAAGAGCAAUAACGAAAAAUG
SEQ ID NO. 439
CAUUUUUCGUUAUUGCUCUUGA


 


rno-miR-336
SEQ ID NO. 134
UCACCCUUCCAUAUCUAGUCUC
SEQ ID NO. 440
GAGACUAGAUAUGGAAGGGUGA


 


rno-miR-343
SEQ ID NO. 135
UCUCCCUCCGUGUGCCCAGUAU
SEQ ID NO. 441
AUACUGGGCACACGGAGGGAGA


 


rno-miR-347
SEQ ID NO. 136
UGUCCCUCUGGGUCGCCCAGCU
SEQ ID NO. 442
AGCUGGGCGACCCAGAGGGACA


 


rno-miR-349
SEQ ID NO. 137
CAGCCCUGCUGUCUUAACCUCU
SEQ ID NO. 443
AGAGGUUAAGACAGCAGGGCUG


 


rno-miR-352
SEQ ID NO. 138
AGAGUAGUAGGUUGCAUAGUAC
SEQ ID NO. 444
GUACUAUGCAACCUACUACUCU
















TABLE 2







Novel Human microRNA and anti-microRNA sequences.









microRNA name
microRNA sequence (5′ to 3′)
Anti-microRNA molecule sequence (5′ to 3′)














hsa-miR-361
SEQ ID NO. 139
UUAUCAGAAUCUCCAGGGGUAC
SEQ ID NO. 445
GUACCCCUGGAGAUUCUGAUAA


 


hsa-miR-362
SEQ ID NO. 140
AAUCCUUGGAACCUAGGUGUGA
SEQ ID NO. 446
UCACACCUAGGUUCCAAGGAUU


 


hsa-miR-363
SEQ ID NO. 141
AUUGCACGGUAUCCAUCUGUAA
SEQ ID NO. 447
UUACAGAUGGAUACCGUGCAAU


 


hsa-miR-364
SEQ ID NO. 142
CGGCGGGGACGGCGAUUGGUCC
SEQ ID NO. 448
GGACCAAUCGCCGUCCCCGCCG


 


hsa-miR-365
SEQ ID NO. 143
UAAUGCCCCUAAAAAUCCUUAU
SEQ ID NO. 449
AUAAGGAUUUUUAGGGGCAUUA


 


hsa-miR-366
SEQ ID NO. 144
UAACUGGUUGAACAACUGAACC
SEQ ID NO. 450
GGUUCAGUUGUUCAACCAGUUA
















TABLE 3







C. elegans microRNA and anti-microRNA sequences.









microRNA name
microRNA sequence (5′ to 3′)
Anti-microRNA molecule sequence (5′ to 3′)














Cel-let-7
SEQ ID NO. 145
UGAGGUAGUAGGUUGUAUAGUU
SEQ ID NO. 451
AACUAUACAACCUACUACCUCA


 


Cel-lin-4
SEQ ID NO. 146
UCCCUGAGACCUCAAGUGUGAG
SEQ ID NO. 452
CUCACACUUGAGGUCUCAGGGA


 


Cel-miR-1
SEQ ID NO. 147
UGGAAUGUAAAGAAGUAUGUAG
SEQ ID NO. 453
CUACAUACUUCUUUACAUUCCA


 


Cel-miR-2
SEQ ID NO. 148
UAUCACAGCCAGCUUUGAUGUG
SEQ ID NO. 454
CACAUCAAAGCUGGCUGUGAUA


 


Cel-miR-34
SEQ ID NO. 149
AGGCAGUGUGGUUAGCUGGUUG
SEQ ID NO. 455
CAACCAGCUAACCACACUGCCU


 


Cel-miR-35
SEQ ID NO. 150
UCACCGGGUGGAAACUAGCAGU
SEQ ID NO. 456
ACUGCUAGUUUCCACCCGGUGA


 


Cel-miR-36
SEQ ID NO. 151
UCACCGGGUGAAAAUUCGCAUG
SEQ ID NO. 457
CAUGCGAAUUUUCACCCGGUGA


 


Cel-miR-37
SEQ ID NO. 152
UCACCGGGUGAACACUUGCAGU
SEQ ID NO. 458
ACUGCAAGUGUUCACCCGGUGA


 


Cel-miR-38
SEQ ID NO. 153
UCACCGGGAGAAAAACUGGAGU
SEQ ID NO. 459
ACUCCAGUUUUUCUCCCGGUGA


 


Cel-miR-39
SEQ ID NO. 154
UCACCGGGUGUAAAUCAGCUUG
SEQ ID NO. 460
CAAGCUGAUUUACACCCGGUGA


 


Cel-miR-40
SEQ ID NO. 155
UCACCGGGUGUACAUCAGCUAA
SEQ ID NO. 461
UUAGCUGAUGUACACCCGGUGA


 


Cel-miR-41
SEQ ID NO. 156
UCACCGGGUGAAAAAUCACCUA
SEQ ID NO. 462
UAGGUGAUUUUUCACCCGGUGA


 


Cel-miR-42
SEQ ID NO. 157
CACCGGGUUAACAUCUACAGAG
SEQ ID NO. 463
CUCUGUAGAUGUUAACCCGGUG


 


Cel-miR-43
SEQ ID NO. 158
UAUCACAGUUUACUUGCUGUCG
SEQ ID NO. 464
CGACAGCAAGUAAACUGUGAUA


 


Cel-miR-44
SEQ ID NO. 159
UGACUAGAGACACAUUCAGCUU
SEQ ID NO. 465
AAGCUGAAUGUGUCUCUAGUCA


 


Cel-miR-45
SEQ ID NO. 160
UGACUAGAGACACAUUCAGCUU
SEQ ID NO. 466
AAGCUGAAUGUGUCUCUAGUCA


 


Cel-miR-46
SEQ ID NO. 161
UGUCAUGGAGUCGCUCUCUUCA
SEQ ID NO. 467
UGAAGAGAGCGACUCCAUGACA


 


Cel-miR-47
SEQ ID NO. 162
UGUCAUGGAGGCGCUCUCUUCA
SEQ ID NO. 468
UGAAGAGAGCGCCUCCAUGACA


 


Cel-miR-48
SEQ ID NO. 163
UGAGGUAGGCUCAGUAGAUGCG
SEQ ID NO. 469
CGCAUCUACUGAGCCUACCUCA


 


Cel-miR-49
SEQ ID NO. 164
AAGCACCACGAGAAGCUGCAGA
SEQ ID NO. 470
UCUGCAGCUUCUCGUGGUGCUU


 


Cel-miR-50
SEQ ID NO. 165
UGAUAUGUCUGGUAUUCUUGGG
SEQ ID NO. 471
CCCAAGAAUACCAGACAUAUCA


 


Cel-miR-51
SEQ ID NO. 166
UACCCGUAGCUCCUAUCCAUGU
SEQ ID NO. 472
ACAUGGAUAGGAGCUACGGGUA


 


Cel-miR-52
SEQ ID NO. 167
CACCCGUACAUAUGUUUCCGUG
SEQ ID NO. 473
CACGGAAACAUAUGUACGGGUG


 


Cel-miR-53
SEQ ID NO. 168
CACCCGUACAUUUGUUUCCGUG
SEQ ID NO. 474
CACGGAAACAAAUGUACGGGUG


 


Cel-miR-54
SEQ ID NO. 169
UACCCGUAAUCUUCAUAAUCCG
SEQ ID NO. 475
CGGAUUAUGAAGAUUACGGGUA


 


Cel-miR-55
SEQ ID NO. 170
UACCCGUAUAAGUUUCUGCUGA
SEQ ID NO. 476
UCAGCAGAAACUUAUACGGGUA


 


Cel-miR-56
SEQ ID NO. 171
UACCCGUAAUGUUUCCGCUGAG
SEQ ID NO. 477
CUCAGCGGAAACAUUACGGGUA


 


Cel-miR-57
SEQ ID NO. 172
UACCCUGUAGAUCGAGCUGUGU
SEQ ID NO. 478
ACACAGCUCGAUCUACAGGGUA


 


Cel-miR-58
SEQ ID NO. 173
UGAGAUCGUUCAGUACGGCAAU
SEQ ID NO. 479
AUUGCCGUACUGAACGAUCUCA


 


Cel-miR-59
SEQ ID NO. 174
UCGAAUCGUUUAUCAGGAUGAU
SEQ ID NO. 480
AUCAUCCUGAUAAACGAUUCGA


 


Cel-miR-60
SEQ ID NO. 175
UAUUAUGCACAUUUUCUAGUUC
SEQ ID NO. 481
GAACUAGAAAAUGUGCAUAAUA


 


Cel-miR-61
SEQ ID NO. 176
UGACUAGAACCGUUACUCAUCU
SEQ ID NO. 482
AGAUGAGUAACGGUUCUAGUCA


 


Cel-miR-62
SEQ ID NO. 177
UGAUAUGUAAUCUAGCUUACAG
SEQ ID NO. 483
CUGUAAGCUAGAUUACAUAUCA


 


Cel-miR-63
SEQ ID NO. 178
AUGACACUGAAGCGAGUUGGAA
SEQ ID NO. 484
UUCCAACUCGCUUCAGUGUCAU


 


Cel-miR-64
SEQ ID NO. 179
UAUGACACUGAAGCGUUACCGA
SEQ ID NO. 485
UCGGUAACGCUUCAGUGUCAUA


 


Cel-miR-65
SEQ ID NO. 180
UAUGACACUGAAGCGUAACCGA
SEQ ID NO. 486
UCGGUUACGCUUCAGUGUCAUA


 


Cel-miR-66
SEQ ID NO. 181
CAUGACACUGAUUAGGGAUGUG
SEQ ID NO. 487
CACAUCCCUAAUCAGUGUCAUG


 


Cel-miR-67
SEQ ID NO. 182
UCACAACCUCCUAGAAAGAGUA
SEQ ID NO. 488
UACUCUUUCUAGGAGGUUGUGA


 


Cel-miR-68
SEQ ID NO. 183
UCGAAGACUCAAAAGUGUAGAC
SEQ ID NO. 489
GUCUACACUUUUGAGUCUUCGA


 


Cel-miR-69
SEQ ID NO. 184
UCGAAAAUUAAAAAGUGUAGAA
SEQ ID NO. 490
UUCUACACUUUUUAAUUUUCGA


 


Cel-miR-70
SEQ ID NO. 185
UAAUACGUCGUUGGUGUUUCCA
SEQ ID NO. 491
UGGAAACACCAACGACGUAUUA


 


Cel-miR-71
SEQ ID NO. 186
UGAAAGACAUGGGUAGUGAACG
SEQ ID NO. 492
CGUUCACUACCCAUGUCUUUCA


 


Cel-miR-72
SEQ ID NO. 187
AGGCAAGAUGUUGGCAUAGCUG
SEQ ID NO. 493
CAGCUAUGCCAACAUCUUGCCU


 


Cel-miR-73
SEQ ID NO. 188
UGGCAAGAUGUAGGCAGUUCAG
SEQ ID NO. 494
CUGAACUGCCUACAUCUUGCCA


 


Cel-miR-74
SEQ ID NO. 189
UGGCAAGAAAUGGCAGUCUACA
SEQ ID NO. 495
UGUAGACUGCCAUUUCUUGCCA


 


Cel-miR-75
SEQ ID NO. 190
UUAAAGCUACCAACCGGCUUCA
SEQ ID NO. 496
UGAAGCCGGUUGGUAGCUUUAA


 


Cel-miR-76
SEQ ID NO. 191
UUCGUUGUUGAUGAAGCCUUGA
SEQ ID NO. 497
UCAAGGCUUCAUCAACAACGAA


 


Cel-miR-77
SEQ ID NO. 192
UUCAUCAGGCCAUAGCUGUCCA
SEQ ID NO. 498
UGGACAGCUAUGGCCUGAUGAA


 


Cel-miR-78
SEQ ID NO. 193
UGGAGGCCUGGUUGUUUGUGCU
SEQ ID NO. 499
AGCACAAACAACCAGGCCUCCA


 


Cel-miR-79
SEQ ID NO. 194
AUAAAGCUAGGUUACCAAAGCU
SEQ ID NO. 500
AGCUUUGGUAACCUAGCUUUAU


 


Cel-miR-227 
SEQ ID NO. 195
AGCUUUCGACAUGAUUCUGAAC
SEQ ID NO. 501
GUUCAGAAUCAUGUCGAAAGCU


 


Cel-miR-80
SEQ ID NO. 196
UGAGAUCAUUAGUUGAAAGCCG
SEQ ID NO. 502
CGGCUUUCAACUAAUGAUCUCA


 


Cel-miR-81
SEQ ID NO. 197
UGAGAUCAUCGUGAAAGCUAGU
SEQ ID NO. 503
ACUAGCUUUCACGAUGAUCUCA


 


Cel-miR-82
SEQ ID NO. 198
UGAGAUCAUCGUGAAAGCCAGU
SEQ ID NO. 504
ACUGGCUUUCACGAUGAUCUCA


 


Cel-miR-83
SEQ ID NO. 199
UAGCACCAUAUAAAUUCAGUAA
SEQ ID NO. 505
UUACUGAAUUUAUAUGGUGCUA


 


Cel-miR-84
SEQ ID NO. 200
UGAGGUAGUAUGUAAUAUUGUA
SEQ ID NO. 506
UACAAUAUUACAUACUACCUCA


 


Cel-miR-85
SEQ ID NO. 201
UACAAAGUAUUUGAAAAGUCGU
SEQ ID NO. 507
ACGACUUUUCAAAUACUUUGUA


 


Cel-miR-86
SEQ ID NO. 202
UAAGUGAAUGCUUUGCCACAGU
SEQ ID NO. 508
ACUGUGGCAAAGCAUUCACUUA


 


Cel-miR-87
SEQ ID NO. 203
GUGAGCAAAGUUUCAGGUGUGC
SEQ ID NO. 509
GCACACCUGAAACUUUGCUCAC


 


Cel-miR-90
SEQ ID NO. 204
UGAUAUGUUGUUUGAAUGCCCC
SEQ ID NO. 510
GGGGCAUUCAAACAACAUAUCA


 


Cel-miR-124
SEQ ID NO. 205
UAAGGCACGCGGUGAAUGCCAC
SEQ ID NO. 511
GUGGCAUUCACCGCGUGCCUUA


 


Cel-miR-228
SEQ ID NO. 206
AAUGGCACUGCAUGAAUUCACG
SEQ ID NO. 512
CGUGAAUUCAUGCAGUGCCAUU


 


CeI-miR-229
SEQ ID NO. 207
AAUGACACUGGUUAUCUUUUCC
SEQ ID NO. 513
GGAAAAGAUAACCAGUGUCAUU


 


Cel-miR-230
SEQ ID NO. 208
GUAUUAGUUGUGCGACCAGGAG
SEQ ID NO. 514
CUCCUGGUCGCACAACUAAUAC


 


Cel-miR-231
SEQ ID NO. 209
UAAGCUCGUGAUCAACAGGCAG
SEQ ID NO. 515
CUGCCUGUUGAUCACGAGCUUA


 


Cel-miR-232
SEQ ID NO. 210
UAAAUGCAUCUUAACUGCGGUG
SEQ ID NO. 516
CACCGCAGUUAAGAUGCAUUUA


 


CeI-miR-233
SEQ ID NO. 211
UUGAGCAAUGCGCAUGUGCGGG
SEQ ID NO. 517
CCCGCACAUGCGCAUUGCUCAA


 


Cel-miR-234
SEQ ID NO. 212
UUAUUGCUCGAGAAUACCCUUU
SEQ ID NO. 518
AAAGGGUAUUCUCGAGCAAUAA


 


Cel-miR-235
SEQ ID NO. 213
UAUUGCACUCUCCCCGGCCUGA
SEQ ID NO. 519
UCAGGCCGGGGAGAGUGCAAUA


 


Cel-miR-236
SEQ ID NO. 214
UAAUACUGUCAGGUAAUGACGC
SEQ ID NO. 520
GCGUCAUUACCUGACAGUAUUA


 


Cel-miR-237
SEQ ID NO. 215
UCCCUGAGAAUUCUCGAACAGC
SEQ ID NO. 521
GCUGUUCGAGAAUUCUCAGGGA


 


Cel-miR-238
SEQ ID NO. 216
UUUGUACUCCGAUGCCAUUCAG
SEQ ID NO. 522
CUGAAUGGCAUCGGAGUACAAA


 


Cel-miR-239a
SEQ ID NO. 217
UUUGUACUACACAUAGGUACUG
SEQ ID NO. 523
CAGUACCUAUGUGUAGUACAAA


 


Cel-miR-239b
SEQ ID NO. 218
UUUGUACUACACAAAAGUACUG
SEQ ID NO. 524
CAGUACUUUUGUGUAGUACAAA


 


Cel-miR-240
SEQ ID NO. 219
UACUGGCCCCCAAAUCUUCGCU
SEQ ID NO. 525
AGCGAAGAUUUGGGGGCCAGUA


 


Cel-miR-241
SEQ ID NO. 220
UGAGGUAGGUGCGAGAAAUGAC
SEQ ID NO. 526
GUCAUUUCUCGCACCUACCUCA


 


Cel-miR-242
SEQ ID NO. 221
UUGCGUAGGCCUUUGCUUCGAG
SEQ ID NO. 527
CUCGAAGCAAAGGCCUACGCAA


 


Cel-miR-243
SEQ ID NO. 222
CGGUACGAUCGCGGCGGGAUAU
SEQ ID NO. 528
AUAUCCCGCCGCGAUCGUACCG


 


Cel-miR-244
SEQ ID NO. 223
UCUUUGGUUGUACAAAGUGGUA
SEQ ID NO. 529
UACCACUUUGUACAACCAAAGA


 


Cel-miR-245
SEQ ID NO. 224
AUUGGUCCCCUCCAAGUAGCUC
SEQ ID NO. 530
GAGCUACUUGGAGGGGACCAAU


 


Cel-miR-246
SEQ ID NO. 225
UUACAUGUUUCGGGUAGGAGCU
SEQ ID NO. 531
AGCUCCUACCCGAAACAUGUAA


 


Cel-miR-247
SEQ ID NO. 226
UGACUAGAGCCUAUUCUCUUCU
SEQ ID NO. 532
AGAAGAGAAUAGGCUCUAGUCA


 


Cel-miR-248
SEQ ID NO. 227
UACACGUGCACGGAUAACGCUC
SEQ ID NO. 533
GAGCGUUAUCCGUGCACGUGUA


 


Cel-miR-249
SEQ ID NO. 228
UCACAGGACUUUUGAGCGUUGC
SEQ ID NO. 534
GCAACGCUCAAAAGUCCUGUGA


 


Cel-miR-250
SEQ ID NO. 229
UCACAGUCAACUGUUGGCAUGG
SEQ ID NO. 535
CCAUGCCAACAGUUGACUGUGA


 


Cel-miR-251
SEQ ID NO. 230
UUAAGUAGUGGUGCCGCUCUUA
SEQ ID NO. 536
UAAGAGCGGCACCACUACUUAA


 


Cel-miR-252
SEQ ID NO. 231
UAAGUAGUAGUGCCGCAGGUAA
SEQ ID NO. 537
UUACCUGCGGCACUACUACUUA


 


Cel-miR-253
SEQ ID NO. 232
CACACCUCACUAACACUGACCA
SEQ ID NO. 538
UGGUCAGUGUUAGUGAGGUGUG


 


Cel-miR-254
SEQ ID NO. 233
UGCAAAUCUUUCGCGACUGUAG
SEQ ID NO. 539
CUACAGUCGCGAAAGAUUUGCA


 


Cel-miR-256
SEQ ID NO. 234
UGGAAUGCAUAGAAGACUGUAC
SEQ ID NO. 540
GUACAGUCUUCUAUGCAUUCCA


 


Cel-miR-257
SEQ ID NO. 235
GAGUAUCAGGAGUACCCAGUGA
SEQ ID NO. 541
UCACUGGGUACUCCUGAUACUC


 


Cel-miR-258
SEQ ID NO. 236
GGUUUUGAGAGGAAUCCUUUUA
SEQ ID NO. 542
UAAAAGGAUUCCUCUCAAAACC


 


Cel-miR-259
SEQ ID NO. 237
AGUAAAUCUCAUCCUAAUCUGG
SEQ ID NO. 543
CCAGAUUAGGAUGAGAUUUACU


 


Cel-miR-260
SEQ ID NO. 238
GUGAUGUCGAACUCUUGUAGGA
SEQ ID NO. 544
UCCUACAAGAGUUCGACAUCAC


 


Cel-miR-261
SEQ ID NO. 239
UAGCUUUUUAGUUUUCACGGUG
SEQ ID NO. 545
CACCGUGAAAACUAAAAAGCUA


 


Cel-miR-262
SEQ ID NO. 240
GUUUCUCGAUGUUUUCUGAUAC
SEQ ID NO. 546
GUAUCAGAAAACAUCGAGAAAC


 


Cel-miR-264
SEQ ID NO. 241
GGCGGGUGGUUGUUGUUAUGGG
SEQ ID NO. 547
CCCAUAACAACAACCACCCGCC


 


Cel-miR-265
SEQ ID NO. 242
UGAGGGAGGAAGGGUGGUAUUU
SEQ ID NO. 548
AAAUACCACCCUUCCUCCCUCA


 


Cel-miR-266
SEQ ID NO. 243
AGGCAAGACUUUGGCAAAGCUU
SEQ ID NO. 549
AAGCUUUGCCAAAGUCUUGCCU


 


Cel-miR-267
SEQ ID NO. 244
CCCGUGAAGUGUCUGCUGCAAU
SEQ ID NO. 550
AUUGCAGCAGACACUUCACGGG


 


Cel-miR-268
SEQ ID NO. 245
GGCAAGAAUUAGAAGCAGUUUG
SEQ ID NO. 551
CAAACUGCUUCUAAUUCUUGCC


 


Cel-miR-269
SEQ ID NO. 246
GGCAAGACUCUGGCAAAACUUG
SEQ ID NO. 552
CAAGUUUUGCCAGAGUCUUGCC


 


Cel-miR-270
SEQ ID NO. 247
GGCAUGAUGUAGCAGUGGAGAU
SEQ ID NO. 553
AUCUCCACUGCUACAUCAUGCC


 


Cel-miR-271
SEQ ID NO. 248
UCGCCGGGUGGGAAAGCAUUCG
SEQ ID NO. 554
CGAAUGCUUUCCCACCCGGCGA


 


Cel-miR-272
SEQ ID NO. 249
UGUAGGCAUGGGUGUUUGGAAG
SEQ ID NO. 555
CUUCCAAACACCCAUGCCUACA


 


Cel-miR-273
SEQ ID NO. 250
UGCCCGUACUGUGUCGGCUGCU
SEQ ID NO. 556
AGCAGCCGACACAGUACGGGCA
















TABLE 4







Drosophila microRNA and anti-microRNA sequences.









microRNA name
microRNA sequence (5′ to 3′)
Anti-microRNA molecule sequence (5′ to 3′)














Dme-miR-263a
SEQ ID NO. 251
GUUAAUGGCACUGGAAGAAUUC
SEQ ID NO. 557
GAAUUCUUCCAGUGCCAUUAAC


 


Dme-miR-184
SEQ ID NO. 252
UGGACGGAGAACUGAUAAGGGC
SEQ ID NO. 558
GCCCUUAUCAGUUCUCCGUCCA


 


Dme-miR-274
SEQ ID NO. 253
UUUUGUGACCGACACUAACGGG
SEQ ID NO. 559
CCCGUUAGUGUCGGUCACAAAA


 


Dme-miR-275
SEQ ID NO. 254
UCAGGUACCUGAAGUAGCGCGC
SEQ ID NO. 560
GCGCGCUACUUCAGGUACCUGA


 


Dme-miR-92a
SEQ ID NO. 255
CAUUGCACUUGUCCCGGCCUAU
SEQ ID NO. 561
AUAGGCCGGGACAAGUGCAAUG


 


Dme-miR-219
SEQ ID NO. 256
UGAUUGUCCAAACGCAAUUCUU
SEQ ID NO. 562
AAGAAUUGCGUUUGGACAAUCA


 


Dme-miR-276a
SEQ ID NO. 257
UAGGAACUUCAUACCGUGCUCU
SEQ ID NO. 563
AGAGCACGGUAUGAAGUUCCUA


 


Dme-miR-277
SEQ ID NO. 258
UAAAUGCACUAUCUGGUACGAC
SEQ ID NO. 564
GUCGUACCAGAUAGUGCAUUUA


 


Dme-miR-278
SEQ ID NO. 259
UCGGUGGGACUUUCGUCCGUUU
SEQ ID NO. 565
AAACGGACGAAAGUCCCACCGA


 


Dme-miR-133
SEQ ID NO. 260
UUGGUCCCCUUCAACCAGCUGU
SEQ ID NO. 566
ACAGCUGGUUGAAGGGGACCAA


 


Dme-miR-279
SEQ ID NO. 261
UGACUAGAUCCACACUCAUUAA
SEQ ID NO. 567
UUAAUGAGUGUGGAUCUAGUCA


 


Dme-miR-33
SEQ ID NO. 262
AGGUGCAUUGUAGUCGCAUUGU
SEQ ID NO. 568
ACAAUGCGACUACAAUGCACCU


 


Dme-miR-280
SEQ ID NO. 263
UGUAUUUACGUUGCAUAUGAAA
SEQ ID NO. 569
UUUCAUAUGCAACGUAAAUACA


 


Dme-miR-281
SEQ ID NO. 264
UGUCAUGGAAUUGCUCUCUUUG
SEQ ID NO. 570
CAAAGAGAGCAAUUCCAUGACA


 


Dme-miR-282
SEQ ID NO. 265
AAUCUAGCCUCUACUAGGCUUU
SEQ ID NO. 571
AAAGCCUAGUAGAGGCUAGAUU


 


Dme-miR-283
SEQ ID NO. 266
UAAAUAUCAGCUGGUAAUUCUG
SEQ ID NO. 572
CAGAAUUACCAGCUGAUAUUUA


 


Dme-miR-284
SEQ ID NO. 267
UGAAGUCAGCAACUUGAUUCCA
SEQ ID NO. 573
UGGAAUCAAGUUGCUGACUUCA


 


Dme-miR-34
SEQ ID NO. 268
UGGCAGUGUGGUUAGCUGGUUG
SEQ ID NO. 574
CAACCAGCUAACCACACUGCCA


 


Dme-miR-124
SEQ ID NO. 269
UAAGGCACGCGGUGAAUGCCAA
SEQ ID NO. 575
UUGGCAUUCACCGCGUGCCUUA


 


Dme-miR-79
SEQ ID NO. 270
UAAAGCUAGAUUACCAAAGCAU
SEQ ID NO. 576
AUGCUUUGGUAAUCUAGCUUUA


 


Dme-miR-276b
SEQ ID NO. 271
UAGGAACUUAAUACCGUGCUCU
SEQ ID NO. 577
AGAGCACGGUAUUAAGUUCCUA


 


Dme-miR-210
SEQ ID NO. 272
UUGUGCGUGUGACAGCGGCUAU
SEQ ID NO. 578
AUAGCCGCUGUCACACGCACAA


 


Dme-miR-285
SEQ ID NO. 273
UAGCACCAUUCGAAAUCAGUGC
SEQ ID NO. 579
GCACUGAUUUCGAAUGGUGCUA


 


Dme-miR-100
SEQ ID NO. 274
AACCCGUAAAUCCGAACUUGUG
SEQ ID NO. 580
CACAAGUUCGGAUUUACGGGUU


 


Dme-miR-92b
SEQ ID NO. 275
AAUUGCACUAGUCCCGGCCUGC
SEQ ID NO. 581
GCAGGCCGGGACUAGUGCAAUU


 


Dme-miR-286
SEQ ID NO. 276
UGACUAGACCGAACACUCGUGC
SEQ ID NO. 582
GCACGAGUGUUCGGUCUAGUCA


 


Dme-miR-287
SEQ ID NO. 277
UGUGUUGAAAAUCGUUUGCACG
SEQ ID NO. 583
CGUGCAAACGAUUUUCAACACA


 


Dme-miR-87
SEQ ID NO. 278
UUGAGCAAAAUUUCAGGUGUGU
SEQ ID NO. 584
ACACACCUGAAAUUUUGCUCAA


 


Dme-miR-263b
SEQ ID NO. 279
CUUGGCACUGGGAGAAUUCACA
SEQ ID NO. 585
UGUGAAUUCUCCCAGUGCCAAG


 


Dme-miR-288
SEQ ID NO. 280
UUUCAUGUCGAUUUCAUUUCAU
SEQ ID NO. 586
AUGAAAUGAAAUCGACAUGAAA


 


Dme-miR-289
SEQ ID NO. 281
UAAAUAUUUAAGUGGAGCCUGC
SEQ ID NO. 587
GCAGGCUCCACUUAAAUAUUUA


 


Dme-bantam
SEQ ID NO. 282
UGAGAUCAUUUUGAAAGCUGAU
SEQ ID NO. 588
AUCAGCUUUCAAAAUGAUCUCA


 


Dme-miR-303
SEQ ID NO. 283
UUUAGGUUUCACAGGAAACUGG
SEQ ID NO. 589
CCAGUUUCCUGUGAAACCUAAA


 


Dme-miR-31b
SEQ ID NO. 284
UGGCAAGAUGUCGGAAUAGCUG
SEQ ID NO. 590
CAGCUAUUCCGACAUCUUGCCA


 


Dme-miR-304
SEQ ID NO. 285
UAAUCUCAAUUUGUAAAUGUGA
SEQ ID NO. 591
UCACAUUUACAAAUUGAGAUUA


 


Dme-miR-305
SEQ ID NO. 286
AUUGUACUUCAUCAGGUGCUCU
SEQ ID NO. 592
AGAGCACCUGAUGAAGUACAAU


 


Dme-miR-9c
SEQ ID NO. 287
UCUUUGGUAUUCUAGCUGUAGA
SEQ ID NO. 593
UCUACAGCUAGAAUACCAAAGA


 


Dme-miR-306
SEQ ID NO. 288
UCAGGUACUUAGUGACUCUCAA
SEQ ID NO. 594
UUGAGAGUCACUAAGUACCUGA


 


Dme-miR-9b
SEQ ID NO. 289
UCUUUGGUGAUUUUAGCUGUAU
SEQ ID NO. 595
AUACAGCUAAAAUCACCAAAGA


 


Dme-miR-125
SEQ ID NO. 290
UCCCUGAGACCCUAACUUGUGA
SEQ ID NO. 596
UCACAAGUUAGGGUCUCAGGGA


 


Dme-miR-307
SEQ ID NO. 291
UCACAACCUCCUUGAGUGAGCG
SEQ ID NO. 597
CGCUCACUCAAGGAGGUUGUGA


 


Dme-miR-308
SEQ ID NO. 292
AAUCACAGGAUUAUACUGUGAG
SEQ ID NO. 598
CUCACAGUAUAAUCCUGUGAUU


 


dme-miR-31a
SEQ ID NO. 293
UGGCAAGAUGUCGGCAUAGCUG
SEQ ID NO. 599
CAGCUAUGCCGACAUCUUGCCA


 


dme-miR-309
SEQ ID NO. 294
GCACUGGGUAAAGUUUGUCCUA
SEQ ID NO. 600
UAGGACAAACUUUACCCAGUGC


 


dme-miR-310
SEQ ID NO. 295
UAUUGCACACUUCCCGGCCUUU
SEQ ID NO. 601
AAAGGCCGGGAAGUGUGCAAUA


 


dme-miR-311
SEQ ID NO. 296
UAUUGCACAUUCACCGGCCUGA
SEQ ID NO. 602
UCAGGCCGGUGAAUGUGCAAUA


 


dme-miR-312
SEQ ID NO. 297
UAUUGCACUUGAGACGGCCUGA
SEQ ID NO. 603
UCAGGCCGUCUCAAGUGCAAUA


 


dme-miR-313
SEQ ID NO. 298
UAUUGCACUUUUCACAGCCCGA
SEQ ID NO. 604
UCGGGCUGUGAAAAGUGCAAUA


 


dme-miR-314
SEQ ID NO. 299
UAUUCGAGCCAAUAAGUUCGG
SEQ ID NO. 605
CCGAACUUAUUGGCUCGAAUA


 


dme-miR-315
SEQ ID NO. 300
UUUUGAUUGUUGCUCAGAAAGC
SEQ ID NO. 606
GCUUUCUGAGCAACAAUCAAAA


 


dme-miR-316
SEQ ID NO. 301
UGUCUUUUUCCGCUUACUGGCG
SEQ ID NO. 607
CGCCAGUAAGCGGAAAAAGACA


 


dme-miR-317
SEQ ID NO. 302
UGAACACAGCUGGUGGUAUCCA
SEQ ID NO. 608
UGGAUACCACCAGCUGUGUUCA


 


dme-miR-318
SEQ ID NO. 303
UCACUGGGCUUUGUUUAUCUCA
SEQ ID NO. 609
UGAGAUAAACAAAGCCCAGUGA


 


dme-miR-2c
SEQ ID NO. 304
UAUCACAGCCAGCUUUGAUGGG
SEQ ID NO. 610
CCCAUCAAAGCUGGCUGUGAUA


 


Dme-miR-iab45p
SEQ ID NO. 305
ACGUAUACUGAAUGUAUCCUGA
SEQ ID NO. 611
UCAGGAUACAUUCAGUAUACGU


 


Dme-miR-iab43p
SEQ ID NO. 306
CGGUAUACCUUCAGUAUACGUA
SEQ ID NO. 612
UACGUAUACUGAAGGUAUACCG









EXAMPLES
Example 1
Materials and Methods
Oligonucleotide Synthesis
MiR-21 were synthesized using 5′-silyl, 2′-ACE phosphoramidites (Dharmacon, Lafayette, Colo., USA) on 0.2 μmol synthesis columns using a modified ABI 394 synthesizer (Foster City, Calif., 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 trihydrate 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 2′-deprotection buffer (400 μl of 100 mM acetate-TEMED, pH 3.8, for a 0.2 μmol 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 μl 5 M NaCl and 1.2 ml of absolute ethanol.
2′-O-Methyl oligoribonucleotides were synthesized using 5′-DMT, 2′-O-methyl phosphoramidites (Proligo, Hamburg, Germany) on 1 μmol synthesis columns loaded with 3′-aminomodifier (TFA) C7 Icaa control pore glass support (Chemgenes, Mass., USA). The aminolinker was added in order to also use the oligonucleotides for conjugation to amino group reactive reagents, such as biotin succinimidyl esters. The synthesis products were deprotected for 16 h at 55° C. in 30% aqueous ammonia and then precipitated by the addition of 12 ml absolute 1-butanol. The full-length product was then gel-purified using a denaturing 20% polyacrylamide gel. 2′-Deoxyoligonucleotides were prepared using 0.2 μmol scale synthesis and standard DNA synthesis reagents (Proligo, Hamburg, Germany).
The sequences of the 2′-O-methyl oligoribonucleotides were 5′-GUCAACAUCAGUCUGAUAAGCUAL (L, 3′ aminolinker) for 2′-OMe miR-21 (SEQ ID NO. 613), and 5′-AAGGCAAGCUGACCCUGAAGUL for EGFP 2′-OMe antisense (SEQ ID NO. 614), 5′-UGAAGUCCCAGUCGAACGGAAL for EGFP 2′-OMe reverse (SEQ ID NO. 615); the sequence of chimeric 2′-OMe/DNA oligonucleotides was 5′-GTCAACATCAGTCTGATAAGCTAGCGL for 2′-deoxy miR-21 (underlined, 2′-OMe residues) (SEQ ID NO. 616), and 5′-AAGGCAAGCTGACCCTGAAGTGCGL for EGFP 2′-deoxy antisense (SEQ ID NO. 617).
The miR-21 cleavage substrate was prepared by PCR-based extension of the partially complementary synthetic DNA oligonucleotides 5′-GAACAATTGCTTTTACAGATGCACATATCGAGGTGAACATCACGTACGTCAACATCA GTCTGATAAGCTATCGGTTGGCAGAAGCTAT (SEQ ID NO. 618) and 5′-GGCATAAAGAATTGAAGAGAGTTTTCACTGCATACGACGATTCTGTGATTTGTATTC AGCCCATATCGTTTCATAGCTTCTGCCAACCGA (SEQ ID NO. 619). The extended dsDNA was then used as template for a new PCR with primers 5′-TAATACGACTCACTATAGAACAATTGCTTTTACAG (SEQ ID NO. 620) and 5′-ATTTAGGTGACACTATAGGCATAAAGAATTGAAGA (SEQ ID NO. 621) 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)). 32P-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 (SEQ ID NO. 622) and 5′-GGCCAGGTACCTAGCTTATCAGACTGATGTTGA (SEQ ID NO. 623) 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)). 5×109 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 10 mM KCl/1.5 mM MgCl2/0.5 mM dithiothreitol/10 mM 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 S100 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 μg of total RNA was isolated from 1 ml of S100 extract using the acidic guanidinium thiocyanate-phenol-chloroform extraction method (Chomczynski 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 μg of total RNA per well (Lagos-Quintana et al., Science 294, 853-858 (2001)).
In Vitro miRNA 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, 1 mM ATP, 0.2 mM GTP, 10 U/ml RNasin (Promega) and 50% HeLa S100 extract in a final reaction volume of 25 μl. The reaction time was 1.5 h at 30° C. The reaction was stopped by addition of 200 μl of 300 mM NaCl/25 mM EDTA/20% w/v SDS/200 mM Tris HCl (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.
Cell Culture and Transfection
HeLa S3 and HeLa S3/GFP were grown in 5% CO2 at 37° C. in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 unit/ml penicillin, and 100 μg/ml streptomycin. One day before transfection, 105 cells were plated in 500 μl DMEM containing 10% FBS per well of a 24-well plate. Plasmid and plasmid/oligonucleotide transfection was carried out with Lipofectamine2000 (Invitrogen). 0.2 μg pEGFP or its derivatives were cotransfected with 0.3 μg pHcRed 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 10×/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 (Hutvágner 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 miRNAs 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 (FIG. 2, lane 1, and data not shown). We therefore decided to interfere with miR-21 guided target RNA cleavage.
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 3′ C7 aminolinker and was complementary to the longest form of the miR-21 was synthesized. The aminolinker was introduced in order to enable post-synthetic conjugation of non-nucleotidic residues such as biotin.
Increasing concentrations of anti miR-21 2′-O-methyl oligoribonucleotide and a control 2′-O-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., Genes & Devel. 17, 991-1008 (2003)).
The control EGFP oligonucleotide did not interfere with miR-21 cleavage even at the highest applied concentration (FIG. 2, lanes 2-3). In contrast, the activity of miR-21 was completely blocked at a concentration of only 3 nM (FIG. 2 lane 5), and a concentration of 0.3 nM showed a substantial 60%-70% reduction of cleavage activity (FIG. 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 (FIG. 2, lane 1, 7).
Antisense 2′-deoxyoligonucleotides (approximately 90% DNA molecules) at concentrations identical to those of 2′-O-methyl oligoribonucleotides, we could not detect blockage of miR-21 induced cleavage (FIG. 2, 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 complementary 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 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-C1 was cotransfected.
Expression of EGFP was observed in HeLa cells transfected with pEGFP and pEGFP-A-21 (FIG. 3, rows 1 and 2), but not from those transfected with pEGFP-S-21 (FIG. 3, row 3). However, expression of EGFP from pEGFP-S-21 was restored upon cotransfection with anti miR-21 2′-O-methyl oligoribonucleotide (FIG. 3, row 4). Consistent with our above observation, the 2′-deoxy anti miR-21 oligonucleotide showed no effect (FIG. 3, row 5). Similarly, cotransfection of the EGFP 2′-O-methyl oligoribonucleotide in sense orientation with respect to the EGFP mRNA (or antisense to EGFP guide siRNA) had no effect (FIG. 3, row 6).
We have demonstrated that miRNP complexes can be effectively and sequence-specifically inhibited with 2′-O-methyl oligoribonucleotides antisense to the guide strand positioned in the RNA silencing complex.
INCORPORATION OF SEQUENCE LISTING
Incorporated herein by reference in its entirety is the Sequence Listing for the application. The Sequence Listing is disclosed on a computer-readable ASCII text file titled, “sequence_listing.txt”, created on Mar. 9, 2011. The sequence_listing.txt file is 103 kb in size.

## Claims

1. An isolated molecule comprising a maximum of fifty moieties, wherein each moiety comprises a base bonded to a backbone unit, said molecule comprising the microRNA molecule identified in SEQ ID NO: 141 or its corresponding anti-micro RNA molecule identified in SEQ ID NO: 447.

2. A molecule according to claim 1, wherein the molecule is modified for increased nuclease resistance.

3. The molecule according to claim 1, wherein at least one of the moieties is a modified ribonucleotide moiety.

4. The molecule according to claim 3, wherein the modified ribonucleotide is substituted at the 2′ position.

5. The molecule according to claim 4, wherein the substituent at the 2′ position is a C1 to C4 alkyl group.

6. The molecule according to claim 5, wherein the alkyl group is methyl.

7. The molecule according to claim 5, wherein the alkyl group is allyl.

8. The molecule according to claim 4, wherein the substituent at the 2′ position is a C1 to C4 alkoxy-C1 to C4 alkyl group.

9. The molecule according to claim 8, wherein the C1 to C4 alkoxy-C1 to C4 alkyl group is methoxyethyl.

10. The molecule according to claim 1, wherein at least one of the moieties is a 2′-fluororibonucleotide moiety.

11. The molecule according to claim 3, wherein the modified ribonucleotide has a methylene bridge between the 2′-oxygen atom and the 4′-carbon atom.

12. The molecule according to claim 1, wherein the molecule comprises at least one modified moiety on the 5′ end.

13. The molecule according to claim 1, wherein the molecule comprises at least two modified moieties at the 5′ end.

14. The molecule according to claim 1, wherein the molecule comprises at least one modified moiety on the 3′ end.

15. The molecule according to claim 1, wherein the molecule comprises at least two modified moieties at the 3′ end.

16. The molecule according to claim 1, wherein the molecule comprises at least two modified moieties at the 5′ end and at least two modified moieties at the 3′ end.

17. The molecule according to claim 1, wherein the molecule comprises a nucleotide cap at the 5′ end, the 3′ end or both.

18. The molecule according to claim 1, wherein the molecule consists of the microRNA molecule identified in SEQ ID NO: 141.

19. The molecule according to claim 1, wherein the molecule consists of the anti-micro RNA molecule identified in SEQ ID NO: 447.

20. A vector comprising an isolated nucleic acid molecule according to claim 1.