Methods and compositions involving mirna and mirna inhibitor molecules

Abstract

The present invention concerns methods and compositions for introducing miRNA activity or function into cells using synthetic nucleic acid molecules. Moreover, the present invention concerns methods and compositions for identifying miRNAs with specific cellular functions that are relevant to therapeutic, diagnostic, and prognostic applications wherein synthetic miRNAs and/or miRNA inhibitors are used in library screening assays.

1 .- 101 . (canceled) 
     
     
         102 . A method for reducing cell proliferation in a cancer cell comprising providing to the cell an effective amount of a synthetic RNA molecule between 22 and 25 nucleotides in length, wherein the molecule comprises
 a) a first strand having an miRNA region comprising a sequence from 5′ to 3′ that is at least 80% identical to mature human miR-34a sequence, wherein the sequence is identical to miR-34b or miR-34c; and   b) a second separate strand having a complementary region whose sequence from 5′ to 3′ is 100% complementary to the sequence of the miRNA region, wherein the second strand comprises at least one chemical modification of the nucleotide at the 5′ end that enhances uptake of the first strand.   
     
     
         103 . The method of  claim 102 , wherein the cancer cell is a lung cancer cell, a cancerous T cell, a prostate cancer cell, or a skin cancer cell. 
     
     
         104 . The method of  claim 102 , wherein the synthetic RNA molecule is 22 or 23 nucleotides in length. 
     
     
         105 . The method of  claim 102 , wherein the synthetic RNA molecule further comprises one or more sugar modifications in the first or last 1 to 6 residues of the second strand. 
     
     
         106 . The method of  claim 105 , wherein the sugar modification is 2′OMe. 
     
     
         107 . The method of  claim 102 , wherein the chemical modification of the nucleotide at the 5′ end is biotin, an amine group, a lower alkylamine group, an acetyl group, 2′ oxygen-methyl (2′O-Me), 4,4′-dimethoxytrityl with oxygen (DMTO), fluoroscein, a thiol, or acridine. 
     
     
         108 . The method of  claim 106 , wherein the chemical modification is a lower alkylamine group. 
     
     
         109 . The method of  claim 102 , wherein the first and second strands are 100% complementary. 
     
     
         110 . A method for inhibiting cell proliferation of a cancer cell comprising administering to the cell an effective amount of a synthetic RNA molecule 21-25 nucleotides in length comprising:
 a) a first polynucleotide comprising an miRNA region whose sequence from 5′ to 3′ is at least 82% identical to a mature human miR-34a sequence, wherein the sequence is miR-34b or miR-34c; and,   b) a second polynucleotide comprising a complementary region whose sequence from 5′ to 3′ is 100% complementary to the sequence of the miRNA region of the first polynucleotide, and further comprising i) a chemical modification on the 5′ terminal nucleotide and/or ii) one or more sugar modifications in the first or last 1 to 6 residues.   
     
     
         111 . The method of  claim 110 , wherein the cancer cell is a lung cancer cell, a cancerous T cell, a prostate cancer cell, or a skin cancer cell. 
     
     
         112 . The method of  claim 110 , wherein the synthetic RNA molecule is 22 or 23 nucleotides in length. 
     
     
         113 . The method of  claim 110 , wherein the synthetic RNA molecule comprises a chemical modification on the 5′ terminal nucleotide of the second polynucleotide. 
     
     
         114 . The method of  claim 110 , wherein the chemical modification of the nucleotide at the 5′ end is biotin, an amine group, a lower alkylamine group, an acetyl group, 2′ oxygen-methyl (2′O-Me), 4,4′-dimethoxytrityl with oxygen (DMTO), fluoroscein, a thiol, or acridine. 
     
     
         115 . The method of  claim 114 , wherein the chemical modification is a lower alkylamine group. 
     
     
         116 . The method of  claim 110 , wherein the synthetic RNA molecule comprises one or more sugar modifications in the first or last 1 to 6 residues of the second strand. 
     
     
         117 . The method of  claim 110 , wherein the sugar modification is 2′OMe. 
     
     
         118 . A method for inducing apoptosis of a cancer cell comprising administering to the cells an effective amount of a synthetic RNA molecule comprising:
 a) an miRNA region whose sequence from 5′ to 3′ is at least 80% identical to a mature human miR-34a sequence on a first strand, wherein the sequence is the sequence of a mature human miR-34b or miR-34c, and   b) a complementary region whose sequence from 5′ to 3′ is 100% complementary to the sequence of the miRNA region on a second strand, wherein the molecule comprises two strands of 22-25 nucleotides in length and wherein the 5′ terminus of the second strand further comprises a nucleotide modification.   
     
     
         119 . The method of  claim 118 , wherein the cancer cell is a lung cancer cell, a cancerous T cell, a prostate cancer cell, or a skin cancer cell. 
     
     
         120 . The method of  claim 118 , wherein the synthetic RNA molecule is 22 or 23 nucleotides in length. 
     
     
         121 . The method of  claim 118 , wherein the synthetic RNA molecule further comprises one or more sugar modifications in the first or last 1 to 6 residues of the second strand. 
     
     
         122 . The method of  claim 105 , wherein the sugar modification is 2′OMe. 
     
     
         123 . The method of  claim 102 , wherein the nucleotide modification is biotin, an amine group, a lower alkylamine group, an acetyl group, 2′ oxygen-methyl (2′O-Me), 4,4′-dimethoxytrityl with oxygen (DMTO), fluoroscein, a thiol, or acridine. 
     
     
         124 . The method of  claim 123 , wherein the chemical modification is a lower alkylamine group.