METHODS AND COMPOSITIONS INVOLVING MIRNA AND MIRNA INHIBITOR MOLECULES
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 synthetic RNA molecule comprising:
a) a first 15-30 residue oligonucleotide having a sequence that is 80-100% identical to a mature microRNA sequence;
b) a second 15-30 residue oligonucleotide having a sequence that is 60-100% identical to a sequence complementary to the mature microRNA sequence; and
c) at least one of:
i) a replacement group for the phosphate or the hydroxyl of the nucleotide at the 5′ end of the second oligonucleotide,
ii) one or more sugar modifications in the first or last 1-6 residues of the second oligonucleotide, and
iii) a noncomplementarity between one or more nucleotides in the last 1-5 residues at the 3′ end of the second oligonucleotide and the corresponding nucleotides of the first oligonucleotide.
103 . The synthetic RNA molecule of claim 102 , wherein the phosphate of the nucleotide at the 5′ end of the second oligonucleotide is replaced and wherein the replacement group is a biotin, an amine group, an acetyl group, 2′O-Me, DMTO, fluoroscein, a lower alkylamine group, a thiol, or acridine.
104 . The synthetic RNA molecule of claim 102 , wherein the hydroxyl of the nucleotide at the 5′ end of the second oligonucleotide is replaced.
105 . The synthetic RNA molecule of claim 102 , comprising one or more sugar modifications in the first 1-6 residues of the second oligonucleotide.
106 . The synthetic RNA molecule of claim 102 , comprising one or more sugar modifications in the last 1-6 residues of the second oligonucleotide.
107 . The synthetic RNA molecule of claim 102 , comprising one or more sugar modifications in the first 2-4 residues of the second oligonucleotide.
108 . The synthetic RNA molecule of claim 102 , comprising one or more sugar modifications in the last 2-4 residues of the second oligonucleotide.
109 . The synthetic RNA molecule of claim 102 , wherein the sugar modification is a 2′O-Me modification
110 . The synthetic RNA molecule of claim 102 , comprising a noncomplementarity between one nucleotide in the last 1-5 residues at the 3′ end of the second oligonucleotide and the corresponding nucleotides of the first oligonucleotide.
111 . The synthetic RNA molecule of claim 102 , comprising a noncomplementarity between two or more nucleotide in the last 1-5 residues at the 3′ end of the second oligonucleotide and the corresponding nucleotides of the first oligonucleotide.
112 . The synthetic RNA molecule of claim 102 , comprising two of i), ii), and iii).
113 . The synthetic RNA molecule of claim 102 , comprising i), ii), and iii).
114 . The synthetic RNA molecule of claim 102 , wherein the first oligonucleotide is 85, 90, 95, or 100% identical to the mature microRNA sequence.
115 . The synthetic RNA molecule of claim 102 , wherein the second oligonucleotide is at least 65, 70, 75, 80, 85, 90, 95, or 100% identical to the sequence complementary to the mature microRNA sequence.
116 . The synthetic RNA molecule of claim 102 , wherein the mature microRNA sequence is a mature miR-1; miR-7; miR-10; miR-15; miR-16; miR-21; miR-24; miR-26; miR-28; miR-29; miR-31; miR-34; miR-92; miR-96; miR-101; miR-105; miR-124; miR-126; miR-133; miR-142; miR-143; miR-147; miR-155; miR-181; miR-192; miR-193; miR-194; miR-200; miR-205; miR-206; miR-215; miR-221; miR-346; miR-449; or let-7 sequence.
117 . A pharmaceutical composition comprising the synthetic RNA of claim 102 and a liposomal delivery vehicle.
118 . A method comprising introducing into a cell an effective amount of the synthetic RNA molecule of claim 102 .
119 . The method of claim 118 , wherein the method is for reducing or inhibiting cell proliferation and wherein the mature microRNA sequence is a miR-15a, miR-16, miR-21, miR-24, miR-34, miR-96, miR-101, miR-105, miR-124, miR-126, miR-142, miR-147, miR-192, miR-194, miR-206, miR-215, or miR-346 sequence.
120 . The method of claim 118 , wherein the method is for inducing apoptosis and wherein the mature microRNA sequence is a let-7b, let-7g, mir-1, mir-1d, mir-7, mir-10a, miR-10b, miR-17-3p, miR-19a, miR-28, miR-28, miR-28, miR-29a, miR-32, miR-34a, miR-122, mir-148, mir-149, mir-154, mir-184, mir-186, mir-188, mir-192, mir-195, mir-196, mir-199a mir-204, mir-208, mir-210, mir-211, mir-212, mir-214, mir-215, mir-216, mir-217, mir-218, mir-293, mir-296, mir-299, mir-321, mir-328, or mir-344 sequence.
121 . The method of claim 118 , wherein the method is for reducing cell viability and wherein the mature microRNA sequence is a let-7a, let-7b, mir-1, mir-7, miR-10b, miR-17-3p, miR-19a, mir-23, mir-24, mir-27a, miR-29a, miR-30a-3p, mir-31, mir-32, miR-34a, miR-101, miR-107, miR-108, miR-122, mir-124, miR-133a, miR-134, miR-135, miR-139, mir-140, miR-141, miR-145, mir-150, mir-192, mir-193, mir-195, mir-206, mir-208, mir-210, mir-210, mir-292-3p, mir-293, mir-297, mir-299, mir-329, mir-337, mir-337, mir-345, mir-346, or mir-409 sequence.