Methods and compositions involving miRNA and miRNA inhibitor molecules
View Patent ↗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. A synthetic RNA molecule comprising two polynucleotides each between 17 and 25 residues in length comprising
a) a first polynucleotide comprising an miRNA region whose sequence from 5′ to 3′ is identical to a mature miRNA sequence, and
b) a second polynucleotide comprising a complementary region whose sequence from 5′ to 3′ is 100% complementary to the miRNA sequence,
wherein i) the 5′ terminus of the second polynucleotide is replaced with a lower alkylamine group and/or ii) there are one or more sugar modifications with a 2′ oxygen-methyl (2′O-Me) in the first or last 1 to 6 residues of the second polynucleotide.
2. The RNA molecule of claim 1 , comprising one or more sugar modifications in the first 1 to 6 residues of the second polynucleotide.
3. The RNA molecule of claim 1 , comprising one or more sugar modifications in the last 1 to 6 residues of the second polynucleotide.
4. The RNA molecule of claim 1 , comprising one or more sugar modifications in the first or last 2 to 4 residues of the second polynucleotide.
5. The RNA molecule of claim 4 , comprising more than one sugar modification in the first or last 2 to 4 residues of the second polynucleotide.
6. The RNA molecule of claim 1 , comprising noncomplementarity between one or more nucleotides in the last 1 to 5 residues at the 3′ end of the complementary region and the corresponding nucleotides in the complementary region.
7. The RNA molecules of claim 6 , wherein there is noncomplementarity with at least 2 nucleotides in the complementary region.
8. The RNA of claim 1 , wherein the RNA molecule further comprises a flanking region of 1-5 nucleotides at either the 5′ or 3′ region of either or both polynucleotides.
9. A synthetic RNA molecule comprising two polynucleotides each between 17 and 25 residues in length comprising
a) a first polynucleotide comprising an miRNA region whose sequence from 5′ to 3′ is identical to a mature miRNA sequence, and
b) a second polynucleotide comprising a complementary region whose sequence from 5′ to 3′ is 100% complementary to the miRNA sequence,
wherein the 5′ terminus of the second polynucleotide is replaced with a lower alkylamine group.