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 method for reducing cell viability of human cancer cells comprising providing miRNA function to the cells by introducing into the cells a synthetic double-stranded RNA molecule 22-30 residues in length comprising:
a) an active strand comprising a sequence identical to human miR-34a and
b) a separate complementary strand comprising a sequence that is 100% complementary to the human miR-34a sequence and at least one chemical modification at the 5′ end that enhances uptake of the active strand,
wherein the human cancer cells are lung cancer cells, cancerous T cells, prostate cancer cells, or skin cancer cells.
2. The method of claim 1 , wherein the human cancer cells are lung cancer cells.
3. The method of claim 1 , wherein the synthetic double-stranded RNA molecule is 22 residues in length.
4. The method of claim 1 , wherein the synthetic double-stranded RNA molecule has a complementary strand that has at least one modified nucleotide that blocks the 5′0H or phosphate at the 5′ terminus.
5. The method of claim 4 , wherein the 5′ terminus is modified with an NH 2 , biotin, an amine group, a lower alkylamine group, or an acetyl group.
6. The method of claim 1 , wherein the synthetic double-stranded RNA molecule has a complementary strand that has a 5′ terminal nucleotide with a sugar modification.
7. The method of claim 6 , wherein the sugar modification is 2′OMe.
8. The method of claim 1 , wherein the chemical modification is a replacement group for the phosphate or hydroxyl of the nucleotide at the 5′ end.
9. The method of claim 1 , wherein the human cancer cells are cancerous T cells.
10. The method of claim 1 , wherein the human cancer cells are prostate cancer cells.
11. The method of claim 1 , wherein the human cancer cells are skin cancer cells.
12. A method for reducing cell viability of human cancer cells comprising providing miRNA function to the cells by introducing into the cells a synthetic double-stranded RNA molecule 22-30 residues in length comprising:
a) an active strand comprising a sequence identical to human miR-34a and
b) a separate complementary strand comprising i) a sequence that is 100% complementary to the human miR-34a sequence and ii) a 5′ blocking agent that enhances uptake of the active strand,
wherein the human cancer cells are lung cancer cells, cancerous T cells, prostate cancer cells, or skin cancer cells.
13. The method of claim 12 , wherein the 5′ blocking agent is an NH 2 , biotin, an amine group, a lower alkylamine group, an acetyl group, or 2′O-Me.
14. The method of claim 12 , wherein the human cancer cells are cancerous T cells.
15. The method of claim 12 , wherein the human cancer cells are lung cancer cells.
16. The method of claim 12 , wherein the human cancer cells are prostate cancer cells.
17. The method of claim 12 , wherein the human cancer cells are skin cancer cells.