Compositions of asymmetric interfering RNA and uses thereof
The present invention provides asymmetrical duplex RNA molecules that are capable of effecting sequence-specific gene silencing. The RNA molecule comprises a first strand and a second strand. The first strand is longer than the second strand. The RNA molecule comprises a double-stranded region formed by the first strand and the second strand, and two ends independently selected from the group consisting of 5′-overhang, 3′-overhang, and blunt end. The RNA molecules of the present invention can be used as research tools and/or therapeutics.
1. An asymmetric interfering RNA duplex molecule comprising:
an antisense strand consisting of 19-23 nucleotides,
wherein the antisense strand includes a 2-4 nucleotide 3′ overhang, and a 2-4 nucleotide 5′ overhang,
wherein the antisense strand at the 3′ overhang and the double-stranded region is complementary to a target RNA,
wherein the 3′ overhang of the antisense strand consists of RNA nucleotides,
and
a sense strand consisting of 14-17 nucleotides,
wherein the sense strand is complementary to the antisense strand, and
wherein the sense strand forms a 14, 15, 16, or 17 base pair double-stranded region with the antisense strand.
2. The RNA duplex molecule of claim 1 , wherein the GC content of the double-stranded region is 30%-70%.
3. The RNA duplex molecule of claim 1 , wherein the antisense strand consists of 21 nucleotides.
4. The RNA duplex molecule of claim 1 , wherein either the sense or antisense strand comprises at least one modified nucleotide or its analogue.
5. The RNA duplex molecule of claim 4 , wherein the at least one modified nucleotide or its analogue is sugar-, backbone-, and/or base-modified ribonucleotide.
6. The RNA duplex molecule of claim 5 , wherein the backbone-modified ribonucleotide comprises a modification in a phosphodiester linkage with another ribonucleotide.
7. The RNA duplex molecule of claim 6 , wherein the phosphodiester linkage is modified to comprises a nitrogen or sulfur heteroatom.
8. The RNA duplex molecule of claim 4 , wherein the at least one modified nucleotide or its analogue comprises inosine or a tritylated base.
9. The RNA duplex molecule of claim 4 , wherein the modified nucleotide or its analogue is a sugar-modified ribonucleotide,
wherein the 2′-OH group is replaced by H, OR, R, halo, SH, SR, NH 2 , NHR, NR 2 , or CN, and wherein each R is independently C 1 -C 6 alkyl, alkenyl or alkynyl, and halo is F, CI, Br, or I.
10. The RNA duplex molecule of claim 4 , wherein the modified nucleotide or its analogue is a backbone-modified ribonucleotide comprising a phosphothioate group.
11. A composition comprising the RNA duplex molecule of claim 1 .
12. A kit comprising the RNA duplex molecule of claim 1 .
13. An expression vector comprising a nucleic acid sequence encoding the RNA duplex molecule of claim 1 .
14. The expression vector of claim 13 , wherein the expression vector is a viral, a eukaryotic, or a bacterial expression vector.
15. An isolated cell comprising the vector of claim 13 .
16. An isolated cell comprising the RNA duplex molecule of claim 1 .
17. The cell of claim 16 , wherein the cell is a mammalian, an avian, or a bacterial cell.
18. The RNA duplex molecule of claim 1 , wherein the sense strand consists of 15 nucleotides.
19. The RNA duplex molecule of claim 9 , wherein the 2′-OH group is replaced by a 2′-O-methyl group.
20. The RNA duplex molecule of claim 1 , wherein at least one nucleotide of the 5′ overhang is A, U, or dT.
21. The RNA duplex molecule of claim 1 , wherein the 5′ overhang of the antisense strand comprises an AA motif.
22. A kit comprising the composition of claim 11 .
23. A method of treating a disease or condition comprising administering to a subject in need thereof the RNA duplex molecule of claim 1 .
24. The method of claim 23 , wherein the RNA duplex molecule is administered systemically or locally.
25. The method of claim 23 , wherein the RNA duplex molecule comprises an antisense strand of 21 nucleotides.
26. The method of claim 23 , wherein the RNA duplex molecule comprises a sense strand of 15 nucleotides.
27. The method of claim 23 , wherein the RNA duplex molecule comprises an antisense strand of 21 nucleotides and a sense strand of 15 nucleotides.
28. A method of treating a malignant disease, comprising administering to a subject in need thereof the RNA duplex molecule of claim 1 .
29. The method of claim 28 , wherein the malignant disease is cancer.
30. The method of claim 28 , wherein the malignant disease is colon cancer.
31. A method of treating an ocular disease comprising administering to a subject in need thereof the RNA duplex molecule of claim 1 .