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. A method for preparing an asymmetric interfering RNA duplex comprising combining a sense oligonucleotide consisting of 14, 15, 16 or 17 nucleotides and an antisense oligonucleotide consisting of 19, 20, 21, 22 or 23 nucleotides,
wherein the antisense oligonucleotide includes a 3′-overhang of 1, 2, 3, 4, 5, 6, 7, 8 or 9 nucleotides and a 5′-overhang of 0, 1, 2, 3, 4, 5, 6, 7 or 8 nucleotides, and having a sequence that is at least 70% complementary to an expressed nucleotide sequence of a target gene and has a last nucleotide at its 3′ end consisting of an A, U, G or C ribonucleotide,
wherein 10, 11, 12, 13, 14, 15, 16, or 17 bases of the sense oligonucleotide are complementary with bases in the antisense oligonucleotide to form a double-stranded region, and at least the first base and last base of the sense oligonucleotide base pair with bases of the antisense oligonucleotide, and
wherein the sense oligonucleotide of the RNA duplex does not substantially mediate off-target silencing.
2. The method of claim 1 , wherein the RNA duplex is more effective at silencing the expressed nucleotide sequence of the target gene than a corresponding 21-mer siRNA duplex targeting the same expressed nucleotide sequence of the target gene.
3. The method of claim 1 , wherein the RNA duplex does not induce an interferon response.
4. The method of claim 1 , wherein the antisense oligonucleotide comprises a 5′-overhang consisting of 0, 1, 2, 3, 4, or 5.
5. The method of claim 1 , wherein the antisense oligonucleotide comprises a 3′-overhang consisting of 2, 3, 4, 5, or 6.
6. The method of claim 1 , wherein the last base of the sense oligonucleotide base pairs with the first base of the antisense oligonucleotide.
7. The method of claim 1 , wherein the bases in the antisense oligonucleotide that are complementary to the expressed nucleotide sequence of the target gene have a GC content ranging from 38.9% to 43%.
8. The method of claim 1 , wherein the bases in the antisense oligonucleotide that are complementary to the expressed nucleotide sequence of the target gene have a GC content of about 43%.
9. The method of claim 1 , wherein the sense oligonucleotide consists of 15 nucleotides.
10. The method of claim 1 , wherein the antisense oligonucleotide consists of 21 nucleotides.
11. The method of claim 1 , wherein the antisense oligonucleotide consists of 19, 20 or 21 nucleotides.
12. The method of claim 1 , wherein the double-stranded region comprises at least one unmatched or mismatched base.
13. The method of claim 1 , wherein 16, 17, 18, 19, 20 or 21 bases of the antisense oligonucleotide are complementary to the expressed nucleotide sequence of the target gene.
14. The method of claim 1 , wherein the last nucleotide at the 3′ end of the antisense oligonucleotide consists of a sugar- and/or backbone-modified A, U, G or C ribonucleotide and/or a base-modified A, G or C ribonucleotide.
15. The method of claim 1 , wherein at least one of the sense oligonucleotide and the antisense oligonucleotide comprises at least one modified nucleotide or its analogue.
16. The method of claim 15 , wherein the at least one modified nucleotide or its analogue is a sugar-, backbone-, and/or base-modified ribonucleotide.
17. The method of claim 16 , wherein the backbone-modified ribonucleotide has a modification in a phosphodiester linkage that links it with another ribonucleotide.
18. The method of claim 17 , wherein the phosphodiester linkage is modified to include at least one of a nitrogen or sulphur heteroatom.
19. The method of claim 15 , wherein the at least one modified nucleotide or its analogue is a modified base.
20. The method of claim 15 , wherein the at least one modified nucleotide or its analogue comprises an inosine or tritylated base.
21. The method of claim 15 , wherein the at least one modified nucleotide or its analogue is a sugar-modified ribonucleotide in which the 2′-OH is replaced by H, OR, R, halo, SH, SR, NH 2 , NHR, NR 2 , or CN, wherein each R is independently C 1 -C 6 alkyl, alkenyl or alkynyl, and the halo is F, Cl, Br or I.
22. The method of claim 15 , wherein the at least one modified nucleotide or its analogue is a backbone-modified ribonucleotide containing a phosphothioate group.
23. The method of claim 15 , wherein at least one of the sense oligonucleotide and the antisense oligonucleotide further comprises at least one deoxynucleotide.
24. The method of claim 1 , wherein the RNA duplex molecule further comprises a peptide, an antibody, a polymer, a lipid, another oligonucleotide, cholesterol, or an aptamer.
25. The method of claim 1 , wherein the sense oligonucleotide and/or antisense oligonucleotide is bound to a lipid or a cholesterol molecule.
26. The method of claim 25 , wherein the lipid or cholesterol molecule is conjugated to the RNA duplex molecule.
27. The method of claim 1 , wherein the antisense oligonucleotide comprises a 3′-overhang of 2-4 nucleotides and a 5′-overhang of 2-4 nucleotides.
28. The method of claim 1 , wherein the antisense oligonucleotide comprises a 3′-overhang of 1-9 nucleotides and a 5′-blunt end.
29. The method of claim 1 , wherein at least one nucleotide of the 5′-overhang of the antisense oligonucleotide is A, U or dT.
30. The method of claim 1 , wherein the 5′-overhang of the antisense oligonucleotide comprises an AA motif.
31. The method of claim 1 , wherein the target gene is:
a) a gene associated with human or animal diseases,
b) a gene of a pathogenic microorganism,
c) a viral gene,
d) a tumor-associated gene, or
e) a gene associated with a disease selected from the group selected from the group consisting of autoimmune diseases, inflammatory diseases, degenerative diseases, infectious diseases, proliferative diseases, metabolic diseases, immune-mediated disorders, allergic diseases, dermatological diseases, malignant diseases, gastrointestinal disorders, respiratory disorders, cardiovascular disorders, renal disorders, rheumatoid disorders, neurological disorders, endocrine disorders, and aging.
32. The method of claim 21 , wherein the 2′-OH is replaced by a 2′-O-methyl group.