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 and a sense strand,
wherein the antisense strand is longer than the sense strand, consists of 19, 20, 21, 22 or 23 nucleotides and includes a 3′-overhang of 2, 3, 4, 5, 6, 7, 8 or 9 nucleotides, as part of the 19-23 nucleotides' length, and a 5′-blunt end when duplexed with the sense strand;
wherein the sense strand consists of 12, 13, 14, 15, 16 or 17 nucleotides, forms a double-stranded region with the antisense strand where the double-stranded region is selected from the group consisting of 10-15 matching base pairs plus 2 mismatch pairs, 11-16 matching base pairs plus 1 mismatch pair, and 12-17 perfectly matching base pairs; and
wherein the antisense strand has a sequence that specifically targets an expressed nucleotide sequence of a target gene through a perfectly complementary section with the target sequence and optionally further comprises a single-nucleotide or dinucleotide non-complementary section between the 5′ end of the antisense strand and the target sequence, the non-complementary section consisting of nucleotide(s) selected from the group consisting of “A,” “U,” and “dT”.
2. The RNA duplex molecule of claim 1 , wherein the sense strand of the RNA duplex does not substantially mediate off-target silencing.
3. The RNA duplex molecule 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.
4. The RNA duplex molecule of claim 1 , wherein the RNA duplex does not induce an interferon response.
5. The RNA duplex molecule of claim 1 , wherein the antisense strand comprises a 3′-overhang consisting of 2, 3, 4, 5, or 6 nucleotides.
6. The RNA duplex molecule of claim 1 , wherein the bases in the antisense strand that are complementary to the expressed nucleotide sequence of the target gene have a GC content ranging from 30% to 50%.
7. The RNA duplex molecule of claim 1 , wherein the sense strand consists of 15 nucleotides.
8. The RNA duplex molecule of claim 1 , wherein the antisense strand consists of 19 or 20 nucleotides.
9. The RNA duplex molecule of claim 1 , wherein the antisense strand consists of 21 nucleotides.
10. The RNA duplex molecule of claim 1 , wherein the double-stranded region between the sense and antisense strands consists of perfectly complementary sequences.
11. The RNA duplex molecule of claim 1 , wherein the double-stranded region comprises one mismatch base pair between the two strands.
12. The RNA duplex molecule of claim 1 , wherein the last nucleotide at the 3′ end of the antisense strand consists of a sugar- and/or backbone-modified A, U, G or C ribonucleotide and/or a base-modified A, G or C ribonucleotide.
13. The RNA duplex molecule of claim 1 , wherein at least one of the sense strand and the antisense strand comprises at least one modified nucleotide or an analogue thereof.
14. The RNA duplex molecule of claim 13 , wherein the at least one modified nucleotide or an analogue thereof is a sugar-, backbone-, and/or base-modified ribonucleotide.
15. The RNA duplex molecule of claim 14 , wherein the backbone-modified ribonucleotide has a modification in a phosphodiester linkage that links it with another ribonucleotide.
16. The RNA duplex molecule of claim 15 , wherein the phosphodiester linkage is modified to include at least one of a nitrogen or sulphur heteroatom.
17. The RNA duplex molecule of claim 13 , wherein the at least one modified nucleotide or an analogue thereof is a modified base.
18. The RNA duplex molecule of claim 13 , wherein the at least one modified nucleotide or an analogue thereof comprises an inosine or tritylated base.
19. The RNA duplex molecule of claim 13 , wherein the at least one modified nucleotide or an analogue thereof is a sugar-modified ribonucleotide in which the 2′-OH is replaced by H, OR, R, halo, SH, SR, NH2, NHR, NR2, or CN, wherein each R is independently C1-C6 alkyl, alkenyl or alkynyl, and the halo is F, Cl, Br or I.
20. The RNA duplex molecule of claim 19 , wherein the 2′-OH is replaced by a 2′-O-methyl group.
21. The RNA duplex molecule of claim 13 , wherein the at least one modified nucleotide or an analogue thereof is a backbone-modified ribonucleotide containing a phosphothioate group.
22. The RNA duplex molecule of claim 1 , wherein at least one of the sense strand and the antisense strand further comprises at least one deoxynucleotide.
23. The RNA duplex molecule of claim 22 , wherein the sense strand comprises 1-5 deoxynucleotides.
24. The RNA duplex molecule of claim 1 , wherein the first and second nucleotides at the 5′ end of the antisense strand form an “AA”, “UU” or “dTdT” motif, thereby forming the non-complementary section with the target sequence.
25. The RNA duplex molecule of claim 1 , wherein the RNA duplex molecule further comprises a peptide, an antibody, a polymer, a lipid, another oligonucleotide, cholesterol, or an aptamer.
26. The RNA duplex molecule of claim 1 , wherein the sense strand and/or antisense strand is bound to a lipid or a cholesterol molecule.
27. The RNA duplex molecule of claim 26 , wherein the lipid or cholesterol molecule is conjugated to the RNA duplex molecule.
28. The RNA duplex molecule 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 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.
29. A composition comprising the RNA duplex molecule of claim 1 .
30. A kit comprising the RNA duplex molecule of claim 1 .
31. An expression vector comprising a nucleic acid sequence encoding the RNA duplex molecule of claim 1 .
32. An isolated cell comprising the expression vector of claim 31 .
33. An isolated cell comprising the RNA duplex molecule of claim 1 .
34. The cell of claim 33 , wherein the cell is a mammalian, avian or bacterial cell.