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 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 when duplexed with the sense strand;
wherein the sense strand consists of 12, 13, 14, 15, 16, 17, 18, or 19 nucleotides and forms a double-stranded region with the antisense strand, and at least the first nucleotide and last nucleotide of the sense strand base pair with nucleotides of the antisense strand, and wherein the double-stranded region has a length of 11, 12, 13, 14, 15, 16, 17, 18, or 19 base pairs; and
wherein the antisense strand is at least 70% complementary to a target mRNA and has the last nucleotide of its 3′ end consisting of an A, U, G or C ribonucleotide.
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 double-stranded region has a length of 12, 13, 14, 15, 16 or 17 base pairs.
6. The RNA duplex molecule of claim 1 , wherein the double-stranded region consists of perfectly complementary sequences.
7. The RNA duplex molecule of claim 1 , wherein the double-stranded region comprises at least one nick, gap or mismatch.
8. The RNA duplex molecule of claim 1 , wherein the sense strand consists of 14 or 15 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 GC content of the double stranded region is 30%-50%.
11. The RNA duplex molecule of claim 1 , wherein the antisense strand comprises a 3′-overhang of 2, 3, 4, 5 or 6 nucleotides.
12. The RNA duplex molecule of claim 1 , wherein the antisense strand comprises a 5′-overhang of 0, 1, 2, 3, 4 or 5 nucleotides.
13. The RNA duplex molecule of claim 1 , wherein the antisense strand comprises a 5′-overhang of 2, 3 or 4 nucleotides and a 3′-overhang of 2, 3 or 4 nucleotides.
14. The RNA duplex molecule of claim 1 , wherein the antisense strand comprises a 5′-overhang of 3 nucleotides and a 3′-overhang of 3 nucleotides.
15. The RNA duplex molecule of claim 1 , wherein the antisense strand comprises a 3′-overhang of 1, 2, 3, 4, 5, 6, 7, 8 or 9 nucleotides and a 5′-blunt end.
16. The RNA duplex molecule of claim 1 , wherein the antisense strand comprises a 3′-overhang of 6 nucleotides and a 5′-blunt end.
17. The RNA duplex molecule of claim 1 , wherein at least one nucleotide of the 5′ end of the antisense strand is selected from the group consisting of A, U, and dT.
18. The RNA duplex molecule of claim 1 , wherein the 5′-end of the antisense oligonucleotide comprises an “AA”, “UU” or “dTdT” motif.
19. The RNA duplex molecule of claim 1 , wherein the 3′-overhang and/or 5′-overhang is stabilized against degradation either through chemical modification or secondary structure.
20. The RNA duplex molecule of claim 1 , wherein the RNA duplex molecule contains at least one modified nucleotide or its analogue.
21. The RNA duplex molecule of claim 20 , wherein the at least one modified nucleotide or its analogue is sugar-, backbone-, and/or base-modified ribonucleotide.
22. The RNA duplex molecule of claim 21 , wherein the backbone-modified ribonucleotide has a modification in a phosphodiester linkage with another ribonucleotide.
23. The RNA duplex molecule of claim 22 , wherein the phosphodiester linkage is modified to include at least one of a nitrogen or sulphur heteroatom.
24. The RNA duplex molecule of claim 20 , wherein the at least one modified nucleotide or its analogue is an unusual base or a modified base.
25. The RNA duplex molecule of claim 20 , wherein the at least one modified nucleotide or its analogue comprises inosine or a tritylated base.
26. The RNA duplex molecule of claim 20 , wherein the nucleotide analogue is a sugar-modified ribonucleotide, wherein the 2′—OH group is replaced by a group selected from H, OR, R, halo, SH, SR, NH2, NHR, NR2 or CN, wherein each R is independently C1-C6 alkyl, alkenyl or alkynyl, and halo is F, Cl, Br or I.
27. The RNA duplex molecule of claim 26 , wherein the 2′-OH is replaced by a 2′-O-methyl group and/or 2′-F.
28. The RNA duplex molecule of claim 20 , wherein the nucleotide analogue is a backbone-modified ribonucleotide containing a phosphothioate group.
29. The RNA duplex molecule of claim 1 , wherein the antisense strand comprises at least one deoxynucleotide.
30. The RNA molecule of claim 1 , wherein at least one nucleotide of the 5′-end of antisense strand is not complementary to the target mRNA sequence.
31. The RNA molecule of claim 1 , wherein the antisense strand and sense strand are joined by a chemical linker.
32. The RNA duplex molecule of claim 1 being conjugated to an entity selected from the group consisting of peptide, antibody, polymer, lipid, oligonucleotide, cholesterol, and aptamer.
33. A composition comprising the RNA duplex molecule of claim 1 and a pharmaceutically acceptable excipient, carrier, or diluent selected from the group consisting of a pharmaceutical carrier, a positive-charge carrier, a liposome, a protein carrier, a polymer, a nanoparticle, a nanoemulsion, a lipid, and a lipoid.
34. A method of treating or preventing a cancer or tumor comprising administering to a subject in need thereof the RNA duplex molecule of claim 1 .
35. The method of claim 34 , wherein the RNA duplex molecule is administered via a route selected from the group consisting of injection, oral administration, inhalation, topical, and regional administration.
36. The method of claim 34 , wherein the cancer is colon cancer.