RNA interference mediating small RNA molecules
Double-stranded RNA (dsRNA) induces sequence-specific post-transcriptional gene silencing in many organisms by a process known as RNA interference (RNAi). Using a Drosophila in vitro system, we demonstrate that 19-23 nt short RNA fragments are the sequence-specific mediators of RNAi. The short interfering RNAs (siRNAs) are generated by an RNase III-like processing reaction from long dsRNA. Chemically synthesized siRNA duplexes with overhanging 3′ ends mediate efficient target RNA cleavage in the lysate, and the cleavage site is located near the center of the region spanned by the guiding siRNA. Furthermore, we provide evidence that the direction of dsRNA processing determines whether sense or antisense target RNA can be cleaved by the produced siRNP complex.
1. An isolated, double-stranded RNA molecule comprising two RNA strands, wherein:
(a) each RNA strand independently consists of 19-25 nucleotides;
(b) at least one RNA strand forms a single-stranded 3′-overhang of 1 to 5 nucleotides at an end of the RNA molecule;
(c) the only single-stranded region in said RNA molecule is the at least one single-stranded 3′-overhang;
(d) a strand of the RNA molecule is at least 85% identical to a target RNA molecule in the double-stranded portion of the RNA molecule;
(e) said RNA molecule mediates target-specific RNA interference;
(f) said RNA molecule is non-enzymatically prepared; and
(g) said RNA molecule comprises at least one modified nucleotide analogue.
2. The RNA molecule according to claim 1 , wherein each strand has a length from 19-23, 20-22, or 21-23 nucleotides.
3. The RNA molecule according to claim 1 , wherein the 3′-overhang is stabilized against degradation.
4. The RNA molecule according to claim 1 , wherein the modified nucleotide analogue is selected from a sugar- or a backbone modified ribonucleotide.
5. The RNA molecule according to claim 4 , wherein the nucleotide analogue is a sugar-modified ribonucleotide, wherein the 2′-OH group is replaced by a group selected from the group consisting of H, OR, R, halo, SH, SR, NH 2 , NHR, N(R) 2 and CN, and wherein R is selected from the group consisting of C 1 -C 6 alkyl, alkenyl and alkynyl, and halo is selected from the group consisting of F, Cl, Br and I.
6. The RNA molecule according to claim 4 , wherein the nucleotide analogue is a backbone-modified ribonucleotide containing a phosphorothioate group.
7. The RNA molecule according to claim 1 , wherein only one RNA strand forms a 3′-overhang of from 1-3 nucleotides at an end of said double-stranded RNA molecule.
8. The RNA molecule according to claim 1 , wherein both RNA strands form 3′-overhangs of from 1 to 5 nucleotides at the ends of said double-stranded RNA molecule.
9. An isolated, synthetically prepared, double-stranded RNA molecule comprising two strands, wherein:
(a) each strand independently consists of 21-23 nucleotides;
(b) the strands form a single-stranded two-nucleotide 3′-overhang at each end of the RNA molecule;
(c) the only single-stranded regions in said RNA molecule are the single-stranded 2-nucleotide 3′-overhangs;
(d) each sugar of the two-nucleotide 3′-overhangs has a 2′H;
(e) the linkage between the two nucleotides of each of said 2-nucleotide 3′-overhangs is a phosphorothioate linkage;
(f) a strand of the RNA molecule is at least 85% identical to a target RNA molecule in the double-stranded portion of the RNA molecule; and
(g) said RNA molecule is capable of mediating target-specific RNA interference.
10. The RNA molecule of claim 9 , wherein the two nucleotides of each of said 3′ overhangs consist of two 2′-deoxythymidine.
11. The RNA molecule of claim 9 , wherein the only modified nucleotides are in the two-nucleotide 3′-overhangs.
12. The RNA molecule of claim 9 , wherein each RNA strand has a length from 21-22 nucleotides.
13. The RNA molecule according to claim 1 , wherein a strand of the RNA molecule is at least 85% identical to the target RNA molecule.
14. The RNA molecule according to claim 1 , wherein a strand of the RNA molecule is identical to the target mRNA molecule in the double-stranded portion of the RNA molecule.
15. The RNA molecule according to claim 1 , wherein one or both strands of the RNA molecule form a 3′-overhang which is 2 nucleotides in length.
16. The RNA molecule according to claim 1 , wherein one end of the RNA molecule is blunt-ended.
17. The RNA molecule according to claim 1 , which comprises a 2′-F sugar modified ribonucleotide, a 2′-OMe sugar modified ribonucleotide, a phosphorothioate backbone modified ribonucleotide, or a combination thereof.
18. The RNA molecule according to claim 1 , wherein the modified nucleotide analogue is located at a position where the target-specific RNA interference is not substantially affected.
19. The RNA molecule according to claim 1 , which comprises at least one modified nucleotide analogue located at the 5′-end, the 3′-end, or both, of the RNA molecule.
20. The RNA molecule according to claim 1 , wherein the length of both strands is the same.
21. The RNA molecule according to claim 1 , wherein the length of both strands is different.
22. The RNA molecule of claim 1 , which is chemically synthesized.
23. The RNA molecule of claim 1 , which mediates RNA interference of a mammalian RNA.
24. The RNA molecule of claim 1 , which mediates RNA interference of a human RNA.
25. The RNA molecule of claim 1 , which mediates RNA interference of a plant RNA.
26. The RNA molecule of claim 1 , which mediates RNA interference of a target gene chosen from a pathogen-associated gene, a viral gene, a tumor-associated gene, or an autoimmune disease-associated gene.
27. A pharmaceutical composition comprising at least one RNA molecule of claim 1 and a pharmaceutical carrier.
28. The RNA molecule according to claim 9 , wherein a strand of the RNA molecule is at least 85% identical to the target RNA molecule.
29. The RNA molecule according to claim 9 , wherein a strand of the RNA molecule is identical to the target RNA molecule in the double-stranded portion of the RNA molecule.
30. The RNA molecule according to claim 9 , which comprises a 2′-F sugar modified ribonucleotide, a 2′-OMe sugar modified ribonucleotide, a phosphorothioate backbone modified ribonucleotide, or a combination thereof.
31. The RNA molecule according to claim 9 , wherein the linkage between the nucleotides of each of said 3′-overhangs is a phosphorothioate linkage.
32. The RNA molecule according to claim 9 , wherein the modified nucleotide analogue is located at a position where the target-specific RNA interference is not substantially affected.
33. The RNA molecule according to claim 9 , which comprises at least one modified nucleotide analogue located at the 5′-end, the 3′-end, or both, of the RNA molecule.
34. The RNA molecule of claim 9 , which mediates RNA interference of a mammalian RNA.
35. The RNA molecule of claim 9 , which mediates RNA interference of a mammalian RNA.
36. The RNA molecule of claim 9 , which mediates RNA interference of a human RNA.
37. The RNA molecule of claim 1 , which mediates RNA interference of a plant RNA.
38. The RNA molecule of claim 1 , which mediates RNA interference of a target gene chosen from a pathogen-associated gene, a viral gene, a tumor-associated gene, or an autoimmune disease-associated gene.
39. A pharmaceutical composition comprising at least one RNA molecule of claim 9 and a pharmaceutical carrier.
40. An isolated, synthetically prepared, double-stranded RNA molecule comprising two strands, wherein:
(a) each strand independently consists of 19-25 nucleotides;
(b) each strand comprises of a sugar- or a backbone modified ribonucleotide, or a combination thereof;
(c) at least one strand forms a single-stranded nucleotide 3′-overhang at the RNA molecule;
(d) said 3′-overhang has been stabilized against degradation;
(e) a strand of the RNA molecule is at least 85% identical to a target RNA molecule in the double-stranded portion of the RNA molecule; and
(f) said RNA molecule is capable of mediating target-specific RNA interference.
41. The RNA molecule of claim 40 , wherein a strand of the RNA molecule is at least 85% identical to the target RNA molecule.
42. The RNA molecule according to claim 40 , wherein a strand of the RNA molecule is identical to the target mRNA molecule in the double-stranded portion of the RNA molecule.
43. The RNA molecule according to claim 40 , wherein the 2′-OH group of said sugar-modified ribonucleotide is replaced by a group selected from the group consisting of H, OR, R, halo, SH, SR, NH 2 , NHR, NR 2 and CN, wherein R is C 1 -C 6 alkyl, C 2 -C 6 alkenyl or alkynyl and halo is F, Cl, Br or I.
44. The RNA molecule according to claim 40 , wherein each strand comprises of a 2′-F sugar modified ribonucleotide, a 2′-OMe sugar modified ribonucleotide or a phosphorothioate backbone modified ribonucleotide, or a combination thereof.
45. The RNA molecule according to claim 40 , wherein the linkage between the nucleotides of said 3′-overhangs is a phosphorothioate linkage.
46. The RNA molecule according to claim 40 , wherein at least one of the RNA strands of the RNA molecule forms a 3′-overhang from 1-3 nucleotides in length.
47. The RNA molecule according to claim 40 , wherein at least one of the strands of the RNA molecule forms a 3′-overhang which is 2 nucleotides in length.
48. The RNA molecule according to claim 40 , wherein each strand of the RNA molecule consists of 19 to 23, 20 to 22, or 21 to 23 nucleotides in length.
49. The RNA molecule according to claim 40 , wherein at least one of the strands consists of 21 nucleotides in length.
50. The RNA molecule according to claim 40 , wherein one strand of the RNA molecule is blunt-ended.
51. The RNA molecule of claim 40 , which mediates RNA interference of a mammalian RNA.
52. The RNA molecule of claim 40 , which mediates RNA interference of a human RNA.
53. The RNA molecule of claim 40 , which mediates RNA interference of a plant RNA.
54. The RNA molecule of claim 40 , which mediates RNA interference of a target gene chosen from a pathogen-associated gene, a viral gene, a tumor-associated gene, or an autoimmune disease-associated gene.
55. A pharmaceutical composition comprising at least one RNA molecule of claim 40 and a pharmaceutical carrier.
56. The RNA molecule according to claim 9 , which is not enzymatically processed.
57. The RNA molecule according to claim 40 , which is not enzymatically processed.