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. A method of cleaving a target mRNA in a cell or an organism in vivo, comprising contacting said cell or organism in vivo with an isolated, non-enzymatically processed double-stranded RNA molecule under conditions in which cleavage of said target mRNA can occur, wherein said double-stranded RNA molecule:
a) has a sense strand and an antisense strand;
b) consists of two strands which are each 19-25 nucleotides in length;
c) consists of a single double stranded region and one or two single stranded regions of 1 to 5 nucleotides each at the 3′ ends of the strands of said double-stranded molecule; and
d) a strand of said double-stranded RNA molecule has an identity of at least 85% to said target mRNA in said single double stranded region,
wherein said double-stranded RNA molecule is administered to said cell or organism by local injection.
2. The method according to claim 1 , wherein said contacting comprises introducing said double-stranded RNA molecule into a target cell in which the cleavage of the target mRNA can occur.
3. The method according to claim 1 , further comprising modulating a function of a gene in a cell in vivo by mediating said cleavage of the target mRNA.
4. The method according to claim 1 , wherein at least one strand of said double-stranded RNA molecule has a 3′-overhang of 1-3 nucleotides.
5. The method according to claim 4 , wherein at least one strand of said double-stranded RNA molecule has a 3′-overhang of 2 nucleotides.
6. The method according to claim 1 , wherein said double-stranded RNA molecule comprises at least one modified nucleotide analogue.
7. The method according to claim 6 , wherein the modified nucleotide analogue is selected from sugar- or backbone modified ribonucleotides.
8. The method according to claim 7 , 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, C 2 -C 6 alkenyl and C 2 -C 6 alkynyl, and halo is selected from the group consisting of F, Cl, Br and I.
9. The method according to claim 1 , wherein said double-stranded RNA molecule consists of two strands which are each 19-23 nucleotides in length.
10. The method according to claim 1 , wherein the function of said target mRNA is unknown.
11. A method for mediating cleavage of a target mRNA in a cell or an organism in vivo, comprising contacting said cell or organism with an isolated, non-enzymatically cleaved double-stranded RNA molecule consisting of 19-25 nucleotides in length under conditions wherein cleavage of a target mRNA can occur, wherein said RNA molecule:
a) has a sense strand and an antisense strand;
b) consists of a single double stranded region of 16-24 nucleotides in length and one or two single stranded regions of 1 to 3 nucleotides each at the 3′ ends of the strands of said double-stranded RNA molecule;
c) a strand of said double-stranded RNA has an identity of at least 85% to said target mRNA in said single double stranded region, and
wherein said double-stranded RNA molecule is administered to said cell or organism by local injection.
12. The method according to claim 11 , wherein said contacting comprises introducing said double-stranded RNA molecule into a target cell in which the cleavage of the target mRNA can occur.
13. The method according to claim 11 , further comprising modulating a function of a gene in a cell in vivo by mediating said cleavage of the target mRNA.
14. The method according to claim 11 , wherein at least one strand of said double-stranded RNA molecule has a 3′-overhang of 1-3 nucleotides.
15. The method according to claim 11 , wherein at least one strand of said double-stranded RNA molecule has a 3′-overhang of 2 nucleotides.
16. The method according to claim 10 , wherein said double-stranded RNA molecule comprises at least one modified nucleotide analogue.
17. The method according to claim 16 , wherein the modified nucleotide analogue is selected from sugar- or backbone modified ribonucleotides.
18. The method according to claim 17 , 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, C 2 -C 6 alkenyl and C 2 -C 6 alkynyl, and halo is selected from the group consisting of F, Cl, Br and I.
19. The method according to claim 11 , wherein said double-stranded RNA molecule consists of two strands which are each 19-23 nucleotides in length.
20. The method according to claim 1 , wherein said cell is a non-embryonic cell.
21. The method according to claim 1 , wherein said double-stranded RNA has an identity of 100% to said target mRNA.
22. A method of cleaving a target mRNA in a cell or an organism in vivo comprising, contacting said cell or organism in vivo with an isolated, non-enzymatically processed double-stranded RNA molecule under conditions in which cleavage of said target mRNA can occur, wherein said double-stranded RNA molecule:
a) has a sense strand and an antisense strand;
b) consists of two strands which are each 19-25 nucleotides in length;
c) consists of a single double stranded region and one or two single stranded regions of 1 to 5 nucleotides each at the 3′ ends of the strands of said double-stranded molecule, and
d) a strand of said double-stranded RNA molecule has an identity of at least 85% to said target mRNA in said single double stranded region,
wherein said double-stranded RNA molecule is administered to said cell or organism using a carrier-mediated delivery.
23. The method according to claim 22 , wherein said carrier-mediated delivery comprises a liposome carrier.
24. The method according to claim 23 , wherein said liposome carrier comprises cationic liposomes.
25. The method according to claim 22 , wherein said contacting comprises introducing said double-stranded RNA molecule into a target cell in which the cleavage of the target mRNA can occur.
26. The method according to claim 22 , further comprising modulating a function of a gene in a cell in vivo by mediating said cleavage of the target mRNA.
27. The method according to claim 22 , wherein at least one strand of said double-stranded RNA molecule has a 3′-overhang of 1-3 nucleotides.
28. The method according to claim 27 , wherein at least one strand of said double-stranded RNA molecule has a 3′-overhang of 2 nucleotides.
29. The method according to claim 22 , wherein said double-stranded RNA molecule comprises at least one modified nucleotide analogue.
30. The method according to claim 29 , wherein the modified nucleotide analogue is selected from sugar- or backbone modified ribonucleotides.
31. The method according to claim 30 , 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, C 2 -C 6 alkenyl and C 2 -C 6 alkynyl, and halo is selected from the group consisting of F, Cl, Br and I.
32. The method according to claim 22 , wherein said double-stranded RNA molecule consists of two strands which are each 19-23 nucleotides in length.
33. A method for mediating cleavage of a target mRNA in a cell in vivo, comprising contacting a cell or an organism with an isolated, non-enzymatically processed double-stranded RNA molecule consisting of 19-25 nucleotides in length under conditions wherein cleavage of a target mRNA can occur, wherein said RNA molecule:
a) has a sense strand and an antisense strand;
b) consists of a single double stranded region of 16-24 nucleotides in length and one or two single stranded regions of 1 to 3 nucleotides each at the 3′ ends of the strands of said double-stranded RNA molecule,
c) a strand of said double-stranded RNA molecule has an identity of at least 85% to said target mRNA in said single double stranded region, and
wherein said RNA molecule is administered to said cell or organism using a carrier-mediated delivery.
34. The method according to claim 33 , wherein said contacting comprises introducing said double-stranded RNA molecule into a target cell in which the cleavage of the target mRNA can occur.
35. The method according to claim 33 , further comprising modulating a function of a gene in a cell in vivo by mediating said cleavage of the target mRNA.
36. The method according to claim 33 , wherein at least one strand of said double-stranded RNA molecule has a 3′-overhang of 1-3 nucleotides.
37. The method according to claim 36 , wherein at least one strand of said double stranded RNA molecule has a 3′-overhang of 2 nucleotides.
38. The method according to claim 33 , wherein said double stranded RNA molecule contains at least one modified nucleotide analogue.
39. The method according to claim 38 , wherein the modified nucleotide analogue is selected from sugar- or backbone modified ribonucleotides.
40. The method according to claim 39 , 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, C 2 -C 6 alkenyl and C 2 -C 6 alkynyl, and halo is selected from the group consisting of F, Cl, Br and I.
41. The method according to claim 33 , wherein said double-stranded RNA molecule consists of two strands which are each 19-23 nucleotides in length.
42. The method according to claim 33 , wherein said cell is a non-embryonic cell.
43. The method according to claim 33 , wherein said double-stranded RNA has an identity of 100% to said target mRNA.
44. The method of claim 1 , wherein the cell or organism is a mammalian cell or a mammal.
45. The method of claim 11 , wherein the cell or organism is a mammalian cell or a mammal.
46. The method of claim 22 , wherein the cell or organism is a mammalian cell or a mammal.
47. The method of claim 33 , wherein the cell or organism is a mammalian cell or a mammal.
48. A method of cleaving a target mRNA in a cell or an organism, comprising:
contacting said cell or organism with an isolated, non-enzymatically cleaved double-stranded RNA molecule under conditions in which cleavage of said target mRNA can occur,
wherein said isolated, RNA molecule:
a) has a sense strand and an antisense strand;
b) consists of two strands which are each 19-23 nucleotides in length;
c) consists of a single double stranded region and one or two single stranded regions of 1 to 3 nucleotides each at the 3′ ends of the strands of said double-stranded molecule, and
d) a strand of said double stranded RNA molecule has an identity of at least 70% to said target mRNA in said single double stranded region,
wherein said double-stranded RNA molecule is administered to said cell or organism by local injection or using a carrier-mediated delivery.
49. The method of claim 48 , wherein the cell or organism is a mammalian cell or a mammal.
50. The method of claim 48 , wherein the contacting step occurs in a cell culture or an organism.
51. A method of mediating RNA interference of a target mRNA in a cell or an organism, comprising:
contacting the cell or organism with an isolated, non-enzymatically processed double-stranded RNA molecule under conditions in which RNA interference of the target mRNA can occur, wherein:
(i) each strand of the RNA molecule independently consists of 19-25 nucleotides in length;
(ii) at least one strand of the RNA molecule forms a 3′-overhang from 1 to 5 nucleotides;
(iii) a strand of the double-stranded RNA molecule has an identity of at least 70% to the target RNA in the double stranded region,
(iv) the double-stranded RNA molecule comprises at least one nucleotide analogue; and
wherein the double-stranded RNA molecule is administered to the cell or organism using a carrier-mediated delivery.
52. The method of claim 51 , wherein the at least one nucleotide analogue is selected from a sugar- or backbone modified ribonucleotide, or a combination thereof.
53. The method of claim 51 , wherein the at least one 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, C 2 -C 6 alkenyl and C 2 -C 6 alkynyl, and halo is selected from the group consisting of F, Cl, Br and I.
54. The method of claim 51 , wherein the at least one nucleotide analogue is a backbone-modified ribonucleotide containing a phosphorothioate group.
55. The method of claim 51 , wherein the double-stranded RNA molecule comprises a 2′-F sugar modified ribonucleotide, a 2′-OMe sugar modified ribonucleotide and a phosphorothioate backbone modified ribonucleotide.
56. The method of claim 51 , wherein the at least one nucleotide analogue is located at the 5′-end, the 3′-end, or both, of the double-stranded RNA molecule.
57. The method of claim 51 , wherein the at least one nucleotide analogue is located in the 3′-overhang of the double-stranded RNA molecule.
58. The method of claim 57 , wherein the nucleotide analogue of the 3′-overhang is selected from a sugar- or a backbone-modified ribonucleotide, or a combination thereof.
59. The method of claim 57 , wherein the nucleotide analogue of the 3′-overhang is a sugar-modified ribonucleotide, wherein the 2′-OH group is replaced by a group selected from H, OR, R, halo, SH, SR, NH 2 , NHR, N(R) 2 or CN, wherein R is C 1 -C 6 alkyl, C 2 -C 6 alkenyl or C 2 -C 6 alkynyl and halo is F, Cl, Br or I.
60. The method of claim 57 , wherein the nucleotide analogue of the 3′-overhang is a backbone-modified ribonucleotide containing a phosphorothioate group.
61. The method of claim 57 , wherein the 3′-overhang of the double-stranded RNA molecule comprises a 2′-deoxythymidine or a nucleotide lacking a 2′-hydroxyl in a sugar.
62. The method of claim 57 , wherein the 3′-overhang of the double-stranded RNA molecule comprises a nucleobase-modified ribonucleotide chosen from a non-naturally-occurring nucleobase modified at the 5-position; an adenosine or a guanosine modified at the 8-position; or an O- or an N-alkylated nucleotide.
63. The method of claim 51 , wherein the 3′-overhang of the RNA molecule has been stabilized against degradation.
64. The method of claim 51 , wherein the 3′-overhang of the RNA molecule is from 1-3 nucleotides in length.
65. The method of claim 51 , wherein the 3′-overhang of the RNA molecule is 2 nucleotides in length.
66. The method of claim 64 , wherein each strand of the RNA molecule consists of 20 to 22, or 21 to 23, nucleotides in length.
67. The method of claim 64 , wherein at least one of the strands of the RNA molecule consists of 21 nucleotides in length.
68. The method of claim 51 , wherein the double-stranded RNA molecule consists of a single double-stranded region and a single-stranded region of 1 to 3 nucleotides at the 3′ end of at least one of the strands of said double-stranded RNA molecule.
69. The method of claim 51 , wherein an end of the double-stranded RNA molecule is blunt-ended.
70. The method of claim 51 , wherein a strand of the RNA molecule is at least 85% identical to the target RNA molecule.
71. The method of claim 51 , wherein a strand of the RNA molecule is identical to the target mRNA molecule in the double-stranded portion of the RNA molecule.
72. The method of claim 51 , wherein the cell or organism is a mammalian cell or organism.
73. The method of claim 51 , wherein the cell is chosen from an embryonic cell, a pluripotent stem cell, a tumor cell or a virus-infected cell.
74. The method of claim 51 , wherein the cell is a human cell.
75. The method of claim 51 , wherein the cell or organism is a plant cell or organism.
76. The method of claim 51 , wherein the RNA molecule mediates RNA interference of a target mRNA encoded by a gene chosen from a pathogen-associated gene, a viral gene, a tumor-associated gene, or an autoimmune disease-associated gene.
77. The method of claim 51 , wherein the RNA molecule is administered by injection.
78. The method of claim 51 , wherein the carrier-mediated delivery is capable of increasing the efficacy of the RNA molecule to enter the cell or organism.
79. The method of claim 78 , wherein the carrier-mediated delivery is a liposomal carrier.
80. The method of claim 51 , wherein the RNA molecule is chemically synthesized.