RNA sequence-specific mediators of RNA interference
View Patent ↗The present invention relates to a Drosophila in vitro system which was used to demonstrate that dsRNA is processed to RNA segments 21-23 nucleotides (nt) in length. Furthermore, when these 21-23 nt fragments are purified and added back to Drosophila extracts, they mediate RNA interference in the absence of long dsRNA. Thus, these 21-23 nt fragments are the sequence-specific mediators of RNA degradation. A molecular signal, which may be their specific length, must be present in these 21-23 nt fragments to recruit cellular factors involved in RNAi. This present invention encompasses these 21-23 nt fragments and their use for specifically inactivating gene function. The use of these fragments (or chemically synthesized oligonucleotides of the same or similar nature) enables the targeting of specific mRNAs for degradation in mammalian cells, where the use of long dsRNAs to elicit RNAi is usually not practical, presumably because of the deleterious effects of the interferon response. This specific targeting of a particular gene function is useful in functional genomic and therapeutic applications.
1. A method of producing knockdown cells, comprising:
introducing into cells in which a gene is to be knocked down an isolated double-stranded RNA molecule of from about 21 nucleotides in length to 23 nucleotides in length in the form of two RNA strands, which are not covalently linked, wherein one strand has sequence correspondence to the mRNA corresponding to the gene;
and maintaining the resulting cells under conditions under which RNA interference (RNAi) occurs, resulting in degradation of the mRNA of the gene,
thereby producing knockdown cells.
2. The method of claim 1 , wherein the gene encodes a cellular or a viral mRNA.
3. The method of claim 1 , wherein the isolated double-stranded RNA molecule is recombinantly produced.
4. The method of claim 3 , wherein the isolated double-stranded RNA molecule comprises a terminal 3′ hydroxyl group.
5. The method of claim 1 , wherein the isolated double-stranded RNA molecule is chemically synthesized.
6. The method of claim 5 , wherein the isolated double-stranded RNA molecule comprises a terminal 3′ hydroxyl group.
7. The method of claim 5 , wherein one or more nucleotides of the isolated double-stranded RNA molecule is a non-naturally occurring nucleotide.
8. The method of claim 5 , wherein one or more nucleotides of the isolated double-stranded RNA molecule is a deoxyribonucleotide.
9. The method of claim 5 , wherein one or more nucleotides of the isolated double-stranded RNA molecule is a non-standard nucleotide.
10. A method of producing a knockdown organism, comprising:
introducing into the organism in which a gene is to be knocked down isolated double-stranded RNA molecule of from about 21 nucleotides in length to 23 nucleotides in length in the form of two RNA strands, which are not covalently linked, wherein one strand has sequence correspondence to the mRNA corresponding to the gene; and
maintaining the resulting organism under conditions under which RNA interference (RNAi) occurs, resulting in degradation of the mRNA of the gene,
thereby producing the knockdown organism.
11. The method of claim 10 , wherein the gene encodes a cellular or a viral mRNA.
12. The method of claim 10 , wherein the isolated double-stranded RNA molecule is recombinantly produced.
13. The method of claim 12 , wherein the isolated double-stranded RNA molecule comprises a terminal 3′ hydroxyl group.
14. The method of claim 10 , wherein the isolated double-stranded RNA molecule is chemically synthesized.
15. The method of claim 14 , wherein the isolated double-stranded RNA molecule comprises a terminal 3′ hydroxyl group.
16. The method of claim 14 , wherein one or more nucleotides of the isolated double-stranded RNA molecule is a non-naturally occurring nucleotide.
17. The method of claim 14 , wherein one or more nucleotides of the isolated double-stranded RNA molecule is a deoxyribonucleotide.
18. The method of claim 14 , wherein one or more nucleotides of the isolated double-stranded RNA molecule is a non-standard nucleotide.
19. The method of claim 2 , wherein the cellular mRNA is mammalian mRNA.
20. The method of claim 1 , wherein the isolated double-stranded RNA molecule consists of from 21 nucleotides in length to 23 nucleotides in length.
21. A method of producing a knockdown cell or organism, comprising:
introducing into the cell or the organism in which a gene is to be knocked down an isolated double-stranded RNA molecule of from about 21 nucleotides in length to 23 nucleotides in length in the form of two strands, which are not covalently linked, wherein one strand has sequence correspondence to an mRNA corresponding to the gene, wherein the double-stranded RNA molecule is chemically synthesized and wherein one or more of the nucleotides are non-naturally occurring nucleotides or deoxyribonucleotides; and
maintaining the resulting cell or organism under conditions under which RNA interference (RNAi) occurs, resulting in degradation of the mRNA of the gene,
thereby producing the knockdown cell or organism.
22. The method of claim 21 , wherein the gene encodes a cellular or a viral mRNA.
23. The method of claim 22 , wherein the cellular mRNA is a mammalian mRNA.
24. The method of claim 23 , wherein the mammalian mRNA is a human mRNA.
25. The method of claim 24 , wherein the human mRNA encodes a protein whose presence is associated with a disease or an undesirable condition.
26. The method of claim 21 , wherein the isolated double-stranded RNA molecule consists of from 21 nucleotides in length to 23 nucleotides in length.
27. The method of claim 21 , wherein the one strand of the double-stranded RNA molecule that has sequence correspondence to the mRNA is perfectly complementary to the mRNA.
28. The method of claim 19 , wherein the mammalian mRNA is a human mRNA.
29. The method of claim 28 , wherein the human mRNA encodes a protein whose presence is associated with a disease or an undesirable condition.
30. The method of claim 1 , wherein the one strand of the double-stranded RNA molecule that has sequence correspondence to the mRNA is perfectly complementary to the mRNA.
31. The method of claim 11 , wherein the cellular mRNA is mammalian mRNA.
32. The method of claim 10 , wherein the isolated double-stranded RNA molecule consists of from 21 nucleotides in length to 23 nucleotides in length.
33. The method of claim 10 , wherein the one strand of the double-stranded RNA molecule that has sequence correspondence to the mRNA is perfectly complementary to the mRNA.
34. A method of producing a knockdown cell or organism, comprising:
introducing into the cell or the organism in which a gene is to be knocked down an isolated double-stranded RNA molecule consisting of from 21 nucleotides in length to 23 nucleotides in length in the form of two strands, which are not covalently linked, wherein one strand has sufficient sequence homology to an mRNA corresponding to the gene to mediate RNA interference (RNAi), and wherein the double-stranded RNA molecule comprises one or more non-naturally occurring nucleotides or non-standard nucleotides; and
maintaining the resulting cell or organism under conditions under which RNAi occurs,
thereby producing the knockdown cell or organism.
35. The method of claim 34 , wherein the gene encodes a cellular or a viral mRNA.
36. The method of claim 35 , wherein the cellular mRNA is a mammalian mRNA.
37. The method of claim 36 , wherein the mammalian mRNA is a human mRNA.
38. The method of claim 34 , wherein the mRNA encodes a protein whose presence is associated with a disease or an undesirable condition.
39. The method of claim 34 , wherein a strand of the double-stranded RNA molecule is 21 nucleotides in length.
40. The method of claim 34 , wherein a strand of the double-stranded RNA molecule is 22 nucleotides in length.
41. The method of claim 34 , wherein a strand of the double-stranded RNA molecule is 23 nucleotides in length.
42. The method of claim 1 , wherein a strand of the double-stranded RNA molecule is about 21 nucleotides in length.
43. The method of claim 10 , wherein a strand of the double-stranded RNA molecule is about 21 nucleotides in length.
44. The method of claim 21 , wherein a strand of the double-stranded RNA molecule is about 21 nucleotides in length.