RNA sequence-specific mediators of RNA interference
원문보기
IPC분류정보
국가/구분
United States(US) Patent
등록
국제특허분류(IPC7판)
C07H-021/04
C07H-021/02
A61K-048/00
출원번호
US-0897740
(2010-10-04)
등록번호
US-8552171
(2013-10-08)
우선권정보
EP-00126325 (2000-12-01)
발명자
/ 주소
Tuschl, Thomas
Zamore, Phillip D.
Sharp, Phillip A.
Bartel, David P.
출원인 / 주소
University of Massachusetts
대리인 / 주소
Lando & Anastasi, LLP
인용정보
피인용 횟수 :
14인용 특허 :
44
초록▼
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 ab
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.
대표청구항▼
1. An isolated double-stranded RNA molecule of from 21 to 23 nucleotides, in the form of two separate RNA strands which are not covalently linked, that is perfectly complementary to an mRNA to mediate RNA interference by directing cleavage of the mRNA within the region that is perfectly complementar
1. An isolated double-stranded RNA molecule of from 21 to 23 nucleotides, in the form of two separate RNA strands which are not covalently linked, that is perfectly complementary to an mRNA to mediate RNA interference by directing cleavage of the mRNA within the region that is perfectly complementary with the isolated RNA molecule, wherein the mRNA is a mammalian cellular mRNA. 2. The isolated double-stranded RNA molecule of claim 1, wherein each RNA strand comprises a terminal 3′ hydroxyl group. 3. An isolated double-stranded RNA molecule of from 21 to 23 nucleotides, which is chemically synthesized, in the form of two separate RNA strands which are not covalently linked, that is perfectly complementary to an mRNA to mediate RNA interference by directing cleavage of the mRNA within the region that is perfectly complementary with the isolated RNA molecule, wherein the mRNA is a mammalian cellular mRNA. 4. A composition comprising the isolated double-stranded RNA molecule of claim 1, and an appropriate carrier. 5. The isolated RNA molecule of claim 3, wherein each RNA strand comprises a terminal 3′ hydroxyl group. 6. The isolated RNA molecule of claim 1, wherein the mRNA is a human mRNA. 7. An isolated double-stranded RNA molecule of from 21 to 23 nucleotides, in the form of two separate RNA strands which are not covalently linked, that is perfectly complementary to a mammalian cellular mRNA to mediate RNA interference by directing cleavage of the mRNA within the region that is perfectly complementary with the isolated RNA molecule, wherein the isolated RNA molecule comprises naturally occurring nucleotides, and wherein one or more nucleotides of the isolated RNA molecule is a deoxyribonucleotide. 8. The isolated RNA molecule of claim 1, wherein a strand of the isolated RNA molecule is 21 nucleotides in length. 9. The isolated RNA molecule of claim 1, wherein a strand of the isolated RNA molecule is 22 nucleotides in length. 10. The isolated RNA molecule of claim 1, wherein a strand of the isolated RNA molecule is 23 nucleotides in length. 11. The isolated RNA molecule of claim 3, wherein the mRNA is a human mRNA. 12. The isolated RNA molecule of claim 7, wherein each RNA strand comprises a terminal 3′ hydroxyl group. 13. A composition comprising an acceptable carrier and comprising the isolated RNA molecule of claim 3. 14. An isolated double-stranded RNA molecule, comprising: two separate RNA strands which are not covalently linked, wherein said double-stranded RNA molecule is sufficiently complementary to a mammalian cellular mRNA to mediate RNA interference by directing cleavage of the mRNA, wherein cleavage is directed within the region of sequence complementarity with the isolated RNA molecule, and wherein at least one strand of the RNA molecule consists of 21 nucleotides in length. 15. The isolated RNA molecule of claim 14, wherein each RNA strand comprises a terminal 3′ hydroxyl group. 16. The isolated RNA molecule of claim 14, wherein the mRNA is a human mRNA. 17. The isolated RNA molecule of claim 15, wherein the mRNA is a human mRNA. 18. An isolated double-stranded RNA molecule, comprising: two separate RNA strands which are not covalently linked, wherein said double-stranded RNA molecule is chemically synthesized and is sufficiently complementary to a mammalian cellular mRNA to mediate RNA interference by directing cleavage of the mRNA, wherein cleavage is directed within the region of sequence complementarity with the isolated RNA molecule, and wherein at least one strand of the RNA molecule consists of 21 nucleotides in length. 19. The isolated RNA molecule of claim 18, wherein each RNA strand comprises a terminal 3′ hydroxyl group. 20. The isolated RNA molecule of claim 18, wherein the mRNA is a human mRNA. 21. The isolated RNA molecule of claim 19, wherein the mRNA is a human mRNA. 22. A composition comprising the isolated double-stranded RNA molecule of claim 14, and an appropriate carrier. 23. The composition of claim 22, wherein each RNA strand of the RNA molecule comprises a terminal 3′ hydroxyl group. 24. The composition of claim 22, wherein the mRNA is a human mRNA. 25. A composition comprising the isolated double-stranded RNA molecule of claim 18 and an appropriate carrier. 26. An isolated double-stranded RNA molecule, in the form of two separate RNA strands which are not covalently linked, that has sequence correspondence to a mammalian cellular mRNA to mediate RNA interference by directing cleavage of the mRNA to which it corresponds, wherein cleavage is directed within the region of sequence correspondence with the isolated RNA molecule, wherein each strand consists of 23 nucleotides in length. 27. The isolated RNA molecule of claim 26, wherein each RNA strand comprises a terminal 3′ hydroxyl group. 28. An isolated double-stranded RNA molecule, which is chemically synthesized RNA in the form of two separate RNA strands which are not covalently linked, that has sequence correspondence to a mammalian cellular mRNA to mediate RNA interference by directing cleavage of the mRNA to which it corresponds, wherein cleavage is directed within the region of sequence correspondence with the isolated RNA molecule, wherein each strand consists of 23 nucleotides in length. 29. The isolated RNA molecule of claim 28, wherein each RNA strand comprises a terminal 3′ hydroxyl group. 30. A composition comprising the isolated double-stranded RNA molecule of claim 26, and an appropriate carrier. 31. The composition of claim 30, wherein each RNA strand of the RNA molecule comprises a terminal 3′ hydroxyl group. 32. The isolated double-stranded RNA molecule of claim 1, which comprises naturally-occurring nucleotides. 33. The isolated double-stranded RNA molecule of claim 2, which comprises naturally-occurring nucleotides. 34. The isolated double-stranded RNA molecule of claim 3, which comprises naturally-occurring nucleotides. 35. The isolated double-stranded RNA molecule of claim 5, which comprises naturally-occurring nucleotides. 36. The isolated double-stranded RNA molecule of claim 1, which consists of naturally-occurring nucleotides. 37. The isolated double-stranded RNA molecule of claim 1, wherein one or more nucleotides of the double-stranded RNA molecule are non-naturally occurring nucleotides or non-standard nucleotides. 38. The isolated double-stranded RNA molecule of claim 2, wherein one or more nucleotides of the double-stranded RNA molecule are non-naturally occurring nucleotides or non-standard nucleotides. 39. The isolated double-stranded RNA molecule of claim 3, wherein one or more nucleotides of the double-stranded RNA molecule are non-naturally occurring nucleotides or non-standard nucleotides. 40. The isolated double-stranded RNA molecule of claim 5, wherein one or more nucleotides of the double-stranded RNA molecule are non-naturally occurring nucleotides or non-standard nucleotides. 41. The isolated double-stranded RNA molecule of claim 1, which comprises naturally-occurring nucleotides and one or more non-naturally occurring nucleotides or non-standard nucleotides. 42. The isolated double-stranded RNA molecule of claim 2, which comprises naturally-occurring nucleotides and one or more non-naturally occurring nucleotides or non-standard nucleotides. 43. The isolated double-stranded RNA molecule of claim 3, which comprises naturally-occurring nucleotides and one or more non-naturally occurring nucleotides or non-standard nucleotides. 44. The isolated double-stranded RNA molecule of claim 5, which comprises naturally-occurring nucleotides and one or more non-naturally occurring nucleotides or non-standard nucleotides. 45. The isolated double-stranded RNA molecule of claim 41, wherein one or more nucleotides of the double-stranded RNA molecule are deoxyribonucleotides. 46. The isolated double-stranded RNA molecule of claim 42, wherein one or more nucleotides of the double-stranded RNA molecule are deoxyribonucleotides. 47. The isolated double-stranded RNA molecule of claim 43, wherein one or more nucleotides of the double-stranded RNA molecule are deoxyribonucleotides. 48. The isolated double-stranded RNA molecule of claim 44, wherein one or more nucleotides of the double-stranded RNA molecule are deoxyribonucleotides. 49. The isolated double-stranded RNA molecule of claim 18, wherein the mRNA is a human mRNA that encodes a protein that is associated with a disease or an undesirable condition. 50. The isolated double-stranded RNA molecule of claim 18, which is perfectly complementary to the mRNA of the target gene. 51. The isolated double-stranded RNA molecule of claim 18, which comprises one or more non-naturally occurring nucleotides or non-standard nucleotides. 52. The isolated double-stranded RNA molecule of claim 18, wherein one or more nucleotides of the double-stranded RNA molecule are deoxyribonucleotides. 53. An isolated double-stranded RNA molecule, comprising: two separate RNA strands which are not covalently linked, wherein a strand of the double-stranded RNA molecule is perfectly complementary to a mammalian cellular mRNA to mediate RNA interference by directing cleavage of the mRNA, wherein cleavage is directed within the region of complementarity with the isolated RNA molecule, and wherein at least one strand consists of 21 or 22 nucleotides in length. 54. The isolated RNA molecule of claim 53, wherein each RNA strand comprises a terminal 3′ hydroxyl group. 55. The isolated RNA molecule of claim 53, which is chemically synthesized. 56. The isolated double-stranded RNA molecule of claim 55, wherein the mRNA is a human mRNA that encodes a protein that is associated with a disease or an undesirable condition. 57. The isolated double-stranded RNA molecule of claim 53, which comprises one or more non-naturally occurring nucleotides or non-standard nucleotides. 58. The isolated double-stranded RNA molecule of claim 53, wherein one or more nucleotides of the double-stranded RNA molecule are deoxyribonucleotides. 59. An isolated double-stranded RNA molecule, each strand consisting of 21 nucleotides in length, in the form of two separate RNA strands which are not covalently linked, wherein: (i) the double-stranded RNA molecule has sequence correspondence to a mammalian cellular mRNA to mediate RNA interference by directing cleavage of the mRNA, wherein cleavage is directed within the region of sequence correspondence with the isolated RNA molecule, and(ii) the double-stranded RNA molecule comprises one or more naturally-occurring nucleotides, or one or more non-naturally occurring nucleotides or non-standard nucleotides. 60. The isolated double-stranded RNA molecule of claim 59, wherein one or more nucleotides of the double-stranded RNA molecule are deoxyribonucleotides. 61. A composition comprising the isolated double-stranded RNA molecule of claim 59 and an appropriate carrier. 62. An isolated double-stranded RNA molecule, in the form of two separate RNA strands which are not covalently linked, comprising a strand that is perfectly complementary to a mammalian cellular mRNA to mediate RNA interference by directing cleavage of the mRNA, wherein cleavage is directed within the region of complementarity with the isolated RNA molecule, and wherein at least one strand consists of 22 nucleotides in length. 63. The isolated RNA molecule of claim 62, wherein each RNA strand comprises a terminal 3′ hydroxyl group. 64. The isolated double-stranded RNA molecule of claim 62, wherein the mRNA is a human mRNA that encodes a protein that is associated with a disease or an undesirable condition. 65. The isolated double-stranded RNA molecule of claim 62, which comprises one or more naturally-occurring nucleotides, one or more deoxyribonucleotides, or one or more non-naturally occurring nucleotides or non-standard nucleotides. 66. The isolated RNA molecule of claim 3, wherein a strand of the isolated RNA molecule is 21 nucleotides in length. 67. The isolated RNA molecule of claim 3, wherein a strand of the isolated RNA molecule is 22 nucleotides in length. 68. The isolated RNA molecule of claim 3, wherein a strand of the isolated RNA molecule is 23 nucleotides in length. 69. The isolated RNA molecule of claim 1, wherein the mammalian cellular mRNA is associated with a disease or an undesirable condition. 70. The isolated RNA molecule of claim 1, wherein the mammalian cellular mRNA encodes an oncoprotein. 71. The isolated RNA molecule of claim 1, which reduces the relative ratio of a target mRNA to a control mRNA by greater than 30-fold. 72. The isolated RNA molecule of claim 3, wherein the mammalian cellular mRNA is associated with a disease or an undesirable condition. 73. The isolated RNA molecule of claim 3, wherein the mammalian cellular mRNA encodes an oncoprotein. 74. The isolated RNA molecule of claim 3, which reduces the relative ratio of a target mRNA to a control mRNA by greater than 30-fold.
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