Methods and compositions relating to labeled RNA molecules that reduce gene expression

• US 20040248094A1
• Filed: 06/12/2002
• Published: 12/09/2004
• Est. Priority Date: 06/12/2002
• Status: Abandoned Application

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Inventors: Byrom, Mike, Ford, Lance P., Pasloske, Brittan L.
• Application Number:
  US10/360,772
• Publication Number:
  US 20040248094A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical

Abstract

The present invention concerns methods and compositions involving labeled, double-stranded RNA (dsRNA), including siRNA, capable of triggering RNA-mediated interference (RNAi) in a cell. Compositions of the invention include labeled dsRNA for RNAi, which may be a single strand of RNA that basepairs with itself or two separate RNA strands. In some embodiments, the label is fluorescent. The present invention further concerns methods for preparing such composition and kits for implementing such methods. Other methods of the invention include ways of using labeled dsRNA for RNAi.

Claims

1. A method for labeling a dsRNA molecule capable of inducing RNA interference comprising:

a) hybridizing sense and antisense regions of at least one RNA strand; and

,

b) labeling at least one RNA strand with a fluorescent dye.

2. The method of claim 1, wherein the sense and antisense regions are on the same strand.

3. The method of claim 1, wherein the sense and antisense regions are on separate strands.

4. The method of claim 1, wherein at least one RNA strand is labeled before the sense and antisense RNA regions are hybridized.

5. The method of claim 1, wherein the hybridized dsRNA molecule is internally labeled.

6. The method of claim 1, wherein the hybridized dsRNA molecule is labeled by incubating the molecule with a) labeling buffer comprising a physiological buffer with a pH of 7.0 to 7.5 and b) a labeling reagent comprising the fluorescent dye.

7. The method of claim 6, wherein the hybridized dsRNA molecule is end-labeled.

8. The method of claim 7, wherein end labeling comprises:

a) incubating a hybridized dsRNA molecule with a 5′

thiophosphate moiety with a thio-reactive fluorescent dye.

9. The method of claim 8, wherein the thio-reactive dye is BODIPY, Alexa Fluor, HEX, ROX, SYPRO, fluorescein, Oregon Green, tetramethylrhodamine, Texas Red, cyanine dye, or derivatives thereof

10. The method of claim 8, wherein the hybridized dsRNA molecule with a 5′

thiophosphate moiety is incubated with the thio-reactive fluorescent dye for at least 5 minutes at a temperature of 25 °

C. to 75°

C.

11. The method of claim 10, wherein the hybridized dsRNA molecule with a 5′

thiophosphate moiety is incubated with the thio-reactive fluorescent dye at a temperature of 60 °

C. to 70°

C.

12. The method of claim 8, wherein the hybridized dsRNA molecule with a 5′

thiophosphate moiety is created by incubating the hybridized dsRNA molecule with a kinase, a kinase buffer, and ATPγ

S.

13. The method of claim 1, wherein the fluorescent dye is fluorescein, Texas red, rhodamine, DAPI, Cy3, or Cy5.

14. The method of claim 1, further comprising isolating the labeled and hybridized dsRNA.

15. The method of claim 14, further comprising measuring the absorbance of the dsRNA.

16. A fluorescently labeled dsRNA molecule capable of inducing RNA interference.

17. The fluorescently labeled dsRNA molecule of claim 16, wherein two strands are labeled.

18. The fluorescently labeled dsRNA molecule of claim 16, wherein the molecule is a single strand comprising at least two regions complementary to each other.

19. The fluorescently labeled dsRNA molecule of claim 18, wherein the complementary regions include at least 5 contiguous bases.

20. The fluorescently labeled dsRNA molecule of claim 16, wherein the antisense strand is labeled.

21. The fluorescently labeled, dsRNA molecule of claim 16, wherein the sense strand is labeled.

22. The fluorescently labeled dsRNA molecule of claim 17, wherein the sense strand is labeled with a different color label than the antisense strand.

23. The fluorescently labeled dsRNA molecule of claim 16, wherein the RNA imolecule is an siRNA.

24. The fluorescently labeled, dsRNA molecule of claim 23, wherein the siRNA is 5 to 25 bases or basepairs in length.

25. The fluorescently labeled, dsRNA molecule of claim 24, wherein the siRNA is 10 to 21 bases or basepairs in length.

26. The fluorescently labeled dsRNA molecule of claim 24, wherein the siRNA is at least 10 bases or basepairs in length.

27. The fluorescently labeled dsRNA molecule of claim 16, wherein the RNA molecule is between 25 and 10,000 bases or basepairs in length.

28. The fluorescently labeled dsRNA molecule of claim 27, wherein the RNA molecule is capable of being processed into at least one siRNA in a cell.

32. The fluorescently labeled dsRNA molecule of claim 16, wherein the molecule is end-labeled.

33. The fluorescently labeled dsRNA molecule of claim 16, wherein the molecule is internally labeled.

34. The fluorescently labeled dsRNA molecule of claim 16, wherein the RNA molecule comprises at least one modified residue.

36. A method for evaluating RNA interference in a cell comprising:

a) introducing into the cell the fluorescently labeled dsRNA molecule of claim 16;

and

b) detecting or visualizing the fluorescently labeled dsRNA molecule.

37. The method of claim 36, wherein the dsRNA molecule comprises a single strand that has at least two region complementary to each other.

38. The method of claim 36, wherein the dsRNA molecule comprises two separate strands in which one strand has a sense region and the other strand has an antisense region.

39. The method of claim 36, wherein both strands of the RNA molecule are labeled.

40. The method of claim 36, wherein the antisense strand of the RNA molecule is labeled.

41. The method of claim 36, wherein the sense strand of the RNA molecule is labeled.

42. The method of claim 39, wherein the sense strand of the RNA molecule is labeled with a different color label than the antisense strand of the RNA molecule.

43. The method of claim 36, wherein the RNA molecule is an siRNA.

44. The method of claim 43, wherein the siRNA is 5 to 25 bases or basepairs in length.

45. The method of claim 44, wherein the siRNA is 10 to 21 bases or basepairs in length.

46. The method of claim 45, wherein the siRNA is at least 10 bases or basepairs in length.

47. The method of claim of claim 46, wherein the RNA molecule is between 25 and 100 bases or basepairs in length.

48. The method of claim 36, wherein the RNA molecule is capable of being processed into at least one siRNA in the cell.

49. The method of claim 36, wherein the RNA molecule is labeled with BODIPY, Alexa Fluor, fluorescein, Oregon Green, tetramethylrhodamine, Texas Red, rhodamine, cyanine dye, or derivatives thereof

50. The method of claim 36, wherein the molecule is end-labeled.

51. The method of claim 36, wherein the molecule is internally labeled.

52. The method of claim 36, wherein the cell is a eukaryotic cell.

53. The method of claim 52, wherein the cell is a mammalian cell.

54. The method of claim 53, wherein the mammalian cell is a primate, rodent, rabbit, or human cell.

55. The method of claim 36, wherein the cell is a prokaryotic cell.

56. The method of claim 36, wherein the cell is alive.

57. The method of claim 56, wherein the cell is in an organism or tissue.

58. The method of claim 36, wherein the cell is dead.

59. The method of claim 58, wherein the cell is fixed.

29. The labeled dsRNA molecule of 16, wherein the label is fluorescent, enzymatic, or radioactive.

30. The labled dsRNA molecule of claim 29, wherein the label is fluorescent.

31. The fluorescently labeled dsRNA molecule of claim 30, wherein the molecule is labeled with BODIPY, Alexa Fluor, fluorescein, Oregon Green, tetramethylrhodamine, Texas Red, rhodamine, cyanine dye, or derivatives thereof

35. A labeled dsRNA molecule capable of associating with an RNA-induced silencing complex.

60. A kit for labeling a dsRNA for RNAi comprising, in a suitable container means,

a) labeling buffer comprising a physiological buffer with a pH range of 7.0 to 7.5;

b) labeling reagent for labeling dsRNA with fluorescent label comprising fluorescent dye;

c) control dsRNA comprising a dsRNA shown to trigger RNAi in a cell when labeled using components a) and b).

61. The kit of claim 60, wherein the labeling reagent further comprises an akylating agent.

62. The kit of claim 60, wherein the labeling buffer is provided at a 10×

concentration.

63. The kit of claim 62, wherein the control dsRNA is single stranded.

64. The kit of claim 62, wherein the control dsRNA comprises separate complementary RNA strands.

65. The kit of claim 60, wherein the labeling reagent comprises Cy3, Cy5, or fluorescein (FAM).

66. The kit of claim 60, further comprising nuclease free water, ethanol, NaCl, Rnase inhibitor, reconstitution solution comprising DMSO, or annealing buffer comprising Hepes, potassium acetate, and magnesium acetate.