Methods and compositions relating to polypeptides with RNase III domains that mediate RNA interference

• US 20040033602A1
• Filed: 06/12/2003
• Published: 02/19/2004
• Est. Priority Date: 06/12/2002
• Status: Abandoned Application

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Ambion Incorporated (Thermo Fisher Scientific Incorporated)
• Inventors: Brown, David, Ford, Lance P.
• Application Number:
  US10/460,775
• Publication Number:
  US 20040033602A1
• Time in Patent Office: Days
• US Class Current: 435/455
• CPC Class Codes: C12N 15/111 General methods applicable to biologically active non-coding nucleic acids
• CPC Class Codes: C12N 15/113 Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
• CPC Class Codes: C12N 15/1135 against oncogenes or tumor suppressor genes
• CPC Class Codes: C12N 15/1136 against growth factors, growth regulators, cytokines, lymphokines or hormones
• CPC Class Codes: C12N 15/1137 against enzymes viral enzymes C12N15/1131; receptors C12N15/1138
• CPC Class Codes: C12N 2310/14 interfering N.A.
• CPC Class Codes: C12N 2330/30 chemically synthesised
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: C12Y 301/26003 Ribonuclease III (3.1.26.3)

Abstract

The present invention concerns methods and compositions involving RNase III and polypeptides containing RNase III domains to generate RNA capable of triggering RNA-mediated interference (RNAi) in a cell. In some embodiments, the RNase III is from a prokaryote. RNase III activity will cleave a double-stranded RNA molecule into short RNA molecules that may trigger or mediate RNAi (siRNA). Compositions of the invention include kits that include an RNase III domain-containing polypeptide. The present invention further concerns methods using polypeptides with RNase III activity for generating RNA molecules that effect RNAi, including the generation of a number of RNA molecules to the same target.

Claims

1. A method of reducing expression of a target gene in a cell comprising:

a) incubating a dsRNA corresponding to part of the target gene with an effective amount of a composition comprising a polypeptide comprising an RNase III domain, under conditions to allow RNase III to cleave the dsRNA into siRNA; and

b) transfecting the siRNA into the cell.

2. The method of claim 1, wherein the polypeptide is chimeric.

3. The method of claim 1, further comprising isolating the siRNA molecules prior to transfection.

4. The method of claim 1, wherein the dsRNA is 25 to 10,000 bases or basepairs in length.

5. The method of claim 4, wherein the dsRNA is 25 to 5,000 bases or basepairs in length.

6. The method of claim 5, wherein the dsRNA is 50 to 1,000 bases or basepairs in length.

7. The method of claim 6, wherein the dsRNA is 100 to 200 bases or basepairs in length.

8. The method of claim 1, wherein the dsRNA is obtained by transcribing each strand of the dsRNA from one or more CDNA encoding the strands in vitro;

isolating the strands; and

, incubating the strands under conditions that allow the strands to hybridize to their complementary strands.

9. The method of claim 1, wherein dsRNA for at least a second targeted gene is included.

10. A method for achieving RNA interference of a target gene in a cell using one or more siRNA molecules comprising:

a) generating at least one double-stranded DNA template corresponding to part of the target gene, wherein the DNA template comprises an SP6, T3, or T7 promoter on at least one strand;

b) transcribing the template, wherein either i) a single RNA strand with a complementarity region, or ii) first and second complementary RNA strands is/are created;

c) hybridizing either the single complementary RNA strand or first and second complementary RNA strands to create a dsRNA molecule corresponding to the target gene;

d) incubating the dsRNA molecule with a polypeptide comprising an RNase III domain, under conditions to allow cleavage of the dsRNA into at least two siRNA; and

e) transfecting at least one siRNA into the cell.

11. The method of claim 10, wherein the polypeptide is RNase III.

12. The method of claim 10, wherein the polypeptide is chimeric.

13. The method of claim 10, wherein multiple siRNA molecules are transfected into the cell.

14. A kit for generating siRNA molecules comprising:

a) recombinant, prokaryotic RNase III;

b) RNase III buffer; and

c) a control nucleic acid.

15. The kit of claim 14, wherein the RNase III is in an enzyme dilution buffer.

16. The kit of claim 14, further comprising an SP6, T3 or T7 RNA polymerase.

17. The kit of claim 16, wherein the polymerase is in an enzyme mix comprising inorganic pyrophosphatase, at least one RNase inhibitor, and about 1% CHAPS.

18. The kit of claim 16, further comprising an SP6, T3, or T7 polymerase buffer.

19. The kit of claim 16, further comprising ATP, CTP, GTP, and UTP.

20. The kit of claim 14, wherein the RNase III buffer comprises Tris and a salt.

21. The kit of claim 14, wherein the control nucleic acid is DNA and comprises an SP6, T3, or T7 promoter.

22. The kit of claim 14, wherein the control nucleic acid is dsRNA.

23. The kit of claim 14, wherein the control nucleic acid is a DNA template capable of being transcribed into a dsRNA.

24. The kit of claim 16, further comprising RNase A.

25. The kit of claim 14, further comprising a cartridge, column, or filter for isolating nucleic acids.

26. The kit of claim 25, further comprising binding buffer comprising NaCl.

27. The kit of claim 25, further comprising wash buffer comprising NaCl.

28. The kit of claim 25, further comprising an elution solution comprising Tris and EDTA.

29. A method for generating siRNA that can reduce expression of a target gene comprising incubating a dsRNA corresponding to part of the target gene with an effective amount of a composition comprising a polypeptide comprising an RNase III domain, under conditions to allow RNase III to cleave the dsRNA into siRNA.

30. The method of claim 29, wherein the polypeptide is chimeric.

31. The method of claim 29, further comprising isolating the siRNA molecules.

32. The method of claim 29, wherein the composition further comprises an RNase III buffer comprising Tris and a salt.

33. The method of claim 29, wherein the dsRNA is 25 to 10,000 bases or basepairs in length.

34. The method of claim 33, wherein the dsRNA is 25 to 5,000 bases or basepairs in length.

35. The method of claim 34, wherein the dsRNA is 50 to 1,000 bases or basepairs in length.

36. The method of claim 35, wherein the dsRNA is 100 to 200 bases or basepairs in length.

37. The method of claim 29, wherein dsRNA for at least a second targeted gene is included.