Methods and compositions for detecting nucleic acids

• US 20050186606A1
• Filed: 02/11/2005
• Published: 08/25/2005
• Est. Priority Date: 02/11/2004
• Status: Abandoned Application

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Inventors: Schroeder, Benjamin G.
• Application Number:
  US11/055,849
• Publication Number:
  US 20050186606A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6813 Hybridisation assays

Abstract

A reporter molecule for detecting a nucleic acid is disclosed. The molecule comprises an enzyme having a k_cat of at least about 200 sec⁻¹, a reversible inhibitor of the enzyme inhibitorily engaging the enzyme, and a nucleobase polymer extending between the enzyme and the inhibitor. The polymer interferes with the engagement between the inhibitor and the enzyme when the nucleic acid contacts the polymer. Methods of making and methods of using the reporter molecule are also disclosed.

Claims

1. A reporter molecule for detecting a nucleic acid, the molecule comprising:

an enzyme having a k_cat of at least about 200 sec<sup>−

1</sup>;

a reversible inhibitor of said enzyme inhibitorily engaging said enzyme; and

a nucleobase polymer extending between said enzyme and said reversible inhibitor;

wherein said polymer is operable to interfere with the engagement between said inhibitor and said enzyme when said nucleic acid contacts said polymer.

2. A reporter molecule according to claim 1 wherein said nucleic acid is selected from the group consisting of a miRNA and a siRNA.

3. A reporter molecule according to claim 1 wherein said enzyme is selected from the group consisting of alkaline phosphatase, β

-galactosidase, chloramphenicol acetyl transferase, β

-glucuronidase, renilla luciferase, firefly luciferase, and horseradish peroxidase.

4. A reporter molecule according to claim 3 wherein said enzyme is an alkaline phosphatase selected from the group consisting of bacterial alkaline phosphatase, shrimp alkaline phosphatase and a mammalian alkaline phosphatase.

5. A reporter molecule according to claim 1 wherein said reversible inhibitor of the enzyme is a transition state mimetic of a substrate of the enzyme.

6. A reporter molecule according to claim 1 wherein said reversible inhibitor of the enzyme is selected from the group consisting of phosphate, phosphonic acid, thiophosphate, vanadate, arsenate, L-phenylalanine, L- homoarginine, L-phenylalanine, levamisole, tetramisole, bromotetramisole, okadaic acid, theophylline, and mixtures thereof.

7. A reporter molecule according to claim 1 wherein said nucleobase polymer is selected from the group consisting of RNA, DNA, peptide nucleic acid, a 2′

-O-Methyl oligoribonucleic acid, and locked nucleic acid.

8. A reporter molecule according to claim 1 wherein said nucleobase polymer comprises at least about 10 bases, said 10 bases comprising a sequence at least about 80% complementary to a contiguous portion of said nucleic acid.

9. A reporter molecule according to claim 1 wherein said nucleobase polymer comprises at least about 10 bases, said 10 bases comprising a sequence about 100% complementary to a contiguous portion of said nucleic acid.

10. A reporter molecule according to claim 1 wherein said nucleobase polymer comprises a sequence of from about 20 to about 24 contiguous bases.

11. A method of detecting a nucleic acid in a sample, the method comprising:

contacting said sample with a reporter molecule for detecting said nucleic acid, wherein said reporter molecule comprises an enzyme having a k_cat of at least about 200 sec<sup>−

1</sup>, a reversible inhibitor of said enzyme inhibitorily engaging said enzyme; and

a nucleobase polymer extending between said enzyme and said reversible inhibitor, said polymer operable to interfere with the engagement between said inhibitor and said enzyme when said nucleic acid contacts said polymer; and

determining activity of said enzyme.

12. A method according to claim 11 wherein said nucleic acid is selected from the group consisting of a miRNA and a siRNA.

13. A method according to claim 11 wherein said enzyme is selected from the group consisting of an alkaline phosphatase, a β

-galactosidase, a chloramphenicol acetyl transferase, a β

-glucuronidase, a renilla luciferase, a firefly luciferase, and a horseradish peroxidase.

14. A method according to claim 13 wherein said enzyme is an alkaline phosphatase selected from the group consisting of a bacterial alkaline phosphatase, a shrimp alkaline phosphatase and a mammalian alkaline phosphatase.

15. A method according to claim 11 wherein said reversible inhibitor of the enzyme is selected from the group consisting of phosphate, phosphonic acid, thiophosphate, vanadate, arsenate, L-phenylalanine, L-homoarginine, L-phenylalanine, levamisole, tetramisole, bromotetramisole, okadaic acid, and theophylline.

16. A method according to claim 11 wherein said nucleobase polymer is selected from the group consisting of RNA, DNA, peptide nucleic acid, 2′

-O-Methyl oligoribonucleic acid, and locked nucleic acid.

17. A method according to claim 11 wherein said nucleobase polymer comprises a sequence of from about 20 to about 24 contiguous bases.

18. A method according to claim 11, further comprising contacting the reporter molecule with a substrate for said enzyme.

19. A method according to claim 18 wherein said substrate is selected from the group consisting of chromogenic substrate, fluorogenic substrate, radioactive substrate and chemiluminescent substrate.

20. A method of making a reporter molecule for detecting a nucleic acid comprising:

covalently attaching both an enzyme having a k_cat of at least about 200 sec<sup>−

1</sup> and a reversible inhibitor of said enzyme to a nucleobase polymer, wherein upon forming said reporter molecule, said reversible inhibitor is engaged to said enzyme inhibitorily and wherein said nucleic acid is operable to interfere with the engagement of said inhibitor and said enzyme upon contacting said polymer.

21. A method according to claim 20 wherein the nucleic acid is selected from the group consisting of a miRNA and a siRNA.

22. A method according to claim 20 wherein said enzyme is selected from the group consisting of alkaline phosphatase, β

-galactosidase, chloramphenicol acetyl transferase, β

-glucuronidase, renilla luciferase, firefly luciferase, and horseradish peroxidase.

23. A method according to claim 20 wherein said enzyme is an alkaline phosphatase selected from the group consisting of bacterial alkaline phosphatase, shrimp alkaline phosphatase and mammalian alkaline phosphatase.

24. A method according to claim 20 wherein said reversible inhibitor of the enzyme is selected from the group consisting of phosphate, phosphonic acid, thiophosphate, vanadate, arsenate, L-phenylalanine, L-homoarginine, L-phenylalanine, levamisole, tetramisole, bromotetramisole, okadaic acid, and theophylline.

25. A method according to claim 20 wherein said nucleobase polymer is selected from the group consisting of RNA, DNA, peptide nucleic acid, 2′

-O-Methyl oligoribonucleic acid, and a locked nucleic acid.

26. A method according to claim 20 wherein said nucleobase polymer comprises a sequence comprising at least about 10 bases, the sequence at least about 80% complementary to a contiguous portion of the nucleic acid.

27. A method according to claim 20 wherein said nucleobase polymer comprises a sequence comprising from about 20 to about 24 contiguous bases.

28. A method according to claim 20, further comprising linking said nucleobase polymer and said enzyme with a chemical linker.

29. A method according to claim 28 wherein said chemical linker comprises at least two reactive moieties, wherein each reactive moiety is independently selected from the group consisting of an amine-reactive moiety, a carboxyl-reactive moiety, a hydroxyl-reactive moiety, and a thiol-reactive moiety.

30. A method according to claim 20, further comprising reacting said enzyme with a chemical precursor of the polymer comprising a protein-reactive moiety.

31. A method according to claim 30 wherein said protein-reactive moiety is selected from the group consisting of an amine-reactive moiety, a carboxyl-reactive moiety, a hydroxyl-reactive moiety, and a thiol-reactive moiety.

32. A method for detecting a small RNA, said method comprising:

providing an enzyme having a k_cat of at least about 200 sec<sup>−

1</sup>;

tethering a reversible inhibitor enzyme to said small RNA;

hybridizing said small RNA with a complementary nucleotide;

determining enzyme activity produced by said hybridizing said small RNA with said complementary nucleotide; and

relating determined enzyme activity to a quantity of said small RNA.

33. A method according to claim 32, further comprising contacting said enzyme to a substrate.

34. A method according to claim 33, further comprising cleaving said enzyme from said substrate during said hybridizing said small RNA with a complementary nucleotide.

35. A method according to claim 34, further comprising producing a fluorescent or a chemiluminescent signal from said cleaving said enzyme from said substrate.

36. A method according to claim 32 wherein said small RNA is selected from the group comprising siRNA and miRNA.

37. A system for detecting a nucleic acid, said system comprising:

a reporter molecule for detecting said nucleic acid, wherein said reporter molecule comprises an enzyme having a k_cat of at least about 200 sec<sup>−

1</sup>, a reversible inhibitor of said enzyme inhibitorily engaging said enzyme; and

a nucleobase polymer extending between said enzyme and said reversible inhibitor, said polymer operable to interfere with the engagement between said inhibitor and said enzyme when said nucleic acid contacts said polymer; and

a substrate for said enzyme.

38. A system according to claim 37 wherein said enzyme is selected from the group consisting of alkaline phosphatase, β

-galactosidase, chloramphenicol acetyl transferase, β

-glucuronidase, renilla luciferase, firefly luciferase, and horseradish peroxidase.

39. A system according to claim 37 wherein said reversible inhibitor of the enzyme is selected from the group consisting of phosphate, phosphonic acid, thiophosphate, vanadate, arsenate, L-phenylalanine, L-homoarginine, L-phenylalanine, levamisole, tetramisole, bromotetramisole, okadaic acid, and theophylline.

40. A system according to claim 37 wherein said nucleobase polymer is selected from the group consisting of RNA, a DNA, peptide nucleic acid, 2′

-O-Methyl oligoribonucleic acid, and locked nucleic acid.

41. A method according to claim 37, wherein said substrate emits a signal which changes depending on whether the reporter molecule contacts said nucleic acid

42. A method according to claim 41 wherein said substrate is selected from the group consisting of chromogenic substrate, fluorogenic substrate, radioactive substrate and chemiluminescent substrate.

43. A system according to claim 41, further comprising a detection system detecting a signal from said enzyme.

44. A system according to claim 43, further comprising a microprocessor collecting and analyzing said signal.

45. A reporter molecule for detecting a nucleic acid, the molecule comprising:

an enzyme moiety having a k_cat of at least about 200 sec<sup>−

1</sup>;

a reversible inhibitor moiety, operable as a reversible inhibitor of said enzyme; and

a nucleobase polymer moiety having at least about 20 bases, covalently bonded to said enzyme moiety and said reversible inhibitor moiety;

wherein the said polymer is operable to allow said inhibitor moiety to reversibly inhibit said enzyme moiety, and to interferes with such inhibition when said nucleic acid contacts said polymer.

46. A reporter molecule according to claim 45 wherein said enzyme is selected from the group consisting of alkaline phosphatase, β

-galactosidase, chloramphenicol acetyl transferase, β

-glucuronidase, renilla luciferase, firefly luciferase, and horseradish peroxidase.

47. A reporter molecule according to claim 45 wherein said enzyme is an alkaline phosphatase selected from the group consisting of bacterial alkaline phosphatase, shrimp alkaline phosphatase and a mammalian alkaline phosphatase.

48. A reporter molecule according to claim 45 wherein said reversible inhibitor of the enzyme is selected from the group consisting of phosphate, phosphonic acid, thiophosphate, vanadate, arsenate, L-phenylalanine, L-homoarginine, L-phenylalanine, levamisole, tetramisole, bromotetramisole, okadaic acid, theophylline, and mixtures thereof.

49. A reporter molecule according to claim 45 wherein said nucleobase polymer is selected from the group consisting of RNA, DNA, peptide nucleic acid, a 2′

-O-Methyl oligoribonucleic acid, and locked nucleic acid.

50. A reporter molecule according to claim 45 wherein said nucleobase polymer comprises at least about 10 bases having a sequence at least about 80% complementary to a contiguous portion of said nucleic acid.

51. A reporter molecule according to claim 45 wherein said nucleobase polymer comprises a sequence of from about 20 to about 40 contiguous bases.