Nuclease inhibitor cocktail

• US 7,264,932 B2
• Filed: 02/25/2004
• Issued: 09/04/2007
• Est. Priority Date: 09/24/1999
• Status: Expired due to Term

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applera Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Latham, Gary J., Pasloske, Brittan L., Kudlicki, W. Antoni, Winkler, Matthew M.
• Primary Examiner(s): WHISENANT, ETHAN C
• Application Number:
  US10/786,875
• Publication Number:
  US 20050014169A1
• Time in Patent Office: 1,287 Days
• Field of Search: 435/6, 530/350, 530/387.1, 530/388.26
• US Class Current: 435/5
• CPC Class Codes: C07K 16/40 against enzymes
• CPC Class Codes: C12N 15/1003 Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
• CPC Class Codes: C12N 15/1096 cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
• CPC Class Codes: C12Q 1/34 involving hydrolase
• CPC Class Codes: G01N 2333/922 Ribonucleases (RNAses); Deoxyribonucleases (DNAses)
• CPC Class Codes: G01N 33/573 for enzymes or isoenzymes

Abstract

Methods and compositions for inhibiting and/or inactivating nucleases by using nuclease inhibitors are provided. The nuclease inhibitors comprise anti-nuclease antibodies and non-antibody nuclease inhibitors.

Claims

1. A method comprising:

a) obtaining at least a first nuclease inhibitor, wherein the first nuclease inhibitor is a RNase inhibitor protein or an anti-nuclease antibody;

b) obtaining at least a second nuclease inhibitor wherein the second nuclease inhibitor is oligovinylsulfonic acid (OVA), aurintricarboxylic acid (ATA), aflatoxin, 2-nitro-5-thiocyanobenzoic acid, iodoacetate, N-bromosuccinimide, p-chloromercuribenzoate, dinitrofluorobenzene, decanavanate, polyvinylsufonic acid, hydrobenzoinphosphate, phenyiphosphate, putrescine, haloacetate, dinitrofluorobenzene, phenyiglyoxal, bromopyruvic, 8-amino-5-(4′

-hydroxy-biphenyl-4-ylazo)-naphthalene-2-sulfonate, 6-hydroxy-5-(2-hydroxy-3,5-dinitro-phenylazo)-naphthalene-2-sulfonate, 3,3′

-dimethylbiphenyl-4,4′

-bis(2-amino-naphthylazo-6-sulfonate), 4,4′

-dicarboxy-3,3-bis(naphthylamido)-diphenylmethanone, 3,3′

-dicarboxy-4,4′

-bis(4-biphenylamido)diphenylmethane, 3,3′

-dicarboxy-4,4′

-bis(3-nitrophenylamido)diphenylmethane or NCI-224131;

c) obtaining a composition; and

d) admixing the first nuclease inhibitor, the second nuclease inhibitor and the composition to form an admixture;

wherein nucleases that may be present in the admixture are inhibited.

2. The method of claim 1, wherein the RNase inhibitor protein is obtained from a human, a chimpanzee, a rat, a mouse, a pig, yeast, or by recombinant means, or derivatives therein.

3. A method of performing an in vitro translation, transcription, reverse transcription or coupled transcription/translation reaction comprising obtaining a composition, the composition comprising the first nuclease inhibitor and a second nuclease inhibitor of claim 1 and placing the composition in an in vitro translation reaction, transcription reaction, reverse transcription reaction or a coupled transcription/translation reaction.

4. A solution comprising the first nuclease inhibitor and the second nuclease inhibitor of claim 1.

5. A kit comprising the first nuclease inhibitor and the second nuclease inhibitor of claim 1 and components for RNA isolation, an in vitro translation reaction, a reverse transcriptase reaction, an RNA amplification reaction, DNA removal, or in vitro transcription.

6. A method comprising:

a) obtaining at least a first nuclease inhibitor, wherein the first nuclease inhibitor is a RNase inhibitor protein;

b) obtaining at least a second nuclease inhibitor wherein the second nuclease inhibitor is purine, pyrimidine, cytidine-N3-oxide 2′

-phosphate, 2′

CMP, ppAp, Ap3A, Ap4A, Ap5A, ATP, 5′

AMP, 5′

ADP, 3′

UMP, 2′

UMP, 2′

CMP, pAp (5′

P-A-3′

P), dUppAp, dUppA2′

p, pdUppAp, pTp, pTppAp, TpdA, TppdA, 4-thiouridine 3′

p, 5-nitro-uracil, 5-aminoethyl-uracil or (Bromoacetamido)nucleoside;

c) obtaining a composition; and

d) admixing the first nuclease inhibitor, the second nuclease inhibitor and the composition to form an admixture;

wherein nucleases that may be present in the admixture are inhibited.

7. A method of performing an in vitro translation, transcription, reverse transcription or coupled transcription/translation reaction comprising obtaining a composition, the composition comprising the first nuclease inhibitor and a second nuclease inhibitor of claim 6 and placing the composition in an in vitro translation reaction, transcription reaction, reverse transcription reaction or a coupled transcription/translation reaction.

8. A solution comprising the first nuclease inhibitor and the second nuclease inhibitor of claim 6.

9. A kit comprising the first nuclease inhibitor and the second nuclease inhibitor of claim 6 and components for RNA isolation, an in vitro translation reaction, a reverse transcriptase reaction, an RNA amplification reaction, DNA removal, or in vitro transcription.

10. A method comprising:

a) obtaining at least a first nuclease inhibitor, wherein the first nuclease inhibitor is an anti-RNase antibody or a RNase inhibitor protein;

b) obtaining at least a second nuclease inhibitor wherein the second nuclease inhibitor is SCN⁻

, Li⁺, ClO₄⁻

, or guanidinium;

c) obtaining a composition; and

d) admixing the first nuclease inhibitor, the second nuclease inhibitor and the composition to form an admixture;

wherein nucleases that may be present in the admixture are inhibited.

11. A method of performing an in vitro translation, transcription, reverse transcription or coupled transcription/translation reaction comprising obtaining a composition, the composition comprising the first nuclease inhibitor and a second nuclease inhibitor of claim 10 and placing the composition in an in vitro translation reaction, transcription reaction, reverse transcription reaction or a coupled transcription/translation reaction.

12. A solution comprising the first nuclease inhibitor and the second nuclease inhibitor of claim 10.

13. A kit comprising the first nuclease inhibitor and the second nuclease inhibitor of claim 10 and components for RNA isolation, an in vitro translation reaction, a reverse transcriptase reaction, an RNA amplification reaction, DNA removal, or in vitro transcription.

14. A method comprising:

a) obtaining at least a first nuclease inhibitor, wherein the first nuclease inhibitor is an anti-RNase antibody or a RNase inhibitor protein;

b) obtaining at least a second nuclease inhibitor wherein the second nuclease inhibitor is an RNA or DNA oligonucleotide;

c) obtaining a composition; and

d) admixing the first nuclease inhibitor, the second nuclease inhibitor and the composition to form an admixture;

wherein nucleases that may be present in the admixture are inhibited.

15. A method of performing an in vitro translation, transcription, reverse transcription or coupled transcription/translation reaction comprising obtaining a composition, the composition comprising the first nuclease inhibitor and a second nuclease inhibitor of claim 14 and placing the composition in an in vitro translation reaction, transcription reaction, reverse transcription reaction or a coupled transcription/translation reaction.

16. A solution comprising the first nuclease inhibitor and the second nuclease inhibitor of claim 14.

17. A kit comprising the first nuclease inhibitor and the second nuclease inhibitor of claim 14 and components for RNA isolation, an in vitro translation reaction, a reverse transcriptase reaction, an RNA amplification reaction, DNA removal, or in vitro transcription.

18. A method comprising:

a) obtaining at least a first nuclease inhibitor, wherein the first nuclease inhibitor is an anti-RNase antibody or a RNase inhibitor protein;

b) obtaining at least a second nuclease inhibitor wherein the second nuclease inhibitor is an aptamer, a competitive inhibitor comprising a ribonucleoside, a deoxyribonucleoside, a dideoxyribonucleoside, a thiol-containing RNA, or a DNP-poly(A);

c) obtaining a composition; and

d) admixing the first nuclease inhibitor, the second nuclease inhibitor and the composition to form an admixture;

wherein nucleases that may be present in the admixture are inhibited.

19. A method of performing an in vitro translation, transcription, reverse transcription or coupled transcription/translation reaction comprising obtaining a composition, the composition comprising the first nuclease inhibitor and a second nuclease inhibitor of claim 18 and placing the composition in an in vitro translation reaction, transcription reaction, reverse transcription reaction or a coupled transcription/translation reaction.

20. A solution comprising the first nuclease inhibitor and the second nuclease inhibitor of claim 18.

21. A kit comprising the first nuclease inhibitor and the second nuclease inhibitor of claim 18 and components for RNA isolation, an in vitro translation reaction, a reverse transcriptase reaction, an RNA amplification reaction, DNA removal, or in vitro transcription.

22. A method comprising:

a) obtaining at least a first nuelease inhibitor, wherein the first nuclease inhibitor is an anti-nuclease antibody;

b) obtaining at least a second nuelease inhibitor wherein the second nuclease inhibitor comprises a non-proteinaceous polycyclic aromatic structure;

c) obtaining a composition; and

d) admixing the first nuclease inhibitor, the second nuclease inhibitor and the composition to form an admixture;

wherein nucleases that may be present in the admixture are inhibited.

23. The method of claim 22, wherein the aromatic structure is:

24. The method of claim 22, wherein the polycyclic aromatic structure is:

25. The method of claim 22, wherein the second nuclease inhibitor comprises the following structure:

26. A method of performing an in vitro translation, transcription, reverse transcription or coupled transcription/translation reaction comprising obtaining a composition, the composition comprising the first nuclease inhibitor and a second nuclease inhibitor of claim 22 and placing the composition in an in vitro translation reaction, transcription reaction, reverse transcription reaction or a coupled transcription/translation reaction.

27. A solution comprising the first nuclease inhibitor and the second nuclease inhibitor of claim 22.

28. A kit comprising the first nuclease inhibitor and the second nuclease inhibitor of claim 22 and components for RNA isolation, an in vitro translation reaction, a reverse transcriptase reaction, an RNA amplification reaction, DNA removal, or in vitro transcription.

29. The method of claim 22, wherein the second nuclease inhibitor comprises a structure selected from the group consisting of NCI-65828, NCI 65845, benzopurpurin B, NCI-65841, NCI 79596, NCI-9617, NCI-16224, suramin, direct red 1, NCI-7815, NCI-45618, NCI-47740, prBZBP, NCI-65568, NCI-79741, NCI-65820, NCI-65553, NCI-58047, NCI-65847, xylidene ponceau 2R, eriochrome black T, amaranth, new coccine, acid red 37, acid violet 7, NCI-45608, NCI-75661, NCI-73416, NCI-724225, orange G, NCI 47755, sunset yellow, NCI-47735, NCI-37176, violamine R, NCI-65844, direct red 13, NCI-45601, NCI 75916, NCI-65546, NCI-65855, NCI-75963, NCI-45612, NCI-8674, NCI-75778, NCI-34933, NCI-1698, NCI-7814, NCI-45550, NCI-77521, cefsulodin, NCI-174066, NCI-12455, NCI-45541, NCI-79744, NCI-42067, NCI-45571, NCI-45538, NCI-45540, NCI-9360, NCI-12857, NCI-D726712, NCI-45542, NCI-7557, S321443, NCI-45557, NCI-1741, NCI-1743, NCI-227726, NCI-16163, NCI-16169, NCI-88947, NCI-17061, NCI-37169, beryllon II, CB-0181431, CB-473872, JLJ-1, JLJ-2, JLJ-3, CB-467929, CB-534510, CB-540408, CB-180582, CB-180553, CB-186847, CB-477474, CB-152591, NCI-37136, NCI-202516, CB-039263, CB-181145, CB-181429, CB-205125, and CB-224197.

30. The method of claim 29, wherein the second nuclease inhibitor is NCI-65828.

31. The method of claim 30, wherein the second nuclease inhibitor is a derivative of NCI-65828.

32. The method of claim 31, wherein the derivative of NCI-65828 comprises at least one modification selected from the group consisting of:

a reduction of the azo to hydrazido, replacement of the azo by an amide, an attachment of a hydroxyl group to position 6 of the naphthalene ring, an attachment of an electron-withdrawing group to position 6 of the naphthalene ring, replacement of a carbon atom in an aromatic ring with a nitrogen or an oxygen, and a replacement of the hydroxyl group on the biphenyl component with a sulfonate.

33. The method of claim 29, wherein the second nuclease inhibitor is CB-473872.

34. The method of claim 33, wherein the second nuclease inhibitor is a derivative of CB-473872.

35. The method of claim 34, wherein the derivative of CB-473872 comprises an addition of at least one of a hydrogen-bonding group selected from the consisting of:

a hydroxyl, an amino, a methyldiamino, a hydroxyethyl, an ethyl-N-formamido, a carboxyamido, a carboxy, a 2-oxo-N-piperidinyl, and a p-benzoyl.

36. The method of claim 34, wherein the derivative of CB-473872 comprises Structure II or Structure III, and wherein:

R₀ is —

H, —

NH₂, or —

OH;

R₃ is —

H, —

CH₂OH, or CONH₂;

R₄ is —

H, —

COOH, or 2-oxo-N-piperidinyl;

R₅ is —

H or p-benzoyl group.

37. The method of claim 34, wherein the derivative of CB-473872 comprises a replacement of a carbon atom in an aromatic ring with a nitrogen or an oxygen.

38. The method of claim 31, wherein the derivative of NCI-65828 comprises at least one modification selected from the group consisting of:

an addition of a hydrogen-bonding group and substitution of a hydroxyl group with an anionic group to the biphenyl component.

39. The method of claim 38, wherein the hydrogen-bonding group is selected from the group consisting of a hydroxyl, an amino, and an amide.

40. The method of claim 38, wherein the anion is selected from the group consisting of a carboxylate, a sulfate, a sulfonate, a phosphate, and a phosphonate.

41. A method comprising

a) obtaining at least a first nuclease inhibitor, wherein the first nuclease inhibitor is benzopurpurin B;

b) obtaining at least a second nuclease inhibitor wherein the second nuclease inhibitor is an anti-nuclease antibody or a RNase inhibitor protein;

c) obtaining a composition; and

d) admixing the first nuclease inhibitor, the second nuclease inhibitor and the composition to form an admixture;

wherein nucleases that may be present in the admixture are inhibited.

42. A method of performing an in vitro translation, transcription, reverse transcription or coupled transcription/translation reaction comprising obtaining a composition, the composition comprising the first nuclease inhibitor and a second nuclease inhibitor of claim 41 and placing the composition in an in vitro translation reaction, transcription reaction, reverse transcription reaction or a coupled transcription/translation reaction.

43. A solution comprising the first nuclease inhibitor and the second nuclease inhibitor of claim 41.

44. A kit comprising the first nuclease inhibitor and the second nuclease inhibitor of claim 41 and components for RNA isolation, an in vitro translation reaction, a reverse transcriptase reaction, an RNA amplification reaction, DNA removal, or in vitro transcription.

45. A method comprising:

a) obtaining at least a first nuclease inhibitor, wherein the first nuclease inhibitor is cytidine-N3-oxide 2′

-phosphate, 2′

CMP, ppAp, Ap3A, Ap4A, Ap5A, ATP, 5′

AMP, 5′

ADP, 3′

UMP, 2′

UMP, 2′

CMP, pAp (5′

P-A-3′

P), dUppAp, dUppA2′

p, pdUppAp, pTp, pTppAp, TpdA, TppdA, 4-thiouridine 3′

p, 5-nitro-uracil, 5-aminoethyl-uracil or (Bromoacetamido)nucleoside;

b) obtaining at least a second nuclease inhibitor wherein the second nuclease inhibitor comprises an anti-nuclease antibody;

c) obtaining a composition; and

d) admixing the first nuclease inhibitor, the second nuclease inhibitor and the composition to form an admixture;

wherein nucleases that may be present in the admixture are inhibited.

46. The method of claim 45, wherein the antibody is a soluble anti-nuclease antibody.

47. The method of claim 45, wherein the antibody is an anti-RNase antibody.

48. The method of claim 47, wherein the anti-RNase antibody is an anti-RNase T1 antibody or an anti-RNase 1 antibody.

49. A method of performing an in vitro translation, transcription, reverse transcription or coupled transcription/translation reaction comprising obtaining a composition, the composition comprising the first nuclease inhibitor and a second nuclease inhibitor of claim 45 and placing the composition in an in vitro translation reaction, transcription reaction, reverse transcription reaction or a coupled transcription/translation reaction.

50. A solution comprising the first nuclease inhibitor and the second nuclease inhibitor of claim 45.

51. A kit comprising the first nuclease inhibitor and the second nuclease inhibitor of claim 45 and components for RNA isolation, an in vitro translation reaction, a reverse transcriptase reaction, an RNA amplification reaction, DNA removal, or in vitro transcription.

52. A method comprising,

a) obtaining at least a first nuclease inhibitor, wherein the first nuclease inhibitor comprises a non-proteinaceous polycyclic aromatic structure;

b) obtaining at least a second nuclease inhibitor wherein the second nuclease inhibitor is a RNase inhibitor protein;

c) obtaining a composition; and

d) admixing the first nuclease inhibitor, the second nuclease inhibitor and the composition to form an admixture;

wherein nucleases that may be present in the admixture are inhibited.

53. A method of performing an in vitro translation, transcription, reverse transcription or coupled transcription/translation reaction comprising obtaining a composition, the composition comprising the first nuclease inhibitor and a second nuclease inhibitor of claim 52 and placing the composition in an in vitro translation reaction, transcription reaction, reverse transcription reaction or a coupled transcription/translation reaction.

54. A solution comprising the first nuclease inhibitor and the second nuclease inhibitor of claim 52.

55. A kit comprising the first nuclease inhibitor and the second nuclease inhibitor of claim 52 and components for RNA isolation, an in vitro translation reaction, a reverse transcriptase reaction, an RNA amplification reaction, DNA removal, or in vitro transcription.

56. The method of claim 52, wherein the aromatic structure is:

57. The method of claim 52, wherein the aromatic structure is:

58. The method of claim 52, wherein the aromatic structure is:

59. The method of claim 52, wherein the aromatic structure is selected from the group consisting of NCI-65828, NCI 65845, benzopurpurin B, NCI-65841, NCI 79596, NCI-9617, NCI-16224, suramin, direct red 1, NCI-7815, NCI-45618, NCI-47740, prBZBP, NCI-65568, NCI-79741, NCI-65820, NCI-65553, NCI-58047, NCI-65847, xylidene ponceau 2R, eriochrome black T, amaranth, new coccine, acid red 37, acid violet 7, NCI-45608, NCI-75661, NCI-73416, NCI-724225, orange G, NCI 47755, sunset yellow, NCI-47735, NCI-37176, violamine R, NCI-65844, direct red 13, NCI-45601, NCI 75916, NCI-65546, NCI-65855, NCI-75963, NCI-45612, NCI-8674, NCI-75778, NCI-34933, NCI-1698, NCI-7814, NCI-45550, NCI-77521, cefsulodin, NCI-174066, NCI-12455, NCI-45541, NCI-79744, NCI-42067, NCI-45571, NCI-45538, NCI-45540, NCI-9360, NCI-12857, NCI-D726712, NCI-45542, NCI-7557, S321443, NCI-45557, NCI-1741, NCI-1743, NCI-227726, NCI-16163, NCI-16169, NCI-88947, NCI-17061, NCI-37169, beryllon II, CB-0181431, CB-473872, JLJ-1, JLJ-2, JLJ-3, CB-467929, CB-534510, CB-540408, CB-180582, CB-180553, CB-186847, CB-477474, CB-152591, NCI-37136, NCI-202516, CB-039263, CB-181145, CB-181429, CB-205125, and CB-224197.

60. The method of claim 59, wherein the aromatic structure is NCI-65828.

61. The method of claim 60, wherein the aromatic structure is a derivative of NCI-65828.

62. The method of claim 61, wherein the derivative of NCI-65828 comprises at least one modification selected from the group consisting of:

a reduction of the azo to hydrazido, replacement of the azo by an amide, an attachment of a hydroxyl group to position 6 of the naphthalene ring, an attachment of an electron-withdrawing group to position 6 of the naphthalene ring, replacement of a carbon atom in an aromatic ring with a nitrogen or an oxygen, and a replacement of the hydroxyl group on the biphenyl component with a sulfonate.

63. The method of claim 61, wherein the derivative of NCI-65828 comprises at least one modification selected from the group consisting of:

an addition of a hydrogen-bonding group and substitution of a hydroxyl group with an anionic group to the biphenyl component.

64. The method of claim 63, wherein the hydrogen-bonding group is selected from the group consisting of a hydroxyl, an amino, and an amide.

65. The method of claim 63, wherein the anion is selected from the group consisting of a carboxylate, a sulfate, a sulfonate, a phosphate, and a phosphonate.

66. The method of claim 59, wherein the aromatic structure is CB-473872.

67. The method of claim 66, wherein the aromatic structure is a derivative of CB-473872.

68. The method of claim 67, wherein the derivative of CB-473872 comprises an addition of at least one of a hydrogen-bonding group selected from the consisting of:

a hydroxyl, an amino, a methyldiamino, a hydroxyethyl, an ethyl-N-formamido, a carboxyamido, a carboxy, a 2-oxo-N-piperidinyl, and a p-benzoyl.

69. The method of claim 67, wherein the derivative of CB-473872 comprises Structure II or Structure III, and wherein:

R₀ is —

H, —

NH₂, or —

OH;

R₃ is —

H, —

CH₂OH, or CONH₂;

R₄ is —

H, —

COOH, or 2-oxo-N-piperidinyl;

R₅ is —

H or p-benzoyl group.

70. The method of claim 67, wherein the derivative of CB-473872 comprises a replacement of a carbon atom in an aromatic ring with a nitrogen or an oxygen.

71. A method comprising:

a) obtaining at least a first nuclease inhibitor, wherein the first nuclease inhibitor is a a salt, wherein the salt is NaCitrate;

b) obtaining at least a second nuclease inhibitor wherein the second nuclease inhibitor comprises an anti-RNase antibody or an RNase inhibitor protein;

c) obtaining a composition; and

d) admixing the first nuclease inhibitor, the second nuclease inhibitor and the composition to form an admixture;

wherein nucleases that may be present in the admixture are inhibited.

72. A method of performing an in vitro translation, transcription, reverse transcription or coupled transcription/translation reaction comprising obtaining a composition, the composition comprising the first nuclease inhibitor and a second nuclease inhibitor of claim 71 and placing the composition in an in vitro translation reaction, transcription reaction, reverse transcription reaction or a coupled transcription/translation reaction.

73. A solution comprising the first nuclease inhibitor and the second nuclease inhibitor of claim 71.

74. A kit comprising the first nuclease inhibitor and the second nuclease inhibitor of claim 71 and components for RNA isolation, an in vitro translation reaction, a reverse transcriptase reaction, an RNA amplification reaction, DNA removal, or in vitro transcription.

75. A method comprising:

a) obtaining at least a first nuclease inhibitor, wherein the first nuclease inhibitor is an RNA or DNA oligonucleotide;

b) obtaining at least a second nuclease inhibitor;

c) obtaining a composition; and

d) admixing the first nuclease inhibitor, the second nuclease inhibitor and the composition to form an admixture;

wherein nucleases that may be present in the admixture are inhibited.

76. A method comprising:

a) obtaining at least a first nuclease inhibitor, wherein the first nuclease inhibitor is an RNA or DNA oligonucleotide, an aptamer, or a competitive inhibitor comprising a deoxyribonucleoside, a dideoxyribonucleoside, a thiol-containing RNA, or a DNP-poly(A).

b) obtaining at least a second nuclease inhibitor;

c) obtaining a composition; and

d) admixing the first nuclease inhibitor, the second nuclease inhibitor and the composition to form an admixture;

wherein nucleases that may be present in the admixture are inhibited.

77. A method comprising:

a) obtaining at least a first nuclease inhibitor, wherein the first nuclease inhibitor is an affinity resin;

b) obtaining at least a second nuclease inhibitor;

c) obtaining a composition; and

d) admixing the first nuclease inhibitor, the second nuclease inhibitor and the composition to form an admixture;

wherein nucleases that may be present in the admixture are inhibited.

78. The method of claim 77, wherein the affinity resin is sulfopropyl sepharose or SP sulfopropyl cation exchange resin.