Nuclease inhibitor cocktail
View Patent ↗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.
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 + , ClO 4 − , 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:
R 0 is —H, —NH 2 , or —OH;
R 3 is —H, —CH 2 OH, or CONH 2 ;
R 4 is —H, —COOH, or 2-oxo-N-piperidinyl;
R 5 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:
R 0 is —H, —NH 2 , or —OH;
R 3 is —H, —CH 2 OH, or CONH 2 ;
R 4 is —H, —COOH, or 2-oxo-N-piperidinyl;
R 5 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.