Multi-Component Inhibitors of Nucleic Acid Polymerases

• US 20100209975A1
• Filed: 12/11/2009
• Published: 08/19/2010
• Est. Priority Date: 01/28/2005
• Status: Active Grant

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: BISHOP, John D., LEE, Jun E.
• Granted Patent: US 9,637,551 B2
• Time in Patent Office: Days
• US Class Current: 435/91.500
• CPC Class Codes: C07K 16/40 against enzymes
• CPC Class Codes: C07K 2317/24 containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• CPC Class Codes: C07K 2317/76 Antagonist effect on antigen, e.g. neutralization or inhibition of binding
• CPC Class Codes: C12N 9/93 Ligases (6)
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6848 characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
• CPC Class Codes: C12Q 2521/101 DNA polymerase
• CPC Class Codes: C12Q 2521/119 RNA polymerase
• CPC Class Codes: C12Q 2527/127 the enzyme inhibitor or activator used

Abstract

The present invention provides multi-component inhibitors of nucleic acid polymerases, methods of making, and methods of using same. One component of the multi-component inhibitor is a molecule that binds to a polymerase (i.e., a polymerase-binding molecule (PBM)), but does not thereby substantially inhibit its polymerase activity. Another component is a molecule or complex of molecules that binds to a PBM (i.e., a PBM-binding molecule). The combination of the PBM and PBM-binding molecule/complex substantially inhibits polymerase activity.

Claims

1. A composition comprising:

(a) a nucleic acid polymerase;

(b) a polymerase-binding molecule (PBM) that binds to said polymerase and does not substantially inhibit the polymerase activity of said polymerase; and

(c) a PBM-binding molecule/complex that binds to said PBM, wherein binding of the PBM and PBM-binding molecule or complex together substantially inhibits the polymerase activity of said polymerase.

2. The composition of claim 1, wherein said PBM is an antibody (PBA).

3. The composition of claim 2, wherein said PBA is a monoclonal antibody.

4. The composition of claim 1, wherein said PBM-binding molecule/complex comprises an antibody, protein G, protein A, a derivatized antibody, a derivatized protein G, a derivatized protein A, IgG, a derivatized protein G, sir22, or sibA.

5. The composition of claim 1, wherein said PBM-binding molecule/complex comprises a detectable label, protein, or a polymer.

6. The composition of claim 5, wherein said detectable label is rhodamine, biotin, fluorescein, horseradish peroxidase, alkaline phosphatase, and AlexaFluor488.

7. The composition of claim 5, wherein said protein is horseradish peroxidase, alkaline phosphatase, and albumin.

8. The composition of claim 5, wherein said polymer is a polyethylene glycol, a polyoxyethylene, a polyoxypropylene, and a polyoxyethylene/polyoxyethylene copolymer.

9. The composition of claim 3, wherein said antibody is selected from the group consisting of (a) IgA;

(b) IgG;

(c) IgM;

(d) IgD;

(e) IgE;

(f) IgY;

(g) a fragment of any of (a)-(f);

(h) a derivative of any of (a)-(f);

(i) a derivatized fragment of any of (a)-(h); and

(j) a complex of any of (a)-(i).

10. The composition of claim 3, wherein said PBM-binding molecule/complex comprises a monoclonal antibody, polyclonal antibody, Fc antibody fragment, chimeric antibody or recombinant antibody.

11. The composition of claim 10, wherein said antibody is derivatized with a detectable label, protein, or polymer.

12. The composition of claim 11, wherein said detectable label is rhodamine, biotin, fluorescein, horseradish peroxidase, alkaline phosphatase, or AlexaFluor488.

13. The composition of claim 11, wherein said protein is horseradish peroxidase, alkaline phosphatase, or albumin.

14. The composition of claim 11, wherein said polymer is a polyethylene glycol, a polyoxyethylene, a polyoxypropylene, or a polyoxyethylene/polyoxypropylene copolymer.

15. The composition of claim 3, wherein said PBM-binding molecule/complex is selected from the group consisting of a goat anti-mouse IgG antibody coupled to horseradish peroxidase, a complex of a goat anti-mouse IgG antibody and protein G-horseradish peroxidase, protein G-AlexaFluor488, a goat anti-mouse IgG antibody, a complex of streptavidin and an antibody to mouse IgG coupled to biotin, and Protein G.

16. The composition of claim 1, wherein said nucleic acid polymerase is a DNA-dependent DNA polymerase or an RNA-dependent DNA polymerase.

17. The composition of claim 16, wherein said polymerase is thermolabile.

18. The composition of claim 16, wherein said polymerase is thermostable.

19. The composition of claim 18, wherein said polymerase is Thermus aquaticus (Taq), Thermus filiformis (Tfi), Tfi strain RT41A, Pyrococcus furiosus (Pfu), Thermococcus zilligi (Tzi), or a mutant of any of the above.

20. The composition of claim 18, wherein said is recombinant.

21. A composition comprising:

(a) a thermostable nucleic acid polymerase;

(b) a polymerase-binding antibody (PBA) that binds to said polymerase and does not substantially inhibit the polymerase activity of said polymerase; and

(c) a PBA-binding molecule/complex that binds to said PBA, wherein binding of the PBA and PBA-binding molecule/complex together substantially inhibits the polymerase activity of said polymerase at a temperature less than about 40°

C., and wherein binding of the PBA and PBA-binding molecule/complex together does not substantially inhibit the polymerase activity of said polymerase at a temperature greater than about 40°

C.

22. The composition of claim 21, wherein said nucleic acid polymerase is a DNA-dependent DNA polymerase or an RNA-dependent DNA polymerase.

23. The composition of claim 22, wherein said polymerase is Thermus aquaticus (Taq), Thermus filiformis (Tfi), Tfi strain RT41A, Pyrococcus furiosus (Pfu), Thermococcus zilligi (Tzi), or a mutant of any of the above.

24. The composition of claim 22, wherein said polymerase is recombinant.

25. A method of inhibiting the polymerase activity of a nucleic acid polymerase comprising contacting said polymerase with a PBM and a PBM-binding molecule/complex, wherein the binding of said PBM does not substantially inhibit polymerase activity of said polymerase, and wherein the binding of said PBM and said PBM-binding molecule/complex together substantially inhibit the polymerase activity of said polymerase.

26. The method of claim 25, wherein said inhibition is reversible.

27. The method of claim 25, wherein said inhibition is reversible by heating to a temperature of at least about 40°

C.

28. The method of claim 25, wherein said PBM is an antibody (PBA).

29. The method of claim 28, wherein said PBA is a monoclonal antibody.

30. The method of claim 25, wherein said nucleic acid polymerase is a DNA-dependent DNA polymerase or an RNA-dependent DNA polymerase.

31. The method of claim 30, wherein said polymerase is Thermus aquaticus (Taq), Thermus filiformis (Tfi), Tfi strain RT41A, Pyrococcus furiosus (Pfu), Thermococcus zilligi (Tzi), or a mutant of any of the above.

32. The method of claim 30, wherein said polymerase is recombinant.

33. A method for synthesizing a nucleic acid molecule, comprising:

(a) contacting a template nucleic acid with a composition comprising a thermostable nucleic acid polymerase, one or more nucleoside and/or deoxynucleoside triphosphates, a polymerase binding molecule (PBM) that does not substantially inhibit the polymerase activity of the polymerase, and a PBM-binding molecule/complex under conditions where the PBM and the PBM-binding molecule/complex together substantially inhibit the polymerase activity of the polymerase; and

(b) bringing the resulting mixture to a temperature sufficient relieve polymerase inhibition.

34. The method of claim 33, wherein said PBM is an antibody (PBA).

35. The method of claim 34, wherein said PBA is a monoclonal antibody.

36. The method of claim 35, wherein said inhibition is reversible by heating to a temperature of at least about 40°

C.

37. The method of claim 33, wherein said nucleic acid polymerase is a DNA-dependent DNA polymerase or an RNA-dependent DNA polymerase.

38. The method of claim 37, wherein said polymerase is Thermus aquaticus (Taq), Thermus filiformis (Tfi), Tfi strain RT41A, Pyrococcus furiosus (Pfu), Thermococcus zilligi (Tzi), or a mutant of any of the above.

39. The method of claim 37, wherein said nucleic acid polymerase is recombinant.