Multi-component inhibitors of nucleic acid polymerases
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.
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 - 9 . (canceled)
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 - 17 . (canceled)
18 . The composition of claim 1 , wherein said polymerase is thermostable.
19 . (canceled)
20 . The composition of claim 18 , wherein said polymerase 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 - 23 . (canceled)
24 . The composition of claim 21 , 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 - 31 . (canceled)
32 . The method of claim 25 , 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 - 38 . (canceled)
39 . The method of claim 33 , wherein said nucleic acid polymerase is recombinant.