Multi-component inhibitors of nucleic acid polymerases

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.

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.