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. The disclosed multi-component inhibitors are useful for DNA sequencing, nucleic acid amplification, cloning and synthesis, and the like.

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 or complex or molecules 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 antibody is a monoclonal antibody. 
   
   
       4 . The composition of  claim 1 , wherein said PBM-binding molecule/complex is selected from the group consisting of: an antibody; protein G; protein A; a derivatized antibody; a derivatized protein G; a derivatized protein A; IgG and a derivatized protein G; sir22; sibA; and a complex comprising any one of the foregoing. 
   
   
       5 . The composition of  claim 2 , wherein said derivatized PBM-binding molecule or complex of molecules comprises a moiety selected from the group consisting of: a detectable label; a protein; and a polymer. 
   
   
       6 . The composition of  claim 5 , wherein said detectable label is selected from the group consisting of: rhodamine; biotin; fluorescein; horseradish peroxidase; alkaline phosphatase;
 and AlexaFluor488.   
   
   
       7 . The composition of  claim 5 , wherein said protein is selected from the group consisting of: horseradish peroxidase; alkaline phosphatase; and albumin. 
   
   
       8 . The composition of  claim 5 , wherein said polymer is selected from the group consisting of: a polyethylene glycol; a polyoxyethylene; a polyoxypropylene; and a polyoxyethylene/polyoxyethylene copolymer. 
   
   
       9 . The composition of  claim 2 , 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 2 , wherein said PBM-binding molecule/complex is a derivatized or underivatized: monoclonal antibody, polyclonal antibody, Fc antibody fragment, chimeric antibody or recombinant antibody. 
   
   
       11 . The composition of  claim 10 , wherein said derivatized antibody is derivatized by attachment of a moiety selected from the group consisting of: a detectable label; a protein; and a polymer. 
   
   
       12 . The composition of  claim 11 , wherein said detectable label is selected from the group consisting of: rhodamine; biotin; fluorescein; horseradish peroxidase; alkaline phosphatase; and AlexaFluor488. 
   
   
       13 . The composition of  claim 11 , wherein said protein is selected from the group consisting of: horseradish peroxidase; alkaline phosphatase; and albumin. 
   
   
       14 . The composition of  claim 11 , wherein said polymer is selected from the group consisting of: a polyethylene glycol; a polyoxyethylene; a polyoxypropylene; and a polyoxyethylene/polyoxypropylene copolymer. 
   
   
       15 . The composition of  claim 2 , wherein said PBM-binding molecule or complex of molecules 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 selected from the group consisting of: a DNA-dependent DNA polymerase; and 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 selected from the group consisting of:  Thermus aquaticus  (Tag);  Thermus thermophilus  (Tth);  Thermus filiformis  (Tfi);  Thermus flavus  (Tfl);  Pyrococcus furiosus  (Pfu);  Thermococcus litoralis  (Tli);  Thermococcus zilligi  (Tzi);  Thermatoga neopolitana  (Tne);  Thermatoga maritime  (Tma); VENT®; DEEPVENT®; THERMOSCRIPT®; SUPERSCRIPT I®; SUPERSCRIPT II®; SUPERSCRIPT III®; and a mutant of any of the above. 
   
   
       20 . The composition of  claim 18 , wherein said nucleic acid 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 or complex or molecules that binds to said PBA, wherein binding of the PBA and PBA-binding molecule or 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 or 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 selected from the group consisting of: a DNA-dependent DNA polymerase; and an RNA-dependent DNA polymerase. 
   
   
       23 . The composition of  claim 22 , wherein said nucleic acid polymerase is selected from the group consisting of:  Thermus aquaticus  (Taq);  Thermus thermophilus  (Tth);  Thermus filiformis  (Tfi);  Thermus flavus  (Tfl);  Pyrococcus furiosus  (Pfu);  Thermococcus litoralis  (Tli);  Thermococcus zilligi  (Tzi);  Thermatoga neopolitana  (Tne);  Thermatoga maritime  (Tma); VENT®; DEEPVENT®; THERMOSCRIPT®; SUPERSCRIPT I®; SUPERSCRIPT II®; SUPERSCRIPT III®; and a mutant of any of the above. 
   
   
       24 . The composition of  claim 21 , wherein said thermostable 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 or complex of molecules, 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 or complex of molecules together substantially inhibits the polymerase activity of said polymerase. 
   
   
       26 . The method of  claim 25 , wherein said inhibition is irreversible. 
   
   
       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 a PBA. 
   
   
       29 . The method of  claim 25 , wherein said PBM-binding molecule is an antibody. 
   
   
       30 . The method of  claim 25 , wherein said nucleic acid polymerase is selected from the group consisting of: a DNA-dependent DNA polymerase; and an RNA-dependent DNA polymerase. 
   
   
       31 . The method of  claim 30 , wherein said nucleic acid polymerase is selected from the group consisting of:  Thermus aquaticus  (Taq);  Thermus thermophilus  (Tth);  Thermus filiformis  (Tfi);  Thermus flavus  (Tfl);  Pyrococcus furiosus  (Pfu);  Thermococcus litoralis  (Tli);  Thermococcus zilligi  (Tzi);  Thermatoga neopolitana  (Tne);  Thermatoga maritime  (Tma); VENT®, DEEPVENT®; THERMOSCRIPT®; SUPERSCRIPT I°; SUPERSCRIPT II®; SUPERSCRIPT III°; and 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 and an inhibitor of said polymerase, wherein said inhibitor comprises:
 (i) a Polymerase binding molecule (PBM) that does not substantially inhibit the polymerase activity of said polymerase; and 
 (ii) a PBM-binding molecule or complex of molecules, wherein the combination of said PBM and said PBM-binding molecule/complex substantially inhibits the polymerase activity of said polymerase; and 
   (b) bringing the resulting mixture to a temperature sufficient to inactivate said inhibitor, but that does not inactivate said polymerase.   
   
   
       34 . The method of  claim 33 , wherein said PBM is a Polymerase Binding Antibody (PBA). 
   
   
       35 . The method of  claim 33 , wherein said PBM-binding molecule is an antibody. 
   
   
       36 . The method of  claim 33 , wherein said inhibition is irreversible. 
   
   
       37 . The method of  claim 33 , wherein said inhibition is reversible by heating to a temperature of at least about 40° C. 
   
   
       38 . The method of  claim 33 , wherein said nucleic acid polymerase is selected from the group consisting of: a DNA-dependent DNA polymerase; and an RNA-dependent DNA polymerase. 
   
   
       39 . The method of  claim 38 , wherein said nucleic acid polymerase is selected from the group consisting of:  Thermus aquaticus  (Tag);  Thermus thermophilus  (Tth);  Thermus filiformis  (Tfi);  Thermus flavus  (Tfl);  Pyrococcus furiosus  (Pfu);  Thermococcus litoralis  (Tli);  Thermococcus zilligi  (Tzi);  Thermatoga neopolitana  (Tne);  Thermatoga maritime  (Tma); VENT®, DEEPVENT®; THERMOSCRIPT®; SUPERSCRIPT I®; SUPERSCRIPT II®; SUPERSCRIPT III®; and a mutant of any of the above. 
   
   
       40 . The method of  claim 38 , wherein said nucleic acid polymerase is recombinant.