Polymerization of nucleic acids using activation by polyphosphorolysis (app) reactions

Abstract

This disclosure relates to methods of performing activation by polyphosphorolysis (APP) reactions using at least one of the polyphosphorylating agents triphosphate, polyphosphate, imidodiphosphate, thiodiphosphate (or μ-monothiopyrophosphate), and related compounds.

1 .- 29 . (canceled) 
     
     
         30 . A composition for performing an activation by polyphosphorolysis (APP) reaction comprising:
 (a) an activatable oligonucleotide;   (b) a target nucleic acid;   (c) one or more polyphosphorolyzing agents selected from the group consisting of:
 (i) a compound of the general Formula I: 
   
       
         
           
           
               
               
           
         
         
           
             wherein n is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; and 
           
           (ii) a compound of general Formula II: 
         
       
       
         
           
           
               
               
           
         
         
           
             wherein n and/or m are selected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10, with the proviso that n and m cannot both be 0, and 
             X is selected from the group consisting of: 
           
         
       
       
         
           
           
               
               
           
         
         and 
         (d) an enzyme having polyphosphorolysis activity. 
       
     
     
         31 . The composition of  claim 30 , wherein the enzyme is a DNA polymerase. 
     
     
         32 . The composition of  claim 31 , wherein the DNA polymerase is thermostable. 
     
     
         33 . The composition of  claim 32 , wherein the thermostable DNA polymerase is selected from the group consisting of thermostable Tfl, Taq, AMPLITAQFS, THERMOSEQUENASE, RQ1, RQY, THERMINATOR I, THERMINATOR II, THERMINATOR III, and THERMINATOR GAMMA. 
     
     
         34 . The composition of  claim 30 , wherein the one or more polyphosphorolyzing agents is a compound of the general Formula I: 
       
         
           
           
               
               
           
         
         wherein n is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. 
       
     
     
         35 . The composition of  claim 31 , wherein the one or more polyphosphorolyzing agents is a compound of the general Formula II: 
       
         
           
           
               
               
           
         
         where n and/or m are selected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10, with the proviso that n and m cannot both be 0, and 
         X is selected from the group consisting of: 
       
       
         
           
           
               
               
           
         
       
     
     
         36 . The composition of  claim 30 , wherein the polyphosphorolyzing agent is a compound selected from the group consisting of: 
       
         
           
           
               
               
           
         
       
     
     
         37 . The composition of  claim 30 , wherein the one or more polyphosphorolyzing agents is a compound selected from the group consisting of triphosphate, polyphosphate, imidodiphosphate, and thiodiphosphate. 
     
     
         38 . The composition of  claim 30 , further comprising one or more nucleoside triphosphates. 
     
     
         39 . The composition of  claim 30 , wherein the one or more polyphosphorolyzing agents is present at a concentration of approximately 1-500 μM. 
     
     
         40 . The composition of  claim 30 , wherein the target nucleic acid is RNA. 
     
     
         41 . The composition of  claim 30 , wherein the enzyme or polymerase has reverse transcriptase activity. 
     
     
         42 . The composition of  claim 30 , wherein the enzyme is RQ1 or its mutant RQY. 
     
     
         43 . The composition of  claim 30 , wherein the one or more polyphosphorolyzing agents is in the form of a salt. 
     
     
         44 . The composition of  claim 30 , wherein the activatable oligonucleotide is hybridized to the target nucleic acid. 
     
     
         45 . The composition of  claim 30 , wherein the 3′ non-extendable terminus (P*) of the activatable oligonucleotide has been removed by polyphosphorolysis. 
     
     
         46 . The composition of  claim 44 , wherein the activatable oligonucleotide has no mismatched nucleotides at or near its 3′ terminus. 
     
     
         47 . The composition of  claim 30 , wherein the activatable oligonucleotide is complementary to the target nucleic acid. 
     
     
         48 . The composition of  claim 47 , wherein the complementary terminal nucleotide of the activatable oligonucleotide has been removed by polyphosphorolysis.