Methods and compositions for nucleoside triphosphate and ribonucleic acid production

Abstract

Provided herein, in some embodiments, are methods and composition for the production of nucleoside triphosphates and ribonucleic acids.

1 . A method for producing one or more nucleoside triphosphates (NTPs), comprising:
 incubating in a reaction mixture one or more nucleoside monophosphates (NMPs), at least one polyphosphate kinase (PPK), and polyphosphate.   
     
     
         2 . (canceled) 
     
     
         3 . The method of  claim 1  further comprising:
 incubating in a reaction mixture cellular ribonucleic acid (RNA) and 
 (a) a polynucleotide phosphorylase (PNPase) and inorganic phosphate; and/or 
 (b) a ribonuclease. 
 
     
     
         4 .- 8 . (canceled) 
     
     
         9 . The method of  claim 1 , wherein the polyphosphate kinase is selected from PPK1 and PPK2 family enzymes. 
     
     
         10 - 26 . (canceled) 
     
     
         27 . The method of  claim 1  further comprising at least one NMP kinase and/or NDP kinase. 
     
     
         28 . The method of  claim 1 , wherein the at least one PPK is a Class III PPK2 enzyme from  Deinococcus geothermalis  (SEQ ID NO: 1). 
     
     
         29 . The method of  claim 1 , wherein the method is a cell-free method. 
     
     
         30 . The method of  claim 1 , wherein the one or more NMPs comprise AMP, GMP, CMP, and/or UMP. 
     
     
         31 . The method of  claim 1 , wherein the one or more NMPs comprise modified nucleoside monophosphates. 
     
     
         32 . The method of  claim 1 , wherein the polyphosphate is selected from the group consisting of tetrapolyphosphates, pentapolyphosphates, and hexametaphosphates. 
     
     
         33 . The method of  claim 27 , wherein:
 (i) the at least one NMP kinase is an AMP kinase, a GMP kinase, a CMP kinase, and/or a UMP kinase; and/or   (ii) the at least one NDP kinase is an ADP kinase, a GDP kinase, a CDP kinase, or a UDP kinase.   
     
     
         34 . The method of  claim 27 , wherein:
 (i) the at least one NMP kinase is AMP kinase from  Thermus thermophilus  (SEQ ID NO: 12), CMP kinase from  Thermus thermophilus  (SEQ ID NO: 13), UMP kinase from  Pyrococcus furiosus  (SEQ ID NO: 14), and/or GMP kinase from  Thermotoga maritima  (SEQ ID NO: 15); and/or   (ii) the at least one NDP kinase is from  Aquifex aeolicus  (SEQ ID NO: 16).   
     
     
         35 . A method for producing one or more nucleoside triphosphates (NTPs), comprising:
 incubating in a reaction mixture one or more nucleoside diphosphates (NDPs), at least one polyphosphate kinase, and polyphosphate.   
     
     
         36 . A method for producing nucleoside triphosphates (NTPs) comprising:
 (a) incubating (i) cellular RNA and (ii) a ribonuclease to produce 5′ nucleoside monophosphates (NMPs);   (b) eliminating the ribonuclease; and   (c) incubating the 5′ NMPs with at least one polyphosphate kinase (PPK), and polyphosphate to produce NTPs.   
     
     
         37 . The method of  claim 36 , wherein step (c) further comprises at least one NMP kinase and/or at least one NDP kinase. 
     
     
         38 . The method of  claim 36 , wherein the cellular RNA comprises ribosomal RNA, messenger RNA, and/or transfer RNA. 
     
     
         39 . The method of  claim 36 , wherein the ribonuclease is Nuclease P1 or RNase R. 
     
     
         40 . The method of  claim 36 , wherein the ribonuclease is eliminated via temperature, pH, salt, detergent, alcohol, chemical inhibitors, separation, precipitation, filtration, capture, and/or chromatography. 
     
     
         41 . The method of  claim 37 , wherein:
 (i) the at least one NMP kinase is an AMP kinase, a GMP kinase, a CMP kinase, and/or a UMP kinase; and/or   (ii) the at least one NDP kinase is an ADP kinase, a GDP kinase, a CDP kinase, or a UDP kinase.   
     
     
         42 . The method of  claim 37 , wherein:
 (i) the at least one NMP kinase is AMP kinase from  Thermus thermophilus  (SEQ ID NO: 12), CMP kinase from  Thermus thermophilus  (SEQ ID NO: 13), UMP kinase from  Pyrococcus furiosus  (SEQ ID NO: 14), and/or GMP kinase from  Thermotoga maritima  (SEQ ID NO: 15); and/or   (ii) the at least one NDP kinase is from  Aquifex aeolicus  (SEQ ID NO: 16).   
     
     
         43 . The method of  claim 36 , wherein the at least one PPK is a PPK1 family enzyme or a PPK2 family enzyme. 
     
     
         44 . The method of  claim 36 , wherein the at least one PPK is a Class III PPK2 enzyme from  Deinococcus geothermalis  (SEQ ID NO: 1). 
     
     
         45 . The method of  claim 36 , wherein the polyphosphate is selected from the group consisting of tertapolyphosphates, pentapolyphosphates, and hexametaphosphates. 
     
     
         46 . The method of  claim 37 , wherein step (c) comprises an enzyme preparation or cell lysate obtained from cells that produce the PPK, the NMP kinase, and/or the NDP kinase. 
     
     
         47 . The method of  claim 46 , wherein the activity of native enzymes in the cells has been eliminated via genetic modification, enzyme secretion from a cell, protease targeting, temperature, pH, salt, detergent, alcohol, chemical inhibitors, separation, precipitation, filtration, capture, and/or chromatography. 
     
     
         48 . The method of  claim 47 , wherein the native enzymes are selected from the group consisting of phosphatases, nucleases, proteases, deaminases, oxidoreductases, and hydrolases. 
     
     
         49 . The method of  claim 1 , further comprising:
 incubating in a reaction mixture cellular ribonucleic acid (RNA), a polynucleotide phosphorylase (PNPase), inorganic phosphate, and a helicase.