Cell-free production of ribonucleic acid

Abstract

Provided herein, in some aspects, are methods and compositions for cell-free production of ribonucleic acid.

1 . A cell-free method of biosynthesizing ribonucleic acid (RNA), the method comprising:
 (a) incubating a cell lysate mixture that comprises (i) cellular RNA and (ii) an enzyme that depolymerizes RNA, and (iii) a thermostable kinase and/or a thermostable RNA polymerase; and producing, a cell lysate mixture that comprises nucleoside monophosphates;   (b) heating the cell lysate mixture produced in step (a) to a temperature that inactivates or partially inactivates the enzymes that depolymerize RNA and enzymes that degrade nucleotides and/or nucleic acids without completely inactivating the thermostable kinase activities and/or thermostable RNA polymerase activities, and producing a cell lysate mixture that comprises nucleoside monophosphates, and the thermostable kinase and/or the RNA polymerase; and   (c) incubating the cell lysate mixture produced in (b) in the presence of an energy source and a deoxyribonucleic acid (DNA) template encoding a RNA of interest, producing nucleoside triphosphates, and producing a cell lysate mixture that comprises the RNA of interest.   
     
     
         2 .- 3 . (canceled) 
     
     
         4 . The method of  claim 1 , wherein the cellular RNA of step (a) is messenger RNA (mRNA), transfer RNA (tRNA), or ribosomal RNA (rRNA). 
     
     
         5 . The method of  claim 1 , wherein the enzyme that depolymerizes RNA comprises a ribonuclease. 
     
     
         6 . The method of  claim 5 , wherein the at least one ribonuclease is selected from the group consisting of S1 nuclease, Nuclease P1, RNase II, RNase III, RNase R, RNase JI, NucA, PNPase, RNase T, RNase E, and RNaseG. 
     
     
         7 . (canceled) 
     
     
         8 . The method of  claim 1 , wherein the thermostable kinase is selected from the group consisting of thermostable nucleoside monophosphate kinases, thermostable nucleoside diphosphate kinases, and thermostable polyphosphate kinases. 
     
     
         9 . The method of  claim 8 , wherein the thermostable nucleoside monophosphate kinases are selected from the group consisting of thermostable uridylate kinases, thermostable cytidylate kinases, thermostable guanylate kinases, and thermostable adenylate kinases. 
     
     
         10 . (canceled) 
     
     
         11 . The method of  claim 8 , wherein the thermostable nucleoside diphosphate kinases are selected from the group consisting of thermostable nucleoside diphosphate kinases encoded by a  Aquifex aeolicus  ndk gene. 
     
     
         12 . The method of  claim 1 , wherein the thermostable polyphosphate kinases are selected from the group consisting of thermostable polyphosphate kinase 1 (PPK1) enzymes and thermostable polyphosphate kinase 2 (PPK2) enzymes. 
     
     
         13 .- 18 . (canceled) 
     
     
         19 . The method of  claim 1 , wherein the at least one thermostable RNA polymerase is selected from the group consisting of thermostable DNA-dependent RNA polymerases. 
     
     
         20 . The method of  claim 19 , wherein the thermostable DNA-dependent RNA polymerases are selected from the group consisting of thermostable T7 RNA polymerases, thermostable SP6 RNA polymerases, and thermostable T3 RNA polymerases. 
     
     
         21 . (canceled) 
     
     
         22 . The method of  claim 1 , wherein the energy source is adenosine triphosphate (ATP) or an ATP regeneration system. 
     
     
         23 .- 27 . (canceled) 
     
     
         28 . The method of  claim 1 , wherein the cell lysate mixture of step (a) comprises the DNA template encoding the RNA of interest. 
     
     
         29 .- 38 . (canceled) 
     
     
         39 . The method of  claim 1 , wherein the temperature of step (b) is 50° C.-80° C. 
     
     
         40 .- 43 . (canceled) 
     
     
         44 . The method of  claim 1 , wherein the cells are bacterial cells or yeast cells. 
     
     
         45 .- 50 . (canceled) 
     
     
         51 . An engineered cell comprising an enzyme that depolymerizes RNA, at least one thermostable kinase, and a thermostable RNA polymerase. 
     
     
         52 . The cell of  claim 51  further comprising an engineered DNA template containing a promoter operably linked to a nucleotide sequence encoding a RNA of interest. 
     
     
         53 . A cell lysate produced by lysing the cultured engineered cell of  claim 51 . 
     
     
         54 . The cell lysate of  claim 53  further comprising an energy source and nucleoside monophosphates. 
     
     
         55 .- 88 . (canceled) 
     
     
         89 . The method of  claim 1 , the method comprising:
 (a) incubating a cell lysate mixture that comprises cellular RNA, an enzyme-that depolymerizes RNA, and a thermostable kinase and producing a cell lysate mixture that comprises nucleoside monophosphates;   (b) heating the cell lysate mixture produced in step (a) to a temperature that inactivates or partially inactivates endogenous nucleases and phosphatases without completely inactivating the thermostable kinase, and producing a cell lysate mixture that comprises nucleoside monophosphates, and the thermostable kinase; and   (c) incubating the cell lysate mixture produced in (b) in the presence of an RNA polymerase, an energy source and a deoxyribonucleic acid (DNA) template encoding a RNA of interest, producing nucleoside triphosphates, and producing a cell lysate mixture that comprises the RNA of interest.   
     
     
         90 . The method of  claim 1 , the method comprising:
 (a) incubating a cell lysate mixture that comprises cellular RNA, an enzyme-that depolymerizes RNA, and a thermostable RNA polymerase and producing a cell lysate mixture that comprises nucleoside monophosphates;   (b) heating the cell lysate mixture produced in step (a) to a temperature that inactivates or partially inactivates endogenous nucleases and phosphatases without completely inactivating the thermostable RNA polymerase, and producing a cell lysate mixture that comprises nucleoside monophosphates, and the thermostable RNA polymerase; and   (c) incubating the cell lysate mixture produced in (b) in the presence of a kinase, an energy source and a deoxyribonucleic acid (DNA) template encoding a RNA of interest, producing nucleoside triphosphates, and producing a cell lysate mixture that comprises the RNA of interest.