Methods and compositions for nucleoside triphosphate and ribonucleic acid production
Provided herein, in some embodiments, are methods and composition for the production of nucleoside triphosphates and ribonucleic acids.
1. A method for producing ribonucleic acid (RNA) comprising:
(a) incubating (i) cellular RNA, wherein the cellular RNA comprises ribosomal RNA, messenger RNA, and/or transfer RNA, and (ii) a ribonuclease to produce 5′ nucleoside monophosphates (NMPs);
(b) eliminating the ribonuclease; and
(c) incubating the 5′ NMPs with polyphosphate kinase (PPK), polyphosphate, deoxyribonucleic acid (DNA) template encoding a RNA of interest, and RNA polymerase to produce the RNA of interest.
2. The method of claim 1 , wherein the ribonuclease is Nuclease P1 or RNase R.
3. The method of claim 1 , wherein the 5′ NMPs include 5′ AMP, 5′ GMP, 5′ CMP, and/or 5′ UMP.
4. The method of claim 1 , wherein the ribonuclease is eliminated via temperature, pH, salt, detergent, alcohol, chemical inhibitors, separation, precipitation, filtration, capture, and/or chromatography.
5. The method of claim 1 , wherein the PPK is a PPK1 family enzyme or a PPK2 family enzyme.
6. The method of claim 1 , wherein the PPK is a Class III PPK2 enzyme from Deinococcus geothermalis (SEQ ID NO: 1).
7. The method of claim 1 , wherein the polyphosphate is selected from the group consisting of tetrapolyphosphates, pentapolyphosphates, and hexametaphosphates.
8. The method of claim 1 , wherein step (c) comprises an enzyme preparation or cell lysate obtained from cells that produce the PPK, the deoxyribonucleic acid (DNA) template, and/or the RNA polymerase.
9. The method of claim 8 , wherein activity of native enzymes in the cell lysate or enzyme preparation 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.
10. The method of claim 9 , wherein the native enzymes are selected from the group consisting of phosphatases, nucleases, proteases, deaminases, oxidoreductases, and hydrolases.
11. A method for producing ribonucleic acid (RNA) 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 NMP kinase, at least one NDP kinase, at least one polyphosphate kinase (PPK), polyphosphate, deoxyribonucleic acid (DNA) template encoding a RNA of interest, and RNA polymerase to produce the RNA of interest.
12. The method of claim 11 , wherein the cellular RNA comprises ribosomal RNA, messenger RNA, and/or transfer RNA.
13. The method of claim 11 , wherein the ribonuclease is Nuclease P1 or RNase R.
14. The method of claim 11 , wherein the 5′ NMPs include 5′ AMP, 5′ GMP, 5′ CMP, and/or 5′ UMP.
15. The method of claim 11 , wherein the ribonuclease is eliminated via temperature, pH, salt, detergent, alcohol, chemical inhibitors, separation, precipitation, filtration, capture, and/or chromatography.
16. The method of claim 11 , wherein the at least one NMP kinase is an AMP kinase, a CMP kinase, a UMP kinase, or a GMP kinase.
17. The method of claim 16 , wherein 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).
18. The method of claim 11 , wherein the at least one NDP kinase is from Aquifex aeolicus (SEQ ID NO: 16).
19. The method of claim 11 , wherein the at least one PPK is a PPK1 family enzyme or a PPK2 family enzyme.
20. The method of claim 19 , wherein the at least one PPK is a Class III PPK2 enzyme from Deinococcus geothermalis (SEQ ID NO: 1).
21. The method of claim 11 , wherein the polyphosphate is selected from the group consisting of tetrapolyphosphates, pentapolyphosphates, and hexametaphosphates.
22. The method of claim 11 , wherein step (c) comprises an enzyme preparation or cell lysate obtained from cells that produce the PPK, the NMP kinase, the NDP kinase, the deoxyribonucleic acid (DNA) template, and/or the RNA polymerase.
23. The method of claim 11 , wherein activity of native enzymes in the cell lysate or enzyme preparation 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.
24. The method of claim 23 , wherein the native enzymes are selected from the group consisting of phosphatases, nucleases, proteases, deaminases, oxidoreductases, and hydrolases.
25. A method for producing ribonucleic acid (RNA) comprising:
(a) incubating (i) cellular RNA and (ii) Nuclease P1 to produce 5′ nucleoside monophosphates (NMPs);
(b) eliminating the Nuclease P1; and
(c) incubating the 5′ NMPs with at least one NMP kinase, at least one NDP kinase, a Class III PPK2 enzyme from Deinococcus geothermalis (SEQ ID NO: 1), polyphosphate, deoxyribonucleic acid (DNA) template encoding a RNA of interest, and RNA polymerase to produce the RNA of interest.
26. The method of claim 25 , wherein 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).
27. The method of claim 25 , wherein the at least one NDP kinase is from Aquifex aeolicus (SEQ ID NO: 16).
28. A method for producing ribonucleic acid (RNA) comprising:
(a) incubating (i) cellular RNA and (ii) a ribonuclease to produce 5′ nucleoside monophosphates (NMPs);
(b) eliminating the ribonuclease, wherein the ribonuclease is eliminated via temperature, pH, salt, detergent, alcohol, chemical inhibitors, separation, precipitation, filtration, capture, and/or chromatography; and
(c) incubating the 5′ NMPs with polyphosphate kinase (PPK), polyphosphate, deoxyribonucleic acid (DNA) template encoding a RNA of interest, and RNA polymerase to produce the RNA of interest.
29. The method of claim 28 , wherein the ribonuclease is Nuclease P1 or RNase R.
30. The method of claim 28 , wherein the PPK is a PPK1 family enzyme or a PPK2 family enzyme.