Method of RNA in vitro transcription using a buffer containing a dicarboxylic acid or tricarboxylic acid or a salt thereof

Patent metadata

Field	Value
Publication number	US-11248223-B2
Application number	US-201616012751-A
Country	US
Kind code	B2
Filing date	Dec 23, 2016
Priority date	Dec 23, 2015
Publication date	Feb 15, 2022
Grant date	Feb 15, 2022

Abstract

The present invention relates to a buffer system comprising a dicarboxylic acid or tricarboxylic acid or a salt thereof for synthesizing RNA molecules as well as a method of RNA in vitro transcription using this buffer system. The present invention also provides the use of this buffer system in RNA in vitro transcription and in the reduction or prevention of precipitates during RNA in vitro transcription.

First claim

The invention claimed is:

1. A method for in vitro transcription of a DNA template into RNA, comprising the steps of:
providing a mixture comprising: citric acid, or a citric acid salt, in a concentration of 10 to 100 mM, a buffer substance, ribonucleoside triphosphates, one or more magnesium salts, said DNA template and a recombinant RNA polymerase, wherein the mixture does not comprise a proteinogenic amino acid or tRNA; and
incubating the reaction mixture under suitable conditions thereby producing the RNA.

2. A method for preparing RNA, comprising the steps of:
a) incubating a mixture comprising: citric acid, or a citric acid salt, in a concentration of 10 to 100 mM, a buffer substance, ribonucleoside triphosphates, one or more magnesium salts, a nucleic acid template and a recombinant RNA polymerase under suitable conditions;
b) adding a DNase to the mixture of a) and incubating the resulting mixture containing DNase under suitable conditions thereby producing the RNA.

3. The method of claim 1 , wherein the citric acid or citric acid salt comprises sodium citrate and/or Tris-citrate.

4. The method according to claim 3 , wherein the concentration of citric acid or citric acid salt is at least half of the concentration of magnesium ions present in the in vitro transcription reaction.

5. The method of claim 1 , wherein the buffer substance is Tris base, HEPES or Tris-HCl.

6. The method of claim 1 , wherein the concentration of the buffer substance is 10 to 100 mM.

7. The method of claim 1 , wherein the reaction mixture comprises Tris-citrate or HEPES-KOH plus sodium citrate, ribonucleoside triphosphates, one or more magnesium salts, said DNA template and RNA polymerase.

8. The method of claim 1 , wherein the magnesium salt is magnesium chloride.

9. The method according to claim 8 , wherein the concentration of magnesium chloride is 1 to 100 mM.

10. The method of claim 1 , wherein the reaction mixture has a pH of 6 to 8.5.

11. The method of claim 1 , wherein the RNA polymerase is T7 RNA polymerase.

12. The method of claim 1 , wherein the total concentration of ribonucleoside triphosphates in the mixture is between 0.1 and 60 mM.

13. The method of claim 1 , wherein the DNA template is a linearized plasmid DNA template.

14. The method of claim 1 , wherein the reaction mixture further comprises one or more of ribonuclease inhibitor, pyrophosphatase, cap analog, one or more antioxidants and one or more amines and/or polyamines.

15. The method according to claim 14 , wherein the antioxidant is DTT.

16. The method of claim 14 , wherein the polyamine is spermidine.

17. The method of claim 1 , further comprising a step (c) of purifying the RNA.

18. The method according to claim 17 , wherein the step of purifying the RNA comprises HPLC using a porous reversed phase as stationary phase and/or tangential flow filtration.

19. A reaction mixture comprising: citric acid, or a citric acid salt, in a concentration of 10 to 100 mM, a buffer substance, ribonucleoside triphosphates, a cap analog, one or more magnesium salts, a DNA template, a RNA molecule comprising a 5′ cap and a recombinant RNA polymerase, but not comprising a proteinogenic amino acid or tRNA, said RNA molecule comprising a heterologous 3′-UTR that comprises a Poly(A) sequence.

20. A method for the reduction of the formation of precipitates in a method of in vitro transcription, the method comprising
providing a mixture comprising: citric acid, or a citric acid salt, in a concentration of 10 to 100 mM, a buffer substance, ribonucleoside triphosphates, one or more magnesium salts, a DNA template and a recombinant RNA polymerase, wherein the mixture does not comprise a proteinogenic amino acid or tRNA; and
incubating the reaction mixture under suitable conditions thereby producing a RNA composition having a reduced amount of precipitates as compared to a method not including said citric acid or citric acid salt.

21. The method of claim 3 , wherein the mixture comprises 10 to 100 mM of sodium citrate.

22. The method of claim 14 , wherein the RNA comprises a 5′ Cap, a 5′-UTR, an open reading frame, a 3′-UTR and a poly(A) sequence.

23. The reaction mixture of claim 19 , wherein the RNA molecule comprises at least one nucleotide analog.

24. The reaction mixture of claim 23 , wherein the heterologous 3′ UTR comprises an alpha-globin 3′-UTR.

25. The method of claim 1 , wherein the RNA comprises at least one nucleotide analog.

26. The method of claim 25 , wherein the at least one nucleotide analog is selected from the group consisting of pseudouridine, pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, 1-taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, and 4-methoxy-2-thio-pseudouridine.

27. The method of claim 26 , wherein the at least one nucleotide analog is pseudouridine or 1-methyl-pseudouridine.

28. The reaction mixture of claim 23 , wherein the at least one nucleotide analog is selected from the group consisting of pseudouridine, pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, 1-taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, and 4-methoxy-2-thio-pseudouridine.

29. The reaction mixture of claim 28 , wherein the at least one nucleotide analog is pseudouridine or 1-methyl-pseudouridine.

Assignees

• Curevac Ag

Inventors

• Yazdan Panah Benyamin
• Wochner Aniela
• Roos Tilmann
• Funkner Andreas
• Kunze Martin

Classifications

• C12Q1/6844 Primary

  Nucleic acid amplification reactions · CPC title

• C12P19/34

  Polynucleotides, e.g. nucleic acids, oligoribonucleotides · CPC title

• C12N15/10 Primary

  Processes for the isolation, preparation or purification of DNA or RNA (chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34) · CPC title

• C12Q2527/125

  Specific component of sample, medium or buffer · CPC title

• C12Q2521/119

  RNA polymerase · CPC title