Non-enzymatic, salt-mediated synthesis of polynucleic acids

• US 10,280,191 B2
• Filed: 09/16/2014
• Issued: 05/07/2019
• Est. Priority Date: 09/17/2013
• Status: Active Grant

• Current Assignee: Sorbonne Universite
• Original Assignee: Sorbonne Universite, Regents of the University of California (University of California)
• Inventors: Deamer, David W., Maurel, Marie-Christine, Da Silva, Laura
• Primary Examiner(s): Crane, Lawrence E
• Application Number:
  US15/022,487
• Publication Number:
  US 20160229882A1
• Time in Patent Office: 1,694 Days
• CPC Class Codes: C07H 1/00 Processes for the preparation of sugar derivatives
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 19/20 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 21/02 with ribosyl as saccharide radical
• CPC Class Codes: C12N 15/111 General methods applicable to biologically active non-coding nucleic acids
• CPC Class Codes: C12N 15/113 Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
• CPC Class Codes: C12N 2310/14 interfering N.A.
• CPC Class Codes: C12N 2330/30 chemically synthesised
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 2523/109 chemical ligation between nucleic acids
• CPC Class Codes: C12Q 2525/207 siRNA, miRNA
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer

Abstract

Provided herein is a method for synthesizing polynucleic acids, comprising the steps of (a) providing an acidic solution substantially free of nucleic acid polymerase and lipids, but containing mononucleotides and a monovalent salt; (b) drying and resolubilizing the mixture of step (a) a plurality of times; and (c) recovering polynucleic acids from a resolubilized mixture of step (b). In certain aspects, the method further uses a low pH, e.g. about 3; it can utilize monophosphates, such as AMP rather than ATP; and it can be used with a polynucleotide template to form a sequence at least partially complementary to said template. Thus, both single-stranded and double-stranded polynucleic acids are provided. Ammonia salts have been used to obtain RNA lengths from 10 to 300 nucleotides after 16 half hour cycles and an effective temperature includes between 80° C. and 100° C.

Claims

1. An in vitro method for preparing a ribonucleic acid (RNA) from a plurality of mononucleotides, comprising:

(a) providing an acidic solution free of polymerase and lipids, but containing a monovalent salt and mononucleotides selected from the group consisting of adenosine 5′

-monophosphate, uridine 5′

-monophosphate, guanosine 5′

-monophosphate, and cytidine-5′

-monophosphate;

(b) drying the acidic solution of step (a);

(c) resolubilizing the solid generated in step (b); and

(d) repeating the drying and resolubilizing steps (b) and (c) a plurality of times causing a reaction linking the mononucleotides thereby synthesizing the RNA,

wherein the monovalent salt is a metal halide salt or an ammonium halide salt.

2. The method of claim 1, wherein the monovalent salt is present in the acidic solution at a present concentration of at least 0.01 M.

3. The method of claim 1, further comprising wherein the drying comprises heating the acidic solution of step (a).

4. The method of claim 3, wherein the heating the acidic solution comprises heating the acidic solution to a temperature between 80°

C. and 90°

C.

5. The method of claim 1, wherein the acidic solution is adjusted to a pH between 2 and 4.

6. The method of claim 1, wherein the monovalent salt concentration is between 0.05 and 2M in solution.

7. The method of claim 3, wherein the mononucleotides are present in the solution at a concentration of at least 1 millimolar.

8. The method of claim 1, wherein the method further comprises recovering the RNA.

9. The method of claim 8, wherein recovering the RNA comprises isolating the RNA via ethanol precipitation.

10. The method of claim 1, wherein the monovalent salt is a metal halide salt.

11. The method of claim 10, wherein the metal halide salt is selected from the group consisting of NaF, CsCl, NaBr, NaClO₄, NaCl, and KCl.

12. The method of claim 11, wherein the metal halide salt is present in the acidic solution at a concentration between 0.05 M and 2M.

13. The method of claim 1, wherein the monovalent salt is an ammonium halide salt.

14. The method of claim 13, wherein the ammonium halide salt is NH₄Cl.

15. The method of claim 14, wherein the NH₄Cl is present in the acidic solution at a concentration between 0.05 M and 2M.

16. The method of claim 1, wherein the RNA has a length of at least 10 bases.

17. The method of claim 1, further comprising wherein the reaction is carried out under anaerobic conditions.

18. The method of claim 17, wherein the anaerobic conditions are formed by adding a CO₂ atmosphere to the reaction.

19. An in vitro method for preparing a ribonucleic acid (RNA) of a desired sequence, comprising:

(a) providing an aqueous solution adjusted to be between pH 2 and 4, free of polymerase and lipids, and comprising;

mononucleotides selected from the group consisting of adenosine 5′

-monophosphate, uridine 5′

-monophosphate, guanosine 5′

-monophosphate, and cytidine-5′

-monophosphate, each at a concentration of from 0.001M to 3M;

a monovalent salt selected from the group consisting of NaF, CsCl, NaBr, NaClO₄, NaCl, KCl, and NH₄Cl at a concentration of at least 0.1 M; and

a template polynucleotide complementary to the desired sequence;

(b) drying the aqueous solution of step (a);

(c) resolubilizing a solid generated in step (b); and

(d) repeating the drying and resolubilizing steps (b) and (c) a plurality of times causing a reaction linking the mononucleotides thereby synthesizing the RNA,

wherein steps (b)-(d) are carried out in an anaerobic environment.

20. An in vitro method for synthesizing a ribonucleic acid (RNA) from a plurality of mononucleotides, comprising:

(a) providing an acidic solution free of polymerase and lipids, but containing mononucleotides selected from the group consisting of adenosine 5′

-monophosphate, uridine 5′

-monophosphate, guanosine 5′

-monophosphate, and cytidine-5′

-monophosphate and a monovalent salt selected from the group consisting of NaF, CsCl, NaBr, NaClO₄, NaCl, KCl, and NH₄Cl and said monovalent salt being at a concentration between 0.05 M and 2M;

(b) drying a solution of step (a) by heating the solution to a temperature between 80°

C. and 90°

C.;

(c) resolubilizing the solid generated in step (b); and

(d) repeating the drying and resolubilizing steps (b) and (c) a plurality of times to generate the RNA.