Method for enzymatic synthesis of oligonucleotides using phosphate precipitation

• US 5,514,569 A
• Filed: 01/23/1995
• Issued: 05/07/1996
• Est. Priority Date: 12/23/1992
• Status: Expired due to Fees

• Current Assignee: Hyman, Edward D.
• Original Assignee: Hyman, Edward D.
• Inventors: Hyman, Edward D.
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): REES, DIANE
• Application Number:
  US08/376,857
• Time in Patent Office: 470 Days
• Field of Search: 435/6, 435/5, 435/91.1, 435/91.2, 536/24.3-24.33, 424/94.1, 424/94.5
• US Class Current: 435/91.2
• CPC Class Codes: B01J 19/0046 Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays synthesis methods per se C40B50/00
• CPC Class Codes: B01J 2219/00283 Reactor vessels with top opening
• CPC Class Codes: B01J 2219/00313 the reactor vessels being formed by arrays of wells in blocks
• CPC Class Codes: B01J 2219/00481 by the use of moving stirrers within the reaction vessels
• CPC Class Codes: B01J 2219/00495 Means for heating or cooling the reaction vessels
• CPC Class Codes: B01J 2219/0059 Sequential processes
• CPC Class Codes: B01J 2219/00599 Solution-phase processes
• CPC Class Codes: B01J 2219/00689 using computers
• CPC Class Codes: B01J 2219/00691 using robots
• CPC Class Codes: B01J 2219/00698 Measurement and control of process parameters
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer
• CPC Class Codes: C12Q 2535/113 Cycle sequencing
• CPC Class Codes: C40B 40/06 Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 50/08 Liquid phase synthesis, i.e. wherein all library building blocks are in liquid phase or in solution during library creation; Particular methods of cleavage from the liquid support
• CPC Class Codes: C40B 60/14 for creating libraries

Abstract

Enzymatic synthesis of a portion of an oligonucleotide is performed by a cycle of synthetic steps: (a) combining an oligonucleotide primer and a blocked nucleotide in a reaction mixture in the presence of a chain extending enzyme, such that a primer-blocked nucleotide product is formed, wherein the blocked nucleotide substrate comprises (i) a nucleotide to be added to form part of the defined sequence and (ii) a blocking group attached to the nucleotide effective to prevent the addition of more than one blocked nucleotide to the primer; and (b) removing the blocking group from the 3'"'"' end of the primer-blocked nucleotide product to form a primer-nucleotide product. Phosphate is generated in at least one synthetic step. A precipitate is formed in the cycle comprising phosphate and at least one precipitation cation. The precipitation of phosphate reduces its unfavorable effect on the method. Preferably, cycles of the method are repeated without intermediate purification of primer-nucleotide product or precursor. The precipitation cation may be a polyvalent elemental cation, spermine, or a cation which forms a poorly soluble salt with phosphate. Preferably, the chain extending enzyme is RNA Ligase, the blocked nucleotide is AppNp, and the blocking group is removed using a phosphatase. There is also provided a method for reducing the inhibitory effect of nucleoside 5'"'"'-monophosphate and of 3'"'"',5'"'"'-nucleoside diphosphate on phosphodiesterase I. This is accomplished by enzymatically converting these inhibitors to less inhibitory products.

Claims

1. A method for synthesizing a portion of an oligonucleotide of defined sequence comprising a cycle of synthetic steps:

(a) combining an oligonucleotide primer and a blocked nucleotide in a reaction mixture in the presence of a chain extending enzyme, such that a primer-blocked nucleotide product is formed, wherein the blocked nucleotide comprises (i) a nucleotide to be added to form part of the defined sequence and (ii) a blocking group attached to the nucleotide effective to prevent the addition of more than one blocked nucleotide to the primer; and

(b) removing the blocking group from the 3'"'"' end of the primer-blocked nucleotide product to form a primer-nucleotide product;

whereby phosphate is generated in at least one synthetic step;

whereby a precipitate is formed; and

whereby said precipitate comprises phosphate and at least one precipitation cation.

2. A method according to claim 1, further comprising the synthetic step (c) of inactivating unreacted blocked nucleotide by converting the unreacted blocked nucleotide to form which is less active as a substrate for the chain extending enzyme, wherein the unreacted blocked nucleotide is blocked nucleotide by converting the unreacted blocked nucleotide to a form which is less active as a substrate for the chain extending enzyme, wherein the unreacted blocked nucleotide is blocked nucleotide, which did not couple to the primer in step (a).

3. A method according to claim 2, wherein a second cycle of synthetic steps (a), (b), and (c) is performed using the primer-nucleotide product of step (b) in the first cycle as the primer of step (a) in the second cycle, and wherein blocked nucleotide is added for coupling in the second cycle, and wherein both cycles are performed without an additional intermediate step of purifying primer-blocked nucleotide or primer-nucleotide.

4. A method according to claim 3, wherein the chain extending enzyme is RNA Ligase.

5. A method according to claim 4, wherein the blocking group is a 3'"'"'-phosphate monoester, and the blocking group is removed using phosphatase.

6. A method according to claim 5, wherein the blocked nucleotide has the formula AppNp, wherein N represents a nucleoside or nucleoside analog, wherein RNA Ligase can couple the AppNp to the oligonucleotide primer, and wherein the primer-pN product is able to serve as a primer for RNA Ligase.

7. A method according to claim 6, wherein the unreacted blocked nucleotide is inactivated using nucleotide pyrophosphatase.

8. A method according to claim 7, wherein the nucleotide pyrophosphatase incubation is performed in the presence of a nucleoside 5'"'"'-monophosphate degrading enzyme, whereby nucleoside 5'"'"'-monophosphate is converted to a form which is less inhibitory to nucleotide pyrophosphatase.

9. A method according to claim 8, wherein the nucleoside 5'"'"'-monophosphate degrading enzyme is 5'"'"'-Nucleotidase.

10. A method according to claim 8, wherein the nucleoside 5'"'"'-monophosphate degrading enzyme is AMP Nucleosidase and the nucleoside 5'"'"'-monophosphate is adenosine 5'"'"'-monophosphate.

11. A method according to claim 9, wherein the incubation is performed in the presence of 3'"'"',5'"'"'-bisphosphate nucleotidase, whereby 3'"'"',5'"'"'-nucleoside diphosphate is converted to a form, nucleoside+two phosphates, which is less inhibitory to nucleotide pyrophosphatase.

12. A method according to claim 2, wherein the precipitation cation is a polyvalent elemental cation.

13. A method according to claim 3, wherein the precipitation cation is a polyvalent elemental cation.

14. A method according to claim 4, wherein the precipitation cation is a polyvalent elemental cation.

15. A method according to claim 5, wherein the precipitation cation is a polyvalent elemental cation.

16. A method according to claim 6, wherein the precipitation cation is a polyvalent elemental cation.

17. A method according to claim 7, wherein the precipitation cation is a polyvalent elemental cation.

18. A method according to claim 2, wherein the precipitation cation is spermine.

19. A method according to claim 3, wherein the precipitation cation is spermine.

20. A method according to claim 4, wherein the precipitation cation is spermine.

21. A method according to claim 5, wherein the precipitation cation is spermine.

22. A method according to claim 6, wherein the precipitation cation is spermine.

23. A method according to claim 7, wherein the precipitation cation is spermine.

24. A method for improving the performance of an enzymatic reaction in a reaction mixture, whereby the reaction mixture comprises:

(a) at least one enzyme,

(b) at least one precipitation cation, and

(c) at least one substrate for said enzyme, whereby phosphate is generated by the action of the enzyme on the substrate;

whereby a precipitate is formed, said precipitate comprising the precipitation cation and phosphate.

25. A method according to claim 24, wherein the precipitation cation is a polyvalent elemental cation.

26. A method according to claim 24, wherein the precipitation cation is spermine.

27. A method for reducing the inhibition of at least one nucleoside 5'"'"'-monophosphate on phosphodiesterase I, comprising incubating phosphodiesterase I in the presence of at least one nucleoside 5'"'"'-monophosphate degrading enzyme, whereby the nucleoside 5'"'"'-monophosphate is converted to a form which is less inhibitory to phosphodiesterase I.

28. A method according to claim 27, wherein the nucleoside 5'"'"'-monophosphate degrading enzyme is 5'"'"'-Nucleotidase.

29. A method according to claim 27, wherein the nucleoside 5'"'"'-monophosphate degrading enzyme is AMP Nucleosidase and the nucleoside 5'"'"'-monophosphate is adenosine 5'"'"'-monophosphate.

30. A method according to claim 27, further comprising the presence of 3'"'"',5'"'"'-bisphosphate nucleotidase, whereby 3'"'"',5'"'"'-nucleoside diphosphate is converted to a form which is less inhibitory to phosphodiesterase I.

31. A method according to claim 30, wherein the nucleoside 5'"'"'-monophosphate degrading enzyme is 5'"'"'-Nucleotidase.

32. A method according to claim 30, wherein the 3'"'"',5'"'"'-nucleoside diphosphate is 3'"'"',5'"'"'adenosine diphosphate and the nucleoside 5'"'"'-monophosphate degrading enzyme is AMP Nucleosidase.