Reusable initiators for synthesizing nucleic acids

• US 10,683,536 B2
• Filed: 08/18/2015
• Issued: 06/16/2020
• Est. Priority Date: 04/02/2013
• Status: Active Grant

• Current Assignee: Molecular Assemblies, Inc.
• Original Assignee: Molecular Assemblies, Inc.
• Inventors: Efcavitch, J. William
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US14/829,269
• Publication Number:
  US 20160046974A1
• Time in Patent Office: 1,764 Days
• Field of Search: 435 61
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: C07H 21/02 with ribosyl as saccharide radical
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 2521/101 DNA polymerase
• CPC Class Codes: C12Q 2525/117 incorporating modified base
• CPC Class Codes: Y02P 20/582 Recycling of unreacted starting or intermediate materials

Abstract

The invention provides improved methods for synthesizing polynucleotides, such as DNA and RNA, using renewable initiators coupled to a solid support. Using the methods of the invention, specific sequences of polynucleotides can be synthesized de novo, base by base, in an aqueous environment, without the use of a nucleic acid template.

Claims

1. A method for reversibly coupling a nucleotide analog to a solid support, the method comprising:

exposing a nucleic acid initiator attached to a solid support to nucleotide analogs in the presence of a polymerase, and in the absence of a template to create a first oligonucleotide;

contacting the nucleic acid initiator with a releasing agent to release the first oligonucleotide from the nucleic acid initiator, wherein the nucleic acid initiator remains a substrate for template-independent nucleic acid synthesis after release of the first oligonucleotide.

2. The method of claim 1, wherein the polymerase is a nucleotidyl transferase or a modified nucleotidyl transferase.

3. The method of claim 1, wherein the polymerase is terminal deoxynucleotidyl transferase (TdT) or a modified TdT.

4. The method of claim 1, wherein the polymerase is poly-A polymerase or a modified poly-A polymerase.

5. The method of claim 1, wherein the polymerase is poly-U polymerase or a modified poly-U polymerase.

6. The method of claim 1, wherein the nucleotide analogs have an unmodified 3′

-OH.

7. The method of claim 1, wherein the nucleotide analogs have an unmodified 3′

-OH and a cleavable terminating group, and wherein the cleavable terminating group blocks subsequent nucleotidyl transferase activity but results in a nucleotide substrate for nucleotidyl transferase upon cleavage of the terminating group.

8. The method of claim 1, wherein the nucleotide analogs comprise a removable blocking moiety protecting the 3′

oxygen.

9. The method of claim 1, wherein said nucleic acid initiator comprises a ribonucleotide.

10. The method of claim 1, wherein said nucleic acid initiator comprises an abasic deoxyribose.

11. The method of claim 1, wherein said nucleic acid initiator comprises an abasic ribose.

12. The method of claim 1, wherein said nucleic acid initiator comprises a non-nucleoside 5′

-monophosphate.

13. The method of claim 1, wherein said releasing agent is a type II restriction nuclease.