Methods and apparatus for synthesizing nucleic acids

• US 10,041,110 B2
• Filed: 07/28/2014
• Issued: 08/07/2018
• Est. Priority Date: 04/02/2013
• Status: Active Grant

• Current Assignee: Molecular Assemblies, Inc.
• Original Assignee: Molecular Assemblies, Inc.
• Inventors: Efcavitch, J. William, Siddiqi, Suhaib
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US14/444,440
• Publication Number:
  US 20140363851A1
• Time in Patent Office: 1,471 Days
• Field of Search: 435 61, 435 911
• CPC Class Codes: C12N 9/1241 Nucleotidyltransferases (2.7.7)
• CPC Class Codes: C12N 9/1264 DNA nucleotidylexotransferase (2.7.7.31), i.e. terminal nucleotidyl transferase
• 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 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Y 207/07019 Polynucleotide adenylyltransferase (2.7.7.19)
• CPC Class Codes: C12Y 207/07031 DNA nucleotidylexotransferase (2.7.7.31), i.e. terminal deoxynucleotidyl transferase
• 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 enzymes and specially designed nucleotide analogs. 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. Because the nucleotide analogs have an unmodified 3′ OH, i.e., as found in “natural” deoxyribose and ribose molecules, the analogs result in natural polynucleotides suitable for incorporation into biological systems.

Claims

1. A method for synthesizing an oligonucleotide, comprising:

exposing a nucleic acid attached to a solid support to a nucleotide analog in the presence of a nucleotidyl transferase enzyme and in the absence of a nucleic acid template, thereby creating an oligonucleotide analog,

wherein the nucleotide analog comprises an unmodified 3′

hydroxyl and a cleavable terminating group comprising a charged moiety,

wherein the oligonucleotide analog is not a substrate for said nucleotidyl transferase enzyme when the cleavable terminating group is attached, but the oligonucleotide analog is a substrate for said nucleotidyl transferase enzyme when the terminating group is not attached.

2. The method of claim 1, wherein the charged moiety comprises a negative charge.

3. The method of claim 1, wherein the charged moiety comprises a net negative charge.

4. The method of claim 3, wherein the charged moiety comprises an amino acid.

5. The method of claim 1, wherein the charged moiety comprises a positive charge.

6. The method of claim 1, wherein the charged moiety comprises a net positive charge.

7. The method of claim 6, wherein the charged moiety comprises an amino acid.

8. The method of claim 1, wherein the nucleotide analog comprises a ribose sugar or a deoxyribose sugar.

9. The method of claim 1, wherein the nucleotide substrate comprises a base selected from the group consisting of adenine, guanine, cytosine, thymine, and uracil.

10. The method of claim 1, wherein the oligonucleotide analog comprising the cleavable terminating group is not a substrate for said nucleotidyl transferase enzyme in an aqueous solution having a pH between about 6.5 and 8.5.

11. The method of claim 1, wherein the oligonucleotide analog comprising the cleavable terminating group is not a substrate for said nucleotidyl transferase because of electrostatic interactions between the charged moiety and said nucleotidyl transferase enzyme.

12. The method of claim 1, wherein said nucleotidyl transferase enzyme comprises a protein sequence that is at least about 90% identical to SEQ ID NO. 1, SEQ ID NO. 3, or SEQ ID NO. 5.

13. The method of claim 1, wherein said nucleotidyl transferase enzyme originates from an organism having a nucleotide sequence that is at least about 90% identical to SEQ ID NO. 2, SEQ ID NO. 4, or SEQ ID NO. 6.

14. A method for oligonucleotide synthesis, the method comprising the steps of:

exposing a support-bound nucleic acid that is free of a nucleic acid template to;

a nucleotide analog that comprises a moiety attached thereto by a cleavable linker and having a free 3′

hydroxyl, and

a nucleotidyl transferase, thereby incorporating said nucleotide analog into said support-bound nucleic acid to create an oligonucleotide analog wherein the oligonucleotide analog is not a substrate for said nucleotidyl transferase when the moiety is attached but is a substrate for said nucleotidyl transferase when the moiety is not attached;

washing said solid support upon incorporation of said nucleotide analog to remove unincorporated nucleotide analog;

cleaving said cleavable linker; and

repeating said exposing, washing, and cleaving steps in order to synthesize an oligonucleotide.

15. The method of claim 14, wherein said nucleotide analog comprises a base selected from the group consisting of adenine, guanine, cytosine, thymine, and uracil.

16. The method of claim 14, wherein said nucleotide analog and said nucleotidyl transferase are present in the same solution, and said solution is substantially recycled between subsequent exposing, washing, and cleaving steps.

17. An apparatus for synthesizing oligonucleotides with a predetermined sequence in an aqueous environment, comprising:

a first, second, third, and fourth source of nucleotide triphosphate (NTP) reagent and template-independent nucleotidyl transferase enzyme solutions in fluid communication with a solid support, wherein the reagent solutions in the first, second, third, and fourth sources are selected from NTP-adenine, NTP-guanine, NTP-cytosine, NTP-thymine,

wherein at least a portion of the nucleotide triphosphates comprise an unmodified 3′

hydroxyl and a cleavable terminating group that, upon incorporation by a nucleotidyl transferase into an oligonucleotide analog, prevents said oligonucleotide analog from being a substrate for said nucleotidyl transferase in the absence of a nucleic acid template and results in the oligonucleotide analog becoming a substrate for said nucleotidyl transferase in the absence of a nucleic acid template upon cleavage of said terminating group.

18. The apparatus of claim 17, wherein the nucleotides are deoxyribonucleotides.

19. The apparatus of claim 17, further comprising a wash reservoir in fluid communication with the solid support.

20. The apparatus of claim 17, further comprising a source of an aqueous deblocking agent in fluid communication with the solid support.

21. The apparatus of claim 17, wherein the nucleotide triphosphate reagents are flowed to the solid support.

22. The apparatus of claim 17, wherein the solid support is moved to the first, second, third, or fourth source of nucleotide triphosphate reagent and enzyme solution.

23. The apparatus of claim 22, further comprising a programmable manipulator configured to move the solid support.