METHODS AND APPARATUS FOR SYNTHESIZING NUCLEIC ACIDS

• US 20160168611A1
• Filed: 02/19/2016
• Published: 06/16/2016
• 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
• Granted Patent: US 9,695,470 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/06 Quantitative determination
• 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 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 an oligonucleotide 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 such that the nucleotide analog is incorporated into the oligonucleotide,

wherein the nucleotide analog comprises a nucleotide, a cleavable linker, and an inhibitor, the nucleotide comprising an unmodified 3′

hydroxyl and coupled, by the cleavable linker, to the inhibitor, wherein the inhbitor prevents the nucleotidyl transferase from catalyzing incorporation of a nucleotide or an additional nucleotide analog into said oligonucleotide until said inhibitor is removed by cleavage of said cleavable linker.

2. The method of claim 1, wherein the nucleotide analog comprises the following NTP structure:

3. The method of claim 1, wherein the nucleotide analog comprises the following NTP structure:

4. The method of claim 1, wherein the nucleotide analog comprises the following cleavable linker structure:

5. The method of claim 1, wherein the nucleotide analog has the following cleavable linker structure:

6. The method of claim 1, wherein the nucleotide analog has the following inhibitor structure:

7. The method of claim 1, wherein the nucleic acid attached to the solid support is exposed to the nucleotide analog in the presence of an aqueous solution having a pH between about 6.5 and 8.5.

8. The method of claim 1, the nucleic acid attached to the solid support is exposed to the nucleotide analog in the presence of an aqueous solution at a temperature between about 35 and 39°

C.

9. The method of claim 1, further comprising:

cleaving the cleavable linker in order to produce a native nucleotide; and

exposing the native nucleotide to a second nucleotide analog in the presence of a transferase enzyme and in the absence of a nucleic acid template, wherein the second nucleotide analog comprises a 3′

hydroxyl on a sugar ring coupled to an inhibitor by a cleavable linker.

10. The method of claim 1, further comprising providing an aqueous solution comprising the nucleotide analog and the nucleotidyl transferase enzyme.

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

12. The method of claim 1, wherein the nucleotidyl transferase 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 the nucleotidyl transferase 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 synthesizing an oligonucleotide, comprising:

exposing an oligonucleotide 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 such that the nucleotide analog is incorporated into the oligonucleotide, the nucleotide analog comprising an unmodified 3′

hydroxyl,

wherein the synthesized oligonucleotide remains attached to the solid support for subsequent use in an application.

15. The method of claim 14 wherein the application is selected from the group consisting of:

hybridization analysis, affinity capture, PCR amplification, and DNA sequencing.

16. The method of claim 14, wherein a bioreactor comprises the solid support.

17. The method of claim 16, wherein the bioreactor comprises an application specific flow cell.

18. The method of claim 17, wherein the application comprises DNA sequencing.

19. The method of claim 16, wherein the bioreactor comprises a reservoir.

20. The method of claim 16, wherein the bioreactor comprises a multi-well plate.

21. The method of claim 20, wherein each well comprises a plurality of oligonucleotides.

22. The method of claim 16, wherein the bioreactor comprises a plurality of oligonucleotides.