Methods and apparatus for synthesizing nucleic acid

• US 9,771,613 B2
• Filed: 08/18/2015
• Issued: 09/26/2017
• 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:
  US14/829,264
• Publication Number:
  US 20160046973A1
• Time in Patent Office: 770 Days
• Field of Search: 435 61, 435 911
• 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 coupled, by a cleavable linker, to an inhibitor that sterically prevents the nucleotidyl transferase from catalyzing incorporation of either a natural nucleotide or a nucleotide analog into said oligonucleotide until said inhibitor is removed by cleavage of said cleavable linker, and

wherein the inhibitor is a macromolecule greater than 50 Å

in diameter.

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

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

10. 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.

11. 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.

12. The method of claim 1, wherein the solid support is a bead, a well, or a peg.

13. The method of claim 1, wherein the nucleic acid is single stranded.

14. The method of claim 1, wherein the cleavable linker comprises a moiety that forms a cyclic by-product when cleaved from the nucleotide analog.

15. 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 nucleotidyl transferase enzyme and in the absence of a nucleic acid template.

16. The method of claim 1, further comprising exposing the oligonucleotide to an exonuclease enzyme.

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

18. The method of claim 1, wherein the inhibitor comprises a charged moiety.

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

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

21. 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.

22. 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.

23. The method of claim 1, wherein the nucleotide analog is delivered in a droplet.

4. 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 coupled, by a cleavable linker, to an inhibitor that sterically prevents the nucleotidyl transferase from catalyzing incorporation of a natural nucleotide or a nucleotide analog into said oligonucleotide until said inhibitor is removed by cleavage of said cleavable linker, and

wherein the inhibitor comprises the following structure;

5. 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 coupled, by a cleavable linker, to an inhibitor that sterically prevents the nucleotidyl transferase from catalyzing incorporation of a natural nucleotide or a nucleotide analog into said oligonucleotide until said inhibitor is removed by cleavage of said cleavable linker, and

wherein the inhibitor comprises a polypeptoid.

6. 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 coupled, by a cleavable linker, to an inhibitor that sterically prevents the nucleotidyl transferase from catalyzing incorporation of a natural nucleotide or a nucleotide analog into said oligonucleotide until said inhibitor is removed by cleavage of said cleavable linker, and

wherein the inhibitor comprises a polymer.

7. The method of claim 6, wherein the polymer comprises polyethylene glycol.

8. 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 coupled, by a cleavable linker, to an inhibitor that sterically prevents the nucleotidyl transferase from catalyzing incorporation of a natural nucleotide or a nucleotide analog into said oligonucleotide until said inhibitor is removed by cleavage of said cleavable linker, and

wherein the inhibitor is a protein.

9. 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 coupled, by a cleavable linker, to an inhibitor that sterically prevents the nucleotidyl transferase from catalyzing incorporation of a natural nucleotide or a nucleotide analog into said oligonucleotide until said inhibitor is removed by cleavage of said cleavable linker, and

wherein the inhibitor is a nanoparticle.