Methods and apparatus for synthesizing nucleic acids

• US 8,808,989 B1
• Filed: 10/17/2013
• Issued: 08/19/2014
• 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:
  US14/056,687
• Time in Patent Office: 306 Days
• Field of Search: 435/6.1, 435/91.1
• US Class Current: 435/6.1
• 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,

wherein the nucleotide analog comprises an unmodified 3′

hydroxyl and a cleavable terminating group comprising an amino acid, wherein the cleavable terminating group blocks nucleotidyl transferase activity but results in a nucleotide substrate for nucleotidyl transferase upon cleavage.

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

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

4. 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.

5. 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.

6. The method of claim 1, wherein the cleavable terminating group inhibits the incorporation of a second nucleotide analog.

7. The method of claim 1, wherein the cleavable terminating group comprises a charged moiety.

8. The method of claim 1, wherein the nucleotide analog has the structure:

9. The method of claim 1, wherein the nucleotide analog has the structure:

10. The method of claim 1, wherein the nucleotide analog has the structure:

11. The method of claim 1, wherein the nucleotide analog has the structure:

12. The method of claim 1, wherein the cleavable terminating group is chemically cleavable, photolytically cleavable, electrochemically cleavable, or biologically cleavable.

13. 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.

14. 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.

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

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

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

18. The method of claim 1, further comprising:

cleaving the cleavable terminating group 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 and a cleavable terminating group.

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