Granted Patent B2

US 12,357,959 · App. 16/726,073 · Granted Jul 15, 2025

Inventors: Bill James Peck (Santa Clara, CA); Mary Noe (San Francisco, CA); Stefan Pitsch (Stein am Rhein, CH); Patrick A. Weiss (Daly City, CA)

Assignee: Twist Bioscience Corporation

B01J19/0046C07H21/04B01J2219/00596B01J2219/00722C07C53/02C07C53/08C07C53/12C07C53/122C07C53/134C07D213/16C07D213/74C07D233/58

Abstract

Claims (40)

1. A method of polynucleotide synthesis comprising:

providing predetermined sequences for a plurality of preselected polynucleotides;

providing a structure comprising a silicon surface;

coupling at least one nucleoside to a polynucleotide attached to the silicon surface;

washing the silicon surface with a wash solvent, wherein the wash solvent consists essentially of acetone;

washing the silicon surface with a capping solution, the capping solution comprising:

(i) at least one base;

(ii) at least one O-nucleophile selected from a group consisting of acetic acid, formic acid, propionic acid, methoxyacetic acid, phenoxyacetic acid, and water; and

(iii) tetrahydrofuran;

oxidizing a linkage between the at least one nucleoside and the polynucleotide with an oxidizing solution; and

repeating the coupling, washing, capping, and oxidizing steps to synthesize a plurality of polynucleotides.

2. The method of claim 1 , wherein the silicon surface is washed with the wash solvent after the coupling step.

3. The method of claim 1 , wherein the at least one base is selected from the group consisting of pyridine, lutidine, and collidine.

4. The method of claim 1 , wherein a concentration of the at least one O-nucleophile is 0.01-3 M.

5. The method of claim 1 , wherein the capping solution further comprises at least one electrophile, and wherein the at least one electrophile is an anhydride, NHS ester, or acid halide.

6. The method of claim 1 , wherein a concentration of the at least one base is 0.01-3 M.

7. The method of claim 1 , wherein the capping solution comprises the at least one base at about 5-30%, the at least one O-nucleophile at about 5-30%, and the tetrahydrofuran at about 60-90%.

8. The method of claim 1 , wherein the oxidizing solution comprises I 2 or iodine salts, and the I 2 or iodine salts have a greater solubility or increased rate of dissolution in the wash solvent compared to acetonitrile.

9. The method of claim 1 , wherein the capping solution further comprises an activator, wherein the activator is N-methylimidazole or DMAP.

10. The method of claim 9 , wherein concentration of the activator in the capping solution is 0.001-0.05 M.

11. The method of claim 1 , wherein oxidizing the linkage between the at least one nucleoside and the polynucleotide with the oxidizing solution includes depositing the oxidizing solution on the silicon surface and the method further comprises a washing step with the wash solvent after depositing the oxidizing solution on the silicon surface.

12. The method of claim 1 , wherein the polynucleotide or nucleoside comprises DNA or RNA.

13. The method of claim 1 , wherein the structure is a plate, a tape, or a belt, and the silicon surface is functionalized with 3-glycidoxypropyltimethoxysilane.

14. A method for polynucleotide synthesis, comprising:

providing predetermined sequences for a plurality of polynucleotides;

providing a structure comprising a silicon surface, wherein the silicon surface comprises a plurality of discrete loci configured for polynucleotide synthesis;

coupling at least one nucleoside to a polynucleotide attached to the silicon surface, according to the predetermined sequences;

oxidizing a linkage between the at least one nucleoside and the polynucleotide with an oxidizing solution; and

washing the silicon surface with a wash solvent, wherein the wash solvent consists essentially of acetone.

15. A method for polynucleotide synthesis, the method comprising repeating the steps of claim 14 to synthesize a plurality of polynucleotides, wherein the plurality of polynucleotides are synthesized at an error rate of about 1/5000 or less.

16. The method of claim 14 , wherein the silicon surface is washed with the wash solvent after the at least one nucleoside is coupled to the polynucleotide.

17. The method of claim 16 , further comprising depositing a capping solution on the silicon surface, wherein the capping solution comprises acetyl chloride, acetic anhydride, or an amine base.

18. The method of claim 17 , wherein washing the silicon surface with the wash solvent occurs after oxidizing the linkage and prior to depositing the capping solution, and where the method further comprises,

after depositing the capping solution, washing the silicon surface with the wash solvent.

19. The method of claim 14 , wherein the silicon surface is functionalized with 3-glycidoxypropyltrimethoxysilane.

20. The method of claim 14 , wherein the oxidizing solution comprises I 2 or iodine salts, and the I 2 or iodine salts have a greater solubility or increased rate of dissolution in the wash solvent compared to acetonitrile.

21. The method of claim 14 , further comprising depositing a deblocking solution on the silicon surface, wherein the deblocking solution includes a deblocking reagent configured to remove a 5′ OH protecting group.

22. The method of claim 21 , wherein the deblocking solution comprises:

acetonitrile, acetone, tetrahydrofuran, or toluene; and

an acid.

Assignments (1)

Continuity (2)

References Cited (400)