Methods of synthesizing oligonucleotides using tethered nucleotides

Patent metadata

Field	Value
Publication number	US-11970697-B2
Application number	US-202117504358-A
Country	US
Kind code	B2
Filing date	Oct 18, 2021
Priority date	Oct 19, 2020
Publication date	Apr 30, 2024
Grant date	Apr 30, 2024

Abstract

Disclosed herein are methods and compositions comprising a polymerase and a phosphorylated nucleoside, wherein the polymerase and the nucleoside are covalently linked by a cleavable linker at the terminal phosphate group. Further disclosed herein are enzymatic polynucleotide synthesis using polymerase and nucleotide conjugation strategies.

First claim

What is claimed is:

1. A composition comprising a complex according to the following formula:

wherein the enzyme comprises TdT.

2. The composition of claim 1 , wherein the linker comprises at least one phosphate.

3. The composition of claim 1 , wherein the enzyme is attached to the linker via a cysteine.

4. The composition of claim 1 , wherein the linker is an acid labile linker, a base labile linker, a pH-sensitive linker, an amine-to-thiol crosslinker, a thiomaleamic acid linker, a photo-cleavable linker, orthonitrobenzyl-based linker, phenacyl linker, alkoxybenzoin linker, chromium arene complex linker, NpSSMpact linker, pivaloylglycol linker, irradiation-cleavable linker, and/or an enzymatically-cleavable linker.

5. The composition of claim 4 , wherein the linker is an enzymatically-cleavable linker that is capable of being cleaved using a peptidase or an esterase.

6. The composition of claim 1 , wherein the linker is selected from the group consisting of a silyl linker, an alkyl linker, a polyether linker, a polysulfonyl linker, a polysulfoxide linker, an amine-to-thiol cross linker, a thiomaleamic acid linker, and any combination thereof.

7. The composition of claim 1 , wherein the enzyme comprises a cysteine mutation.

8. The composition of claim 1 , wherein the template independent polymerase comprises at least one amino acid mutation to a surface-accessible amino acid residue.

9. The composition of claim 1 , wherein the base is selected from an adenine (A), cytosine (C), guanine (G), thymine (T), uracil (U), or a modified base thereof.

10. The composition of claim 1 , further comprising a reversible terminator.

11. A method of synthesizing a polynucleotide, comprising:
(a) contacting a polynucleotide with a composition of claim 1 ;
(b) extending the polynucleotide by covalent addition of an extension nucleotide onto a 3′ hydroxyl of the polynucleotide by the enzyme; and
(c) cleaving the enzyme from the polynucleotide.

12. The method of claim 11 , further comprising repeating (a)-(c) to produce an extended polynucleotide.

13. The method of claim 11 , wherein the enzyme is cleaved using a peptidase or an esterase.

14. A composition comprising a complex according to the following formula:
A-L-B

wherein:
A comprises an enzyme including TdT;
B comprises a nucleotide; and
L comprises a chemical linker that covalently links the enzyme to a terminal phosphate group of the nucleotide, wherein the enzyme is configured to catalyze covalent addition of the nucleotide onto a 3′ hydroxyl of a polynucleotide, and subsequent extension of the polynucleotide.

15. The composition of claim 14 , wherein the linker comprises at least one phosphate.

16. The composition of claim 14 , wherein the linker includes an acid labile linker, a base labile linker, a pH-sensitive linker, an amine-to-thiol crosslinker, a thiomaleamic acid linker, an orthonitrobenzyl-based linker, a phenacyl linker, an alkoxybenzoin linker, a chromium arene complex linker, an NpSSMpact linker, a pivaloylglycol linker, a photo-cleavable linker, an irradiation-cleavable linker, a silyl linker, an alkyl linker, a polyether linker, a polysulfonyl linker, a polysulfoxide linker, an amine-to-thiol crosslinker, a thiomaleamic acid linker, and/or an enzymatically-cleavable linker.

17. The composition of claim 15 , wherein the linker is an enzymatically-cleavable linker that is capable of being cleaved using a peptidase or an esterase.

18. The composition of claim 13 , wherein the enzyme comprises at least one amino acid mutation to a surface-accessible amino acid residue.

19. The composition of claim 13 , wherein the complex further comprises a reversible terminator.

20. The composition of claim 13 , wherein the base is includes an adenine, a cytosine, a guanine, a thymine, a uracil, a modified adenine, a modified cytosine, a modified thymine, and/or a modified uracil.

Assignees

• Twist Bioscience Corp

Inventors

• Lackey Jeremy
• Dodd David

Classifications

• C12N15/1093 Primary

  General methods of preparing gene libraries, not provided for in other subgroups · CPC title

• C12N15/102

  Mutagenizing nucleic acids · CPC title

• C12N15/113

  Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; {Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing (when used in plants C12N15/8218)} · CPC title

• C12N2310/315

  Phosphorothioates · CPC title

• C12N2320/33

  Alteration of splicing · CPC title