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US 2025/0154548 A1Published application
Control of enzymatic nucleic acid synthesis via electrochemical means
AssigneeThe Charles Stark Draper Laboratory, Inc.
InventorsCassie M. Bryan, Melissa Sprachman, Robert Doyle, William C. Records
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Abstract
Provided herein are methods of nucleic acid synthesis. In some embodiments, a method of nucleic acid synthesis comprises selectively activating and deactivating an engineered terminal deoxynucleotidyl transferase (TdT). In some embodiments, a method of nucleic acid synthesis comprises activating a pH sensitive enzyme to digest and remove a TdT.
Claims20
A method of nucleic acid molecule synthesis comprising the steps of:
- (a) providing a single-stranded nucleic acid molecule comprising a 5′ and a 3′ end;
- (b) providing an engineered terminal deoxynucleotidyl transferase (TdT), wherein the engineered TdT comprises a nucleotide molecule covalently attached to the TdT via a cleavable linker;
- (c) providing at least one metal cofactor complexed to at least one cleavable chelating agent, wherein the chelating agent is capable of releasing the metal cofactor upon cleavage;
- (d) selectively activating the TdT by cleaving the chelating agent;
- (e) contacting the TdT and the single-stranded nucleic acid molecule under conditions suitable for the TdT to bind to the 3′ end of the nucleic acid molecule and form a TdT-nucleic acid strand complex, thereby incorporating the nucleotide into the nucleic acid strand;
- (f) exposing the TdT to conditions sufficient to deactivate it and remove it from the nucleic acid molecule;
- and
- (g) repeating steps (b) through (f) thereby synthesizing a nucleic acid molecule.
5 more elements
A method of nucleic acid molecule synthesis in a buffer comprising the steps of:
- (a) providing a single-stranded nucleic acid molecule comprising a 5′ and a 3′ end;
- (b) providing an engineered terminal deoxynucleotidyl transferase (TdT), wherein the engineered TdT comprises a nucleotide molecule covalently attached to the TdT via a cleavable linker and at least one metal cofactor;
- (c) contacting the TdT and the single-stranded nucleic acid molecule under conditions suitable for the TdT to bind to the 3′ end of the nucleic acid molecule and form a TdT-nucleic acid strand complex, thereby incorporating the nucleotide into the nucleic acid strand to create an extended nucleic acid strand;
- (d) providing an inactivated pH sensitive enzyme, wherein the pH sensitive enzyme is inactivated by pH conditions of the buffer;
- (e) activating the pH sensitive enzyme through a change in pH conditions in the buffer, wherein the activated pH sensitive enzyme digests the TdT, thereby removing the TdT from the extended nucleic acid strand;
- (f) inactivating the pH sensitive enzyme through a change in pH conditions;
- and
- (g) repeating steps (c) through (f) at least once thereby synthesizing a nucleic acid molecule.
5 more elements
A system for nucleic acid synthesis,
- wherein the system comprises:
- an engineered TdT, wherein the engineered TdT comprises a nucleotide molecule covalently attached to the TdT via a cleavable linker;
- at least one metal cofactor complexed to at least one cleavable chelating agent;
- a redox shuttle solution;
- and
- two or more electrodes on a surface;
- or
- wherein the system comprises:
- an engineered TdT, wherein the engineered TdT comprises a nucleotide molecule covalently attached to the TdT via a cleavable linker and at least one metal cofactor;
- an inactivated pH sensitive enzyme;
- a redox shuttle solution;
- and
- two or more electrodes on a surface.
10 more elements
A compound represented by:
Dependencies are inferred from the claim text.