Systems and methods using nucleases modified with halogenated amino acids
AssigneeColorado State University Research Foundation
InventorsPui Shing Ho, Cesar G. Mendez, Margaret G. Walker, Alexander Ho, Ryan S. Czarny, Anthony K. Rappe
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Abstract
The present disclosure provides methods, compositions, and systems related to modulating the catalytic activity of nucleases (e.g., endonucleases) through site-specific incorporation of halogenated unnatural amino acids, and cleaving target nucleic acids using such modified nucleases under controlled pH conditions.
Claims30
A method of modulating the catalytic activity of a modified endonuclease, the method comprising:
- (a) contacting the modified endonuclease with a target nucleic acid in a reaction medium, wherein the modified endonuclease comprises a halogenated amino acid substituted at a position corresponding to a divalent metal ion-coordinating residue of a parent endonuclease and the halogenated amino acid forms a halogen bond with the target nucleic acid;
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- (b) adjusting the reaction medium to a pH where the modified endonuclease cleaves the target nucleic acid.
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A method for cleaving a target nucleic acid, the method comprising:
- (a) providing a modified endonuclease comprising a halogenated amino acid substituted at a position corresponding to a divalent metal ion-coordinating residue of a parent endonuclease, wherein the halogenated amino acid forms a halogen bond with a target nucleic acid;
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- (b) contacting the target nucleic acid with the modified endonuclease at a select pH where the modified endonuclease cleaves the target nucleic acid.
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Dependencies are inferred from the claim text.