Methods and compositions for modification and detection of 5-methylcytosine
AssigneeThe University of Chicago
InventorsChuan HE, Weixin TANG, Qinzhe LIU, Pingluan WANG
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Abstract
Disclosed herein are methods and compositions for 5mC modification, detection, sequencing, and analysis. Certain aspects of the disclosure are directed to methods for modification of a 5mC in a nucleic acid molecule, including oxidation, removal, and replacement of a 5mC with a nucleobase derivative. Also disclosed are novel nucleobase derivatives, including thymine derivatives, and other compounds as well as methods for use of such derivatives and compounds in 5mC modification and analysis.
Claims79
A method for modifying a 5-methylcytosine (5mC) in a nucleic acid molecule, the method comprising:
- (a) incubating the nucleic acid molecule with an agent under conditions sufficient to oxidize the 5mC to 5-carboxylcytosine (5caC) or 5-formylcytosine (5fC);
- (b) incubating the nucleic acid molecule with a thymine DNA glycosylase (TDG) enzyme to excise the 5caC or 5fC creating an abasic site;
- and
- (c) incubating the nucleic acid molecule with a nucleobase derivative to attach the nucleobase derivative to the nucleic acid molecule at the abasic site.
A method for modifying a nucleic acid molecule comprising an abasic site, the method comprising incubating the nucleic acid molecule and a nucleobase derivative under conditions sufficient to attach the nucleobase derivative to the nucleic acid molecule at the abasic site.
A method for 5-methylcytosine (5mC) detection, the method comprising:
- (a) incubating a nucleic acid molecule comprising a 5mC with a TET enzyme to oxidize 5mC to 5caC or 5fC;
- (b) incubating the nucleic acid molecule with a TDG enzyme to excise the 5caC or 5fC and generate an abasic site;
- (c) incubating the nucleic acid molecule with a thymine derivative comprising an azide moiety to attach the thymine derivative to the abasic site;
- and
- (d) sequencing the nucleic acid molecule.
A compound of formula (I):
- wherein n is an integer from 0 to 5 and m is an integer from 1 to 5.
A compound having formula (II):
- wherein X is a linker and Y is a click chemistry compatible reactive group selected from alkynes, azides, strained alkynes, dienes, dieneophiles, alkoxyamines, carbonyls, phosphines, hydrazides, thiols, alkenes, tetrazines, tetrazoles, isocyanates, isothiocyanates, and 1,3-nitrones.
A compound having formula (III):
- wherein R is H or alkyl.
Dependencies are inferred from the claim text.