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Selective, efficient, and controllable oxidation of cytosine modifications is valuable for epigenetic analyses, yet only limited progress has been made. Here, we present two modular chemical oxidation reactions: conversion of 5-hydroxymethylcytosine (5hmC) into 5-formylcytosine (5fC) using 4-acetamido-2,2,6,6-tetramethylpiperidine-1-oxoammonium tetrafluoroborate (ACT+BF4-) and further transformation of 5fC into 5-carboxycytosine (5caC) through Pinnick oxidation. Both reactions are mild and efficient on double-stranded DNA. We integrated these two oxidations with borane reduction to develop chemical-assisted pyridine borane sequencing plus (CAPS+), for direct and quantitative mapping of 5hmC. Compared with CAPS, CAPS+ improved the conversion rate and false-positive rate. We applied CAPS+ to mouse embryonic stem cells, human normal brain, and glioblastoma DNA samples and demonstrated its superior sensitivity in analyzing the hydroxymethylome.

Original publication

DOI

10.1021/jacs.3c01663

Type

Journal article

Journal

Journal of the American Chemical Society

Publication Date

04/2023

Volume

145

Pages

7095 - 7100

Addresses

Ludwig Institute for Cancer Research, Nuffield Department of Medicine, University of Oxford, Oxford OX3 7FZ, U.K.

Keywords

Animals, Humans, Mice, Cystine, DNA, DNA Methylation, Oxidation-Reduction