Modular Oxidation of Cytosine Modifications and Their Application in Direct and Quantitative Sequencing of 5-Hydroxymethylcytosine

• DOI: 10.1021/jacs.3c01663
• Publication Date: 2023-03-24T13:06:32Z
• Authors: Haiqi Xu; Jinfeng Chen; Jingfei Cheng; Linzhen Kong; Xiufei Chen; Masato Inoue; Yibin Liu; Skirmantas Kriauc...; Meiping Zhao; Chun-Xiao Song
• Keywords: Mice; Cystine; Humans; Animals; DNA; DNA Methylation; Oxidation-Reduction

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.