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

• Published in: Journal of the American Chemical Society Vol. 145; no. 13; pp. 7095 - 7100
• Main Authors: Xu, Haiqi, Chen, Jinfeng, Cheng, Jingfei, Kong, Linzhen, Chen, Xiufei, Inoue, Masato, Liu, Yibin, Kriaucionis, Skirmantas, Zhao, Meiping, Song, Chun-Xiao
• Format: Journal Article
• Language: English
• Published: WASHINGTON American Chemical Society 05.04.2023; Amer Chemical Soc
• Subjects: Animals; brain; Chemistry; Chemistry, Multidisciplinary; Communication; Cystine - analysis; cytosine; DNA; DNA - genetics; DNA Methylation; epigenetics; glioblastoma; Humans; Mice; oxidation; Oxidation-Reduction; Physical Sciences; pyridines; Science & Technology; ALCOHOLS; REAGENT; 5-METHYLCYTOSINE; MECHANISM; DNA; ALDEHYDES; INSIGHT; SALTS; 5-FORMYLCYTOSINE; OXIDANTS
• ISSN: 0002-7863; 1520-5126; 1520-5126
• DOI: 10.1021/jacs.3c01663

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.