英国牛津大学Chun-Xiao Song团队报道了胞嘧啶修饰的模块化以及他们在5-Hydroxymethylcytosine直接和定量测序方面的应用。相关研究成果发表在2023年3月24日出版的《美国化学会杂志》。

胞嘧啶修饰的选择性、高效和可控氧化对于表观遗传学分析是有价值的，但目前取得的进展有限。

该文中，研究人员提出了两个模块化化学氧化反应：使用4-乙酰氨基-2,2,6,6-四甲基哌啶-1-氧代四氟硼酸铵（ACT+BF4-）将5-羟甲基胞嘧啶（5hmC）转化为5-甲酰基胞嘧啶（5fC），以及通过Pinnick氧化将5fC进一步转化为5-羧基胞嘧啶（5caC）。这两种反应对双链DNA都是温和有效的。研究人员将这两种氧化与硼烷还原结合起来，开发了化学辅助吡啶-硼烷测序+（CAPS+），用于5hmC的直接定量测绘。与CAPS相比，CAPS+提高了转化率和假阳性率。

研究人员将CAPS+应用于小鼠胚胎干细胞、人类正常大脑和胶质母细胞瘤DNA样本，并证明了其在分析羟甲基组方面的优越敏感性。

附：英文原文

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

Author: Haiqi Xu, Jinfeng Chen, Jingfei Cheng, Linzhen Kong, Xiufei Chen, Masato Inoue, Yibin Liu, Skirmantas Kriaucionis, Meiping Zhao, Chun-Xiao Song

Issue&Volume: March 24, 2023

Abstract: 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.

DOI: 10.1021/jacs.3c01663

Source: https://pubs.acs.org/doi/10.1021/jacs.3c01663