美国芝加哥大学何川等研究人员合作实现DNA和RNA中5-甲基胞嘧啶的超快亚硫酸氢盐测序检测。相关论文于2024年1月2日在线发表在《自然—生物技术》杂志上。

研究人员表示，亚硫酸氢盐测序（BS-seq）检测5-甲基胞嘧啶（5mC）的局限性在于反应时间长、DNA损伤严重、5mC水平估计过高以及某些DNA序列的C-U转换不完全。

研究人员提出了超快速亚硫酸氢盐序列分析（UBS-seq），它使用高浓度亚硫酸氢盐试剂和高温反应，将亚硫酸氢盐反应速度提高了约13倍，从而减少了DNA损伤，降低了背景噪音。与传统的BS-seq相比，UBS-seq可从少量纯化的基因组DNA（如无细胞DNA）或直接从1到100个小鼠胚胎干细胞中构建文库，对5mC水平的高估更少，基因组覆盖率更高。

此外，UBS-seq还能从低投入的mRNA中定量绘制RNA 5-甲基胞嘧啶（m⁵C）图谱，并能检测高度结构化的RNA序列中的m⁵C几何结构。UBS-seq结果发现，NSUN2是负责HeLa mRNA中约90% m⁵C位点修饰的主要“写入”蛋白，并揭示了哺乳动物mRNA 5′区域中丰富的m⁵C位点，这些位点可能在mRNA翻译调控中具有功能性作用。

附：英文原文

Title: Ultrafast bisulfite sequencing detection of 5-methylcytosine in DNA and RNA

Author: Dai, Qing, Ye, Chang, Irkliyenko, Iryna, Wang, Yiding, Sun, Hui-Lung, Gao, Yun, Liu, Yushuai, Beadell, Alana, Perea, Jos, Goel, Ajay, He, Chuan

Issue&Volume: 2024-01-02

Abstract: Bisulfite sequencing (BS-seq) to detect 5-methylcytosine (5mC) is limited by lengthy reaction times, severe DNA damage, overestimation of 5mC level and incomplete C-to-U conversion of certain DNA sequences. We present ultrafast BS-seq (UBS-seq), which uses highly concentrated bisulfite reagents and high reaction temperatures to accelerate the bisulfite reaction by ~13-fold, resulting in reduced DNA damage and lower background noise. UBS-seq allows library construction from small amounts of purified genomic DNA, such as from cell-free DNA or directly from 1 to 100mouse embryonic stem cells, with less overestimation of 5mC level and higher genome coverage than conventional BS-seq. Additionally, UBS-seq quantitatively maps RNA 5-methylcytosine (m5C) from low inputs of mRNA and allows the detection of m5C stoichiometry in highly structured RNA sequences. Our UBS-seq results identify NSUN2 as the major ‘writer’ protein responsible for the deposition of ~90% of m5C sites in HeLa mRNA and reveal enriched m5C sites in 5′-regions of mammalian mRNA, which may have functional roles in mRNA translation regulation.

DOI: 10.1038/s41587-023-02034-w

Source: https://www.nature.com/articles/s41587-023-02034-w