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UdgX介导的尿嘧啶单核苷酸分辨测序
作者:小柯机器人 发布时间:2022/1/21 16:36:02

同济大学陈义汉团队开发出了udgx介导的尿嘧啶单核苷酸分辨测序。相关研究成果发表在2022年1月16日出版的《美国化学会杂志》。

尿嘧啶是DNA中的一种异常碱基,由脱氧尿苷(dU)错误结合或胞嘧啶脱氨产生,参与多种生理和病理过程。尿嘧啶的全基因组图谱对于这些过程的研究非常重要。目前用于尿嘧啶全基因组定位的方法都依赖于尿嘧啶DNA N-糖苷酶(UNG),并且在分辨率、特异性和/或敏感性方面受到限制。

该文中,研究人员开发了一种UdgX交联和聚合酶停滞测序(“Ucaps-seq”)方法,以单核苷酸分辨率检测dU。首先,Ucaps-seq在合成DNA上的特异性得到证实。然后在来自不同来源的两个基因组上验证了该方法的有效性。Ucaps-seq不仅检测到尿嘧啶整体升高的培美曲塞治疗的癌细胞dT位点的dU富集,还检测到活化B细胞“WRC”基序内dC位点的dU,其在特定区域的dU增加。

最后,利用Ucaps-seq检测胞嘧啶碱基编辑器(nCas9 APOBEC)引入的dU,并在细胞环境中识别出一个新的非靶点。总之,Ucaps-seq是一个功能强大的工具,具有许多潜在的应用,特别是在评估基本编辑保真度方面。

附:英文原文

Title: UdgX-Mediated Uracil Sequencing at Single-Nucleotide Resolution

Author: Liudan Jiang, Jiayong Yin, Maoxiang Qian, Shaoqin Rong, Shengqi Zhang, Kejing Chen, Chengchen Zhao, Yuanqing Tan, Jiayin Guo, Hao Chen, Siyun Gao, Tingting Liu, Yi Liu, Bin Shen, Jian Yang, Yong Zhang, Fei-Long Meng, Jinchuan Hu, Honghui Ma, Yi-Han Chen

Issue&Volume: January 16, 2022

Abstract: As an aberrant base in DNA, uracil is generated by either deoxyuridine (dU) misincorporation or cytosine deamination, and involved in multiple physiological and pathological processes. Genome-wide profiles of uracil are important for study of these processes. Current methods for whole-genome mapping of uracil all rely on uracil-DNA N-glycosylase (UNG) and are limited in resolution, specificity, and/or sensitivity. Here, we developed a UdgX cross-linking and polymerase stalling sequencing (“Ucaps-seq”) method to detect dU at single-nucleotide resolution. First, the specificity of Ucaps-seq was confirmed on synthetic DNA. Then the effectiveness of the approach was verified on two genomes from different sources. Ucaps-seq not only identified the enrichment of dU at dT sites in pemetrexed-treated cancer cells with globally elevated uracil but also detected dU at dC sites within the “WRC” motif in activated B cells which have increased dU in specific regions. Finally, Ucaps-seq was utilized to detect dU introduced by the cytosine base editor (nCas9-APOBEC) and identified a novel off-target site in cellular context. In conclusion, Ucaps-seq is a powerful tool with many potential applications, especially in evaluation of base editing fidelity.

DOI: 10.1021/jacs.1c11269

Source: https://pubs.acs.org/doi/10.1021/jacs.1c11269

 

期刊信息

JACS:《美国化学会志》,创刊于1879年。隶属于美国化学会,最新IF:14.612
官方网址:https://pubs.acs.org/journal/jacsat
投稿链接:https://acsparagonplus.acs.org/psweb/loginForm?code=1000