Su Li, Zhao Fengjiao, Yu Huahang, Bai Min, Xue Jing, Cao Xiaowen, Chen Feng, Zhao Yongxi
Institute of Analytical Chemistry and Instrument for Life Science, The Key Laboratory of Biomedical Information Engineering of Ministry of Education, School of Life Science and Technology, Xi'an Jiaotong University, Xianning West Road, Xi'an, Shaanxi 710049, China.
Chem Commun (Camb). 2022 Jan 27;58(9):1322-1325. doi: 10.1039/d1cc06080e.
Both sequence enrichment and base resolution are essential for accurate sequencing analysis of low-abundance RNA. Yet they are hindered by the lack of molecular tools. Here we report a bifunctional chemical signature for RNA 4-thiouridine (4sU) enrichment sequencing with single-base resolution. This chemical signature is designed for specific 4sU labeling with two functional parts. One part is a distal alkynyl group for the biotin-assisted pulldown enrichment of target molecules click chemistry crosslinking. The other part is a -NH group proximal to the pyrimidine ring of 4sU. It allows 4sU-to-cytosine transition during the polymerase-catalyzed extension reaction based on altering hydrogen-bonding patterns. Ultimately, the 4sU-containing RNA molecules can be enriched and accurately analyzed by single-base resolution sequencing. The proposed method also holds great potential to investigate transcriptome dynamics integrated with high-throughput sequencing.
序列富集和碱基分辨率对于低丰度RNA的准确测序分析都至关重要。然而，它们受到分子工具缺乏的阻碍。在此，我们报告了一种用于RNA 4-硫尿苷（4sU）富集测序的双功能化学标记，具有单碱基分辨率。这种化学标记通过两个功能部分设计用于特异性4sU标记。一部分是远端炔基，用于生物素辅助的靶分子下拉富集——点击化学交联。另一部分是靠近4sU嘧啶环的-NH基团。它允许在聚合酶催化的延伸反应过程中基于改变氢键模式实现4sU到胞嘧啶的转变。最终，含4sU的RNA分子可以通过单碱基分辨率测序进行富集和准确分析。所提出的方法在结合高通量测序研究转录组动态方面也具有巨大潜力。
