Liu Xingyu, Qi Qianqian, Xiong Wei, Zhang Yuanyuan, Shen Wei, Xu Xinyan, Zhao Yunting, Li Ming, Zhou Enyi, Tian Tian, Zhou Xiang
Key Laboratory of Biomedical Polymers of Ministry of Education, College of Chemistry and Molecular Sciences, Hubei Province Key Laboratory of Allergy and Immunology, The Institute of Molecular Medicine, Wuhan University People's Hospital, Wuhan University, Wuhan 430072, Hubei, China.
Nucleic Acids Res. 2025 Apr 10;53(7). doi: 10.1093/nar/gkaf283.
Many existing methods for post-transcriptional RNA modification rely on a single-step approach, limiting the ability to reversibly control m6A methylation at specific sites. Here, we address this challenge by developing a multi-step system that builds on the concept of sequential RNA bioorthogonal chemistry. Our strategy uses an azide-based reagent (NAI-N3) capable of both cleavage and ligation reactions, thereby allowing iterative and reversible modifications of RNA in living cells. By applying this approach in CRISPR (clustered regularly interspaced short palindromic repeats)-based frameworks, we demonstrate tailored editing of m6A marks at targeted RNA sites, overcoming the one-way restriction of conventional bioorthogonal methods. This sequential protocol not only broadens the scope for fine-tuned RNA regulation but also provides a versatile platform for exploring dynamic m6A function in genetic and epigenetic research.
许多现有的转录后RNA修饰方法依赖于单步操作，限制了在特定位点可逆控制m6A甲基化的能力。在此，我们通过开发一种基于连续RNA生物正交化学概念的多步系统来应对这一挑战。我们的策略使用一种基于叠氮化物的试剂（NAI-N3），它能够进行切割和连接反应，从而允许在活细胞中对RNA进行迭代和可逆修饰。通过在基于CRISPR（成簇规律间隔短回文重复序列）的框架中应用这种方法，我们展示了在靶向RNA位点对m6A标记进行定制编辑，克服了传统生物正交方法的单向限制。这种连续方案不仅拓宽了微调RNA调控的范围，还为在遗传和表观遗传研究中探索动态m6A功能提供了一个通用平台。
