m6A甲基化是一种重要的RNA转录后修饰，分布广泛，在生命过程中的重要性日益凸显。RNA m6A甲基化修饰是METTL3-METTL14甲基转移酶及辅助因子所组成的蛋白质机器介导。我们已解析了其催化结构域的结构，预实验还发现METTL3中的锌指结构域能特异性结合RNA底物，对酶活起关键作用。本项目拟表征核心甲基化酶在RNA动态修饰过程中的结构变化，揭示辅助因子和环境因素对动态结构和催化过程的调控。另一方面，本项目拟分析甲基化动态修饰对RNA结构和功能的影响，确立甲基化修饰与序列、结构的关联。为此，本项目将集成多种稳态表征和动态分析技术，整合单分子测定和系综平均数据，并借助化学标记手段，发现生物大分子动态修饰和动态变化的物理化学本质。两名项目主要申请人有丰富的相关研究经验，并长期合作、技术互补。本项目的实施将阐释RNA m6A甲基化的动态修饰和调控的机制，为发展相应的化学干预提供理论指导。

RNA m6A methylation is an important post-transcriptional modification, which is widely present and is involved in multitude of physiological and pathological processes. A protein machinery consisting of METTL3-METTL14 methyltransferase and other cofactors is responsible for RNA m6A methylation. Previously, we have determined the crystal structure of the catalytic domain of METTL3-METTL14. Our more recent experimental data also found that METTL3 contains a zinc finger domain, which specifically binds to RNA substrate and plays a key role for enzymatic catalysis. This project will characterize the dynamic structure of core methyltransferase in the RNA modification processes, and to reveal the regulatory mechanism by cofactor proteins and environmental factors. On the other hand, this project attempts to analyze the effect of methylation for RNA regarding its ensemble structure and function, and to establish the relationship between RNA methylation and its sequence and structure. To this end, the project will integrate a variety of steady-state characterization and dynamic analysis techniques, and use conjointly single-molecule data and bulk measurements, with additional means of bio-conjugation. Together, the project intends to reveal the physical chemical nature for dynamic modifications and conformational dynamics of biological macromolecules. Two major applicants in this project have a wealth of relevant research experience and long-term collaboration, and are complementary in their expertise. The implementation of this project will account for the dynamic modification and regulation mechanism of RNA m6A modification, and will provide theoretical guidance for future development of relevant chemical interventions.