J Am Chem Soc. 2023 Aug 30;145(34):18773-18777. doi: 10.1021/jacs.3c07736. Epub 2023 Aug 15.
RNA molecules undergo conformational transitions in response to cellular and environmental stimuli. Site-specific protonation, a fundamental chemical property, can alter the conformational landscape of RNA to regulate their functions. However, characterizing protonation-coupled RNA conformational ensembles on a large scale remains challenging. Here, we present pH-differential mutational profiling (PD-MaP) with dimethyl sulfate probing for high-throughput detection of protonation-coupled conformational ensembles in RNA. We demonstrated this approach on microRNA-21 precursor (pre-miR-21) and recapitulated a previously discovered A-G-coupled conformational ensemble. Additionally, we identified a secondary protonation event involving an A-C mismatch. We validated the occurrence of both protonation-coupled ensembles in pre-miR-21 using NMR relaxation dispersion spectroscopy. Furthermore, the application of PD-MaP on a library of well-annotated human primary microRNAs uncovered widespread protonation-coupled conformational ensembles, suggesting their potentially broad functions in biology.
RNA 分子会对细胞和环境刺激做出构象转变。作为一种基本的化学性质，特定位置的质子化作用可以改变 RNA 的构象景观，从而调节其功能。然而，在大规模上对质子化相关 RNA 构象整体进行特征描述仍然具有挑战性。在这里，我们提出了 pH 差异突变分析（PD-MaP）与硫酸二甲酯探测相结合的方法，用于高通量检测 RNA 中的质子化相关构象整体。我们在 microRNA-21 前体（pre-miR-21）上验证了该方法，并重现了先前发现的 A-G 偶联构象整体。此外，我们还鉴定出涉及 A-C 错配的二级质子化事件。我们使用 NMR 弛豫分散光谱学验证了 pre-miR-21 中两种质子化相关构象整体的发生。此外，将 PD-MaP 应用于一系列注释良好的人类初级 microRNAs 文库中，揭示了广泛存在的质子化相关构象整体，这表明它们在生物学中可能具有广泛的功能。
