Department of Chemistry, University of North Carolina, Chapel Hill, NC 27599.
Department of Chemistry, University of North Carolina, Chapel Hill, NC 27599
Proc Natl Acad Sci U S A. 2019 Dec 3;116(49):24574-24582. doi: 10.1073/pnas.1905491116. Epub 2019 Nov 19.
RNA structure and dynamics are critical to biological function. However, strategies for determining RNA structure in vivo are limited, with established chemical probing and newer duplex detection methods each having deficiencies. Here we convert the common reagent dimethyl sulfate into a useful probe of all 4 RNA nucleotides. Building on this advance, we introduce PAIR-MaP, which uses single-molecule correlated chemical probing to directly detect base-pairing interactions in cells. PAIR-MaP has superior resolution compared to alternative experiments, can resolve multiple sets of pairing interactions for structurally dynamic RNAs, and enables highly accurate structure modeling, including of RNAs containing multiple pseudoknots and extensively bound by proteins. Application of PAIR-MaP to human RNase MRP and 2 bacterial messenger RNA 5' untranslated regions reveals functionally important and complex structures undetected by prior analyses. PAIR-MaP is a powerful, experimentally concise, and broadly applicable strategy for directly visualizing RNA base pairs and dynamics in cells.
RNA 的结构和动态对于其生物学功能至关重要。然而，用于在体内确定 RNA 结构的策略受到限制，已建立的化学探测和更新的双链检测方法都有其不足之处。在这里，我们将常见的试剂硫酸二甲酯转化为一种有用的探针，用于探测所有 4 种 RNA 核苷酸。在此基础上，我们引入了 PAIR-MaP，它利用单分子相关化学探测直接检测细胞中的碱基配对相互作用。与替代实验相比，PAIR-MaP 的分辨率更高，可以解析结构动态 RNA 的多组配对相互作用，并能够进行高度准确的结构建模，包括含有多个假结和广泛结合蛋白质的 RNA。将 PAIR-MaP 应用于人类 RNase MRP 和 2 种细菌信使 RNA 5'非翻译区，揭示了先前分析中未检测到的具有重要功能和复杂结构的 RNA。PAIR-MaP 是一种强大、实验简洁且广泛适用于直接可视化细胞中 RNA 碱基对和动态的策略。
