Single-Molecule Correlated Chemical Probing: A Revolution in RNA Structure Analysis.
Single-Molecule Correlated Chemical Probing: A Revolution in RNA Structure Analysis.
复制标题
单分子相关化学探测：RNA 结构分析的革命。
DOI：
10.1021/acs.accounts.2c00782
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发表时间：
2023
影响因子：
18.3
通讯作者：
Weeks,KevinM
中科院分区：
文献类型：
--
作者：
Mustoe,AnthonyM;Weidmann,ChaseA;Weeks,KevinM
伴发RNA分子以其线性序列和碱基配对的二级和三级结构传递生物信息。化学探测实验涉及用修饰构象动态核苷酸的试剂处理RNA，广泛地使在不同环境中（包括活细胞中）检查短程和长程RNA结构成为可能。几十年来，化学探测实验一直以每个核苷酸的方式进行解释，因此在每个核苷酸处测量的反应性报告了样品中所有RNA分子位置的平均结构。然而，有许多重要的情况下，每核苷酸的化学探测福尔斯，包括RNA的结合蛋白质，RNA的形成复杂的高阶结构，和RNA的样品多种conformation.Recent实验和计算创新已经开始了革命的RNA结构分析通过转换成一个大规模的并行，单分子实验的化学探测。通过称为突变谱（MaP）的专门逆转录策略，可以在单个RNA分子内测量多个化学修饰事件。通过直接碱基配对或大规模折叠-解折叠转换进行结构通信的核苷酸将以相关的方式与化学探针反应，从而揭示隐藏在传统方法中的结构复杂性。这些单分子相关化学探测（smCCP）实验可以被解释为直接识别碱基配对的核苷酸（PAIR-MaP策略），并揭示长距离，通过空间结构通信（RING-MaP）。相关探测还可以定义复杂RNA集合的热力学群体（DANCE-MaP）。复杂的RNA-蛋白质网络可以通过将蛋白质与RNA交联并测量交联位置之间的相关性（RNP-MaP）来查询。因此，smCCP以前所未有的准确度可视化RNA二级和高级结构，定义新结构，RNA-蛋白质相互作用网络，时间分辨动力学和变构结构开关。这些策略并不是相互排斥的;在有利的情况下，RNA结构的多个水平-碱基配对，通过空间结构通讯和平衡系综-可以同时解决。smCCP所需的物理实验非常简单，并且可以在细胞中对任何大小的RNA进行实验，包括大型非编码RNA和mRNA。单分子相关化学探测为生命系统中RNA的新一代生物物理研究铺平了道路。
ConspectusRNA molecules convey biological information both in their linear sequence and in their base-paired secondary and tertiary structures. Chemical probing experiments, which involve treating an RNA with a reagent that modifies conformationally dynamic nucleotides, have broadly enabled examination of short- and long-range RNA structure in diverse contexts, including in living cells. For decades, chemical probing experiments have been interpreted in a per-nucleotide way, such that the reactivity measured at each nucleotide reports the average structure at a position over all RNA molecules within a sample. However, there are numerous important cases where per-nucleotide chemical probing falls short, including for RNAs that are bound by proteins, RNAs that form complex higher order structures, and RNAs that sample multiple conformations.Recent experimental and computational innovations have started a revolution in RNA structure analysis by transforming chemical probing into a massively parallel, single-molecule experiment. Enabled by a specialized reverse transcription strategy called mutational profiling (MaP), multiple chemical modification events can be measured within individual RNA molecules. Nucleotides that communicate structurally through direct base pairing or large-scale folding–unfolding transitions will react with chemical probes in a correlated manner, thereby revealing structural complexity hidden to conventional approaches. These single-molecule correlated chemical probing (smCCP) experiments can be interpreted to directly identify nucleotides that base pair (the PAIR-MaP strategy) and to reveal long-range, through-space structural communication (RING-MaP). Correlated probing can also define the thermodynamic populations of complex RNA ensembles (DANCE-MaP). Complex RNA–protein networks can be interrogated by cross-linking proteins to RNA and measuring correlations between cross-linked positions (RNP-MaP).smCCP thus visualizes RNA secondary and higher-order structure with unprecedented accuracy, defining novel structures, RNA–protein interaction networks, time-resolved dynamics, and allosteric structural switches. These strategies are not mutually exclusive; in favorable cases, multiple levels of RNA structure ─ base pairing, through-space structural communication, and equilibrium ensembles ─ can be resolved concurrently. The physical experimentation required for smCCP is profoundly simple, and experiments are readily performed in cells on RNAs of any size, including large noncoding RNAs and mRNAs. Single-molecule correlated chemical probing is paving the way for a new generation of biophysical studies on RNA in living systems.