The discoveries of myriad non-coding RNA molecules, each transiting through multiple flexible states in cells or virions, present major challenges for structure determination. Advances in high-throughput chemical mapping give new routes for characterizing entire transcriptomesin vivo, but the resulting one-dimensional data generally remain too information-poor to allow accuratede novostructure determination. Multidimensional chemical mapping (MCM) methods seek to address this challenge. Mutate-and-map (M2), RNA interaction groups by mutational profiling (RING-MaP and MaP-2D analysis) and multiplexed •OH cleavage analysis (MOHCA) measure how the chemical reactivities of every nucleotide in an RNA molecule change in response to modifications at every other nucleotide. A growing body ofin vitroblind tests and compensatory mutation/rescue experiments indicate that MCM methods give consistently accurate secondary structures and global tertiary structures for ribozymes, ribosomal domains and ligand-bound riboswitch aptamers up to 200 nucleotides in length. Importantly, MCM analyses provide detailed information on structurally heterogeneous RNA states, such as ligand-free riboswitches that are functionally important but difficult to resolve with other approaches. The sequencing requirements of currently available MCM protocols scale at least quadratically with RNA length, precluding general application to transcriptomes or viral genomes at present. We propose a modify-cross-link-map (MXM) expansion to overcome this and other current limitations to resolving thein vivo ‘RNA structurome’.

中文翻译：

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通过多维化学作图的 RNA 结构

无数非编码 RNA 分子的发现，每一个都在细胞或病毒粒子中通过多种柔性状态，对结构确定提出了重大挑战。高通量化学作图的进展为表征整个转录组提供了新途径体内, 但得到的一维数据通常信息量太少而无法准确从头结构确定。多维化学绘图 (MCM) 方法旨在解决这一挑战。变异和映射（M2)，通过突变分析（RING-MaP 和 MaP-2D 分析）和多重•OH 裂解分析 (MOHCA) 的 RNA 相互作用组测量 RNA 分子中每个核苷酸的化学反应性如何随着其他核苷酸的修饰而变化。不断壮大的身体体外盲测和补偿性突变/拯救实验表明，MCM 方法为最长 200 个核苷酸的核酶、核糖体结构域和配体结合的核糖开关适体提供始终如一的准确二级结构和全局三级结构。重要的是，MCM 分析提供了有关结构异质 RNA 状态的详细信息，例如功能重要但难以用其他方法解决的无配体核糖开关。当前可用的 MCM 协议的测序要求至少与 RNA 长度成二次方，目前排除了对转录组或病毒基因组的一般应用。我们提出了一个修改交叉链接图（MXM）扩展来克服这个和其他当前限制来解决体内'RNA 结构组”。