Helmholtz Institute for RNA-based Infection Research, Helmholtz Centre for Infection Research, Würzburg, Germany.
Faculty of Medicine, University of Würzburg, Würzburg, Germany.
Nat Protoc. 2024 Jun;19(6):1835-1865. doi: 10.1038/s41596-024-00959-3. Epub 2024 Feb 12.
RNA structure determination is essential to understand how RNA carries out its diverse biological functions. In cells, RNA isoforms are readily expressed with partial variations within their sequences due, for example, to alternative splicing, heterogeneity in the transcription start site, RNA processing or differential termination/polyadenylation. Nanopore dimethyl sulfate mutational profiling (Nano-DMS-MaP) is a method for in situ isoform-specific RNA structure determination. Unlike similar methods that rely on short sequencing reads, Nano-DMS-MaP employs nanopore sequencing to resolve the structures of long and highly similar RNA molecules to reveal their previously hidden structural differences. This Protocol describes the development and applications of Nano-DMS-MaP and outlines the main considerations for designing and implementing a successful experiment: from bench to data analysis. In cell probing experiments can be carried out by an experienced molecular biologist in 3-4 d. Data analysis requires good knowledge of command line tools and Python scripts and requires a further 3-5 d.
RNA 结构测定对于理解 RNA 如何执行其多种生物学功能至关重要。在细胞中，由于例如选择性剪接、转录起始位点的异质性、RNA 加工或差异终止/多聚腺苷酸化，RNA 异构体很容易在其序列中出现部分变异而被表达。纳米孔硫酸二甲酯突变分析（Nano-DMS-MaP）是一种用于原位异构体特异性 RNA 结构测定的方法。与依赖短测序读长的类似方法不同，Nano-DMS-MaP 采用纳米孔测序来解析长且高度相似的 RNA 分子的结构，从而揭示其先前隐藏的结构差异。本方案描述了 Nano-DMS-MaP 的开发和应用，并概述了设计和实施成功实验的主要注意事项：从实验台到数据分析。在细胞探测实验中，经验丰富的分子生物学家可以在 3-4 天内完成。数据分析需要对命令行工具和 Python 脚本有很好的了解，并且还需要 3-5 天的时间。
