纽约基因组中心技术创新实验室的研究人员在Cell Genomics杂志发表了题为Direct detection of RNA modifications and structure using single-molecule nanopore sequencing的文章。这篇文章介绍了一种新技术：直接RNA纳米孔测序（Direct RNA nanopore sequencing），它能够在单分子水平上检测RNA的修饰和结构。这项技术通过纳米孔测序检测RNA上的内源性和外源性修饰，如2’-O-甲基化（Nm）和假尿苷（𝛹），这通过改变纳米孔传输动力学来实现这些修饰的检测。研究者们开发了一种名为nanoSHAPE的方法，它结合了一种小分子加成反应剂AcIm，能够揭示RNA结构，并通过长读长的测序进行分析。
文章的主要亮点包括：
1. 纳米孔测序技术能够检测单个RNA分子上的内源性RNA修饰。
2. 2’-O-甲基化（Nm）和伪尿苷（𝛹）改变纳米孔传输动力学。
3. AcIm作为SHAPE-MaP中一种小分子加成反应剂
4. AcIm使得全长单分子结构分析成为可能。
这一技术不仅能够提高我们对RNA修饰功能和调控的理解，还有助于揭示RNA在疾病中的作用，为未来的治疗提供新的方向。
论文摘要：
Modifications are present on many classes of RNA, including tRNA, rRNA, and mRNA. These modifications modulate diverse biological processes such as genetic recoding and mRNA export and folding. In addition, modifications can be introduced to RNA molecules using chemical probing strategies that reveal RNA structure and dynamics. Many methods exist to detect RNA modifications by short-read sequencing; however, limitations on read length inherent to short-read-based methods dissociate modifications from their native context, preventing single-molecule modification analysis. Here, we demonstrate direct RNA nanopore sequencing to detect endogenous and exogenous RNA modifications on long RNAs at the single-molecule level. We detect endogenous 2’-O-methyl and base modifications across E. coli and S. cerevisiae ribosomal RNAs as shifts in current signal and dwell times distally through interactions with the helicase motor protein. We further use the 2’-hydroxyl reactive SHAPE reagent acetylimidazole to probe RNA structure at the single-molecule level with readout by direct nanopore sequencing.
详情请阅读原文：https://doi.org/10.1016/j.xgen.2022.100097
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