Whitehead Institute for Biomedical Research, Cambridge, MA, USA; Program in Virology, Harvard Medical School, Boston, MA, USA; Brigham and Women's Hospital, Boston, MA, USA.
Whitehead Institute for Biomedical Research, Cambridge, MA, USA.
Methods. 2020 Nov 1;183:68-75. doi: 10.1016/j.ymeth.2020.04.001. Epub 2020 Apr 3.
RNA structure is critically important to RNA viruses in every part of the replication cycle. RNA structure is also utilized by DNA viruses in order to regulate gene expression and interact with host factors. Advances in next-generation sequencing have greatly enhanced the utility of chemical probing in order to analyze RNA structure. This review will cover some recent viral RNA structural studies using chemical probing and next-generation sequencing as well as the advantages of dimethyl sulfate (DMS)-mutational profiling and sequencing (MaPseq). DMS-MaPseq is a robust assay that can easily modify RNA in vitro, in cell and in virion. A detailed protocol for whole-genome DMS-MaPseq from cells transfected with HIV-1 and the structure of TAR as determined by DMS-MaPseq is presented. DMS-MaPseq has the ability to answer a variety of integral questions about viral RNA, including how they change in different environments and when interacting with different host factors.
RNA 结构对于复制周期各个阶段的 RNA 病毒都至关重要。DNA 病毒也利用 RNA 结构来调节基因表达并与宿主因子相互作用。下一代测序技术的进步极大地增强了化学探测在分析 RNA 结构方面的作用。本综述将介绍一些使用化学探测和下一代测序技术的最新病毒 RNA 结构研究，以及硫酸二甲酯（DMS）-突变分析和测序（MaPseq）的优势。DMS-MaPseq 是一种强大的测定方法，可轻松在体外、细胞中和病毒粒子中修饰 RNA。本文介绍了一种详细的从转染 HIV-1 的细胞中进行全基因组 DMS-MaPseq 的方案，以及通过 DMS-MaPseq 确定的 TAR 的结构。DMS-MaPseq 能够回答有关病毒 RNA 的各种基本问题，包括它们在不同环境中和与不同宿主因子相互作用时如何变化。
