Department of Biological Sciences, The University of North Carolina at Charlotte, Charlotte, NC, USA.
Methods Mol Biol. 2024;2822:311-334. doi: 10.1007/978-1-0716-3918-4_20.
The structure of RNA molecules is absolutely critical to their functions in a biological system. RNA structure is dynamic and changes in response to cellular needs. Within the last few decades, there has been an increased interest in studying the structure of RNA molecules and how they change to support the needs of the cell in different conditions. Selective 2'-hydroxyl acylation-based mutational profiling using high-throughput sequencing is a powerful method to predict the secondary structure of RNA molecules both in vivo and in immunopurified samples. Selective 2'-hydroxyl acylation-based mutational profiling using high-throughput sequencing works by adding bulky groups onto accessible "flexible" bases in an RNA molecule that are not involved in any base-pairing or RNA-protein interactions. When the RNA is reverse transcribed into cDNA, the bulky groups are incorporated as base mutations, which can be compared to an unmodified control to identify the locations of flexible bases. The comparison of sequence data between modified and unmodified samples allows the computer software program (developed to generate reactivity profiles) to generate RNA secondary structure models. These models can be compared in a variety of conditions to determine how specific stimuli influence RNA secondary structures.
RNA 分子的结构对其在生物系统中的功能至关重要。RNA 结构是动态的，会根据细胞的需求而变化。在过去的几十年中，人们越来越关注研究 RNA 分子的结构以及它们如何变化以满足细胞在不同条件下的需求。使用高通量测序的选择性 2'-羟基酰化基于突变分析是一种强大的方法，可以预测 RNA 分子的二级结构，无论是在体内还是在免疫纯化样本中。使用高通量测序的选择性 2'-羟基酰化基于突变分析通过向 RNA 分子中不参与任何碱基配对或 RNA-蛋白质相互作用的可及“灵活”碱基添加大体积基团来工作。当 RNA 被反转录成 cDNA 时，大体积基团被掺入作为碱基突变，可以与未修饰的对照进行比较，以确定灵活碱基的位置。修饰和未修饰样品之间的序列数据比较允许计算机软件程序（用于生成反应性谱）生成 RNA 二级结构模型。可以在各种条件下比较这些模型，以确定特定刺激如何影响 RNA 二级结构。
