Lu Zhipeng, Gong Jing, Zhang Qiangfeng Cliff
Department of Dermatology, Center for Personal Dynamic Regulomes, Stanford University, Stanford, CA, 94305, USA.
MOE Key Laboratory of Bioinformatics, Beijing Advanced Innovation Center for Structural Biology, Center for Synthetic and Systems Biology, Tsinghua-Peking Joint Center for Life Sciences, School of Life Sciences, Tsinghua University, Haidian, Beijing, 100084, China.
Methods Mol Biol. 2018;1649:59-84. doi: 10.1007/978-1-4939-7213-5_4.
RNA has the intrinsic propensity to form base pairs, leading to complex intramolecular and intermolecular helices. Direct measurement of base pairing interactions in living cells is critical to solving transcriptome structure and interactions, and investigating their functions (Lu and Chang, Curr Opin Struct Biol 36:142-148, 2016). Toward this goal, we developed an experimental method, PARIS (Psoralen Analysis of RNA Interactions and Structures), to directly determine transcriptome-wide base pairing interactions (Lu et al., Cell 165(5):1267-1279, 2016). PARIS combines four critical steps, in vivo cross-linking, 2D gel purification, proximity ligation, and high-throughput sequencing to achieve high-throughput and near-base pair resolution determination of the RNA structurome and interactome in living cells. In this chapter, we aim to provide a comprehensive discussion on the principles behind the experimental and computational strategies, and a step-by-step description of the experiment and analysis.
RNA具有形成碱基对的内在倾向，从而导致复杂的分子内和分子间螺旋结构。直接测量活细胞中的碱基配对相互作用对于解析转录组结构和相互作用以及研究它们的功能至关重要（Lu和Chang，《结构生物学当前观点》36:142 - 148，2016年）。为了实现这一目标，我们开发了一种实验方法——PARIS（RNA相互作用和结构的补骨脂素分析），用于直接确定全转录组范围的碱基配对相互作用（Lu等人，《细胞》165(5):1267 - 1279，2016年）。PARIS结合了四个关键步骤，即体内交联、二维凝胶纯化、邻近连接和高通量测序，以实现对活细胞中RNA结构组和相互作用组的高通量且近乎碱基对分辨率的测定。在本章中，我们旨在全面讨论实验和计算策略背后的原理，并对实验和分析进行逐步描述。
