Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06511.
Chemical Biology Institute, Yale University, West Haven, Connecticut 06516.
Cold Spring Harb Perspect Biol. 2019 Nov 1;11(11):a032276. doi: 10.1101/cshperspect.a032276.
The diverse roles of cellular RNAs can be studied by purifying RNAs of interest together with the biomolecules they bind. Biotinylated antisense oligonucleotides that hybridize specifically to the RNA of interest provide a general approach to develop affinity reagents for these experiments. Such oligonucleotides can be used to enrich endogenous RNAs from cross-linked chromatin extracts to study the genomic binding sites of RNAs. These hybridization capture protocols are evolving modular experiments that are compatible with a range of cross-linkers and conditions. This review discusses the principles of these hybridization capture experiments as well as considerations and controls necessary to interpret the resulting data without being misled by artifactual signals.
细胞 RNA 的多种作用可以通过纯化与它们结合的生物分子以及感兴趣的 RNA 来研究。与感兴趣的 RNA 特异性杂交的生物素化反义寡核苷酸为这些实验提供了开发亲和试剂的一般方法。此类寡核苷酸可用于从交联染色质提取物中富集内源性 RNA，以研究 RNA 的基因组结合位点。这些杂交捕获方案正在发展成为模块化实验，与一系列交联剂和条件兼容。本综述讨论了这些杂交捕获实验的原理，以及在没有被人为信号误导的情况下解释结果数据所需的考虑因素和控制。
