Kaczynski Tadeusz, Hussain Ali, Farkas Michael
Department of Ophthalmology, State University of New York at Buffalo, United States.
Research Service, VA Medical Center, United States.
MethodsX. 2019 May 30;6:1292-1304. doi: 10.1016/j.mex.2019.05.014. eCollection 2019.
RNA-binding proteins (RBPs) are instrumental in the biochemical processing and physiological functioning of non-coding RNAs. Therefore, as interest in non-coding RNAs continues to expand, refining the techniques capable of probing protein-RNA interactions will prove ever more valuable in the characterization of these molecules. To identify the RNAs bound by a given RBP, cross-linking and immunoprecipitation (CLIP) and its iterations have been widely utilized, but these approaches can be complex, labor-intensive, and time consuming. Here, we describe a rapid and technically simple method based upon individual nucleotide resolution CLIP (iCLIP) and infrared CLIP (irCLIP). Termed quick-irCLIP, our protocol circumvents confounding steps, can be completed in less than three days, and is capable of interrogating protein-RNA interactions at single nucleotide resolution.
RNA结合蛋白（RBPs）在非编码RNA的生化加工和生理功能中发挥着重要作用。因此，随着对非编码RNA的兴趣不断扩大，完善能够探测蛋白质-RNA相互作用的技术对于这些分子的表征将变得越来越有价值。为了鉴定与特定RBP结合的RNA，交联免疫沉淀（CLIP）及其衍生方法已被广泛应用，但这些方法可能复杂、 labor-intensive且耗时。在这里，我们描述了一种基于单核苷酸分辨率CLIP（iCLIP）和红外CLIP（irCLIP）的快速且技术简单的方法。我们的方案称为快速irCLIP，它规避了混淆步骤，可在不到三天的时间内完成，并且能够在单核苷酸分辨率下研究蛋白质-RNA相互作用。
