SpyCLIP: an easy-to-use and high-throughput compatible CLIP platform for the characterization of protein-RNA interactions with high accuracy
SpyCLIP: an easy-to-use and high-throughput compatible CLIP platform for the characterization of protein-RNA interactions with high accuracy
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SpyCLIP：易于使用且高通量兼容的 CLIP 平台，用于高精度表征蛋白质-RNA 相互作用
DOI：
10.1093/nar/gkz049
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发表时间：
2019
影响因子：
14.9
通讯作者：
Wu Ligang
中科院分区：
文献类型：
--
作者：
Zhao Ya;Zhang Yao;Teng Yilan;Liu Kai;Liu Yanqing;Li Weihua;Wu Ligang
紫外交联和免疫沉淀（CLIP）结合高通量测序是最先进的技术来表征蛋白质-RNA相互作用，但只有一小部分RNA结合蛋白（RBP）已被CLIP研究，由于其复杂的程序和高水平的假阳性信号。在此，我们报告了SpyCLIP方法，该方法采用RBP融合SpyTag和SpyCatcher之间形成的共价键，该共价键可以承受最苛刻的洗涤条件以去除非特异性相互作用。此外，SpyCLIP绕过了放射性标记和PAGE膜纯化步骤，整个过程可以在珠上进行，并且易于自动化。我们通过SpyCLIP研究了多种RBP，并生成了高质量的RNA结合图谱，其重现性和准确性显著提高。因此，SpyCLIP的小标签尺寸和方便的方案为蛋白质-RNA相互作用的常规表征和高通量研究提供了稳健的方法。
UV crosslinking and immunoprecipitation (CLIP) coupled with high-throughput sequencing is the most state-of-the-art technology to characterize protein–RNA interactions, yet only a small portion of RNA-binding proteins (RBPs) have been studied by CLIP due to its complex procedures and high level of false-positive signals. Herein, we report a SpyCLIP method that employs a covalent linkage formed between the RBP-fused SpyTag and SpyCatcher, which can withstand the harshest washing conditions for removing nonspecific interactions. Moreover, SpyCLIP circumvents the radioactive labeling and PAGE-membrane purification steps, and the whole procedure can be performed on beads and is readily amenable to automation. We investigated multiple RBPs by SpyCLIP and generated high-quality RNA binding maps with significantly improved reproductivity and accuracy. Therefore, the small tag size and convenient protocol of SpyCLIP provides a robust method for both routine characterization and high-throughput studies of protein–RNA interactions.