Transcriptome-wide interrogation of RNA secondary structure in living cells with icSHAPE.
Transcriptome-wide interrogation of RNA secondary structure in living cells with icSHAPE.
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DOI：
10.1038/nprot.2016.011
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发表时间：
2016-02
期刊：
影响因子：
14.8
通讯作者：
Chang HY
中科院分区：
文献类型：
--
作者：
Flynn RA;Zhang QC;Spitale RC;Lee B;Mumbach MR;Chang HY
icSHAPE（体内点击选择性2-羟基酰化和分析实验）通过测量碱基分辨率下的核苷酸柔韧性，在转录组范围内捕获RNA二级结构。用icSHAPE化学试剂NAI-N3处理活细胞，然后对NAI-N3修饰的RNA进行选择性化学富集，与利用深度测序的类似方法相比，该方法提供了更高的信噪比。纯化的RNA然后被反向转录生成cDNA，用shape修饰的碱基生成截短的cDNA。在对cDNA进行深度测序后，计算分析得出起始RNA群体中每个碱基的灵活性评分。整个实验过程可以在~5天内完成，测序和生物信息学数据分析需要额外的4-5天，不需要大量的计算技能。比较体内和体外icSHAPE测量值可以揭示体内RNA结合蛋白印记或促进RNA转录后修饰的解剖。icSHAPE反应性还可用于约束和改进RNA二级结构预测模型。
icSHAPE (in vivo click selective 2-hydroxyl acylation and profiling experiment) captures RNA secondary structure at a transcriptome-wide level by measuring nucleotide flexibility at base resolution. Living cells are treated with the icSHAPE chemical NAI-N3 followed by selective chemical enrichment of NAI-N3–modified RNA, which provides an improved signal-to-noise ratio compared with similar methods leveraging deep sequencing. Purified RNA is then reverse-transcribed to produce cDNA, with SHAPE-modified bases leading to truncated cDNA. After deep sequencing of cDNA, computational analysis yields flexibility scores for every base across the starting RNA population. The entire experimental procedure can be completed in ~5 d, and the sequencing and bioinformatics data analysis take an additional 4–5 d with no extensive computational skills required. Comparing in vivo and in vitro icSHAPE measurements can reveal in vivo RNA-binding protein imprints or facilitate the dissection of RNA post-transcriptional modifications. icSHAPE reactivities can additionally be used to constrain and improve RNA secondary structure prediction models.