Mapping RNA Structure In Vitro with SHAPE Chemistry and Next-Generation Sequencing (SHAPE-Seq).
Mapping RNA Structure In Vitro with SHAPE Chemistry and Next-Generation Sequencing (SHAPE-Seq).
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使用 SHAPE 化学和下一代测序 (SHAPE-Seq) 绘制体外 RNA 结构图。
DOI：
10.1007/978-1-4939-6433-8_9
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发表时间：
2016
期刊：
影响因子：
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通讯作者：
Lucks,JuliusB
中科院分区：
文献类型：
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作者：
Watters,KyleE;Lucks,JuliusB
通过引物延伸（SHAPE）化学分析的选择性2′-羟基酰化对RNA结构进行作图已被证明是在各种情况下表征RNA结构的通用方法。SHAPE试剂以结构依赖性方式共价修饰RNA，以在结构灵活的核苷酸上的核糖骨架2 '-OH基团上产生加合物。使用逆转录酶（RT）引物延伸检测这些加合物的位置，所述引物延伸在修饰前终止一个核苷酸，以产生cDNA库，其长度反映SHAPE修饰的位置。cDNA库的定量用于估计RNA分子中每个核苷酸对SHAPE试剂的“反应性”。高反应性表示核苷酸在结构上是柔性的，而低反应性表示核苷酸是非柔性的。然后，这些SHAPE反应性可以用于通过限制RNA结构预测算法来推断RNA结构。在这里，我们提供了一个国家的最先进的协议，描述了如何进行体外RNA结构探测与SHAPE化学使用下一代测序定量cDNA池和估计反应性（SHAPE-Seq）。下一代测序的使用允许更高的通量、更一致的数据分析和多路复用能力。本文所述的技术SHAPE-Seq v2.0使用在SHAPE修饰后与RNA连接的通用逆转录引发位点。引入的引发位点允许独立于其序列的RNA的结构分析。
Mapping RNA structure with selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) chemistry has proven to be a versatile method for characterizing RNA structure in a variety of contexts. SHAPE reagents covalently modify RNAs in a structure-dependent manner to create adducts at the 2′-OH group of the ribose backbone at nucleotides that are structurally flexible. The positions of these adducts are detected using reverse transcriptase (RT) primer extension, which stops one nucleotide before the modification, to create a pool of cDNAs whose lengths reflect the location of SHAPE modification. Quantification of the cDNA pools is used to estimate the “reactivity” of each nucleotide in an RNA molecule to the SHAPE reagent. High reactivities indicate nucleotides that are structurally flexible, while low reactivities indicate nucleotides that are inflexible. These SHAPE reactivities can then be used to infer RNA structures by restraining RNA structure prediction algorithms. Here, we provide a state-of-the-art protocol describing how to perform in vitro RNA structure probing with SHAPE chemistry using next-generation sequencing to quantify cDNA pools and estimate reactivities (SHAPE-Seq). The use of next-generation sequencing allows for higher throughput, more consistent data analysis, and multiplexing capabilities. The technique described herein, SHAPE-Seq v2.0, uses a universal reverse transcription priming site that is ligated to the RNA after SHAPE modification. The introduced priming site allows for the structural analysis of an RNA independent of its sequence.