Selective 2'-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP) for direct, versatile and accurate RNA structure analysis.
Selective 2'-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP) for direct, versatile and accurate RNA structure analysis.
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DOI：
10.1038/nprot.2015.103
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发表时间：
2015-11
期刊：
影响因子：
14.8
通讯作者：
Weeks KM
中科院分区：
文献类型：
--
作者：
Smola MJ;Rice GM;Busan S;Siegfried NA;Weeks KM
SHAPE化学利用与2′-羟基反应的小的亲电试剂以单核苷酸分辨率询问RNA结构。突变谱分析（MaP）基于逆转录酶误读SHAPE修饰的核苷酸的能力来鉴定修饰的残基，然后通过大规模平行测序来计数所产生的突变。SHAPE-MaP方法可以像测量简单的模型RNA一样准确地测量大型和转录组范围系统的结构。该方案描述了在三天内实施的实验步骤，这些步骤是进行SHAPE探测和构建适用于深度测序的多重SHAPE-MaP文库所需的。这些步骤包括RNA折叠和SHAPE结构探测、通过逆转录的突变谱分析、文库构建和测序。使用两个软件包完成MaP测序数据的自动化处理。通常在一小时内，ShapeMapper可将原始测序文件转换为突变图谱，创建SHAPE反应性图，并提供有用的故障排除信息。SuperFold使用这些数据来建模RNA二级结构，识别具有明确结构的区域，并可视化可能的和替代的螺旋，通常在一天之内。我们用E. coli硫胺素焦磷酸核糖开关、E. coli 16 S rRNA和HIV-1基因组RNA。SHAPE-MaP可用于对单个RNA基序、复杂RNA集合的稀有组分和整个转录组进行核苷酸分辨率生物物理测量。简单的MaP策略大大扩展了可分析RNA结构的数量、长度和复杂性。
SHAPE chemistries exploit small electrophilic reagents that react with the 2′-hydroxyl group to interrogate RNA structure at single-nucleotide resolution. Mutational profiling (MaP) identifies modified residues based on the ability of reverse transcriptase to misread a SHAPE-modified nucleotide and then counting the resulting mutations by massively parallel sequencing. The SHAPE-MaP approach measures the structure of large and transcriptome-wide systems as accurately as for simple model RNAs. This protocol describes the experimental steps, implemented over three days, required to perform SHAPE probing and construct multiplexed SHAPE-MaP libraries suitable for deep sequencing. These steps include RNA folding and SHAPE structure probing, mutational profiling by reverse transcription, library construction, and sequencing. Automated processing of MaP sequencing data is accomplished using two software packages. ShapeMapper converts raw sequencing files into mutational profiles, creates SHAPE reactivity plots, and provides useful troubleshooting information, often within an hour. SuperFold uses these data to model RNA secondary structures, identify regions with well-defined structures, and visualize probable and alternative helices, often in under a day. We illustrate these algorithms with the E. coli thiamine pyrophosphate riboswitch, E. coli 16S rRNA, and HIV-1 genomic RNAs. SHAPE-MaP can be used to make nucleotide-resolution biophysical measurements of individual RNA motifs, rare components of complex RNA ensembles, and entire transcriptomes. The straightforward MaP strategy greatly expands the number, length, and complexity of analyzable RNA structures.