RNA Secondary Structure Prediction Using High-throughput SHAPE
RNA Secondary Structure Prediction Using High-throughput SHAPE
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DOI：
10.3791/50243
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发表时间：
2013-05-01
影响因子：
1.2
通讯作者：
Le Grice, Stuart F. J.
中科院分区：
文献类型：
--
作者：
Lusvarghi, Sabrina;Sztuba-Solinska, Joanna;Le Grice, Stuart F. J.
了解RNA在生物过程中的功能需要对RNA结构有透彻的了解。为此，被称为“通过引物延伸分析的高通量选择性2'羟基酰化”或SHAPE的方法允许用单核苷酸分辨率预测RNA二级结构。该方法利用化学探测剂，其优先酰化水溶液中RNA的单链或柔性区域。化学修饰的位点通过修饰的RNA的逆转录来检测，并且该反应的产物通过自动毛细管电泳（CE）分级分离。由于逆转录酶在那些被SHAPE试剂修饰的RNA核苷酸处暂停，因此所得cDNA文库间接映射在折叠RNA的背景下为单链的那些核糖核苷酸。使用Shapetron软件，处理自动CE产生的电泳图并将其转换为核苷酸反应性表，该表本身转换为RNASstructure（v5.3）预测算法中使用的伪能量约束。已经发现通过结合SHAPE探测与计算机RNA二级结构预测获得的二维RNA结构比单独使用任一种方法获得的结构精确得多。
Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed "high-throughput selective 2' hydroxyl acylation analyzed by primer extension", or SHAPE, allows prediction of RNA secondary structure with single nucleotide resolution. This approach utilizes chemical probing agents that preferentially acylate single stranded or flexible regions of RNA in aqueous solution. Sites of chemical modification are detected by reverse transcription of the modified RNA, and the products of this reaction are fractionated by automated capillary electrophoresis (CE). Since reverse transcriptase pauses at those RNA nucleotides modified by the SHAPE reagents, the resulting cDNA library indirectly maps those ribonucleotides that are single stranded in the context of the folded RNA. Using ShapeFinder software, the electropherograms produced by automated CE are processed and converted into nucleotide reactivity tables that are themselves converted into pseudo-energy constraints used in the RNAStructure (v5.3) prediction algorithm. The two-dimensional RNA structures obtained by combining SHAPE probing with in silico RNA secondary structure prediction have been found to be far more accurate than structures obtained using either method alone.