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Author:
Date:
2015-01-20
[Abstract] This protocol describes the methodology for the determination of the secondary structure of an RNA fragment in solution using Selective 2´-Hydroxyl Acylation analyzed by Primer Extension, abbreviation SHAPE. It consists in the very fast chemical modification of flexible and therefore possibly single-stranded nucleotides in a sequence-independent manner using benzoyl cyanide (BzCN), forming 2´-O-adducts. The modifications in the RNA are then analyzed by primer extension. Reverse transcriptase is blocked by the 2´-O-adducts formed. The advantage of the method is, first, that not each RNA molecule studied but the primer used in the extension reaction is labelled and, second, that the resulting cDNA analyzed in sequencing gels is much more stable than the modified RNA.
[摘要] 该方案描述了使用通过引物延伸（缩写为SHAPE）分析的选择性2'-羟基酰化来确定溶液中RNA片段的二级结构的方法。 其包括使用苯甲酰氰（BzCN）以序列非依赖性方式非常快速地化学修饰柔性且因此可能的单链核苷酸，形成2'-O-加合物。 然后通过引物延伸分析RNA中的修饰。 逆转录酶被形成的2'-O-加合物阻断。 该方法的优点是，首先，不是每个RNA分子研究，而是在延伸反应中使用的引物被标记，其次，在测序凝胶中分析得到的cDNA比修饰的RNA更稳定。
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