Site-specific fluorescent probing of RNA molecules by unnatural base-pair transcription for local structural conformation analysis
Site-specific fluorescent probing of RNA molecules by unnatural base-pair transcription for local structural conformation analysis
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DOI：
10.1038/nprot.2010.77
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发表时间：
2010-01-01
期刊：
影响因子：
14.8
通讯作者：
Hirao, Ichiro
中科院分区：
文献类型：
--
作者：
Hikida, Yasushi;Kimoto, Michiko;Hirao, Ichiro
在RNA的特定位置进行荧光探测的方法，通过跟踪荧光变化，为分析功能性RNA分子的局部结构构象提供了有力的工具。在这篇文章中，我们描述了RNA的位点特异性荧光探针转录与扩展的遗传字母表，使用额外的，非自然碱基对之间的2-氨基-6-（2-噻吩基）嘌呤(s)和吡咯-2-乙醛（Pa）。该方案包括含有Pa的模板DNA制备，涉及荧光掺入的转录和转录本的结构分析。s碱基具有很强的荧光性，其核苷5'-三磷酸通过常规T7转录特异性地结合到RNA转录物中，与DNA模板中的Pa相反。s的荧光强度随探针位点周围环境的变化而变化，这为RNA分子的局部结构特征提供了线索。这是第一个在所需位置进行荧光标记的RNA转录物制备方案。含s RNA的制备过程大约需要2-3天。
Methods for fluorescent probing at a defined position of RNA provide powerful tools for analyzing the local structural conformation of functional RNA molecules by tracking fluorescence changes. In this article, we describe the site-specific fluorescent probing of RNA by transcription with an expanded genetic alphabet, using an extra, unnatural base pair between 2-amino-6-(2-thienyl)purine (s) and pyrrole-2-carbaldehyde (Pa). The protocol comprises template DNA preparation containing Pa, transcription involving fluorescent s incorporation and structural analysis of transcripts. The s base is strongly fluorescent, and its nucleoside 5'-triphosphate is site-specifically incorporated into RNA transcripts, opposite Pa in DNA templates, by conventional T7 transcription. The fluorescent intensity of s changes depending on its environment around the probe site, providing clues about the local structural features of RNA molecules. This is the first protocol for RNA transcript preparation with fluorescent labeling at a desired position. The procedure for s-containing RNA preparation takes about 2-3 d.