Fluorescent probing for RNA molecules by an unnatural base-pair system
Fluorescent probing for RNA molecules by an unnatural base-pair system
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DOI：
10.1093/nar/gkm508
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发表时间：
2007-08-01
影响因子：
14.9
通讯作者：
Hirao, Ichiro
中科院分区：
文献类型：
--
作者：
Kimoto, Michiko;Mitsui, Tsuneo;Hirao, Ichiro
核酸的荧光标记广泛用于基础研究和医学应用。我们描述了一种荧光碱基类似物，2-氨基-6-（2-噻吩基）嘌呤（s），通过s和吡咯-2-甲醛（Pa）之间的非天然碱基对介导的转录进入RNA的有效位点特异性掺入。s的核糖核苷5 '-三磷酸通过T7 RNA聚合酶位点特异性地掺入RNA中，与DNA模板中的Pa相对。RNA分子中s的荧光强度根据结构环境而变化。RNA发夹和tRNA分子的位点特异性S标记提供了特征性荧光谱，这取决于标记位点、温度和Mg 2+浓度。含有Pa的DNA模板可以通过使用Pa的另一配对配偶体7-（2-噻吩基）咪唑并[4，5-B]吡啶（Ds）的PCR扩增。通过包括s-Pa和Ds-Pa对的非天然对系统的这种位点特异性荧光探测为研究3D RNA分子的局部结构特征及其分子内和分子间相互作用的动力学提供了有力的工具。
Fluorescent labeling of nucleic acids is widely used in basic research and medical applications. We describe the efficient site-specific incorporation of a fluorescent base analog, 2-amino-6-(2-thienyl) purine (s), into RNA by transcription mediated by an unnatural base pair between s and pyrrole-2-carbaldehyde (Pa). The ribonucleoside 5'-triphosphate of s was site-specifically incorporated into RNA, by T7 RNA polymerase, opposite Pa in DNA templates. The fluorescent intensity of s in RNA molecules changes according to the structural environment. The site- specific s labeling of RNA hairpins and tRNA molecules provided characteristic fluorescent profiles, depending on the labeling sites, temperature and Mg2+ concentration. The Pa- containing DNA templates can be amplified by PCR using 7-(2-thienyl) imidazo[4,5-b] pyridine (Ds), another pairing partner of Pa. This site- specific fluorescent probing by the unnatural pair system including the s-Pa and Ds-Pa pairs provides a powerful tool for studying the dynamics of the local structural features of 3D RNA molecules and their intra- and intermolecular interactions.