Site-specific labeling of RNA by combining genetic alphabet expansion transcription and copper-free click chemistry.
Site-specific labeling of RNA by combining genetic alphabet expansion transcription and copper-free click chemistry.
复制标题
DOI：
10.1093/nar/gkv638
复制
发表时间：
2015-08-18
影响因子：
14.9
通讯作者：
Hirao I
中科院分区：
文献类型：
--
作者：
Someya T;Ando A;Kimoto M;Hirao I
用所需的分子探针对长链RNA进行位点特异性标记是促进功能RNA分子研究的必要技术。通过使用人工第三碱基对（称为非天然碱基对）的遗传字母表扩展，我们提出了一种用于RNA转录物的转录后修饰方法，所述RNA转录物在特定位置含有掺入的叠氮化物连接的非天然碱基，使用无铜点击反应。7-（2-噻吩基）咪唑并[4，5-B]吡啶（Ds）和吡咯-2-甲醛（Pa）之间的非天然碱基对在转录中起作用。因此，我们化学合成了一个三磷酸底物4-（4-叠氮戊基）-吡咯-2-甲醛（N3-PaTP），它可以被引入到RNA的位点特异性，相反的Ds模板T7转录。用二苯并环辛炔（DIBO）衍生物修饰转录物中掺入的N3-Pa。我们展示了17-，76-和260-mer RNA分子的转录及其用Alexa 488，Alexa 594和生物素的位点特异性标记。该方法将用于制备用任何感兴趣的官能团标记的RNA分子，用于体内实验。
Site-specific labeling of long-chain RNAs with desired molecular probes is an imperative technique to facilitate studies of functional RNA molecules. By genetic alphabet expansion using an artificial third base pair, called an unnatural base pair, we present a post-transcriptional modification method for RNA transcripts containing an incorporated azide-linked unnatural base at specific positions, using a copper-free click reaction. The unnatural base pair between 7-(2-thienyl)imidazo[4,5-b]pyridine (Ds) and pyrrole-2-carbaldehyde (Pa) functions in transcription. Thus, we chemically synthesized a triphosphate substrate of 4-(4-azidopentyl)-pyrrole-2-carbaldehyde (N3-PaTP), which can be site-specifically introduced into RNA, opposite Ds in templates by T7 transcription. The N3-Pa incorporated in the transcripts was modified with dibenzocyclooctyne (DIBO) derivatives. We demonstrated the transcription of 17-, 76- and 260-mer RNA molecules and their site-specific labeling with Alexa 488, Alexa 594 and biotin. This method will be useful for preparing RNA molecules labeled with any functional groups of interest, toward in vivo experiments.