Warminski Marcin, Sikorski Pawel J, Warminska Zofia, Lukaszewicz Maciej, Kropiwnicka Anna, Zuberek Joanna, Darzynkiewicz Edward, Kowalska Joanna, Jemielity Jacek
Division of Biophysics, Institute of Experimental Physics, Faculty of Physics, University of Warsaw , 02-093, Warsaw, Poland.
Centre of New Technologies, University of Warsaw , 02-097, Warsaw, Poland.
Bioconjug Chem. 2017 Jul 19;28(7):1978-1992. doi: 10.1021/acs.bioconjchem.7b00291. Epub 2017 Jun 27.
mRNA is a template for protein biosynthesis, and consequently mRNA transport, translation, and turnover are key elements in the overall regulation of gene expression. Along with growing interest in the mechanisms regulating mRNA decay and localization, there is an increasing need for tools enabling convenient fluorescent labeling or affinity tagging of mRNA. We report new mRNA 5' cap analog-based tools that enable site-specific labeling of RNA within the cap using N-hydroxysuccinimide (NHS) chemistry. We explored two complementary methods: a co-transcriptional labeling method, in which the label is first attached to a cap analog and then incorporated into RNA by in vitro transcription, and a post-transcriptional labeling method, in which an amino-functionalized cap analog is incorporated into RNA followed by chemical labeling of the resulting transcript. After testing the biochemical properties of RNAs carrying the novel modified cap structures, we demonstrated the utility of fluorescently labeled RNAs in decapping assays, RNA decay assays, and RNA visualization in cells. Finally, we also demonstrated that mRNAs labeled by the reported method are translationally active. We envisage that the novel analogs will provide an alternative to radiolabeling of mRNA caps for in vitro studies and open possibilities for new applications related to the study of mRNA fates in vivo.
信使核糖核酸（mRNA）是蛋白质生物合成的模板，因此mRNA的运输、翻译和周转是基因表达整体调控中的关键要素。随着人们对调控mRNA降解和定位机制的兴趣日益浓厚，对能够方便地对mRNA进行荧光标记或亲和标记的工具的需求也在增加。我们报告了基于新型mRNA 5'帽类似物的工具，这些工具能够利用N-羟基琥珀酰亚胺（NHS）化学方法对帽内的RNA进行位点特异性标记。我们探索了两种互补的方法：一种是共转录标记方法，其中标记首先连接到帽类似物上，然后通过体外转录整合到RNA中；另一种是转录后标记方法，其中将氨基功能化的帽类似物整合到RNA中，随后对所得转录本进行化学标记。在测试了携带新型修饰帽结构的RNA的生化特性后，我们证明了荧光标记的RNA在脱帽测定、RNA降解测定和细胞内RNA可视化中的实用性。最后，我们还证明了通过所报道方法标记的mRNA具有翻译活性。我们设想，这些新型类似物将为体外研究中mRNA帽的放射性标记提供一种替代方法，并为体内mRNA命运研究的新应用开辟可能性。
