A versatile toolbox for posttranscriptional chemical labeling and imaging of RNA

• Published in: Nucleic acids research 2016-01, Vol.44 (2), p.e16-e16
• Main Authors: Sawant, Anupam A, Tanpure, Arun A, Mukherjee, Progya P, Athavale, Soumitra, Kelkar, Ashwin, Galande, Sanjeev, Srivatsan, Seergazhi G
• Format: Article
• Language: English
• Subjects: Azides - chemistry; Bacteriophage T7 - chemistry; Bacteriophage T7 - enzymology; Click Chemistry; DNA-Directed RNA Polymerases - chemistry; Fluorescent Dyes - chemistry; HeLa Cells; Humans; Methods Online; RNA - chemistry; RNA Processing, Post-Transcriptional; Staining and Labeling - methods; Uridine Triphosphate - analogs & derivatives; Uridine Triphosphate - chemistry; Viral Proteins - chemistry
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/gkv903

Cellular RNA labeling strategies based on bioorthogonal chemical reactions are much less developed in comparison to glycan, protein and DNA due to its inherent instability and lack of effective methods to introduce bioorthogonal reactive functionalities (e.g. azide) into RNA. Here we report the development of a simple and modular posttranscriptional chemical labeling and imaging technique for RNA by using a novel toolbox comprised of azide-modified UTP analogs. These analogs facilitate the enzymatic incorporation of azide groups into RNA, which can be posttranscriptionally labeled with a variety of probes by click and Staudinger reactions. Importantly, we show for the first time the specific incorporation of azide groups into cellular RNA by endogenous RNA polymerases, which enabled the imaging of newly transcribing RNA in fixed and in live cells by click reactions. This labeling method is practical and provides a new platform to study RNA in vitro and in cells.