title: A versatile toolbox for posttranscriptional chemical labeling and imaging of RNA.
abstract: 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.
authors.affiliation: Department of Chemistry, Indian Institute of Science Education and Research, Pune, Dr. Homi Bhabha Road, Pashan, Pune 411008, India.
authors.forename: Anupam A
authors.initials: AA
authors.lastname: Sawant
authors.affiliation: Department of Chemistry, Indian Institute of Science Education and Research, Pune, Dr. Homi Bhabha Road, Pashan, Pune 411008, India.
authors.forename: Arun A
authors.initials: AA
authors.lastname: Tanpure
authors.affiliation: Department of Chemistry, Indian Institute of Science Education and Research, Pune, Dr. Homi Bhabha Road, Pashan, Pune 411008, India.
authors.forename: Progya P
authors.initials: PP
authors.lastname: Mukherjee
authors.affiliation: Center of Excellence in Epigenetics, Indian Institute of Science Education and Research, Pune, Dr. Homi Bhabha Road, Pashan, Pune 411008, India.
authors.forename: Soumitra
authors.initials: S
authors.lastname: Athavale
authors.affiliation: Center of Excellence in Epigenetics, Indian Institute of Science Education and Research, Pune, Dr. Homi Bhabha Road, Pashan, Pune 411008, India.
authors.forename: Ashwin
authors.initials: A
authors.lastname: Kelkar
authors.affiliation: Center of Excellence in Epigenetics, Indian Institute of Science Education and Research, Pune, Dr. Homi Bhabha Road, Pashan, Pune 411008, India National Centre for Cell Science, Ganeshkhind, Pune 411007, India sanjeev@iiserpune.ac.in.
authors.forename: Sanjeev
authors.initials: S
authors.lastname: Galande
authors.affiliation: Department of Chemistry, Indian Institute of Science Education and Research, Pune, Dr. Homi Bhabha Road, Pashan, Pune 411008, India srivatsan@iiserpune.ac.in.
authors.forename: Seergazhi G
authors.initials: SG
authors.lastname: Srivatsan
mesh_terms: D001386:Azides / Q000737:chemistry*; D017123:Bacteriophage T7 / Q000737:chemistry / Q000201:enzymology; D057930:Click Chemistry; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D012313:RNA / Q000737:chemistry*; D012323:RNA Processing, Post-Transcriptional; D013194:Staining and Labeling / Q000379:methods*; D014544:Uridine Triphosphate / Q000031:analogs & derivatives / Q000737:chemistry*; D014764:Viral Proteins / Q000737:chemistry*
chemical_list: D001386:Azides; D005456:Fluorescent Dyes; D014764:Viral Proteins; D012313:RNA; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D014544:Uridine Triphosphate