Imaging Newly Transcribed RNA in Cells by Using a Clickable Azide-Modified UTP Analog

Methods Mol Biol. 2018:1649:359-371. doi: 10.1007/978-1-4939-7213-5_24.


Robust RNA labeling and imaging methods that enable the understanding of cellular RNA biogenesis and function are highly desired. In this context, we describe a practical chemical labeling method based on a bioorthogonal reaction, namely, azide-alkyne cycloaddition reaction, which facilitates the fluorescence imaging of newly transcribed RNA in both fixed and live cells. This strategy involves the transfection of an azide-modified UTP analog (AMUTP) into mammalian cells, which gets specifically incorporated into RNA transcripts by RNA polymerases present inside the cells. Subsequent posttranscriptional click reaction between azide-labeled RNA transcripts and a fluorescent alkyne substrate enables the imaging of newly synthesized RNA in cells by confocal microscopy. Typically, 50 μM to 1 mM of AMUTP and a transfection time of 15-60 min produce significant fluorescence signal from labeled RNA transcripts in cells.

Keywords: Azide–alkyne cycloaddition reaction; Bioorthogonal reaction; Click chemistry; Nucleotide analog; Posttranscriptional chemical modification; RNA imaging; RNA labeling.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Azides / chemistry*
  • Click Chemistry / methods*
  • Cycloaddition Reaction
  • HeLa Cells
  • Humans
  • Imaging, Three-Dimensional / methods*
  • RNA / genetics*
  • Transcription, Genetic*
  • Transfection
  • Uridine Triphosphate / analogs & derivatives*


  • Azides
  • RNA
  • Uridine Triphosphate