Direct Chemical Biotinylation of RNA 5'-Ends Using a Diazo Reagent

Methods Mol Biol. 2019:1870:81-87. doi: 10.1007/978-1-4939-8808-2_6.


Introduction of chemical labels into biomolecules is of utmost importance in chemical biology research. However, methods for selective chemical labeling of in vitro transcribed RNA are scarce. Herein, we describe experimental details for direct labeling of the 5'-phosphate of RNA using a diazo biotin-reagent, as exemplified on a 110 nucleotide RNA obtained via in vitro transcription. The method exploits the fact that, under neutral buffer conditions (~pH 6.8), the 5'-phosphate carries the only mildly acidic proton in the RNA molecule, which allows for selective functionalization at that site using diazo reagents.

Keywords: Biotinylation; Diazo; Labeling; RNA.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biotin / chemistry*
  • Biotinylation* / methods
  • Chromatography, Liquid
  • Diazonium Compounds / chemistry*
  • Mass Spectrometry
  • Molecular Structure
  • RNA / chemistry*
  • RNA / isolation & purification
  • Staining and Labeling


  • Diazonium Compounds
  • RNA
  • Biotin