Chemical RNA digestion enables robust RNA-binding site mapping at single amino acid resolution

Nat Struct Mol Biol. 2020 Jul;27(7):678-682. doi: 10.1038/s41594-020-0436-2. Epub 2020 Jun 8.


RNA-binding sites (RBSs) can be identified by liquid chromatography and tandem mass spectrometry analyses of the protein-RNA conjugates created by crosslinking, but RBS mapping remains highly challenging due to the complexity of the formed RNA adducts. Here, we introduce RBS-ID, a method that uses hydrofluoride to fully cleave RNA into mono-nucleosides, thereby minimizing the search space to drastically enhance coverage and to reach single amino acid resolution. Moreover, the simple mono-nucleoside adducts offer a confident and quantitative measure of direct RNA-protein interaction. Using RBS-ID, we profiled ~2,000 human RBSs and probed Streptococcus pyogenes Cas9 to discover residues important for genome editing.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acids / metabolism
  • Binding Sites
  • CRISPR-Associated Protein 9 / metabolism
  • Chromatography, Liquid
  • Cross-Linking Reagents / chemistry
  • HeLa Cells
  • Humans
  • Hydrofluoric Acid / chemistry
  • Molecular Biology / methods*
  • Proteome / genetics
  • Proteome / metabolism
  • RNA / chemistry*
  • RNA / metabolism*
  • Ribonucleoproteins / metabolism
  • Tandem Mass Spectrometry


  • Amino Acids
  • Cross-Linking Reagents
  • Proteome
  • Ribonucleoproteins
  • RNA
  • CRISPR-Associated Protein 9
  • Cas9 endonuclease Streptococcus pyogenes
  • Hydrofluoric Acid