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Updated: Dec 18, 2025

Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions
Published on: September 28, 2017
Chemical RNA digestion enables robust RNA-binding site mapping at single amino acid resolution
Jong Woo Bae1,2, S Chul Kwon1,2, Yongwoo Na1,2
1Center for RNA Research, Institute for Basic Science, Seoul, Korea.
Abstract:
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.
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