Chemical RNA digestion enables robust RNA-binding site mapping at single amino acid resolution.
Jong Woo BaeS Chul KwonYongwoo NaV Narry KimJong-Seo KimPublished in: Nature structural & molecular biology (2020)
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.
Keyphrases
- amino acid
- genome editing
- liquid chromatography
- tandem mass spectrometry
- crispr cas
- high resolution
- nucleic acid
- mass spectrometry
- ultra high performance liquid chromatography
- endothelial cells
- high performance liquid chromatography
- cystic fibrosis
- escherichia coli
- healthcare
- high resolution mass spectrometry
- staphylococcus aureus
- drug delivery
- solid phase extraction
- molecular dynamics simulations
- anaerobic digestion
- pluripotent stem cells