A Genetically Encoded Diazirine Analogue for RNA–Protein Photo-crosslinking

Dmytro Dziuba, Jan Erik Hoffmann, Matthias W. Hentze, Carsten Schultz

Research output: Contribution to journalArticlepeer-review

3 Scopus citations

Abstract

Ultraviolent crosslinking is a key experimental step in the numerous protocols that have been developed for capturing and dissecting RNA–protein interactions in living cells. UV crosslinking covalently stalls dynamic interactions between RNAs and the directly contacting RNA-binding proteins and enables stringent denaturing downstream purification conditions needed for the enrichment and biochemical analysis of RNA–protein complexes. Despite its popularity, conventional 254 nm UV crosslinking possesses a set of intrinsic drawbacks, with the low photochemical efficiency being the central caveat. Here we show that genetically encoded photoreactive unnatural amino acids bearing a dialkyl diazirine photoreactive group can address this problem. Using the human iron regulatory protein 1 (IRP1) as a model RNA-binding protein, we show that the photoreactive amino acids can be introduced into the protein without diminishing its RNA-binding properties. A sevenfold increase in the crosslinking efficiency compared to conventional 254 nm UV crosslinking was achieved using the diazirine-based unnatural amino acid DiAzKs. This finding opens an avenue for new applications of the unnatural amino acids in studying RNA–protein interactions.

Original languageEnglish (US)
Pages (from-to)88-93
Number of pages6
JournalChemBioChem
Volume21
Issue number1-2
DOIs
StatePublished - Jan 15 2020

Keywords

  • RNA
  • amino acids
  • click chemistry
  • photoaffinity labeling
  • protein modifications

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Medicine
  • Molecular Biology
  • Organic Chemistry

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