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

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.

SUBMITTER: Dziuba D 

PROVIDER: S-EPMC7003851 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature