Genetically encoding an aliphatic diazirine for protein photocrosslinking

Photocrosslinking is an important approach that allows discovery and detailed investigation of protein-protein, protein-oligonucleotide, and protein-small molecule interactions with high temporal and spatial resolution. A major limitation to the universal application of this methodology is the site-specific introduction of efficient aliphatic photocrosslinking probes into proteins of interest. Here, we report a novel aliphatic diazirine amino acid and its genetically encoded, site-specific incorporation into proteins in bacterial and mammalian cells. Furthermore, we demonstrate efficient photocrosslinking of a test protein in vitro and in vivo.