Identification of photocrosslinking peptide ligands by mRNA display

Photoaffinity labelling is a promising method for studying protein-ligand interactions. However, obtaining a specific, efficient crosslinker can require significant optimisation. We report a modified mRNA display strategy, photocrosslinking-RaPID (XL-RaPID), and exploit its ability to accelerate the discovery of cyclic peptides that photocrosslink to a target of interest. As a proof of concept, we generated a benzophenone-containing library and applied XL-RaPID screening against a model target, the second bromodomain of BRD3. This crosslinking screening gave two optimal candidates that selectively labelled the target protein in cell lysate. Overall, this work introduces direct photocrosslinking screening as a versatile technique for identifying covalent peptide ligands from mRNA display libraries incorporating reactive warheads.