Combining mRNA display and photo-crosslinking to discover high-affinity photo-reactive peptide probes for PCSK9

Abstract Photo-crosslinking is a powerful approach for studying complex biological systems. Peptides are an ideal modality for generating photo-crosslinking tools into which photo-reactive moieties can easily be inserted. However, developing a photo-reactive peptide with high-affinity and efficient crosslinking capability can require extensive synthetic optimisation. Here we report a single-round mRNA-based protocol to convert a hit peptide to a high affinity, efficient photo-crosslinking variant. We first identified hit peptides for the LDL-cholesterol regulator PCSK9 through mRNA display. As a proof of concept, we then applied our single-round mRNA display protocol, which enables positional scanning of multiple different photo-reactive amino acids, to two hit peptides. This yielded structurally different, photo-crosslinking efficient hit peptides with high affinity. In cells, our most optimal crosslinking peptide probe was used to track PCSK9 internalisation. Overall, this work optimises and eases the development of photo-crosslinking peptide probes from pre-existing peptide ligands, allowing streamlined generation of matched pairs of crosslinking and reversible peptide probes.