Combining mRNA display and photo-crosslinking to discover high-affinity photoreactive peptide probes for PCSK9
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Cecilie M Jørgensen Christian W Tornøe Thomas Jackson Wei Wang Camille Villequey Louise WalportThis article is a preprint. Preprints have not been peer-reviewed.
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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 photocrosslinking variant. We first identified hit peptides for 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, photocrosslinking 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.
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chemRxiv
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