Diversity Scale of Library Matters: Impact of mRNA Library Diversity Scales on the Discovery of Macrocyclic Peptides Targeting a Protein by the RaPID System

Macrocyclic peptides make up a unique class of modalities
known
for their high affinity, specificity, and ability to modulate protein–protein
interactions, including receptor activation. Messenger RNA display,
including the Random Nonstandard Peptides Integrated Discovery (RaPID)
system, stands out in identifying target-specific macrocyclic peptides,
producing potent binders with low to subnanomolar dissociation constants
against diverse targets. It has often been discussed that this success
is partly attributed to the vast library of over a trillion different
peptide sequences expressed from the corresponding mRNA sequences.
However, the impact of library scales on the identification of various
binders has not been experimentally validated. Here, we report the
RaPID selections against an ectodomain of a receptor tyrosine kinase
MET using peptide libraries ranging from 106 to 1014 unique members of mRNAs. We thoroughly analyzed the outcomes,
including the binding kinetic properties, of the enriched peptide
families. This study provides valuable guidelines for designing libraries
with various numbers of sequences and selection conditions to enrich
macrocyclic peptides with the desired characteristics.