Conserved cysteine frameworks are
essential components
of disulfide-rich
peptides (DRPs), which dominantly define the structural diversity
of both naturally occurring and de novo-designed DRPs. However, there
are only very limited numbers of conserved cysteine frameworks, and
general methods enabling de novo discovery of cysteine frameworks
with robust foldability are still not available. Here, we devised
a “touchstone”-based strategy that relies on chasing
oxidative foldability between two individual disulfide-rich folds
on the phage surface to discover new cysteine frameworks from random
sequences. Unique cysteine frameworks with a high degree of compatibility
with phage display systems and broad sequence tolerance were successfully
identified, which were subsequently exploited for the development
of multicyclic DRP libraries, enabling the rapid discovery of new
peptide ligands with low-nanomolar and picomolar binding affinity.
This study provides an unprecedented method for exploring and exploiting
the sequence and structure space of DRPs that is not readily accessible
by existing strategies, holding the potential to revolutionize the
study of DRPs and significantly advance the design and discovery of
multicyclic peptide ligands and drugs.