Discovery of macrocyclic peptide binders, covalent modifiers, and degraders of a structured RNA by mRNA display

RNA targeting represents a compelling strategy for addressing challenging therapeutic targets that are otherwise intractable through traditional protein targeting. Revolutionary approaches in RNA-focused small molecule libraries have successfully identified RNA-binding ligands, but generally remain limited in diversity and impeded by a dearth of structural insight into RNA and RNA complexes. Cyclic peptides are potential structural mimics of evolutionary RNA-protein interacting motifs and can be massively diversified and selected via genetically encoded libraries, offering a complementary approach. This study introduces genetically encoded thioether cyclic peptide libraries constructed through mRNA display using a dibromoxylene linker and its fluorosulfonyl derivative that can covalently engage RNA nucleophiles. Using an optimized mRNA display workflow for RNA binders, we discovered high affinity, covalent and non-covalent binders for SNCA 5’ UTR IRE, the upstream iron-responsive element that post-transcriptionally regulates the expression of α-synuclein, an intrinsically disordered protein implicated in Parkinsonism and related neurodegenerative diseases. Notably, a stringent selection strategy employing “base-paired” target analog counterselection enhanced specificity by de-enriching nonspecific electrostatic interactions mediated by polycationic residues. Further engineering hit peptides with an imidazole tag yielded selective RNA degraders in which covalent degraders showed noticeably improved potency from non-covalent counterparts. This work provides a prototype framework for evolution-driven, high-throughput RNA-targeted drug discovery using cyclic peptides.