Spacer Length as a Molecular Cue for Self‐Sorting, Living Growth, and Piezoresponse in Peptide Assemblies

ABSTRACT Supramolecular polymerization offers a powerful route to structurally precise and functionally complex materials; however, achieving controlled multicomponent assembly and emergent functionality remains a fundamental challenge. In this study, we show that minimal variations in methylene spacer length between bisurea motifs encode the self‐assembly behavior of peptide amphiphiles. Cy3‐ and Cy5‐labeled peptides undergo nucleation‐elongation‐driven assembly into nanofibers with spacer‐dependent secondary structures. FRET and super‐resolution microscopy reveal that identical spacers promote random co‐assembly, whereas even a single methylene mismatch induces high‐fidelity self‐sorting. Pre‐formed fibers exhibit negligible monomer exchange, indicating nonequilibrium assemblies. Leveraging this, seeded supramolecular polymerization affords well‐defined block copolymers with low dispersity and spatially resolved domains. Furthermore, spacer length and odd‐even parity modulate dipolar alignment, enabling spacer length‐dependent piezoresponse. Collectively, these findings demonstrate how subtle molecular design parameters act as a molecular cue to program multicomponent supramolecular polymerization, offering new opportunities for designing compartmentalized, functional biomaterials and supramolecular bioelectronic systems.