Systemic in vivo phage selection reveals compartment-dependent organization of recoverable peptide repertoires

Abstract

Previous in vivo phage display studies establish the value of the approach for identifying organ-homing and tissue-associated peptides, but whether recovered sequences form diffuse hit lists or structured repertoires remains unclear. This study combines systemic in vivo phage selection, laser-capture microdissection, and high-throughput sequencing to profile recoverable 7-mer peptide repertoires across 41 bulk organs, 39 capillary beds, and 125 parenchymal microenvironments. Across these anatomical scales, the repertoires show non-diffuse, compartment-dependent organization in enrichment-profile space, supported by robustness and null-model analyses. Compartment resolution provides information not apparent in bulk-organ profiles: representative motif-defined neighborhoods exhibit contrasting local sequence-tolerance regimes, ranging from bounded variation to near-single-sequence convergence. Compartment-resolved analysis also nominates EVGTARY, a cortex-associated candidate that is not prioritized by bulk-organ ranking. In an exploratory mouse-level analysis, an EVGTARY-conjugated branched polyethyleneimine (BPEI) polyplex is associated with increased cortical DsRed fluorescence compared with BPEI polyplex alone. Together, these findings shift the analytical unit of in vivo phage display from isolated organ-associated hits toward the organization of recoverable peptide repertoires and support an interpretation in which intact tissue context constrains the range of peptide solutions that remain recoverable after systemic in vivo phage selection.