Engineering Mosaic T7 Bacteriophages for Dual Peptide Display to Enable Cellular Targeting and Ligand‐Directed Protein Capture

Integrating molecular recognition, tracing, and interactome capture within a single nanoparticle could allow for probing, manipulation, and control of complex biological systems. Achieving such multifunctionality within a single well-defined nanostructure remains challenging. T7 bacteriophage-based biological nanoparticles provide a robust and genetically programmable scaffold; however, conventional T7 display systems typically accommodate only one exogenous peptide species per phage particle, limiting multifunctional and combinatorial applications. Here, we describe a mosaic phage platform that enables the simultaneous display of two peptides on separate T7 coat protein subunits. By combining genome-encoded and plasmid-driven expression of peptide-fused capsid proteins, the system provides a modular route for assembling multifunctional phage nanoparticles. We generated mosaic T7 phages co-presenting the neuropilin-1 (NRP-1)-binding peptide RPARPAR alongside either a HA-tag or a Twin-Strep (Tstrep) tag affinity handle. These engineered phages specifically target NRP-1-expressing cells and enable immunofluorescence visualization and pull-down of NRP-1 from complex lysates. Furthermore, a random peptide T7 phage library co-displaying the constant lung-homing peptide RPARPAR shows preferential pulmonary accumulation in vivo, demonstrating utility for combinatorial targeting and tissue-directed phage selection. Collectively, this dual-display approach expands phage display by enabling cell targeting, visualization, molecular interactome probing, and in vivo tissue homing within a single genetically encoded nanoparticle platform.