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

ABSTRACT

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.