Dual “Click-On” Phage Display for Nanoscaffolds with Discretely Tunable Lateral Display Density

Filamentous bacteriophages are monodisperse, high-aspect-ratio protein nanofibers that provide a scalable and genetically addressable platform for biohybrid nanomaterials. However, direct fusion of heterologous proteins to capsid proteins often disrupts assembly and limits high-density functionalization to short peptides. Here, we report a dual-display “type 388” phage vector enabling orthogonal and adjustable surface functionalization. SnoopTag was fused to the minor coat protein p3 for low-valency tip display, while SpyTag variants were displayed on the major coat protein p8 for sidewall presentation. Using fluorescent and immunochemical methods, and high-resolution microscopy techniques, we show that the lateral display density on p8 can be discretely adjusted by selecting distinct signal sequence-SpyTag combinations that modulate the efficiency of bacterial inner membrane integration and capsid incorporation. Replacement of the native p8 signal sequence with a signal recognition particle-compatible counterpart enhances functional display, enabling adjustable multivalent patterning. Orthogonal Tag/Catcher pairs and post-assembly cargo conjugation together establish a plug-and-play, anisotropic nanoscaffold for precision biofunctionalization. As a proof of concept, the phage nanotemplate was functionalized with targeting and reporter modules to generate an antibody-mimetic probe for analyte detection.