Dual “Click-On” phage display for nanoscaffolds with discretely tunable lateral display density

Gnidovec, Klemen (author), Ulčakar, Liza (author), Bozovičar, Krištof (author), Romih, Rok (author), Ljubetič, Ajasja (author), Bratkovič, Tomaž (author)

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.

• Language: English
• Keywords: filamentous phage, nanoscaffold, dual display, signal sequence, split protein, SpyTag/SpyCatcher, SnoopTag/SnoopCatcher
• Work type: Article
• Typology: 1.01 - Original Scientific Article
• Organization: FFA - Faculty of Pharmacy; MF - Faculty of Medicine
• Publication status: Published
• Publication version: Version of Record
• Year: 2026
• Number of pages: Str. 20543–20553
• Numbering: Vol. 9, iss. 44
• PID: 20.500.12556/RUL-190990
• UDC: 66.017-022.513.2:620.3
• ISSN on article: 2574-0970
• DOI: 10.1021/acsanm.6c02379
• COBISS.SI-ID: 304353539
• Publication date in RUL: 10.11.2026

Record is a part of a journal

• Title: ACS applied nano materials
• Shortened title: ACS appl. nano mater.
• Publisher: American Chemical Society
• ISSN: 2574-0970
• COBISS.SI-ID: 32649255

Licences

• License: CC BY 4.0, Creative Commons Attribution 4.0 International
• Link: http://creativecommons.org/licenses/by/4.0/
• Description: This is the standard Creative Commons license that gives others maximum freedom to do what they want with the work as long as they credit the author.

Secondary language

• Language: Slovenian
• Keywords: filamentozni fag, nanogrodje, dvojni prikaz, signalno zaporedje, razdeljeni protein, SpyTag/SpyCatcher, SnoopTag/SnoopCatcher, nanomateriali