Supplementary file 1_Enzyme-mediated site-specific functionalization of mRNA-containing TMV nanorods produced in yeast.docx

The accessibility of the engineered LPETG motif following assembly enabled SrtA mediated conjugation of N-terminal polyglycine-modified ligands, including fluorescent polyG-Alexa probes and a targeting affibody. Successful conjugation was verified by SDS-PAGE and fluorescence analyses, demonstrating efficient post-assembly functionalization while preserving particle integrity. Further, affibody-functionalized TMV nanorods were coupled to gold nanoparticles, and TEM confirmed the surface presentation and accessibility of the conjugated ligands on intact particles.

Together, these results establish K. phaffii as a scalable platform for the production of engineered TMV nanorods and demonstrate that genetically encoded SrtA recognition motifs enable versatile post-assembly surface modification. This approach provides a modular framework for the development of customizable TMV-based nanomaterials for in vivo targeting, imaging, and nanobiotechnology applications.</p>