2026-10-14T05:14:00Z https://api.figshare.com/v2/oai

oai:figshare.com:article/33962056 2026-09-22T05:38:25Z category_134 portal_316 item_type_3 month_year_09_2026

Supplementary file 1_Enzyme-mediated site-specific functionalization of mRNA-containing TMV nanorods produced in yeast.docx Jerrell Tisnado (1904932) Ana Luisa Duran Meza (22822204) David Silverio Moreno Gutierrez (25087792) Nina M. Harpell (25087795) Alyssa Cruz (25087798) Charles M. Knobler (625620) William M. Gelbart (22449) Virology LPETG Sortase A surface conjugation TMV VLPs <p>Virus-like particles (VLPs) derived from Tobacco mosaic virus (TMV) have emerged as attractive platforms for nanotechnology and molecular delivery applications owing to their robust self-assembly, stoichiometric precision, and highly accessible surface architecture. Here we report the development of a yeast-based platform for the production of TMV VLPs – nanorods – that can be site-specific functionalized and that contain RNA of interest. TMV coat proteins engineered with a C-terminal LPETG Sortase A (SrtA) recognition motif were expressed in Komagataella phaffii along with a self-amplifying RNA construct containing the TMV origin of assembly sequence (OAS) – the “packaging signal” – enabling the intracellular formation of nanorods. Transmission electron microscopy (TEM) confirmed the assembly of particles with wildtype TMV virion morphology, while RT-PCR analysis of these purified VLPs preparations demonstrated their packaging of target RNA. 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> 2026-09-22T05:38:25Z Dataset Dataset 10.3389/fviro.2026.1955107.s001 https://figshare.com/articles/dataset/Supplementary_file_1_Enzyme-mediated_site-specific_functionalization_of_mRNA-containing_TMV_nanorods_produced_in_yeast_docx/33962056 CC BY 4.0