Enabling Surface Functionalization of Barley Stripe Mosaic Virus-Like Particles via Click Chemistry

• Nadia Harricharan, Applied Molecular Biology & Biotechnology, University of Delaware

• Kevin Solomon, Chemical engineering, University of Delaware

Virus-like particles (VLPs) have tremendous potential for medical applications including theranostics, vaccines, and drug delivery. Of particular interest are the rod-shaped plant viruses, such as barley stripe mosaic virus (BSMV),  which consist of thousands of coat proteins in each RNA -templated nanoparticle. Our lab was the first to generate bacterially-derived BSMV VLPs, which offer the opportunity for length control via RNA templating and for high-density surface display. However, surface functionalization remains a critical challenge for their development towards these applications. To address this challenge, we inserted cysteine, which is absent in the native coat protein, at the surface exposed C terminus of BSMV. Cysteine contains a thiol group that can be leveraged for selective click chemistries such as thiol-ene and thiol-Michael reactions. We validated the functionality of our cysteine-displaying VLPs via conjugation to maleimide-linked fluorescent dyes. Cysteine-displaying BSMV VLPs can be readily decorated with other functional ligands, but conjugation to peptides and protein ligands containing cysteines would suffer from undesired reactions. To enable more selective functionalization chemistries, we incorporated the unnatural amino acid (p-azido-l-phenylalanine) which was site-specifically incorporated by suppressing the amber stop codon with an additional, engineered aaRS/tRNA pair. This was completed by the addition of two plasmids into a bacterial expression strain which enabled expression and suppression processes, respectively. Furthermore, TEM imaging validated that the rods maintained structural integrity after UAA incorporation. We expect that this platform can be leveraged for facile and modular surface functionalization via copper-catalyzed azide-alkyne cycloaddition (CuAAC) click chemistry. In the immediate future, we will use our fluorescently labeled BSMV VLPs to track uptake and intracellular localization in mammalian cells. Ultimately, these advances establish an opportunity to easily label BSMV VLPs via click chemistry for a plethora of medical applications.