Engineering recombinant barley stripe mosaic virus-like particles for nucleic acid delivery

• Divyashree Thapa, Applied Molecular Biology & Biotechnology, University of Delaware

• Kevin Solomon, Department of Chemical and Biomolecular Engineering, University of Delaware

Surface properties and cargo encapsulation in nanoparticles determine their interactions with cells and successful delivery of cargo. Precise control over these parameters is essential for the success of nanoparticles as delivery vehicles. Rod-shaped plant viruses, such as barley stripe mosaic virus (BSMV), are hierarchically assembled protein nanorods that allow control over these properties. Each nanorod comprises hundreds to thousands of capsid proteins that self-assemble around an RNA template, offering capacity for high-density surface display at the surface-exposed C-terminus and RNA encapsidation. We leverage a bacterial platform to produce non-infectious BSMV virus-like particles (VLPs), in which the viral genome is replaced by a user-defined RNA template for additional control over RNA encapsidation. This flexible engineering platform also allows modification of the solvent-exposed C-terminus to control surface properties. Using Tag/Catcher bioconjugation, SnoopTag fused to the C-terminus of BSMV capsid proteins successfully assembled into rods and enabled the display of hexapeptides on the surface. Introduction of hexapeptides with different properties enabled control over the surface charge of BSMV VLPs, as confirmed by zeta potential measurements. Furthermore, extraction and characterization of encapsidated RNA revealed that the intended full-length RNA sequence was present only in low amounts in the VLPs. RNA analyses indicate that RNA degradation and/or competition with endogenous bacterial RNA may limit encapsidation of the intended RNA during particle assembly. To further investigate these mechanisms, ongoing studies compare RNA encapsidation in wild-type and endonuclease-deficient bacterial strains to determine whether reducing nuclease activity improves the proportion of full-length RNA encapsidated in the VLPs. Future work will include characterizing the effect of surface properties on protein corona composition and tuning the system to encapsidate full-length RNA. These advances will set the stage for the development of BSMV VLPs as a nucleic acid delivery platform.