(767b) Developing Novel Virus-Like Particles As Tunable, Targetable Reporter Platforms

Virus coat and envelope proteins are compelling candidates for nanoparticle technology development because they self-assemble into small, highly symmetric ensembles called virus-like particles (VLP).  These monodisperse VLP particles have a conserved morphology  and can be modified both genetically and chemically in a highly controlled manner, allowing for tuned adaptation for each specific application. The VLP based on bacteriophage Qβ coat proteins (CP) and its maturation protein, A2, are especially appealing for nanoparticle development. The Qβ VLP is highly stabilized by disulfide bonds, which cross-link the CP. Each VLP contains 180 copies of the CP while only incorporating a single A2.

In this work, we explore the development of customized bacteriophage Qβ VLP for targeted photoreporter platforms. Via site-directed genetic mutations and global-methionine replacement, unnatural amino acids (uAA) are inserted into the CP during cell-free protein synthesis. Use of the in vitro cell-free synthesis approach provides ease of access to the synthesis environment, allows for synthesis of highly cytotoxic proteins such as A2, and simplified transport of otherwise less soluble uAA. These uAA provide bio-orthogonal chemical moieties for conjugation of a variety of photoreporters after VLP assembly. The result of photoreporter attachment is a small, stable and densely populated nanoparticle, with upwards of 100 photoreporters per particle.  We genetically modified the A2 protein to contain a single, site-specific insertion for uAA incorporation. This uAA also contains a bio-orthogonal moiety that acts as the targeting-ligand attachment site. Combining the modified CP and A2 proteins, we produce a platform for high density attachment of various photoreporters that can be covalently attached to targeting-ligands of choice.