(713e) Assembly of a Modular and Tunable Worm-like Protein Nanostructure Using a Bottom-up Approach

Protein nanostructures have garnered interest as carriers in drug and intracellular protein delivery for their enhanced biocompatibility and stability compared to most synthetic particle systems. Studies into the design of an effective drug carrier suggest that modulating the shape of the particle is a key parameter in nanoparticle design. Where there is a balance in tuning high-aspect ratio nanoparticle’s maximal lengths between lengths small enough for effective extravasation and internalization by endocytosis and, long enough for an effective circulation half-life through avoiding renal clearance and minimizing macrophage uptake. However, most high-aspect ratio protein nanostructures, like nucleoprotein-based nanorods, either form through self-assembly onto native RNA templates which limits the capacity for tuning length scales or, through a further in vitro disassembly and re-assembly step onto an engineered RNA template. The latter top-down assembly strategy instills the capacity to modify particle lengths but has been shown to regularly result in a heterogenous mixture. Furthermore, these nanostructures commonly lack the capacity for extensive and patterned surface decoration, usually limited to incorporating bioconjugation chemistries at solvent-exposed termini or, randomly reacting solvent-exposed lysine or cysteine side chains. Therefore, we have designed a multi-pot stepwise bottom-up approach for generating a worm-like protein nanostructure with homogenous and tunable control of structure length and, control of the position and valency of multiple protein cargos and a Click-compatible non-standard amino acid (nsAA), using orthogonal Catcher-Tag isopeptide-ligating protein-peptide pairs in tandem with elastin-like polypeptides and the controlled incorporation of 4-azido-L-phenylalanine. With this platform we demonstrate that we can modularly control the valency of multiple protein cargos as well as their position relative to each other within the structure. And that this assembly strategy allows for the incorporation of multiple different alkyne-functionalized ligands with control of their local density and relative positions to each other by virtue of the multi-pot nature of this assembly approach. We anticipate that by using this assembly strategy, high-aspect ratio worm-like protein nanostructures can be assembled for any desired application due to the inherent “plug-and-play” nature of the assembly strategy with respect to protein cargos, Click-compatible nsAA positioning and particle length.