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Recent advances in computational methods have enabled the predictive design of self-assembling protein nanomaterials with atomic-level accuracy. These design strategies focus exclusively on a single target structure, without consideration of the mechanism or dynamics of assembly. However, understanding the assembly process, and in particular its robustness to perturbation, will be critical for translating this class of materials into useful technologies. Here we investigate the assembly of two computationally designed, 120-subunit icosahedral complexes in detail using several complementary biochemical methods. We found that assembly of each material from its two constituent protein building blocks was highly cooperative and yielded exclusively complete, 120-subunit complexes except in one non-stoichiometric regime for one of the materials. Our results suggest that in vitro assembly provides a robust and controllable route for the manufacture of designed protein nanomaterials and confirm that cooperative assembly can be an intrinsic, rather than evolved, feature of hierarchically structured protein complexes.

Self‐assembling protein nanoparticles are a promising class of materials for targeted drug delivery. Here, the use of a computationally designed, two‐component, icosahedral protein nanoparticle is reported to encapsulate multiple macromolecular cargoes via simple and controlled self‐assembly in vitro. Single‐stranded RNA molecules between 200 and 2500 nucleotides in length are encapsulated and protected from enzymatic degradation for up to a month with length‐dependent decay rates. Immunogenicity studies of nanoparticles packaging synthetic polymers carrying a small‐molecule TLR7/8 agonist show that co‐delivery of antigen and adjuvant results in a more than 20‐fold increase in humoral immune responses while minimizing systemic cytokine secretion associated with free adjuvant. Coupled with the precise control over nanoparticle structure offered by computational design, robust and versatile encapsulation via in vitro assembly opens the door to a new generation of cargo‐loaded protein nanoparticles that can combine the therapeutic effects of multiple drug classes.