An engineered closed-shell, two-component, 480-subunit nucleocapsid

ABSTRACT Self-assembling protein cages are powerful nanoscale containers for biotechnology and medical applications. Two-component systems are especially attractive due to their potential for functional complexity. In this study, we demonstrate that the subunits of the 240mer nucleocapsid NC-4, which was previously evolved to package and protect its encoding mRNA, can be split into two fragments without disrupting cage assembly or structure, generating a two-component, 480-subunit capsid. This modification introduces additional termini on the cage’s exterior surface, creating new opportunities for functionalization. We exploited these new sites by genetically appending peptide and protein tags to the exterior surface of split NC-4 (spNC-4), enabling site-specific glycosylation via post-translational modification and cell-specific delivery by targeted antibody recruitment. Our findings broaden the utility of the NC-4 nucleocapsid. By extension, splitting related protein compartments that bind diverse cargoes could offer a robust platform for biotechnological applications requiring simultaneous encapsulation and customizable surface modification. SIGNIFICANCE STATEMENT Natural protein shells such as viral capsids and bacterial microcompartments have inspired efforts to design synthetic compartments that protect and deliver functional molecules. Here we show that a nonviral, artificially evolved nucleocapsid can be split into two fragments that reassemble into a closed, two-component, 480-subunit cage that packages its own mRNA. This redesign preserves the original architecture and selective RNA packaging while creating new engineerable sites on both the interior and exterior surfaces, enabling simultaneous control of internal cargo binding and external modification. The resulting two-component architecture highlights the structural plasticity of synthetic nucleocapsids and provides a general strategy for constructing modular, evolvable protein containers for biotechnology and synthetic biology.