Cargo-Directed Assembly of Nonviral Nucleocapsid with Controlled Size

Precise packaging of diverse cargo within self-assembling protein cages of defined size and shape is essential for many biotechnological applications, yet cellular expression offers limited control over loading. Here, we developed an in vitro cargo-directed reconstitution system of a split, artificial nucleocapsid (spNC-4). Two spNC-4 capsid protein subunits were prepared independently and assembled with cargos cooperatively. As an authentic cargo, a nucleocapsid mRNA is packaged into a 30 nm-spheric nucleocapsid in vitro, closely matching to spNC-4 expressed in cells. In this system, a diverse range of cargos are encapsulated, including noncognate RNA, RNA-positively supercharged fluorescent protein complex, and linear double-stranded DNA. Moreover, by packaging 30 nm-spherical or rod-shaped DNA origamis as templates, the nucleocapsid morphology was altered to an enlarged 60 nm-spherical structure or rod-shaped structure. The developed system accepts versatile composition and programmable control over the artificial nucleocapsid architecture, creating a general platform for enzyme nanoreactors, targeted delivery, and vaccine development.