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 a system for in vitro cargo-directed reconstitution of a split, laboratory evolved nonviral nucleocapsid (spNC-4). Independently expressed and purified spNC- 4 capsid protein subunits were mixed and assembled with cargo molecules in a cooperative manner. As an authentic cargo, 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 cargo molecules, including cognate nucleocapsid mRNA, noncognate RNA, RNA complexed with positively supercharged fluorescent protein, and linear double-stranded DNA are encapsulated within the 30 nm-spheric nucleocapsids in vitro . Moreover, the packaging of 30 nm-spherical or rod-shaped DNA origamis as templates induce morphological alterations of the nucleocapsids, resulting in the formation of enlarged 60 nm-spherical structure or rod-shaped structure, respectively. This split-protein, cargo-dependent system provides versatile and programmable control over both composition and architecture of nonviral protein cages, creating a general platform for enzyme nanoreactors, targeted delivery, and vaccine development.