Cargo-Directed Assembly of Nonviral Nucleocapsid with Controlled Size.

Area of Science:

• Biotechnology
• Nanotechnology
• Structural Biology

Background:

• Precise control over cargo loading into protein cages is crucial for biotechnological applications.
• Current cellular expression methods offer limited control over the encapsulation process.
• Artificial protein cages offer a controllable alternative to natural viral capsids.

Purpose of the Study:

• To develop an in vitro cargo-directed reconstitution system for artificial nucleocapsids.
• To demonstrate programmable control over the size, shape, and cargo diversity of self-assembling protein cages.
• To establish a versatile platform for applications in nanoreactors, drug delivery, and vaccine development.

Main Methods:

• Developed a split, artificial nucleocapsid (spNC-4) system with independently prepared subunits.
• Utilized a cargo-directed assembly approach for cooperative protein subunit assembly with various cargos.
• Employed DNA origami templates to direct the morphology of the assembled nucleocapsids.

Main Results:

• Successfully packaged diverse cargos including mRNA, noncognate RNA, supercharged fluorescent protein complexes, and double-stranded DNA.
• Achieved in vitro assembly of a 30 nm spherical nucleocapsid, matching cellular expression.
• Demonstrated programmable alteration of nucleocapsid morphology to 60 nm spheres or rod shapes using DNA origami templates.

Conclusions:

• The developed in vitro system provides versatile composition and programmable control over artificial nucleocapsid architecture.
• This platform enables precise packaging of diverse cargos within self-assembling protein cages of defined size and shape.
• The system holds significant potential for advancing enzyme nanoreactors, targeted delivery systems, and vaccine development.