In vivo encapsulation of nucleic acids using an engineered nonviral protein capsid

Area of Science:

• Biochemistry and Molecular Biology
• Nanotechnology
• Structural Biology

Background:

• Protein capsids naturally encapsulate molecules and have potential in nanotechnology for carrying non-native guests.
• Charge complementarity is a promising strategy for designing new molecular encapsulation systems.
• Engineering protein capsids can create novel containers for various applications.

Purpose of the Study:

• To explore the use of charge complementarity for encapsulating nucleic acids within protein capsids.
• To engineer the Aquifex aeolicus lumazine synthase (AaLS) capsid to enhance its positive charge for RNA binding.
• To investigate the selectivity and affinity of the engineered capsid for RNA molecules.

Main Methods:

• Site-directed mutagenesis was used to introduce four mutations per subunit in AaLS to increase positive surface charge.
• The engineered mutant capsid (AaLS-pos) was produced and assembled in vivo.
• Characterization of AaLS-pos capsids involved analyzing their encapsulated cargo, specifically RNA content and size.

Main Results:

• The engineered AaLS-pos capsids exhibited increased positive charge on their inner surface.
• In vivo assembly led to the uptake of cellular RNA into the AaLS-pos capsids.
• The encapsulated RNA molecules were predominantly 200-350 bases in length, indicating selective encapsulation.

Conclusions:

• Simple charge complementarity can achieve high-affinity and selective RNA encapsulation within engineered protein capsids.
• Tuning the surface charge of protein capsids offers a method to control RNA loading.
• This approach could provide insights into viral genome recognition and advance RNA delivery system development.