A Nonviral Neo-Nucleocapsid for Cell-Specific RNA Delivery Developed by Pseudo-Cyclic Peptide Grafting and Directed...

Area of Science:

• Biotechnology
• Molecular Biology
• Protein Engineering

Background:

• Nonviral protein vehicles offer scalable, biocompatible RNA delivery for biotechnological uses.
• Directed evolution can enhance protein function, but challenges exist in linking genotype to phenotype for protein vehicles.
• Few nonviral protein vehicles have undergone directed evolution for improved performance.

Purpose of the Study:

• To engineer and evolve a neo-nucleocapsid for specific RNA delivery to cells.
• To develop a versatile system for creating designer protein cages for cargo delivery.

Main Methods:

• Applied the DogTag/DogCatcher protein ligation system to an artificial nucleocapsid.
• Utilized directed evolution to obtain nucleocapsid-displaying proteins.
• Lasso-grafted a pharmacophore sequence targeting the MET receptor into the DogCatcher protein loop.

Main Results:

• Successfully created designer neo-nucleocapsids capable of receptor-specific targeting.
• Enhanced endocytic uptake of neo-nucleocapsids after cellular-based directed evolution.
• Observed no detectable cytosolic release or subsequent protein expression from delivered RNA.

Conclusions:

• The developed DogTag/DogCatcher system provides a versatile platform for engineering protein cages.
• This approach enables the construction of neo-nucleocapsids for targeted delivery of various cargos.
• The study demonstrates a successful strategy for directed evolution of protein vehicles for RNA delivery.