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

Abstract Nonviral protein vehicles for RNA delivery hold significant promises for various biotechnological applications due to their scalability, ease of engineering, and high biocompatibility. Directed evolution is a powerful approach to enhance protein activity, yet few nonviral protein vehicles have been evolved due to the challenges in linking genotype and phenotype. Here, we report engineering and directed evolution of a neo‐nucleocapsid enabling receptor‐specific RNA delivery. We applied the DogTag/DogCatcher protein ligation system to an artificial nucleocapsid and conducted directed evolution to obtain nucleocapsid displaying proteins. To create designer neo‐nucleocapsids, we lasso‐grafted a pharmacophore sequence of a pseudo‐cyclic peptide binding to the hepatocyte growth factor receptor MET into the loop of DogCatcher. After the cellular‐based directed evolution, endocytic uptake of neo‐nucleocapsids was improved whereas cytosolic release and subsequent protein expression from the delivered RNA were not detected. This experimental approach provides a versatile system for constructing protein cages delivering various cargos to specific cells.