Evolution of a designed protein assembly encapsulating its own RNA genome

Computationally designed icosahedral protein-based assemblies can protect their genetic material and evolve in biochemical environments, suggesting a route to the custom design of synthetic nanomaterials for non-viral drug delivery. Synthetic protein shells Viruses use capsids, or protein shells, to envelop and protect their genomes. This tricks the cell's machinery into using their genetic material for their own replication. Several viruses have been used for gene therapy. In this work, David Baker and colleagues design and build icosahedral protein-based assemblies which protect their genetic material and can evolve in biochemical environments, like a synthetic virus capsid. These assemblies are termed synthetic nucleocapsids. Evolution of the nucleocapsid designs improved both their mRNA packaging efficiency, which competes with commonly used viral vectors for gene therapy, and their circulation stability in vivo . This work raises the possibility of designing custom synthetic nucleocapsids for various uses and RNA cargoes, including for drug delivery. The challenges of evolution in a complex biochemical environment, coupling genotype to phenotype and protecting the genetic material, are solved elegantly in biological systems by the encapsulation of nucleic acids. In the simplest examples, viruses use capsids to surround their genomes. Although these naturally occurring systems have been modified to change their tropism 1 and to display proteins or peptides 2 , 3 , 4 , billions of years of evolution have favoured efficiency at the expense of modularity, making viral capsids difficult to engineer. Synthetic systems composed of non-viral proteins could provide a ‘blank slate’ to evolve desired properties for drug delivery and other biomedical applications, while avoiding the safety risks and engineering challenges associated with viruses. Here we create synthetic nucleocapsids, which are computationally designed icosahedral protein assemblies 5 , 6 with positively charged inner surfaces that can package their own full-length mRNA genomes. We explore the ability of these nucleocapsids to evolve virus-like properties by generating diversified populations using Escherichia coli as an expression host. Several generations of evolution resulted in markedly improved genome packaging (more than 133-fold), stability in blood (from less than 3.7% to 71% of packaged RNA protected after 6 hours of treatment), and in vivo circulation time (from less than 5 minutes to