Programmable icosahedral capsids: A layered approach mediated by lipid templates

Self-assembly is a fundamental process in both inanimate and animate systems, where subunits spontaneously organize into structured assemblies. Spanning from lipid membranes to multiprotein filaments, this self-organization is crucial for numerous biological functions. A striking example is the assembly of protein subunits around viral nucleic acids to form protective capsids. Over 50 years ago, the Caspar and Klug theory of quasi-equivalence provided seminal insight into viral capsid structure, observing that most spherical viruses exhibit icosahedral order. While this theory describes the structure of many smaller spherical viruses, larger viruses such as herpes simplex virus assemble in layers and require a template for successful assembly.

Despite extensive studies on the spontaneous assembly of viral proteins into empty capsid shells, the in vitro replication of templated assembly around a rigid spherical template remains unexplored. Here, we aim to construct large capsids with high yield using a minimal number of building blocks, inspired by the layered architecture of large viruses. By utilizing a spherical template to assemble capsids with a single equivalent bonding interaction, we propose an experimental strategy that mimics this natural design. This approach employs engineered lipid vesicles with cholesterol-modified DNA as the inner layer and triangular-shaped DNA origami building blocks as the outer layer.

We developed a single-molecule microscopy assay to investigate the kinetics of DNA origami self-assembly on lipid bilayers using Total Internal Reflection Fluorescence (TIRF) microscopy. Furthermore, we devised a method to obtain quantitative estimates of triangle-lipid binding interactions through flow cytometry on lipid-coated colloids. This comprehensive approach promises to significantly enhance the yield of origami capsids, potentially by several orders of magnitude compared to current methods.