Project Summary The capsids of many RNA viruses spontaneously assemble around the viral RNA in the cytoplasm of an infected host cell. Mapping out the assembly pathway—that is, the set of molecular rearrangements that produce the final infectious structure—is the first step toward developing antiviral drugs that block the process and halt the spread of pathogenic viruses. Available experimental techniques, which measure what happens in a bulk solution of capsids and not in individual capsids, obscure the essential kinetics. As a result, we lack answers to basic questions about how capsids form, such as whether the rate limiting step is nucleation, or growth, or the rearrangement of coat proteins following an initial disordered attachment to the RNA. New tools from nanoscience can tackle these challenges. This proposal describes a plan to measure and understand capsid assembly in vitro at the single-capsid scale: a powerful nanoscale imaging technique called interferometric scattering (iSCAT) microscopy is applied to monitor the growth of individual viral capsids on individual strands of RNA. The data from these measurements can be used to build new models of the assembly pathway that describe the process in unprecedented detail. Special focus is paid to the role of the viral RNA in directing these pathways. New DNA-based techniques are developed to unravel the complex folding patterns of viral RNA and to selectively probe the specific RNA-protein interactions that have been proposed to play a role in capsid assembly. The combined approach of fast nanoscale imaging and DNA-based methods for manipulating specific molecular interactions may reveal unexpected routes for blocking capsid assembly and combating viral disease.