Genetically Engineered, Multichromophore Virus-Like Nanoparticles with Ultranarrow Distribution of Emission Intensity

Variance in the properties of optical mesoscopic probes is often a limiting factor in applications. In the thermodynamic limit, the smaller the probe, the larger the relative variance. However, specific viral protein cages can assemble efficiently outside the bounds of statistical fluctuations at equilibrium through a process that is characterized by intrinsic quality-control and self-limiting capabilities. In this paper, an approach is described that leverages stoichiometric and structural accuracy in the murine polyoma virus capsid assembly to demonstrate bright, narrowly distributed fluorescence intensity from multichromophore particles that surpass state-of-the-art fluorescent nanosphere probes. Charge-detection mass spectrometry analysis demonstrated that proteins resulting from the fusion of superfolding green fluorescent protein (sfGFP) murine polyoma virus coat proteins self-assemble in vitro into virus-like particles that have similar stoichiometry as virus-like particles formed from wild-type virus coat proteins. Single-particle analysis by total internal reflection fluorescence microscopy provided evidence for a narrow fluorescence intensity that reflects stoichiometric accuracy of the construct.