In Singulo Probing of Viral RNA Dynamics by Multichromophore Fluorescence Dequenching

Current understanding of virus life-cycle states and transitions between them is mainly built on knowledge of the protein shell structure encapsulating the genome. Little is known about the genome fate during viral transitions. Here, changes in the fluorescence rate from multilabeled transcript viral RNAs encapsulated in Brome mosaic virus capsids were examined as a function of the RNA state. A simple kinetic model relating chain compactness to single-molecule fluorescence emission suggests that in a dense multichromophore system the rate of energy transfer should scale with distance more gradually than the rate of the Förster energy transfer between two chromophores, which varies sharply as the reciprocal of distance to the sixth power. As a proof-of-principle experiment, we have compared predictions from a numerical model for confined diffusive motion with the fluorescence emission from virus-encapsulated and free single RNA molecules decorated with multiple cyanine dyes and encapsulated inside microscopic emulsion droplets. We found that the effective quantum yield per labeled particle depends on the expansion state, in agreement with theoretical predictions. Since fluorescence single particle tracking is now a well-established methodology for the study of virus life cycle, the findings reported here may pave the way toward reducing the existing gap between in vitro and cellular in singulo studies of the fates of viral RNA.