| It has been observed that icosahedral plant viruses like BMV, CCMV, undergo a swelling transition near neutral pH and by absence of cations. It is widely agreed that swelling is a significant phenomenon in the life cycle of a virus. There has been a long-standing hypothesis that swelling is a mechanism to release the nucleic acid during infection and replication. However, research efforts directed towards investigating this hypothesis have been rare and present conflicting conclusions. Probing for specific RNA-capsid interaction will provide a clearer picture as to how swelling is related to the release of RNA. Fluorescence correlation spectroscopy (FCS) has potential to provide crucial information towards determining the biological role of swelling. In principle, FCS is the time resolved measurement of fluctuations in fluorescence, where different time constants in the process can be isolated by auto-correlating the fluorescence trace. This work aims at utilizing the property of cyanine dyes to possess ON and OFF fluorescent states as a result of the molecule undergoing a cis-trans isomerization. By labeling viral RNA with the Cy5 dye, there is potential to probe the change in the local environment of the RNA molecule as it experiences changing steric forces throughout the swelling transition. This report contains the results from experiments for validation of this method for single molecule fluorescence studies to be performed at a time resolution under 10ms to capture the detailed dynamics of the capsid during the swelling transition. |
