Raster image cross-correlation analysis for spatiotemporal visualization of intracellular degradation activities against exogenous DNAs

Reducing intracellular DNA degradation is critical to enhance the efficiency of gene therapy. Exogenous DNA incorporation into cells is strictly blocked by the defense machinery of intracellular nuclease activity. Raster image correlation spectroscopy (RICS) and raster image cross-correlation spectroscopy (cross-correlation RICS; ccRICS) are image-based correlation methods. These powerful tools allow the study of spatiotemporal molecular dynamics. Here we performed spatiotemporal ccRICS analyses of fluorescent DNA and directly monitored the process of exogenous DNA degradation in living cell cytoplasm. Such direct monitors of DNA degradation allow us to determine the fate of the exogenous DNA in living cells. On comparing the process in living cells, our study shows that cytoplasmic nuclease activity differs between cell lines; therefore, we propose that the difference of nuclease activity in cytoplasm dictates a different resistance to exogenous DNA incorporation. New insight on efficient gene delivery can be provided with our study.