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

Akira Sasaki,Masataka Kinjo,Takashi Jin,Johtaro Yamamoto

doi:10.1038/srep14428

Abstract

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.

Highlights

Example, the expression efficiency of MEF cells is relatively low[10]; in contrast, HEK293 cells can be transfected efficiently and the cell line is usually used for protein factory in mammalian cells[11]
Such direct measurements of DNA degradation allow us to determine the fate of the exogenous DNA in a timely manner in living cells and to monitor the cytoplasmic nuclease activity, which is the critical factor for efficient gene delivery
This study aimed to evaluate when and where exogenous DNAs are degraded in living cells

Summary

Introduction

Example, the expression efficiency of MEF cells is relatively low[10]; in contrast, HEK293 cells can be transfected efficiently and the cell line is usually used for protein factory in mammalian cells[11]. The advantages of RICS/ ccRICS are that dynamic molecular information can be extracted from the image of confocal laser scanning microscopy (LSM), cells can be continuously imaged throughout the measurement, and regions of interest can be selected after image acquisition. This spatiotemporal analysis is ideal for investigating molecular dynamics, reaction kinetics, and molecular interactions in living cells. The dual-color ccRICS technique was employed to visualize when and where the exogenous DNA injected into living cell cytoplasm is degraded Such direct measurements of DNA degradation allow us to determine the fate of the exogenous DNA in a timely manner in living cells and to monitor the cytoplasmic nuclease activity, which is the critical factor for efficient gene delivery

Objectives

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Conclusion