Characterization of exogenous DNA mobility in live cells through fluctuation correlation spectroscopy.
The spatial-temporal dynamics of delivered DNA is a critical aspect influencing successful gene delivery. A comprehensive model of DNA lipoplex trafficking through live cells has yet to be demonstrated. Here the bioimaging approaches Raster Image Correlation Spectroscopy (RICS) and image-Means Square Displacement (iMSD) were applied to quantify DNA mechanical dynamics in live cells. DNA lipoplexes formed from DNA with a range of 21 bp to 5.5 kbp exhibited a similar range of motion within the cytoplasm of myoblast cells regardless of size. However, the rate of motion was dictated by the intracellular location, and DNA cluster size. This analysis demonstrated that the different transport mechanisms either had a size dependent mobility, including random diffusion, whereas other mechanisms were not influenced by the DNA size such as active transport. The transport mechanisms identified followed a spatial dependence comparable to viral trafficking of non-active transport mechanism upon cellular entry, active transport within the cytoplasm and further inactive transportation along the peri-nuclear region. This study provides the first real-time insight into the trafficking of DNA delivered through lipofection using image-based fluctuation correlation spectroscopy approaches. Thereby, gaining information with single particle sensitivity to develop a deeper understanding of DNA lipoplex delivery through the cell.
University of Western Sydney, School of Science and Health, Hawkesbury Campus, Locked Bag 1797, Penrith NSW 2751, Australia.
Stephen
S
Mieruszynski
Department of Developmental and Cell Biology, University of California Irvine, Irvine, California, United States of America.|Department of Biomedical Engineering, Laboratory for Fluorescence Dynamics, University of California Irvine, Irvine, California, United States of America.|Centre for Bioactive Discovery in Health and Ageing, School of Science and Technology, University of New England, Armidale, Australia.
Michelle A
MA
Digman
Department of Developmental and Cell Biology, University of California Irvine, Irvine, California, United States of America.|Department of Biomedical Engineering, Laboratory for Fluorescence Dynamics, University of California Irvine, Irvine, California, United States of America.|Centre for Bioactive Discovery in Health and Ageing, School of Science and Technology, University of New England, Armidale, Australia.
Enrico
E
Gratton
University of Western Sydney, School of Science and Health, Hawkesbury Campus, Locked Bag 1797, Penrith NSW 2751, Australia.
Mark R
MR
Jones
D000818:Animals; D001692:Biological Transport; D002460:Cell Line; D004247:DNA / Q000378:metabolism*; D042541:Intracellular Space / Q000378:metabolism; D018613:Microscopy, Confocal; D051381:Rats; D013057:Spectrum Analysis / Q000379:methods
D004247:DNA