Abstract:

ID: 2260

Abstract
  • Title:
    New Insights into the Mechanism of Concomitant Electrotransfer of Small and Large Nucleic Acids

    Authors:
    Palepšien? R?ta, Vytautas Magnus University, Kaunas Lithuania
    Muralidharan Aswin, Delft University of Technology, Delft, Netherlands
    Maciulevi?ius Martynas, Vytautas Magnus University, Kaunas Lithuania
    Ruzgys Paulius, Vytautas Magnus University, Kaunas Lithuania
    Chopra Sonam, Vytautas Magnus University, Kaunas Lithuania
    Boukany Pouyan E. Delft University of Technology, Delft, Netherlands
    Šatkauskas Saulius, Vytautas Magnus University, Kaunas Lithuania


    Abstract:
    It is well described that electrotransfer of small and large nucleic acids into cells are governed by distinct mechanisms. In both cases the critical steps are cell electroporation and electrophoretic forces that drag the molecules to or through the electroporated membrane. In case of small nucleic acids, it is believed that electrophoretic forces drag the molecules to and through the membrane directly into cytosol, while in case of pDNA, electrophoretic forces drag the molecules to the electroporated membrane where the molecules form pDNA aggregates. These aggregates later are uptaken by the cell employing endocytotic pathways. Here we present a fundamental study of the mechanism of electrotransfer of oligonucleotides and siRNA molecules that occur independently and simultaneously with pDNA by employing confocal fluorescence microscopy. In contrast to the conditional understanding of the mechanism of small nucleic acids electrotransfer, we have shown that the electrotransfer of oligonucleotides and siRNA is driven by both electrophoretic forces and diffusion after EP, followed by subsequent entry into the nucleus within 5 min after treatment. The study also revealed that the efficiency of siRNA electrotransfer decreases in response to an increase in pDNA concentration. Stronger inhibition occurs at the cell side facing the cathode. The analysis shows that this is related to occlusion of electroporated membrane with pDNA aggregates. On the other hand, since inhibition occurs also at the anode-facing membrane side, additional factors are taking place.

    Keywords:
    electroporation, electrotransfer, siRNA, plasmid DNA, oligonuceltides

    Refs:


    Topic 1:
    1. Biological responses (molecular, subcellular, cellular and intercellular)

    Topic 2:
    2. Biophysics and biochemistry of interaction mechanisms


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