New insights into the mechanism of electrotransfer of small nucleic acids
Rūta Palepšienė (Vytautas Magnus university)
Aswin Muralidharan (Kavli institute of nanoscience Delft, TU Delft - BN/Bionanoscience, TU Delft - BN/Stan Brouns Lab)
Martynas Maciulevičius (Vytautas Magnus university)
Paulius Ruzgys (Vytautas Magnus university)
S. Chopra (Vytautas Magnus university, TU Delft - ChemE/Product and Process Engineering)
PE Boukany (TU Delft - ChemE/Product and Process Engineering)
Saulius Šatkauskas (Vytautas Magnus university)
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Abstract
RNA interference (RNAi) is a powerful and rapidly developing technology that enables precise silencing of genes of interest. However, the clinical development of RNAi is hampered by the limited cellular uptake and stability of the transferred molecules. Electroporation (EP) is an efficient and versatile technique for the transfer of both RNA and DNA. Although the mechanism of electrotransfer of small nucleic acids has been studied previously, too little is known about the potential effects of significantly larger pDNA on this process. Here we present a fundamental study of the mechanism of electrotransfer of oligonucleotides and siRNA that occur independently and simultaneously with pDNA by employing confocal fluorescence microscopy. In contrast to the conditional understanding of the mechanism, 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. Overall, the study provides new insights into the mechanism of electrotransfer of small nucleic acids which may have broader implications for the future application of RNAi-based strategies.