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.

中文翻译：

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小核酸电转移机制的新见解

RNA 干扰 (RNAi) 是一种强大且快速发展的技术，可以精确沉默感兴趣的基因。然而，RNAi 的临床开发因转移分子的细胞摄取和稳定性有限而受到阻碍。电穿孔 (EP) 是一种高效且通用的 RNA 和 DNA 转移技术。尽管之前已经研究过小核酸的电转移机制，但对于显着较大的 pDNA 对此过程的潜在影响知之甚少。在这里，我们通过共聚焦荧光显微镜对寡核苷酸和 siRNA 与 pDNA 独立且同时发生的电转移机制进行了基础研究。与对该机制的有条件理解相反，我们已经证明寡核苷酸和 siRNA 的电转移是由电泳力和 EP 后的扩散驱动的，随后在处理后 5 分钟内进入细胞核。研究还表明，随着 pDNA 浓度的增加，siRNA 电转移的效率会降低。总的来说，这项研究为小核酸电转移机制提供了新的见解，这可能对基于 RNAi 的策略的未来应用产生更广泛的影响。