首页> 外文期刊>Nucleic Acid Therapeutics >Sniffing for Gene-Silencing Efficiency of siRNAs in HeLa Cells in Comparison with That in HEK293T Cells: Correlation Between Knockdown Efficiency and Sustainability of siRNAs Revealed by FRET-Based Probing
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Sniffing for Gene-Silencing Efficiency of siRNAs in HeLa Cells in Comparison with That in HEK293T Cells: Correlation Between Knockdown Efficiency and Sustainability of siRNAs Revealed by FRET-Based Probing

机译:与HEK293T细胞相比,嗅探HeLa细胞中siRNA的基因沉默效率:击倒效率与基于FRET的探针揭示的siRNA可持续性之间的相关性

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摘要

Investigation of the intracellular fate of small interfering RNAs (siRNAs) following their delivery into cells is of great importance to elucidate their dynamics in cytoplasm. Here we describe the use of an advanced fluorescence-based method to probe the dissociation and/or degradation of double-labeled siRNAs in HeLa cells in comparison with that in human embryonic kidney 293T (HEK293T) cells. This work was performed with three siRNAs labeled with fluorescence resonance energy transfer (FRET) dyes, allowing a non-destructive and non-invasive assessment of the dissociation and degradation state of siRNAs in cultured cells. Our FRET analysis not only shows the asymmetric degradation as well as the time-dependent dissociation of each siRNA strand during the measured time period, underlining the high intrinsic nuclease resistance of duplex siRNAs, but also reveals the longer sustainability of siRNAs in HeLa cells compared with that in HEK293T cells, explaining the gene silencing in HeLa cells is more efficient than that in HEK293T cells. In addition, our single-molecule FRET assays demonstrate the potential of the delineated fluorescence-based technique for future research on biological behavior of siRNAs even at the single-molecule level. The fluorescence-based method is a straightforward technique to gain direct information on siRNA integrity inside living cells, which can provide a detection tool for dynamics of biological molecules.
机译:小干扰RNA(siRNA)进入细胞后,对其细胞内命运的研究对于阐明其在细胞质中的动态非常重要。在这里,我们描述了使用先进的基于荧光的方法来探测与人胚胎肾293T(HEK293T)细胞相比,HeLa细胞中双标记siRNA的解离和/或降解。这项工作是用标记有荧光共振能量转移(FRET)染料的三种siRNA进行的,从而可以对培养细胞中siRNA的解离和降解状态进行非破坏性和非侵入性评估。我们的FRET分析不仅显示了在测量的时间内每个siRNA链的不对称降解以及时间依赖性的解离,突显了双链siRNA的高固有核酸酶抗性,而且还揭示了siRNA与HeLa细胞相比具有更长的可持续性在HEK293T细胞中,基因沉默的解释比在HEK293T细胞中更有效。此外,我们的单分子FRET分析法证明了基于荧光描述技术的潜力,甚至可以用于单分子水平的siRNA生物学行为的未来研究。基于荧光的方法是一种直接的技术,可直接获得有关活细胞内siRNA完整性的信息,可为生物分子的动力学提供检测工具。

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