Department of Chemistry and Research Institute for Basic Sciences, Kyung Hee University, Seoul, South Korea.
Nucleic Acid Ther. 2013 Apr;23(2):152-9. doi: 10.1089/nat.2012.0396. Epub 2013 Feb 13.
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.
研究小干扰 RNA(siRNAs)在进入细胞后的细胞内命运对于阐明其在细胞质中的动态变化非常重要。在这里,我们描述了一种先进的荧光基础方法,用于探测双标记 siRNA 在 HeLa 细胞中的解离和/或降解情况,并与在人胚肾 293T(HEK293T)细胞中的情况进行比较。这项工作是用三种用荧光共振能量转移(FRET)染料标记的 siRNA 进行的,允许对培养细胞中 siRNA 的解离和降解状态进行非破坏性和非侵入性的评估。我们的 FRET 分析不仅显示了每个 siRNA 链在测量时间段内的不对称降解以及时间依赖性解离,突出了双链 siRNA 的高内在核酸酶抗性,而且还揭示了 siRNA 在 HeLa 细胞中的可持续性比在 HEK293T 细胞中更长,解释了 HeLa 细胞中的基因沉默比 HEK293T 细胞更有效。此外,我们的单分子 FRET 测定还证明了所描述的荧光基础技术在未来研究 siRNA 的生物学行为方面的潜力,甚至可以在单分子水平上进行。基于荧光的方法是一种直接在活细胞内获得 siRNA 完整性的直接信息的技术,可为生物分子的动态变化提供检测工具。