Shin Seonmi, Kwon Hyun-Mi, Yoon Kyung-Sik, Kim Dong-Eun, Hah Sang Soo
Department of Chemistry, Research Institute for Basic Sciences, Research Center for New Nano Bio Fusion Technology, Kyung Hee University, Seoul 130-701, Korea.
Mol Biosyst. 2011 Jul;7(7):2110-3. doi: 10.1039/c1mb05054k. Epub 2011 May 3.
Investigation of the intracellular fate of small interference RNA (siRNA) following their delivery into cells is of great interest to elucidate dynamics of siRNA in cytoplasm. However, its cellular delivery and sustainability should be understood at the molecular level and improved for the successful in vivo application of siRNA. Here we present a fluorescence resonance energy transfer (FRET) based method using oligonucleotide probes to study intracellular dissociation (or melting) and sustainability of siRNAs in live cells. The FRET probes were specifically designed to observe intracellular dissociation (or melting) and degradation of short synthetic RNAs in real-time, thus providing the desired kinetic information in cells. Intracellular FRET analysis shows that siRNA duplex is gradually diffused into cytosol, dissociated, and degraded for a duration of 3.5 h, which is confirmed by confocal microscopy colocalization measurements. In addition, our FRET assays reveal the asymmetric degradation as well as the time-dependent dissociation of each siRNA strand. The application of this FRET technique can allow for direct information on siRNA integrity inside living cells, providing a detection tool for dynamics of biological molecules.
研究小干扰RNA（siRNA）导入细胞后的细胞内命运，对于阐明siRNA在细胞质中的动态变化具有重要意义。然而，为了实现siRNA在体内的成功应用，需要在分子水平上理解其细胞递送和持续性，并加以改进。在此，我们提出一种基于荧光共振能量转移（FRET）的方法，使用寡核苷酸探针来研究活细胞中siRNA的细胞内解离（或解链）及持续性。FRET探针经过专门设计，可实时观察短合成RNA的细胞内解离（或解链）及降解情况，从而在细胞中提供所需的动力学信息。细胞内FRET分析表明，siRNA双链体逐渐扩散到细胞质中，解离并降解，持续时间为3.5小时，这一点通过共聚焦显微镜共定位测量得到了证实。此外，我们的FRET检测揭示了每条siRNA链的不对称降解以及时间依赖性解离。这种FRET技术的应用能够提供活细胞内siRNA完整性的直接信息，为生物分子动态变化提供一种检测工具。
