Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA 30332, USA.
Nucleic Acids Res. 2010 May;38(9):e109. doi: 10.1093/nar/gkq050. Epub 2010 Feb 10.
Molecular beacons (MBs) have the potential to provide a powerful tool for rapid RNA detection in living cells, as well as monitoring the dynamics of RNA expression in response to external stimuli. To exploit this potential, it is necessary to distinguish true signal from background signal due to non-specific interactions. Here, we show that, when cyanine-dye labeled 2'-deoxy and 2'-O-methyl oligonucleotide probes are inside living cells for >5 h, most of their signals co-localize with mitochondrial staining. These probes include random-sequence MB, dye-labeled single-strand linear oligonucleotide and dye-labeled double-stranded oligonucleotide. Using carbonyl cyanide m-chlorophenyl hydrazone treatment, we found that the non-specific accumulation of oligonucleotide probes at mitochondria was driven by mitochondrial membrane potential. We further demonstrated that the dye-labeled oligonucleotide probes were likely on/near the surface of mitochondria but not inside mitochondrial inner membrane. Interestingly, oligonucleotides probes labeled respectively with Alexa Fluor 488 and Alexa Fluor 546 did not accumulate at mitochondria, suggesting that the non-specific interaction between dye-labeled ODN probes and mitochondria is dye-specific. These results may help design and optimize fluorescence imaging probes for long-time RNA detection and monitoring in living cells.
分子信标 (MBs) 有可能成为一种强大的工具，用于在活细胞中快速检测 RNA，以及监测 RNA 表达对外部刺激的动态反应。为了利用这一潜力，有必要区分由于非特异性相互作用而产生的真实信号和背景信号。在这里，我们表明，当氰基染料标记的 2'-脱氧和 2'-O-甲基寡核苷酸探针在活细胞内存在超过 5 小时时，它们的大部分信号与线粒体染色共定位。这些探针包括随机序列 MB、染料标记的单链线性寡核苷酸和染料标记的双链寡核苷酸。使用羰基氰化物 m-氯苯腙处理，我们发现寡核苷酸探针在线粒体中的非特异性积累是由线粒体膜电位驱动的。我们进一步证明，染料标记的寡核苷酸探针可能位于线粒体的表面/附近，但不在线粒体内膜内。有趣的是，分别用 Alexa Fluor 488 和 Alexa Fluor 546 标记的寡核苷酸探针不会在线粒体中积累，这表明染料标记的 ODN 探针与线粒体之间的非特异性相互作用是染料特异性的。这些结果可能有助于设计和优化用于在活细胞中长时间检测和监测 RNA 的荧光成像探针。
