Santangelo Philip J, Nix Brent, Tsourkas Andrew, Bao Gang
Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA 30332, USA.
Nucleic Acids Res. 2004 Apr 14;32(6):e57. doi: 10.1093/nar/gnh062.
The ability to visualize in real-time the expression level and localization of specific endogenous RNAs in living cells can offer tremendous opportunities for biological and disease studies. Here we demonstrate such a capability using a pair of molecular beacons, one with a donor and the other with an acceptor fluorophore that hybridize to adjacent regions on the same mRNA target, resulting in fluorescence resonance energy transfer (FRET). Detection of the FRET signal significantly reduced false positives, leading to sensitive imaging of K-ras and survivin mRNAs in live HDF and MIAPaCa-2 cells. FRET detection gave a ratio of 2.25 of K-ras mRNA expression in stimulated and unstimulated HDF, comparable to the ratio of 1.95 using RT-PCR, and in contrast to the single-beacon result of 1.2. We further revealed intriguing details of K-ras and survivin mRNA localization in living cells. The dual FRET molecular beacons approach provides a novel technique for sensitive RNA detection and quantification in living cells.
实时可视化活细胞中特定内源性RNA的表达水平和定位的能力，可为生物学和疾病研究提供巨大机遇。在此，我们使用一对分子信标展示了这种能力，其中一个带有供体荧光团，另一个带有受体荧光团，它们与同一mRNA靶标的相邻区域杂交，从而产生荧光共振能量转移（FRET）。FRET信号的检测显著减少了假阳性，从而实现了对活的人皮肤成纤维细胞（HDF）和胰腺癌细胞系MIAPaCa-2中K-ras和生存素mRNA的灵敏成像。FRET检测得出，在受刺激和未受刺激的HDF中，K-ras mRNA表达的比率为2.25，与使用逆转录聚合酶链反应（RT-PCR）得出的1.95的比率相当，而单信标检测结果为1.2。我们进一步揭示了活细胞中K-ras和生存素mRNA定位的有趣细节。双FRET分子信标方法为活细胞中RNA的灵敏检测和定量提供了一种新技术。
