Nitin Nitin, Rhee Won Jong, Bao Gang
Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA 30332, USA.
Nucleic Acids Res. 2009 Aug;37(15):4977-86. doi: 10.1093/nar/gkp517. Epub 2009 Jun 15.
Understanding the interaction between oligonucleotide probes and RNA targets in living cells is important for biological and clinical studies of gene expression in vivo. Here, we demonstrate that starvation of cells and translation inhibition by blocking the mTOR or PI-3 kinase pathway could significantly reduce the fluorescence signal from 2'-deoxy molecular beacons (MBs) targeting K-ras and GAPDH mRNAs in living cells. However, the intensity and localization of fluorescence signal from MBs targeting nontranslated 28S rRNA remained the same in normal and translation-inhibited cells. We also found that, in targeting K-ras and GAPDH mRNAs, the signal level from MBs with 2'-O-methyl backbone did not change when translation was repressed. Taken together, our findings suggest that MBs with DNA backbone hybridize preferentially with mRNAs in their translational state in living cells, whereas those with 2'-O-methyl chemistry tend to hybridize to mRNA targets in both translational and nontranslational states. This work may thus provide a significant insight into probe design for detection of RNA molecules in living cells and RNA biology.
了解寡核苷酸探针与活细胞中RNA靶标的相互作用对于体内基因表达的生物学和临床研究至关重要。在此，我们证明细胞饥饿以及通过阻断mTOR或PI-3激酶途径抑制翻译可显著降低活细胞中靶向K-ras和GAPDH mRNA的2'-脱氧分子信标（MBs）的荧光信号。然而，在正常细胞和翻译抑制细胞中，靶向非翻译28S rRNA的MBs的荧光信号强度和定位保持不变。我们还发现，在靶向K-ras和GAPDH mRNA时，当翻译受到抑制时，具有2'-O-甲基骨架的MBs的信号水平没有变化。综上所述，我们的研究结果表明，具有DNA骨架的MBs在活细胞中优先与处于翻译状态的mRNA杂交，而具有2'-O-甲基化学结构的MBs则倾向于与处于翻译和非翻译状态的mRNA靶标杂交。因此，这项工作可能为活细胞中RNA分子检测的探针设计和RNA生物学提供重要的见解。
