In this study, we have analyzed the effect of translation inhibition on hybridization of 2′- O -methyl and 2′-deoxy MBs with target RNA molecules in living cells. Answering these fundamental questions will have a significant impact on both disease detection and fundamental RNA biology studies using MBs. To develop optimal assays for quantitative studies using MBs, we also need to gain an understanding of probe–target hybridization kinetics and thermodynamics in living cells. This information will help develop a relationship between the target RNA concentration and the fluorescent signal from beacon hybridization. In particular, we need to determine the fraction of RNA molecules in a given state that can hybridize with a particular probe design and chemistry. However, to quantifying gene expression in living cells with high sensitivity, we need to have a better understanding of probe–target interactions during various functional stages of RNA. Recently, we were able to detect specific endogenous mRNAs and nuclear RNAs in living cells and image their localization ( 11, 15, 16 ). Hybridization with the target nucleic acid opens the hairpin and physically separates the fluorophore from quencher, allowing a fluorescence signal to be emitted upon excitation. MBs are dual-labeled oligonucleotide hairpin probes with a fluorophore at one end and a quencher at the other end. Molecular beacon (MB)-based methods have been used to image endogenous RNAs in living cells ( 9–14 ). However, these methods are not applicable when detecting gene transcripts within living cells. In situ hybridization methods ( 6–8 ) have been used to address fundamental biological issues such as RNA localization and active transcription sites. Although in vitro approaches provide a powerful tool for studying gene expression ( 5 ), they cannot be used to study the dynamics and localization of gene expression in vivo. Current methods for quantifying gene expression employ either selective amplification (as in polymerase chain reaction (PCR) and serial analysis of gene expression (SAGE)) ( 1–4 ) or saturation binding followed by removal of the excess probes (as in microarrays) to achieve specificity. The ability to detect, localize, quantify and monitor the expression of specific genes in living cells in real time will offer unprecedented opportunities for advancement in molecular biology, disease pathophysiology, drug discovery and medical diagnostics. This work may thus provide a significant insight into probe design for detection of RNA molecules in living cells and RNA biology. 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. 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. However, the intensity and localization of fluorescence signal from MBs targeting nontranslated 28S rRNA remained the same in normal and translation-inhibited cells. 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. Understanding the interaction between oligonucleotide probes and RNA targets in living cells is important for biological and clinical studies of gene expression in vivo.
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