Molecular Beacons There is increasing evidence to suggest that RNA molecules have a wide range of functions, and the real-time visualization of RNA expression and localization in living cells may offer unprecedented opportunities for advancement in molecular biology, disease pathophysiology, drug discovery, and medical diagnostics. The Bao lab has been developing nanoprobes for sensitive RNA detection in living cells, including dual FRET molecular beacons, peptide-linked molecular beacons, and other activatable probes. A molecular beacon is an oligonucleotide probe labeled with a reporter fluorophore at one end and a fluorescence quencher at the other end; it is designed to form a hairpin structure in the absence of target so that fluorescence of the fluorophore is quenched (Fig. 1a). Hybridization with a target mRNA opens the hairpin and separates the fluorophore from quencher, allowing a fluorescence signal to be emitted upon excitation (Fig. 1a). To reduce false-positive signals in detecting RNA in living cells, we developed a novel dual molecular beacons approach in which two molecular beacons, one with a donor and a second with an acceptor fluorophore, hybridize to adjacent regions on the same target, resulting in fluorescence resonance energy transfer (FRET) (Fig. 1b). The detection of a FRET signal enables the discrimination between true and false-positive signals. We have conducted live-cell mRNA imaging using molecular beacons for oncogenes such as K-ras and survivin. We showed that the dual-FRET molecular beacons approach is more quantitative in detecting mRNAs compared with single, unpaired molecular beacons. We demonstrated the ability of molecular beacons in detecting changes in mRNA level. We discovered that both K-ras and GAPDH mRNAs display an intriguing filament-like localization pattern in HDF cells (Fig. 1c), and that these mRNAs co-localize with mitochondria (Fig. 1d). These results may have significant implications to basic RNA biology as well as disease studies. Currently we are addressing additional probe design issues and biological questions, including the ability of molecular beacons in detecting single-base mutations, enhancing the sensitivity of detecting low-abundance genes, RNA transport and localization, and real-time visualization of gene expression dynamics.