Development and optimization of dual FRET-molecular beacons for the detection and visualization of single-stranded nucleic acid targets

The real-time detection of gene expression in living cells has the potential to significantly impact clinical and laboratory studies of mammalian health and disease, as well as studies in basic cell and developmental biology. Molecular beacons (MBs) provide a simple and promising tool for the detection of target mRNA due to their ability to differentiate between bound and unbound probes, their high signal-to-noise ratio, and their improved specificity over linear probes. However, the harsh intracellular environment does limit the detection sensitivity of MBs in vivo. Specifically, MBs bound to target mRNAs cannot be distinguished from those degraded by nucleases, destabilized due to non-specific interactions, or those that simply open due to thermodynamic fluctuations. To overcome this difficulty, we have developed a new methodology that utilizes a pair of molecular beacons. In addition to the fluorescence quenchers, one beacon has a donor fluorophore and the other an acceptor fluorophore, forming a FRET pair. When both MBs are bound to adjacent sites on an mRNA target, there is a sensitized emission of acceptor fluorescence upon donor excitation due to FRET. Since FRET is extremely sensitive to the distance between donor and acceptor fluorophores, it only occurs when both the donor and acceptor MBs are bound to the same target. Therefore, detecting fluorescence due to FRET can significantly reduce signal contamination from beacon degradation and spontaneous opening.;One concern with using dual FRET-MBs to detect mRNA in living cells is the slower hybridization rate of MBs compared with linear probes. To better understand and optimize the performance of MBs we have systematically tested the hybridization kinetics and thermodynamics of MBs with structural variations. It was found that by adjusting the stem length and probe length the kinetics and specificity of the MB could be tailored for any particular application. Various methods were also employed to improve the signal-to-noise ratio of the dual FRET-MBs methodology including two-photon excitation and time-resolved spectroscopy. Time-resolved measurements were particularly promising, demonstrating the ability to potentially remove all false-positives. This gene detection methodology, therefore, has the potential to become a powerful tool in clinical and laboratory studies.