Gold Quenched Molecular Beacons

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Molecular beacons are hairpin loops of DNA, conjugated to a fluorescent label at one end and a quencher at the other. Upon binding to a complementary target, the beacon opens, the fluorophore and quencher move apart, and fluorescence appears. Sensitivity is dependent upon the "signal-to-noise ratio" of fluorescence intensities in the open and closed conformations. Conventional dabcyl-quenched beacons give intensity ratios up to 100. However, the 1.4 nM Nanogold cluster yields intensity ratios up to several thousand, and since it absorbs over a wide range of wavelengths, can quench a variety of fluorophores. Improved gold nanoparticle quenchers will be prepared, using larger gold particles to achieve more effective quenching through surface plasmon coupling. Fluorescence quenching properties of these labels will be made reproducible through the synthesis of modified ligands which increase the solubility, stability and biological compatibility of he beacon, while introducing specific affinities for fluorophores to hold the gold particle and quencher closer together and increase quenching in the closed configuration. Purification methods will be developed and gold quenched DNA and PNA beacons will be compared with conventional analogs for rapid bacterial characterization. Prototype reusable biosensors will be fabricated by immobilizing gold-quenched beacons on fiber optic cables. PROPOSED COMMERCIAL APPLICATIONS: Gold quenchers are expected to improve the signal-to-noise ratio of molecular beacons by more than a order of magnitude, enabling more sensitive and rapid real-time detection of specific DNA sequences in homogeneous specimens. Combined with rapid DNA amplification methods, gold quenched beacons would be used for viral and bacterial typing, diagnosis of infectious disease, and detection of cancer. As components of fiber optic biosensors, they would be used to study events in individual cells, assembled into bundles, these would be used for the genetic screening of tissues and organs for transplantation, and form the basis for more portable, less invasive prenatal testing, screening and genetic profiling technology.