GOLD QUENCHED MOLECULAR BEACONS

分子信标是一段DNA，一端是荧光标记的，另一端连接着猝灭剂。无结合时，它们采用发夹环状构象：荧光团位于猝灭剂旁边，荧光被猝灭。当结合到互补序列时，信标打开，荧光标记物和猝灭剂分开，出现荧光。灵敏度取决于开放构象和闭合构象中的荧光强度之比。在初步实验中，1.4 nm纳米金团簇显示出与通常使用的荧光团相同的猝灭能力，并且由于它在广泛的波长范围内吸收，它猝灭了各种荧光团。纳米金标记的两种配方，以及更大的共价连接金簇，将作为猝灭剂进行研究。金团簇表面的合成修饰、交联剂和荧光团将被用来结合荧光基团和金团簇之间的弱亲和力，使它们更紧密地结合在一起，并提高闭合构象中的猝灭效率。开放构象和闭合构象的荧光强度之比，以及由此产生的灵敏度，将在目前可用的基础上增加。优化的金团簇淬火信标将与光缆交叉连接，以制备高灵敏度和特异性的生物传感器。拟议的商业应用：拟议的技术能够实时检测细胞和其他高度局部化系统中的特定DNA序列。使用新探针的生物传感器可用于移植组织和器官的快速基因筛查，以及其他各种应用，如快速癌症筛查和胎儿测试，这些应用将受益于其高特异性、便携性和低侵袭性。

Molecular beacons are lengths of DNA, fluorescently labeled at one end and linked to a quencher at the other. Unbound, they adopt a hairpin loop conformation: the fluorophore is positioned next to the quencher and fluorescence is quenched. Upon binding to a complementary sequence, the beacon opens, the fluorescent label and quencher move apart and fluorescence appears. Sensitivity is dependent upon the ratio of fluorescence intensities in the open and closed conformations. In preliminary experiments, the 1.4 nm Nanogold gold cluster has demonstrated equal quenching ability to the fluorophores usually used, and since it absorbs at a wide range of wavelengths, it quenches a variety of fluorophores. Two formulations of the Nanogold label, and larger covalently linked gold clusters, will be investigated as quenchers. Synthetic modification of the gold cluster surface, cross-linking protocols and fluorophores will be used to incorporate weak affinities between the fluorophore and gold cluster to bring them closer together and increase quenching efficiency in the closed conformation. The ratio of fluorescence intensities for the open and closed conformations, and hence sensitivity, will be increased beyond that currently available. Optimized gold cluster-quenched beacons will be cross-linked to fiber optic cables to prepare highly sensitive and specific biosensors. PROPOSED COMMERCIAL APPLICATIONS: The proposed technology enables the real-time detection of specific DNA sequences in cells and other highly localized systems. Biosensors using the new probes may be used for rapid genetic screening of tissues and organs for transplantation, and for a variety of other applications such as rapid cancer screening and fetal testing which would benefit from their high specificity, portability and reduced invasiveness.