GOLD QUENCHED MOLECULAR BEACONS

分子信标是一段DNA，一端被荧光标记，另一端与猝灭剂连接。未结合时，它们采用发夹环构象：荧光团位于猝灭剂旁边，荧光被猝灭。在与互补序列结合时，信标打开，荧光标记和猝灭剂分开，并出现荧光。灵敏度取决于开放和闭合构象中的荧光强度的比率。在初步实验中，1.4 nm的纳米金团簇表现出与通常使用的荧光团相同的猝灭能力，并且由于它在宽范围的波长下吸收，它猝灭了各种荧光团。纳米金标记的两种制剂和较大的共价连接的金簇将作为猝灭剂进行研究。金簇表面的合成改性、交联方案和荧光团将用于将荧光团和金簇之间的弱亲和力并入，以使它们更靠近在一起并增加闭合构象中的淬灭效率。开放和封闭构象的荧光强度的比率，因此灵敏度，将增加超过目前可用的。优化的金簇猝灭信标将与光纤电缆交联，以制备高度灵敏和特异的生物传感器。拟议的商业应用：所提出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遗传筛查，以及用于各种其他应用，例如快速癌症筛查和胎儿测试，这将受益于其高特异性，便携性和降低的侵入性。

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.