Nucleic acid-based fluorescence sensors for detecting proteins
Nucleic acid-based fluorescence sensors for detecting proteins
复制标题
DOI：
10.1021/ac0487449
复制
发表时间：
2005-02-15
影响因子：
7.4
通讯作者：
Heyduk, T
中科院分区：
文献类型：
--
作者：
Heyduk, E;Heyduk, T
我们在这里报告的发展，快速，均质，基于适体的荧光检测（“分子信标”）检测蛋白质。该测定涉及识别蛋白质的两个不同表位的两个适体的蛋白质诱导的共缔合。适体含有通过非DNA接头连接到适体的短荧光团标记的互补“信号传导”寡核苷酸。两个适体与蛋白质的共缔合导致两个“信号”寡核苷酸接近，产生荧光团之间荧光共振能量转移的大变化。我们使用凝血酶作为模型系统，以提供验证这种分子信标设计的原理证据。凝血酶信标能够以高选择性（也在复杂的生物混合物中）、皮摩尔灵敏度和高信号背景比检测蛋白质。这是一种不需要样品操作的均相测定。由于这里描述的分子信标的设计不限于任何特定的蛋白质，因此将有可能开发这些信标来检测具有生物医学重要性的各种靶蛋白质。
We report here development of a rapid, homogeneous, aptamer-based fluorescence assay ("molecular beacons") for detecting proteins. The assay involves protein-induced coassociation of two aptamers recognizing two distinct epitopes of the protein. The aptamers contain short fluorophore-labeled complementary "signaling" oligonucleotides attached to the aptamer by non-DNA linker. Coassociation of the two aptarners with the protein results in bringing the two "signaling" oligonucleotides into proximity, producing a large change of fluorescence resonance energy transfer between the fluorophores. We used thrombin as a model system to provide proof-ofprinciple evidence validating this molecular beacon design. Thrombin beacon was capable of detecting the protein with high selectivity (also in complex biological mixtures), picomolar sensitivity, and high signal-to-background ratio. This is a homogeneous assay requiring no sample manipulation. Since the design of molecular beacons described here is not limited to any specific protein, it will be possible to develop these beacons to detect a variety of target proteins of biomedical importance.