Unimolecular beacons for the detection of DNA-binding proteins
Unimolecular beacons for the detection of DNA-binding proteins
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DOI：
10.1021/ac034985p
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发表时间：
2004-02-15
影响因子：
7.4
通讯作者：
Heyduk, T
中科院分区：
文献类型：
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作者：
Knoll, E;Heyduk, T
最近开发了一种用于检测序列特异性DNA结合蛋白的新方法（Heyduk，T.; Heyduk，E.国家生物技术公司2002，20，171）。该方法的核心特征是两个荧光染料标记的DNA片段的蛋白质依赖性缔合，其允许产生报告靶蛋白存在的荧光信号。先前的动力学实验确定了两个DNA片段的缔合作为测定的限速步骤。在这里，我们报告的一个变种的测定，其中的成分的测定荧光DNA片段共价拴在一个非DNA接头的目标是增加的两个片段的协会的速度。我们使用模型DNA结合蛋白研究了在各种条件下系链对测定性能的影响。定量滴定和快速动力学停流实验进行验证的分子模型，描述了两个连接的平衡：开放和封闭状态之间的振荡的拴系结构和排他性协会的蛋白质与封闭状态。还进行了实验以证明这些栓系的构建体在附着于固体表面时发出信号的能力。这种新的分析形式的主要优点是检测的响应时间更快，从而允许更高的分析通量。此外，将有可能将系留信标附着到其他固体表面，从而允许制备包含许多不同DNA结合蛋白的分子信标的阵列。
A new methodology for detecting sequence-specific DNA-binding proteins has been recently developed (Heyduk, T.; Heyduk, E. Nat. Biotechnol. 2002, 20, 171). The core feature of this methodology is protein-dependent association of two fluorochrome-labeled DNA fragments, which allows generation of a fluorescence signal reporting the presence of the target protein. Previous kinetic experiments identified the association of the two DNA fragments as the rate-limiting step of the assay. Here we report on a variant of the assay, in which components of the assay-fluorescent DNA fragments-were covalently tethered by a non-DNA linker with the goal of increasing the rate of association of the two fragments. We investigated the effect of the tether on the performance of the assay under a variety of conditions using a model DNA-binding protein. Quantitative titrations and rapid kinetic stopped-flow experiments were conducted to validate the molecular model that describes the two linked equilibria: oscillation of the tethered construct between the open and closed states and the exclusive association of the protein with the closed state. Experiments were also performed to demonstrate the ability of these tethered constructs to signal when attached to a solid surface. The major advantage of this new assay format is the faster response time for the detection allowing the higher throughput of the analysis. Additionally, it will be possible to attach tethered beacons to other solid surfaces, thus allowing the preparation of arrays containing molecular beacons for many different DNA-binding proteins.