Monitoring Cooperative Binding Using Electrochemical DNA-Based Sensors
Monitoring Cooperative Binding Using Electrochemical DNA-Based Sensors
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DOI：
10.1021/la504083c
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发表时间：
2015-01-20
期刊：
影响因子：
3.9
通讯作者：
White, Ryan J.
中科院分区：
文献类型：
--
作者：
Macazo, Florika C.;Karpel, Richard L.;White, Ryan J.
基于电化学DNA（E-DNA）的传感器用于检测各种目标，包括互补DNA、小分子和蛋白质。这些传感器通常采用表面结合的单链寡核苷酸，其在远端3'末端上用氧化还原活性分子修饰。靶诱导的DNA探针的柔性变化改变了氧化还原活性亚甲基蓝和电极表面之间的电子转移的效率，允许定量检测靶浓度。虽然许多研究已经利用E-DNA传感器的特异性和灵敏度来量化目标浓度，但迄今为止还没有研究证明这类基于碰撞的传感器能够阐明生物化学结合机制，如协同性。在这项研究中，我们证明了E-DNA传感器制造的各种长度的表面结合的寡脱氧胸苷酸[（dT）n]传感探针能够定量区分合作和非合作的单链DNA结合蛋白的结合。具体而言，我们证明，寡（dT）E-DNA传感器能够定量检测nM水平（50 nM-4 μ M）的基因32蛋白（g32 p）。此外，传感器表现出的信号，能够区分全长g32 p的合作结合和非合作结合的核心结构域（*III）片段的单链DNA。最后，我们证明了这种结合是探针长度和离子强度依赖。这项研究说明了这种功能强大的生物传感器的一个新的定量特性，并代表了一个快速，简单的方法来了解蛋白质-DNA结合机制。
Electrochemical DNA-based (E-DNA) sensors are utilized to detect a variety of targets including complementary DNA, small molecules, and proteins. These sensors typically employ surface-bound single-stranded oligonucleotides that are modified with a redox-active molecule on the distal 3' terminus. Target-induced flexibility changes of the DNA probe alter the efficiency of electron transfer between the redox active methylene blue and the electrode surface, allowing for quantitative detection of target concentration. While numerous studies have utilized the specific and sensitive abilities of E-DNA sensors to quantify target concentration, no studies to date have demonstrated the ability of this class of collision-based sensors to elucidate biochemical-binding mechanisms such as cooperativity. In this study, we demonstrate that E-DNA sensors fabricated with various lengths of surface-bound oligodeoxythymidylate [(dT)n] sensing probes are able to quantitatively distinguish between cooperative and noncooperative binding of a single-stranded DNA-binding protein. Specifically, we demonstrate that oligo(dT) E-DNA sensors are able to quantitatively detect nM levels (50 nM-4 mu M) of gene 32 protein (g32p). Furthermore, the sensors exhibit signal that is able to distinguish between the cooperative binding of the full-length g32p and the noncooperative binding of the core domain (*III) fragment to single-stranded DNA. Finally, we demonstrate that this binding is both probe-length- and ionic-strength-dependent. This study illustrates a new quantitative property of this powerful class of biosensor and represents a rapid and simple methodology for understanding protein-DNA binding mechanisms.