Electrochemical DNA-Based Sensors

描述(由申请人提供)：本次续展申请的总体目标是发展DNA介导的电化学作为一种检测DNA结合蛋白质和核酸的新技术。DNA介导的电化学在检测DNA碱基对的扰动方面非常敏感，因此可以用作碱基错配或蛋白质结合引起的碱基堆积变化的传感器。利用这种化学，已经开发出用于突变检测和与DNA结合的碱基翻转蛋白的电子传感器。在拟议的计划中，这种化学的范围将扩大，以开发新的工具来监测与DNA结合的蛋白质。具体地说，DNA修饰的金和石墨电极将被用来探测蛋白质在双链上的相互作用和反应。将测定氧化还原活性转录激活剂与DNA结合的氧化还原电位。对于缺乏氧化还原辅因子的蛋白质，将使用DNA双链上的共价氧化还原探针作为报告，并检测具有不同DNA结合基序的蛋白质。我们还可以监测光解酶和PvuII对DNA的反应，以探测反应过程中发生的DNA构象变化。因此，DNA电化学为探索蛋白质如何与DNA结合和反应提供了一种新的工具。DNA介导的电化学也提供了高灵敏度和多重能力。通过系统地将DNA修饰的电极尺寸减小到100 nm，并利用电催化进行信号放大，我们将构建与DNA结合并激活转录的蛋白质灵敏的电子传感器。微电极提供了更快的动力学，以及在监测蛋白质结合方面的灵敏度。DNA修饰电极的多路阵列将首先在宏观尺度上制造出来进行优化。最初的设计将用于测试结合不同靶标序列的甲基酶。然后，将基于与甲基酶的竞争阵列构建用于测试DNA结合蛋白的多重分析。还将开发蛋白质结合的序列特异性抑制物的分析方法。使用类似的方法，我们将用电化学方法检查RNA检测的范围和限度，如何使DNA介导的检测在低浓度的mRNAs检测中变得稳健和定量。我们还将结合这些检测方法来开发阵列，在这些阵列中可以同时检测到mRNAs和激活其合成的转录因子。这些传感器提供了一种全新技术的潜力，以无标记的形式监测DNA结合蛋白和mRNAs。这些工具可以提供一种灵敏的新诊断方法，同时检测癌症和其他疾病状态的蛋白质和RNA标志物。公共卫生这个项目涉及为RNA和与DNA结合的蛋白质建造新的电子传感器。这些传感器将作为全新的诊断工具，检测与不同疾病状态相关的细胞和组织中的蛋白质和RNA。这些传感器可能代表着癌症和其他疾病早期诊断的一项新技术。

DESCRIPTION (provided by applicant): The overall objective of this renewal application is the development of DNA-mediated electrochemistry as a new technology for sensing DNA-binding proteins and nucleic acids. DNA-mediated electrochemistry is remarkably sensitive in detecting perturbations in the DNA base pairs and hence can be used as a sensor for changes in base stacking that arise with base mismatches or protein binding. Electrical sensors for mutation detection and for base flipping proteins that bind to DNA have been developed using this chemistry. In the program proposed, the scope of this chemistry will be expanded to develop new tools to monitor proteins that bind to DNA. Specifically, DNA-modified gold and graphite electrodes will be used to probe interactions and reactions of proteins on the duplex. DNA-bound redox potentials of redox-active transcriptional activators will be determined. For proteins that lack redox cofactors, covalent redox probes on the DNA duplex will be used as the reporter, and proteins with different DNA binding motifs will be examined. We can also monitor reactions of photolyase and PvuII on DNA to probe the DNA conformational changes that occur during reaction. Thus DNA electrochemistry offers a new tool to probe how proteins bind and react on DNA. DNA-mediated electrochemistry also offers high sensitivity and the ability to multiplex. By systematically reducing the DNA-modified electrode size down to 100 nm, and utilizing electrocatalysis for signal amplification, we will construct sensitive electrical sensors for proteins that bind to DNA and activate transcription. Microelectrodes offer faster kinetics, as well as sensitivity in monitoring protein binding. Multiplexed arrays of DNA-modified electrodes will be fabricated first on the macroscale for optimization. Initial designs will be used to test for methylases that bind different target sequences. A multiplexed assay to test for DNA-binding proteins will then be constructed based upon a competition array with the methylases. Assays for sequence-specific inhibitors of protein binding will also be developed. Using similar approaches, we will examine the scope and limits of RNA detection electrochemically, how DNA-mediated detection can be made robust and quantitative in detecting mRNAs at low concentrations. We will also combine these assays to develop arrays where mRNAs and the transcription factors that activate their synthesis can be detected simultaneously. These sensors offer the potential for a completely new technology to monitor DNA-binding proteins and mRNAs in a label-free format. These tools could provide a sensitive new diagnostic to detect both protein and RNA markers for cancer and other disease states. PUBLIC HEALTHE RELEVANCE This project involves the construction of new electrical sensors for RNA and proteins that bind to DNA. These sensors will serve as completely new diagnostic tools to detect the proteins and RNAs from cells and tissues that are associated with different disease states. These sensors could represent a new technology for the early diagnosis of cancer and other diseases.