ELECTROCHEMICAL DNA-BASED SENSORS

本课题的目标是设计和应用基于DNA的电化学传感器。用DNA寡核苷酸双链修饰电极和非共价结合插入物作为氧化还原探针将被构建为单碱基错配的传感器。该方法不是基于分子识别事件的微分杂交。相反，这些基于DNA的传感器依赖于DNA介导的电荷传输及其对DNA堆叠扰动的敏感性。初步结果已经建立了构建突变分析传感器所需的单个元素：9i)不同的单碱基错配可以通过电化学检测到，并且膜的序列组成可以有很大的变化；(2)单链dna可以通过在电极上重复杂交和变性循环来检测；值得注意的是，通过将dna介导的电荷耦合到电催化循环中，可以实现对单碱基错配的高灵敏度和鉴别。首先提出，与该设计相关的范围和灵敏度可以系统地检查，其中寡核苷酸长度，序列和插入探针是不同的；此外，连杆长度的变化和混合表面的构造将被执行。这些研究不仅将促进错配探针的开发，也将促进dna结合蛋白和小分子电化学探针的开发。这些DNA薄膜将用于开发DNA结合蛋白的电化学分析，例如碱基翻转酶，基于它们对DNA堆叠的扰动。DNA修饰电极还将用于探测DNA修复蛋白的氧化还原化学，如光解酶和Mut Y，以及化疗药物，如烯二炔类和蒽环类抗生素。最后，制作DNA芯片，首先分析p53基因的突变热点，然后分析整个p53基因序列。因此，该计划打算利用dna介导的电荷传输来开发一种全新的基于dna的传感器家族。

The proposed program has as its goal the design and application of DNA- based electrochemical sensors. Electrodes modified with DNA oligonucleotide duplexes and a non-covalently bound intercalator as redox probe will be constructed as sensors for single base mismatches. The approach is not based fundamentally on differential hybridization ir a molecular recognition event. Instead these DNA-based sensors rely upon DNA-mediated charge transport and its sensitivity to perturbations in DNA stacking. Preliminary results have established the individual elements necessary to construct a sensor for mutational analysis: 9i) different single base-mismatches can be detected electrochemically and the sequence composition of the film can be varied substantially; (ii0 single-stranded DNAs can be assayed with repeated cycling of hybridization and denaturation on the electrode; significantly, a high level of sensitivity and discrimination for single base mismatches are achieved by coupling of the DNA-mediated charge transported to an electrocatalytic cycle. It is proposed first that the scope and sensitivity associated with this design can be systematically examined, where oligonucleotide length, sequence, and intercalator probe are varied; additionally variations in linker length and the construction of mixed surfaces will be carried out. These studies will enable not only the development of mismatch probes but also of new electrochemical probes for DNA-binding proteins and small molecules. These DNA films will be utilized to develop electrochemical assays for DNA-binding proteins, for example base flipping enzymes, based upon their perturbations to DNA stacking. The DNA-modified electrodes will also be utilized to probe the redox chemistry of DNA repair proteins, such as photolyase and Mut Y, and chemotherapeutics, such as the enediynes and anthracycline antibiotics. Finally, DNA chips will be fabricated for the analysis first of mutational hot spots in the p53 gene and thereafter for the analysis of the entire p53 gene sequence. The proposed program therefore intends to exploit DNA-mediated charge transport for the development of a completely new family of DNA-based sensors.