ELECTROCHEMICAL DNA-BASED SENSORS

该项目的目标是基于DNA的电化学传感器的设计和应用.修饰有DNA寡核苷酸双链体和非共价结合的嵌入剂作为氧化还原探针的电极将被构建为单碱基错配的传感器。这种方法并不是基于分子识别事件中的差异杂交。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堆叠中的扰动的敏感性。初步结果已经建立了构建用于突变分析的传感器所需的各个元件：9 i）不同的单碱基错配可以被电化学检测，并且膜的序列组成可以显著变化;（ii）单链DNA可以通过在电极上重复杂交和变性的循环来测定;值得注意的是，通过将DNA介导的电荷转移到电催化循环中，实现了对单碱基错配的高水平灵敏度和区分。首先提出，可以系统地检查与这种设计相关的范围和灵敏度，其中寡核苷酸长度、序列和嵌入剂探针是不同的;另外，将进行接头长度和混合表面构建的变化。这些研究将使不仅错配探针的发展，而且新的电化学探针的DNA结合蛋白质和小分子。这些DNA膜将用于开发DNA结合蛋白的电化学测定，例如基于它们对DNA堆积的扰动的碱基翻转酶。DNA修饰电极还将用于探测DNA修复蛋白（如光解酶和Mut Y）和化疗药物（如烯二炔和蒽环类抗生素）的氧化还原化学。最后，将制作DNA芯片，首先分析p53基因中的突变热点，然后分析整个p53基因序列。因此，拟议的计划打算利用DNA介导的电荷传输的一个全新的家庭的DNA为基础的传感器的发展。

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.