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.