Single Molecule Studies of DNA-Cyanine Dye Interactions

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DESCRIPTION (provided by applicant): The cyanine dyes are a class of bicyclic aromatic compounds that are highly fluorescent when associated with DNA. The resulting sensitivity of DNA detection has led to applications ranging from medical diagnosis to molecular biology. The cyanine dyes will also serve as useful models to understand critical features of ligand-DNA interactions because they behave as nonclassical intercalators. This research will use single molecule analysis, as well as macroscopic experimental techniques, to investigate important features of the DNA-cyanine dye interaction from thermodynamic, structural, and kinetic standpoints. I. Equilibrium constant and viscosity measurements will be used to characterize the factors that influence the structure and stability of the DNA-dye complex. Dr. Petty will investigate the role of the structure of the dye, the sequence of the DNA, and the solution environment on the dye-DNA association. II. Dimeric cyanine dyes exhibit strong cooperative interactions when bound to DNA fragments. Atomic force microscopy (AFM) will be used to investigate the structures of these cooperative dye-DNA complexes. The unique capabilities of AFM will allow studies such as the sequence dependence of the cooperativity. III. Because of their favorable fluorescence properties, individual cyanine dye molecules will be studied using single molecule fluorescence microscopy. By eliminating the averaging inherent in bulk studies, new perspectives on the dynamics of DNA-ligand interactions are possible. For example, by measuring the rates of association and dissociation of individual molecules, the role of salt in the mechanism of DNA-ligand interaction will be elucidated. Because of the significance of small molecules and enzymes in DNA regulation, the long-term goal of this project is to use single molecule analysis to provide unique perspectives on these DNA-ligand interactions.