OPTICAL PROBES OF DNA BENDING AND WRAPPING

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DESCRIPTION: This is a proposal designed to understand the factors that determine the interaction of DNA with surfaces, by examining the adsorption of small oligonucleotides to protein-sized (< 200Angstrom in diameter) inorganic substrates. The project will explore the binding of small oligonucleotides with specific sequences and conformations (intrinsically bent, for example) to colloidal cadmium sulfide (CdS), a material that can be made in a variety of sizes (10 - 200 Angstrom diameter) and with a variety of surface groups (cationic, anionic, hydrophobic, hydrophilic, etc.). These colloidal particles emit luminescence when irradiated, and this luminescence is sensitive to the nature and amount of adsorbate bound to the particle surface. Thus, binding of the DNA to CdS will be obtained from the quenching of the luminescence, as a function of added oligonucleotide concentration. Because of the small size (high curvature) of the colloidal particles, the method proposed here is conveniently suited to investigate the effect of pre-existing curvature on the binding affinity of the DNA molecules to the surface. Moreover, surface derivatization with simple cations, small organic molecules, and peptides can influence the binding of the DNA and provide much needed insight into the factors that determine the interaction of the highly charged surface of nucleic acids with different surfaces. The following specific aims are proposed: 1) To characterize the effect of DNA sequence, conformation and length on its binding to the protein-sized particles. In particular, DNA molecules with and without pre-existing bends will be utilized and compared. Changes in luminescence as a function of DNA concentration will be used to extract binding constants. Longer in-phased curved DNAs will be used to determine the influence of curvature on the surface binding. 2) The effect of particle size and functionality on the binding of a particular DNA size will be investigated. The range of the particles will vary in the range of ~ 10 - 200Angstrom. 3) Accessibility of the bound oligo nucleotides to OH-radical-and DNase footprinting will be used to obtain information about the structure of the DNA on the curved surfaces. 4) The effect of DNA structures, either damaged or carrying sequence mismatches, will be assessed to investigate to what degree these factors can play a role in the interaction of the DNA with the colloidal particles.