We propose to continue our thermodynamic characterizations of the molecular forces that control the stability and the conformational preferences of nucleic acid molecules in solution. Our ultimate objective is to establish a comprehensive thermodynamic library that provides the data base needed to evaluate sequence-specific, structure-specific, and solvent-specific conformational preferences of functionally-important domains within naturally-occurring nucleic acids. The thermodynamic data required to achieve these goals will be obtained by applying the techniques of microcalorimetry (both isothermal mixing and temperature scanning) to characterize helix forming and helix disrupting events as well as helix-to-helix conformational transitions in specially designed and synthesized oligomeric and polymeric nucleic acid molecules which possess sequences that will be systematically varied. This approach has allowed us to correlate measured thermodynamic parameters with specific structural and/or conformational features defined by uv and CD spectroscopy as well as by high field NMR. In fact, during the previous budget period, we used this combination of spectroscopic and calorimetric techniques to characterize thermodynamically all ten nearest-neighbor Waston-Crick interactions as well as a variety of DNA secondary structural forms of biological interest (e.g. hairpins, duplexes with dangling ends, duplexes with abasic sites, immobile junctions, """"""""dumbbells,"""""""" etc.). During the next budget period, as described below, we will focus our calorimetric studies on additional nucleic acid nucleic acid structures of biological significate which have yet to be thermodynamically characterized. To be specific, during the requested budget period we propose to determine as a function of base sequence, base modification, and solution conditions the relative stabilities (DeltaGo), the temperature-dependent transitions (DeltaHo, DeltaCp), and the melting cooperativities (DeltaHv.H/DeltaHcal) of the following nucleic acid systems: DNA duplexes with base modified mutagenic lesions (e.g. exocyclic and alkylated adducts); DNA triplexes; DNA duplexes with distortions which when properly phased give rise to """"""""bending""""""""; DNA hairpins; DNA dumbbell-shaped structures; DNA duplexes with dangling ends; and DNA/RNA hybrid duplexes. The thermodynamic data we obtain from these proposed studies will substantially expand our existing library, thereby providing us with a broadened and improved empirical basis for evaluating the relative stabilities and structure, and solution conditions. The thermodynamic data also will assist us in evaluating the degree to which sequence-, structure-, and solvent-induced conformational distortions nad transformations contribute to the overall driving forces of biologically significant processes. Ultimately, we hope to establish a phase diagram for DNA (including DNA/RNA hybrid duplexes) in which we define the relative stabilities and map the temperature- and solvent- induced interconversions of sequence-specific conformational states. Considering the potential roles of base modification and/or conformational heterogeneity in mechanisms for selective, local control of events such as protein-nucleic acid interactions, drug-DNA binding, gene expression, and DNA packaging, an ability to predict sequence-dependent, local conformational preferences and transformations in DNA and in DNA/RNA polymers is of the utmost importance. The calorimetric experiments described in this proposal ar designed to provide the thermodynamic data required to establish this predictive ability so that sequences favoring specific structural forms can be identified and correlated with particular functional roles.
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