DNA POLYMORPHISM IN SOLUTION--A THERMODYNAMIC STUDY

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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. With the impressive volume of sequence data currently being generated by the Human Genome Project, such a capacity is becoming increasingly important. Ultimately, one would like to assess if local sequence domains in the genome favor specific structural motifs which correspond to functional sites of biological action or control. The same thermodynamic data also required for the rational design of solution conditions and third strand oligonucleotide sequences for use in antisense/antigene therapeutic strategies and nucleic-acid based diagnostic protocols. The thermodynamic data needed for these applications will be obtained by using microcalorimetry (both isothermal mixing and temperature scanning) to characterize helix forming events, helix disrupting events, and helix- to-helix transformations in specially designed oligomeric nucleic acid molecules with 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 by high field NMR. In fact, during the previous budget periods, we have used this combination of spectroscopic and calorimetric techniques to characterize thermodynamically a wide range of DNA secondary structural forms of biological interest. During the next budget period, we propose to build and to expand this foundation by focusing our calorimetric studies on recently discovered or rediscovered nucleic acid structures of biological and biomedical significance which have yet to be or are insufficiently 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 (deltaHvH/deltaHcal) of the following nucleic acid systems: DNA duplexes with mutagenic lesions; DNA triplexes; DNA and RNA tetraplexes; DNA/RNA hybrid duplexes; and bent DNA. The resulting data will substantially expand the existing thermodynamic library. Ultimately, we intend to establish nucleic acid phase diagrams which 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 hybrids is of the utmost importance. The calorimetric experiments described in this proposal are 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. In short, the thermodynamic data we propose to obtain will be important for interpreting Human Genome sequence data in terms of structure-function relationships, as well as for developing a rational approach to the design of effective third strand oligomers for use in therapeutic and diagnostic protocols.