9723694 Russu The research will address the question of intramolecular recognition in triple helix formation by mapping the energetics and dynamics of DNA triple helical structures at the level of individual bases. The five DNA triple helices proposed for investigation contain canonical as well as noncanonical base triplets. The structural free energies that stabilize each base in its closed, hydrogen-bonded conformation in the DNA triple helices of interest will be obtained by measuring the thermodynamics and kinetics of opening of individual base pairs using proton exchange and NMR spectroscopy. These parameters will define the stability of individual Hoogsteen base pairs within canonical and noncanonical base triplets, the energetics perturbations of Watson-Crick base pairs in duplex DNA resulting from triple helix formation, and the influence of noncanonical base triplets upon the stability of other triplets in the structures. The resolution of the energetic maps of the DNA triple helices of interest will be enhanced by labeling the molecules with 15N at sites involved in triple helix formation. The isotopic labeling will allow novel heteronuclear 1H - 15N methods to be used to probe conformational fluctuations at these sites, and to correlate them with local stability in the triple helical structures of DNA. Triple helices are alternative DNA structures that involve recognition of base sequences in the double helix by a third strand of DNA oligonucleotide. This recognition reaction has an outstanding base sequence specificity, and thus allows triple helical structures to participate in selective control of gene expression and in chemical modification reactions of the genetic material targeted at specific sites. The molecular mechanisms by which this high degree of specificity is achieved represents a fundamental question in understanding the biological functions of these DNA structures. ***
