This proposal focusses on the structure and stability of DNA, RNA, and mixed DNA/RNA triple helices. These novel DNA structures are of great current interest due to the large number of possible applications of targeting duplex DNA with a third strand as well as the possible in vivo role of such structure in, for example, gene regulation. The long-term goals of this work include understanding the role of nucleotide sequence and composition, cation and ionic strength in determining the stability of both pyrimidine-purine- pyrimidine and purine-purine-pyrimidine triplexes and establishing a high resolution model for the molecular structure of intermolecular triplexes. In particular, we propose to determine the sequence and composition requirements for formation of purine- purine-pyrimidine triplexes using capillary and slab gel electrophoresis, along with circular dichroism and imino proton NMR. Evaluation of the thermodynamic stability of triplexes will be carried out both by UV melting analysis and by calorimetry, thereby permitting assessment of the validity of the all-or-none model for triplex formation. Additional stability studies will consider the effects of RNA strands, mismatches and base modification on triplex stability. Finally, high resolution 2D and 3D NMR methods will be applied to the problem of determining the structure of triplexes at a molecular level. %%% These studies will provide fundamental data on the stability and structure of nucleic acid triplexes, which will be of importance in a large number of potential applications of this unusual nucleic acid structure.
