NSF Award
0315746
This project focuses on the thermodynamics of nucleic acid triple helices, which have been implicated in the control of cellular processes by endogenous or exogenous mechanisms. The long-term goal is to understand the role of nucleotide sequence, strand composition, cations, pH, and hydration on the stability of intramolecular and intermolecular triplexes. A combination of spectroscopic, calorimetric, density and acoustical techniques is used to obtain complete thermodynamic profiles of triplex formation as a function of duplex conformation, pH, salt concentration, and incorporation of mismatches and bulges. Specifically, phase diagrams of the stability of triplexes will be measured as a function of salt concentration and pH; then, pressure-perturbation calorimetric and osmotic-stress techniques will be used to probe their unfolding and hydration changes. The results will be compared with their acoustical and volume measurements, allowing obtaining their hydration properties and the hydration effects in the targeting of nucleic acid single strands or duplexes via duplex and triplex formation, respectively. A comprehensive database will be produced for designing useful triplex reagents for targeting specific nucleic acid sequences and for energy predictions of sequence-specific local conformational rearrangements.<br/><br/>The role of hydration in macromolecular recognition is an important understudied research area in molecular biosciences. Water plays a crucial role in the overall properties of biological macromolecules. A quantitative description of macromolecular hydration is important for correlating sequence with structure, energetics, dynamics and function. The results will improve our current understanding of how hydration controls the stability, conformation and melting behavior of nucleic acid triplexes, providing an insight into global water in solution, useful in molecular modeling and theoretical calculations. <br/><br/>Broader impact: The educational significance of the project is in the mentoring of students; the project will involve training in sophisticated methodology and analysis in biophysical chemistry of nucleic acids and of their interactions with ligands. Furthermore, the research findings will be incorporated into lectures of undergraduate and graduate courses. The database generated in this project will be useful to other investigators for interpreting and analyzing results from many types of structural and biophysical experiments.
