The research focuses on the thermodynamics of nucleic acid intramolecular structures - especially, triplet helices because of their implication in the control of cellular processes by endogenous or exogenous mechanisms. The long term objectives are to understand the molecular forces controlling the overall stability of complex intramolecular DNA structures; to quantify the energetics and hydration contributions governing the association of triplexes, and other unusual structures, with their complementary strands, including the role of sequence and cations; and to determine the thermodynamics for their favorable interaction with polycations for cellular delivery purposes. UV and CD spectroscopies are used to verify that each nucleic acid complex contains the appropriate structural features. Further, a combination of T-dependent UV spectroscopy, differential scanning and pressure perturbation calorimetries will be employed to obtain complete thermodynamic profiles for their unfolding reactions as a function of salt, pH and osmolyte concentration. High sensitivity titration calorimetry and density techniques will be used to measure the heat and volume change of association reactions. The complete thermodynamic characterization of these DNA complexes will provide a fundamental understanding of the physical factors that determine their stability as a function of its sequence and solution conditions. These factors are basic to the rational design of gene-targeting reagents, and for their proper cellular delivery, which can be used in therapeutic, diagnostic and biotechnological applications, and for predicting the energetics of sequence-specific local conformational rearrangements in intracellular processes.
The broader impacts of this research are in carcinogenesis and gene therapy because of the fundamental importance of developing highly specific and stable agents for targeting oncogenes or their transcribed RNA products via triplex or duplex formation. Another impact is the role of water in the overall properties of biological macromolecules and in their interacting behavior towards one another. Specifically, the correlation of energetics with hydration will improve the picture of how hydration controls the stability, conformation and melting behavior of these novel nucleic acid structures. Furthermore, the resulting hydration data can be used in molecular modeling studies and in theoretical calculations, providing an insight into global water. The educational significance is in the mentoring of undergraduate, graduate students and postdoctoral fellows by training them with a wide variety of biophysical techniques and with the fundamental understanding of biophysics. In addition, the research findings are routinely incorporated into lectures of undergraduate, graduate courses and seminars.
This proposal focuses on the biophysical chemistry of nucleic acid intramolecular structures. A combination of spectroscopic and calorimetric techniques was used to determine the energetics for the unfolding of intramolecular DNA structures as a function of the base sequence and solution conditions, and for their interaction with complementary strands and polycations. Specifically, we have studied the unfolding thermodynamics of DNA intramolecular triplexes, the targeting of a variety of non-canonical DNA structures (three-way junctions, pseudoknots and hairpins containing internal loops in their stem) with both their respective complementary strands and polycations. Our general contribution is the on-going demonstrations that nucleic acid intramolecular complexes can be used in the control of cellular processes by endogenous or exogenous mechanisms. For instance, DNA oligonucleotides can be used to block gene expression by targeting mRNA and DNA gene sequences forming duplex and/or triplex structures. Furthermore, unusual DNA intramolecular structures can be used to mimic the structures formed by mRNA; therefore, they can be targeted with complementary strands to mimic the targeting of mRNA. In addition, it is important to investigate the melting behavior/unfolding of these molecules in a variety of solution conditions. The results of this work will allow investigators to make appropriate predictions on how these novel structures respond to changes in nucleotide sequence, strand composition, overall environmental conditions, and in their cellular delivery. The broader impact is in the training of young scientists, especially graduate and undergraduate students. All of these scientists have applied a variety of techniques to improve our understanding of the biophysical chemistry of nucleic acids and interactions. The overall results have been published in 27 articles and 43 abstracts (posters and podiums) presented at local, regional and national meetings. Furthermore, the results have been incorporated into lectures of didactic courses, invited seminars, and conference talks. Outreach activities include attending the annual SACNAS meetings for the recruitment of undergraduates from diverse groups into biophysics and to organize symposia on the "Biophysics of Proteins and Nucleic Acids".
