Sequence Specific Targeting of Nucleic Acids Using Intramolecular Complexes: Energetics, Kinetics and Hydration

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Intellectual MeritThis research focuses on the thermodynamics of nucleic acid intramolecular structures, especially, DNA structures that model the secondary structures of RNA molecules. The broad and long term objectives of this project are to understand the molecular forces controlling the overall stability of complex intramolecular DNA structures; to quantify the energetics, kinetics, and hydration contributions governing the association of these unusual intramolecular structures with their complementary strands, including the role of cations; and to determine the thermodynamics for their favorable interaction with polycations for cellular delivery purposes. The hypothesis is: The presence of unpaired base nucleotides in the loops of nucleic acid secondary structures provides favorable free energy contributions in their reaction with complementary strands and the slightly more hydrophobic surface of these constrained loops contribute favorably towards the interaction with delivery vectors, such as polycations. To test this hypothesis the following aims are proposed: Aim 1: To characterize the melting behavior of stem-loop motifs containing bulges or internal loops, pseudoknots and three-way junctions as a function of sequence, stability of their end loops, and solution conditions. Aim 2: To elucidate and quantify the molecular forces governing the reaction of intramolecular secondary DNA structures with their partially complementary strands. Aim 3: To determine the kinetics of the bimolecular association reactions of aim 2, includes reaction rates and associated activation energies and activation entropies, and to correlate them with their thermodynamics. Aim 4: To elucidate the molecular forces influencing the stability and structure of DNA-polycation complexes, and to quantify the thermodynamics governing their formation; including the role of polycation composition, DNA secondary structure, and solution conditions. The complete thermodynamic characterization of these DNA complexes and their association reactions 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, that can be used in therapeutic, diagnostic and biotechnological applications. Another impact is the global role of water in the physical and chemical properties of biological macromolecules, and their interaction behavior towards one another. The correlation of energetics with hydration should improve our picture of how hydration controls the stability, conformation and melting behavior of unusual nucleic acid structures. In addition, the resulting hydration data can be used in molecular modeling studies and in theoretical calculations, providing an insight into global water that is not available by NMR or X-ray crystallography techniques. Broader ImpactsThe educational significance of this project involves the mentoring of students at all levels underrepresented in the sciences by training them in a wide variety of biophysical techniques. This training will improve their understanding of the molecular forces, kinetics and hydration effects controlling the structure and conformation of macromolecules, and their interaction with other molecules. Furthermore, the research findings generated by this group are routinely incorporated into lectures in Biophysical Chemistry, Quantitative Pharmaceutical Analysis and Biochemistry.