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

本研究的重点是核酸分子内结构的热力学，特别是模拟RNA分子二级结构的DNA结构。这个项目的广泛和长期目标是了解控制复杂分子内DNA结构整体稳定性的分子力；量化这些不寻常的分子内结构及其互补链的能量学、动力学和水合作用，包括阳离子的作用；并确定它们与多阳离子相互作用的热力学，以达到细胞递送的目的。假设是：核酸二级结构环中未配对碱基核苷酸的存在为其与互补链的反应提供了有利的自由能贡献，而这些受限环的稍微疏水的表面有利于与传递载体的相互作用，如聚阳离子。为了验证这一假设，提出了以下目标：目标1：表征包含凸起或内部环、假结和三通结的茎环基序的熔化行为，作为序列、其末端环的稳定性和解决条件的函数。目的2：阐明和量化控制分子内二级DNA结构与其部分互补链反应的分子力。目的3：确定目的2的双分子结合反应的动力学，包括反应速率和相关的活化能和活化熵，并将它们与热力学联系起来。目的4：阐明影响dna -多阳离子配合物的稳定性和结构的分子力，并量化控制其形成的热力学；包括多阳离子组成、DNA二级结构和溶液条件的作用。这些DNA复合物及其结合反应的完整热力学表征将提供对决定其稳定性的物理因素（作为其序列和溶液条件的函数）的基本理解。这些因素对于基因靶向试剂的合理设计及其适当的细胞递送至关重要，可用于治疗、诊断和生物技术应用。另一个影响是水在生物大分子的物理和化学性质中的全球作用，以及它们彼此之间的相互作用行为。能量学与水合作用的相关性有助于我们更好地理解水合作用是如何控制不寻常核酸结构的稳定性、构象和熔化行为的。此外，由此产生的水合作用数据可用于分子建模研究和理论计算，提供了核磁共振或x射线晶体学技术无法获得的全球水的深入了解。更广泛的影响本项目的教育意义包括通过培训各种生物物理技术来指导在科学领域代表性不足的各级学生。这种训练将提高他们对分子力、动力学和水合作用控制大分子的结构和构象，以及它们与其他分子的相互作用的理解。此外，该小组的研究成果经常被纳入生物物理化学，定量药物分析和生物化学的讲座中。

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.