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

使用分子内复合物对核酸进行序列特异性靶向：能量学、动力学和水合

• 批准号: 1122029
• 负责人: Luis Marky
• 金额: $ 106.65万
• 依托单位: University of Nebraska Medical Center
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-15 至 2017-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1122029&HistoricalAwards=false
• 关键词: Sequence; Specific; Targeting; Nucleic; Acids

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.

智力优势本研究重点关注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。该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；量化控制这些不寻常的分子内结构与其互补链的关联的能量学、动力学和水合贡献，包括阳离子的作用；并确定其与用于细胞递送目的的聚阳离子有利的相互作用的热力学。假设是：核酸二级结构的环中存在未配对的碱基核苷酸，在与互补链的反应中提供了有利的自由能贡献，并且这些受限环的稍微疏水的表面有利于与递送载体​​（例如聚阳离子）的相互作用。为了检验这一假设，提出了以下目标： 目标 1：表征包含凸起或内部环、假结和三向连接的茎环基序的解链行为，作为序列、其末端环的稳定性和溶液条件的函数。目标 2：阐明并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。目标 3：确定目标 2 的双分子缔合反应的动力学，包括反应速率和相关的活化能和活化熵，并将它们与其热力学相关联。目标 4：阐明影响 DNA-聚阳离子复合物稳定性和结构的分子力，并量化控制其形成的热力学；包括聚阳离子组成、DNA 二级结构和溶液条件的作用。这些 DNA 复合物及其缔合反应的完整热力学表征将提供对物理因素的基本理解，这些物理因素决定其稳定性作为其序列和溶液条件的函数。这些因素是基因靶向试剂的合理设计及其正确的细胞递送的基础，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整体作用，以及它们之间的相互作用行为。能量学与水合的相关性应该可以改善我们对水合如何控制异常核酸结构的稳定性、构象和熔化行为的了解。此外，所得的水合数据可用于分子建模研究和理论计算，从而提供对全球水的深入了解，这是核磁共振或 X 射线晶体学技术无法获得的。更广泛的影响 该项目的教育意义在于通过对科学领域代表性不足的各个级别的学生进行各种生物物理技术的培训来进行指导。这项培训将提高他们对控制大分子结构和构象的分子力、动力学和水合效应及其与其他分子相互作用的理解。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。