Sequence Specific Targeting of Nucleic Acids Using Intramolecular Triplexes: Energetics and Hydration

这个项目的重点是核酸三螺旋的热力学，这已经涉及控制细胞过程的内源性或外源性机制。长期目标是了解核苷酸序列、链组成、阳离子、pH值和水合作用对分子内和分子间三联体稳定性的影响。结合光谱、量热、密度和声学技术，获得了三重构象、pH值、盐浓度以及不匹配和凸起的函数的完整热力学剖面。具体地说，三络合物稳定性的相图将作为盐浓度和pH的函数来测量；然后，压力摄动量热法和渗透应力技术将用于探测它们的展开和水化变化。结果将与它们的声学和体积测量结果进行比较，从而分别获得它们的水化特性以及通过双链和三链形成的核酸单链或双链的水化效果。将建立一个全面的数据库，用于设计针对特定核酸序列的有用的三重试剂和序列特异性局部构象重排的能量预测。水合作用在大分子识别中的作用是分子生物科学研究的一个重要领域。水在生物大分子的整体特性中起着至关重要的作用。大分子水合反应的定量描述对于研究其结构、能量学、动力学和功能具有重要意义。这些结果将提高我们目前对水合作用如何控制核酸三聚体的稳定性、构象和熔化行为的理解，提供对全球溶液中水的洞察，有助于分子建模和理论计算。更广泛的影响：项目的教育意义在于对学生的指导；该项目将包括培训核酸及其与配体相互作用的生物物理化学的复杂方法和分析。此外，研究成果将纳入本科和研究生课程的讲座。在这个项目中产生的数据库将有助于其他研究人员解释和分析许多类型的结构和生物物理实验的结果。

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.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. 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.