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

研究重点是核酸分子内结构的热力学-特别是三重螺旋，因为它们在内源性或外源性机制控制细胞过程中的意义。长期目标是了解控制复杂的分子内DNA结构的整体稳定性的分子力;以量化的能量和水合作用的贡献，管理协会的三链体，和其他不寻常的结构，与他们的互补链，包括序列和阳离子的作用;并确定热力学为他们有利的相互作用与聚阳离子细胞交付的目的。UV和CD光谱用于验证每个核酸复合物含有适当的结构特征。此外，T-依赖的紫外光谱，差示扫描和压力扰动量热法的组合将被用来获得完整的热力学曲线，其解折叠反应作为盐，pH值和渗透浓度的函数。高灵敏度滴定量热法和密度技术将用于测量缔合反应的热和体积变化。这些DNA复合物的完整的热力学特性将提供一个基本的理解的物理因素，确定其作为其序列和溶液条件的函数的稳定性。这些因素是基因靶向试剂的合理设计的基础，也是它们适当的细胞递送的基础，这些基因靶向试剂可用于治疗、诊断和生物技术应用，以及预测序列的能量学细胞内过程中的特定局部构象重排。由于开发高度特异性和稳定的药物的根本重要性，这项研究在致癌和基因治疗方面具有更广泛的影响用于通过三链体或双链体形成靶向癌基因或其转录的RNA产物。另一个影响是水在生物大分子的整体性质和它们相互作用行为中的作用。具体而言，能量学与水合作用的相关性将改善水合作用如何控制这些新核酸结构的稳定性、构象和熔融行为的图片。此外，由此产生的水化数据可用于分子建模研究和理论计算，提供对全球水的深入了解。其教育意义在于指导本科生、研究生和博士后研究员，培训他们掌握各种生物物理技术和对生物物理学的基本理解。此外，研究结果还经常纳入本科生、研究生课程和研讨会的讲座。

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.