Thermodynamic Origins of Sequence-Recognition in Ligand-DNA Interactions

本研究的重点是深入了解小分子和蛋白质与核酸进行序列选择性结合的机制。我们将致力于将这些相互作用的热力学性质与DNA序列和配体结构联系起来。最近的研究表明，放线菌素D-DNA相互作用所观察到的DNA结合焓和熵不仅受到插入位点碱基序列的显著影响，而且受到插入位点两侧碱基的显著影响。这些观察结果将扩展到探索配体结合位点两侧DNA碱基序列的细微变化的影响及其与热力学结合机制变化的关系。热力学结合性质的分析将与配体与特定DNA碱基序列相互作用所产生的伴随配体-DNA结构的检查相结合，这些配体-DNA结构提供了与复合物形成相关的热力学机制和配体-DNA复合物的结构几何之间的联系。各种生物物理方法，包括吸收光谱法和等温微滴定量热法，将用于检查放线菌素D与寡核苷酸的相互作用，寡核苷酸被专门设计为包含单个中心位置的放线菌素D结合位点和各种相邻和次相邻的侧翼碱基序列。结合位点序列和旁边的碱基将被系统地改变，以确定它们对复合物形成的热力学机制的影响。从这些研究中，洞察DNA序列识别的热力学起源将被辨别出来。配体- dna复合物的结构细节将通过高分辨率核磁共振方法进行检查，并与热力学数据相关联，为序列识别的“热力学起源”提供新的范例。分子识别是理解控制细胞内发生的各种生物化学和分子生物学事件的机制过程的核心问题之一。因此，了解控制小分子和蛋白质与DNA序列特异性相互作用的力量对于全面理解基本生化过程（如复制、转录、重组和DNA修复）至关重要。本研究的重点是确定和表征靶DNA和配体上负责序列选择相互作用的化学和/或结构决定因素。

9728720 Graves The focus of this research is to gain insight into the mechanisms through which small molecules and proteins exert sequence-selective binding to nucleic acids. Efforts will be directed towards correlating the thermodynamic properties associated with these interactions with both DNA sequence and ligand structure. Recent studies have demonstrated the DNA binding enthalpies and entropies observed for actinomycin D-DNA interactions to be markedly influenced not only by the base-sequence at the intercalation site but also by the bases that flank the intercalation site. These observations will be extended to explore the effects of subtle changes in the DNA base sequences that flank the ligand binding site and their relationship to changes in the thermodynamic binding mechanism(s). Analyses of thermodynamic binding properties will be coupled to examination of the concomitant ligand-DNA structures resulting from interactions of ligands with specific DNA base sequences providing linkage between the thermodynamic mechanisms associated with complex formation and the structural geometry of the ligand-DNA complex. Various biophysical methods including absorption spectroscopy and isothermal microtitration calorimetry will be used to examine the interactions of actinomycin D with oligonucleotides that have been designed specifically to harbor a single centrally located actinomycin D binding site and varied neighboring and next-neighboring flanking base sequences. The binding site sequence and bases which flank the binding site will be systematically changed to determine their influence on the thermodynamic mechanism of complex formation. From these studies, insight into the thermodynamic origins of DNA sequence-recognition will be discerned. Structural details of the ligand-DNA complex will be examined by high-resolution nuclear magnetic resonance methods and correlated with the thermodynamic data to provide a novel paradigm for the "thermodynamic origins" of sequence-recognition. Mol ecular recognition is one of the central problems in understanding the mechanistic processes that govern a wide variety of biochemical and molecular biological events that take place in the cell. Hence, an understanding of the forces that govern the sequence specific interactions of small molecules and proteins with DNA is pivotal towards an overall comprehension of basic biochemical processes such as replication, transcription, recombination, and DNA repair. The focus of this research is to determine and characterize the chemical and/or structural determinants on both the target DNA and ligand responsible for sequence-selective interactions.