Solvent-Modulated Energetics of Tetrameric DNA Polymorphism

我们建议继续我们的研究，以表征溶剂在调节生物聚合物的结合亲和力和构象偏好的作用的热力学内容。 在上一个预算期间，我们的研究重点是调查水和水溶性共溶剂在蛋白质和核酸转换以及配体-蛋白质结合的能量学中的作用。 与此同时，我们已经开始建立一个程序，在该程序中，我们表征溶剂和单个溶剂组分对DNA G-四链体和i-基序的能量学的影响。 G-四链体和i-基序形成一类重要的具有生物学重要性的非规范四聚体DNA结构。 富含鸟嘌呤的DNA链易于折叠成各种G-四链体结构，其中鸟嘌呤碱基以稳定的氢键排列彼此缔合，即G-四联体。 富含胞嘧啶的链表现出在弱酸性至接近中性pH下折叠成i-基序构象的倾向。后者代表具有两个平行链双链体以反平行方向插入彼此的四链体排列的DNA构象。 G-四链体和I-基序在拓扑结构和结构上表现出很大的多样性，这被认为是生物学相关的。 核苷酸序列、稳定性抗体的类型和环境条件（例如，例如，在一个实施例中，渗透剂或拥挤剂的存在）导致G-四链体的拓扑结构的显著变化。 在要求的预算期间，我们建议探索水和水混溶性共溶剂（如尿素和甘氨酸甜菜碱）和各种阳离子的浓度和pH值的作用，在指导非典型的四链体DNA基序的多态性倾向。 我们设计了一种实验策略，旨在开发一种预测算法，可用于探索富含鸟嘌呤的序列作为环境因素的函数的构象偏好和它们的互补的富含胞嘧啶的链的存在。 我们将采用差示扫描量热法、紫外/维斯分光光度法、CD光谱法、超声测速法和高精度密度法。 我们已经证明了这些实验方法的可行性和信息含量。 我们的长期目标是开发所需的预测算法，以评估序列特异性，结构特异性和溶剂特异性的构象偏好的功能重要的结构域内天然存在的核酸。 随着越来越多的生物体中基因组序列生成的成功，这种能力变得非常需要。 最后，人们希望评估基因组中有利于特定结构基序的局部序列结构域是否对应于生物作用或控制的功能位点。

We propose to continue our studies to characterize the thermodynamic content of the role of solvent in modulating the binding affinity and conformational preferences of biopolymers. During the previous budget period, our studies were focussed on investigating the role of water and water-soluble cosolvents in the energetics of protein and nucleic acid transitions and ligand-protein binding. In parallel, we have begun to establish a program in which we characterize the influence of solvent and individual solvent components on the energetics of DNA G-quadruplexes and i-motifs. G-quadruplexes and i-motifs form an important class of noncanonical tetrameric DNA structures of biological importance. Guanine-rich DNA strands are prone to fold into various G-quadruplex structures in which guanine bases associate with each other in stable hydrogen-bonded arrangements, G-quartets. Cytosine-rich strands exhibit a propensity to fold into the i-motif conformation at slightly acidic to near neutral pH. The latter represents a DNA conformation with a tetraplex arrangement of two parallel-stranded duplexes intercalated into each other in an antiparallel orientation. G-quadruplexes and i-motifs exhibit a great deal of diversity in topology and structure which is believed to be of biological relevance. Subtle changes in nucleotide sequence, type of the stabilizing counterion, and the environmental conditions (e. g., presence of osmolytes or crowders) results in dramatic changes in the topology of a G-quadruplex. During the requested budget period, we propose to explore the role of water and water-miscible cosolvents (such as urea and glycine betaine) and the concentration of various cations and pH in guiding the polymorphic tendencies of noncanonical tetraplex DNA motifs. We have designed an experimental strategy aimed at developing a predictive algorithm that can be used to explore the conformational preferences of guanine-rich sequences as a function of environmental factors and the presence of their complementary cytosine-rich strands. We will employ differential scanning calorimetry, UV/Vis spectrophotometry, CD spectroscopy, ultrasonic velocimetry, and high precision densimetry. We already have demonstrated the feasibility and information content of these experimental methods. Our long-term objective is to develop predictive algorithms needed to evaluate sequence-specific, structure-specific, and solvent-specific conformational preferences of functionally important domains within naturally occurring nucleic acids. With the success in genomic sequence generation in an increasingly large number of organisms, such a capacity is becoming highly desirable. Ultimately, one would like to assess if local sequence domains in the genome that favor particular structural motifs correspond to functional sites of biological action or control.