DENATURATION STUDIES OF SELF-COMPLEMENTARY DNA LOOPS

这项建议的目标是促进对 序列指导了DNA的热力学行为。一本小说 将采用一种方法来评估热力学参数。 碱基对稳定性、分子内发夹环的形成和 DNA内环的形成与溶剂离子的关系 力量。自动DNA合成将被用于 构建一系列自我互补的DNA环(哑铃)。 这些化合物的吸光度与温度的熔融曲线 分子在广泛的溶剂离子强度范围内将 根据精确的平衡进行测量和分析 已建立的统计热力学模型 预测dna哑铃中的螺旋-螺旋转变。哑铃 在物理研究中与线状碎片相比有几个优势 研究DNA的热稳定性。因为“末日之战”， 链解离及其相关的浓度依赖关系 熔化过程被消除，只有分子内部 自由度对DNA的融化过程有贡献 哑铃。依赖于序列的碱基对稳定性、内环 熔融、熔融协同性与分子内环的形成 所有这些都构成了内部自由度的贡献。 代表这些内部结构的热力学参数 分子自由度与溶剂离子的关系 实力将通过调整它们在 符合实验转变的理论计算。 发夹结构的异源序列DNA限制性片段 还将构建连接到双工末端的环路。 将使用核磁共振和动态光散射来研究 这些“长”DNA哑铃的溶液动力学。这个 分解代谢激活蛋白与EcoRI的相互作用 核酸内切酶及其在DNA上的特定识别序列 哑铃也将被调查。

The goal of this proposal is to contribute to the understanding of sequence directed thermodynamic behavior of DNA. A novel approach will be employed to evaluate thermodynamic parameters of base pair stability, intramolecular hairpin loop formation and internal loop formation in DNA as a function of solvent ionic strength. Automated DNA synthesis will be employed to construct a series of self-complementary DNA loops (dumbbells). Absorbance versus temperature melting profiles of these molecules over a wide range of solvent ionic strengths will be measured and analyzed in terms of an exact equilibrium statistical thermodynamic model that has been formulated for predicting helix-coil transitions in DNA dumbbells. Dumbbells have several advantages over linear fragments in physical studies investigating thermostability of DNA. Because "end-fraying", strand dissociation and the associated concentration dependence of the melting process are eliminated, only the molecular internal degrees of freedom contribute to the melting process of DNA dumbbells. Sequence dependent base pair stability, internal loop melting, melting cooperativity and intramolecular loop formation all constitute contributions from the internal degrees of freedom. Thermodynamic parameters representative of these internal molecular degrees of freedom as a function of solvent ionic strength will be empirically evaluated by adjusting them in the theoretical calculation to fit experimental transitions. Heterogeneous sequence DNA restriction fragments with hairpin loop attached to the ends of the duplex will also be constructed. NMR and dynamic light scattering will be employed to investigate the solution dynamics of these "long" DNA dumbbells. The interactions of the catabolite activator protein and EcoRI endonuclease with their specific recognition sequence on a DNA dumbbell will also be investigated.