DENATURATION OF SELF-COMPLEMENTARY DNA LOOPS

本提案的目的是促进对以下问题的理解： DNA的顺序指导的热力学行为。 一种新型 本文将采用一种新的方法来计算热力学参数 碱基对稳定性，分子内发夹环的形成， DNA内环形成与溶剂离子的关系 实力 自动化DNA合成将被用于 构建一系列自互补DNA环（哑铃）。 这些化合物的吸光度与温度熔融曲线 在宽范围的溶剂离子强度的分子将是 根据精确的平衡来测量和分析 统计热力学模型，已制定为 预测DNA哑铃中的螺旋-卷曲转变。 哑铃 在物理研究中， 研究DNA的热稳定性。 因为“末端磨损”， 链解离和相关的浓度依赖性 的熔化过程被消除，只有分子内部 自由度有助于DNA的解链过程 哑铃 序列依赖性碱基对稳定性，内环 熔融、熔融协同性和分子内环的形成 所有这些都是来自内部自由度的贡献。 代表这些内部的热力学参数 作为溶剂离子的函数的分子自由度 强度将通过调整他们在经验评估， 理论计算以拟合实验跃迁。 具有发夹结构的异源序列DNA限制性片段 还将构建连接到双链体末端的环。 核磁共振和动态光散射将被用来研究 这些“长”DNA哑铃的溶液动力学。 的 分解代谢物激活蛋白和EcoRI的相互作用 在DNA上具有其特异性识别序列核酸内切酶 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.