DNA CONDENSATION BY MULTIVALENT CATIONS

长期目标是了解DNA的物理机制 多价阳离子缩合，以理解影响因素 控制病毒和细胞以及制备期和遗传期的紧凑性 工程应用。了解DNA缩合主要有两个方面 方面：缩合反应的平衡和动力学特征 过程，并阐明凝结背后的作用力。脱氧核糖核酸 凝结似乎要经历四个主要阶段：(L)初期快速 几个链的结合，(2)在某些关键时刻这个复合体的崩溃 大小，(3)临界核的吸积生长，以及(4)长时间 完全形成的凝聚粒子的聚集，以产生更大的 粒子。每个阶段都是可逆的，但整个过程似乎是可逆的 是成核受限的。对DNA的主要能量贡献 凝结是静电、水化和扭曲的双重作用 螺旋。在之前的拨款提案中，我们强调了螺旋的作用 扭曲。我们积累了支持这一观点的重要证据。 的观点，特别是与二价阳离子，但认为证据 这意味着水合力和静电学仍然很强。 具体目标是： *进行早期光散射和EM实验，以表征 导致临界核形成和崩溃的阶段。 *将早期数据与光散射和电子显微镜相结合 后期数据，以获得一组一致的速率和均衡 冷凝过程所有阶段的常量。 *用集群聚集的思想来表征 在最后阶段形成的大集合体，以确定它们是否 在扩散或反应受限的过程中形成。 *研究溶液变量的影响-多价阳离子、盐、 助溶剂、温度和渗透压--关于平衡步骤 在DNA缩合和由此产生的结构的形态中。 *继续开发互补电荷模式的模型 DNA表面的反离子，作为静电的基础 解释DNA分子之间的引力。 *就渗透压力测量进行合作，以开发一个 水合力.作为函数的冷凝剂距离参数 他们的大小和费用。 *研究化学和酶对螺旋扰动的作用 具有Z-DNA形成插入序列的超螺旋质粒的探测， 阳离子-DNA体系的拉曼光谱和圆二色谱。 *对单个拉伸的DNA分子使用力测量来 确定电荷中和是否会导致“皱缩”，或者是否 需要并排的吸引力才能引发凝结。

The long-term objective is to understand the physical mechanism of DNA condensation by multivalent cations, so as to comprehend the factors controlling compaction in viruses and cells and in preparative and genetic engineering applications. Understanding DNA condensation has two main aspects: equilibrium and kinetic characterization of the condensation process, and elucidation of the forces underlying condensation. DNA condensation seems to involve four major stages: (l) initial rapid association of a few chains, (2) collapse of this complex at some critical size, (3) growth of the critical nucleus by accretion, and (4) long-time aggregation of fully-formed condensed particles to produce larger particles. Each stage is reversible, but the process as a whole seems to be nucleation-limited. The major energetic contributions to DNA condensation are electrostatics, hydration, and distortions of the double helix. In the previous grant proposal we emphasized the role of helix distortion. We have accumulated significant evidence supporting this point of view, particularly with divalent cations, but feel that the evidence implicating hydration forces and electrostatics is still very strong. Specific aims are: * Perform early-time light scattering and EM experiments to characterize the stages leading to formation and collapse of the critical nucleus. * Combine early-time data with light scattering and electron microscopic data on later stages, to obtain a consistent set of rate and equilibrium constants for all stages of the condensation process. * Use cluster aggregation ideas to characterize the fractal dimensions of the large aggregates formed in the last stage, to determine whether they are formed in a diffusion- or reaction-limited process. * Study the effects of solution variables - multivalent cations, salt, cosolvents, temperature, and osmotic pressure - on the equilibrium steps in DNA condensation and the morphologies of the resulting structures. * Continue development of a model of complementary charge patterning of counterions on the DNA surface, as the basis for an electrostatic explanation of attractive forces between DNA molecules. * Collaborate on osmotic stress measurements to develop a library of hydration force-distance parameters for condensing agents as functions of their size and charge. * Investigate the role of helix perturbations by chemical and enzymatic probing of supercoiled plasmids with Z-DNA-forming insertion sequences, and Raman and CD spectroscopy of cation-DNA systems. * Use force measurements on single, stretched-out DNA molecules to ascertain whether charge neutralization causes "crumpling", or whether side-by-side attractive forces are required to initiate condensation.