DNA CONDENSATION BY MULTIVALENT CATIONS

长期目标是了解DNA的物理机制 多价阳离子的缩合，从而了解因素 控制病毒和细胞中以及制备和遗传学中的压实 工程应用理解DNA缩合有两个主要的 方面：冷凝的平衡和动力学表征 过程，并阐明了凝聚的基础力量。DNA 冷凝似乎包括四个主要阶段：（1）初始快速 一些链的关联，（2）在某些临界点处该复合体的崩溃 大小，（3）临界核的增长吸积，和（4）长时间 完全形成的凝聚颗粒聚集， 粒子每个阶段都是可逆的，但整个过程似乎 限制成核。对DNA的主要能量贡献 凝结是静电、水合和双重扭曲 螺旋。在之前的拨款申请中我们强调了螺旋的作用 畸变我们已经积累了支持这一点的重要证据 的观点，特别是与二价阳离子，但认为，证据 水化力和静电仍然很强 具体目标是： * 进行早期光散射和EM实验以表征 导致临界核的形成和坍缩的阶段。 * 联合收割机早期数据与光散射和电子显微镜 数据的后期阶段，以获得一个一致的速度和平衡 冷凝过程所有阶段的常数。 * 用聚类聚集的思想刻画了 在最后阶段形成的大聚集体，以确定它们是否 在扩散或反应受限过程中形成。 * 研究溶液变量-多价阳离子，盐， 共溶剂、温度和渗透压-对平衡步骤的影响 在DNA缩合和所产生的结构的形态。 * 继续发展一种互补电荷模式， DNA表面上的抗衡离子，作为静电的基础， 解释DNA分子之间的吸引力。 * 在渗透压测量方面进行合作， 凝结剂的水化力-距离参数作为以下参数的函数： 它们的大小和电荷 * 研究螺旋扰动的作用，通过化学和酶 用Z-DNA形成插入序列探测超螺旋质粒， 以及阳离子-DNA体系的拉曼和圆二色光谱。 * 在单个伸展的DNA分子上使用力测量， 确定电荷中和是否会导致“起皱”，或者 需要并排的吸引力来引发冷凝。

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.