DNA CONDENSATION BY COBALT HEXAAMMINE(III) IN ALCOHOL-WATER MIXTURES - DIELECTRIC-CONSTANT AND OTHER SOLVENT EFFECTS
DNA CONDENSATION BY COBALT HEXAAMMINE(III) IN ALCOHOL-WATER MIXTURES - DIELECTRIC-CONSTANT AND OTHER SOLVENT EFFECTS
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DOI：
10.1002/bip.360360309
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发表时间：
1995-09-01
期刊：
影响因子：
2.9
通讯作者：
BLOOMFIELD, VA
中科院分区：
文献类型：
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作者：
ARSCOTT, PG;MA, CL;BLOOMFIELD, VA
DNA分子在多价阳离子达到临界浓度的情况下凝结成紧凑的结构。为了探究静电力对凝结的贡献，我们使用了水与甲醇（MeOH）、乙醇（EtOH）和异丙醇（iPrOH）的混合物，将介电常数epsilon从80改变到50。六胺钴（III）、Co(NH3)(6)（3+）对pUC18质粒的凝聚过程进行了总散射强度、动态光散射、电子显微镜和CD监测。总散射强度随epsilon从80增加到70，然后随着epsilon的进一步减小而减小。紫外分光光度法证实，低epsilon下的强度损失不是由于粒子沉降出溶液所致。当epsilon从80降低到70时，缩合速率和缩合程度增加，并且与醇的种类（MeOH < EtOH < iPrOH）有关。然而，颗粒的流体动力半径R(H)在300-350埃时大致保持不变，并且与醇的种类无关。R(H)在ε = 70以下增大。当介电常数从80降低到70时，诱导DNA缩聚所需的Co(NH3)(6)（3+）临界浓度从21 μ M降低到16 μ M左右，并随着醇的非极性而适度降低。通过对Manning的双变量反离子凝聚理论的修正，计算出在DNA凝聚开始时被中和的DNA电荷的比例为0.90 +/- 0.001，与ε无关。通过电镜观察，我们观察到凝聚粒子从epsilon = 80时约93%的环面变为epsilon = 70时89%的棒状和epsilon = 65时98%的棒状。当epsilon低于65时，DNA坍塌成多股纤维网络。浓缩DNA颗粒的形态，无论是环状、棒状还是纤维状，都与乙醇种类无关。在乙醇-水混合物中的CD光谱表明，当与Co(NH3)(6)（3+）在大于或等于70的ε ε下缩合时，封闭的环状质粒和线性化的质粒均为B构象，尽管封闭的环状分子表现出弱的Psi-DNA光谱。从B到A的转变发生在epsilon = 70和60之间，远高于该转变的正常介电常数epsilon = 40，表明乙醇和Co(NH3)(6)（3+）协同促进了B-A的转变。我们解释这些结果意味着醇具有静电和结构对DNA的影响，导致三种制度的冷凝。在最低的酒精浓度下，B构象是稳定的，凝结相对缓慢，从而有时间进行必要的包装调整以形成环面。在中等酒精浓度下，缩合速度更快，溶剂和Co(NH3)(6)（3+）的共同作用局部破坏了双螺旋结构的稳定性，允许DNA折叠，从而形成棒状缩合物。当epsilon从80降到65-60时，杆状体变得更短，数量更多，这表明随着酒精的增加，不稳定性增加。在最低的介电常数下，酒精和Co(NH3)6（3+）产生A-DNA，它强烈地自粘并迅速聚集成纤维网络，没有时间形成更紧密的凝聚物。(C) 1995 John Wiley and Sons, Inc。
DNA molecules condense into compact structures in the presence of a critical concentration of multivalent cations. To probe the contribution of electrostatic forces to condensation, we used mixtures of water with methanol (MeOH), ethanol (EtOH), and isopropanol (iPrOH) to vary the dielectric constant epsilon from 80 to 50. The condensation of pUC18 plasmids by hexaammine cobalt (III), Co(NH3)(6)(3+), was monitored by total intensity and dynamic light scattering, electron microscopy, and CD. The total scattering intensity increased as epsilon went from 80 to 70, and then decreased as epsilon decreased further. Ultraviolet spectrophotometry confirmed that the loss of intensity at low epsilon was not due to the particles' settling out of solution. The rate as well as the extent of condensation increased as epsilon was lowered from 80 to 70, and also depended on the species of alcohol (MeOH < EtOH < iPrOH). The hydrodynamic radii R(H) of the particles, however, remained roughly the same at 300-350 Angstrom and was independent of the species of alcohol. R(H) increased below epsilon = 70. The critical concentration of Co(NH3)(6)(3+) required to induce DNA condensation decreased from 21 mu M to about 16 mu M as the dielectric constant decreased from 80 to 70, and decreased moderately with the nonpolarity of the alcohol. The fraction of DNA charge neutralized at the onset of DNA condensation was calculated by a modification of Manning's two-variable counterion condensation theory to be 0.90 +/- 0.001, independent of epsilon. By electron microscopy we observed that the condensed particles changed from about 93% toroids at epsilon = 80 to 89% rods at epsilon = 70 and 98% rods at epsilon = 65. At epsilon lower than 65, DNA collapsed into a network of multistranded fibers. The morphology of condensed DNA particles, whether toroids, rods, or fibers, was independent of the alcohol species. CD spectra in ethanol-water mixtures indicated that both closed circular and linearized plasmids were in the B conformation when condensed with Co(NH3)(6)(3+) at epsilon greater than or equal to 70, although the closed circular molecules exhibited a weak Psi-DNA spectrum. A transition from the B to A form took place between epsilon = 70 and 60, well above the normal dielectric constant of epsilon = 40 for this transition, indicating that ethanol and Co(NH3)(6)(3+) synergistically promote the B-A transition.We interpret these results to mean that alcohols have both electrostatic and structural effects on DNA, leading to three regimes of condensation. At the lowest alcohol concentrations the B conformation is stable and condensation is relatively slow, allowing time for the packing adjustments necessary to form toroids. At intermediate alcohol concentrations condensation is faster, and the combined effects of solvent and Co(NH3)(6)(3+) locally destabilize the double helix, permitting DNA foldbacks that lead to rodlike condensates. Rods become shorter as well as more numerous as epsilon decreased form 80 to 65-60, indicating increasing destabilization as alcohol increases. At the lowest dielectric constants, alcohol and Co(NH3)6(3+) produce A-DNA, which strongly self-adheres and rapidly aggregates into fibrous networks, not allowing time for more compact condensates to form. (C) 1995 John Wiley and Sons, Inc.