DNA condensation by cobalt hexaammine (III) in alcohol-water mixtures: dielectric constant and other solvent effects.
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)(3+)6, 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 the 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 RH of the particles, however, remained roughly the same at 300-350 A and was independent of the species of alcohol. RH increased below epsilon = 70. The critical concentration of Co(NH3)6(3+) required to induce DNA condensation decreased from 21 microM to about 16 microM 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 +/- 0.01, 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 > or = 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.(ABSTRACT TRUNCATED AT 400 WORDS)
Arscott
P G
PG
Department of Biochemistry, University of Minnesota, St. Paul 55108, USA.
Ma
C
C
Wenner
J R
JR
Bloomfield
V A
VA
D000433:1-Propanol; D000438:Alcohols; D001483:Base Sequence; D002942:Circular Dichroism; D003035:Cobalt; D004247:DNA / Q000737:chemistry*; D004563:Electrochemistry; D000431:Ethanol; D000432:Methanol; D008854:Microscopy, Electron; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides; D010957:Plasmids / Q000737:chemistry* / Q000648:ultrastructure; D011838:Radiation-Sensitizing Agents; D012542:Scattering, Radiation; D012997:Solvents; D014867:Water
D000438:Alcohols; D009838:Oligodeoxyribonucleotides; D011838:Radiation-Sensitizing Agents; D012997:Solvents; D014867:Water; C032300:hexaamminecobalt(II); D003035:Cobalt; D000431:Ethanol; D004247:DNA; D000433:1-Propanol; D000432:Methanol