Condensation of DNA by multivalent cations: Experiment studies of condensation kinetics

DNA in viruses and cells exists in highly condensed, tightly packaged state s. We have undertaken an in vitro study of the kinetics of DNA condensation by the trivalent cation hexaammine cobalt (III) with the aim of formulatin g a quantitative, mechanistic model of the condensation process. Experiment al approaches included total intensity and dynamic light scattering, electr on microscopy, and differential sedimentation. We determined the average de gree of condensation, the distribution of condensate sizes, and the fractio n of uncondensed DNA as a function of reaction time for a range of [DNA] an d [Co(NH3)(6)(3+)]. We find the following: (1) DNA condensation occurs only above a critical [Co(NH3)(6)(3+)] for a given DNA and salt concentration. At the onset of condensation, [Co(NH3)(6)(3+)]/[DNA-phosphate] is close to the average value of 0.54, which reflects the 89-90% charge neutralization criterion for condensation. (2) The equilibrium weight average hydrodynamic radius [R-H] of the condensates first decreases, then increases vith incre asing [Co(NH3)(6)(3+)] as they undergo a transition from intramolecular (mo nomolecular): to intermolecular (multimolecular) condensation, However, [R- H] is insensitive to [DNA]. (3) The uncondensed DNA fraction decays approxi mately exponentially with rime. The equilibrium uncondensed DNA fraction an d relaxation time decrease with increasing [Co(NH3)(6)(3+)] but are insensi tive to [DNA]. (4) The condensation rate in its early stages is insensitive to [DNA] but proportional to [Co(NH3)(6)(3+)](xs) = [Co(NH3)(6)(3+)] - [Co (NH3)(6)(3+)](crit). (5) Data for low [DNA] and low [Co(NH3)(6)(3+)] at ear ly stages of condensation are most reliable for kinetic modeling since unde r these conditions there is minimal clumping and network formation among se parate condensates. A mechanism with initial monomolecular nucleation and s ubsequent bimolecular association and unimolecular dissociation steps with rate constants that depend on the number of DNA molecules in the condensate , accounts reasonably well for these observations. (C) 2000 John Wiley & So ns, Inc.