Orientation of large DNA during free solution electrophoresis studied by linear dichroism

Abstract

DNA T7 (40 kbp), T5 (120 kbp) and T2 (170 kbp) have been studied under electrophoretic conditions in free solution (25 mmol dm^–3 ionic strength) regarding chain orientation and field-free decay using phase-modulation detected linear dichroism. The steady-state orientation does not show Kerr-law behaviour for the larger T5 and T2 DNAs but increases roughly linearly with field strength, the orientation factor being around 3 × 10^–3 at 100 V cm^–1. When subjected to the electric field DNA displays within 0.1–5 s an orientation overshoot which increases strongly with DNA size and comes faster the higher the field strength. When the field is switched off a major portion of the DNA orientation has relaxed within 10 ms while a smaller portion decays more slowly: 0.3 s for T2, comparable with the slowest relaxation of a Zimm–Rouse chain. The observed orientation phenomena could be important in free-solution capillary electrophoresis of DNA. The question of the mechanism by which the DNA is aligned (dipole or electrophoretic orientation?) cannot be addressed adequately within existing theories for chain-like macromolecules. Awaiting results from simulations it is speculated that the overshoot phenomenon and the slow decay component of large DNA may represent reptative deformations of the DNA coil similar to those recently observed for large DNA during gel electrophoresis. Rapid field reversal did not lead to any dip in steady-state orientation for the pure DNA; however, in a complex with the recombinase protein RecA which stacks outside DNA, apparently in a polar way to form a large permanent dipole, a pronounced dip suggests that there is a 180° rotation of the particle upon field reversal. The electro-optical cell was designed to give minimal influence from electro-osmosis and convection. The experiments on T2 and T7 DNA (on the ground) were replicated under microgravity conditions during a sounding-rocket flight without any noticeable differences in orientation dynamics.