ORIENTATION OF LARGE DNA DURING FREE SOLUTION ELECTROPHORESIS STUDIEDBY LINEAR DICHROISM

DNA T7 (40 kbp(double dagger)), T5 (120 kbp) and T2 (170 kbp) have bee n studied under electrophoretic conditions in free solution (25 mmol d m-3 ionic strength) regarding chain orientation and field-free decay u sing phase-modulation detected linear dichroism. The steady-state orie ntation does not show Kerr-law behaviour for the larger T5 and T2 DNAs but increases roughly linearly with field strength, the orientation f actor being around 3 x 10(-3) at 100 V cm-1. When subjected to the ele ctric field DNA displays within 0.1-5 s an orientation overshoot which increases strongly with DNA size and comes faster the higher the fiel d strength. When the field is switched off a major portion of the DNA orientation has relaxed within 10 ms while a smaller portion decays mo re slowly: 0.3 s for T2, comparable with the slowest relaxation of a Z imm-Rouse chain. The observed orientation phenomena could be important in free-solution capillary electrophoresis of DNA. The question of th e mechanism by which the DNA is aligned (dipole or electrophoretic ori entation?) cannot be addressed adequately within existing theories for chain-like macromolecules. Awaiting results from simulations it is sp eculated that the overshoot phenomenon and the slow decay component of large DNA may represent reptative deformations of the DNA coil simila r to those recently observed for large DNA during gel electrophoresis. Rapid field reversal did not lead to any dip in steady-state orientat ion for the pure DNA; however, in a complex with the recombinase prote in 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- degrees rotation of the particle upon field reversal. The electro-opti cal cell was designed to give minimal influence from electro-osmosis a nd convection. The experiments on T2 and T7 DNA (on the ground) were r eplicated under microgravity conditions during a sounding-rocket fligh t without any noticeable differences in orientation dynamics.