In vitro characterization of the reaction of four psoralen derivatives with DNA.

Four psoralen derivatives were radiolabeled and used for in vitro DNA binding studies. The derivatives were compared for their dark-binding ability to DNA, photoreactivity, and for unwinding angles. The dark-binding dissociation constants we determined were 1.4 X 10(-3) M for 8-methoxypsoralen (8-MOP), 3.5 X 10(-4) M for 5-methoxypsoralen (5-MOP), and 5.5 X 10(-4) M for 5-methylisopsoralen (5-MIP). We did not detect any dark binding to DNA for 3-carbethoxypsoralen (3-CP). Photoaddition experiments indicated that the relative rates of photoaddition by psoralen to DNA (measured as psoralens bound per base pair per second) are 4.4 X 10(-5) for 5-MIP, 9.2 X 10(-6) for 5-MOP, 7.8 X 10(-6) for 8-MOP, and 4.6 X 10(-6) for 3-CP. We found the peak level of binding (for an initial base pair-to-psoralen ratio of 22) to be 27, 32.2, 31.2, and 1,538 base pairs per psoralen bound for 5-MOP, 8-MOP, 5-MIP, and 3-CP, respectively. In addition, 3-CP adducts could be photoreversed by prolonged irradiation at 360 nm. After 10 hours of irradiation, the amounts of 3-CP bound to DNA had fallen to less than 50% of the peak amount bound. In the same time, the amount of 8-MOP and 5-MOP bound had fallen to 95% of their peak values, and 5-MIP had fallen to 85% of its peak value. We also performed unwinding angle experiments to determine the amount of unwinding of the DNA helix induced per photobound derivative molecule; the unwinding angles +/- 3 degrees were 25 for 5-MOP, 28 for 8-MOP, 26 for 3-CP, and 18 for 5-MIP.