Photofootprinting of drug‐binding sites‐on DNA using diazo‐ and azido‐9‐aminoacridine derivatives

Abstract

It is demonstrated that DNA photofootprinting analysis of the intercalating depsipeptide echinomycin, and the minor groove‐binders distamicyn, 4′,6‐diamidino‐2‐phenylindole (DAPI) and Hoechst 33258 can be performed using 9‐[6‐(2‐diazocyclopentadienylcarbonyloxy)hexylamino]acridine (DHA) [Nielsen et al. (1988) Nucleic Acids Res. 16, 3877–3888] or 2‐methoxy‐6‐azido‐9‐aminoacridine (MAA) [Jeppesen et al. (1988) Nucleic Acids Res. 16, 5755–5770]. Both the extent of the drug‐binding sites and their relative strength can be determined with either reagent. DNA has the advantage of giving virtually sequence‐uniform DNA photocleavage. On the other hand, structural changes in the DNA are detected by MAA. Using the 232‐base‐pair EcoRI–PvuII pUC19 restriction fragment, it is found that cleavage protection by distamycin, DAPI and Hoechst 33258 all require an (A · T)4 sequence, whereas protection by echinomycin was confined to a G+C‐rich 8‐base‐pair region. Copyright © 1989, Wiley Blackwell. All rights reserved