PHOTOAFFINITY APPROACHES TO DETERMINING THE SEQUENCE SELECTIVITIES OFDNA-SMALL MOLECULE INTERACTIONS - ACTINOMYCIN-D AND ETHIDIUM

The DNA photoaffinity ligands, 7-azidoactinomycin D and 8-azidoethidiu m, form DNA adducts that cause chain cleavage upon treatment with pipe ridine. Chemical DNA sequencing techniques were used to detect covalen t binding. The relative preferences for modifications of all possible sites defined by a base pair step (e.g. GC) were determined within all quartet contexts such as (IGCJ). These preferences are described in t erms of 'effective site occupations', which express the ability of a l igand to covalently modify some base in the binding site, Ideally, the effective site occupations measured for photoaffinity agents can also be related to site-specific, non-covalent association constants of th e ligand. The sites most reactive with 7-azidoactinomycin D were those preferred for non-covalent binding of unsubstituted actinomycin D. GC sites were most reactive, but next-nearest neighbors exerted signific ant influences on reactivity. GC sites in 5'-(pyrimidine)GC(purine)-3' contexts, particularly TGCA, were most reactive, while reactivity was strongly suppressed for GC sites with a 5'-flanking G, or a 3'-flanki ng C. High reactivities were also observed for bases in the first (5') GG steps in TOOT TGGG and TGGGT sequences recently shown to bind acti nomycin D with high affinity. Pyrimidine-3',5'-purine steps and GG ste ps flanked by a T were most preferred by 8-azidoethidium, in agreement with the behavior of unsubstituted ethidium. The good correspondence between expected and observed covalent binding preferences of these tw o azide analogs demonstrates that photoaffinity labeling can identify highly preferred sites of non-covalent DNA binding by small molecules.