Demonstration of specific high affinity binding sites in plasmid DNA by photoaffinity labeling with an ethidium...

We have used photoaffinity labeling of pBR322 DNA with 8-azido-3-amino-5-ethyl-6-phenylphenanthridinium chloride to demonstrate high affinity ethidium-binding sites. Plasmid equilibrated with as little as 1 drug/DNA molecule was photoactivated, freed of uncomplexed drug by ethanol precipitation, and subjected to restriction analysis. There was highly specific, rather than random, blockage of HhaI sites (d(GCGC)) at low drug concentrations. Furthermore, the same 7 new digestion fragments were generated at drug to nucleotide ratios ranging from 1:100 to 1:8000. All the new DNA fragments had chain lengths greater than the largest HhaI fragment (393 base pairs). At higher ligand concentrations closely approximating those needed for equilibrium binding studies, detection of the high affinity sites was greatly masked. Drug binding to HhaI restriction fragments which had been prepared prior to the action of drug did not induce new bands. Furthermore, the larger DNA fragments from drug-labeled plasmid were resistant to HhaI digestion over a wide range of enzyme concentrations. These findings suggest that ligand binding can be highly selective even between sites which have the same tetranucleotide sequence. Therefore, selective drug binding must be dictated not only by local base sequence preference, but also by other long range parameters.