We have studied the DNA sequence binding preference of the antitumour antibiotic nogalamycin by DNase-I footprinting using a variety of DNA fragments. The DNA fragments were obtained by cloning synthetic oligonucleotides into longer DNA fragments and were designed to contain isolated ligand-binding sites surrounded by repetitive sequences such as (A),, . (T),, and (AT),. Within regions of (A), . (T),, clear footprints are observed with low concentrations of nogalamycin (< 5 pM), with apparent binding affinities for tetranucleotide sequences which decrease in the order TGCA > AGCT = ACGT > TCGA. In contrast, within regions of (AT),,, the ligand binds best to AGCT; binding to TCGA and TGCA is no stronger than to alternating AT. Within (ATT),, the preference is for ACGT > TCGA. Although each of these binding sites contains all four base pairs, there is no apparent consensus sequence, suggesting that the selectivity is affected by local DNA dynamic and structural effects. At higher drug concentrations ( > 25 pM), nogalamycin prevents DNAse-I cleavage of (AT), but shows no interaction with regions of (AC), . (GT),. Regions of (A), . (T)", which are poorly cut by DNase I, show enhanced rates of cleavage in the presence of low concentrations of nogalamycin, but are protected from cleavage at highcr concentrations. We suggest that this arises because drug binding to adjacent regions distorts the DNA to a structure which is more readily cut by the enzyme and which is better able to bind further ligand molecules.The antitumour antibiotic nogalamycin ( Fig. 1) is unusual in that it possesses bulky groups at both ends of its chromophore [l], yet still binds to DNA by intercalation. Nogalamycin appears to bind by insertion between the base pairs, positioning its sugar residues in both major and minor grooves. Although this model is sterically feasible, it presents considerable dynamic problems, since the minimum width of the antibiotic is 1.2 nm, and it is not possible to open up a potential intercalation site beyond about 1 .0 nm. The DNA therefore needs to be disrupted before the drug can bind, and, as a result, the antibiotic may discriminate between sequences on the basis of their dynamic properties. It binds fastest to those sites that are easiest to disrupt [2], yet dissociates more slowly (half-life, 100-10000 s) from the most stable regions Previous footprinting studies have demonstrated that nogalamycin binds best to regions of alternating purines and pyrimidines, especially when these contain all four DNA bases [4, 51. Regions of alternating AT or GC do not present the best binding sites; the clearest footprints are found around the dinucleotide TpG, especially at the sequence TGC. An in vitro transcription assay also suggested TpG as the preferred binding site [6]. However this selectivity is not absolute, and at higher concentrations the drug binds to almost all sequences, except for poly(dA), in common with many other intercalating drugs. Various NMR [7 -101 and X-ray crystallographic [ll-141 studies have been p...