Fluorescent intercalator displacement analyses of DNA binding by the peptide-derived natural products netropsin, actinomycin, and bleomycin Original Research Article

The response of the high-throughput fluorescent intercalator displacement (HT-FID) assay reported recently by Boger et al. to peptide-based DNA binding intercalators and metal complexes was examined through the study of actinomycin and Co(III) · bleomycin-B2. Along with a validation of netropsin that illustrated the good laboratory-to-laboratory reproducibility of the assay, our examination of actinomycin revealed results for a four base pair cassette library of DNA hairpins that paralleled the known DNA site-selectivity of this agent and also indicated the involvement of the flanking sequences of the hairpin oligonucleotide. In addition, for Co(III) · bleomycin-B2 the established cleavage site-selectivity for 5′-GT and 5′-GC sites was correlated to drug-DNA association in this binding-only assay; our results also suggest a tetranucleotide site-selectivity for metallobleomycin involving cross-strand, ‘back-to-back’ 5′-GT and 5′-GC sites such as 5′-ACGT and 5′-ACGC.