Actinomycin D binding to single-stranded DNA: sequence specificity and hemi-intercalation model from fluorescence and...

We have studied the sequence specificity in the binding of the potent antitumor drug actinomycin D (AMD) to single-stranded DNA (ssDNA) by fluorescence and NMR spectroscopy and by molecular modeling. The significant absorption and emission changes accompanying the interaction of the fluorescent derivative 7-amino-AMD with DNAs varying in length and base composition were used to calculate affinity constants for the drug-DNA complexes. The guanine-containing trinucleotide sequences AGT, AGA, and TGT embedded within 25-base oligonucleotides, constituted favorable binding sites. In contrast, the sequence TGA did not bind the drug appreciably. Among the DNAs studied, the highest affinity was for the tetranucleotide sequence TAGT. The binding was length dependent, an oligonucleotide of at least 14 bases being required for effective complex formation (Ka > 10(4) M1=). AMD also bound to poly(d(AGT)). Gel electrophoresis confirmed that the complex was formed between the drug and individual unstructured DNA strands. The 1H NMR spectra of oligonucleotides containing the TAGT site and their complexes with AMD provided further insight into the mode(s) of interaction. A comparison of the measured chemical shifts with those estimated from ring-current calculations provided strong evidence for a hemi-intercalation of AMD between the A and G purine bases with a preference for one of two possible relative orientations. The latter were modeled as complexes with the sequence T3AGT3 and refined by force field calculations with the AMBER program. The biological implications for this novel form of interaction of AMD with single-stranded DNA are discussed.