ACTINOMYCIN-D-BINDING TO SINGLE-STRANDED-DNA - SEQUENCE SPECIFICITY AND HEMI-INTERCALATION MODEL FROM FLUORESCENCE AND H-1-NMR SPECTROSCOPY

We have studied the sequence specificity in the binding of the potent antitumor drug actinomycin D (AMD) to single-stranded DNA (ssDNA) by f luorescence and NMR spectroscopy and by molecular modeling. The signif icant 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 dr ug-DNA complexes. The guanine-containing trinucleotide sequences AGT, AGA, and TGT embedded within 25-base oligonucleotides, constituted fav orable binding sites. In contrast, the sequence TGA did not bind the d rug appreciably. Among the DNAs studied, the highest affinity was for the tetranucleotide sequence TACT. The binding was length dependent, a n oligonucleotide of at least 14 bases being required for effective co mplex formation (K-a > 10(4) M(-1)). AMD also bound to poly(d(AGT)). G el electrophoresis confirmed that the complex was formed between the d rug and individual unstructured DNA strands. The H-1 NMR spectra of ol igonucleotides containing the TACT site and their complexes with AMD p rovided further insight into the models) 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 pos sible relative orientations. The latter were modeled as complexes with the sequence T(3)AGT(3) 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. (C) 1996 Ac ademic Press Limited