GC-BASE SEQUENCE RECOGNITION BY OLIGO(IMIDAZOLECARBOXAMIDE) AND C-TERMINUS-MODIFIED ANALOGS OF DISTAMYCIN DEDUCED FROM CIRCULAR-DICHROISM, PROTON NUCLEAR-MAGNETIC-RESONANCE, AND METHIDIUMPROPYLETHYLENEDIAMINETETRAACETATE-IRON(II) FOOTPRINTING STUDIES

The DNA binding properties of a series of imidazole-containing and C-terminus-modified analogues 4-7 of distamycin are described. These analogues contain one to four imidazole units, respectively. Data from the ethidium displacement assay showed that these compounds bind in the minor groove of DNA, with the relative order of binding constants of 6 (Im3) > 7 (Im4) > 5 (Im2) > 4 (Im1). The reduced binding constants of these compounds for poly(dA-dT) relative to distamycin, while they still interact strongly with poly(dG-dC), provided evidence of GC sequence acceptance. The preferences for GC-rich sequences by these compounds were established from a combination of circular dichroism (CD) titration, proton nuclear magnetic resonance (H-1-NMR), and methidiumpropylethylenediaminetetraacetate-iron(II) [MPE.Fe-(II)] footprinting studies. In the CD studies, these compounds produced significantly larger DNA-induced ligand bands with poly(dG-dC) than poly(dA-dT) at comparable ligand concentrations. H-1-NMR studies of the binding of 5 to d-[CATGGCCATG] 2 provided further evidence of the recognition of GC sequences by these compounds, and suggested that the ligand was located on the underlined sequence in the minor groove with the C-terminus oriented over the T residue. MPE footprinting studies on a GC-rich BamHI/SalI fragment of pBR322 provided unambiguous evidence for the GC sequence selectivity for some of these compounds. Compounds 4 and 7 produced poor footprints on the gels; however, analogues 5 and 6 gave strong footprints. Detailed densitometric analyses of the gels showed that the preferred binding sites of 5 was at 5'-(G.C)3(A.T), i.e., three GC based pairs followed by an AT base pair, whereas the strongest footprint sites for 6 were within two occurrences of the sequence 5'-TCGGGCT. These results also showed that 5 and 6 bind to different sequences on the same DNA fragment, even though they differ by only one imidazole unit. Molecular modeling experiments were performed to rationalize the lower binding affinity of tetraimidazole analogue 7 compared to triimidazole 6. The radius of curvature of 7 (13 +/- 1 angstrom) was found to be higher than the other compounds (15 +/- 1 and 17 +/- 1 angstrom for 5 and 6, respectively), and this presumably hinders its interaction with the convex surface of the B-DNA conformation of d-[G4.C4], which has a curvature of 19 +/- 1 angstrom.