DNA affinity binding studies using a fluorescent dye displacement technique: the dichotomy of the binding site

Abstract

We have observed a number of discrepancies and contradictions in the use of a fluorescent intercalator displacement assay in surveying the binding affinities of dinuclear polypyridyl ruthenium(II) complexes with DNA. By a modification of the assay using the fluorescent minor-groove binder 4',6-diamidino-2-phenylindole, rather than intercalating dyes (ethidium bromide or thiazole orange), results were obtained for all complexes studied which were consistent with relative affinities and stereoselectivities observed with other techniques, including NMR, affinity chromatography and equilibrium dialysis. It is believed that the difference in binding mode between the minor groove-binding Ru(II) complexes and the intercalating fluorescent dyes they are displacing may contribute to these discrepancies.

Synthesis, characterization and crystal structures of two pentagonal-bipyramidal Fe(III) complexes with dihydrazone of 2,6-diacetylpyridine and Girard's T reagent. Anticancer properties of various metal complexes of the same ligand

“…(2) (see Experimental section).The plots displayed a good linear relationship for the investigated concentration ranges of 4, The quenching constants for 4, 6 and 8, were calculated as K= 0.5979, K = 0.5221 and K = 0.5964, respectively. These data indicate the displacement of ethidium bromide from system EBCT-DNA by the complexes, but in view of UV/Vis absorption spectral results it is most likely not a consequence of intercalation, but of structural changes in DNA arising from electrostatic binding, which reduce the binding affinity of EB[49].…”

Oligonuclear polypyridylruthenium(ii) complexes incorporating flexible polar and non-polar bridges: synthesis, DNA-binding and cytotoxicity

“…The binding affinities of the series of complexes with the single-bulge sequence d(CGCGATATAGCGC)•d(GCGCTA-TATACGCG) {designated SB} and its non-bulged canonical control oligonucleotide sequence (formed from 5¢-CGCGATATATGCGC and 3¢GCGCTATATACGCG) were studied by means of a DAPI fluorescence displacement assay, 9 undertaken at pH 7.8. Under these conditions, it would be expected that the amine groups in the bbN n ligands would be protonated, 14 Scheme 5 whereas the free bpy-N groups in the mononuclear complexes would be unprotonated.…”

Canonical DNA minor groove insertion of bisbenzamidine–Ru(ii) complexes with chiral selectivity

“…2c and S10 in the ESI†). 24 Linear Dichroism (LD) experiments, which provide information about the orientation of the bound complexes with respect to the DNA,25 were also consistent with minor groove insertion. Thus, the LD spectra of a reference intercalative complex Λ-[Ru(bpy) 2 phen] 2+ show a strong positive LD signal in the B(E)-polarized MLCT band at 440 nm, arising from the placement of the phen ligand almost coplanar to the DNA base pairs and the complex two-fold symmetry axis slightly rotated clockwise (by about 10°) from the ideal intercalation geometry.…”