A new 4,9-diazapyrenium intercalator for single- and double-stranded nucleic acids: distinct differences from related diazapyrenium compounds and ethidium bromide

摘要

The new ligand 6 is structurally related to ethidium bromide (EB) and other diazapyrenium compounds (1, 2)nstudied earlier, but exhibits significant differences. In contrast to ligands such as 1 and 2 the new 6 is stable over anlarge pH range. At concentrations > 10u00014 mol dmu00013 6 forms a dimer, which is characterised by NMR analysis. Thenassociation constants of 6 are larger with purine than with pyrimidine nucleotides and are the same for AMP, ADPnor ATP, with an absence of any charge effects. Surprisingly, under conditions close to saturation of intercalationnbinding sites association of 6 with single-stranded poly U and poly A is 100 to 1000 times more efficient than thatnof EB or analogue 2. Also the affinity of 6 toward poly U is significantly higher than toward poly C. CD spectranindicate that stacking of 6 with poly U induces a strong helicity in the usually disordered structure of this singlenstrand. The binding of 6 to poly C is pH dependent, as a consequence of the known formation of a poly CHu0002–polynCHu0002 double strand at pH < 5. With double-stranded polynucleotides the binding affinity of 6 and EB is similarnfor RNA homopolymers. Striking fluorescence differences, however, are observed with complexes of 6–poly GCn(decrease of emission intensity of 6 by 80%), and 6–poly AU (increase by 100%). Similar effects are also observednfor the DNA polynucleotides. At higher ligand to phosphate ratios (r > 0.2) 6 shows with double strands electrostaticnbinding in addition to intercalation, with ensuing opposite effects in fluorescence emission. The biphasicnmelting profiles of poly dA–poly dT with 6 differ sharply from those observed with 1 and EB and are in line withnthe dependence of binding modes obvious from the fluorescence studies. Melting analyses show with calf thymusnDNA and with poly dA–poly dT an increase (ΔΔT) in melting temperature with 6 about twice as high as that withn1 or EB. With the RNA model poly A–poly U the ΔTm values of 6 at low ionic strength are about seven times highernthan those observed with 2 or with EB. In contrast to other 4,9-diazapyrenium compounds which show with polynA–poly U a destabilising and a stabilising step, 6 gives only positive ΔT values at both pH = 5.0 and pH = 7.0.nExtensive supporting data for this paper are available as supplementary material † in electronic form, allowingnre-evaluation of experimental results with suitable programs for curve fitting.