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

The new ligand 6 is structurally related to ethidium bromide (EB) and other diazapyrenium compounds (1, 2) studied earlier, but exhibits significant d ifferences. In contrast to ligands such as 1 and 2 the new 6 is stable over a large pH range. At concentrations >10(-4) mol dm(-3) 6 forms a dimer, wh ich is characterised by NMR analysis. The association constants of 6 are la rger with purine than with pyrimidine nucleotides and are the same for AMP, ADP or ATP, with an absence of any charge effects. Surprisingly, under con ditions close to saturation of intercalation binding sites association of 6 with single-stranded poly U and poly A is 100 to 1000 times more efficient than that of EB or analogue 2. Also the affinity of 6 toward poly U is sig nificantly higher than toward poly C. CD spectra indicate that stacking of 6 with poly U induces a strong helicity in the usually disordered structure of this single strand. The binding of 6 to poly C is pH dependent, as a co nsequence of the known formation of a poly CH+-poly CH+ double strand at pH <5. With double-stranded polynucleotides the binding affinity of 6 and EB i s similar for RNA homopolymers. Striking fluorescence differences, however, are observed with complexes of 6-poly GC (decrease of emission intensity o f 6 by 80%), and 6-poly AU (increase by 100%). Similar effects are also obs erved for the DNA polynucleotides. At higher ligand to phosphate ratios (r> 0.2) 6 shows with double strands electrostatic binding in addition to inter calation, with ensuing opposite effects in fluorescence emission. The bipha sic melting profiles of poly dA-poly dT with 6 differ sharply from those ob served with 1 and EB and are in line with the dependence of binding modes o bvious from the fluorescence studies. Melting analyses show with calf thymu s DNA and with poly dA-poly dm an increase (Delta DeltaT) in melting temper ature with 6 about twice as high as that with 1 or EB. With the RNA model p oly A-poly U the DeltaT(m) values of 6 at low ionic strength are about seve n times higher than those observed with 2 or with EB. In contrast to other 4,9-diazapyrenium compounds which show with poly A-poly U a destabilising a nd a stabilising step, 6 gives only positive DeltaT values at both pH = 5.0 and pH = 7.0. Extensive supporting data for this paper are available as su pplementary material dagger in electronic form, allowing re-evaluation of e xperimental results with suitable programs for curve fitting.