1H NMR study of complex formation between ethidium bromide and palindromic sequence 5′-d(GpCpGpApApGpC) in aqueous solution

Complex formation between the phenanthridine dye ethidium bromide and a palindromic heptadeoxynucleotide sequence 5'-d(GpCpGpApApGpC), which can form a hairpin, in water-salt solution was studied by one-dimensional H-1, and two-dimensional H-1 and P-31 NMR spectroscopy. Two-dimensional homonuclear H-1 NMR spectroscopy (2D TOCSY and 2D NOESY) and heteronuclear H-1-P-31 NMR spectroscopy were used to completely assign the signals of protons and phosphorus nuclei, and to examine the ethidium bromide interaction with heptanucleotide. The concentration (at 298 and 308 K) and temperature (in the range 278-353 K) dependences of the chemical shifts of protons were obtained. Various complexation schemes taking into account different molecular associates in solution were analyzed. The equilibrium constants and the limit chemical shifts of ethidium bromide protons in complexes were determined. The portions of complexes of different types (dye complexes with monomeric, hairpin, and dimeric forms of heptanucleotide) in solution were found, and specific features of the dynamic equilibrium were revealed as functions of the heptanucleotide-to-dye concentration ratio and temperature. From the calculated induced chemical shifts of ethidium bromide protons and the two-dimensional NMR data, the most probable structures of intercalated complexes with ethidium bromide bound to d(GC) sites of the hairpin stem and dimer of heptanucleotide were constructed.