Biopolymers and cell. 1996; 12 (6): 36 - 49

H-NMR investigation of complex fonnation of ethidium bromide with single-stranded non-complementary deoxytetranucleotide S'-d(CpGpApA) in aqueous solution

A. N. Vesetkov, S. F. Baranovsky, L. N. Djimant, N. V. Petrenko, S. G. Osetrov, A. Tucker, D. B. Davies

Complex formation between the aromatic dye ethidium bromide (3,8-diamino-6-phenyl-5-ethyl-phenan-thridine) and single-stranded non-complementary deoxytetranucleotide 5'-d(CpGpApA) in aqueous so¬lution has been studied by one- and two-dimensional H-NMR spectroscopy (500 and 600 MHz). Con¬centration dependences of proton chemical shifts of the interacting molecules were measured at different temperatures Tt - 298 K and T2 = 305 K. Investigations of the self-association of the tetranucleotide molecules have shown that there is a very tow probability of duplex formation at the experimental conditions studied. Itfollows that the complexations of the dye molecules withthe monomer of'thetetranucleotide play the main rote in the complex equilibrium in solution giving an opportunity to analyze the specificity of interactions of aromatic ligand with the single-stranded DNA. Different schemes of complex formation have been examined, equilibrium reaction constants and limiting chemical shifts of dye protons in different complexes have been determined. Analysis of the relative content of different types of complexes was made and special features of the dynamic equilibrium were revealed as a function of both the dye and tetra¬nucleotide concentrations. The analysis has shown that there is a sequence-specific binding of ethidium bromide withthe single-stranded oligonucleotide. The mostfavorablestructures of1:1 complexes of'thedye with the single strand of the tetranucleotide corresponding to the drug binding from the opposite aromatic rings of the phenanthridine chromophore were constructed using the calculated values of induced chemical shifts of ethidium bromide protons.