1H NMR study of the interaction between ethidium bromide and self-complementary and self-complementary deoxytetranucleotide 5′-d(CpGpCpG) in an aqueous solution

Abstract

Complexation of the ethidium bromide (3,8-diamino-6-phenyl-5-ethylphenanthridine) dye with self-complementary deoxytetraribonucleoside triphosphate 5′-d(CpGpCpG) in a water-salt solution is studied by one- and two-dimensional1H NMR spectroscopy (500 MHz). Two-dimensional PMR spectroscopy (2D-COSY and 2D-NOESY) is used for the full assignment of proton signals of the molecules in the solution and for the qualitative analysis of the interaction between ethidium bromide and the tetranucleotide. The concentration dependences of the proton chemical shifts of the molecules at a fixed temperature (T=308 K) are measured. Different schemes of the formation of the dye complexes with the tetranucleotide, taking into account various molecular associations in the solution are considered. The equilibrium constants of the reactions and the limiting chemical shifts of ethidium bromide protons in the complexes are determined. The relative contents of different complexes in the solution are analyzed, and the dynamic equilibrium is studied as a function of the dye-tetranucleotide ratio in the solution. The data obtained suggest that ethidium bromide (like the acridine dye proflavine) is intercalated predominantly into the pyrimidine-purine sections (CG sites) of the tetranucleotide duplex. However, ethidium bromide is intercalated on the side of the narrow slot of the duplex, while proflavine intercalation occurs through the broad slot of the double helix. The most likely structures of the 1∶2 and 2∶2 dye-tetranucleotide complexes in the solution are constructed using the calculated values of the induced chemical shifts of ethidium bromide protons and 2D NOESY spectroscopy data.