H-1-NMR STUDY OF THE INTERACTION BETWEEN ETHIDIUM-BROMIDE AND SELF-COMPLEMENTARY DEOXYTETRANUCLEOTIDE 5'-D(CPGPCPG) IN AN AQUEOUS-SOLUTION

Complexation of the ethidium bromide (3,8-diamino-6-phenyl-5-ethylphen anthridine) dye with self-complementary deoxytetraribonucleoside triph osphate 5'-d(CpGpCpC) in a water-salt solution is studied by one- and two-dimensional H-1 NMR spectroscopy (500 MHz). Two-dimensional PMR sp ectroscopy (2D-COSY and 2D-NOESY) is used for the full assignment of p roton signals of the molecules in the solution and for the qualitative analysis of the interaction between ethidium bromide and the tetranuc leotide. The concentration dependences of the proton chemical shifts o f the molecules at a fired temperature (T = 308 K) are measured Differ ent schemes of the formation of the dye complexes with the tetranucleo tide, taking into account various molecular associations in the soluti on 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 so lution are analyzed and the dynamic equilibrium is studied as a functi on of the dye-tetranucleotide ratio in the solution. The data obtained suggest that ethidium bromide (like the acridine dye proflavine) is i ntercalated predominantly into the pyrimidine-purine sections (CG site s) of the tetranucleotide duplex. However, ethidium bromide is interca lated an the side of the narrow slot of the duplex, while proflavine i ntercalation occurs through the broad slot of the double helix. The mo st likely structures of the 1:2 and 2:2 dye-tetranucleotide complexes in the solution are constructed using the calculated values of the ind uced chemical shifts of ethidium bromide protons and 2D NOESY spectros copy data.