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Synthesis of Wavelength-shifting DNA Hybridization Probes by Using Photostable Cyanine Dyes
Published on: July 6, 2016
1H-NMR structural analysis of ethidium bromide complexation with self-complementary deoxytetranucleotides
D B Davies1, L Karawajew, A N Veselkov
1Department of Chemistry, Birkbeck College, University of London, UK.
Abstract:
Complexation of the trypanocidal drug, ethidium bromide (EB), and the self-complementary deoxytetraribonucleoside triphosphates, 5'-d(ApCpGpT), 5'-d(ApGpCpT), and 5'-d(TpGpCpA), in aqueous salt solution has been investigated using one-dimensional and two-dimensional 500/600 MHz 1H-nmr spectroscopy. Six hundred megahertz two-dimensional homonuclear 1H-nmr spectroscopy (nuclear Overhauser effect spectroscopy) was used for a qualitative determination of the structures of EB binding with the deoxytetranucleotides. Concentration dependencies of proton chemical shifts of the molecules have been measured at constant temperatures (T = 303 or 308 K). Different successive schemes of complex formation between the dye molecule and the tetranucleotides have been examined by taking into account various molecular associations in solution, viz., 1:1, 1:2, 2:1 and 2:2 complexes. Equilibrium reaction constants and the limiting proton chemical shifts in the complexes have been determined. The relative contributions of different types of complexes in the equilibrium mixture have been determined and special features of the dynamic equilibrium have been revealed by analysis of chemical shifts as a function of both the dye and tetranucleotide concentrations. The present analysis leads to the conclusion that EB binds preferentially to the pyrimidine-purine sites of the tetranculeotide duplexes. The results show that the energy of EB binding depends on the base content in the pyrimidine-purine sites of the tetramers and on the nucleotide residuals flanking the preferential site. The most favorable structures of the 1:2 and 2:2 complexes of the dye with the tetranucleotides have been constructed using calculated values of induced chemical shifts of EB protons in conjunction with intermolecular nuclear Overhauser effects. The structures of the EB: tetranucleotide complexes depend on tetramer base sequence and are characterized by differences in helix parameters.
