V. I. Pakhomov

Structural and Thermodynamic Analysis of Daunomycin Binding with Desoxyhexanucleotides with Different Base Sequences by NMR Spectroscopy

Complexation of the anthracycline antibiotic daunomycin (DAU) with self-complementary desoxyhexanucleotides with different base sequences in the chain, 5′-d(CGTACG) and 5′-d(CGCGCG), is studied in aqueous solution. Homonuclear 2D-1H NMR spectroscopy (TOCSY and NOESY) and heteronuclear 1H–31P NMR spectroscopy (HMBC) are used for complete assignment of the nonexchangeable proton resonances and the phosphorus signals and for qualitative determination of preferable DAU binding sites, respectively. Daunomycin is preferably intercalated into the first d(CG)-site of each hexanucleotide, and the aminosaccharide ring of DAU lies in the minor groove of the hexamer duplex, partly overlapping the third base pair. Quantitative analysis of DAU complexation with desoxyhexanucleotides was done by analyzing the concentration and temperature dependences of the DAU proton chemical shifts; the equilibrium reaction constants and the thermodynamic parameters of the formation of the 1:1, 1:2, 2:1, and 2:2 DAU complexes with hexamers, as well as the limiting values of the DAU proton chemical shifts were determined in aqueous solution. The antibiotic preferably binds with the triplet sections of the nucleotide sequence containing two neighboring CG-pairs of nitrogen bases flanked by the AT-pair in 5′-d(CGTACG) compared to the triplets consisting of a sequence of three CG-pairs in the 5′-d(CGCGCG) hexamer. The specific binding of daunomycin depends on the character of the hydrophobic interactions of the aminosaccharide ring of the antibiotic in the minor groove of the DNA double helix. Calculations have been carried out to determine the most probable spatial structures of the 1:2 DAU–desoxyhexanucleotide complexes; the results agree well with X-ray diffraction data.

Analysis of the interaction of ethidium bromide with a DNA octamer 5’-d(GpApCpApTpGpTpC) in aqueous solution using1H NMR data

Complexation of a phenanthridine dye ethidium bromide with a desoxyoligonucleotide 5’-d(GpApCpAp-TpGpTpC) in aqueous salt solution is studied by one- and two-dimensional1H NMR spectroscopy. Two-dimensional correlated homonuclear PMR spectroscopy (2D-TOCSY and 2D-N0ESY) was used for complete assignment of the proton signals of molecules in solution and for qualitative analysis of the character of interaction between ethidium bromide and desoxyoctanucleotide. The concentration dependences of the proton chemical shifts of the molecules were measured at three temperatures (T1 = 298 K, T2 = 308 K, and T3 = 318 K); the temperature dependences were measured in the temperature range 278–358 K. Different schemes of dye complexation with an octamer duplex involving different molecular associates in solution are considered. The equilibrium constants of the reactions, the corresponding thermodynamic parameters (δH0, δS0), and the limiting values of the chemical shifts of ethidium bromide protons in the complexes are determined. The relative contents of complexes of different types in solution (dye complexes with desoxyoctanucleotide in duplex form) are analyzed, and peculiarities of the dynamic equilibrium depending on the ratio of dye and octamer concentrations and temperature are established. The most probable structures of the 1:2 and 2:2 intercalated complexes corresponding to dye intercalation into the pyrimidine-purine sites of the desoxyoctanucleotide duplex are derived using the calculated values of the induced proton chemical shifts of ethidium bromide and two-dimensional PMR data.