500 MHz NMR spectroscopy has been used to investigate the complexation of the anthracycline antibiotic daunomycin (DAU) with self-complementary deoxytetranucleotides, 5'-d(CGCG), 5'-d(GCGC), 5'-d(TGCA), 5'-d(ACGT) and 5'-d(AGCT), of different base sequence in aqueous salt solution. 2D homonuclear 1H NMR spectroscopy (TOCSY and NOESY) and heteronuclear 1H - 31P NMR spectroscopy (HMBC) have been used for complete assignment of the non-exchangeable protons and the phosphorus resonance signals, respectively, and for a qualitative determination of the preferred binding sites of the drug. Analysis shows that DAU intercalates preferentially into the terminal sites of each of the tetranucleotides and that the aminosugar of the antibiotic is situated in the minor groove of the tetramer duplex, partly eclipsing the third base pair. A quantitative determination of the complexation of DAU with the deoxytetranucleotides has been made using the experimental concentration and temperature dependences of the drug proton chemical shifts; these have been analysed in terms of the equilibrium reaction constants, limiting proton chemical shifts and thermodynamical parameters (enthalpies deltaH, entropies deltaS) of different drug-DNA complexes (1:1, 1:2, 2:1, 2:2) in aqueous solution. It is found that DAU interacts with sites containing three adjacent base pairs but does not show any significant sequence specificity of binding with either single or double-stranded tetranucleotides, in contrast with other intercalating drugs such as proflavine, ethidium bromide and actinomycin D. The most favourable structures of the 1:2 complexes have been derived from the induced limiting proton chemical shifts of the drug in the intercalated complexes with the tetranucleotide duplex, in conjunction with 2D NOE data. It has been found that the conformational parameters of the double helix and the orientation of the DAU chromophore in the intercalated complexes depend on base sequence at the binding site of the tetramer duplexes in aqueous solution.

Two component (ethidium bromide-caffeine, ethidium bromide-DNA) and three component (ethidium bromidecaffeine-DNA) systems in aqueous saline (0.01 M NaCl) phosphate buffer solutions (pH 6.86, T=298 K) are studied spectrophotometrically. The equilibrium constants for dimerization of caffeine, K D = 1.22 ± 2 M -1 , and for heteroassociation of ethidium bromide with caffeine, K = 71 ± 8 M -1 , in ethidium bromide-caffeine systems are determined. When the concentration of caffeine is increased, the dynamic equilibrium of the solution shifts toward formation of heterocomplexes which are, presumably, stabilized by dispersive and hydrophobic interactions of chromophores. The equilibrium parameters for ethidium bromide complex formation with DNA are calculated: the coupling constant for the dye with the biopolymer, K 1 = (232 ± 16)⋅10 3 M -1 , and the number of base pairs of the biopolymer participating in bonding with the ligand, n 1 = 3.6 ± 0.2, are calculated. Given these values, it is suggested that under these experimental conditions there are two types of bonding between ethidium bromide and the nucleic acid -intercalation and "external" bonds. A McGhee-von Hippel model for a three component system and the numerical values of the parameters for molecular complex formation in two component systems are used to calculate the bonding constant for caffeine with DNA, K 2 = 127 ± 30 M -1 , and the number of base pairs of DNA which bond with caffeine, n 2 = 1.7 ± 0.2. The concentrations of ethidium bromide and caffeine in the composition of two and three component complexes are calculated as functions of the nucleic acid content in the solution. An analysis of the concentration dependences shows that heteroassociation of ligands has a significant effect on the reduction in the concentration of ethidium bromide-DNA complexes in a three component system for low DNA concentrations, while at high DNA concentrations the bonding of caffeine with the biopolymer has this effect.

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Methods developed for analysing the concentration and temperature dependences of NMR experimental parameters of drugÈ nucleic acid complexation in solution have been used to study the binding of the drug ethidium bromide (EB) with singlestranded (ss) DNA oligomers. Non-self-complementary (nsc) deoxytetranucleotide triphosphates of di †erent base sequence, 5@-d(CpGpApA), 5@-d(ApApGpC), 5@-d(CpTpGpA) and 5@-d(GpApApG) have been used as model systems of ss nucleotide sequences. 1D and 2D 1H NMR spectroscopy (500 and 600 MHz) have been used to investigate self-association of the deoxytetranucleotides, and their complexation with the drug in aqueous solution. 2D homonuclear 1H NMR spectroscopy (2D-TOCSY and 2D-NOESY) was used for complete assignment of the proton signals of the deoxytetranucleotides and to determine qualitatively the binding sites of the dye with tetramers.Experimental results for self-association of the tetranucleotides have been analysed using the dimer model. It has been shown that there is a relatively low probability of dimer formation for nsc compared with self-complementary (sc) tetranucleotides, so that complexation of the drug with ss tetranucleotides is expected to dominate the complex equilibrium in solution. The results show that dimerisation constants for nsc deoxytetranucleotides depend on the base sequences, being higher when there is the possibility of base-pairing in the tetranucleotide sequence. Thermodynamic parameters *G, *H and *S for the dimerisation reactions of nsc tetranucleotides have also been determined and conÐrm the role of base sequences in dimer formation.NMR data for EB complexation with nsc deoxytetranucleotides of di †erent base sequence have been interpreted in terms of equilibrium reaction constants and limiting proton chemical shifts of di †erent complexes (1 : 1, 1 : 2 and 2 : 1) in aqueous solution. Analysis of the relative content of the di †erent complexes has been made and speciÐc features of the dynamic equilibrium have been revealed as a function of the ratio of the drug and tetranucleotide concentrations. The results show that there is a sequence-speciÐc binding of EB with ss DNA and that the pyrimidineÈpurine sequence is preferred, especially the d(CG)-site. However, it is found that the di †erences in binding affinities of EB to di †erent sites containing alternating base sequence in the chain are not as great as for drug intercalation to the duplex. A much lower probability of binding is observed for formation of EB complexes with sites of ss sequence containing identical types of bases in the chain.The experimentally determined induced chemical shifts have been analysed in terms of the structures of the complexes. The most favourable structures of the 1 : 1 drugÈtetranucleotide complexes have been calculated taking into account that two di †erent orientations of the drug chromophore with respect to its longitudinal axis occur with equal probability in the 1 : 1 EBÈ tetranucleotide complexes. The results conÐrm that complexes of the dye with ss sequence...

The thermodynamical parameters (free energy, enthalpy, and entropy) of complex formation between ethidium bromide and single‐stranded and double‐stranded tetranucleotides of different base sequence [5′‐d(TpGpCpA), 5′‐d(ApCpGpT), and 5′‐d(ApGpCpT) have been determined from the temperature dependencies of 500 MHz proton nmr chemical shifts. The analysis enables the contributions to be differentiated for the formation of different types of complexes (1:1, 2:1, 1:2, and 2:2) in aqueous solution. The results have been interpreted in terms of the main types of intermolecular interactions responsible for formation of the different complexes; van der Waals and electrostatic interactions are important for formation of complexes of ethidium bromide with single‐stranded tetranucleotides, whereas van der Waals and hydrophobic interactions play a significant role in the binding of the dye to the tetramer duplexes. © 1997 John Wiley & Sons, Inc. Biopoly 42: 285–295, 1997