STRUCTURAL AND THERMODYNAMICAL ANALYSIS OF DRUG-BINDING TO SINGLE-STRANDED-DNA OLIGOMERS - SELF-ASSOCIATION OF NON-SELF-COMPLEMENTARY DEOXYTETRANUCLEOTIDES OF DIFFERENT BASE SEQUENCE AND THEIR COMPLEXATION WITH ETHIDIUM-BROMIDE IN AQUEOUS-SOLUTION

Methods developed for analysing the concentration and temperature depe ndences of NMR experimental parameters of drug-nucleic acid complexati on in solution have been used to study the binding of the drug ethidiu m bromide (EB) with single-stranded (ss) DNA oligomers. Non-self-compl ementary (nsc) deoxytetranucleotide triphosphates of different base se quence, 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 H-1 NMR spectroscopy (500 and 600 MHz) have been used to investigate s elf-association of the deoxytetranucleotides, and their complexation w ith the drug in aqueous solution. 2D homonuclear H-1 NMR spectroscopy (2D-TOCSY and 2D-NOESY) was used for complete assignment of the proton signals of the deoxytetranucleotides and to determine qualitatively t he binding sites of the dye with tetramers. Experimental results for s elf-association of the tetranucleotides have been analysed using the d imer model. It has been shown that there is a relatively low probabili ty of dimer formation for nsc compared with self-complementary (sc) te tranucleotides, so that complexation of the drug with ss tetranucleoti des is expected to dominate the complex equilibrium in solution. The r esults show that dimerisation constants for nsc deoxytetranucleotides depend on the base sequences, being higher when there is the possibili ty of base-pairing in the tetranucleotide sequence. Thermodynamic para meters Delta G, Delta H and Delta S for the dimerisation reactions of nsc tetranucleotides have also been determined and confirm the role of base sequences in dimer formation. NMR data for EB complexation with nsc deoxytetranucleotides of different base sequence have been interpr eted in terms of equilibrium reaction constants and limiting proton ch emical shifts of different complexes (1:1, 1:2 and 2:1) in aqueous sol ution. Analysis of the relative content of the different complexes has been made and specific features of the dynamic equilibrium have been revealed as a function of the ratio of the drug and tetranucleotide co ncentrations. The results show that there is a sequence-specific bindi ng of EB with ss DNA and that the pyrimidine-purine sequence is prefer red, especially the d(CG)-site. However, it is found that the differen ces in binding affinities of EB to different sites containing alternat ing base sequence in the chain are not as great as for drug intercalat ion to the duplex. A much lower probability of binding is observed for formation of EB complexes with sites of ss sequence containing identi cal 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 drug-tetranucleot ide complexes have been calculated taking into account that two differ ent orientations of the drug chromophore with respect to its longitudi nal axis occur with equal probability in the 1:1 EB-tetranucleotide co mplexes. The results confirm that complexes of the dye with ss sequenc es have substantially higher conformational freedom compared to comple xes with sc tetranucleotide duplexes. The enthalpies and entropies of complex formation between EB and nsc deoxytetranucleotides have been d etermined from the temperature dependence of the 500 MHz H-1 NMR chemi cal shifts. The contributions have been determined for formation of th e different types of complexes (1:1, 2:1 and 1:2) in solution. The nat ure of the intermolecular interactions involved in EB complexation wit h single strands and dimers of nsc tetramers of different base sequenc e is discussed.