Methods developed for analysing the concentration and temperature
dependences of NMR experimental parameters of drug–nucleic acid
complexation in solution have been used to study the binding of the
drug ethidium bromide (EB) with single-stranded (ss) DNA oligomers.
Non-self-complementary (nsc) deoxytetranucleotide triphosphates of
different 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
1
H 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
1
H 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 ΔG,
ΔH and ΔS 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 interpreted in terms of equilibrium
reaction constants and limiting proton chemical shifts of different
complexes (1:1, 1:2 and 2:1) in aqueous solution. 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 concentrations. The
results show that there is a sequence-specific binding of EB with ss
DNA and that the pyrimidine–purine sequence is preferred,
especially the d(CG)-site. However, it is found that the differences
in binding affinities of EB to different 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 drug–tetranucleotide complexes
have been calculated taking into account that two different
orientations of the drug chromophore with respect to its longitudinal
axis occur with equal probability in the 1:1 EB–tetranucleotide
complexes. The results confirm that complexes of the dye with ss
sequences have substantially higher conformational freedom compared to
complexes with sc tetranucleotide duplexes.
The enthalpies and entropies of complex formation between EB and
nsc deoxytetranucleotides have been determined from the temperature
dependence of the 500 MHz
1
H NMR chemical shifts. The
contributions have been determined for formation of the different
types of complexes (1:1, 2:1 and 1:2) in solution. The nature of the
intermolecular interactions involved in EB complexation with single
strands and dimers of nsc tetramers of different base sequence is
discussed.
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