planar to the benzimidazole ring to which it is attached. The O-4 atoms of the deoxyribose sugar of the
DNA backbone are in the correct position to make favourable interactions with the -electron system of
the ligand as it sits within van der Waals contact distance of the groove walls. The differences between
observed structures in NMR and X-ray experiments arise probably because of the different low-energy
conformations that this ligand can adopt when bound to various DNA sequences.
9.7.5.2 Thermodynamics of Hoechst 33258–DNA Interactions. The fluorescent properties of
Hoechst 33258 can be exploited to study the DNA binding character of this dye. Equilibrium experi-
ments show that Hoechst 33258 binds tightly to calf-thymus DNA, poly(dA-dT) poly(dA-dT), and to the
oligonucleotides A2T2 and d(CCGGAATTCCGG) 2 with Kobsvalues in the range 1–3 109 M^1 .45,46
However, this tight binding occurs only once every 100 base pairs of calf-thymus DNA. In fact Hoechst
33258 binds to calf-thymus DNA with a spectrum of different binding modes each characterized by a dif-
ferent affinity, with some values of Kobsin the 106 M^1 range. Similarly with poly(dA-dT) poly(dA-dT),
Job Plots reveal a multiplicity of binding stoichiometries with between 1 and 6 Hoechst 33258 molecules
bound per 5 A T base pairs as well as stoichiometries that suggest 1 or 2 ligand molecules are bound per
DNA phosphate.
Thus Hoechst 33258 binding to DNA is complex and depends strongly on sequence and DNA length.
At least four distinct types of interaction between Hoechst 33258 and DNA are seen:
Sequence-mediated: specific, high affinity, binding observed in X-ray structures and footprinting
studies;
Charge-mediated: non-specific interaction that involves the electrostatic binding of monocationic
Hoechst 33258 with the DNA phosphate backbone;
Ligand-mediated: non-specific type of binding that occurs when a molecule of free Hoechst 33258
in solution binds to other dye molecules that are already associated with the DNA helix, either in
the minor groove or the backbone; and
Structure-mediated: either specific or non-specific binding – a mode that confines Hoechst 33258
to a specific structural region, such as the minor groove, because of some geometric requirement.The differences between these four modes of binding are subtle. They are not totally separate entities because
more than one type of interaction contributes to the overall affinity of each bound ligand.
Sequence mediated (specific) binding of Hoechst 33258 to DNA has been examined in some detail
using d(CGCAAATTTGCG) 2 (A3T3) as the receptor.47–49A detailed partitioning of the overall observed
free energy for the Hoechst 33258–A3T3 interaction (Section 9.6.4) has been carried out by measuring, as
directly as possible, a near complete thermodynamic profile for the interaction. This used a combination
of ITC to measure H and Cpand fluorescence titrations to measure Kobsand hence Gobs. From these
measurements T Sand Gpewere calculated using standard thermodynamic relationships. The measured
binding constant of 3.2 0.6 108 M(duplex)^1 at salt concentration of ca.12 mM is similar to the Hoechst
33258 binding affinity (Kobs3.15 108 M(duplex)^1 ) determined by Clegg under similar conditions for
the less extended A T-tract dodecamer d(CGCGAATTCGCG) 2. Quantification of the salt dependence
yielded a value for SK of 0.99, which is consistent with the monocationic charge of Hoechst 33258. This
allows the overall observed binding free energy ( Gobs) to be broken down into its component polyelectrolyte
and nonpolyelectrolyte terms (Section 9.3). The analysis showed that from a Gobsof 48.9 kJ mol^1 ,
some 7.5 kJ mol^1 (about 15%) is Gpe, i.e.from coupled polyelectrolyte effects such as the release of
condensed counter-ions from the DNA upon ligand binding.
X-ray crystal structures of this complex show a network of hydrogen bonds and other minor groove
interactions involving van der Waals contacts and hydrophobic interactions. The binding free energy
should thus reflect a large nonpolyelectrolyte component. This is borne out by experimental data showing
that out of a total free energy of 48.9 kJ mol^1 , some 41.4 kJ mol^1 is due to non-polyelectrolyte
effects. At each temperature studies, binding enthalpies for the interaction of A3T3 with Hoechst 33258
are positive and their magnitudes decrease with increasing temperature ( H42.7 kJ mol^1 at 9.3°C
368 Chapter 9