Nucleic Acids in Chemistry and Biology

interactions are equivalent, with only subtle differences in hydration and ion binding. However, adri-
amycin has a binding affinity for DNA that is 10-fold greater than that for daunomycin. In addition, binding
of adriamycin to DNA is accompanied by a large favourable entropy term, which is the exact opposite of the
situation for daunomycin. As a consequence, the binding enthalpy of adriamycin is significantly less than
that measured for daunomycin, but the free energy is greater for adriamycin. These clear and unambigu-
ous differences in the binding thermodynamics of two very similar drugs are not predicted from examin-
ation of the structures of the two drug–DNA complexes. Thus isostructural does notmean isoenergetic.

9.6.3 The Neighbour Exclusion Principle

Simple intercalators might be expected to occupy every potential intercalation site, i.e.in between every
base pair in a DNA helix. Therefore, at saturation this would give rise to a binding stoichiometry of one
base pair per bound intercalator. Molecular modelling studies where a drug molecule is bound at every
possible intercalation site have led to energy-minimized structures with reasonable backbone torsion
angles. However, solution studies have generated a different picture. In 1962 Cairns was the first to
observe that even simple intercalators, such as proflavin, show saturation with DNA at a stoichiometry of
one drug molecule per 2 base pairs. More complex intercalators such as daunomycin occupy 1 in 3 sites.
Such empirical observations are a reflection of the neighbour exclusionprinciple, which states that inter-
calators can, at most, only bind at alternate possible base pair sites on DNA. Hence there is only ever a
maximum of one intercalator between every second potential binding site. In the first instance, prior to
addition of any ligand, all inter-base pair spaces have an equal potential to be intercalation sites for a non-
specific compound (ignoring sequence preference). However, when an intercalator binds to a certain site,
the neighbour exclusion principle states that binding of additional ligand molecules at sites immediately
adjacent is inhibited.
Many attempts have been made to account for the molecular basis of neighbour exclusion. One possi-
bility is that intercalator binding induces conformational changes at adjacent sites in the DNA and the new
conformation is structurally or sterically unable to accommodate another mono-intercalator. Friedman and
Manning have advanced an explanation based on counter-ion release and electrostatic effects. Briefly,
binding of cationic intercalators has the effect of neutralization of some of the charge on the DNA back-
bone. In other words, binding of a cationic intercalator lengthens the DNA and hence increases the aver-
age charge spacing, and therefore decreases the linear charge density. In consequence, the binding-induced,
energetically favourable release of condensed counter-ions is reduced and thus the observed equilibrium
binding constant is also diminished. This effect leads to curvature in equilibrium binding isotherms that is
similar to that predicted by neighbour exclusion theory. Since the local release of ions at the intercalation
site would be greater than ion release at some distance from the binding site, then the local release of counter-
ions could also lead to a lower free energy of binding at sites next to an intercalation site. In practice, it is likely
that both conformational and electrostatic factors play a role in the neighbour exclusion phenomena. In
addition, other factors may also be responsible for neighbour exclusion, for example, electrostatic repulsion
between proximally bound drugs.
Various mathematical solutions have been developed that allow drug binding data to be described by the
neighbour exclusion model.20,21In one of these approaches, the DNA is assumed to be a lattice sufficiently
long to allow end effects to be ignored while all potential binding sites are assumed to be equivalent.
McGhee and Von Hippel first wrote the neighbour exclusion modelin this form in 1974:

(9.13)

where ris the binding ratio (defined as the ratio of the concentration of bound drug (Cb) over the total con-
centration of DNA binding sites (S), Cfthe concentration of free drug, Kthe equilibrium binding constant for

r

C

Knr

nr

nr

n

f

   

()

()

1

1

11

1













Reversible Small Molecule–Nucleic Acid Interactions 353