substituent groups must be threaded through the base pair stack to the opposite side of the helix. This more
complicated binding mechanism is reflected in the kinetics of association and dissociation. The sub-
stituents that are threaded through the base pair at the intercalation site are often bulky and/or polar and
hence this may represent a kinetically unfavourable step in the binding mechanism. Examples of interca-
lators that bind to DNA via a threading mechanisminclude naphthalene–bisamides, cationic porphyrins,
and the anticancer antibiotic nogalamycin(Figure 9.11).
The binding mechanism for simple intercalators such as ethidium and proflavin involves just two steps.
First, the cationic intercalator forms a complex with the negatively charged sugar–phosphate backbone of
DNA through electrostatic interactions. Then the intercalator diffuses up and down the helix in the anionic
potential along the surface of the DNA until it comes across gaps in between adjacent base pairs that have
formed due to the normal thermal or “breathing” motions of the DNA. This results in separated base pairs
that form a cavity into which the simple intercalator can bind.
By contrast, threading intercalators, with their large and polar distal-substituted side chains or rings, neces-
sarily involve much larger openings in the DNA base pair stack (i.e.a bubble-type structure), and these may
only form after significant distortions and/or breaking of inter-base hydrogen bonds. These larger and more
drastic dynamic motions in the helix occur with low frequency and hence the association kinetics for threading
intercalators is much slower than for simple intercalators. Thus both association and dissociation rate constants
should decrease relative to classical intercalation. In addition, threading of cationic side chains through a DNA
duplex requires sequential formation and breakage of electrostatic interactions in the threading complex.
Hence the effect of salt concentration on observed rate constants can be used to probe for the threading mode.
Although threading intercalators have more complex binding mechanisms with potentially unfavourable
steps, they can still bind to DNA with high affinity. Once the side chain manages to traverse the intercalation
site, the DNA can adopt a conformation that gives a large and favourable free energy for complex formation.
The side chains present a kinetic barrier to binding but favourable interactions, both in the intercalation site
and in the grooves, give rise to a stable final complex. The nature and extent of the kinetic barrier depends
upon the size of the side chain, its orientation and polarity.
The nogalamycin chromophore is intercalated, with the uncharged nogalose sugar (the aglycone group)
in the minor groove and the positively charged amino sugar in the major groove (Figure 9.11). In the
drug–DNA complex, hydrogen bonds are observed between the drug and N-2 and O-6 atoms of a guanine.
Hence there is simultaneous direct readout of sequence information in both the major and minor grooves.
Nogalamycin has a binding preference for N G or C N sequences, where N can be any base, but the drug
intercalates preferentially at the 5 -side of guanine or at the 3 -side of cytosine. The two sugars point in the
Reversible Small Molecule–Nucleic Acid Interactions 359
Figure 9.11 Structures of three intercalators that bind to DNA by a threading mode. (a) Naphthalene–bisimide.
(b) Nogalamycin. (c) Cationic porphyrin