at temperatures well below the melting temperature to give local, short-range separation of the strands.
This readily reversible process is known as breathing.
The evidence for such dynamic motion comes from chemical reactions, which take place at atoms that are
completely blocked by normal base-pairings. Those used include tritium exchange studies in hydrogen-bonded
protons in base pairs, the reactivity of formaldehyde with base NH groups, and NMR studies of imino–proton
exchange with solvent water. This last technique can be used on a time scale from minutes down to 10 ms. It
shows that in linear DNA the base pairs open singly and transiently with a life time around 10 ms at 15°C.
Because NMR can distinguish between imino- and amino–proton exchange, it can also be used to iden-
tify breathing in specific sequences.^67 Some of the most detailed work of this sort has come from studies
on tRNA molecules, which show that, with increasing temperature, base-triplets (Figure 2.33) are desta-
bilised first followed by the ribothymidine helix and then the dihydrouridine helix. Finally, the acceptor
helix ‘melts’ after the anti-codon helix (Section 7.1.4).
Another possible motion that might be important for the creation of intercalation sites is known as ‘soli-
ton excitation’. The concept here is of a stretching vibration of the DNA chain, which travels like a wave
along the helix axis until, given sufficient energy, it leads to local unstacking of adjacent bases with asso-
ciated deformation of sugar pucker and other bond conformations.
Such pre-melting behaviour may well relate to the process of drug intercalation, to the association of
single-strand specific DNA binding proteins (Section 10.3.8), and to the reaction of small electrophilic
reagents with imino and amino groups such as cytosine-N-3 (Section 8.5).
2.5.3 Energetics of the B–Z Transition
The isomerisation equilibrium between the right-handed B-form and the left-handed Z-form of DNA is
determined by three factors:
- Chemical structure of the polynucleotide (sequence, modified bases)
- Environmental conditions (solvent, pH, temperature, etc.)
- Degree of topological stress (supercoiling, cruciform formation).
Many quantitative data have been obtained from spectroscopic, hydrodynamic and calorimetric studies and
linked to theoretical calculations. Although these have not yet defined the kinetics or complex mechanisms of
the B–Z transition, it is evident that the small transition enthalpies involved lie within the range of the thermal
DNA and RNA Structure 67
Figure 2.44 Renaturation processes (a) for short oligonucleotide and longer homo-polymers and (b) for natural
DNA strands