Modern CD spectrapolarimeters are often equipped with Peltier temperature control, automated titration
and stopped-flow accessories which allows CD to be used to provide information on structure, thermo-
dynamics (binding affinity, van’t Hoff enthalpy, free energy) and kinetics (association and dissociation rate
constants).
A closely related technique to CD is linear dichroism(LD). LD is a type of spectroscopy that yields useful
information on DNA conformation in terms of base inclination and flexibility as well as the binding
geometries of drug–DNA complexes. In this technique, the differential absorption of linearly or plane-
polarised light is measured. A key parameter in LD is the transition moment, which is a vectoral property
of light absorption related to a particular direction of the molecule. Light that is polarised parallel to the
transition moment has a high probability of absorption in the region of spectral interest, whereas if light is
polarised perpendicular to the transition moment, no absorption takes place. In practice, this means that
intercalators that stack closely to base pairs have linear dichroism similar to the base pairs themselves.
However, the dichroism of groove binders is frequently opposite to that of the base pairs, since they bind
along the edges of the base pairs. Hence LD is a useful type of spectroscopy for assessing the binding
mode of a drug to DNA.
11.1.4 Infrared and Raman Spectroscop y
Infrared and Raman spectroscopy are often regarded as closely related techniques in which the vibrational
frequencies of localised parts of the sample molecule are observed.7,8Both techniques are largely non-
destructive and can be used on microscopically small samples. A major advantage is that DNA can be analysed
in crystals, gels or fibres, as well as in solution, and this has supported direct correlations between the
observed vibrational frequencies and 3D structures derived by X-ray diffraction.
Infrared absorptions from nucleic acids are observed in the frequency range between 1800 and 700cm^1.
The problem of strong IR absorption by water near 1600cm^1 and also below 1000cm^1 can be circumvented
using D 2 O as solvent when the water signal at 1600cm^1 is shifted to about 1200cm^1 and the absorption at
1000cm^1 is shifted by an almost equivalent amount (Figure 11.5). So, measuring IR spectra in both solvents
makes it possible to observe the full spectral range of interest. The use of D 2 O also causes small but significant
shifts in nucleic acid absorptions resulting from deuterium exchange, and these can be used to monitor
H–D exchange processes.
Fourier transform infrared absorption (FTIR) is so sensitive that it is possible to make measurements on
very small crystals of nucleic acids and needs about 100g of material. IR spectra are largely unaffected
by the external environment and so FTIR supports the identification of structurally significant bands by
432 Chapter 11
Figure 11.5 Schematic distribution of IR absorption bands of DNA and solvent. (a) 1800–1500 cm^1 corresponding
to stretching of CX double bonds. (b)1500–1250 cm^1 corresponding to base-sugar entities (including
glycosyl torsion angle effects). (c) 1250–1000 cm^1 corresponding to phosphate and sugar absorptions.
(d) Below 1000 cm^1 associated with phosphodiester chain coupled with the sugar vibrations