11.1.3 Circular and Linear Dichroism
Circular dichroism(CD) is a widely used form of chiral spectroscopy which has been applied to the study
of nucleic acids.5,6A CD signal results from the differential absorption of left and right circularly polarised
light. Circularly polarised light has chirality and therefore it will exhibit chiral discrimination. A chiral
molecule will absorb left and right circularly polarised light to differing extents and this difference gives
rise to the phenomenon of CD. Typically, data are presented as plots of versuswavelength. Here is
the difference in molar extinction coefficient for left and right circularly polarised light (L– R).
Isolated purine and pyrimidine bases are planar, intrinsically optically inactive and hence do not exhibit a
CD signal. However when incorporated into nucleosides and nucleotides, the glycosylic bond from the C-1
atom of the sugar to either the N-9 of purines or N-1 of pyrimidines gives rise to a chiral perturbation of the
UV absorption of the base. The CD signal of a nucleic acid increases with length due to the co-operativity
of chiral interactions between contiguous bases. This effect occurs both as a result of sequence effects arising
from nearest-neighbour interactions as well as from overall gross secondary structure.
The information derived from CD spectra is complementary to other types of optical spectroscopy, such
as UV, infrared (IR) and linear dichroism (LD), and it provides a quick, convenient and accurate picture of
the overall conformation and secondary structure of a particular nucleic acid solution. Reference CD spectra
for a B-DNA duplex, single strands and a G3 quadruplex show that each type of secondary structure
exhibits its own signature spectra (Figure 11.4). A- and Z-type DNA (Section 2.2) also have unique CD spec-
tra, as do many nucleic acid–ligand complexes. CD spectroscopy also allows inter-conversions between
different secondary structures to be monitored. For example titration of NaCl into a solution of poly(dG-
dC) to salt concentrations above 4M induces a structural transition in the polynucleotide from a standard
right-handed B-helix to the left-handed Z-form. Increasing the temperature of a nucleic acid solution whilst
measuring the CD signal allows DNA melting to be monitored directly and it is possible to observe the struc-
tural transition from folded duplex to random coil single strands.
Circular dichroism spectroscopy is also a useful tool for studying nucleic acid–ligand interactions. The CD
spectrum of each component in solution is directly proportional to its concentration (Beer’s law), and the
total spectrum arises from the sum of all component spectra. If ligand binding induces extrinsic optical
activity in the chromophores of the bound ligand, an induced CD signal is observed, which is directly pro-
portional to the amount of nucleic acid–ligand complex formed, and hence it can be used to construct a
binding isotherm. Alternatively, ligand binding may result in a conformational change in the nucleic acid,
and the resultant change in the intrinsic CD signal of the macromolecule allows the binding to be quantified.
Physical and Structural Techniques Applied to Nucleic Acids 431
Figure 11.4 Circular dichroism spectra for three different conformations of DNA, a poly(dA) poly(dT) B-type duplex,
a guanine quadruplex (tetraplex) and a single-stranded random coil