11.1 Spectroscopic Techniques
11.1.1 Ultraviolet Absorption
The light absorption characteristics of nucleic acids^1 result from the combination of the strong ultraviolet (UV)
absorption of the purine and pyrimidine bases in the 240–280nm range modulated by the stereochemistry
and conformational influences of a ribose-phosphate backbone that is essentially transparent to light of
that wavelength (Section 2.1.3).
Oligonucleotides exhibit a strong UV absorption maximum maxat approximately 260nm and a molar
extinction coefficient of the order of 10^4 dm^3 mol^1 cm^1 (Table 2.2). This absorption arises almost
entirely from complex electronic transitions in the purine and pyrimidine components. The intensity and
exact position of maxare functions not only of the base composition of the nucleic acid but also of the state
of the base-pairing interactions present, the salt concentration of the solution and its pH. Most importantly,
base–base stacking results in a decrease in – a situation known as hypochromicity. This arises from
dipole–dipole interactions that depend on the three-dimensional (3D) structure of an oligonucleotide and
ranges in magnitude from 1–11% for deoxyribonucleoside phosphates to 30% for most helical polynu-
cleotides. While some degree of stacking is apparent for all dimers, it appears that UpU, UpA, UpC, GpU
and UpU are generally less stacked than other ribodinucleoside phosphates at pH 7. In practice, the effects
of structure on the UV absorption of oligonucleotides are so complex that only the most basic interpretations
can be made (Figure 11.1).
In practical terms it is possible to estimate the molar quantity of an oligonucleotide, which is for example
chemically synthesised (Section 4.1), by measurement of the number of absorbance units at 260nm (A 260 ,
the optical density) of its solution in a UV spectrometer and by relating the value obtained to the sum of
the extinction coefficients of the individual nucleotides (e.g.8.8 for dT, 7.3 for dC, 11.7 for dG and 15.4
for dA for Ain cm^2 mol^1 ). Such estimation makes no allowance for hypochromicity, which will depend
on the particular sequence and whether it forms a secondary structure or not.
Ultraviolet absorption is a sensitive and convenient way to monitor the ‘melting behaviour’ of DNA and
RNA. When the UV absorption of a nucleic acid sample is measured as a function of temperature, the result-
ing plot is known as a melting curve. The midpoint in the melting curve showing the increase in absorbance
with increasing temperature is known as the melting temperature Tm. This is dependent on the base compo-
sition of the sample, the salt concentration of its solution and even the type of counter-ion present (Section
5.5.1). Such melting is a co-operative phenomenon and the observed melting curves become progressively
428 Chapter 11
Figure 11.1 Typical UV absorption curves for equimolar base concentrations of mononucleotides, of single-stranded
(random coil) oligonucleotide and of double-helical DNA