steeper with increasing length of the oligonucleotide. Simple sigmoid melting curves are observed for many
DNA samples, but it is also possible to observe more complex, multi-phase melting in some cases (Figure
11.2). The deconvolution of such multi-phase melting curves makes it possible to examine the effects of base
modification on the stability and nature of nucleic acid secondary structure in more detail.
11.1.2 Fluorescence
Fluorescence is defined as the emission of radiation as a molecule returns to its ground state from an
excited electronic state.^2 In order to characterise the photoexcited emission from a molecule, it is necessary
to determine the spectral distribution, photon yield, excited-state lifetime and polarisation of the emission,
all as functions of excitation wavelength. The precision with which fluorescence intensity can be measured
is very high, since photon-counting techniques eliminate several sources of uncertainty and error. Excitation
and emission occur in timescales of 10 ^15 s, but the lifetime of an excited state has a duration of 10 ^9 s.
Thus any physical process that takes place on a similar timescale as the fluorescence lifetime can be analysed
by examining changes in the emission.
The fluorescence emission from nucleotides and dinucleoside phosphates is very weak at room temperature
and can only be examined in frozen samples at 80K. Fluorescence spectroscopy is nevertheless invaluable
for examining nucleic acid–ligand interactions. Many DNA-binding ligands have little or no fluorescence
in aqueous solution. However, upon binding to a nucleic acid the ligand is in a hydrophobic environment
and the solvent can no longer quench the intrinsic ligand fluorescence. Therefore fluorescence emission is
a direct probe of the concentration of bound ligand.
Fluorescence resonance energy transfer(FRET) is a phenomenon in which the energy of an excited-state
fluorescent donor molecules is transferred to an unexcited acceptor molecule viaa dipole–dipole coupling.
Importantly, the rate of energy transfer is dependent on the distance between donor and acceptor molecules,
the spectral characteristics of the pair and the relative orientations of the donor and acceptor transition
dipoles. FRET experiments have been used widely to determine proximity relationships in protein and nucleic
acid systems since the 1940s when Förster derived the quantitative analysis for FRET. A key parameter in
FRET is the efficiency of depopulation (ET) and this is related to fluorescent lifetimes in the presence (T)
and absence () of resonance energy transfer [Equation (11.1)]:
ET 1 T (11.1)
Physical and Structural Techniques Applied to Nucleic Acids 429
Figure 11.2 An oligonucleotide melting curve (red), the derivative of the curve (black) and the deconvolution of
the derivative into its composite components (red)