calibration with X-ray crystallography of nucleic acid samples. Such ‘marker bands’ are sensitive to
helical type and can be used to show the existence of a conformational transition when different factors,
such as temperature, hydration, concentration, or the nature and amount of cations are varied (B → A,
B → C, B → Z-form, helix → coil, etc.). FTIR is also used to support studies on the recognition of DNA
sequences by a wide variety of molecules, such as oligonucleotides (triple-stranded structures), drugs and
proteins.
Raman spectroscopy also depends on the vibrational frequencies of groups within the molecule and, like
IR, provides information concerning vibrational modes of nucleic acid components that are conformationally
sensitive. The Raman technique has some useful advantages over IR technique. First, the incident radiation
in Raman work is not strongly absorbed and so does little damage to the sample. Second, water has weak
scattering properties and lacks absorption at the irradiation frequencies used for sample irradiation, so its
presence in the sample is not a problem. Third, unlike IR, the intensity of the Raman bands is proportional
to the concentration of the target species. As with IR, the accurate assignment of resonance lines can be
simplified by calibration using samples of known X-ray structure.
Raman spectroscopy has been used to examine nucleic acids in a wide variety of situations including
microcrystals and even within living cells.
11.2 Nuclear Magnetic Resonance
Nuclear magnetic resonance (NMR) spectroscopy is the method of choice for investigation of the conformation
of short (up to about 25 base pairs) nucleic acid fragments in solution. Compared to other spectroscopic
techniques routinely used, NMR is rather insensitive and requires millimolar sample concentrations. However,
the structural information that may be gained from an NMR spectrum is much more detailed than that avail-
able from any other solution-phase technique and is complementary to that available from X-ray diffraction
studies on solid samples. Indeed, interpretation of NMR data often requires an assumption of a structure,
which may based on an X-ray diffraction structure of the specific or a related molecule. Solution-state
NMR data can also reveal flexibility and dynamic behaviour.
The basis of NMR is that atomic nuclei are endowed with a property called nuclear spinand will align
with an applied magnetic field (Bo).^9 The degree of this alignment is dependent not only on the strength of
Bo(magnets with field strengths up to 21.1 T are now commercially available) but also on the type of
nucleus (different elements have different susceptibilities to magnetic fields) and its chemical environment
(e.g.the attached bond and atom types, the 3D arrangement of those bonds, solvent, temperature). The
magnet–nuclear spin alignment may be perturbed by the application of radiation from the radio frequency
region of the electromagnetic spectrum(100–900MHz currently utilised). For example a^1 H in a 14 T
magnet would absorb in the region of 600MHz, over a span of 12,000Hz, and this may be considered as
inducing a spin ‘flip’. This excited state survives for a few seconds in some instances, which is why NMR
spectral lines are sharp. The time taken to regain the ground state depends on the flexibility of the molecule
and how easily it tumbles in solution. Thus, analysis of NMR data can confirm the bonding network in a
molecule and the 3D arrangement of those bonds. It can establish the presence of flexibility and also identify
and quantify molecular interactions.
In nucleic acids, the nuclei that are NMR active are^1 H and^31 P, which have essentially 100% natural
abundance. A proton has the most sensitive, non-radioactive, nuclear spin. Carbon and oxygen in their most
abundant isotopes (^12 C and^16 O) have zero spin and are therefore NMR silent.^15 N is NMR active but is very
insensitive to magnetic fields and produces very broad line, unstructured spectra. It is possible to introduce
chemically or enzymatically^13 C and^15 N isotopes, both of which give an NMR response. Fluorine (^19 F) is
another nucleus that may be introduced into chemically synthesised nucleic acids and is a very good probe of
conformational flexibility. In a 14 T magnet,^31 P nuclei absorb energy around 243MHz,^13 C around 151MHz,
(^15) N around 61MHz and (^19) F around 564MHz.
Whatever be the nuclear type observed, the spectrum has features common to all:^10 an x-axis that contains
the frequency of the applied radiation and a y-axis that reports intensity, which relates to the concentration
Physical and Structural Techniques Applied to Nucleic Acids 433