purpose-developed small cantilevers, which are able to image faster (2s per image). Smaller cantilevers
have high-resonant frequencies, which allow for high-scanning frequencies, but also low-spring constants,
which enable any potential sample damage to be further minimised.
There are two main factors that can influence the level of resolution attainable in AFM images of nucleic
acids. One is the sample preparation method employed for the immobilization of DNA to the underlying
surface, and the other is the sharpness of the AFM probe. To this end, two immobilization methods have
proved to be particularly useful for the analysis of DNA in aqueous environments. One method utilizes an
unmodified mica surface with a buffer containing divalent metal ions (such as Ni^2 , Co^2 , Zn^2 , Mn^2 or
Mg^2 ) or mica presoaked/pretreated with solutions of such ions (in this case a more concentrated divalent
metal ion solution is needed). The other approach uses mica or alternative flat substrates (e.g.silicon) onto
which positive charges have been introduced, for example through aminosilanation. This latter type of sur-
face, however, tends to be more problematic and is more suitable for experiments in which divalent metal
ions cannot be used without perturbing the DNA structure and/or the biomolecular functions under
investigation.
One of the notable drawbacks for AFM is the probe convolution/broadening effect, which manifests as
a size difference between the observed dimensions of the image feature and the actual molecular dimensions
(normally the former is bigger). This effect arises when the radius of curvature of the imaging probe (typically
10–20nm) is greater than the size of the feature to be imaged. Consequently, the apparent width of the
double-stranded DNA helix in AFM images is typically 12–15nm, rather than the 2nm predicted from the
crystal structure. Recent attempts to improve the sharpness of the probe (and thus the image resolution)
have utilised single-walled carbon nanotubes attached to the apexes of AFM probes. Such probes have
decreased the apparent DNA width to around 6–8nm. Commercially available sharpened silicon oxide AFM
probes (radius of curvature ca.2nm) are also available and are helpful in improving the level of attainable
image resolution.
In addition to its imaging function, AFM can also be used to measure forces acting between the probe and
surface. An approach termed single molecule force spectroscopyhas been developed consequently to inves-
tigate the mechanical properties of single biopolymeric molecules, including DNA.^40 Here for example the
opposite ends of a linear double-stranded DNA molecule are tethered between an opposing AFM probe and
substrate, and then the molecule extended/stretched as the probe-substrate separation is increased. Pulling
on single linear DNA molecules (with random sequence) produces force-extension curves (Figure 11.16).
The plateau region at around 65pN corresponds to the overstretching transition of DNA in which DNA is
stretched from its B-DNA state to an overstretched state (around 1.7 times its B-DNA contour length). Upon
further extension, a second transition occurs, during which the DNA can be melted into two single strands.
Researchers have investigated the influence of different base sequences on this ‘mechanical fingerprint’,
and the approach applied to the investigation of more complex molecular architectures including DNA/RNA
hairpins. Recently, the effects of DNA-binding drugs on the mechanical properties of DNA have also been
investigated (Figure 11.16). The observed changes were found to be dependent on the concentration of the
applied drug and its mode of binding.
Although AFM is still a relatively new approach, it overcomes many of the limitations of its STM pre-
decessor. While some improvements are still required before it will be utilised as a routine tool for the inves-
tigation of nucleic acid structure, its advantages over other microscopy approaches have meant that it is
quickly becoming more accepted. The combination of AFM with other rapidly developing experimental
approaches, such as single molecule fluorescence, also holds considerable promise for the future towards
deepening our understanding of the basic processes in which nucleic acids are involved.
11.6 Mass Spectrometr y
The determination of intrinsic molecular masses of nucleic acids using mass spectrometry is widely accepted
as one of the most accurate methods to detect nucleic acids.^41 The key technologies are the second-generation
‘soft ionization’ methods: matrix-assisted laser desorption/ionization time-of-flight(MALDI-TOF) and
Physical and Structural Techniques Applied to Nucleic Acids 449