Figure 1: Mix of relaxed and supercoiled
pTZ18R DNA plasmids adsorbed onto mica
and imaged in air.
DNA length: 2861 base pairs
1.5µm x 1.5µm scan
Z scale: 3nm

Figure 2: DNA minicircles naked (left) orcomplexed to the MC1 protein (right). The
height of naked minicircles is very regularwhereas minicircles bound to MC1 show
important height variations, due to thetorsion induced by the binding of one or
two MC1 per minicircle.
DNA length: 207 base pairs
250nm x 250nm scans
Z scale: 15nm

an atomically flat substrate such as
mica. Adsorption of DNA on thesubstrate is usually promoted byusing a buffer containing Ni2+, Mg2+,
or other divalent cations that favor
the interaction between thenegatively charged DNA and thenegatively charged mica. Under
such conditions of adsorption, themolecules are able to move andequilibrate thermodynamically on thesurface2 and good spreading of DNA
on the surface can be obtained. The
sample is then dried, after which the
DNA or DNA-protein complexes areeasily imaged in air.
As with transmission electron
microscopy (TEM), it is possible toanalyze different kinds of DNA
fragments – linear DNA, supercoiled
or relaxed closed circular DNA suchas plasmids (Figure 1) or minicircles(Figure 2) - and to characterizedifferent types of DNA-protein
complexes. Unlike TEM, however,AFM does not require the use of astaining agent to visualize thebiomolecules. Moreover it provides

topographical information, in contrast
to the purely 2D images produced by
TEM, which can help to distinguishfine structural details. For example,
AFM allowed the unambiguous
identification of binding of Fur protein
to two distinct adjacent promoters asshown in Figure 3 3. In addition tomapping of protein position along
DNA molecules, local DNA curvature,
flexibility 4, or deformation upon
protein binding, such as bending 5,6
or wrapping7-9 of DNA, can bequantitatively analyzed by AFM.
DNA bending induced by proteins on
linear DNA fragments can be
determined by direct angle
measurements. Such deformations
have been illustrated witharchitectural proteins such as MC1,which induces a kink angle of 116° 10
and spatial deformation within DNA
minicircles (Figure 2) 5,11. Recognitionof damage sites by proteins involved
in DNA repair is also frequently
related to DNA bending. DNA
glycosylases such as hOGG1
recognize lesion sites, asa.

b.
Figure 3: Visualization of Fur-DNA complexes
by AFM.
(a.) 3D representation.
400nm x 400nm scan
Z scale: 25nm(b.) High magnification image compared tothat of a similar molecule observed by dark
field Transmission Electron Microscopy; theconstriction observed in the Fur coat by AFM
corresponds to the occupation of the two
adjacent promoters of the aerobactin gene.
Scale bar: 20nm
DNA length: 645 base pairs

Page 2PROBING DNA-PROTEIN INTERACTIONS WITH ATOMIC FORCE MICROSCOPY

20

12

Counts

10

Counts

15

8
6
4
2

10

0

18

36

54

72

90

Bending Angle (degree)
5

0

0

50

100

150

200

250

300

Distance from terminus (nm)Figure 4: hOGG1 is one of the DNA glycosylases responsible for the recognition and removal ofdamaged bases from the genome. Protein position on damaged DNA molecules (as indicated byarrows) is well correlated with the position of the lesion site. Measurements of bending angles
indicate a mean angle value of 70°.
DNA length: 1024 base pairs, with a lesion site 245 base pairs from one end
Scale bar: 50nm

demonstrated by mapping of thecomplexes (Figure 4), and hOGG1
induces a mean bending angleof 70° 6. Refinement of methods used
to analyze bending angles is
described in Cognet et al.12.
The wrapping of DNA bound toproteins is well illustrated in the caseof a chromatin fiber 13, where eachnucleosomal core particle is able towrap 146 base pairs of DNA. DNA
wrapping associated with complex
formation is characterized by ashortening of the apparent DNA
length in AFM images. Interestingly,
a few proteins involved in promoter
regulation have been described to
wrap DNA to form small loops. For
instance, LrpC from B. subtilis wraps
DNA in a right-handed superhelix topromote positive supercoiling. AFM
has been used to demonstrate thatthe formation of these nucleosomelike structures results from apolymerisation of LrpC along DNA
and an oligomerisation induced bycurved DNA (Figure 5) 7. The
implication of wrapping in DNA

regulation has also been illustrated
for DNA gyrase, which promotes
negative supercoiling by wrapping
DNA in a left-handed helix. AFM
has also been used to demonstrate
a complete loss of wrap in the
DNA-gyrase complexes (Figure 6) 9in the presence of ADPNP cofactor.
Numerous other DNA-protein
complexes have also been studied
by AFM in air, and we invite
readers to refer to more complete
reviews 14-16. These examples
demonstrate that AFM imaging in
air, combined with careful choiceand control of biomolecules andimmobilization conditions, canprovide useful qualitative andquantitative characterization of
nucleoproteic complexes. Althoughinferred from DNA molecules andcomplexes immobilized and dried on
the surface, such data already offera good basis for understanding the
behavior of individual proteins along
DNA molecules. However in order toprobe dynamic aspects directly andto better approximate physiological

a.

b.
c.

Figure 5: AFM visualization of LrpC-DNA
complexes; (a) without wrapping, (b) withwrapping of DNA. Interaction of LrpC withflexible/curved DNA is able to induce acomplete wrapping of the DNA around theprotein to form a nucleosome-like structure(radius of curvature: 4.5nm), as schematizedin (c).
DNA length: 331 base pairs
Scale bar: 50nm
Z scale: 5nm

PROBING DNA-PROTEIN INTERACTIONS WITH ATOMIC FORCE MICROSCOPY

Page 3

conditions, it is necessary to study
DNA-protein complexes still able toevolve during the AFM observation,and thus to work in liquid.

PROBING DNA-PROTEININTERACTIONS IN LIQUIDThis is made possible by the use of a
fluid cell, which forms a closed fluid
volume around the cantilever andsample surface (Figure 7). Working
in liquid maintains DNA and proteinsin their native state and allows thepossibility of varying externalparameters such as buffer conditionsin the course of an experiment.
Indeed within the last decade theemergence of single molecule force
measurement techniques and of
single molecule imaging techniques,
including AFM in liquid, has created
new opportunities to analyze thedynamics of DNA-protein

interactions. One can imagine thatthe greatest synergy will be achievedby using complementary methods,
particularly those that allow direct
visualization along with more
traditional biochemical andbiophysical approaches. AFM has a
special preferred position concerning
direct time-resolved visualization of
biological systems in liquid.

SENSING DNA-PROTEININTERACTION FORCESThe same cantilevers that can beused to image with AFM can also be
used to measure nanoscale forces.
The softest cantilevers have spring
constants on the order of 0.01N/m(or 10pN/nm), which allows the AFM
to measure forces in the piconewton
range. These forces can be measured
as the tip approaches the sample,
contacts the surface, and thenretracts from the sample, producing
a “force-distance” curve withpiconewton range force resolution
and sub-nanometer distanceresolution. This force and distance
regime matches well with bothintermolecular interactions (such as
between DNA and proteins) as wellas intramolecular interactions (suchas in protein folding). Readers canfind a detailed overview of the use of
AFM as a force measurement tool inanother Veeco Application Note 17.
In the case of intermolecular DNAprotein interactions18-20, the typicalexperiment involves covalently
attaching the ligand to the cantilever
tip and bringing it into contact withthe surface, where it interacts with
its target to form a complex. Upon
retraction, this complex isincreasingly loaded until it finallyruptures. Repeating this process indifferent conditions with the same
tip and surface produce distributionsof unbinding strength probability,
which allows one to estimate
thermodynamic parameters related
to the energy barriers involved in theinteractions. This methodology has
been used to study the DNA-protein
interaction between thetranscriptional activator ExpG
(S. meliloti bacterium) and threeb.

a.

Laser beam

Unwrapped DNA

Mirror(V)(Occ)
Wrapped DNA

GyrasePhotodiode“Shadow” of the AFM tip
Buffer outlet

Buffer inlet(Rt)

Flexible ringc.
Figure 6: AFM measurements reveal whether DNA-gyrase complexes are (A):unwrapped (in the presence of ADPNP) or (B): wrapped (in the absence of
ADPNP). The red lines show how the read-through DNA length (Rt) and visible
DNA length (V) were respectively measured. Schematic in (C) illustrates thedifference in contour length between wrapped and unwrapped DNA molecules.
DNA length: 1070 base pairs
Scale bar: 100 nm

Page 4PROBING DNA-PROTEIN INTERACTIONS WITH ATOMIC FORCE MICROSCOPY

SamplePiezoelectricscanner
Cantilever and tip

Figure 7: Cross section schematic of the AFM fluid cell setup.

domains of a promoter region19.
In these studies ExpG proteins wereattached to a mica surface treatedwith silanes and DNA wild type ormutated fragments were attachedto the tip. Unbinding force
measurements revealed that only
one binding site within the promoter(core region) determines DNA
recognition by ExpG (Figure 8),
whereas two adjacent domains(box 1 and 2) affect the stability of the
DNA-protein complex.

VISUALIZING DYNAMIC
DNA-PROTEIN INTERACTIONS

Imaging dynamic events at thesingle molecule level is a fascinating
and potentially powerful way toinvestigate DNA-protein interactions.
The use of tapping mode in liquidallows one to observe DNA-protein
complexes in a physiologicalenvironment, and to record theirevolution over the course of time. A
number of pioneering works havedemonstrated the feasibility of thesestudies and their ability to measuredynamics parameters that describe
the interactions. The dynamic
behavior (over a timescale ofminutes) of different types ofcomplexes such as DNA with RNA
polymerase 21, 22, photolyase 23,
p53 24 or DNase I 25, 26 have been
investigated by AFM in liquid. For
example, the RNA polymerase-DNAcomplex has been studied in different
buffer conditions and in different
situations, either where RNAP isstably bound to the mica surface andthe sliding of DNA is characterized(Figure 9) 22 or, conversely, where thesliding of RNAP along immobilized
DNA is analyzed 21.
Efforts are now being made to
improve and rationalize further
sample preparation methods for thiskind of experiment. Indeed thecomplexes must be imaged with
sufficient resolution but also with

Figure 8: Unbinding force histogram between ExpG and (a) the wild-type DNA sequence,(b) the DNA sequence mutated in the core region, (c) and (d) DNA sequences mutatedrespectively in box 1 or box 2. Only the core region appears to be determinant for the
DNA recognition by ExpG.

Figure 9: Characterization of the sliding of DNA along RNA polymerase. The DNA contour
is traced with a thin line and the center of the RNAP is marked with a dot. The elapsed
time after the initial observation of the complex is indicated above each image. Analysisof the sliding of the DNA molecule relatively to RNAP was shown to be consistent with a
1D random walk.
DNA length: 1001 base pairs
Scan size: 350nm x 350nm
Z scale: 5nm

PROBING DNA-PROTEIN INTERACTIONS WITH ATOMIC FORCE MICROSCOPY

Page 5

Figure 10: Streptavidin-biotinylated DNA
complex; one streptavidin protein is bound toeach extremity (arrows).
DNA length: 1444 base pairs
500nm x 500nm scan
Z scale: 3nm

good control of interactions with thesample surface and good knowledgeof their influence on DNA-protein
interactions. Actually each kind of
DNA-protein complex must be
considered as an overall system,
where specific versus non-specificinteractions in the DNA-protein
complex and interactions with thesurface are both very dependent on
the ionic conditions in the buffer.
The main actor of the binding to thesurface is usually the adsorption of
the DNA molecules. The presenceof divalent cations (such as Mg2+ orNi2+) and a low concentration ofmonovalent cations (such as Na+ orK+) in the buffer are known to enable
reliable imaging of DNA moleculeson mica. Recently, the forces acting
between DNA and mica and theirdependence on monovalent anddivalent ion concentrations havebeen investigated and a rationalized
framework describing the issue ofDNA adsorption has been proposed 27.

Page 6

Various attempts have also been
made to design more deliberate andspecific DNA attachment methods.
One approach is to functionalize themica surface with amino groups(APTES), which allows attachment
of DNA molecules on the surface at
random positions along the DNA
chain, without the requirement ofdivalent cations in the buffer. Thismethod has been used to image thedynamics of DNA structures suchas supercoiled plasmids or Holliday
junctions over the course of time 28.
Alternatively, the authors of this noteare developing a strategy to tether
the biotinylated ends of linear DNA
molecules to mica using the biotinstreptavidin complex (Figure 10) andbiotinylated tethers immobilized on
the surface.
Sample preparation for imaging
dynamic DNA-protein interactions inliquid essentially requires satisfying acompromise between the necessity tobring the DNA molecules onto thesubstrate surface and the necessity
to conserve their mobility and theiraccessibility to the proteins. As a tool
to modulate DNA accessibility andreactivity, we have designed a system
in which one can alternately switch
between various DNA adsorption
conditions during a single AFM
experiment. This is done by using
mica pretreated with nickel ions tokeep the molecules on the surface
and varying the monovalent anddivalent ion concentrations in thebuffer to change the adsorption
strength 27, 29. Using this framework,
we have started to examine theinfluence of adsorption strength on
the binding of small ligands(ethidium bromide 30, bleomycin 31) toDNA. Naturally we wish to extend
this work to more challenging DNAprotein complexes and work toward
this goal is currently under way.

PROBING DNA-PROTEIN INTERACTIONS WITH ATOMIC FORCE MICROSCOPYCONCLUSION

The AFM instrument has become
indispensable for probing
DNA-protein interactions at thesingle molecule level. Thisapplication note only covers a few
examples of the knowledge that
AFM has brought forth. Imaging
immobilized DNA-protein complexesin air has essentially shed light on
their architecture. Now, by working inphysiological buffer, it is possible todirectly image dynamic interactions
and to probe the actual interaction
forces. We foresee a whole newrange of fascinating DNA-protein
interaction studies yet to comebased on this solid foundation of
successful investigations andincreasingly rationalized approaches
to sample preparation.

ACKNOWLEDGEMENTS

The authors thank O. Piétrement,
D. Pastré and A. Justome for critical
discussions and reviews during
manuscript preparation and thankB. Ohler (Veeco) for his guidance inthe application note preparation.
F.L. gratefully acknowledges support
from Veeco Instruments and fromthe Centre National de la Recherche
Scientifique during his thesis work.

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