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DNA-intercalator interactions: structural and physical

analysis using atomic force microscopy in solution
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Cite this: Soft Matter, 2013, 9, 11233

M. Maaloum,*a P. Mullera and S. Harleppb

Received 1st August 2013
Accepted 18th September 2013
DOI: 10.1039/c3sm52082j
www.rsc.org/softmatter
DNA has a strong affinity for many heterocyclic aromatic dyes, such as acridine and its derivatives. Lerman
in 1961 first proposed intercalation as the source of this affinity, and this mode of DNA binding has since
attracted considerable research scrutiny. DNA intercalators (molecules that intercalate between DNA base
pairs) have attracted particular attention due to their antitumoral activity and because they are widely used
to fluorescently label DNA. The interaction between double-stranded DNA and bis-intercalators, such as
the fluorescent dye YOYO-1 (dimer of oxazole yellow), has been widely studied. In the literature, there
are contradicting data regarding the structure and the rigidity of the complex DNA–YOYO-1. Here, we
address this problem using high-resolution atomic force microscopy in solution. We directly measured
important structural parameters, such as the helical pitch and the elongation of the complex. By
measuring intercalator-induced DNA elongation at different YOYO-1 concentrations, we determined the
binding constant. We showed that intercalation induces distinct changes in the molecular elasticity
compared to the free double stranded DNA. We found a decrease of the persistence length of DNA
with increasing amount of bound YOYO-1, which contrasts with some previous assumptions. This study
helps in understanding the physicochemical properties of bis-intercalators as well as the mechanism by
which they interact with DNA and this technique can ultimately be applied to a large range of other
DNA binding molecules such as anti-cancer or anti-viral drugs.

Introduction Bisintercalators have two potential intercalating ring systems

Intercalators are one of the most important synthetic groups of
compounds that interact with the DNA double helix. Some of
them are valuable drugs that are currently used for the treat-
ment of some types of cancers, while others are in different
phases of clinical trials.1–3 Intercalating agents share common
structural features. The molecules are usually heterocycles and
are good nucleic acid markers. A number of physical studies on
the interactions of DNA through an intercalation process with
planar aromatic cations were conducted by Lerman in the early
1960s.4 He concluded that planar aromatic molecules could
bind to DNA by a process of intercalation. These molecules can
be intercalated into DNA, in the space between two base pairs.
The rst intercalating agent, a monointercalator, ethidium
bromide, was historically widely used in molecular biology
laboratories to visualize, under a UV lamp, DNA fragments on
gel electrophoresis.5 It is also possible to link two or more ring
systems together with connecting chains of variable length.6
a
Institut Charles Sadron, CNRS – University of Strasbourg, 23 rue du Loess, BP 84087,
67034 Strasbourg Cedex 2, France. E-mail: mounir.maaloum@ics-cnrs.unistra.fr;
pierre.muller@ics-cnrs.unistra.fr; Fax: +33 (0)3-88-41-40-99; +33 (0)3-88-41-40-99;
Tel: +33 (0)3-88-41-40-02; +33 (0)3-88-41-40-07
b
Institut de Physique et Chimie des Matériaux de Strasbourg, 23 rue du Loess BP43,
67034 Strasbourg Cedex 2, France. E-mail: harlepp@ipcms.unistra.fr; Fax: +33 (0)3-
88-10-72-45; Tel: +33 (0)3-88-10-72-13
connected by linkers. The synthesis of these molecules was
originally stimulated by the idea that the pharmacological
activity of intercalating drugs could be enhanced by the signif-
icantly higher DNA binding constants and slower dissociation
rates from DNA expected for bisintercalators relative to mono-
intercalators.6 Special types of intercalators include organome-
tallic complexes with nitrogen containing heterocycles as
ligands, e.g., bipyridyl and phenanthroline complexes with Ru,
Rh, etc. The eld is nicely reviewed in recently published
articles.7,8
Many efforts have addressed the synthesis of mono- and
bisintercalators for various purposes. In this work, we will focus
on the YOYO-1 (YOYO) molecule, which is assumed to act as a
classical bisintercalator. Rye et al.9 reported development of
YOYO dye (for structure, see Fig. 1), for use as a uorescent
probe in DNA analysis. The excellent uorescent properties of
YOYO and its high binding affinity to double-stranded DNA
(ds-DNA) make the dye suitable as a probe in uorescence video
microscopy. However, this intercalating agent may induce
changes in the local structure of the double helix and dynamics
of DNA molecules. So, does YOYO binding alter the DNA
structure and rigidity? From a structural point of view, to our
knowledge, no direct experimental evidence has been presented
verifying this assumption. NMR structures for YOYO and TOTO
bisintercaled into oligonucleotides have been published.10 The

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Fig. 1 Chemical structure of the oxazole yellow homodimer (YOYO-1).
YOYO chromophore contains an oxygen atom instead of the
larger sulphur atom in the corresponding compound, TOTO.
The authors have shown that YOYO intercalation causes the
helix to have a helical repeat of 13 base pairs and requires more
space than TOTO in the intercalation sites.
From a physical point of view, the behaviour of probed DNA
molecules could differ signicantly from that of unstained
DNA, and it is thus important to elucidate the binding mode of
the probe and how the binding affects the length and the ex-
ibility of DNA. The rigidity of DNA is oen characterized by its
persistence length Lp, which measures how far a polymeric
chain persists in a given direction. The persistence length Lp is
about 50 nm for ds-DNA in its B-form and about 1 nm for single
stranded DNA under physiological conditions. In this domain,
widespread experimental values of Lp can be found in the case
of YOYO–DNA complexes. Values ranging from 12 nm to 50 nm
have been reported from single-molecule force-extension
measurements.11,12,14–16 Recently, using magnetic tweezers,
Günther et al.12 showed that the persistence length, estimated
from force measurement by stretching the chain, did not reveal
any change over the whole range of applied molar ratios up to
1.4 dye/base pair. According to the authors, the reasons why
YOYO does not affect the persistence length are still unclear.
Reuter and Dryden13 have investigated the kinetics of binding,
for single, hydrodynamically stretched molecules of lambda
DNA observed via total internal reection uorescence micros-
copy. The maximum DNA extension (36%) and association
constant Ka ¼ 108 M1 have been estimated from a simple
model using a one site binding equation. YOYO molecules, like
all other bis-intercalators, not only affect the rigidity of
the dsDNA, but also the natural length of the DNA. Either from
force-extension measurement or hydrodynamically stretched
molecules, the authors showed that the estimated natural
length (or contour length) of dsDNA with YOYO increases with
increasing staining ratio. However, in these experiments, the
affinity of YOYO was found to be force dependent and only
extrapolations give rise to the affinity at null force. Vladescu
et al.,14 by measuring ligand-induced DNA elongation at
different ligand concentrations, have determined the binding
constant and site size as a function of force for other inter-
calator molecules. Both quantities depend strongly on force
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stretching. The applied force partially relieves normal interca-
lation constraints, which explains the large increase of Ka with
applied force.13
Atomic force microscopy (AFM) in air has been used to
examine perturbations in the tertiary structure of DNA induced
by the binding of ditercalinium, a DNA bis-intercalator.17 The
estimated apparent persistence length of the molecules was
about 100 nm, which is twice larger than of naked DNA.
Here, we show that direct visualization of dsDNA, using AFM
in solution, allows us to directly analyze morphological pro-
perties and structural changes of DNA induced by the presence
of YOYO. Images obtained in solution of DNA molecules
adsorbed onto a at surface provide structural information at
the single molecule level. We have determined how YOYO
binding changes locally the structure and rigidity of dsDNA. In
particular, we systematically investigated the inuence of YOYO
on the persistence and contour length, as well as the YOYO
distribution along the DNA chains over a large range of staining
ratios. Our investigations were carried out in solutions with
moderate ionic strength, which support stable YOYO binding.
Materials and methods
We have worked with two kinds of double stranded DNA
samples, the rst one containing DNA which is polydisperse in
length, the second one containing only one type of DNA. For the
polydisperse case, the cut DNA is of lHindIII type.
The monodisperse DNA made of 4361 base pairs is of type
PBR322 linearized with EcoRI enzyme.
Experiments were performed in a solution containing 10 mM
tris–HCl buffer, pH 7.5, to a nal DNA concentration of 1 mg
ml1. The mixture of YOYO and DNA was incubated, at room
temperature, for 2 hours to achieve homogeneous staining.
200 ml of this DNA–YOYO solution was injected into the AFM
liquid cell and DNA molecules adsorbed onto freshly cleaved
mica at room temperature.
DNA and mica are negatively charged. Only in the case of DNA
solution without YOYO, 1 mM of MgCl2 was added to the DNA
solution in order to visualize the adsorbed DNA on mica.
For each YOYO concentration, we have visualized poly-
disperse and monodisperse DNA. For the monodisperse case,
we analyzed each time about 50 DNA chains, which permits us
to obtain a good statistic. In the polydisperse case, three
experiments have been reproduced for each YOYO concentra-
tion in order to analyse more than 100 molecules to obtain
sufficient statistic.
Images were collected using a Nanoscope 8 (Bruker) oper-
ated in tapping mode in solution. Ultra-sharp non-contact
silicon cantilevers with a nominal tip radius of <10 nm were
driven at oscillation frequencies in the range of 20–26 kHz.
During AFM imaging, the force was reduced in order to avoid
dragging of DNA by the tip. The line scan rate was usually 1.4
Hz. Integral gain was adjusted to give sharp images. Images
were taken without on-line ltering and were subsequently
processed only by attening to remove the background slope.
From AFM images of DNA molecules, we measure end-to-end
distance R and the apparent DNA lengths L.
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Results and discussion
1. Structural analysis
Aer sample preparation and image acquisition in solution, a
quantitative AFM analysis of DNA and DNA–YOYO complexes
was systematically performed in order to gain deeper insight into
the inuence of YOYO intercalators on the DNA secondary
structure and its global conformational properties. Fig. 2A shows
typical AFM images of linear dsDNA deposited on mica (the
presence of a small amount of Mg2+, 1 mM, is necessary to bind
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DNA to the mica surface). YOYO molecules (which bears a 4+
charge) also act as multivalent ions, so that Mg ions are not
required anymore. To conrm that there are no magnesium
specic effects, we did experiments with YOYO–DNA complexes
in a solution containing MgCl2 at the same 1 mM concentration.
As expected, this did not change the results. In Fig. 2B and C,
dsDNA molecules are imaged in the presence of YOYO at a ratio
of 1dye/4 bp and 1dye/bp respectively and show very few cross-
ings, whereas in Fig. 2D, dsDNA molecules are imaged in large
excess of YOYO (ratio 1.5 dye/bp), and show many crossings.
From these images, we can clearly see the conformational change
of dsDNA molecules with YOYO concentration. At saturated
concentration, the dsDNA molecules appear to be more
condensed than those at low ratio of YOYO/bp, indicating that
the dsDNA molecules seem to be stiffer at low intercalator
concentration than at high intercalator concentration. In YOYO
free solution (Fig. 2A), the dsDNA molecules are more extended
(in terms of the end-to-end distance) than those in the presence
of YOYO. In order to provide more reliable results and shed light
on the DNA structure, we have performed a statistical analysis of
Fig. 2 Images of single DNA molecules using AFM in solution. AFM images show
the characteristic conformation of YOYO–DNA complexes using PBR322 DNA
molecules. There is evidence of DNA conformation changing as the concentration
of YOYO increases. Images were captured at molar ratios of YOYO/bp equal to
(A) 0, (B) 0.25, (C) 0.5, (D) 1.5. Height information for all images is color coded
[from dark (low) to light (high)]. Full image size: 4 mm  4 mm.
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a large number of molecules. The analysis of the contour lengths
on a large number of traced molecules as a function of the YOYO/
bp ratio is shown in Fig. 3. For monodisperse DNA molecules in
YOYO free solution, the measured contour length is L0 ¼ 1520 
80 nm which is in excellent agreement with the expected value of
the B-form (Ltheory ¼ 1480 nm) assuming the value of the B-DNA
usual form of DNA base pairs of 3.4 Å. As indicated by the plot of
Fig. 3, the intercalation induces a change in the local structure of
DNA as a lengthening of the DNA strand. The measured contour
length of DNA increases with increasing YOYO/bp ratio. For
YOYO bulk ratios above 0.5, the observed contour length satu-
rates. This plateau indicates an increase of the relative length of
46%. From the contour length measurement, it was possible to
derive the fraction of extension as a function of YOYO concen-
tration per base pair concentration. The intrinsic association
constant Ka of YOYO–DNA interactions was estimated using the
comparison between the experimental data and the equation
derived from the theory developed by McGhee and von Hippel for
ligand binding to a one-dimensional lattice-like macromolecule
in the case of noncooperative ligand binding.18 For the compar-
ison between our experimental data and theory we used the
converted equation of McGhee and von Hippel12 to obtain a
L
relationship between the fractional extension and the ratio
L0
between the total number of YOYO and the number of base pairs.
The obtained equation is

n1
CYOYO Dg 1  nDg þ Dg
¼ Dg þ (1)
CDNA Ka ð1  nDgÞCDNA 1  nDg
where CYOYO and CDNA are the concentration of total dye and
DNA base pairs respectively. g ¼ L/L0  1, n is binding site size
i.e., the number of base pair sites occluded by one bound
YOYO and D ¼ dbp/dYOYO with dbp is the rise parameter per
DNA base pair of 0.34 nm and dYOYO is the DNA elongation per
bound YOYO.
Fig. 3 Change in DNA length in response to the YOYO intercalation. The curve
L
presents the fractional extension defined as the increase in end-to-end distance L due
L0
to the intercalation of YOYO normalized by the natural length of the DNA molecule
(without YOYO), plotted over the YOYO/bp ratio (blue squares). The solid line repre-
sents the fit from the model of McGhee and von Hippel (eqn (1)). The results were best
fit for association constant Ka ¼ 0.8  109 M1 and biding site size n ¼ 3.1 bp.
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Since the equation cannot be inverted analytically, we tted the
equation numerically. Fig. 3 shows the comparison between our
experimental data and theoretical model. The best t of the data is
obtained for the values of binding site size n ¼ 3.1  0.1 bp and
association constant Ka ¼ (0.8  0.4)  109 M1. These values are
in good agreement with the values obtained by Günther et al. using
magnetic tweezers.12 Note that the obtained binding constant of
YOYO to double stranded DNA is, as expected for bisintercalator
molecules, much higher than that of monointercalators which is
of the order of 106 M1 at low salt concentration.6
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At low concentration of YOYO (compared to the base pair
L
concentration), increased linearly with the staining ratio.
L0
Assuming almost complete binding, the slope of the line at the
origin provides DNA elongation per intercalated YOYO mole-
cule of 0.5  0.03 nm.
In Fig. 4, we report high-resolution topographies of DNA and
DNA–YOYO complexes. Regarding the results on high resolu-
tion structures of DNA using AFM, several articles have been
published in this domain.19–23 Fig. 4 corresponds to AFM images

Fig. 4 High-resolution images of DNA molecules in solution. Images were captured at molar ratios of YOYO/bp equal to (A) 0, (B) 0.25, and (C) 1. The helical nature of
the DNA structure can be clearly observed in (A) and (C). Full image size 50 nm  50 nm. (D–F) Cross-sectional profiles of molecules in (A–C), respectively, which are
perpendicular to the DNA axes.

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of shorter strands of DNA molecules (50 nm  50 nm). The
purpose of those high magnication images is to reveal the
periodic pattern that corresponds to the helical nature of
doubled stranded DNA. Therefore these zooms were chosen to
show clearly the height oscillation along the molecular back-
bone, and not to give a typical long-range conformation.
Representative images of DNA conformations are shown in
Fig. 2. The structure of a DNA chain in YOYO free solution is
shown in Fig. 4A. The helical nature of the DNA structure can
clearly be observed. A section of the image along the DNA
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signicantly changed relative to the B-DNA helical structure. In
Fig. 4D–F we plotted the cross-section of single DNA and the
DNA–YOYO complex. The heights of DNA and DNA–YOYO
complexes are 1.8 nm and 2.4 nm respectively. The parts with
increased height correspond to helices with a periodicity
(Fig. 5B and D) characterized by a pitch of 5.2  0.3 nm. This
period length is measured as the peak-to-peak distance
observed along the DNA molecules, as shown in Fig. 5B and D,
and indicates that YOYO molecules cause the helical pitch of
DNA to increase by 53%. This result coincides with what we

molecule shows a periodicity. We have performed a statistical
analysis of the periodicity of ten DNA molecules. The analysis of
the section along the DNA axis (Fig. 5A and C) reveals that the
helical pitch is equal to 3.3  0.2 nm. This value corresponds to
the distance between the major grooves of DNA in its B-form.
In Fig. 4B and C, AFM images of DNA molecules with
different YOYO concentrations show a considerable change in
their structure. Fig. 4B corresponds to DNA with a 1 YOYO/4 bp
ratio. The topography of the complex shows clearly the parts of
the chain that resembles the DNA in YOYO free solution while
others have increased height. For the DNA chains at saturated
YOYO concentration, the conformation of the helix is also
found previously for the contour length measurement of DNA.
However, if one takes into account the DNA contour length, this
result implies that the reduction of the number of turns is 5%,
while that provided by other authors is about 7–8%.12
2. Physical analysis
In this section we have studied the physical behaviour of probed
DNA molecules and how the binding of YOYO molecules affects
the length and the exibility of DNA. The rigidity of DNA is oen
characterized by its persistence length Lp, which is the length
over which the average deection of the polymer axis caused by

Fig. 5 Section profiles along DNA molecules at molar ratios of YOYO/bp equal to (A) 0 and (B) 1 obtained from AFM images previously filtered using a low pass filter.
The periodicity in both cases is clearly visible. (C and D) Histogramms of measured periodicity. In the case of DNA (C), the periodicity is 3.3  0.2 nm, which corresponds
to the helical pitch of usual B-DNA. In the case of saturated YOYO–DNA (D) the periodicity is 5.2  0.3 nm.

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thermal agitation is one radian.24 Herein, we applied a method
for measuring the persistence length of dsDNA under physio-
logical conditions. There are two major methods to estimate the
persistence length of adsorbed macromolecules at surfaces. The
rst method, that we used, is based on the measurements of the
contour length and the end-to-end distance. This method is very
reliable when used on a large number of molecules. The second
method25,26 is based on the measurements of the angle between
two small segments separated by a certain distance. This
method does not require measurements on hundreds of
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where hR2i2D is the mean-square end-to-end distance of the
chain in two dimensions and L is the contour length. For the
long chains hR2i2D ¼ 4LpL.
We beneted from chain polydispersity when calculating the
mean-square end-to-end distance R2 as a function of contour
length (see Fig. 6A). It should be noted that other lengths of
DNA were found in the sample in addition to the expected DNA
fragments ranging from 125 bp to 23 130 bp. Fig. 6A corre-
sponds to the DNA under free YOYO conditions. The images
and measurements are processed with only a simple attening,

molecules, and thus is advantageous for researchers who do not
have a large number of molecules. However, the second method
presents an inconvenient, which is particularly relevant when
using AFM techniques. In fact, this method is very sensitive to
the local bending of the molecules. This local bending can be
easily induced by the AFM tip along the scanning direction.
During adsorption, DNA molecules are transformed from
three dimensional (3D) into two dimensional (2D) objects. Two
extreme cases were reported: one where the molecules freely
equilibrate on the surface, before being trapped in a particular
conformation and one where the molecules adhere without
having equilibrated on the substrate, resembling the true 3D
conformation. In the latter case, the conformation of the
molecules reects the history of their approach to the surface
and it is therefore difficult to distinguish between the intrinsic
conformation and those induced upon adsorption. Recently, we
have shown that in the case of adsorption of DNA in solution,
the molecules freely equilibrate on the surface.27 In this case, it
is possible to obtain an ensemble of 2D conformations, which
can be related to the true 3D DNA structure as
! solutions onto freshly cleaved mica do equilibrate on the
 2 2Lp   L

R 2D ¼ 4Lp L 1  1e 2L p (2)
L dimensions.28 The analysis of the mean-square end-to-end
which does not alter at all the length measurements. This gure
is used only to clarify the method for measuring the contour
length and the end-to-end distance. Fig. 6B shows the curve of
hR2i as a function of the contour length of dsDNA in YOYO free
solution and in solution containing YOYO with a 1 YOYO/bp
ratio. The hR2i averages were obtained by considering chain
lengths within 10% of the error. A quantitative analysis of 50
DNA/fragment traces shows good agreement with the WLC
model. This model is used to t the experimental curve using
one parameter Lp and by taking into account the error bar on
each data point. We observe a linear regime for long DNA chains
with a scaling of hR2i f L, corresponding to a Gaussian chain.
In these experiments, highly positively charged YOYO
molecules also serve as bridges between the negatively charged
mica surface and DNA to anchor DNA onto the surface. We can
think that the anchoring mechanism to be different from that
with Mg2+ in solution, and the measured DNA conguration
is not necessary in equilibrium. We have to note that the
analysis of hR2i and contour length values reveals that DNA
molecules deposited either in 1 mM of Mg2+ or 1 mM of Mg2+
surface and that they behave as ideal worm-like chains in two

Fig. 6 (A) AFM image of a single DNA molecule. The image is used only to clarify the method for measuring the contour length L and the end-to-end distance R. (B)
Typical graph obtained when we plot the mean square value of the end-to-end distance as a function of the contour length for both cases: DNA (black circles) and
YOYO–DNA complex with ratio equal to 1 YOYO/bp (open circles). The hR2i averages were obtained by considering chain lengths within 10% of the error. The fit (solid
lines) corresponds to the worm like chain model in 2 dimensions (eqn (2)) with Lp as a fitting parameter. The results were best fit for Lp (DNA) ¼ 50  1 nm and Lp (1
YOYO/bp) ¼ 28  2 nm.

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L
Fig. 7 Dependence of persistence length Lp on fractional extension . The curve
L0
shows that Lp decreases with increasing YOYO/bp ratio.
distance value of YOYO–DNA fragments deposited onto freshly
cleaved mica shows that in the presence of 200 nM of YOYO in
solution, which is 5000 times smaller than in the case of Mg2+,
YOYO–DNA complexes are also able to equilibrate on the surface,
despite the 4+ charges per molecule. A quantitative analysis of
DNA/fragment traces in Fig. 6B shows good agreement with the
worm like-chain model in two dimensions. The effect of YOYO on
the DNA conguration may occur on a local scale by forming
strong curvatures or kinks. However, theses local distortions
disappear by averaging over the full length of DNA.
The measured value of the persistence length of dsDNA in
YOYO free solution is Lp ¼ 50  1 nm. This value is in excellent
agreement with the expected value obtained from single mole-
cule stretching experiments under physiological conditions.28
However, in solution containing YOYO at 1 YOYO/bp, the
persistence length Lp decreases and is equal to Lp ¼ 28  2 nm.
In Fig. 7, we have plotted the effect of the level of YOYO
intercalation on the persistence length Lp of the DNA–inter-
calator complex. Due to the intercalation, Lp decreases with
increasing staining levels (less base-pairs per YOYO molecule).
This decrease in persistence length is inconsistent with recent
results obtained by Günther et al.12 but consistent with the
previous studies using uorescence intensity as a measure of
the number of bound YOYO molecules.29
Conclusion
In summary, atomic force microscopy in solution, at high reso-
lution, has been used to study the secondary structure of
complexes of YOYO with double stranded DNA and their physical
properties as a function of the ratio of YOYO/base pairs. The
contour length of the DNA and its helical pitch in the presence of
YOYO were measured directly. These quantities increase with
intercalation as a result of intercalation of the dye between the
base pairs of DNA. The binding constant of YOYO was found to
be 0.8  109 M1. We showed that intercalation induces distinct
changes in the molecular elasticity compared to the free double
stranded DNA by decreasing its persistence length.
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