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Article
DNA Compaction and Charge Inversion Induced by
Organic Monovalent Ions
Wenyan Xia, Yanwei Wang, Anthony Yang and Guangcan Yang *
School of Physics and Electronic Information, Wenzhou University, Wenzhou 325035, China;
[email protected] (W.X.); [email protected] (Y.W.); [email protected] (A.Y.)
* Correspondence: [email protected]; Tel.: +86-577-8668-9033; Fax: +86-577-8668-9010
Academic Editor: Helmut Schlaad
Received: 14 January 2017; Accepted: 23 March 2017; Published: 30 March 2017
Abstract: DNA condensation and charge inversion usually occur in solutions of multivalent
counterions. In the present study, we show that the organic monovalent ions of tetraphenyl
chloride arsenic (Ph4 As+ ) can induce DNA compaction and even invert its electrophoretic mobility
by single molecular methods. The morphology of condensed DNA was directly observed by
atomic force microscopy (AFM) in the presence of a low concentration of Ph4 As+ in DNA solution.
The magnetic tweezers (MT) measurements showed that DNA compaction happens at very
low Ph4 As+ concentration (≤1 µM), and the typical step-like structures could be found in the
extension-time curves of tethering DNA. However, when the concentration of Ph4 As+ increased to
1 mM, the steps disappeared in the pulling curves and globular structures could be found in the
corresponding AFM images. Electrophoretic mobility measurement showed that charge inversion
of DNA induced by the monovalent ions happened at 1.6 mM Ph4 As+ , which is consistent with the
prediction based on the strong hydrophobicity of Ph4 As+ . We infer that the hydrophobic effect is the
main driving force of DNA charge inversion and compaction by the organic monovalent ion.
Keywords: DNA compaction; charge inversion; monovalent ions; hydrophobic effect
1. Introduction
DNA is one of the most important biological polyelectrolytes, and is highly negatively charged in
solution. The highly-charged stiff polymer can be condensed into compact structures by multivalent
ions and many other condensing agents [1–3]. The understanding of DNA compaction is not only
important for the study of fundamental biological processes such as chromosome compacting, but
also for the development of new gene carriers in therapeutic applications [4–6]. On the other hand,
DNA compaction is closely related with its charge screening, because structural packaging requires
an effective screening of the negative charges on DNA. The process is generally considered to be
related to the neutralization—or more likely overcompensation—of the DNA electric charge [7–9].
Overcompensation or charge inversion occurs when the charge of counterions surrounding the DNA
surface are greater than the bare charge of polyelectrolyte itself. For DNA systems, the effect seems to be
found almost exclusively in the case of multivalent ions in aqueous solution [7,10–14]. Charge inversion
was also observed in mixtures of charged polyelectrolytes with oppositely charged oligomers [7,15],
polymers [16–24], colloids [18,25–28], or micelles [29]. The related DNA compaction from bulk
solution critically depends on the valence of the counterions, and a valence of three or larger is
required to overcome the inherently large electrostatic repulsive barrier between the like-charged
polyelectrolytes [30–33].
However, the underlying microscopic mechanism of attraction between like-charged macroions
such as DNA and their charge inversion is still controversial. There have been a number of theoretical
studies aimed at elucidating the fundamental physical mechanisms responsible for DNA compaction
Polymers 2017, 9, 128; doi:10.3390/polym9040128
www.mdpi.com/journal/polymers
Polymers 2017, 9, 128
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and charge inversion [34,35]. All these systems are strongly correlated; the electrostatic interactions
are strong and the effects of thermal motions, translational, or conformational entropy of chains is
small. It has been proposed that electrostatic correlations are dominant and lead to charge inversion
in the case of counterions with valences larger than two near strongly-charged interfaces [8]. In the
case of monovalent ions, electrostatic correlations are weak and no charge inversion is expected.
Recently, Martin-Molina et al. [36] proposed a new mechanism for the charge inversion of colloids
in electrolyte solutions based on the hydrophobic effect. They observed charge inversion due to the
organic monovalent ion Ph4 As+ in colloids, and the effect was attributed to the hydrophobic effect.
In the present study, we introduce the organic monovalent ion Ph4 As+ into a DNA system and find
that it not only provokes the charge inversion of DNA, but also leads to DNA compaction, which is the
first experimental evidence for DNA compaction induced by monovalent cations. The phenomenon
seems to be similar to the poor solvent effect of neutral condensing agents such as ethanol in DNA
solution [37].
2. Experimental Procedures
2.1. Materials
Tetraphenylarsonium chloride (Ph4 AsCl) is a tetrahedron consisting of a central As atom
covalently bonded to four hydrophobic groups (the phenyl –C6 H5 rings). This highly hydrophobic
cation is widely employed as a reference cation in electrochemistry because of the relatively small
importance of electrostatics in its hydration free energy in different media; this is due to its large size
(diameter d = 0.94 nm), its symmetry, and its monovalent character. Tetraphenylarsonium chloride
(C24 H20 AsCl·HCl·xH2 O), sodium bromide (NaBr), and MgCl2 were purchased from Sigma-Aldrich,
(Sain Louis, MO, US). Bacterial λ-DNA was purchased from New England Biolabs (Ipswich, MA, US),
and it had a concentration of 500 ng/µL as obtained from the manufacturer. We could use λ-DNA
directly without further treatment in atomic force microscopy (AFM), but DNA modification was
necessary before pulling in magnetic tweezers (MT). The solvent we used was 1 mM sodium bromide
aqueous solution, and water was deionized and purified by a Millipore system and had a conductivity
less than 1 × 10–6 Ω–1 cm–1 . Mica for AFM imaging was cut into approximately 1 cm2 square pieces,
and their surfaces were always freshly cleaved before use. All chemical agents were used as received,
and all measurements were repeated at least twice to obtain consistent results.
2.2. AFM Imaging
The sample preparation procedure is similar to the one in our previous work [36]. It can be
briefly described as follows: mica disks of diameter one centimeter attached to magnetic steel disks
were used as substrates for DNA adsorption. For each sample, the final concentration of DNA was
1 ng/µL, corresponding to 3 µM of phosphate groups, and a drop of about 15 µL of Ph4 As+ mixture
was deposited for 3 min on a freshly-cleaved mica surface. The surface was rinsed with distilled water
and dried with a gentle flow of nitrogen gas.
A multi-mode atomic force microscope (SPM-9600, Shimadzu, Kyoto, Japan) was used for DNA
imaging in the presence of Ph4 As+ . All AFM images were captured in the conventional ambient
tapping mode, with scan speeds of ≈2 Hz and data collection at 512 × 512 pixels. All the images were
processed manually using off-line analysis software equipped with the microscope.
2.3. Magnetic Tweezers Experiment
Magnetic tweezers are common tools for manipulating single molecules, such as tethering a
condensed DNA molecule [38]. A transverse MT system was composed of an inverted microscope
and charge coupled device (CCD) controlled by a personal computer, which is schematically shown in
Figure 1. In our experiments, the ends of DNA were coated with biotin and digoxin by biochemical
method, and the surfaces of paramagnetic beads were covered with streptavidin. A coverslip with one
Polymers 2017, 9, 128
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and functionalized with anti-digoxin in order to link with the dig-end of DNA. The other end of
DNA was linked to a paramagnetic bead by the streptavidin–biotin bond. The force was applied to
the paramagnetic bead by adjusting the permanent magnet which is installed on a
Polymers
2017, 9, 128
3 ofthe
10
micromanipulator.
A video camera was used to monitor the image of the tethered structure, and
positions of paramagnetic beads were recorded in real-time. The analysis of the extension and force
waspolished
determined
a tracking algorithm
based
onslides,
correlation
function
[39].
side
wasby
sandwiched
between two
glass
and was
used as
a flow chamber by sealing
Various
concentrations
of
Ph
4AsCl solution were mixed with NaBr solution (1 mM), then an
the sealing the open sides of the structure. The polished sidewall was silylated and functionalized
equalanti-digoxin
volume of inDNA
containing
10 mM
MgClThe
2 were added for magnetic tweezers
with
ordersolutions
to link with
the dig-end
of DNA.
other end of DNA was linked to a
measurement.
The
solution
was
incubated
for
30
min
at
least
at room
temperature,
and introduced
paramagnetic bead by the streptavidin–biotin bond. The force was
applied
to the paramagnetic
bead
into
the
flow
cell
by
using
a
syringe
pump.
In
a
typical
measurement,
we
moved
the
by adjusting the permanent magnet which is installed on a micromanipulator. A videomagnet
camera from
was
some
distance
to
some
position
close
to
a
paramagnetic
bead,
thus
applying
a
magnetic
force
on
the
used to monitor the image of the tethered structure, and the positions of paramagnetic beads were
suspended
bead.
When
a
fixed
magnetic
force
was
applied
to
the
bead,
we
monitored
the
end-to-end
recorded in real-time. The analysis of the extension and force was determined by a tracking algorithm
lengthon
ofcorrelation
DNA in real-time
measure its conformational change.
based
functionto[39].
Figure
Figure1.1.A
Aschematic
schematicdiagram
diagramof
ofmagnetic
magnetictweezers.
tweezers.
2.4. Electrophoretic
Mobility Measurement
by Dynamic
Scattering
(DLS)
Various concentrations
of Ph4 AsCl solution
were Light
mixed
with NaBr
solution (1 mM), then an equal
volume
of DNA
solutions containingmeasurements
10 mM MgCl2 were
for magnetic
tweezers
measurement.
The
electrophoresis-mobility
wereadded
carried
out by using
a dynamic
light
The
solution
was
incubated
for
30
min
at
least
at
room
temperature,
and
introduced
into
the
flow
cell
scattering device of Malvern Zetasizer Nano ZS90 (Malvern Instruments Ltd., Worcestershire, UK)
by
using
a
syringe
pump.
In
a
typical
measurement,
we
moved
the
magnet
from
some
distance
to
equipped with the patented M3-PALS technique. The laser source is a He-Ne gas laser (λ = 633 nm)
some
position
close
to
a
paramagnetic
bead,
thus
applying
a
magnetic
force
on
the
suspended
bead.
and the light scattering is collected by an avalanche photodiode mounted on the goniometer arm to
When
a fixed magnetic
force was
applied
to DNA
the bead,
we monitored
the end-to-end
length of
in
the direction
of the incident
radiation.
The
samples
were diluted
to a concentration
ofDNA
1 ng/μL
real-time
to
measure
its
conformational
change.
in a buffer solution containing 1 mM NaBr and 5 mM MgCl2; then, different concentrations of Ph4As+
were added. All measurements were carried out after 5 min incubation at room temperature. During
2.4. Electrophoretic Mobility Measurement by Dynamic Light Scattering (DLS)
the measurement, 1 mL of DNA solution was used, and the sample cell was kept at 25 °C
The electrophoresis-mobility measurements were carried out by using a dynamic light scattering
temperature.
device of Malvern Zetasizer Nano ZS90 (Malvern Instruments Ltd., Worcestershire, UK) equipped
3. Results
and Discussion
with
the patented
M3-PALS technique. The laser source is a He-Ne gas laser (λ = 633 nm) and the light
scattering is collected by an avalanche photodiode mounted on the goniometer arm to the direction
3.1.
Observation
by AFM The DNA samples were diluted to a concentration of 1 ng/µL in a buffer
of
the
incident radiation.
+ were
solution
1 mM
NaBr
5 mM
MgCl2 ; then,ofdifferent
concentrations
ofinPh
+ complexes
4 As
We containing
attempted to
observe
theand
change
in morphology
the DNA–As
the
presence
added.
All measurements
carried
out after 5 min incubation at room temperature. During the
of different
concentrationswere
of Ph
4As+ in solution. In our experiment, MgCl2 solution (5 mM) was
◦ C temperature.
measurement,
1
mL
of
DNA
solution
was
used,
andsurface.
the sample
cell was
kept of
at 25
+ complexes
added into the buffer to adsorb DNA on the
mica
The AFM
images
DNA–As
on the mica surface are shown in Figure 2. For reference, we start by imaging DNA alone in the same
3. Results and Discussion
buffer condition as used for the complexes (Figure 2A). We can see that the DNA molecules are well
separated
on the
and have relaxed morphologies with no compaction loops. From Figure
3.1.
Observation
by surface,
AFM
2B–F, the corresponding molar ratio between arsenic cations and the+ phosphate group of DNA
We attempted to observe the change in morphology of the DNA–As complexes in the presence
varies from 3.33 to 333.3. As shown in Figure 2B, there are few intermolecular contacts at lower As+
of different concentrations of Ph4 As+ in solution. In our experiment, MgCl2 solution (5 mM) was
concentrations, but individual molecules have an increased number of intramolecular
loops with
added into the+buffer to adsorb DNA on the mica surface.
The AFM images of DNA–As+ complexes
increasing As concentration. However, when [As+] > 0.1 mM, typical condensed structures were
on the mica surface are shown in Figure 2. For reference, we start by imaging DNA alone in the same
buffer condition as used for the complexes (Figure 2A). We can see that the DNA molecules are well
separated on the surface, and have relaxed morphologies with no compaction loops. From Figure 2B–F,
appeared (Figure 2C), and DNA highly looped around this point, as shown in Figure 2D. The
compaction grew gradually when the concentration of As+ increased further (Figure 2E), and at the
highest concentration (Figure 2F), we can see even more compacting patterns (e.g., globules) on the
mica surface.
AFM may modify the actual morphology of DNA in As+ solution. We have previously shown
Polymers 2017, 9, 128
4 of 10
that the treatment of condensed DNA adsorbed to a surface only reduces the molecules’ heights but
maintains their lateral dimensions [37]. Thus, AFM images flatten the morphology of DNA
condensates
with their
original
compacting
structure.
the condensed
is adsorbed
on
the
corresponding
molar
ratio between
arsenic
cations However,
and the phosphate
groupDNA
of DNA
varies from
a mica
surface
onlyinbinds
thethere
surface
loosely.
Despite thecontacts
interactions
between
the substrate
3.33
to 333.3.
Asand
shown
Figureto2B,
are few
intermolecular
at lower
As+ concentrations,
and individual
the polyelectrolytes
canincreased
modify the
structure
of complexes),loops
there with
is a nice
correlation
but
molecules(which
have an
number
of intramolecular
increasing
As+
+
between the macroscopic
phase [As
diagram
and
thetypical
AFM observations.
We thinkwere
that observed,
the reasonand
for
concentration.
However, when
] > 0.1
mM,
condensed structures
phenomenon
similar towere
the still
mechanism
of alcohol
[37] leading
to DNA compaction.
Since
athis
part
of the DNAisstructures
in the coiled
conformation.
A condensed
center appeared
DNA is2C),
a semiflexible
molecule
andthis
the point,
attractive
forces in
between
segments
are rather
(Figure
and DNA polymer
highly looped
around
as shown
Figure DNA
2D. The
compaction
grew
weak, the when
DNA the
molecules
in poor
tend tofurther
form compact
toroidal
inconcentration
solution due
gradually
concentration
ofsolvent
As+ increased
(Figure 2E),
and atstructures
the highest
to the equilibrium
exclusion
and bending
(Figure
2F), we canbetween
see eventhe
more
compacting
patterns energy.
(e.g., globules) on the mica surface.
2+ solution; Panels (B–F)
Figure 2. AFM observation of DNA complexes.
complexes. Panel (A) λ-DNA
λ-DNA in
in 55 mM
mMMg
Mg2+
+
DNA conformations at different concentrations
concentrations (0.01,
(0.01, 0.05,
0.05, 0.1,
0.1, 0.5
0.5 and
and 11 mM,
mM, respectively)
respectively) of
ofAs
As+ in
2+
5 mM Mg 2+ buffer.
buffer.
+ solution.
To enhance
DNA adsorption
on the micaof
surface
forAs
imaging,
we We
used
a buffer
containing
AFM
may modify
the actual morphology
DNA in
have
previously
showna
2+
high
concentration
of
Mg
(5
mM).
To
rule
out
its
effect
on
DNA
compaction,
we
performed
the
that the treatment of condensed DNA adsorbed to a surface only reduces the molecules’ heights
2+—the lowest concentration to deposit DNA on a mica surface. The
same
experiments
in
1
mM
of
Mg
but maintains their lateral dimensions [37]. Thus, AFM images flatten the morphology of DNA
results are similar,
andoriginal
are shown
in Figure
3. We can
see that
DNA condensed
from loose
condensates
with their
compacting
structure.
However,
thethe
condensed
DNA is adsorbed
on
+ concentration in the low concentration of
structures
to
highly-compact
globules
with
increasing
As
a mica surface and only binds to the surface loosely. Despite the interactions between the substrate
Mg2+the
. Thus,
we can deduce
that the
compaction
is caused
by arsenicthere
ion rather
than
the effect
and
polyelectrolytes
(which
canDNA
modify
the structure
of complexes),
is a nice
correlation
+ is much weaker than spermine,
of
magnesium
ion.
Actually,
the
DNA
condensing
ability
of
Ph
4
As
between the macroscopic phase diagram and the AFM observations. We think that the reason for this
as shown in Figure
4, where
effect of 0.5
As+[37]
is comparable
with 0.01
mM spermine.
phenomenon
is similar
to thethe
mechanism
of mM
alcohol
leading to DNA
compaction.
Since DNA is
a semiflexible polymer molecule and the attractive forces between DNA segments are rather weak,
the DNA molecules in poor solvent tend to form compact toroidal structures in solution due to the
equilibrium between the exclusion and bending energy.
To enhance DNA adsorption on the mica surface for imaging, we used a buffer containing a high
concentration of Mg2+ (5 mM). To rule out its effect on DNA compaction, we performed the same
experiments in 1 mM of Mg2+ —the lowest concentration to deposit DNA on a mica surface. The results
are similar, and are shown in Figure 3. We can see that the DNA condensed from loose structures to
highly-compact globules with increasing As+ concentration in the low concentration of Mg2+ . Thus,
Figure
3. The
structure
of DNA
in mixture
((A)the
0.1 effect
mM; (B)
mM;
we can
deduce
that
the DNA
compaction
is solution
caused of
byvarious
arsenicconcentrations
ion rather than
of 0.5
magnesium
+ and 1 mM Mg2+.
+
(C)
1
mM)
of
As
ion. Actually, the DNA condensing ability of Ph4 As is much weaker than spermine, as shown in
Figure 4, where the effect of 0.5 mM As+ is comparable with 0.01 mM spermine.
same experiments in 1 mM of Mg2+—the lowest concentration to deposit DNA on a mica surface. The
results are similar, and are shown in Figure 3. We can see that the DNA condensed from loose
structures to highly-compact globules with increasing As+ concentration in the low concentration of
Mg2+. Thus, we can deduce that the DNA compaction is caused by arsenic ion rather than the effect
Polymers
2017, 9, 128
5 of 10
of magnesium
ion. Actually, the DNA condensing ability of Ph4As+ is much weaker than spermine,
as shown in Figure 4, where the effect of 0.5 mM As+ is comparable with 0.01 mM spermine.
Figure 3. The
The structure
structure of
of DNA
DNA in
in mixture
mixture solution
solution of
of various
various concentrations
concentrations ((A)
((A) 0.1
0.1 mM;
mM; (B)
(B) 0.5
0.5 mM;
mM;
+ and 1 mM Mg2+
+
2+. .
(C)
1
mM)
of
As
and
1
mM
Mg
Polymers 2017, 9, 128
5 of 9
Figure 4. DNA structures in solution of (A) 0.01 mM spermine and (B) 0.5 mM As+.
Figure 4. DNA structures in solution of (A) 0.01 mM spermine and (B) 0.5 mM As+ .
3.2. Tethering of Single DNA Molecules
3.2. Tethering of Single DNA Molecules
DNA tethering was achieved in a magnetic tweezers (MT) setup, as described above. We first
DNA tethering was achieved in a magnetic tweezers (MT) setup, as described above. We first
put the microsphere-bound DNA molecules in NaBr buffer (1 mM) and magnesium ion (5 mM) into
put the microsphere-bound DNA molecules in NaBr buffer (1 mM) and magnesium ion (5 mM) into
the flow cell and incubated them for 30 min at room temperature. Then, we could find that some
the flow cell and incubated them for 30 min at room temperature. Then, we could find that some
paramagnetic beads had tethered to the surface of the sidewall through a single DNA molecule. In
paramagnetic beads had tethered to the surface of the sidewall through a single DNA molecule. In the
the absence of any condensing agents, we found that the extension of DNA was close to 16 μm under
absence of any condensing agents, we found that the extension of DNA was close to 16 µm under
high extension (>10 pN). Then, about 400 μL of different concentrations of Ph4AsCl solution were
high extension (>10 pN). Then, about 400 µL of different concentrations of Ph4 AsCl solution were
loaded into the flow cell and stretched the DNA by approaching the magnet to the bead, or releasing
loaded into the flow cell and stretched the DNA by approaching the magnet to the bead, or releasing
the bead by moving back the magnet while the extension of the DNA molecule was monitored in
the bead by moving back the magnet while the extension of the DNA molecule was monitored in
real-time. The results of the DNA tethering are shown in Figures 5 and 6. From Figure 5A, we can see
real-time. The results of the DNA tethering are shown in Figures 5 and 6. From Figure 5A, we can see
the stepwise shrinking of the DNA extension when a constant 0.5 pN pulling force was exerted upon
the stepwise shrinking of the DNA extension when a constant 0.5 pN pulling force was exerted upon
the bead at low concentration of Ph4AsCl (1 μM). After the DNA molecule condensed to a compact
the bead at low concentration of Ph4 AsCl (1 µM). After the DNA molecule condensed to a compact
state, we had to apply much larger force (5.4 pN in this case) to unravel the condensed structure, as
state, we had to apply much larger force (5.4 pN in this case) to unravel the condensed structure, as
shown in Figure 5B. We could repeat the shrinking and unraveling many times, and the processes
shown in Figure 5B. We could repeat the shrinking and unraveling many times, and the processes was
was reversible in our experiment. When the concentration of Ph4AsCl in the buffer increased, the
reversible in our experiment. When the concentration of Ph4 AsCl in the buffer increased, the tethering
tethering force increased accordingly to unravel the condensed DNA structures. We could still see
force increased accordingly to unravel the condensed DNA structures. We could still see the stepwise
the stepwise structure in the shrinking or pulling curves of DNA molecules when the concentration
structure in the shrinking or pulling curves of DNA molecules when the concentration of Ph4 AsCl is
of Ph4AsCl is not greater than 0.5 mM, as shown in Figures 6 and 7. However, the stepwise structure
not greater than 0.5 mM, as shown in Figures 6 and 7. However, the stepwise
structure disappeared in
disappeared in the shrinking curves when the concentration
of As+ reached 1 mM, as shown in
the shrinking curves when the concentration of As+ reached 1 mM, as shown in Figure 7C. We infer
Figure 7C. We infer than a more compact structure (e.g., globule) was formed in the high
than a more compact structure (e.g., globule) was formed in the high concentration condition.
concentration condition.
Figure 5. DNA extension–time curves under the influence of Ph4As+. (A) The shrinking of DNA at
pulling force 0.5 pN; (B) unraveling of DNA condensed structures.
tethering force increased accordingly to unravel the condensed DNA structures. We could still see
the stepwise structure in the shrinking or pulling curves of DNA molecules when the concentration
of Ph4AsCl is not greater than 0.5 mM, as shown in Figures 6 and 7. However, the stepwise structure
disappeared in the shrinking curves when the concentration of As+ reached 1 mM, as shown in
Figure
7C. 9,We
Polymers 2017,
128 infer than a more compact structure (e.g., globule) was formed in the 6high
of 10
concentration condition.
+
Figure
Ph44As
Figure 5.
5. DNA
DNA extension–time
extension–time curves
curves under
under the
the influence
influence of
of Ph
As+. .(A)
(A)The
Theshrinking
shrinkingof
of DNA
DNA at
at
pulling
force
0.5
pN;
(B)
unraveling
of
DNA
condensed
structures.
pulling force 0.5 pN; (B) unraveling of DNA condensed structures.
Polymers 2017, 9, 128
6 of 9
Polymers
9, 128 the tethering curves, we concluded that the hydrophobic driving force mechanism
6 of 9
By2017,
analyzing
is similar to the effect of poor solvent. However, the former is much stronger, since the tethering
force is on the order of a few pN, while the shrinking force is difficult to measure in the case of
ethanol solution [35]. For consistency and reproducibility, we repeated our tethering cycle under
each condition at least 10 times. The curve of condensing force is presented in Figure 8. The details of
pulling forces and DNA particle sizes are presented in Tables S1–S3.
Figure
6. DNA
curves when
(A) contracting
(B) stretching
various
Ph4As+ concentrations.
Figure
6. extension–time
DNA extension–time
curves
when (A)and
contracting
andat(B)
stretching
at various
+ concentrations.
Figure
6. DNA
extension–time curves when (A) contracting and (B) stretching at various Ph4As+ concentrations.
Ph4 As
Figure 7. DNA extension–time curves at high concentrations ((A) 0.1 mM; (B) 0.5 mM; (C) 1 mM) of Ph4As+.
Figure7.7.
DNA
extension–time
curves
at high
concentrations
(B) 0.5
Figure
DNA
extension–time
curves
at high
concentrations
((A) 0.1((A)
mM;0.1
(B)mM;
0.5 mM;
(C) mM;
1 mM)(C)
of 1PhmM)
4As+.
+
of Ph4 As .
By analyzing the tethering curves, we concluded that the hydrophobic driving force mechanism
is similar to the effect of poor solvent. However, the former is much stronger, since the tethering force
is on the order of a few pN, while the shrinking force is difficult to measure in the case of ethanol
solution [35]. For consistency and reproducibility, we repeated our tethering cycle under each condition
at least 10 times. The curve of condensing force is presented in Figure 8. The details of pulling forces
and DNA particle sizes are presented in Tables S1–S3.
Figure 8. The curve of condensing force at different concentrations of Ph4As+.
Figure 8. The curve of condensing force at different concentrations of Ph4As+.
3.3. Electrophoretic Mobility of DNA
3.3. Electrophoretic
of DNA
We measuredMobility
the electrophoretic
mobility of DNA in the mixture solution with different
+
concentrations
of Ph
4Aselectrophoretic
and 5 mM MgCl
2 by dynamic
scattering
The different
result is
We measured
the
mobility
of DNA light
in the
mixture technique.
solution with
shown
in
Figure
9.
We
can
see
that
the
mobility
of
DNA
changed
from
negative
to
positive
values
+
concentrations of Ph4As and 5 mM MgCl2 by dynamic light scattering technique. The result
is
+
with the
As can
concentration.
mobility
approached
about 1.75
mM of Ph
4As+.
shown
in increasing
Figure 9. We
see that the The
mobility
of DNA
changedzero
fromatnegative
to positive
values
For cation
concentrations
larger than this,
mobility
reversed from
positive,
with
the increasing
As+ concentration.
Thethe
mobility
approached
zeronegative
at aboutto1.75
mM ofimplying
Ph4As+.
that
charge
inversion
occurred.
Meanwhile,
the
size
of
DNA
complex
decreased
with
As+
For cation concentrations larger than this, the mobility reversed from negative to positive, implying
Polymers 2017, 9, 128
7 of 10
Figure 7. DNA extension–time curves at high concentrations ((A) 0.1 mM; (B) 0.5 mM; (C) 1 mM) of Ph4As+.
Polymers 2017, 9, 128
7 of 9
σ
Figure 8.
8. The
The curve
curve of
of condensing
force0at
differentconcentrations
concentrationsof
ofPh
Ph4As
As++..
I
Figure
force
4
ccondensing
= 0 exp(
Δμ atkdifferent
T)
B
B
ed
(1)
mobility(um*cm/vs)
3.3. Electrophoretic
Mobility of
of DNA
DNA
3.3.
Electrophoretic
Mobility
is the
difference in free energy between the dehydrated ion at close
where
∆ = −
We
measured
the
electrophoretic
mobility
ofelectrolyte,
DNA in
in the
the
mixture
solution
with
different
proximity
to
the
colloid
and
the
hydrate mobility
ion
in bulk
d is mixture
the diameter
of thewith
counter
ions,
We measured the electrophoretic
of
DNA
solution
different
+
concentrations
of charge
Ph4As
and 5 of
mM
MgCl
2 by
light scattering
The result
is
and
σ0 is the bare
DNA.
For
thedynamic
current system,
where d ≈technique.
1 nm, hydration
energy
+density
concentrations
of
Ph
4 As and 5 mM MgCl2 by dynamic light scattering technique. The result is
2changed
shown
in
Figure
9.
We
can
see
that
the
mobility
of
DNA
from
negative
to
positive
values
is
about
−5k
BT, and
bare
surface
charge
is
about
1.0
e/nm
,
one
obtains
=
1.6mM.
The
measured
shown in Figure 9. We can see that the mobility of DNA changed from negative to positive values
+.
with the
the
increasing
As++ concentration.
concentration.
The
mobility
approached
zero
atwhere
about charge
1.75 mM
mM
of Ph
Ph4As
Asis
value
of increasing
charge inversion
is about 1.75
mM,
as shown
in Figure
inversion
+
with
As
The
mobility
approached
zero9,at
about
1.75
of
.
4
For
cation
concentrations
larger
than
this,
the
mobility
reversed
from
negative
to
positive,
implying
observed
for
DNA
in
water
at
298
K
in
the
presence
of
low
concentrations
of
a
monovalent
large
For cation concentrations larger than this, the mobility reversed from negative to positive, implying that
that charge
occurred.
Meanwhile,
the
size
ofagreement
DNA
complex
decreased
with
organic
cationinversion
[36].
The observed
critical
concentration
iscomplex
in
withwith
the
predicted
valueAs
by+
charge
inversion
occurred.
Meanwhile,
the
size of
DNA
decreased
As+ concentration.
+ were
concentration.
When
theofconcentrations
As(1)
0.05,
0.1, 0.5,
1,
3 mM,
the
hydrophobicity.
It is noticeable
be 3applicable
for2,a and
weakly
charged
When
the concentrations
As+ that
wereEquation
0.01,of
0.05,
0.1,might
0.5, 0.01,
1,only
2, and
mM,
the corresponding
particle
corresponding
sizes
of 310,
DNA
condensates
were
310,
210,
180, 182,and
and
180
nm,
system.
DNA condensates
isparticle
a highly
charged
system
where
the
correlation
is significant
and
a errors
more
sizes
of DNA
were
300,
210,
200, 180,
182,
and300,
180effect
nm, 200,
respectively,
the
respectively,
and
the
errors
were
±20
nm.
sophisticated
theory
is
needed.
In
spite
of
the
restriction,
we
can
still
use
it
as
a
preliminary
estimation.
were ±20 nm.
The mechanism of DNA charge reversal is similar to colloid charge inversion, the hydrophobic
effect as a driving force for charge
inversion. This mechanism is able to induce charge inversion at
3
As++5mMMg2+
low concentrations of monovalent ions +containing hydrophobic functional groups, and saturation
As
2
effects appearing at higher concentrations.
Following the similar analysis in Reference [36], the critical concentration of counterions in
1
solution when charge inversion occurs can be expressed as
0
-1
-2
0.0
0.5
1.0
1.5
2.0
2.5
3.0
concentration(mM)
Figure 9.
9. Electrophoretic
Electrophoretic mobility
mobility of
of DNA
DNA vs.
vs. the
the concentration
concentration of
of Ph
Ph44As
As++..
Figure
4. Conclusions
The mechanism of DNA charge reversal is similar to colloid charge inversion, the hydrophobic
we found
for inversion.
the first time
that the organic
ionsinversion
of tetraphenyl
effectInas summary,
a driving force
for charge
This mechanism
is able monovalent
to induce charge
at low
chloride
arsenic
(Ph4As+) can
induce
DNAhydrophobic
compactionfunctional
and its groups,
charge and
inversion,
which
is
concentrations
of monovalent
ions
containing
saturation
effects
contradictory
to theconcentrations.
common understanding that charge inversion occurs only if the valence of
appearing at higher
counterions
is Zthe
≥ 3.similar
These analysis
findings in
were
confirmed
magnetic
tweezers measurements,
atomic
Following
Reference
[36],bythe
critical concentration
of counterions
in
force
microscopy
(AFM)
imaging,
and
dynamic
light
scattering
(DLS).
solution when charge inversion occurs can be expressed as
DNA compaction is usually involved the first-order phase transition between elongated and
|σ0 | cause shrinkage
compact states, although some chemical
species
c BI =
exp(∆µ0 /k B T ) of DNA to be much different from
(1)
ed
the transition [40]. DNA compaction occurs in one mode or mixed modes among all-or-none
compaction,
compaction,
adsorption
and wrapping,
depending
condensing
where
∆µ0 =progressive
µ0S − µ0B is the
differenceand
in free
energy between
the dehydrated
ion at on
close
proximity
agents
and
solvents.
The
DNA
compaction
in
Ph
4As+ solution is in a progressive compaction mode,
to the colloid and the hydrate ion in bulk electrolyte, d is the diameter of the counter ions, and σ0 is
which
directly
shownofinDNA.
their For
AFM
and 3).
electrostatic
interaction
the bareischarge
density
theimages
current(Figures
system, 2where
d ≈Since
1 nm,the
hydration
energy
is about
between
monovalent
ions
and
DNA
is
not
able
to
induce
its
compaction
or
charge
inversion,
we
2
I
–5kB T, and bare surface charge is about 1.0 e/nm , one obtains cB = 1.6 mM. The measured value
infer
that
the
main
driving
force
of
DNA
charge
inversion
and
compaction
by
the
organic
of charge inversion is about 1.75 mM, as shown in Figure 9, where charge inversion is observed for
monovalent ion Ph4As+ is the hydrophobic effect.
Supplementary Materials: The following are available online at www.mdpi.com/2073-4360/9/4/128/s1, Table
S1: The condensing force of DNA at different concentrations of Ph4As+, Table S2: The unravelling force of DNA
at different concentrations of Ph4As+, Table S3: The particle size of DNA at different concentrations of Ph4As+.
Polymers 2017, 9, 128
8 of 10
DNA in water at 298 K in the presence of low concentrations of a monovalent large organic cation [36].
The observed critical concentration is in agreement with the predicted value by hydrophobicity. It is
noticeable that Equation (1) might only be applicable for a weakly charged system. DNA is a highly
charged system where the correlation effect is significant and a more sophisticated theory is needed.
In spite of the restriction, we can still use it as a preliminary estimation.
4. Conclusions
In summary, we found for the first time that the organic monovalent ions of tetraphenyl chloride
arsenic (Ph4 As+ ) can induce DNA compaction and its charge inversion, which is contradictory to
the common understanding that charge inversion occurs only if the valence of counterions is Z ≥ 3.
These findings were confirmed by magnetic tweezers measurements, atomic force microscopy (AFM)
imaging, and dynamic light scattering (DLS).
DNA compaction is usually involved the first-order phase transition between elongated and
compact states, although some chemical species cause shrinkage of DNA to be much different from the
transition [40]. DNA compaction occurs in one mode or mixed modes among all-or-none compaction,
progressive compaction, and adsorption and wrapping, depending on condensing agents and solvents.
The DNA compaction in Ph4 As+ solution is in a progressive compaction mode, which is directly
shown in their AFM images (Figures 2 and 3). Since the electrostatic interaction between monovalent
ions and DNA is not able to induce its compaction or charge inversion, we infer that the main
driving force of DNA charge inversion and compaction by the organic monovalent ion Ph4 As+ is the
hydrophobic effect.
Supplementary Materials: The following are available online at www.mdpi.com/2073-4360/9/4/128/s1,
Table S1: The condensing force of DNA at different concentrations of Ph4 As+ , Table S2: The unravelling force
of DNA at different concentrations of Ph4 As+ , Table S3: The particle size of DNA at different concentrations of
Ph4 As+ .
Acknowledgments: We thank Ruxia Wang and Yang Chen for their help in experiments and helpful discussions.
This work is supported by the National Natural Science Foundation of China (11274245, 10974146, 11304232).
Author Contributions: Guangcan Yang and Yanwei Wang conceived and designed the experiments; Wenyan Xia
and Anthony Yang performed the experiments; Yanwei Wang and Wenyan Xia analyzed the data; Guangcan Yang,
Wenyan Xia and Anthony Yang wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.
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