Coleman, A,

Collard, D

Jalabert, L.

Kim, B,

Montasser, I.

Perret, G

Tarhan, M,

Tauran, Yannick

English

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Direct measurement of the mechanism by which
magnesium specifically modifies the mechanical
properties of DNA
I. Montasser, A Coleman, Yannick Tauran, G Perret, L. Jalabert, D Collard,
B Kim, M Tarhan
To cite this version:
I. Montasser, A Coleman, Yannick Tauran, G Perret, L. Jalabert, et al.. Direct measurement of the
mechanism by which magnesium specifically modifies the mechanical properties of DNA. Biomicroflu-
idics, American Institute of Physics, 2017. ￿hal-02105864￿
Direct measurement of the mechanism by which magnesium specifically modifies the
mechanical properties of DNA
I. Montasser, A. W. Coleman, Y. Tauran, G. Perret, L. Jalabert, D. Collard, B. J. Kim, and M. C. Tarhan
Citation: Biomicrofluidics 11, 051102 (2017);
View online: https://doi.org/10.1063/1.5008622
View Table of Contents: http://aip.scitation.org/toc/bmf/11/5
Published by the American Institute of Physics
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Direct measurement of the mechanism by which
magnesium specifically modifies the mechanical
properties of DNA
I. Montasser,1 A. W. Coleman,2,3,a) Y. Tauran,2,3 G. Perret,2 L. Jalabert,2
D. Collard,2,4 B. J. Kim,2,4 and M. C. Tarhan2,4,5,a)
1INRAP-Technopôle de Sidi Thabet, Sidi Thabet 2020, Tunisia
2LIMMS/CNRS-IIS UMI 2820, Institute of Industrial Science, The University of Tokyo,
Tokyo, Japan
3CNRS UMR 5615, Universite Lyon 1, Villeurbanne 69622, France
4CIRMM, Institute of Industrial Science, The University of Tokyo, Tokyo, Japan
5Univ. Lille, CNRS, Centrale Lille, ISEN, Univ. Valenciennes, UMR 8520 - IEMN,
Lille F59000, France
(Received 8 October 2017; accepted 17 October 2017; published online 27 October 2017)
We examine the effect of physiological cations Naþ, Kþ, Mg2þ, and Ca2þ on the
mechanical properties of bundles of k-phage DNA using silicon nanotweezers
(SNTs). Integrating SNTs with a microfluidic device allows us to perform titration
experiments while measuring the effect in real-time. The results show that only for
Mg2þ and in particular, at the intra-nuclear concentration (100 mM), the interaction
occurs. Published by AIP Publishing. https://doi.org/10.1063/1.5008622
The direct observation of the mechanical properties of biopolymers is clearly a consider-
able task. Simple modifications of proteins or other biopolymers may change interaction con-
stants by one or more orders of magnitude, even for such seemingly trivial modifications such
as fluorescence labeling.1 Thus, modification for surface attachment, as in various force meas-
urements, is not necessarily totally apt under physiological situations.2
Another key point is that any measurements of mechanical properties must be carried out
under the correct biological conditions. Too often, we assume that phosphate-buffered saline
(PBS) is a universal medium. Unfortunately, this is not true. Cation concentrations vary depend-
ing on localization. Extracellular concentrations are quite different from intracellular concentra-
tions, which again differ from ion concentrations in the nucleus (Table I).3 To further compli-
cate matters, ion concentrations may vary under external stress. Thus, there exists a need for a
method capable of detecting effects of local specific ion concentrations on the mechanical prop-
erties of biopolymers.
Besides various force spectroscopy methods, e.g., optical tweezers and magnetic tweezers
suffering from the use of chemical modification and molecular stressing while measuring the
properties of DNA,2,4 the development of microelectromechanical systems (MEMS)
technology-based tweezers targeting biological samples opened up methods for handling biopol-
ymers,5 cells, and even small animals.6 Although silicon nanotweezers (SNTs) showed success-
ful real-time measurements on the mechanical properties of biopolymers,7 such methods were
either performed in air or remained “static” not allowing titration experiments and thus seri-
ously restricting our ability to study the effects of molecular interactions on the mechanical
properties of biopolymers in liquid.8 Therefore, adapting microfluidics to the direct measure-
ment of mechanical properties of biopolymers is a major step forward.9 Here, we describe how
the adaptation of a microfluidic system to the SNT [Fig. 1(a)] allows us to observe the effects
of the common physiological cations Naþ, Kþ, Mg2þ, and Ca2þ on the mechanical properties
of DNA, using the non-coordinating NO3
 anion as the counterion.
a)Authors to whom correspondence should be addressed: cagatay.tarhan@yncrea.fr and anthony.coleman@univ-lyon1.fr
1932-1058/2017/11(5)/051102/4/$30.00 Published by AIP Publishing.11, 051102-1
BIOMICROFLUIDICS 11, 051102 (2017)
Three major points are to be emphasized: first, the tips of the SNT, one of which is fixed
and the other can be oscillated in the horizontal plane, are covered with aluminum, increasing
the strength of the DNA binding by strong Al-Phosphate coordination bonding.3 This allows
mechanical force to be applied to the bridging DNA bundles. By means of a lock-in amplifier,
the SNT can be set to move at the resonant frequency (while monitoring the amplitude value in
real-time) with the biopolymers attached between the two arms (Fig. 1 in the supplementary
material). Second, a fluidic device allows the exchange of the solution in which the SNTs are
immersed [Fig. 1(b)]. In this way, variable concentrations of solutes can be studied and titration
experiments are now possible without the need subjecting DNA bundles to the large mechanical
stresses associated with passage across the water meniscus. Third, the real-time measurements
allow the direct calculation of changes in the biopolymers’ mechanical characteristics, e.g.,
stiffness and viscous losses, using the resonance frequency and amplitude values (supplemen-
tary material).
When working with biomacromolecules under conditions approximating to physiological
conditions, the four main physiological cations sodium, potassium, magnesium, and calcium
with varying cation concentrations (in different physiological locations; Table I) should be
taken into account. Each of these cations is introduced in a microfluidic device (placing a
PDMS slab with a 100-lm channel on a cover slip) to access by SNT tips via a side opening
[Figs. 1(c) and 1(d)]. The dimensions of the side opening (105 lm  90 lm) have to be small
enough to provide a stable air-liquid interface and large enough to allow the insertion of SNT
tips (with a gap of 11 lm) without suffering from the capillary effect. Concentration titration of
each cation is performed using a pressure pump connected to the outlet. A negative pressure is
FIG. 1. (a) A microfludic device with a side opening allows SNTs to access the channel. The solution inside the channel
can be exchanged (by the pressure pump connected to the outlet of the channel) owing to the highly stable air-liquid inter-
face. As a result, titration experiments are possible with high sensitivity, after inserting only the tips of SNTs with a DNA
bundle in between. (b) Successive images demonstrate exchanging solutions inside the channel within seconds. The air-
liquid interface at the side opening was extremely stable due to the surface tension. (c) A DNA bundle between the tweezer
tips enters into a microfluidic channel via the side opening. (d) Using a phase-lock-loop amplifier, the resonance frequency
of the system can be monitored in real-time. SNTs with a DNA bundle between the tips were inserted in liquid at around
t¼ 35 s. Inserting the drier, thus stiffer, the DNA bundle in liquid caused a sudden decrease in the resonance frequency as
DNA became softer due to hydration.
TABLE I. Concentrations of the major physiological cation and the corresponding resonance frequency shift. In the case of
Ca2þ, only free ion concentrations are given. For certain localisations, a range of concentrations are known.
Cation
Concentration (mM) Resonance frequency shift (Hz)
Extracellular Intracellular Nuclear Extracellular Intracellular Nuclear
Naþ 140 10 2 0.03 0.02 0.01
Kþ 3.6–5 150 230 0.02a 0.04 0.00
Mg2þ 0.6–1 5–20 100 0.15b 0.17c 0.22
Ca2þ 2 0.0001 0.001 0.07 0.08 0.08
aKþ at 4 mM.
bMg2þ at 1 mM.
cMg2þ at 10 mM.
051102-2 Montasser et al. Biomicrofluidics 11, 051102 (2017)
applied to withdraw the liquid inside the channel to exchange with a new one fed from the
inlet. The pressure (20 mbar) is controlled to provide a constant liquid flow of 45 ll min1
for 20 s.
The experiments were repeated multiple times using the same DNA bundle for each cation
to verify the reproducibility of the effects on the same sample. Initially, a DNA bundle was
captured between the SNT tips9 and added to a Tris buffer solution (pH 6.8) to yield the base
resonance frequency level (for 3 min). Before exposing the bundles to the cation solutions, the
pH was lowered (3 min) to validate the existence of the bundle and returned back to the Tris
buffer solution (3 min). This pH test was performed between each cycle of cation solution addi-
tion to compare the condition of the DNA bundle. The bundle was incubated in each cation
solution for 6 min. The average resonance frequency values were calculated based on the last
30 s of incubation. The pH values of each cation solution were between 7.0 and 7.5, and thus,
any observed change in resonance frequency was not due to the pH effect. Control experiments
(the same solution sequences without DNA between tips) were also performed to examine the
effect of this procedure on the detection mechanism (red curves, Fig. 2).
In Figs. 2(a) and 2(b), the response curves for interactions are given, respectively: sodium
and magnesium cations. The curves for potassium and calcium are given in Fig. 2 in the sup-
plementary material. The observed frequency changes at the first cation injection cycle are sum-
marized in Table I. For Naþ, Kþ, and Ca2þ, no effects are observed under any of the physio-
logical conditions. The subsequent cycles for these cations show little or no change with
respect to the first cycle. For Mg2þ, the situation is quite different. A non-reversible effect is
seen in each cycle of Mg2þ injection (starting at t¼ 1 min, t¼ 28 min, and t¼ 55 min) as an
increase in the resonance frequency (more dominant at the intra-nuclear concentration). A simi-
lar effect is seen also in the amplitude. Mg2þ causes a decrease in the amplitude, while Naþ
does not show any effect (Fig. 3 in the supplementary material). Monitoring resonance fre-
quency provides the necessary information related to stiffness, and together with the amplitude
value, viscous losses are obtained. Calculated stiffness and viscous losses throughout the experi-
ment are shown in Fig. 4 in the supplementary material. This non-reversible effect tends to pla-
teau by the third cycle. To sum up, in the presence of Mg2þ, the DNA bundle has increased
stiffness and higher viscous losses. The non-reversible characteristic of this increase suggests
that Mg2þ is bound directly to phosphate groups of the DNA bundle. We should note that Naþ,
Kþ, and Ca2þ are water structure breakers, whereas Mg2þ is a structure former. Also, the first
FIG. 2. Real-time resonance frequency changes of the SNT with (blue) and without (red) DNA bundles in (a) Naþ solutions
and (b) Mg2þ solutions. Before injecting each set of the cation solutions, a buffer-acid-buffer cycle was performed to moni-
tor the condition of the DNA bundle. Insets correspond to a close-up at the given time periods. To aid the reader, the solu-
tions used are shown at the top.
051102-3 Montasser et al. Biomicrofluidics 11, 051102 (2017)
three show non-ordered rapidly changing water coordination, whereas Mg2þ is a slow exchang-
ing octahedrally water coordinated cation.10
In conclusion, the use of micro-fluidic coupled SNTs has allowed the in-situ detection of
the binding of Mg2þ cations to DNA more dominantly at the intra-nuclear concentration. These
findings demonstrate the power of this method for the real-time determination of biorelevant
interactions.
See supplementary material for more information on the experimental procedures and sup-
plementary figures referred in the text.
The authors thank the VLSI Design and Education Center (VDEC, The University of Tokyo)
for mask production, M. Kumemura for device preparation, and N. Lafitte for providing software.
Y. Tauran acknowledges financial aid from Universite Lyon 1.
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B. Fenet, B. Kim, and A. W. Coleman, J. Inclusion Phenom. Macrocyclic Chem. 77, 213 (2013).
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051102-4 Montasser et al. Biomicrofluidics 11, 051102 (2017)


Direct measurement of the mechanism by which magnesium specifically modifies the mechanical properties of DNA

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Direct measurement of the mechanism by which magnesium specifically modifies the mechanical properties of DNA

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