Discrimination of Polynucleotide Transport through a Highly

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Discrimination of Polynucleotide Transport Through a Highly Hydrophobic Uncharged Nanopore Fabien Picaud, Guillaume Paris, Tijani Gharbi, Mathilde Lepoitevin, PierreEugene Coulon, Mikhael Bechelany, Jean Marc Janot, and Sebastien Balme J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/acs.jpcc.7b00560 • Publication Date (Web): 10 Mar 2017 Downloaded from http://pubs.acs.org on March 14, 2017

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The Journal of Physical Chemistry

Discrimination of Polynucleotide Transport Through a Highly Hydrophobic Uncharged Nanopore Fabien Picaud1,*, Guillaume Paris1, Tijani Gharbi1, Mathilde Lepoitevin2, Pierre-Eugène Coulon3, Mikhael Bechelany2, Jean Marc Janot2, Sébastien Balme2

1

Laboratoire de Nanomédecine, Imagerie et Thérapeutique, EA 4662, Université Bourgogne FrancheComté, Centre Hospitalier Universitaire de Besançon, 16 route de Gray, 25030 Besançon cedex, France

2

Institut Européen des Membranes, UMR5635 CNRS-UM-ENSCM, University of Montpellier, Place Eugène Bataillon, 34095 Montpellier cedex 5, France 3

Laboratoire des Solides Irradiés, École polytechnique, Université Paris-Saclay, Route de Saclay, 91128 Palaiseau Cedex, France

*Corresponding author: [email protected] Abstract The importance of the DNA structures in medicine forces researchers to develop sequencing methods with low cost and high speed. The potential use of nanofluidics for the analysis of the translocation of polynucleotides through a nanopore is a very interesting solution. Here we prove both by experiments and by explicit solvent molecular dynamics simulations that translocation of Polycytidylic acid and Polyadenylic acid can affect significantly the ionic flux behavior. In particular, the nanochannel enhances the velocity difference between polynucleotides during the translocation process. Our results revealed the important role of the strand structures during their translocation as already observed in biochannel. This folding/unfolding process leads to stronger interaction with the nanopore wall that decreases the velocity of the strands. This could lead to the detection of different polynucleotides in a mixture.

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Introduction Biotechnologies have known an amazing development since the control of electrophoretic transport of biomolecules through single nanopore, which opens the way of multiple applications 1-2. For instance, single nanopore is a promising opportunity to sequence DNA strands while it was so far achieved using biological nanochannel.3-5 Even if solid-state nanopores do not have a resolution at the atomic scale due to their length as opposed to the biological ones which are shorter, they are relevant for the detection of biomacromolecules as well as nanoparticle sensor.6-7 The controls of the solid-state nanopore geometry and its surface state make them a noticeable model to improve the fundamental understanding of macromolecule transport at nanoscale.8-9 This latter requires developing comprehensive model to explain and predict the macromolecule behavior through nanopore.10 This knowledge can serve in many areas from single molecule sensing to nanofiltration because the single nanopores can be considered as the basic element of a multipore membrane. The single molecule sensing using nanopore is performed by resistive pulse experiments. Basically, the macromolecule is placed on a reservoir connected to another one by a single nanopore. Then a voltage is applied between both reservoirs and the corresponding current is recorded as a function of the time. When a macromolecule translocates through a nanopore, it induces a current perturbation,11 due to its intrinsic local charges and to its exclusion volume. More, the encapsulation of additional components such as light sensible elements12-14, potential electrodes15-17 or plasmonic gold prism18-19 increases notably the control of the translocation process or the detection of the translocated molecules. The dynamic processes which govern a macromolecule translocation through a solid state nanopore depend on its intrinsic properties, i.e. the nanopore surface state, the composition of the electrolyte solution and the applied voltage.9, 20-21 As an example, the unspecific adsorption can permit to detect protein in SiN nanopore explaining the low capture rate.22 For charged macromolecules, the driving forces which governs the macromolecule entrance inside a nanopore are the electrophoresis and diffusion. However, in the case of uncharged macromolecule, the electro-osmotic flux can govern the transport.20 In term of surface charges, it is interesting to note that most of the experiments were performed on SiN activated by O2 plasma or piranha solution. In both cases, these treatments induce


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the formation of SiO group at the surface and thus leads to a negative surface charge at neutral pH.20 In this case, the DNA and the nanopore exhibit the same charge. An intriguing effect is that the modification of the nanopore surface charge does not induce the expected effect. Indeed, it has been reported that at low salt concentration, the energy barrier for the DNA strand entrance is reduced for negatively charged nanopore compared to uncharged

8

or positively charged ones.23 For positively

charged nanopore, the complexation between positive amine groups and negative phosphate group of DNA could be at the origin of this phenomenon. In the case of uncharged nanopore, some questions still need debate, namely the nature of the interaction between purine and pyrimidine bases with the nanopore surface.24 Indeed, it is well-known that hydrophobic surfaces are used for DNA combing.25 Note that several recent studies have shown that DNA translocation through charged or uncharged nanopore could be solved through specific current events.26-30 This work aims to investigate the transport of polynucleotide inside uncharged and hydrophobic nanopores. The latter was obtained by the reduction of a SiN nanopore with ZnO/Al2O3 bilayers using atomic layer deposition (ALD). The hydrophobic nature was achieved using trimethylsilane groups coating. The translocations of Polyadenylic acid (Poly(A)) and Polycytidylic acid (Poly(C)) were experimentally investigated at low salt concentration (150 mM). To interpret these results molecular dynamic simulations were conducted in a system which mimicked at best the experimental conditions. Indeed, the translocation of a two different DNA strands were simulated through a hydrophobic nanotube made of neopentane molecule assembly. The influence of the voltage was also investigated through the applications of an electric field in the simulations. This will allow us interpreting the role played by the uncharged nanopore surface during the strand translocation.

Material and methods Experimental Material Diethyl zinc (DEZ) (Zn (CH2CH3)2, 95% purity, CAS: 557-20-0), Trimethylaluminum (TMA) (Al(CH3)3, 97% purity, CAS: 75-24-1), sodium chloride (S9888), Polyadenylic acid potassium salt



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noted poly(A) (P9403) and Polycytidylic acid potassium salt noted poly(C) (P4903) with a polydispersity from 600 to 4000 bases, TRIZMA® BASE (T-6791) and hexamethyldisilazane (HMDS) (reagent grade, ≥99%) were purchased from Sigma Aldrich. Potassium chloride was obtained from VWR (26764.298). Deionized water was produced by Milli-Q system (Millipore). The Tetraflow™ flow cell was bought from Nanopore solutions.

Nanopore design SiN membrane window TEM grids (thickness 30 nm, window 50x50 µm) were purchased from Neyco. By using the focused electron beam of a TEM JEOL 2010F, a nanopore was created, with diameter ranging from 9.2 to 10.2 nm (Figure 1). The ALD technique allows adjusting the nanopore diameter and creating OH function in order to permit the surface modification by silanisation. A custom-made ALD setup31 was used to deposit thin Al2O3/ZnO films. ALD was carried out at 100 °C. The pulses, exposure, and purge times were chosen conservatively to ensure completion of the ALD surface reactions and to prevent mixing of the reactive gases. The growth per cycle was about 2 Å/cycle for Al2O3 and 2.1 Å/cycle for ZnO.

32-35

A sequence of four cycles of Al2O3 preceding four

cycles of ZnO was used to reduce the pore diameter. After ALD deposition, a 24 h HMDS vapour exposure treatment was performed at room temperature to create a hydrophobic pore surface with a −Si(CH3)3 bonds (TriMethyl Silane (TMS)), leading to an uncharged surface. After ALD and surface modification, we obtained nanopore with diameter between 5.2 ± 0.5 nm.


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Figure 1. Initial SiN membrane showing the nanopore with a diameter around 9.7 ± 0.5 nm

Resistive pulse experiments The single nanopore was mounted in a Tetraflow™ (http://www.nanoporesolutions.com) cell containing a buffer solution of 150 mM NaCl, 1 mM KCl, and 10 mM of TRIS at pH~8. The current was measured by two Ag/ AgCl 1M KCl electrode isolated from the solution by an agar salt bridge. One electrode was plugged to the positive end of the amplifier (trans chamber) and the other electrode connected to the ground (cis chamber). Polynucleotides (C = 5 10-12 mg/mL) were added in the cis



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chamber. Positive potentials (1 V) were then applied to the trans chamber. A patch-clamp amplifier (EPC10, HEKA electronics, Germany) at a sampling frequency of 200 kHz recorded the ion current. The data acquisition was performed by Instrutech LIH 8+8 acquisition card using patch master software (HEKA electronics, Germany). A lab-made analyzing software (Matlab, Matworks, USA) was used for the data analysis of the dwell time (∆t) and the difference of current intensity (∆I). Typically, our software detects each current event using derivative method. An event was considered when the current increase was higher than 5 pA during a time longer than 20 µs.

Molecular dynamic simulation Model In our simulation model, the hydrophobic nanopore wall is mimicked using a tubular hexagonal arrangement of neopentane (NEOP) molecules (C-(CH3)4), which remains as close as possible to the trimethylsilane coated (-Si-(CH3)3) nanopore wall used in the experiments. The nanopore is maintained through the fixation of the central carbon atom of each (NEOP) molecule. Then, the electronic degrees of freedom of all other NEOP atoms are leaving free. This movement reflects thus the dynamic changes of the molecular group attached to the nanopore wall. The NEOP particles are periodically distributed within the nanotube, respecting a distance close to equilibrium. Each NEOP particle is located along a hexagonal cell. While the different atoms of each NEOP are partially charged, the net charge of the nanopore wall remains zero. The number of NEOP molecules constituting the nanopore depends on the nanopore radius and length. Here we have modeled a nanopore whose dimensions are 5 nm in length and 3 nm in radius, namely 128 NEOP molecules arranged with a hexagonal nanotubular geometry (Figure 2a). This NEOP nanopore is immersed in a graphite volume in order to limit the water volume around the system.


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a)

b)

c)

Figure 2: Point representation of the two nucleotide bases a) the hydrophobic nanopore (top and front views), b) poly(A), and c) poly(C) after equilibration in a water bath.

Simulations Both NEOP-poly(A) and NEOP-poly(C) systems are composed of a hydrophobic nanotube (Figure 2a) and a homogeneous single stranded polynucleotide fragment composed of 10 deoxyadenosine monophosphate poly(A) (Figure 2b) or 10 deoxycytidine monophosphate poly(C) (Figure 2c) bases with water used as an explicit solvent modeled in the TIP3P scheme. This leads to polybase length equal to 5.4 (3.6) nm for poly(A) (poly(C), respectively). Additional sodium ions were added to neutralize the whole system, plus ionic NaCl salt at 0.1M in concentration. In all the simulations, the DNA strands are initially aligned with the nanopore axis and situated near its entrance in order to have the nearest distance between the poly(A) (or poly(C)) and NEOP atoms as close as 0.2 nm. To allow a rapid insertion and diffusion of the strands, each simulation was performed under a voltage of almost 1V, after the classical equilibration of the systems of 2 ns. Note that, in most cases, the molecules were driven inside the nanopore to ensure a simple one-dimensional translocation. However, some simulations were stopped due to the adsorption of the strands on the nanopore edge, without any possibility for the molecule to be transported inside the nanopore. The system was placed at the center



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of a periodic box whose x-y lengths were set to be 7.8 nm while the z direction was chosen according to the length of the poly(A) (poly(C)) molecules, i.e. 12.7 nm. By doing this, all the fundamental aspects of the experiments are thus taken into account in the simulations. Molecular dynamics simulations were implemented using the NAMD simulation package in the Charmm36 force field. The long-range electrostatic interactions are evaluated by the particle-mesh Ewald method. The integration of the Newton’s equations of motion was performed with a time step of 1 fs. The simulations are first equilibrated for 1 ns in the NPT ensemble at T=300K and P=1bar using the Langevin piston Nosé-Hoover method, in order to ensure a constant volume for the later step. Then, simulation in NVT ensemble is implemented once the external electric field is applied. This field was applied along the axial direction of the nanopore to induce a translocation within the nanosecond time scale.

Results and discussion Experimental As previously mentioned, the goal of our study is to better understand the effect of uncharged and hydrophobic surface on the polynucleotide translocation. In order to conduct this experimental study, the nanopore was drilled by electron beam on a silicon nitride (SiN) thin film (thickness L= 30 nm) using a TEM. The initial diameter of the nanopore (9.7 ± 0.5 nm) was reduced using ALD procedure by addition of successive Al2O3/ZnO layers (4.46 nm). Finally, the nanopore was functionalized with trimethylsilane. At the end of the process, the nanopore presents a mean diameter around 5.2 ± 0.5 nm. The translocation of polynucleotide (c = 5 10-12 mg mL-1) was performed at low salt concentration (NaCl 150 mM, pH =8, EDTA 1mM). The polynucleotides (poly(A) or poly(C)) were placed on the cis chamber and a positive voltage of 1V was applied on the trans chamber (E = 3.33 10-7 V m-1). Under these conditions, the polynucleotide translocation induced a current enhancement (Figure 3) as previously reported elsewhere. 36


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Figure 3: Examples of a current trace recorded with poly(C) (150 mM NaCl, and 10 mM TRIS at pH~8, voltage =1V).

From these current enhancements, we have extracted the dwell time (∆t) of each event and the capture rate (f) which characterize the dynamic of the macromolecule translocation through this single nanopore. The histograms of the different measured dwell times (Figure 4) are centered on 6.06 ms and 3.47 ms for poly(A) and poly(C) respectively and the driving force which governs the translocation is electrophoresis. Based on literature, the velocity ( ) should depend on the competition between the zeta potential of the macromolecule ( ) and the nanopore ( ) as :20

Equation 1

where E represents the electric field, ε is the dielectric permittivity (ε=ε0 εr : εr=78), and η the viscosity of the fluid (10-3 Pa s). If we assume that both, poly(A) and poly(C), present the same zeta potential, we cannot explain the dwell time difference. In order to interpret it, the interactions between the macromolecule and the nanopore surface should be taken into account and compared. Another interesting result is the value of the capture rate (f) which was equal to 4.97 s-1 (13.76 s-1) for poly(A) (poly(C)). This capture rate ( is mainly driven by the diffusion.37 However, the electric field should not be omitted at the nanopore entrance, especially for a macromolecule with a high charge density such as DNA. The capture rate can thus be rewritten as: 38



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2∗

Equation 2

Here D (respectively c) represents the diffusion coefficient (respectively, the concentration of DNA), and rC∗ is the radius at the nanopore vicinity. But this description cannot yet explain the different values obtained for poly(A) and poly(C) since it does not take into account the energy barrier (U) that the macromolecule has to overcome to enter the nanopore. To introduce it, f can be expressed as: 39

exp

Equation 3

where

!"

. exp

$ %& '

Equation 4

In Equation 4, A is the cross section area. V0 is connected to an apparent surface charge (z) as

(

%& ' . )

The energy barrier U should then be decomposed in three terms as follow 9 U = U* + UES – ze|V|.

Equation 5

The term zeV is the energy provided by the applied voltage (V) where z is the apparent charge number of the translocated polynucleotide (e is the elementary charge). UES is the electrostatic potential between the internal surface charge of the nanopore and the charge of the polynucleotide.9 These two terms can be considered similar for both nucleotides. On the contrary, U* is the entropic barrier due to the decrease of degrees of freedom, the adsorption and the unfolding of the polymer when it diffuses through the nanopore. The difference of the capture rate values for the two nucleotides is probably due to this entropy variation, and simulations should be done to underline it.


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Figure 4: Distributions of the dwell time for poly(A) (red – 2987 events) and poly(C) (black – 42420 events) translocations through the nanopore.

Simulations To prepare the system, the poly(C) and poly(A) strands were first equilibrated in the solvent until stabilization of their backbone. This latter is considered as equilibrated if the atomic root mean square displacement (RMSD) and the total energy plot are constant. Of course, the energy should be negative to present a stable hydrated molecule. For each system, the two strands were simulated during 200 ns to obtain a stable structure (Figure 2). This long optimization time comes from the construction method of the strands that was obtained through consecutive attachment of each single base. Since fully human dependent, the structure of these strands were not correctly managed and needed a long stabilization time in the solvent. After these preliminary simulations, the two DNA strands were approached from the hydrophobic nanopore and let relaxing during 1 ns before applying a constant voltage to force their insertion in the nanopore. During the insertion, the modifications of the poly(A) and poly(C) structures were quite low



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since the maximum RMSD values did not exceed 0.8 nm at the end of the simulations, but presented significant differences between each other. Indeed, the final RMSD value for poly(A) (0.8nm) is four times higher than the one for the poly(C) strand. This indicates a stronger modification of the poly(A) backbone compared to the poly(C) one, with a progressive unfolding of poly(A), much more pronounced than for poly(C). Note that similar approach was developed previously using carbon nanotube. Smaller polynucleotides composed of only 5 monobases were used and we observed already a differentiation of the insertion behavior between Poly(A) and Poly(C) due to the folding/unfolding of the bases.40 This could influence the U* term in Equation 5.

Figure 5: Root mean square deviation of the strands during their stay in the nanopore as a function of time. (In black, for poly(A); in red for poly(C)). Poly(A) is clearly more unfolded than poly(C) leading to a lower interaction with the nanopore and with the ions.


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The progressive insertion of each polynucleotide was difficult to obtain due to the configuration of our simulations where one nanopore was embedded in a graphite volume. The most frequent events observed during the molecular dynamic runs were the adsorption of the polynucleotides on the external graphite surface. To circumvent the problem, we have pursued our simulations by placing the molecules inside the nanopore, let the molecules relaxed and then applied the voltage to diffuse them inside the nanopore. Note that the initial position of each strand was roughly the same for all the simulations. To explain the different physical mechanisms of the diffusion process of each strand, we plot for poly(A) and poly(C), the different energy contributions felt by the strand when passing under voltage from the inner part of the nanopore (Fig. 6) to the reservoir.



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Figure 6: Interaction between polynucleotides and NEOP (black) and WATER (green) during polynucleotides diffusion inside the nanopore. Left panels for poly(A) and right panels for poly(C). Snapshots of the simulations are also proposed for the two situations. Instantaneous interactions are shown together with smoothing data curves (full green or black lines).

Our results demonstrate here that poly(A) and poly(C) interact with the nanopore surface during their diffusion. The interaction with NEOP (and ion, not shown here) is almost twice lower for poly(A) compared to poly(C). However, the role of water is clearly undistinguishable in this diffusion process since all the bases feel the same amount of energy with water during their


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diffusion across the nanopore. To better quantify the role of each contribution in the total interaction energy, we performed energy average for each strand during its diffusion inside the NEOP nanopore. Note that the averages were calculated only on frames where the strands were completely inside the nanopore and not in phase of repelling. NEOP

ION

WATER

poly(A)

-68 ± 8

-710 ± 90

-1230 ± 90

poly(C)

-37 ± 6

-400 ± 100

-1200 ± 100

Table 1: Mean energy (in kcal/mol) between the strands and the different elements surrounding them inside the nanopore. The averages were performed when the strands were only in the nanopore.

The values computed in Table 1 confirm our analyses. Poly(A) presents a better affinity for the NEOP nanopore than poly(C). The interaction with ions is also strongly diminished with poly(A) compared to poly(C) strands. Water molecules energy contribution is identical for the two kinds of molecules in the nanopore. In this high confined system, the organisation of the water is strong and could not be too perturbed. To understand the differences in energy of each strands, we plotted in Figure 7 the radial distribution function of the strands with the nanopore (Figure 7a) and the sodium atoms (Figure 7b) respectively.

a)

b)



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Figure 7: a) Radial distribution functions of strands with NEOP molecules of the nanopore and b) with sodium atoms. (In black, for poly(A); in red for poly(C)).

From Figures 7, it is clear that the organization of the NEOP molecules and sodium atoms around the poly(A) molecule is stronger than around poly(C). The structure of the polynucleotides is certainly at the origin of this since the poly(A) tended to unfold progressively on the nanopore walls while poly(C) remains quasi stable during its diffusion across the nanopore (see root mean square deviation plots in Figure 5). Indeed, the unfolding of the poly(A) could be at the origin of the optimized interaction between the carbon-nitride cycles and the NEOP molecules. The different behaviours observed for the encapsulation dynamics of the poly(C) and poly(A) molecules when translocating through the NEOP channel should conduct to differentiated velocity which could lead to a preliminary response for future sequencing method. Besides many studies were focused on the ability of carbon nanotubes to sequence DNA strands depending on their length and on their basis arrangements. The high hydrophobicity proposed by the NEOP nanopore and its short diameter (which can be precisely controlled experimentally) could be a good alternative to separate quickly the DNA bases. Looking into that direction, we have analysed the mean square displacement (MSD) of the strands along the nanopore axis as a function of time. These MSD are linearly related to the diffusion coefficient in a simple mobility scheme and are thus an excellent tool to characterize the strand velocity inside the nanopore.


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Figure 8 depicts the MSD for poly(A) and poly(C). As shown in this figure, the slope of the curve is clearly less for poly(A) than for poly(C) leading to a lower diffusion coefficient along the nanopore axis for poly(A) than for poly(C). Based on the Einstein rules, the estimations of these diffusion coefficients were equal to 0.86 nm²/ns for poly(C) compared to 0.40 nm²/ns for poly(A). The diffusion was thus 2.15 faster for poly(C) than for poly(A), a value which could be qualitatively related to the experimental dwell time difference. The interaction of the two strands with the NEOP molecules combined to their competitive unfolding could explain this large difference.

Figure 8: Mean square displacement of the strands during their stage in the nanopore as a function of time. (In black, for poly(A); in red for poly(C)). The slope of the curve for poly(A) is clearly less than for poly(C).

Discussion



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While spontaneous insertion of poly(A) was difficult to observe as opposed to the one for poly(C), our simulations based on the progressive diffusion of these strands under voltage can be compared to experiments. This problem of insertion was already underlined in ssDNA/(10, 10) single wall nanotube systems where the full encapsulation was not spontaneous for simulation time up to 2 ns (T=400 K and P=3 bars) in aqueous environment. This result was interpreted as due to the water solvated nanotube channel which was first equilibrated before inserting the molecules. Since our protocol was similar, we can conclude that on stronger hydrophobic NEOP nanopores, the structure and energetics of water adsorption inside the channel play an important role for the dynamics of the spontaneous insertion process. Besides, we have shown recently that the adsorption of water onto the hydrophobic NEOP wall showed interesting structural and thermodynamic behavior due to transition between drilled and wet capillary condensation of water confined inside a nanometer-size cylindrical pore41. In particular, at room temperature, the water desorption was characterized by a progressive and irreversible water departure from the nanopore after several nanoseconds with specific confined water organization. Indeed, the formation of water layers is determined by the energy due to the hydrogen bond network formation inside the hydrophobic nanopore. A nice solution to definitively wet the nanopore was to incorporate inside the nanopore a small biochannel able to keep the water confined. The insertion of the strands inside the nanochannel is thus difficult since it would require breaking the tight water structure and force the water molecules to approach the hydrophobic wall, which is generally unfavorable. Once inserted, poly(A) and poly(C) did not show the same structuration with a RMSD value, four times higher for poly(A) than for poly(C). This strong difference in the backbone structure between the two strands allows poly(A) to interact strongly with NEOP wall and with ions. Indeed, it contains one more aromatic cycle that leads to a stronger affinity with the NEOP molecules and with ions when unfolded. These two effects (unfolding + interactions) modify the capture rate (Equation 3) of poly(A) by reducing its velocity inside the nanopore. These observations were already deduced when poly(A) (poly(C)) diffused across a biologic ionic channel (α-hemolysin). In that case, as for our system, it was demonstrated that the biochannel presented important blockade amplitude, and/or blockade time differences. Two possible explanations were used to interpret such differences. The first one dealt with


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a specific interaction of poly(C) with the residues at the aperture of the biochannel which could limit the ionic flux. The second reason came from the structure of the poly(C) which could remain in a helical conformation, producing a greater ionic obstruction than poly(A) which was unfolded.42 Here, the progressive unfolding of poly(A) compared to poly(C) in the NEOP could explain their velocity differences observed both in experiments and in simulations. Indeed, this observable is at the origin of the diffusion of the two strands inside the nanopore. Since poly(A) unfolds and interacts strongly with NEOP molecules, it is slower compared to poly(C) that feels less the hydrophobic wall. The strongest interactions with Na+ ions observed in the poly(A) translocation can also limit its diffusion along the nanopore axis. Besides, the diffusion coefficient obtained through the MSD analyses confirms these deductions. The ratio Dpoly(C)/Dpoly(A) tends to 2.2 which can be related directly to the dwell time ratio obtained in experiments, i.e. ∆tpoly(A)/∆tpoly(C) = 2.6. As already mentioned, the major contribution which could explain these ratios could be originated from the NEOP interaction which is 1.8 times lower for poly(A) than for poly(C). This reduces accordingly the poly(A) velocity.

Conclusion In this article, we have shown both experimentally and by simulations that manufacturing highly hydrophobic single nanopore could be a good solution for DNA sequencing component. We have proven using our technology that the dwell time of poly(A) strands was 2.6 longer than for poly(C) ones and can be connected to the diffusion ratio 2.2 obtained by simulations. The blockade event due to the strand translocation could thus be easily isolated experimentally. Of course, further studies need to be developed to generalize our approach in order to complete our sequence viewer, but the easy manufacture of the hydrophobic nanopore is an important step towards the reduced cost of such sequencer.

Conflict of interest



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The authors declare no competing financial interest. Acknowledgments: Calculations were performed with the supercomputer regional facility Mesocenter of the University of Franche-Comté. The authors gratefully acknowledge the financial support of the Agence

Nationale de la Recherche (ANR Transion ANR-2012-BS08-0023).

References

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TOC Graphic

Manufacturing highly hydrophobic single nanopore appears

7 6

Relative Frequency

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as a good solution for DNA sequencing component. We

5

Poly C

4

proved using our technology that the dwell time of poly(A)

Poly A

3

strands was 2.6 longer than for poly (C).

2 1 0 -4.0

-3.5

-3.0

-2.5

-2.0

-1.5

-1.0

-0.5

0.0

Log (DT)


Discrimination of Polynucleotide Transport through a Highly

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