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Microscopic Mechanics of Hairpin DNA Translocation through Synthetic Nanopores
Abstract
Nanoscale pores have proved useful as a means to assay DNA and are actively being developed as the basis of genome sequencing methods. Hairpin DNA (hpDNA), having both double-helical and overhanging coil portions, can be trapped in a nanopore, giving ample time to execute a sequence measurement. In this article, we provide a detailed account of hpDNA interaction with a synthetic nanopore obtained through extensive all-atom molecular dynamics simulations. For synthetic pores with minimum diameters from 1.3 to 2.2 nm, we find that hpDNA can translocate by three modes: unzipping of the double helix and--in two distinct orientations--stretching/distortion of the double helix. Furthermore, each of these modes can be selected by an appropriate choice of the pore size and voltage applied transverse to the membrane. We demonstrate that the presence of hpDNA can dramatically alter the distribution of ions within the pore, substantially affecting the ionic current through it. In experiments and simulations, the ionic current relative to that in the absence of DNA can drop below 10% and rise beyond 200%. Simulations associate the former with the double helix occupying the constriction and the latter with accumulation of DNA that has passed through the constriction.
... A majority of NFS experiments have involved DNA in some way, due to the importance of the molecule in biology and biotechnology. In addition to the extensive use of NFS for probing the forces involved in unfolding of single DNA molecules [14][15][16][17][18][19][20], protein-DNA interactions have also been explored [21][22][23]. DNA is an especially good subject for NFS because it is highly negatively charged ( À e per nucleotide at physiological pH) and, therefore, can easily be captured by the electric field of the nanopore. ...
... At low bulk ion concentrations, the second effect dominates and the current is enhanced. However, in some cases, both enhancements and reductions are seen under the same macroscopic conditions [18,20,31]. This suggests that the ion current depends on microscopic details of the DNA conformation in the pore. ...
... Abrupt increases in this distance are observed when the enzyme and DNA dissociate. In all cases, a force was applied to reduce the affinity of the DNA for the pore walls [20]; however, for the 2 V simulation this force was not applied until t ¼ 56 nm. Fig. 14.2a. ...
Nanopore force spectroscopy (NFS) has emerged as a convenient method to characterize the behavior of single biomolecules and biomolecular assemblies under force. NFS has many advantages over conventional single molecule techniques, such as being label-free and high throughput; however, NFS lacks direct control over the force applied to the biomolecules and registers the conformational transitions induced by the force only indirectly, by monitoring changes in the ionic current passing through the pore. In this chapter, we describe how all-atom molecular dynamics simulations can complement NFS experiments by providing information inaccessible to experiment. The chapter illustrates applications of the molecular dynamics (MD) method to interpret the results of NFS measurements, characterize the forces involved and determine the microscopic origin of the observed phenomena. Important technical aspects of the method, as well as its pitfalls and limitations are briefly discussed.
... Reflecting the charge, size and shape of a biomolecule, the transport rate can vary over several orders of magnitude [9]. The nanopore transport can also be highly selective1011121314. However, controlling the rate and selectivity of the nanopore transport remains a major challenge. ...
... All simulations reported in this article were performed using the molecular dynamics program NAMD2 [76]. Unless stated otherwise, the simulations employed periodic boundary conditions, CHARMM27 [83] parameter set for water, ions and nucleic acids, and CHARMMcompatible parameters for silicon nitride [11]; ion-pair specific corrections to the Lennard-Jones parameter σ [84], a 2–2–6-fs multiple timestepping, SETTLE algorithm to keep water molecules rigid [85], RATTLE algorithm to keep rigid all other covalent bonds involving hydrogen atoms [86], a 7–8 ˚ A cutoff for van der Waals and short-range electrostatic forces. In the simulations of bulk solutions we instructed NAMD2 to remove net momentum before every full electrostatics calculation with the zeroMomentum [87] feature. ...
... The setup of our dual bath temperature control is shown inFigure 5a . A 3.5 nm-thick Si 3 N 4 mem-brane was built according to procedures described else- where [11]. A double-cone pore of a 3.5 nm-diameter in its center and 4.3 nm-diameter openings at both sides was cut by removing atoms from the membrane. ...
Local modulation of temperature has emerged as a new mechanism for regulation of molecular transport through nanopores. Predicting the effect of such modulations on nanopore transport requires simulation protocols capable of reproducing non-uniform temperature gradients observed in experiment. Conventional molecular dynamics (MD) method typically employs a single thermostat for maintaining a uniform distribution of temperature in the entire simulation domain, and, therefore, can not model local temperature variations. In this article, we describe a set of simulation protocols that enable modeling of nanopore systems featuring non-uniform distributions of temperature. First, we describe a method to impose a temperature gradient in all-atom MD simulations based on a boundary-driven non-equilibrium MD protocol. Then, we use this method to study the effect of temperature gradient on the distribution of ions in bulk solution (the thermophoretic effect). We show that DNA nucleotides exhibit differential response to the same temperature gradient. Next, we describe a method to directly compute the effective force of a thermal gradient on a prototypical biomolecule—a fragment of double-stranded DNA. Following that, we demonstrate an all-atom MD protocol for modeling thermophoretic effects in solid-state nanopores. We show that local heating of a nanopore volume can be used to regulate the nanopore ionic current. Finally, we show how continuum calculations can be coupled to a coarse-grained model of DNA to study the effect of local temperature modulation on electrophoretic motion of DNA through plasmonic nanopores. The computational methods described in this article are expected to find applications in rational design of temperature-responsive nanopore systems.
... The all-atom MD simulations were performed using the program NAMD 30 2.9 with periodic boundary conditions. CHARMM-GUI 28,29 } and Aksimentiev's lab protocols were used to build membrane-inserted pores and the Si 3 N 4 system 39,40 . A Langevin thermostat was applied to the non-hydrogen atoms (Si 3 N 4 ) with a damping constant of 1.0 ps −1 to maintain a temperature of 298.15 K. Interactions between all atoms of the systems, including atoms from the water, ions, and nucleic acids, were calculated using the C27 CHARMM forcefield 31,32 . ...
... After assembly, each system was minimized with the gradual removal of heavy atom constraints, equilibrated for 5 ns in the CPT ensemble and subjected to 60-100 ns runs with applied voltage in the NVT ensemble 12 . The non-bonded parameters were obtained from Comer et al. 40 To maintain the nanopore during the MD simulations, a harmonic potential was applied on the heavy atoms to ensure an appropriate dielectric constant of the surface (ε ∼ 7.5) and bond length. The model pore was generated using the numerical recipe provided by Comer & Aksimentiev for the construction of a phantom pore 16 . ...
... The BROMOC setup for studies of the beta-barrel proteins considered here was analogous to the setup previously reported. 15 In addition to open-pore current-voltage relations, we have considered studies of four different homo-polymeric strands (ss-poly(dA) 40 , ss-poly(dC) 40 , ss-poly(dT) 40 ) with readily available experimental data. Two different orientations were studied. ...
We developed a novel scheme based on the grand-canonical Monte Carlo/Brownian dynamics simulations and have extended it to studies of ion currents across three nanopores with the potential for single-stranded DNA (ssDNA) sequencing: solid-state nanopore Si₃N₄, α-hemolysin, and E111N/M113Y/K147N mutant. To describe nucleotide-specific ion dynamics compatible with ssDNA coarse-grained model, we used the inverse Monte Carlo protocol, which maps the relevant ion-nucleotide distribution functions from all-atom molecular dynamics (MD) simulations. Combined with the previously developed simulation platform for Brownian dynamics simulations of ion transport, it allows for microsecond- and millisecond-long simulations of ssDNA dynamics in the nanopore with a conductance computation accuracy that equals or exceeds that of all-atom MD simulations. In spite of the simplifications, the protocol produces results that agree with the results of previous studies on ion conductance across open channels and provide direct correlations with experimentally measured blockade currents and ion conductances that have been estimated from all-atom MD simulations.
... Second, interactions between DNA and the pore wall may obscure measurements of the strength of a protein-DNA bond, in particular, at low transmembrane biases. For example, our MD simulations of dsDNA translocation in the absence of bound proteins revealed a stick-slip character of dsDNA motion for voltages of 1 V and below [52][53][54][55], which is associated with dsDNA interaction with the pore wall (see also Fig. 6f). Treatment of the pore surface with a coating that eliminates DNA sticking within the nanopore (and protein sticking to the membrane) will be required to perform reliable measurements of the rupture voltage. ...
... In our setup, the z axis is normal to the membrane and parallel to the applied electric field. To minimize thermal noise, the current was computed over a region within the pore (− /2 ≤ z ≤ /2 where = 9 nm) Table 1 [46,52,57] and are due to the increased ion concentration in the vicinity of the DNA. ...
... One could clearly identify steps in the traces shown in Fig. 6f, which reflects the stick-slip character of dsDNA motion through pores smaller in diameter than B-form DNA. Such steps have also been observed in MD simulations DNA hairpin transport through nanopores [52]. ...
Through all-atom molecular dynamics simulations, we explore the use of nanopores in thin synthetic membranes for detection and identification of DNA binding proteins. Reproducing the setup of a typical experiment, we simulate electric field driven transport of DNA-bound proteins through nanopores smaller in diameter than the proteins. As model systems, we use restriction enzymes EcoRI and BamHI specifically and nonspecifically bound to a fragment of dsDNA, and streptavidin and NeutrAvidin proteins bound to dsDNA and ssDNA via a biotin linker. Our simulations elucidate the molecular mechanics of nanopore-induced rupture of a protein-DNA complex, the effective force applied to the DNA-protein bond by the electrophoretic force in a nanopore, and the role of DNA-surface interactions in the rupture process. We evaluate the ability of the nanopore ionic current and the local electrostatic potential measured by an embedded electrode to report capture of DNA, capture of a DNA-bound protein, and rupture of the DNA-protein bond. We find that changes in the strain on dsDNA can reveal the rupture of a protein-DNA complex by altering both the nanopore ionic current and the potential of the embedded electrode. Based on the results of our simulations, we suggest a new method for detection of DNA binding proteins that utilizes peeling of a nicked double strand under the electrophoretic force in a nanopore.
... In a similar spirit, our model system is exclusively informed with the native three-dimensional organization of the Zika xrRNA, thus discounting sequence-dependent effects and specific interactions with nucleases. More in general, it allows for a comparison with other biologically-motivated contexts where the intra-molecular organization has been investigated in connection with the compliance to translocate through enzymes or synthetic pores [6][7][8][9][10][11][12][13][14][15][16]. ...
... Nanopore translocation is a powerful method to probe biopolymers as well as understanding how they interact with processive enzymes [8,15,26,[30][31][32][33][34][35]. Here we used it to clarify the structural determinants of xrRNAs resistance to exonuclease degradation. ...
xrRNAs from flaviviruses survive in host cells for their exceptional dichotomic response to the unfolding action of different enzymes. They can be unwound, and hence copied, by replicases, and yet can resist degradation by exonucleases. How the same stretch of xrRNA can encode such diverse responses is an open question. Here, by using atomistic models and translocation simulations, we uncover an elaborate and directional mechanism for how stress propagates when the two xrRNA ends, 5′ and 3′, are driven through a pore. Pulling the 3′ end, as done by replicases, elicits a progressive unfolding; pulling the 5′ end, as done by exonucleases, triggers a counterintuitive molecular tightening. Thus, in what appears to be a remarkable instance of intra-molecular tensegrity, the very pulling of the 5′ end is what boosts resistance to translocation and consequently to degradation. The uncovered mechanistic principle might be co-opted to design molecular meta-materials.
... 80 A second nanopore system was built and simulated following the exact same procedures as above except that all Ca 2+ ions were replaced by calcium heptahydrate complexes, Ca 2+ (H 2 O) 7 , and that NBFIX corrections, Table 1, were used to describe the interactions between Ca 2+ (H 2 O) 7 In all simulations of the nanopore systems, surface atoms of the silicon nitride membrane were restrained using harmonic potentials of 10 kcal/(mol·Å 2 ) spring constants; the interior atoms were restrained using 1 kcal/(mol·Å 2 ) harmonic potentials to give the membrane a relative bulk permittivity of 7.5. 81 The interactions of silicon nitride atoms with the rest of the system were described by a custom force field. 80 All other interactions were governed by the CHARMM36 force field; previously described NBFIX corrections were applied to describe interactions of potassium ions with chloride ions and DNA phosphates. ...
... 29 A gridbased potential 82 was applied to prevent calcium ions from sticking to the surface of Si 3 N 4 . 81 The DNA was constrained to remain aligned along nanopore axis via a halfharmonic potential acting on the DNA phosphorous atoms; the onset of the half-harmonic potential began at 1.5 nm from the axis of the nanopore, and the spring constant was 1000 pN/nm. ...
Calcium ions (Ca(2+) ) play key roles in various fundamental biological processes such as cell signaling and brain function. Molecular dynamics (MD) simulations have been used to study such interactions, however, the accuracy of the Ca(2+) models provided by the standard MD force fields has not been rigorously tested. Here, we assess the performance of the Ca(2+) models from the most popular classical force fields AMBER and CHARMM by computing the osmotic pressure of model compounds and the free energy of DNA-DNA interactions. In the simulations performed using the two standard models, Ca(2+) ions are seen to form artificial clusters with chloride, acetate, and phosphate species; the osmotic pressure of CaAc2 and CaCl2 solutions is a small fraction of the experimental values for both force fields. Using the standard parameterization of Ca(2+) ions in the simulations of Ca(2+) -mediated DNA-DNA interactions leads to qualitatively wrong outcomes: both AMBER and CHARMM simulations suggest strong inter-DNA attraction whereas, in experiment, DNA molecules repel one another. The artificial attraction of Ca(2+) to DNA phosphate is strong enough to affect the direction of the electric field-driven translocation of DNA through a solid-state nanopore. To address these shortcomings of the standard Ca(2+) model, we introduce a custom model of a hydrated Ca(2+) ion and show that using our model brings the results of the above MD simulations in quantitative agreement with experiment. Our improved model of Ca(2+) can be readily applied to MD simulations of various biomolecular systems, including nucleic acids, proteins and lipid bilayer membranes. This article is protected by copyright. All rights reserved.
... Nanopore-based sensing protocols have found a large number of applications such as nucleic acid sequencing [12,13] and peptide folding analysis [8,14,15]. This experimental activity stimulated several theoretical and computational approaches to describe capture and transport mechanisms [16][17][18][19][20] and to establish a correlation between current values and macromolecule conformations in the pore [5,[21][22][23]. ...
... The pore is generated following the approach proposed in Refs. [21,49,50] and reported in the bionanotechnology-tutorial by Aksimentiev and Comer 3 for the SiN pore. However, in order to keep out surface charge effects, we set the atomic charges of the pore atoms to zero. ...
Several devices for single-molecule detection and analysis employ biological and artificial nanopores as core elements. The performance of such devises strongly depends on the amount of time the analytes spend into the pore. This residence time needs to be long enough to allow the recording of a high signal-to-noise ratio analyte-induced blockade. We propose a simple approach, dubbed nanopore tweezing, for enhancing the trapping time of molecules inside the pore via a proper tuning of the applied voltage. This method requires the creation of a strong dipole that can be generated by adding a positive and a negative tail at the two ends of the molecules to be analyzed. Capture rate is shown to increase with the applied voltage while escape rate decreases. In this paper we rationalize the essential ingredients needed to control the residence time and provide a proof of principle based on atomistic simulations.
... However, the presence of a hpDNA can dramatically change ion distributions in a nanopore and affect ionic current passing through it. As reported by Comer et al. [43] ( Fig. 4c), the ionic current, relative to the hpDNA transport through the nanopore, could drop below 10 % (double helix occupying the constriction) and increased beyond 200 % (accumulation of DNA that had moved away from the constriction). So far, the discrimination of DNA sequences has not been successfully realized using solidstate nanopores. ...
... Color online) Study of current blockage occurring during DNA transport through nanopores. a Current blockage with and without DNA translocation, reprinted with permission from[42], Copyright 2004, Biophysical Society; b the effect of dsDNA conformation on ionic current blockage, reprinted with permission from[22], Copyright 2004, Royal Society of Chemistry; c enhancement of ionic current after hpDNA translocation through the narrowest constriction of the nanopore, reprinted with permission from[43], Copyright 2009, Biophysical Society; d schematic of a graphene nanopore-based device for sequencing DNA, reprinted with permission from[48], Copyright 2012, American Chemical Society; e ionic current discrimination for poly(A-T) ...
DNA sequencing based on nanopore sensors is a promising tool for third-generation sequencing technology because of its special properties, such as revolutionized speed and low cost. With about two decades of nanopore technology development, the pioneering work has demonstrated the ability of nanopores to perform single-molecule detection and DNA sequencing. However, the microscopic mechanisms of DNA transport dynamics through nanopores remain largely unknown. Currently, DNA microscopic transport in a nanopore is difficult to characterize and several unexpected experimental observations are equivocal. This limitation can be resolved using theoretical calculations and simulations. These computational methods can monitor the entire dynamic process that DNA undergoes in solution at a single-atom resolution that can accurately unveil the mystery of DNA transport dynamics and predict certain unexpected phenomena. This paper mainly reports the recent applications of computational and simulation methods applied to the study of DNA transport through both biological and synthetic nanopores. We hope the theoretical calculations and simulations of DNA transport through nanopores can benefit the design of DNA sequencing devices.
... Ubiquitous examples are found in living cells, where double-stranded DNA and folded RNAs are translocated and unzipped by ATP-fueled enzymes [13]. Nanopore-based DNA unzipping is essential in genome sequencing setups [10,25] and DNA capture and threading processes, too [26][27][28][29]. Notably, unzipping experiments combined with advanced theoretical methods allowed to characterize the abrupt unzipping transition thermodynamics of RNA motifs or DNA hairpins of tens of base pairs [30][31][32]. ...
Using theory and simulations, we carried out a first systematic characterization of DNA unzipping via nanopore translocation. Starting from partially unzipped states, we found three dynamical regimes depending on the applied force, f: (i) heterogeneous DNA retraction and rezipping (f < 17pN), (ii) normal (17pN < f < 60pN) and (iii) anomalous (f > 60pN) drift-diffusive behavior. We show that the normal drift-diffusion regime can be effectively modelled as a one-dimensional stochastic process in a tilted periodic potential. We use the theory of stochastic processes to recover the potential from nonequilibrium unzipping trajectories and show that it corresponds to the free-energy landscape for single base-pairs unzipping. Applying this general approach to other single-molecule systems with periodic potentials ought to yield detailed free-energy landscapes from out-of-equilibrium trajectories.
... showed that the strong convective vortices may not be responsible for the overlimiting current. To find out the true mechanism, here, we report all-atom molecular dynamics (MD) simulations [35][36][37][38][39] to understand ion concentration polarization (ICP) and overlimiting current near a nanochannel for the first time. ...
In this paper, we report for the first time overlimiting current near a nanochannel using all-atom molecular dynamics (MD) simulations. Here, the simulated system consists of a silicon nitride nanochannel integrated with two reservoirs. The reservoirs are filled with 0.1M potassium chloride (KCl) solution. A total of ∼1.1 million atoms are simulated with a total simulation time of ∼1μs over ∼ 30000 CPU hours using 128 core processors (Intel(R) E5-2670 2.6 GHz Processor). The origin of overlimiting current is found to be due to an increase in chloride (Cl-) ion concentration inside the nanochannel leading to an increase in ionic conductivity. Such effects are seen due to charge redistribution and focusing of the electric field near the interface of the nanochannel and source reservoir. Also, from the MD simulations, we observe that the earlier theoretical and experimental postulations of strong convective vortices resulting in overlimiting current are not the true origin for overlimiting current. Our study may open up new theories for the mechanism of overlimiting current near the nanochannel interconnect devices.
... Nanopore translocation is a powerful single-molecule probing technique that has been used in diverse contexts: from studying the physical response of homopolymers [19-21, 32, 34, 39], to the topological friction in chains with knots and links [33,[40][41][42][43][44][45][46][47][48], for sequencing and analyzing biopolymers' secondary and tertiary structures [7,11,29,41,[49][50][51][52][53][54][55][56][57], and study RNA too [1][2][3][4][6][7][8]. ...
We use MD simulations to study the pore translocation properties of a pseudoknotted viral RNA. We consider the 71-nucleotide long xrRNA from Zika virus and establish how it responds when driven through a narrow pore by static or periodic forces applied to either one of the two termini. Unlike the case of fluctuating homopolymers, the onset of translocation is significantly delayed with respect to the application of static driving forces. Because of the peculiar xrRNA architecture, activation times can differ by orders of magnitude at the two ends. Instead, translocation duration is much smaller than activation times and occurs on timescales comparable at the two ends. Periodic forces amplify significantly the differences at the two ends, both for activation times and translocation duration. Finally, we use a waiting-times analysis to examine the systematic slowing-downs in xrRNA translocations and associate them to the hindrance of specific secondary and tertiary elements of xrRNA. The findings ought to be useful as a reference to interpret and design future theoretical and experimental studies of RNA translocation.
... Nanopore translocation is a powerful single-molecule probing technique that has been used in diverse contexts: from studying the physical response of homopolymers, [19][20][21]33,35,40 to the topological friction in chains with knots and links, 34,41−49 for sequencing and analyzing biopolymers' secondary and tertiary structures, 7,11,30,42,[50][51][52][54][55][56]58 and study RNA, too. 1−4,6−8 Here, we used nanopore translocation simulations to study the compliance of a viral RNA to be driven through a narrow pore. ...
We use MD simulations to study the pore translocation properties of a pseudoknotted viral RNA. We consider the 71-nucleotide-long xrRNA from the Zika virus and establish how it responds when driven through a narrow pore by static or periodic forces applied to either of the two termini. Unlike the case of fluctuating homopolymers, the onset of translocation is significantly delayed with respect to the application of static driving forces. Because of the peculiar xrRNA architecture, activation times can differ by orders of magnitude at the two ends. Instead, translocation duration is much smaller than activation times and occurs on time scales comparable at the two ends. Periodic forces amplify significantly the differences at the two ends, for both activation times and translocation duration. Finally, we use a waiting-times analysis to examine the systematic slowing downs in xrRNA translocations and associate them to the hindrance of specific secondary and tertiary elements of xrRNA. The findings provide a useful reference to interpret and design future theoretical and experimental studies of RNA translocation.
... The origin of the noise was postulated as surface charge fluctuations [24,25], nanochannel's opening and closing processes in the case of track etched membranes [26], the formation of nanobubble inside the nanopore [27], and cooperative effect on ion motion inside confined geometry [28]. In this paper, we propose allatom molecular dynamics simulations (AA-MD) framework [29][30][31][32] to predict noise inside solid-state nanopores. Any statistical fluctuation of current or voltage may be quantified by the variance, its square root, the standard deviation, or the power spectral density (PSD) which also considers the frequency (f) dependence. ...
In this paper, we perform all-atom molecular dynamics (AA-MD) simulations to predict noise in solid-state nanopores. The simulation system consists of ∼70,000 to ∼350,000 atoms. The simulations are carried out for ∼1.3 µs over ∼6500 CPU hours in 128 processors (Intel® E5-2670 2.6 GHz Processor). We observe low and high frequency noise in solid-state nanopores. The low frequency noise is due to the surface charge density of the nanopore. The high frequency noise is due to the thermal motion of ions and dielectric material of the solid-state nanopore. We propose a generalised noise theory to match both the low and high frequency noise. The study may help ways to study noise in solid-state nanoporous membranes using MD simulations.
... In a similar spirit, our model system is exclusively informed with the native three-dimensional organization of the Zika xrRNA, thus discounting sequence-dependent effects and specific interactions with nucleases. More in general, it allows for a comparison with other biologically-motivated contexts where the intra-molecular organization has been investigated in connection with the compliance to translocate through enzymes or synthetic pores [6][7][8][9][10][11][12][13][14][15][16] . ...
xrRNAs from flaviviruses survive in host cells because of their exceptional dichotomic response to the unfolding action of different enzymes. They can be unwound, and hence copied, by replicases, and yet can resist degradation by exonucleases. How the same stretch of xrRNA can encode such diverse responses is an open question. Here, by using atomistic models and translocation simulations, we uncover an elaborate and directional mechanism for how stress propagates when the two xrRNA ends, 5′ and 3′, are driven through a pore. Pulling the 3′ end, as done by replicases, elicits a progressive unfolding; pulling the 5′ end, as done by exonucleases, triggers a counterintuitive molecular tightening. Thus, in what appears to be a remarkable instance of intra-molecular tensegrity, the very pulling of the 5′ end is what boosts resistance to translocation and consequently to degradation. The uncovered mechanistic principle might be co-opted to design molecular meta-materials. Zika xrRNAs survive in host cells because they can be unwound and copied by replicases, but resist degradation by exonucleases. Here authors use atomistic models and simulations and uncover that pulling into a pore the xrRNA 3′ end, as done by replicases, causes progressive unfolding; pulling the 5′ end, as done by exonucleases, triggers molecular tightening.
... Combinations of experiments and simulations have revealed a wealth of knowledge on DNA translocation through nanopores [41]. Molecular dynamics simulations have been used to understand the role of nucleotide orientation and neighboring nucleotide chemistry on the passage of ions through the nanopore [42,43]. ...
There are over 100 enzyme-catalyzed modifications on transfer RNA (tRNA) molecules. The levels and identity of wobble uridine (U) modifications are affected by environmental conditions and diseased states, making wobble U detection a potential biomarker for exposures and pathological conditions. The current detection of RNA modifications requires working with nucleosides in bulk samples. Nanopore detection technology uses a single-molecule approach that has the potential to detect tRNA modifications. To evaluate the feasibility of this approach, we have performed all-atom molecular dynamics (MD) simulation studies of a five-layered graphene nanopore by localizing canonical and modified uridine nucleosides. We found that in a 1 M KCl solution with applied positive and negative biases not exceeding 2 V, nanopores can distinguish U from 5-carbonylmethyluridine (cm⁵U), 5-methoxycarbonylmethyluridine (mcm5U), 5-methoxycarbonylmethyl-2-thiouridine (mcm⁵s2U), and 5-methoxycarbonylmethyl-2'-Omethyluridine (mcm⁵Um) based on changes in the resistance of the nanopore. Specifically, we observed that in nanopores with dimensions less than 3 nm diameter, a localized mcm⁵Um and mcm⁵U modifications could be clearly distinguished from the canonical uridine, while the other modifications showed a modest yet detectable decrease in their respective nanopore conductance. We have compared the results between nanopores of various sizes to aid in the design, optimization, and fabrication of graphene nanopores devices for tRNA modification detection.
... An external electric field E = -V/LZ was applied along the nanopore axis to produce the target drop of the electric potential, V, over the system's dimension in the direction of the applied field, LZ. 85 In all simulations, a short-range repulsive potential was applied to atoms of NANPs to prevent their permanent binding to the nanopore surface. 86 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 21 The authors declare no competing financial interest. ...
Nucleic acid nanoparticles (NANPs) are an emerging class of programmable structures with tunable shape and function. Their promise as tools for fundamental biophysics studies, molecular sensing, and therapeutic applications, necessitates methods for their detection and characterization at the single-particle level. In this work, we study electrophoretic transport of individual ring-shaped and cube-shaped NANPs through solid-state nanopores. In the optimal nanopore size range, the particles must deform to pass through the nanopore, which considerably increases their residence time within the nanopore. Such anomalously long residence times permit detection of picomolar amounts of NANPs when nanopore measurements are carried out at a high transmembrane bias. In the case of a NANPs mixture, the type of individual particles passing through nanopores can be determined with excellent efficiency from analysis of single electrical pulses. Molecular dynamics simulations provide insight into the mechanical barrier to transport of the NANPs and corroborate the difference in the signal amplitudes observed for the two types of particles. Our study serves as a basis for label-free analysis of soft programmable-shape nanoparticles.
... In general, due to the negative charge of DNA, K + ions have a higher affinity for the nucleotides and are more likely to approach them than Cl − ions. This greater affinity is reflected in the dominant contribution of the K + ions to the blockade current in both solid-state state 60 and biological 61,62 nanopore systems. Furthermore, as K + ions are more likely to come in close contact with DNA than Cl − ions, the sequence dependence of the blockade current is more likely to originate from the modulation in the K + component of the current. ...
Measurements of ionic currents through nanopores partially blocked by DNA have emerged as a powerful method for characterization of the DNA nucleotide sequence. Although the effect of the nucleotide sequence on the nanopore blockade current has been experimentally demonstrated, prediction and interpretation of such measurements remain a formidable challenge. Using atomic resolution computational approaches, here we show how the sequence, molecular conformation, and pore geometry affect the blockade ionic current in model solid-state nanopores. We demonstrate that the blockade current from a DNA molecule is determined by the chemical type and the conformation of at least three consecutive nucleotides. We find the blockade currents produced by the nucleotide triplets to vary considerably with their nucleotide sequence despite having nearly identical molecular conformations. Encouragingly, we find blockade current differences as large as 25% for single-base substitutions in ultra small (1.6 nm x 1.1 nm cross section / 2 nm length) solid-state nanopores. Despite the complex dependence of the blockade current on the sequence and conformation of the DNA triplets, we find that, under many conditions, the number of thymine bases is positively correlated with the current, whereas the number of purine bases and the presence of both purine and pyrimidines in the triplet are negatively correlated with the current. Based on these observations, we construct a simple theoretical model that relates the ion current to the base content of a solid-state nanopore. Furthermore, we show that compact conformations of DNA in narrow pores provide the greatest signal-to-noise ratio for single base detection, whereas reduction of the nanopore length only increases the ionic current noise. Thus, the sequence dependence of a nanopore blockade current can be theoretically rationalized, although doing so for each nanopore type will likely require a custom approach.
... The mean values of t d and ΔI determined from a population of single-molecule transport events (typically at least 1000) reflect properties of the molecules, e.g. their contour length (16)(17)(18)(19), cross-sectional diameter (20,21) and microscopic conformation (17,22). ...
Oxidation of a DNA thymine to 5-hydroxymethyluracil is one of several recently discovered epigenetic modifications. Here,
we report the results of nanopore translocation experiments and molecular dynamics simulations that provide insight into the
impact of this modification on the structure and dynamics of DNA. When transported through ultrathin solid-state nanopores,
short DNA fragments containing thymine modifications were found to exhibit distinct, reproducible features in their transport
characteristics that differentiate them from unmodified molecules. Molecular dynamics simulations suggest that 5-hydroxymethyluracil
alters the flexibility and hydrophilicity of the DNA molecules, which may account for the differences observed in our nanopore
translocation experiments. The altered physico-chemical properties of DNA produced by the thymine modifications may have implications
for recognition and processing of such modifications by regulatory DNA-binding proteins.
... Recently, graphene [6] with the atomic thinness, stability and electrical sensitivity, has been the hotspot of intense research with the potential to characterize single nucleotides of DNA [1,[7][8][9][10]. Since molecular dynamics (MD) simulations can be used to investigate the entire translocation process, the opportunity for molecular modeling arises to play a major role in this research area [11][12][13][14]. Our previous work has shown that small graphene nanopore is very beneficial for 12bp double-stranded DNA (dsDNA) to translocate through because of the strong Van der Waals (VDW) interaction between dsDNA and nanopore [15]. ...
Molecular dynamics simulations are performed to provide valuable information about the translocation of four single-stranded DNAs with ten identical bases through graphene nanopore with diameter of 2 nm. The monolayer graphene nanopore is highly sensitive to ssDNA translocation events and the 10-base resolution detection can be realized by electrophoreticly threading ssDNA through graphene nanopore. Due to the similar sizes of the four nucleotides, the blockage current is unlikely to provide a distinguishable signal. However, by simply monitoring and analyzing the translocation time of poly(dA)10, poly(dC)10, poly(dG)10 and poly(dT)10 though graphene pore, each ssDNA can be identified and characterized.
... In nanopore electrophoresis, DNA is transported from one side of the membrane to the other by a transmembrane electric potential. The translocation events are detected and characterized by measuring the blockades of transmembrane ionic current [303,304]. If the size of the pore constriction is comparable to the diameter of the DNA molecule, the ionic current can be sensitive to the DNA's nucleotide sequence [305,306]. ...
Over the past ten years, the all-atom molecular dynamics method has grown in the scale of both systems and processes amenable to it and in its ability to make quantitative predictions about the behavior of experimental systems. The field of computational DNA research is no exception, witnessing a dramatic increase in the size of systems simulated with atomic resolution, the duration of individual simulations and the realism of the simulation outcomes. In this topical review, we describe the hallmark physical properties of DNA from the perspective of all-atom simulations. We demonstrate the amazing ability of such simulations to reveal the microscopic physical origins of experimentally observed phenomena. We also discuss the frustrating limitations associated with imperfections of present atomic force fields and inadequate sampling. The review is focused on the following four physical properties of DNA: effective electric charge, response to an external mechanical force, interaction with other DNA molecules and behavior in an external electric field.
... Note that this local perturbation does not contradict overall electroneutrality as has been observed in molecular dynamics simulations. 50,51 Rather, the reduction in counterion density local to the sensing region of the nanopore would be accompanied by an equivalent buildup of charge outside the sensing region, as shown schematically in Figure 4 and in more detail in Figure S-6 (Supporting Information). In addition, because polarization will saturate at very high ARTICLE electric field strength, 39 the voltage-dependence would likewise saturate at high voltage. ...
Solid-state nanopore electrical signatures can be convoluted and are thus challenging to interpret. In order to better understand the origin of these conductance changes, we investigate the translocation of DNA over a range of voltage. We observe multiple, discrete populations of conductance blockades that vary with applied voltage. To describe our observations, we develop a simple model that is applicable to solid-state nanopores generally. These results represent an important step towards understanding of the dynamics of the electrokinetic translocation process.
... However, since direct experimental imaging of molecules within nano-pores is extremely difficult, computation plays an important role in associating current with nanoscale phenomenon [12][13][14][15][16][17][18][19] (see [20,21] for recent reviews of the field). ...
The conventional Poisson-Nernst-Planck equations do not account for the finite size of ions explicitly. This leads to solutions featuring unrealistically high ionic concentrations in the regions subject to external potentials, in particular, near highly charged surfaces. A modified form of the Poisson-Nernst-Planck equations accounts for steric effects and results in solutions with finite ion concentrations. Here, we evaluate numerical methods for solving the modified Poisson-Nernst-Planck equations by modeling electric field-driven transport of ions through a nanopore. We describe a novel, robust finite element solver that combines the applications of the Newton’s method to the nonlinear Galerkin form of the equations, augmented with stabilization terms to appropriately handle the drift-diffusion processes.
To make direct comparison with particle-based simulations possible, our method is specifically designed to produce solutions under periodic boundary conditions and to conserve the number of ions in the solution domain. We test our finite element solver on a set of challenging numerical experiments that include calculations of the ion distribution in a volume confined between two charged plates, calculations of the ionic current though a nanopore subject to an external electric field, and modeling the effect of a DNA molecule on the ion concentration and nanopore current.
... All atomistic MD simulations were performed using the program NAMD2, 54 periodic boundary conditions, the CHARMM27 parameter set for water, ions and nucleic acids, 55 CHARMM-compatible parameters for silicon nitride, 56 and ion-pair specific corrections to the Lennard-Jones parameter . 57 All simulations employed a 2-2-6-fs multiple timestepping, SETTLE algorithm to keep water molecules rigid, 58 RATTLE algorithm to keep rigid all other covalent bonds involving hydrogen atoms, 59 a 7-8 Å cutoff for van der Waals and short-range electrostatic forces. ...
Practical applications of solid-state nanopores for DNA detection and sequencing require the electrophoretic motion of DNA through the nanopores to be precisely controlled. Controlling the motion of single-stranded DNA presents a particular challenge, in part because of the multitude of conformations that a DNA strand can adopt in a nanopore. Through continuum, coarse-grained and atomistic modeling, we demonstrate that local heating of the nanopore volume can be used to alter the electrophoretic mobility and conformation of single-stranded DNA. In the nanopore systems considered, the temperature near the nanopore is modulated via a nanometer-size heater element that can be radiatively switched on and off. The local enhancement of temperature produces considerable stretching of the DNA fragment confined within the nanopore. Such stretching is reversible, so that the conformation of DNA can be toggled between compact (local heating is off) and extended (local heating is on) states. The effective thermophoretic force acting on single-stranded DNA in the vicinity of the nanopore is found to be sufficiently large (4-8~pN) to affect such changes in the DNA conformation. The local heating of the nanopore volume is observed to promote single-file translocation of DNA strands at transmembrane biases as low as 10~mV, which opens new avenues for using solid-state nanopores for detection and sequencing of DNA.
The stacking of nanochannels in an array is a promising method for scaling nanoscale phenomena to large-scale systems. However, the scalability of single-nanochannel transport characteristics to a large-scale system (i.e., nanochannel arrays) is limited due to interchannel communications. Here, we report the communication between nanochannels in the array using an all-atom molecular dynamics (MD) simulation. In this simulation, a silicon nitride nanochannel array is integrated with the bulk reservoirs, and an electric field is applied across the reservoirs. Intriguingly, the simulations reveal a distinct pattern of communications between nanochannels and are mapped for the first time in the ohmic and nonohmic regions. In the ohmic region, individual channel current increases from the center channel to the channel near the boundary of the reservoir. Surprisingly, this pattern is reversed for the nonohmic region and the center channel shows a higher current compared to the other channels. This behavior may be attributed to the electro-osmotic instability (EOI) controlling the different length propagation of the extended space charge region into the individual channels. Further, we show the scaling law of the nanochannel conductance with the number of channels in both regions. This study offers useful insights for designing nanochannel arrays to improve the process efficiency of applications such as power generation, desalination, drug delivery, ionic logic gates, and circuits.
Using theory and simulations, we carried out a first systematic characterization of DNA unzipping via nanopore translocation. Starting from partially unzipped states, we found three dynamical regimes depending on the applied force f: (i) heterogeneous DNA retraction and rezipping (f<17 pN), (ii) normal (17 pN<f<60 pN), and (iii) anomalous (f>60 pN) drift-diffusive behavior. We show that the normal drift-diffusion regime can be effectively modeled as a one-dimensional stochastic process in a tilted periodic potential. We use the theory of stochastic processes to recover the potential from nonequilibrium unzipping trajectories and show that it corresponds to the free-energy landscape for single-base-pair unzipping. Applying this general approach to other single-molecule systems with periodic potentials ought to yield detailed free-energy landscapes from out-of-equilibrium trajectories.
Nanopore brings extraordinary properties for a variety of potential applications in various industrial sectors. Since manufacturing of solid‐state nanopore is first reported in 2001, solid‐state nanopore has become a hot topic in the recent years. An increasing number of manufacturing methods have been reported, with continuously decreased sizes from hundreds of nanometers at the beginning to ≈1 nm until recently. To enable more robust, sensitive, and reliable devices required by the industry, researchers have started to explore the possible methods to manufacture nanopore array which presents unprecedented challenges on the fabrication efficiency, accuracy and repeatability, applicable materials, and cost. As a result, the exploration of fabrication of nanopore array is still in the fledging period with various bottlenecks. In this article, a wide range of methods of manufacturing nanopores are summarized along with their achievable morphologies, sizes, inner structures for characterizing the main features, based on which the manufacturing of nanopore array is further addressed. To give a more specific idea on the potential applications of nanopore array, some representative practices are introduced such as DNA/RNA sequencing, energy conversion and storage, water desalination, nanosensors, nanoreactors, and dialysis. Nanopore brings extraordinary properties for a variety of potential applications, such as DNA/RNA sequencing, energy conversion and storage, water desalination, nanosensors, nanoreactors, and dialysis. However, the mass manufacturing of nanopores is still a global challenge. This review systematically summarizes the manufacturing methods for single nanopore and nanopore array, and presents some new possibilities for fabricating large area nanopore array.
Nanopore sensing is nearly synonymous with resistive pulse sensing due to the characteristic occlusion of ions during pore occupancy, particularly at high salt concentrations. Contrarily, conductive pulses are observed under low salt conditions wherein electroosmotic flow is significant. Most literature reports counterions as the dominant mechanism of conductive events (a molecule-centric theory). However, the counterion theory does not fit well with conductive events occurring via net neutral-charged protein translocation, prompting further investigation into translocation mechanics. Herein, we demonstrate theory and experiments underpinning the translocation mechanism (i.e., electroosmosis or electrophoresis), pulse direction (i.e., conductive or resistive) and shape (e.g., monophasic or biphasic) through fine control of chemical, physical, and electronic parameters. Results from these studies predict strong electroosmosis plays a role in driving DNA events and generating conductive events due to polarization effects (i.e., a pore-centric theory).
Knotting is statistically inevitable in long polymer chains, especially under spatial confinement, and tightly packed genomic DNA filaments are no exception. Over several decades, ever more powerful experimental techniques have demonstrated the occurrence of knots and other forms of entanglements in DNA extracted from viruses, bacteria, and eukaryotes. The data have in turn prompted a broad range of theoretical and computational studies of the abundance and complexity of DNA knots, and especially: (i) how it depends on the length and degree of confinement of the filaments (ii) whether it can be used to infer the multiscale spatial organization of genomic DNA, and (iii) its impact on biological processes in vivo. Here, we present an overview and a personal perspective of such theoretical and experimental efforts, from the equilibrium knotting of DNA in bulk to the one observed in various organisms, and concluding with a comparison with RNAs and their entanglement properties.
Nanopore sensing is nearly synonymous with resistive pulse sensing due to the characteristic reduction of ionic flux during molecular occupancy of a pore, particularly at high salt concentrations. However, conductive pulses are widely reported at low salt conditions wherein electroosmotic flow can be quite significant. Aside from transporting molecules like DNA, we investigated whether electroosmotic flow has other potential impacts on sensing attributes such current enhancements due to the analyte molecule. The overwhelming majority of literature reports counterions as the dominant mechanism of conductive events (a molecule-centric theory for conductive events). Conductive events are not well understood due to the complex interplay between (charged) nanopore walls, DNA grooves, ion mobility, and counterion clouds. Yet, the prevailing consensus of counterions being introduced into the pore by the molecule does not fit well with a growing number of experiments including the fact that proteins can generate conductive events despite having a heterogeneous surface charge. Herein, we demonstrate theory and experiments underpinning the translocation mechanism (i.e., electroosmosis or electrophoresis), pulse direction (i.e., conductive or resistive) and shape (e.g., monophasic or biphasic) through fine control of chemical, physical, and electronic parameters. Results from these studies predict strong electroosmosis plays a role in driving DNA events and generating conductive events due to polarization effects (i.e. a pore-centric theory). We believe these findings will stimulate a useful discussion on the nature of conductive events and their impact on molecular sensing in nanoscale pores.
Many small proteins move across cellular compartments through narrow pores. In order to thread a protein through a constriction, free energy must be overcome to either deform or completely unfold the protein. In principle, the diameter of the pore, along with the effective driving force for unfolding the protein, as well as its barrier to translocation, should be critical factors that govern whether the process proceeds via squeezing, unfolding/threading, or both. To probe this for a well-established protein system, we studied the electric-field-driven translocation behavior of cytochrome c (cyt c) through ultrathin silicon nitride (SiN x ) solid-state nanopores of diameters ranging from 1.5 to 5.5 nm. For a 2.5 nm diameter pore we find that, in a threshold electric field regime of ∼30-100 MV/m, cyt c is able to squeeze through the pore. As electric fields inside the pore are increased, the unfolded state of cyt c is thermodynamically stabilized, facilitating its translocation. In contrast, for 1.5 nm and 2.0 nm diameter pores, translocation occurs only by threading of the fully unfolded protein after it transitions through a higher energy unfolding intermediate state at the mouth of the pore. The relative energies between the metastable, intermediate, and unfolded protein states are extracted using a simple thermodynamic model that is dictated by the relatively slow (∼ms) protein translocation times for passing through the nanopore. These experiments map the various modes of protein translocation through a constriction, which opens new avenues for exploring protein folding structures, internal contacts, and electric field-induced deformability.
Significance Statement
Can localized electric fields drive the complete unfolding of a protein molecule? Protein unfolding prior to its translocation through a nanopore constriction is an important step in protein transport across biological membranes and also an important step in nanopore-based protein sequencing. We studied here the electric-field-driven translocation behavior of a model protein (cyt c) through nanopores of diameters ranging from 1.5 to 5.5 nm. These single molecule measurements show that electric fields at the nanopore constriction can select both partially and fully unfolded protein conformations. Zero-field free energy gaps between these conformations, found using a simple thermodynamic model, are in remarkable agreement with previously reported studies of cyt c unfolding energetics.
The nanopore provides a highly electrochemically confined space within which single-molecule characteristics can be efficiently converted into measurable electrochemical signatures with high temporal and current resolution. Aimed at developing the concept of the electrochemical confined space in analysing single molecules, this book serves as a stepping-stone to many exciting discoveries in nanopore-based analysis of biological processes and chemical reactions in confined space.
The field of nanopore sensors is growing rapidly, but there have been no new books on nanopore technology that provide an overview of the research on nanopore-based sensing until now. The book provides a good source of nanopore studies for researchers interested in and working in the general areas of electrochemistry and nanobiotechnology, especially on nanopore sensors.
By applying an electric field to an insulating membrane, movement of charged particles through a nanopore can be induced. The measured ionic current reports on biomolecules passing through the nanopore. In this paper we explore the sequence-dependent dynamics of DNA unzipping using our recently developed coarse-grained (CG) model. We estimated three molecular profiles (the potential of mean force, position-dependent diffusion coefficient and position-dependent effective charge) for the DNA unzipping of four hairpins with different sequences. We found that the molecular profiles are correlated with the ionic current and molecular events. We also explored the unzipping kinetics using Brownian dynamics. We found that the effect of hairpin structure on the unzipping/translocation times is not only energetic (weaker hairpins unzip more quickly), but also kinetic (different unzipping and translocation pathways play an important role).
Herein, the unzipping and translocation of DNA duplexes through a sub-2 nm silicon nitride (SiNx) solid-state nanopore have been demonstrated by well-resolved three-level blockades. In order to examine our observations, we applied a simple model which is applicable to the unzipping and translocation processes of DNA duplexes through solid-state nanopores. The generation of these highly recognizable signatures is an important step towards the real applications of solid-state nanopores in complex samples.
With the development of the transverse tunneling current theory, electrode-embedded nanopore is considered as one of the most promising devices for label-free DNA sequencing. Though there have been a few approaches to fabricate transverse electrode-embedded nanopores, the method universality and nanogap control are still formidable tasks of fabricating a nano-scale electrodenanopore sensor. In this work, a cross-scale approach of fabricating nano-electrodes was proposed, and a 10 nm-width nano-electrode was obtained from 5 μm-width aurum (Au) line. Compared with traditional methods, only electron microscopy was involved in this process, which was a simple and effective method to fabricate electrode-embedded nanopores. Furthermore, the gap of nanoelectrodes was controllable as the nano-electrode was cut off by drilling nanopore. The feasibility of the method was investigated by practice. This article demonstrated a novel approach to fabricate transverse electrode-embedded nanopore which showed great application potential in fabricating nano-electrodes with gap smaller than 2 nm.
Nanopore sensors have developed into powerful tools for single-molecule studies since their inception two decades ago. Nanopore sensors function as nanoscale Coulter counters, by monitoring ionic current modulations as particles pass through a nanopore. While nanopore sensors can be used to study any nanoscale particle, their most notable application is as a low cost, fast alternative to current DNA sequencing technologies. In recent years, signifcant progress has been made toward the goal of nanopore-based DNA sequencing, which requires an ambitious combination of a low-noise and high-bandwidth nanopore measurement system and spatial resolution. In this dissertation, nanopore sensors in thin membranes are developed to improve dimensional resolution, and these membranes are used in parallel with a high-bandwidth amplfier. Using this nanopore sensor system, the signals of three DNA homopolymers are differentiated for the first time in solid-state nanopores. The nanopore noise is also reduced through the addition of a layer of SU8, a spin-on polymer, to the supporting chip structure. By increasing the temporal and spatial resolution of nanopore sensors, studies of shorter molecules are now possible. Nanopore sensors are beginning to be used for the study and characterization of nanoparticles. Nanoparticles have found many uses from biomedical imaging to next-generation solar cells. However, further insights into the formation and characterization of nanoparticles would aid in developing improved synthesis methods leading to more effective and customizable nanoparticles. This dissertation presents two methods of employing nanopore sensors to benet nanoparticle characterization and fabrication. Nanopores were used to study the formation of individual nanoparticles and serve as nanoparticle growth templates that could be exploited to create custom nanoparticle arrays. Additionally, nanopore sensors were used to characterize the surface charge density of anisotropic nanopores, which previously could not be reliably measured. Current nanopore sensor resolution levels have facilitated innovative research on nanoscale systems, including studies of DNA and nanoparticle characterization. Further nanopore system improvements will enable vastly improved DNA sequencing capabilities and open the door to additional nanopore sensing applications.
Recently, protein and synthetic nanopores have been employed extensively as single-molecule probes to illuminate the functional features of proteins, including their binding affinity to different ligands, backbone flexibility, enzymatic activity and folding state. In this chapter, I present a brief overview in this emerging area of biosensing. The underlying principle of detection is that the device is based upon a single nanopore drilled into an insulating membrane, which is immersed in a symmetric chamber containing electrolyte solution. The application of a transmembrane potential across the membrane will enable the recording of a well-defined electric current due to the flow of ions crossing the nanopore. The partitioning of single proteins into the interior of the nanopore is detected by discrete current fluctuations that depend upon the interaction between the proteins and the nanopore. The detection mechanisms include chemical modification and genetic engineering of protein nanopores, electrophoretic capture of proteins via movable nucleic acid arms, and functionalization of the inner surface of synthetic nanopores. This approach holds promise for the exploration of proteins at high temporal and spatial resolution. Moreover, nanopore probe techniques can be employed in high-throughput devices used in biomedical molecular diagnosis and environmental monitoring.
When an electric field is applied to an insulating membrane, movement of charged particles through a nanopore is induced. The measured ionic current reports on biomolecules passing through the nanopore. In this work, we explored the kinetics of DNA unzipping in a nanopore using our coarse-grained model (Stachiewicz and Molski, J. Comput. Chem. 2015, 36, 947). Coarse graining allowed a more detailed analysis for a wider range of parameters than all-atom simulations. Dependence of the translocation mode (unzipping or distortion) on the pore diameter was examined, and the threshold voltages were estimated. We determined the potential of mean force, position-dependent diffusion coefficient, and position-dependent effective charge for the DNA unzipping. The three molecular profiles were correlated with the ionic current and molecular events. On the unzipping/translocation force profile, two energy maxima were found, one of them corresponding to the unzipping, and the other to the translocation barriers. The unzipping kinetics were further explored using Brownian dynamics. © 2015 Wiley Periodicals, Inc.
Despite its successes to probe the chemical reactions and dynamics of macromolecules on sub-millisecond time and nanometer length scales, a major impasse faced by the nanopore technology is the need to cheaply and controllably modulate the macromolecule capture and trafficking across the nanopore. We demonstrate herein that tunable charge separation engineered at the both ends of a macromolecule, modulates very efficiently the dynamics of macromolecules capture and traffic through a nanometer-size pore. In the proof-of-principle approach, we employed a 36 aminoacids long peptide containing at the N- and C-termini uniform patches of glutamic acids and arginines, flanking a central segment of asparagines, and studied its capture by the α-hemolysin (α-HL) and the mean residence time inside the pore, in the presence of a pH gradient across the protein. We propose a solution to effectively control the dynamics of peptide interaction with the nanopore, with both association and dissociation reaction rates of peptide-α-HL interactions spanning orders of magnitude depending upon solution acidity on the peptide addition side and the transmembrane electric potential, while preserving the amplitude of the blockade current signature.
In nanopore force spectroscopy (NFS) a charged polymer is threaded through a channel of molecular dimensions. When an electric field is applied across the insulating membrane, the ionic current through the nanopore reports on polymer translocation, unzipping, dissociation, and so forth. We present a new model that can be applied in molecular dynamics simulations of NFS. Although simplified, it does reproduce experimental trends and all-atom simulations. The scaled conductivities in bulk solution are consistent with experimental results for NaCl for a wide range of electrolyte concentrations and temperatures. The dependence of the ionic current through a nanopore on the applied voltage is symmetric and, in the voltage range used in experiments (up to 2 V), linear and in good agreement with experimental data. The thermal stability and geometry of DNA is well represented. The model was applied to simulations of DNA hairpin unzipping in nanopores. The results are in good agreement with all-atom simulations: the scaled translocation times and unzipping sequence are similar. © 2015 Wiley Periodicals, Inc.
© 2015 Wiley Periodicals, Inc.
Slowing down DNA translocation speed in a nanopore is essential to ensuring reliable resolution of individual bases. Thin membrane materials enhance spatial resolution but simultaneously reduce the temporal resolution as the molecules translocate far too quickly. In this study, the effect of exposed graphene layers on the transport dynamics of both single (ssDNA) and double-stranded DNA (dsDNA) through nanopores is examined. Nanopore devices with various combinations of graphene and Al2O3 dielectric layers in stacked membrane structures are fabricated. Slow translocations of ssDNA in nanopores drilled in membranes with layers of graphene are reported. The increased hydrophobic interactions between the ssDNA and the graphene layers could explain this phenomenon. Further confirmation of the hydrophobic origins of these interactions is obtained through reporting significantly faster translocations of dsDNA through these graphene layered membranes. Molecular dynamics simulations confirm the preferential interactions of DNA with the graphene layers as compared to the dielectric layer verifying the experimental findings. Based on our findings, we propose that the integration of multiple stacked graphene layers could slow down DNA enough to enable the identification of nucleobases.
With the advent of Next-Generation-Sequencing (NGS) technologies, an enormous volume of DNA sequencing data can be generated at low cost, placing genomic science within the grasp of everyday medicine. However, mired in this voluminous data, a new problem has emerged: the assembly of the genome from the short reads. In this chapter we examine the prospects for sequencing DNA using a synthetic nanopore. Nanopore sequencing has the potential for very long reads, reducing the computational burden posed by alignment and genome assembly, while at the same time eliminating logistically challenging and error-prone amplification and library formation due to its exquisite single molecule sensitivity. On the other hand, long high fidelity reads demand stringent control over both the DNA configuration in the pore and the translocation kinetics. We examine the prospects for satisfying these specifications with a synthetic nanopore.
We review the current status of various aspects of biopolymertranslocation through nanopores and the challenges and opportunities it offers. Much of the interest generated by nanopores arises from their potential application to third-generation cheap and fast genome sequencing. Although the ultimate goal of single-nucleotide identification has not yet been reached, great advances have been made both from a fundamental and an applied point of view, particularly in controlling the translocation time, fabricating various kinds of synthetic pores or genetically engineering protein nanopores with tailored properties, and in devising methods (used separately or in combination) aimed at discriminating nucleotides based either on ionic or transverse electron currents, optical readout signatures, or on the capabilities of the cellular machinery. Recently, exciting new applications have emerged, for the detection of specific proteins and toxins (stochastic biosensors), and for the study of protein folding pathways and binding constants of protein–protein and protein–DNA complexes. The combined use of nanopores and advanced micromanipulation techniques involving optical/magnetic tweezers with high spatial resolution offers unique opportunities for improving the basic understanding of the physical behavior of biomolecules in confined geometries, with implications for the control of crucial biological processes such as protein import and protein denaturation. We highlight the key works in these areas along with future prospects. Finally, we review theoretical and simulation studies aimed at improving fundamental understanding of the complex microscopic mechanisms involved in the translocation process. Such understanding is a pre-requisite to fruitful application of nanopore technology in high-throughput devices for molecular biomedical diagnostics.
Solid-state nanopores have been proposed for rapid and inexpensive deoxyribonucleic acid (DNA) sequencing and analysis. This technology is primarily based on characterizing the ionic current flowing through the pore as DNA translocates from one side of the pore to the other side under the influence of an electric field. The magnitude of the DNA-induced current blockade is an important analytical feature for these applications. However, it remains a challenging task to accurately determine the ionic current levels due to small signal-to-noise ratios. In order to facilitate reliable analysis it is necessary to understand the noise statistics and develop effective signal estimation techniques. In this paper, we conduct a molecular dynamics simulation of DNA translocations through a solid-state nanopore and reveal that the simulated ionic current signals contain both thermal and shot noise. We then develop a model for these signals and propose a maximum likelihood estimator (MLE) for estimating the ionic current levels. We show that the MLE has the potential to significantly outperform the classic sample mean estimator.
In the last two decades, new techniques that monitor ionic current modulations as single molecules pass through a nanoscale pore have enabled numerous single-molecule studies. While biological nanopores have recently shown the ability to resolve single nucleotides within individual DNA molecules, similar developments with solid-state nanopores have lagged, due to challenges both in fabricating stable nanopores of similar dimensions as biological nanopores and in achieving sufficiently low-noise and high-bandwidth recordings. Here we show that small silicon nitride nanopores (0.8 to 2-nm-diameter in 5 to 8-nm-thick membranes) can resolve differences between ionic current signals produced by short (30 base) ssDNA homopolymers (poly(dA), poly(dC), poly(dT)), when combined with measurement electronics that allow a signal-to-noise ratio of better than 10 to be achieved at 1 MHz bandwidth. While identifying intramolecular DNA sequences with silicon nitride nanopores will require further improvements in nanopore sensitivity and noise levels, homopolymer differentiation represents an important milestone in the development of solid-state nanopores.
A nanopore is the ultimate analytical tool. It can be used to detect DNA, RNA, oligonucleotides and proteins with sub-molecular sensitivity. This extreme sensitivity is derived from the electric signal associated with the occlusion that develops during the translocation of the analyte across a membrane through a pore immersed in electrolyte. A larger occluded volume results in an improvement in the signal-to-noise ratio and so the pore geometry should be made comparable to the size of the target molecule. However, the pore geometry also affects the electric field, the charge density, the electroosmotic flow, the capture volume and the response time. Seeking an optimal pore geometry, we tracked the molecular motion in three dimensions with high resolution, visualizing with confocal microscopy the fluorescence associated with DNA translocating through nanopores with diameters comparable to the double helix, while simultaneously measuring the pore current. Measurements reveal single molecules translocating across the membrane through the pore commensurate with the observation of a current blockade. To explain the motion of the molecule near the pore, finite-element simulations were employed that account for diffusion, electrophoresis and the electroosmotic flow. According to this analysis, detection using a nanopore comparable in diameter to the double helix represents a compromise between sensitivity, capture volume, the minimum detectable concentration and response time.
A study was conducted to demonstrate modeling and simulation of ion channels. Early phenomenological models of excitable membranes were discussed and the latest developments in this area were reviewed. Early work on phenomenological modeling of ion channels occurred before the existence of ion channels had been established. Researchers were making efforts to understand the mechanism of signal propagation in nerve cells. Early models described the axon as a 'cable', with a conductive core surrounded by a less conductive, capacitive sheath, which was later identified as a membrane in the Hodgkin-Huxley (HH) model. The HH model played a key role in understanding of nerve cells, and excitable membranes, while continuing to influence research work in the field. A team of researchers also demonstrated that millisecond all-atom molecular dynamic (MD) simulation of ion channels was performed by using Anton, a special-purpose hardware designed only for such MD simulations.
We develop an efficient multiple time step (MTS) force splitting scheme for biological applications in the AMBER program in the context of the particle-mesh Ewald (PME) algorithm. Our method applies a symmetric Trotter factorization of the Liouville operator based on the position-Verlet scheme to Newtonian and Langevin dynamics. Following a brief review of the MTS and PME algorithms, we discuss performance speedup and the force balancing involved to maximize accuracy, maintain long-time stability, and accelerate computational times. Compared to prior MTS efforts in the context of the AMBER program, advances are possible by optimizing PME parameters for MTS applications and by using the position-Verlet, rather than velocity-Verlet, scheme for the inner loop. Moreover, ideas from the Langevin/MTS algorithm LN are applied to Newtonian formulations here. The algorithm’s performance is optimized and tested on water, solvated DNA, and solvated protein systems. We find CPU speedup ratios of over 3 for Newtonian formulations when compared to a 1 fs single-step Verlet algorithm using outer time steps of 6 fs in a three-class splitting scheme; accurate conservation of energies is demonstrated over simulations of length several hundred ps. With modest Langevin forces, we obtain stable trajectories for outer time steps up to 12 fs and corresponding speedup ratios approaching 5. We end by suggesting that modified Ewald formulations, using tailored alternatives to the Gaussian screening functions for the Coulombic terms, may allow larger time steps and thus further speedups for both Newtonian and Langevin protocols; such developments are reported separately. © 2001 American Institute of Physics.
A force field (MSXX) for molecular dynamics simulations of silicon nitride is derived using the Hessian biased technique from ab initio calculations on N(SiH3)3 and Si(NH2)4 clusters. This is used to model the nitrogen and silicon centers of the α and β forms of crystalline Si3N4 for prediction of crystal structures, lattice expansion parameters, elastic constants, phonon states, and thermodynamic properties. Experimental measurements on many of these important physical constants are lacking, so that these calculations provide the first reliable data on such fundamental properties of silicon nitride. This MSXX force field is expected to be useful for molecular dynamics simulations of dislocations and grain boundaries and for studying the reconstruction and energetics of clean, reduced, and oxidized surfaces.
Among the variety of roles for nanopores in biology, an important one is enabling polymer transport, for example in gene transfer between bacteria1 and transport of RNA through the nuclear membrane2. Recently, this has inspired the use of protein3, 4, 5 and solid-state6, 7, 8, 9, 10 nanopores as single-molecule sensors for the detection and structural analysis of DNA and RNA by voltage-driven translocation. The magnitude of the force involved is of fundamental importance in understanding and exploiting this translocation mechanism, yet so far it has remained unknown. Here, we demonstrate the first measurements of the force on a single DNA molecule in a solid-state nanopore by combining optical tweezers11 with ionic-current detection. The opposing force exerted by the optical tweezers can be used to slow down and even arrest the translocation of the DNA molecules. We obtain a value of 0.240.02 pN mV-1 for the force on a single DNA molecule, independent of salt concentration from 0.02 to 1 M KCl. This force corresponds to an effective charge of 0.500.05 electrons per base pair equivalent to a 75% reduction of the bare DNA charge.
An electron beam can drill nanopores in SiO2 or silicon nitride membranes and shrink a pore to a smaller diameter. Such nanopores are promising for single molecule detection. The pore formation in a 40 nm thick silicon nitride/SiO2 bilayer using an electron beam with a diameter of 8 nm (full width of half height) was investigated by electron energy loss spectroscopy with silicon nitride facing toward and away from the source. The O loss shows almost linear-independent of which layer faces the source, while N loss is quite complicated. After the formation of a pore, the membrane presents a wedge shape over a 70 nm radius around the nanopore. (c) 2005 American Institute of Physics.
Classical Monte Carlo simulations have been carried out for liquid water in the NPT ensemble at 25 °C and 1 atm using six of the simpler intermolecular potential functions for the water dimer: Bernal–Fowler (BF), SPC, ST2, TIPS2, TIP3P, and TIP4P. Comparisons are made with experimental thermodynamic and structural data including the recent neutron diffraction results of Thiessen and Narten. The computed densities and potential energies are in reasonable accord with experiment except for the original BF model, which yields an 18% overestimate of the density and poor structural results. The TIPS2 and TIP4P potentials yield oxygen–oxygen partial structure functions in good agreement with the neutron diffraction results. The accord with the experimental OH and HH partial structure functions is poorer; however, the computed results for these functions are similar for all the potential functions. Consequently, the discrepancy may be due to the correction terms needed in processing the neutron data or to an effect uniformly neglected in the computations. Comparisons are also made for self‐diffusion coefficients obtained from molecular dynamics simulations. Overall, the SPC, ST2, TIPS2, and TIP4P models give reasonable structural and thermodynamic descriptions of liquid water and they should be useful in simulations of aqueous solutions. The simplicity of the SPC, TIPS2, and TIP4P functions is also attractive from a computational standpoint.
We study the unzipping dynamics of individual DNA hairpins using nanopore force spectroscopy at different voltage ramp
rates and temperatures. At high ramp rates the critical unzipping voltage is proportional to log , where
is the voltage ramp. At low ramp values we observe a crossover to another regime with a weaker dependence on
. Here we report on the dependence of these two regimes on temperature. Remarkably, the unzipping kinetics can
be well described by a simple two-states model that predicts the existence of two asymptotic regimes: quasi-equilibrium
unzipping at low-voltage ramps and irreversible unzipping at high ramp rates.
Solid-state nanopores can be used to detect nucleic acid structures at the
single molecule level. An e-beam has been used to fabricate nanopores in
silicon nitride and silicon dioxide membranes, but the pore formation kinetics,
and hence its final structure, remain poorly understood. With the aid of
high-resolution TEM imaging as well as TEM tomography we examine the effect of
Si3N4
material properties on the nanopore structure. In particular, we study the dependence of
membrane thickness on the nanopore contraction rate for different initial pore sizes. We
explain nanopore formation kinetics as a balance of two opposite processes: (a) material
sputtering and (b) surface-tension-induced shrinking.
We show that an electric field can drive single-stranded RNA and DNA molecules through a 2.6-nm diameter ion channel in a lipid bilayer membrane. Because the channel diameter can accommodate only a single strand of RNA or DNA, each polymer traverses the membrane as an extended chain that partially blocks the channel. The passage of each molecule is detected as a transient decrease of ionic current whose duration is proportional to polymer length. Channel blockades can therefore be used to measure polynucleotide length. With further improvements, the method could in principle provide direct, high-speed detection of the sequence of bases in single molecules of DNA or RNA.
A variety of different DNA polymers were electrophoretically driven through the nanopore of an alpha-hemolysin channel in a lipid bilayer. Single-channel recording of the translocation duration and current flow during traversal of individual polynucleotides yielded a unique pattern of events for each of the several polymers tested. Statistical data derived from this pattern of events demonstrate that in several cases a nanopore can distinguish between polynucleotides of similar length and composition that differ only in sequence. Studies of temperature effects on the translocation process show that translocation duration scales as approximately T(-2). A strong correlation exists between the temperature dependence of the event characteristics and the tendency of some polymers to form secondary structure. Because nanopores can rapidly discriminate and characterize unlabeled DNA molecules at low copy number, refinements of the experimental approach demonstrated here could eventually provide a low-cost high-throughput method of analyzing DNA polynucleotides.
RNA and DNA strands produce ionic current signatures when driven through an alpha-hemolysin channel by an applied voltage. Here we combine this nanopore detector with a support vector machine (SVM) to analyze DNA hairpin molecules on the millisecond time scale. Measurable properties include duplex stem length, base pair mismatches, and loop length. This nanopore instrument can discriminate between individual DNA hairpins that differ by one base pair or by one nucleotide.
Manipulating matter at the nanometre scale is important for many electronic, chemical and biological advances, but present solid-state fabrication methods do not reproducibly achieve dimensional control at the nanometre scale. Here we report a means of fashioning matter at these dimensions that uses low-energy ion beams and reveals surprising atomic transport phenomena that occur in a variety of materials and geometries. The method is implemented in a feedback-controlled sputtering system that provides fine control over ion beam exposure and sample temperature. We call the method "ion-beam sculpting", and apply it to the problem of fabricating a molecular-scale hole, or nanopore, in a thin insulating solid-state membrane. Such pores can serve to localize molecular-scale electrical junctions and switches and function as masks to create other small-scale structures. Nanopores also function as membrane channels in all living systems, where they serve as extremely sensitive electro-mechanical devices that regulate electric potential, ionic flow, and molecular transport across cellular membranes. We show that ion-beam sculpting can be used to fashion an analogous solid-state device: a robust electronic detector consisting of a single nanopore in a Si3N4 membrane, capable of registering single DNA molecules in aqueous solution.
We examined the voltage-driven movement of single-stranded DNA molecules in a membrane channel or "nanopore". Using single channel recording methods and a statistical analysis of many single molecule events, we determined how voltage influences capture and translocation in the nanopore. We verified that the mean time between capture events follows a simple exponential distribution, whereas the translocation times follow a unique distribution that is partly Gaussian and partly exponential. Measurements of polymer sequence effects demonstrated that translocation duration is heavily influenced by specific or nonspecific purine-channel interactions. The single molecule approach we used revealed molecular interactions that can influence both capture rates and translocation velocities in a manner that enriches naive barrier crossing models.
Nanoscale α‐hemolysin pores can be used to analyze individual DNA or RNA molecules. Serial examination of hundreds to thousands
of molecules per minute is possible using ionic current impedance as the measured property. In a recent report, we showed
that a nanopore device coupled with machine learning algorithms could automatically discriminate among the four combinations
of Watson–Crick base pairs and their orientations at the ends of individual DNA hairpin molecules. Here we use kinetic analysis
to demonstrate that ionic current signatures caused by these hairpin molecules depend on the number of hydrogen bonds within
the terminal base pair, stacking between the terminal base pair and its nearest neighbor, and 5′ versus 3′ orientation of
the terminal bases independent of their nearest neighbors. This report constitutes evidence that single Watson–Crick base
pairs can be identified within individual unmodified DNA hairpin molecules based on their dynamic behavior in a nanoscale
pore.
We studied the unzipping of single molecules of double-stranded DNA by pulling one of their two strands through a narrow protein pore. Polymerase chain reaction analysis yielded the first direct proof of DNA unzipping in such a system. The time to unzip each molecule was inferred from the ionic current signature of DNA traversal. The distribution of times to unzip under various experimental conditions fit a simple kinetic model. Using this model, we estimated the enthalpy barriers to unzipping and the effective charge of a nucleotide in the pore, which was considerably smaller than previously assumed.
A nanometre-scale pore in a solid-state membrane provides a new way of electronically probing the structure of single linear polymers, including those of biological interest in their native environments. Previous work with biological protein pores wide enough to let through and sense single-stranded DNA molecules demonstrates the power of using nanopores, but many future tasks and applications call for a robust solid-state pore whose nanometre-scale dimensions and properties may be selected, as one selects the lenses of a microscope. Here we demonstrate a solid-state nanopore microscope capable of observing individual molecules of double-stranded DNA and their folding behaviour. We discuss extensions of the nanopore microscope concept to alternative probing mechanisms and applications, including the study of molecular structure and sequencing.
We have produced single, synthetic nanometer-diameter pores by using a tightly focused, high-energy electron beam to sputter atoms in 10-nm-thick silicon nitride membranes. Subsequently, we measured the ionic conductance as a function of time, bath concentration, and pore diameter to infer the conductivity and ionic mobility through the pores. The pore conductivity is found to be much larger than the bulk conductivity for dilute bath concentrations, where the Debye length is larger than the pore radius, whereas it is comparable with or less than the bulk for high bath concentrations. We interpret these observations by using multiscale simulations of the ion transport through the pores. Molecular dynamics is used to estimate the ion mobility, and ion transport in the pore is described by the coupled Poisson–Nernst–Planck and the Stokes equations that are solved self-consistently for the ion concentration and velocity and electrical potential. We find that the measurements are consistent with the presence of fixed negative charge in the pore wall and a reduction of the ion mobility because of the fixed charge and the ion proximity to the pore wall.
• ion conduction
• nanopore
• nanostructured materials
We report double-strand DNA translocation experiments using silicon oxide nanopores with a diameter of about 10 nm . By monitoring the conductance of a voltage-biased pore, we detect molecules with a length ranging from 6557 to 48 500 base pairs. We find that the molecules can pass the pore both in a straight linear fashion and in a folded state. Experiments on circular DNA further support this picture. We sort the molecular events according to their folding state and estimate the folding position. As a proof-of-principle experiment, we show that a nanopore can be used to distinguish the lengths of DNA fragments present in a mixture. These experiments pave the way for quantitative analytical techniques with solid-state nanopores.
We examined the voltage-driven movement of single-stranded DNA molecules in a membrane channel or “nanopore”. Using single channel recording methods and a statistical analysis of many single molecule events, we determined how voltage influences capture and translocation in the nanopore. We verified that the mean time between capture events follows a simple exponential distribution, whereas the translocation times follow a unique distribution that is partly Gaussian and partly exponential. Measurements of polymer sequence effects demonstrated that translocation duration is heavily influenced by specific or nonspecific purine-channel interactions. The single molecule approach we used revealed molecular interactions that can influence both capture rates and translocation velocities in a manner that enriches naive barrier crossing models.
A force field (MSXX) for molecular dynamics simulations of silicon nitride is derived using the Hessian biased technique from ab initio calculations on N(SiH3)3 and Si(NH2)4 clusters. This is used to model the nitrogen and silicon centers of the α and β forms of crystalline Si3N4 for prediction of crystal structures, lattice expansion parameters, elastic constants, phonon states, and thermodynamic properties. Experimental measurements on many of these important physical constants are lacking, so that these calculations provide the first reliable data on such fundamental properties of silicon nitride. This MSXX force field is expected to be useful for molecular dynamics simulations of dislocations and grain boundaries and for studying the reconstruction and energetics of clean, reduced, and oxidized surfaces.
Modularly invariant equations of motion are derived that generate the isothermal–isobaric ensemble as their phase space averages. Isotropic volume fluctuations and fully flexible simulation cells as well as a hybrid scheme that naturally combines the two motions are considered. The resulting methods are tested on two problems, a particle in a one-dimensional periodic potential and a spherical model of C60 in the solid/fluid phase.
Recent progress in experimental micromanipulation techniques allows single biomolecules, such as DNA, to be mechanically distorted under controlled conditions. As well as providing a wealth of new biochemical information, these experiments are also driving advances in statistical physics and non-equilibrium thermodynamics as theorists extend well established statistical methods to include a description of complex biomolecules at the single molecule level. This article will describe the physical ideas that are necessary to understand the wide range of experiments that stretch individual DNA molecules. These experiments operate in three distinct time regimes relative to thermal noise and therefore require three different branches of physics to understand them. Experiments, which stretch long DNA helices, are well described by equilibrium thermodynamics up until the point at which the molecule is irreversibly distorted, where non-equilibrium ideas apply. Short DNA sequences are only marginally stable at room temperature, therefore their response to an external force is dominated by thermal effects. These experiments are able to probe the kinetics of thermally activated processes in biomolecules in more detail than has been previously possible.
Single molecule sensors in which nanoscale pores within biological or artificial membranes act as mechanical gating elements are very promising devices for the rapid characterization and sequencing of nucleic acid molecules. The two terminal electrical measurements of translocation of polymers through single ion channels and that of ssDNA molecules through protein channels have been demonstrated, and have sparked tremendous interest in such single molecule sensors. The prevailing view regarding the nanopore sensors is that there exists no electrical interaction between the nanopore and the translocating molecule, and that all nanopore sensors reported to-date, whether biological or artificial, operate as a coulter-counter, i.e., the ionic current measured across the pore decreases (is mechanically blocked) when the DNA molecule transverses through the pore. We have fabricated nanopore “channel” sensors with a silicon oxide inner surface, and our results challenge the prevailing view of exclusive mechanical interaction during the translocation of dsDNA molecules through these channels. We demonstrate that the ionic current can actually increase due to electrical gating of surface current in the channel due to the charge on the DNA itself.
In this paper, we evaluate the magnitude of the electrical signals produced by DNA translocation through a 1 nm diameter nanopore in a capacitor membrane with a numerical multi-scale approach, and assess the possibility of resolving individual nucleotides as well as their types in the absence of conformational disorder. We show that the maximum recorded voltage caused by the DNA translocation is about 35 mV, while the maximum voltage signal due to the DNA backbone is about 30 mV, and the maximum voltage of a DNA base is about 8 mV. Signals from individual nucleotides can be identified in the recorded voltage traces, suggesting a 1 nm diameter pore in a capacitor can be used to accurately count the number of nucleotides in a DNA strand. Furthermore, we study the effect of a single base substitution on the voltage trace, and calculate the differences among the voltage traces due to a single base mutation for the sequences C3AC7, C3CC7, C3GC7 and C3TC7. The calculated voltage differences are in the 5–10 mV range. The calculated maximum voltage caused by the translocation of individual bases varies from 2 to 9 mV, which is experimentally detectable.
Extensive detail on the application of the real-time quantitative polymerase chain reaction (QPCR) for the analysis of gene expression is provided in this unit. The protocols are designed for high-throughput, 384-well-format instruments, such as the Applied Biosystems 7900HT, but may be modified to suit any real-time PCR instrument. QPCR primer and probe design and validation are discussed, and three relative quantitation methods are described: the standard curve method, the efficiency-corrected ΔCt method, and the comparative cycle time, or ΔΔCt method. In addition, a method is provided for absolute quantification of RNA in unknown samples. RNA standards are subjected to RT-PCR in the same manner as the experimental samples, thus accounting for the reaction efficiencies of both procedures. This protocol describes the production and quantitation of synthetic RNA molecules for real-time and non-real-time RT-PCR applications.
Keywords:
QPCR;
quantitative PCR;
real-time PCR;
reverse transcription PCR;
RT-PCR;
RNA expression analysis
A multi-scale/multi-material computational model for simulation of the electric signal detected on the electrodes of a metal–oxide–semiconductor (MOS) capacitor forming a nanoscale artificial membrane, and containing a nanopore with translocating DNA, is presented. The multi-scale approach is based on the incorporation of a molecular dynamics description of a translocating DNA molecule in the nanopore within a three-dimensional Poisson equation self-consistent scheme involving electrolytic and semiconductor charges for the electrostatic potential calculation. The voltage signal obtained from the simulation supports the possibility for single nucleotide resolution with a nanopore device. The electric signal predicted on the capacitor electrodes complements ongoing experiments exploring the use of nanopores in a MOS capacitor membrane for DNA sequencing.
We present the derivation of a new molecular mechanical force field for simulating the structures, conformational energies, and interaction energies of proteins, nucleic acids, and many related organic molecules in condensed phases. This effective two-body force field is the successor to the Weiner et al, force field and was developed with some of the same philosophies, such as the use of a simple diagonal potential function and electrostatic potential fit atom centered charges. The need for a 10-12 function for representing hydrogen bonds is no longer necessary due to the improved performance of the new charge model and new van der Waals parameters. These new charges are determined using a 6-31G basis set and restrained electrostatic potential (RESP) fitting and have been shown to reproduce interaction energies, free energies of solvation, and conformational energies of simple small molecules to a good degree of accuracy. Furthermore, the new RESP charges exhibit less variability as a function of the molecular conformation used in the charge determination. The new van der Waals parameters have been derived from liquid simulations and include hydrogen parameters which take into account the effects of any geminal electronegative atoms. The bonded parameters developed by Weiner et al. were modified as necessary to reproduce experimental vibrational frequencies and structures. Most of the simple dihedral parameters have been retained from Weiner et. al., but a complex set of phi and psi parameters which do a good job of reproducing the energies of the low-energy conformations of glycyl and alanyl dipeptides has been developed for the peptide backbone.
Molecular dynamics programs simulate the behavior of biomolecular systems, leading to understanding of their functions. However, the computational complexity of such simulations is enormous. Parallel machines provide the potential to meet this computational challenge. To harness this potential, it is necessary to develop a scalable program. It is also necessary that the program be easily modified by application–domain programmers. The NAMD2 program presented in this paper seeks to provide these desirable features. It uses spatial decomposition combined with force decomposition to enhance scalability. It uses intelligent periodic load balancing, so as to maximally utilize the available compute power. It is modularly organized, and implemented using Charm++, a parallel C++ dialect, so as to enhance its modifiability. It uses a combination of numerical techniques and algorithms to ensure that energy drifts are minimized, ensuring accuracy in long running calculations. NAMD2 uses a portable run-time framework called Converse that also supports interoperability among multiple parallel paradigms. As a result, different components of applications can be written in the most appropriate parallel paradigms. NAMD2 runs on most parallel machines including workstation clusters and has yielded speedups in excess of 180 on 220 processors. This paper also describes the performance obtained on some benchmark applications.
A general method, suitable for fast computing machines, for investigating such properties as equations of state for substances consisting of interacting individual molecules is described. The method consists of a modified Monte Carlo integration over configuration space. Results for the two-dimensional rigid-sphere system have been obtained on the Los Alamos MANIAC and are presented here. These results are compared to the free volume equation of state and to a four-term virial coefficient expansion.
A general method, suitable for fast computing machines, for investigating
such properties as equations of state for substances consisting of
interacting individual molecules is described. The method consists
of a modified Monte Carlo integration over configuration space. Results
for the two-dimensional rigid-sphere system have been obtained on
the Los Alamos MANIAC and are presented here. These results are compared
to the free volume equation of state and to a four-term virial coefficient
expansion. The Journal of Chemical Physics is copyrighted by The
American Institute of Physics.
Atomic force microscope-based single-molecule force spectroscopy was employed to measure sequence-dependent mechanical properties of DNA by stretching individual DNA double strands attached between a gold surface and an AFM tip. We discovered that in lambda-phage DNA the previously reported B-S transition, where 'S' represents an overstretched conformation, at 65 pN is followed by a nonequilibrium melting transition at 150 pN. During this transition the DNA is split into single strands that fully recombine upon relaxation. The sequence dependence was investigated in comparative studies with poly(dG-dC) and poly(dA-dT) DNA. Both the B-S and the melting transition occur at significantly lower forces in poly(dA-dT) compared to poly(dG-dC). We made use of the melting transition to prepare single poly(dG-dC) and poly(dA-dT) DNA strands that upon relaxation reannealed into hairpins as a result of their self-complementary sequence. The unzipping of these hairpins directly revealed the base pair-unbinding forces for G-C to be 20 +/- 3 pN and for A-T to be 9 +/- 3 pN.
Single molecules of DNA or RNA can be detected as they are driven through an alpha-hemolysin channel by an applied electric field. During translocation, nucleotides within the polynucleotide must pass through the channel pore in sequential, single-file order because the limiting diameter of the pore can accommodate only one strand of DNA or RNA at a time. Here we demonstrate that this nanopore behaves as a detector that can rapidly discriminate between pyrimidine and purine segments along an RNA molecule. Nanopore detection and characterization of single molecules represent a new method for directly reading information encoded in linear polymers, and are critical first steps toward direct sequencing of individual DNA and RNA molecules.
We measure current blockade and time distributions for single-stranded DNA polymers during voltage-driven translocations through a single alpha-hemolysin pore. We use these data to determine the velocity of the polymers in the pore. Our measurements imply that, while polymers longer than the pore are translocated at a constant speed, the velocity of shorter polymers increases with decreasing length. This velocity is nonlinear with the applied field. Based on this data, we estimate the effective diffusion coefficient and the energy penalty for extending a molecule into the pore.
Recent single-macromolecule observations revealed that the force/extension characteristics of single-stranded DNA (ssDNA) are closely related to solution ionic concentration and DNA sequence composition. To understand this, we studied the elastic property of ssDNA through the Monte Carlo implementation of a modified freely jointed chain (FJC), with electrostatic, base-pairing, and base-pair stacking interactions all incorporated. The simulated force-extension profiles for both random and designed sequences have attained quantitative agreements with the experimental data. In low-salt solution, electrostatic interaction dominates, and at low forces, the molecule can be more easily aligned than an unmodified FJC. In high-salt solution, secondary hairpin structure appears in ssDNA by the formation of base pairs between complementary bases, and external stretching causes a hairpin-coil structural transition, which is continuous for ssDNA made of random sequences. In designed sequences such as poly(dA-dT) and poly(dG-dC), the stacking potential between base pairs encourages the aggregation of base pairs into bulk hairpins and makes the hairpin-coil transition a discontinuous (first-order) process. The sensitivity of elongation to the base-pairing rule is also investigated. The comparison of modeling calculations and the experimental data suggests that the base pairing of single-stranded polynucleotide molecules tends to form a nested and independent planar hairpin structure rather than a random intersecting pattern.
The dynamics of single-stranded DNA in an alpha-Hemolysin protein pore was studied at the single-molecule level. The escape time for DNA molecules initially drawn into the pore was measured in the absence of an externally applied electric field. These measurements revealed two well-separated timescales, one of which is surprisingly long (on the order of milliseconds). We characterized the long timescale as being associated with the binding and unbinding of DNA from the pore. We have also found that a transmembrane potential as small as 20 mV strongly biased the escape of DNA from the pore. These experiments have been made possible due to the development of a feedback control system, allowing the rapid modulation of the applied force on individual DNA molecules while inside the pore.
We have engineered a nanosensor for sequence-specific detection of single nucleic acid molecules across a lipid bilayer. The sensor is composed of a protein channel nanopore (alpha-hemolysin) housing a DNA probe with an avidin anchor at the 5' end and a nucleotide sequence designed to noncovalently bind a specific single-stranded oligonucleotide at the 3' end. The 3' end of the DNA probe is driven to the opposite side of the pore by an applied electric potential, where it can specifically bind to oligonucleotides. Reversal of the applied potential withdraws the probe from the pore, dissociating it from a bound oligonucleotide. The time required for dissociation of the probe-oligonucleotide duplex under this force yields identifying characteristics of the oligonucleotide. We demonstrate transmembrane detection of individual oligonucleotides, discriminate between molecules differing by a single nucleotide, and investigate the relationship between dissociation time and hybridization energy of the probe and analyte molecules. The detection method presented in this article is a candidate for in vivo single-molecule detection and, through parallelization in a synthetic device, for genotyping and global transcription profiling from small samples.
Each species from bacteria to human has a distinct genetic fingerprint. Therefore, a mechanism that detects a single molecule of DNA represents the ultimate analytical tool. As a first step in the development of such a tool, we have explored using a nanometer-diameter pore, sputtered in a nanometer-thick inorganic membrane with a tightly focused electron beam, as a transducer that detects single molecules of DNA and produces an electrical signature of the structure. When an electric field is applied across the membrane, a DNA molecule immersed in electrolyte is attracted to the pore, blocks the current through it, and eventually translocates across the membrane as verified unequivocally by gel electrophoresis. The relationship between DNA translocation and blocking current has been established through molecular dynamics simulations. By measuring the duration and magnitude of the blocking current transient, we can discriminate single-stranded from double-stranded DNA and resolve the length of the polymer.
We have previously demonstrated that a nanometer-diameter pore in a nanometer-thick metal-oxide-semiconductor-compatible membrane can be used as a molecular sensor for detecting DNA. The prospects for using this type of device for sequencing DNA are avidly being pursued. The key attribute of the sensor is the electric field-induced (voltage-driven) translocation of the DNA molecule in an electrolytic solution across the membrane through the nanopore. To complement ongoing experimental studies developing such pores and measuring signals in response to the presence of DNA, we conducted molecular dynamics simulations of DNA translocation through the nanopore. A typical simulated system included a patch of a silicon nitride membrane dividing water solution of potassium chloride into two compartments connected by the nanopore. External electrical fields induced capturing of the DNA molecules by the pore from the solution and subsequent translocation. Molecular dynamics simulations suggest that 20-basepair segments of double-stranded DNA can transit a nanopore of 2.2 x 2.6 nm(2) cross section in a few microseconds at typical electrical fields. Hydrophobic interactions between DNA bases and the pore surface can slow down translocation of single-stranded DNA and might favor unzipping of double-stranded DNA inside the pore. DNA occluding the pore mouth blocks the electrolytic current through the pore; these current blockades were found to have the same magnitude as the blockade observed when DNA transits the pore. The feasibility of using molecular dynamics simulations to relate the level of the blocked ionic current to the sequence of DNA was investigated.
We have used the nanometer scale alpha-Hemolysin pore to study the unzipping kinetics of individual DNA hairpins under constant force or constant loading rate. Using a dynamic voltage control method, the entry rate of polynucleotides into the pore and the voltage pattern applied to induce hairpin unzipping are independently set. Thus, hundreds of unzipping events can be tested in a short period of time (few minutes), independently of the unzipping voltage amplitude. Because our method does not entail the physical coupling of the molecules under test to a force transducer, very high throughput can be achieved. We used our method to study DNA unzipping kinetics at small forces, which have not been accessed before. We find that in this regime the static unzipping times decrease exponentially with voltage with a characteristic slope that is independent of the duplex region sequence, and that the intercept depends strongly on the duplex region energy. We also present the first nanopore dynamic force measurements (time varying force). Our results are in agreement with the approximately logV dependence at high V (where V is the loading rate) observed by other methods. The extension of these measurements to lower loading rates reveals a much weaker dependence on V.
The capture of single strands of DNA inside the α-hemolysin (α-HL) transmembrane pore protein upon application of a positive potential leads to single α-HL·DNA pseudorotaxane species (see picture). By monitoring the characteristic decreases in the ion conductance of α-HL, a single adenine nucleotide can be identified at a specific location on a strand of DNA.
alpha-Hemolysin of Staphylococcus aureus is a self-assembling toxin that forms a water-filled transmembrane channel upon oligomerization in a lipid membrane. Apart from being one of the best-studied toxins of bacterial origin, alpha-hemolysin is the principal component in several biotechnological applications, including systems for controlled delivery of small solutes across lipid membranes, stochastic sensors for small solutes, and an alternative to conventional technology for DNA sequencing. Through large-scale molecular dynamics simulations, we studied the permeability of the alpha-hemolysin/lipid bilayer complex for water and ions. The studied system, composed of approximately 300,000 atoms, included one copy of the protein, a patch of a DPPC lipid bilayer, and a 1 M water solution of KCl. Monitoring the fluctuations of the pore structure revealed an asymmetric, on average, cross section of the alpha-hemolysin stem. Applying external electrostatic fields produced a transmembrane ionic current; repeating simulations at several voltage biases yielded a current/voltage curve of alpha-hemolysin and a set of electrostatic potential maps. The selectivity of alpha-hemolysin to Cl(-) was found to depend on the direction and the magnitude of the applied voltage bias. The results of our simulations are in excellent quantitative agreement with available experimental data. Analyzing trajectories of all water molecule, we computed the alpha-hemolysin's osmotic permeability for water as well as its electroosmotic effect, and characterized the permeability of its seven side channels. The side channels were found to connect seven His-144 residues surrounding the stem of the protein to the bulk solution; the protonation of these residues was observed to affect the ion conductance, suggesting the seven His-144 to comprise the pH sensor that gates conductance of the alpha-hemolysin channel.
We characterize the voltage-driven motion and the free motion of single-stranded DNA (ssDNA) molecules captured inside the ≈1.5-nm α-hemolysin pore, and show that the DNA–channel interactions depend strongly on the orientation of the ssDNA molecules with respect to the pore. Remarkably, the voltage-free diffusion of the 3′-threaded DNA (in the trans to cis direction) is two times slower than the corresponding 5′-threaded DNA having the same poly(dA) sequence. Moreover, the ion currents flowing through the blocked pore with either a 3′-threaded DNA or 5′ DNA differ by ≈30%. All-atom molecular dynamics simulations of our system reveal a microscopic mechanism for the asymmetric behavior. In a confining pore, the ssDNA straightens and its bases tilt toward the 5′ end, assuming an asymmetric conformation. As a result, the bases of a 5′-threaded DNA experience larger effective friction and forced reorientation that favors co-passing of ions. Our results imply that the translocation process through a narrow pore is more complicated than previously believed and involves base tilting and stretching of ssDNA molecules inside the confining pore.
• asymmetry
• DNA translocation
• DNA hairpin
Reducing a DNA molecule's translocation speed in a solid-state nanopore is a key step toward rapid single molecule identification. Here we demonstrate that DNA translocation speeds can be reduced by an order of magnitude over previous results. By controlling the electrolyte temperature, salt concentration, viscosity, and the electrical bias voltage across the nanopore, we obtain a 3 base/micros translocation speed for 3 kbp double-stranded DNA in a 4-8 nm diameter silicon nitride pore. Our results also indicate that the ionic conductivity inside such a nanopore is smaller than it is in bulk.
We investigate single-molecule electrophoretic translocation of A(50), C(50), A(25)C(50), and C(50)A(25) RNA molecules through the alpha-hemolysin transmembrane protein pore. We observe pronounced bilevel current blockages during translocation of A(25)C(50) and C(50)A(25) molecules. The two current levels observed during these bilevel blockages are very similar to the characteristic current levels observed during A(50) and C(50) translocation. From the temporal ordering of the two levels within the bilevel current blockages, we infer whether individual A(25)C(50) and C(50)A(25) molecules pass through the pore in a 3'-->5' or 5'-->3' orientation. Correlation between the level of current obstruction and the inferred A(25)C(50) or C(50)A(25) orientation indicates that 3'-->5' translocation of a poly C segment causes a significantly deeper current obstruction than 5'-->3' translocation. Our analysis also suggests that the 3' ends of C(50) and A(25)C(50) RNA molecules are more likely to initiate translocation than the 5' ends. Orientation dependent differences in a smaller current blockage that immediately precedes many translocation events suggest that this blockage also contains information about RNA orientation during translocation. These findings emphasize that the directionality of polynucleotide molecules is an important factor in translocation and demonstrate how structure within ionic current signals can give new insights into the translocation process.
The mechanical properties of DNA over segments comparable to the size of a protein-binding site (3-10 nm) are examined using an electric-field-induced translocation of single molecules through a nanometer diameter pore. DNA, immersed in an electrolyte, is forced through synthetic pores ranging from 0.5 to 1.5 nm in radius in a 10 nm thick Si(3)N(4) membrane using an electric field. To account for the stretching and bending, we use molecular dynamics to simulate the translocation. We have found a threshold for translocation that depends on both the dimensions of the pore and the applied transmembrane bias. The voltage threshold coincides with the stretching transition that occurs in double-stranded DNA near 60 pN.
We have explored the electromechanical properties of DNA on a nanometer-length scale using an electric field to force single molecules through synthetic nanopores in ultrathin silicon nitride membranes. At low electric fields, E < 200 mV/10 nm, we observed that single-stranded DNA can permeate pores with a diameter >/=1.0 nm, whereas double-stranded DNA only permeates pores with a diameter >/=3 nm. For pores <3.0 nm diameter, we find a threshold for permeation of double-stranded DNA that depends on the electric field and pH. For a 2 nm diameter pore, the electric field threshold is approximately 3.1 V/10 nm at pH = 8.5; the threshold decreases as pH becomes more acidic or the diameter increases. Molecular dynamics indicates that the field threshold originates from a stretching transition in DNA that occurs under the force gradient in a nanopore. Lowering pH destabilizes the double helix, facilitating DNA translocation at lower fields.
We report experimental measurements of the salt dependence of ion transport and DNA translocation through solid-state nanopores. The ionic conductance shows a three-order-of-magnitude decrease with decreasing salt concentrations from 1 M to 1 muM, strongly deviating from bulk linear behavior. The data are described by a model that accounts for a salt-dependent surface charge of the pore. Subsequently, we measure translocation of 16.5-mum-long dsDNA for 50 mM to 1 M salt concentrations. DNA translocation is shown to result in either a decrease ([KCl] > 0.4 M) or increase of the ionic current ([KCl] < 0.4 M). The data are described by a model where current decreases result from the partial blocking of the pore and current increases are attributed to motion of the counterions that screen the charge of the DNA backbone. We demonstrate that the two competing effects cancel at a KCl concentration of 370 +/- 40 mM.