Voltage-driven Translocation Research Articles

Translocation of Gaussian polymers across a nanometric cylindrical channel

We present an analytical model to study translocation of Gaussian polymers across a cylindrical channel of nanometric size with a chemical potential inside the channel. Results show that polymer conformational entropy generates an entropic M-like free energy barrier for translocation. The presence of a small negative chemical potential reduces the entropic free energy barrier rendering the translocation time to follow a power law τ = AN ν as function of polymer size N. Power law exponents ν found here in varying the channel radius R, run from 1.525 to 1.999 for unforced translocation, and from 1.594 to 2.006 for translocation with small chemical potentials when R = 1 nm. Presence of large negative chemical potentials generate a free energy well rendering the translocation time to follow an exponential growth τ = Ae α N . We show existence of a negative chemical potential μ c that minimizes the translocation time due to an interplay of conformational entropy and channel-polymer interactions. The translocation time as function of channel length L grows exponentially as τ = Ae cL , it runs from milliseconds up to decades in the range of lengths found in biological channels. Interestingly, small negative chemical potentials approaching μ c overcome the effect of large channel lengths reducing the translocation time below seconds. Translocation speeds <v(N) > show a maximum of micrometers per second then it decays with polymer size and channel length, the characteristic decay <v(N) > ∼ N −1 has been observed in previous experiments of voltage-driven DNA translocation.

The translocation dynamics of the polymer through a conical pore: Non-stuck, weak-stuck, and strong-stuck modes.

The external voltage-driven polymer translocation through a conical pore (with a large opening at the entry and a small tip at the exit) is studied by using the Langevin dynamics simulation in this paper. The entire translocation process is divided into an approaching stage and a threading stage. First, the approaching stage starts from the polymer entering the large opening and ends up at a terminal monomer reaching the pore tip. In this stage, the polymer will undergo the conformation adjustment to fit the narrowed cross-sectional area of the pore, leading to three approaching modes: the non-stuck mode with a terminal monomer arriving at the pore tip smoothly, the weak-stuck mode for the polymer stuck inside the pore for a short duration with minor conformational adjustments, and the strong-stuck mode with major conformational changes and a long duration. The approaching times (the duration of the approaching stage) of the three approaching modes show different behavior as a function of the pore apex angle. Second, the threading stage describes that the polymer threads through the pore tip with a linear fashion. In this stage, an increase in the apex angle causes the reduction of the threading time (the duration of the threading stage) due to the increase in the driving force with the apex angle at the tip. Moreover, we also find that with the increase in the apex angle or the polymer length, the polymer threading dynamics will change from the quasi-equilibrium state to the non-equilibrium state.

An efficient kinetic Monte Carlo to study analyte capture by a nanopore: transients, boundary conditions and time-dependent fields.

To better understand the capture process by a nanopore, we introduce an efficient Kinetic Monte Carlo (KMC) algorithm that can simulate long times and large system sizes by mapping the dynamic of a point-like particle in a 3D spherically symmetric system onto the 1D biased random walk. Our algorithm recovers the steady-state analytical solution and allows us to study time-dependent processes such as transients. Simulation results show that the steady-state depletion zone near pore is barely larger than the pore radius and narrows at higher field intensities; as a result, the time to reach steady-state is much smaller than the time required to empty a zone of the size of the capture radius λe. When the sample reservoir has a finite size, a second depletion region propagates inward from the outer wall, and the capture rate starts decreasing when it reaches the capture radius λe. We also note that the flatness of the electric field near the pore, which is often neglected, induces a traffic jam that can increase the transient time by several orders of magnitude. Finally, we propose a new proof-of-concept scheme to separate two analytes of the same mobility but different diffusion coefficients using time-varying fields.

Nanopore device-based fingerprinting of RNA oligos and microRNAs enhanced with an Osmium tag

Protein and solid-state nanopores are used for DNA/RNA sequencing as well as for single molecule analysis. We proposed that selective labeling/tagging may improve base-to-base resolution of nucleic acids via nanopores. We have explored one specific tag, the Osmium tetroxide 2,2′-bipyridine (OsBp), which conjugates to pyrimidines and leaves purines intact. Earlier reports using OsBp-tagged oligodeoxyribonucleotides demonstrated proof-of-principle during unassisted voltage-driven translocation via either alpha-Hemolysin or a solid-state nanopore. Here we extend this work to RNA oligos and a third nanopore by employing the MinION, a commercially available device from Oxford Nanopore Technologies (ONT). Conductance measurements demonstrate that the MinION visibly discriminates oligoriboadenylates with sequence A15PyA15, where Py is an OsBp-tagged pyrimidine. Such resolution rivals traditional chromatography, suggesting that nanopore devices could be exploited for the characterization of RNA oligos and microRNAs enhanced by selective labeling. The data also reveal marked discrimination between a single pyrimidine and two consecutive pyrimidines in OsBp-tagged AnPyAn and AnPyPyAn. This observation leads to the conjecture that the MinION/OsBp platform senses a 2-nucleotide sequence, in contrast to the reported 5-nucleotide sequence with native nucleic acids. Such improvement in sensing, enabled by the presence of OsBp, may enhance base-calling accuracy in enzyme-assisted DNA/RNA sequencing.

Electrostatic Forces in Field-Perturbed Equilibria: Nanopore Analysis of Cage Complexes

Summary Molecular recognition has been widely investigated under equilibrium conditions, but little is known about such processes when perturbed by external forces. Here, we investigate the influence of electric fields on complexes formed between metallosupramolecular cages and a protein nanopore at the single-molecule level. Association rates were dominated by the applied voltage, whereas local electrostatic interactions between the cage and the nanopore more greatly influenced the dissociation kinetics. By exploiting these principles, we showed that the externally applied voltage could be used to selectively bind a specific cage from a mixture containing a large excess of other cages. Moreover, the applied voltage could also be used to drive supramolecular enantio-inversion of the chiral cages or, occasionally, the non-equilibrium capture and disassembly of cages deep within the nanopore. Similar principles might be exploited in the design of other molecular devices that operate within externally applied electric fields or biogenic transmembrane potentials.

Coarse-grained molecular dynamics study of wettability influence on protein translocation through solid nanopores

Protein translocation through nanopores is widely involved in molecular sensing and analyzing devices, whereby nanopore surface properties are crucial. However, fundamental understanding of how these properties affect protein motion inside nanopores remains lacking. In this work, we study the influence of nanopore surface wettability on voltage-driven protein translocation through nanopores with coarse-grained molecular dynamics simulations. The results show that the electrophoretic mobility of protein translocation increases as the contact angle of nanopore surface increases from 0° to 90°, but becomes almost constant as the contact angle is above 90°. This observation can be attributed to the variation of the friction coefficient of protein translocation through the nanopores with different nanopore contact angles. We further show that the interaction between nanopore and water, rather than that between the nanopore and protein, dominates the protein transport in nanopores. These findings provide new insights into protein translocation dynamics across nanopores and will be beneficial to the design of high-efficiency nanopore devices for single molecule protein sensing.

Facilitated polymer capture by charge inverted electroosmotic flow in voltage-driven polymer translocation.

The optimal functioning of nanopore-based biosensing tools necessitates rapid polymer capture from the ion reservoir. We identify an ionic correlation-induced transport mechanism that provides this condition without the chemical modification of the polymer or the pore surface. In the typical experimental configuration where a negatively charged silicon-based pore confines a 1 : 1 electrolyte solution, anionic polymer capture is limited by electrostatic polymer-membrane repulsion and the electroosmotic (EO) flow. Added multivalent cations suppress the electrostatic barrier and reverse the pore charge, inverting the direction of the EO flow that drags the polymer to the trans side. This inverted EO flow can be used to speed up polymer capture from the reservoir and to transport weakly or non-uniformly charged polymers that cannot be controlled by electrophoresis.

Fabrication of Low Noise Borosilicate Glass Nanopores for Single Molecule Sensing.

We show low-cost fabrication and characterization of borosilicate glass nanopores for single molecule sensing. Nanopores with diameters of ~100 nm were fabricated in borosilicate glass capillaries using laser assisted glass puller. We further achieve controlled reduction and nanometer-size control in pore diameter by sculpting them under constant electron beam exposure. We successfully fabricate pore diameters down to 6 nm. We next show electrical characterization and low-noise behavior of these borosilicate nanopores and compare their taper geometries. We show, for the first time, a comprehensive characterization of glass nanopore conductance across six-orders of magnitude (1M-1μM) of salt conditions, highlighting the role of buffer conditions. Finally, we demonstrate single molecule sensing capabilities of these devices with real-time translocation experiments of individual λ-DNA molecules. We observe distinct current blockage signatures of linear as well as folded DNA molecules as they undergo voltage-driven translocation through the glass nanopores. We find increased signal to noise for single molecule detection for higher trans-nanopore driving voltages. We propose these nanopores will expand the realm of applications for nanopore platform.

Fabrication of Low Noise Borosilicate Glass Nanopores for Single Molecule Sensing

We describe low-cost fabrication, size control and characterization of borosilicate glass nanopores for sensing of single biomolecules. Nanopores with pore diameters down to ∼100nm were fabricated in borosilicate glass capillaries using table-top carbon-di-oxide laser assisted glass capillary puller. We further achieve controlled reduction in pore diameter by sculpting of glass capillaries under constant electron beam exposure of a scanning electron microscope. We confirm successful fabrication of sub-10nm pores by SEM imaging. We further show electrical characterization and low-noise behavior of these borosilicate nanopores and compare various geometries of pulled conical glass nanopores. We show, for the first time, a comprehensive characterization of glass nanopore conductance across a wide range (1M-1μM) of salt conditions. We show the conditions for the conical pore conductance model to fit to this extended salt range. We also experimentally show, for the first time, the role of buffer conditions in this wide salt range measurement. Finally, we demonstrate single molecule sensing capabilities of these devices with real-time translocation experiments of individual λ-DNA molecules. We observe distinct current blockage signatures of single and multiply folded DNA molecules as they undergo voltage-driven translocation through the glass nanopores. We find increased signal to noise for single molecule detection for higher trans-nanopore driving voltages.

Single pyrimidine discrimination during voltage-driven translocation of osmylated oligodeoxynucleotides via the α-hemolysin nanopore.

The influence of an electric field on an isolated channel or nanopore separating two compartments filled with electrolytes produces a constant ion flux through the pore. Nucleic acids added to one compartment traverse the pore, and modulate the current in a sequence-dependent manner. While translocation is faster than detection, the α-hemolysin nanopore (α-HL) successfully senses base modifications in ssDNA immobilized within the pore. With the assistance of a processing enzyme to slow down translocation, nanopore-based DNA sequencing is now a commercially available platform. However, accurate base calling is challenging because α-HL senses a sequence, and not a single nucleotide. Osmylated DNA was recently proposed as a surrogate for nanopore-based sequencing. Osmylation is the addition of osmium tetroxide 2,2’-bipyridine (OsBp) to the C5–C6 pyrimidine double bond. The process is simple, selective for deoxythymidine (dT) over deoxycytidine (dC), unreactive towards the purines, practically 100% effective, and strikingly independent of length, sequence, and composition. Translocation of an oligodeoxynucleotide (oligo) dA10XdA9 via α-HL is relatively slow, and exhibits distinct duration as well as distinct residual current when X = dA, dT(OsBp), or dC(OsBp). The data indicate that the α-HL constriction zone/β-barrel interacts strongly with both OsBp and the base. A 23 nucleotide long oligo with four dT(OsBp) traverses 18-times slower, and the same oligo with nine (dT+dC)(OsBp) moieties traverses 84-times slower compared to dA20, suggesting an average rate of 40 or 180 μs/base, respectively. These translocation speeds are well above detection limits, may be further optimized, and clear the way for nanopore-based sequencing using osmylated DNA.

Voltage-driven translocation behaviors of IgG molecule through nanopore arrays

Nanopore-based biosensing has attracted more and more interests in the past years, which is also regarded as an emerging field with major impact on bio-analysis and fundamental understanding of nanoscale interactions down to single-molecule level. In this work, the voltage-driven translocation properties of goat antibody to human immunoglobulin G (IgG) are investigated using nanopore arrays in polycarbonate membranes. Obviously, the background ionic currents are modulated by IgG molecules for their physical place-holding effect. However, the detected ionic currents do ‘not’ continuously decrease as conceived; the currents first decrease, then increase, and finally stabilize with increasing IgG concentration. To understand this phenomenon, a simplified model is suggested, and the calculated results contribute to the understanding of the abnormal phenomenon in the actual ionic current changing tendency.

Voltage-Driven Translocation of DNA through a High Throughput Conical Solid-State Nanopore

Nanopores have become an important tool for molecule detection at single molecular level. With the development of fabrication technology, synthesized solid-state membranes are promising candidate substrates in respect of their exceptional robustness and controllable size and shape. Here, a 30–60 (tip-base) nm conical nanopore fabricated in 100 nm thick silicon nitride (Si3N4) membrane by focused ion beam (FIB) has been employed for the analysis of λ-DNA translocations at different voltage biases from 200 to 450 mV. The distributions of translocation time and current blockage, as well as the events frequencies as a function of voltage are investigated. Similar to previously published work, the presence and configurations of λ-DNA molecules are characterized, also, we find that greater applied voltages markedly increase the events rate, and stretch the coiled λ-DNA molecules into linear form. However, compared to 6–30 nm ultrathin solid-state nanopores, a threshold voltage of 181 mV is found to be necessary to drive DNA molecules through the nanopore due to conical shape and length of the pore. The speed is slowed down ∼5 times, while the capture radius is ∼2 fold larger. The results show that the large nanopore in thick membrane with an improved stability and throughput also has the ability to detect the molecules at a single molecular level, as well as slows down the velocity of molecules passing through the pore. This work will provide more motivations for the development of nanopores as a Multi-functional sensor for a wide range of biopolymers and nano materials.

Translocation of polymers in a lattice model

Voltage-driven polymer translocation is studied by means of a stochastic lattice model. The model incorporates voltage drop over the membrane as a bias in the hopping rate through the pore and exhibits the two main ingredients of the translocation process: driven motion through the pore and diffusive supply of chain length towards the pore on the cis-side and the drift away from the pore on the trans-side. The translocation time is either bias limited or diffusion limited. In the bias-limited regime the translocation time is inversely proportional to the voltage drop over the membrane. In the diffusion-limited regime the translocation time is independent of the applied voltage, but it is rather sensitive to the motion rules of the model. We find that the whole regime is well described by a single curve determined by the initial slope and the saturation value. The dependence of these parameters on the length of the chain, the motion rules and the repton statistics are established. Repulsion of reptons as well as the increase of chain length decrease the throughput of the polymer through the pore. As for free polymers, the inclusion of a mechanism for hernia creations/annihilations leads to the cross-over from Rouse-like behaviour to reptation. For the experimentally most relevant case (Rouse dynamics) the bimodal power law dependence of the translocation time on the chain length is found.

Translocation of reptating chains

Voltage-driven translocation is modeled with the Rubinstein–Duke rules for hoppingreptons in one- and two-dimensional lattices. The chain is driven through the pore by a biaspotential promoting the transition of stored length in one direction. Coupling states give asemi-periodicity of the process that enables us to relate the properties to the stationarystate of the master equation. The exact solution for short chains and Monte Carlosimulations for longer chains are used to calculate displacements, velocities and thetranslocation time.

Protein Transport Through the Anthrax Toxin Channel: Molecular Mechanisms

Bacillus anthracis, the causative agent of anthrax, produced a toxin composed of a translocase heptameric channel, (PA63)7, which allows its two substrate proteins, lethal and edema factor (LF and EF), to translocate across a host cell's endosomal membrane, disrupting the cell's normal function. Protein translocation through the channel, reconstituted in lipid bilayers, is driven (N-terminal end first) by a proton electrochemical potential gradient. The (PA63)7 channel strongly disfavors the entry of negatively charged residues on proteins, and hence the acidic side chains on LFN (the N-terminal 263 residues of LF) enter protonated; these protons are released into the trans solution upon exiting the channel, thereby making this a proton-protein symporter. Consistent with this idea, a single SO3-, which is essentially not titrateable, introduced at most positions in LFN, drastically inhibited voltage-driven LFN translocation. The lumen of the (PA63)7 14-strand β barrel is ∼15 A wide and can barely accommodate an alpha-helix with its side chains. Translocation through the lumen thus requires the substrates to unfold. Here we present an approach using biotin-streptavidin chemistry to determine the length of the translocating polypeptide chain within the channel as it is traversing the (PA63)7 channel lumen, with the goal of shedding light on the structure of the polypeptide chain as it crosses the channel. We created a stopper at the LFN C terminus and attached a biotin at the N terminus. Translocation proceeds until the C terminus reaches the channel's cis entrance, and binding of the N-terminal biotin with streptavidin added to the trans side of the membrane, locks the polypeptide chain within the channel. By reducing the distance between the N-terminal biotin and the C-terminal stopper by deletion constructs, we can determine the minimum length that allows streptavidin to grab the N-terminal biotin.

Reverse DNA translocation through a solid-state nanopore by magnetic tweezers

Voltage-driven DNA translocation through nanopores has attracted wide interest for manypotential applications in molecular biology and biotechnology. However, it is intrinsicallydifficult to control the DNA motion in standard DNA translocation processes in which astrong electric field is required in drawing DNA into the pore, but it also leads touncontrollable fast DNA translocation. Here we explore a new type of DNA translocation.We dub it ‘reverse DNA translocation’, in which the DNA is pulled through a nanoporemechanically by a magnetic bead, driven by a magnetic-field gradient. This technique iscompatible with simultaneous ionic current measurements and is suitable for multiplenanopores, paving the way for large scale applications. We report the first experiment ofreverse DNA translocation through a solid-state nanopore using magnetic tweezers.

Evidence for a Proton–Protein Symport Mechanism in the Anthrax Toxin Channel

The toxin produced by Bacillus anthracis, the causative agent of anthrax, is composed of three proteins: a translocase heptameric channel, (PA63)7, formed from protective antigen (PA), which allows the other two proteins, lethal and edema factors (LF and EF), to translocate across a host cell's endosomal membrane, disrupting cellular homeostasis. It has been shown that (PA63)7 incorporated into planar phospholipid bilayer membranes forms a channel capable of transporting LF and EF. Protein translocation through the channel is driven by a proton electrochemical potential gradient on a time scale of seconds. A paradoxical aspect of this is that although LFN (the N-terminal 263 residues of LF), on which most of our experiments were performed, has a net negative charge, it is driven through the channel by a cis-positive voltage. We have explained this by claiming that the (PA63)7 channel strongly disfavors the entry of negatively charged residues on proteins to be translocated, and hence the aspartates and glutamates on LFN enter protonated (i.e., neutralized). Therefore, the translocated species is positively charged. Upon exiting the channel, the protons that were picked up from the cis solution are released into the trans solution, thereby making this a proton–protein symporter. Here, we provide further evidence of such a mechanism by showing that if only one SO3−, which is essentially not titratable, is introduced at most positions in LFN, through the reaction of an introduced cysteine residue at those positions with 2-sulfonato-ethyl-methanethiosulfonate, voltage-driven LFN translocation is drastically inhibited. We also find that a site that disfavors the entry of negatively charged residues into the (PA63)7 channel resides at or near its Φ-clamp, the ring of seven phenylalanines near the channel's entrance.

Evidence for Proton-Coupled Protein Transport through the Anthrax Toxin Channel

The toxin produced by Bacillus anthracis, the causative agent of anthrax, is composed of a translocase heptameric channel, (PA63)7, formed from protective antigen (PA), which allows its two substrate proteins, lethal and edema factors (LF and EF), to translocate across a host cell's endosomal membrane, disrupting the cellular homeostasis. It has been shown that (PA63)7 incorporated into planar phospholipid bilayer membranes forms a channel capable of transporting LF, EF and other small proteins. Protein translocation through the channel is driven by a proton electrochemical potential gradient, on a time scale of seconds with S-shaped kinetics. A paradoxical aspect of this is that although LFN (the N-terminal 263 residues of LF), on which most of our experiment were performed, has a net negative charge, it is driven through the channel by a cis positive voltage. We have explained this by claiming that the (PA63)7 channel strongly disfavors the entry of negatively charged residues on proteins to be translocated, and hence the aspartates and glutamates on LFN enter protonated (i.e. neutralized), and therefore the translocated species is positively charged. Upon exiting the channel, these protons that were picked up from the cis solution are released into the trans solution, thereby making this a proton-protein symporter. Here, we provide further evidence of such a mechanism by showing that if only one SO3−, which is essentially not titrateable, is introduced at most positions in LFN, through the reaction of a cysteine-modified residue at those positions with (2-Sulfonatoethyl) Methanethiosulfonate (MTS-ES), voltage-driven LFN translocation is drastically inhibited.

DNA Translocation Governed by Interactions with Solid-State Nanopores

We investigate the voltage-driven translocation dynamics of individual DNA molecules through solid-state nanopores in the diameter range 2.7–5 nm. Our studies reveal an order of magnitude increase in the translocation times when the pore diameter is decreased from 5 to 2.7 nm, and steep temperature dependence, nearly threefold larger than would be expected if the dynamics were governed by viscous drag. As previously predicted for an interaction-dominated translocation process, we observe exponential voltage dependence on translocation times. Mean translocation times scale with DNA length by two power laws: for short DNA molecules, in the range 150–3500 bp, we find an exponent of 1.40, whereas for longer molecules, an exponent of 2.28 dominates. Surprisingly, we find a transition in the fraction of ion current blocked by DNA, from a length-independent regime for short DNA molecules to a regime where the longer the DNA, the more current is blocked. Temperature dependence studies reveal that for increasing DNA lengths, additional interactions are responsible for the slower DNA dynamics. Our results can be rationalized by considering DNA/pore interactions as the predominant factor determining DNA translocation dynamics in small pores. These interactions markedly slow down the translocation rate, enabling higher temporal resolution than observed with larger pores. These findings shed light on the transport properties of DNA in small pores, relevant for future nanopore applications, such as DNA sequencing and genotyping.

Scaling Theory of Polymer Translocation into Confined Regions

We examine the voltage-driven polymer translocation from a spacious region into a confined region imposed by two parallel planes, so that the entry is impeded by the entropic confinement but aided by the electric field inside the confined region. Two modes of entry are examined: linear translocation where a chain enters the confined region with chain ends, and hairpin translocation where a chain enters the confined region by forming a hairpin. Our calculation shows that translocation time increases with polymer length for linear entries but decreases with polymer length for hairpin entries. Applying to electrophoresis of DNA molecules through periodic spacious and confined regions, our theory shows that the dominance of hairpin translocations leads to the experimentally observed faster migration of longer DNA molecules. Our theory predicts experimental conditions for the validity of this law in terms of polymer length, size of the confined region, and solution conditions.