Mechanism of How Salt-Gradient-Induced Charges Affect the Translocation of DNA Molecules through a Nanopore

Experiments using nanopores demonstrated that a salt gradient enhances the capture rate of DNA and reduces its translocation speed. These two effects can help to enable electrical DNA sequencing with nanopores. Here, we provide a quantitative theoretical evaluation that shows the positive net charges, which accumulate around the pore entrance due to the salt gradient, are responsible for the two observed effects: they reinforce the electric capture field, resulting in promoted molecule capture rate; and they induce cationic electroosmotic flow through the nanopore, thus significantly retarding the motion of the anionic DNA through the nanopore. Our multiphysical simulation results show that, during the polymer trapping stage, the former effect plays the major role, thus resulting in promoted DNA capture rate, while during the nanopore-penetrating stage the latter effect dominates and consequently reduces the DNA translocation speed significantly. Quantitative agreement with experimental results has been reached by further taking nanopore wall surface charges into account.     

"DNA-Dressed NAnopore" for complementary sequence detection

Single molecule electrical sensing with nanopores is a rapidly developing field with potential revolutionary effects on bioanalytics and diagnostics. The recent success of this technology is in the simplicity of its working principle, which exploits the conductance modulations induced by the electrophoretic translocation of molecules through a nanometric channel. Initially proposed as fast and powerful tools for molecular stochastic sensing, nanopores find now application in a range of different domains, thanks to the possibility of finely tuning their surface properties, thus introducing artificial binding and recognition sites. Here we show the results of DNA translocation and hybridization experiments at the single molecule level by a novel class of selective biosensor devices that we call "DNA-Dressed NAnopore" (DNA(2)), based on solid state nanopore with large initial dimensions, resized and activated by functionalization with DNA molecules. The presented data demonstrate the ability of the DNA(2) to selectively detect complementary target sequences, that is to distinguish between molecules depending on their affinity to the functionalization. The DNA(2) can thus constitute the basis for the design of integrable parallel devices for mutation DNA analysis, diagnostics and bioanalytic investigations.     

Distinguishable populations report on the interactions of single DNA molecules with solid-state nanopores

Solid-state nanopores have received increasing interest over recent years because of their potential for genomic screening and sequencing. In particular, small nanopores (2-5 nm in diameter) allow the detection of local structure along biological molecules, such as proteins bound to DNA or possibly the secondary structure of RNA molecules. In a typical experiment, individual molecules are translocated through a single nanopore, thereby causing a small deviation in the ionic conductance. A correct interpretation of these conductance changes is essential for our understanding of the process of translocation, and for further sophistication of this technique. Here, we present translocation measurements of double-stranded DNA through nanopores down to the diameter of the DNA itself (1.8-7 nm at the narrowest constriction). In contrast to previous findings on such small nanopores, we find that single molecules interacting with these pores can cause three distinct levels of conductance blockades. We attribute the smallest conductance blockades to molecules that briefly skim the nanopore entrance without translocating, the intermediate level of conductance blockade to regular head-to-tail translocations, and the largest conductance blockades to obstruction of the nanopore entrance by one or multiple (duplex) DNA strands. Our measurements are an important step toward understanding the conductance blockade of biomolecules in such small nanopores, which will be essential for future applications involving solid-state nanopores.     

Trapping DNA near a Solid-State Nanopore

We demonstrate that voltage-biased solid-state nanopores can transiently localize DNA in an electrolyte solution. A double-stranded DNA (dsDNA) molecule is trapped when the electric field near the nanopore attracts and immobilizes a nonend segment of the molecule across the nanopore orifice without inducing a folded molecule translocation. In this demonstration of the phenomenon, the ionic current through the nanopore decreases when the dsDNA molecule is trapped by the nanopore. By contrast, a translocating dsDNA molecule under the same conditions causes an ionic current increase. We also present finite-element modeling results that predict this behavior for the conditions of the experiment.     

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.     

Single-Stranded DNA within Nanopores: Conformational Dynamics and Implications for Sequencing; a Molecular Dynamics Simulation Study

Engineered protein nanopores, such as those based on α-hemolysin from Staphylococcus aureus have shown great promise as components of next-generation DNA sequencing devices. However, before such protein nanopores can be used to their full potential, the conformational dynamics and translocation pathway of the DNA within them must be characterized at the individual molecule level. Here, we employ atomistic molecular dynamics simulations of single-stranded DNA movement through a model α-hemolysin pore under an applied electric field. The simulations enable characterization of the conformations adopted by single-stranded DNA, and allow exploration of how the conformations may impact on translocation within the wild-type model pore and a number of mutants. Our results show that specific interactions between the protein nanopore and the DNA can have a significant impact on the DNA conformation often leading to localized coiling, which in turn, can alter the order in which the DNA bases exit the nanopore. Thus, our simulations show that strategies to control the conformation of DNA within a protein nanopore would be a distinct advantage for the purposes of DNA sequencing.     

Hydroxymethyluracil modifications enhance the flexibility and hydrophilicity of double-stranded DNA

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.     

Molecular dynamics simulations of DNA within a nanopore: arginine-phosphate tethering and a binding/sliding mechanism for translocation

Protein nanopores show great potential as low-cost detectors in DNA sequencing devices. To date, research has largely focused on the staphylococcal pore α-hemolysin (αHL). In the present study, we have developed simplified models of the wild-type αHL pore and various mutants in order to study the translocation dynamics of single-stranded DNA under the influence of an applied electric field. The model nanopores reflect the experimentally measured conductance values in planar lipid bilayers. We show that interactions between rings of cationic amino acids and DNA backbone phosphates result in metastable tethering of nucleic acid molecules within the pore, leading us to propose a "binding and sliding" mechanism for translocation. We also observe folding of DNA into nonlinear conformational intermediates during passage through the confined nanopore environment. Despite adopting nonlinear conformations, the DNA hexamer always exits the pore in the same orientation as it enters (3' to 5') in our simulations. The observations from our simulations help to rationalize experimentally determined trends in residual current and translocation efficiency for αHL and its mutants.     

How Nanopore Translocation Experiments Can Measure RNA Unfolding

Electrokinetic translocation of biomolecules through solid-state nanopores represents a label-free single-molecule technique that may be used to measure biomolecular structure and dynamics. Recent investigations have attempted to distinguish individual transfer RNA (tRNA) species based on the associated pore translocation times, ion-current noise, and blockage currents. By manufacturing sufficiently smaller pores, each tRNA is required to undergo a deformation to translocate. Accordingly, differences in nanopore translocation times and distributions may be used to infer the mechanical properties of individual tRNA molecules. To bridge our understanding of tRNA structural dynamics and nanopore measurements, we apply molecular dynamics simulations using a simplified “structure-based” energetic model. Calculating the free-energy landscape for distinct tRNA species implicates transient unfolding of the terminal RNA helix during nanopore translocation. This provides a structural and energetic framework for interpreting current experiments, which can aid the design of methods for identifying macromolecules using nanopores.     

Real-Time Nanopore-Based Recognition of Protein Translocation Success

A growing number of new technologies are supported by a single- or multi-nanopore architecture for capture, sensing, and delivery of polymeric biomolecules. Nanopore-based single-molecule DNA sequencing is the premier example. This method relies on the uniform linear charge density of DNA, so that each DNA strand is overwhelmingly likely to pass through the nanopore and across the separating membrane. For disordered peptides, folded proteins, or block copolymers with heterogeneous charge densities, by contrast, translocation is not assured, and additional strategies to monitor the progress of the polymer molecule through a nanopore are required. Here, we demonstrate a single-molecule method for direct, model-free, real-time monitoring of the translocation of a disordered, heterogeneously charged polypeptide through a nanopore. The crucial elements are two “selectivity tags”—regions of different but uniform charge density—at the ends of the polypeptide. These affect the selectivity of the nanopore differently and enable discrimination between polypeptide translocation and retraction. Our results demonstrate exquisite sensitivity of polypeptide translocation to applied transmembrane potential and prove the principle that nanopore selectivity reports on biopolymer substructure. We anticipate that the selectivity tag technique will be broadly applicable to nanopore-based protein detection, analysis, and separation technologies, and to the elucidation of protein translocation processes in normal cellular function and in disease.     

The electromechanics of DNA in a synthetic nanopore

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.     

An ON/OFF biosensor based on blockade of ionic current passing through a solid-state nanopore

Single nanopores have attracted interest for their use as biosensing devices. In general, methods involve measuring ionic current blockades associated with translocation of analytes through the nanopore, but the detection of such short time lasting events requires complex equipment and setup that are critical for convenient routine biosensing. Here we present a novel biosensing concept based on a single nanopore in a silicon nitride membrane and two anchor-linked DNA species that forms trans-pore hybrids, realizing a stable blockade of ionic current through the pore. Molecular recognition events affecting the DNA hybrids cause a pore opening and the consequent establishment of an ionic current. In the present implementation of the device, we constructed a magnetic bead/streptavidin/biotin-DNA1/DNA2-biotin/streptavidin/Quantumdot-cluster complex (where DNA1 is a mismatched reverse complement of DNA2) through a sub-micrometric pore and monitored DNA strand displacement events occurring after addition of an oligonucleotide complementary to DNA2. The electric and mechanical aspects of the novel device, as well as its potential in biosensing are discussed.     

Nanopore sequencing technology: nanopore preparations

For the past decade, nanometer-scale pores have been developed as a powerful technique for sensing biological macromolecules. Various potential applications using these nanopores have been reported at the proof-of-principle stage, with the eventual aim of using them as an alternative to de novo DNA sequencing. Currently, there have been two general approaches to prepare nanopores for nucleic acid analysis: organic nanopores, such as alpha-hemolysin pores, are commonly used for DNA analysis, whereas synthetic solid-state nanopores have also been developed using various conventional and non-conventional fabrication techniques. In particular, synthetic nanopores with pore sizes smaller than the alpha-hemolysin pores have been prepared, primarily by electron-beam-assisted techniques: these are more robust and have better dimensional adjustability. This review will examine current methods of nanopore preparation, ranging from organic pore preparations to recent developments in synthetic nanopore fabrications.     

Automated forward and reverse ratcheting of DNA in a nanopore at 5-Å precision

An emerging DNA sequencing technique uses protein or solid-state pores to analyze individual strands as they are driven in single-file order past a nanoscale sensor. However, uncontrolled electrophoresis of DNA through these nanopores is too fast for accurate base reads. Here, we describe forward and reverse ratcheting of DNA templates through the α-hemolysin nanopore controlled by phi29 DNA polymerase without the need for active voltage control. DNA strands were ratcheted through the pore at median rates of 2.5-40 nucleotides per second and were examined at one nucleotide spatial precision in real time. Up to 500 molecules were processed at ～130 molecules per hour through one pore. The probability of a registry error (an insertion or deletion) at individual positions during one pass along the template strand ranged from 10% to 24.5% without optimization. This strategy facilitates multiple reads of individual strands and is transferable to other nanopore devices for implementation of DNA sequence analysis.     

The impact of the number of layers of the graphene nanopores and the electrical field on ssDNA translocation

Graphene-based nanopore devices hold great promise for the next generation DNA sequencing because graphene is atomically thin which is extremely important for single base recognition. To understand the fundamental details of DNA translocation through a graphene nanopore, in this work, molecular dynamics simulations of ssDNA translocation through the nanopore were performed to trace the nucleobase trajectories and to investigate the impact of the number of layers of the graphene membrane and the electrical field on ssDNA translocation. We found that the velocity of ssDNA translocation was speeded up with the higher bias voltage, and the two-layered and five-layered graphene membrane with 1.0-nm diameter circular nanopore could discern different DNA strand by the translocation time.     

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.     

Nanopore sensor for fast label-free detection of short double-stranded DNAs

Functionalizing surface enhanced the molecular sensing ability of a fabricated nanopore by increasing the translocation duration time for a short double-stranded DNA. The surface of nanopore was derivatized with γ-aminopropyltriethoxysilane and the positively charged surface attracted DNA molecules when they were in the vicinity of nanopore. The translocation duration time of DNA increased due to the strong electrostatic interaction and it enabled us to detect a short double-stranded DNA (<1 kbp) that is under the size limit of a conventional solid state nanopore sensor. Both 539 and 910 bp double-stranded DNAs were analyzed with the surface functionalized nanopore and their translocation kinetics are presented in this work. The new feature of the surface modified nanopore that can detect short double-stranded DNA molecules could readily be applied for a rapid label-free diagnostic analysis in a Lab-On-a-Chip type DNA sensor.     

204 Polymer translocation

The translocation of a single macromolecule through a protein pore or a solid-state nanopore involves three major stages: (1) approach of the macromolecule towards the pore, (2) capture/recognition of the macromolecule at the pore entrance, and (3) threading through the pore (see the Figure) (Muthukumar, 2011). All of these stages are controlled by conformational entropy of the macromolecule, charge decoration, and the geometry of the pore, hydrodynamics, and electrostatic interactions. Chief among the contributing factors are the entropic barrier presented by the pore to the penetration of the macromolecule, pore–polymer interactions, electro-osmotic flow, and the drift-diffusion of the macromolecule in electrolyte solutions. A unifying theory of these contributing factors will be described in the context of a few illustrative experimental data on DNA translocation and protein translocation through protein pores and solid-state nanopores. Future challenges to specific biological systems will be briefly discussed.     

Mechanisms of pressure-induced water infiltration process through graphene nanopores

Understanding the mechanism of water infiltration through nanopores is essential for wide applications ranging from membrane separation to gene therapy. In this paper, the molecular dynamics simulation method is used to investigate the pressure-assisted water transport process through graphene nanopores. Various factors including the hydrophobicity of nanopore surface, nanopore dimension, temperature as well as external electric field that affect water in permeation into graphene nanopores are discussed. It is found that classic Laplace-Young equation fails and the relationship between pressure and diameter (D) does not follow the 1/D dependence as the characteristic dimension of a nanopore is sufficiently small (smaller than 1?nm). The critical pressure significantly depends on both the pore length and electric field as D is smaller than 5?nm. Besides, enhancing temperature and electric field intensity are obviously beneficial for water infiltration through those nanopores with a diameter smaller than 5?nm.