Session B44: Focus Session: Translocation through Nanopores - Measurements and Theoretical Models

Sequence-dependent ion current modulations in biological and synthetic nanopores

The possibility of DNA sequence detection by measuring the blockade ionic current in nanopores has been the driving force for the spectacular development of the nanopore research field. Nevertheless, fifteen years after the first measurements, the molecular mechanism(s) of ion current modulation by the sequence of DNA nucleotides remains elusive. Here, we report the results of extensive all-atom molecular dynamics and Brownian dynamics simulations of three nanopore systems: a biological nanopore MspA, a solid-state nanopore and a graphene nanopore, aimed at elucidating the microscopic mechanism of the ion current modulation. In the case of solid-state and graphene nanopores, we determined the effect of sequence convolution on the ionic current value by simulating the ionic current blockades produced by all 64 permutations of the DNA nucleotide triplets. In the case of MspA, we determined the effect of the sequence, the global orientation, and the conformation of a DNA strand on the distribution of the ion current blockades. Based on the results of our simulations, we suggest possible routes for increasing the resolution of DNA sequence detection by measuring the nanopore ionic current and describe the inherit limitations of the method.   

Unexpected stop-and-go when DNA is pulled through a network

We perform single-molecule imaging of lambda-DNA chains when DC electric fields drive them through agarose networks in which they are heavily entangled. Velocity is decidedly unsteady. Exhaustive statistics reveal how motion switches between ``mobile'' and ``pause'' states, the latter differing from well-known ``hooking.'' As these observations appear to be inconsistent with the prevailing theories of DNA electrophoresis, we are also engaged in measurements that discriminate between motion of the chain ends and the chain centers, by direct two-color fluorescence imaging.   

Non-Equilibrium DNA Dynamics Probed by Delayed Capture and Recapture by a Solid-State Nanopore

We studied the relaxation of $\lambda $-DNA following its translocation through a voltage-biased solid-state nanopore. The translocation process drives DNA into a non-equilibrium state because the $\sim $2 ms translocation time is roughly fifty times shorter that the polymer's characteristic (Zimm) relaxation time. By reversing the applied voltage at controlled delay times after a translocation event, the nanopore probed the configurations of recaptured molecules at various stages of relaxation. We monitored the disruptions of the ionic current through the nanopore and computed the integrated charge deficits (ECDs) resulting from DNA translocations. As the delay time between voltage reversals was decreased from 50 ms to 5 ms, the distribution of ECDs shifted to lower values. Furthermore, an increasing fraction of recapture events occurred in a shorter interval from the voltage reversal than the delay time. These observations are explained by the expansion of the DNA coil as it approaches equilibrium. Finally, we show that recapturing a molecule multiple times and averaging the ECDs reduces the measurement error, which is useful for molecular diagnostic applications. The variance decreases approximately as the inverse number of passes through the pore.   

Forced Translocation of Polymer through Nanopore: Deterministic Model and Simulations

We propose a new theoretical model of forced translocation of a polymer chain through a nanopore. We assume that DNA translocation at high fields proceeds too fast for the chain to relax, and thus the chain unravels loop by loop in an almost deterministic way. So the distribution of translocation times of a given monomer is controlled by the initial conformation of the chain (the distribution of its loops). Our model predicts the translocation time of each monomer as an explicit function of initial polymer conformation. We refer to this concept as ``fingerprinting''. The width of the translocation time distribution is determined by the loop distribution in initial conformation as well as by the thermal fluctuations of the polymer chain during the translocation process. We show that the conformational broadening \textit{$\Delta $t} of translocation times of $m$-th monomer \textit{$\Delta $t}\textit{$\propto $m}$^{1.5}$ is stronger than the thermal broadening\textit{ $\delta $t}\textit{$\propto $m}$^{1.25}$ The predictions of our deterministic model were verified by extensive molecular dynamics simulations   

Force-Driven Translocation of a Polymer through a Nanopore

We study the far from equilibrium translocation of a DNA molecule through a nanopore. The pore is much narrower than the DNA, so the electrically driven DNA undergoes dramatic deformations during its passage. Using an idealized model in which the DNA is assumed to be a very long and flexible homopolymer driven by a force exerted only in the pore, we modify a previously developed method by introducing the concept of ``iso-flux trumpet''. We show that although the speed of the process is determined by the friction of the trailing part with the solvent, friction dissipates a small portion of the work performed by the electric field on the polymer, and the work is mostly dissipated by the irreversible stretching and destretching of the polymer squeezed into the small pore. Moreover, due to such stretches essentially caused by the membrane, a net heat transfer occurs during translocation from the post-translocation to the pre-translocation side of the membrane. The current theory can be improved by accounting for the nonzero field outside the pore and by considering the coupling between electric and hydrodynamic fields. The forces exerted by such fields on the DNA bulk not only alter the passage dynamics, but also introduce deformations on the initial conformation of the polymer.   

Non-equilibrium tension propagation as a unifying description of driven polymer translocation

We present results from a new Brownian dynamics model of driven polymer translocation$^1$, in which non-equilibrium memory effects due to tension propagation (TP) along the cis side subchain are included as a time-dependent friction. To solve the effective friction, we develop a finite chain length TP formalism, expanding on the work of Sakaue$^{1,2}$. The model yields results in excellent quantitative agreement with molecular dynamics simulations in a wide range of parameters. Our results show that non-equilibrium TP along the cis side subchain dominates the dynamics of driven translocation. In addition, the model explains the different scaling of translocation time with chain length observed both in experiments and simulations as a combined effect of finite chain length and pore-polymer interactions. \\ $^1$T. Ikonen, A. Bhattacharya, T. Ala-Nissila and W. Sung, submitted.\\ $^2$T. Sakaue, Phys. Rev. E {\bf 76}, 021803 (2007)\\ $^3$T. Sakaue, Phys. Rev. E {\bf 81}, 041808 (2010).\\   

Graphene Nano-Electrodes for DNA Sequencing: an Ab initio Perspective

The proposal was made that a graphene nanogap could be used to probe the transverse conductance of individual nucleotides in DNA to rapidly identify the associated base sequence. Experimentally, the characteristic drop in ionic current associated with translocation events of DNA passing through a graphene nanopore was measured. Using first-principles methods, we evaluated the performance of two graphene nano-electrodes configurations for nucleobase identification. In the first study, Nano Lett. 11, 1941 (2011), we investigated the electronic transport properties of the four nucleotides when located in a graphene nanogap by employing density functional theory and the non-equilibrium Green's function method. In particular, we determined the electrical current variation at finite bias due to changes in the nucleotides orientation. Our second study, Adv. Funct. Mater. 21, 2674 (2011), utilized molecular dynamics simulations in conjunction with electronic transport calculations to explore specifically the effect of the hydrogenated graphene edges on the translocating DNA. It is found that edge-hydrogenated graphene electrodes facilitate the temporary formation of weak H-bonds with suitable atomic sites in the nucleotides.   

Preprotein translocation across the endoplasmic reticulum membrane in milieus crowded by proteins

Translocation of preproteins chains between the cytoplasm and the endoplasmic reticulum lumen takes place in a milieu crowded primarily by proteins. We compute translocation and retrotranslocation times for chains of different length in a milieu crowded by spherical agents at volume fractions equivalent to that found in cells. These numerical times obtained from a diffusion-equation model subject to a potential given by the free energy of one chain, indicate that crowding increases the translocation time by up to five times compared to those in dilute conditions for average-size chains and by up to a thousand times for long chains. Retrotranslocation times become smaller than translocation ones, in approximately 75\%. Translocation rates obtained in this work are similar to those found in a theoretical model for Brownian-ratchet translocation and coincide with in vitro experimental results (1-8 aminoacid/s) only in the limit of very long chains; for shorter chains, translocation rates are much faster. Our prediction that for long chains translocation rates would be significantly slowed by crowding can be tested experimentally using vesicles. Discrepancy of time-scales with experiments for short chains indicates that other factors beside crowding must be included in our model.   

Nanopore Mass Spectrometry

We describe a concept for single-DNA analysis called nanopore mass spectrometry, which seeks to combine the benefits of nanopores with the speed, sensitivity, and robustness of single base detection by mass spectrometry. The basic idea is to cleave the individual nucleotides from a DNA polymer as they transit a nanopore in sequence, and to identify each one by determining its charge-to-mass ratio in a mass spectrometer. We describe how nanopore mass spectrometry can addresse the challenges faced by other nanopore-based DNA analysis approaches. We also describe the design, construction, and testing of a prototype instrument that interfaces a nanopore ion source with a quadrupole mass filter and a single ion detector. We are using this new instrument to test the key scientific questions bearing on our analysis strategy: 1) Can DNA nucleotides be reliably transferred from their native liquid phase into the vacuum environment of a mass spectrometer? 2) Can nucleotides be detected with near 100$\%$ efficiency? 3) Can DNA polymers be controllably cleaved to isolate ionized bases or nucleotides in the mass spectrometer?   

Direct observation of DNA translocation influenced by electrically gated nanopores

One of remarkable recent developments in the solid state nanopore based DNA analysis is adding the ability to control electric potential near nanopore as a gate electrode by patterning metal in or on nanopore. In this approach, better control of DNA translocations for example, slowing down the translocation speed might be expected. We have fabricated insulator-metal-insulator nanopores of rather large 100 nm pore in diameter. The 100 nm diameter pores allow us to observe the translocation of lambda-DNA molecules directly by means of fluorescence microscopy without heavy clogging of the DNA molecules into the pores. By controlling ?gate voltage? on metal relative to the cis and trans voltages, the translocation rates of DNA are able to change. Interestingly, applying pulse voltage to the gate metal near 100 ms to reverse the direction of the electric field near the cis side of nanopore reverses the direction of the DNA translocation instantaneously. This in fact provides us a new way to repeat translocation of the same DNA molecule. Furthermore, repeating the pulse tends to clear off the clogged DNA molecules in nanopore. We will present more details of these phenomena caused by the gate voltages.   

Thermophoretic stretch of a polymer confined to a nanofluidic channel

The precise manipulation of macromolecules by thermophoresis is quite promising. Indeed, Thamdrup \textit{et al.} (Nano Lett., 10, 2010) successfully moved double-stranded DNA (dsDNA) filaments in microfluidic geometries and subsequently inserted them into nano-channels using thermophoretic forces originating from highly localized thermal gradients and also showed that once in the channel, DNA can be symmetrically stretched under thermophoresis. This last procedure could be used to better expose the backbone of a nano-confined polymer to study its properties or the binding activity of some enzyme. We present a novel approach to model this symmetric stretch using blobs and the Flory free-energy of a polymer chain. Our model describes the monomer concentration profile reported in the aforementioned study. A value close to what is found in the literature is obtained for the Soret coefficient of the segments of dsDNA, characterizing thermophoresis. We further corroborate the validity of our model using molecular dynamics simulations. In these calculations, excluded volume interactions are shown to play a key role especially when temperatures are close to the solvent's $\theta$ value.   

Small Interfering RNA Transfection Across a Phospholipid Membrane

Small interfering RNA (siRNA) molecules play a pivotal role in silencing gene expression via the RNA interference mechanism. We have performed steered MD simulations to study the transfection of a bare siRNA and siRNA/Oleic Acid (OA) complex across the dipalmitoylphosphatidycholine (DPPC) bilayer at T = 323 K. Bare siRNA induces the formation of frustrated lipid gel domains, whereas in the presence of siRNA/OA complex the membrane is found to be in the liquid-ordered phase. In both cases the stress profiles across the membrane indicate that the membrane is under tension near the head groups and highly compressed at the water-hydrophobic interface. During transfection, the membrane is deformed and the lateral stress is significantly lowered for the bare siRNA and siRNA/OA complex. The bare siRNA transfects through a lipid-nanopore of hydrophilic head-groups and hydrophobic carbon chains, whereas the siRNA/OA complex transfects through a lipid-nanopore of hydrophilic head groups.   

DNA base-specific modulation of $\mu$A transverse edge currents through a metallic graphene nanoribbon with a nanopore

We study two-terminal devices for DNA sequencing which consist of a zigzag graphene nanoribbon (ZGNR) and a nanopore in its interior through which the DNA molecule is translocated. Using the nonequilibrium Green functions combined with density functional theory, we demonstrate that each of the four DNA nucleobases inserted into the nanopore, whose edge carbon atoms are passivated by either hydrogen or nitrogen, will lead to a unique change in the device conductance. Unlike other recent biosensors based on transverse electronic transport through translocated DNA, which utilize small (of the order of pA) tunneling current across a nanogap or a nanopore yielding a poor signal-to-noise ratio, our device concept relies on the fact that in ZGNRs local current density is peaked around the edges so that drilling a nanopore away from the edges will not diminish the conductance. Inserting a nucleobase into the nanopore affects the charge density in the surrounding area, thereby modulating edge conduction currents whose magnitude is of the order of $\mu$A at bias voltage $\simeq 0.1$ V. The proposed biosensors could also be realized with other nanowires supporting transverse edge currents, such as chiral GNRs or wires made of two-dimensional topological insulators.