Deciphering ionic current signatures of DNA transport through a nanopore

Abstract

Within just a decade from the pioneering work demonstrating the utility of nanopores for molecular sensing, nanopores have emerged as versatile systems for single-molecule manipulation and analysis. In a typical setup, a gradient of the electrostatic potential captures charged solutes from the solution and forces them to move through a single nanopore, across an otherwise impermeable membrane. The ionic current blockades resulting from the presence of a solute in a nanopore can reveal the type of the solute, for example, the nucleotide makeup of a DNA strand. Despite great success, the microscopic mechanisms underlying the functionality of such stochastic sensors remain largely unknown, as it is not currently possible to characterize the microscopic conformations of single biomolecules directly in a nanopore and thereby unequivocally establish the causal relationship between the observables and the microscopic events. Such a relationship can be determined using molecular dynamics-a computational method that can accurately predict the time evolution of a molecular system starting from a given microscopic state. This article describes recent applications of this method to the process of DNA transport through biological and synthetic nanopores.

Figure 1

Experimental setup of a single molecule nanopore experiement. Driven by the electrical field between two electrodes, a biomolecule transits the pore in a thin, synthetic membrane (left), inducing a transient blockade of the ionic current (right), measured by the amplifier.

Figure 2

Biological and solid-state nanopores. (a, b) All-atom model of the α-hemolysin (a) and MspA (b) channels suspended in a lipid bilayer membrane [65]. (c) TEM image of a nanopore in a Si₃N₄ membrane [66]. (d) TEM image of a metal-oxide-semiconductor capacitor membrane [8].

Figure 3

Atom-scale models of solid-state (top row) and biological (bottom row) nanopores. To build a microscopic model of a solid-state nanopore, a unit cell (a) of Si₃N₄ or SiO₂ crystal is replicated in 3D to produce a solid-state membrane (b). A nanometer size pore is produced by removing atoms according to the desired shape (c). An optional step is to melt and resolidify the membrane material to obtain an amorphous surface (not shown) [111]. Next, a fragment of DNA is placed near the nanopore’s entrance (d), water molecules are added to fill the volume of the simulated system (e), select water molecules are replaced with the ions of the electrolyte according to the specified concentration (f). Similar steps are required to build a microscopic model of a biological nanopore: an X-ray structure of the pore (g) is combined with the nucleic acid in the desired conformation (h) and embedded in a lipid bilayer membrane (i). The resulting system is solvated (j) and ionized (k). The procedures are described in detail in Refs. [112] and [65, 113] and online tutorials [114].

Figure 4

MD simulation of ionic current. (a) The microscopic model of a nanopore system is subject to an external electric field. Under the action of the field, water and ions rearrange, focusing the electric field to the vicinity of the membrane. (b) The resulting distribution of the electrostatic potential. A transmembrane bias of 0.6 V was imposed in this simulation. (c) Instantaneous currents sampled at 1 (black) and 10 (red) ps. The noise decreases as a square root of the sampling rate. (d) Integrated currents at different voltage biases. A linear cumulative current trace indicates a steady state conductance. (e) The simulated current–voltage curve of α-hemolysin [65].

Figure 5

MD simulation of DNA translocation. (a–c) Electric field-driven transport of ssDNA through α-hemolysin. This proof-of-principle MD trajectory was obtained applying a very high transmembrane bias of 15 V; protein and lipids were restrained. A more realistic trajectory can be obtained using the G-SMD method [163]. (d–h) Transport of dsDNA transport Si₃N₄ pore. The DNA was observed to interact strongly with the surface of the solid-state membrane [112]. (i–m) DNA hairpin permeation through Si₃N₄ pore. The duplex part of the hairpin stretches under the influence of the electric field (panels j–l), which enables the translocation [126].

Figure 6

The effect of dsDNA conformation on the ionic current blockades. (a–c) Snapshots from a MD simulation of dsDNA translocation through a 3.0-nm-diameter pore in Si₃N₄. In this simulation, the Si₃N₄ membrane is 10-nm thick; the transmembrane bias is 1.3 V, the KCl concentration is 0.1 M. (d) The simulated ionic current. The ionic current returns to an open pore value while dsDNA is still in the pore.

Figure 7

Influence of the local ion concentration on the nanopore current. The ion concentration profiles are shown for (a) an empty (no DNA) pore in, (b) an ionic current blockade, and (c) an ionic current enhancement. The pore images are faithfully aligned with the position axes of the corresponding plots. The arrows indicate the direction of the applied electric field. The bulk ion concentration in all simulations was 1 M. Other simulation conditions are described in Ref. [126].

Figure 8

MD simulations of the effective force. (a) Setup of the simulations. A virtual spring limits displacement of the DNA fragment in an external electric field E. The effective force F is the product of the spring constant k and the DNA displacement Δx. (b) The simulated effective force F versus the product of the electrophoretic mobility µ, the friction coefficient ξ and the applied field E; μ and ξ were determined from independent MD simulations. The symbols indicate the results obtained for three different pores and two values of the applied field [179].