Characterizing and controlling the motion of ssDNA in a solid-state nanopore.
ABSTRACT: Sequencing DNA in a synthetic solid-state nanopore is potentially a low-cost and high-throughput method. Essential to the nanopore-based DNA sequencing method is the ability to control the motion of a single-stranded DNA (ssDNA) molecule at single-base resolution. Experimental studies showed that the average translocation speed of DNA driven by a biasing electric field can be affected by ionic concentration, solvent viscosity, or temperature. Even though it is possible to slow down the average translocation speed, instantaneous motion of DNA is too diffusive to allow each DNA base to stay in front of a sensor site for its measurement. Using extensive all-atom molecular dynamics simulations, we study the diffusion constant, friction coefficient, electrophoretic mobility, and effective charge of ssDNA in a solid-state nanopore. Simulation results show that the spatial fluctuation of ssDNA in 1 ns is comparable to the spacing between neighboring nucleotides in ssDNA, which makes the sensing of a DNA base very difficult. We demonstrate that the recently proposed DNA transistor could potentially solve this problem by electrically trapping ssDNA inside the DNA transistor and ratcheting ssDNA base-by-base in a biasing electric field. When increasing the biasing electric field, we observed that the translocation of ssDNA changes from ratcheting to steady-sliding. The simulated translocation of ssDNA in the DNA transistor was theoretically characterized using Fokker-Planck analysis.
Project description:We investigate by means of molecular dynamics simulations stretch-induced stepwise translocation of single-stranded DNA (ssDNA) through graphene nanopores. The intrinsic stepwise DNA motion, found to be largely independent of size and shape of the graphene nanopore, is brought about through alternating conformational changes between spontaneous adhesion of DNA bases to the rim of the graphene nanopore and unbinding due to mechanical force or electric field. The adhesion reduces the DNA bases' vertical conformational fluctuations, facilitating base detection and recognition. A graphene membrane shaped as a quantum point contact permits, by means of transverse electronic conductance measurement, detection of the stepwise translocation of the DNA as predicted through quantum mechanical Green's function-based transport calculations. The measurement scheme described opens a route to enhance the signal-to-noise ratio not only by slowing down DNA translocation to provide sufficient time for base recognition but also by stabilizing single DNA bases and, thereby, reducing thermal noise.
Project description:Nanopores could potentially be used to perform single-molecule DNA sequencing at low cost and with high throughput. Although single base resolution and differentiation have been demonstrated with nanopores using ionic current measurements, direct sequencing has not been achieved because of the difficulties in recording very small (?pA) ionic currents at a bandwidth consistent with fast translocation speeds. Here, we show that solid-state nanopores can be combined with silicon nanowire field-effect transistors to create sensors in which detection is localized and self-aligned at the nanopore. Well-defined field-effect transistor signals associated with DNA translocation are recorded when an ionic strength gradient is imposed across the nanopores. Measurements and modelling show that field-effect transistor signals are generated by highly localized changes in the electrical potential during DNA translocation, and that nanowire-nanopore sensors could enable large-scale integration with a high intrinsic bandwidth.
Project description:Nanopores are now being used not only as an ionic current sensor but also as a means to localize molecules near alternative sensors with higher sensitivity and/or selectivity. One example is a solid-state nanopore embedded in a graphene nanoribbon (GNR) transistor. Such a device possesses the high conductivity needed for higher bandwidth measurements and, because of its single-atomic-layer thickness, can improve the spatial resolution of the measurement. Here measurements of ionic current through the nanopore are shown during double-stranded DNA (dsDNA) translocation, along with the simultaneous response of the neighboring GNR due to changes in the surrounding electric potential. Cross-talk originating from capacitive coupling between the two measurement channels is observed, resulting in a transient response in the GNR during DNA translocation; however, a modulation in device conductivity is not observed via an electric-field-effect response during DNA translocation. A field-effect response would scale with GNR source-drain voltage (Vds), whereas the capacitive coupling does not scale with Vds . In order to take advantage of the high bandwidth potential of such sensors, the field-effect response must be enhanced. Potential field calculations are presented to outline a phase diagram for detection within the device parameter space, charting a roadmap for future optimization of such devices.
Project description:Nanopores are a promising platform in next generation DNA sequencing. In this platform, an individual DNA strand is threaded into nanopore using an electric field, and enzyme-based ratcheting is used to move the strand through the detector. During this process the residual ion current through the pore is measured, which exhibits unique levels for different base combinations inside the pore. While this approach has shown great promise, accuracy is not optimal because the four bases are chemically comparable to one another, leading to small differences in current obstruction. Nucleobase-specific chemical tagging can be a viable approach to enhancing the contrast between different bases in the sequence. Herein we show that covalent modification of one or both of the pyrimidine bases by an osmium bipyridine complex leads to measureable differences in the blockade amplitudes of DNA molecules. We qualitatively determine the degree of osmylation of a DNA strand by passing it through a solid-state nanopore, and are thus able to gauge T and C base content. In addition, we show that osmium bipyridine reacts with dsDNA, leading to substantially different current blockade levels than exhibited for bare dsDNA. This work serves as a proof of principle for nanopore sequencing and mapping via base-specific DNA osmylation.
Project description:DNA sequencing with a solid-state nanopore requires a reduction of the translocation speeds of single-stranded DNA (ssDNA) over 10??s/base. In this study, we report that a nanometre-sized bead structure constructed around a nanopore can reduce the moving speed of ssDNA to 270??s/base by adjusting the diameter of the bead and its surface chemical group. This decelerating effect originates from the strong interaction between ssDNA and the chemical group on the surface of the bead. This nanostructure was simply prepared by dip coating in which a substrate with a nanopore was immersed in a silica bead solution and then dried in an oven. As compared with conventional approaches, our novel method is less laborious, simpler to perform and more effective in reducing ssDNA translocation speed.
Project description:Nanopore analysis is an emerging single-molecule strategy for non-optical and high-throughput DNA sequencing, the principle of which is based on identification of each constituent nucleobase by measuring trans-membrane ionic current blockade or transverse tunnelling current as it moves through the pore. A crucial issue for nanopore sequencing is the fact that DNA translocates a nanopore too fast for addressing sequence with a single base resolution. Here we report that a transverse electric field can be used to slow down the translocation. We find 400-fold decrease in the DNA translocation speed by adding a transverse field of 10?mV/nm in a gold-electrode-embedded silicon dioxide channel. The retarded flow allowed us to map the local folding pattern in individual DNA from trans-pore ionic current profiles. This field dragging approach may provide a new way to control the polynucleotide translocation kinetics.
Project description:We investigate the diffusion and the drift motion of ? DNA molecules near solid-state nanopores prior to their translocation through the nanopores using fluorescence microscopy. The radial dependence of the electric field near a nanopore generated by an applied voltage in ionic solution can be estimated quantitatively in 3D by analyzing the motion of negatively charged DNA molecules. We find that the electric field is approximately spherically symmetric around the nanopore under the conditions investigated. In addition, DNA clogging at the nanopore was directly observed. Surprisingly, the probability of the clogging event increases with increasing external bias voltage. We also find that DNA molecules clogging the nanopore reduce the electric field amplitude at the nanopore membrane surface. To better understand these experimental results, analytical method with Ohm's law and computer simulation with Poisson and Nernst-Planck (PNP) equations are used to calculate the electric field near the nanopore. These results are of great interest in both experimental and theoretical considerations of the motion of DNA molecules near voltage-biased nanopores. These findings will also contribute to the development of solid-state nanopore-based DNA sensing devices.
Project description:Electric field-driven translocation of DNA strands through biological nanopores has been shown to produce blockades of the nanopore ionic current that depend on the nucleotide composition of the strands. Coupling a biological nanopore MspA to a DNA processing enzyme has made DNA sequencing via measurement of ionic current blockades possible. Nevertheless, the physical mechanism enabling the DNA sequence readout has remained undetermined. Here, we report the results of all-atom molecular dynamics simulations that elucidated the physical mechanism of ionic current blockades in the biological nanopore MspA. We find that the amount of water displaced from the nanopore by the DNA strand determines the nanopore ionic current, whereas the steric and base-stacking properties of the DNA nucleotides determine the amount of water displaced. Unexpectedly, we find the effective force on DNA in MspA to undergo large fluctuations, which may produce insertion errors in the DNA sequence readout.
Project description:Protein nanopores may provide a cheap and fast technology to sequence individual DNA molecules. However, the electrophoretic translocation of ssDNA molecules through protein nanopores has been too rapid for base identification. Here, we show that the translocation of DNA molecules through the ?-hemolysin protein nanopore can be slowed controllably by introducing positive charges into the lumen of the pore by site directed mutagenesis. Although the residual ionic current during DNA translocation is insufficient for direct base identification, we propose that the engineered pores might be used to slow down DNA in hybrid systems, for example, in combination with solid-state nanopores.
Project description:Nanopore-based single-molecule detection and analysis have been pursued intensively over the past decade. One of the most promising applications in this regard is DNA sequencing achieved through DNA translocation-induced blockades in ionic current. Recently, nanopores fabricated in graphene sheets were used to detect double-stranded DNA. Due to its subnanometer thickness, graphene nanopores show great potential to realize DNA sequencing at single-base resolution. Resolving at the atomic level electric field-driven DNA translocation through graphene nanopores is crucial to guide the design of graphene-based sequencing devices. Molecular dynamics simulations, in principle, can achieve such resolution and are employed here to investigate the effects of applied voltage, DNA conformation, and sequence as well as pore charge on the translocation characteristics of DNA. We demonstrate that such simulations yield current characteristics consistent with recent measurements and suggest that under suitable bias conditions A-T and G-C base pairs can be discriminated using graphene nanopores.