DNA Dynamics in Dual Nanopore Tug-of-War

Abstract

Solid state nanopores have emerged as powerful tools for single-molecule sensing, yet the rapid uncontrolled translocation of the molecule through the pore remains a key limitation. We have previously demonstrated that an active dual-nanopore system, consisting of two closely spaced pores operated via feedback controlled biasing, shows promise in achieving controlled, slowed-down translocation. Translocation control is achieved via capturing the DNA in a special tug-of-war configuration, whereby opposing electrophoretic forces at each pore are applied to a DNA molecule co-captured at the two pores. Here, we systematically explore translocation physics during DNA tug-of-war focusing on genomically relevant longer dsDNA using a T₄-DNA model (166 kbp). We find that longer molecules can be trapped in tug-of-war states with an asymmetric partitioning of contour between the pores. Secondly, we explore the physics of DNA disengagement from a tug-of-war configuration, focusing on the dynamics of DNA free-end escape, in particular how the free-end velocity depends on pore voltage, DNA size and the presence of additional DNA strands between the pores (i.e. arising in the presence of folded translocation). These findings validate theoretical predictions derived from a first passage model and provide new insight into the physical mechanisms governing molecule disengagement in tug-of-war.

Figure 1:

a) Schematic of the dual-nanopore device. Voltages ${\mathrm{V}}_1$ and ${\mathrm{V}}_2$ are controlled by the FPGA with the common chamber grounded. b) Zoomed in view and c) cross-section of the region of membrane containing the two nanopores. A cartoon DNA (red) is shown in a tug-of-war (TOW) configuration. The voltages ${\mathrm{V}}_1$ and ${\mathrm{V}}_2$ are set to positive polarity to engage the competing force.

Figure 2:

a) The DNA chain is co-captured by the two closely placed nanopores, leading to a TOW event. At the end of the TOW event, b) the DNA free end escapes P1 and then c) escapes P2. d) An example of the DNA TOW events. The grey dashed lines indicate the start and the end of the TOW event; ${\mathrm{T}}_{\mathrm{d}}$ indicates the dwell time of the TOW event. Note that immediately following application of the opposing voltages (i.e. at $t=0$), the capacitive transient leads to an ionic current spike; this stabilizes within 500 μs. The black dashed box indicates the end of the TOW event where the free end escapes from P1 and P2; e) shows a zoom-in view of this portion of the event. The quantity ${\mathrm{T}}_{\mathrm{f}}$ indicates the time-of-flight for the DNA free end between the pores.

Figure 3:

a). Schematic showing asymmetric initial configuration of the T₄-DNA; biasing conditions shown correspond to $V_1<V_2$ (i.e. $\delta V<0$) so the drift velocity points from P1 to P2. The zoomed-in schematic indicates the polymer free ends that exit at P1 and P2 (we call these free-ends P1-exit and P2-exit). b). Histogrammed TOW dwell times for T₄-DNA. The red dashed line indicates fits to the convective diffusion model. The number of events are 84, 48, 260, 104, 98 (for $\delta V$ ranging from −100 mV to +100 mV). c). The cumulative histogram of TOW dwell-times corresponding to the histograms shown in b). The red dashed line indicates the fits to the convective diffusion model. d) The mean TOW dwell time versus pore voltage differential. The round markers correspond to the experiment and the square markers correspond to the model. The error bars for the experimental average dwell time give the standard error of the mean. The error bars for the model values correspond to the fitting covariance; this is smaller than the marker size. e). The drift velocity extracted from the convective diffusion model (blue points, T₄-DNA; red points $\mathrm{\lambda }-\mathrm{D}\mathrm{N}\mathrm{A}$) with accompanying linear fits (dashed lines).

Figure 4:

a) P1 exit probability for all T₄-DNA TOW events as a function of voltage differential. The dashed line is calculated from the first passage model. b) P1 exit probability for the T₄-DNA TOW events showing the exit probability separately for the diffusion peak (dwell time< 30 ms, black triangles) and drift peak (dwell time≥ 30 ms, red dots). The error bars give the standard error on the mean.

Figure 5:

a) Velocity of T₄-DNA free end as it travels between the pores as a function of V₂. The number of events are 185, 279, 294 and 106 for a P2 voltage of 200 mV, 300 mV, 400 mV and 500 mV. b) Velocity of $\mathrm{\lambda }-\mathrm{D}\mathrm{N}\mathrm{A}$ free end as it travels between the pores as a function of P2 voltage. The number of events are 51 and 114 for a P2 voltage of 200 mV and 300 mV c) Mean velocity of free ends of λ- and T₄-DNA during inter-pore travel as a function of voltage applied to P2. The error bars give standard error on the mean. d) Standard deviation of DNA free end velocity normalized to mean velocity plotted versus P2 voltage. The inset gives the velocity standard deviation value. The error bars are propagated using the standard error on the mean velocity.

Figure 6:

a) Schematic of a folded DNA exiting two pores sequentially, with removal of a fold leading to a linearized conformation that then disengages. b) Current trace for P1 and P2 from a folded T₄-DNA chain. c). The double filament DNA free end time delay ${\mathrm{t}}_d$ and d) single filament DNA free end time delay $t_s$. e). The average velocity of the T₄-DNA free end passing from ${\mathrm{P}}_1$ to ${\mathrm{P}}_2$, with or without the presence of the second DNA filament. The number of events are 25, 96, 59, 27 for exiting pore voltages ranging from 200 mV to 500 mV. f) The velocity of the DNA free end with ($v_{\mathrm{d}}$) and without ($v_{\mathrm{s}}$) a second DNA strand. The dashed line shows linear fits used to extract estimated friction factors. The inset gives the friction coefficient difference ${\xi }_d-{\xi }_s$. The error bars give standard error of the mean.