Dynamics of DNA molecules in a membrane channel probed by active control techniques

Abstract

The dynamics of single-stranded DNA in an alpha-Hemolysin protein pore was studied at the single-molecule level. The escape time for DNA molecules initially drawn into the pore was measured in the absence of an externally applied electric field. These measurements revealed two well-separated timescales, one of which is surprisingly long (on the order of milliseconds). We characterized the long timescale as being associated with the binding and unbinding of DNA from the pore. We have also found that a transmembrane potential as small as 20 mV strongly biased the escape of DNA from the pore. These experiments have been made possible due to the development of a feedback control system, allowing the rapid modulation of the applied force on individual DNA molecules while inside the pore.

FIGURE 1

A schematic diagram of the apparatus illustrating the transmembrane voltage control loop. The ion current passing through the α-HL channel is measured at the headstage and continuously monitored at the PC. When recording DNA translocation events, current blockades are continuously identified in software and saved to disk. In the feedback-controlled mode, the start of the current blockade activates a hardware trigger causing the DAQ card to generate transmembrane voltage control signals. In this manner the external electric field is rapidly switched while the DNA is in the channel.

FIGURE 2

Voltage-driven translocations of poly(dA)₆₀ through an α-HL pore, at 15°C and 120 mV. The inset depicts a current trace with three blockade events corresponding to the translocation of three DNA molecules. The event duration, t_D, is measured for each event and the translocation duration histogram is constructed from ∼5000 events (main figure). The most probable translocation duration (the peak of the distribution) is denoted as t_p. The tail of the distribution extends to very long times (not shown). The dashed line (right axis) is the cumulative probability of translocation as a function of t_D.

FIGURE 3

Control of the transmembrane voltage after entry of a DNA molecule into the α-HL channel. The top panel shows two current traces corresponding to events in which the molecule left the pore at different times (low pass filtered at 70 kHz for display purposes). At time t = 0 the molecule enters the pore, triggering the generation of a series of steps in the applied transmembrane voltage as shown in the bottom panel. Initially the transmembrane voltage remains at V_drive (120 mV) for a time t_drive, allowing the DNA to be driven further into the pore. Next, the voltage is switched off (set to 0 mV) for a time t_off, during which the molecule's dynamics is not biased by the external electric field. Finally, the occupancy of the channel is probed by applying a relatively low voltage level V_probe. The black trace corresponds to a molecule that escaped from the pore during the t_off period. The current trace plotted in gray corresponds to a molecule which stayed in the pore for a time denoted t_stay (see arrow). Events in which the molecule escaped the pore during t_drive were excluded from the analysis.

FIGURE 4

A histogram of the pore current levels measured when the probe voltage is applied. The histogram was generated from ∼1000 events similar to those shown in Fig. 3. The pore current was measured 65 μs after the probe voltage was applied (averaged over 20 μs). The two peaks correspond to the two possible states of the pore: “occupied” (by a DNA molecule) or “clear.” The number of events in the upper peak divided by the total number of events is the probability that a DNA molecule will escape the pore during t_off, for a given choice of t_drive and t_off.

FIGURE 5

The DNA escape probability P_escape as a function of t_off, with t_drive fixed at 200 μs. Each data point was evaluated from ∼1000 event recordings as illustrated in Figs. 3 and 4. Error bars were calculated by determining the fraction of events in the overlap region between the two peaks in Fig. 4. The solid line is a three-parameter fit to a sum of two exponents. The fast and slow timescales obtained from the fit are 165 ± 10 μs and 3500 ± 250 μs respectively, with a relative weight factor of 0.49 ± 0.01. The multiple data points at the same t_off values represent measurements repeated using the same parameters.

FIGURE 6

Distributions of the time for which DNA molecules remained in the pore (t_stay) at different transmembrane voltage levels. For these experiments t_drive = 200 μs and t_off = 0 μs, such that the transmembrane voltage was switched directly from V_drive to V_probe 200 μs after the DNA entered the pore. Distributions of t_stay were measured at V_probe levels of 20 mV, 40 mV, and 60 mV. Each of the distributions was approximated by a single exponential decay curve yielding time constants of 1010 μs, 530 μs, and 250 μs respectively. Note that the first bin in each histogram was ignored when calculating the exponential fits (see text).

FIGURE 7

The DNA escape probability P_escape as a function of t_drive, with t_off fixed at 365 μs. Error bars were calculated by determining the fraction of events in the overlap region between the two peaks in Fig. 4. At short t_drive values the probability of escape decreased as a function of t_drive, and for longer t_drive values P_escape continued to decrease, but at a much lower rate. The observation that P_escape continued to decrease over the entire range of t_drive values was unexpected (see text). The dotted curve (right axis) is the fraction of molecules that were fully translocated during t_drive and therefore not included in our analysis (this is the same curve as plotted in Fig. 2).