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The Rowland Institute at Harvard University, Cambridge, Massachusetts 02142
Correspondence: Address reprint requests to Dr. Amit Meller, E-mail: meller{at}rowland.harvard.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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-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. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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). Also, many forms of viral infection rely on the invasion of the plasma membrane followed by import of the viral genome into the cell nucleus through nuclear pore complexes (Kasamatsu and Nakanishi, 1998; Whittaker and Helenius, 1998; Salman et al., 2001). DNA is also known to be translocated through protein channels spanning the bacterial membrane during phage infection (Driselkelmann, 1994). Despite the wide interest in these problems, our understanding of the transport dynamics of biopolymers in pores is limited, and the study of biopolymer dynamics remains an important and fascinating challenge.
The -Hemolysin (-HL) protein complex has recently been used as an in vitro model system for the study of DNA and RNA transport through narrow pores (Kasianowicz et al., 1996; Akeson et al., 1999; Meller et al., 2000, 2001). The -HL protein, secreted by Staphylococcus aureus as a water-soluble monomer, assembles on lipid membranes to form a transmembrane, heptameric channel (Song et al., 1996; Gouax, 1998). The channel conducts ions and can remain open and stable for hours. The channel complex consists of two main parts: a "cap" structure with an internal diameter of 
30 Å that sits outside the membrane, and a narrow ß-barrel region 50 Å in length with an internal diameter that varies between 15 Å and 18 Å. This size is comparable with the mean diameter of single-stranded DNA or RNA molecules (Bezrukov, 2000). In a process termed DNA translocation, single-stranded DNA (a negatively charged polymer) can be driven through the channel upon the application of an electric field. This process has been studied by measuring the blockades in the ion current that occur when individual DNA molecules pass through the pore. Recent experiments have suggested that DNA and RNA molecules interact with the -HL channel and that the dynamics of DNA translocation are sensitive to the magnitude of the applied electric field (Meller et al., 2001; Meller and Branton, 2002). The DNA translocation problem has also been studied theoretically by modeling translocation as a diffusion process across a free energy barrier (Sung and Park, 1996; Muthukumar, 1999; Chuang et al., 2002), or by introducing a simple model potential to describe the DNA-protein interactions in the presence of an electric field (Lubensky and Nelson, 1999).
In this report we present a new experimental approach that allows us to study the dynamics of DNA in the -HL channel. In particular, we find that this approach is sensitive to interactions that occur between the DNA and the channel proteins. To isolate the biasing effects of the electric field we employed two complementary methods. First, we measured the behavior of DNA in the presence of an electric field by characterizing each translocation event in terms of its duration. Next, we measured the behavior of the DNA at different electric field strengths by altering the transmembrane voltage during the passage of each molecule. For example, in some experiments a DNA molecule was driven into the channel and then the applied voltage was set to zero, allowing the unbiased dynamics of the DNA-channel system to govern the motion of the molecule for a fixed time. Our results suggest that polydeoxyadenine molecules (poly-dA) fall into one of two classes: those exhibiting fast escape dynamics and those that remain in the pore for extended periods of time. We measured the timescales associated with the two classes and we propose arguments as to their origin. Finally, the influence of the electric field was characterized by directly measuring the escape times of molecules in the channel at different electric field strengths.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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-HL channel in a horizontally supported planar lipid bilayer has been described previously (Meller et al., 2001). The temperature of the system was maintained at 15.0 ± 0.1°C unless otherwise mentioned, using a custom enclosure design and a thermoelectric controller (Newport 3040, Irvine, CA). The buffer solution was 1 M KCl, 10 mM Tris-HCl with a pH of 8.5. The -HL open pore conductance under these conditions was 8.2 x 10-10 siemens in the forward bias. The diameter of the lipid membrane supporting the channel was 20 µm, with a typical capacitance of 5 ± 1 pF. PAGE-purified polydeoxyadenine 60-mers (Synthetic Genetics, San Diego, CA) were buffered in 10 mM Tris, 1 mM EDTA, pH 8.5 solution, and used without further modification. The ion current was measured using a patch-clamp amplifier (Axopatch 200B, Axon Instruments, Union City, CA) and the signal was filtered using a 100 kHz low-pass four-pole Butterworth filter (Krohn Hite 3302, Avon, MA). We maintained the 100 kHz bandwidth in our measurements to minimize transient delays associated with low-pass filtering. The signal was digitized at 1 MHz/12 bits using a DAQ card (National Instruments PCI-MIO-16E-1, Austin, TX) installed in a Pentium III-based personal computer. All control and acquisition software was written using National Instruments' LabView. Our apparatus incorporates a feedback loop used to control the applied transmembrane voltage. The control loop was used for all experiments with the exception of the translocation duration measurements. In the feedback-controlled configuration the analog trigger circuitry of the DAQ card, programmed to activate when the pore current dropped below a specified level, was used to detect DNA molecules entering the pore. When such an event was detected, a series of control voltages were generated by the DAQ card and routed to the patch-clamp amplifier which maintained the transmembrane voltage at a level proportional to the control voltage. The response time of the DAQ card to a change in the pore current was less than 1 µs. We measured the response time of the membrane potential to a step in the control voltage to be 4 ± 1 µs. Fig. 1 is a schematic representation of our setup, showing the control loop.
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). Recordings of translocation blockades were analyzed and each blockade was characterized in terms of its duration tD, as illustrated in Fig. 2 (inset). The blockade duration was defined as the time during which the ion current remained at the fully blocked state (10% of the open pore current level).
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) and at the same time large enough that the resulting ion current was detectable above the noise level. When the probe voltage was applied the ion current exhibited a transient as the amplifier output was briefly overloaded before settling to a level that depended on whether or not the channel was blocked by DNA. This is illustrated by the two current traces that are plotted in the top panel of Fig. 3. In the trace plotted in black the current rose to a level corresponding to the open state of the channel (32 pA), indicating that the DNA escaped the channel during toff. The second trace (plotted in gray) is an example of a molecule that remained in the channel during toff. In this case the ion current first settled at a low level corresponding to the blocked state of the channel (7 pA) and 700 µs later the ion current rose abruptly to the open channel level, signaling the escape of the molecule. After a fixed time the computer reset the transmembrane voltage to 120 mV, ejecting any molecules that remained in the channel. A typical experiment consisted of 1000 event recordings similar to those shown in Fig. 3.
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Data analysis
The event recordings were analyzed to determine the fraction of DNA molecules that escaped from the channel during toff, for a given choice of tdrive and toff. Recordings in which the current returned to the open pore level during tdrive were excluded from the analysis since they represent molecules that exited the pore before the transmembrane voltage was set to zero. The averaged current level in a window 20 µs wide, measured 65 µs after the application of the probe voltage, was calculated for each recording. A histogram of these values for a typical experiment is plotted in Fig. 4. The two peaks of the histogram illustrate that the two possible states of the channel are easily distinguishable. This allows the determination of the fraction of events in which the channel is clear (no DNA present). This value, denoted Pescape (tdrive, toff), is the probability that a molecule of DNA driven into the channel for a duration tdrive has escaped the channel after a bias-free time duration toff. Note that for the purpose of analysis the 65 µs delay between the application of the probe and the actual measurement was treated as part of toff.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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). The dashed line (right axis) in Fig. 2 represents the cumulative probability of translocation for a given tD. This curve was calculated by integrating over the translocation duration histogram and normalizing to unity at infinite times.
We note that many of the translocation events observed exhibit a step-like feature at the leading edge of the event (for example, the leftmost event shown in Fig. 2, inset) during which the current level drops to approximately half of the open-pore level. The duration of the step was not included in the calculation of the translocation duration tD. We associate these steps with the time the DNA spends in the large "cap" structure of the -HL before entering the narrow part of the channel. We have found that the duration of the steps is not correlated with the translocation duration (Bates et al., unpublished results).
DNA dynamics at zero electric field
The feedback-controlled setup allowed us to investigate the dynamics of DNA in the channel in the absence of an electric field. We measured the number of poly(dA)60 molecules that escaped the pore as a function of time under zero bias conditions. For these measurements we chose tdrive = 200 µs, which is approximately half of the most probable translocation duration (tdrive 
1/2 tp). With this choice of tdrive we expected that the molecules would be threaded through the pore to approximately half their length, such that their initial condition was entropically unfavorable. The probability of escape, Pescape (tdrive, toff), was measured as explained in the Materials and Methods section for toff values ranging from 65 µs to 10065 µs. The results are plotted in Fig. 5.
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DNA escape time distributions
The sensitivity of the DNA dynamics to changes in the transmembrane electric field was studied by recording the time that DNA molecules remained in the channel in the presence of a relatively small field. Specifically, we measured the distributions of tstay (as defined above, see Fig. 3) at different electric field strengths. The initial condition of the molecules was as in the previous experiment (tdrive = 200 µs). Three distributions of tstay, measured at probe voltage levels of 20 mV, 40 mV, and 60 mV, are shown in Fig. 6.
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DNA dynamics at varying initial conditions
In a final set of experiments we studied the effect of changing the initial condition of the molecules with respect to the channel. For these experiments toff was held constant at 365 µs and tdrive was varied between 0 µs and 600 µs. For very short tdrive values (in particular 
) we expected that the probability of escape would approach one because only a small fraction of the molecule would be initially threaded into the pore. By similar reasoning, we expected that Pescape would reach a minimum when the molecules were on average threaded halfway through the channel (tdrive 1/2 tp) and that Pescape would increase for tdrive > 1/2 tp as the molecules' average initial position approached the other side of the membrane.
The escape probability measurements for varying tdrive are shown in Fig. 7. The fraction of molecules that were rejected from the analysis because they exited the pore before toff is plotted in Fig. 7 as a dotted line (right axis). This curve is identical to the cumulative translocation probability shown in Fig. 2. For short tdrive values our data followed the simple prediction discussed above. However, for tdrive > 200 µs our data deviates from the predicted trend. Instead of increasing, Pescape levels off and continues to decrease with a mild slope. In the discussion below we interpret this result in light of the evidence for fast and slow escape timescales presented above (see Fig. 5).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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). Even if these interactions are small in magnitude (on the scale of kBT) they may significantly alter the duration of the escape trajectory if the DNA interacts with the pore at multiple sites along its length.
It was previously found that the most probable translocation duration scales linearly with the DNA contour length (for polynucleotides longer than 12 bases; see Meller et al., 2001), and therefore it is plausible to assume that tdrive determines the extent to which the molecules are on average threaded into the channel. In particular, setting tdrive 1/2 tp should result in an approximately symmetric partitioning of the polymers between the two sides of the membrane. It is therefore unlikely that asymmetric initial partitioning of the polymers was responsible for the appearance of the two timescales in our experiments. On the other hand, DNA-channel interactions have been previously proposed to affect the translocation dynamics (Meller et al., 2000; Henrickson et al., 2000; Lubensky and Nelson, 1999). Our results reinforce these observations.
In the experiments described above we also studied the influence of the electric field on the escape time of molecules in the channel. It was previously realized that the dependence of the translocation time on the electric field is not linear. In particular, a threshold voltage of 47 mV was predicted below which DNA translocation would not occur (Meller et al., 2001). Our technique enabled us to study the influence of the electric field on DNA at levels below this threshold. We found that the escape time distributions of DNA from the channel were sensitive to small changes in the transmembrane electric field (Fig. 6). For example, changing the probing voltage from 20 mV to 40 mV shortened the observed escape timescale by a factor of two.
Do DNA-protein interactions also account for the trend we observe in Pescape when we vary tdrive (Fig. 7)? In the following paragraphs we interpret the dependence of Pescape on tdrive in light of the fast (165 µs) and slow (3500 µs) escape timescales obtained in Fig. 5. Based on the fast timescale, 90% of the fast molecules would have escaped from the pore during the 365-µs bias-free period used in the measurements presented in Fig. 7, regardless of the value of tdrive (we approximate the fraction of molecules that escape by 
). On the other hand, the characteristic 3500 µs escape time of the slow molecules implies that 90% of the slow molecules remain in the channel when we probe the pore. Therefore, in this experiment the lifetime of the slow molecules at zero bias is long enough that they remain in the pore after the fast molecules have escaped. In this sense, Pescape in Fig. 7 can be thought of as the fraction of fast molecules in a population that initially includes fast and slow molecules.
Following this line of reasoning, we interpret Fig. 7 in terms of a binding interaction between the DNA and the channel. For short tdrive values, only a small segment of each molecule will be threaded into the channel, restricting possible DNA-protein interactions associated with the slow molecules. As we increase tdrive longer segments of the DNA are threaded into the pore, gradually increasing the probability of binding. Therefore the relative number of slow molecules increases with tdrive and the escape probability decreases correspondingly, as suggested by our data. For tdrive > 200 µs we observe that the dependence of Pescape on tdrive levels off (with a small negative slope). This trend would be expected if the DNA-channel binding and unbinding rates at 120 mV are fast as compared to the timescale of translocation. This is supported by our data: the trend observed in Fig. 6 suggests that the DNA unbinding time at 120 mV is much smaller than 250 µs, whereas the most probable translocation time is 330 µs (Fig. 2). Thus, the ratio of bound to unbound molecules should reach a steady state for some value of tdrive < tp. Our data suggests that this state is reached 200 µs after the DNA enters the channel.
Based on our results, we propose a three-state picture that relates the observed timescales to the binding kinetics of the DNA. In our model (shown below) all possible configurations of the DNA in the channel are pooled into one of two states: "bound" configurations in which the DNA is bound to the channel (denoted SB), and "free" configurations in which the DNA is not bound (denoted SF). The clear pore state is represented by O:
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diff. The time for DNA to bind and unbind from the pore is denoted reaction and defined as 1/reaction = kB + kF. The clear separation between the slow and the fast timescales observed in Fig. 5 supports the idea that diff is much shorter than reaction at zero bias, and thus diff can be approximated by the fast timescale (165 µs). In this picture, the fast timescale represents molecules that were initially at SF and then diffused out rapidly, without allowing for significant exchange with the bound state. At longer times the situation is different; the rate-limiting step for escaping from the pore is the reaction kinetics. With this assumption, we can approximate reaction at zero bias by the slow component of the escape probability (3500 µs). This value may be specific to the adenine homopolymers used in this experiment. Note that the transition rates can be viewed as the probabilities of DNA binding or unbinding to the pore per unit time (Colquhoun and Hawkes, 1995), and thus have units of s-1. This definition is derived from the fact that we measure individual DNA-channel complexes, as opposed to measuring binding in bulk. |
CONCLUSIONS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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-HL model system allows us to study the dynamics of polynucleotides threaded through membrane channels. In this paper we have extended the capabilities of the system into a new regime: we have demonstrated that the transport of single DNA molecules can be controlled by varying the transmembrane potential during the passage of the molecule. This approach was used to study the escape of DNA molecules from the channel at vanishing electric field strengths and the biasing role of low electric field intensities on the DNA dynamics.
Interactions have been proposed before as an important component of the DNA translocation process (Meller et al., 2000; Henrickson et al., 2000). The data presented in Figs. 5–7 reinforce these observations. We observed two distinct timescales for DNA escape from the pore: a slow timescale which we associate with molecules that bind to the pore, and a fast timescale which we associate with molecules that escape from the pore without binding. In this manner our experiments allow us to decouple the diffusion dynamics of the DNA molecules from their binding kinetics. In the translocation experiments we believe that these timescales overlap due to the constant application of the electric field, yielding a complicated translocation duration histogram (e.g., Fig. 2). Finally, the characterization of the escape time distribution may serve as a method to evaluate the DNA-protein interactions, which were found to be very sensitive to any applied potential.
The implementation of our technique is general and it could be used to extend the scope of DNA hairpin-melting experiments (Vercoutere et al., 2001) and DNA hybridization experiments that make use of modified -HL pores (Howorka et al., 2001). Future experiments will enable us to improve our understanding of DNA dynamics in the pore by experimenting with different DNA lengths and sequences, and to investigate alternative DNA-channel interaction mechanisms.
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ACKNOWLEDGEMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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We acknowledge support from members and staff at the Rowland Institute at Harvard.
Submitted on October 1, 2002; accepted for publication December 26, 2002.
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REFERENCES |
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