Generation of signature events during the translocation of DNA–FcCB[7] through αHL

Translocation of modified 5mC-containing ssDNA through αHL was conducted in a buffer of 3 M KCl and 10 mM Tris (pH 8.0) with the transmembrane potential held at +160 mV. When DNA1–FcCB[7] was placed in the cis compartment, we observed highly characteristic current events at a frequency of around 8.2 min^–1 (Fig. 3a). These events are composed of two consecutive parts, with Level 1 featuring long and deep blockades and Level 2–2′ featuring transient current oscillations (Fig. 3b). The scatter plots of residual current versus duration of Levels 1 and 2 are shown in Fig. 3c.

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Fig. 3

Translocation of the DNA1–FcCB[7] hybrid through αHL. (a) A representative current trace of the translocation of DNA1–FcCB[7] (final concentration 0.25 μM) through αHL. Data were acquired in a buffer of 3 M KCl and 10 mM Tris (pH 8.0) with the transmembrane potential held at +160 mV. Red arrows indicate the multi-level current events. (b) A typical multi-level signature event generated by DNA1–FcCB[7] translocation (marked in (a)). Diagrams above the trace show the molecular mechanism of hybrid dissociation and translocation. Level 0: the open pore current level. Level 1: trapping of DNA1–FcCB[7] in the pore, dissociation of the DNA1–FcCB[7] and translocation of the DNA1–Fc through the pore. Level 2 and 2′: oscillation of CB[7] in the cis vestibule of αHL. (c) Scatter plots showing current blockage versus event durations of Level 1 and Level 2 in the signature events. (d) The dwell-time histogram for Level 1. The solid line in the histogram is a single exponential fit to the data. For Level 1, the mean duration t _D1 = 1376.9 ms. (e) The dwell-time histogram for Level 2. The solid line in the histogram is a single exponential fit to the data. For Level 2, the mean duration t _D2 = 52.4 ms. The sequence of DNA1: 5′-AAAAAAAAAA(5mC)AAAAAAAAAA-3′.

Since none of the control experiments showed any long and deep current blockades (for control experimental traces, see Fig. S5^†), we attributed the Level 1 state to the translocation of DNA1–FcCB[7] and concomitant dissociation of the FcCB[7] complex at the constriction of αHL. There are two features that support this assignment. First, the frequency of the occurrence of signature events is highly dependent on the applied voltage (Fig. S6^†). As the applied potential was increased from 160 to 240 mV, the frequency of multi-level signature events rose exponentially. This clearly indicates that a higher transmembrane potential facilitates the unbinding of the FcCB[7] complex and also promotes DNA translocation from cis to trans. Second, the DNA1–FcCB[7] hybrid also produced single-level long blockades with no residual current that resembles Level 1 of the multi-level signature events (Fig. 3a and b), and the frequency ratio of multi-level to single-level events monotonically increased from 3.30 at 160 mV to 5.26 at 240 mV (Fig. S7^†). The single-level blockade was formed by the trapped hybrid in a vestibule that returned to the cis solution without unbinding and translocation.⁴³ It is conceivable that a higher voltage promotes unbinding of the FcCB[7] complexes and decreases the probability of DNA escaping from the cis side.

The current oscillation between Levels 2 and 2′ is reminiscent of trapping an analyte in the nanocavity of a protein pore (Fig. 3b).⁴⁴ Thus, we speculated that the Level 2–2′ alternation was due to the trapping and oscillation of CB[7] inside the vestibule of αHL. To confirm the association of the signature events with CB[7], we carried out a competition experiment by adding 1-aminoadamantane (final concentration 1.0 μM) to the solution of DNA1–FcCB[7] (final concentration 0.25 μM) during the translocation studies. 1-Aminoadamantane is known to bind with CB[7] as tightly as ferrocene derivatives, and it may replace the ferrocene moiety when present in higher concentrations. After 30 minutes of incubation, we found that all the signature events produced by DNA1–FcCB[7] completely disappeared, and only ssDNA translocation events were observed, whose duration and conductance matched well with that of DNA1–Fc (Fig. S8^†).

The translocation of CB[7] (outer diameter ~1.6 nm) to the trans side would be prevented by the narrow constriction (~1.4 nm) of αHL; however, the cis opening of the vestibule is wide enough (~2.6 nm) for CB[7] to escape to the cis solution. So there must be a mechanism that could trap CB[7] inside the vestibule for tens of milliseconds (the mean duration of Level 2: t _D2 = 52.4 ms, Fig. 3e). It is well known that cucurbiturils have a strong affinity with positively charged organic guests.⁴⁵ We presumed that the side chains of some basic amino acids inside the vestibule might have interactions with CB[7] which provide the driving force for the current oscillation. By examining the crystal structure of αHL in the vestibule region, we found that the two lysine (K) residues at positions 8 and 147 might be crucial for the temporal trapping of CB[7] in the vestibule (Fig. S9^†). To validate this assumption, we carried out translocation of DNA1–FcCB[7] through the αHL mutants K147N and K8L. The mutant pores are homoheptamers, so the mutations appear in all of the 7 subunits. In the K147N mutant, we found that the multi-level signature events completely vanished and the frequency of events dropped to only ~1 s^–1 even though the transmembrane potential was held at +200 mV (Fig. S10^†). It was reported by Bayley and coworkers that elimination of one positively charged residue near the constriction of αHL might significantly reduce the DNA capture.⁴⁶ Interestingly, we observed multi-level events in the K8L mutant (Fig. 4a). Although elimination of positively charged residues at the cis entrance of αHL could also affect the DNA capture, the frequency of the multi-level events was only reduced by ~25% in the K8L mutant compared with the wildtype αHL. However, the feature of the “signature” event has drastically changed. Level 1 remains about the same, but the duration of Level 2 was shortened to ~10 ms and Level 2′ completely disappeared (Fig. 4b). This result provided compelling evidence that the lysines at positions 8 and 147 play important roles in the trapping and oscillation of CB[7] in the vestibule. We also examined the translocation of the hybrid DNA1–adamantaneβ-cyclodextrin (AdβCD) through αHL and found that the events generated did not have Levels 2 and 2′ (Fig. S11^†). This is a further proof that the current oscillation between Levels 2 and 2′ is caused by “host–guest” interactions inside the vestibule of αHL.

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Fig. 4

Translocation of DNA1–FcCB[7] through the engineered αHL mutant K8L. (a) Representative current trace of the translocation of DNA1–FcCB[7] through mutant K8L. Red arrows indicate the multi-level current events. (b) Expanded view of a multi-level signature event. Traces were recorded in 3 M KCl buffered with 10 mM Tris (pH 8.0). DNA1–Fc (final concentration 0.25 μM) was incubated with CB[7] (final concentration 50 μM) at room temperature for 2 hours before measurement. The transmembrane potential was held at +200 mV.

In addition to the singly methylated ssDNA, we also investigated the detection of two 5mC sites in one DNA strand with the above strategy. We observed that the current signature generated by the doubly methylated DNA10 was similar to that of the singly methylated control DNA11 except for a slightly longer Level 1 state (Fig. S12^†). Apparently, the current pattern of the first methylation site was affected by the tandem translocation of the second site. This could be a minor disadvantage when densely methylated DNA samples are being examined. One possible solution to circumvent this problem is to anchor the “host” molecule inside the nanopore, and then thread DNA strands modified with multiple “guest” molecules through the pore. Another problem we encountered was that when longer methylated DNAs (>40 bases) were tested, the Level 1 state in the current signatures became prolonged and sometimes led to long-lived blockades (>10 s). By elevating the temperature to around 46 °C, we nicely solved this clogging problem and obtained a high frequency of current signature around 35.5 min^–1 (Fig. S13^†).

Systematic studies of the factors affecting characteristic signature events

We performed systematic studies of the influence of different experimental conditions on the generation of characteristic signature events. First, we investigated the effects of the transmembrane potential on each current level of the signature events. When the transmembrane potential was relatively low (≤140 mV), only a few characteristic events could be observed. Thus we set the voltage range to between 160 and 240 mV, and found that the duration of the Level 1 state decreased monotonically as the applied voltage was increased (Fig. 5a). This trend is often given as strong evidence to prove the translocation of an analyte through a nanopore.²³ It supports our assumption that Level 1 was caused by the translocation of the DNA1–FcCB[7] hybrid, and the unbinding of the FcCB[7] complex should be mainly responsible for the long duration of the level.

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Fig. 5

Investigation of the influence of the applied potentials and ionic strength on the translocation of DNA1–FcCB[7]. (a) Voltage dependence of the duration of Level 1. All the data points were obtained from a single exponential fit to the corresponding dwell time histograms. Recordings were conducted in a buffer of 3 M KCl and 10 mM Tris (pH 8.0) in the presence of 0.25 μM DNA1–FcCB[7]. The number of individual experiments n = 6. (b) Voltage dependence of the duration of Level 2 as a whole. The recording conditions are the same as in (a). The number of individual experiments n = 6. (c) Representative signature events of the translocation of DNA1–FcCB[7] (0.25 μM) in a buffer of 3 M KCl (i), 2 M KCl (ii), and 1 M KCl (iii) with 10 mM Tris (pH 8.0). The transmembrane potential was held at +200 mV. (d) Expanded views of Levels 2 and 2′ in (c). (e) Normalized all-point current amplitude histograms of Levels 2 and 2′. The current blockage levels are shown in parenthesis. The solid lines are Gaussian fit to the histograms.

The duration of the Level 2 state as a whole increased as the transmembrane potential became more positive (Fig. 5b). This phenomenon is very similar to the interaction of βCD with anion-selective mutant αHL pores from the cis side, where the electroosmotic flow accounted for the enhanced binding of βCD to the sites within αHL.⁴⁷ In the current study, we used wildtype αHL which is also weakly anion-selective at pH 7.5 (P _K⁺/P _Cl^– = 0.79). Therefore, electroosmotic flow from cis to trans should play a crucial role in the voltage-dependent oscillation of CB[7] in the vestibule. In addition, unlike βCD which is neutral under the aforementioned conditions, CB[7] is in equilibrium with CB[7]·K⁺ and CB[7]·K⁺·K⁺ in the electrolyte solutions (K ₁ 600 M^–1, K ₂, 53 M^–1).⁴⁸ The driving force exerted by the electric field on the CB[7]–metal complexes might be in good balance with the electroosmotic force.

In order to clarify the role of electroosmosis in the generation of characteristic signature events, we studied the translocation of DNA1–FcCB[7] in different concentrations of electrolyte. According to ref. 47, the net water flux generated by the electroosmotic flow (J _w) should conform to J _w Vc _KCl, where V is the transmembrane potential and c _KCl is the KCl concentration in moles l^–1 (ESI^† text). Generally, different concentrations of KCl would produce varied electroosmotic force on the DNA1–FcCB[7] hybrid, while the electric field force remains about the same. When the translocation was conducted in 1.0, 2.0 and 3.0 M KCl respectively, we observed prominent changes in the pattern of signature events (Fig. 5c). The Level 1 state only showed minor changes in the duration when c _KCl was lowered from 3.0 M to 1.0 M; however, the Level 2 and 2′ states underwent significant changes along with the decrease of c _KCl. When c _KCl dropped from 3.0 M to 2.0 M, the current signals switched from oscillating between Levels 2 and 2′ to residing mainly on Level 2 with occasional bumps to Level 2′. When c _KCl was further lowered down to 1.0 M, we found that Level 2′ completely disappeared (Fig. 5d and e). Besides, the duration of Level 2 as a whole decreased drastically from 68.4 ms in 3.0 M KCl to 22.9 ms in 2.0 M KCl, and further to 0.81 ms in 1.0 M KCl (Table S2^†). These results clearly indicated that lowering the KCl concentration while maintaining the transmembrane potential would greatly reduce the electroosmotic flow from cis to trans, and thus result in shortened retainment of CB[7] in the vestibule.

We thank Q. S. Liu (IHEP) for providing the αHL mutants used in this work. This project was funded by National Basic Research Program of China (973 program, numbers 2013CB932800), the National Natural Science Foundation of China (numbers 21175135, 21375130, 21205119, 21475132), and the CAS Hundred Talents Program.