Relative stability of phi29 DNAP-DNA binary complexes and KF-DNA binary complexes
To perform nanopore experiments, a single α-HL nanopore is inserted in a lipid bilayer separating two chambers (termed cis
) containing buffer solution, and a patch-clamp amplifier applies voltage and measures ionic current (). To examine binary complexes formed between phi29 DNAP and DNA, we used a 14 base-pair DNA hairpin substrate (). As demonstrated previously 12,15
, when a KF-DNA binary complex formed with this substrate is captured in the α-HL pore, the resulting ionic current signature is characterized by an initial enzyme bound state (EBS). This occurs when KF resides atop the pore 12
, holding the double-strand/single-strand junction of the DNA substrate within the confines of the polymerase active site (). In this KF-bound state, the DNA template strand is suspended through the nanopore lumen, which is wide enough to accommodate single-stranded but not duplex DNA. The amplitude of this state (IEBS
) can be selectively augmented by an insert of abasic (1′,2′-H) residues within the template strand positioned so that it resides in the nanopore lumen when the polymerase-DNA complex is perched atop the pore 14,17
, such as the 5 abasic residues between template positions +12 to +16 in the DNA hairpin shown in . For KF-DNA binary complexes, the EBS typically lasts a few milliseconds at 180 mV applied potential (). It is followed by a shorter lower amplitude state (), which occurs when the force pulling on the template strand causes dissociation of KF from the DNA, and the duplex DNA drops into the nanopore vestibule. When this occurs the abasic block that was positioned in the pore lumen during the EBS is displaced to the trans
side of the pore, where it has negligible effect on the amplitude of this terminal current step (~ 20 pA at 180 mV). Unzipping of the DNA hairpin within the vestibule followed by electrophoresis of the strand to the trans
compartment restores the open channel current ().
Figure 1 Capture of polymerase-DNA binary complexes in the α-HL nanopore. (a) Schematic of the nanopore device. A single α-HL nanopore is inserted in a 30 µm-diameter lipid bilayer that separates two 100 µL wells containing 10 mM (more ...)
Binary complexes between phi29 DNAP and DNA substrates can be formed in the absence of the divalent cations required for both 5′-3′ polymerase and 3′-5′ exonuclease activity 28
. When phi29 DNAP-DNA binary complexes were formed with the hairpin substrate in and captured in the α-HL pore at 180 mV (), the ~ 35 pA IEBS
typically lasted tens of seconds (median = 17.6 s, IQR = 25.6, n = 62). This is approximately 10,000 times longer than KF-DNA binary complexes under the same conditions (median = 1.9 ms, IQR = 2.4 ms, n = 199). In contrast to capture events for KF-DNA complexes, these phi29 DNAP-DNA events did not end in a single terminal step, but instead ended in a series of discrete ionic current steps () that we termed a “terminal cascade”. The 3′-5′ exonuclease of wild type phi29 DNAP is inhibited under the conditions of the experiment (1 mM EDTA, absent added Mg2+
and thus these current steps are not due to digestion of the primer strand. Therefore we reasoned that the DNA duplex may be unzipping while bound within the confines of the enzyme (). In this scenario, as the template threads out of the complex under tension, the abasic block is drawn out of the lumen in single nucleotide increments that give rise to the sequence of discrete amplitude steps in the terminal cascade ().
This model suggests that the interaction between phi29 DNA and the DNA is strong enough that the DNA secondary structure unzips due to the force pulling on the template strand before the bond between phi29 DNAP and DNA can be broken. It furthermore predicts that reducing the applied voltage during the terminal cascade could allow the DNA duplex to re-anneal within the confines of the enzyme and thus reset the phi29 DNAP-DNA complex to its original position on the DNA template strand, indicated by a return to the ~ 35 pA state. To test this prediction, we compared the ability of complexes captured in the presence or absence of Mg2+
to recover their original EBS amplitude at 180 mV following a controlled voltage drop. A prerequisite for this comparison is a means to ensure that DNA molecules captured in the presence of Mg2+
are intact, so that the nanopore assay compares their fate only after capture. Thus exonucleolytic cleavage of the primer strand in the bulk phase must be miminized during the course of the experiment. We tested whether a 3′-H terminus on the DNA substrate inhibited the rate of 3′-5′ exonucleolytic cleavage by phi29 DNAP, in a gel assay comparing degradation of two 67 mer 5′-6-FAM labeled hairpin substrates () bearing either a dCMP (lanes 1–6) or ddCMP (lanes 7–12) terminus. Consistent with the requirement for divalent cations for phi29 DNAP 3′-5′ exonuclease function 29
, no cleavage of either DNA substrate was observed after 45 minutes incubation in nanopore buffer containing 1 mM EDTA absent added Mg2+
(). With 10 mM Mg2+
present, the extent of DNA digestion for the 3′-H substrate was discernably less than for the 3′-OH substrate. After 10 minutes, while only 24.5% full-length DNA molecules remained for the dCMP-terminated hairpin, 90.5% of the ddCMP-terminated substrate remained intact (). After 45 minutes, 4% of the dCMP-terminated substrate and 45% of the ddCMP-terminated substrate remained intact (). The protection against excision afforded by a 3′-H terminus is further evidenced by the extent of primer extension in the presence of all four dNTPs. For the 3′-H terminated substrate, the onset of DNA synthesis requires that the ddCMP residue first be excised. Thus while with the 3′-OH terminated hairpin > 80% of the molecules were extended to the full-length 102 mer product in 45 minutes (), with the 3′-H terminated hairpin, 79.8% of the DNA substrate remained intact, with only 20.1% full-length extension product (). Thus 3′-H terminated DNA substrates afforded a window following the addition of Mg2+
during which phi29 DNAP-DNA complexes could be captured with the DNA substrate intact. We therefore used the ddCMP terminated hairpin shown in in a nanopore experiment designed to assess the potential for hairpin refolding following initiation of the phi29 DNAP terminal cascade.
In this experiment, upon capture of a phi29 DNAP-DNA complex at 180 mV, a finite state machine (FSM, see Methods
) monitored ionic current in real time until the downward current steps of the terminal cascade were detected (). When the ionic current dropped below 31 pA for at least 0.5 ms (red arrow in ), the FSM reduced the applied potential to 70 mV (). After two seconds at 70 mV, the applied potential was restored to 180 mV and the amplitude of the phi29 DNAP-DNA complex was remeasured. In the absence of Mg2+
, the IEBS
level was reproducibly reset to the original 35 pA level in each of 11 molecules tested. This EBS amplitude is indicative of the initial state in which phi29 DNAP is bound to the base-paired duplex with the n = 0 template residue positioned in the polymerase active site (), and is consistent with re-annealing of the DNA template with an intact primer strand. Importantly, the dominant amplitude during the 70 mV intervals was ~ 10.2 pA, with occasional deflections to ~ 8.5 pA, measurably above the 6.8 pA value determined for unbound DNA at 70 mV in a control experiment (Figure S2
). This indicates that the phi29 DNAP complex remained atop the nanopore orifice without dissociating throughout the lower voltage interval, consistent with a model in which hairpin unzipping at 180 mV and refolding at 70 mV occurs within the confines of the enzyme complex atop the pore.
When the refolding experiment was performed in the presence of 10 mM Mg2+, 16 complexes out of 24 captured in the first 12.5 minutes after the addition of Mg2+ had the ~ 35 pA IEBS level indicating they were formed with intact DNA substrate molecules (). This 35 pA state was maintained for several seconds (median = 10.2 s, IQR = 12.7 s, n = 16), before ending with a drop in amplitude (). The features of the steps that occurred following the 35 pA state differed from those that characterized the terminal cascade in the absence of Mg2+ (compare ). For these complexes, when the voltage was reduced to 70 mV for two seconds and then restored to 180 mV, the 35 pA IEBS level did not reset for any of the complexes tested (). This is in contrast to the phi29 DNAP-DNA complexes captured in the absence of Mg2+ and it indicates that the DNA substrates, which had been captured intact, were modified by exonucleolytic cleavage while they were held atop the pore.
Mapping the effect of template abasic insert position on IEBS for DNA substrates bound to phi29 DNAP
Our strategy for detecting DNA synthesis catalyzed by polymerase-DNA complexes held atop the nanopore employs monitoring changes in ionic current as a block of abasic residues in the template strand is drawn into and through the nanopore lumen in single nucleotide increments when the polymerase advances along the template 14,17
. This approach permits the recognition of sequential Angstrom-scale movements driven by the enzyme.
As a prelude to DNA replication experiments with phi29 DNAP, we established a reference map that related IEBS
to the position of a 5 abasic block within the template strand of DNA hairpin substrates (). To construct this map, phi29 DNAP was bound to each of a series of substrates that contained a block of 5 consecutive abasic residues, sequentially displaced by one nucleotide (). We measured the IEBS
in buffer containing 0.3M KCl for captured complexes under two conditions: i) 1 mM EDTA with no added Mg2+
, which permits formation of binary complexes without supporting nucleotide excision or addition (); and ii) 10 mM Mg2+
, 400 µM ddCTP, and 100 µM dGTP. These latter conditions maintained the intact status of 98.2 and 96% of 3′-H terminated hairpin molecules in the bulk phase for 10 and 45 minutes, respectively (). Protection was afforded by ddCTP, which permitted the polymerase function of phi29 DNAP to restore the ddCMP terminus of molecules if it was excised by the exonuclease function (). Protection was enhanced by the presence of dGTP, which is complementary to the template residue at n = 0 and can form a phi29 DNAP-DNA-dGTP ternary complex in the presence of the 3′-H terminated DNA substrate 22
that can increase the proportion of time the primer terminus resides in the polymerase domain rather than in the exonuclease domain (; Fig. S3
). The complex formed in the presence of Mg2+
, ddCTP, and dGTP is therefore operationally defined as a ternary complex
in this study.
Figure 3 EBS amplitudes at 180 mV of phi29 DNAP-DNA complexes as a function of abasic insert position in DNA template strands. (a) DNA hairpins used in phi29 DNAP mapping experiments. In each sequence, red Xs indicate the positions of the abasic (1′,2′-H) (more ...)
maps for phi29 DNAP binary complexes (blue dots) and ternary complexes (red dots) are shown in . Both maps were similar to a map determined for KF(exo-)-DNA-dNTP ternary complexes at 80 mV using a six abasic template insert 17
. In 0.3 M KCl at 180 mV, IEBS
ranged from 22.3 pA for the ternary complex formed with the 5ab(6,10) substrate (abasic block spanning template positions +6 to +10 measured from n = 0 in the polymerase catalytic site), to 35.4 pA for the binary complexes formed with the 5ab(11,15) and 5ab(9,13) substrates (abasic blocks spanning template positions +11 to +15, and +9 to +13, respectively). This gives a dynamic amplitude range of at least 13 pA for the detection of enzyme movements during polymerization or exonucleolytic reactions.
At all positions within the map, IEBS for the binary and ternary complexes were offset from one another. The direction and the scale of the offset depended in part on the position along the map. For example, at position (i) (), the change from a binary complex to a ternary complex caused an IEBS increase from 31.5 pA to 34.5 pA. By comparison, at position (ii) () the binary to ternary change resulted in a relatively small current increase from 34.4 to 35.2 pA, and at position (iii) () the binary to ternary transition caused a large IEBS current decrease from 31.5 pA to 25.5 pA. Interestingly, the direction and magnitude of an ionic current flicker within the binary state often predicted the dominant amplitude observed for the ternary complex formed with the same substrate ().
The results of the mapping experiments permit a prediction based upon the model proposed for the molecular events that give rise to the terminal cascade ( and ): the sequence of current steps in the terminal cascade of binary complex capture events should vary in a manner that is dependent on the initial position of the abasic block in the complex. This was found to be the case. For example, when the duplex segment of the 5ab(6,10) substrate was unzipped during the terminal cascade, the abasic block was drawn from its position proximal to the enzyme towards the trans
chamber. This resulted in a series of current steps with a ~ 36 pA peak as the abasic block traversed the pore lumen (Fig. S4
, a). In contrast, for binary complexes formed with the 5ab(18,22) substrate, the initial position of the abasic block is distal from the enzyme. When this substrate is unzipped in the terminal cascade, no amplitude peak is observed (Fig. S4
Controlled translocation of DNA templates in the nanopore catalyzed by phi29 DNAP
Results from our laboratory have shown that advance of a DNA template in the α-HL nanopore could be detected at single nucleotide precision during replication by T7DNAP(exo-) 17
. However, for the majority of complexes with this enzyme only one or two nucleotide addition cycles could be monitored. To determine if phi29 DNAP was more efficient at catalyzing sequential nucleotide additions on the nanopore, we measured phi29 DNAP-driven displacement of synthetic DNA substrates molecules bearing 5 abasic inserts in their template strands. The map in was used to interpret changes in IEBS
as single nucleotides were enzymatically added to or removed from the DNA 3′ terminus.
The experiment in showed that the slow excision of a ddNMP residue in the bulk phase could be exploited to capture complexes in the presence of Mg2+
in which the primer strand was intact. Importantly, this experiment also showed that excision of the ddNMP residue could be achieved on the pore, exposing the 3′-OH of the -1 residue and thus yielding a substrate that is potentially competent for synthesis reactions atop the pore in the presence of dNTPs. Consistent with previous findings 30,31
, the gel assay in showed that in the presence of dNTPs the polymerization reaction dominated over the exonuclease reaction in bulk phase. These findings were essental to our strategy for DNA replication experiments: capture phi29 DNAP complexes bearing intact 3′-H terminated substrates in the presence of dNTPs, allow the excision reaction to occur on the pore, and use an abasic block marker in the template strand to determine unambiguously whether the polymerization reaction can be observed for complexes held atop the pore. Using this strategy, the majority of complexes captured in the nanopore should initiate replication at the same template position (-1 relative to the original n = 0 position of the starting substrate).
Because dGTP can slow the rate of ddCMP excision due to formation of ternary complexes (, Fig. S3
) we chose to conduct initial nanopore synthesis experiments using 20 µM each of dATP, dCTP, dTTP and 5 µM dGTP. We determined the effect of these conditions on the state of the DNA substrate molecules in bulk phase in a gel assay using the 5′-6-FAM, 3′-H hairpin substrate (). After 10 minutes, 82.5% of the 67 mer starting substrate remained intact, and 13.6% was extended to the 102 mer product. After 20 minutes, these proportions were 69.4 % and 26.1% extension product, and by 45 minutes almost 30% of the fluorescein labeled hairpin had been extended. We therefore confined our measurements in the nanopore experiments to the first 10 minutes following the addition of Mg2+
and dNTP substrates to the cis
Figure 4 DNA replication catalyzed by phi29 DNAP on the nanopore. (a) DNA hairpin substrate for nanopore replication experiments. The starting abasic configuration for this substrate is 5ab(15,19). The onset of primer extension requires exonucleolytic excision (more ...)
In initial nanopore replication experiments under these conditions (), we used a DNA substrate with the starting abasic configuration 5ab(15,19) bearing a 3′ ddCMP terminus (). Typical ionic current traces for capture of phi 29 DNAP-DNA complexes at 180 mV with this substrate in the presence of 10 mM Mg2+, with or without dNTPs, are shown in , respectively. The dominant initial IEBS upon capture was ~ 29 pA under both conditions, with deflections to ~ 26 pA consistent with an oscillation between the map values for 5ab(15,19) binary and ternary complexes (). Under both conditions, there was a delay at this starting IEBS level, afforded by the slow excision of the 3′ ddCMP terminus, after which a series of current changes ensued. We interpret the current changes in the experiment conducted in the absence of dNTPs () as follows: upon ddCMP excision, the phi29 DNAP exonuclease continued to sequentially cleave nucleotides from the primer terminus, resulting in a progressively shorter duplex segment and greater distance between the enzyme and the abasic insert. The abasic segment was thus moved through the pore toward the trans compartment, causing a progressive ionic current decrease. Eventually, the ionic current returned to the open channel state, consistent with dissociation of the DNA molecule from phi29 DNAP and its subsequent electrophoresis into the trans compartment.
In contrast, when the experiment was conducted in the presence of 20 µM each dATP, dCTP, dTTP and 5 µM dGTP a different ionic current pattern resulted, characterized by a peak at 35.4 pA (). We hypothesized that these current changes occurred because, following phi29 DNAP excision of the ddCMP residue protecting the DNA 3′ terminus, the presence of dNTPs favored nucleotide additions catalyzed by phi29 DNAP while atop the pore. The duplex DNA segment was lengthened as phi29 DNAP moved progressively closer to the abasic insert within the DNA template, drawing it through the nanopore lumen with the attendant traversal of the major ionic current peak between abasic configurations 5ab(15,19) to 5ab(6,10) in the map in . Several DNA template replication reactions, catalyzed by phi29 DNAP-DNA complexes captured in series during this experiment are shown in .
In the gel experiment shown in , in addition to the starting 67 mer hairpin substrate and the full length extension products, intermediate bands corresponding to partial extension products accumulated with time (). These products could arise due to depletion of dNTP pools in the bulk phase, as an increasing fraction of the DNA substrate molecules which are present at 1 µM in both the gel and nanopore assays are replicated. Because this has the potential to affect the extent and rate of synthesis catalyzed by phi29 DNAP complexes atop the pore, we examined whether this could be minimized by using a higher concentration of dNTPs.
We measured the extent of primer extension for the 5′-6-FAM, 3′-H terminated hairpin in the presence of 100 µM each of dGTP, dCTP, dTTP and dATP as a function of time (). Under these conditions the rate of accumulation of the full-length product was slower than in the experiment in (using 20 µM each of dCTP, dTTP, dATP and 5 µM dGTP), likely due to the more efficient inhibition of excision of the ddCMP terminus afforded by the higher dGTP concentration. After 20 minutes, 86.3% of the starting DNA substrate remained intact, and 13.6% was fully extended (), compared to 69.4% and 26.1% for these species, respectively, in reactions conducted for the same amount of time with the lower concentrations of dNTPs (). Importantly, even after 30 minutes, accumulation of shorter extension products was below the limit of detection of the assay. We therefore used dNTP substrates at a concentration of 100 µM each in subsequent replication experiments.
Figure 5 Phi29 DNAP-catalyzed replication up to or through a specific template position. (a) Time course of primer extension for a DNA hairpin substrate in bulk phase, in the presence of phi29 DNAP and 100 µM each dGTP, dCTP, dTTP and dATP. A 67 mer, 5′-6-FAM, (more ...)
To test the model proposed for the ionic current signatures observed in the replication experiment in , we used a DNA hairpin substrate in which the first template dTMP residue was at a defined position relative to the abasic insert (). When DNA synthesis reactions are conducted with this substrate in the presence of 100 µM each of dGTP, dCTP, dTTP and ddATP, 12 nucleotides can be added, during which the abasic block will be drawn from its starting position of 5ab(18,22), across the 35.4 pA peak at 5ab(11,15), to position 5ab(6,10). After reaching the dTMP residue at position +12, replication is predicted to stall. In contrast, replication reactions conducted in the presence of 100 µM each dGTP, dCTP, dTTP and dATP should proceed past the +12 position.
When phi29 DNAP complexes formed with this DNA substrate were captured under both of these conditions, an initial period of several seconds occurred during which the dominant current amplitude was ~ 31 A, with oscillations to ~ 27 pA (), similar to the map values for the ternary and binary complexes for this 5ab(18,22) configuration (). After this state ended, the 35.4 pA ionic current peak was rapidly traversed, indicative of the abasic block being drawn through the lumen. If dGTP, dCTP, dTTP and ddATP were present in the cis chamber, after traversing the peak the polymerase stalled in a state in which the current oscillated between a dominant amplitude of ~ 25 pA to 28 pA for several seconds (). In contrast, in the presence of dATP rather than ddATP, the polymerase advanced without stalling through and beyond the 25 pA state (). This establishes that the stalled state observed in the presence of ddATP (which indicates replicating complexes have reached the dTMP residue) is attained after the template segment that causes the amplitude peak traverses the lumen. Because reaching this dTMP template residue requires the nucleotide incorporations necessary to traverse the 5ab(17,21) to 5ab(7,11) abasic configurations, these experiments verify that the characteristic amplitude peak is due to replication that ensues following ddCMP excision on the pore.
The rate of phi29 DNAP catalyzed DNA replication is influenced by applied voltage across the nanopore
Experiments using optical tweezers have shown that the rate of replication catalyzed by phi29 DNAP is slowed by tension on the template at forces between ~ 20 and ~ 37 pN 26
. This result predicts that the rate of phi29 DNAP replication would be influenced by the voltage applied across the nanopore. However, the voltage regime where this would occur is not known. shows representative events during phi29 DNAP replication reactions along a 25 nt template segment of a DNA hairpin substrate (), for experiments in which the applied potential was varied in 40 mV increments in the range between 220 mV and 100 mV. The starting abasic configuration for this substrate was 5ab(25,29); therefore during DNA synthesis, the 5 abasic insert will be drawn through the limiting aperture of the nanopore lumen, spanning abasic configurations 5ab(18,22) to 5ab(6,10) and thus the amplitudes mapped in . These peaks were traversed at each voltage, at rates that appeared to increase as applied voltage was decreased (). We measured the time required to advance between two readily discernible current amplitudes corresponding to positions flanking the major current peak (blue arrows in ), separated by approximately five nucleotides. At 220 mV, the median time required for replication over this distance was 227 ms (IQR = 174 ms, n = 45); at 100 mV, the median time for replication was 67 ms (IQR = 41 ms, n = 59).
Figure 6 Phi29 DNAP-catalyzed replication by complexes held atop the nanopore at different voltages. (a) DNA hairpin substrate for nanopore replication experiments. The starting abasic configuration for this substrate is 5ab(25,29). After the exonucleolytic excision (more ...)
Replication of longer DNA templates by phi29 DNAP on the nanopore
In anticipation of replicating natural DNA templates in the nanopore, we measured phi29 DNAP-dependent replication of a longer segment within a synthetic DNA hairpin substrate. This hairpin substrate had a starting abasic configuration of 5ab(50,54), and up to 50 nucleotides can be added before the enzyme reaches the abasic block (). When phi29 DNAP-DNA complexes formed with this substrate were captured at 180 mV in buffer containing 0.3 M KCl, there was an initial interval of several seconds during which the current oscillated between a dominant amplitude of ~ 23 pA, with transitions to ~ 25 pA. In 27 out of 47 captured complexes that started with this oscillation, when this period ended, the polymerase proceeded to traverse the mapped amplitude peak ().
Figure 7 Processive DNA replication catalyzed by phi29 DNAP on the nanopore. (a) DNA hairpin substrate for nanopore replication experiments. The starting abasic configuration for this substrate is 5ab(50,54). After the exonucleolytic excision of the terminal ddCMP (more ...)
We speculate that this oscillating signature corresponds to complexes captured with the ddCMP terminus intact, prior to the ddCMP excision reaction that permits synthesis to ensue, because i) a similar pattern invariably occurred between capture and synthesis for each successful replication reaction that subsequently traversed the abasic 35.4 pA peak in the experiments shown in , , , and ; ii) the upper and lower amplitude levels of the oscillation differ among those experiments in a manner that depends upon the starting abasic configuration of the DNA substrate; iii) those levels closely approximated the amplitudes for the binary and ternary complexes mapped for the abasic configuration for each substrate; and, iv) the proportion of time spent in the upper or lower amplitude state can be modulated as a function of dGTP concentration (data not shown).
We therefore used the end of this oscillating state as a start point to approximate the time required for phi29 DNAP to traverse the ~ 50 nt template segment. We measured from a small but reproducible current dip that occurred just after the oscillation ended (left blue arrow in ) to a discernible amplitude state on the distal side of the major map peak (right blue arrow in ). The median time required to replicate across this distance in buffer containing 0.3 M KCl was 1.39 s (IQR = 0.57 s; n = 27).
Surprisingly for this mesophilic polymerase, replication of the 5ab(50,54) substrate by phi29 DNAP was also detectable in buffer containing 0.6 M KCl (). Like the replication reactions in 0.3 M KCl, these events began with a state in which the current oscillated between two levels for several seconds before the onset of synthesis (). Under these higher ionic strength conditions, the current oscillated between a dominant level of ~ 32 pA, with transitions to ~ 34 pA. Replication that drew the abasic segment through the nanopore lumen, causing the abasic block to traverse the mapped amplitude peak, ensued in 25 out of 41 events that began with this current oscillation. In 0.6 M KCl, the median time required to traverse the distance between the end of the oscillation period (left blue arrow in ) and the distal side of the major abasic amplitude peak (right blue arrow in ) was 2.41 s (IQR = 1.13 s; n = 25).