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Bilayer-Spanning DNA Nanopores with Voltage-Switching between Open and Closed State

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† Nanion Technologies GmbH, D-80636 Munich, Germany
‡ Cavendish Laboratory, University of Cambridge, Cambridge CB3 0HE, United Kingdom
§ Department of Chemistry, Institute of Structural Molecular Biology, University College London, London WC1H 0AJ, United Kingdom
*Address correspondence to [email protected]; [email protected]
Cite this: ACS Nano 2015, 9, 2, 1117–1126
Publication Date (Web):October 22, 2014
https://doi.org/10.1021/nn5039433

Copyright © 2014 American Chemical Society. This publication is licensed under these Terms of Use.

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Abstract

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Membrane-spanning nanopores from folded DNA are a recent example of biomimetic man-made nanostructures that can open up applications in biosensing, drug delivery, and nanofluidics. In this report, we generate a DNA nanopore based on the archetypal six-helix-bundle architecture and systematically characterize it via single-channel current recordings to address several fundamental scientific questions in this emerging field. We establish that the DNA pores exhibit two voltage-dependent conductance states. Low transmembrane voltages favor a stable high-conductance level, which corresponds to an unobstructed DNA pore. The expected inner width of the open channel is confirmed by measuring the conductance change as a function of poly(ethylene glycol) (PEG) size, whereby smaller PEGs are assumed to enter the pore. PEG sizing also clarifies that the main ion-conducting path runs through the membrane-spanning channel lumen as opposed to any proposed gap between the outer pore wall and the lipid bilayer. At higher voltages, the channel shows a main low-conductance state probably caused by electric-field-induced changes of the DNA pore in its conformation or orientation. This voltage-dependent switching between the open and closed states is observed with planar lipid bilayers as well as bilayers mounted on glass nanopipettes. These findings settle a discrepancy between two previously published conductances. By systematically exploring a large space of parameters and answering key questions, our report supports the development of DNA nanopores for nanobiotechnology.

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Building rationally designed nanoscale porous structures that mediate transport across membranes is of general interest in fundamental and applied science. (1, 2) Biomimetic engineered nanopores (3) that insert into lipid bilayers are of particular relevance for biorelated applications (4) including biosensing. In nanopore analytics, single molecules passing or binding to a nanopore are detected by the associated modulations in ionic current. (5, 2, 6-10) The approach has gained popularity due to its simplicity, the wide range of accessible analytes, (6) and the ability to sequence DNA in a label-free fashion. (11-14) The sensing of large analytes such as proteins has been achieved with dedicated approaches (15-18) and also solid-state nanopores. (6, 19-21) Membrane nanopores are also of interest as research tools in cell biology to regulate ionic flux (22) or in synthetic biology to interconnect cells or proto-cells within artificial tissues. (23) All of these applications rely on engineered pores with tailored dimensions and defined conductance properties.
Engineered or synthetic nanopores can be made from peptides and proteins, but also with organic molecules or polymers (24-29) aided by modeling and simulations. (30) The most recent invention is membrane-spanning channels composed of folded and structurally defined DNA. Predated by nanofunnels (31) and nanoplates with an embedded hole, (32) DNA origami channels carry hydrophobic lipid anchors to overcome the energetic cost of placing negatively charged DNA structures into the hydrophobic lipid bilayer. (33-35) As lipid anchors, cholesterol, (33) charge-neutralized phosphate analogues in the DNA backbone, (34, 36) and porphyrin (35) have been used. The latter is remarkable, as only two separate anchors were required to embed the negatively charged DNA nanopores into the bilayer. (35) The principle of DNA pores has been independently demonstrated with channels of 42 nm length by Langecker etal. (33) and 14 nm by Burns etal. (34) Irrespective of their size, the nominal width of both channels was 2 nm. Their nanoscale architecture also followed the same common honeycomb design based on DNA origami. (37, 38) Accordingly, the central channel was surrounded by six hexagonally arranged DNA duplexes that were interconnected by DNA crossovers. However, the larger pore was formed from a long DNA scaffold and multiple staple oligonucleotides, (33) while the shorter version was assembled solely from oligonucleotides. (34, 35) The latter approach previously used for other small nanostructures (39, 40) benefits from the ease of fabrication, lower synthesis costs, and the production of larger amounts of pores at higher concentration.
DNA nanopores have been characterized in previous studies in terms of assembly, structure, and conductance, (33-35) but several important questions related to the inner channel dimensions and conductance properties remain unresolved. Comprehensive single-channel current analysis is important as it can—in general terms—help distinguish the static and dynamic heterogeneity of samples and probe the channels’ internal structure and properties. In order to advance the field, this report aims to address and settle the following fundamental scientific points about DNA nanopores. At a basic level, (i) it has not been proven that the channels assume the theoretically predicted inner width of 2 nm once embedded inside the bilayer. Conventionally, dimensions of stable membrane-embedded pores are indirectly validated by comparing the experimental conductance values with the theoretical values calculated from the assumed dimensions. In the case of DNA nanopores, the calculations are not suitable because the simple model does not account for the ion permeable (41) and structurally dynamic (42) pore wall. Independent of any model, direct experimental proof such as size-dependent blocking with probe molecules is probably most suited to determine the pore dimensions.
We (ii) lack a fundamental understanding of whether ions indeed flow predominantly inside the section of the channel that spans the lipid bilayer. In a proposed alternative, ions might pass outside the DNA pore through the locally disrupted lipid bilayer membrane. A gap between the outer pore wall and an curved lipid bilayer has been proposed to account for the fact that hydrophobic lipid chains cannot be close to the negatively charged DNA nanopore. (33)
Another important point to be clarified is (iii) the discrepancy between the measured conductance values of the known pores. (33, 34) All published DNA nanochannels follow the six-duplex-bundle design with the same nominal channel diameter. Hence, their pore currents should depend—as a first approximation—solely on the channel height, given the inverse dependence of conductance on conductor length. Yet, contrary to the expectation that a shorter channel should yield a higher current, the conductance for the 14 nm pore of Burns etal. with a value of 0.25–0.4 nS (34, 35) is smaller than the value of 0.89 ± 0.15 nS for the 42 nm origami pore of Langecker etal. (33)
Finally, (iv) DNA pores have been analyzed with different lipid bilayer systems, which might influence the conductance values and could account for the discrepancy. In the case of Langecker etal., (33) the nanostructures were examined with planar lipid bilayer recordings, while Burns etal. (35) inserted nanopores into vesicles, which then adhered to a glass nanopipette. (43) Usually, the bilayer system exerts minimal influence on the conductance of protein pores with some exceptions, (44) but DNA nanostructures could be more flexible (42) or compressible than protein pores, leading to altered conductance.
With the aim to address, settle, and clarify the above questions, we characterize a DNA nanopore of the archetypal six-helix-bundle architecture (Figure 1A) via single-channel current recordings. To establish the (i) experimental width of the DNA channel, we used sizing with poly(ethylene glycol) (PEG). In this approach, the ionic conductance is measured in the presence of PEG of varying length to determine the threshold size under which the organic polymer permeates into the pore to block or affect the ionic current. (45, 46) By probing the diameter of the DNA pore with PEG, it should (ii) be possible to establish that the main ion-conducting path runs through the membrane-spanning channel. Furthermore, we address (iii) the inconsistent conductance values by examining pore current as a function of transmembrane voltage, and (iv) by using planar lipid bilayers as well as vesicle recordings (Figure 1B and C) on a single pore, something that has not been done before. By exploring several tunable parameters we aim to identify conditions that can account for the different conductance values. In brief, our report settles and answers all four points and demonstrates that DNA nanopores are well-defined structures within the selected parameter space.

Results

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Design and Formation of DNA Nanopores

To facilitate the comparison to previous studies, (33-35) we built a DNA nanopore with the archetypal six-helix-bundle architecture. In this design, six DNA duplexes are arranged in a hexagon to enclose an approximately 2 nm-wide channel (Figure 1A). In the channel described by Burns etal. (35) and replicated in this report, the nanobarrel is composed of six DNA oligonucleotides, which thread between the duplexes to form crossovers and add structural stability. The resulting six-helix bundle has a nominal width of 6 nm and a height of 14 nm. Two tetraphenyl porphyrin tags are coupled to one terminus of the nanobarrel to facilitate its insertion into the bilayer (Figure 1A, inset). The synthesis of the tetraphenyl porphyrin, its coupling to form a modified uridine nucleoside, and its incorporation into DNA strands via solid-phase oligonucleotide synthesis have been described. (47, 48)

Figure 1

Figure 1. DNA-based membrane-spanning pore and the two recording setups used to measure the conductance properties of the nanopore. (A) Schematic drawing of a six-duplex-bundle nanopore composed of six DNA oligonucleotides (colored) carrying two tetraphenyl porphyrin tags (black, inset), which anchor the pore into the lipid bilayer. The porphyrin anchor is attached via the acetylene group (inset) to position 5 of a uridine base (not shown). (B) Schematic drawing of a microcavity unit with a suspended planar lipid bilayer. Only one of the 16 units of the recording chip is shown. The nanopores were incorporated into the preformed lipid bilayers. (C) In the second recording setup, the nanopores were initially incorporated into lipid vesicles, which were then spread over the orifice of a 200 nm diameter glass nanopipette (transparent blue). The grounded reference electrodes in B and C are drawn as small, light gray circles, while the other electrode is in dark gray.

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The DNA nanopore was assembled by heating and cooling an equimolar mixture of four native and two porphyrin-modified DNA strands (for sequences see Supporting Information, Table S1, Figure S1). The assembly mixture was analyzed by native agarose gel electrophoresis to confirm the formation of the DNA nanopore. The band migrated to the same height as a control nanopore without the porphyrin tags (Figure 2A, lanes 1 and 2, respectively), suggesting assembly of the correct DNA pore and the absence of misfolded products.

Figure 2

Figure 2. DNA nanopores are self-assembled and embedded into lipid bilayers. (A) Native agarose gel electrophoresis of DNA nanopores without (lane 1) and with the porphyrin lipid anchor (lane 2) 100 bp molecular weight marker (lane 3). (B) Dynamic light scattering trace of a DNA pore with lipid anchor (red line) and without lipid anchor (black dashed line). (C, D) Bright-field (left) and confocal fluorescence images (right) of DPhPC vesicles incubated (C) with DNA pores carrying the fluorescent porphyrin tag and (D) with Cy3-labeled nanopores lacking a lipid anchor. The bright spots in (C) may represent individual or clusters of pores. Excitation wavelength: 532 nm. Scale bar: 5 μm.

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Band streaking has been observed before and is caused by interactions of the hydrophobic tag rather than unfolding. (33, 47, 48) Dynamic light scattering of the nanopore with the lipid anchors revealed a single major peak at a hydrodynamic radius of 5.5 ± 0.2 nm (Figure 2B, red line), which is in agreement with the calculated value of 4.9 nm. (49, 50) The radius for the control pore without an anchor was slightly smaller, at 5.2 ± 0.2 nm (Figure 2B, black dashed line). Fluorescence microscopy established that the pore with the fluorescent porphyrin tag (51, 52) attached to the lipid bilayer of giant unilamellar vesicles (Figure 2C). By contrast, the signal of nanopores carrying a Cy3 tag but no lipid anchor did not localize into the curved membrane (Figure 2D), in line with expectations.

Single-Channel Current Analysis and PEG Sizing Confirm that the DNA Nanopores Adopt, at Low Voltages, the Open-Channel Structure Corresponding to a High-Conductance State

With the aim to settle fundamental questions (i) and (ii) about the DNA channel’s diameter and the main ion-conducting path, respectively, we subjected the porphyrin DNA nanopores to single-channel current recordings. Planar bilayer recordings (Figure 1B) were conducted with the recently developed multicavity parallel analysis platform, Orbit 16, from Nanion (53) using standard electrolyte conditions (1 M KCl, 10 mM HEPES, pH 8.0). Ionic flow through the pore was triggered by applying a transmembrane potential. At +20 mV relative to the grounded cis side (Figure 1B), the trace of the DNA nanopore shown in Figure 3A had a stable current of 33 pA, corresponding to a conductance of 1.65 nS. Averaging the conductance values from over 50 single-channel current traces at +20 mV yielded a value of 1.62 ± 0.07 nS and a narrow distribution in the conductance histogram (Figure 3B). The experimental value is higher than the theoretical prediction of 1.32 nS calculated (54) using the nominal pore geometry. But this calculation assumes a pore with electrically nonpermeable walls, which is most likely not correct for DNA pores. A comparison also has to consider that the diameter of hydrated DNA duplexes may be bigger than 2 nm. Pores with a conductance of around 1.6 nS are referred to as being in the high-conductance state.

Figure 3

Figure 3. High-conductance state of membrane-embedded DNA nanopores at low voltages corresponds to an open pore as shown by PEG sizing. The traces were obtained with planar lipid bilayer recordings. The high conductance state is color-coded in red. (A) Representative ionic current trace at +20 mV. (B) Histogram of channel conductances obtained from measurements at +20 mV. (C) Current traces of individual pores in the absence and presence of PEG molecules of indicated mean molecular mass. (D) Pore blockade as a function of the hydrodynamic diameter of PEG. Between PEG 200 and PEG 600, there is a significant change in relative pore blockade. With PEG 1000 and above the effect is tapering off. This upper turning point indicates that PEG molecules are being excluded from the pore lumen. The hydrodynamic diameter of PEG 1000 (approximately 1.9 nm) is hence assumed to reflect the diameter of the DNA pore. The data present averages and the minima and maxima from five independent recordings (PEG 62 to PEG 400) and three independent recordings (PEG 600 to PEG 3350).

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To address question (i) on channel diameter, the pores in the high-conductance state were subjected to PEG sizing to probe the channel’s diameter. The nominal inner width is 2 nm, but this has not been verified by experimental means. The sizing approach has been used previously to determine the inner width of protein channels. (45, 46) It relies on measuring the pore conductance in the presence of PEG molecules of different hydrodynamic diameters. Molecules smaller than the channel width are expected to permeate into the lumen and reduce or change the pore conductance. By contrast, larger PEG probes unable to move into the pore should have no blocking effect on conductance. For the sizing experiments, PEGs with a mean molecular mass of 62, 200, 300, 400, 600, 1000, and 3350 Da covering a range of hydrodynamic diameters from 0.52 to 3.26 nm were used (see the Methods section for information on the mass dispersity). (45, 46) PEG was added to the cis side of the DNA nanopore to a final concentration of 0.2 g/mL, and the conductance was measured after equilibrating for approximately 10 min. The polymeric probe led to a size-dependent reduction in the pore current, with PEG 200 (Figure 3C) and PEG 62 (Figure S2) marking the biggest drop. The current reduction implies that the polymers enter the pore and reduce ionic flow via steric effects. The blockade levels for PEG 62 and 200 are similar, as they represent the PEG effect on the bulk solution; that is, there is no size discrimination by the pore. By comparison, starting with PEG 300 (Figure 3C) the channel conductance increased (PEG 400, Figure S2) drastically at PEG 600 up to PEG 1000, where it leveled off (Figure 2C) with no major change at PEG 3350 (Figure S2). The saturation indicates that the largest PEG molecules were too big to enter the channel lumen. We note that the current levels for PEG 600 up to PEG 3350 were higher than the pore current in the absence of PEG. This reflects the higher effective concentration of electrolyte ions, or activity, within these PEG solutions, as observed previously. (55, 56)
Plotting the relative current change versus the hydrodynamic diameter of PEG revealed a sigmoidal dependence and an upper transition point with a cutoff at 1.9 nm hydrodynamic diameter (Figure 3D). The diameter reflects the size of PEG which does not enter the pore due to steric exclusion and is in excellent agreement with the expected nominal diameter of the DNA nanopore of 2.0 nm. (35) We are aware that the sizing of pores with PEG relies on the simplifying assumption that the behavior of PEG in the pore is similar to that of bulk PEG. While this is correct in several cases, deviations are known, (57) as also discussed in two reports which size PEG with a protein of known structure. (58, 59) For example, PEG can bind cations, (60, 61) and the threshold size of PEG for permeation into a protein pore was found to depend on the pH-dependent ionization of amino acids inside the channel lumen. (62) Hence, we cannot rule out that our transition point of 1.9 nm might be biased by electrostatic interaction between PEG and the negatively charged DNA pore wall. The size-dependent change of the plot in Figure 3D argues, however, against any possible strong bias.
The constant-current trace for blocked pores suggests that multiple PEG molecules simultaneously reside inside the pore as opposed to individual polymers passing or lodging inside the lumen one at a time. Multiple PEG molecules do not lead to a 100% pore current reduction because the polymer’s loose structure carries electrolytes. Furthermore, ion permeable (41) channel walls may bypass and effectively dilute out the blocking effect of PEG. The major outcome of the PEG sizing is the confirmation of the pore’s diameter once inserted within bilayers.
Sizing the channel width with PEG also settles question (ii) by demonstrating that the main conductance path—at least for the membrane-spanning section of the pore—is inside the channel. The threshold diameter of approximately 1.9 nm is not compatible with a competing model in which ions pass solely through a suggested gap between lipid bilayer and pore; a membrane hole this size would not be energetically stable for our minute-long measurement times used in our PEG sizing experiments. However, it cannot be ruled out that a small proportion of the current runs outside the DNA channel through temporarily occurring gaps between the outer pore wall and the lipid bilayer.

Higher Voltages Lead to a Lower Conductance State Representing a Partially Blocked Channel

In order to clarify point (iii) about the different conductance values reported in the literature, we examined the pore current as a function of transmembrane voltage, which is a key parameter for many membrane channels. As illustrated for a single-channel current trace, increasing the potential’s magnitude in 20 mV steps initially confirmed the presence of the established high-conductance state for the range from −60 to +40 mV (Figure 4A, red section). But applying voltages of <−80 mV or >+60 mV revealed a lower conductance state (Figure 4A, blue section). The voltage-dependent two-state behavior was confirmed in prolonged single-channel current traces for all voltages (Figure S3). When summarized in a current-voltage plot, the high-conductance state was ohmic (Figure 4B, red symbols), in line with the expected cylindrical shape of the DNA channel. By comparison, the low-conductance state was ohmic for a given pore, and slightly varying low-conductance levels were found for different pores (Figure 4B, blue symbols). Pores stayed temporarily or for a longer time in the low-conductance state, as illustrated for several single-channel current traces (Figure 4C, Figure S3). The durations of these blockades, τoff (Figure 4C), were found to spread from 10 ms to several seconds (Figure S4). Possible molecular mechanisms causing the lower conductance state include electric-field-induced fraying of DNA duplexes, flipping of DNA loops at the pore entrances, conformational alterations in the overall duplex structure or the relative position between duplexes, or repositioning of the pore within the bilayer.
To obtain statistically relevant values for the low- and high-conductance states and their voltage-dependent occurrence, we characterized multiple channels via all-point histogram analysis. Data from 38 independent single-channel traces recorded in 20 mV steps from +20 mV up to +100 mV were combined in a series of cumulative all-point histograms (Figure 4D; Figure S5 for −20 to −100 mV). The two-state behavior is clearly demonstrated in the plot by the big peaks for low- and high-conductance states at 0.25–0.5 and 1.6 nS, respectively. Slight variations of conductance within those two dominating states likely represent subconductance levels, which are, however, not discussed further to maintain our focus on the major changes in conductance and pore behavior. The conductance difference between the two levels was also established by independently calculating the blockade magnitude Ab (Figure 4C, Figure S4). The histogram analysis in Figure 4D furthermore confirmed that the low-conductance level first appeared at +60 and −80 mV and dominated at higher voltages (Figure S5), in agreement with a related voltage-dependence for the characteristic inter-event interval τon (Figure S6). To quantify the effect of voltage on pore conductance, we derived the counts for the high-conductance state from the all-point histograms and plotted these probabilities against voltage (Figure 4E, red line). The high-conductance state had a probability of 97% at the lowest recorded voltage of +20 mV. The threshold voltages for 50% probabilities were +64 and −77 mV, which are close to the threshold voltages attained when plotting the frequency of occurrence of blockade events (Figure 4E, black line; Table S2).

Figure 4

Figure 4. Low-conductance state for DNA nanopores occurs at higher voltages. The high conductance state is color-coded in red; the low conductance state is coded in blue. The traces were obtained with planar lipid bilayer recordings. (A) Current traces for 20 mV voltage steps showing that potentials of <−80 mV and >+60 mV lead to a low-conductance state. (B) IV curve displaying the averages and standard deviation from 7 single-channel current traces. (C) Representative single-channel current trace at +100 mV. The amplitude of the blockade, Ab, the event dwell time, τoff, and the inter-event interval, τon, are defined. (D) Cumulative all-point histogram of 38 single-channel current traces for +20 to +100 mV in 20 mV steps illustrating the voltage-dependent switching between the low-voltage open state and the high-voltage partially closed state. (E) Probability of observing the open state as a function of voltage. The probability (red line) is defined as the count for the high-conductance state divided by all counts in the all-point histogram. The black line represents the frequency of occurrence for the low-amplitude events as reported in Table S2. Error bars for the open probability are not given as the conventional way of obtaining averages from Poisson-fits for τon value distributions is not possible when the probabilities are obtained by Gaussian fitting of peaks in all-point histograms. For the frequency of occurrence, there were not enough closing events at small voltages to derive meaningful errors. (F) Traces of a single DNA pore recorded at multiple consecutive voltage ramps running from −100 to +100 mV. The occurrence of the low-conductance state at −20 mV in the third trace rather than at usual voltages of around −80 mV is explained by a memory effect of this particular DNA structure, which had experienced previous voltage ramps and pore closures.

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Our data on the voltage-switching of a DNA nanopore between a high- and a low-conductance state address in part point (iii) on the discrepancy between published conductance values. The high conductance for the 14 nm pore at 1.6 nS is now higher than the conductance of 0.89 nS of the 42 nm pore by Langecker etal., (33) in line with expectations that a pore with a shorter conductance path yields a higher conductance than a pore with a longer channel. In further agreement, the low-conductance state at 0.25–0.4 nS from Burns etal. (35) was well reproduced with a value of 0.36 ± 0.1 nS obtained by our conductance histogram (Figure S7). In our present study, these two states are reversibly interconvertible (Figure 4D) as additionally confirmed by consecutive voltage ramp traces of a single pore (Figure 4F). But the previous report by Burns etal. (35) and another study with a related six-duplex-bundle pore (34) did not describe the interconversion and current values corresponding to the high-conductance state. Hence, question (iii) is not completely resolved. One possible explanation is that the conductance values may be attributed to the two different recording setups, as the data in Figures 3 and 4 were acquired with planar lipid bilayer recordings, while the previous report by Burns etal. used lipid bilayers mounted on a glass nanopipette. (35) This previous study focused on the chemical and structural characterization of the nanopores, and an in-depth conductance analysis with nanopipette membranes could reveal the low-conductance state. However, the related six-duplex-bundle pore was characterized by planar bilayer recordings, but other factors might have favored the preferential recording of the low-conductance state. (34) These include the analysis at a high voltage of 100 mV and the use of the different lipid anchor system composed of 72 ethyl phosphorohioate groups. (34) This may not be comparable to the structurally less intrusive anchoring strategy of the present pore with solely two hydrophobic porphyrin tags.

Nanopores Embedded into Nanopipette-Mounted Membranes Have a Greater Tendency to Show a Lower Conductance State

We examined the porphyrin-based DNA nanopores with a second recording technique to address point (iv) about the lack of comparability between previous studies that examined different pores with different recording techniques. For the current measurements, lipid vesicles with embedded nanopores were mounted onto glass nanopipettes typically under small negative pressure (−1 to −10 PSI) to spread the membrane over the orifice (Figure 1C). (43) Similar to the planar membranes, nanopore recordings with the nanopipette membranes gave rise to the known stable high-conductance state of around 1.5 nS for moderate voltages, as shown for a single-channel current trace at +40 mV (Figure 5A) and an IV curve (Figure 5B). We note that these traces were less frequent than on the planar setup. Furthermore, nanopipette-bilayer traces displayed current fluctuations (Figure 5C and D), which were, however, normally not between the high- and the low-conductance state as found for planar bilayers, but predominantly between either of these levels and a completely closed pore (Figure 5C and D). Indeed, pores in the nanopipette system also permanently closed (Figure 5E). A conductance histogram combining all nonzero current levels recorded at +100 mV (Figure 5F) displayed the major low-conductance state with a value of 0.24 nS, which is close to Burns etal. and the less frequent high-conductance state (Figure 5F, inset).
To determine the influence of voltage, a series of cumulative all-point histograms were plotted to combine the conductance data recorded from +20 to +100 mV at 20 mV steps (Figure 5G). Similar to planar bilayer recordings, the two peaks for the high- and the low-conductance level were clearly visible, and higher voltages shifted the distribution from the former to the latter conductance state (Figure 5G; for a side-by-side comparison of planar and nanopipette bilayer data see Figure S8).

Figure 5

Figure 5. Conductance analysis of DNA nanopores embedded in lipid bilayers mounted across a nanopipette orifice. (A) Single-channel current trace of a pore in the high-conductance state at +40 mV. (B) IV curves for single DNA nanopores in the high and the low-conductance state and a lipid bilayer without pores as reference. The data spread for the two pore conductances can be estimated from the peak width in the all-point histograms in panel D. (C, D) Single-channel current trace fluctuating from the (C) low- and (D) high-conductance state to a completely closed pore. (E) Voltage-stepped current trace of a low-conductance state switching to complete and permanent pore closure at +80 mV. (F) Conductance histogram derived from single-channel current recordings at +100 mV. (G) Cumulative all-point histogram of 21 current traces from +20 to +100 mV in 20 mV steps. (H) Probability of observing the open state as a function of voltage. The probabilities were derived from all-point histograms as described in Figure 4. Filled squares show data from the nanopipette-mounted membrane, while empty triangles are data from planar bilayer recordings.

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As a difference, the distribution of the high conductance level in the nanopipette system was broader, and lower voltage magnitudes were able to shift the conductance distribution to the low-level state (Figure 5G, Figure S8). To quantify and compare the effect of the membrane system on pore conductance, we plotted the voltage-dependent probabilities for the high-conductance state, which represent the corresponding counts in the all-point histograms. For the nanopipette bilayers, the probabilities were 35% and 45% at −20 and +20 mV, respectively (Figure 5H, filled squares). This compares to 88% and 97% for the planar bilayers (Figure 5G, empty triangles; data replotted from Figure 4 to facilitate comparison).

Conclusions

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Our results establish that the DNA pore resides, at low voltages, in a high-conductance state, which is in excellent agreement with an open channel of diameter close to expectations. Directly experimentally validating the diameter of the bilayer-embedded nanopores by probing the lumen with polymers of known size has not been achieved before. Similarly, our report is the first to clarify that the membrane-spanning section of the channel is indeed the main ion-conducting path. Our finding of the voltage-dependent switching between the open and partially closed state is important. It helps reconcile previously reported, conflicting conductance values by showing that the newly discovered open state of the 14 nm long pore has, in line with predictions, a higher conductance than a longer pore with the same six-helix-bundle design. We also pioneer the conductance characterization of DNA nanopores with planar bilayer recordings as well as bilayers mounted on the tip of a nanopipette, thereby establishing a reference standard. The two analysis methods gave consistent and partly overlapping yet different results. Both systems reveal the open and partially closed states and the voltage-induced switching between them. However, the planar bilayer system leads to a higher probability of the open channel at lower voltages and a narrower conductance distribution in the open channel state. Possible reasons for the difference could include, in the case of the nanopipette, the presence of lateral membrane pressure, which might compresses the pore to change the ion-conducting path, or, in the case of the planar bilayers, the use of detergents to facilitate pore insertion. The exact reason for the differences will be examined in future studies, which will also decipher the molecular nature of the partial blockage of the DNA pore at high voltages.

Methods

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Design and Synthesis of Nanopore Structure

The nanobarrel was designed following the honeycomb lattice of caDNAno software (63) including the modification (35) of linking termini of staple and scaffold strands to obtain an architecture with six DNA strands. The positions for attaching porphyrins were selected on two opposite duplexes using a molecular model generated with Macromodel. The tetraphenyl porphyrin-tagged deoxyuridine was synthesized and incorporated into DNA oligonucleotides, (47, 64) the purity of the synthesized DNA strands was confirmed by PAGE, and the yield was determined by UV–vis spectroscopy. (35) Nanopores were assembled by heating at 95 °C for 5 min an equimolar mixture of the six strands at 1 μM each dissolved in buffer A (1 M KCl, 50 mM Tris, pH 8.0; total volume 1000 μL) followed by cooling to 16 °C at a rate of 0.25 °C per min in a Varian Cary 300 Bio UV–vis spectrophotometer equipped with a Peltier cooling element.

Nanopore Characterization with Native Gel Electrophoresis, Dynamic Light Scattering, and Fluorescence Microscopy

The DNA nanopores were analyzed using 1.2% agarose gel electrophoresis in standard TBE buffer supplemented with 11 mM MgCl2. (35) Dynamic light scattering measurements were carried out on a Zetasizer Nano S from Malvern (65) using DNA samples with a concentration of 0.25 μM in buffer A. For fluorescence microscopic imaging of nanopores embedded into lipid bilayers, giant unilamellar vesicles (GUVs) in the range 1–30 μm were prepared using an electroformation unit (Vesicle Prep Pro, Nanion Technologies, Germany) as described. (66, 67) The GUVs (0.5 μL) suspended in 1 M KCl and 50 mM Tris, pH 8.0, were incubated with DNA nanopores at a final concentration of 3 nM for 10 min at RT. The suspension (20 μL) was then imaged using a previously described microscopic setup (68) with a 532 nm laser operating at 2 mW and an EMCCD camera (acquisition time of 5 ms). Data acquisition and image processing were performed as described. (68)

Nanopore Current Recordings

For planar lipid bilayer electrophysiological current measurements, an integrated chip-based, parallel bilayer recording setup (Orbit 16, Nanion Technologies, Munich, Germany) with multielectrode-cavity-array (MECA) chips (IONERA, Freiburg, Germany) was used. Bilayers were automatically formed by remotely actuated spreading 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC, Avanti Polar Lipids, Alabaster, AL, USA) dissolved in octane (10 mg mL–1). The standard electrolyte solution was 1 M KCl and 10 mM HEPES, pH 8, unless stated otherwise. For pore insertion, a 2:1 mixture of porphyin-anchored DNA nanopores and 0.5% OPOE (n-octyloligooxyethylene, in 150 mM KCl, 10 mM HEPES, pH 8) was added to the cis side of the bilayer to a final concentration of 10 nM nanopores. A positive voltage of +40 mV was applied to facilitate pore insertion. Successful incorporation was observed by detecting the current steps to distinct levels. To estimate the size of the pore diameter, currents were recorded in 1 M KCl and 10 mM HEPES, pH 7.5, containing 0.2 g mL–1 poly(ethylene glycol) with an exact molecular mass of 62 and mean molecular masses of 200 (Mn ranging from 190 to 210), 300 (Mn 285–315), 400 (Mn 380–420), 600 (Mn 570–630), 1000 (Mn 950–1050), and 3350 (Mn 3015–3685) (Sigma-Aldrich, Munich, Germany). For equilibration, the solution in the cis chamber was mixed by careful and repeated pipet aspiration. Equilibration was achieved when there was no change in measured current. To measure the conductance of a given single channel with respect to PEG with molar masses of up to 400, polymer in the electrolyte solution in the cis chamber was removed by repeated washing to obtain the original nonblocked current, followed by adding the next PEG polymer and mixing. For PEG 600 and higher, the conductance of pores was measured only for a given polymer size, as washing out of the probe molecules would have taken more than an hour. The data points for PEG 600 to PEG 3350 were obtained using a DNA pore of the same hexagonal six-duplex-bundle scaffold but carrying cholesterol anchors at the outer pore wall. The cholesterol pores had the same conductance values from PEG 62 to PEG 400 as the DNA pores with the porphyrin anchors. The data were Bessel filtered at 2.873 kHz and acquired at 10 kHz with an EPC-10 patch-clamp amplifier (HEKA Elektronik, Lambrecht/Pfalz, Germany) with the PATCHMASTER software (HEKA Elektronik). Single-channel analysis was performed using Clampfit (Molecular Devices, Sunnyvale, CA, USA).
The recordings for nanopores inside lipid vesicles were performed using a nanobilayer setup as previously described. (43) Bilayers were formed reproducibly by bursting GUVs on the tip of a nanopipette (200 nm diameter). GUVs were incubated with 30 nM DNA nanopores for 10 min at RT in buffer A. Bilayers that held DNA nanopores were identified due to their lowered seal resistances. Nanopore incorporation was often triggered by applying a voltage pulse. Ionic current data were acquired using an Axopatch 200B amplifier and analyzed as described. (43, 69)

Supporting Information

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Experimental details on DNA sequences for assembling the DNA nanopores. Experimental results on the conductance properties of DNA nanopores. This material is available free of charge via the Internet at http://pubs.acs.org.

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Author Information

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  • Corresponding Authors
  • Authors
    • Astrid Seifert - Nanion Technologies GmbH, D-80636 Munich, Germany
    • Kerstin Göpfrich - Cavendish Laboratory, University of Cambridge, Cambridge CB3 0HE, United Kingdom
    • Jonathan R. Burns - Department of Chemistry, Institute of Structural Molecular Biology, University College London, London WC1H 0AJ, United Kingdom
    • Niels Fertig - Nanion Technologies GmbH, D-80636 Munich, Germany
  • Notes
    The authors declare no competing financial interest.

Acknowledgment

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We thank Dr. Gerhard Baaken and Dr. Ekaterina Zaitseva from IONERA for their help in screening detergents to facilitate nanopore insertion. The S.H. lab is supported by the Leverhulme Trust (RPG-170), UCL Chemistry, EPSRC (Institutional Sponsorship Award), the National Physical Laboratory, and Oxford Nanopore Technologies. K.G. acknowledges funding from the Winton Program of Physics for Sustainability, Gates Cambridge, and the Oppenheimer Trust. U.F.K. was supported by ERC starting grant no. 261101.

References

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    Current Opinion in Biotechnology (2013), 24 (4), 699-704CODEN: CUOBE3; ISSN:0958-1669. (Elsevier B.V.)
    A review. Nanopore technol. employs a nanoscale hole in an insulating membrane to stochastically sense with high throughput individual biomols. in soln. The generality of the nanopore detection principle and the ease of single-mol. detection suggest many potential applications of nanopores in biotechnol. Recent progress has been made with nanopore fabrication and sophistication, as well as with applications in DNA/protein mapping, biomol. structure anal., protein detection, and DNA sequencing. In addn., concepts for DNA sequencing devices have been suggested, and computational efforts have been made. The state of the nanopore field is maturing and given the right type of nanopore and operating conditions, nearly every application could revolutionize medicine in terms of speed, cost, and quality. In this review, we summarize progress in nanopores for biotechnol. applications over the past 2-3 years.
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  20. 20
    Wei, R. S.; Gatterdam, V.; Wieneke, R.; Tampe, R.; Rant, U. Stochastic Sensing of Proteins with Receptor-Modified Solid-State Nanopores Nat. Nanotechnol. 2012, 7, 257– 263
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    20
    Stochastic sensing of proteins with receptor-modified solid-state nanopores
    Wei, Ruoshan; Gatterdam, Volker; Wieneke, Ralph; Tampe, Robert; Rant, Ulrich
    Nature Nanotechnology (2012), 7 (4), 257-263CODEN: NNAABX; ISSN:1748-3387. (Nature Publishing Group)
    Solid-state nanopores are capable of the label-free anal. of single mols. It is possible to add biochem. selectivity by anchoring a mol. receptor inside the nanopore, but it is difficult to maintain single-mol. sensitivity in these modified nanopores. Here, we show that metalized silicon nitride nanopores chem. modified with nitrilotriacetic acid (NTA) receptors can be used for the stochastic sensing of proteins. The reversible binding and unbinding of the proteins to the receptors is obsd. in real time, and the interaction parameters are statistically analyzed from single-mol. binding events. To demonstrate the versatile nature of this approach, we detect His-tagged proteins and discriminate between the subclasses of rodent IgG antibodies.
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  21. 21
    Firnkes, M.; Pedone, D.; Knezevic, J.; Döblinger, M.; Rant, U. Electrically Facilitated Translocations of Proteins through Silicon Nitride Nanopores: Conjoint and Competitive Action of Diffusion, Electrophoresis, and Electroosmosis Nano Lett. 2010, 10, 2162– 2167
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    21
    Electrically Facilitated Translocations of Proteins through Silicon Nitride Nanopores: Conjoint and Competitive Action of Diffusion, Electrophoresis, and Electroosmosis
    Firnkes, Matthias; Pedone, Daniel; Knezevic, Jelena; Doeblinger, Markus; Rant, Ulrich
    Nano Letters (2010), 10 (6), 2162-2167CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)
    Solid-state nanopores bear great potential to be used to probe single proteins; however, the passage of proteins through nanopores was complex, and unexpected translocation behavior with respect to the passage direction, rate, and duration was obsd. Here the authors study the translocation of a model protein (avidin) through silicon nitride nanopores focusing on the electrokinetic effects that facilitate protein transport across the pore. The nanopore zeta potential ζpore and the protein zeta potential ζprotein are measured independently as a function of soln. pH. The authors' results reveal that electroosmotic transport may enhance or dominate and reverse electrophoretic transport in nanopores. The translocation behavior is rationalized by accounting for the charging states of the protein and the pore, resp.; the resulting translocation direction can be predicted according to the difference in zeta potentials, ζprotein - ζpore. When electrophoresis and electroosmosis cancel each other out, diffusion becomes an effective (and bias-independent) mechanism which facilitates protein transport across the pore at a significant rate.
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  22. 22
    Mourot, A.; Fehrentz, T.; Le Feuvre, Y.; Smith, C. M.; Herold, C.; Dalkara, D.; Nagy, F.; Trauner, D.; Kramer, R. H. Rapid Optical Control of Nociception with an Ion-Channel Photoswitch Nat. Methods 2012, 9, 396– 402
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    22
    Rapid optical control of nociception with an ion-channel photoswitch
    Mourot, Alexandre; Fehrentz, Timm; Le Feuvre, Yves; Smith, Caleb M.; Herold, Christian; Dalkara, Deniz; Nagy, Frederic; Trauner, Dirk; Kramer, Richard H.
    Nature Methods (2012), 9 (4_part1), 396-402CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)
    Local anesthetics effectively suppress pain sensation, but most of these compds. act nonselectively, inhibiting activity of all neurons. Moreover, their actions abate slowly, preventing precise spatial and temporal control of nociception. We developed a photoisomerizable mol., quaternary ammonium-azobenzene-quaternary ammonium (QAQ), that enables rapid and selective optical control of nociception. QAQ is membrane-impermeant and has no effect on most cells, but it infiltrates pain-sensing neurons through endogenous ion channels that are activated by noxious stimuli, primarily TRPV1. After QAQ accumulates intracellularly, it blocks voltage-gated ion channels in the trans form but not the cis form. QAQ enables reversible optical silencing of mouse nociceptive neuron firing without exogenous gene expression and can serve as a light-sensitive analgesic in rats in vivo. Because intracellular QAQ accumulation is a consequence of nociceptive ion-channel activity, QAQ-mediated photosensitization is a platform for understanding signaling mechanisms in acute and chronic pain.
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  23. 23
    Mantri, S.; Sapra, K. T.; Cheley, S.; Sharp, T. H.; Bayley, H. An Engineered Dimeric Protein Pore That Spans Adjacent Lipid Bilayers. Nat. Commun. 2013, 4.
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  24. 24
    Bayley, H.; Jayasinghe, L. Functional Engineered Channels and Pores Mol. Membr. Biol. 2004, 21, 209– 220
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    24
    Functional engineered channels and pores
    Bayley, Hagan; Jayasinghe, Lakmal
    Molecular Membrane Biology (2004), 21 (4), 209-220CODEN: MMEBE7; ISSN:0968-7688. (Taylor & Francis Ltd.)
    A review. Significant progress has been made in membrane protein engineering over the last 5 yr, based largely on the re-design of existing scaffolds. Engineering techniques that have been employed include direct genetic engineering, both covalent and non-covalent modification, unnatural amino acid mutagenesis, and total synthesis aided by chem. ligation of unprotected fragments. Combinatorial mutagenesis and directed evolution remain, by contrast, underemployed. Techniques for assembling and purifying heteromeric multisubunit pores have been improved. Progress in the de novo design of channels and pores has been slower. However, scientists are at the beginning of a new era in membrane protein engineering based on the accelerating acquisition of structural information, a better understanding of mol. motion in membrane proteins, tech. improvements in membrane protein refolding, and the application of computational approaches developed for sol. proteins. In addn., the next 5 yr should see further advances in the applications of engineered channels and pores, notably in therapeutics and sensor technol.
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  25. 25
    Sakai, N.; Matile, S. Synthetic Ion Channels Langmuir 2013, 29, 9031– 9040
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    Synthetic Ion Channels
    Sakai, Naomi; Matile, Stefan
    Langmuir (2013), 29 (29), 9031-9040CODEN: LANGD5; ISSN:0743-7463. (American Chemical Society)
    A review. The objective of this historical review is to recall the development of the field of synthetic ion channels over the past three decades. The most inspiring and influential breakthroughs with regard to structure and function are brought together to give the general reader an easily accessible understanding of the field. Pioneering work in the 1980s is followed by the golden age in the 1990s with structures emphasizing crown ethers, calixarenes, and peptide mimetics. Following the emergence of questions concerning specific functions such as ion selectivity, voltage gating, ligand gating and blockage, and with π-stacks, metal-org. scaffolds, and DNA origami, a new wave of innovative structures has emerged. The perspectives outline promising directions and major challenges waiting to be addressed.
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  26. 26
    Vargas Jentzsch, A.; Hennig, A.; Mareda, J.; Matile, S. Synthetic Ion Transporters That Work with Anion-Pi Interactions, Halogen Bonds, and Anion-Macrodipole Interactions Acc. Chem. Res. 2013, 46, 2791– 2800
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  27. 27
    Gokel, G. W.; Negin, S. Synthetic Ion Channels: From Pores to Biological Applications Acc. Chem. Res. 2013, 46, 2824– 2833
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    27
    Synthetic ion channels: From pores to biological applications
    Gokel, George W.; Negin, Saeedeh
    Accounts of Chemical Research (2013), 46 (12), 2824-2833CODEN: ACHRE4; ISSN:0001-4842. (American Chemical Society)
    A review. The authors describe the development of several diverse families of synthetic, membrane-active amphiphiles that form pores and facilitate transport within membrane bilayers. For the most part, the compds. are amphiphiles that insert into the bilayer and form pores either on their own or by self-assembly. The 1st family of synthetic ion channels prepd. in the authors' lab, the hydraphiles, use crown ethers as head groups and as a polar central element. In a range of biophys. studies, the authors have shown that the hydraphiles form unimol. pores that span the bilayer. They mediate the transport of Na+ and K+, but are blocked by Ag+. The hydraphiles are nonrectifying and disrupt ion homeostasis. As a result, these synthetic ion channels are toxic to various bacteria and yeast, a feature that has been used therapeutically in direct injection chemotherapy. The authors have also developed a family of amphiphilic heptapeptide ion transporters that select Cl- by >10-fold over K+ and show voltage-dependent gating. The formed pores are approx. dimeric, and variations in the N- and C-terminal anchor chains and the acids affect transport rates. Surprisingly, the longer N-terminal anchor chains lead to less transport but greater Cl- selectivity. A Pro residue, which is present in the ClC protein channel conductance pore, has proved to be crit. for Cl- transport selectivity. Pyrogallol[4]arenes are macrocycles formed by acid-catalyzed condensation of 4 1,2,3-trihydroxybenzenes with 4 aldehydes. The combination of 12 OH groups on one face of the macrocycle and 4 pendant alkyl chains has conferred considerable amphiphilicity to these compds. The pyrogallol[4]arenes insert into bilayer membranes and conduct ions. Based on exptl. evidence, the ions pass through a self-assembled pore comprising 4 or 5 amphiphiles rather than passing through the central opening of a single macrocycle. Pyrogallol[4]arenes constructed with branched chains are also amphiphilic and active in membranes. The pyrogallol[4]arene with 3-pentyl side-chains form a unique nanotube assembly and function as an ion channel in bilayer membranes. Finally, the authors have shown that dianilides of either isophthalic or dipicolinic acids, compds. which have been extensively studied as anion binders, can self-assemble to form pores within bilayers. The authors call these dianilides tris-arenes and have shown that they readily bind to phosphate anions. These structures also mediate the transport of DNA plasmids through vital bilayer membranes in Escherichia coli and in Saccharomyces cerevisiae. This transformation or transfection process occurs readily and without any apparent toxicity or mutagenicity.
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  28. 28
    Montenegro, J.; Ghadiri, M. R.; Granja, J. R. Ion Channel Models Based on Self-Assembling Cyclic Peptide Nanotubes Acc. Chem. Res. 2013, 46, 2955– 2965
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    28
    Ion Channel Models Based on Self-Assembling Cyclic Peptide Nanotubes
    Montenegro, Javier; Ghadiri, M. Reza; Granja, Juan R.
    Accounts of Chemical Research (2013), 46 (12), 2955-2965CODEN: ACHRE4; ISSN:0001-4842. (American Chemical Society)
    A review. The lipid bilayer membranes are Nature's dynamic structural motifs that individualize cells and keep ions, proteins, biopolymers and metabolites confined in the appropriate location. The compartmentalization and isolation of these mols. from the external media facilitate the sophisticated functions and connections between the different biol. processes accomplished by living organisms. However, cells require assistance from minimal energy shortcuts for the transport of mols. across membranes so that they can interact with the exterior and regulate their internal environments. Ion channels and pores stand out from all other possible transport mechanisms due to their high selectivity and efficiency in discriminating and transporting ions or mols. across membrane barriers. Nevertheless, the complexity of these smart "membrane holes" has driven researchers to develop simpler artificial structures with comparable performance to the natural systems. As a broad range of supramol. interactions have emerged as efficient tools for the rational design and prepn. of stable 3D superstructures, these results have stimulated the creativity of chemists to design synthetic mimics of natural active macromols. and even to develop artificial structures with functions and properties. In this Account, we highlight results from our labs. on the construction of artificial ion channel models that exploit the self-assembly of conformationally flat cyclic peptides (CPs) into supramol. nanotubes. Because of the straightforward synthesis of the cyclic peptide monomers and the complete control over the internal diam. and external surface properties of the resulting hollow tubular suprastructure, CPs are the optimal candidates for the fabrication of ion channels. The ion channel activity and selective transport of small mols. by these structures are examples of the great potential that cyclic peptide nanotubes show for the construction of functional artificial transmembrane transporters. Our experience to date suggests that the next steps for achieving conceptual devices with better performance and selectivity will derive from the topol. control over cyclic peptide assembly and the functionalization of the lumen.
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  29. 29
    Zhao, Y.; Cho, H.; Widanapathirana, L.; Zhang, S. Conformationally Controlled Oligocholate Membrane Transporters: Learning through Water Play Acc. Chem. Res. 2013, 46, 2763– 2772
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    There is no corresponding record for this reference.
  30. 30
    Maffeo, C.; Bhattacharya, S.; Yoo, J.; Wells, D.; Aksimentiev, A. Modeling and Simulation of Ion Channels Chem. Rev. 2012, 112, 6250– 6284
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    30
    Modeling and Simulation of Ion Channels
    Maffeo, Christopher; Bhattacharya, Swati; Yoo, Jejoong; Wells, David; Aksimentiev, Aleksei
    Chemical Reviews (Washington, DC, United States) (2012), 112 (12), 6250-6284CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)
    A review on recent achievements in modeling and simulation of ion channels.
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  31. 31
    Bell, N. A.; Engst, C. R.; Ablay, M.; Divitini, G.; Ducati, C.; Liedl, T.; Keyser, U. F. DNA Origami Nanopores Nano Lett. 2012, 12, 512– 517
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    31
    DNA Origami Nanopores
    Bell, Nicholas A. W.; Engst, Christian. R.; Ablay, Marc; Divitini, Giorgio; Ducati, Caterina; Liedl, Tim; Keyser, Ulrich F.
    Nano Letters (2012), 12 (1), 512-517CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)
    The authors demonstrate the assembly of functional hybrid nanopores for single mol. sensing by inserting DNA origami structures into solid-state nanopores. In the authors' expts., single artificial nanopores based on DNA origami are repeatedly inserted in and ejected from solid-state nanopores with diams. around 15 nm. The authors show that these hybrid nanopores can be employed for the detection of λ-DNA mols. The authors' approach paves the way for future development of adaptable single-mol. nanopore sensors based on the combination of solid-state nanopores and DNA self-assembly.
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    Wei, R.; Martin, T. G.; Rant, U.; Dietz, H. DNA Origami Gatekeepers for Solid-State Nanopores Angew. Chem., Int. Ed. 2012, 51, 4864– 4867
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    32
    DNA origami gatekeepers for solid-state nanopores
    Wei, Ruoshan; Martin, Thomas G.; Rant, Ulrich; Dietz, Hendrik
    Angewandte Chemie, International Edition (2012), 51 (20), 4864-4867, S4864/1-S4864/31CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
    The authors have presented DNA nanoplates that function with solid-state nanopores, which can be fabricated through std. electron beam lithog. The nanoplates are permeable to small ions, but the passage of macromols. can be controlled by including custom apertures. The chem. addressability of the DNA nanoplates enables bait-prey single-mol. sensing expts., as highlighted here by the sequence-specific detection of DNA snippets and genomic phage DNA. Applications in biomol. interaction screens and for detecting DNA sequences by hybridization are readily conceivable. High-resoln. sensing applications, such as elec. DNA sequencing will require reducing both the leakage current and current fluctuations.
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  33. 33
    Langecker, M.; Arnaut, V.; Martin, T. G.; List, J.; Renner, S.; Mayer, M.; Dietz, H.; Simmel, F. C. Synthetic Lipid Membrane Channels Formed by Designed DNA Nanostructures Science 2012, 338, 932– 936
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    33
    Synthetic Lipid Membrane Channels Formed by Designed DNA Nanostructures
    Langecker, Martin; Arnaut, Vera; Martin, Thomas G.; List, Jonathan; Renner, Stephan; Mayer, Michael; Dietz, Hendrik; Simmel, Friedrich C.
    Science (Washington, DC, United States) (2012), 338 (6109), 932-936CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
    We created nanometer-scale transmembrane channels in lipid bilayers by means of self-assembled DNA-based nanostructures. Scaffolded DNA origami was used to create a stem that penetrated and spanned a lipid membrane, as well as a barrel-shaped cap that adhered to the membrane, in part via 26 cholesterol moieties. In single-channel electrophysiol. measurements, we found similarities to the response of natural ion channels, such as conductances on the order of 1 nanosiemens and channel gating. More pronounced gating was seen for mutations in which a single DNA strand of the stem protruded into the channel. Single-mol. translocation expts. show that the synthetic channels can be used to discriminate single DNA mols.
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    Burns, J.; Stulz, E.; Howorka, S. Self-Assembled DNA Nanopores That Span Lipid Bilayers Nano Lett. 2013, 13, 2351– 2356
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    34
    Self-Assembled DNA Nanopores That Span Lipid Bilayers
    Burns, Jonathan R.; Stulz, Eugen; Howorka, Stefan
    Nano Letters (2013), 13 (6), 2351-2356CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)
    DNA nanotechnol. excels at rationally designing bottom-up structures that can functionally replicate naturally occurring proteins. Here the authors describe the design and generation of a stable DNA-based nanopore that structurally mimics the amphiphilic nature of protein pores and inserts into bilayers to support a steady transmembrane flow of ions. The pore carries an outer hydrophobic belt comprised of small chem. alkyl groups which mask the neg. charged oligonucleotide backbone. This modification overcomes the otherwise inherent energetic mismatch to the hydrophobic environment of the membrane. By merging the fields of nanopores and DNA nanotechnol., the authors expect that the small membrane-spanning DNA pore will help open up the design of entirely new mol. devices for a broad range of applications including sensing, elec. circuits, catalysis, and research into nanofluidics and controlled transmembrane transport.
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  35. 35
    Burns, J. R.; Göpfrich, K.; Wood, J. W.; Thacker, V. V.; Stulz, E.; Keyser, U. F.; Howorka, S. Lipid Bilayer-Spanning DNA Nanopores with a Bifunctional Porphyrin Anchor Angew. Chem., Int. Ed. 2013, 52, 12069– 12072
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    Burns, J. R.; Al-Juffali, N.; Janes, S. M.; Howorka, S. Membrane-Spanning DNA Nanopores with Cytotoxic Effect. Angew. Chem., Int. Ed. 2014, 53, 12466– 12470.
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    36
    Membrane-Spanning DNA Nanopores with Cytotoxic Effect
    Burns, Jonathan R.; Al-Juffali, Noura; Janes, Sam M.; Howorka, Stefan
    Angewandte Chemie, International Edition (2014), 53 (46), 12466-12470CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
    The authors examine whether a membrane-spanning DNA nanopore can interact with cancer cells and potentially trigger cell death. The nanopore was composed of a bundle of six DNA duplexes folded from six DNA strands. The authors' DNA nanopores are the first DNA nanostructures to cause the killing of biol. cells by targeting cellular membranes. A bilayer-spanning hydrophobic belt composed of ethylated phosphorothioate groups on the outside of pore with 2 nm inner width was key to achieve cell death. The cytotoxic nanopores have the potential to be used as novel and valuable research tools and anti-cancer agents.
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  37. 37
    Yin, P.; Hariadi, R. F.; Sahu, S.; Choi, H. M.; Park, S. H.; Labean, T. H.; Reif, J. H. Programming DNA Tube Circumferences Science 2008, 321, 824– 826
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    37
    Programming DNA Tube Circumferences
    Yin, Peng; Hariadi, Rizal F.; Sahu, Sudheer; Choi, Harry M. T.; Park, Sung Ha; LaBean, Thomas H.; Reif, John H.
    Science (Washington, DC, United States) (2008), 321 (5890), 824-826CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
    Synthesizing mol. tubes with monodisperse, programmable circumferences is an important goal shared by nanotechnol., materials science, and supermol. chem. We program mol. tube circumferences by specifying the complementarity relationships between modular domains in a 42-base single-stranded DNA motif. Single-step annealing results in the self-assembly of long tubes displaying monodisperse circumferences of 4, 5, 6, 7, 8, 10, or 20 DNA helixes.
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  38. 38
    Wang, T.; Schiffels, D.; Cuesta, S. M.; Fygenson, D. K.; Seeman, N. C. Design and Characterization of 1d Nanotubes and 2d Periodic Arrays Self-Assembled from DNA Multi-Helix Bundles J. Am. Chem. Soc. 2012, 134, 1606– 1616
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    38
    Design and Characterization of 1D Nanotubes and 2D Periodic Arrays Self-Assembled from DNA Multi-Helix Bundles
    Wang, Tong; Schiffels, Daniel; Martinez Cuesta, Sergio; Kuchnir Fygenson, Deborah; Seeman, Nadrian C.
    Journal of the American Chemical Society (2012), 134 (3), 1606-1616CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Among the key goals of structural DNA nanotechnol. are to build highly ordered structures self-assembled from individual DNA motifs in 1D, 2D, and finally 3D. All three of these goals have been achieved with a variety of motifs. Here, we report the design and characterization of 1D nanotubes and 2D arrays assembled from three novel DNA motifs, the 6-helix bundle (6HB), the 6-helix bundle flanked by two helixes in the same plane (6HB+2), and the 6-helix bundle flanked by three helixes in a trigonal arrangement (6HB+3). Long DNA nanotubes have been assembled from all three motifs. Such nanotubes are likely to have applications in structural DNA nanotechnol., so it is important to characterize their phys. properties. Prominent among these are their rigidities, described by their persistence lengths, which we report here. We find large persistence lengths in all species, around 1-5 μm. The magnitudes of the persistence lengths are clearly related to the designs of the linkages between the unit motifs. Both the 6HB+2 and the 6HB+3 motifs have been successfully used to produce well-ordered 2D periodic arrays via sticky-ended cohesion.
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  39. 39
    Goodman, R. P.; Schaap, I. A.; Tardin, C. F.; Erben, C. M.; Berry, R. M.; Schmidt, C. F.; Turberfield, A. J. Rapid Chiral Assembly of Rigid DNA Building Blocks for Molecular Nanofabrication Science 2005, 310, 1661– 1665
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    39
    Rapid Chiral Assembly of Rigid DNA Building Blocks for Molecular Nanofabrication
    Goodman, R. P.; Schaap, I. A. T.; Tardin, C. F.; Erben, C. M.; Berry, R. M.; Schmidt, C. F.; Turberfield, A. J.
    Science (Washington, DC, United States) (2005), 310 (5754), 1661-1665CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
    Practical components for three-dimensional mol. nanofabrication must be simple to produce, stereopure, rigid, and adaptable. We report a family of DNA tetrahedra, less than 10 nm on a side, that can self-assemble in seconds with near-quant. yield of one diastereomer. They can be connected by programmable DNA linkers. Their triangulated architecture confers structural stability; by compressing a DNA tetrahedron with an at. force microscope, we have measured the axial compressibility of DNA and obsd. the buckling of the double helix under high loads.
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  40. 40
    Mitchell, N.; Schlapak, R.; Kastner, M.; Armitage, D.; Chrzanowski, W.; Riener, J.; Hinterdorfer, P.; Ebner, A.; Howorka, S. A DNA Nanostructure for the Functional Assembly of Chemical Groups at Tuneable Stoichiometry and Defined Nanoscale Geometry Angew. Chem., Int. Ed. 2009, 48, 525– 527
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    Plesa, C.; Ananth, A. N.; Linko, V.; Gülcher, C.; Katan, A. J.; Dietz, H.; Dekker, C. Ionic Permeability and Mechanical Properties of DNA Origami Nanoplates on Solid-State Nanopores ACS Nano 2014, 8, 35– 43
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    41
    Ionic Permeability and Mechanical Properties of DNA Origami Nanoplates on Solid-State Nanopores
    Plesa, Calin; Ananth, Adithya N.; Linko, Veikko; Guelcher, Coen; Katan, Allard J.; Dietz, Hendrik; Dekker, Cees
    ACS Nano (2014), 8 (1), 35-43CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)
    While DNA origami is a popular and versatile platform, its structural properties are still poorly understood. In this study we use solid-state nanopores to investigate the ionic permeability and mech. properties of DNA origami nanoplates. DNA origami nanoplates of various designs are docked onto solid-state nanopores where we subsequently measure their ionic conductance. The ionic permeability is found to be high for all origami nanoplates. We observe the conductance of docked nanoplates, relative to the bare nanopore conductance, to increase as a function of pore diam., as well as to increase upon lowering the ionic strength. The honeycomb lattice nanoplate is found to have slightly better overall performance over other plate designs. After docking, we often observe spontaneous discrete jumps in the current, a process which can be attributed to mech. buckling. All nanoplates show a nonlinear current-voltage dependence with a lower conductance at higher applied voltages, which we attribute to a phys. bending deformation of the nanoplates under the applied force. At sufficiently high voltage (force), the nanoplates are strongly deformed and can be pulled through the nanopore. These data show that DNA origami nanoplates are typically very permeable to ions and exhibit a no. of unexpected mech. properties, which are interesting in their own right, but also need to be considered in the future design of DNA origami nanostructures.
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  42. 42
    Yoo, J.; Aksimentiev, A. In Situ Structure and Dynamics of DNA Origami Determined through Molecular Dynamics Simulations Proc. Natl. Acad. Sci. U.S.A. 2013, 110, 20099– 20104
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    Göpfrich, K.; Kulkarni, C. V.; Pambos, O. J.; Keyser, U. F. Lipid Nanobilayers to Host Biological Nanopores for DNA Translocations Langmuir 2013, 29, 355– 364
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    Perozo, E.; Cortes, D. M.; Sompornpisut, P.; Kloda, A.; Martinac, B. Open Channel Structure of Mscl and the Gating Mechanism of Mechanosensitive Channels Nature 2002, 418, 942– 948
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    44
    Open channel structure of MscL and the gating mechanism of mechanosensitive channels
    Perozo, Eduardo; Cortes, D. Marien; Sompornpisut, Pornthep; Kloda, Anna; Martinac, Boris
    Nature (London, United Kingdom) (2002), 418 (6901), 942-948CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
    Mechanosensitive channels act as membrane-embedded mechano-elec. switches, opening a large water-filled pore in response to lipid bilayer deformations. This process is crit. to the response of living organisms to direct phys. stimulation, such as in touch, hearing and osmoregulation. Here, we have detd. the structural rearrangements that underlie these events in the large prokaryotic mechanosensitive channel (MscL) using ESR spectroscopy and site-directed spin labeling. MscL was trapped in both the open and in an intermediate closed state by modulating bilayer morphol. Transition to the intermediate state is characterized by small movements in the first transmembrane helix (TM1). Subsequent transitions to the open state are accompanied by massive rearrangements in both TM1 and TM2, as shown by large increases in probe dynamics, solvent accessibility and the elimination of all intersubunit spin-spin interactions. The open state is highly dynamic, supporting a water-filled pore of at least 25 Å, lined mostly by TM1. These structures suggest a plausible mol. mechanism of gating in mechanosensitive channels.
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  45. 45
    Merzlyak, P. G.; Yuldasheva, L. N.; Rodrigues, C. G.; Carneiro, C. M. M.; Krasilnikov, O. V.; Bezrukov, S. M. Polymeric Nonelectrolytes to Probe Pore Geometry: Application to the Alpha-Toxin Transmembrane Channel Biophys. J. 1999, 77, 3023– 3033
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    Krasilnikov, O. V.; Rodrigues, C. G.; Bezrukov, S. M. Single Polymer Molecules in a Protein Nanopore in the Limit of a Strong Polymer-Pore Attraction Phys. Rev. Lett. 2006, 97, 018301
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    Nguyen, T.; Brewer, A.; Stulz, E. Duplex Stabilization and Energy Transfer in Zipper Porphyrin-DNA Angew. Chem., Int. Ed. 2009, 48, 1974– 1977
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    Brewer, A.; Siligardi, G.; Neylon, C.; Stulz, E. Introducing Structural Flexibility into Porphyrin–DNA Zipper Arrays Org. Biomol. Chem. 2011, 9, 777– 782
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    Ortega, A.; Amoros, D.; de la Torre, J. G. Prediction of Hydrodynamic and Other Solution Properties of Rigid Proteins from Atomic- and Residue-Level Models Biophys. J. 2011, 101, 892– 898
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    Prediction of Hydrodynamic and Other Solution Properties of Rigid Proteins from Atomic- and Residue-Level Models
    Ortega, A.; Amoros, D.; Garcia de la Torre, J.
    Biophysical Journal (2011), 101 (4), 892-898CODEN: BIOJAU; ISSN:0006-3495. (Cell Press)
    Here the authors extend the ability to predict hydrodynamic coeffs. and other soln. properties of rigid macromol. structures from at.-level structures, implemented in the computer program HYDROPRO, to models with lower, residue-level resoln. Whereas in the former case there is one bead per nonhydrogen atom, the latter contains one bead per amino acid (or nucleotide) residue, thus allowing calcns. when at. resoln. is not available or coarse-grained models are preferred. The authors parameterized the effective hydrodynamic radius of the elements in the at.- and residue-level models using a very large set of exptl. data for translational and rotational coeffs. (intrinsic viscosity and radius of gyration) for >50 proteins. The authors also extended the calcns. to very large proteins and macromol. complexes, such as the whole 70S ribosome. The authors show that with proper parameterization, the two levels of resoln. yield similar and rather good agreement with exptl. data. The new version of HYDROPRO, in addn. to considering various computational and modeling schemes, is far more efficient computationally and can be handled with the use of a graphical interface.
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  50. 50
    Ke, Y. G.; Sharma, J.; Liu, M. H.; Jahn, K.; Liu, Y.; Yan, H. Scaffolded DNA Origami of a DNA Tetrahedron Molecular Container Nano Lett. 2009, 9, 2445– 2447
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    50
    Scaffolded DNA Origami of a DNA Tetrahedron Molecular Container
    Ke, Yonggang; Sharma, Jaswinder; Liu, Minghui; Jahn, Kasper; Liu, Yan; Yan, Hao
    Nano Letters (2009), 9 (6), 2445-2447CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)
    A strategy of scaffolded DNA origami to design and construct 3D mol. cages of tetrahedron geometry with inside vol. closed by triangular faces was described. Each edge of the triangular face is ∼54 nm in dimension. The estd. total external vol. and the internal cavity of the triangular pyramid are about 1.8 × 10-23 and 1.5 × 10-23 m3, resp. Correct formation of the tetrahedron DNA cage was verified by gel electrophoresis, at. force microscopy, transmission electron microscopy, and dynamic light scattering techniques.
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  51. 51
    Börjesson, K.; Wiberg, J.; El-Sagheer, A. H.; Ljungdahl, T.; Mårtensson, J.; Brown, T.; Nordén, B.; Albinsson, B. Functionalized Nanostructures: Redox-Active Porphyrin Anchors for Supramolecular DNA Assemblies ACS Nano 2010, 4, 5037– 5046
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    Woller, J. G.; Börjesson, K.; Svedhem, S.; Albinsson, B. Reversible Hybridization of DNA Anchored to a Lipid Membrane via Porphyrin Langmuir 2012, 28, 1944– 1953
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    Baaken, G.; Ankri, N.; Schuler, A. K.; Ruhe, J.; Behrends, J. C. Nanopore-Based Single-Molecule Mass Spectrometry on a Lipid Membrane Microarray ACS Nano 2011, 5, 8080– 8088
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    53
    Nanopore-Based Single-Molecule Mass Spectrometry on a Lipid Membrane Microarray
    Baaken, Gerhard; Ankri, Norbert; Schuler, Anne-Katrin; Ruhe, Jurgen; Behrends, Jan C.
    ACS Nano (2011), 5 (10), 8080-8088CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)
    We report on parallel high-resoln. elec. single-mol. anal. on a chip-based nanopore microarray. Lipid bilayers of <20 μm diam. contg. single alpha-hemolysin pores were formed on arrays on subpicoliter cavities contg. individual microelectrodes (microelectrode cavity array, MECA), and ion conductance-based single mol. mass spectrometry was performed on mixts. of poly(ethylene glycol) mols. of different length. We thereby demonstrate the function of the MECA device as a chip-based platform for array-format nanopore recordings with a resoln. at least equal to that of established single microbilayer supports. We conclude that devices based on MECAs may enable more widespread anal. use of nanopores by providing the high throughput and ease of operation of a high-d. array format while maintaining or exceeding the precision of state-of-the-art microbilayer recordings.
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  54. 54
    Hille, B. Ion Channels of Excitable Membranes, 3rd ed.; Sinauer Associates, 2001.
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    Rostovtseva, T. K.; Nestorovich, E. M.; Bezrukov, S. M. Partitioning of Differently Sized Poly(ethylene glycol)s into Ompf Porin Biophys. J. 2002, 82, 160– 169
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    Partitioning of differently sized poly(ethylene glycol)s into OmpF porin
    Rostovtseva, Tatiana K.; Nestorovich, Ekaterina M.; Bezrukov, Sergey M.
    Biophysical Journal (2002), 82 (1, Pt. 1), 160-169CODEN: BIOJAU; ISSN:0006-3495. (Biophysical Society)
    To understand the physics of polymer equil. and dynamics in the confines of ion channel pores, we study partitioning of poly(ethylene glycol)s (PEGs) of different mol. wts. into the bacterial porin, OmpF. Thermodn. and kinetic parameters of partitioning are deduced from the effects of polymer addn. on ion currents through single OmpF channels reconstituted into planar lipid bilayer membranes. The equil. partition coeff. is inferred from the av. redn. of channel conductance in the presence of PEG; rates of polymer exchange between the pore and the bulk are estd. from PEG-induced conductance noise. Partition coeff. as a function of polymer wt. is best fitted by a "compressed exponential" with the compression factor of 1.65. This finding demonstrates that PEG partitioning into the OmpF channel pore has sharper dependence on polymer mol. wt. than predictions of hard-sphere, random-flight, or scaling models. A 1360-Da polymer separates regimes of partitioning and exclusion. Comparison of its characteristic size with the size of a 2200-Da polymer previously found to sep. these regimes for the α-toxin shows good agreement with the x-ray structural data for these channels. The PEG-induced conductance noise is compatible with the polymer mobility reduced inside the OmpF pore by an order of magnitude relatively to its value in bulk soln.
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  56. 56
    Bezrukov, S. M.; Vodyanoy, I.; Brutyan, R. A.; Kasianowicz, J. J. Dynamics and Free Energy of Polymers Partitioning into a Nanoscale Pore Macromolecules 1996, 29, 8517– 8522
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    56
    Dynamics and free energy of polymers partitioning into a nanoscale pore
    Bezrukov, Sergey M.; Vodyanoy, Igor; Brutyan, Rafik A.; Kasianowicz, John J.
    Macromolecules (1996), 29 (26), 8517-8522CODEN: MAMOBX; ISSN:0024-9297. (American Chemical Society)
    Membrane-bound proteinaceous nanoscale pores allow us to simultaneously observe the thermodn. and kinetic properties of differently sized polymers within their confines. We det. the dynamic partitioning of polyethylene glycol (PEG) into the pore formed by Staphylococcus aureus α-toxin and evaluate the free energy of polymer confinement by measuring polymer-induced changes to the pore ionic conductance. The free energy deduced from the partition coeff. has a sharper dependence on polymer length (or wt.) than scaling theory predicts. Moreover, the polymer-induced conductance fluctuations show a striking nonmonotonic dependence on the polymer mol. wt. The movement of polymer inside the pore is characterized by a diffusion coeff. that is orders of magnitude smaller than that for polymer in the bulk aq. soln., which suggests that PEG has an attractive interaction with the pore. Using an ad-hoc approach, we show that a simple mol. wt.-dependent modification of the polymer's diffusion coeff. accounts for these results, but only qual. Given that PEG assocs. with hydrophobic regions in proteins, we also conclude that, contrary to the conventional view of ion channels, the aq. cavity of the α-toxin pore's interior is, to some extent, hydrophobic.
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  57. 57
    Merzlyak, P. G.; Capistrano, M. F. P.; Valeva, A.; Kasianowicz, J. J.; Krasilnikov, O. V. Conductance and Ion Selectivity of a Mesoscopic Protein Nanopore Probed with Cysteine Scanning Mutagenesis Biophys. J. 2005, 89, 3059– 3070
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    Robertson, J. W. F.; Rodrigues, C. G.; Stanford, V. M.; Rubinson, K. A.; Krasilnikov, O. V.; Kasianowicz, J. J. Single-Molecule Mass Spectrometry in Solution Using a Solitary Nanopore Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 8207– 8211
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    Single-molecule mass spectrometry in solution using a solitary nanopore
    Robertson, Joseph W. F.; Rodrigues, Claudio G.; Stanford, Vincent M.; Rubinson, Kenneth A.; Krasilnikov, Oleg V.; Kasianowicz, John J.
    Proceedings of the National Academy of Sciences of the United States of America (2007), 104 (20), 8207-8211CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    The authors introduce a 2-dimensional method for mass spectrometry in soln. that is based on the interaction between a nanometer-scale pore and analytes. As an example, poly(ethylene glycol) mols. that enter a single α-hemolysin pore cause distinct mass-dependent conductance states with characteristic mean residence times. The conductance-based mass spectrum clearly resolves the repeat unit of ethylene glycol, and the mean residence time increases monotonically with the poly(ethylene glycol) mass. This technique could prove useful for the real-time characterization of mols. in soln.
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  59. 59
    Reiner, J. E.; Kasianowicz, J. J.; Nablo, B. J.; Robertson, J. W. Theory for Polymer Analysis Using Nanopore-Based Single-Molecule Mass Spectrometry Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 12080– 12085
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    59
    Theory for polymer analysis using nanopore-based single-molecule mass spectrometry
    Reiner, Joseph E.; Kasianowicz, John J.; Nablo, Brian J.; Robertson, Joseph W. F.
    Proceedings of the National Academy of Sciences of the United States of America (2010), 107 (27), 12080-12085, S12080/1-S12080/4CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    Nanometer-scale pores have demonstrated potential for the elec. detection, quantification, and characterization of mols. for biomedical applications and the chem. anal. of polymers. Despite extensive research in the nanopore sensing field, there is a paucity of theor. models that incorporate the interactions between chems. (i.e., solute, solvent, analyte, and nanopore). Here, we develop a model that simultaneously describes both the current blockade depth and residence times caused by individual poly(ethylene glycol) (PEG) mols. in a single α-hemolysin ion channel. Modeling polymer-cation binding leads to a description of two significant effects: a redn. in the mobile cation concn. inside the pore and an increase in the affinity between the polymer and the pore. The model was used to est. the free energy of formation for K+-PEG inside the nanopore (≈ -49.7 meV) and the free energy of PEG partitioning into the nanopore (≈ 0.76 meV per ethylene glycol monomer). The results suggest that rational, phys. models for the anal. of analyte-nanopore interactions will develop the full potential of nanopore-based sensing for chem. and biol. applications.
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  60. 60
    Nablo, B. J.; Halverson, K. M.; Robertson, J. W.; Nguyen, T. L.; Panchal, R. G.; Gussio, R.; Bavari, S.; Krasilnikov, O. V.; Kasianowicz, J. J. Sizing the Bacillus Anthracis Pa63 Channel with Nonelectrolyte Poly(ethylene glycols) Biophys. J. 2008, 95, 1157– 1164
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    Breton, M. F.; Discala, F.; Bacri, L.; Foster, D.; Pelta, J.; Oulchaled, A. Exploration of Neutral Versus Polyelectrolyte Behavior of Poly(ethylene glycol)s in Alkali Ion Solutions Using Single-Nanopore Recording J. Phys. Chem. Lett. 2013, 4, 2202– 2208
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    61
    Exploration of Neutral Versus Polyelectrolyte Behavior of Poly(ethylene glycol)s in Alkali Ion Solutions using Single-Nanopore Recording
    Breton, Marie France; Discala, Francoise; Bacri, Laurent; Foster, Damien; Pelta, Juan; Oukhaled, Abdelghani
    Journal of Physical Chemistry Letters (2013), 4 (13), 2202-2208CODEN: JPCLCD; ISSN:1948-7185. (American Chemical Society)
    We examine the effect of alkali ions (Li+, Na+, K+, Rb+, Cs+) on the partitioning of neutral and flexible poly(ethylene glycol) into the alpha-hemolysin (α-HL) nanopore for a large range of applied voltages at high salt concn. The neutral polymer behaves as if charged, i.e., the event frequency increases with applied voltage, and the residence times decrease with the elec. force for all cations except Li+. In contrast, in the presence of LiCl, we find the classical partitioning behavior of neutral polymers, i.e., the event frequency and the residence times are independent of the applied voltage. Assuming that lithium does not assoc. with PEG enabled us to quantify the relative magnitude of the entropic and enthalpic contribution to the free- energy barrier and the no. of complexed cations using two different arguments; the first est. is based on the balance of forces, and the second is found comparing the blockade ratio in the presence of LiCl (no complexed ions) to the blockade ratio of chains in the presence of the other salts (with complexed ions). This est. is in agreement with recent simulations. These findings demonstrate that the nanopore could prove useful for the rapid probing of the capabilities of different neutral mols. to form complexes with different ions.
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    Bezrukov, S. M.; Kasianowicz, J. J. The Charge State of an Ion Channel Controls Neutral Polymer Entry into Its Pore Eur. Biophys. J. Biophys. 1997, 26, 471– 476
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    62
    The charge state of an ion channel controls neutral polymer entry into its pore
    Bezrukov, Sergey M.; Kasianowicz, John J.
    European Biophysics Journal (1997), 26 (6), 471-476CODEN: EBJOE8; ISSN:0175-7571. (Springer)
    Electrostatic potentials created by fixed or induced charges regulate many cellular phenomena including the rate of ion transport through proteinaceous ion channels. Nanometer-scale pores of these channels also play a crit. role in the transport of charged and neutral macromols. We demonstrate here that, surprisingly, changing the charge state of a channel markedly alters the ability of nonelectrolyte polymers to enter the channel's pore. Specifically, we show that the partitioning of differently-sized linear nonelectrolyte polymers of ethylene glycol into the Staphylococcus aureus α-hemolysin channel is altered by the soln. pH. Protonating some of the channel side chains decreases the characteristic polymer size (mol. wt.) that can enter the pore by ∼25 but increases the ionic current by ∼15. Thus, the "steric" and "elec." size of the channel change in opposite directions. The results suggest that effects due to polymer and channel hydration are crucial for polymer transport through such pores.
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  63. 63
    Douglas, S. M.; Marblestone, A. H.; Teerapittayanon, S.; Vazquez, A.; Church, G. M.; Shih, W. M. Rapid Prototyping of 3d DNA-Origami Shapes with Cadnano Nucleic Acids Res. 2009, 37, 5001– 5006
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    63
    Rapid prototyping of 3D DNA-origami shapes with caDNAno
    Douglas, Shawn M.; Marblestone, Adam H.; Teerapittayanon, Surat; Vazquez, Alejandro; Church, George M.; Shih, William M.
    Nucleic Acids Research (2009), 37 (15), 5001-5006CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)
    DNA nanotechnol. exploits the programmable specificity afforded by base-pairing to produce self-assembling macromol. objects of custom shape. For building megadalton-scale DNA nanostructures, a long scaffold' strand can be employed to template the assembly of hundreds of oligonucleotide staple' strands into a planar antiparallel array of cross-linked helixes. The authors recently adapted this scaffolded DNA origami' method to producing 3-dimensional shapes formed as pleated layers of double helixes constrained to a honeycomb lattice. However, completing the required design steps can be cumbersome and time-consuming. Here the authors present caDNAno, an open-source software package with a graphical user interface that aids in the design of DNA sequences for folding 3-dimensional honeycomb-pleated shapes rectangular-block motifs were designed, assembled, and analyzed to identify a well-behaved motif that could serve as a building block for future studies. The use of caDNAno significantly reduces the effort required to design 3-dimensional DNA-origami structures. The software is available at http://cadnano.org/, along with example designs and video tutorials demonstrating their construction. The source code is released under the MIT license.
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  64. 64
    Fendt, L.-A.; Bouamaied, I.; Thoeni, S.; Amiot, N.; Stulz, E. DNA as Supramolecular Scaffold for Porphyrin Arrays on the Nanometer Scale J. Am. Chem. Soc. 2007, 129, 15319– 15329
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    64
    DNA as Supramolecular Scaffold for Porphyrin Arrays on the Nanometer Scale
    Fendt, Leslie-Anne; Bouamaied, Imenne; Thoeni, Sandra; Amiot, Nicolas; Stulz, Eugen
    Journal of the American Chemical Society (2007), 129 (49), 15319-15329CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Tetra-Ph porphyrin substituted deoxyuridine was used as a building block to create discrete multiporphyrin arrays via site specific incorporation into DNA. The successful covalent attachment of up to 11 tetra-Ph porphyrins in a row onto DNA shows that there is virtually no limitation in the amt. of substituents, and the porphyrin arrays thus obtained reach the nanometer scale (∼10 nm). The porphyrin substituents are located in the major groove of the dsDNA and destabilize the duplex by ΔTm 5-7° per porphyrin modification. Force-field structure minimization shows that the porphyrins are either in-line with the groove in isolated modifications or aligned parallel to the nucleobases in adjacent modifications. The CD signals of the porphyrins are dominated by a neg. peak arising from the intrinsic properties of the building block. In the single strands, the porphyrins induce stabilization of a secondary helical structure which is confined to the porphyrin modified part. This arrangement can be reproduced by force-field minimization and reveals an elongated helical arrangement compared to the double helix of the porphyrin-DNA. This secondary structure is disrupted above ∼55° (Tp) which is shown by various melting expts. Both absorption and emission spectroscopy disclose electronic interactions between the porphyrin units upon stacking along the outer rim of the DNA leading to a broadening of the absorbance and a quenching of the emission. The single-stranded and double-stranded form show different spectroscopic properties due to the different arrangement of the porphyrins. Above Tp the electronic properties (absorption and emission) of the porphyrins change compared to room temp. measurements due to the disruption of the porphyrin stacking at high temp. The covalent attachment of porphyrins to DNA is therefore a suitable way of creating helical stacks of porphyrins on the nanometer scale.
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  65. 65
    Clifton, L. A.; Sanders, M. R.; Castelletto, V.; Rogers, S. E.; Heenan, R. K.; Neylon, C.; Frazier, R. A.; Green, R. J. Puroindoline-a, a Lipid Binding Protein from Common Wheat, Spontaneously Forms Prolate Protein Micelles in Solution Phys. Chem. Chem. Phys. 2011, 13, 8881– 8888
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    Kreir, M.; Farre, C.; Beckler, M.; George, M.; Fertig, N. Rapid Screening of Membrane Protein Activity: Electrophysiological Analysis of Ompf Reconstituted in Proteoliposomes Lab Chip 2008, 8, 587– 595
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    66
    Rapid screening of membrane protein activity: electrophysiological analysis of OmpF reconstituted in proteoliposomes
    Kreir, Mohamed; Farre, Cecilia; Beckler, Matthias; George, Michael; Fertig, Niels
    Lab on a Chip (2008), 8 (4), 587-595CODEN: LCAHAM; ISSN:1473-0197. (Royal Society of Chemistry)
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  • Abstract

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    Figure 1

    Figure 1. DNA-based membrane-spanning pore and the two recording setups used to measure the conductance properties of the nanopore. (A) Schematic drawing of a six-duplex-bundle nanopore composed of six DNA oligonucleotides (colored) carrying two tetraphenyl porphyrin tags (black, inset), which anchor the pore into the lipid bilayer. The porphyrin anchor is attached via the acetylene group (inset) to position 5 of a uridine base (not shown). (B) Schematic drawing of a microcavity unit with a suspended planar lipid bilayer. Only one of the 16 units of the recording chip is shown. The nanopores were incorporated into the preformed lipid bilayers. (C) In the second recording setup, the nanopores were initially incorporated into lipid vesicles, which were then spread over the orifice of a 200 nm diameter glass nanopipette (transparent blue). The grounded reference electrodes in B and C are drawn as small, light gray circles, while the other electrode is in dark gray.

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    Figure 2

    Figure 2. DNA nanopores are self-assembled and embedded into lipid bilayers. (A) Native agarose gel electrophoresis of DNA nanopores without (lane 1) and with the porphyrin lipid anchor (lane 2) 100 bp molecular weight marker (lane 3). (B) Dynamic light scattering trace of a DNA pore with lipid anchor (red line) and without lipid anchor (black dashed line). (C, D) Bright-field (left) and confocal fluorescence images (right) of DPhPC vesicles incubated (C) with DNA pores carrying the fluorescent porphyrin tag and (D) with Cy3-labeled nanopores lacking a lipid anchor. The bright spots in (C) may represent individual or clusters of pores. Excitation wavelength: 532 nm. Scale bar: 5 μm.

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    Figure 3

    Figure 3. High-conductance state of membrane-embedded DNA nanopores at low voltages corresponds to an open pore as shown by PEG sizing. The traces were obtained with planar lipid bilayer recordings. The high conductance state is color-coded in red. (A) Representative ionic current trace at +20 mV. (B) Histogram of channel conductances obtained from measurements at +20 mV. (C) Current traces of individual pores in the absence and presence of PEG molecules of indicated mean molecular mass. (D) Pore blockade as a function of the hydrodynamic diameter of PEG. Between PEG 200 and PEG 600, there is a significant change in relative pore blockade. With PEG 1000 and above the effect is tapering off. This upper turning point indicates that PEG molecules are being excluded from the pore lumen. The hydrodynamic diameter of PEG 1000 (approximately 1.9 nm) is hence assumed to reflect the diameter of the DNA pore. The data present averages and the minima and maxima from five independent recordings (PEG 62 to PEG 400) and three independent recordings (PEG 600 to PEG 3350).

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    Figure 4

    Figure 4. Low-conductance state for DNA nanopores occurs at higher voltages. The high conductance state is color-coded in red; the low conductance state is coded in blue. The traces were obtained with planar lipid bilayer recordings. (A) Current traces for 20 mV voltage steps showing that potentials of <−80 mV and >+60 mV lead to a low-conductance state. (B) IV curve displaying the averages and standard deviation from 7 single-channel current traces. (C) Representative single-channel current trace at +100 mV. The amplitude of the blockade, Ab, the event dwell time, τoff, and the inter-event interval, τon, are defined. (D) Cumulative all-point histogram of 38 single-channel current traces for +20 to +100 mV in 20 mV steps illustrating the voltage-dependent switching between the low-voltage open state and the high-voltage partially closed state. (E) Probability of observing the open state as a function of voltage. The probability (red line) is defined as the count for the high-conductance state divided by all counts in the all-point histogram. The black line represents the frequency of occurrence for the low-amplitude events as reported in Table S2. Error bars for the open probability are not given as the conventional way of obtaining averages from Poisson-fits for τon value distributions is not possible when the probabilities are obtained by Gaussian fitting of peaks in all-point histograms. For the frequency of occurrence, there were not enough closing events at small voltages to derive meaningful errors. (F) Traces of a single DNA pore recorded at multiple consecutive voltage ramps running from −100 to +100 mV. The occurrence of the low-conductance state at −20 mV in the third trace rather than at usual voltages of around −80 mV is explained by a memory effect of this particular DNA structure, which had experienced previous voltage ramps and pore closures.

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    Figure 5

    Figure 5. Conductance analysis of DNA nanopores embedded in lipid bilayers mounted across a nanopipette orifice. (A) Single-channel current trace of a pore in the high-conductance state at +40 mV. (B) IV curves for single DNA nanopores in the high and the low-conductance state and a lipid bilayer without pores as reference. The data spread for the two pore conductances can be estimated from the peak width in the all-point histograms in panel D. (C, D) Single-channel current trace fluctuating from the (C) low- and (D) high-conductance state to a completely closed pore. (E) Voltage-stepped current trace of a low-conductance state switching to complete and permanent pore closure at +80 mV. (F) Conductance histogram derived from single-channel current recordings at +100 mV. (G) Cumulative all-point histogram of 21 current traces from +20 to +100 mV in 20 mV steps. (H) Probability of observing the open state as a function of voltage. The probabilities were derived from all-point histograms as described in Figure 4. Filled squares show data from the nanopipette-mounted membrane, while empty triangles are data from planar bilayer recordings.

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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XksVensbc%253D&md5=5ee259148c2d2f112479e648a619095c
    15. 15
      Movileanu, L.; Howorka, S.; Braha, O.; Bayley, H. Detecting Protein Analytes That Modulate Transmembrane Movement of a Polymer Chain within a Single Protein Pore Nat. Biotechnol. 2000, 18, 1091– 1095
      15
      Detecting protein analytes that modulate transmembrane movement of a polymer chain within a single protein pore
      Movileanu, Liviu; Howorka, Stefan; Braha, Orit; Bayley, Hagan
      Nature Biotechnology (2000), 18 (10), 1091-1095CODEN: NABIF9; ISSN:1087-0156. (Nature America Inc.)
      Here we describe a new type of biosensor element for detecting proteins in soln. at nanomolar concns. We tethered a 3.4 kDa polyethylene glycol chain at a defined site within the lumen of the transmembrane protein pore formed by staphylococcal α-hemolysin. The free end of the polymer was covalently attached to a biotin mol. On incorporation of the modified pore into a lipid bilayer, the biotinyl group moves from one side of the membrane to the other, and is detected by reversible capture with a mutant streptavidin. The capture events are obsd. as changes in ionic current passing through single pores in planar bilayers. Accordingly, the modified pore allows detection of a protein analyte at the single-mol. level, facilitating both quantification and identification through a distinctive current signature. The approach has higher time resoln. compared with other kinetic measurements, such as those obtained by surface plasmon resonance.
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    16. 16
      Howorka, S.; Nam, J.; Bayley, H.; Kahne, D. Stochastic Detection of Monovalent and Multivalent Protein-Ligand Interactions Angew. Chem., Int. Ed. 2004, 43, 842– 846
      There is no corresponding record for this reference.
    17. 17
      Rotem, D.; Jayasinghe, L.; Salichou, M.; Bayley, H. Protein Detection by Nanopores Equipped with Aptamers J. Am. Chem. Soc. 2012, 134, 2781– 2787
      17
      Protein Detection by Nanopores Equipped with Aptamers
      Rotem, Dvir; Jayasinghe, Lakmal; Salichou, Maria; Bayley, Hagan
      Journal of the American Chemical Society (2012), 134 (5), 2781-2787CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Protein nanopores have been used as stochastic sensors for the detection of analytes that range from small mols. to proteins. In this approach, individual analyte mols. modulate the ionic current flowing through a single nanopore. Here, a new type of stochastic sensor based on an αHL pore modified with an aptamer is described. The aptamer is bound to the pore by hybridization to an oligonucleotide that is attached covalently through a disulfide bond to a single cysteine residue near a mouth of the pore. The authors show that the binding of thrombin to a 15-mer DNA aptamer, which forms a cation-stabilized quadruplex, alters the ionic current through the pore. The approach allows the quantification of nanomolar concns. of thrombin, and provides assocn. and dissocn. rate consts. and equil. dissocn. consts. for thrombin·aptamer interactions. Aptamer-based nanopores have the potential to be integrated into arrays for the parallel detection of multiple analytes.
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    18. 18
      Howorka, S.; Siwy, Z. S. Nanopores as Protein Sensors Nat. Biotechnol. 2012, 30, 506– 507
      There is no corresponding record for this reference.
    19. 19
      Stoloff, D. H.; Wanunu, M. Recent Trends in Nanopores for Biotechnology Curr. Opin. Biotechnol. 2013, 24, 699– 704
      19
      Recent trends in nanopores for biotechnology
      Stoloff, Daniel H.; Wanunu, Meni
      Current Opinion in Biotechnology (2013), 24 (4), 699-704CODEN: CUOBE3; ISSN:0958-1669. (Elsevier B.V.)
      A review. Nanopore technol. employs a nanoscale hole in an insulating membrane to stochastically sense with high throughput individual biomols. in soln. The generality of the nanopore detection principle and the ease of single-mol. detection suggest many potential applications of nanopores in biotechnol. Recent progress has been made with nanopore fabrication and sophistication, as well as with applications in DNA/protein mapping, biomol. structure anal., protein detection, and DNA sequencing. In addn., concepts for DNA sequencing devices have been suggested, and computational efforts have been made. The state of the nanopore field is maturing and given the right type of nanopore and operating conditions, nearly every application could revolutionize medicine in terms of speed, cost, and quality. In this review, we summarize progress in nanopores for biotechnol. applications over the past 2-3 years.
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    20. 20
      Wei, R. S.; Gatterdam, V.; Wieneke, R.; Tampe, R.; Rant, U. Stochastic Sensing of Proteins with Receptor-Modified Solid-State Nanopores Nat. Nanotechnol. 2012, 7, 257– 263
      20
      Stochastic sensing of proteins with receptor-modified solid-state nanopores
      Wei, Ruoshan; Gatterdam, Volker; Wieneke, Ralph; Tampe, Robert; Rant, Ulrich
      Nature Nanotechnology (2012), 7 (4), 257-263CODEN: NNAABX; ISSN:1748-3387. (Nature Publishing Group)
      Solid-state nanopores are capable of the label-free anal. of single mols. It is possible to add biochem. selectivity by anchoring a mol. receptor inside the nanopore, but it is difficult to maintain single-mol. sensitivity in these modified nanopores. Here, we show that metalized silicon nitride nanopores chem. modified with nitrilotriacetic acid (NTA) receptors can be used for the stochastic sensing of proteins. The reversible binding and unbinding of the proteins to the receptors is obsd. in real time, and the interaction parameters are statistically analyzed from single-mol. binding events. To demonstrate the versatile nature of this approach, we detect His-tagged proteins and discriminate between the subclasses of rodent IgG antibodies.
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    21. 21
      Firnkes, M.; Pedone, D.; Knezevic, J.; Döblinger, M.; Rant, U. Electrically Facilitated Translocations of Proteins through Silicon Nitride Nanopores: Conjoint and Competitive Action of Diffusion, Electrophoresis, and Electroosmosis Nano Lett. 2010, 10, 2162– 2167
      21
      Electrically Facilitated Translocations of Proteins through Silicon Nitride Nanopores: Conjoint and Competitive Action of Diffusion, Electrophoresis, and Electroosmosis
      Firnkes, Matthias; Pedone, Daniel; Knezevic, Jelena; Doeblinger, Markus; Rant, Ulrich
      Nano Letters (2010), 10 (6), 2162-2167CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)
      Solid-state nanopores bear great potential to be used to probe single proteins; however, the passage of proteins through nanopores was complex, and unexpected translocation behavior with respect to the passage direction, rate, and duration was obsd. Here the authors study the translocation of a model protein (avidin) through silicon nitride nanopores focusing on the electrokinetic effects that facilitate protein transport across the pore. The nanopore zeta potential ζpore and the protein zeta potential ζprotein are measured independently as a function of soln. pH. The authors' results reveal that electroosmotic transport may enhance or dominate and reverse electrophoretic transport in nanopores. The translocation behavior is rationalized by accounting for the charging states of the protein and the pore, resp.; the resulting translocation direction can be predicted according to the difference in zeta potentials, ζprotein - ζpore. When electrophoresis and electroosmosis cancel each other out, diffusion becomes an effective (and bias-independent) mechanism which facilitates protein transport across the pore at a significant rate.
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    22. 22
      Mourot, A.; Fehrentz, T.; Le Feuvre, Y.; Smith, C. M.; Herold, C.; Dalkara, D.; Nagy, F.; Trauner, D.; Kramer, R. H. Rapid Optical Control of Nociception with an Ion-Channel Photoswitch Nat. Methods 2012, 9, 396– 402
      22
      Rapid optical control of nociception with an ion-channel photoswitch
      Mourot, Alexandre; Fehrentz, Timm; Le Feuvre, Yves; Smith, Caleb M.; Herold, Christian; Dalkara, Deniz; Nagy, Frederic; Trauner, Dirk; Kramer, Richard H.
      Nature Methods (2012), 9 (4_part1), 396-402CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)
      Local anesthetics effectively suppress pain sensation, but most of these compds. act nonselectively, inhibiting activity of all neurons. Moreover, their actions abate slowly, preventing precise spatial and temporal control of nociception. We developed a photoisomerizable mol., quaternary ammonium-azobenzene-quaternary ammonium (QAQ), that enables rapid and selective optical control of nociception. QAQ is membrane-impermeant and has no effect on most cells, but it infiltrates pain-sensing neurons through endogenous ion channels that are activated by noxious stimuli, primarily TRPV1. After QAQ accumulates intracellularly, it blocks voltage-gated ion channels in the trans form but not the cis form. QAQ enables reversible optical silencing of mouse nociceptive neuron firing without exogenous gene expression and can serve as a light-sensitive analgesic in rats in vivo. Because intracellular QAQ accumulation is a consequence of nociceptive ion-channel activity, QAQ-mediated photosensitization is a platform for understanding signaling mechanisms in acute and chronic pain.
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    23. 23
      Mantri, S.; Sapra, K. T.; Cheley, S.; Sharp, T. H.; Bayley, H. An Engineered Dimeric Protein Pore That Spans Adjacent Lipid Bilayers. Nat. Commun. 2013, 4.
      There is no corresponding record for this reference.
    24. 24
      Bayley, H.; Jayasinghe, L. Functional Engineered Channels and Pores Mol. Membr. Biol. 2004, 21, 209– 220
      24
      Functional engineered channels and pores
      Bayley, Hagan; Jayasinghe, Lakmal
      Molecular Membrane Biology (2004), 21 (4), 209-220CODEN: MMEBE7; ISSN:0968-7688. (Taylor & Francis Ltd.)
      A review. Significant progress has been made in membrane protein engineering over the last 5 yr, based largely on the re-design of existing scaffolds. Engineering techniques that have been employed include direct genetic engineering, both covalent and non-covalent modification, unnatural amino acid mutagenesis, and total synthesis aided by chem. ligation of unprotected fragments. Combinatorial mutagenesis and directed evolution remain, by contrast, underemployed. Techniques for assembling and purifying heteromeric multisubunit pores have been improved. Progress in the de novo design of channels and pores has been slower. However, scientists are at the beginning of a new era in membrane protein engineering based on the accelerating acquisition of structural information, a better understanding of mol. motion in membrane proteins, tech. improvements in membrane protein refolding, and the application of computational approaches developed for sol. proteins. In addn., the next 5 yr should see further advances in the applications of engineered channels and pores, notably in therapeutics and sensor technol.
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    25. 25
      Sakai, N.; Matile, S. Synthetic Ion Channels Langmuir 2013, 29, 9031– 9040
      25
      Synthetic Ion Channels
      Sakai, Naomi; Matile, Stefan
      Langmuir (2013), 29 (29), 9031-9040CODEN: LANGD5; ISSN:0743-7463. (American Chemical Society)
      A review. The objective of this historical review is to recall the development of the field of synthetic ion channels over the past three decades. The most inspiring and influential breakthroughs with regard to structure and function are brought together to give the general reader an easily accessible understanding of the field. Pioneering work in the 1980s is followed by the golden age in the 1990s with structures emphasizing crown ethers, calixarenes, and peptide mimetics. Following the emergence of questions concerning specific functions such as ion selectivity, voltage gating, ligand gating and blockage, and with π-stacks, metal-org. scaffolds, and DNA origami, a new wave of innovative structures has emerged. The perspectives outline promising directions and major challenges waiting to be addressed.
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    26. 26
      Vargas Jentzsch, A.; Hennig, A.; Mareda, J.; Matile, S. Synthetic Ion Transporters That Work with Anion-Pi Interactions, Halogen Bonds, and Anion-Macrodipole Interactions Acc. Chem. Res. 2013, 46, 2791– 2800
      There is no corresponding record for this reference.
    27. 27
      Gokel, G. W.; Negin, S. Synthetic Ion Channels: From Pores to Biological Applications Acc. Chem. Res. 2013, 46, 2824– 2833
      27
      Synthetic ion channels: From pores to biological applications
      Gokel, George W.; Negin, Saeedeh
      Accounts of Chemical Research (2013), 46 (12), 2824-2833CODEN: ACHRE4; ISSN:0001-4842. (American Chemical Society)
      A review. The authors describe the development of several diverse families of synthetic, membrane-active amphiphiles that form pores and facilitate transport within membrane bilayers. For the most part, the compds. are amphiphiles that insert into the bilayer and form pores either on their own or by self-assembly. The 1st family of synthetic ion channels prepd. in the authors' lab, the hydraphiles, use crown ethers as head groups and as a polar central element. In a range of biophys. studies, the authors have shown that the hydraphiles form unimol. pores that span the bilayer. They mediate the transport of Na+ and K+, but are blocked by Ag+. The hydraphiles are nonrectifying and disrupt ion homeostasis. As a result, these synthetic ion channels are toxic to various bacteria and yeast, a feature that has been used therapeutically in direct injection chemotherapy. The authors have also developed a family of amphiphilic heptapeptide ion transporters that select Cl- by >10-fold over K+ and show voltage-dependent gating. The formed pores are approx. dimeric, and variations in the N- and C-terminal anchor chains and the acids affect transport rates. Surprisingly, the longer N-terminal anchor chains lead to less transport but greater Cl- selectivity. A Pro residue, which is present in the ClC protein channel conductance pore, has proved to be crit. for Cl- transport selectivity. Pyrogallol[4]arenes are macrocycles formed by acid-catalyzed condensation of 4 1,2,3-trihydroxybenzenes with 4 aldehydes. The combination of 12 OH groups on one face of the macrocycle and 4 pendant alkyl chains has conferred considerable amphiphilicity to these compds. The pyrogallol[4]arenes insert into bilayer membranes and conduct ions. Based on exptl. evidence, the ions pass through a self-assembled pore comprising 4 or 5 amphiphiles rather than passing through the central opening of a single macrocycle. Pyrogallol[4]arenes constructed with branched chains are also amphiphilic and active in membranes. The pyrogallol[4]arene with 3-pentyl side-chains form a unique nanotube assembly and function as an ion channel in bilayer membranes. Finally, the authors have shown that dianilides of either isophthalic or dipicolinic acids, compds. which have been extensively studied as anion binders, can self-assemble to form pores within bilayers. The authors call these dianilides tris-arenes and have shown that they readily bind to phosphate anions. These structures also mediate the transport of DNA plasmids through vital bilayer membranes in Escherichia coli and in Saccharomyces cerevisiae. This transformation or transfection process occurs readily and without any apparent toxicity or mutagenicity.
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    28. 28
      Montenegro, J.; Ghadiri, M. R.; Granja, J. R. Ion Channel Models Based on Self-Assembling Cyclic Peptide Nanotubes Acc. Chem. Res. 2013, 46, 2955– 2965
      28
      Ion Channel Models Based on Self-Assembling Cyclic Peptide Nanotubes
      Montenegro, Javier; Ghadiri, M. Reza; Granja, Juan R.
      Accounts of Chemical Research (2013), 46 (12), 2955-2965CODEN: ACHRE4; ISSN:0001-4842. (American Chemical Society)
      A review. The lipid bilayer membranes are Nature's dynamic structural motifs that individualize cells and keep ions, proteins, biopolymers and metabolites confined in the appropriate location. The compartmentalization and isolation of these mols. from the external media facilitate the sophisticated functions and connections between the different biol. processes accomplished by living organisms. However, cells require assistance from minimal energy shortcuts for the transport of mols. across membranes so that they can interact with the exterior and regulate their internal environments. Ion channels and pores stand out from all other possible transport mechanisms due to their high selectivity and efficiency in discriminating and transporting ions or mols. across membrane barriers. Nevertheless, the complexity of these smart "membrane holes" has driven researchers to develop simpler artificial structures with comparable performance to the natural systems. As a broad range of supramol. interactions have emerged as efficient tools for the rational design and prepn. of stable 3D superstructures, these results have stimulated the creativity of chemists to design synthetic mimics of natural active macromols. and even to develop artificial structures with functions and properties. In this Account, we highlight results from our labs. on the construction of artificial ion channel models that exploit the self-assembly of conformationally flat cyclic peptides (CPs) into supramol. nanotubes. Because of the straightforward synthesis of the cyclic peptide monomers and the complete control over the internal diam. and external surface properties of the resulting hollow tubular suprastructure, CPs are the optimal candidates for the fabrication of ion channels. The ion channel activity and selective transport of small mols. by these structures are examples of the great potential that cyclic peptide nanotubes show for the construction of functional artificial transmembrane transporters. Our experience to date suggests that the next steps for achieving conceptual devices with better performance and selectivity will derive from the topol. control over cyclic peptide assembly and the functionalization of the lumen.
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    29. 29
      Zhao, Y.; Cho, H.; Widanapathirana, L.; Zhang, S. Conformationally Controlled Oligocholate Membrane Transporters: Learning through Water Play Acc. Chem. Res. 2013, 46, 2763– 2772
      There is no corresponding record for this reference.
    30. 30
      Maffeo, C.; Bhattacharya, S.; Yoo, J.; Wells, D.; Aksimentiev, A. Modeling and Simulation of Ion Channels Chem. Rev. 2012, 112, 6250– 6284
      30
      Modeling and Simulation of Ion Channels
      Maffeo, Christopher; Bhattacharya, Swati; Yoo, Jejoong; Wells, David; Aksimentiev, Aleksei
      Chemical Reviews (Washington, DC, United States) (2012), 112 (12), 6250-6284CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)
      A review on recent achievements in modeling and simulation of ion channels.
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    31. 31
      Bell, N. A.; Engst, C. R.; Ablay, M.; Divitini, G.; Ducati, C.; Liedl, T.; Keyser, U. F. DNA Origami Nanopores Nano Lett. 2012, 12, 512– 517
      31
      DNA Origami Nanopores
      Bell, Nicholas A. W.; Engst, Christian. R.; Ablay, Marc; Divitini, Giorgio; Ducati, Caterina; Liedl, Tim; Keyser, Ulrich F.
      Nano Letters (2012), 12 (1), 512-517CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)
      The authors demonstrate the assembly of functional hybrid nanopores for single mol. sensing by inserting DNA origami structures into solid-state nanopores. In the authors' expts., single artificial nanopores based on DNA origami are repeatedly inserted in and ejected from solid-state nanopores with diams. around 15 nm. The authors show that these hybrid nanopores can be employed for the detection of λ-DNA mols. The authors' approach paves the way for future development of adaptable single-mol. nanopore sensors based on the combination of solid-state nanopores and DNA self-assembly.
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    32. 32
      Wei, R.; Martin, T. G.; Rant, U.; Dietz, H. DNA Origami Gatekeepers for Solid-State Nanopores Angew. Chem., Int. Ed. 2012, 51, 4864– 4867
      32
      DNA origami gatekeepers for solid-state nanopores
      Wei, Ruoshan; Martin, Thomas G.; Rant, Ulrich; Dietz, Hendrik
      Angewandte Chemie, International Edition (2012), 51 (20), 4864-4867, S4864/1-S4864/31CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
      The authors have presented DNA nanoplates that function with solid-state nanopores, which can be fabricated through std. electron beam lithog. The nanoplates are permeable to small ions, but the passage of macromols. can be controlled by including custom apertures. The chem. addressability of the DNA nanoplates enables bait-prey single-mol. sensing expts., as highlighted here by the sequence-specific detection of DNA snippets and genomic phage DNA. Applications in biomol. interaction screens and for detecting DNA sequences by hybridization are readily conceivable. High-resoln. sensing applications, such as elec. DNA sequencing will require reducing both the leakage current and current fluctuations.
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    33. 33
      Langecker, M.; Arnaut, V.; Martin, T. G.; List, J.; Renner, S.; Mayer, M.; Dietz, H.; Simmel, F. C. Synthetic Lipid Membrane Channels Formed by Designed DNA Nanostructures Science 2012, 338, 932– 936
      33
      Synthetic Lipid Membrane Channels Formed by Designed DNA Nanostructures
      Langecker, Martin; Arnaut, Vera; Martin, Thomas G.; List, Jonathan; Renner, Stephan; Mayer, Michael; Dietz, Hendrik; Simmel, Friedrich C.
      Science (Washington, DC, United States) (2012), 338 (6109), 932-936CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
      We created nanometer-scale transmembrane channels in lipid bilayers by means of self-assembled DNA-based nanostructures. Scaffolded DNA origami was used to create a stem that penetrated and spanned a lipid membrane, as well as a barrel-shaped cap that adhered to the membrane, in part via 26 cholesterol moieties. In single-channel electrophysiol. measurements, we found similarities to the response of natural ion channels, such as conductances on the order of 1 nanosiemens and channel gating. More pronounced gating was seen for mutations in which a single DNA strand of the stem protruded into the channel. Single-mol. translocation expts. show that the synthetic channels can be used to discriminate single DNA mols.
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    34. 34
      Burns, J.; Stulz, E.; Howorka, S. Self-Assembled DNA Nanopores That Span Lipid Bilayers Nano Lett. 2013, 13, 2351– 2356
      34
      Self-Assembled DNA Nanopores That Span Lipid Bilayers
      Burns, Jonathan R.; Stulz, Eugen; Howorka, Stefan
      Nano Letters (2013), 13 (6), 2351-2356CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)
      DNA nanotechnol. excels at rationally designing bottom-up structures that can functionally replicate naturally occurring proteins. Here the authors describe the design and generation of a stable DNA-based nanopore that structurally mimics the amphiphilic nature of protein pores and inserts into bilayers to support a steady transmembrane flow of ions. The pore carries an outer hydrophobic belt comprised of small chem. alkyl groups which mask the neg. charged oligonucleotide backbone. This modification overcomes the otherwise inherent energetic mismatch to the hydrophobic environment of the membrane. By merging the fields of nanopores and DNA nanotechnol., the authors expect that the small membrane-spanning DNA pore will help open up the design of entirely new mol. devices for a broad range of applications including sensing, elec. circuits, catalysis, and research into nanofluidics and controlled transmembrane transport.
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    35. 35
      Burns, J. R.; Göpfrich, K.; Wood, J. W.; Thacker, V. V.; Stulz, E.; Keyser, U. F.; Howorka, S. Lipid Bilayer-Spanning DNA Nanopores with a Bifunctional Porphyrin Anchor Angew. Chem., Int. Ed. 2013, 52, 12069– 12072
      There is no corresponding record for this reference.
    36. 36
      Burns, J. R.; Al-Juffali, N.; Janes, S. M.; Howorka, S. Membrane-Spanning DNA Nanopores with Cytotoxic Effect. Angew. Chem., Int. Ed. 2014, 53, 12466– 12470.
      36
      Membrane-Spanning DNA Nanopores with Cytotoxic Effect
      Burns, Jonathan R.; Al-Juffali, Noura; Janes, Sam M.; Howorka, Stefan
      Angewandte Chemie, International Edition (2014), 53 (46), 12466-12470CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
      The authors examine whether a membrane-spanning DNA nanopore can interact with cancer cells and potentially trigger cell death. The nanopore was composed of a bundle of six DNA duplexes folded from six DNA strands. The authors' DNA nanopores are the first DNA nanostructures to cause the killing of biol. cells by targeting cellular membranes. A bilayer-spanning hydrophobic belt composed of ethylated phosphorothioate groups on the outside of pore with 2 nm inner width was key to achieve cell death. The cytotoxic nanopores have the potential to be used as novel and valuable research tools and anti-cancer agents.
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    37. 37
      Yin, P.; Hariadi, R. F.; Sahu, S.; Choi, H. M.; Park, S. H.; Labean, T. H.; Reif, J. H. Programming DNA Tube Circumferences Science 2008, 321, 824– 826
      37
      Programming DNA Tube Circumferences
      Yin, Peng; Hariadi, Rizal F.; Sahu, Sudheer; Choi, Harry M. T.; Park, Sung Ha; LaBean, Thomas H.; Reif, John H.
      Science (Washington, DC, United States) (2008), 321 (5890), 824-826CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
      Synthesizing mol. tubes with monodisperse, programmable circumferences is an important goal shared by nanotechnol., materials science, and supermol. chem. We program mol. tube circumferences by specifying the complementarity relationships between modular domains in a 42-base single-stranded DNA motif. Single-step annealing results in the self-assembly of long tubes displaying monodisperse circumferences of 4, 5, 6, 7, 8, 10, or 20 DNA helixes.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXptlyhs70%253D&md5=a3fa1ab11537528d1b820aefa3ccc84f
    38. 38
      Wang, T.; Schiffels, D.; Cuesta, S. M.; Fygenson, D. K.; Seeman, N. C. Design and Characterization of 1d Nanotubes and 2d Periodic Arrays Self-Assembled from DNA Multi-Helix Bundles J. Am. Chem. Soc. 2012, 134, 1606– 1616
      38
      Design and Characterization of 1D Nanotubes and 2D Periodic Arrays Self-Assembled from DNA Multi-Helix Bundles
      Wang, Tong; Schiffels, Daniel; Martinez Cuesta, Sergio; Kuchnir Fygenson, Deborah; Seeman, Nadrian C.
      Journal of the American Chemical Society (2012), 134 (3), 1606-1616CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Among the key goals of structural DNA nanotechnol. are to build highly ordered structures self-assembled from individual DNA motifs in 1D, 2D, and finally 3D. All three of these goals have been achieved with a variety of motifs. Here, we report the design and characterization of 1D nanotubes and 2D arrays assembled from three novel DNA motifs, the 6-helix bundle (6HB), the 6-helix bundle flanked by two helixes in the same plane (6HB+2), and the 6-helix bundle flanked by three helixes in a trigonal arrangement (6HB+3). Long DNA nanotubes have been assembled from all three motifs. Such nanotubes are likely to have applications in structural DNA nanotechnol., so it is important to characterize their phys. properties. Prominent among these are their rigidities, described by their persistence lengths, which we report here. We find large persistence lengths in all species, around 1-5 μm. The magnitudes of the persistence lengths are clearly related to the designs of the linkages between the unit motifs. Both the 6HB+2 and the 6HB+3 motifs have been successfully used to produce well-ordered 2D periodic arrays via sticky-ended cohesion.
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    39. 39
      Goodman, R. P.; Schaap, I. A.; Tardin, C. F.; Erben, C. M.; Berry, R. M.; Schmidt, C. F.; Turberfield, A. J. Rapid Chiral Assembly of Rigid DNA Building Blocks for Molecular Nanofabrication Science 2005, 310, 1661– 1665
      39
      Rapid Chiral Assembly of Rigid DNA Building Blocks for Molecular Nanofabrication
      Goodman, R. P.; Schaap, I. A. T.; Tardin, C. F.; Erben, C. M.; Berry, R. M.; Schmidt, C. F.; Turberfield, A. J.
      Science (Washington, DC, United States) (2005), 310 (5754), 1661-1665CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
      Practical components for three-dimensional mol. nanofabrication must be simple to produce, stereopure, rigid, and adaptable. We report a family of DNA tetrahedra, less than 10 nm on a side, that can self-assemble in seconds with near-quant. yield of one diastereomer. They can be connected by programmable DNA linkers. Their triangulated architecture confers structural stability; by compressing a DNA tetrahedron with an at. force microscope, we have measured the axial compressibility of DNA and obsd. the buckling of the double helix under high loads.
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    40. 40
      Mitchell, N.; Schlapak, R.; Kastner, M.; Armitage, D.; Chrzanowski, W.; Riener, J.; Hinterdorfer, P.; Ebner, A.; Howorka, S. A DNA Nanostructure for the Functional Assembly of Chemical Groups at Tuneable Stoichiometry and Defined Nanoscale Geometry Angew. Chem., Int. Ed. 2009, 48, 525– 527
      There is no corresponding record for this reference.
    41. 41
      Plesa, C.; Ananth, A. N.; Linko, V.; Gülcher, C.; Katan, A. J.; Dietz, H.; Dekker, C. Ionic Permeability and Mechanical Properties of DNA Origami Nanoplates on Solid-State Nanopores ACS Nano 2014, 8, 35– 43
      41
      Ionic Permeability and Mechanical Properties of DNA Origami Nanoplates on Solid-State Nanopores
      Plesa, Calin; Ananth, Adithya N.; Linko, Veikko; Guelcher, Coen; Katan, Allard J.; Dietz, Hendrik; Dekker, Cees
      ACS Nano (2014), 8 (1), 35-43CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)
      While DNA origami is a popular and versatile platform, its structural properties are still poorly understood. In this study we use solid-state nanopores to investigate the ionic permeability and mech. properties of DNA origami nanoplates. DNA origami nanoplates of various designs are docked onto solid-state nanopores where we subsequently measure their ionic conductance. The ionic permeability is found to be high for all origami nanoplates. We observe the conductance of docked nanoplates, relative to the bare nanopore conductance, to increase as a function of pore diam., as well as to increase upon lowering the ionic strength. The honeycomb lattice nanoplate is found to have slightly better overall performance over other plate designs. After docking, we often observe spontaneous discrete jumps in the current, a process which can be attributed to mech. buckling. All nanoplates show a nonlinear current-voltage dependence with a lower conductance at higher applied voltages, which we attribute to a phys. bending deformation of the nanoplates under the applied force. At sufficiently high voltage (force), the nanoplates are strongly deformed and can be pulled through the nanopore. These data show that DNA origami nanoplates are typically very permeable to ions and exhibit a no. of unexpected mech. properties, which are interesting in their own right, but also need to be considered in the future design of DNA origami nanostructures.
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    42. 42
      Yoo, J.; Aksimentiev, A. In Situ Structure and Dynamics of DNA Origami Determined through Molecular Dynamics Simulations Proc. Natl. Acad. Sci. U.S.A. 2013, 110, 20099– 20104
      There is no corresponding record for this reference.
    43. 43
      Göpfrich, K.; Kulkarni, C. V.; Pambos, O. J.; Keyser, U. F. Lipid Nanobilayers to Host Biological Nanopores for DNA Translocations Langmuir 2013, 29, 355– 364
      There is no corresponding record for this reference.
    44. 44
      Perozo, E.; Cortes, D. M.; Sompornpisut, P.; Kloda, A.; Martinac, B. Open Channel Structure of Mscl and the Gating Mechanism of Mechanosensitive Channels Nature 2002, 418, 942– 948
      44
      Open channel structure of MscL and the gating mechanism of mechanosensitive channels
      Perozo, Eduardo; Cortes, D. Marien; Sompornpisut, Pornthep; Kloda, Anna; Martinac, Boris
      Nature (London, United Kingdom) (2002), 418 (6901), 942-948CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
      Mechanosensitive channels act as membrane-embedded mechano-elec. switches, opening a large water-filled pore in response to lipid bilayer deformations. This process is crit. to the response of living organisms to direct phys. stimulation, such as in touch, hearing and osmoregulation. Here, we have detd. the structural rearrangements that underlie these events in the large prokaryotic mechanosensitive channel (MscL) using ESR spectroscopy and site-directed spin labeling. MscL was trapped in both the open and in an intermediate closed state by modulating bilayer morphol. Transition to the intermediate state is characterized by small movements in the first transmembrane helix (TM1). Subsequent transitions to the open state are accompanied by massive rearrangements in both TM1 and TM2, as shown by large increases in probe dynamics, solvent accessibility and the elimination of all intersubunit spin-spin interactions. The open state is highly dynamic, supporting a water-filled pore of at least 25 Å, lined mostly by TM1. These structures suggest a plausible mol. mechanism of gating in mechanosensitive channels.
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    45. 45
      Merzlyak, P. G.; Yuldasheva, L. N.; Rodrigues, C. G.; Carneiro, C. M. M.; Krasilnikov, O. V.; Bezrukov, S. M. Polymeric Nonelectrolytes to Probe Pore Geometry: Application to the Alpha-Toxin Transmembrane Channel Biophys. J. 1999, 77, 3023– 3033
      There is no corresponding record for this reference.
    46. 46
      Krasilnikov, O. V.; Rodrigues, C. G.; Bezrukov, S. M. Single Polymer Molecules in a Protein Nanopore in the Limit of a Strong Polymer-Pore Attraction Phys. Rev. Lett. 2006, 97, 018301
      There is no corresponding record for this reference.
    47. 47
      Nguyen, T.; Brewer, A.; Stulz, E. Duplex Stabilization and Energy Transfer in Zipper Porphyrin-DNA Angew. Chem., Int. Ed. 2009, 48, 1974– 1977
      There is no corresponding record for this reference.
    48. 48
      Brewer, A.; Siligardi, G.; Neylon, C.; Stulz, E. Introducing Structural Flexibility into Porphyrin–DNA Zipper Arrays Org. Biomol. Chem. 2011, 9, 777– 782
      There is no corresponding record for this reference.
    49. 49
      Ortega, A.; Amoros, D.; de la Torre, J. G. Prediction of Hydrodynamic and Other Solution Properties of Rigid Proteins from Atomic- and Residue-Level Models Biophys. J. 2011, 101, 892– 898
      49
      Prediction of Hydrodynamic and Other Solution Properties of Rigid Proteins from Atomic- and Residue-Level Models
      Ortega, A.; Amoros, D.; Garcia de la Torre, J.
      Biophysical Journal (2011), 101 (4), 892-898CODEN: BIOJAU; ISSN:0006-3495. (Cell Press)
      Here the authors extend the ability to predict hydrodynamic coeffs. and other soln. properties of rigid macromol. structures from at.-level structures, implemented in the computer program HYDROPRO, to models with lower, residue-level resoln. Whereas in the former case there is one bead per nonhydrogen atom, the latter contains one bead per amino acid (or nucleotide) residue, thus allowing calcns. when at. resoln. is not available or coarse-grained models are preferred. The authors parameterized the effective hydrodynamic radius of the elements in the at.- and residue-level models using a very large set of exptl. data for translational and rotational coeffs. (intrinsic viscosity and radius of gyration) for >50 proteins. The authors also extended the calcns. to very large proteins and macromol. complexes, such as the whole 70S ribosome. The authors show that with proper parameterization, the two levels of resoln. yield similar and rather good agreement with exptl. data. The new version of HYDROPRO, in addn. to considering various computational and modeling schemes, is far more efficient computationally and can be handled with the use of a graphical interface.
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    50. 50
      Ke, Y. G.; Sharma, J.; Liu, M. H.; Jahn, K.; Liu, Y.; Yan, H. Scaffolded DNA Origami of a DNA Tetrahedron Molecular Container Nano Lett. 2009, 9, 2445– 2447
      50
      Scaffolded DNA Origami of a DNA Tetrahedron Molecular Container
      Ke, Yonggang; Sharma, Jaswinder; Liu, Minghui; Jahn, Kasper; Liu, Yan; Yan, Hao
      Nano Letters (2009), 9 (6), 2445-2447CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)
      A strategy of scaffolded DNA origami to design and construct 3D mol. cages of tetrahedron geometry with inside vol. closed by triangular faces was described. Each edge of the triangular face is ∼54 nm in dimension. The estd. total external vol. and the internal cavity of the triangular pyramid are about 1.8 × 10-23 and 1.5 × 10-23 m3, resp. Correct formation of the tetrahedron DNA cage was verified by gel electrophoresis, at. force microscopy, transmission electron microscopy, and dynamic light scattering techniques.
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    51. 51
      Börjesson, K.; Wiberg, J.; El-Sagheer, A. H.; Ljungdahl, T.; Mårtensson, J.; Brown, T.; Nordén, B.; Albinsson, B. Functionalized Nanostructures: Redox-Active Porphyrin Anchors for Supramolecular DNA Assemblies ACS Nano 2010, 4, 5037– 5046
      There is no corresponding record for this reference.
    52. 52
      Woller, J. G.; Börjesson, K.; Svedhem, S.; Albinsson, B. Reversible Hybridization of DNA Anchored to a Lipid Membrane via Porphyrin Langmuir 2012, 28, 1944– 1953
      There is no corresponding record for this reference.
    53. 53
      Baaken, G.; Ankri, N.; Schuler, A. K.; Ruhe, J.; Behrends, J. C. Nanopore-Based Single-Molecule Mass Spectrometry on a Lipid Membrane Microarray ACS Nano 2011, 5, 8080– 8088
      53
      Nanopore-Based Single-Molecule Mass Spectrometry on a Lipid Membrane Microarray
      Baaken, Gerhard; Ankri, Norbert; Schuler, Anne-Katrin; Ruhe, Jurgen; Behrends, Jan C.
      ACS Nano (2011), 5 (10), 8080-8088CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)
      We report on parallel high-resoln. elec. single-mol. anal. on a chip-based nanopore microarray. Lipid bilayers of <20 μm diam. contg. single alpha-hemolysin pores were formed on arrays on subpicoliter cavities contg. individual microelectrodes (microelectrode cavity array, MECA), and ion conductance-based single mol. mass spectrometry was performed on mixts. of poly(ethylene glycol) mols. of different length. We thereby demonstrate the function of the MECA device as a chip-based platform for array-format nanopore recordings with a resoln. at least equal to that of established single microbilayer supports. We conclude that devices based on MECAs may enable more widespread anal. use of nanopores by providing the high throughput and ease of operation of a high-d. array format while maintaining or exceeding the precision of state-of-the-art microbilayer recordings.
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    54. 54
      Hille, B. Ion Channels of Excitable Membranes, 3rd ed.; Sinauer Associates, 2001.
      There is no corresponding record for this reference.
    55. 55
      Rostovtseva, T. K.; Nestorovich, E. M.; Bezrukov, S. M. Partitioning of Differently Sized Poly(ethylene glycol)s into Ompf Porin Biophys. J. 2002, 82, 160– 169
      55
      Partitioning of differently sized poly(ethylene glycol)s into OmpF porin
      Rostovtseva, Tatiana K.; Nestorovich, Ekaterina M.; Bezrukov, Sergey M.
      Biophysical Journal (2002), 82 (1, Pt. 1), 160-169CODEN: BIOJAU; ISSN:0006-3495. (Biophysical Society)
      To understand the physics of polymer equil. and dynamics in the confines of ion channel pores, we study partitioning of poly(ethylene glycol)s (PEGs) of different mol. wts. into the bacterial porin, OmpF. Thermodn. and kinetic parameters of partitioning are deduced from the effects of polymer addn. on ion currents through single OmpF channels reconstituted into planar lipid bilayer membranes. The equil. partition coeff. is inferred from the av. redn. of channel conductance in the presence of PEG; rates of polymer exchange between the pore and the bulk are estd. from PEG-induced conductance noise. Partition coeff. as a function of polymer wt. is best fitted by a "compressed exponential" with the compression factor of 1.65. This finding demonstrates that PEG partitioning into the OmpF channel pore has sharper dependence on polymer mol. wt. than predictions of hard-sphere, random-flight, or scaling models. A 1360-Da polymer separates regimes of partitioning and exclusion. Comparison of its characteristic size with the size of a 2200-Da polymer previously found to sep. these regimes for the α-toxin shows good agreement with the x-ray structural data for these channels. The PEG-induced conductance noise is compatible with the polymer mobility reduced inside the OmpF pore by an order of magnitude relatively to its value in bulk soln.
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    56. 56
      Bezrukov, S. M.; Vodyanoy, I.; Brutyan, R. A.; Kasianowicz, J. J. Dynamics and Free Energy of Polymers Partitioning into a Nanoscale Pore Macromolecules 1996, 29, 8517– 8522
      56
      Dynamics and free energy of polymers partitioning into a nanoscale pore
      Bezrukov, Sergey M.; Vodyanoy, Igor; Brutyan, Rafik A.; Kasianowicz, John J.
      Macromolecules (1996), 29 (26), 8517-8522CODEN: MAMOBX; ISSN:0024-9297. (American Chemical Society)
      Membrane-bound proteinaceous nanoscale pores allow us to simultaneously observe the thermodn. and kinetic properties of differently sized polymers within their confines. We det. the dynamic partitioning of polyethylene glycol (PEG) into the pore formed by Staphylococcus aureus α-toxin and evaluate the free energy of polymer confinement by measuring polymer-induced changes to the pore ionic conductance. The free energy deduced from the partition coeff. has a sharper dependence on polymer length (or wt.) than scaling theory predicts. Moreover, the polymer-induced conductance fluctuations show a striking nonmonotonic dependence on the polymer mol. wt. The movement of polymer inside the pore is characterized by a diffusion coeff. that is orders of magnitude smaller than that for polymer in the bulk aq. soln., which suggests that PEG has an attractive interaction with the pore. Using an ad-hoc approach, we show that a simple mol. wt.-dependent modification of the polymer's diffusion coeff. accounts for these results, but only qual. Given that PEG assocs. with hydrophobic regions in proteins, we also conclude that, contrary to the conventional view of ion channels, the aq. cavity of the α-toxin pore's interior is, to some extent, hydrophobic.
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    57. 57
      Merzlyak, P. G.; Capistrano, M. F. P.; Valeva, A.; Kasianowicz, J. J.; Krasilnikov, O. V. Conductance and Ion Selectivity of a Mesoscopic Protein Nanopore Probed with Cysteine Scanning Mutagenesis Biophys. J. 2005, 89, 3059– 3070
      There is no corresponding record for this reference.
    58. 58
      Robertson, J. W. F.; Rodrigues, C. G.; Stanford, V. M.; Rubinson, K. A.; Krasilnikov, O. V.; Kasianowicz, J. J. Single-Molecule Mass Spectrometry in Solution Using a Solitary Nanopore Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 8207– 8211
      58
      Single-molecule mass spectrometry in solution using a solitary nanopore
      Robertson, Joseph W. F.; Rodrigues, Claudio G.; Stanford, Vincent M.; Rubinson, Kenneth A.; Krasilnikov, Oleg V.; Kasianowicz, John J.
      Proceedings of the National Academy of Sciences of the United States of America (2007), 104 (20), 8207-8211CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      The authors introduce a 2-dimensional method for mass spectrometry in soln. that is based on the interaction between a nanometer-scale pore and analytes. As an example, poly(ethylene glycol) mols. that enter a single α-hemolysin pore cause distinct mass-dependent conductance states with characteristic mean residence times. The conductance-based mass spectrum clearly resolves the repeat unit of ethylene glycol, and the mean residence time increases monotonically with the poly(ethylene glycol) mass. This technique could prove useful for the real-time characterization of mols. in soln.
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    59. 59
      Reiner, J. E.; Kasianowicz, J. J.; Nablo, B. J.; Robertson, J. W. Theory for Polymer Analysis Using Nanopore-Based Single-Molecule Mass Spectrometry Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 12080– 12085
      59
      Theory for polymer analysis using nanopore-based single-molecule mass spectrometry
      Reiner, Joseph E.; Kasianowicz, John J.; Nablo, Brian J.; Robertson, Joseph W. F.
      Proceedings of the National Academy of Sciences of the United States of America (2010), 107 (27), 12080-12085, S12080/1-S12080/4CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      Nanometer-scale pores have demonstrated potential for the elec. detection, quantification, and characterization of mols. for biomedical applications and the chem. anal. of polymers. Despite extensive research in the nanopore sensing field, there is a paucity of theor. models that incorporate the interactions between chems. (i.e., solute, solvent, analyte, and nanopore). Here, we develop a model that simultaneously describes both the current blockade depth and residence times caused by individual poly(ethylene glycol) (PEG) mols. in a single α-hemolysin ion channel. Modeling polymer-cation binding leads to a description of two significant effects: a redn. in the mobile cation concn. inside the pore and an increase in the affinity between the polymer and the pore. The model was used to est. the free energy of formation for K+-PEG inside the nanopore (≈ -49.7 meV) and the free energy of PEG partitioning into the nanopore (≈ 0.76 meV per ethylene glycol monomer). The results suggest that rational, phys. models for the anal. of analyte-nanopore interactions will develop the full potential of nanopore-based sensing for chem. and biol. applications.
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    60. 60
      Nablo, B. J.; Halverson, K. M.; Robertson, J. W.; Nguyen, T. L.; Panchal, R. G.; Gussio, R.; Bavari, S.; Krasilnikov, O. V.; Kasianowicz, J. J. Sizing the Bacillus Anthracis Pa63 Channel with Nonelectrolyte Poly(ethylene glycols) Biophys. J. 2008, 95, 1157– 1164
      There is no corresponding record for this reference.
    61. 61
      Breton, M. F.; Discala, F.; Bacri, L.; Foster, D.; Pelta, J.; Oulchaled, A. Exploration of Neutral Versus Polyelectrolyte Behavior of Poly(ethylene glycol)s in Alkali Ion Solutions Using Single-Nanopore Recording J. Phys. Chem. Lett. 2013, 4, 2202– 2208
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      Exploration of Neutral Versus Polyelectrolyte Behavior of Poly(ethylene glycol)s in Alkali Ion Solutions using Single-Nanopore Recording
      Breton, Marie France; Discala, Francoise; Bacri, Laurent; Foster, Damien; Pelta, Juan; Oukhaled, Abdelghani
      Journal of Physical Chemistry Letters (2013), 4 (13), 2202-2208CODEN: JPCLCD; ISSN:1948-7185. (American Chemical Society)
      We examine the effect of alkali ions (Li+, Na+, K+, Rb+, Cs+) on the partitioning of neutral and flexible poly(ethylene glycol) into the alpha-hemolysin (α-HL) nanopore for a large range of applied voltages at high salt concn. The neutral polymer behaves as if charged, i.e., the event frequency increases with applied voltage, and the residence times decrease with the elec. force for all cations except Li+. In contrast, in the presence of LiCl, we find the classical partitioning behavior of neutral polymers, i.e., the event frequency and the residence times are independent of the applied voltage. Assuming that lithium does not assoc. with PEG enabled us to quantify the relative magnitude of the entropic and enthalpic contribution to the free- energy barrier and the no. of complexed cations using two different arguments; the first est. is based on the balance of forces, and the second is found comparing the blockade ratio in the presence of LiCl (no complexed ions) to the blockade ratio of chains in the presence of the other salts (with complexed ions). This est. is in agreement with recent simulations. These findings demonstrate that the nanopore could prove useful for the rapid probing of the capabilities of different neutral mols. to form complexes with different ions.
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    62. 62
      Bezrukov, S. M.; Kasianowicz, J. J. The Charge State of an Ion Channel Controls Neutral Polymer Entry into Its Pore Eur. Biophys. J. Biophys. 1997, 26, 471– 476
      62
      The charge state of an ion channel controls neutral polymer entry into its pore
      Bezrukov, Sergey M.; Kasianowicz, John J.
      European Biophysics Journal (1997), 26 (6), 471-476CODEN: EBJOE8; ISSN:0175-7571. (Springer)
      Electrostatic potentials created by fixed or induced charges regulate many cellular phenomena including the rate of ion transport through proteinaceous ion channels. Nanometer-scale pores of these channels also play a crit. role in the transport of charged and neutral macromols. We demonstrate here that, surprisingly, changing the charge state of a channel markedly alters the ability of nonelectrolyte polymers to enter the channel's pore. Specifically, we show that the partitioning of differently-sized linear nonelectrolyte polymers of ethylene glycol into the Staphylococcus aureus α-hemolysin channel is altered by the soln. pH. Protonating some of the channel side chains decreases the characteristic polymer size (mol. wt.) that can enter the pore by ∼25 but increases the ionic current by ∼15. Thus, the "steric" and "elec." size of the channel change in opposite directions. The results suggest that effects due to polymer and channel hydration are crucial for polymer transport through such pores.
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    63. 63
      Douglas, S. M.; Marblestone, A. H.; Teerapittayanon, S.; Vazquez, A.; Church, G. M.; Shih, W. M. Rapid Prototyping of 3d DNA-Origami Shapes with Cadnano Nucleic Acids Res. 2009, 37, 5001– 5006
      63
      Rapid prototyping of 3D DNA-origami shapes with caDNAno
      Douglas, Shawn M.; Marblestone, Adam H.; Teerapittayanon, Surat; Vazquez, Alejandro; Church, George M.; Shih, William M.
      Nucleic Acids Research (2009), 37 (15), 5001-5006CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)
      DNA nanotechnol. exploits the programmable specificity afforded by base-pairing to produce self-assembling macromol. objects of custom shape. For building megadalton-scale DNA nanostructures, a long scaffold' strand can be employed to template the assembly of hundreds of oligonucleotide staple' strands into a planar antiparallel array of cross-linked helixes. The authors recently adapted this scaffolded DNA origami' method to producing 3-dimensional shapes formed as pleated layers of double helixes constrained to a honeycomb lattice. However, completing the required design steps can be cumbersome and time-consuming. Here the authors present caDNAno, an open-source software package with a graphical user interface that aids in the design of DNA sequences for folding 3-dimensional honeycomb-pleated shapes rectangular-block motifs were designed, assembled, and analyzed to identify a well-behaved motif that could serve as a building block for future studies. The use of caDNAno significantly reduces the effort required to design 3-dimensional DNA-origami structures. The software is available at http://cadnano.org/, along with example designs and video tutorials demonstrating their construction. The source code is released under the MIT license.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhtVKntbzE&md5=aa99732c1666373a70e9b7b4de6e6d5d
    64. 64
      Fendt, L.-A.; Bouamaied, I.; Thoeni, S.; Amiot, N.; Stulz, E. DNA as Supramolecular Scaffold for Porphyrin Arrays on the Nanometer Scale J. Am. Chem. Soc. 2007, 129, 15319– 15329
      64
      DNA as Supramolecular Scaffold for Porphyrin Arrays on the Nanometer Scale
      Fendt, Leslie-Anne; Bouamaied, Imenne; Thoeni, Sandra; Amiot, Nicolas; Stulz, Eugen
      Journal of the American Chemical Society (2007), 129 (49), 15319-15329CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Tetra-Ph porphyrin substituted deoxyuridine was used as a building block to create discrete multiporphyrin arrays via site specific incorporation into DNA. The successful covalent attachment of up to 11 tetra-Ph porphyrins in a row onto DNA shows that there is virtually no limitation in the amt. of substituents, and the porphyrin arrays thus obtained reach the nanometer scale (∼10 nm). The porphyrin substituents are located in the major groove of the dsDNA and destabilize the duplex by ΔTm 5-7° per porphyrin modification. Force-field structure minimization shows that the porphyrins are either in-line with the groove in isolated modifications or aligned parallel to the nucleobases in adjacent modifications. The CD signals of the porphyrins are dominated by a neg. peak arising from the intrinsic properties of the building block. In the single strands, the porphyrins induce stabilization of a secondary helical structure which is confined to the porphyrin modified part. This arrangement can be reproduced by force-field minimization and reveals an elongated helical arrangement compared to the double helix of the porphyrin-DNA. This secondary structure is disrupted above ∼55° (Tp) which is shown by various melting expts. Both absorption and emission spectroscopy disclose electronic interactions between the porphyrin units upon stacking along the outer rim of the DNA leading to a broadening of the absorbance and a quenching of the emission. The single-stranded and double-stranded form show different spectroscopic properties due to the different arrangement of the porphyrins. Above Tp the electronic properties (absorption and emission) of the porphyrins change compared to room temp. measurements due to the disruption of the porphyrin stacking at high temp. The covalent attachment of porphyrins to DNA is therefore a suitable way of creating helical stacks of porphyrins on the nanometer scale.
      >> More from SciFinder ®
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    65. 65
      Clifton, L. A.; Sanders, M. R.; Castelletto, V.; Rogers, S. E.; Heenan, R. K.; Neylon, C.; Frazier, R. A.; Green, R. J. Puroindoline-a, a Lipid Binding Protein from Common Wheat, Spontaneously Forms Prolate Protein Micelles in Solution Phys. Chem. Chem. Phys. 2011, 13, 8881– 8888
      There is no corresponding record for this reference.
    66. 66
      Kreir, M.; Farre, C.; Beckler, M.; George, M.; Fertig, N. Rapid Screening of Membrane Protein Activity: Electrophysiological Analysis of Ompf Reconstituted in Proteoliposomes Lab Chip 2008, 8, 587– 595
      66
      Rapid screening of membrane protein activity: electrophysiological analysis of OmpF reconstituted in proteoliposomes
      Kreir, Mohamed; Farre, Cecilia; Beckler, Matthias; George, Michael; Fertig, Niels
      Lab on a Chip (2008), 8 (4), 587-595CODEN: LCAHAM; ISSN:1473-0197. (Royal Society of Chemistry)
      Solvent-free planar lipid bilayers were formed in an automatic manner by bursting of giant unilamellar vesicles (GUVs) after gentle suction application through micron-sized apertures in a borosilicate glass substrate. Incubation of GUVs with the purified ion channel protein of interest yielded proteoliposomes. These proteoliposomes allow for immediate recording of channel activity after GUV sealing. This approach reduces the time-consuming, laborious and sometimes difficult protein reconstitution processes normally performed after bilayer formation. Bilayer recordings are attractive for investigations of membrane proteins not accessible to patch clamp anal., like e.g. proteins from organelles. In the presented work, we show the example of the outer membrane protein OmpF from Escherichia coli. We reconstituted OmpF in proteoliposomes and obsd. the characteristic trimeric conductance levels and the typical gating induced by pH and transmembrane voltage. Moreover, OmpF is the main entrance for beta-lactam antibiotics and we investigated translocation processes of antibiotics and modulation of OmpF by spermine. We suggest that the rapid formation of porin contg. lipid bilayers is of potential for the efficient electrophysiol. characterization of the OmpF protein, for studying membrane permeation processes and for the rapid screening of antibiotics.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXjslyksb0%253D&md5=e8f6cb817f1133132a329c8a2404421d
    67. 67
      Gassmann, O.; Kreir, M.; Ambrosi, C.; Pranskevich, J.; Oshima, A.; Roling, C.; Sosinsky, G.; Fertig, N.; Steinem, C. The M34a Mutant of Connexin26 Reveals Active Conductance States in Pore-Suspending Membranes J. Struct. Biol. 2009, 168, 168– 176
      There is no corresponding record for this reference.
    68. 68
      Thacker, V. V.; Ghosal, S.; Hernández-Ainsa, S.; Bell, N. A.; Keyser, U. F. Studying DNA Translocation in Nanocapillaries Using Single Molecule Fluorescence Appl. Phys. Lett. 2012, 101, 223704
      There is no corresponding record for this reference.
    69. 69
      Gornall, J. L.; Mahendran, K. R.; Pambos, O. J.; Steinbock, L. J.; Otto, O.; Chimerel, C.; Winterhalter, M.; Keyser, U. F. Simple Reconstitution of Protein Pores in Nano Lipid Bilayers Nano Lett. 2011, 11, 3334– 3340
      There is no corresponding record for this reference.

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    Experimental details on DNA sequences for assembling the DNA nanopores. Experimental results on the conductance properties of DNA nanopores. This material is available free of charge via the Internet at http://pubs.acs.org.


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