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Bilayer-spanning DNA nanopores with voltage-switching between open and closed state.

Seifert A, Göpfrich K, Burns JR, Fertig N, Keyser UF, Howorka S - ACS Nano (2014)

Bottom Line: 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.This voltage-dependent switching between the open and closed states is observed with planar lipid bilayers as well as bilayers mounted on glass nanopipettes.By systematically exploring a large space of parameters and answering key questions, our report supports the development of DNA nanopores for nanobiotechnology.

View Article: PubMed Central - PubMed

Affiliation: Nanion Technologies GmbH , D-80636 Munich, Germany.

ABSTRACT
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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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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fig4: 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.

Mentions: 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.


Bilayer-spanning DNA nanopores with voltage-switching between open and closed state.

Seifert A, Göpfrich K, Burns JR, Fertig N, Keyser UF, Howorka S - ACS Nano (2014)

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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4508203&req=5

fig4: 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.
Mentions: 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.

Bottom Line: 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.This voltage-dependent switching between the open and closed states is observed with planar lipid bilayers as well as bilayers mounted on glass nanopipettes.By systematically exploring a large space of parameters and answering key questions, our report supports the development of DNA nanopores for nanobiotechnology.

View Article: PubMed Central - PubMed

Affiliation: Nanion Technologies GmbH , D-80636 Munich, Germany.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus