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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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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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fig5: 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.

Mentions: 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).


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)

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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Related In: Results  -  Collection

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

fig5: 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.
Mentions: 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).

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