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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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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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fig3: 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).

Mentions: 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 Nanion53 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 calculated54 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.


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)

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).
© Copyright Policy
Related In: Results  -  Collection

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

fig3: 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).
Mentions: 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 Nanion53 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 calculated54 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.

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