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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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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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fig1: 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.

Mentions: 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.


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: 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.
Mentions: 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.

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