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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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Related in: MedlinePlus

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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fig2: 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.

Mentions: 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.


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 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.
© Copyright Policy
Related In: Results  -  Collection

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

fig2: 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.
Mentions: 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.

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