Single Ion-Channel Recordings Using Glass Nanopore Membranes

White, Raymond P.; Ervin, Eric N.; Yang, Tinglu; Chen, Xin; Daniel, Susan; Cremer, Paul S.; White, Henry S.

doi:10.1021/ja073174q

Cited by 242 publications

(349 citation statements)

References 52 publications

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“…All the aqueous solutions used were prepared with >18 MΩ/cm ultrapure water and filtered with a sterile 0.22-mm Millipore vacuum filter. The wild-type α-HL ion channel was reconstructed across the lipid bilayer supported by a glass nanopore membrane, which was fashioned following procedures described in the literature (50). Data were collected with a 100-kHz low-pass filter and 500-kHz sampling rate.…”

Section: Methodsmentioning

confidence: 99%

Single-molecule investigation of G-quadruplex folds of the human telomere sequence in a protein nanocavity

Fleming

Middleton

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

102

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Human telomeric DNA consists of tandem repeats of the sequence 5′-TTAGGG-3′ that can fold into various G-quadruplexes, including the hybrid, basket, and propeller folds. In this report, we demonstrate use of the α-hemolysin ion channel to analyze these subtle topological changes at a nanometer scale by providing structuredependent electrical signatures through DNA-protein interactions. Whereas the dimensions of hybrid and basket folds allowed them to enter the protein vestibule, the propeller fold exceeds the size of the latch region, producing only brief collisions. After attaching a 25-mer poly-2′-deoxyadenosine extension to these structures, unraveling kinetics also were evaluated. Both the locations where the unfolding processes occur and the molecular shapes of the G-quadruplexes play important roles in determining their unfolding profiles. These results provide insights into the application of α-hemolysin as a molecular sieve to differentiate nanostructures as well as the potential technical hurdles DNA secondary structures may present to nanopore technology.α-hemolysin nanopore | single-molecule detection N ucleic acids can fold into a myriad of secondary structures that depend on the primary sequence as well as the physical conditions in which the structures are prepared and characterized. One prime example of a multistructural sequence is human telomeric DNA comprising the repeat sequence 5′-TTAGGG-3′. This guanine-rich single-stranded sequence is known to fold into highly ordered nanostructures in the form of G-quadruplexes that feature the coordination of two alkali cations to three layers of G-tetrads formed by Hoogsteen hydrogen-bonded assemblies of four guanine bases ( Fig. 1A) (1, 2). G-quadruplexes provide a fascinating case in which the cation and physical context play critical roles in defining the overall structural topology as well as the stability of the fold. Guanine-rich sequences also are known to present challenges to PCR amplification and sequencingby-synthesis methods that require processive analysis of singlestranded DNA (ssDNA) because of the high thermodynamic stability of these folded structures.The following studies highlight the cation and contextdependent conditions in which the topology of the natural human telomere sequence 5′-TAGGG(TTAGGG) 3 TT-3′ is affected. In NaCl solution, NMR studies revealed a structure referred to as the basket fold (Fig. 1A) (4). Key features of this structure include an antiparallel strand arrangement with alternating syn and anti orientations of the guanosine glycosidic bonds and three tetrads linked by two edgewise loops and one diagonal loop. In contrast, NMR-based studies in KCl solution show that the same sequence folds predominantly to the hybrid-1 and hybrid-2 structures (Fig. 1A) (5-8). Characteristics of this structure are an antiparallel strand orientation with a 3+1 core of syn and anti G nucleotides yielding three tetrads. The loop topology of the hybrid fold consists of a double-chain reversal loop and two edgewise loops, in which hybr...

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Section: Methodsmentioning

confidence: 99%

Single-molecule investigation of G-quadruplex folds of the human telomere sequence in a protein nanocavity

Fleming

Middleton

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

102

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“…A great deal of skill and practice is necessary in order to acquire practical data from these channel recordings meaning that although the information acquired is extremely useful, the labour is laborious and frustrating. Thus several proposals have been made for the construction of durable bilayers [1][2][3][4][5]. We have developed a practical method to produce such durable bilayers for channel measurements.…”

Section: Introductionmentioning

confidence: 99%

Lipid Bilayers at Gel/Gel Interface for Ion Channel Recordings

Hirano

Kobayashi²,

Ide

2008

e-J. Surf. Sci. Nanotechnol.

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We have developed a practical method to produce durable artificial lipid bilayers using a hydrogel-hydrogel interface for ion channel measurements. Bilayers were formed by forcing a hydrogel-bead into contact with the hydrogel layer (hydrogel plate) in a lipid solution. The immediate formation of a bilayer was observed (< 1 s). This allows channel recordings to be repeated more easily and quickly as compared to conventional methods. Currents of various types of channel such as gramicidin, hemolysin and BK-channel have been recorded. Our channel property results mirrored those of other techniques and were reproducible. Hydrogel solutions containing gramicidin were extremely stable and could be used months after preparation for bilayer experiments.

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“…These values are comparable to those previously reported in the literature for bilayers formed on similar sized apertures. 29,38,39 Figure 3 shows typical i-V curves between a QRCE in the pipet and one in the bulk solution, before and after bilayer formation, with a typical increase in resistance 15 of 5 orders of magnitude. For these measurements, as shown in Figure 1(c), the potential of one of the QRCEs in the pipet was swept (with respect to ground) whilst the current was recorded at the QRCE in the bulk solution (on the other side of the bilayer) held at ground.…”

Section: Bilayer Characterizationmentioning

confidence: 99%

“…28 The use 10 of small apertures, over which a bilayer is suspended, can also improve electrical and mechanical stability. 29,30 Liposomes have also been used, which do not contain any residual solvent molecules in the interior of the membrane, and are typically stable for considerably longer periods than planar 15 lipid bilayers. 31 However, since the interior of the liposome is inaccessible for sampling, measurement of permeation rates can be difficult.…”

mentioning

confidence: 99%

A new approach for the fabrication of microscale lipid bilayers at glass pipets: application to quantitative passive permeation visualization

2014

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A new method of planar bilayer lipid membrane (BLM) formation is presented that allows stable, solventfree lipid bilayers exhibiting high seal resistances to be formed rapidly, easily and reproducibly. Using these bilayers the passive permeation of a series of carboxylic acids is investigated, to determine quantitatively the trend in permeability with lipophilicity of the acid. BLMs are formed at the tip openings of pulled theta pipets, and the rate of permeation of each carboxylic acid across the bilayer, from 10 within the pipet into the bulk solution is determined. This is achieved through spatially-resolved measurements of the pH change that occurs upon the permeation of the weak acid, visualized using a pHsensitive fluorophore with a confocal laser scanning microscope. The extracted fluorescence profiles are matched to finite element method (FEM) simulations, to allow the associated permeation coefficient for each weak acid to be determined with high accuracy, since this is the only adjustable parameter used to fit 15 the experimental data. For bilayers formed in this way, the weak acids show increasing permeability with lipophilicity. Furthermore, the arrangement allows the effect of a trans-membrane electric field on permeation to be explored. For both propanoic and hexanoic acid it is found that an applied electric field enhances molecular transport, which is attributed to the formation of pores within the membrane.

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Single Ion-Channel Recordings Using Glass Nanopore Membranes

Cited by 242 publications

References 52 publications

Single-molecule investigation of G-quadruplex folds of the human telomere sequence in a protein nanocavity

Single-molecule investigation of G-quadruplex folds of the human telomere sequence in a protein nanocavity

Lipid Bilayers at Gel/Gel Interface for Ion Channel Recordings

A new approach for the fabrication of microscale lipid bilayers at glass pipets: application to quantitative passive permeation visualization

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