ACS Publications. Most Trusted. Most Cited. Most Read
Quick View

OR SEARCH CITATIONS

My Activity
Publications
CONTENT TYPES

Figure 1Loading Img
Download Hi-Res ImageDownload to MS-PowerPointCite This:ACS Nano 2022, 16, 10, 17128-17138
RETURN TO ISSUEPREVArticleNEXT

DNA-Based Optical Quantification of Ion Transport across Giant Vesicles

Cite this: ACS Nano 2022, 16, 10, 17128–17138
Publication Date (Web):October 12, 2022
https://doi.org/10.1021/acsnano.2c07496

Copyright © 2022 The Authors. Published by American Chemical Society. This publication is licensed under

CC-BY 4.0.
  • Open Access

Article Views

2627

Altmetric

-

Citations

-
LEARN ABOUT THESE METRICS
PDF (3 MB)
Get e-Alerts
Supporting Info (1)»
SUBJECTS:

Abstract

High Resolution Image
Download MS PowerPoint Slide

Accurate measurements of ion permeability through cellular membranes remains challenging due to the lack of suitable ion-selective probes. Here we use giant unilamellar vesicles (GUVs) as membrane models for the direct visualization of mass translocation at the single-vesicle level. Ion transport is indicated with a fluorescently adjustable DNA-based sensor that accurately detects sub-millimolar variations in K+ concentration. In combination with microfluidics, we employed our DNA-based K+ sensor for extraction of the permeation coefficient of potassium ions. We measured K+ permeability coefficients at least 1 order of magnitude larger than previously reported values from bulk experiments and show that permeation rates across the lipid bilayer increase in the presence of octanol. In addition, an analysis of the K+ flux in different concentration gradients allows us to estimate the complementary H+ flux that dissipates the charge imbalance across the GUV membrane. Subsequently, we show that our sensor can quantify the K+ transport across prototypical cation-selective ion channels, gramicidin A and OmpF, revealing their relative H+/K+ selectivity. Our results show that gramicidin A is much more selective to protons than OmpF with a H+/K+ permeability ratio of ∼104.

This publication is licensed under

CC-BY 4.0.
  • cc licence
  • by licence
KEYWORDS:
The movement of ions across the lipid membrane is a fundamental process in a cell’s endeavor to maintain homeostasis. Mediated by an array of transport proteins, such as ion pumps and ion channels, ionic flows are harnessed by cells and organelles to induce electrochemical gradients that power vital biochemical activities from nutrient uptake (1,2) to energy conversion. (3) At the same time, however, ions may directly diffuse through the lipid bilayer, acting to dissipate the induced gradient. Therefore, the necessary coupling between multiple translocation pathways for the precise regulation of ionic fluxes in cells makes permeability measurements in living systems highly challenging.
Giant unilamellar vesicles (GUVs), micrometer-sized compartments enclosed by a lipid bilayer, offer a simplified biomimetic membrane model for studying ion transport phenomena at the single-vesicle level and under tightly controlled conditions and membrane compositions. (4,5) Moreover, with dimensions and membrane composition comparable to those of cells, GUVs find applications as protocells and can accommodate different types of biological modules to possess cell-like features. (6−9) Hence, studying ion transport in GUV systems is not only important for the mere interest of resolving transport mechanisms but also for elucidating the energetics of protein machines such as pumps and motors. However, while methods for developing increasingly realistic artificial cell models using GUVs are rapidly growing in number, techniques to investigate ion transport are scarce. (4,5,10) Predominantly, GUVs are analyzed using fluorescence microscopy approaches. Therefore, one of the main reasons for the lack of ion transport studies in GUV systems is the absence of suitable fluorescent probes for sensing physiologically important ions with high sensitivity and selectivity.
Ion-responsive dyes are particularly suited to measurements of ion transport in GUVs, as they can be readily encapsulated within the GUV lumen. (4,10,11) Therefore, by combining microfluidic approaches such as hydrodynamic trapping with real-time fluorescent imaging, large populations of GUVs can be monitored in parallel and at the single-vesicle level, (12−15) enabling the study of one vesicle at a time, (5) unlike the case for whole-GUV patch-clamping. Nevertheless, only a small number of ion-responsive dyes are available commercially and suitable for membrane transport measurements. For instance, pyranine (or HPTS), a pH-sensitive dye, is a well-established fluorescent probe for quantifying proton transport in liposomes, (16−21) and in some cases, it has also been used to indirectly evaluate K+ transport rates from monitoring pH changes. (22) However, for other ions such as K+, the most abundant cation in the intracellular fluid, there is only one commercially available fluorescence indicator, potassium binding benzofuran isophthalate (PBFI). (23) Still, since PBFI has relatively low sensitivity to potassium (Kd = 9–70 mM), (24) it has traditionally been used for monitoring the intracellular level of K+ under physiological electrolyte concentrations (25) and has not been routinely adopted to direct measurements of K+ transport in GUVs. Therefore, although potassium ions play an important role in cellular processes such as action potential induction and osmotic pressure regulation, as far as we are aware, no measurements of potassium ion transport across GUV systems have been reported to date. For these reasons, we sought to develop a fluorescent-based probe suitable for measuring K+ transport in GUV-based synthetic cells.
It is well-known that certain G-rich sequences of single-stranded DNA (ssDNA) fold to G-quadruplex secondary structures (G4) in the presence of specific cations, (26−28) especially potassium ions. (29) While the concept of using G4-based sensors for K+ has already been demonstrated, (30,31) their application in K+ transport studies has not been investigated thus far.
Here we employed a G4-based potassium ion sensor using a human telomere G4 sequence labeled with a fluorophore (FAM) and a quencher (Q) at its two ends (i.e., FAMQ-G4). Upon folding of the telomere G4 sequence, the fluorescence intensity of FAM decreases significantly following its interaction with the quencher. We show that FAMQ-G4 can readily detect sub-millimolar variations in K+ concentration and be used for a quantitative analysis of potassium transport in GUVs. Furthermore, through modifying the fluorophore at one end of our DNA-based sensor, we were able to customize the wavelengths at which K+ is detected, demonstrating its potential use in combination with other fluorescent probes in multi-ion transport studies. By utilizing microfluidic-based techniques to prepare and trap GUVs with encapsulated FAMQ-G4, we directly quantified the permeation coefficient of K+ across the lipid bilayer of GUVs at the single-vesicle level. Taking the same analysis approach, we measured the transport rate of K+ through membranes containing two well-characterized membrane channels, gramicidin A and OmpF, and evaluated the effect of each type of channel on the buildup of transmembrane potential based on their H+/K+ selectivity and intrinsic permeation pathways.

Results and Discussion

ARTICLE SECTIONS
Jump To

Design of a DNA-Based K+ Sensor for Transport Measurements

In our quest to design a suitable ion-responsive sensor for transport studies we have considered the following basic guiding principles: (1) high ion sensitivity, (2) no photobleaching during the time scale of the experiment, (3) negligible permeation through the lipid bilayer, and (4) minimal disruption to membrane properties. DNA nanotechnology offers powerful design techniques for self-assembly of nanostructures with many sensing applications. (31) Ueyama et al. showed that a guanine (G)-rich oligonucleotide (Human Telomere G4-DNA) can be used as an efficient potassium sensor in aqueous media. (32) To detect the presence of K+, G4-DNA was labeled at one end with a FRET donor and at the other end with an acceptor. Thus, upon interaction with potassium it folds to a tetraplex structure, known as G-quadruplex (G4), and generates a FRET signal. Since then, similar concepts have been followed and G4-based sensors have been widely employed for various biosensing applications. (33,34) Nevertheless, the use of G4-based sensors in membrane transport studies has not been demonstrated thus far. We chose the Human Telomere G4-DNA sequence AGGG(TTAGGG)3 (Figure 1A), which retains high specificity for K+ ions and folds into a hybrid type G-quadruplex in the presence of K+. (29,35) To enable the fluorescence detection of potassium, we modified the G4-DNA by attaching a fluorophore (fluorescein, 6-FAM) to its 5′ -end and a quencher (Iowa black FQ, Q) to its 3′-end (Figure 1A). As schematically illustrated in Figure 1A, upon binding two potassium ions, the obtained G4-DNA sensor (FAMQ-G4) folds to a stable tetraplex structure and the distance between the fluorophore and quencher decreases. As a result, the fluorescence intensity drops significantly and plateaus at 0.45 when [K+] > 1 mM (Figure 1B), implying a sensing region of 0 < [K+] < 1 mM for FAMQ-G4.

Figure 1

Figure 1. Design and characterization of G-quadruplex (G4) DNA based K+ probes. (A) Schematic illustrating the K+ sensing principle of G4-DNA probes. A human telomeric DNA (HT G4-DNA), modified with a fluorophore and quencher at opposing ends (5′ and 3′, respectively), folds in response to K+, thereby bringing the fluorophore and quencher into closer contact and decreasing the fluorescence intensity of the probe. (B) Variation of FAMQ-G4 fluorescence intensity with increasing concentration of K+ (blue circles). In the presence of a complementary DNA strand (orange triangles) no reduction in fluorescence is observed for the double-stranded G4-DNA, indicating that folding of the single-stranded G4-DNA in the presence of K+ causes the fluorescent response. (C) Emission spectra of G4 probes modified with different fluorophores at their 5′ end. (D) Fluorescence intensity variation of G4 probes, modified with HEX (green) or Texas Red (red), in response to increasing K+ concentration.

High Resolution Image
Download MS PowerPoint Slide
To quantify the sensitivity of FAMQ-G4 to potassium ions, we investigated the probe’s fluorescent response at different K+ concentrations. Figure 1B shows that the relative fluorescence of FAMQ-G4 (blue circles) gradually decreases with increasing concentrations of K+, indicating that more G-quadruplex structures form when a larger amount of K+ is available to interact with FAMQ-G4. We verified that the fluorescence intensity reduction is due to folding of the G4 probe and is not a result of direct interaction between potassium ions and FAM by first mixing FAMQ-G4 with its complementary sequence (CCCTAA)3CCCT, to form a more rigid double-stranded DNA (dsDNA) structure, and then measuring the fluorescence at increased potassium concentration. As depicted in Figure 1B (orange triangles), no detectable change in fluorescence was observed, confirming that the measured fluorescence reduction is solely due to the folding of FAMQ-G4. By fitting the relevant binding model (Supporting Information) to the fluorescence data (blue line), we evaluated the dissociation constant between potassium and FAMQ-G4 to be Kd=[G4uf][K+]2[G4f]=(1.5±0.3)×10−7M2, where [K+] is the concentration of potassium ions, and [G4uf] and [G4f] are the concentrations of FAMQ-G4 in its unfolded and folded configurations, respectively. The obtained Kd agrees well with a previously reported dissociation constant measured for a similar G4 structure using mass spectrometry, (26) indicating the high affinity between K+ and FAMQ-G4. Correspondingly, we were able to detect sub-millimolar variations in potassium ion concentration (Figure 1B), unlike the case for the only commercially available potassium ion indicator PBFI, which responds to concentration changes in the millimolar range (Kd(PBFI) is between 9 and 70 mM). (24,36) Notably, another benefit of our G-quadruplex-based sensor over conventional ion-sensitive dyes is the ability to tune its affinity to potassium (i.e., Kd) through changing its sequence. (37) In addition, we found that FAMQ-G4 also folds in response to Na+, though with lower sensitivity relative to K+ (Figure S1). Therefore, even in the presence of 5 mM Na+, FAMQ-G4 retains sensitivity to K+ (Figure S2). This result, however, does not necessarily suggest that our G4-DNA sensor may be suitable for quantifying potassium concentrations in living cells, where sodium concentrations are much higher (100–150 mM). Still, we note that in systems where the type and concentration of electrolytes can be carefully chosen and controlled, for instance by using Li+ instead of K+ or Na+ (Figure S3), FAMQ-G4 can be suitably used to detect either sodium or potassium, respectively.
Another important advantage of DNA-based fluorescent probes over conventional molecular dyes is their customizability. Typically, in ion transport studies, the permeation of the examined ion is coupled to the simultaneous flow of other types of ions. Therefore, to understand the combined influence of different ions in processes, such as electrochemical gradient development or the activity of transporters, it is of interest to determine the flux of several types of ions at the same time. We show that FAMQ-G4 can be adjusted using different types of fluorophores and quenchers to enable detection of K+ at various excitation/emission wavelengths while retaining high sensitivity. Figure 1C shows the excitation/emission spectra of three G4-DNA-based designs, FAMQ-G4, HEXQ-G4, and TRQ′-G4, where Q′, HEX, and TR stand for Iowa Black-RQ, hexachlorofluorescein, and Texas Red, respectively. Importantly, regardless of the attachment type, all of the aforementioned DNA probes retain sub-millimolar sensitivity to changes in potassium concentration with comparable Kd(HEXQ-G4) = (7.8 ± 0.2) × 10–7 M2 and Kd(TRQ′-G4) = (6.3 ± 2.0) × 10–7 M2 (Figure 1D and the Supporting Information).

Potassium Ion Permeation across Single GUVs

To confirm that our DNA-based sensor is suitable for ion transport studies in vitro, we utilized FAMQ-G4 to quantify the permeation of K+ across the lipid bilayer of giant unilamellar vesicles (GUVs). For this purpose, we used a droplet-based microfluidic approach, octanol-assisted liposome assembly (OLA), to encapsulate FAMQ-G4 (10 μM), dissolved in buffer A (10 mM Tris, 100 mM sucrose buffer, pH = 7.6), in the interior of negatively charged DOPC/DOPG GUVs (Figure 2A and the Supporting Information). The formed GUVs were then extracted and perfused into a separate microfluidic device, where they were immobilized using hydrodynamic trapping (Figure 2B(i)). We note that the FAMQ-G4 concentration inside the GUVs shows some polydispersity between different experiments, most likely due to extraction of GUVs from the OLA production device to the trapping device. Nevertheless, once trapped, all GUV intensities were stable before perfusion of KCl and no leakage of the DNA fluorescent probe was observed (Figure S4). We account for the polydispersity in fluorescence intensity by normalizing it according to the intensity prior to the transport of K+. In addition, since the quenching time of FAM (∼10 s), due to folding of our DNA sensor following the addition of 100 μM KCl, was found to be comparable to the rate of data acquisition in our experiments (Figure S5), the sensitivity of FAMQ-G4 to K+ influx should not be affected by its folding kinetics.

Figure 2

Figure 2. K+ transport measurements across single GUVs using microfluidic-based approaches. (A) On-chip production of GUVs (DOPC/DOPG, 3:1 w/w) and encapsulation of FAMQ-G4 (10 μM), using octanol-assisted liposome assembly. Scale bar: 20 μm. (B) FAMQ-G4 encapsulated GUV immobilization using microfluidic hydrodynamic trapping (i). Once trapped, a 1 mM KCl solution, containing 500 nM FAMQ-G4, is perfused into the microfluidic chamber (ii), where GUVs can be visualized for >10 h, allowing one to capture the transport process of slow-permeating solutes such as K+. Scale bar: 40 μm. (C) (i) Time lapse of lumenal GUV fluorescence during the K+ transport process, showing the decrease in FAMQ-G4 fluorescence as a result of K+ induced folding. Scale bar: 20 μm. (ii) Analysis of K+ permeation across the lipid bilayer of single GUVs (n = 441) showing the temporal variation of lumenal (black solid line) and background (black dashed line) fluorescence intensity (median). The gray bands represent the lower and upper quartiles of the measured fluorescence at each time point for each measured vesicle.

High Resolution Image
Download MS PowerPoint Slide
As illustrated in Figure 2B(ii), to initiate the permeation of potassium ions across the GUV membrane, we perfused 1 mM KCl solution (also consisting of 500 nM FAMQ-G4 in buffer A) into the microfluidic chamber and monitored the intensity of FAMQ-G4, outside and inside the GUVs, over time. By trapping GUVs in a microfluidic platform, we were able to precisely control the flow rate, monitor the arrival of potassium ions to the trapped GUVs, and analyze their flux across single vesicles over long periods─an essential time frame for determining the membrane transport of ions with very low permeation rates (Figure 2C(i)). As such, we were able to monitor the arrival of K+ precisely and accurately. In addition, no leakage of FAMQ-G4 through the GUV lipid bilayer or photobleaching were observed during the time scale of our experiments (Figure S4). We also verified, using conductance measurements, that FAMQ-G4 does not disrupt the lipid bilayer integrity or form pores that may render the GUV membrane more permeable to ions (Figure S6).
By analyzing the measured time-dependent fluorescence intensities of FAMQ-G4 inside and outside the trapped vesicles (Figure 2C(ii)), we resolved the sub-millimolar variation of lumenal and extravesicular potassium concentrations, [K+]i (solid curve) and [K+]o (dashed curve) respectively, and determined the temporal evolution of potassium concentration gradients across the GUV membrane over the course of more than 13 h following the arrival of K+ (Figure 3A). Consequently, we observed that the gradual increase of [K+]i commences only after [K+]o reached its maximum value, indicating the establishment of a potassium concentration gradient of Δ[K+] = [K+]o – [K+]i ≈ 1 mM prior to the movement of K+ across the GUV membrane (inset to Figure 3A). Furthermore, the significantly slower increase of K+ concentration within the GUVs shows that the rate of potassium permeation is not limited by diffusion through the unstirred layer. Importantly, we note that the ability to investigate the transport of K+ under small concentration gradients (i.e., Δ[K+] ≤ 1 mM) ensures that flux measurements are carried out under low osmotic differences and, thus, with minimal perturbation of the GUV membrane. (38,39)

Figure 3

Figure 3. Quantification of K+ permeability across single GUVs. (A) Variation of K+ concentration inside (solid) and outside (dashed) GUVs during the transport process. The distribution of [K+] among GUVs is represented by the upper and lower quartiles of [K+] at each time point. Inset: magnified view of the transmembrane K+ concentration gradient, Δ[K+], generated in the initial period (typically a few tens of minutes) of our experiments. (B) Variation of K+ flux into GUVs over the measurement time course. Inset: flux profile showing the variation of K+ flux as a function of Δ[K+] during the initial period of Δ[K+] development. The obtained linear flux profile can be represented through J = PΔ[K+], thus enabling the determination of K+ permeability from the slope of the curve. (C) Schematic showing the proposed dissipation mechanism of transmembrane potential by a counter flux of protons across the GUV lipid bilayer in our experiments. (D) Distribution of measured permeability coefficients for negatively charged OLA DOPC/DOPG (3:1) GUVs (N = 441). Inset: measured permeability distribution of electroformed DOPC/DOPG (3:1) GUVs.

High Resolution Image
Download MS PowerPoint Slide
Next, we determined the influx density of potassium from the obtained temporal evolution of [K+]i through JK+ = d[K+]i/dt × r/3, where r is the radius of each detected GUV (Figure 3B). We note that our direct measurement of GUV dimensions at the single-vesicle level allows us to reduce inaccuracies caused by assumptions of radii as in bulk experiments. Under our experimental conditions, the influx of K+ is determined by two opposing driving forces, the concentration difference (Δ[K+]) and transmembrane potential (Δψ) across the membrane, as described by the Goldman–Hodgkin–Katz (GHK) flux equation (20,40)
JK+=PK+ΔψFRT[K+]i−[K+]oe−ΔψF/RT1−e−ΔψF/RT
where [K+]i and [K+]o are the concentrations of K+ (mol/cm3) inside and outside the GUV, respectively, PK+ (cm/s) is the permeability coefficient of K+, and F, R, and T have their usual meanings. However, in cases where Δψ ≈ 0, the flux density is expected to change linearly with concentration gradient according to Fick’s first law:
JK+=−PK+([K+]o−[K+]i)
By plotting the potassium influx as a function of Δ[K+], we found that at the early period of transport (10–20 min) the flux profile JK+(Δ[K+])) across single GUVs changes in a rather linear fashion (inset to Figure 3B), suggesting a negligible development of Δψ during this time frame. One way to justify the insignificant development of Δψ is by considering its dissipation by fluxes of other ions with permeability rates higher than those of potassium, as schematically shown in Figure 3C. For instance, protons (H+) are known to cross the lipid bilayer at rates ∼107 times higher than those of other monovalent ions such as K+. (10,21) In a previous study, we reported that the permeation coefficient of protons across the same composition of OLA-GUVs is PH+ ≈ 0.002 cm/s. (10) Since the concentration gradient of protons during the early period of K+ permeation (i.e., at the linear regime) is negligible and [H+]o ≈ [H+]i (Figure S7), it can be shown that even under a very low transmembrane potential of Δψ = 1 mV the estimated efflux of protons at pH = 7.6 is comparable to that of potassium (inset to Figure 3B). We estimated JH+ = PH+[H+](FΔψ/(RT)) = 2 × 10–15 mol cm–2 s–1 according to the GHK flux equation, where [H+] = 10–pH/1000 is the concentration of protons on both sides of the membrane (mol/cm3).
Such a near 1/1 stoichiometry of the net K+ and H+ fluxes (Figure 3C), which was also observed across small unilamellar vesicles, (23) enables the determination of the potassium permeability coefficient directly from the linear regime of the flux profile using PK+ = JK+/Δ[K+]. Figure 3D shows the measured permeability coefficient distribution of potassium permeation coefficients with a mean value of P̅K+ = (1.5 ± 0.2) × 10–8 cm s–1. Notably, the obtained PK+ values are several orders of magnitude higher in comparison with previously reported K+ permeabilities, which typically fall in the range between 10–10 and 10–12 cm s–1. (16,23) To account for this discrepancy, we considered the possibility that trace amounts of octanol may reside in the lipid bilayer of OLA GUVs, thus rendering it more permeable to cations relative to oil-free membranes. (41) To this end, we encapsulated our DNA-based probe in electroformed GUVs with a lipid composition similar to that of OLA GUVs and measured the permeability of K+ under the same experimental conditions (Figure S8). As shown in the inset to Figure 3D, the obtained potassium permeability coefficients, in the absence of octanol, were found to be 1 order of magnitude lower than in the case of OLA GUVs, with an average value of P̅K+ = (1.3 ± 1.5) × 10–9 cm s–1, suggesting that residual octanol molecules are indeed incorporated in the membrane of OLA GUVs.
An additional contribution to the relatively high measured PK+ values is the negative surface potential of the lipid bilayer. In the case of protons, for instance, a 2-fold increase in permeation coefficient was measured for a negatively charged lipid bilayer (DOPC/DOPG), with a composition similar to that in this study, compared to an undercharged membrane (DOPC). (10) Likewise, Koyanagi et al. compared ion transport rates across small unilamellar vesicles comprising either POPC or POPC/POPG (1/1) lipids and observed a factor of 3 increase in transport rates with negatively charged lipids present. (42) We note, however, that while previously attained PK+ values may still be somewhat lower, the present study signifies a model-free analysis of K+ permeation at the single-vesicle level, unlike earlier bulk studies that measured average PK+ values using an ensemble of nanometric liposomes.

Potassium Transport and H+/K+ Selectivity of Gramicidin A and OmpF

In living cells, the passive movement of ions across the membrane and down the electrochemical gradient is primarily catalyzed by ion-selective channels that discriminate ions mostly based on the atomic composition and size of their binding site. (43) For instance, gramicidin A (gA) is a short peptide (15 amino acids) that forms a cation-selective bilayer-spanning channel with pore dimensions that restrict ions and water to move in single file through it. (44) As a result, protons (H+), which can rapidly hop along the water wire inside the pore, permeate through gA at much higher rates than other monovalent cations such as K+ and Na+17. On the other hand, other cation-selective channels, such as the Gram-negative trimeric porin OmpF (outer membrane protein F), enable higher K+ permeation (45−47) which may approach that of H+. Therefore, it is of interest to evaluate the relative H+/K+ selectivity (i.e., the permeability ratio) of these two prototypical channels and understand how these two distinct channels may affect the development, or dissipation, of the transmembrane potential.
We studied the passive transport of K+ through gA and OmpF using the same basic principles shown in Figure 2. To incorporate the channel proteins (either gA or OmpF) in the lipid bilayer of OLA-GUVs, we perfused the protein solution into the trapping chamber containing the GUVs and then washed away any residual proteins (see Materials and Methods). Importantly, following the incorporation stage we verified that no leakage of FAMQ-G4 occurs through the channels during the period of our measurements (Figure S9). In addition, we note that in each gA experiment we observed two populations of GUVs with different transport rates (Figure S10)─one population with rates comparable to those of OLA GUVs without gA and the other with significantly greater decay rates. Similarly, the occurrence of two populations was also found in the case of OmpF, though to a lesser extent (Figure S11). The observed heterogeneous incorporation of functional gA, which has also been widely observed for different membrane active peptides and proteins, (12,48,49) clearly signifies the advantage of single-vesicle transport measurement over bulk experiments in which contributions from both populations are averaged. Here, for clarity, we removed the slow population of GUVs from further analysis. Furthermore, to verify the successful incorporation of gA into the GUV membrane, we performed the K+ transport experiments following perfusion of different gA concentrations ([gA] = 0.5 or 5 ng/mL) and found that transport rates are [gA] dependent, indicating successful channel insertions (Figure S10).
Figure 4 shows the temporal variation of lumenal and extravesicular potassium concentrations across GUVs containing either gA (Figure 4A, red curves) or OmpF (Figure 4B, blue curves). For comparison, we show the variation of [K+] in a GUV system without incorporated proteins (black curves), measured under the same conditions (see also Figures S10 and S11). As can be seen, the presence of gA and OmpF in the GUV membrane dramatically increases the transport rate of K+ relative to the plain lipid bilayer. We stress, however, that under our experimental conditions K+ movement across the GUV membrane may occur through the lipid bilayer and ion channels at the same time (see insets to Figure 4). Therefore, the membrane permeability coefficient in our system should encompass the contribution from both pathways: PK+m = ϕPK+ch + (1 – ϕ)PK+lb, where ϕ and 1 – ϕ are the membrane area fractions occupied by reconstituted channel proteins and the lipid bilayer, respectively, and PK+ch and PK+lb are their corresponding permeability rates. Nevertheless, since it can be clearly seen from our measured data in Figure 4 that PK+m ≫ PK+lb when gA and OmpF are incorporated in the lipid bilayer, the overall permeability rate of K+ through all reconstituted ion channels in a GUV (PK+ch=ϕPK+ch) can be estimated from the measured membrane permeability as PK+m≈PK+ch. Likewise, the measured flux density of potassium ions across the membrane is determined by passive transport across the ion channels so that JK+m ≈ JK+ch.

Figure 4

Figure 4. K+ transport kinetics across GUVs with reconstituted model ion channels. (A) Time-resolved variation of lumenal (solid lines) and extravesicular (dashed lines) K+ concentration for DOPC/DOPG (3:1) GUVs with (red, n = 46), and without (black, n = 38) reconstituted gramicidin A (gA) (see experimental section). The distribution of permeated [K+] over GUVs is represented by the upper and lower quartiles of [K+] at each time point. Inset: schematic illustrating the two possible transport pathways across gA incorporated GUVs. B. Analysis of lumenal (solid lines) and extravesicular (dashed lines) [K+] across DOPC/DOPG (3:1) GUVs with (blue, n = 83) and without (black, n = 76) reconstituted OmpF (see Materials and Methods). Inset: schematic illustrating the two possible transport pathways across OmpF-incorporated GUVs. (C) Flux profiles obtained for GUVs with reconstituted gA (red) and OmpF (blue). The circles are the mean flux values, and the bands are the lower and upper quartiles for each GUV population. The black dashed line is the best fit of a linear curve to the mean flux data at the linear regime (0 < Δ[K+] < 0.13 mM), using linear regression. The red arrow indicates the Δ[K+] value at which the measured mean flux (red circles) is 0.58 of the flux (black dashed line) at the same Δ[K+] in the absence of transmembrane potential development. (D) Schematic demonstrating the suggested origin for the variance in H+/K+ selectivity between gA and OmpF.

High Resolution Image
Download MS PowerPoint Slide
By comparing the obtained flux profiles JK+m(Δ[K+]) for gA and OmpF at early stages of transport (Figure 4C), i.e. once the flux increase starts to saturate as Δ[K+] increases, it can be seen that in the presence of gA the flux profile deviates from linearity at Δ[K+] ≈ 0.16 mM (or 1.6 × 10–7 mol/cm3), while no such deviation could be detected for OmpF. The absence of a detectable linear regime in the measured OmpF flux profiles implies that the deviation from linearity occurs at very low Δ[K+] values and is faster than the data acquisition rate used in these experiments (6.6 frames/min). Still, the earlier deviation and development of Δψ due to K+ influx across OmpF indicates that compensation of charge imbalance by H+ efflux occurs at much slower rates than in the case of gA. Hence, by considering that Cl– flux through cation-selective channels is negligible, (44,47) the observed variation in flux profiles strongly suggests that the H+/K+ selectivity of gA is higher compared to OmpF.
In the absence of a linear regime in the case of OmpF, we evaluated only the H+/K+ selectivity of gA by estimating the ratio between the overall permeabilities of K+ and H+ across it (PK+gA/PH+gA). To obtain PH+gA, we consider that the influx of K+ through gA and efflux of H+ across both gA and the lipid bilayer are kept approximately similar in the early stage of K+ transport when Δψ < RT/F so that JK+gA ≈ JH+gA + JH+lb (Supporting Information). Also, recalling that [H+]o ≈ [H+]i ≈ 10–7.6/1000 mol/cm3 during the same initial transport period (Supporting Information), PH+gA can be related to the flux of K+ according to JK+gA≈(PH+gA+PH+lb)[H+](FΔψ/RT). Thus, when FΔψ/RT = 1 (or, alternatively, when Δψ = 25 mV at 25 °C), the channel permeability to protons can be approximated through PH+gA≈(JK+gA/[H+])−PH+lb. According to the GHK flux equation, the flux of potassium is expected to drop by ca. 42% relative to its value in the absence of transmembrane potential. By taking PH+lb=0.002 cm/s (10) and evaluating JK+gA (Δ[K+], Δψ = 25 mV) from the measured flux profile (red arrow in Figure 4C), we found that PH+gA=(6.4±2.3)×10−3 cm/s. Additionally, from the linear regime of the gA flux curve we obtained PK+gA=(7.9±1.3)×10−7 cm/s (black dashed line in Figure 4C). Taken together, the evaluated H+/K+ permeability ratio for gA is σgA=PH+gA/PK+gA≈0.8×104, as similarly reported elsewhere. (17) The obtained H+/K+ selectivity value, while approximated, clearly reflects the intrinsic characteristics of the gA pore that contains a single water wire along which protons can “hop” without displacing water molecules as other cations such as K+. (50) For that reason, gA was also observed to effectively dissipate the buildup of Δψ across the GUV membrane. In the case of OmpF, however, the fast generation of charge imbalance at very low K+ gradients clearly indicates that it possesses a much lower H+/K+ selectivity value relative to gA. In addition, the comparable conductances of different cations across OmpF (51) suggest that the permeability rates of K+ and H+ are similar as well (Figure 4D). Overall, as illustrated in Figure 4D, our results suggest that σgA ≫ σOmpF due to the significantly larger H+/K+ permeability ratio of gA relative to OmpF.

Conclusions

ARTICLE SECTIONS
Jump To
To summarize, we utilized a transmembrane ion transport sensing technique by combining a G-quadruplex-forming fluorescent ion sensor with microfluidic GUV handling techniques. We showed that the designed G-quadruplex DNA probe detects sub-millimolar changes in the lumenal concentration of K+ and can be customized to provide a fluorescent signal over a range of wavelengths. By quantifying the fluxes and concentration gradient of potassium ions across single GUVs, we were able to determine the permeation rate of K+ across the lipid bilayer and estimate the complementary H+ fluxes that dissipate the transmembrane potential during the initial stages of K+ transport. In addition, we showed that the presence of oil residues, such as octanol in our case, significantly increases the permeability rate of potassium through the lipid bilayer by 1 order of magnitude. Still, even in the absence of octanol in the membrane of GUVs, the measured K+ permeability coefficients were found to be at least 1 order of magnitude larger than permeability values that were previously obtained from bulk experiments. Employing the same approach, we measured the passive transport of K+ across two archetypical cation-selective ion channels, gramicidin A and OmpF, incorporated in the membrane of GUVs. An analysis of potassium fluxes across the GUV membrane in the presence of these two channel types provided a useful insight into their role in determining the rate of charge accumulation inside the vesicles. Considering that these channels are cation selective, we evaluated the H+/K+ selectivity of gramicidin A and showed that it is much more selective to protons than OmpF. We anticipate that our study will guide future measurements of ion transport across relevant biological systems such as ion channels, ion pumps, and ionophores.

Materials and Methods

ARTICLE SECTIONS
Jump To

Materials

1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phospho-(1-ac-glycerol) (sodium salt; DOPG), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (ammonium salt; 18/1 Liss Rhod PE) were purchased from Avanti Polar Lipids as powders and dissolved in ethanol to final concentrations of 100 mg/mL (DOPC and DOPG) and 0.5 mg/mL (Liss Rhod PE). Fluorophore- and quencher-modified DNA strands (Figure 1A) were synthesized and purified (HPLC) by Integrated DNA Technologies (IDT) and received at 100 μM in IDT TE Buffer pH 8. 1-Octanol was purchased from Sigma and used as received. Polydimethylsiloxane (PDMS; Sylgard 184) was purchased from Dow Corning and used as received. Potassium chloride (KCl) powder was purchased from Sigma and dissolved in a buffer (10 mM Tris, 1 mM EDTA) to a concentration of 1 M. Gramicidin A was purchased as a powder from Sigma and immediately dissolved in ethanol (analytical grade) at 5 mg/mL and stored at 4 °C. Outer membrane protein F (OmpF, 5.5 mg/mL) in a 1% octyl-POE solution was kindly provided by Prof. Mathias Winterhalter and stored at 4 °C.

Fluorescence Examination of the G4-DNA K+ Sensor

Human Telomere G4-DNA (AGGG(TTAGGG)3), modified with a fluorophore (FAM, HEX, or Texas Red) at its 5′-end and a quencher (Iowa Black FQ for FAM and HEX and Iowa Black RQ for Texas Red) at the 3′-end, were slowly brought to room temperature from their storage at −20 °C and diluted to 100 nM with a 10 mM Tris 1 mM EDTA buffer. To study the response of G4-DNA to K+, different concentrations of KCl, varying from 0 to 2 mM, were added to the probe solution. For preparation of the dsDNA, both FAMQ-G4 and its complementary strand were heated to 60 °C for 10 min and then mixed in a 1/1 volume ratio, where the volume of each sample was 300 μL. The fluorescence emission spectra were recorded from 505 to 605 nm at an excitation of 488 nm for FAM, from 545 to 645 nm for HEX at an excitation of 535 nm, and from 575 to 675 nm at an excitation of 559 nm for Texas Red, using a Cary Eclipse fluorescence spectrophotometer (Agilent Technologies, USA). Spectra at each K+ concentration were analyzed using a custom-made Python program. The total fluorescence intensity for each sample was calculated by numerically integrating the spectrum over the range of wavelengths. The relative fluorescence change was then determined by normalizing the fluorescence at each K+ concentration by the fluorescence with no added K+ for each experiment.

Microfluidic Chip Fabrication

Master molds for the microfluidic designs were created by photolithography and soft lithography. A layer of SU-8 2025 (Microchem) was deposited (3800 rpm, 60 s with 100 rpm/s acceleration for the initial 38 s) onto a 4 in. silicon substrate (University Wafers, USA). Then, the wafer was prebaked (2 min at 65 °C, 5 min at 95 °C) and selectively exposed to ultraviolet light (12 s, 365–405 nm, 20 mW cm–2) using a direct laser writer (LPKF ProtoLaser LDI, Germany). The sample was postbaked (2 min at 65 °C, 5 min at 95 °C), developed for 3 min, dried with a gentle stream of nitrogen, and hard baked for 15 min at 125 °C. To create a negative replica from the silicon mold, we used standard soft lithography techniques. Sylgard 184 polydimethylsiloxane (PDMS) was mixed with a curing agent (9/1 w/w, Dow Corning), and the mixture was poured onto the silicon mold and baked at 60 °C for 55 min. The PDMS chip was then peeled off the mold and inlet/outlet columns created using a 0.75 mm biopsy punch (Miltex). The chip was plasma-bonded to a PDMS-coated glass coverslip using an air plasma (10 W, 25 sccm, 10 s exposure, Diener Electronic GmbH & Co. KG, Germany). The OLA formation device was further processed with a PVA coating of the post junction channel, as previously described in detail elsewhere. (52) The microfluidic chip (Figure S12) was plasma-treated for 1 min immediately before device operation.

Microfluidic Experimental Design

Two microfluidic devices were used in this work, one for vesicle formation using the octanol-assisted liposome assembly (OLA) device and another for hydrodynamic trapping of GUVs. The production of GUVs using the OLA has been previously described in detail elsewhere (52−54) and is also elaborated in the Supporting Information. Briefly, OLA produces GUVs at a six-way junction at which three different liquid phases meet, the inner aqueous (IA), lipid-octanol (LO), and outer aqueous (OA). The LO and OA channels both bifurcate from their inlets to create the five flows that meet at the formation junction (Figure S12). By tuning the flow rates of these three phases, double-emulsion (IA/LO/OA) droplets are produced. The octanol spontaneously phase separates, producing a lipid vesicle (GUV) and an octanol droplet.
The trapping device consists of two fully independent networks, each containing four chambers of trap arrays. The trapping principle has been described in previous reports. (52) Separation of the formation and trapping devices into two isolated chips enables simpler user operation of both devices and the ability to remove GUVs from the microfluidics and store with little added operation time. CAD designs for both microfluidic devices are available in the Supporting Information and can be seen in Figure S12.

Microfluidic Device Operation

All microfluidic devices were operated and imaged on an Olympus IX73 inverted microscope. In- and out-of-plane motion was controlled automatically by a motorized XY stage (Prior Scientific Instruments Ltd. UK). Applied pressures were controlled via a pressure-driven pump (MFCS-EZ, Fluigent GmbH, Germany) and were tuned as required using the MAESFLOW software. Four pressure channels were used for the integrated vesicle-formation and filtering device (Figure S12A), while two channels were required to operate the vesicle-trapping device (Figure S12B). All input fluids were connected from their reservoirs (Micrewtube 0.5 or 1.5 mL, Simport) to the microfluidic devices through polymer tubing (Tygon microbore tubing 0.020 in. ID, 0.060 in. OD, Cole-Parmer, UK) and connector tips (isolated from dispensing tips, Gauge 23 blunt end, Intertronics). Camera acquisition was achieved using a Photometrics Evolve 512 delta camera controlled via the open-source software Micro-Manager 1.4, with magnification via a 10× air objective (Olympus UPLFLN). (55) For fluorescence experiments, illumination was supplied by a wLS LED lamp (Q-Imaging) and passed through a FITC filter cube (Chroma).
To immobilize the GUVs during transport experiments, we trapped the vesicles in a microfluidic chip using hydrodynamic traps. The trapping device (Figure S12B) was bonded immediately prior to operation, and the OA solution was then perfused through it to avoid formation of air bubbles. Subsequently, the GUVs were introduced into the device and trapping was stopped once a satisfactory number of vesicles occupied the traps. Before the potassium solution was introduced to the GUVs, the chambers were washed with IA solution for 30 min to wash away residuals such as oil droplets, free protein (gramicidin A or OmpF), etc. In the gramicidin A and OmpF experiments the model ion channel solution (either 0.5 or 5 ng/mL gramicidin A or 0.0125 mg/mL OmpF in 10 mM Tris 1 mM EDTA, pH 7.6) was perfused into one of the inlets (each inlet was connected to four separated chambers), to enable a control measurement in parallel with the transport experiment. IA solution with 500 nM FAMQ-G4 was perfused to the trap chamber to determine the external concentration of potassium as it arrived into the trapping network. In the final perfusion step a 1 mM KCl solution (with 500 nM FAMQ-G4) was introduced to the chamber to initialize the transport of potassium. Data acquisition started prior to the final perfusion step.

Electroformation of Vesicles

Vesicles prepared via electroformation were obtained using a protocol modified from refs (56and57). Briefly, indium tin oxide (ITO) slides were cleaned with 15 min sonication cycles of isopropanol followed by Milli-Q water and subsequently dried under a nitrogen flow. An ITO slide was heated to ∼50 °C, and 45 μL of lipid mixture (4 mg/mL) was gently spread on the conductive side of the slide using a glass coverslip. The slide was placed in a dry silica desiccator and kept under vacuum for 1 h. Electroformation chambers, assembled by coupling ITO slides with an ∼1 mm thick polydimethylsiloxane (PDMS) spacer, were filled with approximately 250 μL of DNA-containing solution (10 μM) rehydration buffer. The chambers were connected with clamps to a frequency generator and subjected to a sinusoidal alternating current with a voltage amplitude of 2 V. Electroformation was carried out using a frequency of 10 Hz for 2 h followed by 1 h at 2 Hz. Finally, vesicles were gently retrieved and stored at room temperature in the dark to prevent photobleaching and photo-oxidation. The electroformed GUVs were diluted 5× in IA and perfused into the hydrodynamic trapping device. To account for multilamellar GUVs, images of all experimental ROIs were taken before and after the ion transport experiment using a filter isolating the membrane fluorescence signal (Liss-Rhodamine-PE). We visually identified subpopulation vesicles at different levels of membrane fluorescence and removed all vesicles that did not belong to the lowest level of fluorescence.

Data Acquisition

The automated stage supporting the microfluidic device and light source was synchronized with the camera exposure being triggered through Micro-Manager 1.4 software. (55) The stage cycled through a number of fields of view, and an image was captured at each. The cycle repeated at user-specified time intervals (9 s for OmpF, 15 s for gramicidin A, and 30 or 60 s for the GUV experiment). The number of positions was chosen according to the number of points of interest in each experiment. The exposure was set to 30 ms in all experiments to avoid bleaching of the fluorescent sensor (Figure S4). The image sequences were stored by field of view into an ImageJ-compatible TIFF Stacks format.

Image Processing

The time-lapse videos were individually processed using an in-house Python program described in detail previously. (14) The program first identified the arrival of the potassium. As the potassium arrived, the background fluorescence smoothly varied over a few video frames to its final background intensity. The time point at which the background reached half its maximum intensity was used to identify the GUVs for measurement. The software automatically identified individual bright objects above a threshold. Because the GUVs moved slightly within their traps (±5 pixels), the software redetected each initially detected GUV center at every time point. The mean intensity within a 4 × 4 pixel region about the GUV center was recorded for the remaining video frames in the experiment. At the same time, the area of each GUV was measured using the Watershed algorithm. (58) The background intensity for each video was also determined by calculating the median intensity over the full field of view. These intensity time series among all experimental videos were collated for further analysis. Filtering was applied to remove GUVs that suddenly escaped their traps, and those below a certain size threshold (<6 μm), which are likely burst and re-formed membranes.

Supporting Information

ARTICLE SECTIONS
Jump To

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsnano.2c07496.

  • GUV formation and filtering, Human Telomere G-quadruplex K+ binding model, Na+ response of FAMQ-G4, sensitivity of FAMQ-G4 to K+ in the presence of Na+, response of FAMQ-G4 to K+ in 100 mM LiCl, FAMQ-HT photobleaching and leakage across the lipid bilayer, folding kinetics of FAMQ-HT following 100 μM KCl, interaction of FAMQ-G4 with the lipid bilayer, change of pH inside GUVs during K+ permeation, measurement of K+ permeation across electroformed GUVs, demonstration of no leakage of FAMQ-G4 from GUVs after perfusion of model ion channels, efficiency of gramicidin A incorporation dependent on its concentration in solution, K+ transport across OmpF containing GUVs, microfluidic device CAD designs, K+ flux density approximately equaling H+ counter flux during the linear regime (PDF)

Terms & Conditions

Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.

Author Information

ARTICLE SECTIONS
Jump To
  • Corresponding Author
  • Authors
  • Author Contributions

    M.F., U.F.K., and R.T. designed the experiments. R.T. and M.F. wrote the manuscript. M.F. conducted the measurements. M.F. and R.T. analyzed the data. K.A.N. assisted with microfluidic device design. J.Z. assisted with the DNA probe design. U.F.K. and R.T. assisted with theoretical aspects of ion transport. R.R.S. produced DNA probe-containing GUVs by electroformation. S.E.S. performed the conductance measurements. All authors discussed the results and commented on the manuscript.

  • Notes
    The authors declare no competing financial interest.

Acknowledgments

ARTICLE SECTIONS
Jump To

The authors thank Prof. Mathias Winterhalter for generously providing the OmpF stock. M.F. acknowledges support from the UK’s Engineering and Physical Sciences Research Council Doctoral Training Programme (EP/R513180/1) and a Cambridge-NPL iCASE studentship. R.T. acknowledges funding from the European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement No. 892333 and from the Blavatnik Family Foundation. R.R.S. and L.D.M. acknowledge funding from the Royal Society Research Fellows Enhanced Research Expenses (RF\ERE\210029). L.D.M. acknowledges support from a Royal Society University Research Fellowship (UF160152) and from the European Research Council (ERC) under the Horizon 2020 Research and Innovation Programme (ERC-STG No. 851667 NANOCELL). K.A.N. acknowledges support from a Cambridge-National Physical Laboratory (U.K.) studentship, the Winton Programme for the Physics of Sustainability, the Trinity-Henry Barlow Scholarship, and the ERC. U.F.K., J.Z., and R.T. acknowledge support from an ERC consolidator grant (Designer-Pores 647144).

References

ARTICLE SECTIONS
Jump To

This article references 58 other publications.

  1. 1
    Clausen, M. V.; Hilbers, F.; Poulsen, H. The Structure and Function of the Na,K-ATPase Isoforms in Health and Disease. Frontiers in Physiology 2017, 8, 1,  DOI: 10.3389/fphys.2017.00371
    Google Scholar
    There is no corresponding record for this reference.
  2. 2
    Navale, A. M.; Paranjape, A. N. Glucose transporters: physiological and pathological roles. Biophysical Reviews 2016, 8 (1), 5– 9,  DOI: 10.1007/s12551-015-0186-2
    Google Scholar
    2
    Glucose transporters: physiological and pathological roles
    Navale, Archana M.; Paranjape, Archana N.
    Biophysical Reviews (2016), 8 (1), 5-9CODEN: BRIECG; ISSN:1867-2450. (Springer)
    A review. Glucose is a primary energy source for most cells and an important substrate for many biochem. reactions. As glucose is a need of each and every cell of the body, so are the glucose transporters. Consequently, all cells express these important proteins on their surface. In recent years developments in genetics have shed new light on the types and physiol. of various glucose transporters, of which there are two main types-sodium-glucose linked transporters (SGLTs) and facilitated diffusion glucose transporters (GLUT)-which can be divided into many more subclasses. Transporters differ in terms of their substrate specificity, distribution and regulatory mechanisms. Glucose transporters have also received much attention as therapeutic targets for various diseases. In this review, we attempt to present a simplified view of this complex topic which may be of interest to researchers involved in biochem. and pharmacol. research.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhtF2isb0%253D&md5=e328a4728ff6bca082764e1d06372066
  3. 3
    Calisto, F.; Sousa, F. M.; Sena, F. V.; Refojo, P. N.; Pereira, M. M. Mechanisms of Energy Transduction by Charge Translocating Membrane Proteins. Chem. Rev. 2021, 121 (3), 1804– 1844,  DOI: 10.1021/acs.chemrev.0c00830
    Google Scholar
    3
    Mechanisms of Energy Transduction by Charge Translocating Membrane Proteins
    Calisto, Filipa; Sousa, Filipe M.; Sena, Filipa V.; Refojo, Patricia N.; Pereira, Manuela M.
    Chemical Reviews (Washington, DC, United States) (2021), 121 (3), 1804-1844CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)
    A review. Life relies on the const. exchange of different forms of energy, i.e., on energy transduction. Therefore, organisms have evolved in a way to be able to harvest the energy made available by external sources (such as light or chem. compds.) and convert these into biol. useable energy forms, such as the transmembrane difference of electrochem. potential (Δ~μ). Membrane proteins contribute to the establishment of Δ~μ by coupling exergonic catalytic reactions to the translocation of charges (electrons/ions) across the membrane. Irresp. of the energy source and consequent type of reaction, all charge-translocating proteins follow two mol. coupling mechanisms: direct- or indirect-coupling, depending on whether the translocated charge is involved in the driving reaction. In this review, we explore these two coupling mechanisms by thoroughly examg. the different types of charge-translocating membrane proteins. For each protein, we analyze the resp. reaction thermodn., electron transfer/catalytic processes, charge-translocating pathways, and ion/substrate stoichiometries.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXitF2jug%253D%253D&md5=9f96c0c718531fa3312a980cbdb9081c
  4. 4
    Valkenier, H.; López Mora, N.; Kros, A.; Davis, A. P. Visualization and quantification of transmembrane ion transport into giant unilamellar vesicles. Angewandte Chemie (International Ed. in English) 2015, 54 (7), 2137– 2141,  DOI: 10.1002/anie.201410200
    Google Scholar
    4
    Visualization and quantification of transmembrane ion transport into giant unilamellar vesicles
    Valkenier Hennie; Lopez Mora Nestor; Kros Alexander; Davis Anthony P
    Angewandte Chemie (International ed. in English) (2015), 54 (7), 2137-41 ISSN:.
    Transmembrane ion transporters (ionophores) are widely investigated as supramolecular agents with potential for biological activity. Tests are usually performed in synthetic membranes that are assembled into large unilamellar vesicles (LUVs). However transport must be followed through bulk properties of the vesicle suspension, because LUVs are too small for individual study. An alternative approach is described whereby ion transport can be revealed and quantified through direct observation. The method employs giant unilamellar vesicles (GUVs), which are 20-60 μm in diameter and readily imaged by light microscopy. This allows characterization of individual GUVs containing transporter molecules, followed by studies of transport through fluorescence emission from encapsulated indicators. The method provides new levels of certainty and relevance, given that the GUVs are similar in size to living cells. It has been demonstrated using a highly active anion carrier, and should aid the development of compounds for treating channelopathies such as cystic fibrosis.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC2MvjtlajsQ%253D%253D&md5=e035e3a1292cea17d96629d2467a570f
  5. 5
    Garten, M.; Mosgaard, L. D.; Bornschlögl, T.; Dieudonné, S.; Bassereau, P.; Toombes, G. E. S. Whole-GUV patch-clamping. Proc. Natl. Acad. Sci. U. S. A. 2017, 114 (2), 328– 333,  DOI: 10.1073/pnas.1609142114
    Google Scholar
    5
    Whole-GUV patch-clamping
    Garten, Matthias; Mosgaard, Lars D.; Bornschlogl, Thomas; Dieudonne, Stephane; Bassereau, Patricia; Toombes, Gilman E. S.
    Proceedings of the National Academy of Sciences of the United States of America (2017), 114 (2), 328-333CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    Studying how the membrane modulates ion channel and transporter activity is challenging because cells actively regulate membrane properties, whereas existing in vitro systems have limitations, such as residual solvent and unphysiol. high membrane tension. Cell-sized giant unilamellar vesicles (GUVs) would be ideal for in vitro electrophysiol., but efforts to measure the membrane current of intact GUVs have been unsuccessful. In this work, two challenges for obtaining the "whole-GUV" patch-clamp configuration were identified and resolved. First, unless the patch pipet and GUV pressures are precisely matched in the GUV-attached configuration, breaking the patch membrane also ruptures the GUV. Second, GUVs shrink irreversibly because the membrane/glass adhesion creating the high-resistance seal (>1 GΩ) continuously pulls membrane into the pipet. In contrast, for cell-derived giant plasma membrane vesicles (GPMVs), breaking the patch membrane allows the GPMV contents to passivate the pipet surface, thereby dynamically blocking membrane spreading in the whole-GMPV mode. To mimic this dynamic passivation mechanism, beta-casein was encapsulated into GUVs, yielding a stable, high-resistance, whole-GUV configuration for a range of membrane compns. Specific membrane capacitance measurements confirmed that the membranes were truly solvent-free and that membrane tension could be controlled over a physiol. range. Finally, the potential for ion transport studies was tested using the model ion channel, gramicidin, and voltage-clamp fluorometry measurements were performed with a voltage-dependent fluorophore/quencher pair. Whole-GUV patch-clamping allows ion transport and other voltage-dependent processes to be studied while controlling membrane compn., tension, and shape.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XitFentrnK&md5=757fba9d98f5630604eb235709f0de83
  6. 6
    Cama, J.; Bajaj, H.; Pagliara, S.; Maier, T.; Braun, Y.; Winterhalter, M.; Keyser, U. F. Quantification of Fluoroquinolone Uptake through the Outer Membrane Channel OmpF of Escherichia coli. J. Am. Chem. Soc. 2015, 137 (43), 13836– 13843,  DOI: 10.1021/jacs.5b08960
    Google Scholar
    6
    Quantification of fluoroquinolone uptake through the outer membrane channel OmpF of Escherichia coli
    Cama, Jehangir; Bajaj, Harsha; Pagliara, Stefano; Maier, Theresa; Braun, Yvonne; Winterhalter, Mathias; Keyser, Ulrich F.
    Journal of the American Chemical Society (2015), 137 (43), 13836-13843CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Decreased drug accumulation is a common cause of antibiotic resistance in microorganisms. However, there are few reliable general techniques capable of quantifying drug uptake through bacterial membranes. The authors present a semiquant. optofluidic assay for studying the uptake of autofluorescent drug mols. in single liposomes. They studied the effect of the Escherichia coli outer membrane channel OmpF on the accumulation of the fluoroquinolone antibiotic, norfloxacin, in proteoliposomes. Measurements were performed at pH 5 and pH 7, corresponding to two different charge states of norfloxacin that bacteria are likely to encounter in the human gastrointestinal tract. At both pH values, the porins significantly enhance drug permeation across the proteoliposome membranes. At pH 5, where norfloxacin permeability across pure phospholipid membranes is low, the porins increase drug permeability by 50-fold on av. The authors est. a flux of about 10 norfloxacin mols. per s per OmpF trimer in the presence of a 1 mM concn. gradient of norfloxacin. They also performed single channel electrophysiol. measurements and found that the application of transmembrane voltages causes an elec. field driven uptake in addn. to concn. driven diffusion. The authors propose a phys. mechanism for the pH mediated change in bacterial susceptibility to fluoroquinolone antibiotics.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhs1Ohur3E&md5=b29d6581fa48b214ad4e5ff617eff944
  7. 7
    Göpfrich, K.; Platzman, I.; Spatz, J. P. Mastering Complexity: Towards Bottom-up Construction of Multifunctional Eukaryotic Synthetic Cells. Trends Biotechnol. 2018, 36 (9), 938– 951,  DOI: 10.1016/j.tibtech.2018.03.008
    Google Scholar
    7
    Mastering Complexity: Towards Bottom-up Construction of Multifunctional Eukaryotic Synthetic Cells
    Gopfrich Kerstin; Platzman Ilia; Spatz Joachim P
    Trends in biotechnology (2018), 36 (9), 938-951 ISSN:.
    With the ultimate aim to construct a living cell, bottom-up synthetic biology strives to reconstitute cellular phenomena in vitro - disentangled from the complex environment of a cell. Recent work towards this ambitious goal has provided new insights into the mechanisms governing life. With the fast-growing library of functional modules for synthetic cells, their classification and integration become increasingly important. We discuss strategies to reverse-engineer and recombine functional parts for synthetic eukaryotes, mimicking the characteristics of nature's own prototype. Particularly, we focus on large outer compartments, complex endomembrane systems with organelles, and versatile cytoskeletons as hallmarks of eukaryotic life. Moreover, we identify microfluidics and DNA nanotechnology as two technologies that can integrate these functional modules into sophisticated multifunctional synthetic cells.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC1MjlslCjsQ%253D%253D&md5=beed6876210fcc29b75bad05443d0ba5
  8. 8
    Ohmann, A.; Li, C.-Y.; Maffeo, C.; Al Nahas, K.; Baumann, K. N.; Göpfrich, K.; Yoo, J.; Keyser, U. F.; Aksimentiev, A. A synthetic enzyme built from DNA flips 107 lipids per second in biological membranes. Nat. Commun. 2018, 9 (1), 1– 9,  DOI: 10.1038/s41467-018-04821-5
    Google Scholar
    8
    A synthetic enzyme built from DNA flips 107 lipids per second in biological membranes
    Ohmann, Alexander; Li, Chen-Yu; Maffeo, Christopher; Al Nahas, Kareem; Baumann, Kevin N.; Gopfrich, Kerstin; Yoo, Jejoong; Keyser, Ulrich F.; Aksimentiev, Aleksei
    Nature Communications (2018), 9 (1), 1-9CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)
    Mimicking enzyme function and increasing performance of naturally evolved proteins is one of the most challenging and intriguing aims of nanoscience. Here, we employ DNA nanotechnol. to design a synthetic enzyme that substantially outperforms its biol. archetypes. Consisting of only eight strands, our DNA nanostructure spontaneously inserts into biol. membranes by forming a toroidal pore that connects the membrane's inner and outer leaflets. The membrane insertion catalyzes spontaneous transport of lipid mols. between the bilayer leaflets, rapidly equilibrating the lipid compn. Through a combination of microscopic simulations and fluorescence microscopy we find the lipid transport rate catalyzed by the DNA nanostructure exceeds 107 mols. per s, which is three orders of magnitude higher than the rate of lipid transport catalyzed by biol. enzymes. Furthermore, we show that our DNA-based enzyme can control the compn. of human cell membranes, which opens new avenues for applications of membrane-interacting DNA systems in medicine.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhtF2jtbzN&md5=9594eb1d972bb6381a9c9d64a5bc4f0f
  9. 9
    Elani, Y.; Trantidou, T.; Wylie, D.; Dekker, L.; Polizzi, K.; Law, R. V.; Ces, O. Constructing vesicle-based artificial cells with embedded living cells as organelle-like modules. Sci. Rep. 2018, 8 (1), 4564,  DOI: 10.1038/s41598-018-22263-3
    Google Scholar
    9
    Constructing vesicle-based artificial cells with embedded living cells as organelle-like modules
    Elani Yuval; Trantidou Tatiana; Wylie Douglas; Law Robert V; Ces Oscar; Elani Yuval; Wylie Douglas; Ces Oscar; Dekker Linda; Polizzi Karen
    Scientific reports (2018), 8 (1), 4564 ISSN:.
    There is increasing interest in constructing artificial cells by functionalising lipid vesicles with biological and synthetic machinery. Due to their reduced complexity and lack of evolved biochemical pathways, the capabilities of artificial cells are limited in comparison to their biological counterparts. We show that encapsulating living cells in vesicles provides a means for artificial cells to leverage cellular biochemistry, with the encapsulated cells serving organelle-like functions as living modules inside a larger synthetic cell assembly. Using microfluidic technologies to construct such hybrid cellular bionic systems, we demonstrate that the vesicle host and the encapsulated cell operate in concert. The external architecture of the vesicle shields the cell from toxic surroundings, while the cell acts as a bioreactor module that processes encapsulated feedstock which is further processed by a synthetic enzymatic metabolism co-encapsulated in the vesicle.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC1Mnht1yisw%253D%253D&md5=8e62e29719807e28d859d2c4d4edfe5b
  10. 10
    Tivony, R.; Fletcher, M.; Keyser, U. F. Quantifying proton-induced membrane polarization in single biomimetic giant vesicles. Biophys. J. 2022, 121, 2223,  DOI: 10.1016/j.bpj.2022.05.041
    Google Scholar
    10
    Quantifying proton-induced membrane polarization in single biomimetic giant vesicles
    Tivony, Ran; Fletcher, Marcus; Keyser, Ulrich F.
    Biophysical Journal (2022), 121 (12), 2223-2232CODEN: BIOJAU; ISSN:0006-3495. (Cell Press)
    Proton gradients are utilized by cells to power the transport activity of many membrane proteins. Synthetic cells, such as proteo-giant unilamellar vesicles, offer an advanced approach for studying the functionality of membrane proteins in isolation. However, understanding of protein-based transport in vitro requires accurate measurements of proton flux and its accompanying electrochem. gradient across the lipid bilayer. We present an approach to directly quantify the flux of protons across single cell-sized lipid vesicles under modulated electrochem. gradients. Our measurements reveal the corresponding assocn. between proton permeation and transmembrane potential development and its relation to the chem. nature of the conjugated anion (base). In the case of formic acid, we showed that, out of the total amt. of permeated protons, a fraction of ≈0.2 traverse the lipid bilayer as H+, with the rest (≈0.8) in the form of a neutral acid. For strong acids (HCl or HNO3), proton permeation was governed by translocation of H+. Accordingly, a larger proton motive force (pmf) was generated for strong acids (pmf = 14.2 mV) relative to formic acid (pmf = 1.3 mV). We anticipate that our approach will guide the development of protein-based transport driven by proton gradient in artificial cell models and enable a deeper understanding of how vital acids, such as fatty acids, amino acids, bile acids, and carboxylic acid-contg. drugs, traverse the lipid bilayer.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XhsVCntr%252FF&md5=f9efcabb59036ea920d68bbf69dff4b3
  11. 11
    Hindley, J. W.; Zheleva, D. G.; Elani, Y.; Charalambous, K.; Barter, L. M.; Booth, P. J.; Bevan, C. L.; Law, R. V.; Ces, O. Building a synthetic mechanosensitive signaling pathway in compartmentalized artificial cells. Proc. Natl. Acad. Sci. U. S. A. 2019, 116 (34), 16711– 16716,  DOI: 10.1073/pnas.1903500116
    Google Scholar
    11
    Building a synthetic mechanosensitive signaling pathway in compartmentalized artificial cells
    Hindley, James W.; Zheleva, Daniela G.; Elani, Yuval; Charalambous, Kalypso; Barter, Laura M. C.; Booth, Paula J.; Bevan, Charlotte L.; Law, Robert V.; Ces, Oscar
    Proceedings of the National Academy of Sciences of the United States of America (2019), 116 (34), 16711-16716CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    To date, reconstitution of one of the fundamental methods of cell communication, the signaling pathway, has been unaddressed in the bottom-up construction of artificial cells (ACs). Such developments are needed to increase the functionality and biomimicry of ACs, accelerating their translation and application in biotechnol. Here, we report the construction of a de novo synthetic signaling pathway in microscale nested vesicles. Vesicle-cell models respond to external calcium signals through activation of an intracellular interaction between phospholipase A2 and a mechanosensitive channel present in the internal membranes, triggering content mixing between compartments and controlling cell fluorescence. Emulsion-based approaches to AC construction are therefore shown to be ideal for the quick design and testing of new signaling networks and can readily include synthetic mols. difficult to introduce to biol. cells. This work represents a foundation for the engineering of multicompartment-spanning designer pathways that can be utilized to control downstream events inside an AC, leading to the assembly of micromachines capable of sensing and responding to changes in their local environment.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhs1alsLrO&md5=c1fba7b047e86a526fba8fc14ac7370c
  12. 12
    Al Nahas, K.; Fletcher, M.; Hammond, K.; Nehls, C.; Cama, J.; Ryadnov, M. G.; Keyser, U. F. Measuring Thousands of Single-Vesicle Leakage Events Reveals the Mode of Action of Antimicrobial Peptides. Anal. Chem. 2022, 94 (27), 9530– 9539,  DOI: 10.1021/acs.analchem.1c03564
    Google Scholar
    12
    Measuring thousands of single-vesicle leakage events reveals mode of action of antimicrobial peptides
    Al Nahas, Kareem; Fletcher, Marcus; Hammond, Katharine; Nehls, Christian; Cama, Jehangir; Ryadnov, Maxim G.; Keyser, Ulrich F.
    Analytical Chemistry (Washington, DC, United States) (2022), 94 (27), 9530-9539CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
    Host defense or antimicrobial peptides hold promise for providing new pipelines of effective antimicrobial agents. Their activity quantified against model phospholipid membranes is fundamental to a detailed understanding of their structure-activity relationships. However, classical characterization assays often lack the ability to achieve this insight. Leveraging a highly parallelized microfluidic platform for trapping and studying thousands of giant unilamellar vesicles, we conducted quant. long-term microscopy studies to monitor the membrane-disruptive activity of archetypal antimicrobial peptides with a high spatiotemporal resoln. We described the modes of action of these peptides via measurements of the disruption of the vesicle population under the conditions of continuous peptide dosing using a range of concns. and related the obsd. modes to the mol. activity mechanisms of these peptides. The study offers an effective approach for characterizing membrane-targeting antimicrobial agents in a standardized manner and for assigning specific modes of action to the corresponding antimicrobial mechanisms.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XhsF2lur7K&md5=367970eacd16a15323d8ca9ca62a0164
  13. 13
    Robinson, T. Microfluidic Handling and Analysis of Giant Vesicles for Use as Artificial Cells: A Review. Advanced Biosystems 2019, 3 (6), 1800318,  DOI: 10.1002/adbi.201800318
    Google Scholar
    There is no corresponding record for this reference.
  14. 14
    Cama, J.; Al Nahas, K.; Fletcher, M.; Hammond, K.; Ryadnov, M. G.; Keyser, U. F.; Pagliara, S. An ultrasensitive microfluidic approach reveals correlations between the physico-chemical and biological activity of experimental peptide antibiotics. Sci. Rep. 2022, 12 (1), 1– 12,  DOI: 10.1038/s41598-022-07973-z
    Google Scholar
    There is no corresponding record for this reference.
  15. 15
    Hammond, K.; Cipcigan, F.; Al Nahas, K.; Losasso, V.; Lewis, H.; Cama, J.; Martelli, F.; Simcock, P. W.; Fletcher, M.; Ravi, J.; Stansfeld, P. J.; Pagliara, S.; Hoogenboom, B. W.; Keyser, U. F.; Sansom, M. S. P.; Crain, J.; Ryadnov, M. G. Switching Cytolytic Nanopores into Antimicrobial Fractal Ruptures by a Single Side Chain Mutation. ACS Nano 2021, 15 (6), 9679– 9689,  DOI: 10.1021/acsnano.1c00218
    Google Scholar
    15
    Switching Cytolytic Nanopores into Antimicrobial Fractal Ruptures by a Single Side Chain Mutation
    Hammond Katharine; Lewis Helen; Ravi Jascindra; Ryadnov Maxim G; Hammond Katharine; Hoogenboom Bart W; Cipcigan Flaviu; Martelli Fausto; Crain Jason; Al Nahas Kareem; Fletcher Marcus; Keyser Ulrich F; Losasso Valeria; Cama Jehangir; Pagliara Stefano; Cama Jehangir; Simcock Patrick W; Stansfeld Phillip J; Sansom Mark S P; Crain Jason; Pagliara Stefano; Hoogenboom Bart W; Ryadnov Maxim G
    ACS nano (2021), 15 (6), 9679-9689 ISSN:.
    Disruption of cell membranes is a fundamental host defense response found in virtually all forms of life. The molecular mechanisms vary but generally lead to energetically favored circular nanopores. Here, we report an elaborate fractal rupture pattern induced by a single side-chain mutation in ultrashort (8-11-mers) helical peptides, which otherwise form transmembrane pores. In contrast to known mechanisms, this mode of membrane disruption is restricted to the upper leaflet of the bilayer where it exhibits propagating fronts of peptide-lipid interfaces that are strikingly similar to viscous instabilities in fluid flow. The two distinct disruption modes, pores and fractal patterns, are both strongly antimicrobial, but only the fractal rupture is nonhemolytic. The results offer wide implications for elucidating differential membrane targeting phenomena defined at the nanoscale.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB3sbktVGruw%253D%253D&md5=1742ac794c7c3de8acc0aa8ee5d1c89c
  16. 16
    Paula, S.; Volkov, A. G.; Van Hoek, A. N.; Haines, T. H.; Deamer, D. W. Permeation of protons, potassium ions, and small polar molecules through phospholipid bilayers as a function of membrane thickness. Biophys. J. 1996, 70 (1), 339– 348,  DOI: 10.1016/S0006-3495(96)79575-9
    Google Scholar
    16
    Permeation of protons, potassium ions, and small polar molecules through phospholipid bilayers as a function of membrane thickness
    Paula, S.; Volkov, A. G.; Van Hoek, A. N.; Haines, T. H.; Deamer, D. W.
    Biophysical Journal (1996), 70 (1), 339-48CODEN: BIOJAU; ISSN:0006-3495. (Biophysical Society)
    Two mechanisms have been proposed to account for solute permeation of lipid bilayers. Partitioning into the hydrophobic phase of the bilayer, followed by diffusion, is accepted by many for the permeation of water and other small neutral solutes, but transient pores have also been proposed to account for both water and ionic solute permeation. These two mechanisms make distinctively different predictions about the permeability coeff. as a function of bilayer thickness. Whereas the soly.-diffusion mechanism predicts only a modest variation related to bilayer thickness, the pore model predicts an exponential relationship. To test these models, we measured the permeability of phospholipid bilayers to protons, potassium ions, water, urea, and glycerol. Bilayers were prepd. as liposomes, and thickness was varied systematically by using unsatd. lipids with chain lengths ranging from 14 to 24 carbon atoms. The permeability coeff. of water and neutral polar solutes displayed a modest dependence on bilayer thickness, with an approx. linear fivefold decrease as the carbon no. varied from 14 to 24 atoms. In contrast, the permeability to protons and potassium ions decreased sharply by two orders of magnitude between 14 and 18 carbon atoms, and leveled off, when the chain length was further extended to 24 carbon atoms. The results for water and the neutral permeating solutes are best explained by the soly.-diffusion mechanism. The results of protons and potassium ions in shorter-chain lipids are consistent with the transient pore model, but better fit the theor. line predicted by the soly.-diffusion model at longer chain lengths.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK28XjslGksQ%253D%253D&md5=e78b1e80f02e1b3942125de80e48dcd9
  17. 17
    Deamer, D. W. Proton permeation of lipid bilayers. Journal of Bioenergetics and Biomembranes 1987, 19 (5), 457– 479,  DOI: 10.1007/BF00770030
    Google Scholar
    17
    Proton permeation of lipid bilayers
    Deamer, D. W.
    Journal of Bioenergetics and Biomembranes (1987), 19 (5), 457-79CODEN: JBBID4; ISSN:0145-479X.
    Two expts. designed to test the tHBC model (transient H-bonded chain model) of H+ permeation in lipid bilayers were performed. These included measurements of relative H+/K+ permeability in the gramicidin channel, and plotting of H+ flux against the magnitude of pH gradients. The relative permeabilities of H+ and K+ through the gramicidin channel, which contains a single strand of H-bonded H2O mols., differed by >4 orders of magnitude when measured at neutral pH, demonstrating that a H-bonded chain of H2O mols. can provide substantial discrimination between H+ and other cations. It was also calcd. that if ∼7% of bilayer H2O was present in a transient configuration similar to that of the gramicidin channel, it could account for the measured proton flux. The plot of proton conductance against pH gradient across liposome membranes was superlinear, a result consistent with 1 of 3 alternative tHBC models for proton conductance described by J. Nagel (1987). A review of the H+ permeability of lipid bilayers is also presented; several proposed models of the process are discussed.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL1cXntVOhtw%253D%253D&md5=64b10ec6566d9e4235f628b30dc39580
  18. 18
    Paula, S.; Volkov, A. G.; Deamer, D. W. Permeation of Halide Anions through Phospholipid Bilayers Occurs by the Solubility-Diffusion Mechanism. Biophys. J. 1998, 74 (1), 319– 327,  DOI: 10.1016/S0006-3495(98)77789-6
    Google Scholar
    18
    Permeation of halide anions through phospholipid bilayers occurs by the solubility-diffusion mechanism
    Paula, S.; Volkov, A. G.; Deamer, D. W.
    Biophysical Journal (1998), 74 (1), 319-327CODEN: BIOJAU; ISSN:0006-3495. (Biophysical Society)
    Two alternative mechanisms are frequently used to describe ionic permeation of lipid bilayers. In the first, ions partition into the hydrophobic phase and then diffuse across (the soly.-diffusion mechanism). The second mechanism assumes that ions traverse the bilayer through transient hydrophilic defects caused by thermal fluctuations (the pore mechanism). The theor. predictions made by both models were tested for halide anions by measuring the permeability coeffs. for chloride, bromide, and iodide as a function of bilayer thickness, ionic radius, and sign of charge. To vary the bilayer thickness systematically, liposomes were prepd. from monounsatd. phosphatidylcholines (PC) with chain lengths between 16 and 24 carbon atoms. The fluorescent dye MQAE (N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide) served as an indicator for halide concn. inside the liposomes and was used to follow the kinetics of halide flux across the bilayer membranes. The obsd. permeability coeffs. ranged from 10-9 to 10-7 cm/s and increased as the bilayer thickness was reduced. Bromide was found to permeate approx. six times faster than chloride through bilayers of identical thickness, and iodide permeated three to four times faster than bromide. The dependence of the halide permeability coeffs. on bilayer thickness and on ionic size were consistent with permeation of hydrated ions by a soly.-diffusion mechanism rather than through transient pores. Halide permeation therefore differs from that of a monovalent cation such as potassium, which has been accounted for by a combination of the two mechanisms depending on bilayer thickness.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1cXhvFSru7o%253D&md5=d4ddbd155fdff8ec0370768a0532c39b
  19. 19
    Shen, Y.; Zhong, Y.; Fei, F.; Sun, J.; Czajkowsky, D. M.; Gong, B.; Shao, Z. Ultrasensitive liposome-based assay for the quantification of fundamental ion channel properties. Anal. Chim. Acta 2020, 1112, 8– 15,  DOI: 10.1016/j.aca.2020.03.044
    Google Scholar
    19
    Ultrasensitive liposome-based assay for the quantification of fundamental ion channel properties
    Shen, Yi; Zhong, Yulong; Fei, Fan; Sun, Jielin; Czajkowsky, Daniel M.; Gong, Bing; Shao, Zhifeng
    Analytica Chimica Acta (2020), 1112 (), 8-15CODEN: ACACAM; ISSN:0003-2670. (Elsevier B.V.)
    One of the most widely used approaches to characterize transmembrane ion transport through nanoscale synthetic or biol. channels is a straightforward, liposome-based assay that monitors changes in ionic flux across the vesicle membrane using pH- or ion-sensitive dyes. However, failure to account for the precise exptl. conditions, in particular the complete ionic compn. on either side of the membrane and the inherent permeability of ions through the lipid bilayer itself, can prevent quantifications and lead to fundamentally incorrect conclusions. Here we present a quant. model for this assay based on the Goldman-Hodgkin-Katz flux theory, which enables accurate measurements and identification of optimal conditions for the detn. of ion channel permeability and selectivity. Based on our model, the detection sensitivity of channel permeability is improved by two orders of magnitude over the commonly used exptl. conditions. Further, rather than obtaining qual. preferences of ion selectivity as is typical, we det. quant. values of these parameters under rigorously controlled conditions even when the exptl. results would otherwise imply (without our model) incorrect behavior. We anticipate that this simply employed ultrasensitive assay will find wide application in the quant. characterization of synthetic or biol. ion channels.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXmsl2qtr8%253D&md5=3a1f9d252c6fcbf2b8a2bb938f9ac640
  20. 20
    Megens, M.; Korman, C. E.; Ajo-Franklin, C. M.; Horsley, D. A. Faster-than-anticipated Na+/Cl- diffusion across lipid bilayers in vesicles. Biochimica et Biophysica Acta (BBA) - Biomembranes 2014, 1838 (10), 2420– 2424,  DOI: 10.1016/j.bbamem.2014.05.010
    Google Scholar
    There is no corresponding record for this reference.
  21. 21
    Kuyper, C. L.; Kuo, J. S.; Mutch, S. A.; Chiu, D. T. Proton Permeation into Single Vesicles Occurs via a Sequential Two-Step Mechanism and Is Heterogeneous. J. Am. Chem. Soc. 2006, 128 (10), 3233– 3240,  DOI: 10.1021/ja057349c
    Google Scholar
    21
    Proton Permeation into Single Vesicles Occurs via a Sequential Two-Step Mechanism and Is Heterogeneous
    Kuyper, Christopher L.; Kuo, Jason S.; Mutch, Sarah A.; Chiu, Daniel T.
    Journal of the American Chemical Society (2006), 128 (10), 3233-3240CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    This article describes the first single-vesicle study of proton permeability across the lipid membrane of small (∼100 nm) uni- and multilamellar vesicles, which were composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). To follow proton permeation into the internal vol. of each vesicle, we encapsulated carboxyfluorescein, a pH-sensitive dye whose fluorescence was quenched in the presence of excess protons. A microfluidic platform was used for easy exchange of high- and low-pH solns., and fluorescence quenching of single vesicles was detected with single-mol. total internal reflection fluorescence (TIRF) microscopy. Upon soln. exchange and acidification of the extravesicular soln. (from pH 9 to 3.5), we obsd. for each vesicle a biphasic decay in fluorescence. Through single-vesicle anal., we found that rate consts. for the first decay followed a Poisson distribution, whereas rate consts. for the second decay followed a normal distribution. We propose that proton permeation into each vesicle first arose from formation of transient pores and then transitioned into the second decay phase, which occurred by the soly.-diffusion mechanism. Furthermore, for the bulk population of vesicles, the decay rate const. and vesicle intensity (dependent on size) correlated to give an av. permeability coeff.; however, for individual vesicles, we found little correlation, which suggested that proton permeability among single vesicles was heterogeneous in our expts.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XhsFarsbs%253D&md5=1f9afa71de10357c3c50f0051d1add8b
  22. 22
    Debnath, M.; Chakraborty, S.; Kumar, Y. P.; Chaudhuri, R.; Jana, B.; Dash, J. Ionophore constructed from non-covalent assembly of a G-quadruplex and liponucleoside transports K+-ion across biological membranes. Nat. Commun. 2020, 11 (1), 1– 12,  DOI: 10.1038/s41467-019-13834-7
    Google Scholar
    There is no corresponding record for this reference.
  23. 23
    Venema, K.; Gibrat, R.; Grouzis, J.-P.; Grignon, C. Quantitative measurement of cationic fluxes, selectivity and membrane potential using liposomes multilabelled with fluorescent probes. Biochimica et Biophysica Acta (BBA) - Biomembranes 1993, 1146 (1), 87– 96,  DOI: 10.1016/0005-2736(93)90342-W
    Google Scholar
    There is no corresponding record for this reference.
  24. 24
    Sambath, K.; Liu, X.; Wan, Z.; Hutnik, L.; Belfield, K. D.; Zhang, Y. Potassium Ion Fluorescence Probes: Structures, Properties and Bioimaging. ChemPhotoChem. 2021, 5 (4), 317– 325,  DOI: 10.1002/cptc.202000236
    Google Scholar
    24
    Potassium Ion Fluorescence Probes: Structures, Properties and Bioimaging
    Sambath, Karthik; Liu, Xiangshan; Wan, Zhaoxiong; Hutnik, Lauren; Belfield, Kevin D.; Zhang, Yuanwei
    ChemPhotoChem (2021), 5 (4), 317-325CODEN: CHEMYH ISSN:. (Wiley-VCH Verlag GmbH & Co. KGaA)
    A review. As one of the most important minerals in the body, potassium is vital for the heart and neurons. Methods that can non-invasively and accurately monitor changes in potassium balances would benefit disease diagnoses as well as offer insight into pathologies. Among the sensing approaches, fluorescent probes serve as a unique detection method for its simplicity, tunable detection range, and bioimaging ability. The design of new probes with highly selective K+ receptors and transduction functionality remains a challenge that is motivated by numerous sensing and detection applications. In this minireview, fluoroionophores are summarized that undergo transduction, producing fluorescence signals when interacting with, e. g., potassium ions. The properties of ionophores (afford selective interaction with potassium) and fluorophores (generate signal read-out) are discussed. Mol. structure design and sensing mechanisms are included along with cell imaging applications. The selectivity toward K+ and the absorption/emission characteristics of the probes are of particular interest.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXislSgsLrO&md5=8d3bbf3a96ee347b797fb363d23636e7
  25. 25
    Kasner, S. E.; Ganz, M. B. Regulation of intracellular potassium in mesangial cells: a fluorescence analysis using the dye, PBFI. American Journal of Physiology 1992, 262 (3), 462– 467,  DOI: 10.1152/ajprenal.1992.262.3.F462
    Google Scholar
    25
    Regulation of intracellular potassium in mesangial cells: a fluorescence analysis using the dye, PBFI
    Kasner, Scott E.; Ganz, Michael B.
    American Journal of Physiology (1992), 262 (3, Pt. 2), F462-F467CODEN: AJPHAP; ISSN:0002-9513.
    Regulatory transport processes were investigated that maintain intracellular K+ homeostasis in cultured rat glomerular mesangial cells (MCs). Intracellular K+ concn. ([K+]i) of quiescent MCs, passages 3-8, grown to subconfluence on glass cover slips, was assessed by spectrofluorometry using the K+-sensitive dye, K+-binding benzofuran isophthalate (PBFI). Serum-starved MCs were incubated at 37° in 5 μM BPFI for 90 min. Excitation ratios of luminescences at 340 and 380 nm, measured at a const. emission at 500 nm, were used to det. [K+]i. Ionophores valinomycin and nigericin were used to clamp [K+]i to known [K+]o and thereby obtain an intracellular calibration of dye. Dependence of fluorescence ratio on [K+]i conformed to Michaelis-Menten behavior, with a Km of 113 mM. PBFI retains its sensitivity to alterations in [K+]i with pH change (pHi from 6.5 to 7.5) but is relatively insensitive when intracellular Na+ is greater than 75 mM and cell osmolarity exceeds 500 mM. Normal resting [K+]i for all expts. was detd. in MCs to be 102 mM in a HCO3--free HEPES-buffered soln. When MCs were exposed to ouabain, [K+]i fell to 48 mM and did not recover, suggesting presence of Na+-K+-ATPase. When MCs were exposed to furosemide, [K+]i transiently declined to 58 mM, which was followed by a rapid recovery to near steady state, indicating addnl. presence of Na+-K+-Cl- cotransporter. Recovery was completely abolished when MCs were exposed to ouabain. Exposure to Ba2+ led to an immediate increase in [K+]i to 124 mM followed by a rapid return to steady-state [K+]i. MCs exposed to tetraethylammonium exhibited a behavior similar to those exposed to Ba2+; resting [K+]i increased to 123 mM. Effects of angiotensin II (ANG II) and serotonin (5-HT) on Na+-K+-ATPase was assessed. None of these agents significantly altered resting [K+]i. ANG II and 5-HT stimulated Na+-K+-ATPase by 28 and 41%, resp., as measured during recovery of [K+]i toward normal after a diuretic-induced fall in [K+]i. The authors conclude that (1) MCs possess ouabain-sensitive Na+-K+-ATPase, loop diuretic-sensitive Na+-K+-Cl- cotransporter, and barium-sensitive K+ channels; (2) ANG II and 5-HT stimulate Na+-K+-ATPase; and (3) a continuous detn. of [K+]i and an examn. of its regulatory mechanism in MCs may be achieved through use of PBFI.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK38XitlamtLo%253D&md5=7e50a45d570e3653f443b34d7d5e40b3
  26. 26
    Marchand, A.; Gabelica, V. Folding and misfolding pathways of G-quadruplex DNA. Nucleic Acids Res. 2016, 44 (22), 10999– 11012,  DOI: 10.1093/nar/gkw970
    Google Scholar
    26
    Folding and misfolding pathways of G-quadruplex DNA
    Marchand, Adrien; Gabelica, Valerie
    Nucleic Acids Research (2016), 44 (22), 10999-11012CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)
    G-quadruplexes adopt various folding topologies, but information on their folding pathways remains scarce. Here, we used electrospray mass spectrometry to detect and quantify the specifically bound potassium ions, and CD to characterize the stacking topol. of each ensemble. For human telomeric (hTel) sequences contg. the d((GGGTTA)3GGG) core, K+ binding affinity and cooperativity strongly depends on the chosen construct. The shortest sequences bind only one K+ at low KCl concn., and this 2-quartet Gquadruplex is antiparallel. Flanking bases increase the K+ binding cooperativity. To decipher the folding pathways, we investigated the kinetics of K+ binding to telomeric (hybrid) and c-myc (parallel) G-quadruplexes. G-quadruplexes fold via branched pathways with multiple parallel reactions. Up to six states (one ensemble without K+, two ensembles with 1-K+ and three ensembles with 2-K+) are sepd. based on their formation rates and ion mobility spectrometry. All G-quadruplexes first form longlived misfolded structures (off-pathway compared to the most stable structures) contg. one K+ and two quartets in an antiparallel stacking arrangement. The results highlight the particular ruggedness of G-quadruplex nucleic acid folding landscapes. Misfolded structures can play important roles for designing artificial G-quadruplex based structures, and for conformational selection by ligands or proteins in a biol. context.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXktlWksbg%253D&md5=4d5b427a02e0a34bc6471ef04fa693ac
  27. 27
    Gray, R. D.; Chaires, J. B. Isothermal Folding of G-quadruplexes. Methods (San Diego, Calif.) 2012, 57 (1), 47– 55,  DOI: 10.1016/j.ymeth.2012.04.006
    Google Scholar
    27
    Isothermal folding of G-quadruplexes
    Gray Robert D; Chaires Jonathan B
    Methods (San Diego, Calif.) (2012), 57 (1), 47-55 ISSN:.
    Thermodynamic studies of G-quadruplex stability are an essential complement to structures obtained by NMR or X-ray crystallography. An understanding of the energetics of quadruplex folding provides a necessary foundation for the physical interpretation of quadruplex formation and reactivity. While thermal denaturation methods are most commonly used to evaluate quadruplex stability, it is also possible to study folding using isothermal titration methods. G-quadruplex folding is tightly coupled to specific cation binding. We describe here protocols for monitoring the cation-driven quadruplex folding transition using circular dichroism or absorbance, and for determination of the distribution of free and bound cation using a fluorescence indicator. Together these approaches provide insight into quadruplex folding at constant temperature, and characterize the linkage between cation binding and folding.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC38rnsVakuw%253D%253D&md5=5accd4d84b3cc305d14a0d33de37370b
  28. 28
    Nishio, M.; Tsukakoshi, K.; Ikebukuro, K. G-quadruplex: Flexible conformational changes by cations, pH, crowding and its applications to biosensing. Biosens. Bioelectron. 2021, 178, 113030,  DOI: 10.1016/j.bios.2021.113030
    Google Scholar
    28
    G-quadruplex: Flexible conformational changes by cations, pH, crowding and its applications to biosensing
    Nishio, Maui; Tsukakoshi, Kaori; Ikebukuro, Kazunori
    Biosensors & Bioelectronics (2021), 178 (), 113030CODEN: BBIOE4; ISSN:0956-5663. (Elsevier B.V.)
    A review. G-quadruplex (G4) is a non-canonical structure that is formed in G-rich sequences of nucleic acids. G4s play important roles in vivo, such as telomere maintenance, transcription, and DNA replication. There are three typical topologies of G4: parallel, anti-parallel, and hybrid. In general, metal cations, such as potassium and sodium, stabilize G4s through coordination in the G-quartet. While G4s have some functions in vivo, there are many reports of developed applications that use G4s. As various conformations of G4s could form from one sequence depending on varying conditions, many researchers have developed G4-based sensors. Furthermore, G4 is a great scaffold of aptamers since many aptamers folded into G4s have also been reported. However, there are some challenges about its practical use due to the difference between practical sample conditions and exptl. ones. G4 conformations are dramatically altered by the surrounding conditions, such as metal cations, pH, and crowding. Many studies have been conducted to characterize G4 conformations under various conditions, not only to use G4s in practical applications but also to reveal its function in vivo. In this review, we summarize recent studies that have investigated the effects of surrounding conditions (e.g., metal cations, pH, and crowding) on G4 conformations and the application of G4s mainly in biosensor fields, and in others.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXis1OhurY%253D&md5=defd5d3aa7abb1cd5a1aa98a3ff5a49d
  29. 29
    Williamson, J. R.; Raghuraman, M. K.; Cech, T. R. Monovalent cation-induced structure of telomeric DNA: the G-quartet model. Cell 1989, 59 (5), 871– 880,  DOI: 10.1016/0092-8674(89)90610-7
    Google Scholar
    29
    Monovalent cation-induced structure of telomeric DNA: the G-quartet model
    Williamson, James R.; Raghuraman, M. K.; Cech, Thomas R.
    Cell (Cambridge, MA, United States) (1989), 59 (5), 871-80CODEN: CELLB5; ISSN:0092-8674.
    Structures formed by oligonucleotides composed of 2 or 4 repeats of the telomeric sequences from Oxytricha and Tetrahymena were investigated. The Oxytricha 4-repeat mol. [d(T4G4)4 = Oxy-4] forms structures with increased electrophoretic mobility in nondenaturing gels contg. Na+, K+, or Cs+, but not in gels contg. Li+ or no added salt. Formation of the folded structure results in protection of a set of dG's from methylation by di-Me sulfate. Efficient UV-induced crosslinks are obsd. in Oxy-4 and the related sequence from Tetrahymena [d(T2G4)4 = Tet-4], and join thymidines in different repeats. Models proposed to account for these data involve G-quartets, H-bonded structures formed from 4 guanosines in a square-planar array. It is proposed that the G-quartet structure must be dealt with in vivo by the telomere replication machinery.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3cXhtFCgsLo%253D&md5=6a86541fc934bcd72b517b75e2dda57b
  30. 30
    Takenaka, S.; Juskowiak, B. Fluorescence Detection of Potassium Ion Using the G-Quadruplex Structure. Anal. Sci. 2011, 27 (12), 1167– 1167,  DOI: 10.2116/analsci.27.1167
    Google Scholar
    30
    Fluorescence detection of potassium ion using the G-quadruplex structure
    Takenaka, Shigeori; Juskowiak, Bernard
    Analytical Sciences (2011), 27 (12), 1167-1172CODEN: ANSCEN; ISSN:0910-6340. (Japan Society for Analytical Chemistry)
    A review. Oligonucleotides with sequences of human telomere DNA or thrombin binding aptamer (TBA) are known to form tetraplex structures upon binding the K+ ion. Structural changes assocd. with the formation of tetraplex assemblies led to the development of potassium-sensing oligonucleotide (PSO) probes, in which two fluorescent dyes were attached to both termini of particular oligonucleotide. The combination of dyes included fluorescence resonance energy transfer (FRET) and excimer emission approaches, and the structural changes upon binding K+ ion could be monitored by a fluorescence technique. These systems showed a very high preference for K+ over Na+ ion, which was suitable for fluorescence imaging of the potassium concn. gradient in a living cell. In the case of human telomere DNA, it was also possible to follow the polymorphism of its tetraplex structures.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xktleiug%253D%253D&md5=d1707097c61dcc0a2c105d30bc73094a
  31. 31
    Juskowiak, B. Analytical potential of the quadruplex DNA-based FRET probes. Anal. Chim. Acta 2006, 568 (1), 171– 180,  DOI: 10.1016/j.aca.2005.12.063
    Google Scholar
    31
    Analytical potential of the quadruplex DNA-based FRET probes
    Juskowiak, Bernard
    Analytica Chimica Acta (2006), 568 (1-2), 171-180CODEN: ACACAM; ISSN:0003-2670. (Elsevier B.V.)
    A review. DNA exhibits structural flexibility and may adopt also tetraplex structures known as guanine-quadruplexes or G-quadruplexes. These G-quadruplexes have recently received great attention because G-rich sequences are often found in genome and because of their potential links to mechanisms that relate to cancer, HIV, and other diseases. The unique structure of quadruplexes has also stimulated development of new anal. and bioanal. assays based on fluorescence resonance energy transfer (FRET). Intramol. folding of a flexible single-stranded DNA mol. into a compact G-quadruplex is a structural transition leading to closer proximity of its 5'- and 3'-ends. Thus, labeling both ends of a DNA strand with donor and acceptor fluorophores enables monitoring the quadruplex formation process by the FRET signal. This review shows how FRET technique contributes to G-quadruplex research and focuses mainly on anal. applications of FRET-labeled quadruplexes. Applications include studies of structural transitions of quadruplexes, FRET-based selection of ligands that bind to quadruplexes, design of mol. probes for protein recognition and development of sensors for detection of potassium ions in aq. soln.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XltVWhu7w%253D&md5=557fa9f26f2aa320cceeccd6a2e22e59
  32. 32
    Ueyama, H.; Takagi, M.; Takenaka, S. A Novel Potassium Sensing in Aqueous Media with a Synthetic Oligonucleotide Derivative. Fluorescence Resonance Energy Transfer Associated with Guanine Quartet-Potassium Ion Complex Formation. J. Am. Chem. Soc. 2002, 124 (48), 14286– 14287,  DOI: 10.1021/ja026892f
    Google Scholar
    32
    A novel potassium sensing in aqueous media with a synthetic oligonucleotide derivative. fluorescence resonance energy transfer associated with guanine quartet-potassium ion complex formation
    Ueyama, Hiroyuki; Takagi, Makoto; Takenaka, Shigeori
    Journal of the American Chemical Society (2002), 124 (48), 14286-14287CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    A novel potassium sensing oligonucleotide (PSO) was constructed by attaching fluorophores 6-FAM and 6-TAMRA to the 5'- and 3'-termini of d(GGG TTA GGG TTA GGG TTA GGG), resp. The affinity of PSO for K+ was 43 000 times greater than that for Na+, high enough selectivity enabling quantitation of K+ specifically in the presence of excess Na+. Fluorescence resonance energy transfer (FRET) to 6-TAMRA from 6-FAM of PSO was obsd. only in the presence of K+. This phenomenon is based on the approxn. of the two fluorophores upon formation of a guanine quartet mediated by K+. Furthermore, the fluorescent color of PSO changes from yellow to red upon formation of the complex, thereby enabling visualization of K+ in aq. media.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XosVCgtLc%253D&md5=7536c6e47b1d3385b1ca0155901ba556
  33. 33
    Xu, J.; Jiang, R.; He, H.; Ma, C.; Tang, Z. Recent advances on G-quadruplex for biosensing, bioimaging and cancer therapy. TrAC Trends in Analytical Chemistry 2021, 139, 116257,  DOI: 10.1016/j.trac.2021.116257
    Google Scholar
    33
    Recent advances on G-quadruplex for biosensing, bioimaging and cancer therapy
    Xu, Jiaqi; Jiang, Rundong; He, Hailun; Ma, Changbei; Tang, Zhenwei
    TrAC, Trends in Analytical Chemistry (2021), 139 (), 116257CODEN: TTAEDJ; ISSN:0165-9936. (Elsevier B.V.)
    A review. G-quadruplex is a three-dimensional secondary structure of nucleic acids formed by the Hoogsteen hydrogen pairing of four guanines. Diverse topologies of G-quadruplex could be employed in biosensing and bioimaging. By intercalating fluorescence dyes into G-quadruplex or forming a horseradish peroxidase (HRP)-mimicking G-quadruplex/hemin DNAzyme, G-quadruplexes based biosensors realized the sensitive and selective detection of nucleic acids, protein, enzyme activity, ions, small mols., exosomes, cells, and microorganisms. The vital role that cellular G-quadruplexes played in genome further facilitated the application of G-quadruplex stabilizing on cancer therapy. Combined with G-quadruplex aptamer, which is an efficient therapeutic tool, a current landscape of the application potential of this fascinate nucleic acids structure from clin. diagnosis to cancer therapy is summarized here.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXntl2qtrs%253D&md5=2a00a67f6c666fe9646e6cb054636210
  34. 34
    Massey, M.; Algar, W. R.; Krull, U. J. Fluorescence resonance energy transfer (FRET) for DNA biosensors: FRET pairs and Förster distances for various dye-DNA conjugates. Anal. Chim. Acta 2006, 568 (1), 181– 189,  DOI: 10.1016/j.aca.2005.12.050
    Google Scholar
    34
    Fluorescence resonance energy transfer (FRET) for DNA biosensors: FRET pairs and Foerster distances for various dye-DNA conjugates
    Massey, Melissa; Algar, W. Russ; Krull, Ulrich J.
    Analytica Chimica Acta (2006), 568 (1-2), 181-189CODEN: ACACAM; ISSN:0003-2670. (Elsevier B.V.)
    Fluorescence resonance energy transfer (FRET) between the extrinsic dye labels Cyanine 3 (Cy3), Cyanine 5 (Cy5), Carboxytetramethyl Rhodamine (TAMRA), Iowa Black Fluorescence Quencher (IabFQ), and Iowa Black RQ (IabRQ) has been studied. The Foerster distances for these FRET-pairs in single- and double-stranded DNA conjugates have been detd. In particular, it should be noted that the quantum yield of the donors Cy3 and TAMRA varies between single- and double-stranded DNA. While this alters the Foerster distance for a donor-acceptor pair, this also allows for detection of thermal denaturation events with a single non-intercalating fluorophore. The utility of FRET in the development of nucleic acid biosensor technol. is illustrated by using TAMRA and IabRQ as a FRET pair in selectivity expts. The differential quenching of TAMRA fluorescence by IabRQ in soln. has been used to discriminate between 0 and 3 base pair mismatches at 60 °C for a 19 base sequence. At room temp., the quenching of TAMRA fluorescence was not an effective indicator of the degree of base pair mismatch. There appears to be a threshold of duplex stability at room temp. which occurs beyond two base pair mismatches and reverses the obsd. trend in TAMRA fluorescence prior to that degree of mismatch. When this exptl. system is transferred to a glass surface through covalent coupling and organosilane chem., the obsd. trend in TAMRA fluorescence at room temp. is similar to that obtained in bulk soln., but without a threshold of duplex stability. In addn. to quenching of fluorescence by FRET, it is believed that several other quenching mechanisms are occurring at the surface.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XltVWhu7s%253D&md5=a1a363321bb23faa6a649a09541dbbb2
  35. 35
    Bianchi, F.; Comez, L.; Biehl, R.; D’Amico, F.; Gessini, A.; Longo, M.; Masciovecchio, C.; Petrillo, C.; Radulescu, A.; Rossi, B.; Sacchetti, F.; Sebastiani, F.; Violini, N.; Paciaroni, A. Structure of human telomere G-quadruplex in the presence of a model drug along the thermal unfolding pathway. Nucleic Acids Res. 2018, 46 (22), 11927– 11938,  DOI: 10.1093/nar/gky1092
    Google Scholar
    35
    Structure of human telomere G-quadruplex in the presence of a model drug along the thermal unfolding pathway
    Bianchi, Federico; Comez, Lucia; Biehl, Ralf; D'Amico, Francesco; Gessini, Alessandro; Longo, Marialucia; Masciovecchio, Claudio; Petrillo, Caterina; Radulescu, Aurel; Rossi, Barbara; Sacchetti, Francesco; Sebastiani, Federico; Violini, Nicolo; Paciaroni, Alessandro
    Nucleic Acids Research (2018), 46 (22), 11927-11938CODEN: NARHAD; ISSN:1362-4962. (Oxford University Press)
    A multi-technique approach, combining CD spectroscopy, UV resonance Raman spectroscopy and small angle scattering techniques, has been deployed to elucidate how the structural features of the human telomeric G-quadruplex d[A(GGGTTA)3GGG] (Tel22) change upon thermal unfolding. The system is studied both in the free form and when it is bound to Actinomycin D (ActD), an anticancer ligand with remarkable conformational flexibility. We find that at room temp. binding of Tel22 with ActD involves end-stacking upon the terminal G-tetrad. Structural evidence for drug-driven dimerization of a significant fraction of the G-quadruplexes is provided. When the temp. is raised, both free and bound Tel22 undergo melting through a multi-state process. We show that in the intermediate states of Tel22 the conformational equil. is shifted toward the (3+1) hybrid-type, while a parallel structure is promoted in the complex. The unfolded state of the free Tel22 is consistent with a self-avoiding random-coil conformation, whereas the high-temp. state of the complex is obsd. to assume a quite compact form. Such an unprecedented high-temp. arrangement is caused by the persistent interaction between Tel22 and ActD, which stabilizes compact conformations even in the presence of large thermal structural fluctuations.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXovFWnt7o%253D&md5=a02f956f569cf716f57c4072fbb50053
  36. 36
    Nagatoishi, S.; Nojima, T.; Juskowiak, B.; Takenaka, S. A Pyrene-Labeled G-Quadruplex Oligonucleotide as a Fluorescent Probe for Potassium Ion Detection in Biological Applications. Angew. Chem. 2005, 117 (32), 5195– 5198,  DOI: 10.1002/ange.200501506
    Google Scholar
    There is no corresponding record for this reference.
  37. 37
    Zhang, A. Y.; Balasubramanian, S. The kinetics and folding pathways of intramolecular G-quadruplex nucleic acids. J. Am. Chem. Soc. 2012, 134 (46), 19297– 19308,  DOI: 10.1021/ja309851t
    Google Scholar
    37
    The Kinetics and Folding Pathways of Intramolecular G-Quadruplex Nucleic Acids
    Zhang, Amy Y. Q.; Balasubramanian, Shankar
    Journal of the American Chemical Society (2012), 134 (46), 19297-19308CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    The folding kinetics of G-quadruplex forming sequences is crit. to their capacity to influence biol. function. While G-quadruplex structure and stability have been relatively well studied, little is known about the kinetics of their folding. We employed a stopped-flow mixing technique to systematically investigate the potassium-dependent folding kinetics of telomeric RNA and DNA G-quadruplexes and RNA G-quadruplexes contg. only two G-quartets formed from sequences r[(GGA)3GG] and r[(GGUUA)3GG]. Our findings suggest a folding mechanism that involves two kinetic steps with initial binding of a single K+, irresp. of the no. of G-quartets involved or whether the G-quadruplex is formed from RNA or DNA. The folding rates for telomeric RNA and DNA G-quadruplexes are comparable at near physiol. [K+] (90 mM) (τ = ∼60 ms). The folding of a 2-quartet RNA G-quadruplex with single nucleotide A loops is considerably slower (τ = ∼700 ms), and we found that the time required to fold a UUA looped variant (τ > 100 s, 500 mM K+) exceeds the lifetimes of some regulatory RNAs. We discuss the implications of these findings with respect to the fundamental properties of G-quadruplexes and their potential functions in biol.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhsF2qt7%252FP&md5=de67961e4c6dbba5edbbe43d7f0a4222
  38. 38
    Deshpande, S.; Wunnava, S.; Hueting, D.; Dekker, C. Membrane Tension-Mediated Growth of Liposomes. Small 2019, 15 (38), 1902898,  DOI: 10.1002/smll.201902898
    Google Scholar
    There is no corresponding record for this reference.
  39. 39
    Bhatia, T.; Christ, S.; Steinkühler, J.; Dimova, R.; Lipowsky, R. Simple sugars shape giant vesicles into multispheres with many membrane necks. Soft Matter 2020, 16 (5), 1246– 1258,  DOI: 10.1039/C9SM01890E
    Google Scholar
    39
    Simple sugars shape giant vesicles into multispheres with many membrane necks
    Bhatia, Tripta; Christ, Simon; Steinkuehler, Jan; Dimova, Rumiana; Lipowsky, Reinhard
    Soft Matter (2020), 16 (5), 1246-1258CODEN: SMOABF; ISSN:1744-6848. (Royal Society of Chemistry)
    Here, we use giant vesicles to investigate fully hydrated lipid membranes in contact with two sugars, glucose and sucrose. The vesicles were osmotically balanced, with the same total sugar concn. in the interior and exterior aq. solns. However, the two solns. differed in their compn.: the interior soln. contained only sucrose whereas the exterior one contained primarily glucose. This sugar asymmetry generated a striking variety of multispherical or "multi-balloon" vesicle shapes. Each multisphere involved only a single membrane that formed several spherical segments, which were connected by narrow, hourglass-shaped membrane necks. These morphologies revealed that the sugar-lipid interactions generated a significant spontaneous curvature with a magnitude of about 1μm-1. Such a spontaneous curvature can be generated both by depletion and by adsorption layers of the sugar mols. arising from effectively repulsive and attractive sugar-lipid interactions. All multispherical shapes are stable over a wide range of parameters, with a substantial overlap between the different stability regimes, reflecting the rugged free energy landscape in shape space. One challenge for future studies is to identify pathways within this landscape that allow us to open and close the membrane necks of these shapes in a controlled and reliable manner. We will then be able to apply these multispheres as metamorphic chambers for chem. reactions and nanoparticle growth.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXitl2gtbrI&md5=5837474231ea5181efec3751e7b46eb2
  40. 40
    Goldman, D. E. Potential, Impedance, and Rectification in membranes. J. Gen. Physiol. 1943, 27 (1), 37– 60,  DOI: 10.1085/jgp.27.1.37
    Google Scholar
    40
    Potential, impedance and rectification in membranes
    Goldman, D. E.
    Journal of General Physiology (1943), 27 (), 37-60CODEN: JGPLAD; ISSN:0022-1295.
    Membranes of collodion, collodion-lecithin, collodion-cephalin, onion cuticle and proteins behave as parallel resistance-capacity combinations when sepg. solns. of electrolytes. The capacities of the above membranes vary slightly with the concn. and nature of the solns. and have phase angles 88-89°, 79-82°, 84-86°, 83-85°, 65-75°, resp. The conductances Λ are approx. proportional to those of the solns. but are much smaller and, like the dielec. consts., are greater than those of the membrane material in bulk. In general Λ varies with current but the capacity is independent of current. Approx. values for the membrane potentials V indicate a linear proportionality with the log of the ratio of the concns. of electrolyte on both sides of the membrane. The amount of rectification produced by the membranes increases with V rather than with the concn. ratio of Λ. By considering the presence of fixed as well as mobile ions, the Planck derivation of liquid-junction potential is extended to membranes, and expressions are obtained for V and for the variation of Λ with current. The calcd. values show qual. agreement with expt.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaH2cXjvVCktw%253D%253D&md5=3d4389a1d16f15567438999f4b5013be
  41. 41
    Gutknecht, J.; Tosteson, D. C. Ionic Permeability of Thin Lipid Membranes: Effects of n-alkyl alcohols, polyvalent cations, and a secondary amine. J. Gen. Physiol. 1970, 55 (3), 359– 374,  DOI: 10.1085/jgp.55.3.359
    Google Scholar
    41
    Ionic permeability of thin lipid membranes. Effects of n-alkyl alcohols, polyvalent cations, and a secondary amine
    Gutknecht, John; Tosteson, D. C.
    Journal of General Physiology (1970), 55 (3), 359-74CODEN: JGPLAD; ISSN:0022-1295.
    Ultrathin (black) lipid membranes were made from n-decane solns. of sheep red cell lipids. Membrane resistance (Rm), voltage (Vm), and ionic transference (Ti) were measured. The presence of each n-alkyl alc., EtOH through n-octanol, in the membrane bath soln. reduced Rm. The concns. required to reduce Rm from >108 to 106 Ω/cm2 were 3.0, 1.4, 0.30, 0.10, 0.028, 0.0054, and >0.00038M EtOH, PrOH, BuOH, n-pentanol, n-hexanol, n-heptanol, and n-octanol, resp. Heptanol increased the permeability of the membrane to K+, with respect to both Na+ and Cl-. The transference nos., Tcation/TCl, increased from 6 to 21; and TK/TNa, from 3 to 21. Other alcs. behaved similarly. Plots of log Rm vs. log alc. concn. were linear over the range of max. change of Rm. The slopes were -3 and -5 for C2C7 alcs. The presence of Th+4 or Fe+3 (10-4M), or of Amberlite LA-2 in the lipid soln. from which the membranes were made, changed the membrane permselectivity from cationic to anionic. When membranes contg. the secondary amine (Amberlite LA-2) were exposed to heptanol, the Rm dropped to 102-105 ohms/cm2, and the membranes became anion selective. Cl diffusion potentials up to 150 millivolts were developed.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaE3cXktVSit7w%253D&md5=f02eb58e4b3a73048b81f74bf2ca33cd
  42. 42
    Koyanagi, T.; Leriche, G.; Yep, A.; Onofrei, D.; Holland, G. P.; Mayer, M.; Yang, J. Effect of Headgroups on Small-Ion Permeability across Archaea-Inspired Tetraether Lipid Membranes. Chem. - Eur. J. 2016, 22 (24), 8074– 8077,  DOI: 10.1002/chem.201601326
    Google Scholar
    42
    Effect of Headgroups on Small-Ion Permeability across Archaea-Inspired Tetraether Lipid Membranes
    Koyanagi, Takaoki; Leriche, Geoffray; Yep, Alvin; Onofrei, David; Holland, Gregory P.; Mayer, Michael; Yang, Jerry
    Chemistry - A European Journal (2016), 22 (24), 8074-8077CODEN: CEUJED; ISSN:0947-6539. (Wiley-VCH Verlag GmbH & Co. KGaA)
    This paper examines the effects of four different polar headgroups on small-ion membrane permeability from liposomes comprised of Archaea-inspired glycerolmonoalkyl glycerol tetraether (GMGT) lipids. We found that the membrane-leakage rate across GMGT lipid membranes varied by a factor of ≤1.6 as a function of headgroup structure. However, the leakage rates of small ions across membranes comprised of com. bilayer-forming 1-palmitoyl-2-oleoyl-sn-glycerol (PO) lipids varied by as much as 32-fold within the same series of headgroups. These results demonstrate that membrane leakage from GMGT lipids is less influenced by headgroup structure, making it possible to tailor the structure of the polar headgroups on GMGT lipids while retaining predictable leakage properties of membranes comprised of these tethered lipids.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XntFGju7c%253D&md5=46df565a8b20019fc696e91038d78751
  43. 43
    Gouaux, E.; Mackinnon, R. Principles of selective ion transport in channels and pumps. Science (New York, N.Y.) 2005, 310 (5753), 1461– 1465,  DOI: 10.1126/science.1113666
    Google Scholar
    43
    Principles of selective ion transport in channels and pumps
    Gouaux Eric; Mackinnon Roderick
    Science (New York, N.Y.) (2005), 310 (5753), 1461-5 ISSN:.
    The transport of ions across the membranes of cells and organelles is a prerequisite for many of life's processes. Transport often involves very precise selectivity for specific ions. Recently, atomic-resolution structures have been determined for channels or pumps that are selective for sodium, potassium, calcium, and chloride: four of the most abundant ions in biology. From these structures we can begin to understand the principles of selective ion transport in terms of the architecture and detailed chemistry of the ion conduction pathways.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BD2MnktFKmsg%253D%253D&md5=822e72453bdb78e41771d74706b1aa0f
  44. 44
    Finkelstein, A.; Andersen, O. S. The gramicidin a channel: A review of its permeability characteristics with special reference to the single-file aspect of transport. J. Membr. Biol. 1981, 59 (3), 155– 171,  DOI: 10.1007/BF01875422
    Google Scholar
    44
    The gramicidin A channel: a review of its permeability characteristics with special reference to the single-file aspect of transport
    Finkelstein, Alan; Andersen, Olaf Sparre
    Journal of Membrane Biology (1981), 59 (3), 155-71CODEN: JMBBBO; ISSN:0022-2631.
    A review with 79 refs.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL3MXktVWqtbg%253D&md5=2bb28f303eefa4c9d36bd1f14e7265fb
  45. 45
    Im, W.; Roux, B. t. Ion Permeation and Selectivity of OmpF Porin: A Theoretical Study Based on Molecular Dynamics, Brownian Dynamics, and Continuum Electrodiffusion Theory. J. Mol. Biol. 2002, 322 (4), 851– 869,  DOI: 10.1016/S0022-2836(02)00778-7
    Google Scholar
    45
    Ion Permeation and Selectivity of OmpF Porin: A Theoretical Study Based on Molecular Dynamics, Brownian Dynamics, and Continuum Electrodiffusion Theory
    Im, Wonpil; Roux, Benoit
    Journal of Molecular Biology (2002), 322 (4), 851-869CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Science Ltd.)
    Three different theor. approaches are used and compared to refine our understanding of ion permeation through the channel formed by OmpF porin from Escherichia coli. Those approaches are all-atom mol. dynamics (MD) in which ions, solvent, and lipids are represented explicitly, Brownian dynamics (BD) in which ions are represented explicitly, while solvent and lipids are represented as featureless dielecs., and Poisson-Nernst-Planck (PNP) electrodiffusion theory in which both solvent and local ion concns. are represented as a continuum. First, the ability of the different theor. approaches in reproducing the equil. av. ion d. distribution in OmpF porin bathed by a 1 M KCl sym. salt soln. is examd. Under those conditions the PNP theory is equiv. to the non-linear Poisson-Boltzmann (PB) theory. Anal. shows that all the three approaches are able to capture the important electrostatic interactions between ions and the charge distribution of the channel that govern ion permeation and selectivity in OmpF. The K+ and Cl- d. distributions obtained from the three approaches are very consistent with one another, which suggests that a treatment on the basis of a rigid protein and continuum dielec. solvent is valid in the case of OmpF. Interestingly, both BD and continuum electrostatics reproduce the distinct left-handed twisted ion pathways for K+ and Cl- extending over the length of the pore which were obsd. previously in MD. Equil. BD simulations in the grand canonical ensemble indicate that the channel is very attractive for cations, particularly at low salt concn. On an av. there is 1.55 K+ inside the pore in 10 mM KCl. Remarkably, there is still 0.17 K+ on av. inside the pore even at a concn. as low as 1 μM KCl. Secondly, non-equil. ion flow through OmpF is calcd. using BD and PNP and compared with exptl. data. The channel conductance in 0.2 M and 1 M KCl calcd. using BD is in excellent accord with the exptl. data. The calcns. reproduce the exptl. well-known conductance-concn. relation and also reveal an asymmetry in the channel conductance (a larger conductance is obsd. under a pos. transmembrane potential). Calcns. of the channel conductance for three mutants (R168A, R132A, and K16A) in 1 M KCl suggest that the asymmetry in the channel conductance arises mostly from the permanent charge distribution of the channel rather than the shape of the pore itself. Lastly, the calcd. reversal potential in a tenfold salt gradient (0.1:1 M KCl) is 27.4(±1.3) mV (BD) and 22.1(±0.6) mV (PNP), in excellent accord with the exptl. value of 24.3 mV. Although most of the results from PNP are qual. reasonable, the calcd. channel conductance is about 50% higher than that calcd. from BD probably because of a lack of some dynamical ion-ion correlations.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XntVGrtrY%253D&md5=5a75370a8388ccf0e69708aa0c1170c4
  46. 46
    Alcaraz, A.; Nestorovich, E. M.; López, M. L.; García-Giménez, E.; Bezrukov, S. M.; Aguilella, V. M. Diffusion, Exclusion, and Specific Binding in a Large Channel: A Study of OmpF Selectivity Inversion. Biophys. J. 2009, 96 (1), 56– 66,  DOI: 10.1016/j.bpj.2008.09.024
    Google Scholar
    46
    Diffusion, exclusion, and specific binding in a large channel: a study of OmpF selectivity inversion
    Alcaraz, Antonio; Nestorovich, Ekaterina M.; Lopez, M. Lidon; Garcia-Gimenez, Elena; Bezrukov, Sergey M.; Aguilella, Vicente M.
    Biophysical Journal (2009), 96 (1), 56-66CODEN: BIOJAU; ISSN:0006-3495. (Cell Press)
    We find that moderate cationic selectivity of the general bacterial porin OmpF in sodium and potassium chloride solns. is inversed to anionic selectivity in concd. solns. of barium, calcium, nickel, and magnesium chlorides. To understand the origin of this phenomenon, we consider several factors, which include the binding of divalent cations, electrostatic and steric exclusion of differently charged and differently sized ions, size-dependent hydrodynamic hindrance, electrokinetic effects, and significant "anionic" diffusion potential for bulk solns. of chlorides of divalent cations. Though all these factors contribute to the measured selectivity of this large channel, the obsd. selectivity inversion is mostly due to the following two. First, binding divalent cations compensates, or even slightly overcompensates, for the neg. charge of the OmpF protein, which is known to be the main cause of cationic selectivity in sodium and potassium chloride solns. Second, the higher anionic (vs. cationic) transport rate expected for bulk solns. of chloride salts of divalent cations is the leading cause of the measured anionic selectivity of the channel. Interestingly, at high concns. the binding of cations does not show any pronounced specificity within the divalent series because the reversal potentials measured in the series correlate well with the corresponding bulk diffusion potentials. Thus our study shows that, in contrast to the highly selective channels of neurophysiol. that employ mostly the exclusion mechanism, quite different factors account for the selectivity of large channels. The elucidation of these factors is essential for understanding large channel selectivity and its regulation in vivo.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXptF2lug%253D%253D&md5=0202c5bf910fe2fb61e64d8babe4d599
  47. 47
    Alcaraz, A.; Nestorovich, E. M.; Aguilella-Arzo, M.; Aguilella, V. M.; Bezrukov, S. M. Salting Out the Ionic Selectivity of a Wide Channel: The Asymmetry of OmpF. Biophys. J. 2004, 87 (2), 943– 957,  DOI: 10.1529/biophysj.104/043414
    Google Scholar
    47
    Salting out the ionic selectivity of a wide channel: The asymmetry of OmpF
    Alcaraz, Antonio; Nestorovich, Ekaterina M.; Aguilella-Arzo, Marcel; Aguilella, Vicente M.; Bezrukov, Sergey M.
    Biophysical Journal (2004), 87 (2), 943-957CODEN: BIOJAU; ISSN:0006-3495. (Biophysical Society)
    Although the crystallog. structure of the bacterial porin OmpF has been known for a decade, the phys. mechanisms of its ionic selectivity are still under investigation. We address this issue in a series of expts. with varied pH, salt concns., inverted salt gradient, and charged and uncharged lipids. Measuring reversal potential, we show that OmpF selectivity (traditionally regarded as slightly cationic) depends strongly on pH and salt concn. and is conditionally asym., i.e., the calcd. selectivity is sensitive to the direction of salt concn. gradient. At neutral pH and subdecimolar salt concns. the channel exhibits nearly ideal cation selectivity (tG+ = 0.98±0.01). Substituting neutral DPhPC with DPhPS, we demonstrate that the fixed charge of the host lipid has a small but measurable effect on the channel reversal potential. The available structural information allows for a qual. explanation of our exptl. findings. These findings now lead us to re-examine the ionization state of 102 titratable sites of the OmpF channel. Using std. methods of continuum electrostatics tailored to our particular purpose, we find the charge distribution in the channel as a function of soln. acidity and relate the pH-dependent asymmetry in channel selectivity to the pH-dependent asymmetry in charge distribution. In an attempt to find a simple phenomenol. description of our results, we also discuss different macroscopic models of electrodiffusion through large channels.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXmvFeitLo%253D&md5=b52955043e257c041a7fe00e63c37762
  48. 48
    Jarczewska, M.; Górski, Ł.; Malinowska, E. Application of DNA aptamers as sensing layers for electrochemical detection of potassium ions. Sens. Actuators, B 2016, 226, 37– 43,  DOI: 10.1016/j.snb.2015.11.139
    Google Scholar
    48
    Application of DNA aptamers as sensing layers for electrochemical detection of potassium ions
    Jarczewska, Marta; Gorski, Lukasz; Malinowska, Elzbieta
    Sensors and Actuators, B: Chemical (2016), 226 (), 37-43CODEN: SABCEB; ISSN:0925-4005. (Elsevier B.V.)
    Detn. of potassium level in organism is of special importance due to its vital role in the maintenance of physiol. activities in organism. Herein, an electrochem. sensor for K+ ions was developed with recognition layer based on DNA aptamers. The elaborated assay was formed by tethering of thiolated aptamer probes to gold disk electrode via Au-S bond. Potassium concn. was detd. with the use of voltammetric techniques by comparison of current change of redox indicators-methylene blue (MB), AQMS and Na4Fe(CN)6. The studies revealed that among aptamer sequences, which were previously shown to bind with potassium ions, 15-mer thrombin binding aptamer (TBA) exhibited highest affinity towards potassium with Kd = 1.13 μmol L-1 when used as a sensing layer. The aptasensor demonstrated linear response within the range from 10-8 to 10-5 mol L-1 with a LOD of 2.31 × 10-9 mol L-1 and good selectivity towards K+ ions with the use of MB electroactive marker.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXitVSqtLbK&md5=b563f0528218b961dc2c52510b0a68fb
  49. 49
    Girard, P.; Pécréaux, J.; Lenoir, G.; Falson, P.; Rigaud, J.-L.; Bassereau, P. A New Method for the Reconstitution of Membrane Proteins into Giant Unilamellar Vesicles. Biophys. J. 2004, 87 (1), 419– 429,  DOI: 10.1529/biophysj.104.040360
    Google Scholar
    49
    A new method for the reconstitution of membrane proteins into giant unilamellar vesicles
    Girard, Philippe; Pecreaux, Jacques; Lenoir, Guillaume; Falson, Pierre; Rigaud, Jean-Louis; Bassereau, Patricia
    Biophysical Journal (2004), 87 (1), 419-429CODEN: BIOJAU; ISSN:0006-3495. (Biophysical Society)
    In this work, we have investigated a new and general method for the reconstitution of membrane proteins into giant unilamellar vesicles (GUVs). We have analyzed systematically the reconstitution of two radically different membrane proteins, the sarcoplasmic reticulum Ca2+-ATPase and the H+ pump bacteriorhodopsin. In a first step, our method involved a detergent-mediated reconstitution of solubilized membrane proteins into proteoliposomes of 0.1-0.2 μm in size. In a second step, these preformed proteoliposomes were partially dried under controlled humidity followed, in a third step, by electroswelling of the partially dried film to give GUVs. The phys. characteristics of GUVs were analyzed in terms of morphol., size, and lamellarity using phase-contrast and differential interference contrast microscopy. The reconstitution process was further characterized by analyzing protein incorporation and biol. activity. Both membrane proteins could be homogeneously incorporated into GUVs at lipid/protein ratios ranging from 5 to 40 (wt./wt.). After reconstitution, both proteins retained their biol. activity as demonstrated by H+ or Ca2+ pumping driven by bacteriorhodopsin or Ca2+-ATPase, resp. This constitutes an efficient new method of reconstitution, leading to the prodn. of large unilamellar membrane protein-contg. vesicles of more than 20 μm in diam., which should prove useful for functional and structural studies through the use of optical microscopy, optical tweezers, microelectrodes, or at. force microscopy.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXmtVels78%253D&md5=0bdd95ffe3a3b4c3686220ec4bd00072
  50. 50
    Pomès, R.; Roux, B. Structure and dynamics of a proton wire: a theoretical study of H+ translocation along the single-file water chain in the gramicidin A channel. Biophys. J. 1996, 71 (1), 19– 39,  DOI: 10.1016/S0006-3495(96)79211-1
    Google Scholar
    50
    Structure and dynamics of a proton wire: a theoretical study of H+ translocation along the single-file water chain in the gramicidin A channel
    Pomes, Regis; Roux, Benoit
    Biophysical Journal (1996), 71 (1), 19-39CODEN: BIOJAU; ISSN:0006-3495. (Biophysical Society)
    The rapid translocation of H+ along a chain of hydrogen-bonded water mols., or proton wire, is thought to be an important mechanism for proton permeation through transmembrane channels. Computer simulations are used to study the properties of the proton wire formed by the single-file waters in the gramicidin A channel. The model includes the polypeptidic dimer, with 22 water mols. and one excess proton. The dissocn. of the water mols. is taken into account by the "polarization model" of Stillinger and co-workers. The importance of quantum effects due to the light mass of hydrogen nuclei is examd. with the use of discretized Feynman path integral mol. dynamics simulations. Results show that the presence of an excess proton in the pore orients the single-file water mols. and affects the geometry of water-water hydrogen bonding interactions. Rather than a well-defined hydronium ion OH3+ in the single-file region, the protonated species is characterized by a strong hydrogen bond resembling that of O2H5+. The quantum dispersion of protons has a small but significant effect on the equil. structure of the hydrogen-bonded water chain. During classical trajectories, proton transfer between consecutive water mols. is a very fast spontaneous process that takes place in the subpicosecond time scale. The translocation along extended regions of the chain takes place neither via a totally concerted mechanism in which the donor-acceptor pattern would flip over the entire chain in a single step, nor via a succession of incoherent hops between well-defined intermediates. Rather, proton transfer in the wire is a semicollective process that results from the subtle interplay of rapid hydrogen-bond length fluctuations along the water chain. These rapid structural fluctuations of the protonated single file of waters around an av. position and the slow movements of the av. position of the excess proton along the channel axis occur on two very different time scales. Ultimately, it is the slow reorganization of hydrogen bonds between single-file water mols. and channel backbone carbonyl groups that, by affecting the connectivity and the dynamics of the single-file water chain, also limits the translocation of the proton across the pore.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK28XktVOhur4%253D&md5=0e6260837d0058a326d2557633533dc1
  51. 51
    Danelon, C.; Suenaga, A.; Winterhalter, M.; Yamato, I. Molecular origin of the cation selectivity in OmpF porin: single channel conductances vs. free energy calculation. Biophys. Chem. 2003, 104 (3), 591– 603,  DOI: 10.1016/S0301-4622(03)00062-0
    Google Scholar
    51
    Molecular origin of the cation selectivity in OmpF porin: single channel conductances vs. free energy calculation
    Danelon, Christophe; Suenaga, Atsushi; Winterhalter, Mathias; Yamato, Ichiro
    Biophysical Chemistry (2003), 104 (3), 591-603CODEN: BICIAZ; ISSN:0301-4622. (Elsevier Science B.V.)
    Ion current through single outer membrane protein F (OmpF) trimers was recorded and compared to mol. dynamics simulation. Unidirectional insertion was revealed from the asymmetry in channel conductance. Single trimer conductance showed particularly high values at low sym. salt soln. The conductance values of various alkali metal ion solns. were proportional to the monovalent cation mobility values in the bulk phase, LiCl<NaCl<KCl<RbCl∼CsCl, but the conductance differences were quant. larger than cond. differences in bulk solns. Selectivity measurements at low concn. showed that OmpF channels favored permeation of alkali metal ions over chloride and suggested size preference for smaller cations. These results suggest that there are specific interactions between the permeating cation and charged residues lining the channel walls. This hypothesis was supported by computational study which predicted that monovalent cations bind to Asp113 at low concn. Here, free energy calcns. revealed that the affinity of the alkali metal ions to its binding site increased with their at. radii, Li+∼Na+<K+∼Rb+∼Cs+. A detailed inspection of both exptl. and computational results suggested that stronger binding at the central constriction of the channel increases the translocation rate of cations under applied voltage by increasing their local concn. relative to the bulk soln.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXmt1GqtL8%253D&md5=1255164202b43106dbf55c3e9b3be939
  52. 52
    Al Nahas, K.; Cama, J.; Schaich, M.; Hammond, K.; Deshpande, S.; Dekker, C.; Ryadnov, M. G.; Keyser, U. F. A microfluidic platform for the characterisation of membrane active antimicrobials. Lab Chip 2019, 19 (5), 837– 844,  DOI: 10.1039/C8LC00932E
    Google Scholar
    52
    A microfluidic platform for the characterisation of membrane active antimicrobials
    Al Nahas, K.; Cama, J.; Schaich, M.; Hammond, K.; Deshpande, S.; Dekker, C.; Ryadnov, M. G.; Keyser, U. F.
    Lab on a Chip (2019), 19 (5), 837-844CODEN: LCAHAM; ISSN:1473-0189. (Royal Society of Chemistry)
    The spread of bacterial resistance against conventional antibiotics generates a great need for the discovery of novel antimicrobials. Polypeptide antibiotics constitute a promising class of antimicrobial agents that favor attack on bacterial membranes. However, efficient measurement platforms for evaluating their mechanisms of action in a systematic manner are lacking. Here we report an integrated lab-on-a-chip multilayer microfluidic platform to quantify the membranolytic efficacy of such antibiotics. The platform is a biomimetic vesicle-based screening assay, which generates giant unilamellar vesicles (GUVs) in physiol. relevant buffers on demand. Hundreds of these GUVs are individually immobilized downstream in phys. traps connected to sep. perfusion inlets that facilitate controlled antibiotic delivery. Antibiotic efficacy is expressed as a function of the time needed for an encapsulated dye to leak out of the GUVs as a result of antibiotic treatment. This proof-of-principle study probes the dose response of an archetypal polypeptide antibiotic cecropin B on GUVs mimicking bacterial membranes. The results of the study provide a foundation for engineering quant., high-throughput microfluidics devices for screening antibiotics.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXitFGntLg%253D&md5=543122ca19b971fde4c274b26f8ed3a4
  53. 53
    Deshpande, S.; Caspi, Y.; Meijering, A. E. C.; Dekker, C. Octanol-assisted liposome assembly on chip. Nat. Commun. 2016, 7 (1), 10447,  DOI: 10.1038/ncomms10447
    Google Scholar
    53
    Octanol-assisted liposome assembly on chip
    Deshpande, Siddharth; Caspi, Yaron; Meijering, Anna E. C.; Dekker, Cees
    Nature Communications (2016), 7 (), 10447CODEN: NCAOBW; ISSN:2041-1723. (Nature Publishing Group)
    Liposomes are versatile supramol. assemblies widely used in basic and applied sciences. Here we present a novel microfluidics-based method, octanol-assisted liposome assembly (OLA), to form monodisperse, cell-sized (5-20μm), unilamellar liposomes with excellent encapsulation efficiency. Akin to bubble blowing, an inner aq. phase and a surrounding lipid-carrying 1-octanol phase is pinched off by outer fluid streams. Such hydrodynamic flow focusing results in double-emulsion droplets that spontaneously develop a side-connected 1-octanol pocket. Owing to interfacial energy minimization, the pocket splits off to yield fully assembled solvent-free liposomes within minutes. This solves the long-standing fundamental problem of prolonged presence of residual oil in the liposome bilayer. We demonstrate the unilamellarity of liposomes with functional α-haemolysin protein pores in the membrane and validate the biocompatibility by inner leaflet localization of bacterial divisome proteins (FtsZ and ZipA). OLA offers a versatile platform for future anal. tools, delivery systems, nanoreactors and synthetic cells.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xht1Cku78%253D&md5=355998fa9e3a32acf032da1d5643cd6d
  54. 54
    Tivony, R.; Fletcher, M.; Al Nahas, K.; Keyser, U. F. A Microfluidic Platform for Sequential Assembly and Separation of Synthetic Cell Models. ACS Synth. Biol. 2021, 10 (11), 3105– 3116,  DOI: 10.1021/acssynbio.1c00371
    Google Scholar
    54
    A Microfluidic Platform for Sequential Assembly and Separation of Synthetic Cell Models
    Tivony, Ran; Fletcher, Marcus; Al Nahas, Kareem; Keyser, Ulrich F.
    ACS Synthetic Biology (2021), 10 (11), 3105-3116CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)
    Cell-sized vesicles like giant unilamellar vesicles (GUVs) are established as a promising biomimetic model for studying cellular phenomena in isolation. However, the presence of residual components and byproducts, generated during vesicles prepn. and manipulation, severely limits the utility of GUVs in applications like synthetic cells. Therefore, with the rapidly growing field of synthetic biol., there is an emergent demand for techniques that can continuously purify cell-like vesicles from diverse residues, while GUVs are being simultaneously synthesized and manipulated. We have developed a microfluidic platform capable of purifying GUVs through stream bifurcation, where a vesicles suspension is partitioned into three fractions: purified GUVs, residual components, and a washing soln. Using our purifn. approach, we show that giant vesicles can be sepd. from various residues-which range in size and chem. compn.-with a very high efficiency (e = 0.99), based on size and deformability of the filtered objects. In addn., by incorporating the purifn. module with a microfluidic-based GUV-formation method, octanol-assisted liposome assembly (OLA), we established an integrated prodn.-purifn. microfluidic unit that sequentially produces, manipulates, and purifies GUVs. We demonstrate the applicability of the integrated device to synthetic biol. through sequentially fusing SUVs with freshly prepd. GUVs and sepg. the fused GUVs from extraneous SUVs and oil droplets at the same time.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXisVGrs7%252FK&md5=f744b0dd616e17f260a55c1a4d18175c
  55. 55
    Edelstein, A. D.; Tsuchida, M. A.; Amodaj, N.; Pinkard, H.; Vale, R. D.; Stuurman, N. Advanced methods of microscope control using μManager software. Journal of Biological Methods 2014, 1 (2), e10  DOI: 10.14440/jbm.2014.36
    Google Scholar
    There is no corresponding record for this reference.
  56. 56
    Morzy, D.; Rubio-Sánchez, R.; Joshi, H.; Aksimentiev, A.; Di Michele, L.; Keyser, U. F. Cations Regulate Membrane Attachment and Functionality of DNA Nanostructures. J. Am. Chem. Soc. 2021, 143 (19), 7358– 7367,  DOI: 10.1021/jacs.1c00166
    Google Scholar
    56
    Cations Regulate Membrane Attachment and Functionality of DNA Nanostructures
    Morzy, Diana; Rubio-Sanchez, Roger; Joshi, Himanshu; Aksimentiev, Aleksei; Di Michele, Lorenzo; Keyser, Ulrich F.
    Journal of the American Chemical Society (2021), 143 (19), 7358-7367CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    The interplay between nucleic acids and lipids underpins several key processes in mol. biol., synthetic biotechnol., vaccine technol., and nanomedicine. These interactions are often electrostatic in nature, and much of their rich phenomenol. remains unexplored in view of the chem. diversity of lipids, the heterogeneity of their phases, and the broad range of relevant solvent conditions. Here we unravel the electrostatic interactions between zwitterionic lipid membranes and DNA nanostructures in the presence of physiol. relevant cations, with the purpose of identifying new routes to program DNA-lipid complexation and membrane-active nanodevices. We demonstrate that this interplay is influenced by both the phase of the lipid membranes and the valency of the ions and observe divalent cation bridging between nucleic acids and gel-phase bilayers. Furthermore, even in the presence of hydrophobic modifications on the DNA, we find that cations are still required to enable DNA adhesion to liq.-phase membranes. We show that the latter mechanism can be exploited to control the degree of attachment of cholesterol-modified DNA nanostructures by modifying their overall hydrophobicity and charge. Besides their biol. relevance, the interaction mechanisms we explored hold great practical potential in the design of biomimetic nanodevices, as we show by constructing an ion-regulated DNA-based synthetic enzyme.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXhtVemsbzP&md5=6f2ca7eebbcbd1077f8ab9f8ecb29d34
  57. 57
    Rubio-Sánchez, R.; Barker, S. E.; Walczak, M.; Cicuta, P.; Michele, L. D. A Modular, Dynamic, DNA-Based Platform for Regulating Cargo Distribution and Transport between Lipid Domains. Nano Lett. 2021, 21 (7), 2800– 2808,  DOI: 10.1021/acs.nanolett.0c04867
    Google Scholar
    57
    A Modular, Dynamic, DNA-Based Platform for Regulating Cargo Distribution and Transport between Lipid Domains
    Rubio-Sanchez, Roger; Barker, Simone Eizagirre; Walczak, Michal; Cicuta, Pietro; Michele, Lorenzo Di
    Nano Letters (2021), 21 (7), 2800-2808CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)
    Cell membranes regulate the distribution of biol. machinery between phase-sepd. lipid domains to facilitate key processes including signaling and transport, which are among the life-like functionalities that bottom-up synthetic biol. aims to replicate in artificial-cellular systems. Here, we introduce a modular approach to program partitioning of amphiphilic DNA nanostructures in coexisting lipid domains. Exploiting the tendency of different hydrophobic "anchors" to enrich different phases, we modulate the lateral distribution of our devices by rationally combining hydrophobes and by changing nanostructure size and topol. We demonstrate the functionality of our strategy with a bioinspired DNA architecture, which dynamically undergoes ligand-induced reconfiguration to mediate cargo transport between domains via lateral redistribution. Our findings pave the way to next-generation biomimetic platforms for sensing, transduction, and communication in synthetic cellular systems.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXmsFOksLo%253D&md5=2b3cb391d463bef6c07fb88a99d6dc15
  58. 58
    Beucher, S. The Watershed Transformation Applied to Image Segmentation. Scanning Microscopy 1992, 1992(6), 1
    Google Scholar
    There is no corresponding record for this reference.

Cited By

ARTICLE SECTIONS
Jump To

This article has not yet been cited by other publications.

Download PDF

  • Abstract

    High Resolution Image
    Download MS PowerPoint Slide

    Figure 1

    Figure 1. Design and characterization of G-quadruplex (G4) DNA based K+ probes. (A) Schematic illustrating the K+ sensing principle of G4-DNA probes. A human telomeric DNA (HT G4-DNA), modified with a fluorophore and quencher at opposing ends (5′ and 3′, respectively), folds in response to K+, thereby bringing the fluorophore and quencher into closer contact and decreasing the fluorescence intensity of the probe. (B) Variation of FAMQ-G4 fluorescence intensity with increasing concentration of K+ (blue circles). In the presence of a complementary DNA strand (orange triangles) no reduction in fluorescence is observed for the double-stranded G4-DNA, indicating that folding of the single-stranded G4-DNA in the presence of K+ causes the fluorescent response. (C) Emission spectra of G4 probes modified with different fluorophores at their 5′ end. (D) Fluorescence intensity variation of G4 probes, modified with HEX (green) or Texas Red (red), in response to increasing K+ concentration.

    High Resolution Image
    Download MS PowerPoint Slide

    Figure 2

    Figure 2. K+ transport measurements across single GUVs using microfluidic-based approaches. (A) On-chip production of GUVs (DOPC/DOPG, 3:1 w/w) and encapsulation of FAMQ-G4 (10 μM), using octanol-assisted liposome assembly. Scale bar: 20 μm. (B) FAMQ-G4 encapsulated GUV immobilization using microfluidic hydrodynamic trapping (i). Once trapped, a 1 mM KCl solution, containing 500 nM FAMQ-G4, is perfused into the microfluidic chamber (ii), where GUVs can be visualized for >10 h, allowing one to capture the transport process of slow-permeating solutes such as K+. Scale bar: 40 μm. (C) (i) Time lapse of lumenal GUV fluorescence during the K+ transport process, showing the decrease in FAMQ-G4 fluorescence as a result of K+ induced folding. Scale bar: 20 μm. (ii) Analysis of K+ permeation across the lipid bilayer of single GUVs (n = 441) showing the temporal variation of lumenal (black solid line) and background (black dashed line) fluorescence intensity (median). The gray bands represent the lower and upper quartiles of the measured fluorescence at each time point for each measured vesicle.

    High Resolution Image
    Download MS PowerPoint Slide

    Figure 3

    Figure 3. Quantification of K+ permeability across single GUVs. (A) Variation of K+ concentration inside (solid) and outside (dashed) GUVs during the transport process. The distribution of [K+] among GUVs is represented by the upper and lower quartiles of [K+] at each time point. Inset: magnified view of the transmembrane K+ concentration gradient, Δ[K+], generated in the initial period (typically a few tens of minutes) of our experiments. (B) Variation of K+ flux into GUVs over the measurement time course. Inset: flux profile showing the variation of K+ flux as a function of Δ[K+] during the initial period of Δ[K+] development. The obtained linear flux profile can be represented through J = PΔ[K+], thus enabling the determination of K+ permeability from the slope of the curve. (C) Schematic showing the proposed dissipation mechanism of transmembrane potential by a counter flux of protons across the GUV lipid bilayer in our experiments. (D) Distribution of measured permeability coefficients for negatively charged OLA DOPC/DOPG (3:1) GUVs (N = 441). Inset: measured permeability distribution of electroformed DOPC/DOPG (3:1) GUVs.

    High Resolution Image
    Download MS PowerPoint Slide

    Figure 4

    Figure 4. K+ transport kinetics across GUVs with reconstituted model ion channels. (A) Time-resolved variation of lumenal (solid lines) and extravesicular (dashed lines) K+ concentration for DOPC/DOPG (3:1) GUVs with (red, n = 46), and without (black, n = 38) reconstituted gramicidin A (gA) (see experimental section). The distribution of permeated [K+] over GUVs is represented by the upper and lower quartiles of [K+] at each time point. Inset: schematic illustrating the two possible transport pathways across gA incorporated GUVs. B. Analysis of lumenal (solid lines) and extravesicular (dashed lines) [K+] across DOPC/DOPG (3:1) GUVs with (blue, n = 83) and without (black, n = 76) reconstituted OmpF (see Materials and Methods). Inset: schematic illustrating the two possible transport pathways across OmpF-incorporated GUVs. (C) Flux profiles obtained for GUVs with reconstituted gA (red) and OmpF (blue). The circles are the mean flux values, and the bands are the lower and upper quartiles for each GUV population. The black dashed line is the best fit of a linear curve to the mean flux data at the linear regime (0 < Δ[K+] < 0.13 mM), using linear regression. The red arrow indicates the Δ[K+] value at which the measured mean flux (red circles) is 0.58 of the flux (black dashed line) at the same Δ[K+] in the absence of transmembrane potential development. (D) Schematic demonstrating the suggested origin for the variance in H+/K+ selectivity between gA and OmpF.

    High Resolution Image
    Download MS PowerPoint Slide

  • References

    ARTICLE SECTIONS
    Jump To

    This article references 58 other publications.

    1. 1
      Clausen, M. V.; Hilbers, F.; Poulsen, H. The Structure and Function of the Na,K-ATPase Isoforms in Health and Disease. Frontiers in Physiology 2017, 8, 1,  DOI: 10.3389/fphys.2017.00371
      Google Scholar
      There is no corresponding record for this reference.
    2. 2
      Navale, A. M.; Paranjape, A. N. Glucose transporters: physiological and pathological roles. Biophysical Reviews 2016, 8 (1), 5– 9,  DOI: 10.1007/s12551-015-0186-2
      Google Scholar
      2
      Glucose transporters: physiological and pathological roles
      Navale, Archana M.; Paranjape, Archana N.
      Biophysical Reviews (2016), 8 (1), 5-9CODEN: BRIECG; ISSN:1867-2450. (Springer)
      A review. Glucose is a primary energy source for most cells and an important substrate for many biochem. reactions. As glucose is a need of each and every cell of the body, so are the glucose transporters. Consequently, all cells express these important proteins on their surface. In recent years developments in genetics have shed new light on the types and physiol. of various glucose transporters, of which there are two main types-sodium-glucose linked transporters (SGLTs) and facilitated diffusion glucose transporters (GLUT)-which can be divided into many more subclasses. Transporters differ in terms of their substrate specificity, distribution and regulatory mechanisms. Glucose transporters have also received much attention as therapeutic targets for various diseases. In this review, we attempt to present a simplified view of this complex topic which may be of interest to researchers involved in biochem. and pharmacol. research.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhtF2isb0%253D&md5=e328a4728ff6bca082764e1d06372066
    3. 3
      Calisto, F.; Sousa, F. M.; Sena, F. V.; Refojo, P. N.; Pereira, M. M. Mechanisms of Energy Transduction by Charge Translocating Membrane Proteins. Chem. Rev. 2021, 121 (3), 1804– 1844,  DOI: 10.1021/acs.chemrev.0c00830
      Google Scholar
      3
      Mechanisms of Energy Transduction by Charge Translocating Membrane Proteins
      Calisto, Filipa; Sousa, Filipe M.; Sena, Filipa V.; Refojo, Patricia N.; Pereira, Manuela M.
      Chemical Reviews (Washington, DC, United States) (2021), 121 (3), 1804-1844CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)
      A review. Life relies on the const. exchange of different forms of energy, i.e., on energy transduction. Therefore, organisms have evolved in a way to be able to harvest the energy made available by external sources (such as light or chem. compds.) and convert these into biol. useable energy forms, such as the transmembrane difference of electrochem. potential (Δ~μ). Membrane proteins contribute to the establishment of Δ~μ by coupling exergonic catalytic reactions to the translocation of charges (electrons/ions) across the membrane. Irresp. of the energy source and consequent type of reaction, all charge-translocating proteins follow two mol. coupling mechanisms: direct- or indirect-coupling, depending on whether the translocated charge is involved in the driving reaction. In this review, we explore these two coupling mechanisms by thoroughly examg. the different types of charge-translocating membrane proteins. For each protein, we analyze the resp. reaction thermodn., electron transfer/catalytic processes, charge-translocating pathways, and ion/substrate stoichiometries.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXitF2jug%253D%253D&md5=9f96c0c718531fa3312a980cbdb9081c
    4. 4
      Valkenier, H.; López Mora, N.; Kros, A.; Davis, A. P. Visualization and quantification of transmembrane ion transport into giant unilamellar vesicles. Angewandte Chemie (International Ed. in English) 2015, 54 (7), 2137– 2141,  DOI: 10.1002/anie.201410200
      Google Scholar
      4
      Visualization and quantification of transmembrane ion transport into giant unilamellar vesicles
      Valkenier Hennie; Lopez Mora Nestor; Kros Alexander; Davis Anthony P
      Angewandte Chemie (International ed. in English) (2015), 54 (7), 2137-41 ISSN:.
      Transmembrane ion transporters (ionophores) are widely investigated as supramolecular agents with potential for biological activity. Tests are usually performed in synthetic membranes that are assembled into large unilamellar vesicles (LUVs). However transport must be followed through bulk properties of the vesicle suspension, because LUVs are too small for individual study. An alternative approach is described whereby ion transport can be revealed and quantified through direct observation. The method employs giant unilamellar vesicles (GUVs), which are 20-60 μm in diameter and readily imaged by light microscopy. This allows characterization of individual GUVs containing transporter molecules, followed by studies of transport through fluorescence emission from encapsulated indicators. The method provides new levels of certainty and relevance, given that the GUVs are similar in size to living cells. It has been demonstrated using a highly active anion carrier, and should aid the development of compounds for treating channelopathies such as cystic fibrosis.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC2MvjtlajsQ%253D%253D&md5=e035e3a1292cea17d96629d2467a570f
    5. 5
      Garten, M.; Mosgaard, L. D.; Bornschlögl, T.; Dieudonné, S.; Bassereau, P.; Toombes, G. E. S. Whole-GUV patch-clamping. Proc. Natl. Acad. Sci. U. S. A. 2017, 114 (2), 328– 333,  DOI: 10.1073/pnas.1609142114
      Google Scholar
      5
      Whole-GUV patch-clamping
      Garten, Matthias; Mosgaard, Lars D.; Bornschlogl, Thomas; Dieudonne, Stephane; Bassereau, Patricia; Toombes, Gilman E. S.
      Proceedings of the National Academy of Sciences of the United States of America (2017), 114 (2), 328-333CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      Studying how the membrane modulates ion channel and transporter activity is challenging because cells actively regulate membrane properties, whereas existing in vitro systems have limitations, such as residual solvent and unphysiol. high membrane tension. Cell-sized giant unilamellar vesicles (GUVs) would be ideal for in vitro electrophysiol., but efforts to measure the membrane current of intact GUVs have been unsuccessful. In this work, two challenges for obtaining the "whole-GUV" patch-clamp configuration were identified and resolved. First, unless the patch pipet and GUV pressures are precisely matched in the GUV-attached configuration, breaking the patch membrane also ruptures the GUV. Second, GUVs shrink irreversibly because the membrane/glass adhesion creating the high-resistance seal (>1 GΩ) continuously pulls membrane into the pipet. In contrast, for cell-derived giant plasma membrane vesicles (GPMVs), breaking the patch membrane allows the GPMV contents to passivate the pipet surface, thereby dynamically blocking membrane spreading in the whole-GMPV mode. To mimic this dynamic passivation mechanism, beta-casein was encapsulated into GUVs, yielding a stable, high-resistance, whole-GUV configuration for a range of membrane compns. Specific membrane capacitance measurements confirmed that the membranes were truly solvent-free and that membrane tension could be controlled over a physiol. range. Finally, the potential for ion transport studies was tested using the model ion channel, gramicidin, and voltage-clamp fluorometry measurements were performed with a voltage-dependent fluorophore/quencher pair. Whole-GUV patch-clamping allows ion transport and other voltage-dependent processes to be studied while controlling membrane compn., tension, and shape.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XitFentrnK&md5=757fba9d98f5630604eb235709f0de83
    6. 6
      Cama, J.; Bajaj, H.; Pagliara, S.; Maier, T.; Braun, Y.; Winterhalter, M.; Keyser, U. F. Quantification of Fluoroquinolone Uptake through the Outer Membrane Channel OmpF of Escherichia coli. J. Am. Chem. Soc. 2015, 137 (43), 13836– 13843,  DOI: 10.1021/jacs.5b08960
      Google Scholar
      6
      Quantification of fluoroquinolone uptake through the outer membrane channel OmpF of Escherichia coli
      Cama, Jehangir; Bajaj, Harsha; Pagliara, Stefano; Maier, Theresa; Braun, Yvonne; Winterhalter, Mathias; Keyser, Ulrich F.
      Journal of the American Chemical Society (2015), 137 (43), 13836-13843CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Decreased drug accumulation is a common cause of antibiotic resistance in microorganisms. However, there are few reliable general techniques capable of quantifying drug uptake through bacterial membranes. The authors present a semiquant. optofluidic assay for studying the uptake of autofluorescent drug mols. in single liposomes. They studied the effect of the Escherichia coli outer membrane channel OmpF on the accumulation of the fluoroquinolone antibiotic, norfloxacin, in proteoliposomes. Measurements were performed at pH 5 and pH 7, corresponding to two different charge states of norfloxacin that bacteria are likely to encounter in the human gastrointestinal tract. At both pH values, the porins significantly enhance drug permeation across the proteoliposome membranes. At pH 5, where norfloxacin permeability across pure phospholipid membranes is low, the porins increase drug permeability by 50-fold on av. The authors est. a flux of about 10 norfloxacin mols. per s per OmpF trimer in the presence of a 1 mM concn. gradient of norfloxacin. They also performed single channel electrophysiol. measurements and found that the application of transmembrane voltages causes an elec. field driven uptake in addn. to concn. driven diffusion. The authors propose a phys. mechanism for the pH mediated change in bacterial susceptibility to fluoroquinolone antibiotics.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhs1Ohur3E&md5=b29d6581fa48b214ad4e5ff617eff944
    7. 7
      Göpfrich, K.; Platzman, I.; Spatz, J. P. Mastering Complexity: Towards Bottom-up Construction of Multifunctional Eukaryotic Synthetic Cells. Trends Biotechnol. 2018, 36 (9), 938– 951,  DOI: 10.1016/j.tibtech.2018.03.008
      Google Scholar
      7
      Mastering Complexity: Towards Bottom-up Construction of Multifunctional Eukaryotic Synthetic Cells
      Gopfrich Kerstin; Platzman Ilia; Spatz Joachim P
      Trends in biotechnology (2018), 36 (9), 938-951 ISSN:.
      With the ultimate aim to construct a living cell, bottom-up synthetic biology strives to reconstitute cellular phenomena in vitro - disentangled from the complex environment of a cell. Recent work towards this ambitious goal has provided new insights into the mechanisms governing life. With the fast-growing library of functional modules for synthetic cells, their classification and integration become increasingly important. We discuss strategies to reverse-engineer and recombine functional parts for synthetic eukaryotes, mimicking the characteristics of nature's own prototype. Particularly, we focus on large outer compartments, complex endomembrane systems with organelles, and versatile cytoskeletons as hallmarks of eukaryotic life. Moreover, we identify microfluidics and DNA nanotechnology as two technologies that can integrate these functional modules into sophisticated multifunctional synthetic cells.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC1MjlslCjsQ%253D%253D&md5=beed6876210fcc29b75bad05443d0ba5
    8. 8
      Ohmann, A.; Li, C.-Y.; Maffeo, C.; Al Nahas, K.; Baumann, K. N.; Göpfrich, K.; Yoo, J.; Keyser, U. F.; Aksimentiev, A. A synthetic enzyme built from DNA flips 107 lipids per second in biological membranes. Nat. Commun. 2018, 9 (1), 1– 9,  DOI: 10.1038/s41467-018-04821-5
      Google Scholar
      8
      A synthetic enzyme built from DNA flips 107 lipids per second in biological membranes
      Ohmann, Alexander; Li, Chen-Yu; Maffeo, Christopher; Al Nahas, Kareem; Baumann, Kevin N.; Gopfrich, Kerstin; Yoo, Jejoong; Keyser, Ulrich F.; Aksimentiev, Aleksei
      Nature Communications (2018), 9 (1), 1-9CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)
      Mimicking enzyme function and increasing performance of naturally evolved proteins is one of the most challenging and intriguing aims of nanoscience. Here, we employ DNA nanotechnol. to design a synthetic enzyme that substantially outperforms its biol. archetypes. Consisting of only eight strands, our DNA nanostructure spontaneously inserts into biol. membranes by forming a toroidal pore that connects the membrane's inner and outer leaflets. The membrane insertion catalyzes spontaneous transport of lipid mols. between the bilayer leaflets, rapidly equilibrating the lipid compn. Through a combination of microscopic simulations and fluorescence microscopy we find the lipid transport rate catalyzed by the DNA nanostructure exceeds 107 mols. per s, which is three orders of magnitude higher than the rate of lipid transport catalyzed by biol. enzymes. Furthermore, we show that our DNA-based enzyme can control the compn. of human cell membranes, which opens new avenues for applications of membrane-interacting DNA systems in medicine.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhtF2jtbzN&md5=9594eb1d972bb6381a9c9d64a5bc4f0f
    9. 9
      Elani, Y.; Trantidou, T.; Wylie, D.; Dekker, L.; Polizzi, K.; Law, R. V.; Ces, O. Constructing vesicle-based artificial cells with embedded living cells as organelle-like modules. Sci. Rep. 2018, 8 (1), 4564,  DOI: 10.1038/s41598-018-22263-3
      Google Scholar
      9
      Constructing vesicle-based artificial cells with embedded living cells as organelle-like modules
      Elani Yuval; Trantidou Tatiana; Wylie Douglas; Law Robert V; Ces Oscar; Elani Yuval; Wylie Douglas; Ces Oscar; Dekker Linda; Polizzi Karen
      Scientific reports (2018), 8 (1), 4564 ISSN:.
      There is increasing interest in constructing artificial cells by functionalising lipid vesicles with biological and synthetic machinery. Due to their reduced complexity and lack of evolved biochemical pathways, the capabilities of artificial cells are limited in comparison to their biological counterparts. We show that encapsulating living cells in vesicles provides a means for artificial cells to leverage cellular biochemistry, with the encapsulated cells serving organelle-like functions as living modules inside a larger synthetic cell assembly. Using microfluidic technologies to construct such hybrid cellular bionic systems, we demonstrate that the vesicle host and the encapsulated cell operate in concert. The external architecture of the vesicle shields the cell from toxic surroundings, while the cell acts as a bioreactor module that processes encapsulated feedstock which is further processed by a synthetic enzymatic metabolism co-encapsulated in the vesicle.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC1Mnht1yisw%253D%253D&md5=8e62e29719807e28d859d2c4d4edfe5b
    10. 10
      Tivony, R.; Fletcher, M.; Keyser, U. F. Quantifying proton-induced membrane polarization in single biomimetic giant vesicles. Biophys. J. 2022, 121, 2223,  DOI: 10.1016/j.bpj.2022.05.041
      Google Scholar
      10
      Quantifying proton-induced membrane polarization in single biomimetic giant vesicles
      Tivony, Ran; Fletcher, Marcus; Keyser, Ulrich F.
      Biophysical Journal (2022), 121 (12), 2223-2232CODEN: BIOJAU; ISSN:0006-3495. (Cell Press)
      Proton gradients are utilized by cells to power the transport activity of many membrane proteins. Synthetic cells, such as proteo-giant unilamellar vesicles, offer an advanced approach for studying the functionality of membrane proteins in isolation. However, understanding of protein-based transport in vitro requires accurate measurements of proton flux and its accompanying electrochem. gradient across the lipid bilayer. We present an approach to directly quantify the flux of protons across single cell-sized lipid vesicles under modulated electrochem. gradients. Our measurements reveal the corresponding assocn. between proton permeation and transmembrane potential development and its relation to the chem. nature of the conjugated anion (base). In the case of formic acid, we showed that, out of the total amt. of permeated protons, a fraction of ≈0.2 traverse the lipid bilayer as H+, with the rest (≈0.8) in the form of a neutral acid. For strong acids (HCl or HNO3), proton permeation was governed by translocation of H+. Accordingly, a larger proton motive force (pmf) was generated for strong acids (pmf = 14.2 mV) relative to formic acid (pmf = 1.3 mV). We anticipate that our approach will guide the development of protein-based transport driven by proton gradient in artificial cell models and enable a deeper understanding of how vital acids, such as fatty acids, amino acids, bile acids, and carboxylic acid-contg. drugs, traverse the lipid bilayer.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XhsVCntr%252FF&md5=f9efcabb59036ea920d68bbf69dff4b3
    11. 11
      Hindley, J. W.; Zheleva, D. G.; Elani, Y.; Charalambous, K.; Barter, L. M.; Booth, P. J.; Bevan, C. L.; Law, R. V.; Ces, O. Building a synthetic mechanosensitive signaling pathway in compartmentalized artificial cells. Proc. Natl. Acad. Sci. U. S. A. 2019, 116 (34), 16711– 16716,  DOI: 10.1073/pnas.1903500116
      Google Scholar
      11
      Building a synthetic mechanosensitive signaling pathway in compartmentalized artificial cells
      Hindley, James W.; Zheleva, Daniela G.; Elani, Yuval; Charalambous, Kalypso; Barter, Laura M. C.; Booth, Paula J.; Bevan, Charlotte L.; Law, Robert V.; Ces, Oscar
      Proceedings of the National Academy of Sciences of the United States of America (2019), 116 (34), 16711-16716CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      To date, reconstitution of one of the fundamental methods of cell communication, the signaling pathway, has been unaddressed in the bottom-up construction of artificial cells (ACs). Such developments are needed to increase the functionality and biomimicry of ACs, accelerating their translation and application in biotechnol. Here, we report the construction of a de novo synthetic signaling pathway in microscale nested vesicles. Vesicle-cell models respond to external calcium signals through activation of an intracellular interaction between phospholipase A2 and a mechanosensitive channel present in the internal membranes, triggering content mixing between compartments and controlling cell fluorescence. Emulsion-based approaches to AC construction are therefore shown to be ideal for the quick design and testing of new signaling networks and can readily include synthetic mols. difficult to introduce to biol. cells. This work represents a foundation for the engineering of multicompartment-spanning designer pathways that can be utilized to control downstream events inside an AC, leading to the assembly of micromachines capable of sensing and responding to changes in their local environment.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhs1alsLrO&md5=c1fba7b047e86a526fba8fc14ac7370c
    12. 12
      Al Nahas, K.; Fletcher, M.; Hammond, K.; Nehls, C.; Cama, J.; Ryadnov, M. G.; Keyser, U. F. Measuring Thousands of Single-Vesicle Leakage Events Reveals the Mode of Action of Antimicrobial Peptides. Anal. Chem. 2022, 94 (27), 9530– 9539,  DOI: 10.1021/acs.analchem.1c03564
      Google Scholar
      12
      Measuring thousands of single-vesicle leakage events reveals mode of action of antimicrobial peptides
      Al Nahas, Kareem; Fletcher, Marcus; Hammond, Katharine; Nehls, Christian; Cama, Jehangir; Ryadnov, Maxim G.; Keyser, Ulrich F.
      Analytical Chemistry (Washington, DC, United States) (2022), 94 (27), 9530-9539CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
      Host defense or antimicrobial peptides hold promise for providing new pipelines of effective antimicrobial agents. Their activity quantified against model phospholipid membranes is fundamental to a detailed understanding of their structure-activity relationships. However, classical characterization assays often lack the ability to achieve this insight. Leveraging a highly parallelized microfluidic platform for trapping and studying thousands of giant unilamellar vesicles, we conducted quant. long-term microscopy studies to monitor the membrane-disruptive activity of archetypal antimicrobial peptides with a high spatiotemporal resoln. We described the modes of action of these peptides via measurements of the disruption of the vesicle population under the conditions of continuous peptide dosing using a range of concns. and related the obsd. modes to the mol. activity mechanisms of these peptides. The study offers an effective approach for characterizing membrane-targeting antimicrobial agents in a standardized manner and for assigning specific modes of action to the corresponding antimicrobial mechanisms.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XhsF2lur7K&md5=367970eacd16a15323d8ca9ca62a0164
    13. 13
      Robinson, T. Microfluidic Handling and Analysis of Giant Vesicles for Use as Artificial Cells: A Review. Advanced Biosystems 2019, 3 (6), 1800318,  DOI: 10.1002/adbi.201800318
      Google Scholar
      There is no corresponding record for this reference.
    14. 14
      Cama, J.; Al Nahas, K.; Fletcher, M.; Hammond, K.; Ryadnov, M. G.; Keyser, U. F.; Pagliara, S. An ultrasensitive microfluidic approach reveals correlations between the physico-chemical and biological activity of experimental peptide antibiotics. Sci. Rep. 2022, 12 (1), 1– 12,  DOI: 10.1038/s41598-022-07973-z
      Google Scholar
      There is no corresponding record for this reference.
    15. 15
      Hammond, K.; Cipcigan, F.; Al Nahas, K.; Losasso, V.; Lewis, H.; Cama, J.; Martelli, F.; Simcock, P. W.; Fletcher, M.; Ravi, J.; Stansfeld, P. J.; Pagliara, S.; Hoogenboom, B. W.; Keyser, U. F.; Sansom, M. S. P.; Crain, J.; Ryadnov, M. G. Switching Cytolytic Nanopores into Antimicrobial Fractal Ruptures by a Single Side Chain Mutation. ACS Nano 2021, 15 (6), 9679– 9689,  DOI: 10.1021/acsnano.1c00218
      Google Scholar
      15
      Switching Cytolytic Nanopores into Antimicrobial Fractal Ruptures by a Single Side Chain Mutation
      Hammond Katharine; Lewis Helen; Ravi Jascindra; Ryadnov Maxim G; Hammond Katharine; Hoogenboom Bart W; Cipcigan Flaviu; Martelli Fausto; Crain Jason; Al Nahas Kareem; Fletcher Marcus; Keyser Ulrich F; Losasso Valeria; Cama Jehangir; Pagliara Stefano; Cama Jehangir; Simcock Patrick W; Stansfeld Phillip J; Sansom Mark S P; Crain Jason; Pagliara Stefano; Hoogenboom Bart W; Ryadnov Maxim G
      ACS nano (2021), 15 (6), 9679-9689 ISSN:.
      Disruption of cell membranes is a fundamental host defense response found in virtually all forms of life. The molecular mechanisms vary but generally lead to energetically favored circular nanopores. Here, we report an elaborate fractal rupture pattern induced by a single side-chain mutation in ultrashort (8-11-mers) helical peptides, which otherwise form transmembrane pores. In contrast to known mechanisms, this mode of membrane disruption is restricted to the upper leaflet of the bilayer where it exhibits propagating fronts of peptide-lipid interfaces that are strikingly similar to viscous instabilities in fluid flow. The two distinct disruption modes, pores and fractal patterns, are both strongly antimicrobial, but only the fractal rupture is nonhemolytic. The results offer wide implications for elucidating differential membrane targeting phenomena defined at the nanoscale.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB3sbktVGruw%253D%253D&md5=1742ac794c7c3de8acc0aa8ee5d1c89c
    16. 16
      Paula, S.; Volkov, A. G.; Van Hoek, A. N.; Haines, T. H.; Deamer, D. W. Permeation of protons, potassium ions, and small polar molecules through phospholipid bilayers as a function of membrane thickness. Biophys. J. 1996, 70 (1), 339– 348,  DOI: 10.1016/S0006-3495(96)79575-9
      Google Scholar
      16
      Permeation of protons, potassium ions, and small polar molecules through phospholipid bilayers as a function of membrane thickness
      Paula, S.; Volkov, A. G.; Van Hoek, A. N.; Haines, T. H.; Deamer, D. W.
      Biophysical Journal (1996), 70 (1), 339-48CODEN: BIOJAU; ISSN:0006-3495. (Biophysical Society)
      Two mechanisms have been proposed to account for solute permeation of lipid bilayers. Partitioning into the hydrophobic phase of the bilayer, followed by diffusion, is accepted by many for the permeation of water and other small neutral solutes, but transient pores have also been proposed to account for both water and ionic solute permeation. These two mechanisms make distinctively different predictions about the permeability coeff. as a function of bilayer thickness. Whereas the soly.-diffusion mechanism predicts only a modest variation related to bilayer thickness, the pore model predicts an exponential relationship. To test these models, we measured the permeability of phospholipid bilayers to protons, potassium ions, water, urea, and glycerol. Bilayers were prepd. as liposomes, and thickness was varied systematically by using unsatd. lipids with chain lengths ranging from 14 to 24 carbon atoms. The permeability coeff. of water and neutral polar solutes displayed a modest dependence on bilayer thickness, with an approx. linear fivefold decrease as the carbon no. varied from 14 to 24 atoms. In contrast, the permeability to protons and potassium ions decreased sharply by two orders of magnitude between 14 and 18 carbon atoms, and leveled off, when the chain length was further extended to 24 carbon atoms. The results for water and the neutral permeating solutes are best explained by the soly.-diffusion mechanism. The results of protons and potassium ions in shorter-chain lipids are consistent with the transient pore model, but better fit the theor. line predicted by the soly.-diffusion model at longer chain lengths.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK28XjslGksQ%253D%253D&md5=e78b1e80f02e1b3942125de80e48dcd9
    17. 17
      Deamer, D. W. Proton permeation of lipid bilayers. Journal of Bioenergetics and Biomembranes 1987, 19 (5), 457– 479,  DOI: 10.1007/BF00770030
      Google Scholar
      17
      Proton permeation of lipid bilayers
      Deamer, D. W.
      Journal of Bioenergetics and Biomembranes (1987), 19 (5), 457-79CODEN: JBBID4; ISSN:0145-479X.
      Two expts. designed to test the tHBC model (transient H-bonded chain model) of H+ permeation in lipid bilayers were performed. These included measurements of relative H+/K+ permeability in the gramicidin channel, and plotting of H+ flux against the magnitude of pH gradients. The relative permeabilities of H+ and K+ through the gramicidin channel, which contains a single strand of H-bonded H2O mols., differed by >4 orders of magnitude when measured at neutral pH, demonstrating that a H-bonded chain of H2O mols. can provide substantial discrimination between H+ and other cations. It was also calcd. that if ∼7% of bilayer H2O was present in a transient configuration similar to that of the gramicidin channel, it could account for the measured proton flux. The plot of proton conductance against pH gradient across liposome membranes was superlinear, a result consistent with 1 of 3 alternative tHBC models for proton conductance described by J. Nagel (1987). A review of the H+ permeability of lipid bilayers is also presented; several proposed models of the process are discussed.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL1cXntVOhtw%253D%253D&md5=64b10ec6566d9e4235f628b30dc39580
    18. 18
      Paula, S.; Volkov, A. G.; Deamer, D. W. Permeation of Halide Anions through Phospholipid Bilayers Occurs by the Solubility-Diffusion Mechanism. Biophys. J. 1998, 74 (1), 319– 327,  DOI: 10.1016/S0006-3495(98)77789-6
      Google Scholar
      18
      Permeation of halide anions through phospholipid bilayers occurs by the solubility-diffusion mechanism
      Paula, S.; Volkov, A. G.; Deamer, D. W.
      Biophysical Journal (1998), 74 (1), 319-327CODEN: BIOJAU; ISSN:0006-3495. (Biophysical Society)
      Two alternative mechanisms are frequently used to describe ionic permeation of lipid bilayers. In the first, ions partition into the hydrophobic phase and then diffuse across (the soly.-diffusion mechanism). The second mechanism assumes that ions traverse the bilayer through transient hydrophilic defects caused by thermal fluctuations (the pore mechanism). The theor. predictions made by both models were tested for halide anions by measuring the permeability coeffs. for chloride, bromide, and iodide as a function of bilayer thickness, ionic radius, and sign of charge. To vary the bilayer thickness systematically, liposomes were prepd. from monounsatd. phosphatidylcholines (PC) with chain lengths between 16 and 24 carbon atoms. The fluorescent dye MQAE (N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide) served as an indicator for halide concn. inside the liposomes and was used to follow the kinetics of halide flux across the bilayer membranes. The obsd. permeability coeffs. ranged from 10-9 to 10-7 cm/s and increased as the bilayer thickness was reduced. Bromide was found to permeate approx. six times faster than chloride through bilayers of identical thickness, and iodide permeated three to four times faster than bromide. The dependence of the halide permeability coeffs. on bilayer thickness and on ionic size were consistent with permeation of hydrated ions by a soly.-diffusion mechanism rather than through transient pores. Halide permeation therefore differs from that of a monovalent cation such as potassium, which has been accounted for by a combination of the two mechanisms depending on bilayer thickness.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1cXhvFSru7o%253D&md5=d4ddbd155fdff8ec0370768a0532c39b
    19. 19
      Shen, Y.; Zhong, Y.; Fei, F.; Sun, J.; Czajkowsky, D. M.; Gong, B.; Shao, Z. Ultrasensitive liposome-based assay for the quantification of fundamental ion channel properties. Anal. Chim. Acta 2020, 1112, 8– 15,  DOI: 10.1016/j.aca.2020.03.044
      Google Scholar
      19
      Ultrasensitive liposome-based assay for the quantification of fundamental ion channel properties
      Shen, Yi; Zhong, Yulong; Fei, Fan; Sun, Jielin; Czajkowsky, Daniel M.; Gong, Bing; Shao, Zhifeng
      Analytica Chimica Acta (2020), 1112 (), 8-15CODEN: ACACAM; ISSN:0003-2670. (Elsevier B.V.)
      One of the most widely used approaches to characterize transmembrane ion transport through nanoscale synthetic or biol. channels is a straightforward, liposome-based assay that monitors changes in ionic flux across the vesicle membrane using pH- or ion-sensitive dyes. However, failure to account for the precise exptl. conditions, in particular the complete ionic compn. on either side of the membrane and the inherent permeability of ions through the lipid bilayer itself, can prevent quantifications and lead to fundamentally incorrect conclusions. Here we present a quant. model for this assay based on the Goldman-Hodgkin-Katz flux theory, which enables accurate measurements and identification of optimal conditions for the detn. of ion channel permeability and selectivity. Based on our model, the detection sensitivity of channel permeability is improved by two orders of magnitude over the commonly used exptl. conditions. Further, rather than obtaining qual. preferences of ion selectivity as is typical, we det. quant. values of these parameters under rigorously controlled conditions even when the exptl. results would otherwise imply (without our model) incorrect behavior. We anticipate that this simply employed ultrasensitive assay will find wide application in the quant. characterization of synthetic or biol. ion channels.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXmsl2qtr8%253D&md5=3a1f9d252c6fcbf2b8a2bb938f9ac640
    20. 20
      Megens, M.; Korman, C. E.; Ajo-Franklin, C. M.; Horsley, D. A. Faster-than-anticipated Na+/Cl- diffusion across lipid bilayers in vesicles. Biochimica et Biophysica Acta (BBA) - Biomembranes 2014, 1838 (10), 2420– 2424,  DOI: 10.1016/j.bbamem.2014.05.010
      Google Scholar
      There is no corresponding record for this reference.
    21. 21
      Kuyper, C. L.; Kuo, J. S.; Mutch, S. A.; Chiu, D. T. Proton Permeation into Single Vesicles Occurs via a Sequential Two-Step Mechanism and Is Heterogeneous. J. Am. Chem. Soc. 2006, 128 (10), 3233– 3240,  DOI: 10.1021/ja057349c
      Google Scholar
      21
      Proton Permeation into Single Vesicles Occurs via a Sequential Two-Step Mechanism and Is Heterogeneous
      Kuyper, Christopher L.; Kuo, Jason S.; Mutch, Sarah A.; Chiu, Daniel T.
      Journal of the American Chemical Society (2006), 128 (10), 3233-3240CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      This article describes the first single-vesicle study of proton permeability across the lipid membrane of small (∼100 nm) uni- and multilamellar vesicles, which were composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). To follow proton permeation into the internal vol. of each vesicle, we encapsulated carboxyfluorescein, a pH-sensitive dye whose fluorescence was quenched in the presence of excess protons. A microfluidic platform was used for easy exchange of high- and low-pH solns., and fluorescence quenching of single vesicles was detected with single-mol. total internal reflection fluorescence (TIRF) microscopy. Upon soln. exchange and acidification of the extravesicular soln. (from pH 9 to 3.5), we obsd. for each vesicle a biphasic decay in fluorescence. Through single-vesicle anal., we found that rate consts. for the first decay followed a Poisson distribution, whereas rate consts. for the second decay followed a normal distribution. We propose that proton permeation into each vesicle first arose from formation of transient pores and then transitioned into the second decay phase, which occurred by the soly.-diffusion mechanism. Furthermore, for the bulk population of vesicles, the decay rate const. and vesicle intensity (dependent on size) correlated to give an av. permeability coeff.; however, for individual vesicles, we found little correlation, which suggested that proton permeability among single vesicles was heterogeneous in our expts.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XhsFarsbs%253D&md5=1f9afa71de10357c3c50f0051d1add8b
    22. 22
      Debnath, M.; Chakraborty, S.; Kumar, Y. P.; Chaudhuri, R.; Jana, B.; Dash, J. Ionophore constructed from non-covalent assembly of a G-quadruplex and liponucleoside transports K+-ion across biological membranes. Nat. Commun. 2020, 11 (1), 1– 12,  DOI: 10.1038/s41467-019-13834-7
      Google Scholar
      There is no corresponding record for this reference.
    23. 23
      Venema, K.; Gibrat, R.; Grouzis, J.-P.; Grignon, C. Quantitative measurement of cationic fluxes, selectivity and membrane potential using liposomes multilabelled with fluorescent probes. Biochimica et Biophysica Acta (BBA) - Biomembranes 1993, 1146 (1), 87– 96,  DOI: 10.1016/0005-2736(93)90342-W
      Google Scholar
      There is no corresponding record for this reference.
    24. 24
      Sambath, K.; Liu, X.; Wan, Z.; Hutnik, L.; Belfield, K. D.; Zhang, Y. Potassium Ion Fluorescence Probes: Structures, Properties and Bioimaging. ChemPhotoChem. 2021, 5 (4), 317– 325,  DOI: 10.1002/cptc.202000236
      Google Scholar
      24
      Potassium Ion Fluorescence Probes: Structures, Properties and Bioimaging
      Sambath, Karthik; Liu, Xiangshan; Wan, Zhaoxiong; Hutnik, Lauren; Belfield, Kevin D.; Zhang, Yuanwei
      ChemPhotoChem (2021), 5 (4), 317-325CODEN: CHEMYH ISSN:. (Wiley-VCH Verlag GmbH & Co. KGaA)
      A review. As one of the most important minerals in the body, potassium is vital for the heart and neurons. Methods that can non-invasively and accurately monitor changes in potassium balances would benefit disease diagnoses as well as offer insight into pathologies. Among the sensing approaches, fluorescent probes serve as a unique detection method for its simplicity, tunable detection range, and bioimaging ability. The design of new probes with highly selective K+ receptors and transduction functionality remains a challenge that is motivated by numerous sensing and detection applications. In this minireview, fluoroionophores are summarized that undergo transduction, producing fluorescence signals when interacting with, e. g., potassium ions. The properties of ionophores (afford selective interaction with potassium) and fluorophores (generate signal read-out) are discussed. Mol. structure design and sensing mechanisms are included along with cell imaging applications. The selectivity toward K+ and the absorption/emission characteristics of the probes are of particular interest.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXislSgsLrO&md5=8d3bbf3a96ee347b797fb363d23636e7
    25. 25
      Kasner, S. E.; Ganz, M. B. Regulation of intracellular potassium in mesangial cells: a fluorescence analysis using the dye, PBFI. American Journal of Physiology 1992, 262 (3), 462– 467,  DOI: 10.1152/ajprenal.1992.262.3.F462
      Google Scholar
      25
      Regulation of intracellular potassium in mesangial cells: a fluorescence analysis using the dye, PBFI
      Kasner, Scott E.; Ganz, Michael B.
      American Journal of Physiology (1992), 262 (3, Pt. 2), F462-F467CODEN: AJPHAP; ISSN:0002-9513.
      Regulatory transport processes were investigated that maintain intracellular K+ homeostasis in cultured rat glomerular mesangial cells (MCs). Intracellular K+ concn. ([K+]i) of quiescent MCs, passages 3-8, grown to subconfluence on glass cover slips, was assessed by spectrofluorometry using the K+-sensitive dye, K+-binding benzofuran isophthalate (PBFI). Serum-starved MCs were incubated at 37° in 5 μM BPFI for 90 min. Excitation ratios of luminescences at 340 and 380 nm, measured at a const. emission at 500 nm, were used to det. [K+]i. Ionophores valinomycin and nigericin were used to clamp [K+]i to known [K+]o and thereby obtain an intracellular calibration of dye. Dependence of fluorescence ratio on [K+]i conformed to Michaelis-Menten behavior, with a Km of 113 mM. PBFI retains its sensitivity to alterations in [K+]i with pH change (pHi from 6.5 to 7.5) but is relatively insensitive when intracellular Na+ is greater than 75 mM and cell osmolarity exceeds 500 mM. Normal resting [K+]i for all expts. was detd. in MCs to be 102 mM in a HCO3--free HEPES-buffered soln. When MCs were exposed to ouabain, [K+]i fell to 48 mM and did not recover, suggesting presence of Na+-K+-ATPase. When MCs were exposed to furosemide, [K+]i transiently declined to 58 mM, which was followed by a rapid recovery to near steady state, indicating addnl. presence of Na+-K+-Cl- cotransporter. Recovery was completely abolished when MCs were exposed to ouabain. Exposure to Ba2+ led to an immediate increase in [K+]i to 124 mM followed by a rapid return to steady-state [K+]i. MCs exposed to tetraethylammonium exhibited a behavior similar to those exposed to Ba2+; resting [K+]i increased to 123 mM. Effects of angiotensin II (ANG II) and serotonin (5-HT) on Na+-K+-ATPase was assessed. None of these agents significantly altered resting [K+]i. ANG II and 5-HT stimulated Na+-K+-ATPase by 28 and 41%, resp., as measured during recovery of [K+]i toward normal after a diuretic-induced fall in [K+]i. The authors conclude that (1) MCs possess ouabain-sensitive Na+-K+-ATPase, loop diuretic-sensitive Na+-K+-Cl- cotransporter, and barium-sensitive K+ channels; (2) ANG II and 5-HT stimulate Na+-K+-ATPase; and (3) a continuous detn. of [K+]i and an examn. of its regulatory mechanism in MCs may be achieved through use of PBFI.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK38XitlamtLo%253D&md5=7e50a45d570e3653f443b34d7d5e40b3
    26. 26
      Marchand, A.; Gabelica, V. Folding and misfolding pathways of G-quadruplex DNA. Nucleic Acids Res. 2016, 44 (22), 10999– 11012,  DOI: 10.1093/nar/gkw970
      Google Scholar
      26
      Folding and misfolding pathways of G-quadruplex DNA
      Marchand, Adrien; Gabelica, Valerie
      Nucleic Acids Research (2016), 44 (22), 10999-11012CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)
      G-quadruplexes adopt various folding topologies, but information on their folding pathways remains scarce. Here, we used electrospray mass spectrometry to detect and quantify the specifically bound potassium ions, and CD to characterize the stacking topol. of each ensemble. For human telomeric (hTel) sequences contg. the d((GGGTTA)3GGG) core, K+ binding affinity and cooperativity strongly depends on the chosen construct. The shortest sequences bind only one K+ at low KCl concn., and this 2-quartet Gquadruplex is antiparallel. Flanking bases increase the K+ binding cooperativity. To decipher the folding pathways, we investigated the kinetics of K+ binding to telomeric (hybrid) and c-myc (parallel) G-quadruplexes. G-quadruplexes fold via branched pathways with multiple parallel reactions. Up to six states (one ensemble without K+, two ensembles with 1-K+ and three ensembles with 2-K+) are sepd. based on their formation rates and ion mobility spectrometry. All G-quadruplexes first form longlived misfolded structures (off-pathway compared to the most stable structures) contg. one K+ and two quartets in an antiparallel stacking arrangement. The results highlight the particular ruggedness of G-quadruplex nucleic acid folding landscapes. Misfolded structures can play important roles for designing artificial G-quadruplex based structures, and for conformational selection by ligands or proteins in a biol. context.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXktlWksbg%253D&md5=4d5b427a02e0a34bc6471ef04fa693ac
    27. 27
      Gray, R. D.; Chaires, J. B. Isothermal Folding of G-quadruplexes. Methods (San Diego, Calif.) 2012, 57 (1), 47– 55,  DOI: 10.1016/j.ymeth.2012.04.006
      Google Scholar
      27
      Isothermal folding of G-quadruplexes
      Gray Robert D; Chaires Jonathan B
      Methods (San Diego, Calif.) (2012), 57 (1), 47-55 ISSN:.
      Thermodynamic studies of G-quadruplex stability are an essential complement to structures obtained by NMR or X-ray crystallography. An understanding of the energetics of quadruplex folding provides a necessary foundation for the physical interpretation of quadruplex formation and reactivity. While thermal denaturation methods are most commonly used to evaluate quadruplex stability, it is also possible to study folding using isothermal titration methods. G-quadruplex folding is tightly coupled to specific cation binding. We describe here protocols for monitoring the cation-driven quadruplex folding transition using circular dichroism or absorbance, and for determination of the distribution of free and bound cation using a fluorescence indicator. Together these approaches provide insight into quadruplex folding at constant temperature, and characterize the linkage between cation binding and folding.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC38rnsVakuw%253D%253D&md5=5accd4d84b3cc305d14a0d33de37370b
    28. 28
      Nishio, M.; Tsukakoshi, K.; Ikebukuro, K. G-quadruplex: Flexible conformational changes by cations, pH, crowding and its applications to biosensing. Biosens. Bioelectron. 2021, 178, 113030,  DOI: 10.1016/j.bios.2021.113030
      Google Scholar
      28
      G-quadruplex: Flexible conformational changes by cations, pH, crowding and its applications to biosensing
      Nishio, Maui; Tsukakoshi, Kaori; Ikebukuro, Kazunori
      Biosensors & Bioelectronics (2021), 178 (), 113030CODEN: BBIOE4; ISSN:0956-5663. (Elsevier B.V.)
      A review. G-quadruplex (G4) is a non-canonical structure that is formed in G-rich sequences of nucleic acids. G4s play important roles in vivo, such as telomere maintenance, transcription, and DNA replication. There are three typical topologies of G4: parallel, anti-parallel, and hybrid. In general, metal cations, such as potassium and sodium, stabilize G4s through coordination in the G-quartet. While G4s have some functions in vivo, there are many reports of developed applications that use G4s. As various conformations of G4s could form from one sequence depending on varying conditions, many researchers have developed G4-based sensors. Furthermore, G4 is a great scaffold of aptamers since many aptamers folded into G4s have also been reported. However, there are some challenges about its practical use due to the difference between practical sample conditions and exptl. ones. G4 conformations are dramatically altered by the surrounding conditions, such as metal cations, pH, and crowding. Many studies have been conducted to characterize G4 conformations under various conditions, not only to use G4s in practical applications but also to reveal its function in vivo. In this review, we summarize recent studies that have investigated the effects of surrounding conditions (e.g., metal cations, pH, and crowding) on G4 conformations and the application of G4s mainly in biosensor fields, and in others.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXis1OhurY%253D&md5=defd5d3aa7abb1cd5a1aa98a3ff5a49d
    29. 29
      Williamson, J. R.; Raghuraman, M. K.; Cech, T. R. Monovalent cation-induced structure of telomeric DNA: the G-quartet model. Cell 1989, 59 (5), 871– 880,  DOI: 10.1016/0092-8674(89)90610-7
      Google Scholar
      29
      Monovalent cation-induced structure of telomeric DNA: the G-quartet model
      Williamson, James R.; Raghuraman, M. K.; Cech, Thomas R.
      Cell (Cambridge, MA, United States) (1989), 59 (5), 871-80CODEN: CELLB5; ISSN:0092-8674.
      Structures formed by oligonucleotides composed of 2 or 4 repeats of the telomeric sequences from Oxytricha and Tetrahymena were investigated. The Oxytricha 4-repeat mol. [d(T4G4)4 = Oxy-4] forms structures with increased electrophoretic mobility in nondenaturing gels contg. Na+, K+, or Cs+, but not in gels contg. Li+ or no added salt. Formation of the folded structure results in protection of a set of dG's from methylation by di-Me sulfate. Efficient UV-induced crosslinks are obsd. in Oxy-4 and the related sequence from Tetrahymena [d(T2G4)4 = Tet-4], and join thymidines in different repeats. Models proposed to account for these data involve G-quartets, H-bonded structures formed from 4 guanosines in a square-planar array. It is proposed that the G-quartet structure must be dealt with in vivo by the telomere replication machinery.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3cXhtFCgsLo%253D&md5=6a86541fc934bcd72b517b75e2dda57b
    30. 30
      Takenaka, S.; Juskowiak, B. Fluorescence Detection of Potassium Ion Using the G-Quadruplex Structure. Anal. Sci. 2011, 27 (12), 1167– 1167,  DOI: 10.2116/analsci.27.1167
      Google Scholar
      30
      Fluorescence detection of potassium ion using the G-quadruplex structure
      Takenaka, Shigeori; Juskowiak, Bernard
      Analytical Sciences (2011), 27 (12), 1167-1172CODEN: ANSCEN; ISSN:0910-6340. (Japan Society for Analytical Chemistry)
      A review. Oligonucleotides with sequences of human telomere DNA or thrombin binding aptamer (TBA) are known to form tetraplex structures upon binding the K+ ion. Structural changes assocd. with the formation of tetraplex assemblies led to the development of potassium-sensing oligonucleotide (PSO) probes, in which two fluorescent dyes were attached to both termini of particular oligonucleotide. The combination of dyes included fluorescence resonance energy transfer (FRET) and excimer emission approaches, and the structural changes upon binding K+ ion could be monitored by a fluorescence technique. These systems showed a very high preference for K+ over Na+ ion, which was suitable for fluorescence imaging of the potassium concn. gradient in a living cell. In the case of human telomere DNA, it was also possible to follow the polymorphism of its tetraplex structures.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xktleiug%253D%253D&md5=d1707097c61dcc0a2c105d30bc73094a
    31. 31
      Juskowiak, B. Analytical potential of the quadruplex DNA-based FRET probes. Anal. Chim. Acta 2006, 568 (1), 171– 180,  DOI: 10.1016/j.aca.2005.12.063
      Google Scholar
      31
      Analytical potential of the quadruplex DNA-based FRET probes
      Juskowiak, Bernard
      Analytica Chimica Acta (2006), 568 (1-2), 171-180CODEN: ACACAM; ISSN:0003-2670. (Elsevier B.V.)
      A review. DNA exhibits structural flexibility and may adopt also tetraplex structures known as guanine-quadruplexes or G-quadruplexes. These G-quadruplexes have recently received great attention because G-rich sequences are often found in genome and because of their potential links to mechanisms that relate to cancer, HIV, and other diseases. The unique structure of quadruplexes has also stimulated development of new anal. and bioanal. assays based on fluorescence resonance energy transfer (FRET). Intramol. folding of a flexible single-stranded DNA mol. into a compact G-quadruplex is a structural transition leading to closer proximity of its 5'- and 3'-ends. Thus, labeling both ends of a DNA strand with donor and acceptor fluorophores enables monitoring the quadruplex formation process by the FRET signal. This review shows how FRET technique contributes to G-quadruplex research and focuses mainly on anal. applications of FRET-labeled quadruplexes. Applications include studies of structural transitions of quadruplexes, FRET-based selection of ligands that bind to quadruplexes, design of mol. probes for protein recognition and development of sensors for detection of potassium ions in aq. soln.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XltVWhu7w%253D&md5=557fa9f26f2aa320cceeccd6a2e22e59
    32. 32
      Ueyama, H.; Takagi, M.; Takenaka, S. A Novel Potassium Sensing in Aqueous Media with a Synthetic Oligonucleotide Derivative. Fluorescence Resonance Energy Transfer Associated with Guanine Quartet-Potassium Ion Complex Formation. J. Am. Chem. Soc. 2002, 124 (48), 14286– 14287,  DOI: 10.1021/ja026892f
      Google Scholar
      32
      A novel potassium sensing in aqueous media with a synthetic oligonucleotide derivative. fluorescence resonance energy transfer associated with guanine quartet-potassium ion complex formation
      Ueyama, Hiroyuki; Takagi, Makoto; Takenaka, Shigeori
      Journal of the American Chemical Society (2002), 124 (48), 14286-14287CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      A novel potassium sensing oligonucleotide (PSO) was constructed by attaching fluorophores 6-FAM and 6-TAMRA to the 5'- and 3'-termini of d(GGG TTA GGG TTA GGG TTA GGG), resp. The affinity of PSO for K+ was 43 000 times greater than that for Na+, high enough selectivity enabling quantitation of K+ specifically in the presence of excess Na+. Fluorescence resonance energy transfer (FRET) to 6-TAMRA from 6-FAM of PSO was obsd. only in the presence of K+. This phenomenon is based on the approxn. of the two fluorophores upon formation of a guanine quartet mediated by K+. Furthermore, the fluorescent color of PSO changes from yellow to red upon formation of the complex, thereby enabling visualization of K+ in aq. media.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XosVCgtLc%253D&md5=7536c6e47b1d3385b1ca0155901ba556
    33. 33
      Xu, J.; Jiang, R.; He, H.; Ma, C.; Tang, Z. Recent advances on G-quadruplex for biosensing, bioimaging and cancer therapy. TrAC Trends in Analytical Chemistry 2021, 139, 116257,  DOI: 10.1016/j.trac.2021.116257
      Google Scholar
      33
      Recent advances on G-quadruplex for biosensing, bioimaging and cancer therapy
      Xu, Jiaqi; Jiang, Rundong; He, Hailun; Ma, Changbei; Tang, Zhenwei
      TrAC, Trends in Analytical Chemistry (2021), 139 (), 116257CODEN: TTAEDJ; ISSN:0165-9936. (Elsevier B.V.)
      A review. G-quadruplex is a three-dimensional secondary structure of nucleic acids formed by the Hoogsteen hydrogen pairing of four guanines. Diverse topologies of G-quadruplex could be employed in biosensing and bioimaging. By intercalating fluorescence dyes into G-quadruplex or forming a horseradish peroxidase (HRP)-mimicking G-quadruplex/hemin DNAzyme, G-quadruplexes based biosensors realized the sensitive and selective detection of nucleic acids, protein, enzyme activity, ions, small mols., exosomes, cells, and microorganisms. The vital role that cellular G-quadruplexes played in genome further facilitated the application of G-quadruplex stabilizing on cancer therapy. Combined with G-quadruplex aptamer, which is an efficient therapeutic tool, a current landscape of the application potential of this fascinate nucleic acids structure from clin. diagnosis to cancer therapy is summarized here.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXntl2qtrs%253D&md5=2a00a67f6c666fe9646e6cb054636210
    34. 34
      Massey, M.; Algar, W. R.; Krull, U. J. Fluorescence resonance energy transfer (FRET) for DNA biosensors: FRET pairs and Förster distances for various dye-DNA conjugates. Anal. Chim. Acta 2006, 568 (1), 181– 189,  DOI: 10.1016/j.aca.2005.12.050
      Google Scholar
      34
      Fluorescence resonance energy transfer (FRET) for DNA biosensors: FRET pairs and Foerster distances for various dye-DNA conjugates
      Massey, Melissa; Algar, W. Russ; Krull, Ulrich J.
      Analytica Chimica Acta (2006), 568 (1-2), 181-189CODEN: ACACAM; ISSN:0003-2670. (Elsevier B.V.)
      Fluorescence resonance energy transfer (FRET) between the extrinsic dye labels Cyanine 3 (Cy3), Cyanine 5 (Cy5), Carboxytetramethyl Rhodamine (TAMRA), Iowa Black Fluorescence Quencher (IabFQ), and Iowa Black RQ (IabRQ) has been studied. The Foerster distances for these FRET-pairs in single- and double-stranded DNA conjugates have been detd. In particular, it should be noted that the quantum yield of the donors Cy3 and TAMRA varies between single- and double-stranded DNA. While this alters the Foerster distance for a donor-acceptor pair, this also allows for detection of thermal denaturation events with a single non-intercalating fluorophore. The utility of FRET in the development of nucleic acid biosensor technol. is illustrated by using TAMRA and IabRQ as a FRET pair in selectivity expts. The differential quenching of TAMRA fluorescence by IabRQ in soln. has been used to discriminate between 0 and 3 base pair mismatches at 60 °C for a 19 base sequence. At room temp., the quenching of TAMRA fluorescence was not an effective indicator of the degree of base pair mismatch. There appears to be a threshold of duplex stability at room temp. which occurs beyond two base pair mismatches and reverses the obsd. trend in TAMRA fluorescence prior to that degree of mismatch. When this exptl. system is transferred to a glass surface through covalent coupling and organosilane chem., the obsd. trend in TAMRA fluorescence at room temp. is similar to that obtained in bulk soln., but without a threshold of duplex stability. In addn. to quenching of fluorescence by FRET, it is believed that several other quenching mechanisms are occurring at the surface.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XltVWhu7s%253D&md5=a1a363321bb23faa6a649a09541dbbb2
    35. 35
      Bianchi, F.; Comez, L.; Biehl, R.; D’Amico, F.; Gessini, A.; Longo, M.; Masciovecchio, C.; Petrillo, C.; Radulescu, A.; Rossi, B.; Sacchetti, F.; Sebastiani, F.; Violini, N.; Paciaroni, A. Structure of human telomere G-quadruplex in the presence of a model drug along the thermal unfolding pathway. Nucleic Acids Res. 2018, 46 (22), 11927– 11938,  DOI: 10.1093/nar/gky1092
      Google Scholar
      35
      Structure of human telomere G-quadruplex in the presence of a model drug along the thermal unfolding pathway
      Bianchi, Federico; Comez, Lucia; Biehl, Ralf; D'Amico, Francesco; Gessini, Alessandro; Longo, Marialucia; Masciovecchio, Claudio; Petrillo, Caterina; Radulescu, Aurel; Rossi, Barbara; Sacchetti, Francesco; Sebastiani, Federico; Violini, Nicolo; Paciaroni, Alessandro
      Nucleic Acids Research (2018), 46 (22), 11927-11938CODEN: NARHAD; ISSN:1362-4962. (Oxford University Press)
      A multi-technique approach, combining CD spectroscopy, UV resonance Raman spectroscopy and small angle scattering techniques, has been deployed to elucidate how the structural features of the human telomeric G-quadruplex d[A(GGGTTA)3GGG] (Tel22) change upon thermal unfolding. The system is studied both in the free form and when it is bound to Actinomycin D (ActD), an anticancer ligand with remarkable conformational flexibility. We find that at room temp. binding of Tel22 with ActD involves end-stacking upon the terminal G-tetrad. Structural evidence for drug-driven dimerization of a significant fraction of the G-quadruplexes is provided. When the temp. is raised, both free and bound Tel22 undergo melting through a multi-state process. We show that in the intermediate states of Tel22 the conformational equil. is shifted toward the (3+1) hybrid-type, while a parallel structure is promoted in the complex. The unfolded state of the free Tel22 is consistent with a self-avoiding random-coil conformation, whereas the high-temp. state of the complex is obsd. to assume a quite compact form. Such an unprecedented high-temp. arrangement is caused by the persistent interaction between Tel22 and ActD, which stabilizes compact conformations even in the presence of large thermal structural fluctuations.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXovFWnt7o%253D&md5=a02f956f569cf716f57c4072fbb50053
    36. 36
      Nagatoishi, S.; Nojima, T.; Juskowiak, B.; Takenaka, S. A Pyrene-Labeled G-Quadruplex Oligonucleotide as a Fluorescent Probe for Potassium Ion Detection in Biological Applications. Angew. Chem. 2005, 117 (32), 5195– 5198,  DOI: 10.1002/ange.200501506
      Google Scholar
      There is no corresponding record for this reference.
    37. 37
      Zhang, A. Y.; Balasubramanian, S. The kinetics and folding pathways of intramolecular G-quadruplex nucleic acids. J. Am. Chem. Soc. 2012, 134 (46), 19297– 19308,  DOI: 10.1021/ja309851t
      Google Scholar
      37
      The Kinetics and Folding Pathways of Intramolecular G-Quadruplex Nucleic Acids
      Zhang, Amy Y. Q.; Balasubramanian, Shankar
      Journal of the American Chemical Society (2012), 134 (46), 19297-19308CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      The folding kinetics of G-quadruplex forming sequences is crit. to their capacity to influence biol. function. While G-quadruplex structure and stability have been relatively well studied, little is known about the kinetics of their folding. We employed a stopped-flow mixing technique to systematically investigate the potassium-dependent folding kinetics of telomeric RNA and DNA G-quadruplexes and RNA G-quadruplexes contg. only two G-quartets formed from sequences r[(GGA)3GG] and r[(GGUUA)3GG]. Our findings suggest a folding mechanism that involves two kinetic steps with initial binding of a single K+, irresp. of the no. of G-quartets involved or whether the G-quadruplex is formed from RNA or DNA. The folding rates for telomeric RNA and DNA G-quadruplexes are comparable at near physiol. [K+] (90 mM) (τ = ∼60 ms). The folding of a 2-quartet RNA G-quadruplex with single nucleotide A loops is considerably slower (τ = ∼700 ms), and we found that the time required to fold a UUA looped variant (τ > 100 s, 500 mM K+) exceeds the lifetimes of some regulatory RNAs. We discuss the implications of these findings with respect to the fundamental properties of G-quadruplexes and their potential functions in biol.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhsF2qt7%252FP&md5=de67961e4c6dbba5edbbe43d7f0a4222
    38. 38
      Deshpande, S.; Wunnava, S.; Hueting, D.; Dekker, C. Membrane Tension-Mediated Growth of Liposomes. Small 2019, 15 (38), 1902898,  DOI: 10.1002/smll.201902898
      Google Scholar
      There is no corresponding record for this reference.
    39. 39
      Bhatia, T.; Christ, S.; Steinkühler, J.; Dimova, R.; Lipowsky, R. Simple sugars shape giant vesicles into multispheres with many membrane necks. Soft Matter 2020, 16 (5), 1246– 1258,  DOI: 10.1039/C9SM01890E
      Google Scholar
      39
      Simple sugars shape giant vesicles into multispheres with many membrane necks
      Bhatia, Tripta; Christ, Simon; Steinkuehler, Jan; Dimova, Rumiana; Lipowsky, Reinhard
      Soft Matter (2020), 16 (5), 1246-1258CODEN: SMOABF; ISSN:1744-6848. (Royal Society of Chemistry)
      Here, we use giant vesicles to investigate fully hydrated lipid membranes in contact with two sugars, glucose and sucrose. The vesicles were osmotically balanced, with the same total sugar concn. in the interior and exterior aq. solns. However, the two solns. differed in their compn.: the interior soln. contained only sucrose whereas the exterior one contained primarily glucose. This sugar asymmetry generated a striking variety of multispherical or "multi-balloon" vesicle shapes. Each multisphere involved only a single membrane that formed several spherical segments, which were connected by narrow, hourglass-shaped membrane necks. These morphologies revealed that the sugar-lipid interactions generated a significant spontaneous curvature with a magnitude of about 1μm-1. Such a spontaneous curvature can be generated both by depletion and by adsorption layers of the sugar mols. arising from effectively repulsive and attractive sugar-lipid interactions. All multispherical shapes are stable over a wide range of parameters, with a substantial overlap between the different stability regimes, reflecting the rugged free energy landscape in shape space. One challenge for future studies is to identify pathways within this landscape that allow us to open and close the membrane necks of these shapes in a controlled and reliable manner. We will then be able to apply these multispheres as metamorphic chambers for chem. reactions and nanoparticle growth.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXitl2gtbrI&md5=5837474231ea5181efec3751e7b46eb2
    40. 40
      Goldman, D. E. Potential, Impedance, and Rectification in membranes. J. Gen. Physiol. 1943, 27 (1), 37– 60,  DOI: 10.1085/jgp.27.1.37
      Google Scholar
      40
      Potential, impedance and rectification in membranes
      Goldman, D. E.
      Journal of General Physiology (1943), 27 (), 37-60CODEN: JGPLAD; ISSN:0022-1295.
      Membranes of collodion, collodion-lecithin, collodion-cephalin, onion cuticle and proteins behave as parallel resistance-capacity combinations when sepg. solns. of electrolytes. The capacities of the above membranes vary slightly with the concn. and nature of the solns. and have phase angles 88-89°, 79-82°, 84-86°, 83-85°, 65-75°, resp. The conductances Λ are approx. proportional to those of the solns. but are much smaller and, like the dielec. consts., are greater than those of the membrane material in bulk. In general Λ varies with current but the capacity is independent of current. Approx. values for the membrane potentials V indicate a linear proportionality with the log of the ratio of the concns. of electrolyte on both sides of the membrane. The amount of rectification produced by the membranes increases with V rather than with the concn. ratio of Λ. By considering the presence of fixed as well as mobile ions, the Planck derivation of liquid-junction potential is extended to membranes, and expressions are obtained for V and for the variation of Λ with current. The calcd. values show qual. agreement with expt.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaH2cXjvVCktw%253D%253D&md5=3d4389a1d16f15567438999f4b5013be
    41. 41
      Gutknecht, J.; Tosteson, D. C. Ionic Permeability of Thin Lipid Membranes: Effects of n-alkyl alcohols, polyvalent cations, and a secondary amine. J. Gen. Physiol. 1970, 55 (3), 359– 374,  DOI: 10.1085/jgp.55.3.359
      Google Scholar
      41
      Ionic permeability of thin lipid membranes. Effects of n-alkyl alcohols, polyvalent cations, and a secondary amine
      Gutknecht, John; Tosteson, D. C.
      Journal of General Physiology (1970), 55 (3), 359-74CODEN: JGPLAD; ISSN:0022-1295.
      Ultrathin (black) lipid membranes were made from n-decane solns. of sheep red cell lipids. Membrane resistance (Rm), voltage (Vm), and ionic transference (Ti) were measured. The presence of each n-alkyl alc., EtOH through n-octanol, in the membrane bath soln. reduced Rm. The concns. required to reduce Rm from >108 to 106 Ω/cm2 were 3.0, 1.4, 0.30, 0.10, 0.028, 0.0054, and >0.00038M EtOH, PrOH, BuOH, n-pentanol, n-hexanol, n-heptanol, and n-octanol, resp. Heptanol increased the permeability of the membrane to K+, with respect to both Na+ and Cl-. The transference nos., Tcation/TCl, increased from 6 to 21; and TK/TNa, from 3 to 21. Other alcs. behaved similarly. Plots of log Rm vs. log alc. concn. were linear over the range of max. change of Rm. The slopes were -3 and -5 for C2C7 alcs. The presence of Th+4 or Fe+3 (10-4M), or of Amberlite LA-2 in the lipid soln. from which the membranes were made, changed the membrane permselectivity from cationic to anionic. When membranes contg. the secondary amine (Amberlite LA-2) were exposed to heptanol, the Rm dropped to 102-105 ohms/cm2, and the membranes became anion selective. Cl diffusion potentials up to 150 millivolts were developed.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaE3cXktVSit7w%253D&md5=f02eb58e4b3a73048b81f74bf2ca33cd
    42. 42
      Koyanagi, T.; Leriche, G.; Yep, A.; Onofrei, D.; Holland, G. P.; Mayer, M.; Yang, J. Effect of Headgroups on Small-Ion Permeability across Archaea-Inspired Tetraether Lipid Membranes. Chem. - Eur. J. 2016, 22 (24), 8074– 8077,  DOI: 10.1002/chem.201601326
      Google Scholar
      42
      Effect of Headgroups on Small-Ion Permeability across Archaea-Inspired Tetraether Lipid Membranes
      Koyanagi, Takaoki; Leriche, Geoffray; Yep, Alvin; Onofrei, David; Holland, Gregory P.; Mayer, Michael; Yang, Jerry
      Chemistry - A European Journal (2016), 22 (24), 8074-8077CODEN: CEUJED; ISSN:0947-6539. (Wiley-VCH Verlag GmbH & Co. KGaA)
      This paper examines the effects of four different polar headgroups on small-ion membrane permeability from liposomes comprised of Archaea-inspired glycerolmonoalkyl glycerol tetraether (GMGT) lipids. We found that the membrane-leakage rate across GMGT lipid membranes varied by a factor of ≤1.6 as a function of headgroup structure. However, the leakage rates of small ions across membranes comprised of com. bilayer-forming 1-palmitoyl-2-oleoyl-sn-glycerol (PO) lipids varied by as much as 32-fold within the same series of headgroups. These results demonstrate that membrane leakage from GMGT lipids is less influenced by headgroup structure, making it possible to tailor the structure of the polar headgroups on GMGT lipids while retaining predictable leakage properties of membranes comprised of these tethered lipids.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XntFGju7c%253D&md5=46df565a8b20019fc696e91038d78751
    43. 43
      Gouaux, E.; Mackinnon, R. Principles of selective ion transport in channels and pumps. Science (New York, N.Y.) 2005, 310 (5753), 1461– 1465,  DOI: 10.1126/science.1113666
      Google Scholar
      43
      Principles of selective ion transport in channels and pumps
      Gouaux Eric; Mackinnon Roderick
      Science (New York, N.Y.) (2005), 310 (5753), 1461-5 ISSN:.
      The transport of ions across the membranes of cells and organelles is a prerequisite for many of life's processes. Transport often involves very precise selectivity for specific ions. Recently, atomic-resolution structures have been determined for channels or pumps that are selective for sodium, potassium, calcium, and chloride: four of the most abundant ions in biology. From these structures we can begin to understand the principles of selective ion transport in terms of the architecture and detailed chemistry of the ion conduction pathways.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BD2MnktFKmsg%253D%253D&md5=822e72453bdb78e41771d74706b1aa0f
    44. 44
      Finkelstein, A.; Andersen, O. S. The gramicidin a channel: A review of its permeability characteristics with special reference to the single-file aspect of transport. J. Membr. Biol. 1981, 59 (3), 155– 171,  DOI: 10.1007/BF01875422
      Google Scholar
      44
      The gramicidin A channel: a review of its permeability characteristics with special reference to the single-file aspect of transport
      Finkelstein, Alan; Andersen, Olaf Sparre
      Journal of Membrane Biology (1981), 59 (3), 155-71CODEN: JMBBBO; ISSN:0022-2631.
      A review with 79 refs.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL3MXktVWqtbg%253D&md5=2bb28f303eefa4c9d36bd1f14e7265fb
    45. 45
      Im, W.; Roux, B. t. Ion Permeation and Selectivity of OmpF Porin: A Theoretical Study Based on Molecular Dynamics, Brownian Dynamics, and Continuum Electrodiffusion Theory. J. Mol. Biol. 2002, 322 (4), 851– 869,  DOI: 10.1016/S0022-2836(02)00778-7
      Google Scholar
      45
      Ion Permeation and Selectivity of OmpF Porin: A Theoretical Study Based on Molecular Dynamics, Brownian Dynamics, and Continuum Electrodiffusion Theory
      Im, Wonpil; Roux, Benoit
      Journal of Molecular Biology (2002), 322 (4), 851-869CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Science Ltd.)
      Three different theor. approaches are used and compared to refine our understanding of ion permeation through the channel formed by OmpF porin from Escherichia coli. Those approaches are all-atom mol. dynamics (MD) in which ions, solvent, and lipids are represented explicitly, Brownian dynamics (BD) in which ions are represented explicitly, while solvent and lipids are represented as featureless dielecs., and Poisson-Nernst-Planck (PNP) electrodiffusion theory in which both solvent and local ion concns. are represented as a continuum. First, the ability of the different theor. approaches in reproducing the equil. av. ion d. distribution in OmpF porin bathed by a 1 M KCl sym. salt soln. is examd. Under those conditions the PNP theory is equiv. to the non-linear Poisson-Boltzmann (PB) theory. Anal. shows that all the three approaches are able to capture the important electrostatic interactions between ions and the charge distribution of the channel that govern ion permeation and selectivity in OmpF. The K+ and Cl- d. distributions obtained from the three approaches are very consistent with one another, which suggests that a treatment on the basis of a rigid protein and continuum dielec. solvent is valid in the case of OmpF. Interestingly, both BD and continuum electrostatics reproduce the distinct left-handed twisted ion pathways for K+ and Cl- extending over the length of the pore which were obsd. previously in MD. Equil. BD simulations in the grand canonical ensemble indicate that the channel is very attractive for cations, particularly at low salt concn. On an av. there is 1.55 K+ inside the pore in 10 mM KCl. Remarkably, there is still 0.17 K+ on av. inside the pore even at a concn. as low as 1 μM KCl. Secondly, non-equil. ion flow through OmpF is calcd. using BD and PNP and compared with exptl. data. The channel conductance in 0.2 M and 1 M KCl calcd. using BD is in excellent accord with the exptl. data. The calcns. reproduce the exptl. well-known conductance-concn. relation and also reveal an asymmetry in the channel conductance (a larger conductance is obsd. under a pos. transmembrane potential). Calcns. of the channel conductance for three mutants (R168A, R132A, and K16A) in 1 M KCl suggest that the asymmetry in the channel conductance arises mostly from the permanent charge distribution of the channel rather than the shape of the pore itself. Lastly, the calcd. reversal potential in a tenfold salt gradient (0.1:1 M KCl) is 27.4(±1.3) mV (BD) and 22.1(±0.6) mV (PNP), in excellent accord with the exptl. value of 24.3 mV. Although most of the results from PNP are qual. reasonable, the calcd. channel conductance is about 50% higher than that calcd. from BD probably because of a lack of some dynamical ion-ion correlations.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XntVGrtrY%253D&md5=5a75370a8388ccf0e69708aa0c1170c4
    46. 46
      Alcaraz, A.; Nestorovich, E. M.; López, M. L.; García-Giménez, E.; Bezrukov, S. M.; Aguilella, V. M. Diffusion, Exclusion, and Specific Binding in a Large Channel: A Study of OmpF Selectivity Inversion. Biophys. J. 2009, 96 (1), 56– 66,  DOI: 10.1016/j.bpj.2008.09.024
      Google Scholar
      46
      Diffusion, exclusion, and specific binding in a large channel: a study of OmpF selectivity inversion
      Alcaraz, Antonio; Nestorovich, Ekaterina M.; Lopez, M. Lidon; Garcia-Gimenez, Elena; Bezrukov, Sergey M.; Aguilella, Vicente M.
      Biophysical Journal (2009), 96 (1), 56-66CODEN: BIOJAU; ISSN:0006-3495. (Cell Press)
      We find that moderate cationic selectivity of the general bacterial porin OmpF in sodium and potassium chloride solns. is inversed to anionic selectivity in concd. solns. of barium, calcium, nickel, and magnesium chlorides. To understand the origin of this phenomenon, we consider several factors, which include the binding of divalent cations, electrostatic and steric exclusion of differently charged and differently sized ions, size-dependent hydrodynamic hindrance, electrokinetic effects, and significant "anionic" diffusion potential for bulk solns. of chlorides of divalent cations. Though all these factors contribute to the measured selectivity of this large channel, the obsd. selectivity inversion is mostly due to the following two. First, binding divalent cations compensates, or even slightly overcompensates, for the neg. charge of the OmpF protein, which is known to be the main cause of cationic selectivity in sodium and potassium chloride solns. Second, the higher anionic (vs. cationic) transport rate expected for bulk solns. of chloride salts of divalent cations is the leading cause of the measured anionic selectivity of the channel. Interestingly, at high concns. the binding of cations does not show any pronounced specificity within the divalent series because the reversal potentials measured in the series correlate well with the corresponding bulk diffusion potentials. Thus our study shows that, in contrast to the highly selective channels of neurophysiol. that employ mostly the exclusion mechanism, quite different factors account for the selectivity of large channels. The elucidation of these factors is essential for understanding large channel selectivity and its regulation in vivo.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXptF2lug%253D%253D&md5=0202c5bf910fe2fb61e64d8babe4d599
    47. 47
      Alcaraz, A.; Nestorovich, E. M.; Aguilella-Arzo, M.; Aguilella, V. M.; Bezrukov, S. M. Salting Out the Ionic Selectivity of a Wide Channel: The Asymmetry of OmpF. Biophys. J. 2004, 87 (2), 943– 957,  DOI: 10.1529/biophysj.104/043414
      Google Scholar
      47
      Salting out the ionic selectivity of a wide channel: The asymmetry of OmpF
      Alcaraz, Antonio; Nestorovich, Ekaterina M.; Aguilella-Arzo, Marcel; Aguilella, Vicente M.; Bezrukov, Sergey M.
      Biophysical Journal (2004), 87 (2), 943-957CODEN: BIOJAU; ISSN:0006-3495. (Biophysical Society)
      Although the crystallog. structure of the bacterial porin OmpF has been known for a decade, the phys. mechanisms of its ionic selectivity are still under investigation. We address this issue in a series of expts. with varied pH, salt concns., inverted salt gradient, and charged and uncharged lipids. Measuring reversal potential, we show that OmpF selectivity (traditionally regarded as slightly cationic) depends strongly on pH and salt concn. and is conditionally asym., i.e., the calcd. selectivity is sensitive to the direction of salt concn. gradient. At neutral pH and subdecimolar salt concns. the channel exhibits nearly ideal cation selectivity (tG+ = 0.98±0.01). Substituting neutral DPhPC with DPhPS, we demonstrate that the fixed charge of the host lipid has a small but measurable effect on the channel reversal potential. The available structural information allows for a qual. explanation of our exptl. findings. These findings now lead us to re-examine the ionization state of 102 titratable sites of the OmpF channel. Using std. methods of continuum electrostatics tailored to our particular purpose, we find the charge distribution in the channel as a function of soln. acidity and relate the pH-dependent asymmetry in channel selectivity to the pH-dependent asymmetry in charge distribution. In an attempt to find a simple phenomenol. description of our results, we also discuss different macroscopic models of electrodiffusion through large channels.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXmvFeitLo%253D&md5=b52955043e257c041a7fe00e63c37762
    48. 48
      Jarczewska, M.; Górski, Ł.; Malinowska, E. Application of DNA aptamers as sensing layers for electrochemical detection of potassium ions. Sens. Actuators, B 2016, 226, 37– 43,  DOI: 10.1016/j.snb.2015.11.139
      Google Scholar
      48
      Application of DNA aptamers as sensing layers for electrochemical detection of potassium ions
      Jarczewska, Marta; Gorski, Lukasz; Malinowska, Elzbieta
      Sensors and Actuators, B: Chemical (2016), 226 (), 37-43CODEN: SABCEB; ISSN:0925-4005. (Elsevier B.V.)
      Detn. of potassium level in organism is of special importance due to its vital role in the maintenance of physiol. activities in organism. Herein, an electrochem. sensor for K+ ions was developed with recognition layer based on DNA aptamers. The elaborated assay was formed by tethering of thiolated aptamer probes to gold disk electrode via Au-S bond. Potassium concn. was detd. with the use of voltammetric techniques by comparison of current change of redox indicators-methylene blue (MB), AQMS and Na4Fe(CN)6. The studies revealed that among aptamer sequences, which were previously shown to bind with potassium ions, 15-mer thrombin binding aptamer (TBA) exhibited highest affinity towards potassium with Kd = 1.13 μmol L-1 when used as a sensing layer. The aptasensor demonstrated linear response within the range from 10-8 to 10-5 mol L-1 with a LOD of 2.31 × 10-9 mol L-1 and good selectivity towards K+ ions with the use of MB electroactive marker.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXitVSqtLbK&md5=b563f0528218b961dc2c52510b0a68fb
    49. 49
      Girard, P.; Pécréaux, J.; Lenoir, G.; Falson, P.; Rigaud, J.-L.; Bassereau, P. A New Method for the Reconstitution of Membrane Proteins into Giant Unilamellar Vesicles. Biophys. J. 2004, 87 (1), 419– 429,  DOI: 10.1529/biophysj.104.040360
      Google Scholar
      49
      A new method for the reconstitution of membrane proteins into giant unilamellar vesicles
      Girard, Philippe; Pecreaux, Jacques; Lenoir, Guillaume; Falson, Pierre; Rigaud, Jean-Louis; Bassereau, Patricia
      Biophysical Journal (2004), 87 (1), 419-429CODEN: BIOJAU; ISSN:0006-3495. (Biophysical Society)
      In this work, we have investigated a new and general method for the reconstitution of membrane proteins into giant unilamellar vesicles (GUVs). We have analyzed systematically the reconstitution of two radically different membrane proteins, the sarcoplasmic reticulum Ca2+-ATPase and the H+ pump bacteriorhodopsin. In a first step, our method involved a detergent-mediated reconstitution of solubilized membrane proteins into proteoliposomes of 0.1-0.2 μm in size. In a second step, these preformed proteoliposomes were partially dried under controlled humidity followed, in a third step, by electroswelling of the partially dried film to give GUVs. The phys. characteristics of GUVs were analyzed in terms of morphol., size, and lamellarity using phase-contrast and differential interference contrast microscopy. The reconstitution process was further characterized by analyzing protein incorporation and biol. activity. Both membrane proteins could be homogeneously incorporated into GUVs at lipid/protein ratios ranging from 5 to 40 (wt./wt.). After reconstitution, both proteins retained their biol. activity as demonstrated by H+ or Ca2+ pumping driven by bacteriorhodopsin or Ca2+-ATPase, resp. This constitutes an efficient new method of reconstitution, leading to the prodn. of large unilamellar membrane protein-contg. vesicles of more than 20 μm in diam., which should prove useful for functional and structural studies through the use of optical microscopy, optical tweezers, microelectrodes, or at. force microscopy.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXmtVels78%253D&md5=0bdd95ffe3a3b4c3686220ec4bd00072
    50. 50
      Pomès, R.; Roux, B. Structure and dynamics of a proton wire: a theoretical study of H+ translocation along the single-file water chain in the gramicidin A channel. Biophys. J. 1996, 71 (1), 19– 39,  DOI: 10.1016/S0006-3495(96)79211-1
      Google Scholar
      50
      Structure and dynamics of a proton wire: a theoretical study of H+ translocation along the single-file water chain in the gramicidin A channel
      Pomes, Regis; Roux, Benoit
      Biophysical Journal (1996), 71 (1), 19-39CODEN: BIOJAU; ISSN:0006-3495. (Biophysical Society)
      The rapid translocation of H+ along a chain of hydrogen-bonded water mols., or proton wire, is thought to be an important mechanism for proton permeation through transmembrane channels. Computer simulations are used to study the properties of the proton wire formed by the single-file waters in the gramicidin A channel. The model includes the polypeptidic dimer, with 22 water mols. and one excess proton. The dissocn. of the water mols. is taken into account by the "polarization model" of Stillinger and co-workers. The importance of quantum effects due to the light mass of hydrogen nuclei is examd. with the use of discretized Feynman path integral mol. dynamics simulations. Results show that the presence of an excess proton in the pore orients the single-file water mols. and affects the geometry of water-water hydrogen bonding interactions. Rather than a well-defined hydronium ion OH3+ in the single-file region, the protonated species is characterized by a strong hydrogen bond resembling that of O2H5+. The quantum dispersion of protons has a small but significant effect on the equil. structure of the hydrogen-bonded water chain. During classical trajectories, proton transfer between consecutive water mols. is a very fast spontaneous process that takes place in the subpicosecond time scale. The translocation along extended regions of the chain takes place neither via a totally concerted mechanism in which the donor-acceptor pattern would flip over the entire chain in a single step, nor via a succession of incoherent hops between well-defined intermediates. Rather, proton transfer in the wire is a semicollective process that results from the subtle interplay of rapid hydrogen-bond length fluctuations along the water chain. These rapid structural fluctuations of the protonated single file of waters around an av. position and the slow movements of the av. position of the excess proton along the channel axis occur on two very different time scales. Ultimately, it is the slow reorganization of hydrogen bonds between single-file water mols. and channel backbone carbonyl groups that, by affecting the connectivity and the dynamics of the single-file water chain, also limits the translocation of the proton across the pore.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK28XktVOhur4%253D&md5=0e6260837d0058a326d2557633533dc1
    51. 51
      Danelon, C.; Suenaga, A.; Winterhalter, M.; Yamato, I. Molecular origin of the cation selectivity in OmpF porin: single channel conductances vs. free energy calculation. Biophys. Chem. 2003, 104 (3), 591– 603,  DOI: 10.1016/S0301-4622(03)00062-0
      Google Scholar
      51
      Molecular origin of the cation selectivity in OmpF porin: single channel conductances vs. free energy calculation
      Danelon, Christophe; Suenaga, Atsushi; Winterhalter, Mathias; Yamato, Ichiro
      Biophysical Chemistry (2003), 104 (3), 591-603CODEN: BICIAZ; ISSN:0301-4622. (Elsevier Science B.V.)
      Ion current through single outer membrane protein F (OmpF) trimers was recorded and compared to mol. dynamics simulation. Unidirectional insertion was revealed from the asymmetry in channel conductance. Single trimer conductance showed particularly high values at low sym. salt soln. The conductance values of various alkali metal ion solns. were proportional to the monovalent cation mobility values in the bulk phase, LiCl<NaCl<KCl<RbCl∼CsCl, but the conductance differences were quant. larger than cond. differences in bulk solns. Selectivity measurements at low concn. showed that OmpF channels favored permeation of alkali metal ions over chloride and suggested size preference for smaller cations. These results suggest that there are specific interactions between the permeating cation and charged residues lining the channel walls. This hypothesis was supported by computational study which predicted that monovalent cations bind to Asp113 at low concn. Here, free energy calcns. revealed that the affinity of the alkali metal ions to its binding site increased with their at. radii, Li+∼Na+<K+∼Rb+∼Cs+. A detailed inspection of both exptl. and computational results suggested that stronger binding at the central constriction of the channel increases the translocation rate of cations under applied voltage by increasing their local concn. relative to the bulk soln.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXmt1GqtL8%253D&md5=1255164202b43106dbf55c3e9b3be939
    52. 52
      Al Nahas, K.; Cama, J.; Schaich, M.; Hammond, K.; Deshpande, S.; Dekker, C.; Ryadnov, M. G.; Keyser, U. F. A microfluidic platform for the characterisation of membrane active antimicrobials. Lab Chip 2019, 19 (5), 837– 844,  DOI: 10.1039/C8LC00932E
      Google Scholar
      52
      A microfluidic platform for the characterisation of membrane active antimicrobials
      Al Nahas, K.; Cama, J.; Schaich, M.; Hammond, K.; Deshpande, S.; Dekker, C.; Ryadnov, M. G.; Keyser, U. F.
      Lab on a Chip (2019), 19 (5), 837-844CODEN: LCAHAM; ISSN:1473-0189. (Royal Society of Chemistry)
      The spread of bacterial resistance against conventional antibiotics generates a great need for the discovery of novel antimicrobials. Polypeptide antibiotics constitute a promising class of antimicrobial agents that favor attack on bacterial membranes. However, efficient measurement platforms for evaluating their mechanisms of action in a systematic manner are lacking. Here we report an integrated lab-on-a-chip multilayer microfluidic platform to quantify the membranolytic efficacy of such antibiotics. The platform is a biomimetic vesicle-based screening assay, which generates giant unilamellar vesicles (GUVs) in physiol. relevant buffers on demand. Hundreds of these GUVs are individually immobilized downstream in phys. traps connected to sep. perfusion inlets that facilitate controlled antibiotic delivery. Antibiotic efficacy is expressed as a function of the time needed for an encapsulated dye to leak out of the GUVs as a result of antibiotic treatment. This proof-of-principle study probes the dose response of an archetypal polypeptide antibiotic cecropin B on GUVs mimicking bacterial membranes. The results of the study provide a foundation for engineering quant., high-throughput microfluidics devices for screening antibiotics.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXitFGntLg%253D&md5=543122ca19b971fde4c274b26f8ed3a4
    53. 53
      Deshpande, S.; Caspi, Y.; Meijering, A. E. C.; Dekker, C. Octanol-assisted liposome assembly on chip. Nat. Commun. 2016, 7 (1), 10447,  DOI: 10.1038/ncomms10447
      Google Scholar
      53
      Octanol-assisted liposome assembly on chip
      Deshpande, Siddharth; Caspi, Yaron; Meijering, Anna E. C.; Dekker, Cees
      Nature Communications (2016), 7 (), 10447CODEN: NCAOBW; ISSN:2041-1723. (Nature Publishing Group)
      Liposomes are versatile supramol. assemblies widely used in basic and applied sciences. Here we present a novel microfluidics-based method, octanol-assisted liposome assembly (OLA), to form monodisperse, cell-sized (5-20μm), unilamellar liposomes with excellent encapsulation efficiency. Akin to bubble blowing, an inner aq. phase and a surrounding lipid-carrying 1-octanol phase is pinched off by outer fluid streams. Such hydrodynamic flow focusing results in double-emulsion droplets that spontaneously develop a side-connected 1-octanol pocket. Owing to interfacial energy minimization, the pocket splits off to yield fully assembled solvent-free liposomes within minutes. This solves the long-standing fundamental problem of prolonged presence of residual oil in the liposome bilayer. We demonstrate the unilamellarity of liposomes with functional α-haemolysin protein pores in the membrane and validate the biocompatibility by inner leaflet localization of bacterial divisome proteins (FtsZ and ZipA). OLA offers a versatile platform for future anal. tools, delivery systems, nanoreactors and synthetic cells.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xht1Cku78%253D&md5=355998fa9e3a32acf032da1d5643cd6d
    54. 54
      Tivony, R.; Fletcher, M.; Al Nahas, K.; Keyser, U. F. A Microfluidic Platform for Sequential Assembly and Separation of Synthetic Cell Models. ACS Synth. Biol. 2021, 10 (11), 3105– 3116,  DOI: 10.1021/acssynbio.1c00371
      Google Scholar
      54
      A Microfluidic Platform for Sequential Assembly and Separation of Synthetic Cell Models
      Tivony, Ran; Fletcher, Marcus; Al Nahas, Kareem; Keyser, Ulrich F.
      ACS Synthetic Biology (2021), 10 (11), 3105-3116CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)
      Cell-sized vesicles like giant unilamellar vesicles (GUVs) are established as a promising biomimetic model for studying cellular phenomena in isolation. However, the presence of residual components and byproducts, generated during vesicles prepn. and manipulation, severely limits the utility of GUVs in applications like synthetic cells. Therefore, with the rapidly growing field of synthetic biol., there is an emergent demand for techniques that can continuously purify cell-like vesicles from diverse residues, while GUVs are being simultaneously synthesized and manipulated. We have developed a microfluidic platform capable of purifying GUVs through stream bifurcation, where a vesicles suspension is partitioned into three fractions: purified GUVs, residual components, and a washing soln. Using our purifn. approach, we show that giant vesicles can be sepd. from various residues-which range in size and chem. compn.-with a very high efficiency (e = 0.99), based on size and deformability of the filtered objects. In addn., by incorporating the purifn. module with a microfluidic-based GUV-formation method, octanol-assisted liposome assembly (OLA), we established an integrated prodn.-purifn. microfluidic unit that sequentially produces, manipulates, and purifies GUVs. We demonstrate the applicability of the integrated device to synthetic biol. through sequentially fusing SUVs with freshly prepd. GUVs and sepg. the fused GUVs from extraneous SUVs and oil droplets at the same time.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXisVGrs7%252FK&md5=f744b0dd616e17f260a55c1a4d18175c
    55. 55
      Edelstein, A. D.; Tsuchida, M. A.; Amodaj, N.; Pinkard, H.; Vale, R. D.; Stuurman, N. Advanced methods of microscope control using μManager software. Journal of Biological Methods 2014, 1 (2), e10  DOI: 10.14440/jbm.2014.36
      Google Scholar
      There is no corresponding record for this reference.
    56. 56
      Morzy, D.; Rubio-Sánchez, R.; Joshi, H.; Aksimentiev, A.; Di Michele, L.; Keyser, U. F. Cations Regulate Membrane Attachment and Functionality of DNA Nanostructures. J. Am. Chem. Soc. 2021, 143 (19), 7358– 7367,  DOI: 10.1021/jacs.1c00166
      Google Scholar
      56
      Cations Regulate Membrane Attachment and Functionality of DNA Nanostructures
      Morzy, Diana; Rubio-Sanchez, Roger; Joshi, Himanshu; Aksimentiev, Aleksei; Di Michele, Lorenzo; Keyser, Ulrich F.
      Journal of the American Chemical Society (2021), 143 (19), 7358-7367CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      The interplay between nucleic acids and lipids underpins several key processes in mol. biol., synthetic biotechnol., vaccine technol., and nanomedicine. These interactions are often electrostatic in nature, and much of their rich phenomenol. remains unexplored in view of the chem. diversity of lipids, the heterogeneity of their phases, and the broad range of relevant solvent conditions. Here we unravel the electrostatic interactions between zwitterionic lipid membranes and DNA nanostructures in the presence of physiol. relevant cations, with the purpose of identifying new routes to program DNA-lipid complexation and membrane-active nanodevices. We demonstrate that this interplay is influenced by both the phase of the lipid membranes and the valency of the ions and observe divalent cation bridging between nucleic acids and gel-phase bilayers. Furthermore, even in the presence of hydrophobic modifications on the DNA, we find that cations are still required to enable DNA adhesion to liq.-phase membranes. We show that the latter mechanism can be exploited to control the degree of attachment of cholesterol-modified DNA nanostructures by modifying their overall hydrophobicity and charge. Besides their biol. relevance, the interaction mechanisms we explored hold great practical potential in the design of biomimetic nanodevices, as we show by constructing an ion-regulated DNA-based synthetic enzyme.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXhtVemsbzP&md5=6f2ca7eebbcbd1077f8ab9f8ecb29d34
    57. 57
      Rubio-Sánchez, R.; Barker, S. E.; Walczak, M.; Cicuta, P.; Michele, L. D. A Modular, Dynamic, DNA-Based Platform for Regulating Cargo Distribution and Transport between Lipid Domains. Nano Lett. 2021, 21 (7), 2800– 2808,  DOI: 10.1021/acs.nanolett.0c04867
      Google Scholar
      57
      A Modular, Dynamic, DNA-Based Platform for Regulating Cargo Distribution and Transport between Lipid Domains
      Rubio-Sanchez, Roger; Barker, Simone Eizagirre; Walczak, Michal; Cicuta, Pietro; Michele, Lorenzo Di
      Nano Letters (2021), 21 (7), 2800-2808CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)
      Cell membranes regulate the distribution of biol. machinery between phase-sepd. lipid domains to facilitate key processes including signaling and transport, which are among the life-like functionalities that bottom-up synthetic biol. aims to replicate in artificial-cellular systems. Here, we introduce a modular approach to program partitioning of amphiphilic DNA nanostructures in coexisting lipid domains. Exploiting the tendency of different hydrophobic "anchors" to enrich different phases, we modulate the lateral distribution of our devices by rationally combining hydrophobes and by changing nanostructure size and topol. We demonstrate the functionality of our strategy with a bioinspired DNA architecture, which dynamically undergoes ligand-induced reconfiguration to mediate cargo transport between domains via lateral redistribution. Our findings pave the way to next-generation biomimetic platforms for sensing, transduction, and communication in synthetic cellular systems.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXmsFOksLo%253D&md5=2b3cb391d463bef6c07fb88a99d6dc15
    58. 58
      Beucher, S. The Watershed Transformation Applied to Image Segmentation. Scanning Microscopy 1992, 1992(6), 1
      Google Scholar
      There is no corresponding record for this reference.

  • Supporting Information

    Supporting Information

    ARTICLE SECTIONS
    Jump To

    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsnano.2c07496.

    • GUV formation and filtering, Human Telomere G-quadruplex K+ binding model, Na+ response of FAMQ-G4, sensitivity of FAMQ-G4 to K+ in the presence of Na+, response of FAMQ-G4 to K+ in 100 mM LiCl, FAMQ-HT photobleaching and leakage across the lipid bilayer, folding kinetics of FAMQ-HT following 100 μM KCl, interaction of FAMQ-G4 with the lipid bilayer, change of pH inside GUVs during K+ permeation, measurement of K+ permeation across electroformed GUVs, demonstration of no leakage of FAMQ-G4 from GUVs after perfusion of model ion channels, efficiency of gramicidin A incorporation dependent on its concentration in solution, K+ transport across OmpF containing GUVs, microfluidic device CAD designs, K+ flux density approximately equaling H+ counter flux during the linear regime (PDF)


    Terms & Conditions

    Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.

PDF [ 3MB]

Pair your accounts.

Export articles to Mendeley

Get article recommendations from ACS based on references in your Mendeley library.

Pair your accounts.

Export articles to Mendeley

Get article recommendations from ACS based on references in your Mendeley library.

You’ve supercharged your research process with ACS and Mendeley!

STEP 1:
Click to create an ACS ID

Please note: If you switch to a different device, you may be asked to login again with only your ACS ID.

Please note: If you switch to a different device, you may be asked to login again with only your ACS ID.

Please note: If you switch to a different device, you may be asked to login again with only your ACS ID.

MENDELEY PAIRING EXPIRED
Your Mendeley pairing has expired. Please reconnect