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Macrocycles and dyes used as reporter pairs as well as the analytes investigated. b,c) Working principle of a supramolecular tandem membrane assay. Illustration of macrocyclic host–dye complexes encapsulated inside a liposome before (left) and after (right) translocation of an analyte (blue) through b) a channel protein (green) or c) directly through the biomembrane; the analyte binds to the macrocycle, thereby displaces the dye, which in turn becomes either b) strongly fluorescent in its uncomplexed form (switch-on fluorescence response) or c) weakly fluorescent (switch-off response).
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The efficacy of drugs and biomolecules relies on their ability to pass through the bilayer. The development of methods to directly and sensitively monitor these membrane transport processes has remained an experimental challenge. A macrocyclic host (p-sulfonatocalix[4]arene or cucurbit[7]uril) and a fluorescent dye (lucigenin or berberine) are enca...
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Context 1
... the analyte concentrations resulted in both a faster kinetics and a higher final fluores- cence intensity, which reached a plateau at high protamine concentration (Figure 3 b). This demonstrated that the trans- location rate reached a limiting value and that the displace- ment became quantitative at high analyte concentration (see the Supporting Information and Figure S11). Hill analysis of the initial rates yielded a half-saturation constant (EC 50 ) of 450 nm with a Hill slope of approximately 6. [15] The tandem membrane assays therefore complement the electrophysio- logical measurements, in which the differentiation between analyte binding to the channel or translocation through it, as well as the determination of actual kinetics becomes difficult, in particular for high-molecular-weight analytes and slow translocation rates (see the Supporting Information). ...
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... the method is based on an unselective molecular recognition, it is not limited to protamine, but transferable to other organic analytes (Figure 1 a). For instance, we have unambiguously demonstrated the successful translocation of heptaarginine, a membrane transduction peptide, and of acetylcholine, a low-molecular-weight neurotransmitter, both through OmpF ( Figure S12). ...
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... the method is based on an unselective molecular recognition, it is not limited to protamine, but transferable to other organic analytes (Figure 1 a). For instance, we have unambiguously demonstrated the successful translocation of heptaarginine, a membrane transduction peptide, and of acetylcholine, a low-molecular-weight neurotransmitter, both through OmpF ( Figure S12). Direct permeation (without channel) of analytes can also be conveniently monitored, for example, of amantadine, an anti-Alzheimers drug (Fig- ure S13); this assay was additionally performed in liposomes of different lipid composition and by utilizing an alternative chemosensing ensemble composed of cucurbit [7]uril (CB7) as macrocyclic receptor and berberine (BE) as fluorescent dye (Figure 1 a). ...
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... instance, we have unambiguously demonstrated the successful translocation of heptaarginine, a membrane transduction peptide, and of acetylcholine, a low-molecular-weight neurotransmitter, both through OmpF ( Figure S12). Direct permeation (without channel) of analytes can also be conveniently monitored, for example, of amantadine, an anti-Alzheimers drug (Fig- ure S13); this assay was additionally performed in liposomes of different lipid composition and by utilizing an alternative chemosensing ensemble composed of cucurbit [7]uril (CB7) as macrocyclic receptor and berberine (BE) as fluorescent dye (Figure 1 a). ...
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... instance, we have unambiguously demonstrated the successful translocation of heptaarginine, a membrane transduction peptide, and of acetylcholine, a low-molecular-weight neurotransmitter, both through OmpF ( Figure S12). Direct permeation (without channel) of analytes can also be conveniently monitored, for example, of amantadine, an anti-Alzheimers drug (Fig- ure S13); this assay was additionally performed in liposomes of different lipid composition and by utilizing an alternative chemosensing ensemble composed of cucurbit [7]uril (CB7) as macrocyclic receptor and berberine (BE) as fluorescent dye (Figure 1 a). ...
Citations
... This is because living cells have highly compartmentalized organelles that optimize reactions and activities by occupying independent spaces or locations isolated from the external environment. Another important property of artificial compartments is the ability to control the transport of substances of interest, which is achieved by incorporating membrane proteins, transporters, and channels, such as AqpZ, [14] OmpF, [15] and FhuA [16] for bacteria, into phospholipid membranes. These components allow the transport of molecules and ions of interest, such as substrates and reaction products, while selectively filtering them based on their intrinsic properties. ...
The cellular compartment plays an essential role in organizing the complex and diverse biochemical reactions within the cell. By mimicking the function of such cellular compartments, the challenge of constructing artificial compartments has been taken up to develop new biochemical tools for efficient material production and diagnostics. The important features required for the artificial compartment are that it isolates the interior from the external environment and is further functionalized to control the transport of target chemicals to regulate the interior concentration of both substrate and reaction products. In this study, an artificial compartment with size‐selective molecular transport function was constructed by using a DNA origami‐guided liposome prepared by modifying the method reported by Perrault et al. This completely isolates the liposome interior, including the DNA origami skeleton, from the external environment and allows the assembly of a defined number of molecules of interest inside and/or outside the compartment. By incorporating a bacterial membrane protein, OmpF, into the liposome, the resulting artificial compartment was shown to transport only the molecule of interest with a molecular weight below 600 Da from the external environment into the interior of the compartment.
... The first type of sensor array was constructed by changing both the receptors and the corresponding dyes ( Supplementary Fig. 27a). In order to explain the discrimination principle of the sensor array more clearly, we first present a theoretical model based on host-guest recognition 59 . The discriminative ability of this sensor array comes from different binding strengths between the receptors/ dyes and the receptors/analytes. ...
Differential sensing, which discriminates analytes via pattern recognition by sensor arrays, plays an important role in our understanding of many chemical and biological systems. However, it remains challenging to develop new methods to build a sensor unit library without incurring a high workload of synthesis. Herein, we propose a supramolecular approach to construct a sensor unit library by taking full advantage of recognition and assembly. Ten sensor arrays are developed by replacing the building block combinations, adjusting the ratio between system components, and changing the environment. Using proteins as model analytes, we examine the discriminative abilities of these supramolecular sensor arrays. Then the practical applicability for discriminating complex analytes is further demonstrated using honey as an example. This sensor array construction strategy is simple, tunable, and capable of developing many sensor units with as few syntheses as possible. The development of sensor unit libraries that require minimal synthetic effort is challenging. Here, the authors report the preparation of sensor array libraries based on a supramolecular approach that relies on the recognition and assembly of macrocyclic amphiphiles.
... Remarkably, B 12 Br 12 2− transported many types of cargo, ranging from positive to non-charged and zwitterionic molecules, or from small ones, such as acetylcholine (molecular mass of 146 Da), to larger polypeptides, such as protamine (molecular mass of 4,500 Da), with the exception of the negatively charged molecules glutamate and albumin, for which no carrier-cargo charge attenuation can occur (Fig. 2d, Extended Data Fig. 3b and Extended Data Table 1). The very fast transport kinetics were in the range of seconds for most cargo types (Fig. 2f), which becomes competitive with uptake through membrane pores or channels 40 . The insensitivity towards the chemical nature of the diverse functional groups in the transported cargos (Extended Data Fig. 9) confirmed that the carrier activity of the boron clusters is not limited to residues that entertain salt bridges or specific intermolecular interactions, Article and is much less restrictive than for amphiphilic activators. ...
The membrane translocation of hydrophilic substances constitutes a challenge for their application as therapeutic compounds and labelling probes1–4. To remedy this, charged amphiphilic molecules have been classically used as carriers3,5. However, such amphiphilic carriers may cause aggregation and non-specific membrane lysis6,7. Here we show that globular dodecaborate clusters, and prominently B12Br122−, can function as anionic inorganic membrane carriers for a broad range of hydrophilic cargo molecules (with molecular mass of 146–4,500 Da). We show that cationic and neutral peptides, amino acids, neurotransmitters, vitamins, antibiotics and drugs can be carried across liposomal membranes. Mechanistic transport studies reveal that the carrier activity is related to the superchaotropic nature of these cluster anions8–12. We demonstrate that B12Br122− affects cytosolic uptake of different small bioactive molecules, including the antineoplastic monomethyl auristatin F, the proteolysis targeting chimera dBET1 and the phalloidin toxin, which has been successfully delivered in living cells for cytoskeleton labelling. We anticipate the broad and distinct delivery spectrum of our superchaotropic carriers to be the starting point of conceptually distinct cell-biological, neurobiological, physiological and pharmaceutical studies. The superchaotropic nature of globular boron cluster anions enables direct passage of a wide range of molecules across lipid membranes.
... Having looked at stationary properties of diffusion with resetting, we now turn attention to firstpassage properties which have numerous applications. [4][5][6][7][8][9]28,29,[33][34][35][36][37][38][39][40][53][54][55][56][57][58][59][60][61][62][63][64][65] We recall that while the mean first-passage time (MFPT) of a Brownian particle to a stationary target diverges, 28,29 resetting will render it finite, 2 even if returns are non-instantaneous. [5][6][7]20,26,40 To experimentally show this, we consider the setup in Fig. 5a. ...
... Ghale et al. have introduced supramolecular tandem membrane assays, where the complex of the macrocyclic host (p-sulfonatocalix [4]arene or cucurbit [7]uril) and fluorescence dye was encapsulated inside the liposomes having OmpF porins. 249 Further addition of the antimicrobial peptide protamine led to porin-mediated uptake in liposomes, followed by displacement and dequenching of the fluorophores due to a stronger binding to the host. The change in the fluorescence intensity was provided as proof for porinmediated transport of protamine. ...
Despite tremendous successes in the field of antibiotic discovery seen in the previous century, infectious diseases have remained a leading cause of death. More specifically, pathogenic Gram-negative bacteria have become a global threat due to their extraordinary ability to acquire resistance against any clinically available antibiotic, thus urging for the discovery of novel antibacterial agents. One major challenge is to design new antibiotics molecules able to rapidly penetrate Gram-negative bacteria in order to achieve a lethal intracellular drug accumulation. Protein channels in the outer membrane are known to form an entry route for many antibiotics into bacterial cells. Up until today, there has been a lack of simple experimental techniques to measure the antibiotic uptake and the local concentration in subcellular compartments. Hence, rules for translocation directly into the various Gram-negative bacteria via the outer membrane or via channels have remained elusive, hindering the design of new or the improvement of existing antibiotics. In this review, we will discuss the recent progress, both experimentally as well as computationally, in understanding the structure-function relationship of outer-membrane channels of Gram-negative pathogens, mainly focusing on the transport of antibiotics.
... Ghale et al. [127] also used p-sulfonatocalix [4]arene for the detection of arginine-rich peptides, using an indicator displacement method. In this study, this receptor was applied for the detection of several cationic analytes in liposomes (with more focus on protamine, a natural occurring peptide with 21 arginine residues) and to study their transport across membranes, mediated by the membrane protein OmpF. ...
To this day, the recognition and high affinity binding of biomolecules in water by synthetic receptors remains challenging, while the necessity for systems for their sensing, transport and modulation persists. This problematic is prevalent for the recognition of peptides, which not only have key roles in many biochemical pathways, as well as having pharmacological and biotechnological applications, but also frequently serve as models for the study of proteins. Taking inspiration in nature and on the interactions that occur between several receptors and peptide sequences, many researchers have developed and applied a variety of different synthetic receptors, as is the case of macrocyclic compounds, molecular imprinted polymers, organometallic cages, among others, to bind amino acids, small peptides and proteins. In this critical review, we present and discuss selected examples of synthetic receptors for amino acids and peptides, with a greater focus on supramolecular receptors, which show great promise for the selective recognition of these biomolecules in physiological conditions. We decided to focus preferentially on small synthetic receptors (leaving out of this review high molecular weight polymeric systems) for which more detailed and accurate molecular level information regarding the main structural and thermodynamic features of the receptor biomolecule assemblies is available.
... The most studied homologs with n = 6, 7, and 8 are known to bind to various amino acids, peptides, and proteins with affinities in the range of 10 3 -10 9 M −1 , and the molecular recognition properties indicated a preference for hydrophobic and cationic amino acid residues (Urbach and Ramalingam, 2011;Gamal-Eldin and Macartney, 2013;Logsdon and Urbach, 2013;Lee et al., 2015a,b;Smith et al., 2015;Li et al., 2016;Webber et al., 2016;Hirani et al., 2018). In our own work, we have exploited the interactions of cucurbiturils with amino acids, peptides, and proteins for the sensing of enzyme activity, membrane transport, and chirality Bailey et al., 2008;Nau et al., 2009;Dsouza et al., 2012;Biedermann and Nau, 2014;Ghale et al., 2014;Schnurr et al., 2015;Nilam et al., 2017;Barba-Bon et al., 2019;Liu et al., 2019;Biedermann et al., 2020). It was therefore considered to be very likely that CB7 will bind to several sites of the polyfunctional substrate H-(NO 2 )Tyr-Gly-Ser-Gly-Phe-Arg-Gly-Dbo-NH 2 with different affinity, in particular because DBO is also known to strongly bind with CB7 (Marquez et al., 2004). ...
We report the use of the macrocyclic host cucurbit[7]uril (CB7) as a supramolecular additive in nanosecond time-resolved fluorescence (Nano-TRF) assays for proteases to enhance the discrimination of substrates and products and, thereby, the sensitivity. A peptide substrate was labeled with 2,3-diazabicyclo[2.2.2]oct-2-ene (DBO) as a long-lived (>300 ns) fluorescent probe and 3-nitrotyrosine was established as a non-fluorescent fluorescence resonance energy transfer (FRET) acceptor that acts as a “dark quencher.” The substrate was cleaved by the model proteases trypsin and chymotrypsin and the effects of the addition of CB7 to the enzyme assay mixture were investigated in detail using UV/VIS absorption as well as steady-state and time-resolved fluorescence spectroscopy. This also allowed us to identify the DBO and nitrotyrosine residues as preferential binding sites for CB7 and suggested a hairpin conformation of the peptide, in which the guanidinium side chain of an arginine residue is additionally bound to a vacant ureido rim of one of the CB7 hosts.
... For instance, dynamic fluorescence quenching of an encapsulated reporter dye was used for permeation monitoring of redox-active analytes at very high (millimolar) concentrations 18 . We showed that an indicator displacement assay 19 can be adopted to monitor membrane translocation of a label-free highly charged antimicrobial peptide and selected charged amino acid derivatives through membrane pores 20,21 . For this purpose, an environmentresponsive dye is precomplexed with a host to yield a chromophoric or emissive reporter pair. ...
The spatiotemporally resolved monitoring of membrane translocation, e.g., of drugs or toxins, has been a long-standing goal. Herein, we introduce the fluorescent artificial receptor-based membrane assay (FARMA), a facile, label-free method. With FARMA, the permeation of more than hundred organic compounds (drugs, toxins, pesticides, neurotransmitters, peptides, etc.) through vesicular phospholipid bilayer membranes has been monitored in real time (µs-h time scale) and with high sensitivity (nM-µM concentration), affording permeability coefficients across an exceptionally large range from 10–9–10–3 cm s–1. From a fundamental point of view, FARMA constitutes a powerful tool to assess structure-permeability relationships and to test biophysical models for membrane passage. From an applied perspective, FARMA can be extended to high-throughput screening by adaption of the microplate reader format, to spatial monitoring of membrane permeation by microscopy imaging, and to the compartmentalized monitoring of enzymatic activity. Biedermann et al develop a tool to monitor transport across membranes using fluorescent artificial receptors, thereby bypassing the need for analyte labelling. They show the utility of the method for a large number of compounds suggesting it can be widely applied to both basic research and drug screening.
... Having looked at stationary properties of diffusion with resetting, we now turn attention to firstpassage properties which have numerous applications. [4][5][6][7][8][9]28,29,[33][34][35][36][37][38][39][40][53][54][55][56][57][58][59][60][61][62][63][64][65] We recall that while the mean first-passage time (MFPT) of a Brownian particle to a stationary target diverges, 28,29 resetting will render it finite, 2 even if returns are non-instantaneous. [5][6][7]20,26,40 To experimentally show this, we consider the setup in Fig. 5a. ...
Stochastic resetting is prevalent in natural and man-made systems giving rise to a long series of non-equilibrium phenomena. Diffusion with stochastic resetting serves as a paradigmatic model to study these phenomena, but the lack of a well-controlled platform by which this process can be studied experimentally has been a major impediment to research in the field. Here, we report the experimental realization of colloidal particle diffusion and resetting via holographic optical tweezers. This setup serves as a proof-of-concept which opens the door to experimental study of resetting phenomena. It also vividly illustrates why existing theoretical models must be improved and revised to better capture the real-world physics of stochastic resetting.
... Reporter Pair Selection and Assay Design. Supramolecular host/dye reporter pairs were previously used by others and us to assay enzyme activity 37−45 for enantiomeric excess determination and analyte sensing, 46 chirality sensing, 47 and drug delivery, 48 and they were recently introduced to follow translocation through the bacterial transmembrane protein OmpF, 36 in vesicle fusion assays, 49 in the visualization of autophagy processes, 50 and as chemosensors in cells. 51 In the present work, we encapsulate membrane-impermeable macrocyclic host/dye reporter pairs in liposomes (Scheme 2). ...
... The combination of the negatively charged p-sulfonatocalix [4]arene (CX4) and the fluorescent dye lucigenin (LCG) was previously established as a versatile and membrane-impermeable reporter pair for polycationic peptides. 36,44,51 Because most CPPs contain lysine and arginine side chains, the cation-selective CX4/LCG reporter pair was expected to serve as a broadly applicable fluorescence sensor for polycationic CPPs. The second host− dye reporter pair is composed of cucurbit [7]uril (CB7) and berberin (BE) 52 and is expected to selectively respond to Nterminal aromatic amino acids. ...
... 53 A third reporter pair, cucurbit [8]uril (CB8) and N,N-dimethyl-2,7-diazapyrenium (MDAP), was used, which is different from the former two reporter pairs; instead of acting by dye displacement, CB8/ MDAP forms a ternary charge-transfer complex with cavitybound aromatic amino acid side chains 54−57 in which MDAP fluorescence is decreased. 41,58 All three reporter pairs can be encapsulated in stable liposomes to afford CX4/LCG-LUVs, CB7/BE-LUVs, 36 and CB8/MDAP-LUVs 54 (see the Supporting Information). ...
The membrane transport mechanisms of cell-penetrating peptides (CPPs) are still controversial and reliable assays to report on their internalization in model membranes are required. Herein, we introduce a label-free, fluorescence-based method to monitor membrane transport of peptides in real time. For this purpose, a macrocyclic host and a fluorescent dye forming a host-dye reporter pair are encapsulated inside phospholipid vesicles. Internalization of peptides, which can bind to the supramolecular host, leads to displacement of the dye from the host, resulting in a fluorescence change that signals the peptide uptake and, thus, provides unambiguous evidence for their transport through the membrane. The method was successfully validated with various established CPPs, including the elusive peptide TP2, in the presence of counterion activators of CPPs, and with a calixarene-based supramolecular membrane transport system. In addition, transport experiments with encapsulated host-dye reporter pairs are not limited to large unilamellar vesicles (LUVs), but can also be used with giant unilamellar vesicles (GUVs) and fluorescence microscopy imaging.
