RESUMEN
DNA nanotechnology has seen large developments over the last 30 years through the combination of detection and discovery of DNAs, and solid phase synthesis to increase the chemical functionalities on nucleic acids, leading to the emergence of novel and sophisticated in features, nucleic acids-based biopolymers. Arguably, nanopores developed for fast and direct detection of a large variety of molecules, are part of a revolutionary technological evolution which led to cheaper, smaller and considerably easier to use devices enabling DNA detection and sequencing at the single-molecule level. Through their versatility, the nanopore-based tools proved useful biomedicine, nanoscale chemistry, biology and physics, as well as other disciplines spanning materials science to ecology and anthropology. This mini-review discusses the progress of nanopore- and hybridization-based DNA detection, and explores a range of state-of-the-art applications afforded through the combination of certain synthetically-derived polymers mimicking nucleic acids and nanopores, for the single-molecule biophysics on short DNA structures.
Asunto(s)
Nanoporos , Ácidos Nucleicos , ADN/química , NanotecnologíaRESUMEN
Fast, cheap, and easy to implement point-of-care testing for various pathogens constituted a game changer in past years due to its potential for early disease diagnosis. Herein, we report on the proof-of-concept of a simple method enabling in vitro detection of a structural spike protein subunit from the SARS-CoV-2 (S1 ) in aqueous samples. At the core of this discovery lies the well-known paradigm of monitoring the capacitive current across a reconstituted zwitterionic lipid membrane subjected to a periodic transmembrane potential, followed by the real-time spectral analysis enabling the extraction of the second harmonic of the capacitive current. Subsequent changes in the amplitude of this harmonic recorded during lipid membrane-S1 interactions were correlated with alterations induced in the inner membrane potential profile by the S1 protein subunit adsorption, and were shown to be augmented by ionic strength, the presence of a specific monoclonal antibody designed against the S1 subunit and the angiotensin-converting enzyme 2 (ACE2) protein receptor, and uninhibited by the presence of other human serum proteins.
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Técnicas Biosensibles , COVID-19 , Humanos , Inmunoensayo , Lípidos , Subunidades de Proteína/metabolismo , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/metabolismoRESUMEN
In this work, comparative studies on DNA-PNA and polyarginine-conjugated DNA-PNA duplexes unzipping inside the α-hemolysin nanopore (α-HL) are presented. We identified significant differences in the blockade currents, as the applied voltage across the nanopore facilitated the duplex capture inside the nanopore's vestibule against the constriction region, subsequent cDNA strand insertion inside the nanopore's ß-barrel past the constriction site, its complete unzip from the duplex, and translocation. We observed that inside the voltage-biased nanopore, polyarginine-conjugated DNA-PNA duplexes dehybridize faster than their DNA-PNA counterparts and proposed a model to describe the duplex unzipping. This study identifies key particularities of DNA-PNA duplex unzipping as it takes place inside the nanopore and being preceded by entrapment in the vestibule domain of the α-HL. Our results are a crucial step toward understanding the nucleic acids duplexes unzipping kinetics variability, in confined, variable geometries.
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ADN/química , Proteínas Hemolisinas/análisis , Nanoporos , Ácidos Nucleicos de Péptidos/química , Péptidos/química , CinéticaRESUMEN
The decades long advances in nanotechnology, biomolecular sciences, and protein engineering ushered the introduction of groundbreaking technologies devoted to understanding how matter behaves at single particle level. Arguably, one of the simplest in concept is the nanopore-based paradigm, with deep roots in what is originally known as the Coulter counter, resistive-pulse technique. Historically, a nanopore system comprising the oligomeric protein generated by Staphylococcus aureus toxin α-hemolysin (α-HL) was first applied to detecting polynucleotides, as revealed in 1996 by John J. Kasianowicz, Eric Brandin, Daniel Branton, and David W. Deamer, in the Proceedings of the National Academy of Sciences. Nowadays, a wide variety of other solid-state or protein-based nanopores have emerged as efficient tools for stochastic sensing of analytes as small as single metal ions, handling single molecules, or real-time, label-free probing of chemical reactions at single-molecule level. In this Account, we demonstrate the usefulness of the α-HL nanopore on probing metal-induced folding of peptides, and to investigating the reversible binding of various metals to physiologically relevant amyloid fragments. The widely recognized Achilles heel of the approach, is the relatively short dwell time of the analytes inside the nanopore. This hinders the collection of sufficient data required to infer statistically meaningful conclusions about the physical or chemical state of the studied analyte. To mitigate this, various approaches were successfully applied in particular experiments, including but not restricted to altering physical parameters of the aqueous solution, downsizing the nanopore geometry, the controlled tuning of the balance between the electrostatic and electro-osmotic forces, coating nanopores with a fluid lipid bilayer, employing a pressure-voltage biased pore. From our perspective, in this Account, we will present two strategies aimed at controlling the analyte passage across the α-HL. First, we will reveal how the electroosmotic flow can be harnessed to control residence time, direction, and the sequence of spatiotemporal dynamics of a single peptide along the nanopore. This also allows one to identify the mesoscopic trajectory of a peptide exiting the nanopore through either the vestibule or ß-barrel moiety. Second, we lay out the principles of an approach dubbed "nanopore tweezing", enabling simultaneous capture rate increase and escape rate decrease of a peptide from the α-HL, with the applied voltage. At its core, this method requires the creation of an electrical dipole on the peptide under study, via engineering positive and negative amino acid residues at the two ends of the peptide. Concise applications of this approach are being demonstrated, as in proof-of-concept experiments we probed the primary structure exploration of polypeptides, via discrimination between selected neutral amino acid residues. Another useful venue provided by the nanopores is represented by single-molecule force experiments on captured analytes inside the nanopore, which proved useful in exploring force-induced rupture of nucleic acids duplexes, hairpins, or various nucleic acids-ligand conjugates. We will show that when applied to oppositely charged, polypeptide-functionalized PNA-DNA duplexes, the nanopore tweezing introduces a new generation of force-spectroscopy nanopore-based platforms, facilitating unzipping of a captured duplex and enabling the duplex hybridization energy estimation.
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Péptidos beta-Amiloides/química , ADN/química , Proteínas Hemolisinas/química , Nanoporos , Fragmentos de Péptidos/química , Ácidos Nucleicos de Péptidos/química , Secuencia de Aminoácidos , Péptidos beta-Amiloides/metabolismo , Cobre/metabolismo , Humanos , Fragmentos de Péptidos/metabolismo , Unión ProteicaRESUMEN
We report here on the ability of the α-hemolysin (α-HL) nanopore to achieve label-free, selective, and real-time detection of 15 nt long ssDNA fragments in solution, by exploiting their hybridization with freely added, polycationic peptides-functionalized PNAs. At the core of our work lies the paradigm that when PNAs and ssDNA are mixed together, the bulk concentration of free PNA decreases, depending upon the (mis)match degree between complementary strands and their relative concentrations. We demonstrate that the ssDNA sensing principle and throughput of the method are determined by the rate at which nonhybridized, polycationic peptides-functionalized PNA molecules arrive at the α-HL's vestibule entrance and thread into the nanopore. We found that with the application of a 30-fold salt gradient across the nanopore, the method enhances single-molecule detection sensitivity in the nanomolar range of ssDNA concentrations. This study demonstrates that the transmembrane potential-dependent unzip of single PNA-DNA duplexes at the α-HL's ß-barrel entry permits discrimination between sequences that differ by one base pair.
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Técnicas Biosensibles/métodos , ADN de Cadena Simple/análisis , Proteínas Hemolisinas/química , Nanoporos , Ácidos Nucleicos de Péptidos/análisis , Imagen Individual de Molécula/métodos , ADN de Cadena Simple/química , Proteínas Hemolisinas/genética , Humanos , Hibridación de Ácido Nucleico , Ácidos Nucleicos de Péptidos/químicaRESUMEN
Peptide nucleic acids (PNAs) are artificial, oligonucleotides analogues, where the sugar-phosphate backbone has been substituted with a peptide-like N-(2-aminoethyl)glycine backbone. Because of their inherent benefits, such as increased stability and enhanced binding affinity toward DNA or RNA substrates, PNAs are intensively studied and considered beneficial for the fields of materials and nanotechnology science. Herein, we designed cationic polypeptide-functionalized, 10-mer PNAs, and demonstrated the feasible detection of hybridization with short, complementary DNA substrates, following analytes interaction with the vestibule entry of an α-hemolysin (α-HL) nanopore. The opposite charged state at the polypeptide-functionalized PNA-DNA duplex extremities, facilitated unzipping of a captured duplex at the lumen entry of a voltage-biased nanopore, followed by monomers threading. These processes were resolvable and identifiable in real-time, from the temporal profile of the ionic current through a nanopore accompanying conformational changes of a single PNA-DNA duplex inside the α-HL nanopore. By employing a kinetic description within the discrete Markov chains theory, we proposed a minimalist kinetic model to successfully describe the electric force-induced strand separation in the duplex. The distinct interactions of the duplex at either end of the nanopore present powerful opportunities for introducing new generations of force-spectroscopy nanopore-based platforms, enabling from the same experiment duplex detection and assessment of interstrand base pairing energy.
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ADN/análisis , ADN/química , Proteínas Hemolisinas/química , Nanoporos , Ácidos Nucleicos de Péptidos/análisis , Ácidos Nucleicos de Péptidos/química , Factores de TiempoRESUMEN
Herein, we report uni-molecular observations of electric potential- and electrolyte-dependent elasticity of poly(amidoamine) (PAMAM)-G1.5 dendrimers containing sodium carboxylate surface groups, using the electric field-assisted migration through the α-hemolysin nanopore (α-HL). Although at moderate transmembrane potentials the dendrimer (~ 2.5 nm in diameter) is sterically excluded from translocation across the constriction region of the nanopore (~ 1.5 nm in diameter), we found a threshold for its translocation that depends on both the electrolyte pH and ionic strength. We posit that the decreased repulsive intramolecular interactions among dendrimer's branches at low when compared to neutral pH, caused mainly by the protonation of surface groups on the dendrimer, determine a larger propensity of the dendrimer to collapse and deform. This in turns enables the dendrimer to adopt more favorably conformations that facilitate its optimal squeezing through the α-HL's constriction region at low pH, despite the fact that the estimated net force acting on it becomes approximately one order of magnitude lower than at neutral pH. Experiments performed in a low ionic strength buffer, which decreases Coulombic screening, enhance the intramolecular forces on the dendrimer and renders the dendrimer stiffer than in high ionic strength buffer, confirming the dendrimer elastic properties-dependent threshold for deformation inside the nanopore.
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Dendrímeros/química , Nanoporos , Electrofisiología , Concentración de Iones de Hidrógeno , Conformación Molecular , Concentración OsmolarRESUMEN
Stereochemistry is an essential theme for a number of industries and applications, constructed around discriminating various chiral enantiomers, including amino acids, chiral metal complexes, and drugs. In this work, we designed a set of peptide mutants of the human amyloidic Aß1-16 sequence, known to display an effective Cu(2+) coordinating pocket provided mainly by the intramolecular His-6, His-13, and His-14 residues, that were engineered to contain L- and D-His enantiomers in positions 6 and 13 and provide a local coordination environment with distinct Cu(2+) binding geometries and affinities. We examined the mechanism of selective chiral recognition of Cu(2+) by such mutant peptides, by quantifying their stochastic sensing in real time with a single α-hemolysin (α-HL) protein immobilized in a planar lipid membrane, while incubated in various concentrations of Cu(2+). Our data reveal that the Cu(2+)-binding affinity lies within the micromolar range, and decreases by orders of magnitude as L-His is replaced with its Denantiomer, with the effect being prevalent when such changes were inflicted on the His-6 residue. The presented results demonstrate the feasibility of tuning the metal selectivity in a relatively simple peptide substrate by enantiomeric replacement of key metal binding residues and illustrates the potential of the protein nanopores as a promising approach to quantify the chiral recognition of l/d amino acids by metals.
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Aminoácidos/química , Péptidos beta-Amiloides/química , Amiloide/química , Cobre/química , Histidina/química , Modelos Biológicos , Nanoporos , Aminoácidos/metabolismo , Humanos , Ingeniería de Proteínas , EstereoisomerismoRESUMEN
One of the prevailing paradigms regarding the onset of Alzheimer's disease endows metal ions with key roles in certain steps of the amyloid-ß (Aß) peptide aggregation cascade, through peptide conformational changes induced by metal binding. Herein, we focused on the truncated, more soluble Aß1-16 peptide fragment from the human Aß1-40, and demonstrated the utility of a sensing element based on the α-hemolysin (α-HL) protein to examine and compare at single-molecule level the interactions between such peptides and various metals. By using the same approach, we quantified Cu(2+) and Zn(2+) binding affinities to the Aß1-16 fragment, whereas the statistical analysis of blockages induced by a single Aß1-16 peptide on the current flow through an open α-HL pore show that the metal propensity to interacting with the peptide and entailing conformational changes obey the following order: Cu(2+) > Zn(2+) > Fe(3+) > Al(3+).
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Péptidos beta-Amiloides/química , Nanoporos , Óxido de Aluminio/química , Cobre/química , Hierro/química , Zinc/químicaRESUMEN
The exploration of single-strand DNA-binding protein (SSB)-ssDNA interactions and their crucial roles in essential biological processes lagged behind other types of protein-nucleic acid interactions, such as protein-dsDNA and protein-RNA interactions. The ssDNA binding protein gene product 32 (gp32) of the T4 bacteriophage is a central integrating component of the replication complex that must continuously bind to and unbind from transiently exposed template strands during the DNA synthesis. To gain deeper insights into the electrostatic conditions influencing the stability of the ssDNA-gp32 molecular complex, like the salt concentration or some metal ions proven to specifically bind to gp32, we employed a method that performs rapid measurements of the DNA-protein stability using an α-Hemolysin (α-HL) protein nanopore. We indirectly probed the stability of a protein-nucleic acid complex by monitoring the dissociation process between the gp32 protein and the ssDNA molecular complex in single-molecular electrophysiology experiments, but also through fluorescence spectroscopy techniques. We have shown that the complex is more stable in 0.5 M KCl solution than in 2 M KCl solution and that the presence of Zn2+ ions further increases this stability for any salt used in the present study. This method can be applied to other nucleic acid-protein molecular complexes, as well as for an accurate determination of the drug-protein carrier stability.
RESUMEN
Recent evidence shows that metal coordination by amyloid beta peptides (Aß) determines structural alterations of peptides, and His-13 from Aß is crucial for Cu(2+) binding. This study used the truncated, more soluble Aß1-16 isoforms derived from human and rat amyloid peptides to explore their interaction with Cu(2+) by employing the membrane-immobilized α-hemolysin (α-HL) protein as a nanoscopic probe in conjunction with single-molecule electrophysiology techniques. Unexpectedly, the experimental data suggest that unlike the case of the human Aß1-16 peptide, Cu(2+) complexation by its rat counterpart leads to an augmented association and dissociation kinetics of the peptide reversible interaction with the protein pore, as compared to the Cu(2+)-free peptide. Single-molecule electrophysiology data reveal that both human and rat Cu(2+)-complexed Aß peptides induce a higher degree of current flow obstruction through the α-HL pore, as compared to the Cu(2+)-free peptides. It is suggested that morphology changes brought by Cu(2+) binding to such amyloidic fragments depend crucially upon the presence of the His-13 residue on the primary sequence of such peptide fragments, and the α-HL protein-based approach provides unique opportunities and challenges to probing metal-induced folding of peptides.
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Péptidos beta-Amiloides/química , Péptidos beta-Amiloides/metabolismo , Cobre/química , Cobre/metabolismo , Nanoporos , Animales , Humanos , Unión Proteica , RatasRESUMEN
Nanopores offer highly sensitive, low-cost, and single-molecule sensing capabilities, and the societal impact of this approach is best captured by the advent of nanopore-based DNA detection and sequencing technologies, which extract genomic information without amplification. To address a critical difficulty plaguing such undertakings involving especially protein-based nanopores isolated in lipid bilayers, namely, the formation of a stable, long-lasting single nanopore, we pioneer herein an approach for generating functional nanostructures enabling small single-stranded DNA (ssDNA) detection. We designed a dynamic hybrid construct by appending extramembrane peptide nucleic acid (PNA) segments to the C-terminus of modified ion channel-forming alamethicin monomers. We found that the resulting chimeric molecules successfully coassemble in a voltage-dependent manner in planar lipid membranes generating diameter-variable oligomers. The subsequent interaction at the flexible extramembrane segment of such formed dynamic nanopores with aqueously added complementary ssDNA fragments leads to overall conformational alterations affecting the peptide assembly state kinetics and mediated ionic current. Such recognition events were found specific to the primary structure of target ssDNA and uninhibited the presence of serum. Our platform demonstrates the feasibility of designing an entirely new class of versatile chimeric biosensors, for which, dependent upon the nature of the attached receptor moiety and underlying recognition chemistry, the applicability area may extend to other analytes.
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Nanoporos , Receptores Artificiales , Antibacterianos/farmacología , Péptidos/genética , Hibridación de Ácido Nucleico , ADN de Cadena SimpleRESUMEN
Metal ions binding exert a crucial influence upon the aggregation properties and stability of peptides, and the propensity of folding in various substates. Herein, we demonstrate the use of the α-HL protein as a powerful nanoscopic tool to probe Cu(2+)-triggered physicochemical changes of a 20 aminoacids long, antimicrobial-derived chimera peptide with a His residue as metal-binding site, and simultaneously dissect the kinetics of the free- and Cu(2+)-bound peptide interaction to the α-HL pore. Combining single-molecule electrophysiology on reconstituted lipid membranes and fluorescence spectroscopy, we show that the association rate constant between the α-HL pore and a Cu(2+)-free peptide is higher than that of a Cu(2+)-complexed peptide. We posit that mainly due to conformational changes induced by the bound Cu(2+) on the peptide, the resulting complex encounters a higher energy barrier toward its association with the protein pore, stemming most likely from an extra entropy cost needed to fit the Cu(2+)-complexed peptide within the α-HL lumen region. The lower dissociation rate constant of the Cu(2+)-complexed peptide from α-HL pore, as compared to that of Cu(2+)-free peptide, supports the existence of a deeper free energy well for the protein interaction with a Cu(2+)-complexed peptide, which may be indicative of specific Cu(2+)-mediated contributions to the binding of the Cu(2+)-complexed peptide within the pore lumen.
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Toxinas Bacterianas/química , Cobre/química , Proteínas Hemolisinas/química , Histidina/química , Membrana Dobles de Lípidos/química , Péptidos/química , Proteínas Recombinantes de Fusión/química , Péptidos Catiónicos Antimicrobianos/química , Sitios de Unión , Cinética , Magaininas/química , Potenciales de la Membrana , Nanoporos , Péptidos/síntesis química , Unión Proteica , Conformación Proteica , Proteínas Recombinantes de Fusión/síntesis química , Espectrometría de Fluorescencia , TermodinámicaRESUMEN
The implication of nanopores as versatile components in dedicated biosensors, nanoreactors, or miniaturized sequencers has considerably advanced single-molecule investigative science in a wide range of disciplines, ranging from molecular medicine and nanoscale chemistry to biophysics and ecology. Here, we employed the nanopore tweezing technique to capture amino acid-functionalized peptide nucleic acids (PNAs) with α-hemolysin-based nanopores and correlated the ensuing stochastic fluctuations of the ionic current through the nanopore with the composition and order of bases in the PNAs primary structure. We demonstrated that while the system enables the detection of distinct bases on homopolymeric PNA or triplet bases on heteropolymeric strands, it also reveals rich insights into the conformational dynamics of the entrapped PNA within the nanopore, relevant for perfecting the recognition capability of single-molecule sequencing.
RESUMEN
Due to the pressing need to generate specific drugs or vaccines for COVID-19 and management of its outbreak, detailed knowledge regarding the SARS-CoV-2 entry into host cells and timely, cheap, and easy-to-use detection methods are of critical importance for containing the SARS-CoV-2 epidemic. Through electrophysiology and fluorescence spectroscopy experiments, we show that even in the absence of the angiotensin-converting enzyme 2 receptor, the S1 subunit from SARS-CoV-2 spike protein binding to neutral phospholipid membranes leads to their mechanical destabilization and permeabilization. A similar cytotoxic effect of the protein was seen in human lung epithelial cells. A monoclonal antibody generated toward the S1 subunit alleviates to a considerable extent the destabilizing potential of the protein in such model membranes. Finally, we demonstrate the proof-of-concept capability of an α-hemolysin (α-HL) protein nanopore to detect in aqueous buffer and real time the region-binding domain of the S1 subunit from SARS-CoV-2 spike protein by monitoring its immunological interaction with a target antibody. Our results may offer new perspectives in understanding the pathogenesis of the SARS-CoV-2 infection, its treatment, and real-time detection.
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COVID-19/genética , Lípidos/genética , SARS-CoV-2/genética , Glicoproteína de la Espiga del Coronavirus/genética , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , COVID-19/virología , Vacunas contra la COVID-19/genética , Vacunas contra la COVID-19/inmunología , Humanos , SARS-CoV-2/patogenicidad , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/metabolismoRESUMEN
Formaldehyde has become a ubiquitous contaminant in the air, and people are exposed to it worldwide. However, few studies have evaluated the temporal-spatial levels/changes of formaldehyde exposure at residences, and the relationship between its outdoor and indoor levels has been rarely examined. The aim of this study was to assess community formaldehyde exposure in Sebes and Aiud, Romania to identify: (1) home environment characteristics that may play an important role in exposure; and understand: (2) if there were differences in formaldehyde levels between the two cities; (3) if there were temporal variations within each city; and (4) whether outdoor formaldehyde levels influence indoor levels. We simultaneously performed indoor and outdoor active air sampling for formaldehyde at each investigated residential location over a 3-year period and analyzed the samples by gas chromatography with flame ionization detector (GC-FID). The mean values of indoor and outdoor formaldehyde levels in both cities fell in the range 0.014-0.035 mg/m3. The correlation analysis indicated mostly positive but not significant (p > 0.05) correlations between indoor formaldehyde and microclimate factors (temperature, humidity, pressure). Notably, home insulation was found to be significantly correlated with increased indoor formaldehyde levels. There were no significant differences in mean indoor or outdoor formaldehyde levels between Sebes and Aiud over the 3-year study period. When comparing the formaldehyde levels in both cities over the 3-year period, only outdoor formaldehyde levels were significantly higher in 2016, as compared to those in 2017 and 2018.
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Contaminantes Atmosféricos/análisis , Contaminación del Aire Interior/análisis , Monitoreo del Ambiente , Formaldehído/análisis , Características de la Residencia , Ciudades , RumaníaRESUMEN
Herein, we describe at uni-molecular level the interactions between poly(amidoamine) (PAMAM) dendrimers of generation 1 and the α-hemolysin protein nanopore, at acidic and neutral pH, and ionic strengths of 0.5 M and 1 M KCl, via single-molecule electrical recordings. The results indicate that kinetics of dendrimer-α-hemolysin reversible interactions is faster at neutral as compared to acidic pH, and we propose as a putative explanation the fine interplay among conformational and rigidity changes on the dendrimer structure, and the ionization state of the dendrimer and the α-hemolysin. From the analysis of the dendrimer's residence time inside the nanopore, we posit that the pH- and salt-dependent, long-range electrostatic interactions experienced by the dendrimer inside the ion-selective α-hemolysin, induce a non-Stokesian diffusive behavior of the analyte inside the nanopore. We also show that the ability of dendrimer molecules to adapt their structure to nanoscopic spaces, and control the flow of matter through the α-hemolysin nanopore, depends non-trivially on the pH- and salt-induced conformational changes of the dendrimer.
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Dendrímeros/química , Proteínas Hemolisinas/química , Concentración de Iones de Hidrógeno , Cinética , Conformación Molecular , Nanoporos , Electricidad EstáticaRESUMEN
We report on the ability to control the dynamics of a single peptide capture and passage across a voltage-biased, α-hemolysin nanopore (α-HL), under conditions that the electroosmotic force exerted on the analyte dominates the electrophoretic transport. We demonstrate that by extending outside the nanopore, the electroosmotic force is able to capture a peptide at either the lumen or vestibule entry of the nanopore, and transiently traps it inside the nanopore, against the electrophoretic force. Statistical analysis of the resolvable dwell-times of a metastable trapped peptide, as it occupies either the ß-barrel or vestibule domain of the α-HL nanopore, reveals rich kinetic details regarding the direction and rates of stochastic movement of a peptide inside the nanopore. The presented approach demonstrates the ability to shuttle and study molecules along the passage pathway inside the nanopore, allows to identify the mesoscopic trajectory of a peptide exiting the nanopore through either the vestibule or ß-barrel moiety, thus providing convincing proof of a molecule translocating the pore. The kinetic analysis of a peptide fluctuating between various microstates inside the nanopore, enabled a detailed picture of the free energy description of its interaction with the α-HL nanopore. When studied at the limit of vanishingly low transmembrane potentials, this provided a thermodynamic description of peptide reversible binding to and within the α-HL nanopore, under equilibrium conditions devoid of electric and electroosmotic contributions.
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Electroósmosis , Nanoporos , Péptidos/metabolismo , Transporte de Proteínas/fisiología , Proteínas Hemolisinas/metabolismo , CinéticaRESUMEN
Herein we explored the role of topological distribution of aromatic amino acids in peptide-membrane interfacial interactions. The membrane activity of closely related peptides and their binding energy is sensitive to the positioning of minimum two tryptophans, and by the degree of flanking at the membrane interface mediated by aromatic amino acids.