Thermally Switchable Nanogate Based on Polymer Phase Transition

Mimicking and extending the gating properties of biological pores is of paramount interest for the fabrication of membranes that could be used in filtration or drug processing. Here, we build a selective and switchable nanopore for macromolecular cargo transport. Our approach exploits polymer graftings within artificial nanopores to control the translocation of biomolecules. To measure transport at the scale of individual biomolecules, we use fluorescence microscopy with a zero-mode waveguide set up. We show that grafting polymers that exhibit a lower critical solution temperature creates a toggle switch between an open and closed state of the nanopore depending on the temperature. We demonstrate tight control over the transport of DNA and viral capsids with a sharp transition (∼1 °C) and present a simple physical model that predicts key features of this transition. Our approach provides the potential for controllable and responsive nanopores in a range of applications

Interaction de polymères naturels et synthétiques avec des pores protéiques

The technique of detection with nanopores at the single molecule level, is one of the most powerful method for the analysis of various molecules, of which biological and synthetic polymers, proteins and peptides, sugar molecules or metal nanoparticles. These pores can also serve as a platform for the study of fundamental physical and biological phenomenons. In the context of molecule analysis, this work, which is experimented using the technique of planar lipid bilayer painting, focuses mainly on the detection of polymers and their utility to portray fundamental processes of the α-hemolysin and aerolysin biological nanopores.The first results chapter described the probing of flows through α-hemolysin and aerolysin using polyethylene glycols (PEGs) and α-cyclodextrines, and the effects of KCl and LiCl salts on the interaction of PEGs with these pores. One main finding was that there exists a stronger electoosmotic flow in aerolysin, responsible for the transport of the neutral molecules α-cyclodextrines. The second finding was that the dynamics of PEGs with the nanopores are strongly dependent on the salt, showing drastic differences of frequency and dwell times vs. voltage for the two salts, although, the results of detection of mass of PEGs pointed to the fact that the nature of the interaction with the pore is similar in both salts.The aim of the work presented in the second results chapter, was to detect precision polymers, and find the best conditions, which can lead to their sequencing with nanopores. The homo- an copolymers of poly(phosphodiester)s were probed using α-hemolysin, aerolysin and MspA. The first type of polymers investigated which contained a 3-polythymidine primer and a sequence of comonomers of type (0) showed a strong interaction with the pores that was interpreted as the promotion of ssDNA-primer to the binding with the pore, combined to a high flexibility of the first type of polymers. The polymers which contained alkyne and triazole side chains, were found to have more complex interactions, but interacted for shorter durations with the pore indicating them to be stiffer. The second type of polymers seemed to be clustering in solution due the interaction between side chains, which proved the importance of performing characterization of these molecules in solution using wave scattering in the context of detection and ultimately sequencing.The study of the third result chapter, focused on the dynamics of small oligonucleotides with the aerolysin pore. The interaction of polyadenines (A3, A4, A5) showed complex dynamics and kinetics with pore, which was investigated via analysis of the events pattern. The whole process was found to be governed by two binding sites and energy barriers inside the pore that the molecules have to overcome. These results were combined to a developed kinetic model which allowed a complete description of the binding and translocation (or failure of it) of these polyadenines.The last results chapter described the interaction of bigger polyadenines (A6-A7-A8-A9-A10) with the aerolysin nanopore. The analysis of amplitude of currents of the adenine-induced blocks inside this pore showed an orientation dependent interaction of the molecules with the pore. This orientation dependent interaction started to be apparent for the A7 molecule and became the dominant effect for A9 and A10. Due to the flexibility of ssDNA, this effect is not observed for smaller sized molecules (A6 and below) because of their possibility of reorientation while inside the pore.La technique de détection à l'aide de nanopores au niveau de la molécule unique est l'une des plus puissantes pour l'analyse de diverses molécules, dont les polymères biologiques et synthétiques, les protéines et les peptides, les molécules de sucre ou les nanoparticules métalliques. Ces pores peuvent également servir de plate-forme pour l'étude de phénomènes physiques et biologiques fondamentaux. Dans le cadre de l'analyse de molécules, ce travail, expérimenté en utilisant la technique de la peinture de bicouche lipidique, porte principalement sur la détection des polymères et leur utilité pour sonder les processus fondamentaux des de l'α-hémolysine et de l'aérolysine.Le premier chapitre de résultats décrit l’analyse des flux à travers l'hémolysine et l'aérolysine à l’aide des polyéthylèneglycols (PEG) et des α-cyclodextrines, ainsi que les effets des sels de KCl et de LiCl sur l'interaction des PEG avec ces pores. L'une des principales conclusions est qu'il existe un flux électoosmotique plus fort dans l'aérolysine, responsable du transport des molécules neutres, les α-cyclodextrines. La seconde constatation concerne la dynamique des PEG avec les nanopores qui semblait être fortement dépendante du sel, montrant des différences drastiques de fréquence et de durée d’interaction en fonction de la tension pour les deux sels, bien que la détection de la masse de PEG dans les deux conditions indique que la nature de l'interaction avec le pore est similaire dans les deux types de sels.Le but des travaux présentés dans le deuxième chapitre de résultats était de détecter les polymères de précision et à trouver les meilleures conditions pouvant conduire à leur séquençage avec des nanopores. Des homopolymères et copolymères de poly(phosphodiester)s ont été sondés en utilisant l'hémolysine, l'aérolysine et MspA. Le premier type de polymères étudiés contenant une amorce 3-polythymidine et une suite de comonomères de type (0) a montré une forte interaction avec les pores qui a été interprétée comme la promotion de la liaison avec les pores, due à l'amorce d’ADN simple brin, combinée à une grande flexibilité du premier type de polymères. Les polymères qui contenaient des chaînes latérales alcyne et triazole se sont révélés avoir des interactions plus complexes, mais ont interagi pendant des durées plus courtes avec les pores indiquant qu'ils étaient plus rigides. Le second type de polymères semble s’agréger en solution du fait de l’interaction entre les chaînes latérales, ce qui prouve l’importance de la caractérisation de ces molécules en solution par diffusion de rayons, dans le cadre de la détection et finalement de leur séquençage.L'étude du troisième chapitre de résultats, a porté sur la dynamique de petits oligonucléotides avec le pore d’aerolysine. Les polyadénines (A3, A4, A5) ont montré une dynamique complexe d’interaction avec le pore, qui a été étudiée par l'analyse et la quantification des différentes propriétés des événements. L'ensemble du processus s'est avéré être régi par deux sites de liaison et des barrières énergétiques à l'intérieur du pore que les molécules doivent surmonter. Ces résultats ont été combinés à un modèle cinétique qui a permis une description complète de la liaison et de la translocation (ou son non succès) des polyadénines.Le dernier chapitre des résultats décrit l’interaction de plus grandes polyadénines (A6-A7-A8-A9-A10) avec l’aérolysine. L’analyse de l'amplitude des courants des blocs induits par l'adénine à l'intérieur de ce pore montre une interaction dépendante de l'orientation des molécules avec le pore. Cette interaction dépendante de l'orientation a commencé à apparaître pour la molécule A7 et est devenue l'effet dominant pour A9 et A10. En raison de la flexibilité de l'ANDsb, cet effet n'est pas observé pour les molécules de plus petite taille (A6 et inférieures) en raison de leur possibilité de réorientation à l'intérieur du pore

Electrophoresis and Electroosmosis in Aerolysin and Hemolysin Nanopores

Voltage-dependent interaction of macromolecules with biological nanopores can arise from electroosmotic (EOF) or electrophoretic (EPF) forces. Both poly(ethylene-glycol) (PEG), and cyclodextrins (β- and α-CD, respectively) block the ionic current through α-hemolysin (αHL) and Aerolysin (AeL) pore-forming proteins with well-documented voltage-dependence. Here, we attempt to relate differential effects of the electrolytes KCl and LiCl on the voltage-dependence of frequency and dwell-time of blocks to the relative contributions of EOF and EPF.For AeL, irrespective of electrolyte, blocks by α-CD only occurred from the pore's cis-side and with trans-positive voltages. Blocks in LiCl were much longer than those in KCl (11ms vs. 760μs at 90mV) but dwell times showed a positive, linear voltage dependence in both cases (LiCl:6fold/100mV; KCl:3fold/100mV). In KCl, cis-side PEG blocked AeL with trans-negative voltages only.1 Surprisingly, in LiCl, blocks occurred only at trans-positive voltages and at more than 1000 fold PEG-concentration but we still are in a very diluted regime. Blocks were shorter than in KCl by a factor of 5 (60 vs. 320μs at 100mV), but frequency (3fold/100mV) and dwell time (2fold/100mV) depended linearly on voltage, reminiscent of the behavior of α-CD in both electrolytes.Linear voltage dependence of block frequency might indicate dominance of EOF for pore entry of PEG in LiCl and for α-CD generally, while an exponential voltage-dependence would be expected for an electrophoretic force. Indeed, for PEG in KCl, which may be positively charged due to K+-chelation (see Ref.1 for discussion), we found an exponential increase in frequency for blocks of AeL and αHL with increasing trans-negative and trans-positive voltages, respectively. We cannot, however, exclude voltage and ion-dependent effects on the binding reaction, which, rather than partitioning, may be rate-limiting for the electrophysiologically detectable blockages

Kinetic Analysis of Single Molecule Electrodiffusion in a Biological Nanopore with Two Binding Sites

International audienceWhen single short adenine oligonucleotides (d.p. 3-5) block the pore formed by the bacterial toxin aerolysin, the resultant resistive pulses are complex with two levels (short and deep vs. long and shallow) in a variety of configurations, the probability of which depends on voltage. We account for this by a four-state kinetic mechanism with two undistinguishable open states i,o and two blocked states m1 (short-deep) and m2 (long-shallow) linked as om1m2->i. M1 is directly accessible from o which designates the presence of the nucleotide at the pore0s cis-side mouth, while m2 is inaccessible from state i, as nucleotides are unable to enter from the trans-side irrespective of voltage. In this framework, several experimentally accessible statistical quantities such as the frequency or probability of returns to m1 from m2, the fraction of restive pulses ending in m1and the mean dwell times in m2 as well as the mean total duration of resistive pulses acquire mechanistic significance and allow direct kinetic predictions using a Q-matrix approach. For A3, the data are consistent with a charged particle moving through a one-dimensional energy landscape with two minimain an electrically biased random walk. For longer nucleotides (e.g. A5) the success rate for translocation is higher than predicted by the rate constants determined from the other observables. It appears likely that this is due to an ability of the longer oligomers to simultaneously link with the two binding sites, producing an excess of returns from m2 to m1, which, however, does not entail a propensity to result in translocation failures. Such double tethering of DNA might promote translocation of longer chains by producing an extended conformation and may also contribute to the strong rectification of transport observed for aerolysin

Probing driving forces in aerolysin and α-hemolysin biological nanopores: electrophoresis versus electroosmosis

International audienceThe transport of macromolecules through nanopores is involved in many biological functions and is today at the basis of promising technological applications. Nevertheless the interpretation of the dynamics of the macromolecule/nanopore interaction is still misunderstood and under debate. At the nanoscale, inside biomimetic channels under an external applied voltage, electrophoresis, which is the electric force acting on electrically charged molecules, and electroosmotic flow (EOF), which is the fluid transport associated with ions, contribute to the direction and magnitude of the molecular transport. In order to decipher the contribution of the electrophoresis and electroosmotic flow, we explored the interaction of small, rigid, neutral molecules (cyclodextrins) and flexible, non-ionic polymers (poly(ethylene glycol), PEG) that can coordinate cations under appropriate experimental conditions, with two biological nanopores: aerolysin (AeL) and α-hemolysin (aHL). We performed experiments using two electrolytes with different ionic hydration (KCl and LiCl). Regardless of the nature of the nanopore and of the electrolyte, cyclodextrins behaved as neutral analytes. The dominant driving force was attributed to EOF, acting in the direction of the anion flow and stronger in LiCl than in KCl. The same qualitative behaviour was observed for PEGs in LiCl. In contrast, in KCl, PEGs behaved as positively charged polyelectrolytes through both AeL and aHL. Our results are in agreement with theoretical predictions about the injection of polymers inside a confined geometry (ESI). We believe our results to be of significant importance for better control of the dynamics of analytes of different nature through biological nanopores

Translocation of Sequence-Controlled Synthetic Polymers through Biological Nanopores

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Thermally Switchable Nanogate Based on Polymer Phase Transition

International audienceMimicking and extending the gating properties of biological pores is of paramount interest for the fabrication of membranes that could be used in filtration or drug processing. Here, we build a selective and switchable nanopore for macromolecular cargo transport. Our approach exploits polymer graftings within artificial nanopores to control the translocation of biomolecules. To measure transport at the scale of individual biomolecules, we use fluorescence microscopy with a zero-mode waveguide set up. We show that grafting polymers that exhibit a lower critical solution temperature creates a toggle switch between an open and closed state of the nanopore depending on the temperature. We demonstrate tight control over the transport of DNA and viral capsids with a sharp transition (∼1 °C) and present a simple physical model that predicts key features of this transition. Our approach provides the potential for controllable and responsive nanopores in a range of applications