From current trace to the understanding of confined media.

Nanopores constitute devices for the sensing of nano-objects such as ions, polymer chains, proteins or nanoparticles. We describe what information we can extract from the current trace. We consider the entrance of polydisperse chains into the nanopore, which leads to a conductance drop. We describe the detection of these current blockades according to their shape. Finally, we explain how data analysis can be used to enhance our understanding of physical processes in confined media.

Uncooked spaghetti in a colander: Injection of semiflexible polymers in a nanopore.

We study the flow injection of semiflexible polymers in a nanopore with a diameter smaller than the persistence length of the macromolecules. The suction model from de Gennes and Brochard is modified to take into account the effect of the rigidity of the polymer in the Odijk regime. We show that in this case of extreme confinement the flow threshold vanishes slowly and that in the limit of infinitely small nanopore the free energy barrier eventually disappears.

Dynamics of a polyelectrolyte through aerolysin channel as a function of applied voltage and concentration⋆.

We describe the behaviour of a polyelectrolyte in confined geometry. The transport of a polyelectrolyte, dextran sulfate, through a recombinant protein channel, aerolysin, inserted into a planar lipid bilayer is studied as a function of applied voltage and polyelectrolyte concentration and chain length. The aerolysin pore has a weak geometry asymmetry, a high number of charged residues and the polyelectrolyte is strongly negatively charged. The resulting current blockades were characterized by short and long dwelling times. Their frequency varies exponentially as a function of applied voltage and linearly as a function of polyelectrolyte concentration. The long blockade duration decreases exponentially when the electrical force increases. The ratio of the population of short events to the one of long events decreases when the applied voltage increases and displays an exponential variation. The long residence time increases with the polyelectrolyte chain length. We measure a reduction of the effective charge of the polyelectrolyte at the pore entry and inside the channel. For a fixed applied voltage, + / - 100 mV, at both sides of the protein pore entrance, the events frequency is similar as a function of dextran sulfate concentration. The mean blockade durations are independent of polyelectrolyte concentration and are similar for both entrances of the pore and remain constant as a function of the electrical force.

Polynucleotide transport through lipid membrane in the presence of starburst cyclodextrin-based poly(ethylene glycol)s.

Symmetrical cyclodextrin-based 14-arm star polymers with poly(ethylene glycol) PEG branches were synthesized and characterized. Interactions of the star polymers with lipid bilayers were studied by the "black lipid membrane" technique in order to demonstrate the formation of monomolecular artificial channels. The conditions for the insertion are mainly based on dimensions and amphiphilic properties of the star polymers, in particular the molar mass of the water-soluble polymer branches. Translocation of single-strand DNA (ssDNA) through those synthetic nanopores was investigated, and the close dimension between the cross-section of ssDNA and the cyclodextrin cavity led to an energy barrier that slowed down the translocation process.

Temperature Effect on Ionic Current and ssDNA Transport through Nanopores.

We have investigated the role of electrostatic interactions in the transport of nucleic acids and ions through nanopores. The passage of DNA through nanopores has so far been conjectured to involve a free-energy barrier for entry, followed by a downhill translocation where the driving voltage accelerates the polymer. We have tested the validity of this conjecture by using two toxins, α-hemolysin and aerolysin, which differ in their shape, size, and charge. The characteristic timescales in each toxin as a function of temperature show that the entry barrier is ∼15 kBT and the translocation barrier is ∼35 kBT, although the electrical force in the latter step is much stronger. Resolution of this fact, using a theoretical model, reveals that the attraction between DNA and the charges inside the barrel of the pore is the most dominant factor in determining the translocation speed and not merely the driving electrochemical potential gradient.

Zero-mode waveguide detection of flow-driven DNA translocation through nanopores.

We directly measure the flow-driven injection of DNA through nanopores at the level of single molecule and single pore using a modified zero-mode waveguide method. We observe a flow threshold independent of the pore radius, the DNA concentration, and length. We demonstrate that the flow injection of DNA in nanopores is controlled by an energy barrier as proposed in the de Gennes-Brochard suction model. Finally, we show that the height of the energy barrier is modulated by functionalizing the nanopores.

Kinetics of enzymatic degradation of high molecular weight polysaccharides through a nanopore: experiments and data-modeling.

The enzymatic degradation of long polysaccharide chains is monitored by nanopore detection. It follows a Michaelis-Menten mechanism. We measure the corresponding kinetic constants at the single molecule level. The simulation results of the degradation process allowed one to account for the oligosaccharide size distribution detected by a nanopore.

Mapping the conformational stability of maltose binding protein at the residue scale using nuclear magnetic resonance hydrogen exchange experiments.

Being able to differentiate local fluctuations from global folding-unfolding dynamics of a protein is of major interest for improving our understanding of structure-function determinants. The maltose binding protein (MBP), a protein that belongs to the maltose transport system, has a structure composed of two globular domains separated by a rigid-body "hinge bending". Here we determined, by using hydrogen exchange (HX) nuclear magnetic resonance experiments, the apparent stabilization free energies of 101 residues of MBP bound to ß-cyclodextrin (MBP-ßCD) under native conditions. We observed that the last helix of MBP (helix α14) has a lower protection factor than the rest of the protein. Further, HX experiments were performed using guanidine hydrochloride under subdenaturing conditions to discriminate between local fluctuations and global unfolding events and to determine the MBP-ßCD energy landscape. The results show that helix α4 and a part of helices α5 and α6 are clearly grouped into a subdenaturing folding unit and represent a partially folded intermediate under native conditions. In addition, we observed that amide protons located in the hinge between the two globular domains share similar ΔG(gu)(app) and m values and should unfold simultaneously. These observations provide new points of view for improving our understanding of the thermodynamic stability and the mechanisms that drive folding-unfolding dynamics of proteins.

Thermal unfolding of proteins probed at the single molecule level using nanopores.

The nanopore technique has great potential to discriminate conformations of proteins. It is a very interesting system to mimic and understand the process of translocation of biomacromolecules through a cellular membrane. In particular, the unfolding and folding of proteins before and after going through the nanopore are not well understood. We study the thermal unfolding of a protein, probed by two protein nanopores: aerolysin and α-hemolysin. At room temperature, the native folded protein does not enter into the pore. When we increase the temperature from 25 to 50 °C, the molecules unfold and the event frequency of current blockade increases. A similar sigmoid function fits the normalized event frequency evolution for both nanopores, thus the unfolding curve does not depend on the structure and the net charge of the nanopore. We performed also a circular dichroism bulk experiment. We obtain the same melting temperature (around 45 °C) using the bulk and single molecule techniques.

Transport of long neutral polymers in the semidilute regime through a protein nanopore.

We investigate the entrance of single poly(ethylene glycol) chains into an α-hemolysin channel. We detect the frequency and duration of the current blockades induced by large neutral polymers, where chain radius is larger than pore diameter. In the semidilute regime, these chains pass only if the monomer concentration is larger than a well-defined threshold. Experiments are performed in a very large domain of concentration and molecular mass, up to 35% and 200 kDa, respectively, which was previously unexplored. The variation of the dwell time as a function of molecular mass shows that the chains are extracted from the semidilute solution in contact with the pore by a reptation mechanism.

Dynamics of unfolded protein transport through an aerolysin pore.

Protein export is an essential mechanism in living cells and exported proteins are usually translocated through a protein-conducting channel in an unfolded state. Here we analyze, by electrical detection, the entry and transport of unfolded proteins, at the single molecule level, with different stabilities through an aerolysin pore, as a function of the applied voltage and protein concentration. The frequency of ionic current blockades varies exponentially as a function of the applied voltage and linearly as a function of protein concentration. The transport time of unfolded proteins decreases exponentially when the applied voltage increases. We prove that the ionic current blockade duration of a double-sized protein is longer than that assessed for a single protein supporting the transport phenomenon. Our results fit with the theory of confined polyelectrolyte and with some experimental results about DNA or synthetic polyelectrolyte translocation through protein channels as a function of applied voltage. We discuss the potential of the aerolysin nanopore as a tool for protein folding studies as it has already been done for α-hemolysin.

Discrimination of neutral oligosaccharides through a nanopore.

The detection of oligosaccharides at the single-molecule level was investigated using a protein nanopore device. Neutral oligosaccharides of various molecular weights were translocated through a single α-hemolysin nanopore and their nano-transit recorded at the single-molecule level. The translocation of maltose and dextran oligosaccharides featured by 1→4 and 1→6 glycosidic bonds respectively was studied in an attempt to discriminate oligosaccharides according to their polymerization degree and glycosidic linkages. Oligosaccharides were translocated through a free diffusion regime indicating that they adopted an extended conformation during their translocation in the nanopore. The dwell time increased with molecular mass, suggesting the usefulness of nanopore as a molecular sizing device.

Polyelectrolyte entry and transport through an asymmetric alpha-hemolysin channel.

We study the entry and transport of a polyelectrolyte, dextran sulfate (DS), through an asymmetric alpha-hemolysin protein channel inserted into a planar lipid bilayer. We compare the dynamics of the DS chains as they enter the channel at the opposite stem or vestibule sides. Experiments are performed at the single-molecule level by using an electrical method. The frequency of current blockades varies exponentially as a function of applied voltage. This frequency is smaller for the stem entrance than for the vestibule one, due to a smaller coupling with the electric field and a larger activation energy for entry. The value of the activation energy is quantitatively interpreted as an entropic effect of chain confinement. The translocation time decreases when the applied voltage increases and displays an exponential variation which is independent of the stem or vestibule sides.

Two independent ways of preparing hypercharged hydrolyzable polyaminorotaxane.

The aim of this work is to synthesize new PEO-based polyrotaxanes from modified cyclodextrins. Two strategies are discussed and compared. In the first, a pseudopolyrotaxane was formed between alpha,omega- PEO dimethacrylate and alpha-cyclodextrin. A coupling reaction between 1-pyrenebutyric acid N-hydroxysuccinimide ester was carried out to block the cyclic molecules onto the PEO. Cyclodextrins of the supramolecular assemblies were then oxidized using sodium periodate and reacted with spermine to form a potentially highly charged polyrotaxane. In the second strategy, cyclodextrins were first modified, and used to form the polyrotaxane through the pseudopolyrotaxane synthesis followed by the blocking reaction. Acidic titration allowed quantifying the number of amine functions borne by the supramolecular assemblies through two variables: the number of rings per polymer chain and the number of spermine groups per cyclic molecule. The supramolecules obtained by both strategies are discussed.

Urea denaturation of alpha-hemolysin pore inserted in planar lipid bilayer detected by single nanopore recording: loss of structural asymmetry.

The aim of this work is to study pore protein denaturation inside a lipid bilayer and to probe current asymmetry as a function of the channel conformation. We describe the urea denaturation of alpha-hemolysin channel and the channel formation of alpha-hemolysin monomer incubated with urea prior to insertion into a lipid bilayer. Analysis of single-channel recordings of current traces reveals a sigmoid curve of current intensity as a function of urea concentration. The normalized current asymmetry at 29+/-4% is observed between 0 and 3.56M concentrations and vanishes abruptly down to 0 concentration exceeds 4M. The loss of current asymmetry through alpha-hemolysin is due to the denaturation of the channel's cap. We also show that the alpha-hemolysin pore inserted into a lipid bilayer is much more resistant to urea denaturation than the alpha-hemolysin monomer in solution: The pore remains in the lipid bilayer up to 7.2M urea. The pore formation is possible up to 4.66M urea when protein monomers were previously incubated in urea.

PEO-PPO block copolymer vectors do not interact directly with DNA but with lipid membranes.

Small angle neutron (SANS) and light scattering was used to study the interaction between fragments of double stranded deoxyribonucleic acid (DNA) and a synthetic triblock [poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide)] amphiphilic polymer, known as L64, a potential vector for gene therapy. The mechanism of action of this vector is yet unknown. The contrast variation method was used to separate the partial structure factors of the different components in mixtures of triblock and DNA. It has been found that the copolymer and DNA molecules exhibit repulsive interactions. Further, the interaction between the copolymer and a model lipid membrane was investigated in order to explain the action of the vector. Electrical measurements on black lipid membranes indicated that the main effect of L64 as a vector is to permeabilize the cell's membrane.

Nuclear pore complex plasticity during developmental process as revealed by super-resolution microscopy.

Nuclear Pore Complex (NPC) is of paramount importance for cellular processes since it is the unique gateway for molecular exchange through the nucleus. Unraveling the modifications of the NPC structure in response to physiological cues, also called nuclear pore plasticity, is key to the understanding of the selectivity of this molecular machinery. As a step towards this goal, we use the optical super-resolution microscopy method called direct Stochastic Optical Reconstruction Microscopy (dSTORM), to analyze oocyte development impact on the internal structure and large-scale organization of the NPC. Staining of the FG-Nups proteins and the gp210 proteins allowed us to pinpoint a decrease of the global diameter by measuring the mean diameter of the central channel and the luminal ring of the NPC via autocorrelation image processing. Moreover, by using an angular and radial density function we show that development of the Xenopus laevis oocyte is correlated with a progressive decrease of the density of NPC and an ordering on a square lattice.

Long-living channels of well defined radius opened in lipid bilayers by polydisperse, hydrophobically-modified polyacrylic acids.

In the presence of random amphiphilic polymers devoid of secondary structure, lipid membranes become leaky to albumin and dextran (fluorescence of giant unilamellar vesicles) and their conductance (black lipid films) shows the transient opening of channels of typical radius 1-5 nm.

Ternary complex formation in aqueous solution between a beta-cyclodextrin polymer, a cationic surfactant and DNA.

Polyelectrolyte complexes have been elaborated by mixing in water neutral poly(beta-CD), a cationic surfactant (DTAC) and herring sperm DNA fragments. The driving forces for the poly(beta-CD)/DTAC/DNA association in aqueous solution are, on the one hand, reversible inclusion interactions between the CD cavities of poly(beta-CD) and the alkyl group of DTAC, leading to the formation of a polycation and, on the other hand, electrostatic interactions between the opposite charges of the cationic surfactant and anionic DNA. Viscometry and SANS have been used to prove the occurrence of such ternary complexes in dilute aqueous solutions.