Single Molecule Detection of Glycosaminoglycan Hyaluronic Acid Oligosaccharides and Depolymerization Enzyme Activity Using a Protein Nanopore

Glycosaminoglycans are biologically active anionic carbohydrates that are among the most challenging biopolymers with regards to their structural analysis and functional assessment. The potential of newly introduced biosensors using protein nanopores that have been mainly described for nucleic acids and protein analysis to date, has been here applied to this polysaccharide-based third class of bioactive biopolymer. This nanopore approach has been harnessed in this study to analyze the hyaluronic acid glycosamiglycan and its depolymerization-derived oligosaccharides. The translocation of a glycosaminoglycan is reported using aerolysin protein nanopore. Nanopore translocation of hyaluronic acid oligosaccharides was evidenced by the direct detection of translocated molecules accumulated into the arrival compartment using high-resolution mass spectrometry. Anionic oligosaccharides of various polymerization degrees were discriminated through measurement of the dwelling time and translocation frequency. This molecular sizing capability of the protein nanopore device allowed the real-time recording of the enzymatic cleavage of hyaluronic acid polysaccharide. The time-resolved detection of enzymatically produced oligosaccharides was carried out to monitor the depolymerization enzyme reaction at the single-molecule level

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

Exploration of Neutral Versus Polyelectrolyte Behavior of Poly(ethylene glycol)s in Alkali Ion Solutions using Single-Nanopore Recording

We examine the effect of alkali ions (Li⁺, Na⁺, K⁺, Rb⁺, Cs⁺) on the partitioning of neutral and flexible poly­(ethylene glycol) into the alpha-hemolysin (α-HL) nanopore for a large range of applied voltages at high salt concentration. The neutral polymer behaves as if charged, that is, the event frequency increases with applied voltage, and the residence times decrease with the electric force for all cations except Li⁺. In contrast, in the presence of LiCl, we find the classical partitioning behavior of neutral polymers, that is, the event frequency and the residence times are independent of the applied voltage. Assuming that lithium does not associate with PEG enabled us to quantify the relative magnitude of the entropic and enthalpic contribution to the free- energy barrier and the number of complexed cations using two different arguments; the first estimate is based on the balance of forces, and the second is found comparing the blockade ratio in the presence of LiCl (no complexed ions) to the blockade ratio of chains in the presence of the other salts (with complexed ions). This estimate is in agreement with recent simulations. These findings demonstrate that the nanopore could prove useful for the rapid probing of the capabilities of different neutral molecules to form complexes with different ions

Protein Transport through a Narrow Solid-State Nanopore at High Voltage: Experiments and Theory

We report experimentally the transport of an unfolded protein through a narrow solid-state nanopore of 3 nm diameter as a function of applied voltage. The random coil polypeptide chain is larger than the nanopore. The event frequency dependency of current blockades from 200 to 750 mV follows a van’t Hoff–Arrhenius law due to the confinement of the unfolded chain. The protein is an extended conformation inside the pore at high voltage. We observe that the protein dwell time decreases exponentially at medium voltage and is inversely proportional to voltage for higher values. This is consistent with the translocation mechanism where the protein is confined in the pore, creating an entropic barrier, followed by electrophoretic transport. We compare these results to our previous work with a larger pore of 20 nm diameter. Our data suggest that electro-osmotic flow and protein adsorption on the narrowest nanopore wall are minimized. We discuss the experimental data obtained as compared with recent theory for the polyelectrolyte translocation process. This theory reproduces clearly the experimental crossover between the entropic barrier regime with medium voltage and the electrophoretic regime with higher voltage