title: Single molecule detection of glycosaminoglycan hyaluronic acid oligosaccharides and depolymerization enzyme activity using a protein nanopore.
abstract: 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.
authors.affiliation: CNRS UMR 8587, Laboratoire Analyse et Modélisation pour la Biologie et l'Environnement, Université d'Evry Val d'Essonne, Bd François Mitterrand, 91025 Evry Cedex, France.
authors.forename: Aziz
authors.initials: A
authors.lastname: Fennouri
authors.forename: Cédric
authors.initials: C
authors.lastname: Przybylski
authors.forename: Manuela
authors.initials: M
authors.lastname: Pastoriza-Gallego
authors.forename: Laurent
authors.initials: L
authors.lastname: Bacri
authors.forename: Loïc
authors.initials: L
authors.lastname: Auvray
authors.forename: Régis
authors.initials: R
authors.lastname: Daniel
mesh_terms: D001427:Bacterial Toxins / Q000737:chemistry*; D015374:Biosensing Techniques / Q000295:instrumentation*; D004789:Enzyme Activation; D004867:Equipment Design; D019544:Equipment Failure Analysis; D006820:Hyaluronic Acid / Q000032:analysis* / Q000737:chemistry; D015336:Molecular Probe Techniques / Q000295:instrumentation*; D049329:Nanostructures / Q000737:chemistry*; D036103:Nanotechnology / Q000295:instrumentation*; D052899:Pore Forming Cytotoxic Proteins / Q000737:chemistry*; D016062:Porosity
chemical_list: D001427:Bacterial Toxins; D052899:Pore Forming Cytotoxic Proteins; C032676:aerolysin; D006820:Hyaluronic Acid