This study describes a single-molecule, nanopore-based approach for the sequence deconvolution of diverse glycosaminoglycans (GAGs). The workflow is designed to analyse complex mixtures of GAG di- and oligosaccharides derived from heterogeneous polysaccharide chains without the need for fractionation. By combining targeted chemical transformations with nanopore sensing of reversible covalent adducts, the authors achieve discrete clustering of electrical event amplitudes that report on sugar size and fine structural features.
Key to the workflow is a modular chemical preprocessing pipeline. Native GAG chains are subjected to deacylation followed by an amino-selective, ring-contractive transformation that generates electrophilic aldehydes (anhydrosugars) from aminosugar residues. These chemical conversions simplify the structural diversity of GAG libraries into a set of reactive species that are compatible with downstream nanopore readout via reversible covalent adduct formation. The article reports these steps as operationally simple and integral to enabling single-molecule analysis of otherwise intractable mixtures.
Following chemical conversion, the anhydrosugars form reversible covalent adducts that interact with the nanopore sensor. Nanopore current traces are parsed into events whose amplitudes cluster into discrete populations. These amplitude clusters serve as the primary observables for deconvolution: distinct amplitude levels correspond to specific features of the modified sugars. The authors exploit these amplitude distinctions to count molecules and to infer compositional and structural attributes at single-molecule resolution.
Amplitude changes permit several layers of structural discrimination. The authors report three empirical step-change scales observed in amplitude clustering:
These resolution metrics underpin the method’s ability to resolve both coarse and fine features of GAG structure from nanopore signals.
Using chemical logic to interpret the observed anhydrosugar signals, the authors perform reverse mapping to infer the precursor disaccharide identities. Applied to natural heparan sulfate (HS), this reverse mapping covers roughly 84–100% of all disaccharides and their eliminative digestion variants reported in the sample set. The reported coverage indicates broad applicability of the mapping strategy to the diverse disaccharide repertoire present in HS.
The authors demonstrate practical utility by analysing a panel of HS polysaccharides chosen to span natural GAG structural diversity. The workflow distinguished compositional differences across that panel. In addition, the method detected contaminants in a pharmaceutical heparin sample, specifically identifying oversulfated chondroitin sulfate, a contaminant previously implicated in a global healthcare safety incident. These demonstrations illustrate applicability for both research-grade compositional profiling and forensic or quality-control screening of complex glycan-containing products.
A central advantage highlighted is that the workflow requires no fractionation and is operationally simple. Because it relies on nanopore sensing chemistry that is compatible with widely used, portable nanopore sequencing platforms, the approach is presented as a potential route toward broader access to precise glycan analysis. The authors suggest that adaptation to portable devices may contribute to the long-sought democratization of glycan sequencing and compositional diagnostics.
The preprint discloses a competing interest: one author is founder, consultant and shareholder of a company engaged in nanopore technology (Oxford Nanopore Technologies), and a patent on ring-contractive glycan sensing has been filed that could generate royalties if licensed. Funders reported in the article include Oxford Nanopore Technologies, the Gates Foundation, UK Research and Innovation, and the Engineering and Physical Sciences Research Council.
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