Nanopore-based sequence deconvolution of diverse glycosaminoglycans

Glycosaminoglycan (GAG) polysaccharides play vital roles in animal physiology and disease1. Their diverse and intricate patterns of sulfation and epimerization endow them with an extensive potential to encode functional information2,3. GAG characterization, however, remains a formidable challenge for state-of-the-art ensemble-based techniques4,5. Single-molecule techniques are uniquely suited for analysing complex mixtures6,7. Here, we report the single-molecule resolution and counting of diverse GAG di- and oligosaccharides derived from longer heterogeneous chains as part of a deconvolutive nanopore-based workflow that requires no fractionation and is operationally simple. Modular chemical deacylation and amino-selective ring-contractive formation of electrophilic aldehydes enable the parsing of libraries of GAG structures into simplified sets of reactive anhydrosugars for nanopore readout via reversible covalent adduct formation. Discrete clustering of event amplitudes enables direct sugar sizing ([~]10 % step change per residue), which can be coupled to precisely resolved amplitude differences that further reveal sugar fine structure--including the number and position of sulfate groups ([~]2 % step change per sulfate) alongside single- atom stereochemistry ([~]0.5 % step change between epimers). Guided by chemical logic, the reverse mapping of resolved anhydrosugars to their precursors covers [~]84-100 % of all disaccharides and their eliminative digestion variants in natural heparan sulfate (HS). We demonstrate the practical utility and scope of our approach through the compositional analysis of a panel of HS polysaccharides that together encompass natural GAG structural diversity. Moreover, we detect contaminants in heparin, including oversulfated chondroitin sulfate found in an authentic pharmaceutical heparin sample previously implicated in a global healthcare crisis. Together, our results suggest a general chemo-biophysical framework for the precise and sensitive characterization of GAGs that extends to other aminosugar biopolymers. When adapted for portable, widely used nanopore sequencing devices, our approach may offer a path towards the long-sought democratization of glycan analysis.