Structurally defined synthetic heparin oligosaccharides reveal unique signatures for nanopore structural analysis of...

Efforts to fully understand the structure-to-function relationships of glycosaminoglycans (GAGs) have been hampered by the complexity and polydispersity of GAGs and, more importantly, by the need for sensitive analytical techniques to discern their subtle structural motifs. Conventional analytical methods often fail to provide information on the sparse motifs of GAGs. Concurrently, nanopore-based analysis has shown great potential in GAGs structural analysis. Still, until now, it has been limited by the difficulty of relating ionic current changes to specific motifs. To address this, the present study allowed for obtaining reference signals by employing carefully designed synthetic sulfated tetra- to dodecasaccharides to allow precise sequence control and purity. Reference signals were obtained for specific motifs, paving the way for accurate motif recognition. The outcomes of the study demonstrated unequivocal recognition of specific GAG structures by monitoring the evolution of nanopore signals in response to the translocation of synthetic heparin (HP)-like oligosaccharides with controlled structures, allowing extensive structural discrimination. The findings highlight the nanopore's capability to detect subtle structural features, such as differences in sulfation patterns and epimerization of uronic acid. These results open the way to establishing detailed heparin (HP)-specific reference signals and to nanopore fingerprinting and sequencing of GAGs.