Motor protein-assisted glycan translocation and sequencing using nanopores

The glycan sequencing remains a significant bottleneck in glycoscience. While nanopore platforms have achieved substantial progress in single-molecule nucleic acid sequencing, their application to glycan sequencing has faced considerable challenges, with limited advancements to date. In this study, we propose a novel strategy for controlling glycan translocation through the MspA nanopore as an initial step toward glycan sequencing. By conjugating the target glycan with a helicase-controlled single-stranded DNA, we successfully achieved sequencing reads of up to eleven glycans. For the first time, we isolated glycan-associated electrical signals, enabling the translocation, stretching, and controlled speed of neutral glycans through the nanopore. This method provides a platform for obtaining glycan read lengths and identifying different glycan modifications, demonstrating the capability to resolve monosaccharide composition and glycosidic linkages. To further improve resolution, we propose engineered M2-MspA to reduce the pore constriction size and enhance precision by minimizing the random thermal motion of the translocating glycan. These modifications are expected to increase the sequencing accuracy and reliability. This work represents the first proof-of-concept demonstration of glycan chain nanopore sequencing and lays a promising foundation for the development of single-molecule glycan fingerprinting and sequencing technologies. We anticipate that this approach will significantly advance the development and commercialization of nanopore-based glycan sequencing techniques.