Single-Feature Identification of α2-8 Linked Sialoglycans Using Engineered Aerolysin Nanopores: A Paradigm for Glycan...

Area of Science:

• Glycoscience
• Nanotechnology
• Biophysics

Background:

• Nanopore sensing offers high-sensitivity, single-molecule analysis of glycans.
• Current methods face limitations in glycan profiling and sequencing.
• Advancements are needed to enhance nanopore resolution and applicability for diverse glycans.

Purpose of the Study:

• To develop a novel single-feature paradigm for nanopore glycan linkage analysis.
• To engineer aerolysin nanopores for specific recognition of glycan structures.
• To establish a method for glycan characterization and sequencing in complex biological samples.

Main Methods:

• Engineered aerolysin nanopore variants (K238Q and K238N) were designed for selective glycan binding.
• A dual-pore logic-gate assay combined readouts from different nanopore variants.
• Nanopore-compatible preprocessing and machine learning were employed for data analysis.

Main Results:

• The K238Q nanopore specifically recognized and decelerated α2-8 glycosidic linkages in sialoglycans.
• Submonosaccharide-level characterization and fingerprinting of α2-8 sialoglycans were achieved.
• A dual-pore assay with K238Q and K238N variants extracted structural information from unknown glycans.
• Proof-of-concept identification and quantification of sialoglycans in serum were demonstrated.

Conclusions:

• Engineered aerolysin nanopores provide a powerful platform for selective sialoglycan discrimination.
• This approach enables characterization of glycans within complex biological matrices.
• The study opens new avenues for nanopore-based glycan sequencing.