In Silico Study on a Binding Mechanism of ssDNA Aptamers Targeting Glycosidic Bond-Containing Small Molecules

Aptamer-based detection targeting glycoconjugates has attracted significant attention for its remarkable potential in identifying structural changes in saccharides in different stages of various diseases. However, the challenges in screening aptamers for small carbohydrates or glycoconjugates, which contain highly flexible and diverse glycosidic bonds, have hindered their application and commercialization. In this study, we investigated the binding conformations between three glycosidic bond-containing small molecules (GlySMs; glucose, N-acetylneuraminic acid, and neomycin) and their corresponding aptamers in silico, and analyzed factors contributing to their binding affinities. Based on the findings, a novel binding mechanism was proposed, highlighting the central role of the stem structure of the aptamer in binding and recognizing GlySMs and the auxiliary role of the mismatched bases in the adjacent loop. Guided by this binding mechanism, an aptamer with a higher 6'-sialyllactose binding affinity was designed, achieving a K_D value of 4.54 ± 0.64 μM in vitro through a single shear and one mutation. The binding mechanism offers crucial guidance for designing high-affinity aptamers, enhancing the virtual screening efficiency for GlySMs. This streamlined workflow filters out ineffective binding sites, accelerating aptamer development and providing novel insights into glycan-nucleic acid interactions.