Structure-guided engineering of aptamers to enhanced structural stability and application performance in alleviating β-lactoglobulin allergenicity

Aptamers are synthetic single-strand oligonucleotide-based ligands with great application potential due to their intriguing properties of specific and high affinity target binding and inhibition. However, the excessive flexibility of aptamer conformation will lead to instability and diversity of the folded structure. It remains a great challenge to keep high binding performance in real-world applications. Herein, we rational engineered a structurally enhanced aptamer (Cb-#T4M8) through hairpin structural enhancement guided by structural information combined with cyclization strategies, that possesses excellent binding affinity (5.1 ± 1.2 nM) against β-lactoglobulin (β-Lg), as well as highest stability. Notably, molecular simulation and competitive experimental analysis revealed that Cb-#T4M8 shares the same binding epitopes on β-Lg with IgE. Using Cb-#T4M8 as an inhibitor, Cb-#T4M8 was successfully demonstrated to interfere with IgE binding to β-Lg in vitro experiments. Furthermore, The application of Cb-#T4M8 in allergy models of cells and mice significantly reduced the allergic response induced by β-Lg. Overall, our study not only provided a reference for the engineering of aptamers to obtain a good application in the real world but also proposed a new direction for developing treatment against food allergy.