Direct Evolution of Matrix-Resistant Circular Bivalent DNA Aptamers for Ara h1

Peanut allergenic protein Ara h1 is a serious food allergen due to its potentially life-threatening effects, and its accurate detection holds significant importance. While several selections were previously reported to isolate DNA aptamers for Ara h1, little success was achieved for binding in a complex food matrix. In this work, circular bivalent aptamers for Ara h1 were obtained by employing a dumbell-shaped DNA library with dual random domains for selection. The highest affinity aptamer, named as CB-APT1, exhibited a dissociation constant (K_d) of 36.3 nM, as determined by microscale thermophoresis. Notably, a similar binding affinity was observed even in a complex matrix that contained 80% w/w total peanut proteins. Further analysis indicates that each random domain acts as a unique binding moiety, working together to enhance the overall affinity. Subsequently, a label-free fluorescent aptasensor was developed for Ara h1, which demonstrated a low detection limit of 1.3 nM and performed well, even in food samples. Our evolution-based approach for developing circular bivalent DNA aptamers does not rely on structural information on the target protein, making it applicable to a wide range of protein targets. We believe this strategy can be leveraged to generate a diverse set of high-quality circular bivalent DNA aptamers that are both stable and functional in real biological samples, thus enhancing the practical applications of DNA aptamers in real-world applications.