Engineering Robust Aptamers with High Affinity by Key Fragment Evolution and Terminal Fixation

Researchers have been looking for ways to fix the structural stability of aptamers so as to achieve the high affinity of aptamers and thus the high sensitivity of analytical methods. Herein, we report a post-selection strategy to facilitate the formation of aptameric structures and enhance their affinity. Key fragments containing crucial bases of parent aptamers were identified and evolved by iterative embedding to form chimeras. The termini of the optimized chimera were then fixed by hybridization to limit their flexibility. Robust aptamers with more stable structures and higher affinity were thus engineered. An anti-okadaic acid aptamer, anti-dinophysistoxin aptamer, and anti-phosphatidylserine (PS) aptamer were engineered in this way, with the affinity enhanced by 160.5-fold, 50.36-fold, and 39.28-fold over that of the parent aptamers, respectively. Furthermore, the practicability of the anti-PS aptamer was validated with a polyA-nanotetrahedron-assisted electrochemical aptasensor. The aptasensor achieved high sensitivity, with the limit of detection as low as 1.741 nM, good accuracy, and good selectivity when monitoring PS in real biosynthesis samples. This study offers a facile and efficient approach to generate robust aptamers and aptasensors for real-world applications.