Binding‐Site Directed Selection and Large‐Scale Click‐Synthesis of a Coagulation Factor XIa‐Inhibiting Circular DNA Aptamer

Abstract Factor XIa (FXIa) is a plasma protease that catalyzes the intrinsic pathway of blood coagulation, thus being regarded as a promising antithrombotic target. Circular DNA aptamers, with their dramatically enhanced biological and structural stability, hold great potential as new‐generation DNA‐based anticoagulants. However, the functional selection of circular aptamers and large‐scale synthesis of them remains a substantial challenge. In this study, we explored a binding‐site directed selection strategy to enrich circular DNA aptamers that can target the catalytic domain of FXIa and inhibit its function. An asymmetrical dumbbell structured DNA library with self‐shielding primer‐binding site was utilized for selection. This approach has resulted in the identification of a circular DNA aptamer, named FICAPT1, which demonstrates a high binding affinity ( K d =20 nM), notable stability (with a half‐life of 16 h in human plasma) and outstanding anticoagulation activity (with significantly prolonged aPTT), showing great promise as a novel anticoagulant. Moreover, a refined version of FICAPT1, named as CC‐FICAPT1, was quantitatively synthesized (~2 mg/mL) by taking advantage of the pre‐designed structure and high cyclization efficiency of click chemistry. The approach presented in this study enables the efficient generation of functional circular DNA aptamers and allows for their synthesis in a satisfactory scale, making it adaptable for the production of various circular aptamers for therapeutic applications.