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

Factor XIa (FXIa) is a plasma protease that plays a crucial role in the intrinsic pathway of blood coagulation, making it a promising target for antithrombotic therapy. Circular DNA aptamers, with their dramatically enhanced biological and structural stability, hold great potential as new-generation DNA-based anticoagulants. However, the functional selection and large-scale synthesis of them remains a substantial challenge. Here, we explored a 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 featuring a hairpin-like primer-binding site was utilized for selection. This approach has resulted in the identification of a circular DNA aptamer, named FICAPT1, which exhibits high binding affinity (Kd = 20 nM), notable stability (half-life of 16 hours in human plasma) and outstanding anticoagulation activity (significantly prolonged aPTT), showing great promise as a novel anticoagulant. The hairpin-like constant region further served as a scaffold for the large-scale click-synthesis ( ~ 2 mg/mL) of the refined circular aptamer. The approach presented in this study enables the efficient generation of functional circular DNA aptamers and their synthesis at a satisfactory scale, making it adaptable for the production of various circular aptamers for therapeutic applications.