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 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 (Kd=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.