2′,4′-BNA/LNA aptamers: CE-SELEX using a DNA-based library of full-length 2′-O,4′-C-methylene-bridged/linked bicyclic ribonucleotides

DNA-based aptamers that contain 2′-O,4′-C-methylene-bridged/linked bicyclic ribonucleotides (B/L nucleotides) over the entire length were successfully obtained using a capillary electrophoresis systematic evolution of ligands by exponential enrichment (CE-SELEX) method. A modified DNA library was prepared with an enzyme mix of KOD Dash and KOD mutant DNA polymerases. Forty 2′-O,4′-C-methylene bridged/locked nucleic acid (2′,4′-BNA/LNA) aptamers were isolated from an enriched pool and classified into six groups according to their sequence. 2′,4′-BNA/LNA aptamers of groups V and VI bound human thrombin with Kd values in the range of several 10 nanomolar levels.