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 K(d) values in the range of several 10 nanomolar levels.
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