Capillary Electrophoresis–Systematic Evolution of Ligands by Exponential Enrichment Selection of Base- and Sugar-Modified DNA Aptamers: Target Binding Dominated by 2′‑O,4′‑C‑Methylene-Bridged/Locked Nucleic Acid Primer

Chemically modified DNA aptamers specific to human α-thrombin were obtained from oligodeoxyribonucleotide (ODN) libraries by using a capillary electrophoresis–systematic evolution of ligands by exponential enrichment (CE–SELEX) method. These libraries contained 2′-O,4′-C-methylene-bridged/linked bicyclic ribonucleotides (B/L nucleotides) in the primer region and/or C5-modified thymidine bearing N 6-ethyladenine (t) in the nonprimer region. Modified DNA aptamers showed high binding affinities to the target, with dissociation constants (K d) values in the range of subnanomolar to several ten nanomolar levels. The introduction of base modification significantly suppressed the frequency of G-quadruplex motifs, which are often seen in thrombin-binding DNA aptamers. The resulting alternatives contained the 10-mer consensus sequence t 5Gt 2G2, which is frequently found in modified DNA aptamers with subnanomolar protein binding affinities. Furthermore, some base- and sugar-modified DNA aptamers with the 12-mer consensus sequence t 2G2 tC(A/G)A2G2 t displayed binding activities that were dependent on the presence of B/L nucleotides in the primer region. Such aptamers were interestingly not recovered from a natural DNA library or from DNA libraries modified with either B/L nucleotides or t’s. This emerging characteristic binding property will enable the creation of a direct selection methodology for DNA-based molecular switches that are triggered by chemical conversion of B/L nucleotides introduced to constant sequence regions in ODN libraries.