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

摘要

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_2G_2, 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_2G_2tC(A/G)A_2G_2t 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.