title: 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.
abstract: 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 (Kd) 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 t5Gt2G2, 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 t2G2tC(A/G)A2G2t 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.
authors.affiliation: Graduate School of Science and Technology, Gunma University, Kiryu, Gunma, Japan.
authors.forename: Yuuya
authors.initials: Y
authors.lastname: Kasahara
authors.forename: Yuuta
authors.initials: Y
authors.lastname: Irisawa
authors.forename: Hiroto
authors.initials: H
authors.lastname: Fujita
authors.forename: Aiko
authors.initials: A
authors.lastname: Yahara
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Ozaki
authors.forename: Satoshi
authors.initials: S
authors.lastname: Obika
authors.forename: Masayasu
authors.initials: M
authors.lastname: Kuwahara
mesh_terms: D052157:Aptamers, Nucleotide / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D017931:DNA Primers / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019075:Electrophoresis, Capillary / Q000379:methods*; D054856:G-Quadruplexes; D006801:Humans; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D052156:SELEX Aptamer Technique / Q000379:methods*; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity
chemical_list: D052157:Aptamers, Nucleotide; D017931:DNA Primers; D009841:Oligonucleotides; C477371:locked nucleic acid