ABSTRACT
We successfully generated chimeric DNA aptamers that contained six nucleoside analogs of 2'-O,4'-C-methylene bridged/locked nucleic acid (2',4'-BNA/LNA) in the primer region and multiple guanosine analogs of 2'-deoxy-2'-fluoro-ribonucleic acid (FNA) in the non-primer region using capillary electrophoresis-systematic evolution of ligands by exponential enrichment (CE-SELEX). Active species enrichment became saturated only after five selection rounds, and we obtained DNA-based xeno-nucleic acid (XNA) aptamers that had high binding affinities for the target human thrombin, with dissociation constant (Kd) values of ≥10 nanomolar. Based on sequence and circular dichroism (CD) analyses, these XNA aptamers exhibited RNA-like conformations, which could cause DNA-based strands to adopt structurally diverse conformations.
Subject(s)
Aptamers, Nucleotide/chemistry , Gene Library , Nucleic Acids/chemistry , Oligonucleotides/chemistry , RNA/chemistry , SELEX Aptamer Technique , Aptamers, Nucleotide/genetics , Aptamers, Nucleotide/pharmacokinetics , Binding Sites , Humans , Kinetics , Nucleic Acid Conformation , Nucleic Acids/genetics , Oligonucleotides/genetics , Protein Binding , Structure-Activity Relationship , Thrombin/metabolismABSTRACT
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.
Subject(s)
Aptamers, Nucleotide/chemistry , Aptamers, Nucleotide/metabolism , DNA Primers/metabolism , Electrophoresis, Capillary/methods , Oligonucleotides/metabolism , SELEX Aptamer Technique/methods , Aptamers, Nucleotide/genetics , Base Sequence , DNA Primers/chemistry , DNA Primers/genetics , G-Quadruplexes , Humans , Molecular Sequence Data , Oligonucleotides/chemistry , Oligonucleotides/genetics , Sequence Analysis, DNA , Substrate SpecificityABSTRACT
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.
