title: Selection, Characterization and Application of Artificial DNA Aptamer Containing Appended Bases with Sub-nanomolar Affinity for a Salivary Biomarker.
abstract: We have attained a chemically modified DNA aptamer against salivary α-amylase (sAA), which attracts researchers' attention as a useful biomarker for assessing human psychobiological and social behavioural processes, although high affinity aptamers have not been isolated from a random natural DNA library to date. For the selection, we used the base-appended base (BAB) modification, that is, a modified-base DNA library containing (E)-5-(2-(N-(2-(N6-adeninyl)ethyl))carbamylvinyl)-uracil in place of thymine. After eight rounds of selection, a 75 mer aptamer, AMYm1, which binds to sAA with extremely high affinity (Kd< 1 nM), was isolated. Furthermore, we have successfully determined the 36-mer minimum fragment, AMYm1-3, which retains target binding activity comparable to the full-length AMYm1, by surface plasmon resonance assays. Nuclear magnetic resonance spectral analysis indicated that the minimum fragment forms a specific stable conformation, whereas the predicted secondary structures were suggested to be disordered forms. Thus, DNA libraries with BAB-modifications can achieve more diverse conformations for fitness to various targets compared with natural DNA libraries, which is an important advantage for aptamer development. Furthermore, using AMYm1, a capillary gel electrophoresis assay and lateral flow assay with human saliva were conducted, and its feasibility was demonstrated.
authors.affiliation: Innovation Laboratory, NEC Solution Innovators, Ltd., 1-18-7, Shinkiba, Koto-Ku, Tokyo 136-8627, Japan.
authors.forename: Hirotaka
authors.initials: H
authors.lastname: Minagawa
authors.affiliation: Graduate School of Science and Technology, Gunma University, 1-5-1 Tenjin-cho, Kiryu, Gunma 376-8515, Japan.
authors.forename: Kentaro
authors.initials: K
authors.lastname: Onodera
authors.affiliation: Graduate School of Science and Technology, Gunma University, 1-5-1 Tenjin-cho, Kiryu, Gunma 376-8515, Japan.
authors.forename: Hiroto
authors.initials: H
authors.lastname: Fujita
authors.affiliation: Department of Life and Environmental Sciences, Chiba Institute of Technology, 2-17-1 Tsudanuma, Narashino 275-0016, Japan.
authors.forename: Taiichi
authors.initials: T
authors.lastname: Sakamoto
authors.affiliation: Innovation Laboratory, NEC Solution Innovators, Ltd., 1-18-7, Shinkiba, Koto-Ku, Tokyo 136-8627, Japan.
authors.forename: Joe
authors.initials: J
authors.lastname: Akitomi
authors.affiliation: Innovation Laboratory, NEC Solution Innovators, Ltd., 1-18-7, Shinkiba, Koto-Ku, Tokyo 136-8627, Japan.
authors.forename: Naoto
authors.initials: N
authors.lastname: Kaneko
authors.affiliation: Innovation Laboratory, NEC Solution Innovators, Ltd., 1-18-7, Shinkiba, Koto-Ku, Tokyo 136-8627, Japan.
authors.forename: Ikuo
authors.initials: I
authors.lastname: Shiratori
authors.affiliation: Graduate School of Science and Technology, Gunma University, 1-5-1 Tenjin-cho, Kiryu, Gunma 376-8515, Japan.
authors.forename: Masayasu
authors.initials: M
authors.lastname: Kuwahara
authors.affiliation: Innovation Laboratory, NEC Solution Innovators, Ltd., 1-18-7, Shinkiba, Koto-Ku, Tokyo 136-8627, Japan.
authors.forename: Katsunori
authors.initials: K
authors.lastname: Horii
authors.affiliation: Innovation Laboratory, NEC Solution Innovators, Ltd., 1-18-7, Shinkiba, Koto-Ku, Tokyo 136-8627, Japan.
authors.forename: Iwao
authors.initials: I
authors.lastname: Waga
mesh_terms: D052157:Aptamers, Nucleotide / Q000737:chemistry*; D015415:Biomarkers / Q000032:analysis; D006801:Humans; D052156:SELEX Aptamer Technique / Q000379:methods; D012463:Saliva / Q000737:chemistry*; D014498:Uracil / Q000031:analogs & derivatives
chemical_list: D052157:Aptamers, Nucleotide; D015415:Biomarkers; D014498:Uracil