Selection, Characterization and Application of Artificial DNA Aptamer Containing Appended Bases with Sub-nanomolar Affinity for a Salivary Biomarker.
Sci Rep
; 7: 42716, 2017 03 03.
Article
em En
| MEDLINE
| ID: mdl-28256555
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.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Saliva
/
Aptâmeros de Nucleotídeos
Tipo de estudo:
Prognostic_studies
Limite:
Humans
Idioma:
En
Revista:
Sci Rep
Ano de publicação:
2017
Tipo de documento:
Article
País de afiliação:
Japão