title: Identification of the target binding site of ethanolamine-binding aptamers and its exploitation for ethanolamine detection.
abstract: Aptamers are promising recognition elements for sensitive and specific detection of small molecules. We have previously selected ssDNA aptamers for ethanolamine, one of the smallest aptamer targets so far. The work presented here focuses on the determination of the binding region within the aptamer structure and its exploitation for the development of an aptamer-based assay for detection of ethanolamine. Sequence analysis of the aptamers resulted in the identification of a G-rich consensus sequence, which was able to fold in a typical two- or three-layered G-quartet structure. Experiments with stepwise truncated variants of the aptamers revealed that the consensus sequence is responsible and sufficient for binding to the target. On the basis of the knowledge of the aptamers binding site, we developed an aptamer-based microarray assay relying on competition between ethanolamine and an oligonucleotide complementary to the consensus sequence. Competitive binding of ethanolamine and fluorescently labeled complementary oligonucleotides resulted in fluorescence intensities dependent on ethanolamine concentration with a limit of detection of 10 pM. This method enables detection of small molecules without any labeling of analytes. The competitive assay could potentially be transferred to other aptamers and thus provides a promising system for aptamer-based detection of diverse small molecules.
authors.affiliation: Gottfried Wilhelm Leibniz Universität Hannover, Institut für Technische Chemie, Callinstr. 5, D-30167 Hannover, Germany.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Heilkenbrinker
authors.forename: Christine
authors.initials: C
authors.lastname: Reinemann
authors.forename: Regina
authors.initials: R
authors.lastname: Stoltenburg
authors.forename: Johanna-Gabriela
authors.initials: JG
authors.lastname: Walter
authors.forename: André
authors.initials: A
authors.lastname: Jochums
authors.forename: Frank
authors.initials: F
authors.lastname: Stahl
authors.forename: Stefan
authors.initials: S
authors.lastname: Zimmermann
authors.forename: Beate
authors.initials: B
authors.lastname: Strehlitz
authors.forename: Thomas
authors.initials: T
authors.lastname: Scheper
mesh_terms: D052157:Aptamers, Nucleotide / Q000737:chemistry / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D015374:Biosensing Techniques / Q000379:methods*; D004983:Ethanolamines / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000737:chemistry*; D008969:Molecular Sequence Data
chemical_list: D052157:Aptamers, Nucleotide; D004983:Ethanolamines; D005456:Fluorescent Dyes