title: Selection and characterization of single stranded DNA aptamers for the hormone abscisic Acid.
abstract: The hormone abscisic acid (ABA) is a small molecule involved in pivotal physiological functions in higher plants. Recently, ABA has been also identified as an endogenous hormone in mammals, regulating different cell functions including inflammatory processes, stem cell expansion, insulin release, and glucose uptake. Aptamers are short, single-stranded (ss) oligonucleotidesable to recognize target molecules with high affinity. The small size of the ABA molecule represented a challenge for aptamer development and the aim of this study was to develop specific anti-ABA DNA aptamers. Biotinylated abscisic acid (bio-ABA) was immobilized on streptavidin-coated magnetic beads. DNA aptamers against bio-ABA were selected with 7 iterative rounds of the systematic evolution of ligands by exponential enrichment method (SELEX), each round comprising incubation of the ABA-binding beads with the ssDNA sequences, DNA elution, electrophoresis, and polymerase chain reaction (PCR) amplification. The PCR product was cloned and sequenced. The binding affinity of several clones was determined using bio-ABA immobilized on streptavidin-coated plates. Aptamer 2 and aptamer 9 showed the highest binding affinity, with dissociation constants values of 0.98 ± 0.14 μM and 0.80 ± 0.07 μM, respectively. Aptamers 2 and 9 were also able to bind free, unmodified ABA and to discriminate between different ABA enantiomers and isomers. Our findings indicate that ssDNA aptamers can selectively bind ABA and could be used for the development of ABA quantitation assays.
authors: Department of Experimental Medicine, Section of Biochemistry, University of Genova, Italy.
authors: Alessia
authors: A
authors: Grozio
authors: Victor M
authors: VM
authors: Gonzalez
authors: Enrico
authors: E
authors: Millo
authors: Laura
authors: L
authors: Sturla
authors: Tiziana
authors: T
authors: Vigliarolo
authors: Luca
authors: L
authors: Bagnasco
authors: Lucrezia
authors: L
authors: Guida
authors: Cristina
authors: C
authors: D'Arrigo
authors: Antonio
authors: A
authors: De Flora
authors: Annalisa
authors: A
authors: Salis
authors: Elena M
authors: EM
authors: Martin
authors: Marta
authors: M
authors: Bellotti
authors: Elena
authors: E
authors: Zocchi
mesh_terms: D000040:Abscisic Acid / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism; D052157:Aptamers, Nucleotide / Q000737:chemistry* / Q000235:genetics; D001483:Base Sequence; D001710:Biotin / Q000737:chemistry; D019921:Biotinylation; D002478:Cells, Cultured; D003001:Cloning, Molecular; D000242:Cyclic AMP / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000737:chemistry* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D006098:Granulocytes / Q000166:cytology / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008024:Ligands; D059346:Magnets; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D016133:Polymerase Chain Reaction; D052156:SELEX Aptamer Technique; D019809:Streptavidin / Q000737:chemistry
chemical_list: D052157:Aptamers, Nucleotide; D004277:DNA, Single-Stranded; D008024:Ligands; D001710:Biotin; D000040:Abscisic Acid; D019809:Streptavidin; D000242:Cyclic AMP
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