Selection and Characterization of Single Stranded DNA Aptamers for the Hormone Abscisic Acid
| Autor: | Tiziana Vigliarolo, Annalisa Salis, Víctor M. González, Enrico Millo, Cristina D'Arrigo, Elena Zocchi, Lucrezia Guida, Luca Bagnasco, Elena M. Martin, Marta Bellotti, Antonio De Flora, Laura Sturla, Alessia Grozio |
|---|---|
| Rok vydání: | 2013 |
| Předmět: |
Aptamer
Molecular Sequence Data Biotin DNA Single-Stranded Biology Ligands Polymerase Chain Reaction Biochemistry law.invention chemistry.chemical_compound Original Research Reports law Drug Discovery Cyclic AMP Escherichia coli Genetics Humans Biotinylation Cloning Molecular Molecular Biology Abscisic acid Cells Cultured Polymerase chain reaction single stranded DNA Base Sequence organic chemicals SELEX Aptamer Technique fungi aptamer food and beverages streptavidin Aptamers Nucleotide Small molecule Kinetics chemistry Magnets Nucleic Acid Conformation Molecular Medicine Streptavidin DNA Systematic evolution of ligands by exponential enrichment Abscisic Acid Granulocytes |
| Zdroj: | Nucleic acid therapeutics 23 (2013): 322–331. doi:10.1089/nat.2013.0418 info:cnr-pdr/source/autori:Grozio A.; Gonzalez VM.; Millo E.; Sturla L.; Vigliarolo T.; Bagnasco L.; Guida L.; D'Arrigo C.; De Flora A.; Salis A.; Martin EM.; Bellotti M.; Zocchi E./titolo:Selection and characterization of single stranded DNA aptamers for the hormone abscisic acid/doi:10.1089%2Fnat.2013.0418/rivista:Nucleic acid therapeutics (Print)/anno:2013/pagina_da:322/pagina_a:331/intervallo_pagine:322–331/volume:23 |
| ISSN: | 2159-3345 2159-3337 |
| DOI: | 10.1089/nat.2013.0418 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|