title: Evaluation of Staphylococcus aureus DNA aptamer by enzyme-linked aptamer assay and isothermal titration calorimetry.
abstract: To monitor the specificity of Staphylococcus aureus aptamer (SA-31) against its target cell, we used enzyme-linked aptamer assay. In the presence of target cell, horseradish peroxidase-conjugated streptavidin bound to biotin-labeled SA-31 showed specific binding to S  aureus among 3 different bacteria with limit of detection of 103colony-forming unit per milliliter. The apparent Kawas 1.39 μM-1± 0.3 μM-1. The binding of SA-31 to membrane proteins extracted from cell surface was characterized using isothermal titration calorimetry, and the effect of changes in binding temperature and salt concentrations of binding buffer was evaluated based on thermodynamic parameters (Ka, ΔH, and ΔG). Since binding of aptamer to its targets solely depends on its 3-dimensional structure under experimental conditions used in selection process, the change in temperature and ion concentration changed the affinity of SA-31 to its target on surface of bacteria. At 4°C, SA-31 did not show an affinity to its target with poor heat change upon injection of membrane fraction to aptamer solution. However, the apparent association constants of SA-31 slightly varied from Ka= 1.56 μM-1± 0.69 μM-1at 25°C to Ka= 1.03 μM-1± 0.9 μM-1at 37°C. At spontaneously occurring exothermic binding reactions, affinities of S aureus aptamer to its target were also 9.44 μM-1± 0.38 μM-1at 50mM, 1.60 μM-1± 0.11 μM-1at 137mM, and 3.28 μM-1± 0.46 μM-1at 200 mM of salt concentration. In this study, it was demonstrated that enzyme-linked aptamer assay and isothermal titration calorimetry were useful tools for studying the fundamental binding mechanism between a DNA aptamer and its target on the outer surface of S aureus.
authors.affiliation: Bioengineering Department, Karamanoğlu Mehmetbey University, Yunus Emre Campus, Karaman, Turkey.|Nanobiotechnology Research and Development Group, Department of Biotechnology, Middle East Technical University, Ankara, Turkey.
authors.forename: Ceren
authors.initials: C
authors.lastname: Bayraç
authors.affiliation: Konya Food & Agriculture University, Dedekorkut Mah. Beyşehir Cad. No. 9, Konya, Turkey.|Nanobiotechnology Research and Development Group, Department of Biotechnology, Middle East Technical University, Ankara, Turkey.
authors.forename: Hüseyin Avni
authors.initials: HA
authors.lastname: Öktem
mesh_terms: D052157:Aptamers, Nucleotide / Q000737:chemistry* / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry*; D002151:Calorimetry / Q000379:methods; D006735:Horseradish Peroxidase / Q000737:chemistry*; D007700:Kinetics; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D013211:Staphylococcus aureus / Q000235:genetics / Q000254:growth & development*
chemical_list: D052157:Aptamers, Nucleotide; D001426:Bacterial Proteins; C058865:Strep-avidin conjugated horseradish peroxidase; D006735:Horseradish Peroxidase
keywords: DNA aptamer; S aureus; enzyme-linked aptamer assay; isothermal titration calorimetry