Evaluation of Staphylococcus aureus DNA aptamer by enzyme‐linked aptamer assay and isothermal titration calorimetry

• Published in: Journal of molecular recognition 2017-02, Vol.30 (2), p.np-n/a
• Main Authors: Bayrac, Ceren, Oktem, Huseyin Avni
• Format: Article
• Language: English
• Subjects: Affinity; Aptamers; Aptamers, Nucleotide - chemistry; Aptamers, Nucleotide - metabolism; Assaying; Bacteria; Bacterial Proteins - chemistry; Binding; Biotin; Calorimetry - methods; Deoxyribonucleic acid; DNA; DNA aptamer; Enzymes; enzyme‐linked aptamer assay; Exothermic reactions; Heat measurement; Horseradish Peroxidase - chemistry; Ion concentration; isothermal titration calorimetry; Kinetics; Models, Molecular; Nucleic Acid Conformation; Peroxidase; Proteins; Recognition; Staphylococcus aureus; Staphylococcus aureus - genetics; Staphylococcus aureus - growth & development; S aureus; Titration calorimetry
• ISSN: 0952-3499; 1099-1352
• DOI: 10.1002/jmr.2583

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 103 colony‐forming unit per milliliter. The apparent Ka was 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−1 at 25°C to Ka = 1.03 μM−1 ± 0.9 μM−1 at 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−1 at 50mM, 1.60 μM−1 ± 0.11 μM−1 at 137mM, and 3.28 μM−1 ± 0.46 μM−1 at 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. In this study, Staphylococcus aureus DNA aptamer showed higher specificity against its target based on enzyme‐linked aptamer assay, and also the potential use of enzyme‐linked aptamer assay as a detection platform for S aureus was demonstrated with the use of DNA aptamer as recognition agent. Besides, without prior knowledge of exact target, identification of aptamer binding characteristics has been possible with isothermal titration calorimetry. Temperature and salt concentrations influenced directly 3‐dimensional structure of DNA aptamer along with the affinity. Isothermal titration calorimetry was used first for studying the fundamental binding mechanism between aptamer and its extracted target.