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

Date

2017-02-01

Author

BAYRAÇ, CEREN
Öktem, Hüseyin Avni

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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 K-a was 1.39 mu M-1 +/- 0.3 mu M-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 (K-a, Delta H, and Delta G). 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 degrees 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 K-a = 1.56 mu M-1 +/- 0.69 mu M-1 at 25 degrees C to K-a = 1.03 mu M-1 +/- 0.9 mu M-1 at 37 degrees C. At spontaneously occurring exothermic binding reactions, affinities of S aureus aptamer to its target were also 9.44 mu M-1 +/- 0.38 mu M-1 at 50mM, 1.60 mu M-1 +/- 0.11 mu M-1 at 137mM, and 3.28 mu M-1 +/- 0.46 mu M-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.

Subject Keywords

DNA aptamer, Enzyme-linked aptamer assay, Isothermal titration calorimetry, S aureus

URI

https://hdl.handle.net/11511/36076

Journal

JOURNAL OF MOLECULAR RECOGNITION

DOI

https://doi.org/10.1002/jmr.2583

Collections

Department of Biology, Article

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Citation Formats

C. BAYRAÇ and H. A. Öktem, “Evaluation of Staphylococcus aureus DNA aptamer by enzyme-linked aptamer assay and isothermal titration calorimetry,” JOURNAL OF MOLECULAR RECOGNITION, pp. 0–0, 2017, Accessed: 00, 2020. [Online]. Available: https://hdl.handle.net/11511/36076.