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Evaluation of Staphylococcus aureus DNA aptamer by enzyme‐linked aptamer assay and isothermal titration calorimetry
Author(s) -
Bayraç Ceren,
Öktem Hüseyin Avni
Publication year - 2017
Publication title -
journal of molecular recognition
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.401
H-Index - 79
eISSN - 1099-1352
pISSN - 0952-3499
DOI - 10.1002/jmr.2583
Subject(s) - aptamer , isothermal titration calorimetry , chemistry , binding constant , chromatography , staphylococcus aureus , lysis , horseradish peroxidase , dna , titration , dissociation constant , binding site , enzyme , biochemistry , bacteria , microbiology and biotechnology , inorganic chemistry , biology , genetics , receptor
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 10 3 colony‐forming unit per milliliter. The apparent K a 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 ( K a , Δ H , and Δ 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°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 μM −1 ± 0.69 μM −1 at 25°C to K a = 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 .