Measurement of (Aptamer–Small Target) KD Using the Competition between Fluorescently Labeled and Unlabeled Targets and the Detection of Fluorescence Anisotropy

Samokhvalov, A.V.; Safenkova, I.V.; Eremin, S.A.; Zherdev, A.V.; Dzantiev, B.B.

Авторы: Samokhvalov Alexey V., Safenkova Irina V., Eremin Sergei A., Zherdev Anatoly V., Dzantiev Boris B.
Журнал: ANALYTICAL CHEMISTRY
Том: 90
Номер: 15
Год издания: 2018
Первая страница: 9189
Последняя страница: 9198
DOI: 10.1021/acs.analchem.8b01699
Аннотация: Registration of fluorescence anisotropy (FA) allows for characterizing the interactions of ligands with aptamers and other receptors under homogeneous conditions without reagents immobilization, prolonged incubations and products separation. We proposed approach for aptamer’s affinity determination by FA taking into account the difference in label fluorescence before and after complexation. The detailed step by step scheme using a native and fluorescently labeled ligand was described and justified in the paper. The scheme ensures the exclusion of data with low reliability and establishes valid criteria for selecting optimal concentrations of reagents (labeled ligand and aptamer) used in the experiments. The approach was experimentally tested using ochratoxin A (OTA), its fluorescein-labeled derivative (OTA-Flu), and the aptamer binding them. We demonstrated that it allows minimizing the influence of fluorescence change to accurately determine the dissociation constant. Based on FA registration, the binding constants of the aptamer–OTA-Flu and the aptamer–OTA complexes were found to be equal 245+33 and 63+18 nM, respectively. The value for the aptamer–OTA complexes was confirmed by the equilibrium dialysis technique. The resulting constant was 80±9 nM. The versatility and methodological simplicity of the proposed protocol, as well as the short implementation time, are why it can be recommended as an effective tool for characterizing
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Measurement of (Aptamer–Small Target) KD Using the Competition between Fluorescently Labeled and Unlabeled Targets and the Detection of Fluorescence Anisotropyстатья

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