The Role of Surfactants in Stabilizing Fluorescence Anisotropy for Protein-Aptamer Binding Affinity Measurements (Dataset)

Samarakoon, Bhagya Roshani; Bilderback, Susan; Whelan, Rebecca J.

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The Role of Surfactants in Stabilizing Fluorescence Anisotropy for Protein-Aptamer Binding Affinity Measurements (Dataset)

Samarakoon, Bhagya Roshani
;
Bilderback, Susan
;
Whelan, Rebecca

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Abstract

Fluorescence Anisotropy (FA) is a sensitive, efficient technique for quantifying biomolecular interactions, offering advantages such as minimal sample requirements and elimination of separation of bound from unbound species. Thus, it is well suited for aptamer–protein binding affinity studies. However, accurately determining equilibrium dissociation constants (Kd) in FA requires low concentrations of fluorescently labeled aptamers to prevent ligand depletion. A significant challenge arises at low aptamer concentrations due to an unexpected increase in anisotropy, which introduces artifacts in the calibration curve and impairs accurate data fitting. This anomaly may arise from non-specific adsorption of aptamers to surfaces. In this study we investigated the use of non-ionic surfactants to mitigate these effects and stabilize anisotropy signal at low aptamer concentrations using the thrombin aptamer as a model system. We evaluated the impact of varying concentrations of Tween 20 and Triton X-100 on calibration curves and determined binding affinities. Addition of 0.1% Tween suppresses the anomalous increase in anisotropy at low aptamer concentrations, enabling the use of aptamer concentrations as low as 5 nM. Triton X-100 was less effective. By incorporating optimized concentrations of Tween 20, we demonstrated improved assay reproducibility and accuracy in Kd determination, expanding the dynamic range of usable aptamer concentrations in FA based binding affinity studies. Similar benefits were observed with the clinically relevant aptamer s10yh2 and human serum albumin. These findings provide a practical strategy for enhancing the robustness of FA measurements and may be applicable to other aptamer–target systems and high-throughput assay formats.