Registration of fluorescence anisotropy (FA) allows for characterizing the interactions of ligands with aptamers and other receptors under homogeneous conditions without reagent immobilization, prolonged incubations, and product separation. We proposed an approach for aptamer 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. On the basis of FA registration, the binding constants of the aptamer–OTA-Flu and the aptamer–OTA complexes were found to be equal to 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 aptamer–ligand complexes.

中文翻译：

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使用荧光标记的和未标记的靶标之间的竞争以及荧光各向异性的检测来测量（适体–小靶标）K _D

荧光各向异性（FA）的配准可表征配体与适体和其他受体在均相条件下的相互作用，而无需固定试剂，延长孵育时间和分离产物。考虑到复合之前和之后标记荧光的差异，我们提出了一种通过FA确定适体亲和力的方法。本文描述并证明了使用天然和荧光标记的配体的详细分步方案。该方案可确保以低可靠性排除数据，并建立有效标准以选择实验中使用的最佳试剂浓度（标记的配体和适体）。该方法使用to曲霉毒素A（OTA），其荧光素标记的衍生物（OTA-Flu）以及与它们结合的适体进行了实验测试。我们证明了它可以最大程度地减少荧光变化的影响，从而准确确定解离常数。基于FA配准，发现适体-OTA-Flu和适体-OTA复合物的结合常数分别等于245 + 33和63 + 18 nM。适体-OTA复合物的值已通过平衡透析技术确认。所得常数为80±9 nM。拟议协议的多功能性和方法简单性以及较短的实施时间，这就是为什么可以将其推荐为表征适体-配体复合物的有效工具的原因。发现适体-OTA-Flu和适体-OTA复合物的结合常数分别等于245 + 33和63 + 18 nM。适体-OTA复合物的值已通过平衡透析技术确认。所得常数为80±9 nM。拟议协议的多功能性和方法简单性以及较短的实施时间，这就是为什么可以将其推荐为表征适体-配体复合物的有效工具的原因。发现适体-OTA-Flu和适体-OTA复合物的结合常数分别等于245 + 33和63 + 18 nM。适体-OTA复合物的值已通过平衡透析技术确认。所得常数为80±9 nM。拟议协议的多功能性和方法简单性以及较短的实施时间，这就是为什么可以将其推荐为表征适体-配体复合物的有效工具的原因。