Integrated Biomedical Sciences Graduate Program, University of Notre Dame, Notre Dame, IN, USA.
Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN, USA.
Anal Methods. 2021 Mar 18;13(10):1302-1307. doi: 10.1039/d0ay02256j.
Aptamers have many useful attributes including specific binding to molecular targets. After aptamers are identified, their target binding must be characterized. Fluorescence anisotropy (FA) is one technique that can be used to characterize affinity and to optimize aptamer-target interactions. Efforts to make FA assays more efficient by reducing assay volume and time from mixing to measurement may save time and resources by minimizing consumption of costly reagents. Here, we use thrombin and two thrombin-binding aptamers as a model system to show that plate-based FA experiments can be performed in volumes as low as 2 μL per well with 20 minute incubations with minimal loss in assay precision. We demonstrate that the aptamer-thrombin interaction is best modelled with the Hill equation, indicating cooperative binding. The miniaturization of this assay has implications in drug development, as well as in the efficiency of aptamer selection workflows by allowing for higher throughput aptamer analysis.
适配体具有许多有用的属性，包括与分子靶标的特异性结合。在鉴定出适配体后，必须对其靶标结合进行特征分析。荧光各向异性（FA）是一种可用于表征亲和力和优化适配体-靶标相互作用的技术。通过减少从混合到测量的检测体积和时间来提高 FA 检测效率的努力，可以通过最小化昂贵试剂的消耗来节省时间和资源。在这里，我们使用凝血酶和两个凝血酶结合适配体作为模型系统，表明板载 FA 实验可以在每孔 2 μL 的体积下进行，孵育 20 分钟，在不损失检测精度的情况下，最小化试剂消耗。我们证明，适配体-凝血酶相互作用最适合用 Hill 方程来建模，表明存在协同结合。该检测方法的小型化在药物开发以及通过允许更高通量的适配体分析来提高适配体选择工作流程的效率方面具有重要意义。
