Environment and Natural Sciences, Institute of Biotechnology, Brandenburg Technical University Cottbus-Senftenberg, Universitätsplatz 1, 01968, Senftenberg, Germany.
Faculty of Health Brandenburg, Brandenburg University of Technology Cottbus-Senftenberg, Senftenberg, Germany.
Sci Rep. 2022 Feb 22;12(1):2961. doi: 10.1038/s41598-022-06817-0.
For improving aptamer-ligand binding we have developed a screening system that defines optimal binding buffer composition. Using multiplex assays, one buffer system is needed which guarantees the specific binding of all aptamers. We investigated nine peer-reviewed DNA aptamers. Non-specific binding of aptamers is an obstacle. To address this, we investigated 16 proteins as specificity controls bound covalently to encoded microbeads in a multiplex assay. Increasing the NaCl concentration decreased the binding for all aptamers. Changing pH values by one unit higher or lower did not influence the aptamer binding significantly. However, pH < 5 led to non-specific binding for all aptamers. The PfLDH-aptamer selected in the absence of divalent cations exhibited doubling of its binding signal by the addition of Ca and Mg. We confirmed Ca and Mg dependency of the aptamers for streptavidin and thrombin by observing a 90% and 50% binding decrease, respectively. We also achieved a doubling of binding for the streptavidin aptamer when replacing Ca and Mg by Mn. A buffer suitable for all aptamers can have considerable variations in pH or ionic strength, but divalent cations (Ca, Mg, Mn) are essential.
为了提高适配体-配体的结合能力，我们开发了一种筛选系统，可以确定最佳的结合缓冲液组成。使用多重分析，需要有一种缓冲液系统来保证所有适配体的特异性结合。我们研究了 9 种经过同行评审的 DNA 适配体。适配体的非特异性结合是一个障碍。为了解决这个问题，我们研究了 16 种蛋白质作为特异性对照物，它们通过共价结合固定在编码微珠上，进行多重分析。增加 NaCl 浓度会降低所有适配体的结合。将 pH 值改变一个单位不会显著影响适配体的结合。然而，pH 值<5 会导致所有适配体的非特异性结合。在没有二价阳离子的情况下选择的 PfLDH-适配体，通过添加 Ca 和 Mg，其结合信号增加了一倍。我们通过观察到分别为 90%和 50%的结合减少，证实了 Ca 和 Mg 对链霉亲和素和凝血酶适配体的依赖性。当用 Mn 替代 Ca 和 Mg 时，我们还使链霉亲和素适配体的结合增加了一倍。适合所有适配体的缓冲液在 pH 值或离子强度上可能有很大的变化，但二价阳离子（Ca、Mg、Mn）是必需的。
