A multiparametric fluorescence assay for screening aptamer-protein interactions based on microbeads

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 Ca2+ and Mg2+. We confirmed Ca2+ and Mg2+ 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 Ca2+ and Mg2+ by Mn2+. A buffer suitable for all aptamers can have considerable variations in pH or ionic strength, but divalent cations (Ca2+, Mg2+, Mn2+) are essential.

Automated summary

In this study, we developed a screening system to identify the optimal binding buffer composition for improving aptamer-ligand binding. Using multiplex assays, we determined that a specific binding buffer system is required to ensure the specific binding of all aptamers. We found that increasing NaCl concentration decreased binding for all aptamers, while pH values did not significantly affect the aptamer binding. However, pH < 5 resulted in non-specific binding for all aptamers. Additionally, we discovered that the presence of divalent cations (Ca2+, Mg2+, Mn2+) is essential for the binding of all aptamers.