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

Abstract

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.