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

Area of Science:

• Biochemistry
• Molecular Biology
• Analytical Chemistry

Background:

• Aptamer-ligand binding is crucial for various applications.
• Non-specific binding of aptamers hinders assay performance.
• A universal buffer system is needed for multiplex aptamer assays.

Purpose of the Study:

• To develop a screening system for optimal aptamer-ligand binding buffer composition.
• To identify buffer conditions that ensure specific binding for multiple DNA aptamers.
• To investigate the impact of ionic strength, pH, and divalent cations on aptamer binding.

Main Methods:

• Development of a multiplex assay system.
• Investigation of nine peer-reviewed DNA aptamers.
• Use of 16 proteins as specificity controls bound to microbeads.
• Systematic variation of NaCl concentration, pH, and divalent cation presence (Ca2+, Mg2+, Mn2+).

Main Results:

• Increased NaCl concentration reduced binding for all aptamers.
• pH variations within one unit did not significantly affect binding, but pH < 5 induced non-specific binding.
• Divalent cations (Ca2+, Mg2+) significantly enhanced binding for the PfLDH-aptamer.
• Ca2+ and Mg2+ were confirmed as essential for streptavidin and thrombin aptamer binding.
• Mn2+ could substitute Ca2+ and Mg2+ for streptavidin aptamer binding, doubling its signal.

Conclusions:

• A single buffer system can accommodate various aptamers with controlled variations in pH and ionic strength.
• Divalent cations (Ca2+, Mg2+, Mn2+) are critical components for achieving specific and enhanced aptamer-ligand binding.
• The developed screening system aids in defining optimal buffer conditions for aptamer-based assays.