High-Throughput Aptamer Characterization via Real-Time Nuclease Digestion

Area of Science:

• Biotechnology and Molecular Biology
• Analytical Chemistry
• Biochemistry

Background:

• Aptamers are valuable biorecognition elements for various applications due to their high-affinity binding.
• Characterizing aptamer-ligand interactions accurately and at high throughput remains a significant challenge.
• Previous exonuclease digestion assays offered parallel, label-free screening but lacked sufficient throughput.

Purpose of the Study:

• To develop a next-generation, high-throughput assay for screening aptamer-ligand interactions.
• To improve the efficiency, precision, and simplicity of aptamer characterization.
• To enable label-free, real-time monitoring of aptamer binding.

Main Methods:

• Development of a real-time exonuclease (RT-Exo) assay utilizing a fluorogenic aptamer reporter.
• Monitoring exonucleolytic digestion of aptamers in real time using automated readers.
• Validation of the RT-Exo assay with diverse aptamers and targets under various conditions.

Main Results:

• The RT-Exo assay achieves throughput orders of magnitude higher than previous methods with reduced labor and resources.
• The assay demonstrated robust performance across different aptamer structures and target properties.
• Successful application in screening mutants for structure-activity studies and profiling binding to 120 fentanyl analogs in a single run.

Conclusions:

• The RT-Exo assay significantly enhances the throughput, precision, and simplicity of aptamer characterization.
• This method streamlines the process of screening aptamer-ligand interactions.
• The RT-Exo assay is a powerful tool for advancing aptamer-based applications and research.