Hydrogel-Bead Particles Enable Activity-Based Identification of Nucleic Acid Aptamer Enzyme Inhibitors.

Area of Science:

• Biotechnology
• Molecular Biology
• Biochemistry

Background:

• Nucleic acid aptamers offer advantages over antibodies as ligands due to their stability, selectivity, and low immunogenicity.
• Current aptamer discovery methods rely on affinity selection, which only identifies sequences based on target binding, not functional activity.

Purpose of the Study:

• To develop an activity-based aptamer screening system for enhanced aptamer discovery.
• To enable the selection of aptamers based on both target binding and functional activity, moving beyond traditional affinity-based methods.

Main Methods:

• Developed a system using magnetic beads templated with aptamer-encoding DNAs encapsulated in polyacrylamide hydrogel droplets.
• Employed transcription-reverse transcription amplification to copy magnetic bead-bound DNAs to the gel periphery.
• Created an in-gel activity assay using a model enzyme (trypsin) and a fluorescently labeled substrate peptide, inhibited by specific aptamers.

Main Results:

• Successfully demonstrated proof-of-concept screens that enriched positive controls by 100,000-fold over multiple rounds.
• The activity-based assay allowed for sorting based on high Cy5 fluorescence, indicating functional aptamer presence.
• Validated the system's ability to select functional aptamers, not just binders.

Conclusions:

• The developed activity-based aptamer screening system significantly enhances the efficiency and scope of aptamer discovery.
• This technology can select aptamers based on functional activity, overcoming limitations of traditional affinity-based methods.
• The platform is adaptable for DNA aptamers, non-natural nucleic acids, and other encoded library formats.