Selection-by-function: efficient enrichment of cathepsin E inhibitors from a DNA library

Area of Science:

• Biochemistry
• Molecular Biology
• Biotechnology

Background:

• Protease inhibitors are crucial therapeutic agents.
• Developing efficient methods for aptamer selection is essential.
• Cathepsin E (CE) is a target protease implicated in various diseases.

Purpose of the Study:

• To develop an efficient method for enriching protease inhibitors from DNA libraries.
• To identify novel aptamers with inhibitory activity against Cathepsin E.
• To introduce and validate the SF-link technology for aptamer screening.

Main Methods:

• Development of SF-link technology combining aptamer binding and functional inhibition.
• A two-step selection strategy involving initial aptamer enrichment and subsequent functional selection.
• Covalent linking of a biotinylated peptide substrate to ssDNA molecules for SF-link generation.
• Characterization of selected aptamers, including IC50, Kd, and selectivity assays.

Main Results:

• The SF-link technology enabled gradual enrichment of inhibitory DNA molecules.
• A specific aptamer, SFR-6-3, was identified with an IC50 of approximately 30 nM and Kd of approximately 15 nM for CE.
• SFR-6-3 demonstrated high selectivity for Cathepsin E.
• Sequence analysis revealed a novel G-free, C-rich sequence potentially forming an i-motif structure.

Conclusions:

• SF-link technology is a novel and effective screening tool for identifying specific protease inhibitors.
• The identified aptamer SFR-6-3 is a potent and selective inhibitor of Cathepsin E.
• The developed method has potential for broader applications in aptamer discovery and development.