Hi-Fi SELEX: A High-Fidelity Digital-PCR Based Therapeutic Aptamer Discovery Platform

Area of Science:

• Biotechnology
• Molecular Biology
• Drug Discovery

Background:

• Systematic Evolution of Ligands by Exponential Enrichment (SELEX) is standard for aptamer discovery.
• Traditional SELEX faces limitations including non-specific aptamer retention and amplification artifacts.
• Current screening criteria (binding affinity) may not correlate with therapeutic efficacy.

Purpose of the Study:

• Introduce High-Fidelity (Hi-Fi) SELEX, a novel platform to improve aptamer selection stringency and efficiency.
• Address limitations of conventional SELEX for discovering high-affinity and therapeutically relevant aptamers.
• Demonstrate the platform's capability in identifying therapeutic aptamer candidates.

Main Methods:

• Hi-Fi SELEX incorporates fixed-region blocking elements to preserve library diversity.
• Engineered target-display surface chemistry and solvent composition minimize non-specific aptamer retention.
• Digital PCR is used for amplification, eliminating artifacts and ensuring stoichiometric conversion to single-stranded DNA.

Main Results:

• Achieved partition efficiencies approaching 10^6.
• Demonstrated rapid enrichment of DNA aptamers with mean equilibrium dissociation constants (Kd) in the low nanomolar range within three rounds.
• Successfully identified therapeutic aptamer candidates against human factor D.

Conclusions:

• Hi-Fi SELEX significantly improves aptamer selection efficiency and specificity compared to traditional methods.
• The platform enables rapid discovery of high-affinity aptamers with therapeutic potential.
• This technology offers a robust approach for identifying novel aptamer therapeutics.