Combining capillary electrophoresis and next-generation sequencing for aptamer selection

Area of Science:

• Biotechnology
• Molecular Biology
• Analytical Chemistry

Background:

• Next-generation sequencing (NGS) enables high-throughput analysis of complex biological molecules.
• Aptamers are short DNA or RNA molecules that bind to specific targets.
• Selecting functional aptamers from large libraries is a critical challenge in molecular biology.

Purpose of the Study:

• To evaluate capillary electrophoresis (CE) as a method for aptamer selection.
• To assess the efficiency of capillary transient isotachophoresis (ctITP)-based nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM) for aptamer enrichment.
• To quantify the enrichment of thrombin-binding aptamers using CE.

Main Methods:

• Utilized ctITP-NECEEM for aptamer selection from a randomized library.
• Employed capillary electrophoresis with minimal sample volumes (100 nL).
• Sequenced selected aptamer libraries using Ion Torrent Personal Genome Machine (PGM) for analysis.

Main Results:

• Demonstrated a significant enrichment of thrombin-binding aptamers after one round of CE selection.
• Increased aptamer content from 0.4% to over 15% in the selected DNA oligo mixture.
• Validated the quantitative determination of target-binding aptamer enrichment in a solution-phase environment.

Conclusions:

• CE, specifically ctITP-NECEEM, is a highly efficient method for aptamer selection.
• This technique offers a rapid and quantitative approach to enrich functional aptamers.
• CE-based selection is a valuable tool for accelerating aptamer discovery and development.