Selection of aptamers targeting small molecules by capillary electrophoresis: Advances, challenges, and prospects

Aptamers, as novel recognition molecules, hold immense potential across various domains such as biosensing, nucleic acid drugs, medical diagnostics, as well as environmental and food analysis. The majority of aptamer selection processes targeting small molecules and protein commonly employ magnetic bead-based methodologies, wherein the target is initially immobilized on magnetic beads, followed by magnetic separation. The Evolutionary Systematic Evolution of Ligands by Exponential Enrichment technique based on capillary electrophoresis (CE-SELEX) is acknowledged as one of the most efficient screening methods. Our research group has achieved breakthroughs in employing CE-SELEX for the selection of aptamers targeting small molecules. This paper outlines specific methodologies utilized from 2005 to 2023 for CE-SELEX screening for small-molecule targets. It summarizes the methods for the separation of small molecules and oligonucleotide complexes, as well as the identification of candidate aptamers. Drawing upon our research group's extensive experience in CESELEX for selecting aptamers targeting multi-scale targets, we offer strategic guidance specifically tailored to the screening of aptamers for small-molecule targets using CE-SELEX. This includes systematic insights into each technical aspect of the screening process: analysis of the structure of small-molecule targets and characteristics of ssDNA libraries, patterns of CE separation and collection of complexes, screening strategies, and CE-based methods for the affinity and specificity characterization of aptamers. This comprehensive review aims to contribute to the widespread adoption of CE-SELEX technology, enhancing the efficiency and success rate of selecting aptamers for small-molecule targets.