A beginners guide to SELEX and DNA aptamers

Abstract

SELEX stands for "Systematic Evolution of Ligands by Exponential Enrichment." It is an in vitro, iterative, PCR-based, target-specific selection strategy used to generate single-stranded DNA (ssDNA) aptamers that bind a target of interest. Properly selected aptamers bind their targets with high affinity and specificity and have utility in a multitude of detection assays. They are thus similar to antibodies but have the advantage of being more stable and cheaper to produce. The SELEX process encompasses several steps, some of which are critical to the successful isolation of an aptamer. Careful analysis and optimization of the SELEX process are thus important. This review summarizes our own experience when we, as complete novices, were setting up the SELEX system in our lab. It is thus meant to give some general and practical but concise pointers for anyone interested in initiating their own SELEX experiments. As such, the review covers key elements of the SELEX process, including library design, target selection and immobilization strategies, aptamer binding conditions, partitioning techniques, and PCR optimization. We also discuss common pitfalls such as by-product formation and single-stranded DNA recovery challenges, along with practical strategies to overcome them. Emerging trends and post-SELEX considerations, such as sequencing, structure prediction, and chemical modifications, are included to guide beginners through every stage of aptamer development.

Keywords: DNA aptamers; High-affinity binding; Molecular recognition; Primer design; SELEX optimization; Target-specific selection.