From SELEX to Sequencing: Uncovering enriched Aptamers with Next-Generation Sequencing and Bioinformatics

The combinatorial chemistry process called systematic evolution of ligands by exponential enrichment (SELEX) enables the selection of aptamers against different targets. The integration of next-generation sequencing (NGS) into SELEX workflows represents a powerful strategy to enhance the identification and prioritization of target-binding aptamers. In this study, we present an optimized computational approach for the NGS-based analysis of enriched aptamer pools generated through SELEX. It encompasses sequencing library preparation, sequence data preprocessing, clustering, multiple sequence alignment, phylogenetic analysis, and secondary structure prediction.

To identify target-specific aptamers, two comparative analysis strategies were implemented. In the first strategy, sequences detected in the negative selection pool were removed from the positive selection pool to eliminate nonspecific binders, resulting in six distinct sequence clusters. In the second approach, all sequences were retained, and cluster distributions were compared between the positive and negative selection pool, identifying seven clusters in the positive and six in the negative pool. Phylogenetic relationships and structural predictions enabled the selection of promising candidates, which were prioritized based on secondary structure, sequence abundance, and genetic distance from non-specific binders. Binding specificity was assessed through dot blot assays, confirming that the selected aptamers exhibited strong specific binding to the target protein. These results demonstrate the effectiveness of the NGS-guided workflow for aptamer discovery and highlight its value in streamlining candidate selection for downstream functional validation.