Improving Aptamer Affinity and Determining Sequence–Activity Relationships via Motif-SELEX

via Systematic Evolution of Ligands by Exponential Enrichment (SELEX) is often limited because the entire potential sequence space cannot be screened. In this study, we introduce Motif-SELEX, a novel method that enables the optimization of existing underperforming aptamers by generating libraries that broadly represent both the sequence and length variations of the parent sequence. This approach enables the isolation of sequences with improved affinity without the biases and limitations of traditional mutagenesis methods like doped SELEX and error-prone PCR. As a demonstration, we applied Motif-SELEX to a DNA-based morphine aptamer and a 2' fluoro- and methoxy-RNA-based apixaban aptamer, discovering new, better-performing sequences with differing random domain lengths from their parents and up to 10-fold improvements in affinity. These new sequences would be inaccessible to traditional post-SELEX methods. Critically, our analysis of Motif-SELEX pools also enabled us to identify sequence and structural elements crucial for target binding and to predict secondary and tertiary structures for a given aptamer family─even when those structures involve noncanonical nucleotide interactions. We believe that Motif-SELEX offers an effective and generalizable solution for optimizing the structure and binding properties of functional nucleic acid molecules for diverse applications.