From Theophylline to Adenine or preQ ₁ : Repurposing a DNA Aptamer Revealed by Crystal Structure Analysis

Abstract Aptamers, which are short, single‐stranded DNA or RNA, are capable of binding to a wide array of targets with exceptional selectivity. Those with high affinity for theophylline have the potential to serve as biosensors, crucial for tracking theophylline levels in the treatment of respiratory conditions. Despite the extensive structural characterization of the RNA theophylline aptamer, the DNA counterpart's ligand‐recognition mechanism has remained unclear. Here, we elucidate the DNA theophylline aptamer's ligand‐binding mechanism through high‐resolution crystal structures of its complexes with theophylline, 3‐methylxanthine, and hypoxanthine. Guided by these structural insights, we computationally redesigned the theophylline‐binding pocket via rational mutagenesis of key nucleotides, generating novel aptamers selective for adenine and prequeuosine (preQ 1 ) ligands. These engineered aptamers were validated through biochemical and crystallographic analyses. By integrating structural biology with computational design, our work provides a relatively simple and effective method for developing new aptamers. While this strategy does not supplant SELEX, it serves as a beneficial complement to it.