New insights into a classic aptamer: binding sites, cooperativity and more sensitive adenosine detection.

The DNA aptamer for adenosine (also for AMP and ATP) is a highly conserved sequence that has recurred in a few selections. It it a widely used model aptamer for biosensor development, and its nuclear magnetic resonance structure shows that each aptamer binds two AMP molecules. In this work, each binding site was individually removed by rational sequence design, while the remaining site still retained a similar binding affinity and specificity as confirmed by isothermal titration calorimetry. The thermodynamic parameters of binding are presented, and its biochemical implications are discussed. The number of binding sites can also be increased, and up to four sites are introduced in a single DNA sequence. Finally, the different sequences are made into fluorescent biosensors based on the structure-switching signaling aptamer design. The one-site aptamer has 3.8-fold higher sensitivity at lower adenosine concentration with a limit of detection of 9.1 μM adenosine, but weaker fluorescence signal at higher adenosine concentrations, consistent with a moderate cooperativity in the original aptamer. This work has offered insights into a classic aptamer for the relationship between the number of binding sites and sensitivity, and a shorter aptamer for improved biosensor design.