An engineered one-site aptamer with higher sensitivity for label-free detection of adenosine on graphene oxide

The 27-nucleotide DNA aptamer for adenosine and ATP, originally selected by the Szostak lab in 1995, has been a very popular model system for biosensor development. This unique aptamer has two target binding sites, and we recently showed that it is possible to remove either site while the other one still retains binding. From an analytical perspective, tuning the number of binding sites has important implications in modulating sensitivity of the resulting biosensors. In this work, we report that the engineered one-site aptamer showed excellent signaling properties with a 2.6-fold stronger signal intensity and also a 4.2-fold increased detection limit compared with the wild-type two-site aptamer. The aptamer has a hairpin structure, and the length of the hairpin stem was systematically varied for the one-site aptamers. Isothermal titration calorimetry and a label-free fluorescence signaling method with graphene oxide and SYBR Green I were respectively used to evaluate binding and sensor performance. Although longer stemmed aptamers produced better adenosine binding affinity, the signaling was quite independent of the stem length as long as more than three base pairs were left. This was explained by the higher affinity of binding to GO by the longer aptamers, cancelling out the higher affinity for adenosine binding. This work further confirms the analytical applications of such one-site adenosine aptamers, which are potentially useful for improved ATP imaging and for developing new biosensors.