Reagentless Real‐Time ATP Monitoring with New DNA Aptamers

Abstract Adenosine triphosphate (ATP) is essential to numerous biological processes, and there is considerable interest in methods for measuring this molecule in real time. Current ATP sensors have fundamental shortcomings that limit their utility, such as poor specificity, the need for exogenous reagents, or assay complexity. Aptamer‐based biosensors have shown great promise for ATP detection, but existing aptamers have modest affinity and poor specificity that limit their practical utility. Here, the systematic evolution of ligands by exponential enrichment (SELEX) process is used to discover new DNA aptamers that bind ATP with unprecedentedly high affinity and specificity. The best‐performance aptamer, ATP18‐13, displays at least tenfold greater affinity for ATP relative to previously reported aptamers, with far superior capacity to discriminate against analogs including adenosine diphosphate (ADP), adenosine monophosphate (AMP), and adenosine. ATP18‐13 is used to develop a fluorescent beacon sensor that achieves a limit of detection of 125 n m ATP in buffer and 1 µ m ATP in cell culture medium. The capability of this sensor is demonstrated to achieve real‐time, seconds‐scale monitoring of deoxyATP consumption during DNA polymerase‐mediated template extension. The aptamers and sensors developed here can thus prove useful for monitoring ATP in a variety of biological contexts.