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Using Exonucleases for Aptamer Characterization, Engineering, and Sensing

NCJ Number
Accounts of Chemical Research Volume: 56 Issue: 13 Dated: 2023 Pages: 1731–1743
Date Published
13 pages

The authors describe their research in the use of nuclease enzymes to address the problems of adequate sensitivity, bottlenecks in aptamer binding characterization, and the cost and labor associated with aptamer engineering; they also discuss their serendipitous discovery that the digestion of DNA aptamers by exonucleases is inhibited when an aptamer is bound to a ligand.


Aptamers, selected from random libraries to bind specific molecules with high affinity via an in vitro method termed systematic evolution of ligands by exponential enrichment (SELEX), have been generated for diverse targets ranging from metal ions to small molecules to proteins and have demonstrated considerable promise as biorecognition elements in sensors for applications including medical diagnostics, environmental monitoring, food safety, and forensic analysis. While aptamer sensors have made great strides in terms of sensitivity, specificity, turnaround time, and ease of use, several challenges have hindered their broader adoption. In this report, the authors describe their successes in using nuclease enzymes to address these problems. While working with nucleases to enhance the sensitivity of split aptamer sensors via enzyme-assisted target recycling, the authors serendipitously discovered that the digestion of DNA aptamers by exonucleases is inhibited when an aptamer is bound to a ligand. This finding served as the foundation for the development of three novel aptamer-related methodologies in their laboratory, and through their approach, they were able to detect analytes at nanomolar levels in biological samples, with the capacity for achieving multiplexed detection by using molecular beacons. Their efforts have enabled more comprehensive analysis of aptamers by greatly increasing the number of aptamer candidates and aptamer-ligand pairs that can be tested in a single experiment. The authors have also demonstrated the success of this method as a means for identifying new mutant aptamers with augmented binding properties and for quantifying aptamer-target affinity. The authors suggest that their enzymatic technologies can streamline the aptamer characterization and sensor development process, and suggest that, in the future, it should be possible to rapidly identify the most suitable aptamers for a particular application from hundreds to thousands of candidates. Publisher Abstract Provided

Date Published: January 1, 2023