Using Exonucleases for Aptamer Characterization, Engineering, and Sensing

Conspectus Aptamers are short, single-strandednucleic acids that have beenselected from random libraries to bind specific molecules with highaffinity via an in vitro method termed systematicevolution of ligands by exponential enrichment (SELEX). They havebeen generated for diverse targets ranging from metal ions to smallmolecules to proteins and have demonstrated considerable promise asbiorecognition elements in sensors for applications including medicaldiagnostics, 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 challengeshave hindered their broader adoption. These include inadequate sensitivity,bottlenecks in aptamer binding characterization, and the cost andlabor associated with aptamer engineering. In this Account, we describeour successes in using nuclease enzymes to address these problems.While working with nucleases to enhance the sensitivity of split aptamersensors via enzyme-assisted target recycling, we serendipitously discoveredthat the digestion of DNA aptamers by exonucleases is inhibited when an aptamer is bound to a ligand. This finding served as the foundationfor the development of three novel aptamer-related methodologies inour laboratory. First, we used exonucleases to truncate nonessentialnucleotides from aptamers to generate structure-switching aptamersin a single step, greatly simplifying the aptamer engineering process.Second, we used exonucleases to develop a label-free aptamer-baseddetection platform that can utilize aptamers directly obtained from in vitro selection to detect analytes with ultralow backgroundand high sensitivity. Through this approach, we were able to detectanalytes at nanomolar levels in biological samples, with the capacityfor achieving multiplexed detection by using molecular beacons. Finally,we used exonucleases to develop a high throughput means of characterizingaptamer affinity and specificity for a variety of ligands. This approachhas enabled more comprehensive analysis of aptamers by greatly increasingthe number of aptamer candidates and aptamer-ligand pairs that canbe tested in a single experiment. We have also demonstrated the successof this method as a means for identifying new mutant aptamers withaugmented binding properties and for quantifying aptamer-target affinity.Our enzymatic technologies can greatly streamline the aptamer characterizationand sensor development process, and with the adoption of roboticsor liquid handling systems in the future, it should be possible torapidly identify the most suitable aptamers for a particular applicationfrom hundreds to thousands of candidates.

Automated summary

This article introduces the research results of using nucleases to improve the sensitivity, specificity, and cost-effectiveness of aptamer sensors. The author serendipitously discovered that the digestion of DNA aptamers by exonucleases is inhibited when an aptamer is bound to a ligand, which laid the foundation for the development of three novel aptamer-related methodologies. The enzymatic technologies described in this article can greatly streamline the aptamer characterization and sensor development process.