Label-free profiling of DNA aptamer-small molecule binding using T5 exonuclease

Abstract

In vitro aptamer isolation methods can yield hundreds of potential candidates, but selecting the optimal aptamer for a given application is challenging and laborious. Existing aptamer characterization methods either entail low-throughput analysis with sophisticated instrumentation, or offer the potential for higher throughput at the cost of providing a relatively increased risk of false-positive or -negative results. Here, we describe a novel method for accurately and sensitively evaluating the binding between DNA aptamers and small-molecule ligands in a high-throughput format without any aptamer engineering or labeling requirements. This approach is based on our new finding that ligand binding inhibits aptamer digestion by T5 exonuclease, where the extent of this inhibition correlates closely with the strength of aptamer-ligand binding. Our assay enables accurate and efficient screening of the ligand-binding profiles of individual aptamers, as well as the identification of the best target binders from a batch of aptamer candidates, independent of the ligands in question or the aptamer sequence and structure. We demonstrate the general applicability of this assay with a total of 106 aptamer-ligand pairs and validate these results with a gold-standard method. We expect that our assay can be readily expanded to characterize small-molecule-binding aptamers in an automated, high-throughput fashion.

Accelerating Post-SELEX Aptamer Engineering Using Exonuclease Digestion

“…Rate reduction was calculated by the expression 1 – ( k ligand / k blank ). Finally, resistance values were calculated as previously described . The area under the curve (AUC) of the fluorescence digestion plots was determined using Origin 2019 software, and the resistance value was calculated using the expression (AUC ligand /AUC blank ) – 1.…”

DNA Aptamer–Cyanine Complexes as Generic Colorimetric Small‐Molecule Sensors

“…Finally, we evaluated the binding and target‐induced displacement of five more small‐molecule‐binding DNA aptamers with MTC. As seen in Figure S29, these aptamers are diverse with regard to their size, sequence, and three‐dimensional structure: a 19‐nt ochratoxin A‐binding aptamer containing a triple‐stem structure (OBA3, K D =1.4 μM), [33] a stem‐loop structured 46‐nt aptamer for mephedrone (MMC1, K D =15 μM), [34] 44‐nt dopamine‐ ( K D =150 nM) and serotonin‐binding aptamers ( K D =30 nM) with G‐quadruplex features, [35] and a 40‐nt glucose‐binding aptamer ( K D =10 mM) [35] . All five aptamers could bind to MTC with varying levels of complexation, as evaluated based on their capability to monomerize/dimerize MTC and reduce J‐aggregate concentrations (SI, Figures S29–34).…”

Advances and Challenges in Small‐Molecule DNA Aptamer Isolation, Characterization, and Sensor Development

“…We have recently developed exonuclease-based assays based on the inhibition of aptamer digestion by enzymes upon ligand binding to characterize the binding between multiple aptamer-ligand pairs simultaneously in as ingle-pot reaction. [235] Based on our preliminary success in characterizing avariety of aptamers with diverse structure and many ligands with wide-ranging physicochemical properties,w eb elieve that this assay can be readily scaled up to characterize aptamer binding profile in ah igh-throughput, automated fashion. After narrowing down the aptamer candidates, precise quantification of target-binding affinity of aptamers is recommended.…”

“…Methods such as ITC and SPR can determine quantitative binding thermodynamics and kinetics, but are ill‐suited for extensively screening aptamer specificity due to low throughput. We have recently developed exonuclease‐based assays based on the inhibition of aptamer digestion by enzymes upon ligand binding to characterize the binding between multiple aptamer–ligand pairs simultaneously in a single‐pot reaction [235] . Based on our preliminary success in characterizing a variety of aptamers with diverse structure and many ligands with wide‐ranging physicochemical properties, we believe that this assay can be readily scaled up to characterize aptamer binding profile in a high‐throughput, automated fashion.…”