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Conventional directed evolution methods offer the ability to select bioreceptors with high binding affinity for a specific target in terms of thermodynamic properties. However, there is a lack of analogous approaches for kinetic selection, which could yield affinity reagents that exhibit slow off-rates and thus remain tightly bound to targets for extended periods. Here, we describe an in vitro directed evolution methodology that uses the nuclease flap endonuclease 1 to achieve the efficient discovery of aptamers that have slow dissociation rates. Our nuclease-assisted selection strategy can yield specific aptamers for both small molecules and proteins with off-rates that are an order of magnitude slower relative to those obtained with conventional selection methods while still retaining excellent overall target affinity in terms of thermodynamics. This new methodology provides a generalizable approach for generating slow off-rate aptamers for diverse targets, which could, in turn, prove valuable for applications including molecular devices, bioimaging, and therapy. 

Abstract Aptamers are nucleic acid bioreceptors that have been used in various applications including medical diagnostics and as therapeutic agents. Identifying the most optimal aptamer for a particular application is very challenging. Here, we for the first time have developed a high-throughput method for accurately quantifying aptamer binding affinity, specificity, and cross-reactivity via the kinetics of aptamer digestion by exonucleases. We demonstrate the utility of this approach by isolating a set of new aptamers for fentanyl and its analogs, and then characterizing the binding properties of 655 aptamer–ligand pairs using our exonuclease digestion assay and validating the results with gold-standard methodologies. These data were used to select optimal aptamers for the development of new sensors that detect fentanyl and its analogs in different analytical contexts. Our approach dramatically accelerates the aptamer characterization process and streamlines sensor development, and if coupled with robotics, could enable high-throughput quantitative analysis of thousands of aptamer–ligand pairs. 

Abstract Aptamers are short oligonucleotides isolated in vitro from randomized libraries that can bind to specific molecules with high affinity, and offer a number of advantages relative to antibodies as biorecognition elements in biosensors. However, it remains difficult and labor‐intensive to develop aptamer‐based sensors for small‐molecule detection. Here, we review the challenges and advances in the isolation and characterization of small‐molecule‐binding DNA aptamers and their use in sensors. First, we discuss in vitro methodologies for the isolation of aptamers, and provide guidance on selecting the appropriate strategy for generating aptamers with optimal binding properties for a given application. We next examine techniques for characterizing aptamer–target binding and structure. Afterwards, we discuss various small‐molecule sensing platforms based on original or engineered aptamers, and their detection applications. Finally, we conclude with a general workflow to develop aptamer‐based small‐molecule sensors for real‐world applications. 

Abstract Aptamers are promising biorecognition elements for sensors. However, aptamer‐based assays often lack the requisite levels of sensitivity and/or selectivity because they typically employ structure‐switching aptamers with attenuated affinity and/or utilize reporters that require aptamer labeling or which are susceptible to false positives. Dye‐displacement assays offer a label‐free, sensitive means for overcoming these issues, wherein target binding liberates a dye that is complexed with the aptamer, producing an optical readout. However, broad utilization of these assays has been limited. Here, we demonstrate a rational approach to develop colorimetric cyanine dye‐displacement assays that can be broadly applied to DNA aptamers regardless of their structure, sequence, affinity, or the physicochemical properties of their targets. Our approach should accelerate the development of mix‐and‐measure assays that could be applied for diverse analytical applications.