High-throughput quantitative binding analysis of DNA aptamers using exonucleases.

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.

High-Affinity Aptamers for In Vitro and In Vivo Cocaine Sensing.

The ability to quantify cocaine in biological fluids is crucial for both the diagnosis of intoxication and overdose in the clinic as well as investigation of the drug's pharmacological and toxicological effects in the laboratory. To this end, we have performed high-stringency in vitro selection to generate DNA aptamers that bind cocaine with nanomolar affinity and clinically relevant specificity, thus representing a dramatic improvement over the current-generation, micromolar-affinity, low-specificity cocaine aptamers. Using these novel aptamers, we then developed two sensors for cocaine detection. The first, an in vitro fluorescent sensor, successfully detects cocaine at clinically relevant levels in 50% human serum without responding significantly to other drugs of abuse, endogenous substances, or a diverse range of therapeutic agents. The second, an electrochemical aptamer-based sensor, supports the real-time, seconds-resolved measurement of cocaine concentrations in vivo in the circulation of live animals. We believe the aptamers and sensors developed here could prove valuable for both point-of-care and on-site clinical cocaine detection as well as fundamental studies of cocaine neuropharmacology.

Generation of High-Affinity Aptamers for Indazole Synthetic Cannabinoids.

Synthetic cannabinoids are a widely abused class of dangerous psychoactive substances, especially among youths and young adults. Dozens of such drugs have been identified to date, and new ones continue to emerge. The ability to detect these drugs is important for interdiction efforts and the diagnosis of drug overdose, but existing analytical methods lack broad cross-reactivity to diverse members of this drug family. Here, we have utilized library-immobilized SELEX to generate DNA aptamers that can broadly recognize various members of the indazole-3-carboxamide synthetic cannabinoid family. Using two representatives of this family, AB-FUBINACA and 5F-AMB, we identify two aptamers FUB4 and AMB2F with respective dissociation constants (KDs) of 138 ± 15 and 411 ± 20 nM for their targets. These aptamers can recognize many indazole-based synthetic cannabinoids with high affinity and excellent specificity against natural cannabinoids as well as other structurally similar interferents like serotonin and tryptophan. We use these two aptamers to develop fluorescence strand-displacement sensors that successfully detect these synthetic cannabinoids at concentrations as low as 50 nM in human serum. The sensors can also detect up to 14 different drugs from this family─a major improvement over the six recognized by an existing commercial immunoassay.

Using Exonucleases for Aptamer Characterization, Engineering, and Sensing.

Aptamers are short, single-stranded nucleic acids that have been 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). They 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. These include inadequate sensitivity, bottlenecks in aptamer binding characterization, and the cost and labor associated with aptamer engineering. In this Account, we describe our 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, we 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 our laboratory. First, we used exonucleases to truncate nonessential nucleotides from aptamers to generate structure-switching aptamers in a single step, greatly simplifying the aptamer engineering process. Second, we used exonucleases to develop a label-free aptamer-based detection platform that can utilize aptamers directly obtained from in vitro selection to detect analytes with ultralow background and high sensitivity. Through this approach, we were able to detect analytes at nanomolar levels in biological samples, with the capacity for achieving multiplexed detection by using molecular beacons. Finally, we used exonucleases to develop a high throughput means of characterizing aptamer affinity and specificity for a variety of ligands. This approach has 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. We 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. Our enzymatic technologies can greatly streamline the aptamer characterization and sensor development process, and with the adoption of robotics or liquid handling systems in the future, it should be possible to rapidly identify the most suitable aptamers for a particular application from hundreds to thousands of candidates.

Comparison of Aptamer Signaling Mechanisms Reveals Disparities in Sensor Response and Strategies to Eliminate False Signals.

Aptamers are nucleic acid-based affinity reagents that have been incorporated into a variety of molecular sensor formats. However, many aptamer sensors exhibit insufficient sensitivity and specificity for real-world applications, and although considerable effort has been dedicated to improving sensitivity, sensor specificity has remained largely neglected and understudied. In this work, we have developed a series of sensors using aptamers for the small-molecule drugs flunixin, fentanyl, and furanyl fentanyl and compare their performance─in particular, focusing on their specificity. Contrary to expectations, we observe that sensors using the same aptamer operating under the same physicochemical conditions produce divergent responses to interferents depending on their signal transduction mechanism. For instance, aptamer beacon sensors are susceptible to false-positives from interferents that weakly associate with DNA, while strand-displacement sensors suffer from false-negatives due to interferent-associated signal suppression when both the target and interferent are present. Biophysical analyses suggest that these effects arise from aptamer-interferent interactions that are either nonspecific or induce aptamer conformational changes that are distinct from those induced by true target-binding events. We also demonstrate strategies for improving the sensitivity and specificity of aptamer sensors with the development of a "hybrid beacon," wherein the incorporation of a complementary DNA competitor into an aptamer beacon selectively hinders interferent─but not target─binding and signaling, while simultaneously overcoming signal suppression by interferents. Our results highlight the need for systematic and thorough testing of aptamer sensor response and new aptamer selection methods that optimize specificity more effectively than traditional counter-SELEX.

Aptamer-Based Fentanyl Detection in Biological Fluids.

Fentanyl is a widely abused analgesic and anesthetic drug with a narrow therapeutic window that creates easy opportunities for overdose and death. Rapid, accurate, and sensitive fentanyl detection in biosamples is crucial for therapeutic drug monitoring and overdose diagnosis. Unfortunately, current methods are limited to either sophisticated laboratory-based tests or antibody-based immunoassays, which are prone to false results and are mainly used with urine samples. Here, we have utilized library-immobilized SELEX to isolate new aptamers─nucleic acid-based bioreceptors that are well-suited for biosensing─that can specifically bind fentanyl under physiological conditions. We isolated multiple aptamers with nanomolar affinity and excellent specificity against dozens of interferents and incorporated one of these into an electrochemical aptamer-based sensor that can rapidly detect fentanyl at clinically relevant concentrations in 50% diluted serum, urine, and saliva. Given the excellent performance of these sensors, we believe that they could serve as the basis for point-of-care devices for monitoring fentanyl during medical procedures and determining fentanyl overdose.

Near-Infrared Dye-Aptamer Assay for Small Molecule Detection in Complex Specimens.

Aptamers are single-stranded oligonucleotides isolated in vitro that bind specific targets with high affinity and are commonly used as receptors in biosensors. Aptamer-based dye-displacement assays are a promising sensing platform because they are label-free, sensitive, simple, and rapid. However, these assays can exhibit impaired sensitivity in biospecimens, which contain numerous interferents that cause unwanted absorbance, scattering, and fluorescence in the UV-vis region. Here, this problem is overcome by utilizing near-infrared (NIR) signatures of the dye 3,3'-diethylthiadicarbocyanine iodide (Cy5). Cy5 initially complexes with aptamers as monomers and dimers; aptamer-target binding displaces the dye into solution, resulting in the formation of J-aggregates that provide a detectable NIR signal. The generality of our assay is demonstrated by detecting three different small-molecule analytes with their respective DNA aptamers at clinically relevant concentrations in serum and urine. These successful demonstrations show the utility of dye-aptamer NIR biosensors for high-throughput detection of analytes in clinical specimens.

DNA Aptamer-Cyanine Complexes as Generic Colorimetric Small-Molecule Sensors.

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.

Accelerating Post-SELEX Aptamer Engineering Using Exonuclease Digestion.

The systematic evolution of ligands by exponential enrichment (SELEX) process enables the isolation of aptamers from random oligonucleotide libraries. However, it is generally difficult to identify the best aptamer from the resulting sequences, and the selected aptamers often exhibit suboptimal affinity and specificity. Post-SELEX aptamer engineering can improve aptamer performance, but current methods exhibit inherent bias and variable rates of success or require specialized instruments. Here, we describe a generalizable method that utilizes exonuclease III and exonuclease I to interrogate the binding properties of small-molecule-binding aptamers in a rapid, label-free assay. By analyzing an ochratoxin-binding DNA aptamer and six of its mutants, we determined that ligand binding alters the exonuclease digestion kinetics to an extent that closely correlates with the aptamer's ligand affinity. We then utilized this assay to enhance the binding characteristics of a DNA aptamer which binds indiscriminately to ATP, ADP, AMP, and adenosine. We screened 13 mutants derived from this aptamer against all these analogues and identified two new high-affinity aptamers that solely bind to adenosine. We incorporated these two aptamers directly into an electrochemical aptamer-based sensor, which achieved a detection limit of 1 µM adenosine in 50% serum. We also confirmed the generality of our method to characterize target-binding affinities of protein-binding aptamers. We believe our approach is generalizable for DNA aptamers regardless of sequence, structure, and length and could be readily adapted into an automated format for high-throughput engineering of small-molecule-binding aptamers to acquire those with improved binding properties suitable for various applications.

Isolation of Natural DNA Aptamers for Challenging Small-Molecule Targets, Cannabinoids.

Aptamers are nucleic acid-based affinity reagents that are isolated via an in vitro process known as systematic evolution of ligands by exponential enrichment (SELEX). Despite their great potential for a wide range of analytical applications, there are relatively few high-quality small-molecule binding aptamers, especially for "challenging" targets that have low water solubility and/or limited moieties for aptamer recognition. The use of libraries containing chemically modified bases may improve the outcome of some SELEX experiments, but this approach is costly and yields inconsistent results. Here, we demonstrate that a thoughtfully designed SELEX procedure with natural DNA libraries can isolate aptamers with high affinity and specificity for challenging small molecules, including targets for which such selections have previously failed. We first isolate a DNA aptamer with nanomolar affinity and high specificity for (-)-trans-Δ9-tetrahydrocannabinol (THC), a target previously thought to be unsuitable for SELEX with natural DNA libraries. We subsequently isolate aptamers that exhibit high affinity and cross-reactivity to two other challenging targets, synthetic cannabinoids UR-144 and XLR-11, while maintaining excellent specificity against a wide range of non-target interferents. Our findings demonstrate that natural nucleic acid libraries can yield high-quality aptamers for small-molecule targets, and we outline a robust workflow for isolating other such aptamers in future selection efforts.

In vitro isolation of class-specific oligonucleotide-based small-molecule receptors.

Class-specific bioreceptors are highly desirable for recognizing structurally similar small molecules, but the generation of such affinity elements has proven challenging. We here develop a novel 'parallel-and-serial' selection strategy for isolating class-specific oligonucleotide-based receptors (aptamers) in vitro. This strategy first entails parallel selection to selectively enrich cross-reactive binding sequences, followed by serial selection that enriches aptamers binding to a designated target family. As a demonstration, we isolate a class-specific DNA aptamer against a family of designer drugs known as synthetic cathinones. The aptamer binds to 12 diverse synthetic cathinones with nanomolar affinity and does not respond to 11 structurally similar non-target compounds, some of which differ from the cathinone targets by a single atom. This is the first account of an aptamer exhibiting a combination of broad target cross-reactivity, high affinity and remarkable specificity. Leveraging the qualities of this aptamer, instantaneous colorimetric detection of synthetic cathinones at nanomolar concentrations in biological samples is achieved. Our findings significantly expand the binding capabilities of aptamers as class-specific bioreceptors and further demonstrate the power of rationally designed selection strategies for isolating customized aptamers with desired binding profiles. We believe that our aptamer isolation approach can be broadly applied to isolate class-specific aptamers for various small molecule families.

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

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.

In vitro isolation of small-molecule-binding aptamers with intrinsic dye-displacement functionality.

Aptamer-based sensors offer a powerful tool for molecular detection, but the practical implementation of these biosensors is hindered by costly and laborious sequence engineering and chemical modification procedures. We report a simple strategy for directly isolating signal-reporting aptamers in vitro through systematic evolution of ligands by exponential enrichment (SELEX) that transduce binding events into a detectable change of absorbance via target-induced displacement of a small-molecule dye. We first demonstrate that diethylthiatricarbocyanine (Cy7) can stack into DNA three-way junctions (TWJs) in a sequence-independent fashion, greatly altering the dye's absorbance spectrum. We then design a TWJ-containing structured library and isolate an aptamer against 3,4-methylenedioxypyrovalerone (MDPV), a synthetic cathinone that is an emerging drug of abuse. This aptamer intrinsically binds Cy7 within its TWJ domain, but MDPV efficiently displaces the dye, resulting in a change in absorbance within seconds. This assay is label-free, and detects nanomolar concentrations of MDPV. It also recognizes other synthetic cathinones, offering the potential to detect newly-emerging designer drugs, but does not detect structurally-similar non-cathinone compounds or common cutting agents. Moreover, we demonstrate that the Cy7-displacement colorimetric assay is more sensitive than a conventional strand-displacement fluorescence assay. We believe our strategy offers an effective generalized approach for the development of sensitive dye-displacement colorimetric assays for other small-molecule targets.

Introducing structure-switching functionality into small-molecule-binding aptamers via nuclease-directed truncation.

We report a broadly applicable enzyme digestion strategy for introducing structure-switching functionality into small-molecule-binding aptamers. This procedure is based on our discovery that exonuclease III (Exo III) digestion of aptamers is greatly inhibited by target binding. As a demonstration, we perform Exo III digestion of a pre-folded three-way-junction (TWJ)-structured cocaine-binding aptamer and a stem-loop-structured ATP-binding aptamer. In the absence of target, Exo III catalyzes 3'-to-5' digestion of both aptamers to form short, single-stranded products. Upon addition of target, Exo III digestion is halted four bases prior to the target-binding domain, forming a major target-bound aptamer digestion product. We demonstrated that target-binding is crucial for Exo III inhibition. We then determine that the resulting digestion products of both aptamers exhibit a target-induced structure-switching functionality that is absent in the parent aptamer, while still retaining high target-binding affinity. We confirm that these truncated aptamers have this functionality by using an exonuclease I-based digestion assay and further evaluate this characteristic in an electrochemical aptamer-based cocaine sensor and a fluorophore-quencher ATP assay. We believe our Exo III-digestion method should be applicable for the generation of structure-switching aptamers from other TWJ- or stem-loop-containing small-molecule-binding aptamers, greatly simplifying the generation of functionalized sensor elements for folding-based aptasensors.

Innovative engineering and sensing strategies for aptamer-based small-molecule detection.

Aptamers are nucleic acid-based affinity reagents that have gained widespread attention as biorecognition elements for the detection of targets such as ions, small molecules, and proteins. Over the past three decades, the field of aptamer-based sensing has grown considerably. However, the advancement of aptamer-based small-molecule detection has fallen short of the high demand for such sensors in applications such as diagnostics, environmental monitoring, and forensics. This is due to two challenges: the complexity of developing generalized sensing platforms and the poor sensitivities of assays targeting small molecules. This paper will review new approaches for the streamlined development of high-performance aptamer-based sensors for small-molecule detection. We here provide historical context, explore the current state-of-the art, and offer future directions-with emphasis placed on new aptamer engineering methods, the use of cooperative binding, and label-free approaches using fully-folded, high-affinity aptamers for small-molecule sensing.

No Structure-Switching Required: A Generalizable Exonuclease-Mediated Aptamer-Based Assay for Small-Molecule Detection.

The binding of small molecules to double-stranded DNA can modulate its susceptibility to digestion by exonucleases. Here, we show that the digestion of aptamers by exonuclease III can likewise be inhibited upon binding of small-molecule targets and exploit this finding for the first time to achieve sensitive, label-free small-molecule detection. This approach does not require any sequence engineering and employs prefolded aptamers which have higher target-binding affinities than structure-switching aptamers widely used in current small-molecule detecting assays. We first use a dehydroisoandrosterone-3-sulfate-binding aptamer to show that target binding halts exonuclease III digestion four bases prior to the binding site. This leaves behind a double-stranded product that retains strong target affinity, whereas digestion of nontarget-bound aptamer produces a single-stranded product incapable of target binding. Exonuclease I efficiently eliminates these single-stranded products but is unable to digest the target-bound double-stranded product. The remaining products can be fluorescently quantified with SYBR Gold to determine target concentrations. We demonstrate that this dual-exonuclease-mediated approach can be broadly applied to other aptamers with differing secondary structures to achieve sensitive detection of various targets, even in biological matrices. Importantly, each aptamer digestion product has a unique sequence, enabling the creation of multiplex assays, and we successfully demonstrate simultaneous detection of cocaine and ATP in a single microliter volume sample in 25 min via sequence-specific molecular beacons. Due to the generality and simplicity of this assay, we believe that different DNA signal-reporting or amplification strategies can be adopted into our assay for target detection in diverse analytical contexts.

Sensitive Detection of Small-Molecule Targets Using Cooperative Binding Split Aptamers and Enzyme-Assisted Target Recycling.

Signal amplification via enzyme-assisted target recycling (EATR) offers a powerful means for improving the sensitivity of DNA detection assays, but it has proven challenging to employ EATR with aptamer-based assays for small-molecule detection due to insensitive target response of aptamers. Here, we describe a general approach for the development of rapid and sensitive EATR-amplified small-molecule sensors based on cooperative binding split aptamers (CBSAs). CBSAs contain two target-binding domains and exhibit enhanced target response compared with single-domain split aptamers. We introduced a duplexed C3 spacer abasic site between the two binding domains, enabling EATR signal amplification through exonuclease III's apurinic endonuclease activity. As a demonstration, we engineered a CBSA-based EATR-amplified fluorescence assay to detect dehydroisoandrosterone-3-sulfate. This assay achieved 100-fold enhanced target sensitivity relative to a non-EATR-based assay, with a detection limit of 1 µM in 50% urine. We further developed an instrument-free colorimetric assay employing EATR-mediated aggregation of CBSA-modified gold nanoparticles for the visual detection of low-micromolar concentrations of cocaine. On the basis of the generalizability of CBSA engineering and the robust performance of EATR in complex samples, we believe that such assays should prove valuable for detecting small-molecule targets in diverse fields.

Nuclease-assisted selection of slow-off rate aptamers.

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.

Developing Aptamer-Based Colorimetric Opioid Tests.

Opioids collectively cause over 80,000 deaths in the United States annually. The ability to rapidly identify these compounds in seized drug samples on-site will be essential for curtailing trafficking and distribution. Chemical reagent-based tests are fast and simple but also notorious for giving false results due to poor specificity, whereas portable Raman spectrometers have excellent selectivity but often face interference challenges with impure drug samples. In this work, we develop on-site sensors for morphine and structurally related opioid compounds based on in vitro-selected oligonucleotide affinity reagents known as aptamers. We employ a parallel-and-serial selection strategy to isolate aptamers that recognize heroin, morphine, codeine, hydrocodone, and hydromorphone, along with a toggle-selection approach to isolate aptamers that bind oxycodone and oxymorphone. We then utilize a new high-throughput sequencing-based approach to examine aptamer growth patterns over the course of selection and a high-throughput exonuclease-based screening assay to identify optimal aptamer candidates. Finally, we use two high-performance aptamers with KD of ∼1 µM to develop colorimetric dye-displacement assays that can specifically detect opioids like heroin and oxycodone at concentrations as low as 0.5 µM with a linear range of 0-16 µM. Importantly, our assays can detect opioids in complex chemical matrices, including pharmaceutical tablets and drug mixtures; in contrast, the conventional Marquis test completely fails in this context. These aptamer-based colorimetric assays enable the naked-eye identification of specific opioids within seconds and will play an important role in combatting opioid abuse.

Exploring the Landscape of Aptamers: From Cross-Reactive to Selective to Specific, High-Affinity Receptors for Cocaine.

We reported over 20 years ago MNS-4.1, the first DNA aptamer with a micromolar affinity for cocaine. MNS-4.1 is based on a structural motif that is very common in any random pool of oligonucleotides, and it is actually a nonspecific hydrophobic receptor with wide cross-reactivity with alkaloids and steroids. Despite such weaknesses preventing broad applications, this aptamer became widely used in proof-of-concept demonstrations of new formats of biosensors. We now report a series of progressively improved DNA aptamers recognizing cocaine, with the final optimized receptors having low nanomolar affinity and over a thousand-fold selectivity over the initial cross-reactants. In the process of optimization, we tested different methods to eliminate cross-reactivities and improve affinity, eventually achieving properties that are comparable to those of the reported monoclonal antibody candidates for the therapy of overdose. Multiple aptamers that we now report share structural motifs with the previously reported receptor for serotonin. Further mutagenesis studies revealed a palindromic, highly adaptable, broadly cross-reactive hydrophobic motif that could be rebuilt through mutagenesis, expansion of linker regions, and selections into receptors with exceptional affinities and varying specificities.