@article{canoura_alkhamis_byrd_wang_bryant_xiao_2024, title={Determining the Precision of High-Throughput Sequencing and Its Influence on Aptamer Selection}, volume={10}, ISSN={["1520-6882"]}, DOI={10.1021/acs.analchem.4c03972}, abstractNote={selection offers a means of discovering functional nucleic acids from randomized libraries, and high-throughput sequencing (HTS) is a powerful tool for monitoring the evolution of oligonucleotide pools over many cycles of enrichment. Many groups now use HTS-derived measures of sequence enrichment across different rounds of}, journal={ANALYTICAL CHEMISTRY}, author={Canoura, Juan and Alkhamis, Obtin and Byrd, Caleb and Wang, Linlin and Bryant, Alexandra and Xiao, Yi}, year={2024}, month={Oct} }
 @article{canoura_alkhamis_venzke_ly_xiao_2024, title={Developing Aptamer-Based Colorimetric Opioid Tests}, volume={3}, ISSN={["2691-3704"]}, DOI={10.1021/jacsau.3c00801}, abstractNote={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.}, journal={JACS AU}, author={Canoura, Juan and Alkhamis, Obtin and Venzke, Matthew and Ly, Phuong T. and Xiao, Yi}, year={2024}, month={Mar} }
 @article{wang_canoura_byrd_nguyen_alkhamis_ly_xiao_2024, title={Examining the Relationship between Aptamer Complexity and Molecular Discrimination of a Low-Epitope Target}, volume={11}, ISSN={["2374-7951"]}, DOI={10.1021/acscentsci.4c01377}, abstractNote={Aptamers are oligonucleotide-based affinity reagents that are increasingly being used in various applications. Systematic evolution of ligands by exponential enrichment (SELEX) has been widely used to isolate aptamers for small-molecule targets, but it remains challenging to generate aptamers with high affinity and specificity for targets with few functional groups. To address this challenge, we have systematically evaluated strategies for optimizing the isolation of aptamers for (+)-methamphetamine, a target for which previously reported aptamers have weak or no binding affinity. We perform four trials of library-immobilized SELEX against (+)-methamphetamine and demonstrate that N30 libraries do not yield high-quality aptamers. However, by using a more complex N40 library design, stringent counter-SELEX, and fine-tuned selection conditions, we identify aptamers with high affinity for (+)-methamphetamine and better selectivity relative to existing antibodies. Bioinformatic analysis from our selections reveals that high-quality aptamers contain long conserved motifs and are more informationally dense. Finally, we demonstrate that our best aptamer can rapidly detect (+)-methamphetamine at toxicologically relevant concentrations in saliva in a colorimetric dye-displacement assay. The insights provided here demonstrate the challenges in generating high-quality aptamers for low complexity small-molecule targets and will help guide the design of more efficient future selection efforts.}, journal={ACS CENTRAL SCIENCE}, author={Wang, Linlin and Canoura, Juan and Byrd, Caleb and Nguyen, Thinh and Alkhamis, Obtin and Ly, Phuong and Xiao, Yi}, year={2024}, month={Nov} }
 @article{yang_alkhamis_canoura_bryant_gong_barbu_taylor_nikic_banerjee_xiao_et al._2024, title={Exploring the Landscape of Aptamers: From Cross-Reactive to Selective to Specific, High-Affinity Receptors for Cocaine}, volume={2}, ISSN={["2691-3704"]}, DOI={10.1021/jacsau.3c00781}, abstractNote={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.}, journal={JACS AU}, author={Yang, Kyungae and Alkhamis, Obtin and Canoura, Juan and Bryant, Alexandra and Gong, Edward M. and Barbu, Mihaela and Taylor, Steven and Nikic, Dragan and Banerjee, Saswata and Xiao, Yi and et al.}, year={2024}, month={Feb} }
 @article{alkhamis_canoura_wu_emmons_wang_honeywell_plaxco_kippin_xiao_2024, title={High-Affinity Aptamers for <i>In Vitro</i> and <i>In Vivo</i> Cocaine Sensing}, volume={146}, ISSN={["1520-5126"]}, DOI={10.1021/jacs.3c11350}, abstractNote={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}, number={5}, journal={JOURNAL OF THE AMERICAN CHEMICAL SOCIETY}, author={Alkhamis, Obtin and Canoura, Juan and Wu, Yuyang and Emmons, Nicole A. and Wang, Yuting and Honeywell, Kevin M. and Plaxco, Kevin W. and Kippin, Tod E. and Xiao, Yi}, year={2024}, month={Jan}, pages={3230–3240} }
 @article{alkhamis_canoura_wang_xiao_2024, title={Nuclease-assisted selection of slow-off rate aptamers}, volume={10}, ISSN={["2375-2548"]}, DOI={10.1126/sciadv.adl3426}, abstractNote={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.}, number={24}, journal={SCIENCE ADVANCES}, author={Alkhamis, Obtin and Canoura, Juan and Wang, Linlin and Xiao, Yi}, year={2024}, month={Jun} }
 @article{wang_alkhamis_canoura_yu_xiao_2024, title={Rapid Nuclease-Assisted Selection of High-Affinity Small-Molecule Aptamers}, volume={7}, ISSN={["1520-5126"]}, DOI={10.1021/jacs.4c00748}, abstractNote={Aptamers are nucleic acid bioreceptors that have been widely utilized for a variety of biosensing applications, including}, journal={JOURNAL OF THE AMERICAN CHEMICAL SOCIETY}, author={Wang, Linlin and Alkhamis, Obtin and Canoura, Juan and Yu, Haixiang and Xiao, Yi}, year={2024}, month={Jul} }
 @article{canoura_liu_alkhamis_xiao_2023, title={Aptamer-Based Fentanyl Detection in Biological Fluids}, volume={95}, ISSN={["1520-6882"]}, DOI={10.1021/acs.analchem.3c04104}, abstractNote={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.}, number={49}, journal={ANALYTICAL CHEMISTRY}, author={Canoura, Juan and Liu, Yingzhu and Alkhamis, Obtin and Xiao, Yi}, year={2023}, month={Nov}, pages={18258–18267} }
 @article{alkhamis_canoura_willis_wang_perry_xiao_2023, title={Comparison of Aptamer Signaling Mechanisms Reveals Disparities in Sensor Response and Strategies to Eliminate False Signals}, volume={145}, ISSN={["1520-5126"]}, DOI={10.1021/jacs.3c03640}, abstractNote={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.}, number={22}, journal={JOURNAL OF THE AMERICAN CHEMICAL SOCIETY}, author={Alkhamis, Obtin and Canoura, Juan and Willis, Connor and Wang, Linlin and Perry, Jacob and Xiao, Yi}, year={2023}, month={May}, pages={12407–12422} }
 @article{alkhamis_canoura_ly_xiao_2023, title={Using Exonucleases for Aptamer Characterization, Engineering, and Sensing}, volume={6}, ISSN={["1520-4898"]}, DOI={10.1021/acs.accounts.3c00113}, abstractNote={ConspectusAptamers 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.}, journal={ACCOUNTS OF CHEMICAL RESEARCH}, author={Alkhamis, Obtin and Canoura, Juan and Ly, Phuong T. and Xiao, Yi}, year={2023}, month={Jun} }
 @article{alkhamis_canoura_bukhryakov_tarifa_decaprio_xiao_2022, title={DNA Aptamer-Cyanine Complexes as Generic Colorimetric Small-Molecule Sensors}, volume={61}, ISSN={["1521-3773"]}, DOI={10.1002/anie.202112305}, abstractNote={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.}, number={3}, journal={ANGEWANDTE CHEMIE-INTERNATIONAL EDITION}, author={Alkhamis, Obtin and Canoura, Juan and Bukhryakov, Konstantin V and Tarifa, Anamary and DeCaprio, Anthony P. and Xiao, Yi}, year={2022}, month={Jan} }
 @article{canoura_alkhamis_liu_willis_xiao_2022, title={High-throughput quantitative binding analysis of DNA aptamers using exonucleases}, volume={12}, ISSN={["1362-4962"]}, DOI={10.1093/nar/gkac1210}, abstractNote={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.}, journal={NUCLEIC ACIDS RESEARCH}, author={Canoura, Juan and Alkhamis, Obtin and Liu, Yingzhu and Willis, Connor and Xiao, Yi}, year={2022}, month={Dec} }
 @article{jin_liu_alkhamis_canoura_bacon_xu_fu_xiao_2022, title={Near-Infrared Dye-Aptamer Assay for Small Molecule Detection in Complex Specimens}, volume={7}, ISSN={["1520-6882"]}, DOI={10.1021/acs.analchem.2c01095}, abstractNote={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.}, journal={ANALYTICAL CHEMISTRY}, author={Jin, Xin and Liu, Yingzhu and Alkhamis, Obtin and Canoura, Juan and Bacon, Adara and Xu, Ruyi and Fu, Fengfu and Xiao, Yi}, year={2022}, month={Jul} }
 @article{alkhamis_xiao_2022, title={Systematic Study of in Vitro Selection Stringency Reveals How To Enrich High-Affinity Aptamers}, volume={12}, ISSN={["1520-5126"]}, DOI={10.1021/jacs.2c09522}, abstractNote={Aptamers are oligonucleotide receptors with great potential for sensing and therapeutic applications. They are isolated from random libraries through an in vitro method termed systematic evolution of ligands by exponential enrichment (SELEX). Although SELEX-based methods have been widely employed over several decades, many aspects of the experimental process remain poorly understood in terms of how to adjust the selection conditions to obtain aptamers with the desired set of binding characteristics. As a result, SELEX is often performed with arbitrary parameters that tend to produce aptamers with insufficient affinity and/or specificity. Having a better understanding of these basic principles could increase the likelihood of obtaining high-quality aptamers. Here, we have systematically investigated how altering the selection stringency in terms of target concentration─which is essentially the root source of selection pressure for aptamer isolation─affects the outcome of SELEX. By performing four separate trials of SELEX for the same small-molecule target, we experimentally prove that the use of excessively high target concentrations promotes enrichment of low-affinity binders while also suppressing the enrichment of high-affinity aptamers. These findings should be broadly applicable across SELEX methods, given that they share the same core operating principle, and will be crucial for guiding selections to obtain high-quality aptamers in the future.}, journal={JOURNAL OF THE AMERICAN CHEMICAL SOCIETY}, author={Alkhamis, Obtin and Xiao, Yi}, year={2022}, month={Dec} }