@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{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}, 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{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 in vivo detection methods that would not be possible with antibody-based systems. However, it remains challenging to generate high-quality aptamers for small molecule targets, particularly for use under physiological conditions. We present a highly effective aptamer selection technology for small-molecule targets that utilizes the nuclease EcoRI to remove nonspecific or weakly binding sequences in solution phase, rapidly enriching high-affinity target binders within just a few rounds of selection. As proof-of-concept, we used our nuclease-assisted SELEX (NA-SELEX) method to isolate aptamers for a synthetic cannabinoid, AB-FUBINACA. Within five rounds, we identified two highly specific aptamers that exhibit nanomolar affinity at physiological temperature. We also demonstrate the robustness and reproducibility of NA-SELEX by performing the same selection experiment with fresh reagents and libraries, obtaining the same two aptamers as well as two other high-quality aptamer candidates. Finally, we compare NA-SELEX against a conventional library-immobilized SELEX screen for AB-FUBINACA using the same screening conditions, identifying aptamers with 25–100-fold weaker affinity after 11 rounds of selection. NA-SELEX therefore could be an effective selection method for the isolation of high-quality aptamers for small-molecule targets.}, 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{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} }