@article{alkhamis_byrd_canoura_bacon_hill_xiao_2025, title={Exploring the relationship between aptamer binding thermodynamics, affinity, and specificity}, DOI={10.1093/nar/gkaf219}, abstractNote={Aptamers are oligonucleotide-based bioreceptors that are selected in vitro from randomized libraries to bind specific molecules with high affinity, and are proving popular for applications in diagnostics, bioimaging, and therapeutics. A better understanding of aptamer-ligand interactions could facilitate sequence engineering efforts to improve aptamer binding properties, and perhaps eventually allow for the direct design of high-quality aptamers. To date, however, there have been very few comprehensive studies exploring the relationship between aptamer binding properties and thermodynamics. Isothermal titration calorimetry (ITC) is a gold-standard method for studying the thermodynamics of ligand-receptor interactions. In this work, we have compiled ITC-derived thermodynamic binding data from 317 small-molecule-binding DNA aptamers, along with specificity profiles for ∼6000 aptamer-ligand pairs, and performed systematic analysis of the resulting datasets. This analysis revealed a variety of interesting patterns and trends. For example, ligand binding for most aptamers is generally driven solely by enthalpy, and aptamers with the highest binding enthalpy and greatest entropic binding penalties consistently have high specificity. We envision that the expansion and further analysis of such datasets will yield a far better understanding of the complex interplay between the various non-covalent interactions underlying aptamer-ligand recognition.}, journal={Nucleic Acids Research}, author={Alkhamis, Obtin and Byrd, Caleb and Canoura, Juan and Bacon, Adara and Hill, Ransom and Xiao, Yi}, year={2025}, month={Mar} }
 @article{nguyen_alkhamis_bacon_bukhryakov_vinueza_xiao_2025, title={High-Contrast Aptamer-Based Merocyanine Displacement Assays for Sensitive Small Molecule Detection}, volume={10}, DOI={10.1021/acssensors.5c02194}, abstractNote={Aptamer-based colorimetric dye-displacement assays offer a promising means for on-site detection due to their single-step mix-and-read procedure and rapid naked-eye readout. Here, analyte binding to noncovalent aptamer-dye complexes triggers the displacement of the dye into solution, resulting in the formation of aggregates with divergent optical absorbance properties from the monomeric aptamer-bound dye. However, these assays suffer from low sensitivity, poor color contrast, and small absorbance wavelength shifts between the dye monomers and aggregates, as well as the large spectral overlaps between these two forms. Here, we introduce the solvatochromic coumarin-based merocyanine dye X-19539-107 as a novel indicator that enables the highly sensitive detection of small-molecule targets in dye-displacement assays. In aqueous solution, the dye forms H-aggregates that absorb at 445 nm, whereas aptamer-bound dye monomers absorb at 610-625 nm─a difference of up to 180 nm, with minimal spectral overlap. We utilize X-19539-107 in dye-displacement assays with diverse aptamers to detect various small-molecule analytes with unprecedentedly high target-induced dye displacement efficiency and greater sensitivity than conventional cyanine indicators and excellent color contrast. We also demonstrate cocaine detection at toxicologically relevant levels in saliva, highlighting the real-world utility of these assays. We conclude that our merocyanine dye offers a powerful alternative to cyanine dyes for colorimetric aptamer-based sensing.}, journal={ACS Sensors}, author={Nguyen, Thinh and Alkhamis, Obtin and Bacon, Adara and Bukhryakov, Konstantin V. and Vinueza, Nelson R. and Xiao, Yi}, year={2025}, month={Oct} }
 @article{yu_canoura_byrd_alkhamis_bacon_yan_sullenger_xiao_2025, title={Improving Aptamer Affinity and Determining Sequence–Activity Relationships via Motif-SELEX}, volume={147}, DOI={10.1021/jacs.4c17041}, abstractNote={The affinity of nucleic acid aptamers isolated <i>in vitro</i> via Systematic Evolution of Ligands by Exponential Enrichment (SELEX) is often limited because the entire potential sequence space cannot be screened. In this study, we introduce Motif-SELEX, a novel method that enables the optimization of existing underperforming aptamers by generating libraries that broadly represent both the sequence and length variations of the parent sequence. This approach enables the isolation of sequences with improved affinity without the biases and limitations of traditional mutagenesis methods like doped SELEX and error-prone PCR. As a demonstration, we applied Motif-SELEX to a DNA-based morphine aptamer and a 2' fluoro- and methoxy-RNA-based apixaban aptamer, discovering new, better-performing sequences with differing random domain lengths from their parents and up to 10-fold improvements in affinity. These new sequences would be inaccessible to traditional post-SELEX methods. Critically, our analysis of Motif-SELEX pools also enabled us to identify sequence and structural elements crucial for target binding and to predict secondary and tertiary structures for a given aptamer family─even when those structures involve noncanonical nucleotide interactions. We believe that Motif-SELEX offers an effective and generalizable solution for optimizing the structure and binding properties of functional nucleic acid molecules for diverse applications.}, number={11}, journal={Journal of the American Chemical Society}, author={Yu, Haixiang and Canoura, Juan and Byrd, Caleb and Alkhamis, Obtin and Bacon, Adara and Yan, Amy and Sullenger, Bruce A and Xiao, Yi}, year={2025}, month={Mar}, pages={9472–9486} }