A High-Affinity but Low-Abundance Kanamycin Aptamer Reveals Hybridization-Limited Capture-SELEX

Biochemistry · 2026-03-31

Kanamycin A, or simply referred to as kanamycin, is an aminoglycoside antibiotic with a narrow therapeutic window. Aptamers are useful recognition molecules for their detection and continuous monitoring. However, a short and high-affinity kanamycin aptamer that works under physiological conditions is still lacking. In this work, we revisited a previous aptamer selection done at pH 8, which had been abandoned due to poor sequence enrichment. Its top sequence, named KAN8–1, shows a Kd of 51 nM at pH 7.5 for kanamycin as measured by isothermal titration calorimetry, and its affinities to kanamycin A and B are similar. Using NMR spectroscopy methods, the KAN8–1 aptamer undergoes ligand-induced folding and likely has a better-defined structure compared to the KAN6–1 aptamer, which was highly enriched in the pH 6 selection. Using the KAN8–1 aptamer, a strand-displacement biosensor was developed, and it has a limit of detection of 0.9 μM with excellent selectivity. This sensor also has a similar performance in serum. The reason for the poor enrichment of KAN8–1 was attributed to its low hybridization efficiency and poor hybridization stability to the capture strand as demonstrated by a fluorescence titration assay and melting analysis, which indicated a limitation of the capture-SELEX method.

High-Affinity Three-Way Junction Aptamers for Sensing Cationic Polymyxin Antibiotics

Analytical Chemistry · 2026-03-14

Polymyxins B and E (colistin) were historically the last-resort antibiotics for treating infections caused by multidrug-resistant Gram-negative bacteria. However, their narrow therapeutic windows and significant nephrotoxicity necessitate rapid and precise detection methods. In this study, we employed a mixture of polymyxins B and E as targets to isolate DNA aptamers via the capture-SELEX method. Sequence analysis revealed two primary families, both characterized by three-way junction structures. The first family, represented by PM-2, exhibits comparable low-micromolar affinity for both polymyxins B and E and features a loopless three-way junction. In contrast, the PM-3 family demonstrates higher potency and selectivity, binding polymyxin B with a dissociation constant of 440 nM, approximately 10-fold stronger than its binding to polymyxin E, as determined by isothermal titration calorimetry. Binding assays under varying ionic strengths showed that affinity decreased with increasing salt concentration, suggesting that electrostatic interactions drive molecular recognition. Furthermore, thioflavin T fluorescence spectroscopy confirmed target-specific binding. Leveraging these distinct binding profiles, we developed a sensor array capable of discriminating between these two closely related antibiotics using a principal component analysis. These aptamers provide a robust foundation for the analytical detection and monitoring of polymyxin antibiotics in clinical settings.

Reconsidering molecular docking practices in aptamer research

ChemRxiv · 2026-04-08

Molecular docking is increasingly used to study aptamer–target interactions, yet most studies rely on computationally generated aptamer structures rather than experimentally determined ones. This approach presents fundamental limitations, including structure prediction without the target molecule and the common conversion of RNA models into DNA by deleting the 2′-hydroxyl group, while neglecting the intrinsic differences between A-form RNA and B-form DNA. Using the theophylline aptamer, for which crystal structures are available for both RNA and DNA forms, we systematically examine these issues through a step-by-step analysis of the standard docking workflow. Docking-based selectivity predictions against caffeine, together with molecular dynamics simulations, further demonstrate the inconsistency and limited reliability of this strategy. Overall, current docking practices, when applied without experimental structures, can produce unreliable and potentially misleading interpretations of aptamer binding mechanisms.

Thioflavin T Is a More General and Less Interfering Probe for Aptamer Binding Assays Than SYBR Green I

Analytical Chemistry · 2025-09-04 · 19 citations

Label-free fluorescent binding assays employing DNA staining dyes as probes are widely adopted techniques in the aptamer field. While many dyes have been used, thioflavin T (ThT) did not receive much attention for this purpose until recently, since it has long been perceived primarily as a G-quadruplex staining dye. Based on recent studies, ThT appears to serve as a reliable probe for evaluating the binding of non-G-quadruplex aptamers, and we seek to clarify the underlying mechanisms responsible for the exceptional performance of ThT. We investigated three non-G-quadruplex aptamers targeting adenosine, kanamycin, and Tris(hydroxymethyl)aminomethane (Tris). Consistent Kd values and fluorescence reductions were observed in the presence of ThT regardless of the aptamer-to-dye ratio. In contrast, SYBR Green I (SGI) exhibited responses with apparent Kd values that were far away from the true Kd in most cases. Additional assays demonstrated that ThT exhibits a weaker affinity for aptamers and random DNA sequences compared to SGI. In addition, ThT has weaker affinities to double-stranded DNA than to single-stranded DNA, which is opposite to that for SGI. Lastly, testing the T30695 DNA binding with Pb2+ confirmed that ThT can influence the binding of G-quadruplex aptamers. These findings explain ThT’s reliability for probing non-G-quadruplex aptamers.

A High-affinity but Low-abundance Kanamycin Aptamer Reveals Hybridization-limited Capture-SELEX

ChemRxiv · 2025-12-16

Kanamycin A, or simply referred to as kanamycin, is an aminoglycoside antibiotic with a narrow therapeutic window. Aptamers are useful recognition molecules for its detection and continuous monitoring. However, a high-quality kanamycin aptamer that works under physiological condition is still lacking. In this work, we revisited a previous aptamer selection done at pH 8, which was abandoned due to poor sequence enrichment. Its top sequence named KAN8-1 shows a Kd of 51 nM at pH 7.5 for kanamycin as measured by isothermal titration calorimetry, and its affinities to kanamycin A and B are similar. Using NMR spectroscopy methods, the KAN8-1 aptamer undergoes ligand-induced folding and likely has a better defined structure compared to the KAN6-1 aptamer, which was highly enriched at the pH 6 selection. Using the KAN8-1 aptamer, a strand-displacement biosensor was developed and it has a limit of detection of 0.9 µM with excellent selectivity. This sensor also has a similar performance in serum. The reason for the poor enrichment of KAN8-1 was attributed to its low hybridization efficiency and poor hybridization stability to the capture strand as demonstrated by a fluorescence titration assay and melting analysis, which indicated a limitation of the capture-SELEX method.

Thermal drying synthesized density-tunable and high stability nonthiolated spherical nucleic acids for split aptamer and lateral flow biosensors

Biosensors and Bioelectronics · 2025-08-14 · 7 citations

Spherical nucleic acids (SNAs) have attracted considerable interest in designing biosensors. Our group recently developed a thermal drying method to fabricate SNAs with an ultrahigh density of cost-effective nonthiolated DNA containing a polyadenine block. In this study, functional properties of such SNAs in two types of biosensors were investigated using aptamer and hybridization-based model detection systems. For aptamer-based target recognition, we observed that a high DNA density on SNAs hindered target binding. To address this, short dilution strands were introduced to decrease probe density. Using oxytetracycline as a target, a plasmonic colorimetric sensor with a detection limit of 0.5 μM was achieved. For hybridization-based recognition, we identified key parameters for avoiding false positives in lateral flow assays (LFA) and demonstrated that high DNA density did not compromise sensitivity. Using genetically modified soybean MON87705 as a target, we developed a portable LFA platform by integrating loop-mediated isothermal amplification with CRISPR/Cas12a, achieving a detection limit of 0.08 wt%. Overall, this study provides specific guidance for the practical application of nonthiolated SNA probes in biosensors.

A Non‐G‐Quadruplex Hemin Aptamer Forms a Better Peroxidase Mimicking DNAzyme

ChemBioChem · 2025-10-28 · 1 citations

G‐quadruplex DNA is known to bind to hemin, forming a complex that exhibits peroxidase‐like activity. A non‐G‐quadruplex aptamer named Hem1‐2T also exhibits horseradish peroxidase (HRP) like activity upon binding to hemin. Herein, the catalytic characteristics of the Hem1‐2T aptamer are studied and compared with PS2.M, an extensively studied G‐quadruplex. From pH 6–8, the activity of Hem1‐2T decreases with the increase in pH, which is similar to HRP, whereas the activity of PS2.M increases with pH, suggesting that Hem1‐2T might be a better mechanistic mimic of HRP. Additionally, Hem1‐2T is more effective at protecting hemin from degradation by H 2 O 2 , as evidenced by a slower decrease in the absorbance at 404 nm compared to PS2.M and more sustained catalysis. NMR spectroscopy indicates that hemin promotes ligand‐induced structure formation in the Hem1‐2T aptamer and forms a specific complex, whereas hemin interacts with the PS2.M G‐quadruplex in a way leading to the disappearance of NMR peaks. Overall, the Hem1‐2T‐hemin complex is a better and more stable HRP mimic, supporting its potential applications in bioanalysis and biocatalysis.

Green Fluorescent Protein SELEX: Immobilization Chemistry and His‐Tag Epitope Bias

Angewandte Chemie · 2025-08-21

Abstract Despite numerous DNA aptamers for proteins having been reported, a model system allowing the use of cost‐effective proteins, unmodified DNA, and convenient homogeneous assays is still lacking, which has in turn limited not only fundamental studies of aptamers but also their translation to practical applications. Herein, three separate green fluorescent protein (GFP) selections were carried out using both non‐tagged and His‐tagged GFP immobilized on either NHS‐activated resin or Co 2+ affinity resin. Only the GFP/NHS system resulted in aptamers that consistently bind to unmodified GFP, whereas the His‐tagged GFP yielded aptamers biased toward the His‐tag epitope. Sequence alignment and fluorescence polarization assays indicate that many previously published aptamers bound to the His‐tag instead of the intended protein. This work not only obtained a model aptamer for proteins but also revealed critical information on bias toward His‐tags during aptamer selections.

Machine Learning-Assisted 3D SERS Chip with Acoustic Enrichment for High-Accuracy Diagnosis of Respiratory Viruses and Emerging Pathogens

ACS Sensors · 2025-10-01 · 2 citations

PFU/mL under acoustic enrichment. This approach holds significant promise for the early screening and detection of emerging and known respiratory pathogens.

Au@Ag, Au@Pd, Au@Pt and Au@Ir Nanoparticles as Colorimetric and Peroxidase-Like Labels for Lateral Flow Assays

ACS Applied Nano Materials · 2025-08-28 · 6 citations

Lateral flow assays (LFAs) are an indispensable point-of-care (POC) diagnostic tool. While it is fast, accessible, and user-friendly, LFAs often suffer from limited sensitivity (high limit of detection – LOD). One widely adopted strategy to overcome this limitation involves replacing conventional gold nanoparticle (Au NP) labels with alternative nanomaterials. Utilizing more efficient colorimetric labels preserves all the advantages of LFA as a POC method while improving LOD. Alternative nanomaterial-based labels may exhibit catalytic activities, allowing for signal amplification and further improvement in sensitivity. In this study, we investigated the optical and catalytic properties of core@shell NPs to evaluate their performance as labels for LFAs. A total of 67 core@shell NPs, comprising Au cores and Ag, Pd, Pt, or Ir shells with varying thicknesses (0.5–50 nm) were synthesized. For the first time, we comprehensively examined the relationship between composition, morphology, optical behavior, and catalytic performance across these core@shell NPs. We used two parameters for evaluating nanoparticles as LFA labels: integrated extinction in the visible range (380–750 nm) and specific peroxidase-like activity. Based on these parameters, we concluded that their LOD values on nitrocellulose membranes do not improve significantly through optical enhancement alone. A substantial improvement in sensitivity of two to 3 orders of magnitude is associated with NPs exhibiting high peroxidase-like activity. This work has demonstrated the universal applicability of the proposed parameters as reliable descriptors for nanoparticle performance in LFAs.