Search Results (10)

The Arabidopsis transcription factor WUSCHEL-related homeobox 14 (AtWOX14) plays versatile roles in plant growth and development. However, its biochemical specificity of DNA binding, its genome-wide regulatory targets, and how these are affected by DNA methylation remain uncharacterized. To clarify the biochemistry underlying the regulatory function of AtWOX14, using the recently developed 5mC-incorporation strategy, this study performed SELEX and DAP-seq for AtWOX14 both in the presence and absence of cytosine methylation, systematically curated 65 motif models and identified 51,039 genomic binding sites for AtWOX14, and examined how 5mC affects DNA binding of AtWOX14 through bioinformatic analyses. Overall, 5mC represses the DNA binding of AtWOX14 monomers but facilitates the binding of its dimers, and the methylation effect on a cytosine’s affinity to AtWOX14 is position-dependent. Notably, we found that the most preferred homodimeric configuration of AtWOX14 has changed from ER1 to ER0 upon methylation. This change has the potential to rewire the regulatory network downstream of AtWOX14, as suggested by the GO analyses and the strength changes in the DAP-seq peaks upon methylation. Therefore, this work comprehensively illustrates the specificity and targets of AtWOX14 and reports a previously unrecognized effect of DNA methylation on transcription factor binding. Full article

Background/Objectives: Ricin’s high toxicity and potential as a bioweapon underscore the need for effective antidotes. Monoclonal antibodies, though effective, are limited by complex production. This study aimed to develop a graphene oxide-based aptamer nanoarray (ARMAN) for improved neutralization and protection against ricin. Methods: High-affinity aptamers targeting ricin’s RTA and RTB subunits were selected using SELEX technology and conjugated to graphene oxide (GO) via click chemistry. ARMAN’s characteristics, including morphology, stability, and biosecurity, were assessed. Its performance was evaluated in terms of affinity for ricin, neutralization capacity, and therapeutic effects in cellular assays and a mouse model of ricin poisoning. Results: ARMAN exhibited a uniform morphology with an average particle size of 217 nm and demonstrated significantly enhanced affinity for ricin compared to free aptamers. ARMAN showed rapid and effective neutralization ability, significantly increasing cell viability in BEAS-2B, GES-1, and HL7702 cell lines exposed to ricin. In vivo, ARMAN treatment led to a notable prolongation of survival in ricin-poisoned mice, highlighting its potential for both pre- and post-exposure treatment. These findings indicate that ARMAN not only neutralizes ricin effectively but also provides a therapeutic window for treatment. Conclusions: ARMAN’s superior binding affinity, serum stability, biocompatibility, and broad therapeutic efficacy make it a promising new antidote against ricin poisoning. This study’s findings represent significant progress in the development of rapid-response antidotes, with ARMAN offering a potential solution for both military and civilian emergency response scenarios. Full article

Ofloxacin (OFL) is widely used in animal husbandry and aquaculture due to its low price and broad spectrum of bacterial inhibition, etc. However, it is difficult to degrade and is retained in animal-derived food products, which are hazardous to human health. In this study, a simple and efficient method was developed for the detection of OFL residues in meat products. OFL coupled with amino magnetic beads by an amination reaction was used as a stationary phase. Aptamer AWO-06, which showed high affinity and specificity for OFL, was screened using the exponential enrichment (SELEX) technique. A fluorescent biosensor was developed by using AWO-06 as a probe and graphene oxide (GO) as a quencher. The OFL detection results could be obtained within 6 min. The linear range was observed in the range of 10–300 nM of the OFL concentration, and the limit of the detection of the sensor was 0.61 nM. Furthermore, the biosensor was stored at room temperature for more than 2 months, and its performance did not change. The developed biosensor in this study is easy to operate and rapid in response, and it is suitable for on-site detection. This study provided a novel method for the detection of OFL residues in meat products. Full article

Thiamethoxam, a nicotinic pesticide used worldwide, can cause great harm to the environment and even to human health, and aptamers, known as chemical antibodies, have high affinity and specificity for the target, as well as great potential in detecting small molecules such as pesticides. In this paper, we report a highly sensitive biosensor system for thiamethoxam residue detection based on aptamer technology. After 15 rounds of screening with the pressurized GO-SELEX technology, we found that the aptamer libraries of the 5th and 9th rounds showed high affinity by the capture method. Four candidate aptamers were obtained by high-throughput sequencing and secondary structure prediction. Among them, the aptamer named Thi-5R-18 from the 5th round was demonstrated to possess the highest affinity by isothermal titration calorimetry, with a dissociation constant (Kd) of 4.935 × 10⁻⁵ M. The results of molecular docking showed that thiamethoxam and Thi-5R-18 were combined with bases G-15, A-19, and T-71 through hydrogen bonding and π–π interaction.Thi-5R-18 was used as a recognition element to construct a AuNPs colorimetric aptasensor, achieving an ultralow detection limit of 0.37 nM. More importantly, this colorimetric aptasensor can be used for quantitative detection of thiamethoxam on tea leaves, with a recovery of 96.94%~105.86%. This study provides a highly sensitive biosensor for detection of thiamethoxam residue. Full article

Bacterial kidney disease (BKD) is a major health problem of salmonids, affecting both wild and cultured salmon. The disease is caused by Renibacterium salmoninarum (Rs), a fastidious, slow-growing and strongly Gram-positive diplobacillus that produces chronic, systemic infection characterized by granulomatous lesions in the kidney and other organs, often resulting in death. Fast detection of the pathogen is important to limit the spread of the disease, particularly in hatcheries or aquaculture facilities. Aptamers are increasingly replacing conventional antibodies as platforms for the development of rapid diagnostic tools. In this work, we describe the first instance of isolating and characterizing a ssDNA aptamer that binds with high affinity to p57 or major soluble antigen (MSA), the principal antigen found on the cell wall surface of Rs. Specifically, in this study a construct of the full-length protein containing a DNA binding domain (MSA-R2c) was utilized as target. Aptamers were isolated from a pool of random sequences using GO-SELEX (graphene oxide-systematic evolution of ligands by exponential enrichment) protocol. The selection generated multiple aptamers with conserved motifs in the random region. One aptamer with high frequency of occurrence in different clones was characterized and found to display a strong binding affinity to MSA-R2c with a K_d of 3.0 ± 0.6 nM. The aptamer could be potentially utilized for the future development of a sensor for rapid and onsite detection of Rs in water or in infected salmonids, replacing time-consuming and costly lab analyses. Full article

Malachite green oxalate (MG) is a kind of veterinary drug, which is freely soluble in water and hazardous to aquatic products, resulting in food toxicity and human health problems. The demand for effective and sensitive detection of MG residues is increasing in food safety. In this work, three DNA aptamers MG-36-12/16/17 targeting MG with good affinity (K_d values were 169.78, 71.94, and 102.46 μM, respectively) were obtained by Capture-SELEX. Furthermore, MG-36-12, MG-76-16-6A, and MG-36-17 were found to perform sensitively and specifically to detect MG as a sensing probe in a FRET fluorescent aptasensor, where the FAM-labeled aptamer and GO were employed as efficient energy donor–acceptor pair. The linear range of this aptasensor using aptamer MG-36-12 was from 1.71 to 514.29 ng/mL and the LOD was as low as 0.79 ng/mL. Additionally, the fluorescent assay using aptamer MG-36-17 to detect MG exhibited a linear relationship from 1.71 to 857.14 ng/mL and a LOD of 2.13 ng/mL. Meanwhile, the aptasensor showed high specificity to MG with no cross-reactivity to other veterinary drugs and had a mean recovery of 81.54% to 100.96% in actual water samples from the aquatic product market. Full article

Diabetes is one of the top 10 global causes of death. About one in 11 global adults have diabetes. As the disease progresses, the mortality rate increases, and complications can develop. Thus, early detection and effective management of diabetes are especially important. Herein, we present a novel glycated human serum albumin (GHSA) aptamer, i.e., GABAS-01, which has high affinity and specificity. The aptamer was selected by reduced graphene oxide-based systematic evolution of ligands by exponential enrichement (rGO-based SELEX) against GHSA. After five rounds of selection through gradually harsher conditions, GABAS-01 with high affinity and specificity for the target was obtained. GABAS-01 was labeled by FAM at the 5′-end and characterized by measuring the recovery of a fluorescence signal that is the result of fluorescence quenching effect of rGO. As a result, GABAS-01 had low-nanomolar Kd values of 1.748 ± 0.227 nM and showed a low limit of detection of 16.40 μg/mL against GHSA. This result shows the potential application of GABAS-01 as an effective on-site detection probe of GHSA. In addition, these properties of GABAS-01 are expected to contribute to detection of GHSA in diagnostic fields. Full article

Seafood is an emerging health food, and interest in improving the quality of seafood is increasing. Saxitoxin (STX) is a neurotoxin produced by marine dinoflagellates that is accumulated in seafood. It can block the neuronal transmission between nerves and muscle cell membranes, resulting in the disturbance of neuromuscular transmission and subsequent voluntary muscle paralysis. Here, we developed a new aptamer for the detection of STX using graphene oxide–systematic evolution of ligands by exponential enrichment (GO-SELEX). Furthermore, we confirmed sensitivity and selectivity of the developed aptamer specific to STX using a localized surface plasmon resonance (LSPR) sensor. The sensing chip was fabricated by fixing the new STX aptamer immobilized on the gold nanorod (GNR) substrate. The STX LSPR aptasensor showed a broad, linear detection range from 5 to 10,000 μg/L, with a limit of detection (LOD) of 2.46 μg/L (3σ). Moreover, it was suitable for the detection of STX (10, 100, and 2000 μg/L) in spiked mussel samples and showed a good recovery rate (96.13–116.05%). The results demonstrated that the new STX aptamer-modified GNR chip was sufficiently sensitive and selective to detect STX and can be applied to real samples as well. This LSPR aptasensor is a simple, label-free, cost-effective sensing system with a wide detectable range. Full article

Aptamers are small non-coding RNAs capable of recognizing, with high specificity and affinity, a wide variety of molecules in a manner that resembles antibodies. This class of nucleic acids is the resulting product of applying a well-established screening method known as SELEX. First developed in 1990, the SELEX process has become a powerful tool to select structured oligonucleotides for the recognition of targets, starting with small molecules, going through protein complexes until whole cells. SELEX has also evolved along with new technologies positioning itself as an alternative in the design of a new class of therapeutic agents in modern molecular medicine. This review is an historical follow-up of SELEX method over the two decades since its first appearance. Full article

DNA aptamers are synthetic, single-stranded DNA oligonucleotides selectedby SELEX methods for their binding with specific ligands. Here we present ethidiumbinding results for three related DNA aptamers (PDB code: 1OLD, 1DB6, and 2ARG)that bind L-argininamide (L-Arm). The ligand bound form of each aptamer's structurehas been reported and each are found to be composed primarily of two domainsconsisting of a stem helical region and a loop domain that forms a binding pocket for thecognate ligand. Previous thermodynamic experiments demonstrated that the DNAaptamer 1OLD undergoes a large conformational ordering upon binding to L-Arm. Herewe extend those linkage binding studies by examining the binding of the heterocyclicintercalator ethidium to each of the three aptamers by fluorescence and absorptionspectrophotometric titrations. Our results reveal that ethidium binds to each aptamer with∆G^o's in the range of -8.7 to -9.4 kcal/mol. The stoichiometry of binding is 2:1 for eachaptamer and is quantitatively diminished in the presence of L-Arm as is the overallfluorescence intensity of ethidium. Together, these results demonstrate that a portion ofthe bound ethidium is excluded from the aptamer in the presence of a saturating amountof L-Arm. These results demonstrate the utility of ethidium and related compounds forthe probing of non-conventional DNA structures and reveal an interesting fundamentalthermodynamic linkage in DNA aptamers. Results are discussed in the context of thethermodynamic stability and structure of each of the aptamers examined. Full article