Search Results (265)

Chronic and non-communicable diseases (NCDs) account for a large proportion of global disorders and mortality, posing significant burdens on healthcare systems. Early diagnosis and timely interference are critical for effective management and disease prevention. However, the traditional methods of diagnosis still suffer from high costs, time delays in processing, and infrastructure requirements that are usually unaffordable in resource-constrained settings. Aptamer-based biosensors have emerged as promising alternatives to offer enhanced specificity, stability, and cost-effectiveness for disease biomarker detection. The SELEX (Systematic Evolution of Ligands by Exponential Enrichment) methodology allows developing aptamers with high-affinity binding capabilities to a variety of targets, for instance proteins, cells, or even small molecules, hence rendering them suitable for NCD diagnosis. Aptasensors—recent developments in the electrochemical and optical dominion—offer much enhanced sensitivity, selectivity, and stability of detection across a diverse range of diseases from lung cancer and leukemia to diabetes and chronic respiratory disorders. This study provides a comprehensive review of progress in aptamer-based sensors, focusing on their role in point-of-care diagnostics and adaptability in a real-world environment with future directions in overcoming current limitations. 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

Chronic immunoinflammatory rheumatic diseases, such as axial spondyloarthritis (AxSpA), are accompanied by a dysregulation of bone remodeling. Among potential biomarkers of bone metabolism, the Wnt pathway antagonist, Dickkopf-1 (DKK-1), is of particular interest because of its potential to reflect a shift towards joint ossification or osteoporosis, but its diagnostic value needs validation. There is still a lack of stable and efficient methods of measuring serum DKK-1 levels suitable for longitude studies. The use of aptamer-based diagnostic assays could be very promising for this purpose. We generated novel anti-DKK-1 DNA aptamers from a combinatorial library with a pre-defined sequence pattern in the randomized region. This approach showed high efficacy, as only four SELEX rounds of selection produced high-affinity aptamers with dissociation constants ranging from 1.3 to 3.7 nM. A family of their truncated versions was also developed by rational design. Novel DNA aptamers functioned as capture components in a microplate ELISA-like assay with HRP-conjugated anti-DKK-1 antibody as a reporter component. We succeeded in revealing the aptamer/aptamer sandwich pairs that provided an aptamer-only sandwich colorimetric assay. The aptamer/antibody colorimetric test systems were also examined in the analyses of blood serum from AxSpA patients and shown sufficient workability. However, in a number of cases we registered significant differences between assays based on TD10 and DK4 aptamers and made some suggestions about the origin of this effect. Full article

Vibrio harveyi is a serious bacterial pathogen which can infect a wide range of marine organisms, such as marine fish, invertebrates, and shrimp, in aquaculture, causing severe losses. In addition, V. harveyi can be transmitted through food and water, infecting humans and posing a serious threat to public safety. Therefore, rapid and accurate detection of this pathogen is key for the prevention and control of related diseases. In this study, nine rounds of in vitro screening were conducted with Systematic Evolution of Ligands by Exponential Enrichment (SELEX) technology using unmodified DNA libraries, targeting the crude extracellular matrix (CEM) of V. harveyi. Two DNAzymes, named DVh₁ and DVh₃, with high activity and specificity were obtained. Furthermore, a fluorescent biosensor with dual DNAzymes was constructed which exhibited improved detection efficiency. The sensor showed a good fluorescence response to multiple aquatic products (i.e., fish, shrimp, and shellfish) infected with V. harveyi, with a detection limit below 11 CFU/mL. The fluorescence signal was observed within 30 min of reaction after target addition. This simple, inexpensive, highly effective, and easy to operate DNAzymes biosensor can be used for field detection of V. harveyi. Full article

Lactopontin (LPN) is a highly phosphorylated O-glycosylated acidic protein closely associated with infant gut, brain, and immune development, and its recognition is urgent due to its rising application in fortified dairy products and infant formula. In this study, an ssDNA aptamer against LPN was obtained, among which two kinds of matrix-background-assisted systematic evolution of ligands via exponential enrichment (SELEX) approaches were performed and compared. The direct approach was to utilize the sample matrix as the mixing-incubation background between the ssDNA library and LPN that can theoretically increase screening pressure and simulate practical application scenarios. The indirect approach was to utilize a PBS buffer as a screening background and to include counter-screening steps that adopt the “sample matrix” as a whole as the counter-screening target. Their screening evolutions were monitored through qPCR assays from sequence diversity convergences of each sub-library based on the change in the proportion of hetero- and homo-duplexes from the dissociation curve and melting temperature, which were also verified from the sequence statistics of high-throughput sequencing. The common sequence of Seq.I1II3 from the two approaches was finally fished out as the aptamer through multiple analyses of combining the sequence frequency, secondary structures, homology, and binding assessments, which was demonstrated good specificity and low-nanomolar affinity by qPCR assay (K_D, 5.9 nM). In addition, molecular docking and a dynamics simulation were performed for their binding site prediction and affinity confirmation. This study provides a potential identifying element and a basis for accelerating the development of methods for LPN detection in dairy products. Full article

Aptamer-based biosensors have been widely constructed and applied to detect diverse targets. Glutathione S-transferase (GST), a pivotal phase II metabolic enzyme, plays a critical role in biotransformation in vivo, and aberrant GST expression is associated with various health risks. Herein, aptamers targeting GST were systematically selected from a randomized single-stranded DNA (ssDNA) library of 79 nucleotides (nt) using a biotinylated GST-immobilized streptavidin agarose (SA) bead SELEX technology. Following rigorous screening across eight rounds, four aptamers with strikingly similar secondary structures emerged. Among these, Seq3 exhibited the highest affinity towards GST and was selected for further optimization. A semi-rational post-SELEX truncation strategy was then employed based on base composition analysis, secondary structure analysis and affinity assessment. This strategy enabled the systematic removal of redundant nucleotides in Seq3 without compromising its affinity, ultimately yielding a truncated aptamer, Seq3-3, which retains its specificity with a compact 39nt length. Building upon Seq3-3, a double-stranded fluorescent aptamer probe was ingeniously designed for the in vitro detection of GST. The detection mechanism hinges on the competitive displacement of the complementary chain from the probe, mediated by the target protein, leading to the separation of the antisense oligonucleotide from the double-stranded complex. This process triggers the restoration of the fluorescence signal, enabling sensitive detection, and the probe exhibits excellent response within a linear range of GST activity ranging from 0 to 1500 U/L. The results show that not only an efficient strategy for screening robust and practicable aptamers but also an ultrahighly sensitive detection platform for GST was established. Full article

Tumor cell-induced platelet aggregation (TCIPA) is a mechanism for the protection of tumor cells in the bloodstream and the promotion of tumor progression and metastases. The platelet C-type lectin-like receptor 2 (CLEC-2) can bind podoplanin (PDPN) on a cancer cell surface to facilitate TCIPA. Selective blockage of PDPN-mediated platelet–tumor cell interaction is a plausible strategy for inhibiting metastases. In this study, we aimed to screen for aptamers, which are the single-stranded DNA oligonucleotides that form a specific three-dimensional structure, bind to specific molecular targets with high affinity and specificity, bind to PDPN, and interfere with PDPN/CLEC-2 interactions. The systematic evolution of ligands by exponential enrichment (SELEX) was employed to enrich aptamers that recognize PDPN. The initial characterization of ssDNA pools enriched by SELEX revealed a PDPN aptamer designated as A1 displaying parallel-type G-quadruplexes and long stem-and-loop structures and binding PDPN with a material with a dissociation constant (K_d) of 1.3 ± 1.2 nM. The A1 aptamer recognized both the native and denatured form of PDPN. Notably, the A1 aptamer was able to quantitatively detect PDPN proteins in Western blot analysis. The A1 aptamer could interfere with the interaction between PDPN and CLEC-2 and inhibit PDPN-induced platelet aggregation in a concentration-dependent manner. These findings indicated that the A1 aptamer is a candidate for the development of biosensors in detecting the levels of PDPN expression. The action by A1 aptamer could result in the prevention of tumor cell metastases, and if so, could become an effective pharmacological agent in treating cancer patients. Full article

Pearl millet is a major cereal crop that feeds more than 90 million people worldwide in arid and semi-arid regions. The stalk phenotypes of Poaceous grasses are critical for their productivity and stress tolerance; however, the molecular mechanisms governing stalk development in pearl millet remain to be deciphered. In this study, we spatiotemporally measured 19 transcriptomes for stalk internodes of four different early developmental stages. Data analysis of the transcriptomes defined four developmental zones on the stalks and identified 12 specific gene sets with specific expression patterns across the zones. Using weighted gene co-expression network analysis (WGCNA), we found that two co-expression modules together with candidate genes were involved in stalk elongation and the thickening of pearl millet. Among the elongation-related candidate genes, we established by SELEX that a MYB-family transcription factor PMF7G02448 can bind to the promoters of three cell wall synthases genes (CesAs). In summary, these findings provide insights into stalk development and offer potential targets for future genetic improvement in pearl millet. Full article

This review article is focused on the progress made in the synthesis of 5′-α-P-modified nucleoside triphosphates (α-phosphate mimetics). A variety of α-P-modified nucleoside triphosphates (NTPαXYs, Y = O, S; X = S, Se, BH₃, alkyl, amine, N-alkyl, imido, or others) have been developed. There is a unique class of nucleoside triphosphate analogs with different properties. The main chemical approaches to the synthesis of NTPαXYs are analyzed and systematized here. Using the data presented here on the diversity of NTPαXYs and their synthesis protocols, it is possible to select an appropriate method for obtaining a desired α-phosphate mimetic. Triphosphates’ substrate properties toward nucleic acid metabolism enzymes are highlighted too. We reviewed some of the most prominent applications of NTPαXYs including the use of modified dNTPs in studies on mechanisms of action of polymerases or in systematic evolution of ligands by exponential enrichment (SELEX). The presence of heteroatoms such as sulfur, selenium, or boron in α-phosphate makes modified triphosphates nuclease resistant. The most distinctive feature of NTPαXYs is that they can be recognized by polymerases. As a result, S-, Se-, or BH₃-modified phosphate residues can be incorporated into DNA or RNA. This property has made NTPαXYs a multifunctional tool in molecular biology. This review will be of interest to synthetic chemists, biochemists, biotechnologists, or biologists engaged in basic or applied research. Full article

Aptamers are nucleic acid sequences that specifically bind with target molecules and are vital to applications such as biosensing, drug development, disease diagnostics, etc. The traditional selection procedure of aptamers is based on the Systematic Evolution of Ligands by an Exponential Enrichment (SELEX) process, which relies on repeating cycles of screening and amplification. With the rapid development of aptamer applications, RNA and XNA aptamers draw more attention than before. But their selection is troublesome due to the necessary reverse transcription and transcription process (RNA) or low efficiency and accuracy of enzymes for amplification (XNA). In light of this, we review the recent advances in aptamer selection methods and give an outlook on future development in a non-SELEX approach, which simplifies the procedure and reduces the experimental costs. We first provide an overview of the traditional SELEX methods mostly designed for screening DNA aptamers to introduce the common tools and methods. Then a section on the current screening methods for RNA and XNA is prepared to demonstrate the efforts put into screening these aptamers and the current difficulties. We further predict that the future trend of aptamer selection lies in non-SELEX methods that do not require nucleic acid amplification. We divide non-SELEX methods into an immobilized format and non-immobilized format and discuss how high-resolution partitioning methods could facilitate the further improvement of selection efficiency and accuracy. Full article

Aptamers are short oligonucleotides with single-stranded regions or peptides that recently started to transform the field of diagnostics. Their unique ability to bind to specific target molecules with high affinity and specificity is at least comparable to many traditional biorecognition elements. Aptamers are synthetically produced, with a compact size that facilitates deeper tissue penetration and improved cellular targeting. Furthermore, they can be easily modified with various labels or functional groups, tailoring them for diverse applications. Even more uniquely, aptamers can be regenerated after use, making aptasensors a cost-effective and sustainable alternative compared to disposable biosensors. This review delves into the inherent properties of aptamers that make them advantageous in established diagnostic methods. Furthermore, we will examine some of the limitations of aptamers, such as the need to engage in bioinformatics procedures in order to understand the relationship between the structure of the aptamer and its binding abilities. The objective is to develop a targeted design for specific targets. We analyse the process of aptamer selection and design by exploring the current landscape of aptamer utilisation across various industries. Here, we illuminate the potential advantages and applications of aptamers in a range of diagnostic techniques, with a specific focus on quartz crystal microbalance (QCM) aptasensors and their integration into the well-established ELISA method. This review serves as a comprehensive resource, summarising the latest knowledge and applications of aptamers, particularly highlighting their potential to revolutionise diagnostic approaches. Full article

Affinity reagents, or target-binding molecules, are quite versatile and are major workhorses in molecular biology and medicine. Antibodies are the most famous and frequently used type and they have been used for a wide range of applications, including laboratory techniques, diagnostics, and therapeutics. However, antibodies are not the only available affinity reagents and they do have significant drawbacks, including laborious and costly production. Aptamers are one potential alternative that have a variety of unique advantages. They are single stranded DNA or RNA molecules that can be selected for binding to many targets including proteins, carbohydrates, and small molecules—for which antibodies typically have low affinity. There are also a variety of cost-effective methods for producing and modifying nucleic acids in vitro without cells, whereas antibodies typically require cells or even whole animals. While there are also significant drawbacks to using aptamers in therapeutic applications, including low in vivo stability, aptamers have had success in clinical trials for treating a variety of diseases and two aptamer-based drugs have gained FDA approval. Aptamer development is still ongoing, which could lead to additional applications of aptamer therapeutics, including antitoxins, and combinatorial approaches with nanoparticles and other nucleic acid therapeutics that could improve efficacy. Full article

Selenium is an essential inorganic compound in human and animal nutrition, involved in the proper functioning of the body. As a micronutrient, it actively contributes to the regulation of various metabolic activities, i.e., thyroid hormone, and protection against oxidative stress. However, Se exhibits a narrow concentration window between having a positive effect and exerting a toxic effect. In higher doses, it negatively affects living organisms and causes DNA damage through the formation of free radicals. Increased reactivity of Se anions can also disrupt the integrity and function of DNA-repairing proteins. As the permissible concentration of Se in drinking water is 10 µg/L, it is vital to develop sensitive and robust methods of Se detection in aqueous samples. In this study, for the first time, we proposed a selective aptamer for selenate ion detection, chosen following the SELEX process, and its application in the construction of an electrochemical aptasensor towards SeO₄²⁻ ions. Measurement conditions such as the used redox marker and pH value of the measurement solution were chosen. The proposed aptasensor is characterized by good selectivity and an LOD of 1 nM. Conditions for biosensor regeneration and storage were also investigated in this research. Full article

EGFRvIII is expressed only in tumor cells and strongly in glioblastoma and is considered a promising target in cancer diagnosis and therapy. Aptamers are synthetic single-stranded oligonucleotides that bind to biochemical target molecules with high binding affinity and specificity. This study examined the potential of the ⁶⁸Ga-NOTA-EGFRvIII aptamer as a nuclear imaging probe for visualizing EGFRvIII-expressing glioblastoma by positron emission tomography (PET). EGFRvIII aptamer was selected using the SELEX technology, and flow cytometry and fluorescence microscopy verified the high binding affinity to EGFRvIII positive U87MG vIII 4.12 glioma cells but not to EGFRvIII negative U87MG cells. The EGFRvIII aptamer was conjugated with a chelator (1,4,7-triazanonane-1,4,7-triyl)triacetic acid (NOTA) for ⁶⁸Ga-labeling. The ⁶⁸Ga-NOTA-EGFRvIII aptamer was prepared using the preconcentration-based labeling method with a high radiolabeling yield at room temperature. Ex vivo biodistribution analyses confirmed the significantly higher tumor uptake of the ⁶⁸Ga-NOTA-EGFRvIII aptamer in EGFRvIII-expressing xenograft tumors than that in EGFRvIII negative tumors, confirming the specific tumor uptake of the ⁶⁸Ga-NOTA-EGFRvIII aptamer in vivo. PET imaging studies revealed a high retention rate of the ⁶⁸Ga-NOTA-EGFRvIII aptamer in U87MG vIII 4.12 tumors but only low uptake levels in U87-MG tumors, suggesting that the ⁶⁸Ga-NOTA-EGFRvIII aptamer may be used as a PET imaging agent for EGFRvIII-expressing glioblastoma. Full article

There is a significant need to develop new environmentally friendly, extraction-free sample collection mediums that can effectively preserve and protect genetic material for point-of-care and/or self-collection, home-collection, and mail-back testing. Systematic evolution of ligands by exponential enrichment (SELEX) was used to create anti-ribonuclease (RNase) deoxyribonucleic acid (DNA) aptamers against purified RNase A conjugated to paramagnetic carboxylated beads. Following eight rounds of SELEX carried out under various stringency conditions, e.g., selection using Xtract-Free™ (XF) specimen collection medium and elevated ambient temperature of 28 °C, a panel of five aptamers was chosen following bioinformatic analysis using next-generation sequencing. The efficacy of aptamer inactivation of RNase was assessed by monitoring ribonucleic acid (RNA) integrity via fluorometric and real-time RT-PCR analysis. Inclusion of aptamers in reaction incubations resulted in an 8800- to 11,200-fold reduction in RNase activity, i.e., digestion of viral RNA compared to control. Thus, anti-RNase aptamers integrated into XF collection medium as well as other commercial reagents and kits have great potential for ensuring quality intact RNA for subsequent genomic analyses. Full article