Search Results (242)

Aptamers are single-stranded DNA or RNA oligonucleotides screened by systematic evolution of ligands by exponential enrichment (SELEX) methods, which are widely used in food analysis. Aptamers have the advantages of low molecular weight, ease of preparation, simplicity of chemical modification, and structural stability. Aptamers generated by SELEX are typically 80–100 bases in length, and the affinity of the aptamer can be improved by sequence optimization. Methods of aptamer optimization commonly include truncation, mutation, and chemical modification, and molecular docking, molecular dynamics, circular dichroism, and isothermal titration to assess often the binding performance of the aptamer to the target. Optimized aptamers usually enhance the affinity of the aptamer for the target and increase its sensitivity in the detection of pesticides, heavy metals, fungal toxins, pathogenic bacteria, and other objects. This paper focuses on truncation, mutation, chemical modification, the introduction of rare nucleotides, and computer-aided design. It provides an overview of non-immobilized optimization metrics. Full article

Tumor immunotherapy has revolutionized cancer treatment by harnessing the immune system to recognize and eliminate malignant cells, with immune checkpoint inhibitors targeting programmed death receptor 1 (PD-1), programmed death-ligand 1 (PD-L1), and cytotoxic T-lymphocyte-associated protein-4 (CTLA-4) demonstrating remarkable clinical success. However, challenges such as treatment resistance, immune-related adverse effects, and high costs highlight the need for novel therapeutic approaches. Aptamers, short, single-stranded oligonucleotides with high specificity and affinity for target molecules, have emerged as promising alternatives to conventional antibody-based therapies. This review provides a comprehensive analysis of aptamer-based strategies targeting immune checkpoints, with a particular focus on PD-1/PD-L1 and CTLA-4. We summarize recent advances in aptamer design, including bispecific and multifunctional aptamers, and explore their potential in overcoming immune resistance and improving therapeutic efficacy. Additionally, we discuss strategies to enhance aptamer stability, bioavailability, and tumor penetration through chemical modifications and nanoparticle conjugation. Preclinical and early clinical studies have demonstrated that aptamers can effectively block immune checkpoint pathways, restore T-cell activity, and synergize with other immunotherapeutic agents to achieve superior anti-tumor responses. By systematically reviewing the current research landscape and identifying key challenges, this review aims to provide valuable insights into the future directions of aptamer-based cancer immunotherapy, paving the way for more effective and personalized treatment strategies. Full article

Shaking off the forgetfulness towards the methodological power of inosine-mediated error-prone PCR (epPCR), this study reintroduces an often-underappreciated method as a considerably powerful approach for generating aptamer libraries from a single decameric ATCG-repeat-oligonucleotide. The aim was to demonstrate that this simple way of creating sequence diversity was suitable for delivering functional starting libraries for a set of ten whole-cell-SELEX (Systematic Evolution of Ligands by Exponential Enrichment) processes. This epPCR method uses inosine to introduce targeted mutations, avoiding the need for commercial oligo pools or large-scale synthesis. We applied this method to a “universal aptamer” and subjected the three resulting libraries to two rounds of selection against ten diverse targets including probiotic and pathogenic bacteria (Gram-negative and -positive) as well as human cell lines. The enriched aptamers exhibited new binding specificities, demonstrating that the approach supports functional selection. Much like dusting off an old tool and finding it perfectly suited for a modern task, this work shows that revisiting established techniques can address current challenges in aptamer development. Our main finding is that epPCR provides a robust, cost-effective strategy for generating starting libraries and lowers the barrier for initiating successful SELEX campaigns. Full article

Background: Cell membrane proteins play crucial roles in signal transduction and nutrient transport. Many membrane proteins are reportedly overexpressed in cancer cells, which is closely related to cancer progression. The targeted degradation of these membrane proteins has been demonstrated to be a promising strategy for tumor treatment. Several strategies using aptamers to mediate membrane protein lysis, such as lysosomal-mediated lysis and proteasome-mediated lysis, have been reported, but their efficiency is limited by the binding affinity of the aptamer to a single target. Methods: We constructed DNA tweezers with replaceable clamps, which can lyse different proteins upon clamp replacement. Moreover, the clamp improved the degradation efficiency of the target proteins by enhancing the specificity and improving the binding affinity. Results: Lysis was verified in different tumor cell lines and the antitumor activity was confirmed in zebrafish. Conclusions: Overall, these DNA tweezers improve the efficiency of the targeted delivery of functional nucleic acids, provide an efficient and versatile strategy for the degradation of disease-causing proteins, and expand the approach to antitumor therapy. Full article

Despite various methods for detecting and treating SARS-CoV-2, affordable and easily applicable solutions are still needed. Aptamers can potentially fill this gap. Here, we establish a workflow to identify aptamers that bind to the spike proteins of SARS-CoV-2, a process applicable to other targets as well. The spike protein is crucial for the virus’s entry into host cells. The aptamer development process for the spike protein’s receptor binding domain (RBD) begins with splitting the SARS-CoV-2’s genome into 40 nucleotide-long sequences, predicting their two-dimensional structure, and sorting based on the free energy. Selected oligomers undergo three-dimensional structure prediction and docking onto the viral spike protein’s RBD. Six RNA oligomers were identified as top candidates based on the RNA docking with the SARS-CoV-2 wild-type (WT) (Wuhan-Hu-1 strain) and Omicron variant BA.1 RBD and molecular dynamics simulations. Three oligomers also demonstrated strong predicted binding affinity with other SARS-CoV-2 variants, including BA.2, XBB.1.5, and EG.5, based on the protein–aptamer docking followed by stability evaluation using the MD simulations. The aptamer with the best fit for the spike protein RBD was later validated using biolayer interferometry. The process has resulted in identifying a single aptamer from a library of 29,000 RNA oligomers, which exhibited affinity in the submicromolar range and the potential to develop into a viral screen or therapeutic. Full article

T40214 (STAT) and its recently investigated analogue STATB are G-quadruplex (G4) forming aptamers characterized by an unusually high percentage of C. The therapeutic potential of T40214 relies on its ability to inhibit the signalling pathway of STAT3, a protein frequently overexpressed in tumor cells. STAT adopts a dimeric 5′-5′ end-stacked quadruplex structure, characterized by parallel strands, three G-tetrads and three propeller-shaped loops formed by a cytidine residue. STATB folds in a very similar structure, apart from an additional cytidine bulge loop. Many studies suggest that thermal stability and topology of G4 can be significantly affected by C methylation, thus resulting in altered interaction of G4-binding proteins with these structures. Considering this, two series of STAT and STATB analogues containing a single 5-methyl-2′-deoxycytidine (mC) residue instead of canonical C nucleotide in the loop have been prepared and investigated by a combination of spectroscopic and electrophoretic techniques. CD, NMR and PAGE data clearly indicate that all derivatives adopt dimeric G4 strictly similar to that assumed by parent aptamers, but with higher stabilities. Furthermore, the resistance to nucleases and the antiproliferative activity of these mC-containing derivatives against HCT116 (human colorectal carcinoma) and T24 (human bladder carcinoma) cell lines have been evaluated. In most of the cases, STAT and STATB derivatives inhibit cell proliferation to different extents, although to a lesser degree than the unmodified parent sequences. All the data highlight the key role of the loops and indicate mC as a useful tool to contribute favorably to the stability of G4-forming aptamers without alteration of their topology, required for the biological activity. Full article

The dual-signal output self-calibration mode reduces the false positive and negative signals of electrochemiluminescence (ECL) aptamer sensors. A competitive dual-signal ECL platform was designed for the ultrasensitive detection of kanamycin (KAN) using a zirconium metal–organic framework (Zr MOF) and Luminol as ECL emitters. To enhance the ECL efficiency, a co-reactant (polyethyleneimine, PEI) was covalently bound to the Zr MOF to achieve self-enhanced ECL. Based on the selective interaction between KAN and its aptamer, the Luminol/KAN/Zr MOF-PEI “sandwich” structure was immobilized on the electrode surface. The competition for PEI between emitters increased the Luminol ECL signal and decreased the Zr MOF’s ECL signal. The ratio in ECL signals between the two competitive emitters enabled the quantitative analysis of KAN, achieving a detection limit as low as 7.86 × 10⁻⁴ ng/mL. This study elucidated the synergistic mechanism between self-enhanced ECL and ECL competition, offering a novel approach for constructing dual-signal ECL sensors using a single co-reactant. Full article

Background: Synthetic DNA aptamers are a class of molecules with potential applications in medicine, serving as molecular sensors or ligands for targeted drug delivery. Systematic evolution of ligands by exponential enrichment (SELEX) is a technology for selecting functional aptamers that was first reported three decades ago and has been actively developed since. SELEX involves multiple iterations of two fundamental steps: (i) target affinity-based partitioning of aptamers from a random library and (ii) amplification of selected aptamers by PCR, followed by isolation of single-stranded DNA (ssDNA). SELEX protocols have diversified considerably, with numerous variations possible for each step. This heterogeneity makes it challenging to identify optimal methods. Comparative analysis of different approaches for the major stages of SELEX is therefore of considerable practical importance. Methods: Four widely used methods for ssDNA generation were performed in parallel: (a) PCR followed by digestion of the antisense strand with exonuclease lambda, (b) PCR with an extended primer followed by size-dependent strand separation using denaturing PAGE, (c) asymmetric PCR, and (d) asymmetric PCR with a primer-blocker. Results: The specificity, efficiency, reproducibility, and duration of each method were compared. Conclusions: Asymmetric PCR with a primer-blocker yielded the most favorable results. Full article

Nano-luciferase binary technology (NanoBiT)-based pseudoviral sensors are innovative tools for monitoring viral infection dynamics. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infects host cells via its trimeric surface spike protein, which binds to the human angiotensin-converting enzyme II (hACE2) receptor. This interaction is crucial for viral entry and serves as a key target for therapeutic interventions against coronavirus disease 2019 (COVID-19). Aptamers, short single-stranded DNA (ssDNA) or RNA molecules, are highly specific, high-affinity biorecognition elements for detecting infective pathogens. Despite their potential, optimizing viral infection assays using traditional protein–protein interaction (PPI) methods often face challenges in optimizing viral infection assays. In this study, we selected and evaluated aptamers for their ability to interact with viral proteins, enabling the dynamic visualization of infection progression. The NanoBiT-based pseudoviral sensor demonstrated a rapid increase in luminescence within 3 h, offering a real-time measure of viral infection. A comparison of detection technologies, including green fluorescent protein (GFP), luciferase, and NanoBiT technologies for detecting PPI between the pseudoviral spike protein and hACE2, highlighted NanoBiT’s superior sensitivity and performance, particularly in aptamer selection. This bioluminescent system provides a robust, sensitive, and early-stage quantitative approach to studying viral infection dynamics. Full article

Understanding brain function requires advanced neural probes to monitor electrical and chemical signaling across multiple timescales and brain regions. Microelectrode arrays (MEAs) are widely used to record neurophysiological activity across various depths and brain regions, providing single-unit resolution for extended periods. Recent advancements in flexible MEAs, built on micrometer-thick polymer substrates, have improved integration with brain tissue by mimicking the brain’s soft nature, reducing mechanical trauma and inflammation. These flexible, subcellular-scale MEAs can record stable neural signals for months, making them ideal for long-term studies. In addition to electrical recording, MEAs have been functionalized for electrochemical neurotransmitter detection. Electroactive neurotransmitters, such as dopamine, serotonin, and adenosine, can be directly measured via electrochemical methods, particularly on carbon-based surfaces. For non-electroactive neurotransmitters like acetylcholine, glutamate, and γ-aminobutyric acid, alternative strategies, such as enzyme immobilization and aptamer-based recognition, are employed to generate electrochemical signals. This review highlights recent developments in flexible MEA fabrication and functionalization to achieve both electrochemical and electrophysiological recordings, minimizing sensor fowling and brain damage when implanted long-term. It covers multi-time scale neurotransmitter detection, development of conducting polymer and nanomaterial composite coatings to enhance sensitivity, incorporation of enzyme and aptamer-based recognition methods, and the integration of carbon electrodes on flexible MEAs. Finally, it summarizes strategies to acquire electrochemical and electrophysiological measurements from the same device. Full article

Exosomes carry diverse tumor-associated molecular information that can reflect real-time tumor progression, making them a promising tool for liquid biopsy. However, traditional methods for exosome isolation and detection often rely on large, expensive equipment and are time-consuming, limiting their practical applicability in clinical settings. Microfluidic technology offers a versatile platform for exosome analysis, with advantages such as seamless integration, portability and reduced sample volumes. Aptamers, which are single-stranded oligonucleotides with high affinity and specificity for target molecules, have been frequently employed in the development of aptamer-based microfluidics for the isolation, signal amplification, and quantitative detection of exosomes. This review summarizes recent advances in aptamer-based microfluidic strategies for exosome analysis, including (1) strategies for on-chip exosome capture mediated by aptamers combined with nanomaterials or nanointerfaces; (2) aptamer-based on-chip signal amplification techniques, such as enzyme-free hybridization chain reaction (HCR), rolling circle amplification (RCA), and DNA machine-assisted amplification; and (3) various aptamer-assisted detection methods, such as fluorescence, electrochemistry, surface-enhanced Raman scattering (SERS), and magnetism. The limitations and advantages of these methods are also summarized. Finally, future challenges and directions for the clinical analysis of exosomes based on aptamer-based microfluidics are discussed. Full article

Lateral flow immunoassays (LFIAs) are widely used for their low cost, simplicity, and rapid results; however, enhancing their reliability requires the meticulous selection of ligands and nanoparticles (NPs). SiO₂@QD@SiO₂ (QD²) nanoparticles, which consist of quantum dots (QDs) embedded in a silica (SiO₂) core and surrounded by an outer SiO₂ shell, exhibit significantly higher fluorescence intensity (FI) compared to single QDs. In this study, we prepared QD²@PEG@Aptamer, an aptamer conjugated with QD² using succinimidyl-[(N-maleimidopropionamido)-hexaethyleneglycol]ester, which is 130 times brighter than single QDs, for detecting carbohydrate antigen (CA) 19-9 through LFIA. For LFIA optimization, we determined the optimal conditions as a 1.0:2.0 × 10⁻² ratio of polyethylene glycol (PEG) to aptamer by adjusting the amounts of PEG and aptamer, phosphate-buffered saline containing 0.5% Tween^® 20 as a developing solution, and 0.15 μg NPs by setting the NP weight during development. Under these conditions, QD²@PEG@Aptamer selectively detected CA19-9, achieving a detection limit of 1.74 × 10⁻² mg·mL⁻¹. Moreover, FI remained stable for 10 days after detection. These results highlight the potential of QD² and aptamer conjugation technology as a reliable and versatile sensing platform for various diagnostic applications. Full article

Nucleic acid aptamers are single-stranded oligonucleotides that are selected through exponential enrichment (SELEX) technology from synthetic DNA/RNA libraries. These aptamers can specifically recognize and bind to target molecules, serving as specific recognition elements. Surface-enhanced Raman scattering (SERS) spectroscopy is an ultra-sensitive, non-destructive analytical technique that can rapidly acquire the “fingerprint information” of the measured molecules. It has been widely applied in qualitative and trace analysis across various fields, including food safety, environmental monitoring, and biomedical applications. Small molecules, such as toxins, antibiotics, and pesticides, have significant biological effects and are harmful to both human health and the environment. In this paper, we mainly introduced the application and the research progress of SERS detection with aptamers (aptamer-based SERS techniques) in the field of small-molecule detection, particularly in the analysis of pesticide (animal) residues, antibiotics, and toxins. And the progress and prospect of combining the two methods in detection were reviewed. Full article

With the goal of fast and accurate diagnosis of infectious diseases, this study presents a novel electrochemical biosensor that employs a refined aptamer (C9t) for the detection of spike (S) protein SARS-CoV-2 variants in a flexible multielectrode aptasensor array with PoC capabilities. Two aptamer modifications were employed: removing the primer binding sites and including two dithiol phosphoramidite anchor molecules. Thus, reducing fabrication time from 24 to 3 h and increasing the stability and sparseness for multi-thiol aptasensors compared to a standard aptasensor using single thiols, without a reduction in aptamer density. The biosensor fabrication, optimization, and detection were verified in detail by electrochemistry, QCM-D, SPR, and XPS. The analyte–receptor binding was further confirmed spectroscopically at the level of individual molecules by AFM-IR. The aptasensor possesses a low limit of detection (8.0 fg/mL), the highest sensitivity reported for S protein (209.5 signal per concentration decade), and a wide dynamic detection range (8.0 fg/mL–38 ng/mL) in nasopharyngeal samples, covering the clinically relevant range. Furthermore, the C9t aptasensor showed high selectivity for SARS-CoV-2 S proteins over biomarkers for MERS-CoV, RSV, and Influenza. Even more, it showed a three times higher sensitivity for the Omicron in comparison to the Wuhan strain (wild type), alpha, and beta variants of the SARS-CoV-2 virus. Those results demonstrate the creation of an affordable and variant-selective refined C9t aptasensor that outperformed current rapid diagnosis tests. Full article

RNA nanoparticles, derived from the packaging RNA three-way junction motif (pRNA-3WJ) of the bacteriophage phi29 DNA packaging motor, have been demonstrated to be thermodynamically and chemically stable, with promise as a nanodelivery system. Background/Objectives: A previous study showed that RNA nanoparticles with antiangiogenic aptamers (anti-vascular endothelial growth factor (VEGF) and anti-angiopoietin-2 (Ang2) aptamers) inhibited cell proliferation via WST-1 assay. To further investigate the antiangiogenic potential of these RNA nanoparticles, a modified three-dimensional (3D) spheroid sprouting assay model of human umbilical vein endothelial cells was utilized in the present study. Methods: Three groups of RNA nanoparticles were evaluated, namely, pRNA-3WJ series, RNA square series (polygon-type RNA nanoparticles), and 8WJ series (multiple-way junction RNA nanoparticles), which were conjugated with a single anti-VEGF, the combination of one anti-VEGF and one anti-Ang2, or multiple anti-VEGF aptamers. The core scaffold RNA nanoparticles (without aptamers) were used as the references, and bevacizumab was used as the positive control. Results: The results demonstrated the inhibition effects of the RNA nanoparticles on endothelial cell tube formation at 67 nM in a 3D spheroid sprouting model. The results in the 3D spheroid sprouting assay are consistent with those of the WST-1 proliferation assays. Conclusions: Among the RNA nanoparticles evaluated, 3WJ-3VEGF and SQR-VEGF-Ang2 had inhibition effects equivalent to bevacizumab and were promising for anti-angiogenesis treatment. Full article