AptaDB: a comprehensive database integrating aptamer-target interactions.

Aptamers have emerged as research hotspots of the next generation due to excellent performance benefits and application potentials in pharmacology, medicine, and analytical chemistry. Despite the numerous aptamer investigations, the lack of comprehensive data integration has hindered the development of computational methods for aptamers and the reuse of aptamers. A public access database named AptaDB, derived from experimentally validated data manually collected from the literature, was hence developed, integrating comprehensive aptamer-related data, which include six key components: (i) experimentally validated aptamer-target interaction information, (ii) aptamer property information, (iii) structure information of aptamer, (iv) target information, (v) experimental activity information, and (vi) algorithmically calculated similar aptamers. AptaDB currently contains 1350 experimentally validated aptamer-target interactions, 1230 binding affinity constants, 1293 aptamer sequences, and more. Compared to other aptamer databases, it contains twice the number of entries found in available databases. The collection and integration of the above information categories is unique among available aptamer databases and provides a user-friendly interface. AptaDB will also be continuously updated as aptamer research evolves. We expect that AptaDB will become a powerful source for aptamer rational design and a valuable tool for aptamer screening in the future. For access to AptaDB, please visit http://lmmd.ecust.edu.cn/aptadb/.

DNA Reaction Circuits to Establish Designated Biological Functions in Multicellular Community.

In multicellular organisms, individual cells are coordinated through complex communication networks to accomplish various physiological tasks. Aiming to establish new biological functions in the multicellular community, we used DNA as the building block to develop a cascade of nongenetic reaction circuits to establish a dynamic cell-cell communication network. Utilizing membrane-anchored amphiphilic DNA tetrahedra (TDN) as the nanoscaffold, reaction circuits were incorporated into three unrelated cells in order to uniquely regulate their sense-and-response behaviors. As a proof-of-concept, this step enabled these cells to simulate significant biological events involved in T cell-mediated anticancer immunity. Such events included cancer-associated antigen recognition and the presentation of antigen-presenting cells (APCs), APC-facilitated T cell activation and dissociation, and T cell-mediated cancer targeting and killing. By combining the excellent programmability and molecular recognition ability of DNA, our cell-surface reaction circuits hold promise for mimicking and manipulating many biological processes.

Nanopore Blockade Sensors for Quantitative Analysis Using an Optical Nanopore Assay.

Nanopore sensing is a popular biosensing strategy that is being explored for the quantitative analysis of biomarkers. With low concentrations of analytes, nanopore sensors face challenges related to slow response times and selectivity. Here, we demonstrate an approach to rapidly detect species at ultralow concentrations using an optical nanopore blockade sensor for quantitative detection of the protein vascular endothelial growth factor (VEGF). This sensor relies on monitoring fluorescent polystyrene nanoparticles blocking nanopores in a nanopore array of 676 nanopores. The fluorescent signal is read out using a wide-field fluorescence microscope. Nonspecific blockade events are then distinguished from specific blockade events based on the ability to pull the particles out of the pore using an applied electric field. This allows the detection of VEGF at sub-picomolar concentration in less than 15 min.

Selection of internalizing RNA aptamers into human breast cancer cells derived from primary sites.

Breast cancer is the most common cancer in women. Although chemotherapy is still broadly used in its treatment, adverse effects remain a challenge. In this scenario, aptamers emerge as a promising alternative for theranostic applications. Studies using breast cancer cell lines provide useful information in laboratory and preclinical investigations, most of which use cell lines established from metastatic sites. However, these cell lines correspond to cell populations of the late stage of tumor progression. On the other hand, studies using breast cancer cells established from primary sites make it possible to search for new theranostic approaches in the early stages of the disease. Therefore, this work aimed to select RNA aptamers internalized by MGSO-3 cells, a human breast cancer cell line, derived from a primary site previously established in our laboratory. Using the Cell-Internalization SELEX method, we have selected two candidate aptamers (ApBC1 and ApBC2). We evaluated their internalization efficiencies, specificities, cellular localization by Reverse Transcription-qPCR (RT-qPCR) and confocal microscopy assays. The results suggest that both aptamers were efficiently internalized by human breast cancer cells, MACL-1, MDA-MB-231, and especially by MGSO-3 cells. Furthermore, both aptamers could effectively distinguish human breast cancer cells derived from normal human mammary cell (MCF 10A) and prostate cancer cell (PC3) lines. Therefore, ApBC1 and ApBC2 could be promising candidate molecules for theranostic applications, even in the early stages of tumor progression.

Aptamer-Functionalized Magnetic Ti3C2 Based Nanoplatform for Simultaneous Enrichment and Detection of Exosomes.

Exosomes are nanovesicles secreted by cells, which play a crucial role in various pathological processes. Exosomes have shown great promise as tumor biomarkers because of the abundant secretion during tumor formation. The development of a convenient, efficient, and cost-effective method for simultaneously enriching and detecting exosomes is of utmost importance for both basic research and clinical applications. In this study, an aptamer-functionalized magnetic Ti3C2 composite material (Fe3O4@Ti3C2@PEI@DSP@aptamer@FAM-ssDNA) is prepared for the simultaneous enrichment and detection of exosomes. CD63 aptamers are utilized to recognize and capture the exosomes, followed by magnetic separation. The exosomes are then released by cleaving the disulfide bonds of DSP. Compared to traditional methods, Fe3O4@Ti3C2@PEI@DSP@aptamer@FAM-ssDNA exhibited superior efficiency in enriching exosomes while preserving their structural and functional integrity. Detection of exosome concentration is achieved through the fluorescence quenching of Ti3C2 and the competitive binding between the exosomes and a fluorescently labeled probe. This method exhibited a low detection limit of 4.21 × 104 particles mL-1, a number that is comparable to the state-of-the-art method in the detection of exosomes. The present study demonstrates a method of simultaneous enrichment and detection of exosomes with a high sensitivity, accuracy, specificity, and cost-effectiveness providing significant potential for clinical research and diagnosis.

Enhancing Neuronal Cell Uptake of Therapeutic Nucleic Acids with Tetrahedral DNA Nanostructures.

The successful translation of therapeutic nucleic acids (NAs) for the treatment of neurological disorders depends on their safe and efficient delivery to neural cells, in particular neurons. DNA nanostructures can be a promising NAs delivery vehicle. Nonetheless, the potential of DNA nanostructures for neuronal cell delivery of therapeutic NAs is unexplored. Here, tetrahedral DNA nanostructures (TDN) as siRNA delivery scaffolds to neuronal cells, exploring the influence of functionalization with two different reported neuronal targeting ligands: C4-3 RNA aptamer and Tet1 peptide are investigated. Nanostructures are characterized in vitro, as well as in silico using molecular dynamic simulations to better understand the overall TDN structural stability. Enhancement of neuronal cell uptake of TDN functionalized with the C4-3 Aptamer (TDN-Apt), not only in neuronal cell lines but also in primary neuronal cell cultures is demonstrated. Additionally, TDN and TDN-Apt nanostructures carrying siRNA are shown to promote silencing in a process aided by chloroquine-induced endosomal disruption. This work presents a thorough workflow for the structural and functional characterization of the proposed TDN as a nano-scaffold for neuronal delivery of therapeutic NAs and for targeting ligands evaluation, contributing to the future development of new neuronal drug delivery systems based on DNA nanostructures.

Kinetic ITC of DNA Aptamers Binding for Small Molecules and Implications for Binding Assays and Biosensors.

The determination of kon and koff values through kinetic analysis is crucial for understanding the intricacies of aptamer-target binding interactions. By employing kinetic ITC, we systematically analyzed a range of ITC data of various aptamers. Upon plotting their kon and koff values as a function of their Kd values, a notable trend emerged. Across a range of Kd values spanning from 28 nM to 864 µM, the kon value decreased from 2×105 M-1 s-1 to 96 M-1 s-1, whereas the koff value increased from 1.03×10-3 s-1 to 0.012 s-1. Thus, both kon and koff contributed to the change of Kd in the same direction, although the range of kon change was larger. Since experiments are often run at close to the Kd value, this concentration effect also played an important role in the observed binding kinetics. The effect of these kinetic parameters on two common sensing mechanisms, including aptamer beacons and strand-displacement assays, are discussed. This work has provided the kinetic values of small molecule binding aptamers and offered insights into aptamer-based biosensors.

DNA Aptamer Integrated Hydrogel Nanocomposites on Screen Printed Gold Electrodes for Point-of-Care Detection of Testosterone in Human Serum.

This work describes the development and evaluation of a novel electrochemical aptasensor for testosterone detection. The sensor utilizes a specifically designed DNA immobilized on a screen-printed gold electrode (SPGE) modified with a conductive hydrogel and gold nanoparticles (HG/NP) composite. The aptasensor exhibited a dose-dependent response to testosterone (0.05 to 50 ng/mL) with a detection limit of 0.14 ng/mL and a good sensitivity of 0.23 µA ng-1 mL cm-2. The sensor displayed excellent selectivity towards testosterone compared to structurally similar steroid hormones. Importantly, the incorporation of HG/NP not only improved the sensor's conductivity but also acted as an antifouling layer, minimizing signal interference from non-specific biomolecule interactions in complex biological samples like human serum. The results obtained from the aptasensor showed good correlation with a standard ELISA method, demonstrating its effectiveness in real-world scenarios.

The effect of magnetic bead size on the isolation efficiency of lung cancer cells in a serpentine microchannel with added cavities.

An investigation was conducted to examine the effect of magnetic bead (MB) size on the effectiveness of isolating lung cancer cells using the immunomagnetic separation (IMS) method in a serpentine microchannel with added cavities (SMAC) structure. Carboxylated magnetic beads were specifically conjugated to target cells through a modification procedure using aptamer materials. Cells immobilized with different sizes (in micrometers) of MBs were captured and isolated in the proposed device for comparison and analysis. The study yields significance regarding the clarification of device working principles by using a computational model. Furthermore, an accurate evaluation of the MB size impact on capture efficiency was achieved, including the issue of MB-cell accumulation at the inlet-channel interface, despite it being overlooked in many previous studies. As a result, our findings demonstrated an increasing trend in binding efficiency as the MB size decreased, evidenced by coverages of 50.5%, 60.1%, and 73.4% for sizes of 1.36 µm, 3.00 µm, and 4.50 µm, respectively. Additionally, the overall capture efficiency (without considering the inlet accumulation) was also higher for smaller MBs. However, when accounting for the actual number of cells entering the channel (i.e., the effective capture), larger MBs showed higher capture efficiency. The highest effective capture achieved was 88.4% for the size of 4.50 µm. This research provides an extensive insight into the impact of MB size on the performance of IMS-based devices and holds promise for the efficient separation of circulating cancer cells (CTCs) in practical applications.

Nanobiosensors based on on-site detection approaches for rapid pesticide sensing in the agricultural arena: A systematic review of the current status and perspectives.

The extensive use of chemical pesticides has significantly boosted agricultural food crop yields. Nevertheless, their excessive and unregulated application has resulted in food contamination and pollution in environmental, aquatic, and agricultural ecosystems. Consequently, the on-site monitoring of pesticide residues in agricultural practices is paramount to safeguard global food and conservational safety. Traditional pesticide detection methods are cumbersome and ill-suited for on-site pesticide finding. The systematic review provides an in-depth analysis of the current status and perspectives of nanobiosensors (NBS) for pesticide detection in the agricultural arena. Furthermore, the study encompasses the fundamental principles of NBS, the various transduction mechanisms employed, and their incorporation into on-site detection platforms. Conversely, the assortment of transduction mechanisms, including optical, electrochemical, and piezoelectric tactics, is deliberated in detail, emphasizing its advantages and limitations in pesticide perception. Incorporating NBS into on-site detection platforms confirms a vital feature of their pertinence. The evaluation reflects the integration of NBS into lab-on-a-chip systems, handheld devices, and wireless sensor networks, permitting real-time monitoring and data-driven decision-making in agronomic settings. The potential for robotics and automation in pesticide detection is also scrutinized, highlighting their role in improving competence and accuracy. Finally, this systematic review provides a complete understanding of the current landscape of NBS for on-site pesticide sensing. Consequently, we anticipate that this review offers valuable insights that could form the foundation for creating innovative NBS applicable in various fields such as materials science, nanoscience, food technology and environmental science.

RNA nanostructures for targeted drug delivery and imaging.

The RNA molecule plays a pivotal role in many biological processes by relaying genetic information, regulating gene expression, and serving as molecular machines and catalyzers. This inherent versatility of RNA has fueled significant advancements in the field of RNA nanotechnology, driving the engineering of complex nanoscale architectures toward biomedical applications, including targeted drug delivery and bioimaging. RNA polymers, serving as building blocks, offer programmability and predictability of Watson-Crick base pairing, as well as non-canonical base pairing, for the construction of nanostructures with high precision and stoichiometry. Leveraging the ease of chemical modifications to protect the RNA from degradation, researchers have developed highly functional and biocompatible RNA architectures and integrated them into preclinical studies for the delivery of payloads and imaging agents. This review offers an educational introduction to the use of RNA as a biopolymer in the design of multifunctional nanostructures applied to targeted delivery in vivo, summarizing physical and biological barriers along with strategies to overcome them. Furthermore, we highlight the most recent progress in the development of both small and larger RNA nanostructures, with a particular focus on imaging reagents and targeted cancer therapeutics in pre-clinical models and provide insights into the prospects of this rapidly evolving field.

Synthetic Biology Approaches to Posttranslational Regulation in Plants.

To date synthetic biology approaches involving creation of functional genetic modules are used in a wide range of organisms. In plants, such approaches are used both for research in the field of functional genomics and to increase the yield of agricultural crops. Of particular interest are methods that allow controlling genetic apparatus of the plants at post-translational level, which allow reducing non-targeted effects from interference with the plant genome. This review discusses recent advances in the plant synthetic biology for regulation of the plant metabolism at posttranslational level and highlights their future directions.

Aptamer-functionalized MOFs and AI-driven strategies for early cancer diagnosis and therapeutics.

Metal-Organic Frameworks (MOFs) have exceptional inherent properties that make them highly suitable for diverse applications, such as catalysis, storage, optics, chemo sensing, and biomedical science and technology. Over the past decades, researchers have utilized various techniques, including solvothermal, hydrothermal, mechanochemical, electrochemical, and ultrasonic, to synthesize MOFs with tailored properties. Post-synthetic modification of linkers, nodal components, and crystallite domain size and morphology can functionalize MOFs to improve their aptamer applications. Advancements in AI and machine learning led to the development of nonporous MOFs and nanoscale MOFs for medical purposes. MOFs have exhibited promise in cancer therapy, with the successful accumulation of a photosensitizer in cancer cells representing a significant breakthrough. This perspective is focused on MOFs' use as advanced materials and systems for cancer therapy, exploring the challenging aspects and promising features of MOF-based cancer diagnosis and treatment. The paper concludes by emphasizing the potential of MOFs as a transformative technology for cancer treatment and diagnosis.

Structure-based insights into fluorogenic RNA aptamers.

Fluorogenic RNA aptamers are in vitro-selected RNA molecules capable of binding to specific fluorophores, significantly increasing their intrinsic fluorescence. Over the past decade, the color palette of fluorescent RNA aptamers has greatly expanded. The emergence and development of these fluorogenic RNA aptamers has introduced a powerful approach for visualizing RNA localization and transport with high spatiotemporal resolution in live cells. To date, a variety of tertiary structures of fluorogenic RNA aptamers have been determined using X-ray crystallography or NMR spectroscopy. Many of these fluorogenic RNA aptamers feature base quadruples or base triples in their fluorophore-binding sites. This review summarizes the structure-based investigations of fluorogenic RNA aptamers, with a focus on their overall folds, ligand-binding pockets and fluorescence activation mechanisms. Additionally, the exploration of how structures guide rational optimization to enhance RNA visualization techniques is discussed.

Exploring aptamers for targeted enrichment of X sperm in bovine: unraveling selective potential.

Nucleic acid aptamers have been used in the past for the development of diagnostic methods against a number of targets such as bacteria, pesticides, cancer cells etc. In the present study, six rounds of Cell-SELEX were performed on a ssDNA aptamer library against X-enriched sperm cells from Sahiwal breed cattle. Sequencing was used to examine the aptamer sequences that shown affinity for sperm carrying the X chromosome in order to find any possible X-sperm-specific sequences. Out of 35 identified sequences, 14 were selected based on bioinformatics analysis like G-Score and Mfold structures. Further validation of their specificity was done via fluorescence microscopy. The interaction of biotinylated-aptamer with sperm was also determined by visualizing the binding of streptavidin coated magnetic beads on the head region of the sperm under bright field microscopy. Finally, a real-time experiment was designed for the validation of X-sperm enrichment by synthesized aptamer sequences. Among the studied sequences, aptamer 29a exhibited a higher affinity for X sperm compared to Y sperm in a mixed population of sperm cells. By using aptamer sequence 29a, we obtained an enrichment of 70% for X chromosome bearing sperm cells.

Development of aptamer surface-enhanced Raman spectroscopy sensor based on Fe3O4@Pt and Au@Ag nanoparticles for the determination of acetamiprid.

As a common chlorinated nicotinic pesticide with high insecticidal activity, acetamiprid has been widely used for pest control. However, the irrational use of acetamiprid will pollute the environment and thus affect human health. Therefore, it is crucial to develop a simple, highly sensitive, and rapid method for acetamiprid residue detection. In this study, the capture probe (Fe3O4@Pt-Aptamer) was connected with the signal probe (Au@DTNB@Ag CS-cDNA) to form an assembly with multiple SERS-enhanced effects. Combined with magnetic separation technology, a SERS sensor with high sensitivity and stability was constructed to detect acetamiprid residue. Based on the optimal conditions, the SERS intensity measured at 1333 cm-1 is in relation to the concentration of acetamiprid in the range 2.25 × 10-9-2.25 × 10-5 M, and the calculated limit of detection (LOD) was 2.87 × 10-10 M. There was no cross-reactivity with thiacloprid, clothianidin, nitenpyram, imidacloprid, and chlorpyrifos, indicating that this method has good sensitivity and specificity. Finally, the method was applied to the detection of acetamiprid in cucumber samples, and the average recoveries were 94.19-103.58%, with RSD < 2.32%. The sensor can be used to analyse real samples with fast detection speed, high sensitivity, and high selectivity.

A comprehensive review of the application of Zr-based metal-organic frameworks for electrochemical sensors and biosensors.

A family of inorganic-organic hybrid crystalline materials made up of organic ligands and metal cations or clusters is known as metal-organic frameworks (MOFs). Because of their unique stability, intriguing characteristics, and structural diversity, zirconium-based MOFs (Zr-MOFs) are regarded as one of the most interesting families of MOF materials for real-world applications. Zr-MOFs that have the ligands, metal nodes, and guest molecules enclosed show distinct electrochemical reactions. They can successfully and sensitively identify a wide range of substances, which is important for both environmental preservation and human health. The rational design and synthesis of Zr-MOF electrochemical sensors and biosensors, as well as their applications in the detection of drugs, biomarkers, pesticides, food additives, hydrogen peroxide, and other materials, are the main topics of this comprehensive review. We also touch on the current issues and potential future paths for Zr-MOF electrochemical sensor research.

Micromotor-based dual aptassay for early cost-effective diagnosis of neonatal sepsis.

Given the long-life expectancy of the newborn, research aimed at improving sepsis diagnosis and management in this population has been recognized as cost-effective, which at early stages continues to be a tremendous challenge. Despite there is not an ideal-specific biomarker, the simultaneous detection of biomarkers with different behavior during an infection such as procalcitonin (PCT) as high specificity biomarker with one of the earliest biomarkers in sepsis as interleukin-6 (IL-6) increases diagnostic performance. This is not only due to their high positive predictive value but also, since it can also help the clinician to rule out infection and thus avoid the use of antibiotics, due to their high negative predictive value. To this end, we explore a cutting-edge micromotor (MM)-based OFF-ON dual aptassay for simultaneous determination of both biomarkers in 15 min using just 2 µL of sample from low-birth-weight neonates with gestational age less than 32 weeks and birthweight below 1000 g with clinical suspicion of late-onset sepsis. The approach reached the high sensitivities demanded in the clinical scenario (LODPCT = 0.003 ng/mL, LODIL6 = 0.15 pg/mL) with excellent correlation performance (r > 0.9990, p < 0.05) of the MM-based approach with the Hospital method for both biomarkers during the analysis of diagnosed samples and reliability (Er < 6% for PCT, and Er < 4% for IL-6). The proposed approach also encompasses distinctive technical attributes in a clinical scenario since its minimal sample volume requirements and expeditious results compatible with few easy-to-obtain drops of heel stick blood samples from newborns admitted to the neonatal intensive care unit. This would enable the monitoring of both sepsis biomarkers within the initial hours after the manifestation of symptoms in high-risk neonates as a valuable tool in facilitating prompt and well-informed decisions about the initiation of antibiotic therapy.These results revealed the asset behind micromotor technology for multiplexing analysis in diagnosing neonatal sepsis, opening new avenues in low sample volume-based diagnostics.

Detection of Dopamine Based on Aptamer-Modified Graphene Microelectrode.

In this paper, a novel aptamer-modified nitrogen-doped graphene microelectrode (Apt-Au-N-RGOF) was fabricated and used to specifically identify and detect dopamine (DA). During the synthetic process, gold nanoparticles were loaded onto the active sites of nitrogen-doped graphene fibers. Then, aptamers were modified on the microelectrode depending on Au-S bonds to prepare Apt-Au-N-RGOF. The prepared microelectrode can specifically identify DA, avoiding interference with other molecules and improving its selectivity. Compared with the N-RGOF microelectrode, the Apt-Au-N-RGOF microelectrode exhibited higher sensitivity, a lower detection limit (0.5 µM), and a wider linear range (1~100 µM) and could be applied in electrochemical analysis fields.

Combining the Benefits of Biotin-Streptavidin Aptamer Immobilization with the Versatility of Ni-NTA Regeneration Strategies for SPR.

The high affinity of the biotin-streptavidin interaction has made this non-covalent coupling an indispensable strategy for the immobilization and enrichment of biomolecular affinity reagents. However, the irreversible nature of the biotin-streptavidin bond renders surfaces functionalized using this strategy permanently modified and not amenable to regeneration strategies that could increase assay reusability and throughput. To increase the utility of biotinylated targets, we here introduce a method for reversibly immobilizing biotinylated thrombin-binding aptamers onto a Ni-nitrilotriacetic acid (Ni-NTA) sensor chip using 6xHis-tagged streptavidin as a regenerable capture ligand. This approach enabled the reproducible immobilization of aptamers and measurements of aptamer-protein interaction in a surface plasmon resonance assay. The immobilized aptamer surface was stable during five experiments over two days, despite the reversible attachment of 6xHis-streptavidin to the Ni-NTA surface. In addition, we demonstrate the reproducibility of this immobilization method and the affinity assays performed using it. Finally, we verify the specificity of the biotin tag-streptavidin interaction and assess the efficiency of a straightforward method to regenerate and reuse the surface. The method described here will allow researchers to leverage the versatility and stability of the biotin-streptavidin interaction while increasing throughput and improving assay efficiency.