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Lipids in Health and Disease May 2024The degradation of low-density lipoprotein receptor (LDLR) is induced by proprotein convertase subtilisin/kexin type 9 (PCSK9), resulting in elevated plasma... (Review)
Review
The degradation of low-density lipoprotein receptor (LDLR) is induced by proprotein convertase subtilisin/kexin type 9 (PCSK9), resulting in elevated plasma concentrations of LDL cholesterol. Therefore, inhibiting the interactions between PCSK9 and LDLR is a desirable therapeutic goal for managing hypercholesterolemia. Aptamers, which are RNA or single-stranded DNA sequences, can recognize their targets based on their secondary structure. Aptamers exhibit high selectivity and affinity for binding to target molecules. The systematic evolution of ligands by exponential enrichment (SELEX), a combination of biological approaches, is used to screen most aptamers in vitro. Due to their unique advantages, aptamers have garnered significant interest since their discovery and have found extensive applications in various fields. Aptamers have been increasingly utilized in the development of biosensors for sensitive detection of pathogens, analytes, toxins, drug residues, and malignant cells. Furthermore, similar to monoclonal antibodies, aptamers can serve as therapeutic tools. Unlike certain protein therapeutics, aptamers do not elicit antibody responses, and their modified sugars at the 2'-positions generally prevent toll-like receptor-mediated innate immune responses. The focus of this review is on aptamer-based targeting of PCSK9 and the application of aptamers both as biosensors and therapeutic agents.
Topics: Proprotein Convertase 9; Humans; Aptamers, Nucleotide; Biosensing Techniques; Lipid Metabolism; Receptors, LDL; SELEX Aptamer Technique; Hypercholesterolemia; Animals; PCSK9 Inhibitors
PubMed: 38796450
DOI: 10.1186/s12944-024-02151-8 -
Pharmaceutics May 2024In recent years, biopolymer-based nano-drug delivery systems with antioxidative properties have gained significant attention in the field of pharmaceutical research.... (Review)
Review
In recent years, biopolymer-based nano-drug delivery systems with antioxidative properties have gained significant attention in the field of pharmaceutical research. These systems offer promising strategies for targeted and controlled drug delivery while also providing antioxidant effects that can mitigate oxidative stress-related diseases. Generally, the healthcare landscape is constantly evolving, necessitating the continual development of innovative therapeutic approaches and drug delivery systems (DDSs). DDSs play a pivotal role in enhancing treatment efficacy, minimizing adverse effects, and optimizing patient compliance. Among these, nanotechnology-driven delivery approaches have garnered significant attention due to their unique properties, such as improved solubility, controlled release, and targeted delivery. Nanomaterials, including nanoparticles, nanocapsules, nanotubes, etc., offer versatile platforms for drug delivery and tissue engineering applications. Additionally, biopolymer-based DDSs hold immense promise, leveraging natural or synthetic biopolymers to encapsulate drugs and enable targeted and controlled release. These systems offer numerous advantages, including biocompatibility, biodegradability, and low immunogenicity. The utilization of polysaccharides, polynucleotides, proteins, and polyesters as biopolymer matrices further enhances the versatility and applicability of DDSs. Moreover, substances with antioxidative properties have emerged as key players in combating oxidative stress-related diseases, offering protection against cellular damage and chronic illnesses. The development of biopolymer-based nanoformulations with antioxidative properties represents a burgeoning research area, with a substantial increase in publications in recent years. This review provides a comprehensive overview of the recent developments within this area over the past five years. It discusses various biopolymer materials, fabrication techniques, stabilizers, factors influencing degradation, and drug release. Additionally, it highlights emerging trends, challenges, and prospects in this rapidly evolving field.
PubMed: 38794332
DOI: 10.3390/pharmaceutics16050670 -
Sensors (Basel, Switzerland) May 2024Fluorescence induced by the excitation of a fluorophore with plane-polarized light has a different polarization depending on the size of the fluorophore-containing...
Fluorescence induced by the excitation of a fluorophore with plane-polarized light has a different polarization depending on the size of the fluorophore-containing reagent and the rate of its rotation. Based on this effect, many analytical systems have been implemented in which an analyte contained in a sample and labeled with a fluorophore (usually fluorescein) competes to bind to antibodies. Replacing antibodies in such assays with aptamers, low-cost and stable oligonucleotide receptors, is complicated because binding a fluorophore to them causes a less significant change in the polarization of emissions. This work proposes and characterizes the compounds of the reaction medium that improve analyte binding and reduce the mobility of the aptamer-fluorophore complex, providing a higher analytical signal and a lower detection limit. This study was conducted on aflatoxin B1 (AFB1), a ubiquitous toxicant contaminating foods of plant origins. Eight aptamers specific to AFB1 with the same binding site and different regions stabilizing their structures were compared for affinity, based on which the aptamer with 38 nucleotides in length was selected. The polymers that interact reversibly with oligonucleotides, such as poly-L-lysine and polyethylene glycol, were tested. It was found that they provide the desired reduction in the depolarization of emitted light as well as high concentrations of magnesium cations. In the selected optimal medium, AFB1 detection reached a limit of 1 ng/mL, which was 12 times lower than in the tris buffer commonly used for anti-AFB1 aptamers. The assay time was 30 min. This method is suitable for controlling almond samples according to the maximum permissible levels of their contamination by AFB1. The proposed approach could be applied to improve other aptamer-based analytical systems.
Topics: Aflatoxin B1; Aptamers, Nucleotide; Fluorescence Polarization; Polyelectrolytes; Biosensing Techniques; Polyamines; Limit of Detection; Fluorescent Dyes
PubMed: 38794084
DOI: 10.3390/s24103230 -
Sensors (Basel, Switzerland) May 2024Liquid biopsy is expected to become widespread in the coming years thanks to point of care devices, which can include label-free biosensors. The surface...
Liquid biopsy is expected to become widespread in the coming years thanks to point of care devices, which can include label-free biosensors. The surface functionalization of biosensors is a crucial aspect that influences their overall performance, resulting in the accurate, sensitive, and specific detection of target molecules. Here, the surface of a microring resonator (MRR)-based biosensor was functionalized for the detection of protein biomarkers. Among the several existing functionalization methods, a strategy based on aptamers and mercaptosilanes was selected as the most highly performing approach. All steps of the functionalization protocol were carefully characterized and optimized to obtain a suitable protocol to be transferred to the final biosensor. The functionalization protocol comprised a preliminary plasma treatment aimed at cleaning and activating the surface for the subsequent silanization step. Different plasma treatments as well as different silanes were tested in order to covalently bind aptamers specific to different biomarker targets, i.e., C-reactive protein, SARS-CoV-2 spike protein, and thrombin. Argon plasma and 1% / mercaptosilane were found as the most suitable for obtaining a homogeneous layer apt to aptamer conjugation. The aptamer concentration and time for immobilization were optimized, resulting in 1 µM and 3 h, respectively. A final passivation step based on mercaptohexanol was also implemented. The functionalization protocol was then evaluated for the detection of thrombin with a photonic biosensor based on microring resonators. The preliminary results identified the successful recognition of the correct target as well as some limitations of the developed protocol in real measurement conditions.
Topics: Biosensing Techniques; Aptamers, Nucleotide; Silanes; Humans; Thrombin; C-Reactive Protein; Spike Glycoprotein, Coronavirus; SARS-CoV-2; Biomarkers; Surface Properties; COVID-19
PubMed: 38793970
DOI: 10.3390/s24103107 -
Sensors (Basel, Switzerland) May 2024Biosensors based on ion-sensitive field effect transistors (ISFETs) combined with aptamers offer a promising and convenient solution for point-of-care testing...
Biosensors based on ion-sensitive field effect transistors (ISFETs) combined with aptamers offer a promising and convenient solution for point-of-care testing applications due to the ability for fast and label-free detection of a wide range of biomarkers. Mobile and easy-to-use readout devices for the ISFET aptasensors would contribute to further development of the field. In this paper, the development of a portable PC-controlled device for detecting aptamer-target interactions using ISFETs is described. The device assembly allows selective modification of individual ISFETs with different oligonucleotides. TaO-gated ISFET structures were optimized to minimize trapped charge and capacitive attenuation. Integrated CMOS readout circuits with linear transfer function were used to minimize the distortion of the original ISFET signal. An external analog signal digitizer with constant voltage and superimposed high-frequency sine wave reference voltage capabilities was designed to increase sensitivity when reading ISFET signals. The device performance was demonstrated with the aptamer-driven detection of troponin I in both reference voltage setting modes. The sine wave reference voltage measurement method reduced the level of drift over time and enabled a lowering of the minimum detectable analyte concentration. In this mode (constant voltage 2.4 V and 10 kHz 0.1Vp-p), the device allowed the detection of troponin I with a limit of detection of 3.27 ng/mL. Discrimination of acute myocardial infarction was demonstrated with the developed device. The ISFET device provides a platform for the multiplexed detection of different biomarkers in point-of-care testing.
Topics: Aptamers, Nucleotide; Transistors, Electronic; Biosensing Techniques; Biomarkers; Humans; Troponin I
PubMed: 38793863
DOI: 10.3390/s24103008 -
Molecules (Basel, Switzerland) May 2024The ongoing SARS-CoV-2 pandemic has underscored the urgent need for versatile and rapidly deployable antiviral strategies. While vaccines have been pivotal in...
Molecular Mechanism of Interaction between DNA Aptamer and Receptor-Binding Domain of Severe Acute Respiratory Syndrome Coronavirus 2 Variants Revealed by Steered Molecular Dynamics Simulations.
The ongoing SARS-CoV-2 pandemic has underscored the urgent need for versatile and rapidly deployable antiviral strategies. While vaccines have been pivotal in controlling the spread of the virus, the emergence of new variants continues to pose significant challenges to global health. Here, our study focuses on a novel approach to antiviral therapy using DNA aptamers, short oligonucleotides with high specificity and affinity for their targets, as potential inhibitors against the spike protein of SARS-CoV-2 variants Omicron and JN.1. Our research utilizes steered molecular dynamics (SMD) simulations to elucidate the binding mechanisms of a specifically designed DNA aptamer, AM032-4, to the receptor-binding domain (RBD) of the aforementioned variants. The simulations reveal detailed molecular insights into the aptamer-RBD interaction, demonstrating the aptamer's potential to maintain effective binding in the face of rapid viral evolution. Our work not only demonstrates the dynamic interaction between aptamer-RBD for possible antiviral therapy but also introduces a computational method to study aptamer-protein interactions.
Topics: Aptamers, Nucleotide; Molecular Dynamics Simulation; SARS-CoV-2; Spike Glycoprotein, Coronavirus; Protein Binding; Humans; Binding Sites; Antiviral Agents; Protein Domains; COVID-19; COVID-19 Drug Treatment
PubMed: 38792076
DOI: 10.3390/molecules29102215 -
International Journal of Molecular... May 2024Maternal immune activation (MIA) is a risk factor for multiple neurodevelopmental disorders; however, animal models developed to explore MIA mechanisms are sensitive to...
Maternal immune activation (MIA) is a risk factor for multiple neurodevelopmental disorders; however, animal models developed to explore MIA mechanisms are sensitive to experimental factors, which has led to complexity in previous reports of the MIA phenotype. We sought to characterize an MIA protocol throughout development to understand how prenatal immune insult alters the trajectory of important neurodevelopmental processes, including the microglial regulation of synaptic spines and complement signaling. We used polyinosinic:polycytidylic acid (polyI:C) to induce MIA on gestational day 9.5 in CD-1 mice, and measured their synaptic spine density, microglial synaptic pruning, and complement protein expression. We found reduced dendritic spine density in the somatosensory cortex starting at 3-weeks-of-age with requisite increases in microglial synaptic pruning and phagocytosis, suggesting spine density loss was caused by increased microglial synaptic pruning. Additionally, we showed dysregulation in complement protein expression persisting into adulthood. Our findings highlight disruptions in the prenatal environment leading to alterations in multiple dynamic processes through to postnatal development. This could potentially suggest developmental time points during which synaptic processes could be measured as risk factors or targeted with therapeutics for neurodevelopmental disorders.
Topics: Animals; Microglia; Mice; Female; Pregnancy; Dendritic Spines; Poly I-C; Complement System Proteins; Prenatal Exposure Delayed Effects; Phagocytosis; Disease Models, Animal; Somatosensory Cortex; Synapses; Neuronal Plasticity
PubMed: 38791517
DOI: 10.3390/ijms25105480 -
International Journal of Molecular... May 2024Orthodontic space closure following tooth extraction is often hindered by alveolar bone deficiency. This study investigates the therapeutic use of nuclear factor-kappa B...
Orthodontic space closure following tooth extraction is often hindered by alveolar bone deficiency. This study investigates the therapeutic use of nuclear factor-kappa B (NF-κB) decoy oligodeoxynucleotides loaded with polylactic-co-glycolic acid nanospheres (PLGA-NfDs) to mitigate alveolar bone loss during orthodontic tooth movement (OTM) following the bilateral extraction of maxillary first molars in a controlled experiment involving forty rats of OTM model with ethics approved. The decreased tendency of the OTM distance and inclination angle with increased bone volume and improved trabecular bone structure indicated minimized alveolar bone destruction. Reverse transcription-quantitative polymerase chain reaction and histomorphometric analysis demonstrated the suppression of inflammation and bone resorption by downregulating the expression of tartrate-resistant acid phosphatase, tumor necrosis factor-α, interleukin-1β, cathepsin K, NF-κB p65, and receptor activator of NF-κB ligand while provoking periodontal regeneration by upregulating the expression of alkaline phosphatase, transforming growth factor-β1, osteopontin, and fibroblast growth factor-2. Importantly, relative gene expression over the maxillary second molar compression side in proximity to the alveolus highlighted the pharmacological effect of intra-socket PLGA-NfD administration, as evidenced by elevated osteocalcin expression, indicative of enhanced osteocytogenesis. These findings emphasize that locally administered PLGA-NfD serves as an effective inflammatory suppressor and yields periodontal regenerative responses following tooth extraction.
Topics: Animals; Polylactic Acid-Polyglycolic Acid Copolymer; Rats; Nanospheres; Tooth Movement Techniques; Oligodeoxyribonucleotides; Tooth Socket; Male; NF-kappa B; Wound Healing; Alveolar Bone Loss; Tooth Extraction
PubMed: 38791262
DOI: 10.3390/ijms25105223 -
Communications Biology May 2024Generating genetic diversity lies at the heart of directed evolution which has been widely used to engineer genetic parts and gene circuits in synthetic biology. With...
Generating genetic diversity lies at the heart of directed evolution which has been widely used to engineer genetic parts and gene circuits in synthetic biology. With the ever-expanding application of directed evolution, different approaches of generating genetic diversity are required to enrich the traditional toolbox. Here we show in vitro generation of genetic diversity for directed evolution by error-prone artificial DNA synthesis (epADS). This approach comprises a three-step process which incorporates base errors randomly generated during chemical synthesis of oligonucleotides under specific conditions into the target DNA. Through this method, 200 ~ 4000 folds of diversification in fluorescent strength have been achieved in genes encoding fluorescent proteins. EpADS has also been successfully used to diversify regulatory genetic parts, synthetic gene circuits and even increase microbial tolerance to carbenicillin in a short time period. EpADS would be an alternative tool for directed evolution which may have useful applications in synthetic biology.
Topics: Directed Molecular Evolution; Genetic Variation; DNA; Synthetic Biology; Oligonucleotides; Escherichia coli
PubMed: 38789612
DOI: 10.1038/s42003-024-06340-0 -
SLAS Discovery : Advancing Life... Jun 2024Methylation of proteins and nucleic acids plays a fundamental role in epigenetic regulation, and discovery of methyltransferase (MT) inhibitors is an area of intense...
Methylation of proteins and nucleic acids plays a fundamental role in epigenetic regulation, and discovery of methyltransferase (MT) inhibitors is an area of intense activity. Because of the diversity of MTs and their products, assay methods that detect S-adenosylhomocysteine (SAH) - the invariant product of S-adenosylmethionine (SAM)-dependent methylation reactions - offer some advantages over methods that detect specific methylation events. However, direct, homogenous detection of SAH requires a reagent capable of discriminating between SAH and SAM, which differ by a single methyl group. Moreover, MTs are slow enzymes and many have submicromolar affinities for SAM; these properties translate to a need for detection of SAH at low nanomolar concentrations in the presence of excess SAM. To meet these needs, we leveraged the exquisite molecular recognition properties of a naturally occurring SAH-sensing RNA aptamer, or riboswitch. By splitting the riboswitch into two fragments, such that SAH binding induces assembly of a trimeric complex, we engineered sensors that transduce binding of SAH into positive fluorescence polarization (FP) and time resolved Förster resonance energy transfer (TR-FRET) signals. The split riboswitch configuration, called the AptaFluor™ SAH Methyltransferase Assay, allows robust detection of SAH (Z' > 0.7) at concentrations below 10 nM, with overnight signal stability in the presence of typical MT assay components. The AptaFluor assay tolerates diverse MT substrates, including histones, nucleosomes, DNA and RNA, and we demonstrated its utility as a robust, enzymatic assay method for several methyltransferases with SAM K values < 1 µM. The assay was validated for HTS by performing a pilot screen of 1,280 compounds against the SARS-CoV-2 RNA capping enzyme, nsp14. By enabling direct, homogenous detection of SAH at low nanomolar concentrations, the AptaFluor assay provides a universal platform for screening and profiling MTs at physiologically relevant SAM concentrations.
Topics: S-Adenosylhomocysteine; Riboswitch; Methyltransferases; Enzyme Assays; S-Adenosylmethionine; Fluorescence Resonance Energy Transfer; Methylation; Humans; Fluorescence Polarization; Aptamers, Nucleotide
PubMed: 38788976
DOI: 10.1016/j.slasd.2024.100161