Utilizing the DNA Aptamer to Determine Lethal α-Amanitin in Mushroom Samples and Urine by Magnetic Bead-ELISA (MELISA)

Amanita poisoning is one of the most deadly types of mushroom poisoning. α-Amanitin is the main lethal toxin in amanita, and the human-lethal dose is about 0.1 mg/kg. Most of the commonly used detection techniques for α-amanitin require expensive instruments. In this study, the α-amanitin aptamer was selected as the research object, and the stem-loop structure of the original aptamer was not damaged by truncating the redundant bases, in order to improve the affinity and specificity of the aptamer. The specificity and affinity of the truncated aptamers were determined using isothermal titration calorimetry (ITC) and gold nanoparticles (AuNPs), and the affinity and specificity of the aptamers decreased after truncation. Therefore, the original aptamer was selected to establish a simple and specific magnetic bead-based enzyme linked immunoassay (MELISA) method for α-amanitin. The detection limit was 0.369 μg/mL, while, in mushroom it was 0.372 μg/mL and in urine 0.337 μg/mL. Recovery studies were performed by spiking urine and mushroom samples with α-amanitin, and these confirmed the desirable accuracy and practical applicability of our method. The α-amanitin and aptamer recognition sites and binding pockets were investigated in an in vitro molecular docking environment, and the main binding bases of both were T3, G4, C5, T6, T7, C67, and A68. This study truncated the α-amanitin aptamer and proposes a method of detecting α-amanitin.

DNA binding by the antimalarial compound artemisinin

Artemisinin (ART) is a vital medicinal compound that is used alone or as part of a combination therapy against malaria. ART is thought to function by attaching to heme covalently and alkylating a range of proteins. Using a combination of biophysical methods, we demonstrate that ART is bound by three-way junction and duplex containing DNA molecules. Binding of ART by DNA is first shown for the cocaine-binding DNA aptamer and extensively studied using this DNA molecule. Isothermal titration calorimetry methods show that the binding of ART is both entropically and enthalpically driven at physiological NaCl concentration. Native mass spectrometry methods confirm DNA binding and show that a non-covalent complex is formed. Nuclear magnetic resonance spectroscopy shows that ART binds at the three-way junction of the cocaine-binding aptamer, and that binding results in the folding of the structure-switching variant of this aptamer. This structure-switching ability was exploited using the photochrome aptamer switch assay to demonstrate that ART can be detected using this biosensing assay. This study is the first to demonstrate the DNA binding ability of ART and should lay the foundation for further work to study implications of DNA binding for the antimalarial activity of ART.

NXP032 Ameliorates Aging-Induced Oxidative Stress and Cognitive Impairment in Mice through Activation of Nrf2 Signaling

Aging is a neurodegenerative disease that leads to cognitive impairment, and an increase in oxidative stress as a major cause is an important factor. It has been reported that aging-related cognitive impairment is associated with increased oxidative damage in several brain regions during aging. As a powerful antioxidant, vitamin C plays an important role in preventing oxidative stress, but due to its unstable chemical properties, it is easily oxidized and thus the activity of antioxidants is reduced. In order to overcome this easily oxidized vulnerability, we developed NXP032 (vitamin C/DNA aptamer complex) that can enhance the antioxidant efficacy of vitamin C using an aptamer. We developed NXP032 (vitamin C/DNA Aptamin C320 complex) that can enhance the antioxidant efficacy of vitamin C using an aptamer. In the present study, we evaluated the neuroprotective effects of NXP032 on aging-induced cognitive decline, oxidative stress, and neuronal damage in 17-month-old female mice. NXP032 was orally administered at 200 mg/kg of ascorbic acid and 4 mg/kg of DNA aptamer daily for eight weeks. Before the sacrifice, a cognitive behavioral test was performed. Administration of NXP032 alleviated cognitive impairment, neuronal damage, microglia activity, and oxidative stress due to aging. We found that although aging decreases the Nrf2-ARE pathway, NXP032 administration activates the Nrf2-ARE pathway to increase the expression of SOD-1 and GSTO1/2. The results suggest that the new aptamer complex NXP032 may be a therapeutic intervention to alleviate aging-induced cognitive impairment and oxidative stress.

Detecting Redox Potentials Using Porous Boron Nitride/ATP-DNA Aptamer/Methylene Blue Biosensor to Monitor Microbial Activities

Microbial activity has gained attention because of its impact on the environment and the quality of people’s lives. Most of today’s methods, which include genome sequencing and electrochemistry, are costly and difficult to manage. Our group proposed a method using the redox potential change to detect microbial activity, which is rooted in the concept that metabolic activity can change the redox potential of a microbial community. The redox potential change was captured by a biosensor consisting of porous boron nitride, ATP-DNA aptamer, and methylene blue as the fluorophore. This assembly can switch on or off when there is a redox potential change, and this change leads to a fluorescence change that can be examined using a multipurpose microplate reader. The results show that this biosensor can detect microbial community changes when its composition is changed or toxic metals are ingested.

Biophysical Characterization of Novel DNA Aptamers against K103N/Y181C Double Mutant HIV-1 Reverse Transcriptase

The human immunodeficiency virus type-1 Reverse Transcriptase (HIV-1 RT) plays a pivotal role in essential viral replication and is the main target for antiviral therapy. The anti-HIV-1 RT drugs address resistance-associated mutations. This research focused on isolating the potential specific DNA aptamers against K103N/Y181C double mutant HIV-1 RT. Five DNA aptamers showed low IC50 values against both the KY-mutant HIV-1 RT and wildtype (WT) HIV-1 RT. The kinetic binding affinity forms surface plasmon resonance of both KY-mutant and WT HIV-1 RTs in the range of 0.06–2 μM and 0.15–2 μM, respectively. Among these aptamers, the KY44 aptamer was chosen to study the interaction of HIV-1 RTs-DNA aptamer complex by NMR experiments. The NMR results indicate that the aptamer could interact with both WT and KY-mutant HIV-1 RT at the NNRTI drug binding pocket by inducing a chemical shift at methionine residues. Furthermore, KY44 could inhibit pseudo-HIV particle infection in HEK293 cells with nearly 80% inhibition and showed low cytotoxicity on HEK293 cells. These together indicated that the KY44 aptamer could be a potential inhibitor of both WT and KY-mutant HIV-RT.

Development of DNA Aptamers to Visualize Release of Mycobacterial Membrane-Derived Extracellular Vesicles in Infected Macrophages

Extracellular vesicles (EVs) have emerged into a novel vaccine platform, a biomarker and a nano-carrier for approved drugs. Their accurate detection and visualization are central to their utility in varied biomedical fields. Owing to the limitations of fluorescent dyes and antibodies, here, we describe DNA aptamer as a promising tool for visualizing mycobacterial EVs in vitro. Employing SELEX from a large DNA aptamer library, we identified a best-performing aptamer that is highly specific and binds at nanomolar affinity to EVs derived from three diverse mycobacterial strains (pathogenic, attenuated and avirulent). Confocal microscopy revealed that this aptamer was not only bound to in vitro-enriched mycobacterial EVs but also detected EVs that were internalized by THP-1 macrophages and released by infecting mycobacteria. To the best of our knowledge, this is the first study that detects EVs released by mycobacteria during infection in host macrophages. Within 4 h, most released mycobacterial EVs spread to other parts of the host cell. We predict that this tool will soon hold huge potential in not only delineating mycobacterial EVs-driven pathogenic functions but also in harboring immense propensity to act as a non-invasive diagnostic tool against tuberculosis in general, and extra-pulmonary tuberculosis in particular.