RESUMO
Sterigmatocystin (ST) is a known toxin whose aptamer has rarely been reported because ST is a water-insoluble small-molecule target with few active sites, leading to difficulty in obtaining its aptamer using traditional target fixation screening methods. To obtain aptamer for ST, we incorporated FAM tag size separation into the capture-systematic evolution of ligands by exponential enrichment and combined it with molecular activation for aptamer screening. The screening process was monitored using a quantitative polymerase chain reaction fluorescence amplification curve and recovery of negative-, counter-, and positive-selected ssDNA. The affinity and specificity of the aptamer were verified by constructing an aptamer-affinity column, and the binding sites were predicted using molecular docking simulations. The results showed that the Kd value of the H Seq02 aptamer was 25.3 nM. The aptamer-affinity column based on 2.3 nmol of H Seq02 exhibited a capacity of about 80 ng, demonstrating better specificity than commercially available antibody affinity columns. Molecular simulation docking predicted the binding sites for H Seq02 and ST, further explaining the improved specificity. In addition, circular dichroism and isothermal titration calorimetry were used to verify the interaction between the aptamer and target ST. This study lays the foundation for the development of a new ST detection method.
Assuntos
Aptâmeros de Nucleotídeos , Aptâmeros de Nucleotídeos/química , Esterigmatocistina , Técnica de Seleção de Aptâmeros/métodos , Simulação de Acoplamento Molecular , LigantesRESUMO
Fat mass and obesity-associated protein (FTO) plays a crucial role in regulating the dynamic modification of N6-methyladenosine (m6A) in eukaryotic mRNA. Sensitive detection of the FTO level and efficient evaluation of the FTO demethylase activity are of great importance to early cancer diagnosis and anticancer drug discovery, which are currently challenged by limited sensitivity/precision and low throughput. Herein, a robust strategy based on the dephosphorylation switch DNAzyme-rolling circle amplification (RCA) circuit, termed DSD-RCA, is developed for highly sensitive detection of FTO and inhibitor screening. Initially, the catalytic activity of DNAzyme is silenced by engineering with an m6A modification in its catalytic core. Only in the presence of target FTO can the methyl group on DNAzyme be eliminated, resulting in the activation of the catalytic activity of DNAzyme and thus cleaving the hairpin substrate to release numerous primers. Different from the conventional methods that use the downstream cleavage primer with the original 3'-hydroxyl end directly as the RCA primer with the problem of high background signal, which should be compensated by additional separation and wash steps in heterogeneous format, our DSD-RCA assay uses the upstream cleavage primer with a 2',3'-cyclic phosphate terminus at the 3'-end serving as an intrinsically blocked 3' end. Only after a dephosphorylation reaction mediated by T4 polynucleotide kinase can the upstream cleavage primers with a resultant 3'-hydroxyl end be extended by RCA. With the high signal-to-noise ratio and homogeneous property, the proposed platform can sensitively detect FTO with a limit of detection of 31.4 pM, and the relative standard deviations (RSDs %) ranging from 0.8 to 2.0% were much lower than the heterogeneous methods. The DSD-RCA method was applied for analyzing FTO in cytoplasmic lysates from different cell lines and tissues of breast cancer patients and further used for screening FTO inhibitors without the need for separation or cleaning, providing an opportunity for achieving high throughput and demonstrating the potential applications of this strategy in disease diagnostics, drug discovery, and biological applications.
Assuntos
Técnicas Biossensoriais , DNA Catalítico , Humanos , DNA Catalítico/química , Técnicas Biossensoriais/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Linhagem Celular , Polinucleotídeo 5'-Hidroxiquinase , Limite de Detecção , Dioxigenase FTO Dependente de alfa-CetoglutaratoRESUMO
The ability to specifically image cancer cells is essential for cancer diagnosis; however, this ability is limited by the false positive associated with single-biomarker sensors and off-site activation of "always active" nucleic acid probes. Herein, we propose an on-site, activatable, transmembrane logic DNA (TLD) nanodevice that enables dual-biomarker sensing of tumor-related nucleolin and intracellular microRNA for highly specific cancer cell imaging. The TLD nanodevice is constructed by assembling a tetrahedral DNA nanostructure containing a linker (L)-blocker (B)-DNAzyme (D)-substrate (S) unit. AS-apt, a DNA strand containing an elongated segment and the AS1411 aptamer, is pre-anchored to nucleolin protein, which is specifically expressed on the membrane of cancer cells. Initially, the TLD nanodevice is firmly sealed by the blocker containing an AS-apt recognition zone, which prevents off-site activation. When the nanodevice encounters a target cancer cell, AS-apt (input 1) binds to the blocker and unlocks the sensing ability of the nanodevice for miR-21 (input 2). The TLD nanodevice achieves dual-biomarker sensing from the cell membrane to the cytoplasm, thereby ensuring cancer cell-specific imaging. This TLD nanodevice represents a promising strategy for the highly reliable analysis of intracellular biomarkers and a promising platform for cancer diagnosis and related biomedical applications.
Assuntos
Aptâmeros de Nucleotídeos , MicroRNAs , Neoplasias , Humanos , MicroRNAs/genética , Neoplasias/diagnóstico por imagem , DNA/química , Fosfoproteínas , NucleolinaRESUMO
Sensitive imaging of microRNAs (miRNAs) in living cells is significant for accurate cancer clinical diagnosis and prognosis research studies, but it is challenged by inefficient intracellular delivery, instability of nucleic acid probes, and limited amplification efficiency. Herein, we engineered a DNAzyme-amplified cascade catalytic hairpin assembly (CHA)-based nanosystem (DCC) that overcomes these challenges and improves the imaging sensitivity. This enzyme-free amplification nanosystem is based on the sequential activation of DNAzyme amplification and CHA. MnO2 nanosheets were used as nanocarriers for the delivery of nucleic acid probes, which can resist the degradation by nucleases and supply Mn2+ for the DNAzyme reaction. After entering into living cells, the MnO2 nanosheets can be decomposed by intracellular glutathione (GSH) and release the loaded nucleic acid probes. In the presence of target miRNA, the locking strand (L) was hybridized with target miRNA, and the DNAzyme was released, which then cleaved the substrate hairpin (H1). This cleavage reaction resulted in the formation of a trigger sequence (TS) that can activate CHA and recover the fluorescence readout. Meanwhile, the DNAzyme was released from the cleaved H1 and bound to other H1 for new rounds of DNAzyme-based amplification. The TS was also released from CHA and involved in the new cycle of CHA. By this DCC nanosystem, low-abundance target miRNA can activate many DNAzyme and generate numerous TS for CHA, resulting in sensitive and selective analysis of miRNAs with a limit of detection of 5.4 pM, which is 18-fold lower than that of the traditional CHA system. This stable, sensitive, and selective nanosystem holds great potential for miRNA analysis, clinical diagnosis, and other related biomedical applications.
Assuntos
Técnicas Biossensoriais , DNA Catalítico , MicroRNAs , MicroRNAs/genética , MicroRNAs/análise , DNA Catalítico/metabolismo , Compostos de Manganês , Óxidos , Catálise , Técnicas Biossensoriais/métodos , Limite de DetecçãoRESUMO
Ammonium (NH4+) enrichment of riverbank filtration (RBF) systems is gaining popularity. However, most previous research has concentrated on NO3- removal efficiencies, while the mechanisms of NH4+ enrichment remain unknown. A nitrogen biogeochemical process model was developed for the quantitative analysis of NH4+ enrichment in the Kaladian well field in northwest Songyuan City, NE China. Data from laboratory experiments and in-situ monitoring were used to determine initial values and calibrate the thermodynamic/kinetic parameters representing nitrogen (N) biogeochemical reactions. (1) The NO3- from river was subjected to denitrification (DNF) and dissimilatory nitrate reduction to ammonium (DNRA) within 10-14 m of the shore, whereas the NH4+ in groundwater was caused by DNRA, organic nitrogen mineralization (MIN), and mixing with laterally recharged high NH4+ groundwater. (2) DNF and DNRA were regulated by hydrodynamic processes, with the ranges of these processes being more significant in the wet season due to a higher hydraulic gradient. MIN occurred widely throughout the water flow path, with temperature primarily controlling the rates of the three reactions. (3) DNRA activity was relatively higher in the wet season when the water temperature was higher within 10-14 m of the shore. In the wet season, DNRA contributed 25%-30% to NO3- reduction, which was higher than in the dry season (5%-10%). DNRA contributed at least 40% and 15% to NH4+ enrichment in the wet and dry seasons, respectively. (4). Organic N in media gradually released NH4+ into groundwater via MIN and desorption across the entire flow path, with contributions to NH4+ enrichment reaching 75% and 85%, respectively, in the wet and dry seasons.
Assuntos
Compostos de Amônio , Nitrogênio , Desnitrificação , Nitratos/análise , Óxidos de Nitrogênio , Compostos Orgânicos , ÁguaRESUMO
Imaging of tumor-associated microRNAs (miRNAs) can provide abundant information for cancer diagnosis, whereas the occurrence of trace amounts of miRNAs in normal cells inevitably causes an undesired false-positive signal in the discrimination of cancer cells during miRNA imaging. In this study, we propose a dual-locked (D-locked) platform consisting of the enzyme/miRNA-D-locked DNAzyme sensor and the honeycomb MnO2 nanosponge (hMNS) nanocarrier for highly specific cancer cell imaging. For a proof-of-concept demonstration, apurinic/apyrimidinic endonuclease 1 (APE1) and miR-21 were chosen as key models. The hMNS nanocarrier can efficiently release the D-locked DNAzyme sensor in living cells due to the decomposition of hMNS by glutathione, which can also supply Mn2+ for DNAzyme cleavage. Ascribing to the smart design of the D-locked DNAzyme sensor, the fluorescence signal can only be generated by the synergistic response of APE1 and miR-21 that are overexpressed in cancer cells. Compared with the miRNA single-locked DNAzyme sensor and the small-molecule (ATP)/miRNA D-locked DNAzyme sensor, the proposed enzyme (APE1)/miRNA D-locked DNAzyme sensor exhibited 2.6-fold and 2.4-fold higher discrimination ratio (Fcancer/Fnormal) for cancer cell discrimination, respectively. Owing to the superior performance, the D-locked strategy can selectively generate a fluorescence signal in cancer cells, facilitating accurate discrimination of cancer both in vitro and in vivo. Furthermore, this D-locked platform is easily adaptable toward other target molecules by redesigning the DNA sequences. The outstanding performance and expansibility of this D-locked platform holds promising prospects for cancer diagnosis and related biomedical applications.
Assuntos
Técnicas Biossensoriais , DNA Catalítico , MicroRNAs , Neoplasias , DNA Catalítico/genética , Compostos de Manganês , MicroRNAs/genética , Microscopia de Fluorescência/métodos , ÓxidosRESUMO
Cumulative research reveals that microRNAs (miRNAs) are involved in many critical biological processes including cell proliferation, differentiation and apoptosis. It is of great significance to figure out the associations between miRNAs and human diseases that are the basis for finding biomarkers for diagnosis and targets for treatment. To overcome the time-consuming and labor-intensive problems faced by traditional experiments, a computational method was developed to identify potential associations between miRNAs and diseases based on the graph attention network (GAT) with different meta-path mode and support vector (SVM). Firstly, we constructed a multi-module heterogeneous network based on the meta-path and learned the latent features of different modules by GAT. Secondly, we found the average of the latent features with weight to obtain a final node representation. Finally, we characterized miRNA-disease-association pairs with the node representation and trained an SVM to recognize potential associations. Based on the five-fold cross-validation and benchmark datasets, the proposed method achieved an area under the precision-recall curve (AUPR) of 0.9379 and an area under the receiver-operating characteristic curve (AUC) of 0.9472. The results demonstrate that our method has an outstanding practical application performance and can provide a reference for the discovery of new biomarkers and therapeutic targets.
Assuntos
MicroRNAs , Algoritmos , Área Sob a Curva , Biologia Computacional/métodos , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Curva ROCRESUMO
River bank filtration can effectively reduce the number of pathogenic microorganisms infiltrating into groundwater from surface water. Groundwater seepage velocity and temperature are considered to be important factors affecting the process, but the magnitude and mechanism of their impacts have not been clear for a long time. Based on the actual monitoring data of the Escherichia coli concentrations and soil samples of Second Songhua riverside source area, the migration of E. coli in saturated porous media under different velocities and different temperatures was studied using saturated soil column transport experiments. Concurrently, the migration characteristics of E. coli in the riverside source area were replicated by mathematical simulation. According to the field monitoring results, the concentration of E. coli decreased in the riverbank infiltration zone, and the removal rate was greater than 96%. The column experimental results showed that the lower the flow velocity was and the higher the temperature was, the greater the removal rate of E. coli was. And the flow velocity was the main factor affecting the removal of E. Coli. The mathematical simulation results showed that under the conditions of the largest hydraulic gradient (20%) and the highest concentration of E. coli (2500 MPN/100 mL) in river water, the safe exploitation distance of groundwater that did not cause a risk of E. coli pollution was more than 7 m away from the river bank. These findings are expected to provide a scientific basis for the design of water intake schemes and the optimization of mining technology.
Assuntos
Escherichia coli , Água Doce/microbiologia , Rios , China , Filtração/métodos , Água Subterrânea/microbiologia , Porosidade , Solo/química , Temperatura , Microbiologia da ÁguaRESUMO
To investigate the protective effect of glutamine (Gln) against obstructive cholestasis in association with farnesoid X receptor (FXR) activation, an obstructive cholestasis model was established in male Sprague-Dawley rats by bile duct ligation (BDL). Serum biomarkers and hematoxylin plus eosin staining were used to identify the degree of hepatic injury in the rats with obstructive cholestasis after Gln treatment. Immunohistochemistry, real-time PCR, Western blot, cultured primary rat hepatocytes with FXR knockdown, and dual-luciferase reporter assay were performed to elucidate the mechanisms underlying Gln hepatoprotection. We found that Gln treatment protected against obstructive cholestasis induced by BDL through reducing hepatocyte injury. Upregulation of the hepatic efflux transporters small heterodimer partner (Shp), bile salt export pump (Bsep), and multidrug resistance-associated protein 2 (Mrp2), and inhibition of the hepatic uptake transporter Na+/taurocholate cotransporting polypeptide (Ntcp) and the bile acid synthesis enzyme cholesterol 7α-hydroxylase (Cyp7a1) expression were observed in rats with BDL treated with Gln in vivo. Furthermore, the regulatory effect of Gln on Bsep and Mrp2 expression was abrogated after FXR knockdown in rat primary cultured hepatocytes. Luciferase assay HepG2 cells also illustrated FXR was a direct target for Gln treatment. In conclusion, the regulation of Bsep and Mrp2 expression mediated by FXR might be an important mechanism for Gln against obstructive cholestasis.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Colestase/metabolismo , Glutamina/farmacologia , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Receptores Citoplasmáticos e Nucleares/metabolismo , Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Animais , Colestase/patologia , Colesterol 7-alfa-Hidroxilase/antagonistas & inibidores , Glutamina/efeitos adversos , Hepatócitos/metabolismo , Testes de Função Hepática , Masculino , Proteína 2 Associada à Farmacorresistência Múltipla , Transportadores de Ânions Orgânicos Dependentes de Sódio/antagonistas & inibidores , Cultura Primária de Células , Substâncias Protetoras/efeitos adversos , Substâncias Protetoras/farmacologia , RNA Interferente Pequeno/farmacologia , Ratos , Simportadores/antagonistas & inibidoresRESUMO
CRISPR-based genomic-imaging systems have been utilized for spatiotemporal imaging of the repetitive genomic loci in living cells, but they are still challenged by limited signal-to-noise ratio (SNR) at a non-repetitive genomic locus. Here, an efficient genomic-imaging system is proposed, termed CRISPR/Pepper-tDeg, by engineering the CRISPR sgRNA scaffolds with the degron-binding Pepper aptamers for binding fluorogenic proteins fused with Tat peptide derived degron domain (tDeg). The target-dependent stability switches of both sgRNA and fluorogenic protein allow this system to image repetitive telomeres sensitively with a 5-fold higher SNR than conventional CRISPR/MS2-MCP system using "always-on" fluorescent protein tag. Subsequently, CRISPR/Pepper-tDeg is applied to simultaneously label and track two different genomic loci, telomeres and centromeres, in living cells by combining two systems. Given a further improved SNR by the split fluorescent protein design, CRISPR/Pepper-tDeg system is extended to non-repetitive sequence imaging using only one sgRNA with two aptamer insertions. Neither complex sgRNA design nor difficult plasmid construction is required, greatly reducing the technical barriers to define spatiotemporal organization and dynamics of both repetitive and non-repetitive genomic loci in living cells, and thus demonstrating the large application potential of this genomic-imaging system in biological research, clinical diagnosis and therapy.
RESUMO
Autocatalytic biocircuit are powerful tools for analysing intracellular biomarkers, but these tools are constrained by limitations in amplification capacity and intracellular delivery efficiency. In this work, we developed a DNAzyme-based dual-feedback autocatalytic exponential amplification biocircuit sustained by a honeycomb MnO2 nanosponge (EDA2@hMNS) for live-cell imaging of intracellular low-abundance microRNAs (miRNA). The EDA2 biocircuit comprises a blocked DNAzyme (b-DNAzyme), a Fuel strand and a Substrate strand. In the EDA2 biocircuit, target miRNAs are recycled and feedback for rounds of DNAzymatic amplification, and the DNAzymatic reactions continuously generate target miRNA analogues for dual-feedback to achieve multiple parallel cascade DNAzymatic reactions that improve amplification capacity substantially. In addition, the hMNS ensures high loading and delivery efficiency of biocircuit probes into living cells and also provides sufficient Mn2+ DNAzyme cofactor from in situ decomposition by intracellular glutathione (GSH). The EDA2@hMNS realized a detection limit of 17 pM, which is 288-fold lower than the b-DNAzyme lacking the DNAzymatic amplification. These results demonstrate the great promise for this critical tool in analysing low-abundance biomarkers and cancer diagnostics.
Assuntos
Técnicas Biossensoriais , DNA Catalítico , MicroRNAs , MicroRNAs/análise , DNA Catalítico/química , Retroalimentação , Compostos de Manganês/química , Técnicas Biossensoriais/métodos , Óxidos/química , Biomarcadores , Técnicas de Amplificação de Ácido Nucleico/métodosRESUMO
BACKGROUND: The present study aimed to explore the effects of the active form of vitamin D (calcitriol, 1α,25-dihydroxyvitamin D3 , 1α,25 (OH)2 D3 , 1,25D) on live Porphyromonas gingivalis internalized into KB cells and U937 cells. METHODS: Quantitative real-time polymerase chain reaction method was used to evaluate the number of surviving P. gingivalis internalized into KB cells and U937 cells. Transmission electron microscopy was used to detect P. gingivalis in cells. A western blot analysis was performed to observe LC3 expressions. RESULTS: 1) Treatment with 1,25D decreased the number of live P. gingivalis in KB cells and U937 cells in a dose-dependent manner. 2) Dividing P. gingivalis were found only in KB cells but not in U937 cells. The cell walls of most P. gingivalis in KB cells were intact, while those in U937 cells were disrupted. Treatment with 1,25D promoted the encapsulation of P. gingivalis in autophagosomes in both KB and U937 cells. 3) Both 1,25D treatment and P. gingivalis infection increased the LC3 II/I ratio. Furthermore, 1,25D treatment increased the P. gingivalis-upregulated LC3 II/I ratio. 4) Treatment with 3-methyladenine (3-MA) decreased the number of P. gingivalis by 11.41% in KB cells, while increased that by 121.51% in U937 cells. Under 1,25D treatment conditions, 3-MA treatment increased the number of P. gingivalis by 88.71% in KB cells and by 284.70% in U937 cells. CONCLUSIONS: Autophagy may facilitate P. gingivalis survival in KB cells and eliminate P. gingivalis in U937 cells. Treatment with 1,25D may help decrease the number of live P. gingivalis in KB cells and U937 cells by promoting functional autophagy.