RESUMEN

Silica aerogels exhibit a unique nanostructure with low thermal conductivity and low density, making them attractive materials for thermal isolation under extreme conditions. The TiO2 particle is one of the common industrial additives used to reduce the thermal radiation of aerogel composites under high-temperature environments, but its influence on thermal resistance is almost unknown. Herein, we report the effect of TiO2 nanoparticles with different crystal phases and different sizes on the thermal stability of silica aerogel composites. By adding TiO2 nanoparticles, the aerogel can significantly resist collapse at high temperatures (up to 1000 °C). And compared with the rutile phase TiO2, the anatase phase TiO2 shows much higher temperature resistance performance, with shrinkage of only one-sixth of the rutile phase after 800 °C treatment. Interestingly, energy-dispersive spectrometer mapping results show that after 800 °C treatment, silica nanoparticles (NPs) are squeezed out in between anatase TiO2 particles, which resists the coarsening of silica NPs and ultimately enhances the stability of aerogel composites. The optimal anatase phase TiO2-doped silica aerogel demonstrates the integrated properties of crack-free morphology (2.84% shrinkage), low thermal conductivity (29.30 mW/(m·K)) and low density (149.4 mg/cm3) after 800 °C treatment. This study may provide new insights for developing oxide-doped silica aerogels with both high-temperature resistance and low thermal radiation.

RESUMEN

Biological and clinical studies have indicated that aberrant expression of circMTO1 served as a crucial biomarker for the diagnosis and prognosis of hepatocellular carcinoma (HCC) patients as well as a potential therapeutic target. However, the detection of circRNAs currently faces challenges such as homologous linear RNA interference and low-expression abundance of certain circRNAs. Therefore, we developed a triple amplification method based on catalytic hairpin assembly (CHA) activation by back-splice junction (BSJ), resulting in CHA products that triggered primer exchange reaction to generate DNAzyme. Subsequently, DNAzyme cleaved the fluorescent reporter chain, enabling ultrasensitive detection of hepatocellular carcinoma-associated circMTO1 through the output fluorescence signal. The catalytic hairpin opening sequence within CHA specifically targeted the BSJ sequence in circRNA, thereby avoiding false positive signals observed in circRNA assays due to the recognition of homologous linear RNA molecules. Moreover, this triple amplification method facilitated sensitive detection of circRNA and addressed the issue of low-abundance expression levels associated with circMTO1 in HCC samples. Notably, our newly designed assay for detecting circRNA exhibited a linear range from 1 fM to 100 nM with a detection limit of 0.265 fM. Furthermore, it demonstrated excellent and consistent performance even within complex systems. Our proposed method enabled the specific and sensitive detection of circMTO1 in various cancer cells and blood samples from HCC patients, providing an innovative approach for investigating the role of circRNA in tumorigenesis and development while promoting its clinical application.

Asunto(s)

Carcinoma Hepatocelular , ADN Catalítico , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/diagnóstico , Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/genética , ARN Circular , Bioensayo

RESUMEN

Exosomal surface proteins are potentially useful for breast cancer diagnosis and awareness of risk. However, some detection techniques involving complex operations and expensive instrumentation are limited to advance to clinical applications. To solve this problem, we develop a dual-modal sensor combining naked-eye detection and electrochemical assay of exosomal surface proteins from breast cancer. Most of existing sensors rely on aptamers recognizing exosomes and generating amplified signals at the same time, which require well-designed aptamer probes to avoid difficulties in identifying exosomes. In our work, aptamers not bound by the exosomes can serve as complete templates to induce formation of G quadruplexes. The peroxidase activity of the G-quadruplex/hemin DNAzyme catalyze substrates can generate both color and electrochemical signals. The developed dual-modal sensor offers a remarkable capability to differentiate nonmetastatic, metastatic breast cancer patients, and healthy individuals through the analysis of exosomal surface proteins. The sensor's distinctive features, including its universality, simplicity, and cost-effectiveness, position it as a promising diagnostic tool in breast cancer research and clinical practice.

Asunto(s)

Técnicas Biosensibles , Neoplasias de la Mama , Colorimetría , Técnicas Electroquímicas , Humanos , Línea Celular , Neoplasias de la Mama/diagnóstico por imagen , Técnicas Biosensibles/instrumentación , Técnicas Biosensibles/métodos , Colorimetría/instrumentación , Colorimetría/métodos , ADN Catalítico/química , ADN Catalítico/metabolismo , Técnicas Electroquímicas/instrumentación , Técnicas Electroquímicas/métodos , Exosomas/química , Exosomas/metabolismo , G-Cuádruplex

RESUMEN

Due to its high efficiency and selectivity, cell-free biosynthesis has found broad utility in the fields of bioproduction, environment monitoring, and disease diagnostics. However, the practical application is limited by its low productivity. Here, we introduce the entropy-driven assembly of transcription templates as dynamic amplifying modules to accelerate the cell-free transcription process. The catalytic DNA circuit with high sensitivity and enzyme-free format contributes to the production of large amounts of transcription templates, drastically accelerating the as-designed cell-free transcription system without interference from multiple enzymes. The proposed approach was successfully applied to the ultrasensitive detection of SARS-CoV-2, improving the sensitivity by 3 orders of magnitude. Thanks to the high programmability and diverse light-up RNA pairs, the method can be adapted to multiplexing detection, successfully demonstrated by the analysis of two different sites of the SARS-CoV-2 gene in parallel. Further, the flexibility of the entropy-driven circuit enables a dynamic responding range by tuning the circuit layers, which is beneficial for responding to targets with different concentration ranges. The strategy was also applied to the analysis of clinical samples, providing an alternative for sensitively detecting the current SARS-CoV-2 RNA that quickly mutates.

Asunto(s)

Técnicas Biosensibles , COVID-19 , Humanos , ADN/análisis , Entropía , ARN Viral , SARS-CoV-2/genética , Técnicas Biosensibles/métodos

RESUMEN

Heteromultivalent scaffolds with different repeated monomers have great potential in biomedicine, but convenient construction strategies for integrating various functional modules to achieve multiple biological functions are still lacking. Here, taking advantage of the heteromultivalent effect of dendritic nucleic acids and the specific biochemical properties of microRNAs (miRNAs), we assembled novel heteromultivalent nucleic acid scaffolds by biomimetic co-assembly of DNA-RNA building blocks. In our approach, two miRNAs were used to initiate and maintain dendritic structures in an interdependent manner; so, the heteromultivalent nanostructure can only form in the presence of both miRNAs. The proposed nanostructure can be used for one-step analysis of two miRNAs in an AND logic format. Taking miR-18b-5p and miR-342-3p which are associated with Alzheimer's disease as an example, a FRET sensing system was fabricated for the simultaneous analysis of two miRNAs within one hour at picomolar concentration. Further studies show that the designed device may have the potential to distinguish between AD patients and the healthy population by analysis of two miRNAs in CSF (cerebrospinal fluid) samples, suggesting its possible applicability in clinics.

Asunto(s)

Enfermedad de Alzheimer , MicroARNs , Humanos , MicroARNs/genética , Biomimética , Perfilación de la Expresión Génica , ADN

RESUMEN

Long non-coding RNAs (lncRNAs) played vital roles in physiological and pathological conditions. Consistent results from cell experiments, animal experiments, and clinical studies suggested that lncRNA HULC was an oncogenic lncRNA serving as a potential diagnostic and prognostic marker of hepatocellular carcinoma. In this study, we developed a fluorescent biosensor for lncRNA HULC detection based on rolling circle amplification (RCA) induced by multi-primer probes. Multiple primer probes can not only combine with lncRNA to break its secondary structure, which was conducive to lncRNA captured by Y-shaped probes, but also trigger multiple RCA reactions to achieve signal amplification and the goal of sensitive detection of lncRNA. Compared to previous detection methods, in this scheme, we took advantage of the long sequence characteristics of lncRNA to make it a carrier that can bind multiple primers to initiate RCA. This newly designed biosensor provided a linear range from 1 pM to 100 nM with a detection limit of 0.06 pM. This method can provide a new idea for the application of isothermal amplification in detecting lncRNA. Furthermore, the application of the biosensor in liver cancer cell lines and whole blood samples from hepatocellular carcinomatosis patients also confirmed that the method had good selectivity and sensitivity to lncRNA HULC. This method offered a new way for transforming specific lncRNA into clinical application for diagnosis, prognosis, or predicting treatment response.

Asunto(s)

Técnicas Biosensibles , Carcinoma Hepatocelular , Neoplasias Hepáticas , ARN Largo no Codificante , Animales , Carcinoma Hepatocelular/diagnóstico , Carcinoma Hepatocelular/genética , ARN Largo no Codificante/genética , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/genética , Detección Precoz del Cáncer , Cartilla de ADN/metabolismo , Técnicas Biosensibles/métodos , Técnicas de Amplificación de Ácido Nucleico/métodos , Límite de Detección

RESUMEN

Exosome-based liquid biopsy technologies play an increasingly prominent role in tumor diagnosis. However, the simple and sensitive method for counting exosomes still faces considerable challenges. In this work, the CD63 aptamer-modified DNA tetrahedrons on the gold electrode were used as recognition elements for the specific capture of exosomes. Partially complementary DNA probes act as bridges linking trapped exosomes and three AuNP-DNA signal probes. This clover-like structure can tackle the recognition and sensitivity issues arising from the undesired AuNP aggregation event. When cancerous exosomes are present in the system, the high accumulation of methylene blue molecules from DNA-AuNP nanocomposites on the surface of the electrode leads to an intense current signal. According to the results, the aptasensor responds to MCF-7 cell-derived exosomes in the concentration range from 1.0 × 103 to 1.0 × 108 particles·µL-1, with the detection limit of 158 particles·µL-1. Furthermore, the aptasensor has been extended to serum samples from breast cancer patients and exhibited excellent specificity. To sum it up, the aptasensor is sensitive, straightforward, less expensive, and fully capable of receiving widespread application in clinics for tumor monitoring.

Asunto(s)

Aptámeros de Nucleótidos , Técnicas Biosensibles , Neoplasias de la Mama , Exosomas , Nanopartículas del Metal , Humanos , Femenino , Exosomas/química , Oro/química , Neoplasias de la Mama/diagnóstico , ADN/análisis , Células MCF-7 , Aptámeros de Nucleótidos/química , Técnicas Biosensibles/métodos , Nanopartículas del Metal/química

RESUMEN

A simple method is proposed in this work for the detection of SARS-CoV-2 RNA based on a functional RNA/DNA circuit. By ingeniously integrating the nucleic acid circuit technology and CRISPR/cas12a system, this method can achieve femtomolar detection of the target RNA in one step and successfully distinguish COVID-19 positive cases from clinical samples, proving its great potential for clinical application.

Asunto(s)

COVID-19 , ARN Viral , Humanos , ARN Viral/genética , SARS-CoV-2/genética , COVID-19/diagnóstico , ARN no Traducido , ADN

RESUMEN

Aptamer-based methods have attracted increasing interest due to flexible engineering, but their generality is limited by the heterogeneity of signal transduction mechanisms. Given the fact that nonlinear and large molecules are more likely to make the nanosurface overloaded, we investigated a novel signal transduction process to extend the application of aptasensors. In this work, an aptamer complementary element (ACE) is designed with a primer region to serve as the signal probe, which can fully hybridize with an aptamer and be separated by magnetic beads (MBs). Upon target binding, the formed aptamer/target complex is much larger than the linear aptamer/ACE-primer dimer, causing overload of MBs on account of steric hindrance. An extra aptamer/ACE-primer can escape from the surface to the supernatant, which can be amplified by a catalytic hairpin assembly (CHA) circle. The size-dependent signal transduction and the modular design endow the method with high generality and flexibility for protein analysis. The proposed aptasensor was successfully applied to the detection of tau proteins ranging from 0.5 to 1000 ng mL-1 with a limit of detection (LOD) as low as 0.254 ng mL-1. The recovery tests in both human serum and cerebra spinal fluid confirmed the high accuracy and stability. Furthermore, a successful distinction was made between AD patients and healthy controls by the method, suggesting the possible applicability for practical analysis of tau proteins.

Asunto(s)

Aptámeros de Nucleótidos , Técnicas Biosensibles , Aptámeros de Nucleótidos/química , Técnicas Biosensibles/métodos , Humanos , Límite de Detección , Transducción de Señal , Proteínas tau/metabolismo

RESUMEN

Programmed-death ligand 1 (PD-L1), as one of major immune checkpoints, is highly expressed on cancer cells and participates in the immune escape process of tumor cells. The level of PD-L1 in patients is closely related to the efficacy of anti-PD-L1 immunotherapy, and patients with a high level have better response to immunotherapy. Therefore, PD-L1 can be an indicator of patient classification and medication guidance. In this work, we have developed a novel strategy for detecting PD-L1-positive circulating tumor cells based on steric hindrance generated after cell capture, using the primer exchange reaction (PER) amplification method. The principle is to modify a single strand containing the PD-L1 aptamer and the PER primer on the electrode surface. When PD-L1-positive circulating tumor cells exist, the aptamer will capture them. The steric hindrance generated by the captured cells due to their large volume hinders the subsequent approach of PER materials, thus hindering the occurrence of PER signal amplification. The number of HRP bound to the electrode surface is reduced, and the current signal output is inversely proportional to the number of captured cells. This method realizes convenient and sensitive detection of PD-L1-positive tumor cells and provides a new means for clinical judgment of whether patients should adopt immunotherapy.

Asunto(s)

Células Neoplásicas Circulantes , Receptor de Muerte Celular Programada 1 , Antígeno B7-H1/metabolismo , Humanos , Factores Inmunológicos , Inmunoterapia/métodos , Microambiente Tumoral

RESUMEN

A simple method is proposed in this work for the detection of SARS-CoV-2 RNA based on a primer exchange reaction (PER). By ingeniously integrating the PER cascade and CRISPR/cas12a system, this method can achieve convenient detection of the target RNA in 40 min and distinguish a single-base mutation from the target sequence, demonstrating its superior analytical performance.

Asunto(s)

COVID-19 , SARS-CoV-2 , COVID-19/diagnóstico , Sistemas CRISPR-Cas/genética , Humanos , Técnicas de Amplificación de Ácido Nucleico/métodos , ARN Viral/genética , SARS-CoV-2/genética

RESUMEN

Cancer-derived exosomes have emerged as a valuable biomarker for cancer diagnosis and prognosis. However, the heterogeneity of exosomes often leads to low selectivity based on the single recognition method. Given this, we have developed a dual-aptamer recognition strategy based on G-quadruplex nanowires for selective analysis of exosomes. In this work, target exosomes were first captured by CD63 aptamers modified on magnetic beads (MBs) and then combined with AS1411 aptamer, which shows high binding affinity to nucleolin when forming stable G-quadruplex structure. Then the free myc monomer can spontaneously assemble into higher order G-wire superstructures on the allosteric AS1411, and resulting enhanced fluorescence signal, which can realize sensitive and specific analysis of the target exosomes. This dual-aptamer recognition-based method is simple and universal for different types of exosomes, which is of great significance for clinical cancer diagnosis.

Asunto(s)

Aptámeros de Nucleótidos , Exosomas , G-Cuádruplex , Nanocables , Neoplasias , Humanos

RESUMEN

Efficient capture and release of circulating tumor cells play an important role in cancer diagnosis, but the limited affinity of monovalent adhesion molecules in existing capture technologies leads to low capture efficiency, and the captured cells are difficult to be separated. Inspired by the phenomenon that the long tentacles of jellyfish contain multiple adhesion domains and can effectively capture moving food, we have constructed a biomimetic recognition strategy to capture and release tumor cells. In details, gold-coated magnetic nanomaterials (Au@Fe3O4 NPs) were first prepared and characterized by scanning electron microscopy, UV-vis absorption spectra, and Zeta potential. Then, the DNA primers modified on Au@Fe3O4 nanoparticles can be extended to form many radialized DNA products by rolling circle amplification. These long DNA products resemble jellyfish tentacles and contain multivalent aptamers that can be extended into three dimensions to increase the accessibility of target cells, resulting in efficient, simple, rapid, and specific cells capture. The capture efficiencies are no less than 92% in PBS buffer and 77% in blood. Subsequently, DNase I was selected to degrade biomimetic tentacles to release the captured tumor cells with high viability. This release strategy can not only improve cell viability, but also reduce a tedious release process and unnecessary costs. We believe that the proposed method can be expanded for the capture and release of various tumor cells and will inspire the development of circulating tumor cells analysis. A biomimetic recognition strategy for capture and release of circulating tumor cells has been developed. This method modified specific P1 DNA primers on Au@Fe3O4 NPs to form many radialized DNA products by rolling circle amplification. These products can efficiently capture CTCs since it contains multiple aptamers with a multivalent binding capacity. This make it a promising tool to capture and release of other tumor cells, and will inspire the development of CTC analysis.

Asunto(s)

Biomimética/métodos , Células Neoplásicas Circulantes/metabolismo , Aptámeros de Nucleótidos/química , Aptámeros de Nucleótidos/metabolismo , Supervivencia Celular/efectos de los fármacos , Óxido Ferrosoférrico/química , Oro/química , Células HeLa , Humanos , Nanopartículas de Magnetita/química , Nanopartículas de Magnetita/toxicidad , Técnicas de Amplificación de Ácido Nucleico

RESUMEN

Emerging evidence reveals that the epitranscriptomic mark N6-methyladenosine (m6A) plays vital roles in organisms, including gene regulation and disease progression. However, developing sensitive methods to detect m6A modification, especially the identification of m6A marks at the single-site level, remains a challenge. Therefore, based on target-specific triggered signal amplification, we developed a highly sensitive electrochemical method to detect site-specific m6A modifications in DNA. In this work, the m6A site in DNA can restrict the ligation assisted by Ag+, and this restriction effect can activate the subsequent strand displacement reaction and hybridization chain reaction (HCR), thus achieving signal amplification from the m6A site, and finally realizing high sensitivity analysis of m6A methylation. Benefiting from the high specificity of base pairs and the extremely weak binding affinity between Ag+ and m6A, the proposed method was used for not only detecting the target DNA with a putative m6A site, but also identifying m6A marks at the single-site level in DNA. In addition, this study does not rely on antibodies and radiolabeling, so it has the advantage of cost-effectiveness. Therefore, we believe that the proposed strategy may provide a new perspective for methylation research, which can be used to test more clinical samples in further research.

Asunto(s)

Adenosina , Plata , Adenosina/análogos & derivados , Técnicas Electroquímicas , Metilación

RESUMEN

The competitive endogenous RNA hypothesis is a new mechanism of RNA dialogue, in which circRNA-miRNA interaction (cmRRI) is found to be widely involved in the regulation of gene expression in tumors and other diseases. It is urgent but challenging to develop a convenient and efficient method to study the interaction between target circRNA and the candidate miRNAs. In this work, a biosensing method that allows directly analyzing cmRRI has been developed, so as to reveal the RNA dialogue strategy. The sensing system uses a bifunctional magnetic bead for the capture of target circRNA/miRNA complex as well as the signal amplification. Based on the nature of circRNA as a miRNA sponge, only if the target circRNAs and its regulatory miRNAs coexist as a complex, can the rolling circle amplification reaction be initiated to give a fluorescent signal as the output. Compared with traditional methods where the circRNA and its regulatory miRNAs have been separately analyzed, our design allows the integrated profiling of specific cmRRI by correlation characterization of two correlative RNAs, which represents a function-oriented method. The presented method also shows the analysis of the potential binding affinity of candidate miRNAs to target circRNAs. Furthermore, we have verified the ability of the sensor to directly detect cmRRI in biological samples, which reveals the promising applicability of this method for biomedical and clinical researches in the future.

Asunto(s)

Técnicas Biosensibles , MicroARNs , Perfilación de la Expresión Génica , Redes Reguladoras de Genes , MicroARNs/genética , ARN/genética , ARN Circular

RESUMEN

Biomimetic construction of artificial scaffolds has attracted increasing attention. However, the construction methods usually require redundant materials and procedures, which is inconvenient for further application. Herein, inspired by the polyvalent multifunctional structure in nature, we have designed a polyvalent biotinylated aptamer scaffold (PBAS) which can conduct analytical performance with high sensitivity and simplified procedures. To construct a PBAS, the aptamers are designed to hybridize with prepared linker probes to form polyvalent biotinylated scaffolds, which contain both multiple aptamers and signal labels. Therefore, multifunctional scaffolds can be constructed with high recognition and capture efficiency as well as significant signal amplification. Furthermore, the scaffold can be used for the assay of some disease marker proteins. By taking tau proteins as an example, the proposed aptasensor can exhibit excellent performance with a low detection limit of 153 pg mL-1 and a short assay time of 50 min, which is much better than most of the previous methods. By assays of tau proteins in both serum and artificial cerebro spinal fluid, the PBAS-based aptasensor can work well. Therefore, the scaffold may be expected to be a powerful analytical tool which may have wide applications in the detection of a variety of analytes.

Asunto(s)

Aptámeros de Nucleótidos/metabolismo , Técnicas Biosensibles/métodos , Límite de Detección , Proteínas tau/metabolismo , Biotinilación , Humanos , Factores de Tiempo , Proteínas tau/sangre

RESUMEN

Pectins are a major dietary nutrient source for the human gut microbiota. The prominent gut microbe Bacteroides thetaiotaomicron was recently shown to encode the founding member (BT1017) of a new family of pectin methylesterases essential for the metabolism of the complex pectin rhamnogalacturonan-II (RG-II). However, biochemical and structural knowledge of this family is lacking. Here, we showed that BT1017 is critical for the metabolism of an RG-II-derived oligosaccharide ΔBT1017oligoB generated by a BT1017 deletion mutant (ΔBT1017) during growth on carbohydrate extract from apple juice. Structural analyses of ΔBT1017oligoB using a combination of enzymatic, mass spectrometric, and NMR approaches revealed that it is a bimethylated nonaoligosaccharide (GlcA-ß1,4-(2-O-Me-Xyl-α1,3)-Fuc-α1,4-(GalA-ß1,3)-Rha-α1,3-Api-ß1,2-(Araf-α1,3)-(GalA-α1,4)-GalA) containing components of the RG-II backbone and its side chains. We showed that the catalytic module of BT1017 adopts an α/ß-hydrolase fold, consisting of a central twisted 10-stranded ß-sheet sandwiched by several α-helices. This constitutes a new fold for pectin methylesterases, which are predominantly right-handed ß-helical proteins. Bioinformatic analyses revealed that the family is dominated by sequences from prominent genera of the human gut microbiota, including Bacteroides and Prevotella Our re-sults not only highlight the critical role played by this family of enzymes in pectin metabolism but also provide new insights into the molecular basis of the adaptation of B. thetaiotaomicron to the human gut.

Asunto(s)

Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Bacteroides thetaiotaomicron/enzimología , Hidrolasas de Éster Carboxílico/química , Hidrolasas de Éster Carboxílico/metabolismo , Microbioma Gastrointestinal , Oligosacáridos/metabolismo , Bacteroides thetaiotaomicron/crecimiento & desarrollo , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Filogenia , Conformación Proteica

RESUMEN

COVID-19 pandemic outbreak is the most astounding scene ever experienced in the 21st century. It has been determined to be caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). With the global pandemic, the lack of efficient rapid and accurate molecular diagnostic testing tools has hindered the public opportunely response to the emerging viral threat. Herein, a DNA nanoscaffold hybrid chain reaction (DNHCR)-based nucleic acid assay strategy is reported for rapid detection of SARS-CoV-2 RNA. In this method, the DNA nanoscaffolds have been first constructed by the self-assembly of long DNA strands and self-quenching probes (H1). Then, the SARS-CoV-2 RNA will initiate the hybridization of H1 and free H2 DNA probes along the nanoscaffold, and an illuminated DNA nanostring is instantly obtained. By taking advantages of the localization design of the H1 probes and the temperature tolerance of the isothermal amplification, the proposed DNHCR method can detect target at short responding time (within 10 min) and mild condition (15 °C-35 °C). Moreover, the reliability of DNHCR method in serum and saliva samples have also been validated. Therefore, DNHCR-based method is expected to provide a simple and faster alternative to the traditional SARS-CoV-2 qRT-PCR assay.

Asunto(s)

Betacoronavirus , Técnicas Biosensibles/métodos , Técnicas de Laboratorio Clínico/métodos , Infecciones por Coronavirus/diagnóstico , Infecciones por Coronavirus/virología , Neumonía Viral/diagnóstico , Neumonía Viral/virología , Betacoronavirus/genética , Betacoronavirus/aislamiento & purificación , COVID-19 , Prueba de COVID-19 , Técnicas de Laboratorio Clínico/estadística & datos numéricos , Infecciones por Coronavirus/epidemiología , ADN/síntesis química , ADN/química , ADN/genética , Estudios de Factibilidad , Humanos , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Diagnóstico Molecular/estadística & datos numéricos , Nanoestructuras/química , Nanotecnología , Técnicas de Amplificación de Ácido Nucleico/métodos , Pandemias , Neumonía Viral/epidemiología , ARN Viral/análisis , ARN Viral/genética , Reproducibilidad de los Resultados , SARS-CoV-2 , Sensibilidad y Especificidad

RESUMEN

Accurate analysis of circular RNA (circRNA) is essential for the elucidation of circRNA-mediated signaling pathways and its association with diseases. Here we present a new tool, namely, linear DNA nanostructure (LDN), for efficient assay of circRNA. In this assay, target circRNA initiates cascade displacement reaction between two hairpin probes sequentially assembled along the LDN and finally lights up the whole LDN. Meanwhile, the target circRNA can be recycled and serve as a catalyst for multiple LDNs lighting, leading to the signal switch from "OFF" to "ON". Systematic studies reveal that our LDN-based method can strictly recognize and rapid respond to circRNA with high signal gain. Furthermore, by using human tumor cells it has been verified that the LDN-based method can also be used for easy and high-sensitive imaging of intracellular circRNA, which provides a useful platform for assaying low-abundance circRNA in cells.

Asunto(s)

ADN/química , Nanoestructuras/química , ARN Circular/análisis , Citometría de Flujo , Humanos , Microscopía Confocal , Células Tumorales Cultivadas

RESUMEN

Cytophaga hutchinsonii is an aerobic cellulolytic soil bacterium that rapidly digests crystalline cellulose. The predicted mechanism by which C. hutchinsonii digests cellulose differs from that of other known cellulolytic bacteria and fungi. The genome of C. hutchinsonii contains 22 glycoside hydrolase (GH) genes, which may be involved in cellulose degradation. One predicted GH with uncertain specificity, CHU_0961, is a modular enzyme with several modules. In this study, phylogenetic tree of the catalytic modules of the GH9 enzymes showed that CHU_0961 and its homologues formed a new group (group C) of GH9 enzymes. The catalytic module of CHU_0961 (CHU_0961B) was identified as a 1,4-ß-D-glucan glucohydrolase (EC 3.2.1.74) that has unique properties compared with known GH9 cellulases. CHU_0961B showed highest activity against barley glucan, but low activity against other polysaccharides. Interestingly, CHU_0961B showed similar activity against ρ-nitrophenyl ß-D-cellobioside (ρ-NPC) and ρ-nitrophenyl ß-D-glucopyranoside. CHU_0961B released glucose from the nonreducing end of cello-oligosaccharides, ρ-NPC, and barley glucan in a nonprocessive exo-type mode. CHU_0961B also showed same hydrolysis mode against deacetyl-chitooligosaccharides as against cello-oligosaccharides. The kcat/Km values for CHU_0961B against cello-oligosaccharides increased as the degree of polymerization increased, and its kcat/Km for cellohexose was 750 times higher than that for cellobiose. Site-directed mutagenesis showed that threonine 321 in CHU_0961 played a role in hydrolyzing cellobiose to glucose. CHU_0961 may act synergistically with other cellulases to convert cellulose to glucose on the bacterial cell surface. The end product, glucose, may initiate cellulose degradation to provide nutrients for bacterial proliferation in the early stage of C. hutchinsonii growth. KEY POINTS: • CHU_0961 and its homologues formed a novel group (group C) of GH9 enzymes. • CHU_0961 was identified as a 1,4-ß-d-glucan glucohydrolase with unique properties. • CHU_0961 may play an important role in the early stage of C. hutchinsonii growth.

Asunto(s)

Proteínas Bacterianas/metabolismo , Cytophaga/enzimología , Glucano 1,4-beta-Glucosidasa/metabolismo , Filogenia , Proteínas Bacterianas/genética , Celulosa/metabolismo , Cytophaga/genética , Genoma Bacteriano , Glucano 1,4-beta-Glucosidasa/genética , Cinética , Alineación de Secuencia