RESUMO
Microbial surface transmission has aroused great attention since the pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Developing a simple in situ detection method for viruses on solid surfaces is of great significance for timely public health surveillance. Taking advantage of the natural structure of SARS-CoV-2, we reported the assembly of Au@AgNPs on the surface of a single virus by the specific aptamer-spike protein interaction. Multiple hotspots can be created between the neighboring Au@AgNPs for the highly sensitive surface-enhanced Raman scattering (SERS) detection of SARS-CoV-2. Using two different aptamers labeled with Cy3 and Au@AgNPs, in situ SERS detection of pseudotyped SARS-CoV-2 (PSV) on packaging surfaces was achieved within 20 min, with a detection limit of 5.26 TCID50/mL. For the blind testing of 20 PSV-contaminated packaging samples, this SERS aptasensor had a sensitivity of 100% and an accuracy of 100%. This assay has been successfully applied to in situ detection of PSV on the surfaces of different packaging materials, suggesting its potential applicability.
Assuntos
Aptâmeros de Nucleotídeos , COVID-19 , Ouro , Limite de Detecção , Nanopartículas Metálicas , SARS-CoV-2 , Prata , Análise Espectral Raman , SARS-CoV-2/isolamento & purificação , Análise Espectral Raman/métodos , Ouro/química , Nanopartículas Metálicas/química , COVID-19/diagnóstico , COVID-19/virologia , Prata/química , Aptâmeros de Nucleotídeos/química , Humanos , Glicoproteína da Espícula de Coronavírus/análise , Propriedades de SuperfícieRESUMO
Binary (also known as split) nucleic acid enzymes have emerged as novel tools in biosensors. We report a new split strategy to split the DNAzyme kinase into two independent and non-functional fragments, denoted DK1sub and DK1enz. In the presence of the specific target, their free ends are brought sufficiently close to interact with each other without the formation of Watson-Crick base pairings between Dk1sub and Dk1enz, thus allowing the DNA phosphorylation reaction. We term this approach proximity-dependent activation of split DNAzyme kinase (ProxSDK). The utility of ProxSDK is demonstrated by engineering a biosensing system that is capable of measuring specific DNA-protein interactions. We envision that the approach described herein will ï¬nd useful applications in biosensing, imaging, and clinical diagnosis.
RESUMO
We report on the first efforts to isolate acidic RNA-cleaving DNAzymes (aRCDs) from a random-sequence DNA pool by in vitro selection that are activated by a microbe Escherichia coli (E. coli), at pH 5.3. Importantly, these E. coli-responsive aRCDs only require monovalent metal ions as cofactors for cleaving a fluorogenic chimeric DNA/RNA substrate. Such characteristics can be used to efficiently protect RCDs from both intrinsic chemical instability and external enzymatic degradation. One remarkable DNAzyme, aRCD-EC1, is specific for E. coli, and its target is likely a protein. Furthermore, truncated aRCD-EC1 had significantly improved catalytic activity with an observed rate constant (kobs) of 1.18 min-1, making it the fastest bacteria-responding RCD reported to date. Clinical evaluation of this aRCD-based fluorescent assay using 40 patient urine samples demonstrated a diagnostic sensitivity of 100% and a specificity of 100% at a total analysis time of 50 min without a bacterial culture. This work can expand the repertoire of DNAzymes that are active under nonphysiological conditions, thus facilitating the development of diverse DNAzyme-based biosensors in clinical diagnosis.
Assuntos
Técnicas Biossensoriais , DNA Catalítico , Humanos , DNA Catalítico/química , Escherichia coli/metabolismo , DNA/química , RNA/química , MetaisRESUMO
Functional nucleic acids (FNAs), such as DNAzymes and DNA aptamers, can be engineered into circular forms for improved performance. Circular FNAs are promising candidates for bioanalytical and biomedical applications due to their intriguing properties of enhanced biological stability and compatibility with rolling circle amplification. They are typically made from linear single-stranded (ss) DNA molecules via ligase-mediated ligation. However, it remains a great challenge to synthesize circular ssDNA molecules in high yield due to inherent side reactions where two or more of the same ssDNA molecules are ligated. Herein, we present a strategy to overcome this issue by first using in vitro selection to search from a random-sequence DNA library a ligatable DNA aptamer that binds a DNA ligase and then by engineering this aptamer into a general-purpose templating DNA scaffold to guide the ligase to execute selective intramolecular circularization. We demonstrate the broad utility of this approach via the creation of several species of circular DNA molecules, including a circular DNAzyme sensor for a bacterium and a circular DNA aptamer sensor for a protein target with excellent detection sensitivity and specificity.
Assuntos
Aptâmeros de Nucleotídeos , Técnicas Biossensoriais , DNA Catalítico , DNA Catalítico/metabolismo , Aptâmeros de Nucleotídeos/química , Ligases/metabolismo , Técnicas de Amplificação de Ácido Nucleico , DNA/química , DNA Circular , DNA de Cadeia SimplesRESUMO
Rapid detection of single nucleotide polymorphisms (SNPs) in the CYP2C19 gene is of great significance for clopidogrel-accurate medicine. CRISPR/Cas systems have been increasingly used in SNP detection due to their single-nucleotide mismatch specificity. PCR, as a powerful amplification tool, has been incorporated into the CRISPR/Cas system to improve the sensitivity. However, the complicated three-step temperature control of the conventional PCR impeded rapid detection. The "V" shape PCR can shorten about 2/3 of the amplification time compared with conventional PCR. Herein, we present a new system termed the "V" shape PCR-coupled CRISPR/Cas13a (denoted as VPC) system, achieving the rapid, sensitive, and specific genotyping of CYP2C19 gene polymorphisms. The wild- and mutant-type alleles in CYP2C19*2, CYP2C19*3, and CYP2C19*17 genes can be discriminated by using the rationally programmed crRNA. A limit of detection (LOD) of 102 copies/µL was obtained within 45 min. In addition, the clinical applicability was demonstrated by genotyping SNPs in CYP2C19*2, CYP2C19*3, and CYP2C19*17 genes from clinical blood samples and buccal swabs within 1 h. Finally, we conducted the HPV16 and HPV18 detections to validate the generality of the VPC strategy.
Assuntos
Técnicas de Amplificação de Ácido Nucleico , Polimorfismo de Nucleotídeo Único , Genótipo , Citocromo P-450 CYP2C19/genética , Reação em Cadeia da PolimeraseRESUMO
clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems are increasingly used in biosensor development. However, directly translating recognition events for non-nucleic acid targets by CRISPR into effective measurable signals represents an important ongoing challenge. Herein, it is hypothesized and confirmed that CRISPR RNAs (crRNAs) in a circular topology efficiently render Cas12a incapable of both site-specific double-stranded DNA cutting and nonspecific single-stranded DNA trans cleavage. Importantly, it is shown that nucleic acid enzymes (NAzymes) with RNA-cleaving activity can linearize the circular crRNAs, activating CRISPR-Cas12a functions. Using ligand-responsive ribozymes and DNAzymes as molecular recognition elements, it is demonstrated that target-triggered linearization of circular crRNAs offers great versatility for biosensing. This strategy is termed as "NAzyme-Activated CRISPR-Cas12a with Circular CRISPR RNA (NA3C)." Use of NA3C for clinical evaluation of urinary tract infections using an Escherichia coli-responsive RNA-cleaving DNAzyme to test 40 patient urine samples, providing a diagnostic sensitivity of 100% and specificity of 90%, is further demonstrated.
Assuntos
Técnicas Biossensoriais , Ácidos Nucleicos , Humanos , Sistemas CRISPR-Cas/genética , RNA Circular , DNA de Cadeia Simples , RNARESUMO
Inspired by intrinsically disordered proteins in nature, DNA aptamers can be engineered to display strongly homotropic allosteric (or cooperative) ligand binding, representing a unique feature that could be of great utility in applications such as biosensing, imaging and drug delivery. The use of an intrinsic disorder mechanism, however, comes with an inherent drawback of significantly reduced overall binding affinity. We hypothesize that it could be addressed via the design of multivalent supramolecular aptamers. We built functional DNA superstructures (denoted as 3D DNA), made of long-chain DNA containing tandem repeating DNA aptamers (or concatemeric aptamers). The 3D DNA systems exhibit highly cooperative binding to both small molecules and proteins, without loss of binding affinities of their parent aptamers. We further produced a highly responsive sensor for fluorescence imaging of glutamate stimulation-evoked adenosine triphosphate (ATP) release in neurons, as well as force stimulus-triggered ATP release in astrocytes.
Assuntos
Aptâmeros de Nucleotídeos , Técnicas Biossensoriais , Aptâmeros de Nucleotídeos/química , Ligantes , DNA/química , Trifosfato de Adenosina/metabolismo , Técnicas Biossensoriais/métodosRESUMO
Superoxide and other reactive oxygen species (ROS) shape microbial communities and drive the transformation of metals and inorganic/organic matter. Taxonomically diverse bacteria and phytoplankton produce extracellular superoxide during laboratory cultivation. Understanding the physiological reasons for extracellular superoxide production by aerobes in the environment is a crucial question yet not fully solved. Here, we showed that iron-starving Arthrobacter sp. QXT-31 (A. QXT-31) secreted a type of siderophore [deferoxamine (DFO)], which provoked extracellular superoxide production by A. QXT-31 during carbon sources-level fluctuation. Several other siderophores also demonstrated similar effects to A. QXT-31. RNA-Seq data hinted that DFO stripped iron from iron-bearing proteins in electron transfer chain (ETC) of metabolically active A. QXT-31, resulting in electron leakage from the electron-rich (resulting from carbon sources metabolism by A. QXT-31) ETC and superoxide production. Considering that most aerobes secrete siderophore(s) and undergo carbon sources-level fluctuation, the superoxide-generation pathway is likely a common pathway by which aerobes produce extracellular superoxide in the environment, thus influencing the microbial community and cycling of elements. Our results pointed that the ubiquitous siderophore might be the potential driving force for the microbial generation of superoxide and other ROS and revealed the important role of iron physiology in microbial ROS generation.
Assuntos
Arthrobacter , Sideróforos , Arthrobacter/genética , Arthrobacter/metabolismo , Carbono/metabolismo , Ferro/metabolismo , Sideróforos/metabolismo , Superóxidos/metabolismoRESUMO
Bioaerosols could carry and spread harmful microorganisms, thus posing a continuous threat to human beings and livestock health. Early warning and management are crucial for controlling the spread of bioaerosols. Herein, we developed a split aptamer (SA)-based electrochemical nanosensor chip (denoted SAE-nChip) for rapid and sensitive detection of adenosine triphosphate (ATP) in bioaerosols. The platform features two components: split DNA aptamers for their ability to bind ATP and undergo target-induced assembly on the chip surface and ZIF-8@MXene composites for their ability to provide a high surface density of aptamer-binding sites and facilitate the electron transfer at the biointerface. The SAE-nChip was capable of detecting ATP with a detection limit of 10 pM. Furthermore, this assay allowed the detection of ATP in cultured microorganisms and collected real bioaerosols. Overall, this strategy of interfacing DNA aptamers with MXene-based composite materials represents a versatile approach for the ubiquitous detection of biochemical targets in bioaerosols.
Assuntos
Aptâmeros de Nucleotídeos , Técnicas Biossensoriais , Humanos , Aptâmeros de Nucleotídeos/química , Limite de Detecção , Trifosfato de Adenosina/metabolismoRESUMO
Pathogens have long presented a significant threat to human lives, and hence the rapid detection of infectious pathogens is vital for improving human health. Current detection methods lack the means to detect infectious pathogens in a simple, rapid, and reliable manner at the time and point of need. Functional nucleic acids (FNAs) have the potential to overcome these limitations by acting as key components for point-of-care (POC) biosensors due to their distinctive advantages that include high binding affinities and specificities, excellent chemical stability, ease of synthesis and modification, and compatibility with a variety of signal-amplification and signal-transduction mechanisms.This Account summarizes the work completed in our groups toward developing FNA-based biosensors for detecting bacteria. In vitro selection has led to the isolation of many RNA-cleaving fluorogenic DNAzymes (RFDs) and DNA aptamers that can recognize infectious pathogens, including Escherichia coli, Clostridium difficile, Helicobacter pylori, and Legionella pneumophila. In most cases, a "many-against-many" approach was employed using a DNA library against a crude cellular mixture of an infectious pathogen containing diverse biomarkers as the target to isolate RFDs, with combined counter and positive selections ensuring high specificity toward the desired target. This procedure allows for the isolation of pathogen-specific FNAs without first identifying a suitable biomarker. Multiple target-specific DNA aptamers, including anti-glutamate dehydrogenase (GDH) circular aptamers, anti-degraded toxin B aptamers, and anti-RNase HII aptamers, have also been isolated for the detection of bacteria such as Clostridium difficile. The isolated FNAs have been integrated into fluorescent, colorimetric, and electrochemical biosensors using various signal transduction mechanisms. Both simple-to-use paper-based analytical devices and hand-held electrical devices with integrated FNAs have been developed for POC applications. In addition, signal-amplification strategies, including DNA catenane enabled rolling circle amplification (RCA), DNAzyme feedback RCA, and an all-DNA amplification system using a four-way junction and catalytic hairpin assembly (CHA), have been designed and applied to these systems to further increase their detection sensitivity. The use of these FNA-based biosensors to detect pathogens directly in clinical samples, such as urine, blood, and stool, has now been demonstrated with an outstanding sensitivity of as low as 10 cells per milliliter, highlighting the tremendous potential of using FNA-based sensors in clinical applications. We further describe strategies to overcome the challenges of using FNA-based biosensors in clinical applications, including strategies to improve the stability of FNAs in biological samples and prevent their nonspecific degradation from nucleases and strategies to deal with issues such as signal loss caused by nonspecific binding and biofouling. Finally, the remaining roadblocks for employing FNA-based biosensors in clinical applications are discussed.
Assuntos
Aptâmeros de Nucleotídeos/metabolismo , Bactérias/genética , Técnicas Biossensoriais/métodos , DNA Catalítico/metabolismo , Aptâmeros de Nucleotídeos/química , Bactérias/isolamento & purificação , DNA Catalítico/química , Escherichia coli/genética , Escherichia coli/isolamento & purificação , Técnicas de Amplificação de Ácido Nucleico , Sistemas Automatizados de Assistência Junto ao LeitoRESUMO
Adenosine 5'-triphosphate (ATP) is a central extracellular signaling agent involved in various physiological and pathological processes. However, precise measurements of the temporal and spatial components of ATP dynamics are lacking due primarily to the limitations of available methods for ATP detection. Here, we report on the first effort to design a self-phosphorylating DNAzyme (SPDz) sensor for fluorescence imaging of ATP. In response to ATP, SPDz sensors exhibit subsecond response kinetics, extremely high specificity, and micromolar affinities. In particular, we demonstrate cell-surface-anchored SPDz sensors for fluorescence imaging of both stress-induced endogenous ATP release in astrocytes and mechanical stimulation-evoked ATP release at the single-cell level. We also validated their utility for visualizing the rapid dynamic properties of ATP signaling upon electrical stimulation in astrocytes. Thus, SPDz sensors are robust tools for monitoring ATP signaling underlying diverse cellular processes.
Assuntos
Trifosfato de Adenosina/química , DNA Catalítico/química , DNA Catalítico/metabolismo , Imagem Óptica/métodos , Imagem Individual de Molécula/métodos , Astrócitos , Técnicas Biossensoriais , Humanos , Células MCF-7 , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Fosforilação , Sensibilidade e Especificidade , Estresse FisiológicoRESUMO
There is currently a great need for developing a simple and effective biosensing platform for the detection of single biomolecules (e.g., DNAs, RNAs, or proteins) in the biological, medical, and environmental fields. Here, we show a versatile and sensitive fluorescence counting strategy for quantifying proteins and microRNAs by employing functional DNA superstructures (denoted as 3D DNA). A 3D DNA biolabel was first engineered to become highly fluorescent and carry recognition elements for the target of interest. The presence of a target cross-links the resultant of the 3D DNA biolabel and a surface-bound capturing antibody or DNA oligonucleotide, thus forming a sandwich complex that can be easily resolved using traditional fluorescence microscopy. The broad utility of this platform is illustrated by engineering two different 3D DNA biolabels that enable the quantification of ß-lactamase (one secreted bacterial hydrolase) and miR-21 (one overexpressed microRNA in cancer cells) with detection limits of 100 aM and 1 fM, respectively. We envision that the approach described herein will find useful applications in chemical biology, medical diagnostics, and biosensing.
Assuntos
Técnicas Biossensoriais , MicroRNAs , DNA , MicroRNAs/genética , Oligonucleotídeos , ProteínasRESUMO
Phosphorus is a key element responsible for eutrophication, and its measurement and speciation is a critical topic in analytical chemistry. Most research efforts have been devoted to detecting phosphate (P(V)), while few reports on phosphite (P(III)) are available, making it difficult for sensor-based understanding of the phosphorus cycle. This study presents a fluorescent "turn-on" sensor for quantitative and highly selective analysis of phosphite based on the different coordination strength of N and P lone-pair electrons toward nickel oxide (NiO). A few N-containing compounds (mainly Good's buffers) were screened as inhibitors for the oxidase-like activity of NiO nanoparticles for the oxidation of Amplex red (AR). HEPES was found to be most effective for inhibiting the formation of fluorescent resorufin, the oxidation product of AR. Among various phosphorus-, arsenic-, selenium-, and sulfur-containing species, along with various cations, phosphite was the only one that could restore the activity, likely due to its stronger affinity with the surface, and it is not an inhibitor. Under the optimum condition, the sensor detected P(III) up to 1 mM with a detection limit of 1.46 µM. The phosphite analysis with recovery rates ranged from 74.2 ± 2.6% to 107.5 ± 0.5% in real water and biological samples, suggesting the potential applicability of this sensor.
Assuntos
Materiais Biomiméticos/química , Enzimas/metabolismo , Limite de Detecção , Nanopartículas/química , Níquel/química , Fosfitos/análise , Oxazinas/química , Fosfitos/química , Espectrometria de FluorescênciaRESUMO
A large number of nanomaterials can strongly adsorb DNA and quench fluorescence, such as graphene oxide, gold nanoparticles, and most metal oxides. On the other hand, noncationic nanomaterials that adsorb DNA but cannot quench fluorescence are less known. These materials are attractive for studying the mechanism of DNA-based surface reactions. Y2O3 was found to have this property. Herein, we used fluorescently labeled oligonucleotides as probes to study the mechanism of DNA adsorption. The fluorescence was quenched at low concentrations of Y2O3 and then recovered and even enhanced with higher Y2O3 concentrations. The reason was attributed to the intermolecular quenching by the DNA bases of the neighboring strands. The fluorescence enhancement was due to breaking of the intramolecular fluorophore/DNA interactions, and the most enhancement was observed with a Cy3-labeled DNA. DNA adsorption followed the Langmuir isotherm on Y2O3. Desorption experiments suggested that DNA was adsorbed through the phosphate backbone, with FAM-G15 and FAM-C15 adsorbed more strongly than the other two DNA homopolymers. With a high salt concentration, no fluorescence change was observed, suggesting that the DNA adsorbed in a folded state reducing intermolecular quenching. Overall, Y2O3 might be useful as a model surface for investigating DNA hybridization on a surface.
Assuntos
DNA/química , Oligonucleotídeos/química , Ítrio/química , Adsorção , Propriedades de SuperfícieRESUMO
Protein biomarkers often exist as degradation fragments in biological samples, and affinity agents derived using a purified protein may not recognize them, limiting their value for clinical diagnosis. Herein, we present a method to overcome this issue, by selecting aptamers against a degraded form of the toxinâ B protein, which is a marker for diagnosing toxigenic Clostridium difficile infections. This approach has led to isolation of a DNA aptamer that recognizes degraded toxinâ B, fresh toxinâ B, and toxinâ B spiked into human stool samples. DNA aptamers selected using intact recombinant toxinâ B failed to recognize degraded toxinâ B, which is the form present in stored stool samples. Using this new aptamer, we produced a simple paper-based analytical device for colorimetric detection of toxinâ B in stool samples, or in the NAP1 strain of Clostridium difficile. The combined aptamer-selection and paper-sensing strategy can expand the practical utility of DNA aptamers in clinical diagnosis.
Assuntos
Aptâmeros de Nucleotídeos/metabolismo , Técnicas Biossensoriais/métodos , Fragmentos de Peptídeos/metabolismo , Proteólise , Fezes/química , Humanos , Papel , Toxinas Biológicas/análise , Toxinas Biológicas/química , Toxinas Biológicas/metabolismoRESUMO
φ29 DNA polymerase (Polφ29) is capable of synthesizing long-chain single-stranded (ss) DNA molecules by copying the sequence of a small ss circular DNA template (ssCDT) in a process known as rolling circle amplification (RCA). The use of a ssCDT in RCA, however, comes with a key drawback: the rate of DNA synthesis is significantly reduced. We hypothesize that this issue can be overcome using a very long linear ssDNA template with a repeating sequence. To test this idea, we engineered a DNA assembly, which we denote "micrometer-sized DNA track" (µDT). This µDT, with an average length of ≈13.5â µm, is made of a long chain DNA with a primer-binding domain at its 3' end and ≈1000 repeating sequence units at its 5' end, each carrying a DNA anchor. We find that Polφ29 copies µDT at a speed ≈5-time faster than it does a related ssCDT. We use this to design a simple all-in-one printed paper device for rapid and sensitive detection of microRNA let-7. This paper sensor is capable of detecting 1â pM let-7a in 10â minutes.
Assuntos
Técnicas Biossensoriais , DNA Polimerase Dirigida por DNA/metabolismo , DNA/biossíntese , Técnicas de Amplificação de Ácido Nucleico , DNA/químicaRESUMO
Fluorescent metal sensors based on DNA often rely on changes in end-to-end distance or local environmental near fluorophore labels. Because metal ions can also nonspecifically interact with DNA through various mechanisms, such as charge screening, base binding, and increase or decrease in duplex stability, robust and specific sensing of metal ions has been quite challenging. In this work, a side-by-side comparison of two signaling strategies on a Na+ -specific DNAzyme that contained a Na+ -binding aptamer was performed. The duplex regions of the DNAzyme was systematically shortened and its effect was studied by using a 2-aminopurine (2AP)-labeled substrate strand. Na+ binding affected the local environmental of the 2AP label and increased its fluorescence. A synergistic process of Na+ binding and forming the duplex on the 5'-end of the enzyme strand was observed, and this end was close to the aptamer loop. Effective Na+ binding was achieved with a five base-pair stem. The effect on the 3'-end is more continuous, and the stem needs to form first before Na+ can bind. With an optimized substrate binding arm, a FRET-based sensor has been designed by labeling the two ends of a cis form of the DNAzyme with two fluorophores. In this case, Na+ failed to show a distinct difference from that of Li+ or K+ ; thus indicating that probing changes to the local environment allows more robust sensing of metal ions.
Assuntos
DNA Catalítico/química , Metais/análise , Sódio/metabolismo , 2-Aminopurina/metabolismo , Aptâmeros de Nucleotídeos , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes , Íons , Conformação de Ácido NucleicoRESUMO
We report on the first effort to select DNA aptamers from a circular DNA library, which resulted in the discovery of two high-affinity circular DNA aptamers that recognize the glutamate dehydrogenase (GDH) from Clostridium difficile, an established antigen for diagnosing Clostridium difficile infection (CDI). One aptamer binds effectively in both the circular and linear forms, the other is functional only in the circular configuration. Interestingly, these two aptamers recognize different epitopes on GDH, demonstrating the advantage of selecting aptamers from circular DNA libraries. A sensitive diagnostic test was developed to take advantage of the high stability of circular DNA aptamers in biological samples and their compatibility with rolling circle amplification. This test is capable of identifying patients with active CDI using stool samples. This work represents a significant step forward towards demonstrating the practical utility of DNA aptamers in clinical diagnosis.
Assuntos
Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais/métodos , DNA Circular/química , Aptâmeros de Nucleotídeos/genética , Aptâmeros de Nucleotídeos/metabolismo , Clostridioides difficile/enzimologia , Infecções por Clostridium/diagnóstico , Infecções por Clostridium/microbiologia , DNA Circular/genética , Glutamato Desidrogenase/análise , Glutamato Desidrogenase/genética , Glutamato Desidrogenase/metabolismo , Humanos , Técnica de Seleção de Aptâmeros/métodosRESUMO
Technologies capable of linking DNA amplification to molecular recognition are very desirable for ultrasensitive biosensing applications. We have developed a simple but powerful isothermal DNA amplification method, termed DNAzyme feedback amplification (DFA), that is capable of relaying molecular recognition to exponential DNA amplification. The method incorporates both an RNA-cleaving DNAzyme (RCD) and rolling circle amplification (RCA) carried out by a special DNA polymerase using a circular DNA template. DFA begins with a stimulus-dependent RCA reaction, producing tandemly linked RCDs in long-chain DNA products. These RCDs cleave an RNA-containing DNA sequence to form additional primers that hybridize to the circular DNA molecule, giving rise to DNA assemblies that act as the new inputs for RCA. The RCA reaction and the cleavage event keep on feeding each other autonomously, resulting in exponential growth of repetitive DNA sequences that can be easily detected. This method can be used for the detection of both nucleic acid based targets and non-nucleic acid analytes. In this article, we discuss the conceptual framework of the feedback amplification approach, the essential features of this method as well as remaining challenges and possible solutions.
Assuntos
DNA Catalítico/metabolismo , Técnicas Biossensoriais/métodos , DNA , Primers do DNA , DNA Circular , DNA Polimerase Dirigida por DNA , Estrutura Molecular , Técnicas de Amplificação de Ácido Nucleico/métodos , Hibridização de Ácido Nucleico , RNA/efeitos dos fármacosRESUMO
Interactions between microbes and micropollutants (MPs) play a crucial role in water purification or treatment. Current studies have generally focused on the direct degradation or cometabolism of MPs. Considering the increasing interest in and importance of the roles of MPs in microbial metabolism, we adopted an Mn(II)-oxidizing Pseudomonas sp. QJX-1 using tyrosine (Tyr) as the sole carbon and nitrogen source to investigate the effects of seven MPs on its growth and function. Six MPs exhibited an inhibition effect on bacterial growth and Mn(II) oxidation. Only benzophenone-4 (BP-4) promoted the growth of QJX-1 and biogenic oxidation Mn(II), but its concentration was not directly coupled to growth, which was unexpected. RNA-seq data suggested that the addition of BP-4 did not significantly change the basic metabolic function of QJX-1, but stimulated the upregulation of the pyruvate and gluconeogenesis metabolic pathways of Tyr for QJX-1 growth. Furthermore, protein identification and extracellular superoxide detection indicated that Mn(II) oxidation was largely driven by the formation of superoxide in response to Tyr starvation; the acceleration of superoxide production, due to BP-4 accelerating Tyr consumption, was responsible for the promotion effect of BP-4 on QJX-1 Mn(II) oxidation. Our findings highlight the dual effects that MPs can have on the growth and function of a single strain in aquatic ecosystem, i.e., the coexistence of inhibition and promotion.