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1.
Anal Chem ; 96(11): 4597-4604, 2024 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-38456210

RESUMO

DNA assemblies are commonly used in biosensing, particularly for the detection and imaging of microRNAs (miRNAs), which are biomarkers associated with tumor progression. However, the difficulty lies in the exploration of high-sensitivity analytical techniques for miRNA due to its limited presence in living cells. In this study, we introduced a DNA nanosphere (DS) enhanced catalytic hairpin assembly (CHA) system for the detection and imaging of intracellular miR-21. The single-stranded DNA with four palindromic portions and extending sequences at the terminal was annealed for assembling DS, which avoided the complex sequence design and high cost of long DNA strands. Benefiting from the multiple modification sites of DS, functional hairpins H1 (modified with Cy3 and BHQ2) and H2 were grafted onto the surface of DS for assembling DS-H1-H2 using a hybridization reaction. The DS-H1-H2 system utilized spatial confinement and the CHA reaction to amplify fluorescence signals of Cy3. This enabled highly sensitive and rapid detection of miR-21 in the range from 0.05 to 3.5 nM. The system achieved a limit of determination (LOD) of 2.0 pM, which was 56 times lower than that of the control CHA circuit with freedom hairpins. Additionally, the sensitivity was improved by 8 times. Moreover, DS-H1-H2 also showed an excellent imaging capability for endogenous miR-21 in tumor cells. This was due to enhanced cell internalization efficiency, accelerated reaction kinetics, and improved biostability. The imaging strategy was shown to effectively monitor the dynamic content of miR-21 in live cancer cells and differentiate various cells. In general, the simple nanostructure DS not only enhanced the detection and imaging capability of the conventional probe but also could be easily integrated with the reported DNA-free probe, indicating a wide range of potential applications.


Assuntos
Técnicas Biossensoriais , DNA Catalítico , MicroRNAs , Nanosferas , Neoplasias , MicroRNAs/genética , MicroRNAs/química , DNA/genética , DNA/química , Hibridização de Ácido Nucleico , Sondas de DNA/química , Técnicas Biossensoriais/métodos , Limite de Detecção
2.
Anal Chem ; 96(11): 4402-4409, 2024 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-38457775

RESUMO

The ultrasensitive DNA methyltransferase (Dam MTase) assay is of high significance for biomedical research and clinical diagnosis because of its profound effect on gene regulation. However, detection sensitivity is still limited by shortcomings, including photobleaching and weak signal intensities of conventional fluorophores at low concentrations. Plasmonic nanostructures with ultrastrong electromagnetic fields and fluorescence enhancement capability that can overcome these intrinsic defects hold great potential for ultrasensitive bioanalysis. Herein, a silica-coated gold nanostars (Au NSTs@SiO2)-based plasmon-enhanced fluorescence (PEF) probe with 20 "hot spots" was developed for ultrasensitive detection of Dam MTase. Here, the Dam Mtase assay was achieved by detecting the byproduct PPi of the rolling circle amplification reaction. It is worth noting that, benefiting from the excellent fluorescence enhancement capability of Au NSTs originating from their 20 "hot spots", the detection limit of Dam Mtase was reduced by nearly 105 times. Moreover, the proposed Au NST-based PEF probe enabled versatile evaluation of Dam MTase inhibitors as well as endogenous Dam MTase detection in GW5100 and JM110 Escherichia coli cell lysates, demonstrating its potential in biomedical analysis.


Assuntos
Técnicas Biossensoriais , DNA Metiltransferases Sítio Específica (Adenina-Específica) , DNA Metiltransferases Sítio Específica (Adenina-Específica)/análise , Dióxido de Silício , Ouro/química , Metilases de Modificação do DNA , Escherichia coli , Corantes Fluorescentes/química , DNA , Sondas de DNA/química
3.
Biosens Bioelectron ; 253: 116185, 2024 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-38457863

RESUMO

Mechanical forces play an important role in cellular communication and signaling. We developed in this study novel electrochemical DNA-based force sensors for measuring cell-generated adhesion forces. Two types of DNA probes, i.e., tension gauge tether and DNA hairpin, were constructed on the surface of a smartphone-based electrochemical device to detect piconewton-scale cellular forces at tunable levels. Upon experiencing cellular tension, the unfolding of DNA probes induces the separation of redox reporters from the surface of the electrode, which results in detectable electrochemical signals. Using integrin-mediated cell adhesion as an example, our results indicated that these electrochemical sensors can be used for highly sensitive, robust, simple, and portable measurements of cell-generated forces.


Assuntos
Técnicas Biossensoriais , Técnicas Biossensoriais/métodos , DNA/genética , Adesão Celular , Sondas de DNA , Integrinas/metabolismo
4.
Methods Mol Biol ; 2784: 215-225, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38502489

RESUMO

DNA fluorescence in situ hybridization (FISH) enables the visualization of chromatin architecture and the interactions between genomic loci at a single-cell level, complementary to genome-wide methods such as Hi-C. DNA FISH uses fluorescent-labeled DNA probes targeted to the loci of interest, allowing for the analysis of their spatial positioning and proximity with microscopy. Here, we describe an optimized experimental procedure for DNA FISH, from probe design and sample preparation through imaging and image quantification. This protocol can be readily applied to querying the spatial positioning of genomic loci of interest.


Assuntos
Cromatina , DNA , Hibridização in Situ Fluorescente/métodos , DNA/genética , Cromatina/genética , Cromossomos , Sondas de DNA/genética , Corantes Fluorescentes
5.
Methods Mol Biol ; 2784: 271-284, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38502492

RESUMO

Genomic instability is an important biomarker in the progression of cervical carcinoma. DBD-FISH (DNA breakage detection-fluorescence in situ hybridization) is a sensitive method that detects strand breaks, alkali-labile sites, and incomplete DNA excision repair in cells of the cervical epithelium. This technique integrates the microgel immersion of cells from a vaginal lesion scraping and the DNA unwinding treatment with the capacity of FISH integrated into digital image analysis. Cells captured within an agarose matrix are lysed and submerged in an alkaline unwinding solution that generates single-stranded DNA motifs at the ends of internal DNA strand breaks. After neutralization, the microgel is dehydrated and the cells are incubated with DNA-labeled probes. The quantity of a hybridized probe at a target sequence corresponds to the measure of the single-stranded DNA produced during the unwinding step, which is equivalent to the degree of local DNA breakage. DNA damage does not show uniformly throughout the entire DNA of a cell; rather, it is confined to specific chromosomal sites. In this chapter, an overview of the technique is supplied, focusing on its ability for assessing the association between DNA damage in specific sequences and in the progressive stages of cervical carcinoma.


Assuntos
Carcinoma , Microgéis , Neoplasias do Colo do Útero , Feminino , Humanos , DNA de Cadeia Simples , Hibridização in Situ Fluorescente/métodos , DNA , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/patologia , Dano ao DNA , Sondas de DNA/genética
6.
Sci Rep ; 14(1): 5229, 2024 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-38433255

RESUMO

In the Anthropocene, plastic pollution has become a new environmental biotope, the so-called plastisphere. In the oceans, nano- and micro-sized plastics are omnipresent and found in huge quantities throughout the water column and sediment, and their large surface area-to-volume ratio offers an excellent surface to which hydrophobic chemical pollutants (e.g. petrochemicals and POPs) can readily sorb to. Our understanding of the microbial communities that breakdown plastic-sorbed chemical pollutants, however, remains poor. Here, we investigated the formation of 500 nm and 1000 nm polystyrene (PS) agglomerations in natural seawater from a coastal environment, and we applied DNA-based stable isotope probing (DNA-SIP) with the 500 nm PS sorbed with isotopically-labelled phenanthrene to identify the bacterial members in the seawater community capable of degrading the hydrocarbon. Whilst we observed no significant impact of nanoplastic size on the microbial communities associated with agglomerates that formed in these experiments, these communities were, however, significantly different to those in the surrounding seawater. By DNA-SIP, we identified Arcobacteraceae, Brevundimonas, Comamonas, uncultured Comamonadaceae, Delftia, Sphingomonas and Staphylococcus, as well as the first member of the genera Acidiphilum and Pelomonas to degrade phenanthrene, and of the genera Aquabacterium, Paracoccus and Polymorphobacter to degrade a hydrocarbon. This work provides new information that feeds into our growing understanding on the fate of co-pollutants associated with nano- and microplastics in the ocean.


Assuntos
Comamonadaceae , Poluentes Ambientais , Microbiota , Fenantrenos , Microplásticos , Plásticos , Poliestirenos , Sondas de DNA , Isótopos , DNA
7.
Nat Commun ; 15(1): 1027, 2024 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-38310092

RESUMO

Fluorescent in situ hybridization (FISH) is a powerful method for the targeted visualization of nucleic acids in their native contexts. Recent technological advances have leveraged computationally designed oligonucleotide (oligo) probes to interrogate > 100 distinct targets in the same sample, pushing the boundaries of FISH-based assays. However, even in the most highly multiplexed experiments, repetitive DNA regions are typically not included as targets, as the computational design of specific probes against such regions presents significant technical challenges. Consequently, many open questions remain about the organization and function of highly repetitive sequences. Here, we introduce Tigerfish, a software tool for the genome-scale design of oligo probes against repetitive DNA intervals. We showcase Tigerfish by designing a panel of 24 interval-specific repeat probes specific to each of the 24 human chromosomes and imaging this panel on metaphase spreads and in interphase nuclei. Tigerfish extends the powerful toolkit of oligo-based FISH to highly repetitive DNA.


Assuntos
DNA , Sequências Repetitivas de Ácido Nucleico , Humanos , Hibridização in Situ Fluorescente/métodos , DNA/genética , Sequências Repetitivas de Ácido Nucleico/genética , Sondas de Oligonucleotídeos/genética , Sondas de DNA/genética , Oligonucleotídeos/genética
8.
J Am Chem Soc ; 146(10): 6516-6521, 2024 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-38411013

RESUMO

Regulatory modules for controlling the kinetics of toehold-mediated strand displacement (TMSD) play critical roles in designing dynamic and dissipative DNA chemical reaction networks (CRNs) but are hardwired into sequence designs. Herein, we introduce antitoehold (At), a plug-and-play module for reversible and continuous tuning of TMSD kinetics by temporarily occupying the toehold domain via a metastable duplex and base stacking. We demonstrate that kinetic control can be readily activated or deactivated in real time for any TMSD by simply adding At or anti-At. Continuous tuning of TMSD kinetics can also be achieved by altering the concentration of At. Moreover, the simple addition of At could readily reprogram existing TMSDs into a pulse-generation DNA CRN with continuous tunability. Our At approach also offers a new way for engineering continuously tunable DNA hybridization probes, which may find practical uses for discriminating clinically important mutations. Because of the simplicity, we anticipate that At will find wide applications for engineering DNA CRNs with diverse dynamic and dissipative behaviors, and DNA hybridization probes with tunable affinity and selectivity.


Assuntos
DNA , DNA/química , Hibridização de Ácido Nucleico , Cinética , Sondas de DNA
9.
Talanta ; 272: 125777, 2024 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-38364565

RESUMO

Tyramine signal amplification (TSA) has made its mark in immunoassay due to its excellent signal amplification ability and short reaction time, but its application in nucleic acid detection is still very limited. Herein, an ultrasensitive microRNA (miRNA) biosensor by coupling hybridization-initiated exonuclease I (Exo I) protection and TSA strategy was established. Target miRNA is complementarily hybridized to the biotin-modified DNA probe to form a double strand, which protects the DNA probe from Exo I hydrolysis. Subsequently, horseradish peroxidase (HRP) is attached to the duplex via the biotin-streptavidin reaction and catalyzes the deposition of large amounts of biotin-tyramine in the presence of hydrogen peroxide (H2O2), followed by the conjugation of signal molecule streptavidin-phycoerythrin (SA-PE), which generates an intense fluorescence signal upon laser excitation. This method gave broad linearity in the range of 0.1 fM - 10 pM, yielding a detection limit as low as 74 aM. An increase in sensitivity of 4 orders of magnitude was observed compared to the miRNA detection without TSA amplification. This biosensor was successfully applied to the determination of miR-21 in breast cancer cells and human serum. By further design of specific DNA probes and coupling with the Luminex xMAP technology, it could be easily extended to multiplex miRNA assay, which possesses great application potential in clinical diagnosis.


Assuntos
Técnicas Biossensoriais , Exodesoxirribonucleases , MicroRNAs , Humanos , MicroRNAs/genética , Biotina , Estreptavidina , Peróxido de Hidrogênio , Técnicas Biossensoriais/métodos , Sondas de DNA/genética , Tiramina , Limite de Detecção , Técnicas de Amplificação de Ácido Nucleico/métodos
10.
Biosens Bioelectron ; 251: 116065, 2024 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-38330772

RESUMO

Lipid nanoparticles (LNPs) containing ionizable cationic lipids are proven delivery systems for therapeutic nucleic acids, such as small interfering RNA (siRNA). It is important to understand the relationship between the interior pH of LNPs and the pH of the external environment to understand LNP formulation and function. Here, we developed a simple and rapid approach for determining the pH of the LNP core using a pH-sensitive fluorescent dye-based DNA probe. LNP siRNA systems containing pH-responsive DNA probes (LNP-siRNA&DNA) were generated by rapid mixing of lipids in ethanol and pH 4 aqueous buffer containing siRNA and DNA probes. We demonstrated that DNA probes were readily encapsulated in LNP systems and were sequestered into an environment at a high concentration as evidenced by an inter-probe FRET signal. It was shown that the pH of LNP encapsulated probes closely follows the pH increase or decrease of the external environment. This indicates that the clinically approved LNP RNA systems with similar lipid compositions (e.g., Onpattro and Comirnaty) are highly permeable to protons and that the pH of the interior environment closely mirrors the external environment. The pH-dependent response of the probe in LNPs was also confirmed under buffer conditions at various pHs. Furthermore, we showed that the pH-sensitive DNA probe can be incorporated into LNP systems at levels that allow the pH response to be monitored at a single LNP level using convex lens-induced confinement (CLiC) confocal microscopy. Direct visualization of the internal pH of single particles with the fluorescent DNA probe was achieved by CLiC for LNP-siRNA&DNA systems formulated under both high and normal ionic strength conditions.


Assuntos
Técnicas Biossensoriais , Lipossomos , Nanopartículas , Corantes Fluorescentes , Lipídeos/química , Nanopartículas/química , RNA Interferente Pequeno/química , DNA , Sondas de DNA
11.
Brief Bioinform ; 25(2)2024 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-38305453

RESUMO

Target enrichment sequencing techniques are gaining widespread use in the field of genomics, prized for their economic efficiency and swift processing times. However, their success depends on the performance of probes and the evenness of sequencing depth among each probe. To accurately predict probe coverage depth, a model called Deqformer is proposed in this study. Deqformer utilizes the oligonucleotides sequence of each probe, drawing inspiration from Watson-Crick base pairing and incorporating two BERT encoders to capture the underlying information from the forward and reverse probe strands, respectively. The encoded data are combined with a feed-forward network to make precise predictions of sequencing depth. The performance of Deqformer is evaluated on four different datasets: SNP panel with 38 200 probes, lncRNA panel with 2000 probes, synthetic panel with 5899 probes and HD-Marker panel for Yesso scallop with 11 000 probes. The SNP and synthetic panels achieve impressive factor 3 of accuracy (F3acc) of 96.24% and 99.66% in 5-fold cross-validation. F3acc rates of over 87.33% and 72.56% are obtained when training on the SNP panel and evaluating performance on the lncRNA and HD-Marker datasets, respectively. Our analysis reveals that Deqformer effectively captures hybridization patterns, making it robust for accurate predictions in various scenarios. Deqformer leads to a novel perspective for probe design pipeline, aiming to enhance efficiency and effectiveness in probe design tasks.


Assuntos
Aprendizado Profundo , RNA Longo não Codificante , Sondas de DNA/genética , Hibridização de Ácido Nucleico , Genômica
12.
Biosens Bioelectron ; 250: 116055, 2024 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-38266617

RESUMO

This study introduces an innovative detection system for multiple cancer biomarkers, employing transcription isothermal amplification methods in conjunction with a tetrahedral DNA nanostructure (TDN). We demonstrate that TDN enhances various transcription isothermal amplification methods by placing DNA probes in proximity. Notably, the TDN-enhanced split T7 promoter-based isothermal transcription amplification with light-up RNA aptamer (STAR) system stands out for its optimal performance and operational simplicity, especially in identifying non-coding RNAs such as microRNAs and long non-coding RNAs (lncRNAs). Multiplex detection of lncRNAs was also achieved by generating distinct light-up RNA aptamers, each emitting unique fluorescence signals. The system effectively identified the target lncRNAs, demonstrating high sensitivity and selectivity in both cell lines and clinical samples. The system, utilizing the single enzyme T7 RNA polymerase, can be easily tailored for alternative targets by substituting target-specific sequences in DNA probes and seamlessly integrated with other isothermal amplification methods for greater sensitivity and accuracy in the detection of multiple cancer biomarkers.


Assuntos
Aptâmeros de Nucleotídeos , Técnicas Biossensoriais , Nanoestruturas , Neoplasias , RNA Longo não Codificante , Humanos , RNA Longo não Codificante/genética , Técnicas Biossensoriais/métodos , DNA/genética , DNA/química , Aptâmeros de Nucleotídeos/química , Biomarcadores Tumorais/genética , Sondas de DNA , Técnicas de Amplificação de Ácido Nucleico/métodos
13.
Anal Chem ; 96(2): 866-875, 2024 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-38164718

RESUMO

Despite extensive efforts, point-of-care testing (POCT) of protein markers with high sensitivity and specificity and at a low cost remains challenging. In this work, we developed an aptamer-CRISPR/Cas12a-regulated liquid crystal sensor (ALICS), which achieved ultrasensitive protein detection using a smartphone-coupled portable device. Specifically, a DNA probe that contained an aptamer sequence for the protein target and an activation sequence for the Cas12a-crRNA complex was prefixed on a substrate and was released in the presence of target. The activation sequence of the DNA probe then bound to the Cas12a-crRNA complex to activate the collateral cleavage reaction, producing a bright-to-dark optical change in a DNA-functionalized liquid crystal interface. The optical image was captured by a smartphone for quantification of the target concentration. For the two model proteins, SARS-CoV-2 nucleocapsid protein (N protein) and carcino-embryonic antigen (CEA), ALICS achieved detection limits of 0.4 and 20 pg/mL, respectively, which are higher than the typical sensitivity of the SARS-CoV-2 test and the clinical CEA test. In the clinical sample tests, ALICS also exhibited superior performances compared to those of the commercial ELISA and lateral flow test kits. Overall, ALICS represents an ultrasensitive and cost-effective platform for POCT, showing a great potential for pathogen detection and disease monitoring under resource-limited conditions.


Assuntos
Técnicas Biossensoriais , Cristais Líquidos , Sistemas Automatizados de Assistência Junto ao Leito , Sistemas CRISPR-Cas , RNA Guia de Sistemas CRISPR-Cas , Oligonucleotídeos , Sondas de DNA
14.
Anal Chem ; 96(3): 1054-1063, 2024 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-38190445

RESUMO

In this work, an integrated strategy with excellent accuracy and high throughput is proposed for the precise indication of single nucleotide polymorphism (SNP) in nonsmall cell lung cancer diseases. Two types of point mutations (L858R and T790M) and the corresponding wild types could be identified together in a single high-performance liquid chromatographic run. Signal amplification was achieved through a series of enzyme ligation, primer extension, and enzyme cleavage strategies, and a large number of DNA probes with different fluorescence signals were finally generated. The factors affecting the spatiotemporal separation efficiency of four DNA probes were systematically investigated. The limits of detection of wild types (WTs) or mutant types (MTs) abbreviated as L858R-MT, L858R-WT, T790M-MT, and T790M-WT were 26, 24, 19, and 22 aM, respectively. In addition, the levels of mutant types and wild types in the serum of 40 nonsmall cell lung cancer patients at different stages were detected using the method, and the correlation between the mutation ratios and cancer stages was preliminarily verified. The proposed highly selective and sensitive method may serve as an alternative approach for early diagnosis and staging of nonsmall cell lung cancer.


Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Humanos , Carcinoma Pulmonar de Células não Pequenas/diagnóstico , Carcinoma Pulmonar de Células não Pequenas/genética , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/genética , Receptores ErbB/metabolismo , Polimorfismo de Nucleotídeo Único , Mutação , Inibidores de Proteínas Quinases , Cromatografia Líquida , Sondas de DNA
15.
Anal Chem ; 96(4): 1556-1564, 2024 01 30.
Artigo em Inglês | MEDLINE | ID: mdl-38214216

RESUMO

Fluorescence in situ hybridization (FISH) is a widely used technique for detecting intracellular nucleic acids. However, its effectiveness in detecting low-copy nucleic acids is limited due to its low fluorescence intensity and background autofluorescence. To address these challenges, we present here an approach of lanthanide-complex-enhanced bioorthogonal-branched DNA amplification (LEBODA) with high sensitivity for in situ nuclear acid detection in single cells. The approach capitalizes on two levels of signal amplification. First, it utilizes click chemistry to directly link a substantial number of bridge probes to target-recognizing probes, providing an initial boost in signal intensity. Second, it incorporates high-density lanthanide complexes into each bridge probe, enabling secondary amplifications. Compared to the traditional "double Z" probes used in the RNAscope method, LEBODA exhibits 4 times the single enhancement for RNA detection signal with the click chemistry approach. Using SARS-CoV-2 pseudovirus-infected HeLa cells, we demonstrate the superiority in the detection of viral-infected cells in rare populations as low as 20% infectious rate. More encouragingly, the LEBODA approach can be adapted for DNA-FISH and single-molecule RNA-FISH, as well as other hybridization-based signal amplification methods. This adaptability broadens the potential applications of LEBODA in the sensitive detection of biomolecules, indicating promising prospects for future research and practical use.


Assuntos
Elementos da Série dos Lantanídeos , Humanos , Hibridização in Situ Fluorescente/métodos , Células HeLa , RNA , DNA/genética , Sondas de DNA/genética
16.
Anal Chem ; 96(5): 2253-2263, 2024 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-38277203

RESUMO

Current study in the heterogeneity and physiological behavior of tumor cells is limited by the fluorescence in situ hybridization technology in terms of probe assembly efficiency, background suppression capability, and target compatibility. In a typically well-designed assay, hybridization probes are constructed in a confined nanostructure to achieve a rapid assembly for efficient signal response, while the excessively high local concentration between different probes inevitably leads to nonspecific background leakage. Inspired by the fabric zipper, we propose a novel confinement reaction pattern in a zipper-confined DNA nanoframe (ZCDN), where two kinds of hairpin probes are independently anchored respective tracks. The metastable states of the dual tracks can well avoid signal leakage caused by the nonspecific probe configuration change. Biomarker-mediated proximity ligation reduces the local distance of dual tracks, kinetically triggering an efficient allosteric chain reaction between the hairpin probes. This method circumvents nonspecific background leakage while maintaining a high efficiency in responding to targets. ZCDN is employed to track different cancer biomarkers located in both the cytoplasm and cytomembrane, of which the expression level and oligomerization behavior can provide crucial information regarding intratumoral heterogeneity. ZCDN exhibits high target response efficiency and strong background suppression capabilities and is compatible with various types of biological targets, thus providing a desirable tool for advanced molecular diagnostics.


Assuntos
Técnicas Biossensoriais , Nanoestruturas , Hibridização in Situ Fluorescente , DNA/química , Diagnóstico por Imagem , Nanoestruturas/química , Sondas de DNA/genética , Sondas de DNA/química , Técnicas Biossensoriais/métodos
17.
J Biotechnol ; 382: 8-20, 2024 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-38211667

RESUMO

Photoacoustic imaging(PAI) is a widely developing imaging modality that has seen tremendous evolvement in the last decade. PAI has gained the upper hand in the imaging field as it takes advantage of optical absorption and ultrasound detection that imparts higher resolution, rich contrast and elevated penetration depth. Unlike other imaging techniques, PAI does not use ionising radiation and is a better, cost-effective and healthier alternative to other imaging techniques. It offers greater specificity than conventional ultrasound imaging with the ability to detect haemoglobin, lipids, water and other light-absorbing chromophores. These properties of PAI have led to its extended applications in the biomedical field in the treatment of diseases such as cancer. This paper reviews how DNA probes have been used in PAI, the various techniques by which it has been modified, and their role in the process. We also focus on different nanocomposites containing DNA having PAI and photothermal therapy(PTT) properties for detection, diagnosis and therapy, its constituents and the role of DNA in it.


Assuntos
Neoplasias , Técnicas Fotoacústicas , Humanos , Técnicas Fotoacústicas/métodos , Fototerapia/métodos , Neoplasias/diagnóstico por imagem , Neoplasias/terapia , Sondas de DNA
18.
RNA ; 30(4): 448-462, 2024 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-38282416

RESUMO

This report describes a chemiluminescence-based detection method for RNAs on northern blots, designated Chemi-Northern. This approach builds on the simplicity and versatility of northern blotting, while dispensing of the need for expensive and cumbersome radioactivity. RNAs are first separated by denaturing gel electrophoresis, transferred to a nylon membrane, and then hybridized to a biotinylated RNA or DNA antisense probe. Streptavidin conjugated with horseradish peroxidase and enhanced chemiluminescence substrate are then used to detect the probe bound to the target RNA. Our results demonstrate the versatility of this method in detecting natural and engineered RNAs expressed in cells, including messenger and noncoding RNAs. We show that Chemi-Northern detection is sensitive and fast, detecting attomole amounts of RNA in as little as 1 sec, with high signal intensity and low background. The dynamic response displays excellent linearity. Using Chemi-Northern, we measure the reproducible, statistically significant reduction of mRNA levels by human sequence-specific RNA-binding proteins, PUM1 and PUM2. Additionally, we measure the interaction of the poly(A) binding protein, PABPC1, with polyadenylated mRNA. Thus, the Chemi-Northern method provides a versatile, simple, and cost-effective method to enable researchers to analyze expression, processing, binding, and decay of RNAs.


Assuntos
Proteínas de Ligação a RNA , RNA , Humanos , Northern Blotting , RNA Mensageiro/metabolismo , RNA/química , Sondas de Oligonucleotídeos , Sequência de Bases , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Sondas de DNA
19.
Anal Chim Acta ; 1287: 342053, 2024 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-38182365

RESUMO

Rapid and specific detection of virulent bacterial strains is a great challenge for food safety regarding large amounts of contaminated samples. Herein, a dual-mode hydrogel array biosensor was constructed to simultaneously rapidly screen and precisely quantitatively detect virulent Escherichia coli O157:H7 (E. coli O157:H7) based on a novel DNA-modified phage probe. First, E. coli O157:H7 was incubated with alginate to form the E. coli O157:H7/hydrogel premix complex. Subsequently, hydrogel formation by cross-linking upon the addition of calcium ions and phages for E. coli O157:H7 modified with a DNA primer (phage-DNA) was added to the alginate hydrogel. The DNA on the complex could trigger rolling circle amplification (RCA) to form a phage probe containing a long-chain DNA skeleton (phage@RCA-DNA). The RCA-DNA was then hybridized with the complementary DNA (cDNA) to form double-stranded DNA fragments (phage@RCA-dsDNA), which could be stained by the SYBR Green dye to emit visual green fluorescence (FL) and determined by a smartphone for rapid screening. Meanwhile, the unreacted cDNA in the supernatant could be quantitatively detected by microfluidic chip electrophoresis (MCE). The signal decrement was also proportional to the bacterial concentration. The detection limit values of E. coli O157:H7 were 50 CFU mL-1 by the FL signal and 6 CFU mL-1 by the MCE signal. The two results could be mutually corrected to decrease the false-positive results. This assay was also employed to detect virulent Salmonella Typhimurium (S. Typhimurium) using the corresponding S. Typhimurium phage@RCA-DNA probe. All these results demonstrated that the universal bioassay was suitable for simultaneous rapid screening and precisely quantitative detection of virulent bacterial strains.


Assuntos
Bacteriófagos , Escherichia coli O157 , DNA Complementar , Hidrogéis , Microfluídica , Sondas de DNA , Alginatos , Corantes , Eletroforese
20.
Anal Chim Acta ; 1289: 342187, 2024 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-38245202

RESUMO

The abnormal expression levels of miRNAs have been proven to be highly related to the generation of various diseases and are also closely associated with the stages and types of disease development. The novel RNA aptamers-based homogenous fluorescent methods were simple, with low background signal and high signal-to-noise ratio, but lacked effective signal amplification technology to achieve sensitive detection of trace miRNA markers. There is an urgent need for combining effective nucleic acid amplification technology with RNA aptamer to achieve highly sensitive and accurate detection of miRNA. For this purpose, a new DNA multi-arm nanostructure-based dual rolling circle transcription machinery for the generation of lighting-up MG RNA aptamers is constructed for label-free and highly sensitive sensing of miRNA-21. In this system, the target miRNA-21 induces a structural transformation of the DNA multi-arm nanostructure probe to recycle miRNA-21 and trigger two independent rolling circle transcription reactions to generate two long RNAs, which can partially hybridize with each other to generate large amounts of complete MG RNA aptamers. These RNA aptamers can associate with organic MG dye to produce significantly enhanced fluorescence signals to accomplish ultrasensitive miRNA-21 detection down to 0.9 fM. In addition, this method exhibits high selectivity to distinguish miRNA-21 even with single nucleotide mismatch, and also has potential application capability to monitor different expression levels of miRNA-21 from different cancer cells. The effective collaboration between MG RNA aptamer and rolling circle transcription reaction makes this fluorescent method show the significant advantages of low background signal, high signal-to-noise ratio and high detection sensitivity. It has great potential to be a promising means to achieve label-free and highly sensitive monitoring of other trace biological markers via a simple change of target sequence.


Assuntos
Aptâmeros de Nucleotídeos , MicroRNAs , Neoplasias , MicroRNAs/genética , Aptâmeros de Nucleotídeos/genética , Aptâmeros de Nucleotídeos/química , Limite de Detecção , DNA/química , Sondas de DNA/química , Corantes Fluorescentes/química , Neoplasias/diagnóstico , Neoplasias/genética
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