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Scientific Reports Feb 2018Functional nanomaterials with fluorescent or quenching abilities are important for the development of molecular probes for detection and studies of nucleic acids. Here,...
Functional nanomaterials with fluorescent or quenching abilities are important for the development of molecular probes for detection and studies of nucleic acids. Here, we describe a new class of molecular nanoprobes, the NanoCeracQ that uses nanoceria particles as a nanoquencher of fluorescent oligonucleotides for rapid and sensitive detection of DNA sequences and hybridization events. We show that nanoceria forms stable and reversible bionanoconjugates with oligonucleotides and can specifically recognize and detect DNA sequences in a single step. In absence of the target DNA, the nanoprobe produced minimal background fluorescence due to the high quenching efficiency of nanoceria. Competitive binding of the target induced a concentration dependent increase in the fluorescence signal due to hybridization and release of the fluorescent tag from the nanoparticle surface. The nanoprobe enabled sensitive detection of the complementary strand with a detection limit of 0.12 nM, using a single step procedure. The results show that biofunctionalized nanoceria can be used as a universal nanoquencher and nanosensing platform for fluorescent DNA detection and studies of nucleic acid interactions. This approach can find broad applications in molecular diagnostics, sensor development, gene expression profiling, imaging and forensic analysis.
Topics: Base Pairing; Base Sequence; Binding, Competitive; Biological Assay; Biosensing Techniques; Cerium; DNA; DNA Probes; Fluorescent Dyes; Limit of Detection; Nanostructures; Nucleic Acid Hybridization; Oligonucleotides; Spectrometry, Fluorescence
PubMed: 29402996
DOI: 10.1038/s41598-018-20659-9 -
Nucleic Acids Research Sep 2017Quantitative measurement of mRNA levels in single cells is necessary to understand phenotypic variability within an otherwise isogenic population of cells....
Quantitative measurement of mRNA levels in single cells is necessary to understand phenotypic variability within an otherwise isogenic population of cells. Single-molecule mRNA Fluorescence In Situ Hybridization (FISH) has been established as the standard method for this purpose, but current protocols require a long region of mRNA to be targeted by multiple DNA probes. Here, we introduce a new single-probe FISH protocol termed sFISH for budding yeast, Saccharomyces cerevisiae using a single DNA probe labeled with a single fluorophore. In sFISH, we markedly improved probe specificity and signal-to-background ratio by using methanol fixation and inclined laser illumination. We show that sFISH reports mRNA changes that correspond to protein levels and gene copy number. Using this new FISH protocol, we can detect >50% of the total target mRNA. We also demonstrate the versatility of sFISH using FRET detection and mRNA isoform profiling as examples. Our FISH protocol with single-fluorophore sensitivity significantly reduces cost and time compared to the conventional FISH protocols and opens up new opportunities to investigate small changes in RNA at the single cell level.
Topics: Carbocyanines; DNA Probes; Fluorescent Dyes; Gene Expression Regulation, Fungal; In Situ Hybridization, Fluorescence; RNA, Messenger; Saccharomyces cerevisiae; Sensitivity and Specificity; Single Molecule Imaging
PubMed: 28666354
DOI: 10.1093/nar/gkx568 -
Nucleic Acids Research Sep 2016A new class of modified oligonucleotides (combination probes) has been designed and synthesised for use in genetic analysis and RNA detection. Their chemical structure...
A new class of modified oligonucleotides (combination probes) has been designed and synthesised for use in genetic analysis and RNA detection. Their chemical structure combines an intercalating anchor with a reporter fluorophore on the same thymine nucleobase. The intercalator (thiazole orange or benzothiazole orange) provides an anchor, which upon hybridisation of the probe to its target becomes fluorescent and simultaneously stabilizes the duplex. The anchor is able to communicate via FRET to a proximal reporter dye (e.g. ROX, HEX, ATTO647N, FAM) whose fluorescence signal can be monitored on a range of analytical devices. Direct excitation of the reporter dye provides an alternative signalling mechanism. In both signalling modes, fluorescence in the unhybridised probe is switched off by collisional quenching between adjacent intercalator and reporter dyes. Single nucleotide polymorphisms in DNA and RNA targets are identified by differences in the duplex melting temperature, and the use of short hybridization probes, made possible by the stabilisation provided by the intercalator, enhances mismatch discrimination. Unlike other fluorogenic probe systems, placing the fluorophore and quencher on the same nucleobase facilitates the design of short probes containing multiple modifications. The ability to detect both DNA and RNA sequences suggests applications in cellular imaging and diagnostics.
Topics: Base Pair Mismatch; Base Sequence; Benzothiazoles; DNA; DNA Probes; DNA, Complementary; Fluorescent Dyes; Genes, Reporter; Intercalating Agents; Methylation; Mutation; Nucleic Acid Heteroduplexes; Nucleic Acid Hybridization; Oligonucleotides; Quinolines; RNA; Spectrometry, Fluorescence
PubMed: 27369379
DOI: 10.1093/nar/gkw579 -
Bioinformatics (Oxford, England) Feb 2019Recombinase polymerase amplification (RPA), an isothermal nucleic acid amplification method, is enhancing our ability to detect a diverse array of pathogens, thereby...
SUMMARY
Recombinase polymerase amplification (RPA), an isothermal nucleic acid amplification method, is enhancing our ability to detect a diverse array of pathogens, thereby assisting the diagnosis of infectious diseases and the detection of microorganisms in food and water. However, new bioinformatics tools are needed to automate and improve the design of the primers and probes sets to be used in RPA, particularly to account for the high genetic diversity of circulating pathogens and cross detection of genetically similar organisms. PrimedRPA is a python-based package that automates the creation and filtering of RPA primers and probe sets. It aligns several sequences to identify conserved targets, and filters regions that cross react with possible background organisms.
AVAILABILITY AND IMPLEMENTATION
PrimedRPA was implemented in Python 3 and supported on Linux and MacOS and is freely available from http://pathogenseq.lshtm.ac.uk/PrimedRPA.html.
SUPPLEMENTARY INFORMATION
Supplementary data are available at Bioinformatics online.
Topics: Computational Biology; DNA Primers; Nucleic Acid Amplification Techniques; Recombinases; Software
PubMed: 30101342
DOI: 10.1093/bioinformatics/bty701 -
Accounts of Chemical Research Jun 2019Protein-DNA interactions are important in replication, transcription, repair, as well as epigenetic modifications of DNA, which involve methylation and demethylation of...
Protein-DNA interactions are important in replication, transcription, repair, as well as epigenetic modifications of DNA, which involve methylation and demethylation of DNA resulting in regulation of gene expression. Understanding of these processes and chemical tools for studying and perhaps even modulating them could be of great relevance and importance not only in chemical biology but also in real diagnostics and treatment of diseases. In the past decade, we have been working on development of synthesis of base-modified 2'-deoxyribo- or ribonucleoside triphosphates (dNTPs or NTPs) and their use in enzymatic synthesis of modified nucleic acids using DNA or RNA polymerases. These synthetic and enzymatic methods are briefly summarized with focus on recent development and outlining of scope, limitations, and further challenges. The main focus of this Account is on applications of base-modified nucleic acids in sensing of protein-DNA interactions, in covalent cross-linking to DNA-binding proteins ,and in modulation of protein-DNA binding and transcription. Several environment-sensitive fluorescent nucleotides were incorporated to DNA probes which responded to protein binding by light-up, changing of color, or lifetime of fluorescence. Using a cyclodextrin-peptide transporter, fluorescent nucleotides can be transported through the cell membrane and incorporated to genomic DNA. Several dNTPs bearing reactive groups (i.e., vinylsulfonamide or chloroacetamide) were used for polymerase synthesis of DNA reactive probes which cross-link to Cys, His, or Lys in peptides or proteins. An attractive challenge is to use DNA modifications and bioorthogonal reactions in the major groove of DNA for modulation and switching of protein-DNA interactions. We have systematically explored the influence of major-groove modifications on recognition and cleavage of DNA by restriction endonucleases and constructed simple chemical switches of DNA cleavage. Systematic study of the influence of major-groove modifications on transcription with bacterial RNA polymerases revealed not only that some modified bases are tolerated, but also that the presence of 5-hydroxymethyluracil or -cytosine can even enhance the transcription (350 or 250% compared to native DNA). Based on these results, we have constructed the first chemical switch of transcription based on photocaging of hydroxymethylpyrimidines in DNA by 2-nitrobenzyl protection (transcription off), photochemical deprotection of the DNA (transcription on), and enzymatic phosphorylation (only for 5-hydroxymethyluracil, transcription off). Although it has been so far demonstrated only in vitro, it is the proof-of-principle first step toward chemical epigenetics.
Topics: DNA; DNA Probes; DNA-Binding Proteins; DNA-Directed DNA Polymerase; DNA-Directed RNA Polymerases; Light; Protein Binding; Ribonucleotides; Transcription, Genetic
PubMed: 31181911
DOI: 10.1021/acs.accounts.9b00195 -
Scientific Reports Oct 2020Mycobacterium tuberculosis (Mtb) is an insidious scourge that has afflicted millions of people worldwide. Although there are many rapid methods to detect it based on...
Ultrasensitive detection of Mycobacterium tuberculosis by a rapid and specific probe-triggered one-step, simultaneous DNA hybridization and isothermal amplification combined with a lateral flow dipstick.
Mycobacterium tuberculosis (Mtb) is an insidious scourge that has afflicted millions of people worldwide. Although there are many rapid methods to detect it based on loop-mediated isothermal amplification (LAMP) and a lateral flow dipstick (LFD), this study made further improvements using a new set of primers to enhance LAMP performance and a novel DNA probe system to simplify detection and increase specificity. The new probe system eliminates the post-LAMP hybridization step typically required for LFD assays by allowing co-hybridization and amplification of target DNA in one reaction while preventing self-polymerization that could lead to false-positive results. The improved assay was named Probe-Triggered, One-Step, Simultaneous DNA Hybridization and LAMP Integrated with LFD (SH-LAMP-LFD). SH-LAMP-LFD was simpler to perform and more sensitive than previously reported LAMP-LFD and PCR methods by 100 and 1000 times, respectively. It could detect a single cell of Mtb. The absence of cross-reactivity with 23 non-TB bacteria, and accurate test results with all 104 blind clinical samples have highlighted its accuracy. Its robustness and portability make SH-LAMP-LFD suitable for users in both low and high resource settings.
Topics: DNA Probes; DNA, Bacterial; Humans; Molecular Diagnostic Techniques; Mycobacterium tuberculosis; Nucleic Acid Amplification Techniques; Nucleic Acid Hybridization; Polymerase Chain Reaction; Sensitivity and Specificity; Tuberculosis
PubMed: 33046776
DOI: 10.1038/s41598-020-73981-6 -
ACS Applied Materials & Interfaces Jul 2023Cells sense and respond to the physical properties of their environment through receptor-mediated signaling, a process known as mechanotransduction, which can modulate...
Cells sense and respond to the physical properties of their environment through receptor-mediated signaling, a process known as mechanotransduction, which can modulate critical cellular functions such as proliferation, differentiation, and survival. At the molecular level, cell adhesion receptors, such as integrins, transmit piconewton (pN)-scale forces to the extracellular matrix, and the magnitude of the force plays a critical role in cell signaling. The most sensitive approach to measuring integrin forces involves DNA hairpin-based sensors, which are used to quantify and map forces in living cells. Despite the broad use of DNA hairpin sensors to study a variety of mechanotransduction processes, these sensors are typically anchored to rigid glass slides, which are orders of magnitude stiffer than the extracellular matrix and hence modulate native biological responses. Here, we have developed nuclease-resistant DNA hairpin probes that are all covalently tethered to PEG hydrogels to image cell traction forces on physiologically relevant substrate stiffness. Using HeLa cells as a model cell line, we show that the molecular forces transmitted by integrins are highly sensitive to the bulk modulus of the substrate, and cells cultured on the 6 and 13 kPa gels produced a greater number of hairpin unfolding events compared to the 2 kPa substrates. Tension signals are spatially colocalized with pY118-paxillin, confirming focal adhesion-mediated probe opening. Additionally, we found that integrin forces are greater than 5.8 pN but less than 19 pN on 13 kPa gels. This work provides a general strategy to integrate molecular tension probes into hydrogels, which can better mimic in vivo mechanotransduction.
Topics: Humans; Mechanotransduction, Cellular; Hydrogels; HeLa Cells; Traction; DNA Probes; Cell Adhesion; DNA; Integrins; Receptors, Cell Surface
PubMed: 37409737
DOI: 10.1021/acsami.3c04826 -
Nature Communications Sep 2017Analysis of the spatial arrangement of molecular features enables the engineering of synthetic nanostructures and the understanding of natural ones. The ability to...
Analysis of the spatial arrangement of molecular features enables the engineering of synthetic nanostructures and the understanding of natural ones. The ability to acquire a comprehensive set of pairwise proximities between components would satisfy an increasing interest in investigating individual macromolecules and their interactions, but current biochemical techniques detect only a single proximity partner per probe. Here, we present a biochemical DNA nanoscopy method that records nanostructure features in situ and in detail for later readout. Based on a conceptually novel auto-cycling proximity recording (APR) mechanism, it continuously and repeatedly produces proximity records of any nearby pairs of DNA-barcoded probes, at physiological temperature, without altering the probes themselves. We demonstrate the production of dozens of records per probe, decode the spatial arrangements of 7 unique probes in a homogeneous sample, and repeatedly sample the same probes in different states.The spatial organisation of nanostructures is fundamental to their function. Here, the authors develop a non-destructive, proximity-based method to record extensive spatial organization information in DNA molecules for later readout.
Topics: Base Sequence; DNA; DNA Probes; Models, Molecular; Nanostructures; Nanotechnology; Nucleic Acid Conformation; Streptavidin; Thermodynamics
PubMed: 28947733
DOI: 10.1038/s41467-017-00542-3 -
Sensors (Basel, Switzerland) Dec 2022The present paper describes an alternative approach to the traditionally used covalent immobilization methods that require cost-intensive and complicated chemistry...
Electrochemical DNA Biosensor Based on Immobilization of a Non-Modified ssDNA Using Phosphoramidate-Bonding Strategy and Pencil Graphite Electrode Modified with AuNPs/CB and Self-Assembled Cysteamine Monolayer.
The present paper describes an alternative approach to the traditionally used covalent immobilization methods that require cost-intensive and complicated chemistry modification of a single-stranded DNA (ssDNA) capture probe. The low-cost pencil graphite electrode (PGE) modified with carbon black (CB) and gold nanoparticles (AuNPs) was used as an electrochemical platform and the non-modified ssDNA was immobilized on a self-assembled cysteamine modified AuNPs/CB-PGE through a phosphoramidate bond between the 5'-terminal phosphate group of ssDNA and the primary amine group of cysteamine. The microRNA-21 was used as a target model in the fabrication of this electrochemical DNA biosensor and the hybridization process with the complementary probe was monitored by differential pulse voltammetry using methylene blue (MB) as an electrochemical hybridization indicator. The decreased reduction peak current of MB shows a good linear correlation with the increased concentration of microRNA-21 target sequences because the MB signal is determined by the amount of exposed guanine bases. The linear range of the fabricated DNA biosensor was from 1.0 × 10 to 5.0 × 10 M with a detection limit of 1.0 × 10 M. These results show that the covalent immobilization of a non-modified ssDNA capture probe through a phosphoramidate-bonding strategy could serve as a cost-effective and versatile approach for the fabrication of DNA biosensors related to a wide range of applications that cover the fields of medical diagnostic and environmental monitoring. The fabricated electrochemical DNA biosensor was used to analyze microRNA-21 in a (spiked) human serum sample and it showed satisfactory and encouraging results as an electrochemical DNA biosensor platform.
Topics: Humans; Gold; Graphite; DNA, Single-Stranded; Soot; DNA Probes; Cysteamine; Metal Nanoparticles; Biosensing Techniques; Electrodes; DNA; Methylene Blue; MicroRNAs; Electrochemical Techniques
PubMed: 36502122
DOI: 10.3390/s22239420 -
RNA (New York, N.Y.) Jun 2023Although RNA plays a vital role in gene expression, it is less used as an in situ biomarker for clinical diagnostics than DNA and protein. This is mainly due to...
Although RNA plays a vital role in gene expression, it is less used as an in situ biomarker for clinical diagnostics than DNA and protein. This is mainly due to technical challenges caused by the low expression level and easy degradation of RNA molecules. To tackle this issue, methods that are sensitive and specific are needed. Here, we present an RNA single-molecule chromogenic in situ hybridization assay based on DNA probe proximity ligation and rolling circle amplification. When the DNA probes hybridize into close proximity to the RNA molecules, they form a V-shape structure and mediate the circularization of circle probes. Thus, our method was termed vsmCISH. We successfully applied our method to assess mRNA expression status in invasive breast cancer tissue and investigated the utility of albumin mRNA ISH for differentiating primary from metastatic liver cancer. The promising results on clinical samples indicate that our method has great potential for application in diagnosing diseases using RNA biomarkers.
Topics: RNA; In Situ Hybridization; RNA, Messenger; DNA; DNA Probes
PubMed: 36813533
DOI: 10.1261/rna.079482.122