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Methods in Molecular Biology (Clifton,... 2022Sensitive quantification of RNA transcripts via fluorescence in situ hybridization (FISH) is a ubiquitous part of understanding quantitative gene expression in single...
Sensitive quantification of RNA transcripts via fluorescence in situ hybridization (FISH) is a ubiquitous part of understanding quantitative gene expression in single cells. Many techniques exist to identify and localize transcripts inside the cell, but often they are costly and labor intensive. Here we present a method to use a singly labeled short DNA oligo probe to perform FISH in yeast cells. This method is effective for highly constrained FISH applications where the target length is limited (<200 nucleotides). This method can quantify different RNA isoforms or enable the use of fluorescence resonance energy transfer (FRET) to detect co-transcription of neighboring sequence blocks. Since this method relies on a single probe, it is also more cost-effective than a multiple probe labeling strategy.
Topics: DNA Probes; Fluorescence Resonance Energy Transfer; In Situ Hybridization, Fluorescence; RNA
PubMed: 34718992
DOI: 10.1007/978-1-0716-1585-0_5 -
Bioorganic & Medicinal Chemistry Letters Nov 2021Micro RNAs (miRNAs) are involved in a variety of biological functions and are attracting attention as diagnostic and prognostic markers for various diseases. Highly...
Micro RNAs (miRNAs) are involved in a variety of biological functions and are attracting attention as diagnostic and prognostic markers for various diseases. Highly sensitive RNA detection methods are required to determine miRNA expression levels and intracellular localization. In this study, we designed new double-stranded peptide nucleic acid (PNA)/DNA probes consisting of a fluorophore-PNA-quencher (fPq) and a quencher-DNA (qD) for miR-221 detection. We optimized the fPq structure, PNA-DNA hybrid length, and hybrid position. The resultant fPq-2/qD-6b probe was a 6-bp hybrid probe with a 10-base fPq and a 6-base qD. The signal-to-background ratios of the probes showed that fPq-2/qD-6b had a higher target sensitivity than fPq (PNA beacon)-type and fP/qD-type probes. The results of the detection limit and target specificity indicate that the fPq/qD probe is promising for RNA detection in both cells and cell extracts as well as for miRNA diagnosis.
Topics: DNA Probes; Fluorescent Dyes; Humans; MicroRNAs; Peptide Nucleic Acids
PubMed: 34534675
DOI: 10.1016/j.bmcl.2021.128359 -
Methods in Molecular Biology (Clifton,... 2022High-throughput DNA fluorescence in situ hybridization (hiFISH) combines multicolor combinatorial DNA FISH staining with automated image acquisition and analysis to...
High-throughput DNA fluorescence in situ hybridization (hiFISH) combines multicolor combinatorial DNA FISH staining with automated image acquisition and analysis to visualize and localize tens to hundreds of genomic loci in up to millions of cells. hiFISH can be used to measure physical distances between pairs of genomic loci, radial distances from genomic loci to the nuclear edge or center, and distances between genomic loci and nuclear structures defined by protein or RNA markers. The resulting large datasets of 3D spatial distances can be used to study cellular heterogeneity in genome architecture and the molecular mechanisms underlying this phenomenon in a variety of cellular systems. In this chapter we provide detailed protocols for hiFISH to measure distances between genomic loci, including all steps involved in DNA FISH probe design and preparation, cell culture, DNA FISH staining in 384-well imaging plates, automated image acquisition and analysis, and, finally, statistical analysis.
Topics: Cell Nucleus; DNA; DNA Probes; Genome; In Situ Hybridization, Fluorescence
PubMed: 35867253
DOI: 10.1007/978-1-0716-2497-5_12 -
Angewandte Chemie (International Ed. in... Sep 2021Threading intercalators bind DNA with high affinities. Here, we describe single-molecule studies on a cell-permeant luminescent dinuclear ruthenium(II) complex that has...
Threading intercalators bind DNA with high affinities. Here, we describe single-molecule studies on a cell-permeant luminescent dinuclear ruthenium(II) complex that has been previously shown to thread only into short, unstable duplex structures. Using optical tweezers and confocal microscopy, we show that this complex threads and locks into force-extended duplex DNA in a two-step mechanism. Detailed kinetic studies reveal that an individual stereoisomer of the complex exhibits the highest binding affinity reported for such a mono-intercalator. This stereoisomer better preserves the biophysical properties of DNA than the widely used SYTOX Orange. Interestingly, threading into torsionally constrained DNA decreases dramatically, but is rescued on negatively supercoiled DNA. Given the "light-switch" properties of this complex on binding DNA, it can be readily used as a long-lived luminescent label for duplex or negatively supercoiled DNA through a unique "load-and-lock" protocol.
Topics: Coordination Complexes; DNA; DNA Probes; Molecular Structure; Ruthenium
PubMed: 34378843
DOI: 10.1002/anie.202108077 -
Chemistry (Weinheim An Der Bergstrasse,... Aug 2023Accurate cancer diagnosis especially early diagnosis is of great importance for prompt therapy and elevated survival rate. mRNAs are widely used as biomarkers for cancer...
Accurate cancer diagnosis especially early diagnosis is of great importance for prompt therapy and elevated survival rate. mRNAs are widely used as biomarkers for cancer identification and treatment. mRNA expression levels are highly associated with cancer stage and malignant progression. Nevertheless, single type mRNA detection is insufficient and unreliable. Herein, we developed a DNA nano-windmill probe for in situ multiplexed mRNAs detection and imaging in this paper. The probe is designed to simultaneously target four types of mRNA through wind blades. Importantly, recognition of targets is independent from each other, which further facilitate cell type discrimination. The probe can specifically distinguish cancer cell lines from normal cells. In addition, it can identify changes in mRNA expression levels in living cells. The current strategy enriches the toolbox for improving the accuracy of cancer diagnosis and therapeutic solutions.
Topics: RNA, Messenger; DNA Probes; Cell Line, Tumor; DNA
PubMed: 37314386
DOI: 10.1002/chem.202301300 -
Analytical Chemistry Nov 2022The investigation on electrochemiluminescence (ECL) multiplexing bioassays mainly focuses on simultaneously detecting either proteins or nucleic acids. To overcome the...
The investigation on electrochemiluminescence (ECL) multiplexing bioassays mainly focuses on simultaneously detecting either proteins or nucleic acids. To overcome the limitation of a short waveband for spectrum-resolved ECL multiplexing bioassays, herein, a highly monochromatic (FWHM <40 nm) and bandgap-engineered ECL luminophore, that is, mercaptopropionic acid-capped and Zn-mediated aggregation-induced emission (AIE) assembly of Au nanocrystals (NCs) (Zn-AIE-AuNCs), of strong emission and the maximum emission wavelength at 485 nm is developed. The highly monochromatic and bandgap-engineered ECL (485 nm) of Zn-AIE-AuNCs can multiplex with the single-waveband and surface-defect-involved ECL (775 nm) of dual-stabilizer-capped CuInS@ZnS NCs (CIS@ZnS-NCs), enabling a spectrum-resolved ECL multiplexing strategy with different NCs luminophores of a similar particle size as tags. This ECL multiplexing strategy can be utilized to simultaneously detect antigen and DNA probe together without any additional signal amplification procedure and obvious spectroscopic cross-talk, in which the highly monochromatic ECL from Zn-AIE-AuNCs is utilized to dynamically determine human carcinoembryonic antigen from 1 pg/mL to 50 ng/mL with a limit of detection (LOD) of 0.3 pg/mL, while the single-waveband ECL from CIS@ZnS-NCs is employed to linearly detect wild-type p53 from 1 pM to 50 nM with a LOD of 0.5 pM. The ECL immunoassay of the proposed strategy is free from the interference of the synchronously conducted DNA probe assay and vice versa, which would open an avenue to couple the immunoassay and DNA probe assay together for clinical colon and breast cancer identification.
Topics: Humans; Luminescent Measurements; Electrochemical Techniques; Immunoassay; Limit of Detection; DNA Probes; Biological Assay; Biosensing Techniques
PubMed: 36334096
DOI: 10.1021/acs.analchem.2c03579 -
Journal of Visualized Experiments : JoVE Feb 2022Current single-cell epigenome analyses are designed for single use. The cell is discarded after a single use, preventing analysis of multiple epigenetic marks in a...
Current single-cell epigenome analyses are designed for single use. The cell is discarded after a single use, preventing analysis of multiple epigenetic marks in a single cell and requiring data from other cells to distinguish signal from experimental background noise in a single cell. This paper describes a method to reuse the same single cell for iterative epigenomic analyses. In this experimental method, cellular proteins are first anchored to a polyacrylamide polymer instead of crosslinking them to protein and DNA, alleviating structural bias. This critical step allows repeated experiments with the same single cell. Next, a random primer with a scaffold sequence for proximity ligation is annealed to the genomic DNA, and the genomic sequence is added to the primer by extension using a DNA polymerase. Subsequently, an antibody against an epigenetic marker and control IgG, each labeled with different DNA probes, are bound to the respective targets in the same single cell. Proximity ligation is induced between the random primer and the antibody by adding a connector DNA with complementary sequences to the scaffold sequence of the random primer and the antibody-DNA probe. This approach integrates antibody information and nearby genome sequences in a single DNA product of proximity ligation. By enabling repeated experiments with the same single cell, this method allows an increase in data density from a rare cell and statistical analysis using only IgG and antibody data from the same cell. The reusable single cells prepared by this method can be stored for at least a few months and reused later to broaden epigenetic characterization and increase data density. This method provides flexibility to researchers and their projects.
Topics: DNA; DNA Probes; DNA-Directed DNA Polymerase; Epigenome; Epigenomics
PubMed: 35225292
DOI: 10.3791/63456 -
ACS Synthetic Biology Jul 2020DNA is now well-established as a nanoscale building material with applications in fields such as biosensing and molecular computation. Molecular processes such as logic... (Review)
Review
DNA is now well-established as a nanoscale building material with applications in fields such as biosensing and molecular computation. Molecular processes such as logic gates, nucleic acid circuits, and multiplexed detection have used different readout strategies to measure the output signal. In biosensing, this output can be the diagnosis of a disease biomarker, whereas in molecular computation, the output can be the result of a mathematical operation carried out using DNA. Recent developments have shown that the output of such processes can be displayed graphically as a macroscopic symbol or an alphanumeric character on multiwell plates, microarray chips, gels, lateral flow devices, and DNA origami surfaces. This review discusses the concepts behind such graphical readouts of molecular events, available display platforms, and the advantages and challenges in adapting such methods for practical use. Graphical display systems have the potential to be used in the creation of intelligent computing and sensing devices by which nanoscale binding events are translated into macroscopic visual readouts.
Topics: Biosensing Techniques; DNA; DNA Probes; Logic; Nanostructures; Nanotechnology; Nucleic Acid Hybridization; Signal Processing, Computer-Assisted
PubMed: 32584557
DOI: 10.1021/acssynbio.0c00246 -
The Journal of Membrane Biology Dec 2020Continuous, dynamic, and controlled membrane remodeling creates flow of information and materials across membranes to sustain life in all biological systems. Multiple... (Review)
Review
Continuous, dynamic, and controlled membrane remodeling creates flow of information and materials across membranes to sustain life in all biological systems. Multiple nanoscale phenomena of membranes regulate mesoscale processes in cells, which in turn control macro-scale processes in living organisms. Understanding the molecular mechanisms that cells use for membrane homeostasis, i.e., to generate, maintain, and deform the membrane structures has therefore been the mammoth's task in biology. Using the principles of DNA nanotechnology, researchers can now precisely recapitulate the functional interactions of the biomolecules that can now probe, program, and re-program membrane remodeling and associated phenomena. The molecular mechanisms for membrane dynamics developing in vitro conditions in which the membrane modulating components are precisely organized and modulated by DNA nanoscaffolds are adding new chapters in the field of DNA nanotechnology. In this review, we discuss DNA nanodevices-based membrane remodeling and trafficking machineries and their applications in biological systems.
Topics: Cell Membrane; DNA; DNA Probes; Molecular Dynamics Simulation; Nanostructures; Nanotechnology
PubMed: 33200237
DOI: 10.1007/s00232-020-00154-x -
Analytical Methods : Advancing Methods... Dec 2022Herein, a new allosteric DNA switch-mediated catalytic DNA circuit reaction strategy has been proposed for ratiometric and sensitive nucleic acid detection. The sensing...
Herein, a new allosteric DNA switch-mediated catalytic DNA circuit reaction strategy has been proposed for ratiometric and sensitive nucleic acid detection. The sensing system was based on two DNA hybrid probes, each of which was constructed by annealing a reconfigurable DNA hairpin with single-stranded DNA. Upon target recognition by the first DNA hybrid probe, a reconfigurable DNA switch was liberated, triggering a toehold-mediated strand displacement reaction (TSDR) with the second DNA hybrid probe, which was accompanied by the release of another reconfigurable DNA switch. This released allosteric DNA switch could further interact with the first hybrid DNA probe the TSDR strategy to form a reciprocal strand displacement network between the two DNA hybrid probes. Theoretically, this reciprocal strand displacement reaction would continue till the complete consumption of the reaction substrates. Thus, it provides a new signal amplification method leading toward target recognition. More interestingly, it creates a ratiometric signal response mode for target recognition, which involves the fluorescence increment of one fluorophore (Cy5) and concurrent decrement of another fluorophore (Cy3) accompanied by the target-triggered reciprocal strand displacement reaction. This process could achieve a low detection limit of about 0.1 pM toward the target nucleic acid and selective discrimination toward different mismatched targets. It could also be applied for detection in a serum sample. Thus, the developed catalytic DNA circuit reaction strategy together with ratiometric signal readout provides a new avenue for programmable, reliable and sensitive detection of nucleic acids and might also pave the way for developing more advanced DNA circuits or biosensors.
Topics: DNA, Catalytic; Nucleic Acid Hybridization; Limit of Detection; DNA; DNA Probes
PubMed: 36504112
DOI: 10.1039/d2ay01751b