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Angewandte Chemie (International Ed. in... Feb 2022The cell membrane is a dynamic and heterogeneous structure composed of distinct sub-compartments. Within these compartments, preferential interactions occur among...
The cell membrane is a dynamic and heterogeneous structure composed of distinct sub-compartments. Within these compartments, preferential interactions occur among various lipids and proteins. Currently, it is still challenging to image these short-lived membrane complexes, especially in living cells. In this work, we present a DNA-based probe, termed "DNA Zipper", which allows the membrane order and pattern of transient interactions to be imaged in living cells using standard fluorescence microscopes. By fine-tuning the length and binding affinity of DNA duplex, these probes can precisely extend the duration of membrane lipid interactions via dynamic DNA hybridization. The correlation between membrane order and the activation of T-cell receptor signaling has also been studied. These programmable DNA probes function after a brief cell incubation, which can be easily adapted to study lipid interactions and membrane order during different membrane signaling events.
Topics: Animals; Cell Membrane; DNA Probes; Dogs; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Madin Darby Canine Kidney Cells
PubMed: 34767659
DOI: 10.1002/anie.202112033 -
Sensors (Basel, Switzerland) Nov 2014The highly programmable positioning of molecules (biomolecules, nanoparticles, nanobeads, nanocomposites materials) on surfaces has potential applications in the fields... (Review)
Review
The highly programmable positioning of molecules (biomolecules, nanoparticles, nanobeads, nanocomposites materials) on surfaces has potential applications in the fields of biosensors, biomolecular electronics, and nanodevices. However, the conventional techniques including self-assembled monolayers fail to position the molecules on the nanometer scale to produce highly organized monolayers on the surface. The present article elaborates different techniques for the immobilization of the biomolecules on the surface to produce microarrays and their diagnostic applications. The advantages and the drawbacks of various methods are compared. This article also sheds light on the applications of the different technologies for the detection and discrimination of viral/bacterial genotypes and the detection of the biomarkers. A brief survey with 115 references covering the last 10 years on the biological applications of microarrays in various fields is also provided.
Topics: Adsorption; Biocompatible Materials; Biosensing Techniques; Coated Materials, Biocompatible; DNA Probes; Equipment Design; Equipment Failure Analysis; In Situ Hybridization; Oligonucleotide Array Sequence Analysis
PubMed: 25429408
DOI: 10.3390/s141222208 -
Nature Communications Aug 2019The selective amplification of DNA in the polymerase chain reaction is used to exponentially increase the signal in molecular diagnostics for nucleic acids, but there...
The selective amplification of DNA in the polymerase chain reaction is used to exponentially increase the signal in molecular diagnostics for nucleic acids, but there are no analogous techniques for signal enhancement in clinical tests for proteins or cells. Instead, the signal from affinity-based measurements of these biomolecules depends linearly on the probe concentration. Substituting antibody-based probes tagged for fluorescent quantification with lasing detection probes would create a new platform for biomarker quantification based on optical rather than enzymatic amplification. Here, we construct a virus laser which bridges synthetic biology and laser physics, and demonstrate virus-lasing probes for biosensing. Our virus-lasing probes display an unprecedented > 10,000 times increase in signal from only a 50% increase in probe concentration, using fluorimeter-compatible optics, and can detect biomolecules at sub-100 fmol mL concentrations.
Topics: Antibodies, Monoclonal; Bacteriophage M13; Biophysical Phenomena; Biosensing Techniques; DNA; DNA Probes; Electrons; Fluorescent Dyes; Humans; Lasers; Ligands; Models, Chemical; Nucleic Acids; Oligonucleotide Probes; Polymerase Chain Reaction; Viruses
PubMed: 31399594
DOI: 10.1038/s41467-019-11604-z -
Scientific Reports Oct 2021Fluorescently labeled antibody and aptamer probes are used in biological studies to characterize binding interactions, measure concentrations of analytes, and sort...
Fluorescently labeled antibody and aptamer probes are used in biological studies to characterize binding interactions, measure concentrations of analytes, and sort cells. Fluorescent nanoparticle labels offer an excellent alternative to standard fluorescent labeling strategies due to their enhanced brightness, stability and multivalency; however, challenges in functionalization and characterization have impeded their use. This work introduces a straightforward approach for preparation of fluorescent nanoparticle probes using commercially available reagents and common laboratory equipment. Fluorescent polystyrene nanoparticles, Thermo Fisher Scientific FluoSpheres, were used in these proof-of-principle studies. Particle passivation was achieved by covalent attachment of amine-PEG-azide to carboxylated particles, neutralizing the surface charge from - 43 to - 15 mV. A conjugation-annealing handle and DNA aptamer probe were attached to the azide-PEG nanoparticle surface either through reaction of pre-annealed handle and probe or through a stepwise reaction of the nanoparticles with the handle followed by aptamer annealing. Nanoparticles functionalized with DNA aptamers targeting histidine tags and VEGF protein had high affinity (ECs ranging from 3 to 12 nM) and specificity, and were more stable than conventional labels. This protocol for preparation of nanoparticle probes relies solely on commercially available reagents and common equipment, breaking down the barriers to use nanoparticles in biological experiments.
Topics: Amino Acid Sequence; Aptamers, Nucleotide; Base Sequence; Biosensing Techniques; DNA Probes; Fluorescent Dyes; Humans; Nanoparticles; Nanotechnology; Peptides; Polyethylene Glycols; Proteins; Quantum Dots; Staining and Labeling
PubMed: 34620912
DOI: 10.1038/s41598-021-99084-4 -
Micromachines Feb 2021The emergence of the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a zoonotic pathogen, has led to the outbreak of coronavirus disease 2019...
The emergence of the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a zoonotic pathogen, has led to the outbreak of coronavirus disease 2019 (COVID-19) pandemic and brought serious threats to public health worldwide. The gold standard method for SARS-CoV-2 detection requires both reverse transcription (RT) of the virus RNA to cDNA and then polymerase chain reaction (PCR) for the cDNA amplification, which involves multiple enzymes, multiple reactions and a complicated assay optimization process. Here, we developed a duplex-specific nuclease (DSN)-based signal amplification method for SARS-CoV-2 detection directly from the virus RNA utilizing two specific DNA probes. These specific DNA probes can hybridize to the target RNA at different locations in the nucleocapsid protein gene (N gene) of SARS-CoV-2 to form a DNA/RNA heteroduplex. DSN cleaves the DNA probe to release fluorescence, while leaving the RNA strand intact to be bound to another available probe molecule for further cleavage and fluorescent signal amplification. The optimized DSN amount, incubation temperature and incubation time were investigated in this work. Proof-of-principle SARS-CoV-2 detection was demonstrated with a detection sensitivity of 500 pM virus RNA. This simple, rapid, and direct RNA detection method is expected to provide a complementary method for the detection of viruses mutated at the PCR primer-binding regions for a more precise detection.
PubMed: 33672890
DOI: 10.3390/mi12020197 -
ACS Combinatorial Science Nov 2020Emulsions offer the means to miniaturize and parallelize high-throughput screening but require a robust method to localize activity-based fluorescent probes in each...
Emulsions offer the means to miniaturize and parallelize high-throughput screening but require a robust method to localize activity-based fluorescent probes in each droplet. Multiplexing probes in droplets is impractical, though highly desirable for identifying library members that possess very specific activity. Here, we present multiplexed probe immobilization on library beads for emulsion screening. During library bead preparation, we quantitated ∼10 primers per bead by fluorescence in situ hybridization, however emulsion PCR yielded only ∼10 gene copies per bead. We leveraged the unextended bead-bound primers to hybridize complementary probe-oligonucleotide heteroconjugates to the library beads. The probe-hybridized bead libraries were then used to program emulsion in vitro transcription/translation reactions and analyzed by FACS to perform multiplexed activity-based screening of trypsin and chymotrypsin mutant libraries for novel proteolytic specificity. The approach's modularity should permit a high degree of probe multiplexing and appears extensible to other enzyme classes and library types.
Topics: Chymotrypsin; Combinatorial Chemistry Techniques; DNA; DNA Primers; Emulsions; Enzyme Activation; Flow Cytometry; Fluorescent Dyes; Gene Library; High-Throughput Screening Assays; In Situ Hybridization, Fluorescence; Multiplex Polymerase Chain Reaction; Mutation; Surface Properties; Trypsin
PubMed: 32803953
DOI: 10.1021/acscombsci.0c00116 -
Clinical Epigenetics 2017The screening and diagnosis of colorectal cancer (CRC) currently relies heavily on invasive endoscopic techniques as well as imaging and antigen detection tools. More... (Review)
Review
The screening and diagnosis of colorectal cancer (CRC) currently relies heavily on invasive endoscopic techniques as well as imaging and antigen detection tools. More accessible and reliable biomarkers are necessary for early detection in order to expedite treatment and improve patient outcomes. Recent studies have indicated that levels of specific microRNA (miRNA) are altered in CRC; however, measuring miRNA in biological samples has proven difficult, given the complicated and lengthy PCR-based procedures used by most laboratories. In this manuscript, we examine the potential of miRNA as CRC biomarkers, summarize the methods that have commonly been employed to quantify miRNA, and focus on novel strategies that can improve or replace existing technology for feasible implementation in a clinical setting. These include isothermal amplification techniques that can potentially eliminate the need for specialized thermocycling equipment. Additionally, we propose the use of near-infrared (NIR) probes which can minimize autofluorescence and photobleaching and streamline quantification without tedious sample processing. We suggest that novel miRNA quantification tools will be necessary to encourage new discoveries and facilitate their translation to clinical practice.
Topics: Biomarkers, Tumor; Colorectal Neoplasms; DNA Methylation; DNA Probes; Early Detection of Cancer; Genetic Predisposition to Disease; High-Throughput Nucleotide Sequencing; Humans; MicroRNAs; Oligonucleotide Array Sequence Analysis; Sequence Analysis, DNA
PubMed: 29090038
DOI: 10.1186/s13148-017-0420-9 -
BMC Biotechnology Jul 2019Long Adapter Single-Stranded Oligonucleotide (LASSO) probes were developed as a novel tool for massively parallel cloning of kilobase-long genomic DNA sequences. LASSO...
BACKGROUND
Long Adapter Single-Stranded Oligonucleotide (LASSO) probes were developed as a novel tool for massively parallel cloning of kilobase-long genomic DNA sequences. LASSO dramatically improves the capture length limit of current DNA padlock probe technology from approximately 150 bps to several kbps. High-throughput LASSO capture involves the parallel assembly of thousands of probes. However, malformed probes are indiscernible from properly formed probes using gel electrophoretic techniques. Therefore, we used next-generation sequencing (NGS) to assess the efficiency of LASSO probe assembly and how it relates to the nature of DNA capture and amplification. Additionally, we introduce a simplified single target LASSO protocol using classic molecular biology techniques for qualitative and quantitative assessment of probe specificity.
RESULTS
A LASSO probe library targeting 3164 unique E. coli ORFs was assembled using two different probe assembly reaction conditions with a 40-fold difference in DNA concentration. Unique probe sequences are located within the first 50 bps of the 5' and 3' ends, therefore we used paired-end NGS to assess probe library quality. Properly mapped read pairs, representing correctly formed probes, accounted for 10.81 and 0.65% of total reads, corresponding to ~ 80% and ~ 20% coverage of the total probe library for the lower and higher DNA concentration conditions, respectively. Subsequently, we used single-end NGS to correlate probe assembly efficiency and capture quality. Significant enrichment of LASSO targets over non-targets was only observed for captures done using probes assembled with a lower DNA concentration. Additionally, semi-quantitative polyacrylamide gel electrophoresis revealed a ~ 10-fold signal-to-noise ratio of LASSO capture in a simplified system.
CONCLUSIONS
These results suggest that LASSO probe coverage for target sequences is more predictive of successful capture than probe assembly depth-enrichment. Concomitantly, these results demonstrate that DNA concentration at a critical step in the probe assembly reaction significantly impacts probe formation. Additionally, we show that a simplified LASSO capture protocol coupled to PAGE (polyacrylamide gel electrophoresis) is highly specific and more amenable to small-scale LASSO approaches, such as screening novel probes and templates.
Topics: Cloning, Molecular; DNA; DNA Primers; DNA Probes; DNA, Single-Stranded; Electrophoresis, Polyacrylamide Gel; Escherichia coli Proteins; Gene Amplification; Gene Library; High-Throughput Nucleotide Sequencing; Oligonucleotides; Open Reading Frames; Polymerase Chain Reaction; Reproducibility of Results
PubMed: 31340783
DOI: 10.1186/s12896-019-0547-1 -
Analytical Chemistry Dec 2022Reliable characterization of binding affinities is crucial for selected aptamers. However, the limited repertoire of universal approaches to obtain the dissociation...
Reliable characterization of binding affinities is crucial for selected aptamers. However, the limited repertoire of universal approaches to obtain the dissociation constant () values often hinders the further development of aptamer-based applications. Herein, we present a competitive hybridization-based strategy to characterize aptamers using DNA origami-based chiral plasmonic assemblies as optical reporters. We incorporated aptamers and partial complementary strands blocking different regions of the aptamers into the reporters and measured the kinetic behaviors of the target binding to gain insights on aptamers' functional domains. We introduced a reference analyte and developed a thermodynamic model to obtain a relative dissociation constant of an aptamer-target pair. With this approach, we characterized RNA and DNA aptamers binding to small molecules with low and high affinities.
Topics: Aptamers, Nucleotide; RNA; DNA; DNA Probes; Nucleic Acid Hybridization; SELEX Aptamer Technique
PubMed: 36480745
DOI: 10.1021/acs.analchem.2c04034 -
ACS Chemical Biology Dec 2019Hybridization probes have become an indispensable tool for nucleic acid analysis. Systematic efforts in probe optimization resulted in their improved binding affinity,...
Hybridization probes have become an indispensable tool for nucleic acid analysis. Systematic efforts in probe optimization resulted in their improved binding affinity, turn-on ratios, and ability to discriminate single nucleotide substitutions (SNSs). The use of split (or multicomponent) probes is a promising strategy to improve probe selectivity and enable an analysis of folded analytes. Here, we developed criteria for the rational design of a split G-quadruplex (G4) peroxidase-like deoxyribozyme (sPDz) probe that provides a visual output signal. The sPDz probe consists of two DNA strands that hybridize to the abutting positions of a DNA/RNA target and form a G4 structure catalyzing, in the presence of a hemin cofactor, HO-mediated oxidation of organic compounds into their colored oxidation products. We have demonstrated that probe design becomes complicated in the case of target sequences containing clusters (two or more) of cytosine residues and developed strategies to overcome the challenges to achieving high signal-to-noise and excellent SNS discrimination. Specifically, to improve selectivity, a conformational constraint that stabilizes the probe's dissociated state is beneficial. If the signal intensity is compromised, introduction of flexible non-nucleotide linkers between the G4-forming and target-recognizing elements of the probe helps to decrease the steric hindrance for G4 PDz formation observed as a signal increase. Varying the modes of G4 core splitting is another instrument for the optimal sPDz design. The suggested algorithm was successfully utilized for the design of the sPDz probe interrogating a fragment of the Influenza A virus genome (subtype H1N1), which can be of practical use for flu diagnostics and surveillance.
Topics: Algorithms; Cytosine; DNA Probes; G-Quadruplexes; Hemin; Hydrogen Peroxide; Nucleic Acid Conformation; Nucleic Acid Hybridization; Oxidation-Reduction; RNA Probes
PubMed: 31599573
DOI: 10.1021/acschembio.9b00634