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Biotechnology and Applied Biochemistry Aug 2022An oligonucleotide DNA probe has been developed for the application in the DNA electrochemical biosensor for the early diagnosis of coronavirus disease (COVID-19). Here,...
An oligonucleotide DNA probe has been developed for the application in the DNA electrochemical biosensor for the early diagnosis of coronavirus disease (COVID-19). Here, the virus microRNA from the N-gene of severe acute respiratory syndrome-2 (SARS-CoV-2) was used for the first time as a specific target for detecting the virus and became a framework for developing the complementary DNA probe. The sequence analysis of the virus microRNA was carried out using bioinformatics tools including basic local alignment search tools, multiple sequence alignment from CLUSTLW, microRNA database (miRbase), microRNA target database, and gene analysis. Cross-validation of distinct strains of coronavirus and human microRNA sequences was completed to validate the percentage of identical and consent regions. The percent identity parameter from the bioinformatics tools revealed the virus microRNAs' sequence has a 100% match with the genome of SARS-CoV-2 compared with other coronavirus strains, hence improving the selectivity of the complementary DNA probe. The 30 mer with 53.0% GC content of complementary DNA probe 5' GCC TGA GTT GAG TCA GCA CTG CTC ATG GAT 3' was designed and could be used as a bioreceptor for the biosensor development in the clinical and environmental diagnosis of COVID-19.
Topics: Biosensing Techniques; COVID-19; DNA Probes; DNA, Complementary; Genome, Viral; Humans; MicroRNAs; SARS-CoV-2
PubMed: 34378814
DOI: 10.1002/bab.2239 -
Scientific Reports Oct 2017The aggregation of gold nanoparticles (AuNPs) is known to induce an enhancement of localized surface plasmon resonance due to the coupling of plasmonic fields of...
The aggregation of gold nanoparticles (AuNPs) is known to induce an enhancement of localized surface plasmon resonance due to the coupling of plasmonic fields of adjacent nanoparticles. Here we show that AuNPs aggregation also causes a significant enhancement of chemiluminescence in the presence of luminophores. The phenomenon is used to introduce a rapid and sensitive DNA detection method that does not require amplification. DNA probes conjugated to AuNPs were used to detect a DNA target sequence specific to the fungus Ceratocystis fagacearum, causal agent of oak wilt. The hybridization of the DNA target with the DNA probes results in instantaneous aggregation of AuNPs into nanoballs, leading to a significant enhancement of luminol chemiluminescence. The enhancement reveals a linear correlation (R = 0.98) to the target DNA concentration, with a limit of detection down to 260 fM (260 × 10 M), two orders of magnitude higher than the performance obtained with plasmonic colorimetry and absorption spectrometry of single gold nanoparticles. Furthermore, the detection can be performed within 22 min using only a portable luminometer.
Topics: Ascomycota; Biosensing Techniques; Colorimetry; DNA Probes; DNA, Fungal; DNA, Single-Stranded; Gold; Luminescence; Luminescent Measurements; Luminol; Metal Nanoparticles; Nucleic Acid Hybridization; Polymerase Chain Reaction
PubMed: 29070890
DOI: 10.1038/s41598-017-14580-w -
Accounts of Chemical Research Dec 2022DNA repair enzymes continuously provide surveillance throughout our cells, protecting the enclosed DNA from the damage that is constantly arising from oxidation,...
DNA repair enzymes continuously provide surveillance throughout our cells, protecting the enclosed DNA from the damage that is constantly arising from oxidation, alkylating species, and radiation. Members of this enzyme class are intimately linked to pathways controlling cancer and inflammation and are promising targets for diagnostics and future therapies. Their study is benefiting widely from the development of new tools and methods aimed at measuring their activities. Here, we provide an Account of our laboratory's work on developing chemical tools to study DNA repair processes , as well as in cells and tissues, and what we have learned by applying them.We first outline early work probing how DNA repair enzymes recognize specific forms of damage by use of chemical analogs of the damage with altered shapes and H-bonding abilities. One outcome of this was the development of an unnatural DNA base that is incorporated selectively by polymerase enzymes opposite sites of missing bases (abasic sites) in DNA, a very common form of damage.We then describe strategies for design of fluorescent probes targeted to base excision repair (BER) enzymes; these were built from small synthetic DNAs incorporating fluorescent moieties to engender light-up signals as the enzymatic reaction proceeds. Examples of targets for these DNA probes include UDG, SMUG1, Fpg, OGG1, MutYH, ALKBH2, ALKBH3, MTH1, and NTH1. Several such strategies were successful and were applied both and in cellular settings; moreover, some were used to discover small-molecule modulators of specific repair enzymes. One of these is the compound SU0268, a potent OGG1 inhibitor that is under investigation in animal models for inhibiting hyperinflammatory responses.To investigate cellular nucleotide sanitation pathways, we designed a series of "two-headed" nucleotides containing a damaged DNA nucleotide at one end and ATP at the other; these were applied to studying the three human sanitation enzymes MTH1, dUTPase, and dITPase, some of which are therapeutic targets. The MTH1 probe (ARGO) was used in collaboration with oncologists to measure the enzyme in tumors as a disease marker and also to develop the first small-molecule activators of the enzyme.We proceed to discuss the development of a "universal" probe of base excision repair processes (UBER), which reacts covalently with abasic site intermediates of base excision repair. UBER probes light up in real time as the reaction occurs, enabling the observation of base excision repair as it occurs in live cells and tissues. UBER probes can also be used in efficient and simple methods for fluorescent labeling of DNA. Finally, we suggest interesting directions for the future of this field in biomedicine and human health.
Topics: Animals; Humans; DNA; DNA Damage; DNA Probes; DNA Repair; Nucleotides
PubMed: 36355579
DOI: 10.1021/acs.accounts.2c00608 -
ACS Sensors Apr 2022MicroRNAs (miRNAs) play a crucial role in regulating gene expression and have been linked to many diseases. Therefore, sensitive and accurate detection of disease-linked...
MicroRNAs (miRNAs) play a crucial role in regulating gene expression and have been linked to many diseases. Therefore, sensitive and accurate detection of disease-linked miRNAs is vital to the emerging revolution in early diagnosis of diseases. While the detection of miRNAs is a challenge due to their intrinsic properties such as small size, high sequence similarity among miRNAs and low abundance in biological fluids, the majority of miRNA-detection strategies involve either target/signal amplification or involve complex sensing designs. In this study, we have developed and tested a DNA-based fluorescence resonance energy transfer (FRET) sensor that enables ultrasensitive detection of a miRNA biomarker (miRNA-342-3p) expressed by triple-negative breast cancer (TNBC) cells. The sensor shows a relatively low FRET state in the absence of a target but it undergoes continuous FRET transitions between low- and high-FRET states in the presence of the target. The sensor is highly specific, has a detection limit down to low femtomolar (fM) without having to amplify the target, and has a large dynamic range (3 orders of magnitude) extending to 300 000 fM. Using this strategy, we demonstrated that the sensor allows detection of miRNA-342-3p in the miRNA-extracts from cancer cell lines and TNBC patient-derived xenografts. Given the simple-to-design hybridization-based detection, the sensing platform developed here can be used to detect a wide range of miRNAs enabling early diagnosis and screening of other genetic disorders.
Topics: DNA Probes; Fluorescence Resonance Energy Transfer; Humans; MicroRNAs; Nucleic Acid Hybridization; Triple Negative Breast Neoplasms
PubMed: 35312280
DOI: 10.1021/acssensors.1c02748 -
The Journal of Molecular Diagnostics :... Jan 2019Quantitative real-time PCR methods are commonly used to monitor BCR-ABL transcript levels in patients with chronic myelogenous leukemia. However, standard techniques...
Quantitative real-time PCR methods are commonly used to monitor BCR-ABL transcript levels in patients with chronic myelogenous leukemia. However, standard techniques involve separate measurements of target and reference DNAs, require standard curves, and are susceptible to PCR inhibition. An optimized duplex droplet digital PCR (ddPCR) should provide absolute quantification without the need for standard curves. The combination of high sensitivity and low background is particularly important for reliable monitoring of minimal residual disease. In this report, we describe primer probe set testing and step by step optimization of a duplex ddPCR for BCR-ABL/ABL. The optimization of ddPCR parameters increased ABL and BCR-ABL fluorescence signals by two- and fivefold, respectively, and enhanced the resolution between positive and negative drops. The optimized procedure generates a background false-positive rate of 5% of samples and reliably detects BCR-ABL/ABL down to 1/100,000 (CV <10%), with a single BCR-ABL copy being detected in 54% of reactions performed in duplicate from a molecular remission (MR) 5 sample (limit of detection = 1). Assay of duplicates resulted in a detection rate of 100% and 92% for MR4 and MR4.5, respectively. Detection of MR4.5 was increased to 100% by analyzing quadruplicates. Selection of an optimal primer and probe combination and stepwise optimization of the ddPCR conditions has yielded a robust low-background duplex ddPCR procedure for BCR-ABL/ABL.
Topics: Cell Line, Tumor; DNA Primers; DNA Probes; Fusion Proteins, bcr-abl; Gene Dosage; Humans; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Neoplasm, Residual; Polymerase Chain Reaction
PubMed: 30347270
DOI: 10.1016/j.jmoldx.2018.08.012 -
Sensors (Basel, Switzerland) Mar 2021The pH drop in the hindgut of the horse is caused by lactic acid-producing bacteria which are abundant when a horse's feeding regime is excessively carbohydrate rich....
The pH drop in the hindgut of the horse is caused by lactic acid-producing bacteria which are abundant when a horse's feeding regime is excessively carbohydrate rich. This drop in pH below six causes hindgut acidosis and may lead to laminitis. Lactic acid-producing bacteria and have been found to produce high amounts of L-lactate and D-lactate, respectively. Early detection of increased levels of these bacteria could allow the horse owner to tailor the horse's diet to avoid hindgut acidosis and subsequent laminitis. Therefore, 16s ribosomal ribonucleic acid (rRNA) sequences were identified and modified to obtain target single stranded deoxyribonucleic acid (DNA) from these bacteria. Complementary single stranded DNAs were designed from the modified target sequences to form capture probes. Binding between capture probe and target single stranded deoxyribonucleic acid (ssDNA) in solution has been studied by gel electrophoresis. Among pairs of different capture probes and target single stranded DNA, hybridization of capture probe 1 (SECP1) and target 1 (SET1) was portrayed as gel electrophoresis. Adsorptive stripping voltammetry was utilized to study the binding of thiol modified SECP1 over gold on glass substrates and these studies showed a consistent binding signal of thiol modified SECP1 and their hybridization with SET1 over the gold working electrode. Cyclic voltammetry and electrochemical impedance spectroscopy were employed to examine the binding of thiol modified SECP1 on the gold working electrode and hybridization of thiol modified SECP1 with the target single stranded DNA. Both demonstrated the gold working electrode surface was modified with a capture probe layer and hybridization of the thiol bound ssDNA probe with target DNA was indicated. Therefore, the proposed electrochemical biosensor has the potential to be used for the detection of the non-synthetic bacterial DNA target responsible for equine hindgut acidosis.
Topics: Acidosis; Animals; Biosensing Techniques; DNA; DNA Probes; Electrochemical Techniques; Electrodes; Firmicutes; Gold; Horses; Nucleic Acid Hybridization; Streptococcus bovis
PubMed: 33810389
DOI: 10.3390/s21072319 -
ACS Sensors Mar 2021Selective and sensitive detection of nucleic acid biomarkers is of great significance in early-stage diagnosis and targeted therapy. Therefore, the development of...
Selective and sensitive detection of nucleic acid biomarkers is of great significance in early-stage diagnosis and targeted therapy. Therefore, the development of diagnostic methods capable of detecting diseases at the molecular level in biological fluids is vital to the emerging revolution in the early diagnosis of diseases. However, the vast majority of the currently available ultrasensitive detection strategies involve either target/signal amplification or involve complex designs. Here, using a p53 tumor suppressor gene whose mutation has been implicated in more than 50% of human cancers, we show a background-free ultrasensitive detection of this gene on a simple platform. The sensor exhibits a relatively static mid-FRET state in the absence of a target that can be attributed to the time-averaged fluorescence intensity of fast transitions among multiple states, but it undergoes continuous dynamic switching between a low- and a high-FRET state in the presence of a target, allowing a high-confidence detection. In addition to its simple design, the sensor has a detection limit down to low femtomolar (fM) concentration without the need for target amplification. We also show that this sensor is highly effective in discriminating against single-nucleotide polymorphisms (SNPs). Given the generic hybridization-based detection platform, the sensing strategy developed here can be used to detect a wide range of nucleic acid sequences enabling early diagnosis of diseases and screening genetic disorders.
Topics: Base Sequence; DNA; DNA Probes; Fluorescence Resonance Energy Transfer; Humans; Nucleic Acid Hybridization
PubMed: 33720708
DOI: 10.1021/acssensors.1c00002 -
Molecules (Basel, Switzerland) Dec 2022Sensitive detection methods for T4 polynucleotide kinase/phosphatase (T4 PNKPP) are urgently required to obtain information on malignancy and thereby to provide better...
Sensitive detection methods for T4 polynucleotide kinase/phosphatase (T4 PNKPP) are urgently required to obtain information on malignancy and thereby to provide better guidance in PNKP-related diagnostics and drug screening. Although the CRISPR/Cas12a system shows great promise in DNA-based signal amplification protocols, its guide RNAs with small molecular weight often suffer nuclease degradation during storage and utilization, resulting in reduced activation efficiency. Herein, we proposed a self-supplying guide RNA-mediated CRISPR/Cas12a system for the sensitive detection of T4 PNKP in cancer cells, in which multiple copies of guide RNA were generated by in situ transcription. In this assay, T4 PNKP was chosen as a model, and a dsDNA probe with T7 promoter region and the transcription region of guide RNA were involved. Under the action of T4 PNKP, the 5'-hydroxyl group of the dsDNA probe was converted to a phosphate group, which can be recognized and digested by Lambda Exo, resulting in dsDNA hydrolysis. The transcription template was destroyed, which resulted in the failure to generate guide RNA by the transcription pathway. Therefore, the CRISPR/Cas12a system could not be activated to effectively cleavage the F-Q-reporter, and the fluorescence signal was turned off. In the absence of T4 PNKP, the 5'-hydroxyl group of the substrate DNA cannot be digested by Lambda Exo. The intact dsDNA acts as the transcription template to generate a large amount of guide RNA. Finally, the formed Cas12a/gRNA complex triggered the reverse cleavage of Cas12a on the F-Q-reporter, resulting in a "turn-on" fluorescence signal. This strategy displayed sharp sensitivity of T4 PNKP with the limit of detection (LOD) down to 0.0017 mU/mL, which was mainly due to the multiple regulation effect of transcription amplification. In our system, the dsDNA simultaneously serves as the T4 PNKP substrate, transcription template, and Lambda Exo substrate, avoiding the need for multiple probe designs and saving costs. By integrating the target recognition, Lambda Exo activity, and trans-cleavage activity of Cas12a, CRISPR/Cas12a catalyzed the cleavage of fluorescent-labeled short-stranded DNA probes and enabled synergetic signal amplification for sensitive T4 PNKP detection. Furthermore, the T4 PNKP in cancer cells has been evaluated as a powerful tool for biomedical research and clinical diagnosis, proving a good practical application capacity.
Topics: CRISPR-Cas Systems; DNA; Polynucleotide 5'-Hydroxyl-Kinase; DNA Probes; Fluorescent Dyes; RNA
PubMed: 36558152
DOI: 10.3390/molecules27249019 -
International Journal of Molecular... Jul 2021Oligonucleotides fluorescence in situ hybridization (Oligo-FISH) is an emerging technology and is an important tool in research areas such as detection of chromosome... (Review)
Review
Oligonucleotides fluorescence in situ hybridization (Oligo-FISH) is an emerging technology and is an important tool in research areas such as detection of chromosome variation, identification of allopolyploid, and deciphering of three-dimensional (3D) genome structures. Based on the demand for highly efficient oligo probes for oligo-FISH experiments, increasing numbers of tools have been developed for probe design in recent years. Obsolete oligonucleotide design tools have been adapted for oligo-FISH probe design because of their similar considerations. With the development of DNA sequencing and large-scale synthesis, novel tools have been designed to increase the specificity of designed oligo probes and enable genome-scale oligo probe design, which has greatly improved the application of single copy oligo-FISH. Despite this, few studies have introduced the development of the oligo-FISH probe design tools and their application in FISH experiments systematically. Besides, a comprehensive comparison and evaluation is lacking for the available tools. In this review, we provide an overview of the oligo-FISH probe design process, summarize the development and application of the available tools, evaluate several state-of-art tools, and eventually provide guidance for single copy oligo-FISH probe design.
Topics: Chromosomes, Plant; DNA Probes; Fluorescence; Genome; In Situ Hybridization, Fluorescence; Oligonucleotide Probes; Oligonucleotides; Repetitive Sequences, Nucleic Acid; Research Design; Sequence Analysis, DNA
PubMed: 34281175
DOI: 10.3390/ijms22137124 -
Organic & Biomolecular Chemistry Nov 2022Three probe chemistries are evaluated with respect to thermal denaturation temperatures, UV-Vis and fluorescence characteristics, recognition of complementary and...
Three probe chemistries are evaluated with respect to thermal denaturation temperatures, UV-Vis and fluorescence characteristics, recognition of complementary and mismatched DNA hairpin targets, and recognition of chromosomal DNA targets in the context of non-denaturing fluorescence hybridization (nd-FISH) experiments: (i) serine-γPNAs (SγPNAs), , single-stranded peptide nucleic acid (PNA) probes that are modified at the γ-position with ()-hydroxymethyl moieties, (ii) Invader probes, , DNA duplexes modified with +1 interstrand zippers of 2'--(pyren-1-yl)methyl-RNA monomers, a molecular arrangement that results in a violation of the neighbor exclusion principle, and (iii) double-stranded chimeric SγPNAs:Invader probes, , duplexes between complementary SγPNA and Invader strands, which are destabilized due to the poor compatibility between intercalators and PNA:DNA duplexes. Invader probes resulted in efficient, highly specific, albeit comparatively slow recognition of the model DNA hairpin targets. Recognition was equally efficient and faster with the single-stranded SγPNA probes but far less specific, whilst the double-stranded chimeric SγPNAs:Invader probes displayed recognition characteristics that were intermediate of the parent probes. All three probe chemistries demonstrated the capacity to target chromosomal DNA in nd-FISH experiments, with Invader probes resulting in the most favorable and consistent characteristics (signals in >90% of interphase nuclei against a low background and no signal in negative control experiments). These probe chemistries constitute valuable additions to the molecular toolbox needed for DNA-targeting applications.
Topics: Serine; In Situ Hybridization, Fluorescence; DNA; Peptide Nucleic Acids; RNA; DNA Probes
PubMed: 36285843
DOI: 10.1039/d2ob01567f