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Analytica Chimica Acta May 2024MicroRNAs (miRNAs) are potential biomarkers for cancer diagnosis and prognosis, methods for detecting miRNAs with high sensitivity, selectivity, and stability are...
MicroRNAs (miRNAs) are potential biomarkers for cancer diagnosis and prognosis, methods for detecting miRNAs with high sensitivity, selectivity, and stability are urgently needed. Various nucleic acid probes that have traditionally been for this purpose suffer several drawbacks, including inefficient signal-to-noise ratios and intensities, high cost, and time-consuming method establishment. Computing tools used for investigating the thermodynamics of DNA hybridization reactions can accurately predict the secondary structure of DNA and the interactions between DNA molecules. Herein, NUPACK was used to design a series of nucleic acid probes and develop a phosphorothioated-terminal hairpin formation and self-priming extension (PS-THSP) signal amplification strategy, which enabled the ultrasensitive detection of miR-200a in serum samples. The free and binding energies of the DNA detection probes calculated using NUPACK, as well as the biological experimental results, were considered synthetically to select the best sequence and experimental conditions. A unified dynamic programming framework, NUPACK analysis and the experimental data, were complementary and improved the designed model in all respects. Our study demonstrates the feasibility of using computer technology such as NUPACK to simplify the experimental process and provide intuitive results.
Topics: DNA Probes; MicroRNAs; Nucleic Acids; Signal-To-Noise Ratio; Thermodynamics
PubMed: 38609269
DOI: 10.1016/j.aca.2024.342530 -
Andrology Sep 2023The sperm retrieval rate of microdissection testicular sperm extraction varies from 25% to 60%. Therefore, it is necessary to establish objective selection criteria for...
BACKGROUND
The sperm retrieval rate of microdissection testicular sperm extraction varies from 25% to 60%. Therefore, it is necessary to establish objective selection criteria for identifying seminiferous tubules with spermatozoa.
OBJECTIVES
Our aim was to develop a method for identifying spermatogenesis without sectioning testicular tissues.
MATERIALS AND METHODS
Testicular tissues of 10-week-old normal rats were fixed with 4% paraformaldehyde. Fluorescent labeling of seminiferous tubule nuclei and F-actin was performed, and the specimens were observed without sectioning using a multiphoton microscope. Cryptorchid rats were used as a model lacking elongated spermatids. Multiphoton images were compared with images of normal seminiferous tubules. In addition, seminiferous tubules of 10-week-old normal rats were labeled by testicular interstitial injection of fluorescent probes and observed by a multiphoton microscope without fixation. Terminal deoxynucleotidyl transferase dUTP nick end labeling-stained images of normal and probe-injected testes were compared.
RESULTS
In fixed seminiferous tubules, elongated spermatids were identified. In addition, F-actin of apical ectoplasmic specialization was observed around elongated spermatids. Furthermore, spermatogenic stages were identified by an array of nuclei or F-actin. In cryptorchid testes, elongated spermatids and F-actin of the apical ectoplasmic specialization were not observed. In testes injected with fluorescent probes, F-actin of the apical ectoplasmic specialization was observed, and spermatogenic stages were identified without fixation. There was no significant difference in the number of terminal deoxynucleotidyl transferase dUTP nick end labeling-positive cells per seminiferous tubule between normal and probe-injected testes.
CONCLUSIONS
Seminiferous epithelium could be observed without sectioning of tissues by fluorescent probes and a multiphoton microscope. Active spermatogenesis was observed by labeling F-actin with and without fixation. Moreover, the toxicity of fluorescent probes was limited. Our method has a potential for live imaging of testicular tissue.
Topics: Male; Rats; Animals; Actins; DNA Nucleotidylexotransferase; Fluorescent Dyes; Semen; Spermatogenesis; Testis; Spermatids; Seminiferous Tubules
PubMed: 36597184
DOI: 10.1111/andr.13379 -
PloS One 2023During development of a novel detection method for the UDP-glucuronosyl transferase 1A1 (UGT1A1)*28, the fluorescence intensity of a dye conjugated to cytosine (C) at...
During development of a novel detection method for the UDP-glucuronosyl transferase 1A1 (UGT1A1)*28, the fluorescence intensity of a dye conjugated to cytosine (C) at the end of a DNA strand decreased upon hybridization with guanine (G). This phenomenon is referred to as photoinduced electron transfer (PeT). Using this phenomenon, we devised a method for the naked-eye detection of UGT1A1*28 (thymine-adenine (TA)-repeat polymorphism). Fluorescently labeled single-stranded DNA (ssDNA) oligonucleotides (probes) were designed and hybridized with complementary strand DNAs (target DNAs). Base pair formation at the blunt end between fluorescently labeled C (probe side) and G (target side), induced dramatic fluorescence quenching. Additionally, when the labeled-CG pair formed near the TA-repeat sequence, different TA-repeat numbers were discriminated. However, obtaining enough target DNA for this probe by typical polymerase chain reaction (PCR) was difficult. To enable the practical use of the probe, producing sufficient target DNA remains problematic.
Topics: Electrons; DNA; Nucleic Acid Hybridization; DNA, Complementary; DNA, Single-Stranded; Microsatellite Repeats
PubMed: 37535593
DOI: 10.1371/journal.pone.0289506 -
Nature Nanotechnology Dec 2023There is an unmet need to develop low-cost, rapid and highly multiplexed diagnostic technology platforms for quantitatively detecting blood biomarkers to advance...
There is an unmet need to develop low-cost, rapid and highly multiplexed diagnostic technology platforms for quantitatively detecting blood biomarkers to advance clinical diagnostics beyond the single biomarker model. Here we perform nanopore sequencing of DNA-barcoded molecular probes engineered to recognize a panel of analytes. This allows for highly multiplexed and simultaneous quantitative detection of at least 40 targets, such as microRNAs, proteins and neurotransmitters, on the basis of the translocation dynamics of each probe as it passes through a nanopore. Our workflow is built around a commercially available MinION sequencing device, offering a one-hour turnaround time from sample preparation to results. We also demonstrate that the strategy can directly detect cardiovascular disease-associated microRNA from human serum without extraction or amplification. Due to the modularity of barcoded probes, the number and type of targets detected can be significantly expanded.
Topics: Humans; MicroRNAs; Nanopore Sequencing; DNA; DNA Probes; Sequence Analysis, DNA; Biomarkers; Nanopores; High-Throughput Nucleotide Sequencing
PubMed: 37749222
DOI: 10.1038/s41565-023-01479-z -
Analytica Chimica Acta May 2024Size selectivity is crucial in highly accurate preparation of biosensors. Herein, we described an innovative electrochemiluminescence (ECL) sensing platform based on the...
Size selectivity is crucial in highly accurate preparation of biosensors. Herein, we described an innovative electrochemiluminescence (ECL) sensing platform based on the confined DNA tetrahedral molecular sieve (DTMS) for size-selective recognition of nucleic acids and small biological molecule. Firstly, DNA template (T) was encapsulated into the inner cavity of DNA tetrahedral scaffold (DTS) and hybridized with quencher (Fc) labeled probe DNA to prepare DTMS, accordingly inducing Ru(bpy) and Fc closely proximate, resulting the sensor in a "signal-off" state. Afterwards, target molecules entered the cavity of DTMS to realize the size-selective molecular recognition while prohibiting large molecules outside of the DTMS, resulting the sensor in a "signal-on" state due to the release of Fc. The rigid framework structure of DTS and the anchor of DNA probe inside the DTS effectively avoided the nuclease degradation of DNA probe, and nonspecific protein adsorption, making the sensor possess potential application prospect for size-selective molecular recognition in diagnostic analysis with high accuracy and specificity.
Topics: Luminescent Measurements; Photometry; Biosensing Techniques; DNA; DNA Probes; Electrochemical Techniques
PubMed: 38637057
DOI: 10.1016/j.aca.2024.342561 -
Biosensors Dec 2023CRISPR/Cas12a is a potent biosensing tool known for its high specificity in DNA analysis. Cas12a recognizes the target DNA and acquires nuclease activity toward...
CRISPR/Cas12a is a potent biosensing tool known for its high specificity in DNA analysis. Cas12a recognizes the target DNA and acquires nuclease activity toward single-stranded DNA (ssDNA) probes. We present a straightforward and versatile approach to transforming common Cas12a-cleavable DNA probes into enhancing tools for fluorescence anisotropy (FA) measurements. Our study involved investigating 13 ssDNA probes with linear and hairpin structures, each featuring fluorescein at one end and a rotation-slowing tool (anchor) at the other. All anchors induced FA changes compared to fluorescein, ranging from 24 to 110 mr. Significant FA increases (up to 180 mr) were obtained by adding divalent metal salts (Mg, Ca, Ba), which influenced the rigidity and compactness of the DNA probes. The specific Cas12a-based recognition of double-stranded DNA (dsDNA) fragments of the bacterial phytopathogen allowed us to determine the optimal set (probe structure, anchor, concentration of divalent ion) for FA-based detection. The best sensitivity was obtained using a hairpin structure with dC10 in the loop and streptavidin located near the fluorescein at the stem in the presence of 100 mM Mg. The detection limit of the dsDNA target was equal to 0.8 pM, which was eight times more sensitive compared to the common fluorescence-based method. The enhancing set ensured detection of single cells of per reaction in an analysis based on CRISPR/Cas12a with recombinase polymerase amplification. Our approach is universal and easy to implement. Combining FA with Cas12a offers enhanced sensitivity and signal reliability and could be applied to different DNA and RNA analytes.
Topics: Salts; CRISPR-Cas Systems; Reproducibility of Results; DNA; DNA, Single-Stranded; Fluorescein; Biosensing Techniques
PubMed: 38131794
DOI: 10.3390/bios13121034 -
Viruses Aug 2023The story of the discovery of hepatitis E originated in the late 1970s with my extreme belief that there was a hidden saga in the relationship between jaundice and... (Review)
Review
The story of the discovery of hepatitis E originated in the late 1970s with my extreme belief that there was a hidden saga in the relationship between jaundice and pregnancy in developing countries and the opportunity for a massive epidemic of viral hepatitis, which hit the Gulmarg Kashmir region in November 1978. Based on data collected from a door-to-door survey, the existence of a new disease, epidemic non-A, non-B hepatitis, caused by a hitherto unknown hepatitis virus, was announced. This news was received by the world community with hype and skepticism. In the early 1980s, the world watched in awe as an extreme example of human self-experimentation led to the identification of VLP. In 1990, a cDNA clone from the virus responsible for epidemic non-A, non-B hepatitis was isolated. Over the years, we traversed three eras of ambiguity, hope, and hype of hepatitis E research and conducted several seminal studies to understand the biology of HEV and manifestations of hepatitis E. Many milestones have been reached on the long and winding road of hepatitis E research to understand the structure, biology, and diversity of the agent, changing the behavior of the pathogen in developed countries, and the discovery of a highly effective vaccine.
Topics: Female; Pregnancy; Humans; Hepatitis E; Global Health; Hepatitis C; DNA, Complementary; Epidemics
PubMed: 37632090
DOI: 10.3390/v15081745 -
Biosensors & Bioelectronics Oct 2023Following the recent pandemic and with the emergence of cell-free nucleic acids in liquid biopsies as promising biomarkers for a broad range of pathologies, there is an... (Review)
Review
Following the recent pandemic and with the emergence of cell-free nucleic acids in liquid biopsies as promising biomarkers for a broad range of pathologies, there is an increasing demand for a new generation of nucleic acid tests, with a particular focus on cost-effective, highly sensitive and specific biosensors. Easily miniaturized electrochemical sensors show the greatest promise and most typically rely on the chemical functionalization of conductive materials or electrodes with sequence-specific hybridization probes made of standard oligonucleotides (DNA or RNA) or synthetic analogues (e.g. Peptide Nucleic Acids or PNAs). The robustness of such sensors is mostly influenced by the ability to control the density and orientation of the probe at the surface of the electrode, making the chemistry used for this immobilization a key parameter. This exhaustive review will cover the various strategies to immobilize nucleic acid probes onto different solid electrode materials. Both physical and chemical immobilization techniques will be presented. Their applicability to specific electrode materials and surfaces will also be discussed as well as strategies for passivation of the electrode surface as a way of preventing electrode fouling and reducing nonspecific binding.
Topics: Nucleic Acids; Biosensing Techniques; DNA; Peptide Nucleic Acids; Oligonucleotides; Electrodes; Electrochemical Techniques; Nucleic Acid Hybridization
PubMed: 37406480
DOI: 10.1016/j.bios.2023.115440 -
Biosensors Sep 2023DNA ligases are essential enzymes involved in DNA replication and repair processes in all organisms. These enzymes seal DNA breaks by catalyzing the formation of...
DNA ligases are essential enzymes involved in DNA replication and repair processes in all organisms. These enzymes seal DNA breaks by catalyzing the formation of phosphodiester bonds between juxtaposed 5' phosphate and 3' hydroxyl termini in double-stranded DNA. In addition to their critical roles in maintaining genomic integrity, DNA ligases have been recently identified as diagnostic biomarkers for several types of cancers and recognized as potential drug targets for the treatment of various diseases. Although DNA ligases are significant in basic research and medical applications, developing strategies for efficiently detecting and precisely quantifying these crucial enzymes is still challenging. Here, we report our design and fabrication of a highly sensitive and specific biosensor in which a stable DNA hairpin is utilized to stimulate the generation of fluorescence signals. This probe is verified to be stable under a wide range of experimental conditions and exhibits promising performance in detecting DNA ligases. We anticipate that this hairpin-based biosensor will significantly benefit the development of new targeting strategies and diagnostic tools for certain diseases.
PubMed: 37754109
DOI: 10.3390/bios13090875 -
Chembiochem : a European Journal of... Apr 2024Non-enzymatic template-directed primer extension is increasingly being studied for the production of RNA and DNA. These reactions benefit from producing RNA or DNA in an... (Review)
Review
Non-enzymatic template-directed primer extension is increasingly being studied for the production of RNA and DNA. These reactions benefit from producing RNA or DNA in an aqueous, protecting group free system, without the need for expensive enzymes. However, these primer extension reactions suffer from a lack of fidelity, low reaction rates, low overall yields, and short primer extension lengths. This review outlines a detailed mechanistic pathway for non-enzymatic template-directed primer extension and presents a review of the thermodynamic driving forces involved in entropic templating. Through the lens of entropic templating, the rate and fidelity of a reaction are shown to be intrinsically linked to the reactivity of the activating agent used. Thus, a strategy is discussed for the optimization of non-enzymatic template-directed primer extension, providing a path towards cost-effective in vitro synthesis of RNA and DNA.
Topics: DNA Primers; Nucleic Acids; DNA; RNA; Thermodynamics; Templates, Genetic
PubMed: 38282207
DOI: 10.1002/cbic.202300859