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Science (New York, N.Y.) Aug 2016Single-cell RNA sequencing (RNA-Seq) provides rich information about cell types and states. However, it is difficult to capture rare dynamic processes, such as adult...
Single-cell RNA sequencing (RNA-Seq) provides rich information about cell types and states. However, it is difficult to capture rare dynamic processes, such as adult neurogenesis, because isolation of rare neurons from adult tissue is challenging and markers for each phase are limited. Here, we develop Div-Seq, which combines scalable single-nucleus RNA-Seq (sNuc-Seq) with pulse labeling of proliferating cells by 5-ethynyl-2'-deoxyuridine (EdU) to profile individual dividing cells. sNuc-Seq and Div-Seq can sensitively identify closely related hippocampal cell types and track transcriptional dynamics of newborn neurons within the adult hippocampal neurogenic niche, respectively. We also apply Div-Seq to identify and profile rare newborn neurons in the adult spinal cord, a noncanonical neurogenic region. sNuc-Seq and Div-Seq open the way for unbiased analysis of diverse complex tissues.
Topics: Animals; Cell Division; Cell Nucleus; Deoxyuridine; Hippocampus; Isotope Labeling; Mice; Neurogenesis; Neurons; Sequence Analysis, RNA; Single-Cell Analysis; Spinal Cord; Transcription, Genetic; Transcriptome
PubMed: 27471252
DOI: 10.1126/science.aad7038 -
Proceedings of the National Academy of... Feb 2008We have developed a method to detect DNA synthesis in proliferating cells, based on the incorporation of 5-ethynyl-2'-deoxyuridine (EdU) and its subsequent detection by...
We have developed a method to detect DNA synthesis in proliferating cells, based on the incorporation of 5-ethynyl-2'-deoxyuridine (EdU) and its subsequent detection by a fluorescent azide through a Cu(I)-catalyzed [3 + 2] cycloaddition reaction ("click" chemistry). Detection of the EdU label is highly sensitive and can be accomplished in minutes. The small size of the fluorescent azides used for detection results in a high degree of specimen penetration, allowing the staining of whole-mount preparations of large tissue and organ explants. In contrast to BrdU, the method does not require sample fixation or DNA denaturation and permits good structural preservation. We demonstrate the use of the method in cultured cells and in the intestine and brain of whole animals.
Topics: Animals; DNA; DNA Replication; Deoxyuridine; Mice; Molecular Structure; NIH 3T3 Cells; Reproducibility of Results; Sensitivity and Specificity; Time Factors
PubMed: 18272492
DOI: 10.1073/pnas.0712168105 -
Archives of Biochemistry and Biophysics Oct 2022Alzheimer's disease (AD) is a progressive disease with a long duration and complicated pathogenesis. Thymidine (Thy) and 2'-deoxyuridine (2'-De) are pyrimidines...
Alzheimer's disease (AD) is a progressive disease with a long duration and complicated pathogenesis. Thymidine (Thy) and 2'-deoxyuridine (2'-De) are pyrimidines nucleotides that are associated with nervous system diseases. However, it remains unclear whether Thy and 2'-De exert neuroprotective effects in AD. Therefore, this study was conducted to explore the interventional effects and mechanisms of Thy and 2'-De on the Aβ-induced brain injury. Donepezil (Do, 10 mg/kg/d), Thy (20 mg/kg/d), and 2'-De (20 mg/kg/d) were administered for 4 weeks after the injection of Aβ peptides (200 μM, i.c.v.) to mice. UPLC-MS/MS method was performed to quantify Thy and 2'-De in the hippocampus of mice brain. The cognition ability, neuronal and mitochondria damage, and levels of Aβ/Aβ, p-Tau, Na K-ATPase, apoptosis, oxidative stress, immune cells, and Iba 1 were measured in Aβ-induced mice. The oxygen consumption (OCR) and extracellular acidification rate (ECAR) were measured using a seahorse analyzer in Aβ-induced N9 cells. Moreover, 2-Deoxy-D-glucose (2-DG), a glycolysis inhibitor, was added to explore the mechanisms underlying the effects of Thy and 2'-De on Aβ-induced N9 cells. The expression of Iba 1 and levels of CD11b and reactive oxygen species (ROS) were measured after treatment with Thy (5 μM) and 2'-De (10 μM) against 2-DG (5 mM) in Aβ-induced N9 cells. The results suggested that Do, Thy, and 2'-De improved the cognition ability, attenuated the damage to hippocampus and mitochondria, downregulated the levels of Aβ/Aβ, p-Tau, Na K-ATPase, apoptosis, oxidative stress, and Iba 1, and regulated the immune response induced by Aβ against the brain injury. Furthermore, Do, Thy, and 2'-De increased ATP production and inhibited glycolysis in Aβ-induced N9 cells. Moreover, 2-DG enhanced the effects of drugs, reduced microglial activation, and attenuated oxidative stress to interfere with Aβ-induced N9 cells. In conclusion, Thy and 2'-De reduced microglial activation and improved oxidative stress damage by modulating glycolytic metabolism on the Aβ-induced brain injury.
Topics: Adenosine Triphosphatases; Adenosine Triphosphate; Alzheimer Disease; Amyloid beta-Peptides; Animals; Apoptosis; Brain Injuries; Chromatography, Liquid; Deoxyglucose; Deoxyuridine; Donepezil; Glycolysis; Mice; Microglia; Neuroprotective Agents; Nucleotides; Oxidative Stress; Peptide Fragments; Pyrimidines; Reactive Oxygen Species; Tandem Mass Spectrometry; Thymidine
PubMed: 35998686
DOI: 10.1016/j.abb.2022.109377 -
Molecules (Basel, Switzerland) Sep 2011Replicating cells undergo DNA synthesis in the highly regulated, S-phase of the cell cycle. Analogues of the pyrimidine deoxynucleoside thymidine may be inserted into... (Review)
Review
Replicating cells undergo DNA synthesis in the highly regulated, S-phase of the cell cycle. Analogues of the pyrimidine deoxynucleoside thymidine may be inserted into replicating DNA, effectively tagging dividing cells allowing their characterisation. Tritiated thymidine, targeted using autoradiography was technically demanding and superseded by 5-bromo-2-deoxyuridine (BrdU) and related halogenated analogues, detected using antibodies. Their detection required the denaturation of DNA, often constraining the outcome of investigations. Despite these limitations BrdU alone has been used to target newly synthesised DNA in over 20,000 reviewed biomedical studies. A recent breakthrough in "tagging DNA synthesis" is the thymidine analogue 5-ethynyl-2'-deoxyuridine (EdU). The alkyne group in EdU is readily detected using a fluorescent azide probe and copper catalysis using 'Huisgen's reaction' (1,3-dipolar cycloaddition or 'click chemistry'). This rapid, two-step biolabelling approach allows the tagging and imaging of DNA within cells whilst preserving the structural and molecular integrity of the cells. The bio-orthogonal detection of EdU allows its application in more experimental assays than previously possible with other "unnatural bases". These include physiological, anatomical and molecular biological experimentation in multiple fields including, stem cell research, cancer biology, and parasitology. The full potential of EdU and related molecules in biomedical research remains to be explored.
Topics: Animals; Bromodeoxyuridine; Cell Proliferation; Cells, Cultured; Click Chemistry; DNA; Deoxyuridine; Fluorescent Dyes; Humans; Staining and Labeling; Stem Cell Niche; Thymidine
PubMed: 21921870
DOI: 10.3390/molecules16097980 -
The Biochemical Journal Jul 1986
Review
Topics: Binding Sites; DNA, Single-Stranded; Deoxyuridine; Genetic Vectors; Mutation; Nucleic Acid Hybridization; Oligonucleotides; Templates, Genetic
PubMed: 3541892
DOI: 10.1042/bj2370001 -
Chemphyschem : a European Journal of... Aug 2011Fluorescent nucleoside analogs, commonly used to explore nucleic acid dynamics, recognition and damage, frequently respond to a single environmental parameter. Herein we...
Fluorescent nucleoside analogs, commonly used to explore nucleic acid dynamics, recognition and damage, frequently respond to a single environmental parameter. Herein we address the development of chromophores that can simultaneously probe more than one environmental factor while having each associated with a unique spectroscopic signature. We demonstrate that an isomorphic emissive pyridine-modified 2-deoxy-uridine 1, containing multiple sensory elements, responds to changes in acidity, viscosity, and polarity. Protonation of the pyridine moiety (pK(a) 4.4) leads to enhanced emission (λ(em) =388 nm) and red-shifted absorption spectra (λ(abs) =319 nm), suggesting the formation of an intramolecular hydrogen bond with the neighboring pyrimidine carbonyl. This "locked" conformation can also be mimicked by increasing solvent viscosity, resulting in a stark enhancement of emission quantum yield. Finally, increasing solvent polarity substantially impacts the chromophore's Stokes shift [from 5.8×10(3) cm(-1) at E(T) (30)=36.4 kcal mol(-1) to 9.3 ×10(3) cm(-1) at E(T) (30)=63.1 kcal mol(-1)]. The opposite effect is seen for the impact of solvent polarity of the protonated form. The characteristic photophysical signature induced by each parameter facilitates the exploration of these environmental factors both individually and simultaneously.
Topics: Absorption; Chemistry Techniques, Analytical; DNA; Deoxyuridine; Fluorescent Dyes; Hydrogen Bonding; Hydrogen-Ion Concentration; Models, Molecular; Protons; Pyridines; Spectrometry, Fluorescence; Viscosity
PubMed: 21698743
DOI: 10.1002/cphc.201100315 -
Angewandte Chemie (International Ed. in... Mar 2022Nucleosidic diarylethenes (DAEs) are an emerging class of photochromes but have rarely been used in materials science. Here, we have developed doubly methylated DAEs...
Nucleosidic diarylethenes (DAEs) are an emerging class of photochromes but have rarely been used in materials science. Here, we have developed doubly methylated DAEs derived from 2'-deoxyuridine with high thermal stability and fatigue resistance. These new photoswitches not only outperform their predecessors but also rival classical non-nucleosidic DAEs. To demonstrate the utility of these new DAEs, we have designed an all-optical excitonic switch consisting of two oligonucleotides: one strand containing a fluorogenic double-methylated 2'-deoxyuridine as a fluorescence donor and the other a tricyclic cytidine (tC) as acceptor, which together form a highly efficient conditional Förster-Resonance-Energy-Transfer (FRET) pair. The system was operated in liquid and solid phases and showed both strong distance- and orientation-dependent photochromic FRET. The superior ON/OFF contrast was maintained over up to 100 switching cycles, with no detectable fatigue.
Topics: DNA; Deoxyuridine; Fluorescence Resonance Energy Transfer; Nucleosides; Oligonucleotides
PubMed: 35076154
DOI: 10.1002/anie.202117735 -
Chemistry (Weinheim An Der Bergstrasse,... Feb 2022Anomeric base pairs in heterochiral DNA with strands in the α-d and β-d configurations and homochiral DNA with both strands in α-d configuration were functionalized....
Anomeric DNA: Functionalization of α-d Anomers of 7-Deaza-2'-deoxyadenosine and 2'-Deoxyuridine with Clickable Side Chains and Click Adducts in Homochiral and Heterochiral Double Helices.
Anomeric base pairs in heterochiral DNA with strands in the α-d and β-d configurations and homochiral DNA with both strands in α-d configuration were functionalized. The α-d anomers of 2'-deoxyuridine and 7-deaza-2'-deoxyadenosine were synthesized and functionalized with clickable octadiynyl side chains. Nucleosides were protected and converted to phosphoramidites. Solid-phase synthesis furnished 12-mer oligonucleotides, which were hybridized. Pyrene click adducts display fluorescence, a few of them with excimer emission. T values and thermodynamic data revealed the following order of duplex stability α/α-d≫β/β-d≥α/β-d. CD spectra disclosed that conformational changes occur during hybridization. Functionalized DNAs were modeled and energy minimized. Clickable side chains and bulky click adducts are well accommodated in the grooves of anomeric DNA. The investigation shows for the first time that anomeric DNAs can be functionalized in the same way as canonical DNA for potential applications in nucleic acid chemistry, chemical biology, and DNA material science.
Topics: Base Pairing; DNA; Deoxyuridine; Tubercidin
PubMed: 34878201
DOI: 10.1002/chem.202103872 -
International Journal of Molecular... Sep 2020BrdU (bromodeoxyuridine) and EdU (ethynyldeoxyuridine) have been largely utilized as the means of monitoring DNA replication and cellular division. Although BrdU induces...
BrdU (bromodeoxyuridine) and EdU (ethynyldeoxyuridine) have been largely utilized as the means of monitoring DNA replication and cellular division. Although BrdU induces gene and chromosomal mutations and induces sensitization to photons, EdU's effects have not been extensively studied yet. Therefore, we investigated EdU's potential cytotoxic and mutagenic effects and its related underlying mechanisms when administered to Chinese hamster ovary (CHO) wild type and DNA repair-deficient cells. EdU treatment displayed a higher cytotoxicity and genotoxicity than BrdU treatment. Cells with defective homologous recombination repair displayed a greater growth delay and severe inhibition of clonogenicity with EdU compared to wild type and other DNA repair-deficient cells. Inductions of sister chromatid exchange and hypoxanthine phosphorybosyl transferase (HPRT) mutation were observed in EdU-incorporated cells as well. Interestingly, on the other hand, EdU did not induce sensitization to photons to the same degree as BrdU. Our results demonstrate that elevated concentrations (similar to manufacturers suggested concentration; >5-10 μM) of EdU treatment were toxic to the cell cultures, particularly in cells with a defect in homologous recombination repair. Therefore, EdU should be administered with additional precautions.
Topics: A549 Cells; Animals; Bromodeoxyuridine; CHO Cells; Cricetulus; DNA Repair; Deoxyuridine; Genes, BRCA2; Humans; Mutagenicity Tests
PubMed: 32927807
DOI: 10.3390/ijms21186631 -
Open Biology Jan 20165-Ethynyl-2'-deoxyuridine (EdU) and 5-ethynyl-2'-deoxycytidine (EdC) are mainly used as markers of cellular replicational activity. Although EdU is employed as a...
5-Ethynyl-2'-deoxyuridine (EdU) and 5-ethynyl-2'-deoxycytidine (EdC) are mainly used as markers of cellular replicational activity. Although EdU is employed as a replicational marker more frequently than EdC, its cytotoxicity is commonly much higher than the toxicity of EdC. To reveal the reason of the lower cytotoxicity of EdC, we performed a DNA analysis of five EdC-treated human cell lines. Surprisingly, not a single one of the tested cell lines contained a detectable amount of EdC in their DNA. Instead, the DNA of all the cell lines contained EdU. The content of incorporated EdU differed in particular cells and EdC-related cytotoxicity was directly proportional to the content of EdU. The results of experiments with the targeted inhibition of the cytidine deaminase (CDD) and dCMP deaminase activities indicated that the dominant role in the conversion pathway of EdC to EdUTP is played by CDD in HeLa cells. Our results also showed that the deamination itself was not able to effectively prevent the conversion of EdC to EdCTP, the conversion of EdC to EdCTP occurs with much lesser effectivity than the conversion of EdU to EdUTP and the EdCTP is not effectively recognized by the replication complex as a substrate for the synthesis of nuclear DNA.
Topics: Antibodies; Bromodeoxyuridine; Cell Death; Cell Line, Tumor; Cell Nucleus; Cytidine Deaminase; DNA; DNA Replication; Deoxycytidine; Deoxyuridine; Humans; Metabolome; RNA, Small Interfering
PubMed: 26740587
DOI: 10.1098/rsob.150172