-
The Journal of Biological Chemistry Jan 2015Because of their high mutation rates, RNA viruses and retroviruses replicate close to the threshold of viability. Their existence as quasi-species has pioneered the...
Because of their high mutation rates, RNA viruses and retroviruses replicate close to the threshold of viability. Their existence as quasi-species has pioneered the concept of "lethal mutagenesis" that prompted us to synthesize pyrimidine nucleoside analogues with antiviral activity in cell culture consistent with an accumulation of deleterious mutations in the HIV-1 genome. However, testing all potentially mutagenic compounds in cell-based assays is tedious and costly. Here, we describe two simple in vitro biophysical/biochemical assays that allow prediction of the mutagenic potential of deoxyribonucleoside analogues. The first assay compares the thermal stabilities of matched and mismatched base pairs in DNA duplexes containing or not the nucleoside analogues as follows. A promising candidate should display a small destabilization of the matched base pair compared with the natural nucleoside and the smallest gap possible between the stabilities of the matched and mismatched base pairs. From this assay, we predicted that two of our compounds, 5-hydroxymethyl-2'-deoxyuridine and 5-hydroxymethyl-2'-deoxycytidine, should be mutagenic. The second in vitro reverse transcription assay assesses DNA synthesis opposite nucleoside analogues inserted into a template strand and subsequent extension of the newly synthesized base pairs. Once again, only 5-hydroxymethyl-2'-deoxyuridine and 5-hydroxymethyl-2'-deoxycytidine are predicted to be efficient mutagens. The predictive potential of our fast and easy first line screens was confirmed by detailed analysis of the mutation spectrum induced by the compounds in cell culture because only compounds 5-hydroxymethyl-2'-deoxyuridine and 5-hydroxymethyl-2'-deoxycytidine were found to increase the mutation frequency by 3.1- and 3.4-fold, respectively.
Topics: Anti-HIV Agents; Base Pair Mismatch; Base Pairing; Base Sequence; Deoxycytidine; Drug Design; HIV Reverse Transcriptase; HIV-1; High-Throughput Screening Assays; Molecular Sequence Data; Mutagenesis; Mutagens; Nucleic Acid Denaturation; Predictive Value of Tests; Reverse Transcriptase Inhibitors; Reverse Transcription; Thermodynamics; Thymidine; Time Factors
PubMed: 25398876
DOI: 10.1074/jbc.M114.616383 -
Nucleic Acids Research Sep 2019A new approach to single-molecule DNA sequencing in which dNTPs, released by pyrophosphorolysis from the strand to be sequenced, are captured in microdroplets and read...
A new approach to single-molecule DNA sequencing in which dNTPs, released by pyrophosphorolysis from the strand to be sequenced, are captured in microdroplets and read directly could have substantial advantages over current sequence-by-synthesis methods; however, there is no existing method sensitive enough to detect a single nucleotide in a microdroplet. We have developed a method for dNTP detection based on an enzymatic two-stage reaction which produces a robust fluorescent signal that is easy to detect and process. By taking advantage of the inherent specificity of DNA polymerases and ligases, coupled with volume restriction in microdroplets, this method allows us to simultaneously detect the presence of and distinguish between, the four natural dNTPs at the single-molecule level, with negligible cross-talk.
Topics: DNA-Directed DNA Polymerase; Deoxyribonucleosides; Deoxyribonucleotides; High-Throughput Nucleotide Sequencing; Limit of Detection; Microscopy, Fluorescence; Oligodeoxyribonucleotides; Sensitivity and Specificity; Sequence Analysis, DNA
PubMed: 31318971
DOI: 10.1093/nar/gkz611 -
Bioorganic & Medicinal Chemistry Feb 2019We report herein the synthesis and evaluation of a series of β-d-2'-deoxy-2'-α-chloro-2'-β-fluoro and β-d-2'-deoxy-2'-α-bromo-2'-β-fluoro nucleosides along with...
We report herein the synthesis and evaluation of a series of β-d-2'-deoxy-2'-α-chloro-2'-β-fluoro and β-d-2'-deoxy-2'-α-bromo-2'-β-fluoro nucleosides along with their corresponding phosphoramidate prodrugs. Key intermediates, lactols 11 and 12, were obtained by a diastereoselective fluorination of protected 2-deoxy-2-chloro/bromo-ribonolactones 7 and 8. All synthesized nucleosides and prodrugs were evaluated with a hepatitis C virus (HCV) subgenomic replicon system.
Topics: Animals; Antiviral Agents; Cell Line, Tumor; Chlorocebus aethiops; Deoxyribonucleosides; Hepacivirus; Humans; Prodrugs; Stereoisomerism; Vero Cells
PubMed: 30655167
DOI: 10.1016/j.bmc.2019.01.005 -
Journal of the American Chemical Society Sep 2021Oxidative stress produces a variety of radicals in DNA, including pyrimidine nucleobase radicals. The nitrogen-centered DNA radical 2'-deoxycytidin-4-yl radical (dC·)...
Oxidative stress produces a variety of radicals in DNA, including pyrimidine nucleobase radicals. The nitrogen-centered DNA radical 2'-deoxycytidin-4-yl radical (dC·) plays a role in DNA damage mediated by one electron oxidants, such as HOCl and ionizing radiation. However, the reactivity of dC· is not well understood. To reduce this knowledge gap, we photochemically generated dC· from a nitrophenyl oxime nucleoside and within chemically synthesized oligonucleotides from the same precursor. dC· formation is confirmed by transient UV-absorption spectroscopy in laser flash photolysis (LFP) experiments. LFP and duplex DNA cleavage experiments indicate that dC· oxidizes dG. Transient formation of the dG radical cation (dG) is observed in LFP experiments. Oxidation of the opposing dG in DNA results in hole transfer when the opposing dG is part of a dGGG sequence. The sequence dependence is attributed to a competition between rapid proton transfer from dG to the opposing dC anion formed and hole transfer. Enhanced hole transfer when less acidic 6-methyl-2'-deoxyguanosine is opposite dC· supports this proposal. dC· produces tandem lesions in sequences containing thymidine at the 5'-position by abstracting a hydrogen atom from the thymine methyl group. The corresponding thymidine peroxyl radical completes tandem lesion formation by reacting with the 5'-adjacent nucleotide. As dC· is reduced to dC, its role in the process is traceless and is only detectable because of the ability to independently generate it from a stable precursor. These experiments reveal that dC· oxidizes neighboring nucleotides, resulting in deleterious tandem lesions and hole transfer in appropriate sequences.
Topics: DNA; DNA Damage; Deoxycytidine; Deoxyguanosine; Free Radicals; Oximes; Photolysis; Ultraviolet Rays
PubMed: 34467764
DOI: 10.1021/jacs.1c06425 -
PloS One 2020Bacterial production has been often estimated from DNA synthesis rates by using tritium-labeled thymidine. Some bacteria species cannot incorporate extracellular...
Bacterial production has been often estimated from DNA synthesis rates by using tritium-labeled thymidine. Some bacteria species cannot incorporate extracellular thymidine into their DNA, suggesting their biomass production might be overlooked when using the conventional method. In the present study, to evaluate appropriateness of deoxyribonucleosides for evaluating bacterial production of natural bacterial communities from the viewpoint of DNA synthesis, incorporation rates of four deoxyribonucleosides (thymidine, deoxyadenosine, deoxyguanosine and deoxycytidine) labeled by nitrogen stable isotope (15N) into bacterial DNA were examined in both ocean (Sagami Bay) and freshwater (Lake Kasumigaura) ecosystems in July 2015 and January 2016. In most stations in Sagami Bay and Lake Kasumigaura, we found that incorporation rates of deoxyguanosine were the highest among those of the four deoxyribonucleosides, and the incorporation rate of deoxyguanosine was approximately 2.5 times higher than that of thymidine. Whereas, incorporation rates of deoxyadenosine and deoxycytidine were 0.9 and 0.2 times higher than that of thymidine. These results clearly suggest that the numbers of bacterial species which can incorporate exogenous deoxyguanosine into their DNA are relatively greater as compared to the other deoxyribonucleosides, and measurement of bacterial production using deoxyguanosine more likely reflects larger numbers of bacterial species productions.
Topics: Bays; Biomass; DNA, Bacterial; Deoxyadenosines; Deoxycytidine; Deoxyguanosine; Ecosystem; Japan; Kinetics; Lakes; Microbial Consortia; Nitrogen Isotopes; Thymidine
PubMed: 32106263
DOI: 10.1371/journal.pone.0229740 -
Nucleic Acids Research Mar 198513C-enriched deoxyribonucleosides have been isolated from the DNA of Algal cells grown in an atmosphere of 90% 13C-labelled carbon dioxide. The 13C enriched DNA was...
13C-enriched deoxyribonucleosides have been isolated from the DNA of Algal cells grown in an atmosphere of 90% 13C-labelled carbon dioxide. The 13C enriched DNA was quantitatively hydrolysed with DNase I, snake venom phosphodiesterase I and alkaline phosphatase of intestinal mucosa. The resulting deoxyribonucleosides were separated by preparative reversed-phase high pressure liquid chromatography in 60 minutes with detection by ultraviolet absorption at 254 nm. The final products were obtained in milligram quantities in high purity and in high yield. The 1H resonances of the base and sugar protons of these deoxyribonucleosides appear as well resolved multiplets in the 600 MHz NMR spectrum, due to the extensive 1H-13C couplings. Similarly, the 13C resonances of these deoxyribonucleosides appear as multiplets in the 75.5 MHz 13C NMR spectrum, due to 13C-13C couplings. The 1H-13C and 13C-13C coupling constants were also measured and tabulated. The isotopic enrichment of 13C these deoxyribonucleosides was obtained by integration of the 1H and/or 13C NMR spectra. It was found that the enrichment varied from carbon to carbon and species to species in the range of 70-89%, suggesting differential uptake and assimilation of 90% 13CO2 during metabolism pathways. This protocol provides experimentally useful quantities of 13C-enriched deoxyribonucleosides, which may be incorporated into site-specifically labeled oligonucleotides by chemical synthesis.
Topics: Carbon Isotopes; Chromatography, High Pressure Liquid; Deoxyribonucleosides; Eukaryota; Magnetic Resonance Spectroscopy
PubMed: 4000954
DOI: 10.1093/nar/13.6.2097 -
Journal of the American Chemical Society Oct 2022A new approach for synthesizing polycyclic heterofused 7-deazapurine heterocycles and the corresponding nucleosides was developed based on C-H functionalization of...
A new approach for synthesizing polycyclic heterofused 7-deazapurine heterocycles and the corresponding nucleosides was developed based on C-H functionalization of diverse (hetero)aromatics with dibenzothiophene--oxide followed by the Negishi cross-cooupling with bis(4,6-dichloropyrimidin-5-yl)zinc. This cross-coupling afforded a series of (het)aryl-pyrimidines that were converted to fused deazapurine heterocycles through azidation and thermal cyclization. The fused heterocycles were glycosylated to the corresponding 2'-deoxy- and ribonucleosides, and a series of derivatives were prepared by nucleophilic substitutions at position 4. Four series of new polycyclic thieno-fused 7-deazapurine nucleosides were synthesized using this strategy. Most of the deoxyribonucleosides showed good cytotoxic activity, especially for the CCRF-CEM cell line. Phenyl- and thienyl-substituted thieno-fused 7-deazapurine nucleosides were fluorescent, and the former one was converted to 2'-deoxyribonucleoside triphosphate for enzymatic synthesis of labeled oligonucleotides.
Topics: Nucleosides; Cell Line, Tumor; Ribonucleosides; Pyrimidines; Oxides; Zinc; Oligonucleotides; Deoxyribonucleosides; Purine Nucleosides
PubMed: 36245092
DOI: 10.1021/jacs.2c07517 -
Nucleic Acids Research Jan 2003DNA and DNA precursors (deoxyribonucleotides) suffer damage by reactive oxygen/nitrogen species. They are important mutagens for organisms, due to their endogenous... (Review)
Review
Mutagenic potentials of damaged nucleic acids produced by reactive oxygen/nitrogen species: approaches using synthetic oligonucleotides and nucleotides: survey and summary.
DNA and DNA precursors (deoxyribonucleotides) suffer damage by reactive oxygen/nitrogen species. They are important mutagens for organisms, due to their endogenous formation. Damaged DNA and nucleotides cause alterations of the genetic information by the mispairing properties of the damaged bases, such as 8-hydroxyguanine (7,8-dihydro-8-oxoguanine) and 2-hydroxyadenine. Here, the author reviews the mutagenic potentials of damaged bases in DNA and of damaged DNA precursors formed by reactive oxygen/nitrogen species, focusing on the results obtained with synthetic oligonucleotides and 2'-deoxyribonucleoside 5'-triphosphates.
Topics: 8-Hydroxy-2'-Deoxyguanosine; Animals; DNA; DNA Damage; DNA Repair; DNA-Directed DNA Polymerase; Deoxyguanosine; Deoxyuracil Nucleotides; Guanine; Guanosine Monophosphate; Humans; Mutation; Reactive Nitrogen Species; Reactive Oxygen Species
PubMed: 12527759
DOI: 10.1093/nar/gkg137 -
Journal of Computational Chemistry Jun 2017Locked nucleic acid (LNA), a modified nucleoside which contains a bridging group across the ribose ring, improves the stability of DNA/RNA duplexes significantly, and...
Locked nucleic acid (LNA), a modified nucleoside which contains a bridging group across the ribose ring, improves the stability of DNA/RNA duplexes significantly, and therefore is of interest in biotechnology and gene therapy applications. In this study, we investigate the free energy change between LNA and DNA nucleosides. The transformation requires the breaking of the bridging group across the ribose ring, a problematic transformation in free energy calculations. To address this, we have developed a 3-step (easy to implement) and a 1-step protocol (more efficient, but more complicated to setup), for single and dual topologies in classical molecular dynamics simulations, using the Bennett Acceptance Ratio method to calculate the free energy. We validate the approach on the solvation free energy difference for the nucleosides thymidine, cytosine, and 5-methyl-cytosine. © 2017 The Authors. Journal of Computational Chemistry Published by Wiley Periodicals, Inc.
Topics: Cytosine; DNA; Deoxyribonucleosides; Molecular Dynamics Simulation; Nucleic Acid Conformation; Oligonucleotides; Ribose; Thermodynamics; Thymidine
PubMed: 28101966
DOI: 10.1002/jcc.24692 -
International Journal of Molecular... Mar 2023Nucleoside analogues are important compounds for the treatment of viral infections or cancers. While (chemo-)enzymatic synthesis is a valuable alternative to traditional...
Nucleoside analogues are important compounds for the treatment of viral infections or cancers. While (chemo-)enzymatic synthesis is a valuable alternative to traditional chemical methods, the feasibility of such processes is lowered by the high production cost of the biocatalyst. As continuous enzyme membrane reactors (EMR) allow the use of biocatalysts until their full inactivation, they offer a valuable alternative to batch enzymatic reactions with freely dissolved enzymes. In EMRs, the enzymes are retained in the reactor by a suitable membrane. Immobilization on carrier materials, and the associated losses in enzyme activity, can thus be avoided. Therefore, we validated the applicability of EMRs for the synthesis of natural and dihalogenated nucleosides, using one-pot transglycosylation reactions. Over a period of 55 days, 2'-deoxyadenosine was produced continuously, with a product yield >90%. The dihalogenated nucleoside analogues 2,6-dichloropurine-2'-deoxyribonucleoside and 6-chloro-2-fluoro-2'-deoxyribonucleoside were also produced, with high conversion, but for shorter operation times, of 14 and 5.5 days, respectively. The EMR performed with specific productivities comparable to batch reactions. However, in the EMR, 220, 40, and 9 times more product per enzymatic unit was produced, for 2'-deoxyadenosine, 2,6-dichloropurine-2'-deoxyribonucleoside, and 6-chloro-2-fluoro-2'-deoxyribonucleoside, respectively. The application of the EMR using freely dissolved enzymes, facilitates a continuous process with integrated biocatalyst separation, which reduces the overall cost of the biocatalyst and enhances the downstream processing of nucleoside production.
Topics: Nucleosides; Pentosyltransferases; Enzymes, Immobilized; Biocatalysis; Deoxyribonucleosides; Purine-Nucleoside Phosphorylase
PubMed: 37047056
DOI: 10.3390/ijms24076081