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Synthesis of N2-alkyl-8-oxo-7,8-dihydro-2'-deoxyguanosine derivatives and effects of these modifications on RNA duplex stability.

ABSTRACT: N(2)-alkyl analogues of 8-oxo-7,8-dihydro-2'-deoxyguanosine (OG) were synthesized (alkyl = propyl, benzyl) via reductive amination of the protected OG nucleoside and incorporated into various positions of an RNA strand. Thermal stability studies of duplexes containing A or C opposite a single modified base revealed only moderate destabilization. Both OG as well as its N(2)-alkyl analogues can pair opposite A or C with nearly equal stability, potentially offering a new means of modulating RNA-protein interactions in the minor vs major grooves.

SUBMITTER: Kannan A

PROVIDER: S-EPMC3025262 | biostudies-literature | 2011 Jan

REPOSITORIES: biostudies-literature

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Synthesis of N2-alkyl-8-oxo-7,8-dihydro-2'-deoxyguanosine derivatives and effects of these modifications on RNA duplex stability.

Kannan Arunkumar A Burrows Cynthia J CJ

The Journal of organic chemistry 20101230 2

N(2)-alkyl analogues of 8-oxo-7,8-dihydro-2'-deoxyguanosine (OG) were synthesized (alkyl = propyl, benzyl) via reductive amination of the protected OG nucleoside and incorporated into various positions of an RNA strand. Thermal stability studies of duplexes containing A or C opposite a single modified base revealed only moderate destabilization. Both OG as well as its N(2)-alkyl analogues can pair opposite A or C with nearly equal stability, potentially offering a new means of modulating RNA-pro ...[more]

PMID: 21192638

Similar Datasets

The effect of the 2-amino group of 7,8-dihydro-8-oxo-2'-deoxyguanosine on translesion synthesis and duplex stability.

Project description:Replication of DNA containing 7,8-dihydro-8-oxo-2'-deoxyguanosine (OxodG) gives rise to G --> T transversions. The syn-isomer of the lesion directs misincorporation of 2'-deoxyadenosine (dA) opposite it. We investigated the role of the 2-amino substituent on duplex thermal stability and in replication using 7,8-dihydro-8-oxo-2'-deoxyinosine (OxodI). Oligonucleotides containing OxodI at defined sites were chemically synthesized via solid phase synthesis. Translesion incorporation opposite OxodI was compared with 7,8-dihydro-8-oxo-2'-deoxyguanosine (OxodG), 2'-deoxyinosine (dI) and 2'-deoxyguanosine (dG) in otherwise identical templates. The Klenow exo- fragment of Escherichia coli DNA polymerase I incorporated 2'-deoxyadenosine (dA) six times more frequently than 2'-deoxycytidine (dC) opposite OxodI. Preferential translesion incorporation of dA was unique to OxodI. UV-melting experiments revealed that DNA containing OxodI opposite dA is more stable than when the modified nucleotide is opposed by dC. These data suggest that while duplex DNA accommodates the 2-amino group in syn-OxodG, this substituent is thermally destabilizing and does not provide a kinetic inducement for replication by Klenow exo-.

| S-EPMC1067523 | biostudies-literature

Short-term biological variation and Reference change values of urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine and 8-oxo-7,8-dihydroguanosine.

Project description:AimThe DNA and RNA oxidative damage products urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-odGsn) and 8-oxo-7,8-dihydroguanosine (8-oGsn) have potential use in clinical practice. However, biological variation (BV) and reference change values (RCVs) have not been established. The aim of this study was to establish the short-term between-subject BV(CVG ), within-subject BV(CVI ), and RCVs for urinary 8-odGsn and 8-oGsn.MethodsFirst-morning midstream urine specimens were collected from 20 apparently healthy subjects(ten males and ten females) on five consecutive days. 8-odGsn and 8-oGsn were measured using LC-MS/MS, while urine creatinine (U-Cr) was also measured to correct their results. A two-level nested ANOVA was used to estimate the CVI and CVG. RESULTS: The values of CVG for 8-odGsn, 8-odGsn/U-Cr, 8-oGsn, and 8-oGsn/U-Cr were 31.2%, 39.6%, 35.3%, and 28.8%, respectively, while CVI for them were 40.5%, 9.0%, 33.5%, and 12.1%, respectively. The RCVs for 8-odGsn, 8-odGsn/U-Cr, 8-oGsn, and 8-oGsn/U-Cr were 112.5%, 26.7%, 93.7%, and 36.5%, respectively.ConclusionBV and RCVs were firstly established for 8-oxo-dGsn and 8-oGsn, and can be used in clinical practice.

| S-EPMC7755802 | biostudies-literature

8-Oxo-7,8-dihydro-2'-deoxyguanosine and abasic site tandem lesions are oxidation prone yielding hydantoin products that strongly destabilize duplex DNA.

Project description:In DNA, 2'-deoxyguanosine (dG) is susceptible to oxidative modification by reactive oxygen species (ROS) yielding many products, one of which is 8-oxo-7,8-dihydro-2'-deoxyguanosine (dOG). Interestingly, dOG is stable but much more labile toward oxidation than dG, furnishing 5-guanidinohydantoin-2'-deoxyribose (dGh) that is favored in the duplex context or spiroiminodihydantoin-2'-deoxyribose (dSp) that is favored in the oxidation of single-stranded contexts. Previously, exposure of DNA to ionizing radiation found ?50% of the dOG exists as a tandem lesion with an adjacent formamide site. The present work explored oxidation of dOG in a tandem lesion with a THF abasic site analog (F) that models the formamide on either the 5' or 3' side. When dOG was in a tandem lesion, both dGh and dSp were observed as oxidation products. The 5' versus 3' side in which F resided influenced the stereochemistry of the dSp formed. Further, tandem lesions with dOG were found to be up to two orders of magnitude more reactive to oxidation than dOG in an intact duplex. When dOG is in a tandem lesion it is up to fivefold more prone to formation of spermine cross-links during oxidation compared to dOG in an intact duplex. Lastly, dOG, dGh, and each dSp diastereomer were synthesized as part of a tandem lesion in a duplex DNA to establish that dOG tandem lesions decrease the thermal stability by 12-13 °C, while dGh or either dSp diastereomer in a tandem lesion decrease the stability by >20 °C. The biological consequences of these results are discussed.

| S-EPMC5636683 | biostudies-literature

Quantitative detection of 8-Oxo-7,8-dihydro-2'-deoxyguanosine using chemical tagging and qPCR.

Project description:8-Oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) is a commonly formed DNA lesion that is useful as a biomarker for oxidative stress. Although methods for selective quantification of 8-oxodGuo exist, there is room for additional methods that are sensitive and utilize instrumentation that is widely available. We previously took advantage of the reported reactivity of 8-oxodGuo to develop a method for detecting the lesion by selectively covalently tagging it with a molecule equipped with a biotin label that can be used subsequently with a reporting method ( Xue , L. and Greenberg , M. M. ( 2007 ) J. Am. Chem. Soc. 129 , 7010 ). We now report a method that can detect as little as 14 amol of 8-oxodGuo by tagging DNA with a reagent containing a disulfide that reduces background due to nonspecific binding. The reagent also contains biotin that enables capturing target DNA on streptavidin-coated magnetic beads. The captured DNA is quantified using quantitative PCR. The method is validated by comparing the amount of 8-oxodGuo detected as a function of Fe(2+)/H2O2/ascorbate-dose to that reported previously using mass spectrometry.

| S-EPMC4106692 | biostudies-literature

Is pre-heat necessary for the measurement of 8-oxo-7,8-dihydroguanosine and 8-oxo-7,8-dihydro-2'-deoxyguanosine in urine samples.

Project description:BackgroundIt is currently unclear for the necessary of pre-heating urine samples for the accurate determination of 8-oxo-7,8-dihydroguanosine (8-oxoG) and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG). Thus, we conducted this study to evaluate the effect of pre-heat (i.e., to 37°C) on the accurate measurement of 8-oxoG and 8-oxodG in frozen urine samples.MethodsRandom urine samples from six healthy volunteers, six patients with renal dysfunction, and six patients with systematic diseases such as diabetes were collected, split, and stored at -80°C for up to 1 month. The frozen samples were thawed at room temperature (RT) or 37°C for different time, 10-fold diluted with ddH2O containing 1% formic acid, and determined by self-established LC-MS/MS method coupled with an ACQUITY™ Primer HSS T3 column.ResultsThawing the samples at RT for 30 or 120 min, or at 37°C for 15 or 90 min did not affect the determination of 8-oxoG and 8-oxodG in urine samples. Moreover, no significant difference between thawing the urine samples at RT and 37°C was found after storing at -80°C for 1-3 months.ConclusionIt is not always necessary to pre-heat the frozen urine samples to release 8-oxoG and 8-oxodG from precipitates, which is associated with different pre-treatment and determination methods.

| S-EPMC9550956 | biostudies-literature

Sequence-dependent variation in the reactivity of 8-Oxo-7,8-dihydro-2'-deoxyguanosine toward oxidation.

Project description:The goal of this study was to define the effect of DNA sequence on the reactivity of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) toward oxidation. To this end, we developed a quadrupole/time-of-flight (QTOF) mass spectrometric method to quantify the reactivity of site specifically modified oligodeoxyribonucleotides with two model oxidants: nitrosoperoxycarbonate (ONOOCO(2)(-)), a chemical mediator of inflammation, and photoactivated riboflavin, a classical one-electron oxidant widely studied in mutagenesis and charge transport in DNA. In contrast to previous observations with guanine [ Margolin , Y. , ( 2006 ) Nat. Chem. Biol. 2 , 365 ], sequence context did not affect the reactivity of ONOOCO(2)(-) with 8-oxodG, but photosensitized riboflavin showed a strong sequence preference in its reactivity with the following order (8-oxodG = O): COA ? AOG > GOG ? COT > TOC > AOC. That the COA context was the most reactive was unexpected and suggests a new sequence context where mutation hotspots might occur. These results point to both sequence- and agent-specific effects on 8-oxodG oxidation.

| S-EPMC3288455 | biostudies-literature

Kinetics, structure, and mechanism of 8-Oxo-7,8-dihydro-2'-deoxyguanosine bypass by human DNA polymerase ?.

Project description:DNA damage incurred by a multitude of endogenous and exogenous factors constitutes an inevitable challenge for the replication machinery. Cells rely on various mechanisms to either remove lesions or bypass them in a more or less error-prone fashion. The latter pathway involves the Y-family polymerases that catalyze trans-lesion synthesis across sites of damaged DNA. 7,8-Dihydro-8-oxo-2'-deoxyguanosine (8-oxoG) is a major lesion that is a consequence of oxidative stress and is associated with cancer, aging, hepatitis, and infertility. We have used steady-state and transient-state kinetics in conjunction with mass spectrometry to analyze in vitro bypass of 8-oxoG by human DNA polymerase ? (hpol ?). Unlike the high fidelity polymerases that show preferential insertion of A opposite 8-oxoG, hpol ? is capable of bypassing 8-oxoG in a mostly error-free fashion, thus preventing GC?AT transversion mutations. Crystal structures of ternary hpol ?-DNA complexes and incoming dCTP, dATP, or dGTP opposite 8-oxoG reveal that an arginine from the finger domain assumes a key role in avoiding formation of the nascent 8-oxoG:A pair. That hpol ? discriminates against dATP exclusively at the insertion stage is confirmed by structures of ternary complexes that allow visualization of the extension step. These structures with G:dCTP following either 8-oxoG:C or 8-oxoG:A pairs exhibit virtually identical active site conformations. Our combined data provide a detailed understanding of hpol ? bypass of the most common oxidative DNA lesion.

| S-EPMC4059130 | biostudies-literature

Genome-wide analysis of 8-oxo-7,8-dihydro-2'-deoxyguanosine at single-nucleotide resolution unveils reduced occurrence of oxidative damage at G-quadruplex sites

Project description:8-Oxo-7,8-dihydro-2’-deoxyguanosine (OG), one of the most common oxidative DNA damages, causes genome instability and is associated with cancer, neurological diseases and aging. In addition, OG and its repair intermediates can regulate gene transcription, and thus play a role in sensing cellular oxidative stress. However, the lack of methods to precisely map OG has hindered the study of its biological roles. Here we developed a single-nucleotide resolution OG-sequencing method, named CLAPS-seq (Chemical Labeling And Polymerase Stalling Sequencing), to measure the genome-wide distribution of both exogenous and endogenous OGs with high specificity. Our data identified decreased OG occurrence at G-quadruplexes (G4s), in association with underrepresentation of OGs in promoters which have high GC content. Furthermore, we discovered that potential quadruplex sequences (PQSs) were hotspots of OGs which may in turn stimulate the formation of G4s at PQSs, implying a role of non-G4-PQSs in OG-mediated oxidative stress response.

2021-10-09 | GSE181312 | GEO

Mutagenic potential of 8-oxo-7,8-dihydro-2'-deoxyguanosine bypass catalyzed by human Y-family DNA polymerases.

Project description:One of the most common lesions induced by oxidative DNA damage is 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG). Replicative DNA polymerases poorly traverse this highly mutagenic lesion, suggesting that the replication fork may switch to a polymerase specialized for translesion DNA synthesis (TLS) to catalyze 8-oxodG bypass in vivo. Here, we systematically compared the 8-oxodG bypass efficiencies and fidelities of the TLS-specialized, human Y-family DNA polymerases eta (hPol?), iota (hPol?), kappa (hPol?), and Rev1 (hRev1) either alone or in combination. Primer extension assays revealed that the times required for hPol?, hRev1, hPol?, and hPol? to bypass 50% of the 8-oxodG lesions encountered (t50(bypass)) were 0.58, 0.86, 108, and 670 s, respectively. Although hRev1 bypassed 8-oxodG efficiently, hRev1 failed to catalyze the extension step of TLS, and a second polymerase was required to extend the lesion bypass products. A high-throughput short oligonucleotide sequencing assay (HT-SOSA) was used to quantify the types and frequencies of incorporation errors produced by the human Y-family DNA polymerases at and near the 8-oxodG site. Although hPol? bypassed 8-oxodG most efficiently, hPol? correctly incorporated dCTP opposite 8-oxodG within only 54.5% of the sequences analyzed. In contrast, hPol? bypassed the lesion least efficiently but correctly incorporated dCTP at a frequency of 65.8% opposite the lesion. The combination of hRev1 and hPol? was most accurate opposite 8-oxodG (92.3%), whereas hPol? alone was the least accurate (18.5%). The t50(bypass) value and correct dCTP incorporation frequency in the presence of an equal molar concentration of all four Y-family enzymes were 0.60 s and 43.5%, respectively. These values are most similar to those of hPol? alone, suggesting that hPol? outcompetes the other three Y-family polymerases to catalyze 8-oxodG bypass in vitro and possibly in vivo.

| S-EPMC4033635 | biostudies-literature

Roles of Residues Arg-61 and Gln-38 of Human DNA Polymerase ? in Bypass of Deoxyguanosine and 7,8-Dihydro-8-oxo-2'-deoxyguanosine.

Project description:Like the other Y-family DNA polymerases, human DNA polymerase ? (hpol ?) has relatively low fidelity and is able to tolerate damage during DNA synthesis, including 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxoG), one of the most abundant DNA lesions in the genome. Crystal structures show that Arg-61 and Gln-38 are located near the active site and may play important roles in the fidelity and efficiency of hpol ?. Site-directed mutagenesis was used to replace these side chains either alone or together, and the wild type or mutant proteins were purified and tested by replicating DNA past deoxyguanosine (G) or 8-oxoG. The catalytic activity of hpol ? was dramatically disrupted by the R61M and Q38A/R61A mutations, as opposed to the R61A and Q38A single mutants. Crystal structures of hpol ? mutant ternary complexes reveal that polarized water molecules can mimic and partially compensate for the missing side chains of Arg-61 and Gln-38 in the Q38A/R61A mutant. The combined data indicate that the positioning and positive charge of Arg-61 synergistically contribute to the nucleotidyl transfer reaction, with additional influence exerted by Gln-38. In addition, gel filtration chromatography separated multimeric and monomeric forms of wild type and mutant hpol ?, indicating the possibility that hpol ? forms multimers in vivo.

| S-EPMC4481197 | biostudies-literature

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