A novel and specific fluorescence reaction for uracil (original) (raw)
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Chemical & pharmaceutical bulletin, 2021
A facile and reliable fluorescence method for the quantification of urinary uracil concentration is proposed herein. The assay utilizes a specific fluorescence (FL) derivatization reaction for uracil using 3-methylbenzamidoxime as a fluorogenic reagent. Although the presence of urine inhibited the FL reaction, 10 µL of urine was sufficient for the detection of urinary uracil. The uracil derivative was successfully separated from other fluorescent impurities using simple reversed-phase LC with FL detection. Urinary uracil concentrations from 16 people were compared with the concentrations obtained by the traditional column-switching liquid chromatographic analysis with UV detection. The FL derivative of uracil appeared as a single peak in the chromatograms of all samples. However, several samples showed an additional peak overlapping the uracil peak when using the column-switching method because of UV-active impurities. These results indicated that that the present method is not affe...
Nucleic Acids Research, 2015
The role of uracil in genomic DNA has been recently re-evaluated. It is now widely accepted to be a physiologically important DNA element in diverse systems from specific phages to antibody maturation and Drosophila development. Further relevant investigations would largely benefit from a novel reliable and fast method to gain quantitative and qualitative information on uracil levels in DNA both in vitro and in situ, especially since current techniques does not allow in situ cellular detection. Here, starting from a catalytically inactive uracil-DNA glycosylase protein, we have designed several uracil sensor fusion proteins. The designed constructs can be applied as molecular recognition tools that can be detected with conventional antibodies in dot-blot applications and may also serve as in situ uracil-DNA sensors in cellular techniques. Our method is verified on numerous prokaryotic and eukaryotic cellular systems. The method is easy to use and can be applied in a highthroughput manner. It does not require expensive equipment or complex know-how, facilitating its easy implementation in any basic molecular biology laboratory. Elevated genomic uracil levels from cells of diverse genetic backgrounds and/or treated with different drugs can be demonstrated also in situ, within the cell.
Analytical Chemistry, 2002
A sensitive and selective method for determination of the uracil content in human DNA was first developed on the basis of high-performance liquid chromatography-tandem mass spectrometry. Uracil was excised from DNA using uracil DNA glycosylase. The released uracil was derivatized with 4-bromomethyl-7-methoxycoumarin, thereby forming bis-N,N′-(4-methylene-7-methoxycoumaryl)-uracil (uracil-MMC). 15 N 2 -Uracil was used as an internal standard. The analytes were separated on an Adsorbsphere XL ODS column. A SCIEX API III tandem mass spectrometer equipped with a turbo ion-spray interface was used as the detector. Multiple reaction monitoring using the parent f product ion combinations of m/z 489 f 232 and 491 f 233 were used to detect uracil-MMC and the internal standard, respectively. The detection limit for this assay is <1.0 × 10 -10 mol/L uracil, and the linearity is from 1.0 × 10 -10 to 2.5 × 10 -6 mol/ L. The method was used for determination of uracil in human DNA. Our data show that the uracil levels in human DNA isolated from peripheral white blood cells did not differ between subjects with folate deficiency and subjects with normal red cell folate levels. Mack, M. M.; Wehr, C. M.; MacGregor, J. T.; Hiatt, R. A.; Wang, G.; Wickramasinghe, S. N.; Everson, R. B.; Ames, B. N.
Photochemistry and Photobiology, 2007
We report a comparison of the steady-state absorption and fluorescence spectra of three representative uracil derivatives (uracil, thymine and 5-fluorouracil) in alcoholic solutions. The present results are compared with those from our previous experimental and computational studies of the same compounds in water and acetonitrile. The effects of solvent polarity and hydrogen bonding on the spectra are discussed in the light of theoretical predictions. This comparative analysis provides a more complete picture of the solvent effects on the absorption and fluorescence properties of pyrimidine nucleobases, with special emphasis on the mechanism of the excited state deactivation. 11/02/2010
Highly Sensitive Nuclease Assays Based on Chemically Modified DNA or RNA
Sensors, 2014
Nucleolytic enzymes are associated with various diseases, and several methods have been developed for their detection. DNase expression is modulated in such diseases as acute myocardial infarction, transient myocardial ischemia, oral cancer, stomach cancer, and malignant lymphoma, and DNase I is used in cystic fibroma therapy. RNase is used to treat mesothelial cancer because of its antiproliferative, cytotoxic, and antineoplastic activities. Angiogenin, an angiogenic factor, is a member of the RNase A family. Angiogenin inhibitors are being developed as anticancer drugs. In this review, we describe fluorometric and electrochemical techniques for detecting DNase and RNase in disease. Oligonucleotides having fluorescence resonance energy transfer (FRET)-causing chromophores are non-fluorescent by themselves, yet become fluorescent upon cleavage by DNase or RNase. These oligonucleotides serve as a powerful tool to detect activities of these enzymes and provide a basis for drug discovery. In electrochemical techniques, ferrocenyl oligonucleotides with or without a ribonucleoside unit are used for the detection of RNase or DNase. This technique has been used to monitor blood or serum samples in several diseases associated with DNase and RNase and is unaffected by interferents in these sample types.
Photochemistry and EC of uracils
The redox mechanism and electronic absorption behavior of a commercial anticancer drug, 5-fluorouracil (5-FU) and two potential anticancer drugs, 2-thiouracil (2-TU) and dithiouracil (DTU) have been investigated in a wide pH range by UV-Vis spectroscopy, cyclic voltammetry and differential pulse voltammetry. The effect of electrolytes, substituents, successive sweeps and potential scan rate on the voltammetric response of uracils was examined. Organized structure-activity relationships of these drugs were established on the basis of the results obtained from electronic absorption spectroscopy and cyclic voltammetry. The electrode reaction mechanism was suggested using the experimentally determined electrochemical parameters. The DNA binding propensity of uracils was found greater than the classical intercalator, proflavin and clinically used drug, epirubicin. Moreover, the results obtained through ab initio calculations for the oxidation potential of the three uracil derivatives were found in good agreement with the electrochemical results.
Indian Journal of Clinical Biochemistry, 2015
The most important objectives that are frequently found in bio-analytical chemistry involve applying tools to relevant medical/biological problems and refining these applications. Developing a reliable sample preparation step, for the medical and biological fields is another primary objective in analytical chemistry, in order to extract and isolate the analytes of interest from complex biological matrices. Since, main inborn errors of metabolism (IEM) diagnosable through uracil analysis and the therapeutic monitoring of toxic 5-fluoruracil (an important anticancerous drug) in dihydropyrimidine dehydrogenase deficient patients, require an ultra-sensitive, reproducible, selective, and accurate analytical techniques for their measurements. Therefore, keeping in view, the diagnostic value of uracil and 5-fluoruracil measurements, this article refines several analytical techniques involved in selective recognition and quantification of uracil and 5-fluoruracil from biological and pharmaceutical samples. The prospective study revealed that implementation of molecularly imprinted polymer as a solid-phase material for sample preparation and preconcentration of uracil and 5-fluoruracil had proven to be effective as it could obviates problems related to tedious separation techniques, owing to protein binding and drastic interferences, from the complex matrices in real samples such as blood plasma, serum samples.
DNA Repair, 2013
Considerable progress has been made in understanding the origins of genomic uracil and its role in genome stability and host defense; however, the main question concerning the basal level of uracil in DNA remains disputed. Results from assays designed to quantify genomic uracil vary by almost three orders of magnitude. To address the issues leading to this inconsistency, we explored possible shortcomings with existing methods and developed a sensitive LC/MS/MS-based method for the absolute quantification of genomic 2 -deoxyuridine (dUrd). To this end, DNA was enzymatically hydrolyzed to 2 -deoxyribonucleosides and dUrd was purified in a preparative HPLC step and analyzed by LC/MS/MS. The standard curve was linear over four orders of magnitude with a quantification limit of 5 fmol dUrd. Control samples demonstrated high inter-experimental accuracy (94.3%) and precision (CV 9.7%). An alternative method that employed UNG2 to excise uracil from DNA for LC/MS/MS analysis gave similar results, but the intra-assay variability was significantly greater. We quantified genomic dUrd in Ung +/+ and Ung −/− mouse embryonic fibroblasts and human lymphoblastoid cell lines carrying UNG mutations. DNA-dUrd is 5-fold higher in Ung −/− than in Ung +/+ fibroblasts and 11-fold higher in UNG2 dysfunctional than in UNG2 functional lymphoblastoid cells. We report approximately 400-600 dUrd per human or murine genome in repair-proficient cells, which is lower than results using other methods and suggests that genomic uracil levels may have previously been overestimated.