Real time monitoring uracil excision using uracil-containing molecular beacons
| Autor: | Haizhen Zhu, Dan Xiang, Chen Li, Bin Liu, Ying Long |
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| Rok vydání: | 2013 |
| Předmět: |
Time Factors
Chemistry Uracil Biochemistry Molecular biology Fluorescence Analytical Chemistry Enzyme Activation chemistry.chemical_compound Spectrometry Fluorescence Molecular beacon DNA glycosylase Uracil-DNA glycosylase Molecular Probes Damage repair Environmental Chemistry Nuclease inhibitor Uracil-DNA Glycosidase Spectroscopy |
| Zdroj: | Analytica chimica acta. 819 |
| ISSN: | 1873-4324 |
| Popis: | As a highly conserved damage repair protein, UDG excises uracil bases through its glycosylase activity. We report here an alternative fluorescence method for UDG assay with high accuracy and sensitivity by applying uracil-modified molecular beacons as substrates. The detection limit of UDG is 0.005 U mL −1 . The K M and k cat are 0.89 ± 0.1 μM and 210 ± 10 min −1 , respectively. The method is applied to screening inhibitors and the results indicate that both of the 5-FU and cisplatin can inhibit UDG activity with the IC 50 values of 6.1 ± 0.52 mM and 3.2 ± 0.24 mM, respectively. Furthermore, the combination of uracil-modified molecular beacons and nuclease inhibitor makes the new method possible to specifically detect UDG activity in cell-free extracts and serum. Taken together, the simple, rapid and sensitive method has potential relevance for a variety of applications, such as molecular diagnosis and screening of UDG inhibitors. |
| Databáze: | OpenAIRE |
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