title: Real time monitoring uracil excision using uracil-containing molecular beacons.
abstract: 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 KM and kcat 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 IC50 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.
authors.affiliation: College of Biology, Hunan University, Changsha, Hunan 410082, People's Republic of China.
authors.forename: Chen
authors.initials: C
authors.lastname: Li
authors.affiliation: College of Biology, Hunan University, Changsha, Hunan 410082, People's Republic of China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Long
authors.affiliation: College of Biology, Hunan University, Changsha, Hunan 410082, People's Republic of China. Electronic address: binliu2001@hotmail.com.
authors.forename: Bin
authors.initials: B
authors.lastname: Liu
authors.affiliation: College of Biology, Hunan University, Changsha, Hunan 410082, People's Republic of China.
authors.forename: Dan
authors.initials: D
authors.lastname: Xiang
authors.affiliation: College of Biology, Hunan University, Changsha, Hunan 410082, People's Republic of China.
authors.forename: Haizhen
authors.initials: H
authors.lastname: Zhu
mesh_terms: D004789:Enzyme Activation; D005453:Fluorescence; D015335:Molecular Probes / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013997:Time Factors; D014498:Uracil / Q000032:analysis / Q000378:metabolism*; D051981:Uracil-DNA Glycosidase / Q000378:metabolism*
chemical_list: D015335:Molecular Probes; D014498:Uracil; D051981:Uracil-DNA Glycosidase
keywords: Fluorescence; Modified molecular beacon; Real time; Uracil-DNA glycosylase