Real time monitoring uracil excision using uracil-containing molecular beacons

• A sensitive and low-cost fluorescence detection system for UDG assay was constructed.
• A fluorescence strategy for sensing UDG activity and kinetics study was proposed.
• A low detection limit (0.005 U mL−1) was obtained without any amplification.
• The strategy can be reliably applied for quantitative assay of UDG in complicated biological samples.

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.

A real time fluorescence method with promising applications is developed for UDG assay with high accuracy and specificity using modified molecular beacons as substrates.Figure optionsDownload as PowerPoint slide