Sensitive evaluation of the uracil-DNA glycosylase (UDG) activity is greatly significant in both fundamental biochemical process studies and disease prognosis. In this study, a simple but sensitive UDG activity-sensing strategy was designed on the basis of UDG-triggered rolling circle amplification (RCA) reaction. In this strategy, two oligonucleotides were used. The hairpin-like structure of the oligonucleotide containing a uracil nucleotide is destroyed in the presence of UDG, and then can be employed to form the circular template of RCA and initiate the subsequent RCA reaction. The participation of a nicking endonuclease makes the RCA reaction proceed in an exponential amplification mode. The amplification product may fold into a G-quadruplex structure, which can be specifically combined with thioflavin T to generate a fluorescence signal without any extra label. This UDG activity-sensing strategy was demonstrated to work well in both end-point and real-time detection modes with high sensitivity and excellent specificity. As low as 5.5 × 10⁻⁵ U mL⁻¹ UDG could be detected. The advantages of simple operation, short RCA time and automatic measurement using commercial instruments make the real-time detection mode suitable for high-throughput detection with reduced risk of amplification product carryover contamination, and its application feasibility in real samples was demonstrated by UDG activity analysis of cell lysate.

中文翻译：

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使用指数实时滚环扩增技术对尿嘧啶DNA糖基化酶进行无标记灵敏检测†

在基本的生化过程研究和疾病预后中，对尿嘧啶DNA糖基化酶（UDG）活性的敏感评估都非常重要。在这项研究中，基于UDG触发的滚环扩增（RCA）反应，设计了一种简单而敏感的UDG活性传感策略。在这种策略中，使用了两个寡核苷酸。在UDG的存在下，含有尿嘧啶核苷酸的寡核苷酸的发夹状结构被破坏，然后可用于形成RCA的环状模板并引发随后的RCA反应。切口内切核酸酶的参与使得RCA反应以指数扩增模式进行。扩增产物可能会折叠成G-四链体结构，可以与硫黄素T特异性结合，产生荧光信号，而无需任何额外的标记。该UDG活动感测策略已证明在终点和实时检测模式下均具有良好的灵敏度和出色的特异性，可以很好地工作。低至5.5×10可以检测到^-5 U mL ^-1 UDG。操作简单，RCA时间短以及使用商业仪器进行自动测量的优点使实时检测模式适用于高通量检测，并降低了扩增产物残留污染的风险，并且通过UDG活性分析证明了其在实际样品中的应用可行性细胞裂解液。