Nie Huaijun, Huang Huifen, Li Wang, Yang Tao
State Environmental Protection Key Laboratory of Drinking Water Source Management and Technology, Shenzhen Key Laboratory of Drinking Water Source Safety Control, Shenzhen Research Academy of Environmental Sciences.
Anal Sci. 2016;32(11):1245-1250. doi: 10.2116/analsci.32.1245.
Endonuclease V (EndoV) plays the important role of nucleotide excision repair (NER) in the maintenance of genomic stability. Highly sensitive detection of EndoV was achieved through an oligonucleotides sensitizing Tb luminescent technique. We found that although both guanine-rich (G-rich) single-stranded DNA and dGMP could enhance the time-resolved luminescence of Tb, their efficiencies of enhancement were considerably different. Employing such interesting phenomenon, a label-free and time-resolved luminescent strategy for the sensitive detection of EndoV activity was developed based on DNA-enhanced time-resolved luminescence (TRL) of Tb. The EndoV was used to cut off the deoxyinosine site (dI) and convert the 3'-protruding termini to a recessed end, and Exonuclease III (Exo III) was used to enhance the signal contrast via digestion of G-rich DNA to dNTP. Combining with the natural advantages of the TRL, the proposed method exhibited a good linear response to EndoV ranging from 0.005 to 0.4 U/mL, with a low limit of detection of 0.005 U/mL.
核酸内切酶V（EndoV）在维持基因组稳定性方面发挥着核苷酸切除修复（NER）的重要作用。通过寡核苷酸敏化Tb发光技术实现了对EndoV的高灵敏度检测。我们发现，尽管富含鸟嘌呤的（G-rich）单链DNA和dGMP都能增强Tb的时间分辨发光，但它们的增强效率却有很大差异。利用这一有趣现象，基于Tb的DNA增强时间分辨发光（TRL），开发了一种用于灵敏检测EndoV活性的无标记时间分辨发光策略。EndoV用于切割脱氧肌苷位点（dI）并将3'突出末端转化为凹陷末端，而核酸外切酶III（Exo III）用于通过将富含G的DNA消化为dNTP来增强信号对比度。结合TRL的天然优势，所提出的方法对EndoV在0.005至0.4 U/mL范围内表现出良好的线性响应，检测限低至0.005 U/mL。
