Ji Lijuan, Qian Yingdan, Wu Ping, Zhang Hui, Cai Chenxin
Jiangsu Key Laboratory of New Power Batteries, Jiangsu Collaborative Innovation Center of Biomedical Functional Materials, National and Local Joint Engineering Research Center of Biomedical Functional Materials, College of Chemistry and Materials Science, Nanjing Normal University, Nanjing 210097, PR China.
Jiangsu Key Laboratory of New Power Batteries, Jiangsu Collaborative Innovation Center of Biomedical Functional Materials, National and Local Joint Engineering Research Center of Biomedical Functional Materials, College of Chemistry and Materials Science, Nanjing Normal University, Nanjing 210097, PR China.
Anal Chim Acta. 2015 Apr 15;869:74-80. doi: 10.1016/j.aca.2015.02.021. Epub 2015 Feb 12.
We report on the development of a sensitive and selective deoxyribonucleic acid (DNA) demethylase (using MBD2 as an example) activity assay by coupling the fluorescence quenching of graphene oxide (GO) with the site-specific cleavage of HpaII endonuclease to improve the selectivity. This approach was developed by designing a single-stranded probe (P1) that carries a binding region to facilitate the interaction with GO, which induces fluorescence quenching of the labeled fluorophore (FAM, 6-carboxyfluorescein), and a sensing region, which contains a hemi-methylated site of 5'-CmCGG-3', to specifically recognize the target (T1, a 32-mer DNA from the promoter region of p53 gene) and hybridize with it to form a P1/T1 duplex. After demethylation with MBD2, the duplex can be specifically cleaved using HpaII, which releases the labeled FAM from the GO surface and results in the recovery of fluorescence. However, this cleavage is blocked by the hemi-methylation of this site. Thus, the magnitude of the recovered fluorescence signal is related to the MBD2 activity, which establishes the basis of the DNA demethylase activity assay. This assay can determine as low as ∼(0.05±0.01) ng mL(-1) (at a signal/noise of 3) of MBD2 with a linear range of 0.2-300 ng mL(-1) and recognize MBD2 from other possibly coexisting proteins and cancer cell extracts. The advantage of this assay is its ability to avoid false signals and no requirement of bisulfite conversion, PCR amplification, radioisotope labeling, or separation.
我们报道了一种灵敏且具选择性的脱氧核糖核酸（DNA）脱甲基酶（以MBD2为例）活性测定方法的开发，该方法通过将氧化石墨烯（GO）的荧光猝灭与HpaII核酸内切酶的位点特异性切割相结合来提高选择性。此方法是通过设计一条单链探针（P1）来实现的，该探针带有一个结合区域以促进与GO的相互作用，GO会诱导标记荧光团（FAM，6-羧基荧光素）的荧光猝灭，同时还带有一个传感区域，该区域包含一个5'-CmCGG-3'的半甲基化位点，用于特异性识别靶标（T1，一段来自p53基因启动子区域的32聚体DNA）并与之杂交形成P1/T1双链体。用MBD2进行脱甲基后，该双链体可被HpaII特异性切割，从而使标记的FAM从GO表面释放出来并导致荧光恢复。然而，该位点的半甲基化会阻止这种切割。因此，恢复的荧光信号强度与MBD2活性相关，这奠定了DNA脱甲基酶活性测定的基础。该测定方法能够检测低至约(0.05±0.01) ng mL(-1)（信噪比为3时）的MBD2，线性范围为0.2 - 300 ng mL(-1)，并且能够从其他可能共存的蛋白质和癌细胞提取物中识别出MBD2。该测定方法的优点在于能够避免假信号，且无需亚硫酸氢盐转化、PCR扩增、放射性同位素标记或分离。
