Highly Sensitive Assay of Methyltransferase Activity Based on an Autonomous Concatenated DNA Circuit.
Highly Sensitive Assay of Methyltransferase Activity Based on an Autonomous Concatenated DNA Circuit.
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基于自主串联 DNA 电路的甲基转移酶活性高灵敏测定。
DOI：
10.1021/acssensors.8b00738
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发表时间：
2018-10
期刊：
影响因子：
--
通讯作者：
Wang Fuan
中科院分区：
文献类型：
--
作者：
Li Chunxiao;Wang Huimin;Shang Jinhua;Liu Xiaoqing;Yuan Bifeng;Wang Fuan
甲基转移酶参与的DNA甲基化是最重要的表观遗传过程之一，使得超灵敏的MTase检测在临床诊断和生物医学研究中非常理想。传统的单级放大手段往往实现线性放大，难以满足日益增长的检测痕量目标的需求。我们希望构建多级级联放大器，以增强信号放大。本文成功设计了一个基于双层DNA电路的强大的非酶促mase传感平台，其中上游的催化发夹组装（CHA）电路依次产生DNA产物，该产物可用于激活下游的杂交链反应（HCR）电路，从而产生显着放大的荧光信号。在缺乏m.s sssi MTase的情况下，HpaII核酸内切酶可以特异性识别辅助发夹底物，然后催化切割相应的识别位点，释放DNA片段，触发cha - hcr介导的FRET转导。然而，msssi甲基化的发夹底物不能被HpaII酶切割，从而阻止cha - hcr介导的FRET产生，这与mtase缺失的系统提供了实质性的信号差异。利用MTase/核酸内切酶多重保证识别的高特异性和连接CHA-HCR电路的协同扩增特性，该方法可以在血清和大肠杆菌细胞中超灵敏地检测MTase及其抑制剂。此外，合理组装的CHA-HCR还允许通过相应底物的简单设计来探测其他不同的生物转化。预计无限层的多层DNA电路可以进一步提高系统的信号增益，以准确检测其他重要的生物标志物，从而在癌症治疗和生物医学研究中具有很大的前景。
Methyltransferase-involved DNA methylation is one of the most important epigenetic processes, making the ultrasensitive MTase assay highly desirable in clinical diagnosis as well as biomedical research. Traditional single-stage amplification means often achieve linear amplification that might not fulfill the increasing demands for detecting trace amount of target. It is desirable to construct multistage cascaded amplifiers that allow for enhanced signal amplifications. Herein, a powerful nonenzymatic MTase-sensing platform is successfully engineered based on a two-layered DNA circuit, in which the upstream catalytic hairpin assembly (CHA) circuit successively generates DNA product that could be used to activate the downstream hybridization chain reaction (HCR) circuit, resulting in the generation of a dramatically amplified fluorescence signal. In the absence of M.SssI MTase, HpaII endonuclease could specifically recognize the auxiliary hairpin substrate and then catalytically cleave the corresponding recognition site, releasing a DNA fragment that triggers the CHA-HCR-mediated FRET transduction. Yet the M.SssI-methylated hairpin substrate could not be cleaved by HpaII enzyme, and thus prohibits the CHA-HCR-mediated FRET generation, providing a substantial signal difference with that of MTase-absent system. Taking advantage of the high specificity of multiple-guaranteed recognitions of MTase/endonuclease and the synergistic amplification features of concatenated CHA-HCR circuit, this method enables an ultrasensitive detection of MTase and its inhibitors in serum and E. coli cells. Furthermore, the rationally assembled CHA-HCR also allows for probing other different biotransformations through a facile design of the corresponding substrates. It is anticipated that the infinite layer of multilayered DNA circuit could further improve the signal gain of the system for accurately detecting other important biomarkers, and thus holds great promise for cancerous treatment and biomedical research.
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影响因子：
7.4
作者：
Zhan Wu;Zhenkun Wu;Hao Tang;Li-Juan Tang;Jian-hui Jiang
通讯作者：
Zhan Wu;Zhenkun Wu;Hao Tang;Li-Juan Tang;Jian-hui Jiang
DOI：
10.1073/pnas.0407024101
发表时间：
2004-10-26
影响因子：
11.1
作者：
Dirks, RM;Pierce, NA
通讯作者：
Pierce, NA
影响因子：
7.4
作者：
Lopez Torres, Adolfo;Yanez Barrientos, Eunice;Wrobel, Kazimierz
通讯作者：
Wrobel, Kazimierz
影响因子：
2.9
作者：
Kim, BY;Kwon, OS;Ahn, JS
通讯作者：
Ahn, JS
影响因子：
7.4
作者：
Ke Quan;Jin Huang;Xiaohai Yang;Yanjing Yang;Le Ying;He Wang;Nuli Xie;Min Ou;Kemin Wang
通讯作者：
Ke Quan;Jin Huang;Xiaohai Yang;Yanjing Yang;Le Ying;He Wang;Nuli Xie;Min Ou;Kemin Wang