Zhang Siqi, Shao Huahao, Li Kai-Bin, Shi Wei, Wang Yichao, Han De-Man, Mo Jinggang
Department of Hepatobiliary Surgery, Taizhou Central Hospital (Taizhou University Hospital), Taizhou University, Jiaojiang, Zhejiang, 318000, China; School of Pharmaceutical and Chemical Engineering, Taizhou University, Jiaojiang, Zhejiang, 318000, China.
Zhijiang College of Zhejiang University of Technology, Shaoxing, Zhejiang, 312000, China.
Anal Chim Acta. 2023 Apr 29;1252:341057. doi: 10.1016/j.aca.2023.341057. Epub 2023 Mar 8.
The accurate and ultrasensitive detection of multiple methyltransferases was in great request for clinical diagnosis and epigenetic therapy. Here, a novel fluorescence assay was proposed for ultrasensitive CpG methyltransferase (M.SssI) and DNA adenine methyltransferase (Dam) activity detection based on hyperbranched rolling circle amplification (HRCA) and DNA walkers. The biosensor showed an extremely high sensitivity due to the dual-amplification strategy of HRCA and DNA walker. The LOD of the biosensor for M.SssI and Dam methyltransferase was estimated at 0.0004 U/mL and 0.001 U/mL, respectively. Without the presence of M.SssI methyltransferase, the corresponding recognition site of hairpin H was cleaved by HpaII endonuclease, generating a DNA fragment (T-DNA) and inducing the DNA walker-HRCA reaction. Since the HRCA products contained numerous double-strand DNA (dsDNA), SYBR Green I could be embedded in the dsDNA, leading to a high fluorescent signal. In the presence of M.SssI methyltransferase, the corresponding recognition site of hairpin H was methylated and the HpaII endonuclease-catalyzed stem of hairpin H dissociation was hindered, leading to no DNA fragment (T-DNA) present. Hence, the DNA walker-HRCA reaction was not initiated and the fluorescent signal of SYBR Green I remained at a low level. Similarly, DNA adenine methyltransferase (Dam) and its inhibitors could also be detected by redesigning hairpin H with the Dam recognition sequences. Furthermore, the sensing system was applied to analyze the endogenic Dam methyltransferase in the real samples such as E. coli cell lysate.
对多种甲基转移酶进行准确且超灵敏的检测在临床诊断和表观遗传治疗中具有迫切需求。在此，基于超支化滚环扩增（HRCA）和DNA步行器，提出了一种用于超灵敏检测CpG甲基转移酶（M.SssI）和DNA腺嘌呤甲基转移酶（Dam）活性的新型荧光测定法。由于HRCA和DNA步行器的双重扩增策略，该生物传感器显示出极高的灵敏度。该生物传感器对M.SssI和Dam甲基转移酶的检测限分别估计为0.0004 U/mL和0.001 U/mL。在不存在M.SssI甲基转移酶的情况下，发夹H的相应识别位点被HpaII核酸内切酶切割，产生一个DNA片段（T-DNA）并诱导DNA步行器-HRCA反应。由于HRCA产物包含大量双链DNA（dsDNA），SYBR Green I可嵌入dsDNA中，导致高荧光信号。在存在M.SssI甲基转移酶的情况下，发夹H的相应识别位点被甲基化，HpaII核酸内切酶催化的发夹H茎解离受到阻碍，导致不存在DNA片段（T-DNA）。因此，DNA步行器-HRCA反应未启动，SYBR Green I的荧光信号保持在低水平。同样，通过重新设计带有Dam识别序列的发夹H，也可以检测DNA腺嘌呤甲基转移酶（Dam）及其抑制剂。此外，该传感系统被应用于分析实际样品（如大肠杆菌细胞裂解物）中的内源性Dam甲基转移酶。
