ANALYST
·
2014
·Vol. 139
·期 24
·6387-6392
·
IF 3.906
Chunyan Gao,Henan Li,Yuanjian Liu,Wei Wei,Yuanjian Zhang,Songqin Liu
Strategies to detect the methylation of site specific DNA and assay of M.SssI methyltransferase (M.SssI MTase) activity are important in determining human cancers due to aberrant methylation linked to cancer initiation and progression. Herein, we report a label-free fluorescence detection method for DNA methylation and MTase activity based on restriction endonuclease HpaII and exonuclease III (Exo III). A label-free probe DNA was designed, which hybridized with target DNA (one 32-mer DNA from the exon 8 promoter region of the Homo sapiens p53 gene) to form double stranded DNA (dsDNA). Upon the cleavage action of HpaII and degradation reaction of Exo III, dsDNA changed to single stranded DNA (ssDNA) and the fluorescence intensity of thiazole orange (TO) is weak. After the resulting dsDNA was methylated by M.SssI MTase, the action of HpaII and Exo III was prevented, then TO intercalates into the dsDNA and emits strong fluorescence. This method can determine DNA methylation at the site of CpG and distinguish a one-base mismatched target sequence. The fluorescence intensity has a linear relationship with M.SssI MTase activities in the range of 1–10 U mL−1 with a detection limit of 0.16 U mL−1 in terms of 3 times deviation of the blank sample. The methylation of DNA by a hydroxyl radical triggered by DMSO and CH3CHO was also measured. These results show that the proposed method can specifically and selectively detect DNA methylation and M.SssI MTase activity. Human serum has no obvious effects on the assay performance, indicating that the method has great potential for further application in complex samples.
研究领域:
生物化学
·
分子生物学
·
遗传学
·
癌症研究
✓ 已验证
DOI
PubMed
高影响因子
BIOSENSORS & BIOELECTRONICS
·
2015
·
IF 6.409
Linqun Zhang,Min Wei,Chunyan Gao,Wei Wei,Yuanjian Zhang,Songqin Liu
Detection of DNA methylation and methyltransferase (MTase) activity are important in determining human cancer because aberrant methylation was linked to cancer initiation and progression. In this work, we proposed an electrochemical method for sensitive detection of DNA methylation and MTase activity based on methylation sensitive restriction endonuclease HpaII and the deposition of polyaniline (PANI) catalyzed by HRP-mimicking DNAzyme. In the presence of methylated DNA, HRP-mimicking DNAzyme catalyzed the polymerization of aniline on the dsDNA template, producing huge DPV current. In the presence of non-methylated DNA, dsDNA are cleaved and digested by HpaII and exonuclease III , as a result, no PANI are deposited. This method can be used to determine DNA methylation at the site of CpG. It exhibits a wide linear response toward M.SssI MTase activity in the range of 0.5–0.6 U mL −1 with the detection limit of 0.12 U mL −1 . G-rich DNA forms HRP mimicking DNAzyme, which avoids complex labeling procedures and is robust. The method is simple, reliable, sensitive and specific, which has been successfully applied in human serum samples and been used to screen the inhibitors. Thus, the proposed method may be a potential and powerful tool for clinical diagnosis and drug development in the future.
研究领域:
生物化学
·
医学诊断
·
分子生物学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
ANALYTICAL AND BIOANALYTICAL CHEMISTRY
·
2023
·Vol. 415
·期 12
·2271-2280
·
IF 4.3
An Yaqian,Yu Zhiqi,Liu Di,Han Lirong,Zhang Xian,Xin Xuelian,Li Cuiping
The detection of methyltransferase (MTase) activity is of great significance in methylation-related disease diagnosis and drug screening. Herein, a HpaII-assisted and linear amplification-enhanced exponential amplification strategy is proposed for sensitive and label-free detection of M.SssI MTase activity. The P1 probe contains self-complementary sequence 5′-CTAGCCGGCTAG-3′ at 3′-terminal. After denaturation and annealing, P1 probes hybridize with itself to generate P1 duplexes. M.SssI MTase induces methylation of cytosine at 5′-CG-3′ in P1 duplexes, and thus, HpaII fails to cleave at 5′-CCGG-3′ due to methylation sensitivity, leaving P1 duplex intact. Then, these intact P1 duplexes are extended along 3′-terminal through Vent (exo − ) DNA polymerase to generate dsDNA, which is recognized and nicked at the recognition sites by Nt.BstNBI, releasing two copies of primer X. Primer X hybridizes with X′ at the amplification template T1 (X′-Y′-X′) and then serves as primers to trigger the exponential amplification reaction (EXPAR). The point of inflection (POI) values of real-time fluorescence curves is linearly correlated with the logarithm of M.SssI MTase concentration in the range of 0.125 \(-\) 8 U mL −1 with a low detection limit of 0.034 U mL −1 . In the absence of M.SssI, P1 duplexes are cut by HpaII and separated into ssDNA under the executed temperature of EXPAR and thus unable to trigger the amplification. The strategy provides good selectivity against other types of MTases and protein and is able to detect M.SssI activity in human serum. Furthermore, the analytical method has the generality and can be extended to the analysis of other types of DNA MTases. Graphical Abstract
研究领域:
生物化学
·
分子生物学
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分析化学
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生物技术
✓ 已验证
产品关联
DOI
PubMed
高影响因子
SENSORS AND ACTUATORS B-CHEMICAL
·
2017
·
IF 5.401
Haiyan Wang,Pei Liu,Wenjing Jiang,Xue Li,Huanshun Yin,Shiyun Ai
DNA methylation is one of the important epigenetics events, and it is responsible for transcription, genomic imprinting and cellular differentiation. Aberrant DNA methylation is always contacted with various diseases. Herein, a simple and low-cost photoelectrochemical (PEC) biosensing method is proposed for detection of DNA methylation, assay of DNA methyltransferase (MTase) activity and screening of MTase inhibitor, which is based on M. SssI MTase- HpaII endonuclease system. The graphite-like C 3 N 4 (g-C 3 N 4 ) and CdS quantum dots (CdS QDs) are used as photoactive materials. After the double-stranded DNA (dsDNA) was treated with M. SssI MTase in the presence of S -adenosylmethionine, the methylated dsDNA cannot be digested by HpaII endonuclease and dithiol group at the terminal of dsDNA can be retained. As a result, CdS QDs could be coupled successfully with methylated dsDNA between the reaction of CdS QDs with dithiol of dsDNA, achieving the amplification of PEC response. Thus, the photocurrent of PEC biosensor is consistent with the M. SssI activity. The increased photocurrent was in proportion to M. SssI activity in the linear range from 1 to 80 U/mL with a detection limit of 0.316 U/mL. Furthermore, the effects of atrazine and azamethipos on M. SssI MTase activity were investigated, which might provide useful information on carcinogenic mechanism of pesticide. The PEC biosensor has potential to screen of DNA MTase inhibitor, providing valuable information for anti-cancer drug research and also for cancer therapy.
研究领域:
生物化学
·
分子生物学
·
分析化学
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产品关联
DOI
高影响因子
JOURNAL OF ELECTROANALYTICAL CHEMISTRY
·
2018
·
IF 3.235
Jing Bao,Xintong Geng,Changjun Hou,Yanan Zhao,Danqun Huo,You Wang,Zhaonan Wang,Yan Zeng,Mei Yang,Huanbao Fa
Many studies have confirmed that DNA methylation is highly correlated with the occurrence and development of various diseases including cancers. In this work, we developed a simple, sensitive, selective, and universal electrochemical biosensor for detection of DNA methylation and assay of DNA methyltransferase (MTase) activity using M.SssI MTase as an example. The thiolated single-stranded DNA S1 was self-assembled on the surface of gold nanoparticles deposition modified glassy carbon electrode via Au S bonding, then hybridization between the DNA S1 and its complementary DNA S2 formed a double-stranded target sequence for both M.SssI MTase and restriction endonuclease Hpa II. HpaII could not cleave the target sequence after it was methylated by M.SssI MTase, while the sequence without methylation could be cleaved. Here, we used methylene blue (MB) as electrochemical indicator. The electrochemical signal of MB increased linearly with increasing M.SssI MTase concentration from 0.5 to 25 U/mL and from 25 to 400 U/mL with a detection limit of 0.04 U/mL. Moreover, screening of M.SssI MTase inhibitors 5-azacytidine (5-Aza) and 5-Aza-2′-deoxycytidine (5-Aza-dC) were successfully investigated using the fabricated electrochemical biosensor and showed that the two classic drugs could both inhibit the M.SssI MTase activity with the IC 50 of 2.8 μM and 0.37 μM, respectively, indicating potential application in discovery of new anticancer drugs.
研究领域:
生物化学
·
分子生物学
·
分析化学
·
癌症研究
✓ 已验证
产品关联
DOI
高影响因子
ANALYTICAL CHEMISTRY
·
2012
·
IF 5.856
Mo Wang,Zhenning Xu,Lijian Chen,Huanshun Yin,Shiyun Ai
In this paper, we developed a novel electrochemical method to quantify DNA methyltransferase (MTase) and analyze DNA MTase activity. After the double DNA helix structure was assembled on the surface of gold nanoparticle modified glassy carbon electrode, it was first methylated by M. SssI MTase and then digested by restriction endonuclease HpaII, which could not recognize the methylated CpG site. Successively, anti-5-methylcytosine antibody was specifically conjugated on the CpG methylation site and horseradish peroxidase labeled goat antimouse IgG (HRP-IgG) was conjugated on anti-5-methylcytosine antibody. In the detection buffer solution containing H2O2 and hydroquinone, HRP-IgG can catalyze hydroquinone oxidation by H2O2 to generate benzoquinone, resulting in a highly electrochemical reduction signal. Consequently, the activity of M. SssI MTase was assayed, and DNA methylation was detected using the signal change with and without methylation. Furthermore, the inhibition investigation demonstrated that, in the presence of 160 μM S-adenosyl-l-methionine as methyl donor, 5-aza-2′-deoxycytidine, procaine, epicatechin, and caffeic acid could inhibit the M. SssI MTase activity with the IC50 values of 45.77, 410.3, 129.03, and 124.2 μM, respectively. Therefore, this study may provide a sensitive platform for screening DNA MTase inhibitors.
研究领域:
生物化学
·
分析化学
·
分子生物学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
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