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1.
Anal Chem ; 92(19): 13573-13580, 2020 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-32927942

RESUMO

DNA methylation plays important roles in various biological processes, and the alteration of DNA methyltransferase activity can induce the aberrant DNA methylation patterns. Despite the progress in methyltransferase activity assays, few methods enable the detection of both bacteria and human methyltransferases. Herein, we construct a universal and label-free chemiluminescent sensor for accurate quantification of both bacteria methyltransferases (e.g., M. SssI methyltransferase (M.SssI MTase)) and human methyltransferases (e.g., DNA (cytosine-5)-methyltransferase 1, (Dnmt1)) by integrating a dumbbell probe with BssHII endonuclease-mediated rolling circle amplification (RCA). We ingeniously design a structure-switchable dumbbell probe which integrates target-recognition, BssHII endonuclease-cleavage, RCA amplification and signal transduction in one probe for the detection of both M.SssI MTase and Dnmt1. Moreover, the introduction of two BssHII endonuclease recognition sites in a dumbbell probe can greatly reduce the false positivity resulting from the incomplete cleavage of dumbbell probe by BssHII, because once one of two recognition sites is identified by BssHII, the dumbbell probe can be completely digested by Exonuclease III (Exo III) and Exonuclease I (Exo I) to prevent the nonspecific RCA. This chemiluminescent sensor can accurately quantify M.SssI MTase in both 10% serum and various cell lysis buffers, and even sensitively detect Dnmt1 activity in MCF-7 cells. Furthermore, this chemiluminescent sensor can be used to screen the inhibitors of Dnmt1 and M.SssI MTase, with promising applications in disease diagnosis and drug discovery.


Assuntos
DNA (Citosina-5-)-Metiltransferase 1/análise , DNA-Citosina Metilases/análise , Medições Luminescentes , Spiroplasma/enzimologia , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , DNA-Citosina Metilases/metabolismo , Humanos
2.
Nanoscale ; 12(7): 4519-4526, 2020 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-32039424

RESUMO

CpG island methylation plays an important role in diverse biological processes including the regulation of imprinted genes, X chromosome inactivation, and tumor suppressor gene silencing in human cancer. Due to the dependence of DNA methylation on DNA methyltransferase (MTase) activity, DNA MTases have become the potential targets in anticancer therapy. Herein we demonstrate for the first time the construction of a single quantum dot (QD) nanosensor with the capability of sensing methylcytosine sites for sensitive quantification of M.SssI CpG methyltransferase (M.SssI MTase). We design a biotin-/phosphate-modified double-stranded DNA (dsDNA) substrate with a 5'-G-C-G-mC-3'/3'-mC-G-mC-G-5' site for sensing M.SssI MTase. In the presence of M.SssI MTase, the methylation-responsive sequence of the dsDNA substrate is methylated and cleaved by GlaI endonuclease, producing two dsDNA fragments with a free 3'-OH terminus. In the presence of terminal deoxynucleotidyl transferase (TdT), multiple Cy5-dATPs can be sequentially added to the free 3'-OH terminus of dsDNA fragments to obtain biotin-/multiple Cy5-labeled dsDNAs. The resultant biotin-/multiple Cy5-labeled dsDNAs can assemble on the surface of the streptavidin-coated QD to obtain a QD-dsDNA-Cy5 nanostructure in which the fluorescence resonance energy transfer (FRET) from the QD to Cy5 can occur. The emission of Cy5 can be simply quantified by single-molecule detection. By the integration of sensing methylcytosine sites and enzymatic polymerization, the sensitivity of this nanosensor has been significantly enhanced. This nanosensor can detect as low as 2.1 × 10-7 U µL-1 M.SssI MTase with good selectivity against other cytosine MTases, and it can be further applied for the screening of MTase inhibitors and complex biological sample analysis, holding great potential in clinical diagnosis and drug discovery.


Assuntos
Técnicas Biossensoriais , Metilação de DNA , DNA-Citosina Metilases/análise , DNA/química , Pontos Quânticos/química
3.
ACS Sens ; 3(11): 2359-2366, 2018 11 26.
Artigo em Inglês | MEDLINE | ID: mdl-30350594

RESUMO

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.


Assuntos
DNA Concatenado/química , DNA-Citosina Metilases/análise , Ensaios Enzimáticos/métodos , Técnicas Biossensoriais/métodos , Metilação de DNA , DNA Concatenado/genética , DNA-Citosina Metilases/química , Desoxirribonuclease HpaII/química , Escherichia coli/enzimologia , Fluoresceínas/química , Fluorescência , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/química , Haemophilus parainfluenzae/enzimologia , Sequências Repetidas Invertidas , Limite de Detecção , Técnicas de Amplificação de Ácido Nucleico/métodos , Hibridização de Ácido Nucleico , Spiroplasma/enzimologia
4.
Talanta ; 189: 579-584, 2018 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-30086963

RESUMO

DNA methyltransferase (MTase) has a crucial role in many biological processes, its abnormal expression level has been regarded as a predictive cancer biomarker. Herein, a sensitive fluorescence method based on thermosensitive poly (N-isopr-opylacrylamide) was developed to assay of M.SssI activity. When the M.SssI was introduced, dsDNA was methylated at palindromic sequence 5'-CmCGG-3' and became resistant to cleavage by the endonuclease HpaII. Therefore, a biotin modified ssDNA and a FAM modified ssDNA were designed including the recognized sites for both methyltransferase M.SssI and endonuclease HpaII. By SA-biotin intereaction, the DNA was conjugated to thermosensitive poly (N-isopropylacrylamide) modified by SA, the methylated substrate fluorescence was increased with the concentration of M.SssI increasing. The proposed method has a low detection limit of 0.18 U/mL. This simple method can be a useful tool to apply in diagnosis and biomedical research, which was successfully investigated in the serum sample.


Assuntos
Resinas Acrílicas/química , Técnicas Biossensoriais/métodos , DNA-Citosina Metilases/análise , Limite de Detecção , Temperatura , Biotina/metabolismo , DNA-Citosina Metilases/química , DNA-Citosina Metilases/metabolismo , Espectrometria de Fluorescência , Estreptavidina/metabolismo
5.
Methods Mol Biol ; 1811: 173-182, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29926453

RESUMO

DNA methylation entails the covalent addition of a methyl group to C-5 position of cytosine by a family of DNA methyltransferase enzymes and has a significant role in gene regulation. Epigenetic changes such as DNA methylation of CpG islands located in the promoter region of some tumor suppressor genes are very common in human diseases such as cancer. Detection of aberrant methylation pattern could serve as an excellent diagnostic approach. It is key to develop methods for the direct and simple detection of methylated DNA or of methyltransferase activity without using antibodies, chemical modification, labeling and enzymatic treatments. In this study, we employ DNA-templated silver nanoclusters for detection of DNA methylation. This method entails use of cytosine rich DNA sequence as an effective template. By monitoring changes in fluorescence intensity, DNA methylation and DNA methyltransferase activity is detected. Upon DNA methylation, the fluorescence intensity of DNA templated Ag/NCs is decreased in a linear range when the concentration of methylated DNA is increased.


Assuntos
Metilação de DNA , DNA-Citosina Metilases/análise , Prata/química , Técnicas Biossensoriais/métodos , Ilhas de CpG , Epigênese Genética , Fluorescência , Nanopartículas Metálicas/química , Microscopia Eletrônica de Transmissão
6.
Biosens Bioelectron ; 91: 417-423, 2017 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-28063390

RESUMO

Sensitive and reliable detection of DNA methyltransferase (MTase) is of great significance for both early tumor diagnosis and therapy. In this study, a simple, label-free and sensitive DNA MTase-sensing method was developed on the basis of a nicking endonuclease-mediated multiple primers-like rolling circle amplification (RCA) strategy. In this method, a dumbbell RCA template was prepared by blunt-end ligation of two molecules of hairpin DNA. In addition to the primer-binding sequence, the dumbbell template contained another three important parts: 5'-CCGG-3' sequences in double-stranded stems, nicking endonuclease recognition sites and C-rich sequences in single-stranded loops. The introduction of 5'-CCGG-3' sequences allows the dumbbell template to be destroyed by the restriction endonuclease, HpaII, but is not destroyed in the presence of the target MTase-M.SssI MTase. The introduction of nicking endonuclease recognition sites makes the M.SssI MTase-protected dumbbell template-mediated RCA proceed in a multiple primers-like exponential mode, thus providing the RCA with high amplification efficiency. The introduction of C-rich sequences may promote the folding of amplification products into a G-quadruplex structure, which is specifically recognized by the commercially available fluorescent probe thioflavin T. Improved RCA amplification efficiency and specific fluorescent recognition of RCA products provide the M.SssI MTase-sensing platform with high sensitivity. When a dumbbell template containing four nicking endonuclease sites is used, highly specific M.SssI MTase activity detection can be achieved in the range of 0.008-50U/mL with a detection limit as low as 0.0011U/mL. Simple experimental operation and mix-and-detection fluorescent sensing mode ensures that M.SssI MTase quantitation works well in a real-time RCA mode, thus further simplifying the sensing performance and making high throughput detection possible. The proposed MTase-sensing strategy was also demonstrated to be applicable for screening and evaluating the inhibitory activity of MTase inhibitors.


Assuntos
Técnicas Biossensoriais/métodos , DNA (Citosina-5-)-Metiltransferases/análise , DNA-Citosina Metilases/análise , Espectrometria de Fluorescência/métodos , DNA/química , DNA/metabolismo , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/metabolismo , Primers do DNA/química , Primers do DNA/metabolismo , DNA-Citosina Metilases/metabolismo , Endonucleases/metabolismo , Ensaios Enzimáticos/métodos , Quadruplex G , Células HEK293 , Células HeLa , Humanos , Células MCF-7 , Técnicas de Amplificação de Ácido Nucleico/métodos
7.
Anal Bioanal Chem ; 408(21): 5867-5872, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27311957

RESUMO

A simple method for highly sensitive and selective detection of M.SssI CpG methyltransferase (M.SssI MTase) activity is developed, leveraging on the portability and ease of use of a personal glucose meter (PGM). Briefly, DNA-invertase conjugates are hybridized with their complementary DNA strands pre-immobilized on magnetic beads. The 5'-CCGG-3' sequence present in the DNA duplexes serves as the recognition site for both Hpa II restriction enzyme and M.SssI MTase (5'-CG-3'). Hpa II restriction enzyme specifically cleaves at unmethylated 5'-CCGG-3' sequence, and the invertase that remains on the methylated DNA catalyzes the hydrolysis of sucrose to glucose and fructose. It is found that the amount of glucose is proportional to the M.SssI MTase methylation activity in the range of 0.5 to 80 U/mL with a detection limit of 0.37 U/mL. Due to the specific recognition sequence present in the DNA strands, this method also shows high selectivity for M.SssI MTase. In addition, inhibition studies with 5'-azacytidine demonstrate the capability of inhibition screening using this method. Graphical abstract Deteciton of M.SssI DNA methyltransferase activity by a personal glucose meter.


Assuntos
Técnicas Biossensoriais/métodos , Automonitorização da Glicemia/métodos , DNA-Citosina Metilases/análise , Técnicas Eletroquímicas/métodos , Glucose/análise , DNA-Citosina Metilases/metabolismo , Ensaios Enzimáticos/métodos , Glucose/metabolismo , Células HeLa , Humanos , Ácidos Nucleicos Imobilizados/química , Ácidos Nucleicos Imobilizados/metabolismo , Limite de Detecção , Células MCF-7 , Imãs/química
8.
Biosens Bioelectron ; 73: 188-194, 2015 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-26070170

RESUMO

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.


Assuntos
Técnicas Biossensoriais/métodos , Metilação de DNA , DNA-Citosina Metilases/análise , Técnicas Eletroquímicas/métodos , Compostos de Anilina , Sondas de DNA , DNA Catalítico , DNA-Citosina Metilases/antagonistas & inibidores , DNA-Citosina Metilases/sangue , Desoxirribonuclease HpaII , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/farmacologia , Humanos , Limite de Detecção
9.
Biosens Bioelectron ; 73: 228-233, 2015 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-26086442

RESUMO

In this manuscript, a nanosilver enhanced SERS strategy was successfully constructed for the determination of DNA methyltransferase activity in soulution combined with hybridization chain reaction (HCR). The proposed method was mainly on the basis of excellent separation ability of magnetic microparticles (MMPs), HCR as signal amplification unit and assembled AgNPs as enhancement substrate. In the presence of M. SssI MTase, the duplex sequence (5'-CCGG-3') tethered to MMPs was methylated, which cannot be cleaved by HpaII endonuclease. The resulted DNA skeleton captured on MMPs then triggered the HCR reaction, generated a polymerized and extended symmetrical sequence, in which more biotin terminal was available for the conjugation of AgNPs-SA, leading to significantly amplified SERS response. When it was used to analyze M. SssI activity, a linear equation ∆ISERS=1215.32+446.80 cM.SssI was obtained with the M. SssI activity ranged from 0.1 to 10.0 U with the correlation coefficient (r(2)) of 0.97. The most important advantage of this method is the combination of SERS and HCR in solution for the first time and its good selectivity, which enabled the detection of even one-base mismatched sequence. The new assay method holds great promising application to be a versatile platform for sensitive, high-throughput detection, and the screening of new anticancer drugs on DNA MTase.


Assuntos
Metilases de Modificação do DNA/análise , Análise Espectral Raman/métodos , Técnicas Biossensoriais/métodos , Técnicas Biossensoriais/estatística & dados numéricos , Sondas de DNA , DNA-Citosina Metilases/análise , Nanopartículas Metálicas , Prata
10.
Biosens Bioelectron ; 49: 39-45, 2013 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-23708816

RESUMO

In this work, we fabricated a novel electrochemical immunosensor for detection of DNA methylation, analysis of DNA MTase activity and screening of MTase inhibitor. The immunosensor was on the basis of methyl binding domain protein of MeCP2 as DNA CpG methylation recognization unit, anti-His tag antibody as "immuno-bridge" and horseradish peroxidase labeled immuneglobulin G functionalized gold nanoparticles (AuNPs-IgG-HRP) as signal amplification unit. In the presence of M. SssI MTase, the symmetrical sequence of 5'-CCGG-3' was methylated and then recognized by MeCP2 protein. By the immunoreactions, anti-His tag antibody and AuNPs-IgG-HRP was captured on the electrode surface successively. Under the catalysis effect of HRP towards hydroquinone oxidized by H2O2, the electrochemical reduction signal of benzoquinone was used to analyze M. SssI MTase activity. The electrochemical reduction signal demonstrated a wide linear relationship with M. SssI concentration ranging from 0.05 unit/mL to 90 unit/mL, achieving a detection limit of 0.017 unit/mL (S/N=3). The most important advantages of this method were its high sensitivity and good selectivity, which enabled the detection of even one-base mismatched sequence. In addition, we also verified that the developed method could be applied for screening the inhibitors of DNA MTase and for developing new anticancer drugs.


Assuntos
Técnicas Biossensoriais/métodos , Metilação de DNA , DNA-Citosina Metilases/antagonistas & inibidores , DNA-Citosina Metilases/metabolismo , DNA-Citosina Metilases/análise , Técnicas Eletroquímicas/métodos , Inibidores Enzimáticos/farmacologia , Peroxidase do Rábano Silvestre/química , Humanos , Imunoensaio/métodos , Imunoglobulina G/química , Limite de Detecção , Proteína 2 de Ligação a Metil-CpG/metabolismo
11.
J Intern Med ; 261(5): 488-99, 2007 May.
Artigo em Inglês | MEDLINE | ID: mdl-17444888

RESUMO

OBJECTIVE: The lifespan of dialysis patients is as short as in patients with metastatic cancer disease, mainly due to cardiovascular disease (CVD). DNA methylation is an important cellular mechanism modulating gene expression associated with ageing, inflammation and atherosclerotic processes. DESIGN: DNA methylation was analysed in peripheral blood leucocytes from three different groups of chronic kidney disease (CKD) populations (37 CKD stages 3 and 4 patients, 98 CKD stage 5 patients and 20 prevalent haemodialysis patients). Thirty-six healthy subjects served as controls. Clinical characteristics (diabetes mellitus, nutritional status and presence of clinical CVD), inflammation and oxidative stress biomarkers, homocysteine and global DNA methylation in peripheral blood leucocytes (defined as HpaII/MspI ratio by the Luminometric Methylation Assay method) were evaluated. CKD stage 5 patients (n=98) starting dialysis treatment were followed for a period of 36 +/- 2 months. RESULTS: Inflamed patients had lower ratios of HpaII/MspI, indicating global DNA hypermethylation. Analysis by the Cox regression model demonstrated that DNA hypermethylation (HpaII/MspI ratio

Assuntos
Doenças Cardiovasculares/genética , Metilação de DNA , Epigênese Genética/genética , Nefropatias/genética , Biomarcadores/sangue , Doenças Cardiovasculares/metabolismo , Doenças Cardiovasculares/mortalidade , Doença Crônica , DNA-Citosina Metilases/análise , DNA-Citosina Metilases/metabolismo , Feminino , Ácido Fólico/sangue , Homocisteína/sangue , Humanos , Inflamação/genética , Inflamação/metabolismo , Nefropatias/metabolismo , Nefropatias/mortalidade , Masculino , Pessoa de Meia-Idade , Estresse Oxidativo/genética , Reprodutibilidade dos Testes , Fatores de Risco
12.
Cell ; 71(7): 1073-80, 1992 Dec 24.
Artigo em Inglês | MEDLINE | ID: mdl-1473145

RESUMO

HpaII methylase (M. HpaII), an example of a DNA (cytosine-5)-methyltransferase, was found to induce directly a high frequency of C-->U transition mutations in double-stranded DNA. A mutant pSV2-neo plasmid, constructed with an inactivating T-->C transition mutation creating a CCGG site, was incubated with M. HpaII in the absence of S-adenosylmethionine (SAM). This caused an approximately 10(4)-fold increase in the rate of reversion when the mutant neo plasmid was transformed into bacteria lacking uracil-DNA glycosylase. The mutation frequency was very sensitive to SAM concentration and was reduced to background when the concentration of the methyl donor exceeded 300 nM. The data support current models for the formation of a covalent complex between the methyltransferase and cytosine. They also suggest that the occurrence of mutational hot spots at CpG sites may not always be due to spontaneous deamination of 5-methylcytosine, but might also be initiated by enzymatic deamination of cytosine and proceed through a C-->U-->T pathway.


Assuntos
Citosina/metabolismo , DNA-Citosina Metilases/metabolismo , DNA/metabolismo , S-Adenosil-Homocisteína/farmacologia , Sequência de Bases , DNA-Citosina Metilases/análise , Desaminação , Metilação , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação
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