Methylated dsDNA Research Articles

Hybridization-based CpG methylation level detection using methyl-CpG-binding domain–fused luciferase

Hypermethylation of tumor-suppressor genes and global hypomethylation, which is related to methylation level at the retroelement, have been recognized as features of the cancer genome. In this study, we developed a hybridization-based CpG methylation level detection method using methyl-CpG-binding domain-fused firefly luciferase (MBD-Fluc). In this method, methylated probe oligonucleotides were used to capture target oligonucleotides. Fully methylated and hemimethylated double-stranded DNA (dsDNA) was formed by hybridization of the methylated captured oligonucleotides with methylated or unmethylated target oligonucleotides, respectively. MBD-Fluc specifically binds to fully methylated dsDNA but not to hemimethylated dsDNA; therefore, methylated target oligonucleotides can be detected by measuring the luciferase activity of the bound MBD-Fluc. Using the corresponding methylated probe oligonucleotides, the CpG methylation levels of SEPT9, BRCA1, and long interspersed nuclear element-1 (LINE-1) oligonucleotides were quantified. Moreover, we demonstrated that the emission detection signal was not affected by the methylation state of the overhang region of the target oligonucleotide, which was not hybridized to the probe oligonucleotide, indicating that methylated CpG of the target region could be accurately detected. Unmethylated-CpG-binding domain-fused luciferases and 5-hydroxymethyl-CpG-binding domain-fused luciferases have been constructed, suggesting that other modified bases can be detected by the same platform.

Dual-signal amplified photoelectrochemical assay for DNA methyltransferase activity based on RGO-CdS:Mn nanoparticles and a CdTe@DNA network

DNA methyltransferase (DNA MTase) has been shown to catalyze DNA methylation, which is closely associated with cancers. Therefore, developing detection technology for DNA MTase activity is significant for early diagnosis of cancer. Herein, a new and ultrasensitive photoelectrochemical (PEC) biosensor was fabricated to analyze DNA MTase activity using a dual-signal amplified system, where RGO-CdS:Mn nanoparticles and a CdTe@DNA network were used as photoelectric materials. RGO-CdS:Mn nanoparticles were first modified on a gold electrode as substrate materials. Then, probe DNA and target DNA with a recognition sequence for DNA methylation were assembled on the electrode. After the process of methylation and digestion, the methylated dsDNA could be reserved and further hybridized with a CdTe@DNA network, leading to a stronger photocurrent. As expected, the PEC responses were in proportion to the logarithm of M.SssI MTase concentration from 0.01 to 80 U/mL with a detection limit of 0.0071 U/mL. This proposed method showed ideal detection ability for DNA MTase activity and could provide a new idea as well as technology in cancer diagnosis.

Single-Molecule Fluorescence Imaging for Ultrasensitive DNA Methyltransferase Activity Measurement and Inhibitor Screening.

Aberrant DNA methylation by DNA methyltransferases (MTase) is related to the initiation and progression of many diseases. Thus, site-specific identification of DNA methylation and detection of MTase activity are very important to diagnose and treat methylation-related diseases. Herein, a single-molecule counting based ultrasensitive assay was developed for facile and direct detection of MTase activity and inhibitor screening without the assistance of restriction endonuclease. A double-strand DNA (dsDNA) was designed with the recognition site of M. SssI MTase and assembled on the coverslip surface. After the dsDNA was methylated by M. SssI, the biotin conjugated anti-5-methylcytosine antibody (5mC Ab) would specifically bind the CpG methylation site, and subsequently, the streptavidin-labeled quantum dots (QS585) bind the biotins. By taking and counting the image spots of fluorescently labeled methylated dsDNA molecules, the single-molecule imaging of methylated dsDNA molecules was recorded to quantify the DNA MTase activity. The spot number shows a linear relation with the logarithm of M. SssI concentration in the concentration range of 0.001-1 U/mL. Compared with most of the state of the art methods, the proposed assay displays a lower detection limit of 0.0005 U/mL and can detect the DNA MTase more directly. Moreover, it can selectively detect M. SssI in more complex samples. In addition, it is further demonstrated that the protocol could be successfully applied to evaluate the inhibition efficiency of M. SssI inhibitors. This assay is anticipated to provide a new approach for clinical diagnosis of methylation-related diseases and screening of new anticancer drugs.

Fluorescent copper nanoclusters as a nano-dye for DNA methyltransferase activity analysis and inhibitor screening

Fluorescent copper nanoslusters (CuNCs) as a new class of fluorophores have attracted more and more attention due to their ease of synthesis, excellent optical properties, and low cost. In this study, a novel label-free fluorescent method was developed for the detection of DNA methyltransferases based on template length-dependent of dsDNA-CuNCs. In the absence of DNA adenine methylation methyltransferase (Dam MTase), the dsDNA containing the methylation-responsive sequence could effectively template the formation of fluorescent CuNCs with bright fluorescence. When the dsDNA substrate is methylated by Dam MTase, the methylation-sensitive restriction endonuclease Dpn I cleaves the methylated dsDNA and produces shorter dsDNA product, which fails to template fluorescent CuNCs. So, the Dam MTase activity could be identified by the changes of CuNCs' fluorescence. Based on this method, a linear range of 0.5–10 U/mL was achieved with high sensitivity and selectivity. Moreover, we also demonstrate the proposed method can be applied to evaluation and screening of inhibitors for Dam MTase.

A microchip electrophoretic assay for DNA methyltransferase activity based on methylation-sensitive endonuclease DpnⅡ.

DNA methylation is a significant epigenetic modification and the methods for the detection of DNA methyltransferase (MTase) activity are important due to aberrant methylation closely related to the occurrence of cancer. In this study, a simple and rapid microchip electrophoresis (ME) coupled with LED-induced fluorescence (LEDIF) method was presented for the detection of Dam MTase activity. This strategy was based on methylation-sensitive endonuclease DpnⅡ which could recognize the same specific site 5'-GATC-3' with Dam MTase in double-stranded DNA (dsDNA). The adenines in the specific site could be methylated by Dam MTase, then the special site could not be digested by DpnⅡ. Both methylated dsDNA and unmethylated dsDNA could be analyzed by ME-LEDIF after stained by SYBR gold. The results showed the fluorescence intensities of methylated dsDNA were directly proportional to Dam MTase activities in the range of 0.5-20U/mL with a detection limit of 0.12U/mL. Furthermore, the method could successfully be applied to evaluation experiments of Dam MTase inhibitors. The results confirmed the ME-LEDIF method is a promising approach for inhibitors screening of DNA MTase and development of anticancer drugs.

Commercial glucometer as signal transducer for simple evaluation of DNA methyltransferase activity and inhibitors screening

DNA methyltransferase (MTase) plays an important role in many biological processes and has been recognized as a predictive cancer biomarker far before other signs of malignancy and a therapeutic target in cancer treatment. Thus simple and sensitive determination of DNA MTase activity is urgently required. The commercially available glucometer is considered as the most successful point-of-care (POC) sensor up to date, and researchers extend its application in monitoring different types of targets rather than only glucose. Here, we developed a simple strategy for the sensitive detection of the DNA MTase (using M.SssI as an example) activity by using a glucometer as the signal transducer. A S1/S2 hybrid probe was designed including a specific recognition sequence for both DNA MTase and restriction endonuclease, and a complementary sequence for biotin-S3. Firstly, the S1/S2 hybrid probe was self-assembled on the gold electrode and methylated by M.SssI MTase to form the methylated dsDNA. Then, HpaII endonuclease specifically cleaved the residue of the unmethylated dsDNA. Subsequently, biotin-S3 hybridized with the overhang sequence of the methylated dsDNA. Finally, the biotin tag was successively combined with streptavidin (STV) and biotin-invertase. The invertase efficiently catalyzed the hydrolysis of sucrose to generate abundant glucose, which led to an amplified response of glucometer. This strategy could detect DNA MTase activity as low as 0.3 U mL−1 with good selectivity against other two cytosine MTases (HaeIII MTase and AluI MTase), and be successfully applied for screening the DNA MTase inhibitors (5-azacytidine and 5-aza-2′-deoxycytidine), implying our proposed method holds great promising application in early cancer diagnosis and therapeutics.

Study on the Interaction of the CpG Alternating DNA with CdTe Quantum Dots.

A novel sensitive method for detection of DNA methylation was developed with thioglycollic acid (TGA)-capped CdTe quantum dots (QDs) as fluorescence probes. Recognition of methylated DNA sites would be useful strategy due to the important roles of methylation in disease occurrence and developmental processes. DNA methylation occurs most often at cytosine-guanine sites (CpG dinucleotides) of gene promoters. The QDs significantly interacted with hybridized unmethylated and methylated DNA. The interaction of CpG rich methylated and unmethylated DNA hybrid with quantum dots as an optical probe has been investigated by fluorescence spectroscopy and electrophoresis assay. The fluorescence intensity of QDs was highly dependent to unmethylated and methylated DNA. Specific site of CpG islands of Adenomatous polyposis coli (APC), a well-studied tumor suppressor gene, was used as the detection target. Under optimum conditions, upon the addition of unmethylated dsDNA, the fluorescence intensity increased in linear range from 1.0 × 10- 10 to 1.0 × 10- 6M with detection limit of 6.2 × 10- 11 M and on the other hand, the intensity of QDs showed no changes with addition of methylated dsDNA. We also demonstrated that the unmethylated and methylated DNA and QDs complexes showed different mobility in electrophoresis assay. This easy and reliable method could distinguish between methylated and unmethylated DNA sequences.

Ultrasensitive and Accurate Assay of Human Methyltransferase Activity at the Single-Cell Level Based on a Single Integrated Magnetic Microprobe.

Human DNA methyltransferase (MTase) activity expression patterns and inhibition response are linked to related cancer initiation, progression, and therapeutic responses. Sensitive and accurate human MTase activity assay in cancer cells, especially at the single-cell level, is essential for biological study, clinical diagnosis, and therapy. Here, we developed an ultrasensitive and accurate DNA (cytosine-5)-methyltransferase 1 (Dnmt1) activity assay at the single-cell level based on a single integrated magnetic microprobe of functionalized double-stranded DNA (dsDNA) anchored to a single magnetic microbead surface. Functionalized dsDNA is designed with a hemimethylated DNA site for Dnmt1 recognition and a single-stranded tail to trigger in situ rolling circle amplification (RCA). Under the action of Dnmt1, hemimethylated dsDNA could be recognized and catalyzed to fully methylated dsDNA, which would protect them from the cleavage of BssHII. However, the dsDNA without full methylation would be cut by BssHII, making single-stranded tail separated from the single integrated microprobe. Subsequently, full methylation-protected in situ RCA could be performed, and multiple signal probes were hybridized to the single integrated microprobe for amplified signal accumulation. Finally, Dnmt1 activity could be evaluated by reading the fluorescence of the single integrated microprobe. Meanwhile, to minimize matrix interferences, magnetic separation was performed in the process. In this strategy, the single integrated magnetic microprobe was provided with integrated capacities of target recognition, signal amplification, signal accumulation, and matrix isolation. Therefore, an ultralow detection limit of 0.007 U/mL Dnmt1 was obtained, and accurate Dnmt1 activity assays in multiple cell lysates at the single-cell level were achieved. Furthermore, the inhibition effect of RG108 was evaluated conveniently. These results indicate that the single integrated magnetic microprobe-based strategy is an excellent candidate for sensitive monitoring of Dnmt1 activity and screening of anticancer drugs.

Photoelectrochemical immunosensing platform for M. SssI methyltransferase activity analysis and inhibitor screening based on g-C3N4 and CdS quantum dots

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 C3N4 (g-C3N4) 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 80U/mL with a detection limit of 0.316U/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.

Spectroscopic Study of CpG Alternating DNA-Methylene Blue Interaction for Methylation Detection.

Recognition of methylated DNA sites would be useful strategy due to the important roles of methylation in disease occurrence and developmental processes. The interaction of CpG rich methylated and unmethylated DNA hybrid with methylene blue (MB) as an optical probe has been investigated by absorption, emission, circular dichorism and fluorescence anisotropy analysis. Titration of MB with both sequences caused a hypsochromism and decreased the absorption of MB that indicating an intercalative mode of interaction. The experimental results revealed that MB as the optical indicator could distinguish between the methylated and unmethylated DNA sequences. Under optimum conditions, upon the addition of methylated dsDNA, the fluorescence intensity increased in linear range from 1.0×10(-9) to 1.0×10(-6)M with detection limit of 7.2×10(-10)M and on the other hand, the intensity of MB showed no change with addition of unmethylated dsDNA.

Structural and Functional Analyses of IDPs by High-Speed AFM Imaging

The structure of intrinsically disordered proteins (IDPs) is highly flexible and dynamically samples a multitude of conformational states. Therefore, the structural characterization of IDPs is considerably challenging. Here, we developed a single-molecule method to analyze their structure, dynamics and function, using high-speed atomic force microscopy (HS-AFM). A mechanical property of constantly or fully disordered regions within IDPs was found to be independent of their amino-acid sequences. Thanks to the invariance of this property, HS-AFM imaging of IDPs allowed us to estimate the number of amino acids contained in these regions as well as to identify and characterize at the residue level other regions undergoing order-to-disorder transitions. As a test sample for functional visualization by HS-AFM, we imaged the methyl-CpG-binding protein MeCP2, whose gene mutations cause neurodevelopmental disorder called Rett syndrome. Its methyl-CpG binding domain (MBD) was observed to undergo order-to-disorder transitions, whereas other regions were mostly or constantly disordered. In MBDs of several mutated MeCP2, this two-state equilibrium was shifted to the disordered state with different degrees. This disorder propensity was found to be tightly correlated to the lower activity of MeCP2 to condense methylated dsDNA, and hence, their lower activity to condense DNA must be directly involved in causing Rett syndrome. Thus, HS-AFM imaging of single molecules opens a new opportunity to readily acquire site-resolved, quantitative and dynamic structural information on IDPs in real space as well as to study how minute changes in the structure and dynamics of IDPs are connected to diseases.

A dual amplification fluorescent strategy for sensitive detection of DNA methyltransferase activity based on strand displacement amplification and DNAzyme amplification.

DNA methyltransferase (MTase) plays a critical role in many biological processes and has been regarded as a predictive cancer biomarker and a therapeutic target in cancer treatment. Sensitive detection of DNA MTase activity is essential for early cancer diagnosis and therapeutics. Here, we developed a dual amplification fluorescent strategy for sensitive detection of DNA MTase activity based on strand displacement amplification (SDA) and DNAzyme amplification. A trifunctional double-stranded DNA (dsDNA) probe was designed including a methylation site for DNA MTase recognition, a complementary sequence of 8-17 DNAzyme for synthesizing DNAzyme, and a nicking site for nicking enzyme cleavage. Firstly, the trifunctional dsDNA probe was methylated by DNA MTase to form the methylated dsDNA. Subsequently, HpaII restriction endonuclease specifically cleaved the residue of unmethylated dsDNA. Next, under the action of polymerase and nicking enzyme, the methylared dsDNA initiated SDA, releasing numbers of 8-17 DNAzymes. Finally, the released 8-17 DNAzymes triggered DNAzyme amplification reaction to induce a significant fluorescence enhancement. This strategy could detect DNA MTase activity as low as 0.0082U/mL. Additionally, the strategy was successfully applied for evaluating the inhibitions of DNA MTase using two anticancer drugs, 5-azacytidine and 5-aza-2'-deoxycytidine. The results indicate the proposed strategy has a potential application in early cancer diagnosis and therapeutics.

Proximity-based electrochemical biosensor for highly sensitive determination of methyltransferase activity using gold nanoparticle-based cooperative signal amplification

We describe a turn-on electrochemical biosensor for the detection of methyltransferases (MTases) causing DNA adenine methylation. This biosensor is based on insertion, methylation-resistant cleavage, signal enrichment caused by gold nanoparticles (AuNPs), and a signal probe-dragging strategy. A double-stranded DNA (dsDNA) containing identical MTase and methylation-resistant endonuclease (Mbo I) sites was immobilized on the surface of a gold electrode via Au-S covalent binding. The surface was subsequently treated with MTase and Mbo I and then washed. Results revealed that the surface of the electrode contains methylated dsDNA and 12-base nucleotides residual. Depending on biotin-streptavidin interactions that enabled signal probes and nucleotide residue hybridization and AuNP enrichment, a large number of signal probes labeled with ferrocene (Fc) are captured by the electrode. Under optimal conditions, the differential pulse voltammetry signals of Fc tags (at a working voltage of 0.24 V vs. Ag/AgCl) are linearly related to the log of the MTase activity in the 0.1 to 40 U·mL−1 range. The dynamic range extends from 0.05 to 50 U·mL−1, and the limit of detection is 0.024 U·mL−1 (at an S/N ratio of 3). The assay is well reproducible and highly selective. In our perception, this strategy provides a promising approach for simple, sensitive and selective detection of Dam MTase and may be extended to the determination of other MTase by exchanging the corresponding DNA.

An electrochemical one-step system for assaying methyltransferase activity based on transport of a quantum dot signaling tracer

A one-step, electrochemical method for assaying methyltransferase (MTase) activity, based on the convective transport of a quantum dot (QD) signaling tracer, has been developed. The assay chip used in this system was prepared by modifying a gold matrix with CdSe/ZnS QD-tagged dsDNA, which contains a specific methylation site (5′–GATC-3′) recognized by MTase. Treatment of the chip with DNA adenine methylation (Dam) MTase, generates a methylated sequence (5′-GAmTC-3′) within the dsDNA. The methylated dsDNA is then subjected to a cleavage reaction, induced by DpnI, which leads to release from the gold matrix of a DNA fragment tethered to a QD. Detection of the released QD, using square wave anodic stripping voltammetry (SWASV) on a glassy carbon (GC) electrode, enables the reliable quantitation of the methylated DNA. Because it is accomplished in a simple and convenient one step and does not require any complicated secondary or tedious washing steps, the new assay method holds great promise for epigenetic analysis in facility-limited environments or point-of-care testing (POCT) applications.

Effect of Methylation on the Nanomechanical Properties of Double-Stranded DNA

In mammalian cells 60-90% of cytosines in the genome are methylated. The methylation sites are unevenly distributed and are often found in clusters called “CpG islands”. Approximately 70% of promoters in the human genome contain or are preceeded by CG-rich regions, suggesting that methylation may be important in gene regulation. Cyclization-kinetic and nucleosome-binding assays suggest that methylation may significantly affect DNA flexibility. However, a direct effect of methylation on the mechanics of DNA is yet unknown. To investigate the impact of methylation on DNA mechanics, here we manipulated single molecules of methylated dsDNA and compared their nanomechanical properties with those of unmethylated DNA.A 3500-base-pair sequence of lambda-phage DNA composed almost entirely of CpG islands was cloned by using PCR containing dm5CTP to produce the fully methylated product. Individual DNA molecules were mechanically manipulated in stretch and relaxation cycles by using custom-built dual-beam counter-propagating optical tweezers. Force versus extension data were fitted with the extensible wormlike-chain model to obtain the contour length, the persistence length (entropic component of rigidity) and the stretch modulus (enthalpic component of rigidity) of dsDNA. Methylation reduced the contour length and stretch modulus of dsDNA from 1036±22 nm to 966±8 nm and from 1225±115 pN to 373±30 pN, respectively. Persistence length was 34±2 nm for the non-methylated and 35±2 nm in case of the hypermethylated DNA. The observed changes may be caused by a complex shift in tertiary structure, accounting for both the reduction of the contour length and the increase of the intrinsic compliance of the dsDNA chain. The methylation-induced effects on the nanomechanical properties of dsDNA may play an important role in the regulation of steric access to its sequence-specific sites.

Cloning of Nt.CviQII nicking endonuclease and its cognate methyltransferase: M.CviQII methylates AG sequences

Chlorella virus NY-2A has a large, highly methylated dsDNA genome (45% of the cytosines are 5-methylcytosine and 37% of the adenines are N 6-methyladenine). Here, we report the cloning, expression, and characterization of the NY-2A-encoded CviQII nicking-modification (N-M) system. The nicking endonuclease, Nt.CviQII, recognizes R ↓ AG (R = A or G, ↓ indicating cleavage site) sequences and cleaves the phosphodiester bond 5′ to the adenosine. Because of the difficulty in cloning and expressing the wild-type Nt.CviQII, C-terminal truncation mutants were generated and full-length Nt.CviQII was reconstructed by intein-mediated peptide ligation. The truncation mutants and the reconstructed full-length Nt.CviQII have the same recognition and cleavage specificity as the native enzyme. Full-length and truncated Nt.CviQII produced by a cell-free transcription/translation system have similar reaction rates. The methyltransferase, M.CviQII, was also cloned and expressed. It modifies the adenine in AG doublets of DNA in vitro and in vivo in Escherichia coli. To our knowledge, M.CviQII is the first adenine methyltransferase that recognizes a dinucleotide. Therefore, M.CviQII may be a useful reagent for blocking endonuclease cleavage when restriction sites overlap with AG sequences.