Methyltransferase Activity Assay Research Articles

Real-time monitoring of DNA methyltransferase activity using a hemimethylated smart probe

A real-time assay for DNA methyltransferase (MTase) activity has been developed. A hemimethylated smart probe is used as the substrate for DNA MTase. Cleavage of the methylated product leads to separation of fluorophore from quencher, giving a proportional increase in fluorescence. The method permits real-time monitoring of DNA methylation process and makes it easy to characterize the activity of DNA MTase. It also has the potential to screen suitable inhibitor drugs for DNA MTase.

A colorimetric assay of DNA methyltransferase activity based on the keypad lock of duplex DNA modified meso-SiO2@Fe3O4

Abnormal level of DNA methyltransferase (MTase) – mediated DNA methylation is closely related with cancer and bacterial diseases. Herein, a novel strategy based on the keypad lock of duplex DNA modified meso-SiO2@Fe3O4 was developed for colorimetric assay of Dam MTase activity. When the Dam MTase was introduced, the duplex DNA can be methylated at a palindrome sequence of 5′-GATC-3′ and cleaved by the methylation-sensitive restriction endonuclease Dpn I. Due to the instability of the newly formed DNA fragment, the hybrid will separated into a single-stranded DNA. Then the keypad lock will open, and the catalytic reaction of TMB and H2O2 can be initiated through the pores of meso-SiO2@Fe3O4, and a high color signal can be clearly observed by the naked eye. Contrarily, without Dam MTase, the catalytic reaction will not be initiated, and result no color signal. The proposed method exhibited a wide dynamic range with a low detection limit of 0.73 U/mL. Additionally, this way can be performed in human serum with satisfying recovery. And the inhibition of Dam MTase can also be well demonstrated by using paclitaxel as a model. Therefore, the designed way not only provides a platform for monitoring Dam MTase activity, but also useful for further application in disease diagnosis and drug discovery.

Sensitive SERS detection of DNA methyltransferase by target triggering primer generation-based multiple signal amplification strategy

A novel and sensitive surface-enhanced Raman scattering (SERS) method is proposed for the assay of DNA methyltransferase (MTase) activity and evaluation of inhibitors by developing a target triggering primer generation-based multiple signal amplification strategy. By using of a duplex substrate for Dam MTase, two hairpin templates and a Raman probe, multiple signal amplification mode is achieved. Once recognized by Dam MTase, the duplex substrate can be cleaved by Dpn I endonuclease and two primers are released for triggering the multiple signal amplification reaction. Consequently, a wide dynamic range and remarkably high sensitivity are obtained under isothermal conditions. The detection limit is 2.57×10−4UmL−1. This assay exhibits an excellent selectivity and is successfully applied in the screening of inhibitors for Dam MTase. In addition, this novel sensing system is potentially universal as the recognition element can be conveniently designed for other target analytes by changing the substrate of DNA MTase.

Efficient Enhancement of Electrochemiluminescence from Cadmium Sulfide Quantum Dots by Glucose Oxidase Mimicking Gold Nanoparticles for Highly Sensitive Assay of Methyltransferase Activity.

Herein, an original electrochemiluminescence (ECL) method for the detection of DNA methyltransferase (MTase) activity is presented based on the efficient enhanced ECL of CdS quantum dots (QDs) through catalytic generation of coreactant and energy transfer by glucose oxidase mimicking gold nanoparticles (Au NPs). Briefly, a double-stranded DNA (ds-DNA) containing the symmetric sequence of 5'-CCGG-3' was bonded to the CdS QDs modified glassy carbon electrode (GCE). After that, the electrode was incubated with M.SssI CpG MTase which catalyzed the methylation of the specific CpG dinucleotides. Subsequently, the electrode was treated with a restriction endonuclease HpaII which could recognize and cut off the 5'-CCGG-3' sequence. Once the CpG site in the 5'-CCGG-3' was methylated, the recognition function of HpaII was blocked, and it could not cut off the ds-DNA. Later, Au NPs were combined with the end of the ds-DNA section which was not cut off and has -SH groups. Therefore, the higher M.SssI MTase activity could lead to more Au NPs immobilized on ds-DNA. Au NPs could not only catalyze the oxidation of glucose with cosubstrate oxygen, producing gluconate and hydrogen peroxide (H2O2) which served as the ECL coreactant of CdS QDs, but also enhanced CdS QDs ECL via energy transfer (ET). Thus, the methylation event corresponding to the MTase activity could be monitored and amplified by this method. Finally, a logarithmic linear correlation between the ECL intensity of CdS QDs and the activity of M.SssI MTase that ranged from 1.0 to 120 U mL(-1) with the detection limit of 0.05 U mL(-1) was obtained.

Abstract A34: Decline in Arylsulfatase B activates Wnt signaling by effects on DKK-3 methylation

Abstract Immunostaining and activity of the enzyme Arylsulfatase B (ARSB; N-acetylgalactosamine 4-sulfatase) were reduced in prostatic malignancies, and decline in ARSB was associated with higher Gleason score and biochemical recurrence in two prostate cancer tissue microarrays. Since the only known function of ARSB is to remove 4-sulfate groups from chondroitin 4-sulfate (C4S) and dermatan sulfate, a mechanism for this association of ARSB with more aggressive prostate cancer was unknown. Subsequent studies showed transcriptional effects of ARSB, mediated by reduced binding of galectin-3 to the more highly sulfated C4S present when ARSB was reduced. Decline in ARSB induced increased expression of the extracellular matrix proteoglycan versican, which has chondroitin 4-sulfate attachments and has been shown to be increased in more aggressive prostate cancers. The increase in versican was mediated by increased nuclear galectin-3 and AP-1 effects on the versican promoter. Other experiments following decline in ARSB showed increased expression of Wnt9A, mediated by increased nuclear galectin-3 and Sp1. These findings suggested that ARSB acted as a tumor suppressor gene and decline in ARSB enhanced tumor progression. Experiments were performed to address transcriptional mechanisms by which decline in ARSB and the accompanying increase in chondroitin 4-sulfation could activate Wnt signaling and lead to more aggressive prostate cancers. The impact of ARSB on Wnt signaling was evaluated in prostate tissue from the ARSB-null mouse, and human prostate stromal (WPMY-1, ATCC), epithelial (RWPE-1, ATCC), and stem (WPE-stem, ATCC) cell lines. The effects of ARSB on Wnt signaling were assessed by ELISA, Western blot, silencing by siRNA, ARSB overexpression, DNA methyltransferase (DNMT) activity assays, QRT-PCR, and measurements of promoter methylation. Decline in ARSB increased Wnt signaling, manifested by increases in nuclear beta-catenin, DNA-bound TCF-LEF, nuclear c-myc and cyclin D1. To determine if increased Wnt signaling was attributable to reduction of the inhibitory effect of Dickkopf (DKK), an established negative regulator of Wnt signaling and tumor suppressor, the expression of DKK 1-4 was determined by QRT-PCR. DKK-3 expression in the prostate cells following ARSB silencing and in the ARSB-null tissue was significantly reduced. ARSB overexpression increased the DKK-3 expression. DNA methyltransferase (DNMT) activity was significantly increased when ARSB was silenced, and reduced when ARSB was overexpressed. Expression of DNMT 1 and 3a was increased in the ARSB-null mouse prostate tissue and prostate cells, consistent with the decline in DKK-3 expression. Determinations of DKK-3 promoter methylation showed increased methylation following ARSB silencing and in the malignant prostate tissue. Hence, decline in ARSB activated Wnt signaling by increased methylation of DKK-3, leading to disinhibition of Wnt signaling, attributable to reduced binding of DKK-3 with the Frizzled - LRP5/6 receptor complex. These findings demonstrate an epigenetic mechanism whereby decline in ARSB increases Wnt signaling in prostate tissue and can lead to more aggressive prostate malignancy. Also, the role of ARSB as a tumor suppressor gene is supported by this demonstration of inverse effects of decline and overexpression of ARSB on Wnt signaling and on DNMT expression and activity. Experiments are ongoing to clarify the intermediates in the pathway by which ARSB affects DNMT expression. Note: This abstract was not presented at the conference. Citation Format: Sumit Bhattacharyya, Leo Feferman, Joanne Kramer Tobacman. Decline in Arylsulfatase B activates Wnt signaling by effects on DKK-3 methylation. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr A34.

DNA–gold nanoparticles network based electrochemical biosensors for DNA MTase activity

In this work, a highly sensitive electrochemical DNA methyltransferase (MTase) activity assay was fabricated with DNA–gold nanoparticles (Au NPs) network as signal amplification unit and an easy assembly method by the linkage of benzenedithiol bridge. By two complementary AuNPs modified single-stranded DNA, DNA–gold nanoparticles network was self-assembled. With the linkage of benzenedithiol bridge, the DNA network structure was immobilized on the surface of gold electrode through the covalent Au–S bond. In the presence of Dam MTase, the special sites of DNA–AuNPs network were methylated and could not be digested by restriction endonuclease Mbo I. Thus the loaded electrochemical indicator Methylene blue (MB) was MB molecules still remained on the DNA–Au NPs network. The electrochemical response depended on the methylated degree, which could be used to detect MTase activity. By the differential pulse voltammetry (DPV), it was demonstrated that a linear relationship between the DPV response and logarithm of Dam concentration ranged from 0.075 to 30U/mL, achieving a low detection limit of 0.02U/mL. The use of benzenedithiol avoided the direct incubation of the solid electrode with the capture DNA probe under complex and harsh conditions. Therefore the immobilization of DNA–AuNPs network was easy to be carried out, which is favorable for the specially high stability and reproducibility of the electrochemical biosensor.

Target-protected dumbbell molecular probe mediated cascade rolling circle amplification strategy for the sensitive assay of DNA methyltransferase activity.

A novel fluorescence detection system was developed for DNA methyltransferase (MTase) activity assay based on a target-protected dumbbell molecular probe mediated cascade rolling circle amplification. It showed excellent specificity and sensitivity with a detection limit of 0.0024 U mL(-1), and potential application in quantitatively monitoring MTase activity and screening of anticancer drugs.

A novel label-free fluorescence strategy for methyltransferase activity assay based on dsDNA-templated copper nanoparticles coupled with an endonuclease-assisted signal transduction system.

Evaluating DNA methyltransferase (MTase) activity has received considerable attention due to its significance in the fields of early cancer clinical diagnostics and drug discovery. Herein, we proposed a novel label-free fluorescence method for MTase activity assay by coupling double-stranded DNA (dsDNA)-templated copper nanoparticles (CuNPs) with an endonuclease-assisted signal transduction system. In this strategy, dsDNA molecules were first methylated by DNA adenine methylation (Dam) MTase and then cleaved by the methylation-sensitive restriction endonuclease DpnI. The cleaved DNA fragments could not act as efficient templates for the formation of fluorescent CuNPs and thus no fluorescence signal was produced. Under optimized experimental conditions, the developed strategy exhibited a sensitive fluorescence response to Dam MTase activity. This strategy was also demonstrated to provide an excellent platform to the inhibitor screening for Dam MTase. These results demonstrated the great potential for the practical applications of the proposed strategy for Dam MTase activity assay.

Methyltransferase activity assay based on the use of exonuclease III, the hemin/G-quadruplex system and reduced graphene oxide on a gold electrode, and a study on enzyme inhibition

We describe an electrochemical bioassay for the detection of the activity of methyltransferase (MTase), and for screening this enzyme’s inhibitors. The assay is based on the conjugation of a hemin to a G-quadruplex that enables enzymatic signal amplification with the aid of exonuclease III (ExoIII). In the first step, double-stranded DNA containing the quadruplex-forming oligomer is assembled on the surface of a gold electrode and then methylated by DNA adenine methyltransferase (DAM). After cleaved by endonuclease DpnI, the methylated DNA is digested by ExoIII and the quadruplex-forming oligomers are liberated. This leads to the formation of a hemin/G-quadruplex (in presence of hemin and of potassium ions). The hemin/G-quadruplex catalyzes the oxidization of hydroquinone by H2O2 and the benzoquinone was formed to generate electrochemical signal. Finally, the gold electrode modified with reduced graphene oxide was used as working electrode for performing differential pulse voltammetry. The method has a detection limit of 0.31 unit · mL−1. A study on the inhibition of MTase showed it was inhibited by epicatechin with an IC50 value of 157 μM.

Ultrasensitive homogeneous electrochemical strategy for DNA methyltransferase activity assay based on autonomous exonuclease III-assisted isothermal cycling signal amplification.

DNA methylation catalyzed by methyltransferase (MTase) plays an important role in many biological processes, including gene transcription, genomic imprinting and cellular differentiation. Herein, a simple and novel homogeneous electrochemical strategy for ultrasensitive DNA MTase activity assay has been successfully developed, which is based on methylation-triggered exonuclease (Exo) III-assisted autonomous isothermal cycling signal amplification. A duplex DNA (P1-P2 hybrid) containing the methylation-responsive sequence is ingeniously designed. In the presence of DNA adenine methylation (Dam) methyltransferase (MTase), P1-P2 hybrid is methylated and subsequently recognized and cleaved by Dpn I endonuclease, which triggers the Exo III-catalyzed autonomous cycling cleavage processes. Therefore, a large amount of methylene blue-labeled mononucleotides are released, generating a significantly amplified electrochemical signal toward the Dam MTase activity assay. The directly measured detection limit down to 0.004 U/mL is obtained, which is one or two orders magnitude lower than that of the approaches reported in literature. Since this assay is carried out in homogeneous solution phase under isothermal condition and sophisticated probe immobilization processes are avoided, it is very simple and easy to implement. Due to its advantages of ultrahigh sensitivity, excellent selectivity and simple operation, the as-proposed strategy has great potential in the applications in DNA methylation related clinical practices and biochemical researches.

A highly sensitive fluorescence assay for methyltransferase activity by exonuclease-aided signal amplification.

DNA methylation, catalyzed by methyltransferases, plays critical roles in various biological processes in both prokaryotes and eukaryotes. Bacterial DNA adenine methyltransferases (DAM) are associated with bacterial pathogenesis and essential for bacterial virulence and viability. Since mammals do not methylate DNA at adenine, bacterial DAM is considered to be a great candidate target for developing new therapeutics for diseases. In the current study, we developed a simple, rapid and highly sensitive fluorescence method for the detection of DAM based on exonuclease-aided signal amplification. In the proposed strategy, a liberated amplifier upon DAM methylation and Dpn I digestion of the substrate can hybridize with a reporter (FT) that contains a quencher (TAMRA) at the second base of the 3' end and a fluorophore (FAM) at the fifth base. Upon hybridization, exonuclease III degrades the reporter in the formed duplex DNA from the 3' end successively, releasing the fluorophore from the quencher and resulting in an intensive appearance of the fluorescent signal. The amplifier will hybridize with another reporter and enter a new cycle, which therefore can amplify the signal and dramatically increase the detection sensitivity even with an extremely low amount of amplifier. Using this strategy, the detection limit down to 0.0025 U mL(-1) of DAM was achieved within a short assay time of 30 min. Furthermore, the assay was applied to evaluate endogenous DAM activity in E. coli cell at different growth stages as well as the effects of inhibitors on DAM activity. Given the attractive analytical performance, the sensing strategy may find many important applications in biomedical research and clinical diagnosis.

MoS2 nanosheets as an effective fluorescence quencher for DNA methyltransferase activity detection.

As one of the inorganic graphene analogues, two dimensional MoS2 nanosheets have been drawing extensive attention in the past few years due to their remarkable structural and electronic properties. Herein, a simple signal-on fluorescence DNA methyltransferase (MTase) activity assay using a MoS2 nanosheet mediated fluorescence quenching strategy is described. Briefly, substrate DNA is designed to possess a double-stranded DNA (ds-DNA) segment containing the recognition sequence of DNA adenosine methyltransferase (Dam) and a single-stranded DNA (ss-DNA) segment for anchoring the substrate DNA to MoS2 nanosheets via van der Waals interactions. Once the substrate DNA is absorbed onto MoS2 nanosheets, the fluorescence of the fluorophores labeled to the end of the ds-DNA region is quenched. When the substrate DNA is methylated by Dam, fluorescence is recovered resulting from the release of the fluorophore labeled segment cleaved by the methylation sensitive restriction endonuclease DpnI. Since the fluorescence recovery directly reflects the methylation level, the Dam MTase activity can be quantified accordingly. Based on this assay, a linear range of 0.2-20 U mL(-1) is achieved with high sensitivity and selectivity. Furthermore, the inhibitor screening ability is well demonstrated.

A label-free electrochemical strategy for highly sensitive methyltransferase activity assays.

A new electrochemical strategy for the simple and ultrasensitive evaluation of DNA methyltransferase (MTase) activity was developed based on electrocatalytic oxidation of ascorbic acid by graphene. In addition, the suitability of this sensing platform for MTase inhibitor screening was also demonstrated.

Highly sensitive fluorescence assay of DNA methyltransferase activity by methylation-sensitive cleavage-based primer generation exponential isothermal amplification-induced G-quadruplex formation

Site-specific identification of DNA methylation and assay of MTase activity are imperative for determining specific cancer types, provide insights into the mechanism of gene repression, and develop novel drugs to treat methylation-related diseases. Herein, we developed a highly sensitive fluorescence assay of DNA methyltransferase by methylation-sensitive cleavage-based primer generation exponential isothermal amplification (PG-EXPA) coupled with supramolecular fluorescent Zinc(II)-protoporphyrin IX (ZnPPIX)/G-quadruplex. In the presence of DNA adenine methylation (Dam) MTase, the methylation-responsive sequence of hairpin probe is methylated and cleaved by the methylation-sensitive restriction endonuclease Dpn I. The cleaved hairpin probe then functions as a signal primer to initiate the exponential isothermal amplification reaction (EXPAR) by hybridizing with a unimolecular DNA containing three functional domains as the amplification template, producing a large number of G-quadruplex nanostructures by utilizing polymerases and nicking enzymes as mechanical activators. The G-quadruplex nanostructures act as host for ZnPPIX that lead to supramolecular complexes ZnPPIX/G-quadruplex, which provides optical labels for amplified fluorescence detection of Dam MTase. While in the absence of Dam MTase, neither methylation/cleavage nor PG-EXPA reaction can be initiated and no fluorescence signal is observed. The proposed method exhibits a wide dynamic range from 0.0002 to 20U/mL and an extremely low detection limit of 8.6×10−5U/mL, which is superior to most conventional approaches for the MTase assay. Owing to the specific site recognition of MTase toward its substrate, the proposed sensing system was able to readily discriminate Dam MTase from other MTase such as M.SssI and even detect the target in a complex biological matrix. Furthermore, the application of the proposed sensing strategy for screening Dam MTase inhibitors was also demonstrated with satisfactory results. This novel method not only provides a promising platform for monitoring activity and inhibition of DNA MTases, but also shows great potentials in biological process researches, drugs discovery and clinical diagnostics.

Highly sensitive homogeneous electrochemical assay for methyltransferase activity based on methylation-responsive exonuclease III-assisted signal amplification

DNA methylation catalyzed by methyltransferase (MTase) plays a critical role in many biological processes. In this paper, a novel and highly sensitive homogeneous electrochemical assay was developed for the detection of DNA MTase based on methylation-responsive exonuclease III-assisted signal amplification. Upon the action of MTase/endonuclease on hairpin probe 1 (HP 1) containing the methylation-responsive sequence, single-stranded DNA segments are generated to hybridize with methylene blue (MB)-labeled hairpin probe 2 (HP 2). Then the digestion of HP 2 from the blunt 3′ terminus by exonuclease III is activated, resulting in the release of MB-labeled mononucleotides and the complementary DNA segment which could hybridize with another HP 2 to initiate the signal amplification process. The MB-labeled mononucleotide, due to its less negative charge and smaller size, diffuses easily to the negatively charged indium tin oxide (ITO) electrode, generating an amplified electrochemical signal. The detection limit of the proposed assay was estimated to be 0.04U/mL, which was better than or comparable to that of the biosensors previously reported. To the best of our knowledge, it is the first time to adopt exonuclease III-assisted signal amplification for homogeneous electrochemical assay of MTase activity, and this strategy exhibits the advantages of high sensitivity as well as simplicity. Since this assay is carried out in a homogeneous solution phase instead of on an electrode/solution interface, sophisticated probe immobilization processes could be avoided. The as-proposed strategy exhibits promising potential for MTase functional studies and related researches.

Highly sensitive photoelectrochemical assay for DNA methyltransferase activity and inhibitor screening by exciton energy transfer coupled with enzyme cleavage biosensing strategy

Highly sensitive DNA methyltransferase (MTase) activity and inhibitor screening photoelectrochemical (PEC) assay was developed based on the exciton energy transfer (EET) effect coupled with site-specific cleavage of restriction endonuclease (HpaII). The assay was designed by integrating the Au nanoparticles (NPs) labeled probe DNA (pDNA-Au) with CdSe quantum dots (QDs). The strong EET effect between Au NPs and CdSe QDs resulted in the dramatic decrease of photocurrent signal. The pDNA carried a sensing region for specifically recognizing target DNA (tDNA) and hybridizing with it to form a DNA duplex. With the site-specific cleavage of HpaII, the DNA duplex could be cleaved and Au NPs would be released, which broke the EET and resulted in the restoration of photocurrent signal. However, when the DNA duplex was methylated by M.SssI MTase, this cleavage of HpaII was blocked, and therefore the unbroken EET effect kept the lower photocurrent signal. That was, the restored photocurrent was inversely proportional to the MTase activity. Based on this strategy, the PEC assay could determine as low as ~0.0042U/mL of M.SssI MTase with a linear range from 0.01 to 150U/mL. In addition, the assay could be used for the screening of the inhibitors of MTase. This PEC assay provides a promising platform for monitoring the activity and inhibition of DNA MTase, and thus shows a great potential in cancer diagnostics and anti-cancer drugs discovery.

Gold nanorods-based FRET assay for ultrasensitive detection of DNA methylation and DNA methyltransferase activity.

A fluorescence method for the detection of DNA methylation and the assay of methyltransferase activity is proposed using gold nanorods as a fluorescence quencher on the basis of fluorescence resonance energy transfer. It is demonstrated that this method is capable of detecting methyltransferase with a detection limit of 0.25 U mL(-1), which might make this method a good candidate for monitoring DNA methylation in the future.

Antroquinonol D, isolated from Antrodia camphorata, with DNA demethylation and anticancer potential.

DNA methyltransferase 1 (DNMT1) catalyzes DNA methylation and is overexpressed in various human diseases, including cancer. A rational approach to preventing tumorigenesis involves the use of pharmacologic inhibitors of DNA methylation; these inhibitors should reactivate tumor suppressor genes (TSGs) in tumor cells and restore tumor suppressor pathways. Antroquinonol D (3-demethoxyl antroquinonol), a new DNMT1 inhibitor, was isolated from Antrodia camphorata and identified using nuclear magnetic resonance. Antroquinonol D inhibited the growth of MCF7, T47D, and MDA-MB-231 breast cancer cells without harming normal MCF10A and IMR-90 cells. The SRB assay showed that the 50% growth inhibition (GI50) in MCF7, T47D, and MDA-MB-231 breast cancer cells following treatment with antroquinonol D was 8.01, 3.57, and 25.08 μM, respectively. d-Antroquinonol also inhibited the migratory ability of MDA-MB-231 breast cancer cells in wound healing and Transwell assays. In addition, antroquinonol D inhibited DNMT1 activity, as assessed by the DNMT1 methyltransferase activity assay. As the cofactor SAM level increased, the inhibitory effects of d-antroquinonol on DNMT1 gradually decreased. An enzyme activity assay and molecular modeling revealed that antroquinonol D is bound to the catalytic domain of DNMT1 and competes for the same binding pocket in the DNMT1 enzyme as the cofactor SAM, but does not compete for the binding pocket in the DNMT3B enzyme. An Illumina Methylation 450 K array-based assay and real-time PCR assay revealed that antroquinonol D decreased the methylation status and reactivated the expression of multiple TSGs in MDA-MB-231 breast cancer cells. In conclusion, we showed that antroquinonol D induces DNA demethylation and the recovery of multiple tumor suppressor genes, while inhibiting breast cancer growth and migration potential.

Highly Sensitive Electrochemical Methyltransferase Activity Assay

A simple and highly sensitive electrochemical DNA methyltransferase (MTase) activity assay is presented in this report. The assay employs the electrocatalytic oxidation of ascorbic acid (AA) by a threading intercalator (N,N'-bis(3-propylimidazole)-1,4,5,8-naphthalene diimide (PIND) functionalized with electrocatalytic redox Os(bpy)2Cl(+) moieties (PIND-Os)). Briefly, a double-stranded DNA (ds-DNA) containing the symmetric sequence of 5'-CCGG-3' is first immobilized on a gold electrode. The electrode is then incubated with M.SssI CpG methyltransferase (M.SssI MTase) which catalyzes the methylation of the specific CpG dinucleotides, and the electrode is subsequently treated with a restriction endonuclease HpaII which recognizes the 5'-CCGG-3' sequence. Once the CpG site in the 5'-CCGG-3' is methylated, HpaII recognition is blocked. Higher M.SssI MTase activity leads to more CpG sites being methylated and consequently impedes more the restriction endonuclease HpaII digestion process. Thus, a larger amount of ds-DNA remains on the electrode surface after the HpaII treatment. Thereafter, the electrode is incubated with PIND-Os during which PIND-Os specifically inserts itself between base pairs of ds-DNA and catalyzes the electrooxidation of AA. The methylation event corresponding to the MTase activity can therefore be monitored and amplified by the electrocatalytic oxidation of AA. A linear correlation between the catalytic oxidation current of AA and the activity of M.SssI MTase ranged from 0 to 120 U/mL with a current sensitivity of 0.046 μA mL U(-1) is obtained. The inhibitor screening ability of the developed MTase activity assay is also demonstrated.

Highly sensitive methyltransferase activity assay and inhibitor screening based on fluorescence quenching of graphene oxide integrated with the site-specific cleavage of restriction endonuclease.

We report a sensitive and selective approach for the DNA methyltransferase (MTase) activity assay and MTase inhibitor screening by coupling the fluorescence quenching of graphene oxide with site-specific cleavage of a restriction endonuclease.