Detection Of DNA Methyltransferase Activity Research Articles

A High-Affinity Methyl-CpG-Binding Protein for Endonuclease-Free and Label-Free DNA Methyltransferase Activity Detection

A High-Affinity Methyl-CpG-Binding Protein for Endonuclease-Free and Label-Free DNA Methyltransferase Activity Detection

Ratiometric fluorescent method for highly sensitive detection of DNA methyltransferase activity and inhibitor screening based on the DNA strand replacement reaction initiated by “toehold”

DNA methylation has been generally regarded as predictive biomarkers for tumor diagnosis and potential biotarget for the treatment of tumors. Herein, we present dual-emission ratiometric fluorescent system primarily based on DNA strand replacement reaction initiated by“toehold”for accurately assay of Dam MTase activity. This new strategy involved thiolated double-loop hairpin DNA probe with a specific recognition sites for Dam MTase and DpnI endonucleases which was labeled by fluorescent dye FAM at 5′ end (DH-DNA), a DNA labeled by fluorescent dye Cy5 at 5′ end (T-DNA). The DH-DNA probe was previously attached on Au nanoparticles (Au NPs) surface at 5′ end (Au-HP-DNA), resulting in the fluoresence quencher of the FAM via extinction of Au NPs. Upon addition of Dam MTase, Au-HP-DNA will be methylated and cleaved into two fragments. One of the Au NPs-labeled DNA fragment can be served as“toehold”to hybridization with T-DNA and thus displacement of FAM-labeled DNA fragment, resulting in significantly fluoresence quencher of Cy5 and obvious increase of fluorescent signal of FAM. Benefiting from the built-in correlation that eliminates for external interferential effects and the large range of emission ratios, the protocol allows improved sensitivity and accurate assay for Dam MTase with a detection limit of 0.0036 U/mL. Furthermore, the protocol can be extended to screen suitable inhibitor of Dam. Thus, the present protocol might be help for further application in the therapeutic associated with DNA methylation.

Fluorescence turn-off strategy for sensitive detection of DNA methyltransferase activity based on DNA-templated gold nanoclusters

DNA methylation results in a variety of human diseases and the DNA methylation process is mediated by DNA methyltransferases, which have therefore become potential targets for disease treatment. In this study, a turn-off nanogold biological probe system was successfully created for determining the activity of DNA methyltransferases (M.SssI MTase). A dumbbell-shaped DNA probe with a site-recognizable region of M. SssI MTase and a fluorescent signal probe based on a DNA-templated gold nanocluster (DNA-AuNC) probe combined for the quantitative detection of M. SssI MTase. This dumbbell-shaped DNA probe was methylated by M. SssI MTase, and the dumbbell-shaped DNA probe with a methyl group was recognized by an endonuclease (GlaI) and cleaved into hairpin DNA. The dGTP was added to the 3′-OH terminus of hairpin DNA fragments in the presence of terminal deoxynucleotidyl transferase (TdT), and the hairpin DNA was extended with a G-rich sequence that can be used as an inactivation probe. When the inactivation probe was combined with the signal probe, the fluorescent signal disappeared due to the photoinduced electron transfer effect. Methyltransferase activity was then detected based on the turn-off principle of the fluorescence signal from the DNA-AuNCs. The bioprobe enabled sensitive detection of M. SssI MTase with a detection limit of 0.178 U mL−1 and good specificity. The bioprobe demonstrated good detection efficiency in both human serum and cell lysates, and its unique fluorescence turn-off mechanism provided good resistance to interference, thus increasing its potential application in complex biological samples. Moreover, it is suitable for screening and assessing the inhibitory activity of M. SssI MTase inhibitors, and therefore has significant potential for disease diagnosis and drug discovery.

A programmable DNA nanodevice for colorimetric detection of DNA methyltransferase activity using functionalized hemin/G-quadruplex DNAzyme

The measurement of DNA methyltransferase (MTase) activity and screening of DNA MTase inhibitors holds significant importance for the diagnosis and therapy of methylation-related illness. Herein, we developed a colorimetric biosensor (PER-FHGD nanodevice) to detect DNA MTase activity by integrating the primer exchange reaction (PER) amplification and functionalized hemin/G-quadruplex DNAzyme (FHGD). By replacing the native hemin cofactor into the functionalized cofactor mimics, FHGD has exhibited significantly improved catalytic efficiency, thereby enhancing the detection performance of the FHGD-based system. The proposed PER-FHGD system is capable of detecting Dam MTase with excellent sensitivity, exhibiting a limit of detection (LOD) as low as 0.3 U/mL. Additionally, this assay demonstrates remarkable selectivity and ability for Dam MTase inhibitors screening. Furthermore, using this assay, we successfully detect the Dam MTase activity both in serum and in E. coli cell extracts. Importantly, this system has the potential to serve as a universal strategy for FHGD-based diagnosis in point-of-care (POC) tests, by simply altering the recognition sequence of the substrate for other analytes.

HpaII-assisted and linear amplification-enhanced isothermal exponential amplification fluorescent strategy for rapid and sensitive detection of DNA methyltransferase activity.

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 [Formula: see text] 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.

Dual-modal biosensor for highly sensitive and selective DNA methyltransferase activity detection based on a porous organic polymer-inorganic nanocomposite (Cu2O@FePPOPBADE) with high laccase-like activity

Abnormal DNA Methyltransferase (MTase) activity is closely related with multiple human diseases, which triggered its accurate and sensitive detecting demands. Here, an electrochemical and colorimetric, dual-modal biosensor for Dam MTase (Dam) assay was developed based on an organic-inorganic nanocomposite (Cu2O@FePPOPBADE). Stemming from the laccase-like activity of Cu2O, the catalytic activity of ferriporphyrin and excellent protective effect of the FePPOPBADE shell, Cu2O@FePPOPBADE not only exhibited significantly improved laccase-like activity to catalyze the chromogenic reaction of 2,4-dichlorophenol (2,4-DP) with 4-aminoantipyrine (4-AP), but possessed a strong electrochemical signal. Cu2O@FePPOPBADE was further functionalize with Au nanoparticles and served as a signal probe (Cu2O@FePPOPBADE@AuNPs). Also, magnetic bead (MB)/dsDNA was designed by assembly of streptavidin-modified MB with biotinylated-terminated ssDNA1 and thiolated-terminated ssDNA2, both of which contained specific sequences 5′-G-A-T-C-3′. When Dam and Dpn I were present, the specific sequence of dsDNA is methylated by Dam and then is cut by Dpn I, causing the leaving of thiolated-terminated ssDNA2. Sequentially, the residual MB/dsDNA is linked to the Cu2O@FePPOPBADE@AuNPs probe via Au-S bonds. After another magnetic separation, the unlinked Cu2O@FePPOPBADE@AuNPs left in the supernatant were transferred to accomplish the “dual signal-on” analysis of Dam with lower detection limits and high specificity.

Methylation-blocked cascade strand displacement amplification for rapid and sensitive fluorescence detection of DNA methyltransferase activity

DNA methylation catalyzed by DNA adenine methylation methyltransferase (Dam MTase) is strongly connected with a variety of biological processes, hence, monitoring Dam MTase activity is of great importance. Here, we developed a rapid and sensitive fluorescence sensing strategy for the detection of Dam MTase activity based on methylation-blocked enzymatic recycling amplification. In this fluorescence sensing system, Dam MTase-induced methylation blocked the subsequent reactions. In contrast, in the absence of Dam MTase, the unmethylated probe initiated the cascade strand displacement amplification for significant signal amplification. Under optimized conditions, this method has a lower detection limit of 0.67 U/mL and a shorter assay time (90 min) compared with previously reported similar methodologies.

Detection of DNA Methyltransferase Activity via Fluorescence Resonance Energy Transfer and Exonuclease-Mediated Target Recycling.

In this study, a sensitive method for detecting DNA methyltransferase (MTase) activity was developed by combining the effective fluorescence resonance energy transfer (FRET) of cationic conjugated polymers and exonuclease (Exo) III–mediated signal amplification. DNA adenine MTase targets the GATC sequence within a substrate and converts the adenine in this sequence into N6-methyladenine. In the method developed in this study, the methylated substrate is cleaved using Dpn I, whereby a single-stranded oligodeoxynucleotide (oligo) is released. Afterward, the oligo is hybridized to the 3ʹ protruding end of the F-DNA probe to form a double-stranded DNA, which is then digested by Exo III. Subsequently, due to weak electrostatic interactions, only a weak FRET signal is observed. The introduction of the Exo-III–mediated target-recycling reaction improved the sensitivity for detecting MTase. This detection method was found to be sensitive for MTase detection, with the lowest detection limit of 0.045 U/mL, and was also suitable for MTase-inhibitor screening, whereby such inhibitors can be identified for disease treatment.

A cascade amplification strategy for the detection of DNA methyltransferase activity by elemental labeling inductively coupled plasma mass spectrometry

A cascade amplification strategy combining strand displacement amplification (SDA) and multicomponent nucleic acid enzyme (MNAzyme) was proposed for detecting DNA MTase activity by AuNPs labeling inductively coupled plasma mass spectrometry (ICP-MS). With the absence of Dam MTase, the hairpin strand containing the recognition site of nicking endonuclease performed SDA reaction and generated abundant short strands. These strands hybridized with partzyme A to block the progress of the MNAzyme reaction. In the presence of Dam MTase, the action of methylation-sensitive nicking endonuclease was blocked and SDA process was inhibited, leading to activation of subsequent MNAzyme reaction. The completely assembled MNAzyme cleaved the substrates on the magnetic beads and the released AuNPs were detected by ICP-MS. The method shows an ultra-high sensitivity with a limit of detection of 1.51 × 10−4 U mL−1, around 1–2 orders of magnitude lower than most reported methods. It was applied to the screening of inhibitors of Dam MTase as well as Dam MTase analysis in serum samples. Recoveries of 94.6–104% were obtained for Dam MTase in spiked serum samples, demonstrating good potential of the method for highly sensitive detecting DNA MTase activity in complex biological samples.

Ultrasensitive and Label-Free Detection of Multiple DNA Methyltransferases by Asymmetric Nanopore Biosensor.

DNA methylation is catalyzed by a family of DNA methyltransferases that play crucial roles in various biological processes. Therefore, an ultrasensitive methyltransferase assay is highly desirable in biomedical research and clinical diagnosis. However, conventional assays for the detection of DNA methyltransferase activity often involve radioactive labeling, costly equipment, and laborious operation. In this study, an ultrasensitive and label-free method for detecting DNA adenine methyltransferase (Dam) and CpG methyltransferase (M.SssI) was developed using the nanopore technique coupled with DNA cascade signal amplification reactions. A hairpin DNA (HD) comprising of the methylation-responsive sequences was skillfully designed. In the presence of Dam methyltransferase, the corresponding recognition site of hairpin HD was methylated and specifically cleaved by DpnI endonuclease, thus forming a DNA fragment that induces the catalytic hairpin assembly and hybridization chain reaction (CHA-HCR). The generated products could be absorbed onto the Zr4+-coated nanopore, resulting in an ion current rectification signal change. Considering the high sensitivity of the nanopore and excellent specificity toward the recognition of methyltransferase/endonuclease, our developed method could detect both Dam and M.SssI methyltransferases in the same sensing platform. Furthermore, the designed nanopore sensor could realize the multiplex detection of Dam and M.SssI methyltransferases after integration with the cascaded INHIBIT-AND logic gate. This ultrasensitive methyltransferase assay holds great promise in the field of cancer diagnosis.

A Cascade Amplification Strategy for the Detection of DNA Methyltransferase Activity by Elemental Labeling Inductively Coupled Plasma Mass Spectrometry

A Cascade Amplification Strategy for the Detection of DNA Methyltransferase Activity by Elemental Labeling Inductively Coupled Plasma Mass Spectrometry

Ultrasensitive detection of DNA methyltransferase activity: a novel dual-amplification fluorescence technique.

DNA methyltransferase (MTase) is an important regulatory enzyme in various biological processes. However, current methods for investigating MTase activity are still limited in terms of sensitivity and/or generality. Herein, we proposed a dual amplification fluorescence strategy for the ultrasensitive detection of DNA adenine methylation methyltransferase (Dam MTase) activity based on strand displacement amplification (SDA) coupled with rolling circle amplification (RCA). In this study, the hairpin probe could not be cleaved by Nt.AlwI nicking endonuclease (Nt.AlwI) in the presence of Dam MTase, and the subsequent SDA-RCA reaction was blocked, resulting in a weak fluorescence signal. Moreover, the blocking effect was more pronounced at a higher concentration of Dam MTase. This assay provides a very low detection limit (down to 0.0067 U ml-1), as well as good selectivity against other types of MTases (e.g., CpG methyltransferase (M.SssI MTase)). In addition, the analytical mode improves the generality and can be extended to the detection of other types of DNA MTases.

Dual-Modal Biosensor for Highly Sensitive and Selective DNA Methyltransferase Activity Detection Based on a Porous Organic Polymer-Inorganic Nanocomposite (Cu2o@Feppopbade) with High Laccase-Like Activity

Dual-Modal Biosensor for Highly Sensitive and Selective DNA Methyltransferase Activity Detection Based on a Porous Organic Polymer-Inorganic Nanocomposite (Cu2o@Feppopbade) with High Laccase-Like Activity

DNA-functionalized biosensor for amplifying signal detection of DNA methyltransferase activity

Abstract DNA methylation is catalyzed by DNA methyltransferase (DAM) and plays an important role in many biological processes. The monitoring of DAM activity is of great significance in disease diagnosis. In this paper, an electrochemical DNA biosensor was developed for DAM detection via recognition of DNA tetrahedron and SiO2@Au core/shell nanoparticles-based amplification technique. We immobilize the DNA tetrahedron on the gold electrode surface to construct a well sensing interface. SiO2@Au core/shell nanoparticles was selected to achieve signal amplification for improving the sensitivity. The detection limit of DAM activity by the proposed method was reduced down to 0.0005 U mL−1. The proposed sensing system also showed ideal specificity, repeatability and stability. Moreover, the novel sensor developed could serve as a highly sensitive probe for DAM detection and shows great potentials in designed drug therapy, clinical diagnostics.

Highly sensitive detection of DNA methyltransferase activity and its inhibitor screening by coupling fluorescence correlation spectroscopy with polystyrene polymer dots.

DNA methylation is a critical part of epigenetics and plays a vital role in maintaining normal cell function, genetic imprinting, and human tumorigenesis. Thus, it is important to develop a sensitive method for the determination of DNA methyltransferase (MTase) activity. Here, we present a simple and sensitive method based on single molecule fluorescence correlation spectroscopy (FCS) and polystyrene polymer dots (PS Pdots) for the quantitative detection of DNA adenine methylation (Dam) MTase activity and its inhibitor screening in homogeneous solution without separation. Its principle is based on the measurement of the characteristic diffusion time (τD) of unmethylated and methylated DNA-fluorescent probes by FCS. A hairpin DNA probe including the 5'-GATC-3' sequence is used by doubly labelling fluorophore Alexa Fluor 488 (Alexa 488) and biotin at the 5'- and 3'-terminus, respectively. Dam MTase catalyzed the methylation of the sequence of 5'-GATC-3', and DpnI cleaved the sequence of 5'-G-Am-TC-3'. Streptavidin conjugated PS Pdots were used to react with DNA probes without methylation to further increase the difference in τD values between methylated and unmethylated DNA-Alexa 488 probes. We used the FCS method to measure the τD values of DNA-Alexa 488 probes and further obtained the activity of Dam MTase. It is found that the τD value of the methylated DNA probe is negatively correlated with the logarithm of Dam MTase concentration in the range from 0.025 U mL-1 to 3 U mL-1. The detection limit is as low as 0.025 U mL-1. Furthermore, we evaluated the inhibition effect of drug-related DNA methylation and the half-maximal inhibitory concentration (IC50) value is consistent with a previous study. The results demonstrated that our proposed method will become a promising platform for the determination of Dam MTase activity and inhibitor screening.

A novel DNA biosensor for the ultrasensitive detection of DNA methyltransferase activity based on a high-density "hot spot" SERS substrate and rolling circle amplification strategy.

Herein, we proposed a novel biosensor based on a high-density "hot spot" Au@SiO2 array substrate and rolling circle amplification (RCA) strategy for the ultrasensitive detection of CpG methyltransferase (M.SssI) activity. In the presence of M.SssI, the RCA process can be triggered, causing the augmentation of the single-stranded DNA (ssDNA) at the tail of the double-stranded DNA (dsDNA), and the ssDNA can be hybridized with numerous DNA probes labeled with Raman reporters in the next steps. Afterwards, the resultant ssDNA can be modified to the Au@SiO2 array substrate with the SERS enhancement factor of 7.49 × 106. The substrate was synthesized by using a monolayer SiO2 array to pick up the Au nanoparticle (AuNP) array and finite-difference time-domain (FDTD) simulation showed its excellent SERS effect. Particularly, the developed biosensor displayed a significant sensitivity with a broad detection range covering from 0.005 to 50 U mL-1, and the limits of detection (LODs) in PBS buffer and human serum were 2.37 × 10-4 U mL-1 and 2.51 × 10-4 U mL-1, respectively. Finally, in order to verify the feasibility of its clinical application, the serum samples of healthy subjects and breast cancer, prostate cancer, gastric cancer and cervical cancer patients were analyzed, and the reliability of the results was also confirmed by western blot (WB) experiments. Taking advantage of these merits, the proposed biosensor can be a very promising alternative tool for the detection of M.SssI activity, which is of vital importance in the early detection and prevention of tumors.

A cascade strand displacement amplification strategy for highly sensitive and label-free detection of DNA methyltransferase activity

DNA methyltransferase (MTase) is regarded as a promising biomarker and therapeutic target, which plays an important regulatory role in many biological processes. Here, a cascade strand displacement amplification strategy was explored for highly sensitive and label-free fluorescent detection of DNA MTase activity. Firstly, the hairpin recognition probe (RP) with a long stem was designed. DNA MTase catalyzed the RP methylation, and then DpnI endonuclease cleaved the methylated RP, releasing a trigger strand. Subsequently, it as a primer hybridized with hairpin probe 1 and triggered strand displacement amplification reaction (SDAR), producing numerous replaced strands. And the replaced strands hybridized with hairpin probe 2 and triggered next SDAR, producing a larger number of G-rich sequences. After that, N-methylmesoporphyrin IX was selectively intercalated into the G-quadruplex structures which were folded from the G-rich sequences, obtaining a significantly enhanced fluorescent signal. However, RP was stable, and could not produce primer for the subsequent cascade SDAR in the absence of DNA MTase. The effective amplification efficiency of cascade SDAR improved the detection sensitivity (detection limit: 0.0073 U/mL). Moreover, this strategy well evaluated the inhibitions of 5-fluorouracil, benzylpenicillin and gentamycin on DNA MTase activity. The results indicated this method held great potential in new method exploration and early disease diagnosis.

A renewable DNA biosensor for sensitive detection of DNA methyltransferase activity based on cascade signal amplification

DNA methyltransferase catalyzes the methylation process of DNA. As a potential biomarker, methyltransferase activity assay has attracted more and more attention. Herein, we present a new biosensor based on cascade invasive reactions for DNA methyltransferase activity analysis. The sensing probes on the biosensor were first methylated by the Dam MTase and then cleaved by the DpnI endonuclease. The cleaved probe acted as upstream probe for the invasion reaction, triggering a highly sensitive signal amplification reaction that produced a strong fluorescent signal. After detection, the cleaved probes were extended by DNA polymerase, and regenerated to be an intact sensor. Meanwhile, as the invasion reaction was a sequence-independent signal amplification method, our proposed biosensor could be a universal strategy for other enzyme activity analysis, simply by replacing the recognition sequence.

Detection of DNA methyltransferase activity using template-free DNA polymerization amplification based on aggregation-induced emission

A sensitive and selective fluorescence assay for DNA methyltransferase (MTase) activity detection was designed based on aggregation-induced emission (AIE) and target initiated template-free DNA polymerization. Quaternized tetraphenylethene salt was synthesized as the AIE probe, which binds to single-stranded DNA by electrostatic interaction. A hairpin probe was designed with a specific sequence for DNA MTase. In the presence of DNA MTase, the methylation reaction initiated DNA polymerization with terminal deoxynucleotidyl transferase (TdT), which activated the fluorescence intensity through AIE. The designed DNA sensor displayed a linear response to concentrations of DNA adenine methyltransferase (Dam) MTase from 0.5 U·mL−1 to 100 U mL−1, with a limit of detection of 0.16 U mL−1. The assay was also effective for detection of DNA MTase activity in human serum and for showing the inhibitory effect of 5-fluorouracil on Dam MTase.

A dual-response biosensor for electrochemical and glucometer detection of DNA methyltransferase activity based on functionalized metal-organic framework amplification

DNA methylation is catalyzed by DNA methyltransferase (MTase) and concerned with many biological processes including pathogenesis of various human diseases. The monitoring of MTase activity is thus of great significance in disease diagnosis and drug screening. Herein, we developed a facile way to synthesize biocompatible invertase enzyme modified metal-organic framework (Invertase/MOF) materials, and explored its application in constructing a dual-response Dam MTase sensor for the first time. By using them as signal probes, in which high density of metal sites could be electrochemically detected and invertase could hydrolyze sucrose into glucose for generation of glucometer signal output, dual-response for accurate detection of Dam MTase was realized. In the presence of Dam MTase, the methylation of hairpin probe 1 (HP1) occurred and thus caused the cleavage of HP1 assisted by a restriction endonuclease (DpnI) to produce the binding sequences. The binding sequences then hybridized with the electrode-assembled HP2 to expose their sticky termini which sequentially hybridized with the Invertase/MOFs-tethered capture probes. Finally, the electrodes were incubated with a sucrose solution, followed by the separate electrochemical and glucometer detection. The present assay brought good performance which could detect Dam MTase activity as low as 0.001 U mL−1 with wide linear range and good selectivity against other cytosine MTase (M.SssI MTase). Moreover, it also owns ability to be potentially applied for the inhibitors screening by utilization of 5-fluorouracil as an inhibitor model. The results imply that our proposed method provides a convenient platform for early cancer diagnosis and therapeutic applications.