Methyltransferase Activity Assay Research Articles

Ultrasensitive electrochemical detection and inhibition evaluation of DNA methyltransferase based on cascade strand displacement amplification.

An electrochemical sensing approach for ultrasensitive DNA methyltransferase (MTase) activity assay is proposed. After specific cleavage reaction in the presence of a methylated state, strand displacement polymerization (SDP) is initiated in the solution. The product of upstream SDP further triggers downstream SDP, which enriches abundant electrochemical species at the electrode. The whole process is quite convenient with shared enzymes. Due to the cascade signal amplification, ultrahigh sensitivity is promised. Inhibitor screening results are also demonstrated to be good. Besides, target MTase can be accurately determined in human serum samples, confirming excellent practical utility. This work provides a reliable approach for the analysis of MTase activity, which is of vital importance for related biological studies and clinical applications.

Tetrahedral DNA nanostructure-corbelled click chemistry-based large-scale assembly of nanozymes for ratiometric fluorescence assay of DNA methyltransferase activity.

Ligation efficiency in a surface-based DNA click chemistry (CuAAC) reaction is extremely restricted by the orientation and density of probes arranged on a heterogeneous surface. Herein, we engineer DNA tetrahedral nanostructure (DTN)-corbelled click chemistry to trigger a hybridization chain reaction (HCR) assembling a large-scale of nanozymes for ratiometric fluorescence detection of DNA adenine methyltransferase (Dam). In this study, a DNA tetrahedron structure with an alkynyl modifying pendant DNA probe (Alk-DTN) is designed and assembled on a magnetic bead (MB) as a scaffold for click chemistry. When a CuO NP-encoded magnetic nanoparticle (CuO-MNP) substrate was methylated by Dam, CuO NPs were released and turned into a mass of Cu+. The Cu+ droves azido modifying lDNA (azide-lDNA) to connect with the Alk-DTN probe on the MB through the click reaction, forming an intact primer to initiate the HCR. The HCR product, a rigid structure double-stranded DNA, periodically assembles glucose oxidase mimicking gold nanoparticles (GNPs) into a large-scale of nanozymes for catalyzing the oxidation of glucose to H2O2. NH2-MIL-101 MOFs, a fluorescent indicator and a biomimetic catalyst, activated the product H2O2 to oxidize o-phenylenediamine (oPD) into visually detectable 2,3-diaminophenazine (DAP). The change of the signal ratio between DAP and NH2-MIL-101 is proportional to the methylation event corresponding to the MTase activity. In this study, the DTN enhances the efficiency of the surface-based DNA click reaction and maintains the catalytic activities of gold nanoparticle nanozymes due to the intrinsic nature of mechanical rigidity and well-controlled orientation and well-adjusted size. Large-scale assembly of nanozymes circumvents the loss of natural enzyme activity caused by chemical modification and greatly improves the amplification efficiency. The proposed biosensor displayed a low detection limit of 0.001 U mL-1 for Dam MTase due to multiple amplification and was effective in real samples and methylation inhibitor screening, providing a promising modular platform for bioanalysis.

Cryo-EM structures of human m6A writer complexes

N6-methyladenosine (m6A) is the most abundant ribonucleotide modification among eukaryotic messenger RNAs. The m6A “writer” consists of the catalytic subunit m6A-METTL complex (MAC) and the regulatory subunit m6A-METTL-associated complex (MACOM), the latter being essential for enzymatic activity. Here, we report the cryo-electron microscopy (cryo-EM) structures of MACOM at a 3.0-Å resolution, uncovering that WTAP and VIRMA form the core structure of MACOM and that ZC3H13 stretches the conformation by binding VIRMA. Furthermore, the 4.4-Å resolution cryo-EM map of the MACOM–MAC complex, combined with crosslinking mass spectrometry and GST pull-down analysis, elucidates a plausible model of the m6A writer complex, in which MACOM binds to MAC mainly through WTAP and METTL3 interactions. In combination with in vitro RNA substrate binding and m6A methyltransferase activity assays, our results illustrate the molecular basis of how MACOM assembles and interacts with MAC to form an active m6A writer complex.

Modified Adenosines Sensitize Glioblastoma Cells to Temozolomide by Affecting DNA Methyltransferases.

Glioblastoma (GBM) is the most common and lethal primary malignant brain tumor, and due to its unique features, its management is certainly one of the most challenging ones among all cancers. N6-isopentenyladenosine (IPA) and its analog N6-benzyladenosine (N6-BA) are modified nucleosides endowed with potent antitumor activity on different types of human cancers, including GBM. Corroborating our previous finding, we demonstrated that IPA and N6-BA affect GBM cell line proliferation by modulating the expression of the F-box WD repeat domain-containing-7 (FBXW7), a tumor suppressor with a crucial role in the turnover of many proteins, such as SREBPs and Mcl1, involved in malignant progression and chemoresistance. Luciferase assay revealed that IPA-mediated upregulation of FBXW7 translates in transcriptional inactivation of its oncogenic substrates (Myc, NFkB, or HIF-1α). Moreover, downregulating MGMT expression, IPA strongly enhances the killing effect of temozolomide (TMZ), producing a favorable sensitizing effect starting from a concentration range much lower than TMZ EC50. Through DNA methyltransferase (DNMT) activity assay, analysis of the global DNA methylation, and the histone modification profiles, we demonstrated that the modified adenosines behave similar to 5-AZA-dC, known DNMT inhibitor. Overall, our results provide new perspectives for the first time, suggesting the modified adenosines as epigenetic tools able to improve chemo- and radiotherapy efficacy in glioblastoma and potentially other cancers.

Induction of m6A methylation in adipocyte exosomal LncRNAs mediates myeloma drug resistance

BackgroundTherapeutic resistance occurs in most patients with multiple myeloma (MM). One of the key mechanisms for MM drug resistance comes from the interaction between MM cells and adipocytes that inhibits drug-induced apoptosis in MM cells; MM cells reprogram adipocytes to morph into different characterizations, including exosomes, which are important for tumor-stroma cellular communication. However, the mechanism by which exosomes mediate the cellular machinery of the vicious cycle between MM cells and adipocytes remains unclear.MethodsAdipocytes were either isolated from bone marrow aspirates of healthy donors or MM patients or derived from mesenchymal stem cells. Co-culturing normal adipocytes with MM cells was used to generate MM-associated adipocytes. Exosomes were collected from the culture medium of adipocytes. Annexin V-binding and TUNEL assays were performed to assess MM cell apoptosis. Methyltransferase activity assay and dot blotting were used to access the m6A methylation activity of methyltransferase like 7A (METTL7A). RIP, MeRIP-seq, and RNA–protein pull down for assessing the interaction between long non-cording RNAs (LncRNAs) and RNA binding proteins were performed. Adipocyte-specific enhancer of zeste homolog 2 (EZH2) knockout mice and MM-xenografted mice were used for evaluating MM therapeutic response in vivo.ResultsExosomes collected from MM patient adipocytes protect MM cells from chemotherapy-induced apoptosis. Two LncRNAs in particular, LOC606724 and SNHG1, are significantly upregulated in MM cells after exposure to adipocyte exosomes. The raised LncRNA levels in MM cells are positively correlated to worse outcomes in patients, indicating their clinical relevancy in MM. The functional roles of adipocyte exosomal LOC606724 or SNHG1 in inhibition of MM cell apoptosis are determined by knockdown in adipocytes or overexpression in MM cells. We discovered the interactions between LncRNAs and RNA binding proteins and identified methyltransferase like 7A (METTL7A) as an RNA methyltransferase. MM cells promote LncRNA package into adipocyte exosomes through METTL7A-mediated LncRNA m6A methylation. Exposure of adipocytes to MM cells enhances METTL7A activity in m6A methylation through EZH2-mediated protein methylation.ConclusionThis study elucidates an unexplored mechanism of how adipocyte-rich microenvironment exacerbates MM therapeutic resistance and indicates a potential strategy to improve therapeutic efficacy by blocking this vicious exosome-mediated cycle.

Construction of a Dye-Sensitized and Gold Plasmon-Enhanced Cathodic Photoelectrochemical Biosensor for Methyltransferase Activity Assay.

DNA methyltransferases may function as important biomarkers of cancers and genetic diseases. Herein, we develop a dye-sensitized and gold plasmon-enhanced cathodic photoelectrochemical (PEC) biosensor on the basis of p-type covalent organic polymers (COPs) for the signal-on measurement of M.SssI methyltransferase (M.SssI MTase). The cathodic PEC biosensor is constructed by the in situ growth of p-type COP films onto a glass coated with indium tin oxide and the subsequent assembly of biotin- and HS-labeled double-stranded DNA (dsDNA) probes onto the COP film via biotin-streptavidin interaction. The dsDNA probe contains the recognition sequence of M.SssI MTase. The COP thin films possess a porous ultrathin nanosheet structure with abundant active sites, facilitating the generation of a high photocurrent compared with the hydrothermally synthesized ones. The presence of DNA methyltransferases can prevent the digestion of restriction endonuclease HpaII, consequently inducing the introduction of gold nanoparticles (AuNPs) to the dsDNA probes via the S-Au bond and the intercalation of rhodamine B (RhB) into the DNA grooves to produce a high photocurrent due to the dye-photosensitized enhancement and AuNP-mediated surface plasmon resonance. However, in the absence of M.SssI MTase, HpaII digests the dsDNA probes, and neither AuNPs nor RhB can be introduced onto the electrode surface, leading to a low photocurrent. This cathodic PEC biosensor possesses high sensitivity and good selectivity, and it can screen the inhibitors and detect M.SssI MTase in serum as well.

Effect of gold nanoparticles on the structure and neuroprotective function of protein L-isoaspartyl methyltransferase (PIMT)

Fibrillation of peptides and proteins is implicated in various neurodegenerative diseases and is a global concern. Aging leads to the formation of abnormal isoaspartate (isoAsp) residues from isomerization of normal aspartates in proteins, triggering fibril formation that leads to neurodegenerative diseases. Protein L-isoaspartyl methyltransferase (PIMT) is a repair enzyme which recognizes and converts altered isoAsp residues back to normal aspartate. Here we report the effect of gold nanoparticles (AuNPs) of different sizes on the structure and function of PIMT. Spherical AuNPs, viz. AuNS5, AuNS50 and AuNS100 (the number indicating the diameter in nm) stabilize PIMT, with AuNS100 exhibiting the best efficacy, as evident from various biophysical experiments. Isothermal titration calorimetry (ITC) revealed endothermic, but entropy driven mode of binding of PIMT with all the three AuNSs. Methyltransferase activity assay showed enhanced activity of PIMT in presence of all AuNSs, the maximum being with AuNS100. The efficacy of PIMT in presence of AuNS100 was further demonstrated by the reduction of fibrillation of Aβ42, the peptide that is implicated in Alzheimer’s disease. The enhancement of anti-fibrillation activity of PIMT with AuNS100 was confirmed from cell survival assay with PC12 derived neuronal cells against Aβ42 induced neurotoxicity.

Structural model of the M7G46 Methyltransferase TrmB in complex with tRNA

ABSTRACT TrmB belongs to the class I S-adenosylmethionine (SAM)-dependent methyltransferases (MTases) and introduces a methyl group to guanine at position 7 (m7G) in tRNA. In tRNAs m7G is most frequently found at position 46 in the variable loop and forms a tertiary base pair with C13 and U22, introducing a positive charge at G46. The TrmB/Trm8 enzyme family is structurally diverse, as TrmB proteins exist in a monomeric, homodimeric, and heterodimeric form. So far, the exact enzymatic mechanism, as well as the tRNA-TrmB crystal structure is not known. Here we present the first crystal structures of B. subtilis TrmB in complex with SAM and SAH. The crystal structures of TrmB apo and in complex with SAM and SAH have been determined by X-ray crystallography to 1.9 Å (apo), 2.5 Å (SAM), and 3.1 Å (SAH). The obtained crystal structures revealed Tyr193 to be important during SAM binding and MTase activity. Applying fluorescence polarization, the dissociation constant Kd of TrmB and tRNAPhe was determined to be 0.12 µM ± 0.002 µM. Luminescence-based methyltransferase activity assays revealed cooperative effects during TrmB catalysis with half-of-the-site reactivity at physiological SAM concentrations. Structural data retrieved from small-angle x-ray scattering (SAXS), mass-spectrometry of cross-linked complexes, and molecular docking experiments led to the determination of the TrmB-tRNAPhe complex structure.

Label-free fluorescence strategy for methyltransferase activity assay based on poly-thymine copper nanoclusters engineered by terminal deoxynucleotidyl transferase

The assay of detecting DNA methyltransferase activity has been identified as one of the central challenges in cancer diagnostics as DNA methylation is closely related to the diagnosis and treatment of tumors. In this study, a label-free fluorescence probe based on poly-thymine copper nanoclusters engineered by terminal deoxynucleotidyl transferase is proposed for methyltransferase activity assay. Taking advantage of the efficient polymerization extension reaction catalyzed by terminal deoxynucleotidyl transferase and the copper nanoclusters with large Stokes shift instead of labeling fluorescent dyes, the strategy exhibits a broader linear scope from 1 to 300 U mL−1 with a detection limit of 0.176 U mL−1. The economical method is specificity for M.SssI and also provides a convenient and high-throughput platform for screening the DNA methylation inhibitors, which displays a great potential for the practical applications of the drug development and clinical cancer diagnosis in the future.

Construction of a Universal and Label-Free Chemiluminescent Sensor for Accurate Quantification of Both Bacteria and Human Methyltransferases.

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.

Direct High-Throughput Screening Assay for mRNA Cap Guanine-N7 Methyltransferase Activity.

In eukaryotes, mature mRNA is formed through modifications of precursor mRNA, one of which is 5’ cap biosynthesis, involving RNA cap guanine‐N7 methyltransferase (N7‐MTase). N7‐MTases are also encoded by some eukaryotic viruses and facilitate their replication. N7‐MTase inhibitors have therapeutic potential, but their discovery is difficult because long RNA substrates are usually required for activity. Herein, we report a universal N7‐MTase activity assay based on small‐molecule fluorescent probes. We synthesized 12 fluorescent substrate analogues (GpppA and GpppG derivatives) varying in the dye type, dye attachment site, and linker length. GpppA labeled with pyrene at the 3’‐O position of adenosine acted as an artificial substrate with the properties of a turn‐off probe for all three tested N7‐MTases (human, parasite, and viral). Using this compound, a N7‐MTase inhibitor assay adaptable to high‐throughput screening was developed and used to screen synthetic substrate analogues and a commercial library. Several inhibitors with nanomolar activities were identified.

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.

Label-free and near-zero-background-noise photoelectrochemical assay of methyltransferase activity based on a Bi2S3/Ti3C2 Schottky junction.

Herein, a novel label-free photoelectrochemical (PEC) sensing platform with near-zero background noise was developed for M.SssI CpG methyltransferase (M.SssI MTase) activity assay based on a new Schottky junction of Bi2S3/Ti3C2 nanosheets. The proposed PEC sensor exhibited a low detection limit and a high signal-to-noise ratio for M.SssI MTase assay.

Immuno-DNA binding directed template-free DNA extension and enzyme catalysis for sensitive electrochemical DNA methyltransferase activity assay and inhibitor screening.

Sensitive and accurate determination of DNA methyltransferase (DNA Mtase) activity is highly pursued for understanding fundamental biological processes related to DNA methylation, clinical disease diagnosis and drug discovery. Herein, we propose a new electrochemical immuno-DNA sensing platform for DNA Mtase activity assay and inhibitor screening. After homogeneous DNA methylation by CpG methyltransferase (M.SssI Mtase), the methylated DNA can be specifically recruited onto an electrode via its immunological binding with the immobilized anti-5-methylcytosine antibody. The recruited methylated DNA was simultaneously used as a substrate to facilitate successive template-free DNA extension and enzyme catalysis for the dual-step signal amplification of DNA Mtase activity. The developed immuno-DNA sensing strategy effectively integrates solution-phase DNA methylation, surface affinity binding recognition, and successive template-free DNA extension and enzyme catalysis-based signal amplification, rendering a highly specific, sensitive and accurate assay of DNA Mtase activity. A low detection limit of 0.039 U mL-1 could be achieved with a high selectivity. It was also applied for efficient evaluation of various inhibitors. Current affinity recognition of the immobilized antibody with methylated DNA switches the sensing platform into a DNA operation interface, facilitating the opportunity for combining various DNA-based signal amplification strategies to improve the detection performance. It would be used as a general strategy for the analysis of DNA Mtase activity, inhibitors and more analytes, and is anticipated to show potential for applications in disease diagnosis and drug discovery.

Substrate recognition by the Pseudomonas aeruginosa EF-Tu–modifying methyltransferase EftM

The opportunistic bacterial pathogen Pseudomonas aeruginosa is a leading cause of serious infections in individuals with cystic fibrosis, compromised immune systems, or severe burns. P. aeruginosa adhesion to host epithelial cells is enhanced by surface-exposed translation elongation factor EF-Tu carrying a Lys-5 trimethylation, incorporated by the methyltransferase EftM. Thus, the EF-Tu modification by EftM may represent a target to prevent P. aeruginosa infections in vulnerable individuals. Here, we extend our understanding of EftM activity by defining the molecular mechanism by which it recognizes EF-Tu. Acting on the observation that EftM can bind to EF-Tu lacking its N-terminal peptide (encompassing the Lys-5 target site), we generated an EftM homology model and used it in protein/protein docking studies to predict EftM/EF-Tu interactions. Using site-directed mutagenesis of residues in both proteins, coupled with binding and methyltransferase activity assays, we experimentally validated the predicted protein/protein interface. We also show that EftM cannot methylate the isolated N-terminal EF-Tu peptide and that binding-induced conformational changes in EftM are likely needed to enable placement of the first 5-6 amino acids of EF-Tu into a conserved peptide-binding channel in EftM. In this channel, a group of residues that are highly conserved in EftM proteins position the N-terminal sequence to facilitate Lys-5 modification. Our findings reveal that EftM employs molecular strategies for substrate recognition common among both class I (Rossmann fold) and class II (SET domain) methyltransferases and pave the way for studies seeking a deeper understanding of EftM's mechanism of action on EF-Tu.

Beyond a Ribosomal RNA Methyltransferase, the Wider Role of MraW in DNA Methylation, Motility and Colonization in Escherichia coli O157:H7.

MraW is a 16S rRNA methyltransferase and plays a role in the fine-tuning of the ribosomal decoding center. It was recently found to contribute to the virulence of Staphylococcus aureus. In this study, we examined the function of MraW in Escherichia coli O157:H7 and found that the deletion of mraW led to decreased motility, flagellar production and DNA methylation. Whole-genome bisulfite sequencing showed a genome wide decrease of methylation of 336 genes and 219 promoters in the mraW mutant including flagellar genes. The methylation level of flagellar genes was confirmed by bisulfite PCR sequencing. Quantitative reverse transcription PCR results indicated that the transcription of these genes was also affected. MraW was furtherly observed to directly bind to the four flagellar gene sequences by electrophoretic mobility shift assay (EMSA). A common flexible motif in differentially methylated regions (DMRs) of promoters and coding regions of the four flagellar genes was identified. Reduced methylation was correlated with altered expression of 21 of the 24 genes tested. DNA methylation activity of MraW was confirmed by DNA methyltransferase activity assay in vitro and repressed by DNA methylation inhibitor 5-aza-2′-deoxycytidine (5-aza). In addition, the mraW mutant colonized poorer than wild type in mice. We also found that the expression of mraZ in the mraW mutant was increased confirming the antagonistic effect of mraW on mraZ. In conclusion, mraW was found to be a DNA methylase and have a wide-ranging effect on E. coli O157:H7 including motility and virulence in vivo via genome wide methylation and mraZ antagonism.

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.

Pharmacological Modulation of Flavivirus Methyltransferase Activity of Dengue Virus (DEV)

Abstract: Background: The occurrence of dengue fever plagues has expanded significantly in the course of the most recent couple of decades. Be that as it may, no immunization or antiviral treatments are accessible. In this manner, the requirement for protected and successful antiviral medications have turned out to be essential. Aim: Methyltransferases, with their altered enzymatic action, have been found associated with several diseases or infections. It has been observed that the dengue virus infection is correlated with the hyperactivity of methyltransferase. In the current study, we proposed to investigate the inhibitory effects of commercially available compounds on the methyltransferase activity. Materials and Methods: Evaluation of methyltransferase activity of the purified enzyme and their inhibitors was carried out using Methyltransferase Activity Assay Kit (Colorimetric). Results: Among the screened components, we identify two effective compounds that revealed significant effect against flavivirus methyltransferase activity. Further, time-dependent inhibition profiles for both compounds revealed that preincubation of fractions of Azacitidine and Zebularine for different time windows showed time-dependent inactivation of methyltransferase as the degree of inhibition increased with time. Conclusion: We conclude that both Azacitidine and Zebularine may be emerging as possible suitable drugs for the treatment of dengue virus infection. Key words: Methyltransferase, Dengue virus, Inhibitor, Concentration, Time course.

Highly selective ratiometric electrogenerated chemiluminescence assay of DNA methyltransferase activity via polyaniline and anti-fouling peptide modified electrode

In this work, an antifouling electrochemiluminescent (ECL) ratiometric biosensor is designed for the accurate, selective and sensitive detection of DNA methyltransferase (MTase) activity based on a dual-signaling strategy. Briefly, an ITO electrode is used to construct the anti-fouling interfaces with the modification of polyaniline (PANI), AuNPs and peptide. Hairpin DNA molecules containing the symmetric sequence of 5′-CATC-3′ are attached onto the modified ITO electrode and the ds-DNA can be cut off in the presence of Dam MTase and DpnI. The residual DNA and two hairpin DNA could lead to the extension of ds-DNA due to the Hybridization Chain Reaction (HCR). ECL signal is amplified significantly with the insertion of PTC-NH2 molecules into the dsDNA grooves. The ECLPTC-NH2/ECLAu@luminol is found in a logarithmic linear relation with the concentration of Dam MTase. Moreover, owing to the presence of antifouling peptide on the sensing interface, the ECL biosensor was capable of sensing MTase activity in complex biological media, such as FBS samples and human serum with significantly reduced nonspecific adsorption effect. Assaying Dam MTase in complex sample mixture containing 5% calf serum and 5% human serum further proved the feasibility of this ECL biosensor for early clinical diagnosis.

Homogeneous fluorescent biosensing method for DNA methyltransferase activity analysis and inhibitor screening based on highly efficient isothermal amplification

DNA methyltransferase (MTase) activity assay and its inhibitor screening are important for the diagnosis and treatment of methylation-related diseases. Herein, a label-free and highly sensitive biosensing method based on entropy-driven reaction and toehold-initiated rolling circle amplification (TIRCA) was developed for DNA MTase activity detection and inhibitor screening. Briefly, in the presence of MTase, the triple-stranded complex (TSC) could be methylated to avoid cleaving by MboI endonuclease. The complete TSC was able to initiate downstream entropy-driven reaction and TIRCA to produce G-quadruplex that can bind with Thioflavin T (ThT) to output fluorescent signal. Under the optimal experimental conditions, the established biosensing strategy could detect Dam MTase down to 0.06 U/mL with a linear range from 0.1 U/mL to 40 U/mL. Importantly, the signal-to-noise (S/N) of this biosensing strategy was extremely high. This strategy could discriminate target Dam MTase from another two MTases efficiently. Moreover, this developed biosensing method was applied to screen DNA MTase inhibitors. Therefore, we anticipate this unique strategy will serve as an alternative tool to detect DNA MTase activity and screen its inhibitors in clinical diagnosis and therapeutics.