Rapid restriction enzyme free detection of DNA methyltransferase activity based on DNA-templated silver nanoclusters

Abstract

DNA methylation has significant roles in gene regulation. DNA methyltransferase (MTase) enzyme characterizes DNA methylation and also induces an aberrant methylation pattern that is related to many diseases, especially cancers. Thus, it is required to develop a method to detect the DNA MTase activity. In this study, we developed a new sensitive and reliable method for methyltransferase activity assay by employing DNA-templated silver nanoclusters (DNA/Ag NCs) without using restriction enzymes. The Ag NCs have been utilized for the determination of M.SssI MTase activity and its inhibition. We designed an oligonucleotide probe which contained an inserted six-cytosine loop as Ag NCs formation template. The changes in fluorescence intensity were monitored to quantify the M.SssI activity. The fluorescence spectra showed a linear decrease in the range of 0.4 to 20 U/ml with a detection limit of 0.1 U/ml, which was significant compared with previous reports. The proposed method was applied successfully for demonstrating the Gentamicin effect as MTase inhibitor. The proposed method showed convenient reproducibility and sensitivity indicating its potential for the determination of methyltransferase activity.

Cytosine-5 methylation-directed construction of a Au nanoparticle-based nanosensor for simultaneous detection of multiple DNA methyltransferases at the single-molecule level

“…The detection limit is calculated to be 2.01 Â 10 À3 U mL À1 by evaluating three times the standard deviation over the signal of negative control without M.SssI MTase. The sensitivity of this method has been improved by more than 497.5-fold compared with that of the colorimetric assay based on enzymelinkage-mediated AuNP aggregation (1.0 U mL À1 ), 18 164.2-fold compared with that of the electrochemical assay based on a photoelectrochemical (PEC) immunosensor (0.33 U mL À1 ), 51 49.8-fold compared with that of the luminescence assay based on DNA-templated silver nanoclusters (0.1 U mL À1 ), 52 and 29.9fold compared with that of the uorescence assay based on methylation-sensitive cleavage coupled with the nicking enzyme-assisted signal amplication (0.06 U mL À1 ), 30 and even is comparable to that of the uorescence assay based on SDA and DNAzyme amplication (0.0082 U mL À1 ). 53 As shown in Fig.…”

New Colorimetric DNA Sensor for Detection of Campylobacter jejuni in Milk Sample Based on Peroxidase‐Like Activity of Gold/Platinium Nanocluster

“…Conjugation of peroxidase mimic nanostructure with bio‐receptor not only decreased catalytic activity but also diminish binding affinity of bio‐receptor . Nowadays DNA has been attractive for its potency as a scaffold for synthesis of nanoclusters . This capacity resolved bio receptor conjugation in designing DNA biosensor.…”

Sensitive detection of methylated DNA and methyltransferase activity based on the lighting up of FAM-labeled DNA quenched fluorescence by gold nanoparticles

“…Previous studies showed that exploiting the advantages of nanomaterials for biosensor fabrication while enhancing their performance improved their detection limit as demonstrated for DNA detection. [7][8][9][10] The conventional methods for the detection of MTase enzyme activity and methylated DNA include bisulte treatment, 11 high performance liquid chromatography (HPLC), 12 electrochemistry, [13][14][15][16][17] enzyme-linked immunosorbent assay (ELISA), 18 surface plasmon resonance (SPR) spectroscopy, 19,20 surface-enhanced Raman scattering (SERS), 21,22 and use of microuidic based biosensors 23,24 and uorescence based biosensors. [25][26][27][28] Although several attempts have been made for the detection of methylated DNA in recent years, there are still some drawbacks that limit their sensitivity and efficacy in clinical applications.…”