Target-mediated consecutive endonuclease reactions for specific and sensitive homogeneous fluorescence assay of O6-methylguanine-DNA methyltransferase

A simple and rapid mix-and-read assay for sensitive detection of O6-methylguanine DNA methyltransferase

“…Despite their good performance, these methods often require long analysis times, have poor sensitivity, and require complex probe synthesis/purification processes. To improve the sensitivity, polymerase chain reaction (PCR) amplification 15 and nicking enzyme-assisted cyclical cleavage 16 can be utilized, but this requires expensive PCR thermal cyclers and precise temperature control. 17 The development of a simple and rapid method for the sensitive detection of MGMT activity and for inhibitor screening is urgently required.…”

“…We have developed a simple mix-and-detection assay for sensitive and rapid detection of O 6 -methylguanine DNA methyltransferase (MGMT) activity based on exonuclease III-assisted signal amplification at completely isothermal conditions (37 1C). This assay has distinct advantages: (1) it is an isothermal assay with completely isothermal conditions (37 1C), avoiding the requirement for a sophisticated thermal cycler and precise temperature control that are required in PCR and nicking enzymeassisted cyclical cleavage assays; 15,16 (2) it is a simple mix-anddetection assay without the involvement of any washing and separation steps, and it can be completed within 60 min, much shorter than other reported assays (Table S3, ESI †); and (3) the integration of dumbbell probe with the Exo III-assisted signal amplification can effectively eliminate false positive results and greatly improves the detection sensitivity. This method is very sensitive with a limit of detection of 3.6 Â 10 À8 ng mL À1 (1.34 fM), and it can detect MGMT activity at the single-cell level.…”

“…The sensitivity of this method is 6 orders of magnitude higher than those of fluorescent methods based on fluorescence-switchable chemical probes (5 nM) 22 and DNAzymes (2 nM), 14 and 4 orders of magnitude higher than those of fluorescent methods based on graphene oxide (0.15 ng mL −1 ) 23 and hairpin probes (0.5 ng mL −1 ). 24 The high sensitivity of the proposed method can be ascribed to: (1) the efficient transduction of the MGMT enzyme signal into a nucleic acid signal via the PvuII-mediated cleavage of the MGMT reaction product; (2) the low background noise resulting from the good resistance of the dumbbell probe against nonspecific digestion; and (3) the high signal amplification induced by the Exo III-mediated cyclical digestion of the signal probes. Moreover, this method exhibits high specificity toward MGMT with no response to other kinds of demethylases and DNA repair enzymes including fat mass and obesity-associated protein (FTO), ten-eleven translocation enzyme II (TeT2), uracil-DNA glycosylase (UDG), apyrimidinic endonuclease 1 (APE1) and formamidopyrimidine DNA glucosylase (FPG) (Fig.…”

G-quadruplex functionalized nano mesoporous silica for assay of the DNA methyltransferase activity

“…Up to now, a few methods for DNA MTase activity detection have been developed, mainly including methylation-specific polymerase chain reaction [7][8][9], fluorescence methods [10,11], immunochemical [12,13], restriction enzymes [14,15], colorimetric [16] and high-performance liquid chromatography [17,18]. As these above-mentioned approaches have the disadvantage of timeconsuming, costly and tedious process, it is in great need to develop new ways to monitor the process of DNA MTase activity.…”