Construction of an APE1-Mediated Cascade Signal Amplification Platform for Homogeneously Sensitive and Rapid Measurement of DNA Methyltransferase in Escherichia coli Cells

Abstract

DNA methylation is an essential genomic epigenetic behavior in both eukaryotes and prokaryotes. Deregulation of DNA methyltransferase (Dam MTase) can change the DNA methylation level and cause various diseases. Herein, we develop an apurinic/apyrimidinic endonuclease 1 (APE1)-mediated cascade signal amplification platform for homogeneously sensitive and rapid measurement of Dam MTase in Escherichia coli cells. This assay involves a partial double-stranded DNA (dsDNA) substrate and two hairpin signal probes (HP1 and HP2) that are modified with Cy5 and BHQ2 at two ends, respectively. When Dam MTase is present, it methylates the dsDNA substrate, and subsequently, endonuclease DpnI cleaves the methylated substrate, yielding trigger probe 1. Hybridization of trigger probe 1 with HP1 forms a partial dsDNA containing an apurinic/apyrimidinic (AP) site, which is cleaved by APE1 to induce the cyclic cleavage of HP1 and the production of abundant trigger probe 2. Subsequent hybridization of trigger probe 2 with HP2 forms a partial dsDNA with an AP site, inducing the cyclic cleavage of HP2 by APE1. Consequently, cyclic cleavage of HP1 and HP2 induces the generation of abundant Cy5 molecules, which are easily measured by single-molecule imaging. This assay can be performed homogeneously and rapidly within 2 h, which is the shortest among the reported amplification-based assays. Moreover, it exhibits good selectivity and high sensitivity, and it can discriminate Dam MTase from other enzymes and screen inhibitors. Importantly, it can accurately measure the Dam MTase activity in serum and E. coli cells, with promising applications in clinical diagnosis and drug discovery.

Fluorogenic RNA Aptamer‐Based Amplification and Transcription Strategy for Label‐free Sensing of Methyltransferase Activity in Complex Matrixes

“…Besides, we further examined the real‐time fluorescence results from the proposed program processed by other DNA adenine MTase (Dam). [ 24 ] We found that the fluorescence intensity gradually increased upon adding M.SssI and reached a plateau at 100 min, while it remained very low when M.SssI was replaced with Dam (Figure 2C). Altogether, these results demonstrated that the present circuit was viable for accurate and selective M.SssI detection.…”

Plasmonic Gold Nanostar-Based Probes with Distance-Dependent Plasmon-Enhanced Fluorescence for Ultrasensitive DNA Methyltransferase Assay

“…3−5 Therefore, sensitively measuring Dam MTase is of high significance for methylation detection and early clinical diagnosis. 6 To date, various conventional strategies have been developed for Dam MTase detection, including high-performance liquid chromatography, radioactive labeling-based gel electrophoresis, enzyme-based immunoassay, and capillary electrophoresis. 7−10 Nevertheless, the involvement of radiolabeled substrates, timeconsuming procedures, and costly antibodies severely limits their wide applications.…”

“…DNA methylation is an important epigenetic modification catalyzed by DNA methyltransferase (Dam MTase), which plays a crucial role in the regulation of gene transcription, gene silencing, genome integrity, and cell development , Aberrant DNA methylation is usually caused by abnormal expression of Dam MTase, resulting in various human diseases, including genetic diseases, autoimmune disease, and cancers. − Therefore, sensitively measuring Dam MTase is of high significance for methylation detection and early clinical diagnosis . To date, various conventional strategies have been developed for Dam MTase detection, including high-performance liquid chromatography, radioactive labeling-based gel electrophoresis, enzyme-based immunoassay, and capillary electrophoresis. − Nevertheless, the involvement of radiolabeled substrates, time-consuming procedures, and costly antibodies severely limits their wide applications .…”