Abstract
Methylcytosines in mammalian genomes are the main epigenetic molecular codes that switch off the repertoire of genes in cell-type and cell-stage dependent manners. DNA methyltransferases (DMT) are dedicated to managing the status of cytosine methylation. DNA methylation is not only critical in normal development, but it is also implicated in cancers, degeneration, and senescence. Thus, the chemicals to control DMT have been suggested as anticancer drugs by reprogramming the gene expression profile in malignant cells. Here, we report a new optical technique to characterize the activity of DMT and the effect of inhibitors, utilizing the methylation-sensitive B-Z transition of DNA without bisulfite conversion, methylation-sensing proteins, and polymerase chain reaction amplification. With the high sensitivity of single-molecule FRET, this method detects the event of DNA methylation in a single DNA molecule and circumvents the need for amplification steps, permitting direct interpretation. This method also responds to hemi-methylated DNA. Dispensing with methylation-sensitive nucleases, this method preserves the molecular integrity and methylation state of target molecules. Sparing methylation-sensing nucleases and antibodies helps to avoid errors introduced by the antibody’s incomplete specificity or variable activity of nucleases. With this new method, we demonstrated the inhibitory effect of several natural bio-active compounds on DMT. All taken together, our method offers quantitative assays for DMT and DMT-related anticancer drugs.
Highlights
DNA methylation is an important bio-marker for cancers [7,9] and the detection of DNA methylation is significant in epigenetic analysis and cancer biology [3,4,8,10,12]
We demonstrated that the single-molecule FRET experiment is an ideal technique to detect the B-Z transition and the methylated CpG repeat undergoes the transition at lower monovalent (Na+ ) or divalent (Mg2+ ) cation concentrations than the non-methylated, otherwise identical sequence [49]
Our method adopts the singlemolecule FRET technique, which is sensitive enough to detect the structural transition of DNA at the level of a single molecule
Summary
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Recognition of methylcytosine at a CpG site by such nucleases is the first step in a broad range of DMT assays In this way, one can check whether a certain locus is methylated, and detect the activity of DMT on a well-defined (cognate) sequence. We report a new method to characterize the activity of DMT and the effect of inhibitor drugs, utilizing a methylation-sensitive conformational transition of DNA. We established the quantitative dependence of Z-DNA formation on the degree of cytosine methylation, the sensitivity of which is the logic behind this BZ transition-based DMT assay. This method can distinguish different degrees of cytosine methylation in a single DNA molecule. The technique would certainly find excellent applications with distinct benefits unavailable so far in other traditional approaches
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
