Simultaneous and Sensitive Detection of Multisite 5‑Methylcytosine Including Non-CpG Sites at Single-5mC-Resolution

The methylation status of multiplexed methylcytosine sites can be simultaneously monitored by ligation-depended PCR assay. The ability of quantitative detection of multiplexed sites in one PCR reaction makes it a good choice for detecting methylation at both CpG and non-CpG sites for research and diagnosis of disease compared with others. The assay can determine as low as 20 aM methylated DNA and has been successfully applied to the genomic DNA sample derived from cancer cell lines

Fluorescent Strategy Based on Cationic Conjugated Polymer Fluorescence Resonance Energy Transfer for the Quantification of 5‑(Hydroxymethyl)cytosine in Genomic DNA

DNA methylation is dynamically reprogrammed during early embryonic development in mammals. It can be explained partially by the discovery of 5-(hydroxymethyl)­cytosine (5-hmC), 5-formylcytosine (5-fC), and 5-carboxylcytosine (5-caC), which are identified as key players involved in both active and passive demethylation pathways. As one of the ten–eleven translocation oxidation products, 5-hmC was found relatively abundant in neuron cells and embryonic stem cells. Herein we report a new method for 5-hmC quantification in genomic DNA based on CCP-FRET (cationic conjugated polymers act as the energy donor and induce fluorescence resonance energy transfer) assay combined with KRuO₄ oxidation. 5-hmC in genomic DNA can be selectively transformed into 5-fC by the oxidation of KRuO₄ and then labeled with hydroxylamine-BODIPY (BODIPY = 4,4-difluoro-4-bora-3a,4a-diaza-<i>s</i>-indacene) fluorophore through the reaction between 5-fC and hydroxylamine-BODIPY. After the fluorescently labeled DNA was captured by CCP through electrostatic interactions, a significant FRET between CCP and hydroxylamine-BODIPY fluorophore was observed. This CCP-FRET-based assay benefits from light-harvesting, large Stokes shift, and optical signal amplification properties of the CCP. Furthermore, this CCP-FRET-based assay was quite successfully demonstrated for the 5-hmC quantification in three types of cells (mESc, HeLa, HEK 293T), providing a much more convenient choice for 5-hmC quantification in genomic DNA

Highly Selective Detection of 5‑Methylcytosine in Genomic DNA Based on Asymmetric PCR and Specific DNA Damaging Reagents

DNA methylation is a significant epigenetic modification of the genome that is involved in regulating many cellular processes. An increasing number of human diseases have been discovered to be associated with aberrant DNA methylation, and aberrant DNA methylation has been deemed to be a potential biomarker for diseases such as cancers. A safe, nontoxic, and sensitive method for accurate detection of 5-methylcytosine in genomic DNA is extremely useful for early diagnosis and therapy of cancers. In this paper, we established a novel system to detect 5-methylcytosine, which is based on bisulfite treatment, asymmetric PCR, and specific DNA damaging reagents. Our method could be used for identifying the loci of 5mC in genomic DNA and detecting the DNA methylation levels in tissues as well

Precise Antibody-Independent m6A Identification via 4SedTTP-Involved and FTO-Assisted Strategy at Single-Nucleotide Resolution

Innovative detection techniques to achieve precise m6A distribution within mammalian transcriptome can advance our understanding of its biological functions. We specifically introduced the atom-specific replacement of oxygen with progressively larger atoms (sulfur and selenium) at 4-position of deoxythymidine triphosphate to weaken its ability to base pair with m6A, while maintaining A-T* base pair virtually the same as the natural one. 4SedTTP turned out to be an outstanding candidate that endowed m6A with a specific signature of RT truncation, thereby making this “RT-silent” modification detectable with the assistance of m6A demethylase FTO through next-generation sequencing. This antibody-independent, 4SedTTP-involved and FTO-assisted strategy is applicable in m6A identification, even for two closely gathered m6A sites, within an unknown region at single-nucleotide resolution

Application of <i>N</i>‑Halogeno‑<i>N</i>‑sodiobenzenesulfonamide Reagents to the Selective Detection of 5‑Methylcytosine in DNA Sequences

To surmount the challenges of the locus determination and accurate quantification of 5-methyl-2′-deoxycytidine (^5MedC) in DNA fragments that contain multiple ^5MedC residues, we designed and synthesized two <i>N</i>-halogeno-<i>N</i>-sodiobenzenesulfonamide reagents that provide a new chemical method for probing ^5MedC in DNA sequences. When the strategy we provided was combined with β-glucosyltransferase, ^5MedC could be distinguished from 5-hydroxymethyl-2′-deoxycytidine (^5hmdC) and deoxycytidine (dC) through the introduction of a glucose moiety to the hydroxyl group of ^5hmdC

Fluorescein Derivatives as Bifunctional Molecules for the Simultaneous Inhibiting and Labeling of FTO Protein

The FTO protein is unequivocally reported to play a critical role in human obesity and in the regulation of cellular levels of m⁶A modification, which makes FTO a significant and worthy subject of study. Here, we identified that fluorescein derivatives can selectively inhibit FTO demethylation, and the mechanisms behind these activities were elucidated after we determined the X-ray crystal structures of FTO/fluorescein and FTO/5-aminofluorescein. Furthermore, these inhibitors can also be applied to the direct labeling and enrichment of FTO protein combined with photoaffinity labeling assay