The chemical modifications of DNA are of pivotal importance in the epigenetic regulation of cellular processes. Although the function of 5-methylcytosine (5mC) has been extensively investigated, the significance of 5-hydroxymethylcytosine (5hmC) has only recently been acknowledged. Conventional methods for the detection of DNA methylation frequently lack the capacity to distinguish between 5mC and 5hmC, resulting in the combined reporting of both. The growing importance of 5hmC has prompted the development of a multitude of methods for the qualitative and quantitative analysis of 5hmC in recent years, thereby facilitating researchers’ understanding of the mechanisms underlying the onset and progression of numerous diseases. This review covers both established and novel methods for the detection of cytosine modifications, including 5mC, 5hmC, 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC), with a particular focus on those that allow for accurate mapping and detection, particularly with third-generation sequencing. The review aims to help researchers choose the most appropriate methods based on their specific research goals and budget.

Olivomycin A is a highly active antitumor drug that belongs to the family of aureolic acid antibiotics. The antitumor effect of olivomycin A is related to its ability to bind to the DNA minor groove...

Background: The methylation of cytosine residues at CpG sites within the O6-methylguanine-DNA methyltransferase (MGMT) promoter is a key biomarker in glioblastoma therapy. The MGMT promoter (MGMTp) contains multiple guanine-rich sequences capable of folding into G-quadruplexes (G4s), but their relevance for MGMTp methylation is poorly understood. Objectives: Our study explores the impact of potential G-quadruplex-forming sequences (PQS) in the MGMT promoter CpG island on the activity of de novo DNA methyltransferase Dnmt3a. Additionally, we investigate their influence on the accuracy of methylation pattern detection using nanopore sequencing. Methods: Nanopore sequencing was employed to analyze the methylation of 94 clinically significant CpG sites in the human MGMTp using an in vitro de novo methylation system. Circular dichroism spectroscopy was used to identify G4 structures within the MGMTp CpG island. Interactions between the catalytic domain of Dnmt3a and the PQS from the MGMTp were examined by biolayer interferometry. Results: Guanine-rich DNA strands of the PQSs in the MGMTp were hypomethylated, while the complementary cytosine-rich strands were methylated by DNA methyltransferase Dnmt3a with higher efficiency. The accuracy of detecting modified bases in the PQS was significantly lower compared to surrounding sequences. Single-stranded guanine-rich DNA sequences from the MGMTp exhibited strong binding to Dnmt3a-CD, with an affinity approximately 10 times higher than their cytosine-rich complements (K_d = 3 × 10⁻⁸ M and 3 × 10⁻⁷ M, respectively). By binding to Dnmt3a, G4-forming oligonucleotides from MGMTp effectively inhibited the methylation reaction (IC₅₀ 6 × 10⁻⁷ M). Conclusions: The obtained data indicate the role of PQSs in establishing de novo methylation of the MGMT promoter. They also highlight the challenges of sequencing guanine-rich regions and the impact of specific de novo methylation patterns on clinical data interpretation.

S-Adenosyl-L-methionine (SAM) is a central cofactor in cellular methylation, donating methyl groups to a wide range of biological substrates. SAM analogues are promising tools for selective modulation of methyltransferase activity. Here, we investigated phosphorus-containing analogues of SAM and S-adenosyl-L-homocysteine (SAH), focusing on the H-phosphinic SAM analogue ((R,S)-SAM-P_H) with the HO(H)(O)P group replacing the carboxyl group of SAM. We examined the interaction of (R,S)-SAM-P_H with three representative methyltransferases: Dnmt1, responsible for maintenance of DNA methylation; Dnmt3a, which establishes de novo DNA methylation; and catechol-O-methyltransferase (COMT), which methylates protocatechuic aldehyde to yield vanillin and isovanillin. (R,S)-SAM-P_H is a methyl group donor for Dnmt3a and COMT, but not for Dnmt1, despite the high structural similarity of the Dnmt1 and Dnmt3a catalytic domains. These results demonstrate that targeted modification of the carboxyl group of SAM can yield analogues with specific activity towards various methyltransferases. The different recognition of (R,S)-SAM-P_H by Dnmt3a and Dnmt1 highlights its potential as a molecular probe for distinguishing de novo from maintenance DNA methylation. This work enriches our understanding of methyltransferase substrate specificity and provides a new tool for selective modulation of epigenetic processes.

The methylation of cytosines at CpG sites in DNA, carried out de novo by DNA methyltransferase Dnmt3a, is a basic epigenetic modification involved in gene regulation and genome stability. Aberrant CpG methylation in gene promoters leads to oncogenesis. In oncogene promoters, CpG sites often colocalize with guanine-rich sequences capable of folding into G-quadruplexes (G4s). Our in vitro study aimed to investigate how parallel G4s formed by a sequence derived from the c-MYC oncogene promoter region affect the activity of the Dnmt3a catalytic domain (Dnmt3a-CD). For this purpose, we designed synthetic oligonucleotide constructs: a c-MYC G4-forming oligonucleotide and linear double-stranded DNA containing an embedded stable extrahelical c-MYC G4. The topology and thermal stability of G4 structures in these DNA models were analyzed using physicochemical techniques. We showed that Dnmt3a-CD specifically binds to an oligonucleotide containing c-MYC G4, resulting in inhibition of its methylation activity. c-MYC G4 formation in a double-stranded context significantly reduces Dnmt3a-CD-induced methylation of a CpG site located in close proximity to the quadruplex structure; this effect depends on the distance between the non-canonical structure and the specific CpG site. One would expect DNA hypomethylation near the G4 structure, while regions distant from this non-canonical form would maintain a regular pattern of high methylation levels. We hypothesize that the G4 structure sequesters the Dnmt3a-CD and impedes its proper binding to B-DNA, resulting in hypomethylation and activation of c-MYC transcription.