Orthologous Mammalian A3A-Mediated Single-Nucleotide Resolution Sequencing of DNA Epigenetic Modification 5-Hydroxymethylcytosine

Chemical Science, Год журнала: 2025, Номер unknown

Опубликована: Янв. 1, 2025

Epigenetic modifications in genomes play a crucial role regulating gene expression mammals. Among these modifications, 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) are recognized as the fifth sixth nucleobases genomes, respectively, two most significant epigenetic marks 5hmC serves both an intermediate active DNA demethylation stable modification involved various biological processes. Analyzing location of is essential for understanding its functions. In this study, we introduce orthologous mammalian A3A-mediated sequencing (OMA-seq) method quantitative detection genomic at single-nucleotide resolution. OMA-seq relies on deamination properties naturally occurring A3A proteins: green monkey (gmA3A) dog (dogA3A). The combined use gmA3A dogA3A effectively deaminates cytosine (C) 5mC, but not 5hmC. As result, original C 5mC deaminated read thymine (T) during sequencing, while remains unchanged C. Consequently, remaining sequence indicates presence Using OMA-seq, successfully quantified from lung cancer tissue corresponding normal tissue. enables accurate mapping resolution, utilizing pioneering single-step protocol that leverages high specificity natural deaminases. This approach eliminates need bisulfite conversion, glycosylation, chemical oxidation, or screening engineered protein variants, thereby streamlining analysis utilization enzymes expands toolkit research, enabling precise modified nucleosides uncovering new insights into regulation.

Язык: Английский

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Persistent organic pollutant perfluorooctanoic acid induces alterations in epigenetic modifications of DNA and RNA

Chinese Chemical Letters, Год журнала: 2025, Номер unknown, С. 110957 - 110957

Опубликована: Фев. 1, 2025

Язык: Английский

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Bisulfite-Free and Quantitative Detection of DNA Methylation at Single-Base Resolution by eROS1-seq

Analytical Chemistry, Год журнала: 2024, Номер unknown

Опубликована: Дек. 16, 2024

5-Methylcytosine (5mC) is the most significant DNA modification present in mammalian genomes. Understanding roles of 5mC diverse biological processes requires quantitative detection at single-base resolution. In this study, we engineered repressor silencing 1 (ROS1) protein derived from

Язык: Английский

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Research advances in the transplacental transfer efficiencies of environmental pollutants

Chinese Journal of Chromatography, Год журнала: 2025, Номер 43(1), С. 13 - 21

Опубликована: Янв. 1, 2025

Industrialization has led to significant increases in the types and quantities of pollutants, with environmental pollutants widely present various media, including air, food, everyday items. These can enter human body via multiple pathways, ingestion through food absorption skin; this intrusion disrupt production, release, circulation hormones body, resulting a range illnesses that affect reproductive, endocrine, nervous systems. Consequently, these pose substantial risks health. In particular, fetuses are highly sensitive during critical stages development, exposure periods growth development result more-obvious severe health hazards lead preterm birth, low birth weight, fetal malformations. The placenta acts as barrier between mother fetus, selectively filters certain pollutants. While some remain maternal bloodstream, others cross placental into umbilical blood passive diffusion, transport proteins, or endocytosis. transplacental transfer efficiency (TTE) is ratio level pollutant blood, valuable metric for evaluating ability breach barrier. A higher TTE implies larger proportion transferred from thereby amplifying potential fetus. Mass spectrometry-based detection methods extensively used chemical sciences because they exceptionally resolving. This analytical technique involves ionizing compounds within sample identifying them based on their distinct mass-to-charge ratios; it enables both qualitative quantitative analyses Current methodologies examining include in-vitro experiments, animal studies, epidemiologic model calculation; approaches help evaluate fetus placenta. Analyzing TTEs different chemicals high-risk be identified provides an understanding abilities Research focused mainly per- polyfluoroalkyl substances (PFASs), polybrominated diphenyl ethers (PBDEs), polychlorinated biphenyls (PCBs), organochlorine pesticides (OCPs), relatively few studies other reported. Pollutant complex process influenced by factors physical properties (e.g., molecular mass, solubility, lipophilicity), lifestyle, genetics, conditions, socioeconomic status), characteristics maturity, flow, metabolic enzymes). review summarizes recent advances research focusing methods, PFASs, PBDEs, PCBs, OCPs, pivotal influence TTEs. Studying elucidated, providing support data exposure, transfer, accumulation well theoretical framework mechanism expected play vital role assessing impact pregnant women fetuses.

Язык: Английский

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Label-Free and Sequence-Independent Isothermal Amplification Strategy for the Simultaneous Detection of Genomic 5-Methylcytosine and 5-Hydroxymethylcytosine

Analytical Chemistry, Год журнала: 2025, Номер unknown

Опубликована: Янв. 27, 2025

5-Methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) are crucial epigenetic modifications in eukaryotic genomic DNA that regulate gene expression associated with the occurrence of various cancers. Here, we combined bisulfite conversion 4-acetamido-2,2,6,6-tetramethyl-1-oxopiperridinium tetrafluoroborate (ACT+BF4-, TCI) oxidation to develop a label-free sequence-independent isothermal amplification (BTIA) assay for genome-wide 5mC 5hmC analysis. The BTIA strategy can distinguish signatures from other bases high sensitivity good specificity, avoiding sophisticated chemical expensive protein labeling. Moreover, utilization terminal deoxynucleotidyl transferase (TdT) enables proposed detect global without sequence dependence. With only 78 ng input DNA, levels were accurately quantified cells (including cancer A549, T47D, K562 normal HEK-293T, CHO, NRK-52E) clinical whole blood samples healthy control, precancerous cervical cancer, confirmed cancer) within 18 h. detection results suggested was highly expressed cells. More importantly, significant increase observed further upregulation suggesting correlation between development. However, showed reverse result these tested samples. Collectively, be easily performed on ordinary heating apparatus almost all research medical laboratories, showing application early screening clinic.

Язык: Английский

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Glycosidase-Enabled Locus-Specific Detection of 8-Oxo-7,8-dihydroguanine in Genomes under Oxidative Stress

Analytical Chemistry, Год журнала: 2025, Номер unknown

Опубликована: Апрель 12, 2025

Oxidative DNA damage is closely linked to the onset of various age-related diseases. A significant oxidation product, 8-oxo-7,8-dihydroguanine (8OG), has been considered an important epigenetic-like marker in regulating gene expression. Accurately quantifying locus-specific 8OG levels crucial for understanding its functional roles disease induction and regulation. In this study, we developed a glycosylase cleavage-mediated extension stalling (GCES) method detection quantification genomic DNA. This utilizes 8OG-DNA Pab-AGOG, which induces single-strand breaks containing 8OG. The resulting cleavage then assessed quantified by using quantitative real-time PCR (qPCR). We successfully applied strategy evaluate synthesized from HEK293T, HeLa, HepG2 cell lines under oxidative stress or triclosan treatment. results demonstrate that GCES accurate suitable site-specific both biological samples. observed increased level samples treated with H2O2 triclosan, indicating agents can elevate Overall, provides valuable, straightforward, cost-effective tool at base resolution, facilitates investigation as marker.

Язык: Английский

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Deaminase-Driven Random Mutation Enables Efficient DNA Mutagenesis for Protein Evolution

Chemical Science, Год журнала: 2025, Номер unknown

Опубликована: Янв. 1, 2025

Protein evolution has emerged as a crucial tool for generating proteins with novel characteristics. A key step in protein is the mutagenesis of protein-coding DNA. Error-prone PCR (epPCR) is...

Язык: Английский

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Direct Single-Nucleotide Resolution Sequencing of DNA 5-Methylcytosine Using Engineered DNA Methyltransferase-Mediated CMD-seq

Chemical Science, Год журнала: 2025, Номер unknown

Опубликована: Янв. 1, 2025

Utilizing the newly identified M.MedI-N377K methyltransferase, we developed a novel method of CMD-seq for stoichiometric detection 5mC in DNA at single-nucleotide resolution.

Язык: Английский

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Precise Recognition and Quantification of Locus‐Specific DNA Methylation Using Engineered ROS1^†

Chinese Journal of Chemistry, Год журнала: 2025, Номер unknown

Опубликована: Апрель 24, 2025

Comprehensive Summary 5‐Methylcytosine (5mC) plays pivotal roles in numerous biological processes. To gain a deeper understanding of the functions 5mC, it is essential to develop methods for its quantitative analysis. Herein, we engineered repressor silencing 1 (ROS1) protein enhance glycosylase/lyase activity towards resulting an ROS1 (eROS1) that can effectively excise 5mC from DNA. Using eROS1, developed method termed ROS1‐mediated (eRMQ) analysis, locus‐specific quantification genomic This capitalizes on ability eROS1 selectively cleave which creates one‐nucleotide gap. The presence this gap hinders extension DNA polymerase, leading reduction products be evaluated using real‐time PCR (qPCR). limit detection eRMQ was as low fM. method, achieved analysis at individual sites within and demonstrated significant levels lung cancer tissues compared adjacent normal tissues. Taken together, study introduces DNA, offering valuable tool exploring epigenetic regulation human diseases.

Язык: Английский

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Bisulfite-free whole-genome mapping of 5-methylcytosine at single-base resolution by NTD-seq

Science China Life Sciences, Год журнала: 2025, Номер unknown

Опубликована: Май 22, 2025

Язык: Английский

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