Whole-Genome Mapping of Epigenetic Modification of 5-Formylcytosine at Single-Base Resolution by Chemical Labeling Enrichment and Deamination Sequencing

Analytical Chemistry, Journal Year: 2024, Volume and Issue: 96(11), P. 4726 - 4735

Published: March 7, 2024

DNA cytosine methylation (5-methylcytosine, 5mC) is a predominant epigenetic modification that plays critical role in variety of biological and pathological processes mammals. In active demethylation, the 10-11 translocation (TET) dioxygenases can sequentially oxidize 5mC to generate three modified forms cytosine, 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), 5-carboxylcytosine (5caC). Beyond being demethylation intermediate, recent studies have shown 5fC has regulatory functions gene expression chromatin organization. While some methods been developed detect 5fC, genome-wide mapping at base resolution still highly desirable. Herein, we propose chemical labeling enrichment deamination sequencing (CLED-seq) method for detecting genomic single-base resolution. The CLED-seq utilizes selective 5fC-containing fragments, followed by mediated apolipoprotein B mRNA-editing catalytic polypeptide-like 3A (APOBEC3A or A3A) sequencing. process, while all C, 5mC, 5hmC are interpreted as T during sequencing, read enabling precise detection DNA. Using proposed method, accomplished mouse embryonic stem cells. study revealed promoter regions enriched with overlapped H3K4me1, H3K4me3, H3K27ac marks. These findings suggest correlation between marks mESCs. conclusion, straightforward, bisulfite-free offers valuable tool genomes

Language: Английский

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Quantification and mapping of RNA modifications

TrAC Trends in Analytical Chemistry, Journal Year: 2024, Volume and Issue: 172, P. 117606 - 117606

Published: Feb. 21, 2024

Language: Английский

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Simultaneous Detection of Adenosine-to-Inosine Editing and N⁶-Methyladenosine at Identical RNA Sites through Deamination-Assisted Reverse Transcription Stalling

Analytical Chemistry, Journal Year: 2024, Volume and Issue: 96(21), P. 8730 - 8739

Published: May 14, 2024

Adenosine-to-inosine (A-to-I) editing and N6-methyladenosine (m6A) modifications are pivotal RNA with widespread functional significance in physiological pathological processes. Although significant effort has been dedicated to developing methodologies for identifying quantifying these modifications, traditional approaches have often focused on each modification independently, neglecting the potential co-occurrence of A-to-I m6A at same adenosine residues. This limitation constrained our understanding intricate regulatory mechanisms governing function interplay between different types modifications. To address this gap, we introduced an innovative technique called deamination-assisted reverse transcription stalling (DARTS), specifically designed simultaneous quantification sites. DARTS leverages selective deamination activity engineered TadA–TadA8e protein, which converts residues inosine, combination unique property Bst 2.0 DNA polymerase, stalls when encountering inosine during transcription. approach enables accurate editing, m6A, unmodified identical The method is remarkable its ability directly quantify two distinct simultaneously, a capability that remained largely unexplored field biology. By facilitating comprehensive analysis interaction opens new avenues exploring complex networks modulated by

Language: Английский

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Accurate Quantification of Ten Methylated Purine Nucleosides by Highly Sensitive and Stable Isotope-Diluted UHPLC–MS/MS

Analytical Chemistry, Journal Year: 2024, Volume and Issue: 96(28), P. 11366 - 11373

Published: July 6, 2024

The dynamic landscape of cellular nucleotides/nucleosides associated with RNA metabolism, particularly in diseases like cancer, has spurred intensive interest. Here, we report a robust stable isotope-diluted UHPLC-ESI-MS/MS method for accurate quantification 12 purine ribonucleosides, including 10 methylated nucleosides. By the use thermally decomposable ammonium bicarbonate (NH

Language: Английский

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Single-Base Resolution Detection of N⁶-Methyladenosine in RNA by Adenosine Deamination Sequencing

Analytical Chemistry, Journal Year: 2023, Volume and Issue: 95(28), P. 10588 - 10594

Published: July 4, 2023

N6-Methyladenosine (m6A) is one of the most abundant and prevalent natural modifications occurring in diverse RNA species. m6A plays a wide range roles physiological pathological processes. Revealing functions relies on faithful detection individual sites RNA. However, developing simple method for single-base resolution still challenging task. Herein, we report an adenosine deamination sequencing (AD-seq) technique facile at resolution. The AD-seq approach capitalizes selective adenosine, but not m6A, by evolved tRNA deaminase (TadA) variant TadA8e or dimer protein TadA-TadA8e. In AD-seq, deaminated TadA-TadA8e to form inosine, which pairs with cytidine read as guanosine sequencing. resists due interference methyl group N6 position adenosine. Thus, base thymine differential readouts from A can achieve Application proposed successfully identified Escherichia coli 23S rRNA. Taken together, allows cost-effective RNA, provides valuable tool decipher

Language: Английский

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Site-specific quantification of Adenosine-to-Inosine RNA editing by Endonuclease-Mediated qPCR

Bioorganic & Medicinal Chemistry, Journal Year: 2024, Volume and Issue: 110, P. 117837 - 117837

Published: July 11, 2024

Language: Английский

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

Analytical Chemistry, Journal Year: 2024, Volume and Issue: unknown

Published: Dec. 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

Language: Английский

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

Analytical Chemistry, Journal Year: 2025, Volume and Issue: unknown

Published: April 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.

Language: Английский

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Quantification of Epigenetic DNA and RNA Modifications by UHPLC–MS/MS Technologies: New Concepts and New Improvements for the Special Collections

Journal of Separation Science, Journal Year: 2025, Volume and Issue: 48(5)

Published: May 1, 2025

ABSTRACT Dynamic and reversible DNA RNA modifications are essential for cell differentiation development. Aberrant epigenetic closely associated with the occurrence progression of diseases, serving as potential markers cancer diagnosis prognosis. Ultra‐high‐performance liquid chromatography coupled tandem mass spectrometry (UHPLC–MS/MS) offers distinct advantages in qualitative quantitative analysis various due to its sensitivity, specificity, accuracy. This review provides a comprehensive overview current knowledge regarding chromatography–mass (LC–MS) modifications, including analytical procedures, advancements, biological applications, focus on tracing source (N6‐2′‐deoxy‐adenosine) 6mdA eukaryotes. Additionally, we examine integration UHPLC–MS/MS other separation techniques achieve accurate quantification specific regions, certain fragments, free nucleosides.

Language: Английский

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

Science China Life Sciences, Journal Year: 2025, Volume and Issue: unknown

Published: May 22, 2025

Language: Английский

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