Detection Of 5hmC Research Articles

Methods for Detection and Mapping of Methylated and Hydroxymethylated Cytosine in DNA

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.

Cell-Free DNA Hydroxymethylation in Cancer: Current and Emerging Detection Methods and Clinical Applications.

In the era of precision oncology, identifying abnormal genetic and epigenetic alterations has transformed the way cancer is diagnosed, managed, and treated. 5-hydroxymethylcytosine (5hmC) is an emerging epigenetic modification formed through the oxidation of 5-methylcytosine (5mC) by ten-eleven translocase (TET) enzymes. DNA hydroxymethylation exhibits tissue- and cancer-specific patterns and is essential in DNA demethylation and gene regulation. Recent advancements in 5hmC detection methods and the discovery of 5hmC in cell-free DNA (cfDNA) have highlighted the potential for cell-free 5hmC as a cancer biomarker. This review explores the current and emerging techniques and applications of DNA hydroxymethylation in cancer, particularly in the context of cfDNA.

Abstract 7008: NEBNext® E5hmC-seqTM: Direct detection of 5-hydroxymethylcytosine at single base resolution

Abstract DNA methylation is a critical epigenetic regulator of gene expression during development and in diseases such as cancer. The modified cytosines, 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC), can be detected by sequencing libraries generated using existing methods: NEBNext® EM-seq™ or bisulfite sequencing. However, these methods cannot differentiate between 5mC and 5hmC. Interest in the role of 5hmC in regulating gene expression and as a cancer biomarker has increased, resulting in the need for robust methods for the identification of specific 5hmC sites. Methods currently exist to enable discrimination of 5mC and 5hmC (for example, oxBS-seq and TAB-seq), however these are based on modifications of bisulfite sequencing, and suffer from reduced data quality due to fragmentation and loss of DNA. Here we describe an enzymatic method that enables specific detection of 5hmC, termed NEBNext® Enzymatic 5hmC-seq (E5hmC-seqTM). E5hmC-seq libraries were generated for 0.1 ng to 200 ng DNA isolated from human brain and E14 mouse embryonic stem cells. Libraries were prepared using NEBNext® Ultra IITM reagents followed by two enzymatic steps to detect 5hmC. In the first step, 5hmCs are glucosylated, which protects them from subsequent deamination by APOBEC. In contrast, cytosines and 5mCs are deaminated resulting in their conversion to uracil and thymine, respectively. This conversion allows discrimination of 5hmC from cytosine and 5mC. Finally, libraries were PCR amplified using NEBNext® Q5U® and sequenced on the Illumina platform. The E5hmC-seq libraries have similar characteristics to EM-seq libraries, including expected insert sizes due to intact DNA molecules, low duplication rates and minimal GC bias. Moreover, E5hmC-seq libraries were well-correlated between inputs and replicates at higher sequencing depths. Fully hydroxymethylated T4147 phage DNA was used as an internal control, with 98-99% of cytosines identified as 5hmC. 5mC and 5hmC levels were also profiled during E14 cell differentiation for a period of 10 days. Interestingly, 5hmC levels decreased whereas 5mC levels increased particularly during the first five days of differentiation. LC-MS/MS quantification of this same DNA mirrored the changes observed by sequencing. E5hmC-seq libraries provide accurate measurements of 5hmC across a wide input range with expected insert sizes and minimal GC bias. Additionally, subtracting E5hmC-seq data from EM-seq data, which detects both 5mC and 5hmC, enables the precise location of individual 5mC and 5hmC sites. The ability to discriminate between 5mC and 5hmC will allow key insights to be made into the role of cytosine modifications in development and cancer. Citation Format: Garrett M. Cammarata, Daniel J. Evanich, V. K. Chaithanya Ponnaluri, Vaishnavi Panchapakesa, Ariel Erijman, Matt A. Campbell, Nan Dai, Bradley W. Langhorst, Louise Williams. NEBNext® E5hmC-seqTM: Direct detection of 5-hydroxymethylcytosine at single base resolution [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7008.

Association of tet methylcytosine dioxygenase 2 and 5-hydroxymethylcytosine in endometrioid adenocarcinoma and its clinical significance

BackgroundAberrant DNA methylation is a vital molecular alteration commonly detected in type I endometrial cancers (EC), and tet methylcytosine dioxygenase 2 (TET2) and 5-hydroxymethylcytosine (5hmC) play significant roles in DNA demethylation. However, little is known about the function and correlation of TET2 and 5hmC co-expressed in EC. This study intended to investigate the clinical significance of TET2 and 5hmC in EC.MethodsThe levels of TET2 and 5hmC were detected in 326 endometrial tissues by immumohistochemistry, and the correlation of their level was detected by Pearson analysis. The association between the levels of TET2 and 5hmC and clinicopathologic characteristics was analyzed. Prognostic value of TET2 and 5hmC was explored by Kaplan–Meier analysis. The Cox proportional hazard regression model was used for univariate and multivariate analyses.ResultsBased on the analysis results, TET2 protein level was positively correlated with 5hmC level in EC tissues (r = 0.801, P < 0.001). TET2+5hmC+ (high TET2 and high 5hmC) association was significantly associated with well differentiation, myometrial invasion, negative lymph node metastasis, and tumor stage in EC. Association of TET2 and 5hmC was confirmed as a prognostic factor (HR = 2.843, 95%CI = 1.226–3.605, P = 0.007) for EC patients, and EC patients with TET2−5hmC− level had poor overall survival.ConclusionsIn summary, the association of TET2 and 5hmC was downregulated in EC tissues, and may be a potential poor prognostic indicator for EC patients. Combined detection of TET2 and 5hmC may be valuable for the diagnosis and prognosis of EC.

Construction of a glycosylation-mediated fluorescent biosensor for label-free measurement of site-specific 5-hydroxymethylcytosine in cancer cells with zero background signal

Background5-hydroxymethylcytosine (5hmC) as an epigenetic modification can regulate gene expression, and its abnormal level is related with various tumor invasiveness and poor prognosis. Nevertheless, the current methods for 5hmC assay usually involve expensive instruments/antibodies, radioactive risk, high background, laborious bisulfite treatment procedures, and non-specific/long amplification time. ResultsWe develop a glycosylation-mediated fluorescent biosensor based on helicase-dependent amplification (HDA) for label-free detection of site-specific 5hmC in cancer cells with zero background signal. The glycosylated 5hmC-DNA (5ghmC) catalyzed by β-glucosyltransferase (β-GT) can be cleaved by AbaSI restriction endonuclease to generate two dsDNA fragments with sticky ends. The resultant dsDNA fragments are complementary to the biotinylated probes and ligated by DNA ligases, followed by being captured by magnetic beads. After magnetic separation, the eluted ligation products act as the templates to initiate HDA reaction, generating abundant double-stranded DNA (dsDNA) products within 20 min. The dsDNA products are measured in a label-free manner with SYBR Green I as an indicator. This biosensor can measure 5hmC with a detection limit of 2.75 fM and a wide linear range from 1 × 10−14 to 1 × 10−8 M, and it can discriminate as low as 0.001% 5hmC level in complex mixture. Moreover, this biosensor can measure site-specific 5hmC in cancer cells, and distinguish tumor cells from normal cells. SignificanceThis biosensor can achieve a zero-background signal without the need of either 5hmC specific antibody or bisulfite treatment, and it holds potential applications in biological research and disease diagnosis.

An Overview of Global, Local, and Base-Resolution Methods for the Detection of 5-Hydroxymethylcytosine in Genomic DNA.

The discovery of 5-hydroxymethylcytosine (5hmC) as a common DNA modification in mammalian genomes has ushered in new areas of inquiry regarding the dynamic epigenome. The balance between 5hmC and its precursor, 5-methylcytosine (5mC), has emerged as a determinant of key processes including cell fate specification, and alterations involving these bases have been implicated in the pathogenesis of various diseases. The identification of 5hmC separately from 5mC initially posed a challenge given that legacy epigenetic sequencing technologies could not discriminate between these two most abundant modifications, a significant blind spot considering their potentially functionally opposing roles. The growing interest in 5hmC, as well as in the Ten-Eleven Translocation (TET) familyenzymes that catalyze its generation and further oxidation to 5-formylcytosine (5fC) and 5-carboxycytosine (5caC), has spurred the development of versatile methods for 5hmC detection. These methods enable the quantification and localization of 5hmC in diverse biological samples and, in some cases, at the resolution of individual nucleotides. However, navigating this growing toolbox of methods for 5hmC detection can be challenging. Here, we detail existing and emerging methods for the detection, quantification, and localization of 5hmC at global, locus-specific, and base resolutionlevels. These methods are discussed in the context of their advantages and limitations, with the goal of providing a framework to help guide researchers in choosing the level of resolution and the associated method that could be most suitable for specific needs.

Simultaneous detection of 5-methylcytosine and 5-hydroxymethylcytosine at specific genomic loci by engineered deaminase-assisted sequencing.

Cytosine modifications, particularly 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC), play crucial roles in numerous biological processes. Current analytical methods are often constrained to the separate detection of either 5mC or 5hmC, or the combination of both modifications. The ability to simultaneously detect C, 5mC, and 5hmC at the same genomic locations with precise stoichiometry is highly desirable. Herein, we introduce a method termed engineered deaminase-assisted sequencing (EDA-seq) for the simultaneous quantification of C, 5mC, and 5hmC at the same genomic sites. EDA-seq utilizes a specially engineered protein, derived from human APOBEC3A (A3A), known as eA3A-M5. eA3A-M5 exhibits distinct deamination capabilities for C, 5mC, and 5hmC. In EDA-seq, C undergoes complete deamination and is sequenced as T. 5mC is partially deaminated resulting in a mixed readout of T and C, and 5hmC remains undeaminated and is read as C. Consequently, the proportion of T readouts (P T) reflects the collective occurrences of C and 5mC, regulated by the deamination rate of 5mC (R 5mC). By determining R 5mC and P T values, we can deduce the precise levels of C, 5mC, and 5hmC at particular genomic locations. We successfully used EDA-seq to simultaneously measure C, 5mC, and 5hmC at specific loci within human lung cancer tissue and their normal counterpart. The results from EDA-seq demonstrated a strong concordance with those obtained from the combined application of BS-seq and ACE-seq methods. EDA-seq eliminates the need for bisulfite treatment, DNA oxidation or glycosylation and uniquely enables simultaneous quantification of C, 5mC and 5hmC at the same genomic locations.

Abstract 6018: Sequencing of 5-hydroxymethylcytosine to single base resolution

Abstract DNA methylation is an epigenetic regulator of gene expression with important functions in development and diseases such as cancer. The modified cytosines, 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) are identified using Illumina sequencing. Libraries are generated using either NEBNext® EM-seq™, an enzyme-based workflow, or by using sodium bisulfite conversion. These methods, however, cannot differentiate between 5mC and 5hmC. Distinguishing these modifications is important as there is increased interest in 5hmCs role in regulating gene expression. Methods currently exist to enable discrimination of 5mC and 5hmC, for example, oxBS-seq and TAB-seq. These methods still rely upon sodium bisulfite conversion and have reduced data quality due to fragmentation and loss of DNA. Here we describe an enzymatic method that enables specific detection of 5hmC. 5hmC is detected using two enzymatic steps. Firstly, 5hmCs are glucosylated, which protects them from subsequent deamination by APOBEC. In contrast, cytosines and 5mCs are deaminated resulting in their conversion to uracil and thymine, respectively. During Illumina sequencing deaminated cytosine and 5mC are read as thymine and the glucosylated 5hmC are read as cytosine. Subtractive analysis of 5hmC data from EM-seq data, which detects both 5mC and 5hmC, permits the identification of individual 5mC and 5hmC sites. 5hmC data were generated for inputs of 0.1 ng to 200 ng DNA isolated from E14 mouse embryonic stem cells and human brain. The 5hmC libraries had similar characteristics to EM-seq libraries, including expected insert sizes due to intact DNA molecules, low duplication rates and minimal GC bias. T4147 phage DNA was used as an internal control, as all cytosines are 5-hydroxymethylated, with 98-99% of cytosines identified as 5hmC. 5mC and 5hmC levels were also profiled during E14 cell differentiation for 10 days. Interestingly, 5hmC levels decreased whereas 5mC levels increased particularly during the first five days of differentiation. LC-MS/MS quantification of this same DNA mirrored the changes observed by sequencing. The ability to discriminate between 5mC and 5hmC will provide key insights into the role of these cytosine modifications in development and disease. Citation Format: Daniel J. Evanich, V. K. Chaithanya Ponnaluri, Vaishnavi Panchapakesa, Ariel Erijman, Matthew A. Campbell, Nan Dai, Bradley W. Langhorst, Romualdas Vaisvila, Louise Williams. Sequencing of 5-hydroxymethylcytosine to single base resolution [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6018.

EBS-seq: enrichment-based method for accurate analysis of 5-hydroxymethylcytosine at single-base resolution

BackgroundA growing body of research has emphasized 5-hydroxymethylcytosine (5hmC) as an important epigenetic mark. High-resolution methods to detect 5hmC require high sequencing depth and are therefore expensive. Many studies have used enrichment-based methods to detect 5hmC; however, conventional enrichment-based methods have limited resolution. To overcome these limitations, we developed EBS-seq, a cost-efficient method for 5hmC detection with single-base resolution that combines the advantages of high-resolution methods and enrichment-based methods.ResultsEBS-seq uses selective labeling of 5hmC, deamination of cytosine and 5-methylcytosine, pull-down of labeled 5hmC, and C-to-T conversion during DNA amplification. Using this method, we profiled 5hmC in HEK293T cells and two colorectal cancer samples. Compared with conventional enrichment-based 5hmC detection, EBS-seq improved 5hmC signals by localizing them at single-base resolution. Furthermore, EBS-seq was able to determine 5hmC levels in CpG-dense regions where distortion of signals can occur, such as CpG islands and CpG shores. Comparing EBS-seq and conventional high-resolution 5hmC detection by ACE-seq, we showed that EBS-seq is more effective at finding 5hmC sites. Using EBS-seq, we found strong associations between gene expression and gene-body 5hmC content in both HEK293T cells and colorectal cancer samples.ConclusionsEBS-seq is a reliable and cost-efficient method for 5hmC detection because it simultaneously enriches 5hmC-containing DNA fragments and localizes 5hmC signals at single-base resolution. This method is a promising choice for 5hmC detection in challenging clinical samples with low 5hmC levels, such as cancer tissues.

Methylation of TET2 Promoter Is Associated with Global Hypomethylation and Hypohydroxymethylation in Peripheral Blood Mononuclear Cells of Systemic Lupus Erythematosus Patients.

(1) Background: It is widely accepted that aberrant methylation patterns contribute to the development of systemic lupus erythematosus (SLE). Ten-eleven translocation (TET) methylcytosine dioxygenase is an essential enzyme of which there are three members, TET1, 2, and 3, involved in hydroxymethylation, a newly uncovered mechanism of active DNA methylation. The epigenomes of gene transcription are regulated by 5-hydroxymethylcytocine (5-hmC) and TETs, leading to dysregulation of the immune system in SLE. The purpose of this study was to investigate the global hydroxymethylation status in SLE peripheral blood mononuclear cells (PBMCs) and to explore the role of TETs in changing the patterns of methylation. (2) Methods: We collected PBMCs from 101 SLE patients and 100 healthy donors. TaqMan real-time polymerase chain-reaction assay was performed for the detection of 5-methylcytosine (5-mC), 5-hmC, and TET2 mRNA expression and single-nucleotide polymorphism genotyping. The methylation rates in different CpG sites of TET2 promoters were examined using next-generation sequencing-based deep bisulfite sequencing. Putative transcription factors were investigated using the UCSC Genome Browser on the Human Dec. 2013 (GRCh38/hg38) Assembly. (3) Results: 5-mC and 5-hmC were both decreased in SLE. The mRNA expression level of TET2 was notably high and found to be correlated with the levels of immunologic biomarkers that are indicative of SLE disease activity. The analysis of methylation rates in the TET2 promoter revealed that SLE patients had significantly higher and lower rates of methylation in TET2 105146072-154 and TET2 105146218-331, respectively. (4) Conclusions: TET2 may play an important role in 5-mC/5-hmC dynamics in the PBMCs of SLE patients. The epigenetic modification of TET2 promoters could contribute to the pathogenesis of SLE and the intensity of the immunologic reaction.

Identifying 5-Hydroxymethylcytosine without Sequence Specificity Using MOF-Derived MnOxSy Nanoflowers for Boosting Electrochemiluminescence.

It is universally recognized that the quantification of DNA hydroxymethylation at random gene sequences still remains challenging. Herein, the highly sensitive identifying strategy of 5-hydroxymethylcytosine (5-hmC) without sequence specificity was achieved with a novel electrochemiluminescence (ECL) biosensor, which deftly integrated metal-organic framework (MOF)-derived amorphous MnOxSy nanoflowers (MnOxSy NFs) as a bifunctional co-reaction accelerator and cross-shaped DNA tracks as a well-regulated signal switch. Specifically, the target recognition process of 5-hmC was performed through specific chemical modification, where the hydroxymethyl sites were first aminated and then labeled with a 5'-carboxyl-functioned DNA walker, thus forming the target labeled DNA walker (5-ghmC-walker). Subsequently, the cross-shaped DNA tracks were ingeniously designed to endow the 5-ghmC-walker with continuous mechanical motion due to the long periodic linear alignment structure and well-regulated highly ordered interfaces. Furthermore, as a bifunctional co-reaction accelerator synthesized by in situ Mn-MOF template-sacrificing strategy, the MnOxSy NFs could promote the reduction of both dissolved O2 and S2O82-, remarkably boosting the ECL intensity of a peroxydisulfate (S2O82-) solution by 5.2 times compared to the pure S2O82- solution. Benefiting from specific target recognition and a dual-pathway strategy for boosting ECL, the proposed ECL platform can quantify 5-hmC with a wide linear range of 1 fM-1 nM and a low detection limit of 0.29 fM. This simple, highly sensitive strategy without sequence specificity provides a powerful platform for 5-hmC detection in the epigenetic study and disease pathogenesis.

5-Hydroxymethylation alterations in cell-free DNA reflect molecular distinctions of diffuse large B cell lymphoma at different primary sites

Background5-Hydroxymethylcytosine (5hmC), an important DNA epigenetic modification, plays a vital role in tumorigenesis, progression and prognosis in many cancers. Diffuse large B cell lymphoma (DLBCL) can involve almost any organ, but the prognosis of patients with DLBCL at different primary sites varies greatly. Previous studies have shown that 5hmC displays a tissue-specific atlas, but its role in DLBCLs at different primary sites remains unknown.ResultsWe found that primary gastric DLBCL (PG-DLBCL) and lymph node-involved DLBCL (LN-DLBCL) patients had a favorable prognosis, while primary central nervous system DLBCL (PCNS-DLBCL) patients faced the worst prognosis, followed by primary testicular DLBCL (PT-DLBCL) and primary intestinal DLBCL (PI-DLBCL) patients. Thus, we used hmC-CATCH, a bisulfite-free and cost-effective 5hmC detection technology, to first generate the 5hmC profiles from plasma cell-free DNA (cfDNA) of DLBCL patients at these five different primary sites. Specifically, we found robust cancer-associated features that could be used to distinguish healthy individuals from DLBCL patients and distinguish among different primary sites. Through functional enrichment analysis of the differentially 5hmC-enriched genes, almost all DLBCL patients were enriched in tumor-related pathways, and DLBCL patients at different primary sites had unique characteristics. Moreover, 5hmC-based biomarkers can also highly reflect clinical features.ConclusionsCollectively, we revealed the primary site differential 5hmC regions of DLBCL at different primary sites. This new strategy may help develop minimally invasive and effective methods to diagnose and determine the primary sites of DLBCL.

A fast and label-free detection of hydroxymethylated DNA using a nozzle-jet printed AuNPs@Ti3C2 MXene-based electrochemical sensor

5-hydroxymethylcytosine (5hmC) is a key epigenetic mark in the mammalian genome that has been proposed as a promising cancer biomarker with diagnostic and prognostic potentials. A new type of two-dimensional (2D) material called MXene includes transition metal carbides and nitrides and possesses unique physico-chemical properties suitable for diverse applications, including electrochemical sensors. Here, we report a new nozzle-jet printed electrochemical sensor using gold nanoparticles (AuNPs)@Ti3C2 MXene nanocomposite for the real-time and label-free detection of 5hmC in the genome. We utilized Ti3C2 MXene as a platform to immobilize AuNPs, which have been shown to exhibit different affinity interactions toward 5-methylcytosine (5 mC) and 5hmC, and thus produce distinct electrochemical responses. To fabricate the electrode, a highly conductive and adhesive silver ink was prepared to generate a silver line onto polyethylene terephthalate (PET) substrate using nozzle-jet printing, followed by deposition of AuNPs@Ti3C2 MXene ink at one end via dropcasting. Analyses of morphology and chemical composition showed that all steps of the sensor fabrication were successful. The fabricated sensor coupled with cyclic voltammetry showed excellent performance in distinguishing 5 mC- or 5hmC-enriched cellular genomic DNAs. As a proof-of-concept investigation, we confirmed that our sensor readily and consistently detected 5hmC diminution in multiple tumors, compared to the paired normal tissues. Thus, our simple and cost-effective sensing strategy using printable AuNPs@Ti3C2 MXene ink holds promise for a wide range of practical applications in epigenetic studies as well as clinical settings.

Bisulfite-free and single-nucleotide resolution sequencing of DNA epigenetic modification of 5-hydroxymethylcytosine using engineered deaminase.

The discovery of 5-hydroxymethylcytosine (5hmC) in mammalian genomes is a landmark in epigenomics study. Similar to 5-methylcytosine (5mC), 5hmC is viewed as a critical epigenetic modification. Deciphering the functions of 5hmC necessitates the location analysis of 5hmC in genomes. Here, we proposed an engineered deaminase-mediated sequencing (EDM-seq) method for the quantitative detection of 5hmC in DNA at single-nucleotide resolution. This method capitalizes on the engineered human apolipoprotein B mRNA-editing catalytic polypeptide-like 3A (A3A) protein to produce differential deamination activity toward cytosine, 5mC, and 5hmC. In EDM-seq, the engineered A3A (eA3A) protein can deaminate C and 5mC but not 5hmC. The original C and 5mC in DNA are deaminated by eA3A to form U and T, both of which are read as T during sequencing, while 5hmC is resistant to deamination by eA3A and is still read as C during sequencing. Therefore, the remaining C in the sequence manifests the original 5hmC. By EDM-seq, we achieved the quantitative detection of 5hmC in genomic DNA of lung cancer tissue. The EDM-seq method is bisulfite-free and does not require DNA glycosylation or chemical treatment, which offers a valuable tool for the straightforward and quantitative detection of 5hmC in DNA at single-nucleotide resolution.

A digital immuno-PCR assay for simultaneous determination of 5-methylcytosine and 5-hydroxymethylcytosine in human serum

This work aimed to develop an ultrasensitive and specific immunosorbent assay for simultaneous detection of double DNA methylation marks. Being considered the most important indicators in disease diagnosis, clinical treatment, and prognosis, 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) were chosen as the proof-of-concept targets. The described strategy consisted of Phos-tag Biotin anchoring at streptavidin-magnetic nanoparticles, specific immune recognition of anti-5mC antibody and anti-5hmC antibody and labeling of Barcode-antibody, signal amplification of immune PCR and digital PCR machine. Under optimal conditions, the digital immuno-PCR assay showed a board dynamic range from 2.7 × 10−13 mol/L to 2.7 × 10−9 mol/L and the detection limits were 61.7 fmol/L for 5mC, and of 0.111 pmol/L for 5hmC. A 16-fold and 186-fold improvement of LOD were obtained by the proposed approach for 5mC and 5hmC detection compared with real-time immune PCR. The approach also showed ideal specificity, repeatability and stability. The recovery test demonstrated that the digital immuno-PCR assay is a promising platform for the simultaneous determination of the two DNA methylation marks in human serum sample.

Photoelectrochemical biosensor for DNA hydroxymethylation detection based on the enhanced photoactivity of in-situ synthesized Bi4NbO8Cl@Bi2S3 heterojunction

As an important epigenetic modification, 5-hydroxymethylcytosine (5hmC) aroused wide concern about the distribution and the function. Due to the necessity of 5hmC detection, a novel photoelectrochemical (PEC) biosensor was established based on the in-situ generated heterojunction of Bi4NbO8Cl@Bi2S3, which was employed as the substrate material with excellent photoelectric property. The specific recognition of 5hmC relied on the covalent reaction between –CH2OH of 5hmC and –SH on the substrate electrode under the catalysis of M.HhaI methyltransferase. Afterwards, ZrO2 was used as signal amplification unit capturing by the specific reaction of Zr with the phosphate group of 5hmC. The experimental results demonstrated well specificity and sensitivity of this biosensor. Under optimal conditions, the linear relationship between the photocurrent and the logarithm value of 5hmC concentration was constructed with the range from 0.3 to 300 nM and the detection limit of 0.0779 nM (S/N = 3). The procedures of constructing this biosensor were compact and convenient, and this biosensor realized actual detection of 5hmC level in wheat sample. Significantly, this biosensor was applied to a preliminary study that the heavy metal Pb2+ and the perfluorooctanoic acid influence the expression of 5hmC in the genomic DNA of wheat seedling roots and leaves.

Label-Free and Template-Free Chemiluminescent Biosensor for Sensitive Detection of 5-Hydroxymethylcytosine in Genomic DNA.

5-Hydroxymethylcytosine (5hmC) is a modified base present at low levels in various mammalian cells, and it plays essential roles in gene expression, DNA demethylation, and genomic reprogramming. Herein, we develop a label-free and template-free chemiluminescent biosensor for sensitive detection of 5hmC in genomic DNAs based on 5hmC-specific glucosylation, periodate (IO4+) oxidation, biotinylation, and terminal deoxynucleotidyl transferase (TdT)-assisted isothermal amplification strategy, which we term hmC-GLIB-IAS. This hmC-GLIB-IAS exhibits distinct advantages of bisulfite-free, improved sensitivity, and genome-wide analysis of 5hmC at constant reaction temperature without the involvement of either specially labeled nucleic acid probes or specific templates for signal amplification. This method can sensitively detect 5hmC with a detection limit of 2.07 × 10-13 M, and it can detect 5hmC in the whole genome DNA with a detection limit of 3.92 × 10-5 ng/μL. Moreover, this method can distinguish 5hmC from 5-methylcytosine (5mC) and cytosine (C) and even discriminate 0.1% 5hmC in the mixture of 5hmC-DNA and 5mC-DNA. Importantly, this hmC-GLIB-IAS strategy enables genome-wide analysis without the involvement of either isotope-labeled substrates or specific antibodies, providing a powerful platform to detect 5hmC in real genomic DNA with high reproducibility and accuracy.

Aba-Seq: High-Resolution Enzymatic Mapping of Genomic 5-Hydroxymethylcytosine.

Aba-Seq (DNA modification-dependent restriction endonuclease AbaSI coupled with sequencing) provides a cost-effective and non-chemical based method for the high-resolution mapping of genomic 5-hydroxymethylcytosine (5hmC). The high specificity of the AbaSI enzyme allows sensitive detection of 5hmC in the genome. Here, we describe the Aba-Seq technique that was used for the high-resolution mapping of 5hmC in the genome of mouse embryonic stem cells (E14).

ELISA-Based Quantitation of Global 5hmC Levels.

5-Hydroxymethylcytosine (5hmC) is an abundant DNA modification in human and mouse brain, as well as in embryonic stem cells, while severely depleted in multiple types of cancer. Assays for 5hmC detection and quantification, both on a locus-specific and global level, are limited in number and often resource-intensive. Immunodetection of 5hmC through antibodies remains a cost-effective and widely accessible approach. This chapter describes an ELISA-based protocol for 5hmC detection and quantification in genomic or in vitro modified DNA. It is based on the passive adsorption of DNA onto a solid polystyrene surface and the specific detection of 5hmC, which generates a measurable chemiluminescent signal, proportional to the amount of immobilized 5hmC. The assay utilizes a standard curve for interpolation of 5hmC percentage and a loading standard for monitoring loading precision.

Electrochemiluminescence immunosensor for 5-hydroxymethylcytosine detection based on PAMAM-nanosilver‑nitrogen doped graphene nanocomposite

As a significant potential biomarker for early disease diagnostics, the detection of 5-hydroxymethylcytosine (5hmC) has garnered widespread attention. In this work, a simple and sensitive electrochemiluminescence (ECL) was developed for 5hmC detection. After substrate electrode preparation, anti-5hmC antibody was captured on electrode surface by the formation of amide bond. Then, through immunoreaction, 5hmC was specifically recognized and captured. Utilizing the crosslink effect of Zr 4+ with phosphoric acid recognition, aminomethyl phosphate was modified on electrode to further capture PAMAM-silver nanoclusters/Nitrogen-doped graphene nanocomposite (PAMAM-Ag-NG), where this nanocomposite was employed as ECL signal probe. The designed immunosensor showed a linear concentration range from 10 pM to 30 nM with a low detection limit of 2.47 pM (S/N = 3). This method presented good selectivity and reproducibility. To prove the applicability, this method was further used to study the influence of phytohormone and avian leukemia virus on 5hmC content in maize seedling leaves and chicken fibroblast cells, respectively. • A novel ECL strategy was developed for sensitive detection of 5hmC. • PAMAM-silver nanoclusters/Nitrogen-doped graphene nanocomposite was employed as ECL probe. • Avian leukemia virus decreased 5hmC content in chicken fibroblast cells. • Abscisic acid and gibberellinon decreased 5hmC content in maize seedling leaves. • The low detection limits of 2.47 pM was achieved.