液相色谱-串联质谱法测定生物样本全基因组DNA甲基化

建立了基于液相色谱-电喷雾串联质谱的分析方法,对生物样本中全基因组DNA甲基化水平进行定量测定.首先将DNA从生物样本中提取出来,将DNA片段酶解为单核苷,利用液相色谱-串联质谱测定胞嘧啶核苷和5-甲基胞嘧啶核苷的含量,从而计算出其全基因组DNA甲基化率.利用该法研究了暴露于全氟辛烷磺酸的L-02细胞、10例原发性肝癌病例血浆样本和10例对照血浆样本的全基因组DNA甲基化水平,得出了它们的总甲基化率变化的初步结果.本方法操作简单,具有很高的灵敏度和稳定性,为研究生物样本,尤其是临床上易得但DNA含量极低的血浆样本的总甲基化水平提供了思路.     

Determination of the DNA methylation level in tumor cells by capillary electrophoresis and laser-induced fluorescence detection

An analytical method to determine the genome-wide DNA methylation in only 100 ng DNA is presented. The analysis is based on DNA isolation and hydrolysis followed by derivatization of the 2'-desoxyribonucleoside-3'-monophosphates with a fluorescence dye (4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl ethylene diamine hydrochloride, Bodipy FL EDA). The separation of the derivatives was carried out by micellar electrokinetic chromatography, and laser-induced fluorescence was used for detection. To calculate the methylation level, the derivatization factor and the quantum yields of the Bodipy conjugates of 2'-desoxycytidine-3'-monophosphate (dCMP) and 2'-desoxy-5-methylcytidine-3'-monophosphate (5m-dCMP) were determined by measurement of methylated Lambda DNA. The assignment was made by cochromatography with the synthesized and characterized standard compound 5m-dCMP. After optimization of the method it was possible to determine the methylation level in 100-ng DNA samples with a standard deviation of less than 5%.     

Identification of CpG methylation of the SNRPN gene by methylation‐specific multiplex PCR

In this article, we show that methylation‐specific multiplex PCR (MS‐multiplex PCR) is a sensitive and specific single assay for detecting CpG methylation status as well as copy number aberrations. We used MS‐multiplex PCR to simultaneously amplify three sequences: the 3′ ends of the SNRPN gene (for unmethylated sequences), the KRITI gene (as internal control), and the promoter of the SNRPN gene containing CpG islands (for methylated sequences) after digestion with a methylation‐sensitive restriction enzyme (HhaI). We established this duplex assay for the analysis of 38 individuals with Prader–Willi syndrome, 2 individuals with Angelman syndrome, and 28 unaffected individuals. By comparing the copy number of the three regions, the methylation status and the copy number changes can be easily distinguished by MS‐multiplex PCR without the need of bisulfite treatment of the DNA. The data showed that MS‐multiplex PCR allows for the estimation of the methylation level by comparing the copy number aberrations of unknown samples to the standards with a known methylated status. The in‐house‐designed MS‐multiplex PCR protocol is a relatively simple, cost‐effective, and highly reproducible approach as a significant strategy in clinical applications for epigenetics in a routine laboratory.     

面向DNA甲基化修饰分析的质谱技术

DNA甲基化作为表观遗传修饰中一种重要的调控方式,通过调控基因的表达,从而影响机体内一系列的生物学过程。色谱-质谱法是研究DNA甲基化修饰的重要研究手段。随着对哺乳动物DNA甲基化的生物学功能的深入研究,应用于研究表观遗传修饰的手段与仪器设备越来越先进。为了对DNA修饰进行定性与定量的分析检测,除了高效液相色谱整合不同种类质量分析器的质谱联用（HPLC-MS）技术外,目前还开发应用了基质辅助激光解析质谱技术（MALDI-ToF-MS）和气相色谱-质谱联用技术（GC-MS）,从而极大拓展了DNA甲基化修饰研究的手段。本文对分析表观遗传DNA甲基化修饰的质谱技术发展进行综述,希望为DNA甲基化修饰分析提供有价值的研究策略。     

Boronic acid-based fluorescent receptors for selective recognition of thymine glycol

Thymine glycol is the major oxidation product of thymine. The amount of thymine glycol present within cellular DNA is one marker of the extent of oxidative damage, and chemosensors for thymine glycol have therefore a number of potential applications. In continuation of our studies devoted to the detection of modified nucleosides, we report herein our results toward the fluorescence detection of thymine glycol at both the nucleoside and the oligonucleotide levels using boronic acid sensors. These receptors show significant fluorescence enhancements and high selectivities in aqueous conditions upon binding with thymine glycol.     

Sensitive detection of DNA methyltransferase activity based on supercharged fluorescent protein and template-free DNA polymerization

DNA methylation, catalyzed by DNA methyltransferases(MTases), is a key component of genetic regulation, and DNA MTases have been regarded as potential targets in anticancer therapy. Herein, based on our previously developed DNA-mediated supercharged green fluorescent protein(Sc GFP)/graphene oxide(GO) interaction, coupled with methylation-initiated template-free DNA polymerization, we propose a novel fluorescence assay strategy for sensitive detection of DNA MTase activity. A hairpin DNA with a methylation-sensitive site and an amino-modified 3′-terminal(DNA-1) was designed and worked as a starting molecule. In the presence of DNA MTase, methylation-sensitive restriction endonuclease, and terminal deoxynucleotidyl transferase(Td T), DNA-1 can be sequentially methylated, cleaved, and further elongated. The resulting long DNA fragments quickly bind with Sc GFP and form the Sc GFP/DNA nanocomplex. Such nanocomplex can effectively protect Sc GFP from being adsorbed and quenched by GO. Without the methylation-initiated DNA polymerization, the fluorescence of Sc GFP will be quenched by GO. Thus, the DNA MTase activity, which is proportional to the amount of DNA polymerization products, can be measured by reading the fluorescence of Sc GFP/GO. The method was successfully used to detect the activity of DNA adenine methylation(Dam) MTase with a wide linear range(0.1–100 U/m L) and a low detection limit of 0.1 U/m L. In addition, the method showed high selectivity and the potential to be applied in a complex sample. Furthermore, this study was successfully extended to evaluate the inhibition effect of 5-fluorouracil on Dam MTase activity and detect Td T activity.     

DNA tetrahedron-based split aptamer probes for reliable imaging of ATP in living cells

Accurate detection and imaging of adenosine triphosphate(ATP) expression levels in living cells is of great value for understanding cell metabolism, physiological activities, and pathologic mechanisms. Here, we developed a DNA tetrahedron-based split aptamer probe(TD probe) for ratiometric fluorescence imaging of ATP in living cells. The TD probe is constructed by hybridizing two split ATP aptamer probes(Apt-a and Apt-b) to a DNA tetrahedron assembled by four DNA oligonucleotides(T1, T2, T3 and ...     

Bisulfite-free analysis of 5MeC-binding proteins and locus-specific methylation density using a microparticle-based flow cytometry assay

DNA methylation analysis is emerging as a new technique with potential capabilities for early cancer detection. However, current state-of-the-art techniques are not easily translatable into routine clinical methods. Herein we describe a bead-based flow cytometry assay which combines DNA hybridization to microparticles with 5MeC-specific proteins/antibodies. These assays can be used to study the binding properties of current and emerging 5MeC-binding proteins and may also have potential in the measurement of 5MeC density in clinical samples for cancer detection.     

Universal optical assays based on multi-component nanoprobes for genomic deoxyribonucleic acid and proteins

In this report, we developed a universal assay method for both genomic DNA and proteins by using enzyme-based multi-component optical nanoprobes. The nanoprobes are gold nanoparticles assembled with bio-recognizing and signaling elements. We firstly demonstrated that the nanoprobes could detect unpurified asymmetric polymerase chain reaction (PCR) product from genomic DNA of Escherichia coli, with the sensitivity approximately 10 times higher than that of quantitative real-time PCR assay. The limit of detection (LOD) of our nanoprobe-based method is less than 10 pg template DNA (target DNA). Using DNA aptamers as recognition elements, we also showed that as few as 0.1 nM thrombin could be colorimetrically detected with high specificity. These results indicated that the enzyme-based multi-component nanoprobes have the capability to work with real biological samples, and have the potential in various biological and clinical applications.     

Sensitive detection of DNA methyltransferase activity based on rolling circle amplification technology

This work develops a fluorescence approach for sensitive detection of DNA methyltransferase activity based on endonuclease and rolling circle amplification （RCA） technique. In the presence of DNA adenine methylation （Dam） MTase, the methylation-responsive sequence of hairpin probe is methylated and cleaved by the methylation-sensitive restriction endonuclease Dpn 1. The products cleaved by restriction endonuclease Dpn I then function as a signal primer to initiate RCA reaction by hybridizing with the circular DNA template. Each RCA product containing thousands of repeated sequences might hybridize with a large number of molecular beacons （detection probes）, resulting in an enhanced fluorescence signal. In the absence of Dam MTase, neither methylation/cleavage nor RCA reaction can be initiated and no fluorescence signal is observed. The proposed method exhibits a dynamic range from 0.5 U/mL to 30 U/mL and a detection limit of 0.18 U/mL. This method can be used for the screening of antimicrobial drugs and has a great potential to be further applied in early clinical diagnosis.     

Quantitative analysis of DNA methylation in the promoter region of the methylguanine‐O6‐DNA‐methyltransferase gene by COBRA and subsequent native capillary gel electrophoresis

Along with histone modifications, RNA interference and delayed replication timing, DNA methylation belongs to the key processes in epigenetic regulation of gene expression. Therefore, reliable information about the methylation level of particular DNA fragments is of major interest. Herein the methylation level at two positions of the promoter region of the gene methylguanine‐O⁶‐DNA‐Methyltransferase (MGMT) was investigated. Previously, it was demonstrated that the epigenetic status of this DNA region correlates with response to alkylating anticancer agents. An automated CGE method with LIF detection was established to separate the six DNA fragments resulting from combined bisulfite restriction analysis of the methylated and non‐methylated MGMT promoter. In COBRA, the DNA was treated with bisulfite converting cytosine into uracil. During PCR uracil pairs with adenine, which changes the original recognition site of the restriction enzyme Taql. Artificial probes generated by mixing appropriate amounts of DNA after bisulfite treatment and PCR amplification were used for validation of the method. The methylation levels of these samples could be determined with high accuracy and precision. DNA samples prepared by mixing the corresponding clones first and then performing PCR amplification led to non‐linear correlation between the corrected peak areas and the methylation levels. This effect is explained by slightly different PCR amplification of DNA with different sequences present in the mixture. The superiority of CGE over PAGE was clearly demonstrated. Finally, the established method was used to analyze the methylation levels of human brain tumor tissue samples.     

Fluorescent conjugated polyelectrolyte as an indicator for convenient detection of DNA methylation

A convenient, sensitive, and label-free method to determine the DNA methylation status of CpG sites of plasmid and human colon cancer cell has been developed. The system relies on highly selective single base extension reaction and significant optical amplification of cationic conjugated polyelectrolytes (CCP-1). The higher fluorescence resonance energy transfer efficiency between CCP-1 and fluorescein-labeled dGTP (dGTP-Fl) is correlated to the incorporation of dGTP-Fl into the probe DNA by single base extension reaction when the target/probe pair is complementary at the methylation site. As low as 1% methylation status can be determined by this new assay method. Because of the optical amplification property of CCP-1, the method exhibited high sensitivity with a concentration of analyte DNA at the picomolar level. The CCP-1 can form a complex with negatively charged DNA through electrostatic interactions, avoiding labeling the DNA target and probe by covalent linking. The isolation steps employed in other typical assays were avoided to simplify operations and increase repeatability. These features make the system promising for future use for early cancer diagnosis.     

Fast and stable photochromic oxazines for fluorescence switching

The stringent limitations imposed by diffraction on the spatial resolution of fluorescence microscopes demand the identification of viable strategies to switch fluorescence under optical control. In this context, the photoinduced and reversible transformations of photochromic compounds are particularly valuable. In fact, these molecules can be engineered to regulate the emission intensities of complementary fluorophores in response to optical stimulations. On the basis of this general design logic, we assembled a functional molecular construct consisting of a borondipyrromethene fluorophore and a nitrospiropyran photochrome and demonstrated that the emission of the former can be modulated with the interconversion of the latter. This fluorophore-photochrome dyad, however, has a slow switching speed and poor fatigue resistance. To improve both parameters, we developed a new family of photochromic switches based on the photoinduced opening and thermal closing of an oxazine ring. These compounds switch back and forth between ring-closed and -open isomers on nanosecond-microsecond timescales and tolerate thousands of switching cycles with no sign of degradation. In addition, the attachment of appropriate chromophoric fragments to their switchable oxazine ring can be exploited to either deactivate or activate fluorescence reversibly in response to illumination with a pair of exciting beams. Specifically, we assembled three dyads, each based on either a borondipyrromethene or a coumarin fluorophore and an oxazine photochrome, and modulated their fluorescence in a few microseconds with outstanding fatigue resistance. The unique photochemical and photophysical properties of our fluorophore-photochrome dyads can facilitate the development of switchable fluorophores for superresolution imaging and, ultimately, provide valuable molecular probes for the visualization of biological samples on the nanometer level.     

DNA methylation-based age prediction with bloodstains using pyrosequencing and random forest regression

The use of DNA methylation to predict chronological age has shown promising potential for obtaining additional information in forensic investigations. To date, several studies have reported age prediction models based on DNA methylation in body fluids with high DNA content. However, it is often difficult to apply these existing methods in practice due to the low amount of DNA present in stains of body fluids that are part of a trace material. In this study, we present a sensitive and rapid test for age prediction with bloodstains based on pyrosequencing and random forest regression. This assay requires only 0.1 ng of genomic DNA and the entire procedure can be completed within 10 h, making it practical for forensic investigations that require a short turnaround time. We examined the methylation levels of 46 CpG sites from six genes using bloodstain samples from 128 males and 113 females aged 10–79 years. A random forest regression model was then used to construct an age prediction model for males and females separately. The final age prediction models were developed with seven CpG sites (three for males and four for females) based on the performance of the random forest regression. The mean absolute deviation was less than 3 years for each model. Our results demonstrate that DNA methylation-based age prediction using pyrosequencing and random forest regression has potential applications in forensics to accurately predict the biological age of a bloodstain donor.     

Electrochemical analysis of nucleic acids as potential cancer biomarkers

In recent years, a huge progress has been made regarding the development of electrochemical (EC) assays for detection of nucleic acids — DNA or RNA — as potential cancer biomarkers. Various ingenious strategies for determination of DNA methylation of gene promoters, circulating tumor DNAs, viral nucleic acids, or short noncoding microRNAs were presented, many of them showing remarkable sensitivities. However, a majority of these assays were not applied into clinical samples from patients, which is crucial should the electrochemistry compete with conventional, routinely used techniques. In this review, we critically evaluate strengths and weaknesses of EC assays that recognized this necessity and successfully determined endogenous DNA or RNA in patient samples with various forms of tumors.     

From Natural Products to Drugs for Epimutation Computer-Aided Drug Design

The epimutational event, i.e., ectopic methylation in tumor suppressor genes, can lead to gene silencing, thus promoting prognosis of cancer. The progression of DNA methylation is a cycle of demethylation, de novo methylation, and maintenance methylation. The enzyme responsible for maintenance of methylation status is DNA methyltransferase 1 (DNMT1), the continuous activity of which is required to maintain the pattern of epimutation; thus, its inhibition is a promising strategy for the treatment of cancer. To the best of our knowledge, this study is the first to focus on the recently developed crystal structure of the catalytic site of DNMT1. Here in this study, we have used the crystal structure for the development of non-nucleoside DNMT1 inhibitors using virtual screening (VS), absorption, distribution, metabolism, elimination/toxicology analysis, and molecular docking studies. In this study, VS was carried out on 48,531 natural products to create a subset of lead-like natural products. Three of them were found to form hydrogen bonds with the catalytic site of the DNMT1 (Cys 1226). Thus, this study adumbrates potential lead compounds for treatment of epimutation.     

p16基因甲基化的芯片定量检测

p16基因的失活与多种肿瘤相关,但p16基因缺失率较低,突变更为罕见,p16基因启动子区CpG岛甲基化与其蛋白表达密切相关．DNA甲基化已成为目前研究的热点,现有的技术包括：Southernblot法、限制性内切酶-PCR法、DNA测序法、甲基化特异性PCR(MSP)、     

DNA microarray: a high throughput approach for methylation detection

We described a DNA microarray-based method combined with bisulphite treatment of DNA and regular PCR to examine hyper-methylation in promoter 1A of APC gene. A set of oligonucleotide probes were designed and immobilized on the aldehyde-coated glass slides for detecting the methylation pattern of 15 selected CpG sites in the region. The methylation status of 30 colorectal tumor samples have been examined by both of methylation-specific PCR (MS-PCR) and the present microarray method. The methylation pattern of the 15 CpG sites for the samples have been obtained with the microarray. A total of 19 samples out of 30 were methylated by microarray, in which five samples cannot be detected by MS-PCR due to the methylated CpG patterns not accordant to the MS-PCR primers. The detecting ratio for methylation of APC gene of colorectal tumor samples increased from 46.7% with MS-PCR to 63.3% with the microarray, which successfully demonstrated that DNA microarray-based method not only can obtained the methylation patterns for the related genes, but also decrease the false-negative results of methylation status by the conventional MS-PCR for the investigated genes.     

Hypermethylation of PDX1, EN2, and MSX1 predicts the prognosis of colorectal cancer

Despite numerous observations regarding the relationship between DNA methylation changes and cancer progression, only a few genes have been verified as diagnostic biomarkers of colorectal cancer (CRC). To more practically detect methylation changes, we performed targeted bisulfite sequencing. Through co-analysis of RNA-seq, we identified cohort-specific DNA methylation markers: CpG islands of the intragenic regions of PDX1, EN2, and MSX1. We validated that these genes have oncogenic features in CRC and that their expression levels are increased in correlation with the hypermethylation of intragenic regions. The reliable depth of the targeted bisulfite sequencing data enabled us to design highly optimized quantitative methylation-specific PCR primer sets that can successfully detect subtle changes in the methylation levels of candidate regions. Furthermore, these methylation levels can divide CRC patients into two groups denoting good and poor prognoses. In this study, we present a streamlined workflow for screening clinically significant differentially methylated regions. Our discovery of methylation markers in the PDX1, EN2, and MSX1 genes suggests their promising performance as prognostic markers and their clinical application in CRC patients.Subject terms: Prognostic markers, Methylation analysis