A standardized protocol for sample preparation for scanning electron microscopy to visualize extrachromosomal DNA.

Extrachromosomal DNA (ecDNA) are circular DNA structures associated with cancer and drug resistance. One specific type, double minute (DM) chromosomes, has been studied since the 1960s using imaging techniques like cytogenetics and fluorescence microscopy. Specialized techniques such as atomic force microscopy (AFM) and scanning electron microscopy (SEM) offer micro to nano-scale visualization, but current sample preparation methods may not optimally preserve ecDNA structure. Our study introduces a systematic protocol using SEM for high-resolution ecDNA visualization. We have optimized the end-to-end procedure, providing a standardized approach to explore the circular architecture of ecDNA and address the urgent need for better understanding in cancer research.

Despite advances in extrachromosomal DNA (ecDNA) detection, current methods struggle to reveal ecDNA's architecture within cells. Specialized techniques like scanning electron microscopy (SEM) provide the needed resolution, but existing sample preparation may not preserve ecDNA well. Our study introduces a systematic method using SEM, optimizing procedures for preparing and visualizing metaphase spread samples. This offers a standardized approach to study ecDNA's circular architecture, addressing a pressing need in cancer research.

Predicting gestational diabetes mellitus risk at 11-13 weeks' gestation: the role of extrachromosomal circular DNA.

BACKGROUND: Gestational diabetes mellitus (GDM) significantly impacts maternal and infant health both immediately and over the long term, yet effective early diagnostic biomarkers are currently lacking. Thus, it is essential to identify early diagnostic biomarkers for GDM risk screening. Extrachromosomal circular DNA (eccDNA), being more stable than linear DNA and involved in disease pathologies, is a viable biomarker candidate for diverse conditions. In this study, eccDNA biomarkers identified for early diagnosis and assessment of GDM risk were explored. METHODS: Using Circle-seq, we identified plasma eccDNA profiles in five pregnant women who later developed GDM and five matched healthy controls at 11-13 weeks of gestation. These profiles were subsequently analyzed through bioinformatics and validated through outward PCR combined with Sanger sequencing. Furthermore, candidate eccDNA was validated by quantitative PCR (qPCR) in a larger cohort of 70 women who developed GDM and 70 normal glucose-tolerant (NGT) subjects. A ROC curve assessed the eccDNA's diagnostic potential for GDM. RESULTS: 2217 eccDNAs were differentially detected between future GDM patients and controls, with 1289 increased and 928 decreased in abundance. KEGG analysis linked eccDNA genes mainly to GDM-related pathways such as Rap1, MAPK, and PI3K-Akt, and Insulin resistance, among others. Validation confirmed a significant decrease in eccDNA PRDM16circle in the plasma of 70 women who developed GDM compared to 70 NGT women, consistent with the eccDNA-seq results. PRDM16circle showed significant diagnostic value in 11-13 weeks of gestation (AUC = 0.941, p < 0.001). CONCLUSIONS: Our study first demonstrats that eccDNAs are aberrantly produced in women who develop GDM, including PRDM16circle, which can predict GDM at an early stage of pregnancy, indicating its potential as a biomarker. TRIAL REGISTRATION: ChiCTR2300075971, http://www.chictr.org.cn . Registered 20 September 2023.

Analysis of HBV cccDNA Minichromosome Accessibility by MNase-qPCR and High-Throughput Sequencing.

The covalently closed circular DNA (cccDNA) of the hepatitis B virus (HBV) is organized as a minichromosome structure in the nucleus of infected hepatocytes and considered the major obstacle to the discovery of a cure for HBV. Until now, no strategies directly targeting cccDNA have been advanced to clinical stages as much is unknown about the accessibility and activity regulation of the cccDNA minichromosome. We have described the method for evaluation of the cccDNA minichromosome accessibility using micrococcal nuclease-quantitative polymerase chain reaction and high-throughput sequencing, which could be useful tools for cccDNA research and HBV cure studies.

Circular Chromosome Conformation Capture (4C-Seq) Analysis of HBV-Host Chromosome DNA Interactome.

Hepatitis B virus (HBV) infects hepatocytes that are in the G0/G1 phase with intact nuclear membrane and organized chromosome architecture. In the nucleus of the infected cells, HBV covalently closed circular (ccc) DNA, an episomal minichromosome, serves as the template for all viral transcripts and the reservoir of persistent infection. Nuclear positioning of cccDNA can be assessed by the spatial distance between viral DNA and host chromosomal DNA through Circular Chromosome Conformation Capture (4C) combined with high-throughput sequencing (4C-seq). The 4C-seq analysis relies on proximity ligation and is commonly used for mapping genomic DNA regions that communicate within a host chromosome. The method has been tailored for studying nuclear localization of HBV episomal cccDNA in relation to the host chromosomes. In this study, we present a step-by-step protocol for 4C-seq analysis of HBV infection, including sample collection and fixation, 4C DNA library preparation, sequence library preparation, and data analysis. Although limited by proximity ligation of DNA fragments, 4C-seq analysis provides useful information of HBV localization in 3D genome, and aids the understanding of viral transcription in light of host chromatin conformation.

Rapid and Reliable Protein-Free HBV DNA Extraction and Sensitive Branched DNA Southern Blot Assay.

HBV covalently closed circular DNA (cccDNA) plays an important role in the persistence of hepatitis B virus (HBV) infection by serving as the template for transcription of viral RNAs. To cure HBV infection, it is expected that cccDNA needs either to be eliminated or silenced. Hence, precise cccDNA quantification is essential. Sample preparation is crucial to specifically detect cccDNA. Southern blot is regarded as the "gold standard" for specific cccDNA detection but lacks sensitivity. Here, we describe a rapid and reliable modified kit-based, HBV protein-free DNA extraction method as well as a novel enhanced sensitivity Southern blot that uses branched DNA technology to detect HBV DNA in cell culture and liver tissue samples. It is useful for both HBV molecular biology and antiviral research.

A Fluorescent In Situ Hybridization (FISH) Assay for Detection of HBV RNA, DNA, and cccDNA in Cell Culture.

Hepatitis B virus (HBV) is undoubtedly a master in exploiting host resources while evading host defense for its multiplication within a constrained genetic coding capacity. To further unravel these cunning strategies, a clear picture of virus-host interaction with key subcellular and molecular contexts is needed. Here, we describe a FISH protocol modified from the ViewRNA assay that allows direct visualization of HBV RNA, DNA, and cccDNA in cell culture models (e.g., HepAD38, HepG2-NTCP). It can be coupled with immunofluorescence staining of viral or host proteins or other fluorescent tagging systems which could illuminate numerous aspects of virus-host interactions.

A Chromogenic In Situ Hybridization (CISH) Assay for Detection of HBV RNA, DNA, and cccDNA in Liver Tissue.

Hepatitis B virus (HBV) developed highly intricates mechanisms exploiting host resources for its multiplication within a constrained genetic coding capacity. With the aid of a series of classical analytical methods such as ultrafiltration, and Southern and Northern blots, a general framework of HBV life cycle has been established. However, this picture still lacks many key histological contexts which involves pathophysiological changes of hepatocytes, non-parenchymal cells, infiltrated leukocytes, and associated extracellular matrix. Here, we describe a CISH protocol modified from the ViewRNA assay that allows direct visualization of HBV RNA, DNA, and cccDNA in liver tissue of chronic hepatitis B patients. By coupling it with immunohistochemistry and other histological stains, much richer information regarding the HBV-induced pathological changes can be harvested.

Detection of Hepatitis B Virus Covalently Closed Circular DNA and Intermediates in Its Formation.

Hepatitis B virus (HBV) infection remains a global public health issue, and approximately 294 million individuals worldwide are chronically infected with HBV. Approved antivirals rarely cure chronic HBV infection due to their inability to eliminate the HBV covalently closed circular DNA (cccDNA), the viral episome, in the nucleus of infected hepatocytes. The persistence of cccDNA underlies the chronic nature of HBV infection and the frequent relapse after the cessation of antiviral treatment. However, drug development targeting cccDNA formation and maintenance is hindered by the lack of sufficient biological knowledge on cccDNA, and of its reliable detection due to its low abundance and the presence of high levels of HBV DNA species similar to cccDNA. Here, we describe a Southern blot method for reliably detecting the HBV cccDNA even in the presence of high levels of plasmid DNA and other HBV DNA species, based on the efficient removal of plasmid DNA and all DNA species with free 3' ends. This approach also allows the detection of certain potential intermediates during cccDNA formation.

One-Step Reverse Transcriptase qPCR Method for Serum Hepatitis B Virus RNA Quantification.

In recent years, serum hepatitis B virus (HBV) RNA has been identified as a promising noninvasive surrogate biomarker of intrahepatic covalently closed circular DNA (cccDNA), detection of which requires an invasive liver biopsy in patients with chronic HBV infection. It is impractical to detect intrahepatic cccDNA as a routine diagnosis for chronic hepatitis B (CHB) patients in clinical management. Here, we describe a detailed protocol for serum HBV RNA quantification, which can reflect the activity of intrahepatic cccDNA. The procedure includes three major steps: (1) Simultaneous isolation of HBV DNA and RNA from patients' serum, (2) DNase I digestion for removing HBV DNA contamination, and (3) HBV RNA quantification by one-step reverse transcription qPCR.

The Cre/loxP-Based Recombinant HBV cccDNA System In Vitro and In Vivo.

Covalently closed circular DNA (cccDNA) exists as a stable episomal minichromosome in the nucleus of hepatocytes and is responsible for hepatitis B virus (HBV) persistence. We recently reported a technique involving recombinant cccDNA (rcccDNA) of HBV by site-specific DNA recombination. A floxed monomeric HBV genome was engineered into a precursor plasmid (prcccDNA) which was excised via Cre/loxP-mediated DNA recombination to form a 3.3-kb rcccDNA bearing a loxP-chimeric intron. The foreign sequence was efficiently removed during RNA splicing, rendering a functionally seamless insertion. We characterized rcccDNA formation, effective viral transcription, and replication induced by rcccDNA both in vitro and in vivo. Furthermore, we closely simulated chronic hepatitis by using a replication-defective recombinant adenoviral vector to deliver rcccDNA to the transgenic mice expressing Cre recombinase, which led to prominent HBV persistence. Here, we describe a detailed protocol about how to construct and evaluate Cre/loxP-based recombinant HBV cccDNA system both in vitro and in vivo.

Large bubble drives circular DNA melting.

We investigate the melting transition of non-supercoiled circular DNA of different lengths, employing Brownian dynamics simulations. In the absence of supercoiling, we find that melting of circular DNA is driven by a large bubble, which agrees with the previous predictions of circular DNA melting in the presence of supercoiling. By analyzing sector-wise changes in average base-pair distance, our study reveals that the melting behavior of circular DNA closely resembles that of linear DNA. Additionally, we find a marked difference in the thermal stability of circular DNA over linear DNA at very short length scales, an effect that diminishes as the length of circular DNA increases. The stability of smaller circular DNA is linked to the occurrence of transient small bubbles, characterized by a lower probability of growth.

Hepatitis B Virus Covalently Closed Circular DNA Chromatin Immunoprecipitation Assay.

Hepatitis B virus (HBV) is an obligate human hepatotropic DNA virus causing both transient and chronic infection. The livers of chronic hepatitis B patients have a high risk of developing liver fibrosis, cirrhosis, and hepatocellular carcinoma. The nuclear episomal viral DNA intermediate, covalently closed circular DNA (cccDNA), forms a highly stable complex with host and viral proteins to serve as a transcription template and support HBV infection chronicity. Thus, characterization of the composition and dynamics of cccDNA nucleoprotein complexes providing cccDNA stability and gene regulation is of high importance for both basic and medical research. The presented method for chromatin immunoprecipitation coupled with qPCR (ChIP-qPCR) allows to assess provisional physical interaction of the protein of interest (POI) with cccDNA using POI-specific antibody, the level of enrichment of a POI on cccDNA versus control/background is characterized quantitatively using qPCR.

Progress in application of cyclic single-stranded nucleic acids.

Cyclic nucleic acids are biologically stable against nucleic acid exonucleases due to the absence of 5' and 3' termini. Studies of cyclic nucleic acids mainly focus on cyclic single-stranded nucleic acids. Cyclic single-stranded nucleic acids are further divided into circular RNA (circRNA) and circular single-stranded DNA (cssDNA). The synthesis methods of circRNA include lasso-driven cyclization, intron-paired cyclization, intron cyclization, intron complementary pairing-driven cyclization, RNA-binding protein-driven cyclization, and artificial synthesis depending on the source. Its main role is to participate in gene expression and the treatment of some diseases. Circular single-stranded DNA is mainly synthesized by chemical ligation, template-directed enzyme ligation, and new techniques for the efficient preparation of DNA single loops and topologies based on CircLigase. It is mainly used in rolling circle amplification (RCA) technology and in the bioprotection of circular aptamers and second messengers. This review focuses on the types, synthesis methods, and applications of cyclic single-stranded nucleic acids, providing a reference for further research on cyclic single-stranded nucleic acids.

Horizontally transferred mitochondrial DNA tracts become circular by microhomology-mediated repair pathways.

The holoparasitic plant Lophophytum mirabile exhibits remarkable levels of mitochondrial horizontal gene transfer (HGT). Gathering comparative data from other individuals and host plants can provide insights into the HGT process. We sequenced the mitochondrial genome (mtDNA) from individuals of two species of Lophophytum and from mimosoid hosts. We applied a stringent phylogenomic approach to elucidate the origin of the whole mtDNAs, estimate the timing of the transfers, and understand the molecular mechanisms involved. Ancestral and recent HGT events replaced and enlarged the multichromosomal mtDNA of Lophophytum spp., with the foreign DNA ascending to 74%. A total of 14 foreign mitochondrial chromosomes originated from continuous regions in the host mtDNA flanked by short direct repeats. These foreign tracts are circularized by microhomology-mediated repair pathways and replicate independently until they are lost or they eventually recombine with other chromosomes. The foreign noncoding chromosomes are variably present in the population and likely evolve by genetic drift. We present the 'circle-mediated HGT' model in which foreign mitochondrial DNA tracts become circular and are maintained as plasmid-like molecules. This model challenges the conventional belief that foreign DNA must be integrated into the recipient genome for successful HGT.

Extrachromosomal circular DNA promotes prostate cancer progression through the FAM84B/CDKN1B/MYC/WWP1 axis.

BACKGROUND: Extrachromosomal circular DNA (eccDNA), a kind of circular DNA that originates from chromosomes, carries complete gene information, particularly the oncogenic genes. This study aimed to examine the contributions of FAM84B induced by eccDNA to prostate cancer (PCa) development and the biomolecules involved. METHODS: The presence of eccDNA in PCa cells and the FAM84B transcripts that eccDNA carries were verified by outward and inward PCR. The effect of inhibition of eccDNA synthesis on FAM84B expression in PCa cells was analyzed by knocking down Lig3. The impact of FAM84B on the growth and metastases of PCa cells was verified by Cell Counting Kit-8 (CCK8), EdU, transwell assays, and a xenograft mouse model. Chromatin immunoprecipitation quantitative PCR (ChIP-qPCR) and dual-luciferase reporter assays were carried out to examine the effect of FAM84B/MYC on WWP1 transcription, and a co-immunoprecipitation (Co-IP) assay was conducted to verify the modification of CDKN1B by WWP1. The function of this molecular axis in PCa was explored by rescue assays. RESULTS: The inhibited eccDNA synthesis significantly downregulated FAM84B in PCa cells, thereby attenuating the growth and metastasis of PCa. FAM84B promoted the transcription of WWP1 by MYC by activating the expression of MYC coterminous with the 8q24.21 gene desert in a beta catenin-dependent approach. WWP1 transcription promoted by MYC facilitated the ubiquitination and degradation of CDKN1B protein and inversely attenuated the repressive effect of CDKN1B on MYC expression. Exogenous overexpression of CDKN1B blocked FAM84B-activated MYC/WWP1 expression, thereby inhibiting PCa progression. CONCLUSIONS: FAM84B promoted by eccDNA mediates degradation of CDKN1B via MYC/WWP1, thereby accelerating PCa progression.

CRISPR/Cas9 System with Dual gRNAs Synergically Inhibit Hepatitis B Virus Replication.

BACKGROUND: In recent years, a gene-editing technology known as clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 has been developed and is progressively advancing into clinical trials. While current antiviral therapies are unable to eliminate the Hepatitis B virus (HBV), it stands as a prime target for the CRISPR/Cas9 technology. The objective of this study was to enhance the efficacy of CRISPR/Cas9 in suppressing HBV replication, lowering HBsAg and HBeAg levels, and eliminating covalently closed circular DNA (cccDNA). METHODS: To enhance the anti-HBV effectiveness of CRISPR/Cas9, our study delved into a dual-guide RNA (gRNA) strategy. After evaluating the antiviral activities of multiple gRNAs that effectively impeded HBV replication, we identified three specific gRNAs-namely 10, 4, and 21. These gRNAs were selected for their targeting of distinct yet conserved regions within the HBV genome. RESULTS: In HBV-stable cell lines, namely HepAD38, and HBV infection models of HepG2-NTCP cells, our investigation revealed that the co-application of gRNA-10 with either gRNA-4 or gRNA-21 within the CRISPR/Cas9 system demonstrated heightened efficacy in impeding HBV replication, reducing the levels of HBsAg, HBeAg, and cccDNA levels, along with a more pronounced promotion of HBsAg clearance when compared to the use of a single gRNA. CONCLUSIONS: The CRISPR/Cas9 system employing dual gRNAs has proven highly effective in both suppressing HBV replication and facilitating HBsAg clearance. This promising outcome suggests that it holds potential to emerge as a novel approach for achieving the functional cure of patients with HBV infection.

Current Research on Small Circular Molecules: A Comprehensive Overview on SPHINX/BMMF.

Several years of research into the small circular DNA molecules called SPHINX and BMMF (SPHINX/BMMF) have provided information on several areas of research, medicine, microbiology and nutritional science. But there are still open questions that have not yet been addressed. Due to the unclear classification, evolution and sources of SPHINX/BMMF, a risk assessment is currently not possible. However, risk assessment is necessary as SPHINX/BMMF are suspected to be involved in the development of cancer and neurodegenerative diseases. In order to obtain an overview of the current state of research and to identify research gaps, a review of all the publications on this topic to date was carried out. The focus was primarily on the SPHINX/BMMF group 1 and 2 members, which is the topic of most of the research. It was discovered that the SPHINX/BMMF molecules could be integral components of mammalian cells, and are also inherited. However, their involvement in neurodegenerative and carcinogenic diseases is still unclear. Furthermore, they are probably ubiquitous in food and they resemble bacterial plasmids in parts of their DNA and protein (Rep) sequence. In addition, a connection with bacterial viruses is also suspected. Ultimately, it is still unclear whether SPHINX/BMMF have an infectious capacity and what their host or target is.

Landscape of extrachromosomal circular DNAs, transcriptome, and proteome analysis reveals insights into alcoholic liver cirrhosis.

Alcoholic liver cirrhosis (ALC) is a result of excessive and chronic alcohol consumption. Because alchol can cause DNA damage, extrachromosomal circular DNA (eccDNA) was investigated in ALC liver due to it can be a result of DNA damage. Considering eccDNA has ability to lead to genomic instability as an enhancer of gene transcription, we utilized Circle-Seq to identify differences in eccDNA profiles and gene expression patterns in liver samples obtained from ALC patients (n = 3) and healthy controls (n = 3) to investigate the role of eccDNA in the development of ALC. The abundance of eccDNA in ALC (mean = 13,349) were higher than the healthy control (mean = 11,557) without significant difference (pvalue = 0.6530). We observed 1,032 eccDNA containing genes showed higher expression in ALC patients compared to healthy controls (p < 0.05, log2FC > 1). Notably, we discovered seven genes that exhibited a significant positive correlation between eccDNA abundance and gene expression levels. These genes include A disintegrin and metalloproteinase with thrombospondin motifs 2 (ADAMTS2), Voltage-dependent L-type calcium channel subunit alpha-1C (CACNA1C), Protein TANC1 (TANC1), Integrin alpha-2 (ITGA2), EH domain-containing protein 4 (EHD4), Phosphofurin acidic cluster sorting protein 1 (PACS1), and Neuron navigator 2 (NAV2). Through mass spectrometry proteomics, ITGA2 were found to have significantly higher abbudance in ALC. Integrins are a family of proteins plays key roles in the fibrosis development of liver. Thus, our study opens a new perspective for liver fibrosis development.