Differential expression and analysis of extrachromosomal circular DNAs as serum biomarkers in pulmonary arterial hypertension.

BACKGROUND: Extrachromosomal circular DNAs (eccDNAs) have been reported to play a key role in the occurrence and development of various diseases. However, the characterization and role of eccDNAs in pulmonary arterial hypertension (PAH) remain unclear. METHODS: In the discovery cohort, we first explored eccDNA expression profiles by Circle-sequencing analysis. The candidate eccDNAs were validated by routine polymerase chain reaction (PCR), TOPO-TA cloning and Sanger sequencing. In the validation cohort, 30 patients with PAH and 10 healthy controls were recruited for qPCR amplification to detect the candidate eccDNAs. Datas at the baseline were collected, including clinical background, biochemical variables, echocardiography and hemodynamic factors. Receiver operating characteristic curve was used to investigate the diagnostic effect of the eccDNA. RESULTS: We identified a total of 21,741 eccDNAs in plasma samples of 3 IPAH patients and 3 individuals in good health, and the expression frequency, GC content, length distribution, and genome distribution of the eccDNAs were thoroughly characterized and analyzed. In the validation cohort, 687 eccDNAs were differentially expressed in patients with IPAH compared with healthy controls (screening threshold: |FC|≥2 and P < 0.05). Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that the specific eccDNAs in IPAH were significantly enriched in calcium channel activity, the mitogen-activated protein kinase pathway, and the wnt signaling pathway. Verification queue found that the expression of eccDNA-chr2:131208878-131,424,362 in PAH was considerably higher than that in healthy controls and exhibited a high level of accuracy in predicting PAH with a sensitivity of 86.67% and a specificity of 90%. Furthermore, correlation analysis disclosed a significant association between serum eccDNA-chr2:131208878-131,424,362 and mean pulmonary artery pressure (mPAP) (r = 0.396, P = 0.03), 6 min walking distance (6MWD) (r = -0.399, P = 0.029), N-terminal pro-B-type natriuretic peptide (NT-proBNP) (r = 0.685, P < 0.001) and cardiac index (CI) (r = - 0.419, P = 0.021). CONCLUSIONS: This is the first study to identify and characterize eccDNAs in patients with PAH. We revealed that serum eccDNA-chr2:131208878-131,424,362 is significantly overexpressed and can be used in the diagnosis of PAH, indicating its potential as a novel non-invasive biomarker.

Untying relaxed circular DNA of hepatitis B virus by polymerase reaction provides a new option for accurate quantification and visualization of covalently closed circular DNA.

Hepatitis B virus (HBV) is a small hepatotropic DNA virus that replicates via an RNA intermediate. After entry, the virus capsid carries relaxed circular DNA (rcDNA) into the nucleus where the viral genome is converted into covalently closed circular DNA (cccDNA), which serves as the template for all viral transcripts. To monitor cccDNA levels, preprocessing methods to eliminate rcDNA have emerged for quantitative PCR, although Southern blotting is still the only method to discriminate cccDNA from other DNA intermediates. In this study, we have established a robust method for untying mature rcDNA into double stranded linear DNA using specific polymerases. Untying rcDNA provides not only an alternative method for cccDNA quantification but also a sensitive method for visualizing cccDNA. We combined this method with plasmid-safe DNase and T5 exonuclease preprocessing and revealed that accurate quantification requires cccDNA digestion by a restriction enzyme because heat stability of cccDNA increases after T5 exonuclease treatment. In digital PCR using duplex TaqMan probes, fewer than 1000 copies of cccDNA were successfully visualized as double positive spots that were distinct from single positives derived from untied rcDNA. This method was further applied to the infection model of primary hepatocytes treated with nucleoside analogues and a core protein allosteric modulator to monitor cccDNA levels. Relative quantification of cccDNA by human genome copy demonstrated the possibility of precise evaluation of cccDNA level per nucleus. These results clearly indicate that the sequential reaction from untying rcDNA is useful to investigate cccDNA fates in a small fraction of nuclei.

Droplet digital PCR assay provides intrahepatic HBV cccDNA quantification tool for clinical application.

The persistence of covalently closed circular DNA (cccDNA) poses a major obstacle to curing chronic hepatitis B (CHB). Here, we used droplet digital PCR (ddPCR) for cccDNA quantitation. The cccDNA-specific ddPCR showed high accuracy with the dynamic range of cccDNA detection from 101 to 105 copies/assay. The ddPCR had higher sensitivity, specificity and precisely than qPCR. The results of ddPCR correlated closely with serum HB core-related antigen and HB surface antigen (HBsAg) in 24 HBV-infected human-liver-chimeric mice (PXB-mice). We demonstrated that in 2 PXB-mice after entecavir treatment, the total cccDNA content did not change during liver repopulation, although the cccDNA content per hepatocyte was reduced after the treatment. In the 6 patients with HBV-related hepatocellular carcinoma, ddPCR detected cccDNA in both tumor and non-tumor tissues. In 13 HBeAg-negative CHB patients with pegylated interferon alpha-2a, cccDNA contents from paired biopsies were more significantly reduced in virological response (VR) than in non-VR at week 48 (p = 0.0051). Interestingly, cccDNA levels were the lowest in VR with HBsAg clearance but remained detectable after the treatment. Collectively, ddPCR revealed that cccDNA content is stable during hepatocyte proliferation and persists at quantifiable levels, even after serum HBsAg clearance.

Serum HBV RNA correlated with intrahepatic cccDNA more strongly than other HBV markers during peg-interferon treatment.

BACKGROUND: Serum hepatitis B virus RNA (HBV RNA) has been reported to be a surrogate marker of intrahepatic cccDNA during nucleos(t)ide analogs therapy. However, in HBeAg-positive patients treated with peg-interferon (peg-IFN), whether HBV RNA is superior to other HBV markers in reflecting cccDNA profile is still unclear. METHODS: Serum HBV RNA, HBcrAg, HBV DNA, and HBsAg were longitudinally assessed among 30 HBeAg-positive patients during 48-week peg-IFN treatment. Besides, intrahepatic cccDNA was detected at baseline and week 48 respectively. Then, the individual correlations between HBV RNA, HBcrAg, HBV DNA, HBsAg, and cccDNA were statistically analyzed. RESULTS: HBV RNA levels decreased more rapidly in patients with HBeAg seroconversion than those without HBeAg seroconversion. Among all patients, cccDNA correlated better with HBV RNA than with HBcrAg, HBV DNA, and HBsAg at baseline. After 48 weeks peg-IFN treatment, cccDNA still correlated more strongly with HBV RNA than other HBV markers. Further analysis indicated that in patients with HBeAg seroconversion cccDNA strongly correlated with HBV RNA and HBcrAg, whereas not correlate with HBV DNA and HBsAg. While in patients without HBeAg seroconversion, cccDNA highly correlated with HBV RNA and HBV DNA, moderately correlated with HBcrAg, and not correlated with HBsAg. CONCLUSION: Compared to HBcrAg, HBV DNA, and HBsAg, serum HBV RNA correlated more strongly with intrahepatic cccDNA levels before and after 48-week peg-IFN treatment. The level of serum HBV RNA may be a superior surrogate marker in reflecting the intrahepatic cccDNA profile in HBeAg-positive patients during peg-IFN treatment. Trial registration ClinicalTrials, NCT03546530. Registered 1 January 2015. https://clinicaltrials.gov/ct2/results?cond=&term=NCT03546530 .

Transition to HBeAg-negative chronic hepatitis B virus infection is associated with reduced cccDNA transcriptional activity.

BACKGROUND & AIMS: HBeAg seroconversion during the natural history of chronic hepatitis B (CHB) is associated with a strong drop in serum HBV DNA levels and a reduction of intrahepatic covalently closed circular DNA (cccDNA) content. Of particular interest is the transition to HBeAg-negative chronic infection (ENCI). ENCI, previously known as inactive carrier state, is characterized by very low or negative viremia and the absence of liver disease. The molecular mechanisms responsible for the transition to ENCI and for the control of viral replication in ENCI are still poorly understood. METHODS: To identify which step(s) in the viral life cycle are controlled during the transition to ENCI, we quantified cccDNA, pre-genomic RNA (pgRNA), total HBV RNA and DNA replicative intermediates in 68 biopsies from patients in different phases of CHB. RESULTS: HBeAg seroconversion is associated with a reduction of cccDNA amounts as well as transcriptional activity. Silencing of cccDNA is particularly pronounced in ENCI, where there was ~46 times less pgRNA per cccDNA compared to HBeAg-negative CHB. Furthermore, a subgroup of patients with HBeAg-negative CHB can be characterized by reduced replication efficiency downstream of pgRNA. CONCLUSIONS: The reduction in serum viral load during the transition to ENCI seems to primarily result from strong inhibition of the transcriptional activity of cccDNA which can be maintained in the absence of liver disease. LAY SUMMARY: During the natural course of chronic hepatitis B virus infections, the immune response can gain control of viral replication. Quantification of viral DNA and RNA in liver biopsies of patients in different stages of chronic hepatitis B allowed us to identify the steps in the viral life cycle that are affected during the transition from active to inactive disease. Therapeutic targeting of these steps might induce sustained inhibition of viral transcription.

Scalable Synthesis, In Vitro cccDNA Reduction, and In Vivo Antihepatitis B Virus Activity of a Phosphonomethoxydeoxythreosyl Adenine Prodrug.

Standard literature procedures for the chemical synthesis of l-threose nucleosides generally employ l-ascorbic acid as starting material. Herein, we have explored two alternative routes that start from either l-arabitol or l-diethyl tartrate, both affording 2-O-methyl-l-threofuranose as a key building block for nucleobase incorporation. The access to multigram quantities of this glycosyl donor in a reproducible fashion allows for the preparation of 2'-deoxy-α-l-threofuranosyl phosphonate nucleosides on a large scale. This methodology was applied to the gram scale synthesis of an aryloxy amidate prodrug of phosphonomethoxydeoxythreosyl adenine. This prodrug exerted potent activity against an entecavir-resistant hepatitis B virus (HBV) strain, while leading to a significant reduction in the levels of HBV covalently closed circular DNA in a cellular assay. Furthermore, its remarkable anti-HBV efficacy was also confirmed in vivo using a hydrodynamic injection-based HBV mouse model, without relevant toxicity and systemic exposure occurring.

Single hepatocytes show persistence and transcriptional inactivity of hepatitis B.

There is no cure for the more than 270 million people chronically infected with HBV. Nucleos(t)ide analogs (NUCs), the mainstay of anti-HBV treatment, block HBV reverse transcription. NUCs do not eliminate the intranuclear covalently closed circular DNA (cccDNA), from which viral RNAs, including pregenomic RNA (pgRNA), are transcribed. A key gap in designing a cure is understanding how NUCs affect HBV replication and transcription because serum markers yield an incomplete view of intrahepatic HBV. We applied single-cell laser capture microdissection and droplet digital PCR to paired liver biopsies collected from 5 HBV/HIV-coinfected persons who took NUCs over 2-4 years. From biopsy 1 to 2, proportions of HBV-infected hepatocytes declined with adherence to NUC treatment (P < 0.05); we extrapolated that eradication of HBV will take over 10 decades with NUCs in these participants. In individual hepatocytes, pgRNA levels diminished 28- to 73-fold during NUC treatment, corresponding with decreased tissue HBV core antigen staining (P < 0.01). In 4 out of 5 participants, hepatocytes with cccDNA but undetectable pgRNA (transcriptionally inactive) were present, and these were enriched in 3 participants during NUC treatment. Further work to unravel mechanisms of cccDNA transcriptional inactivation may lead to therapies that can achieve this in all hepatocytes, resulting in a functional cure.

Novel insights into extrachromosomal DNA: redefining the onco-drivers of tumor progression.

Extrachromosomal DNA (ecDNA), gene-encoding extrachromosomal particles of DNA, is often present in tumor cells. Recent studies have revealed that oncogene amplification via ecDNA is widespread across a diverse range of cancers. ecDNA is involved in increasing tumor heterogeneity, reverting tumor phenotypes, and enhancing gene expression and tumor resistance to chemotherapy, indicating that it plays a significant role in tumorigenesis. In this review, we summarize the characteristics and genesis of ecDNA, connect these characteristics with their concomitant influences on tumorigenesis, enumerate the oncogenes encoded by ecDNA in multiple cancers, elaborate the roles of ecDNA in tumor pathogenesis and progression, and propose the considerable research and therapeutic prospects of ecDNA in cancer.

Current understanding of extrachromosomal circular DNA in cancer pathogenesis and therapeutic resistance.

Extrachromosomal circular DNA was recently found to be particularly abundant in multiple human cancer cells, although its frequency varies among different tumor types. Elevated levels of extrachromosomal circular DNA have been considered an effective biomarker of cancer pathogenesis. Multiple reports have demonstrated that the amplification of oncogenes and therapeutic resistance genes located on extrachromosomal DNA is a frequent event that drives intratumoral genetic heterogeneity and provides a potential evolutionary advantage. This review highlights the current understanding of the extrachromosomal circular DNA present in the tissues and circulation of patients with advanced cancers and provides a detailed discussion of their substantial roles in tumor regulation. Confirming the presence of cancer-related extrachromosomal circular DNA would provide a putative testing strategy for the precision diagnosis and treatment of human malignancies in clinical practice.

Replicative aging is associated with loss of genetic heterogeneity from extrachromosomal circular DNA in Saccharomyces cerevisiae.

Circular DNA can arise from all parts of eukaryotic chromosomes. In yeast, circular ribosomal DNA (rDNA) accumulates dramatically as cells age, however little is known about the accumulation of other chromosome-derived circles or the contribution of such circles to genetic variation in aged cells. We profiled circular DNA in Saccharomyces cerevisiae populations sampled when young and after extensive aging. Young cells possessed highly diverse circular DNA populations but 94% of the circular DNA were lost after ∼15 divisions, whereas rDNA circles underwent massive accumulation to >95% of circular DNA. Circles present in both young and old cells were characterized by replication origins including circles from unique regions of the genome and repetitive regions: rDNA and telomeric Y' regions. We further observed that circles can have flexible inheritance patterns: [HXT6/7circle] normally segregates to mother cells but in low glucose is present in up to 50% of cells, the majority of which must have inherited this circle from their mother. Interestingly, [HXT6/7circle] cells are eventually replaced by cells carrying stable chromosomal HXT6 HXT6/7 HXT7 amplifications, suggesting circular DNAs are intermediates in chromosomal amplifications. In conclusion, the heterogeneity of circular DNA offers flexibility in adaptation, but this heterogeneity is remarkably diminished with age.

A multiplex platform for digital measurement of circular DNA reaction products.

Digital PCR provides high sensitivity and unprecedented accuracy in DNA quantification, but current approaches require dedicated instrumentation and have limited opportunities for multiplexing. Here, we present an isothermal platform for digital enumeration of DNA reaction products in multiplex via standard fluorescence microscopy. Circular DNA strands, which may result from a wide range of molecular detection reactions, are captured on streptavidin-coated surfaces via hybridized biotinylated primers, followed by rolling circle amplification (RCA). The addition of 15% polyethylene glycol 4000 during RCA resulted in uniform, easily recorded reaction products. Immobilized DNA circles were visualized as RCA products with 100% efficiency, as determined by droplet digital PCR. We confirmed previous reports about the influence on RCA by sequence composition and size of RCA templates, and we developed an efficient one-step restaining procedure for sequential multiplexing using toehold-triggered DNA strand displacement. Finally, we exemplify applications of this digital readout platform by demonstrating more than three orders of magnitude improved sensitivity by digital measurement of prostate specific antigen (PSA) (detection threshold ∼100 pg/l), compared to a commercial enzyme-linked immunosorbent assay (ELISA) with analogue readout (detection threshold ∼500 ng/l), using the same antibody pair.

Full-length sequencing of circular DNA viruses and extrachromosomal circular DNA using CIDER-Seq.

Circular DNA is ubiquitous in nature in the form of plasmids, circular DNA viruses, and extrachromosomal circular DNA (eccDNA) in eukaryotes. Sequencing of such molecules is essential to profiling virus distributions, discovering new viruses and understanding the roles of eccDNAs in eukaryotic cells. Circular DNA enrichment sequencing (CIDER-Seq) is a technique to enrich and accurately sequence circular DNA without the need for polymerase chain reaction amplification, cloning, and computational sequence assembly. The approach is based on randomly primed circular DNA amplification, which is followed by several enzymatic DNA repair steps and then by long-read sequencing. CIDER-Seq includes a custom data analysis package (CIDER-Seq Data Analysis Software 2) that implements the DeConcat algorithm to deconcatenate the long sequencing products of random circular DNA amplification into the intact sequences of the input circular DNA. The CIDER-Seq data analysis package can generate full-length annotated virus genomes, as well as circular DNA sequences of novel viruses. Applications of CIDER-Seq also include profiling of eccDNA molecules such as transposable elements (TEs) from biological samples. The method takes ~2 weeks to complete, depending on the computational resources available. Owing to the present constraints of long-read single-molecule sequencing, the accuracy of circular virus and eccDNA sequences generated by the CIDER-Seq method scales with sequence length, and the greatest accuracy is obtained for molecules <10 kb long.

Requirement of cyclin-dependent kinase function for hepatitis B virus cccDNA synthesis as measured by digital PCR.

INTRODUCTION AND OBJECTIVES: HBV covalently closed circular (ccc) DNA is the key player in viral persistence and an important predictive biomarker for hepatitis relapse. Precise quantification of intracellular cccDNA is challenging because cccDNA is present in very low levels in hepatocytes, where it also co-exists with a large excess amount of relaxed circular (rc) DNA. We aimed to develop a highly sensitive cccDNA detection method for cccDNA quantification by digital PCR (dPCR). PATIENTS OR MATERIALS AND METHODS: A standard plasmid containing the whole HBV genome in the closed circular conformation was employed to characterize the performance of dPCR. rcDNA in the growth medium of HBV-producing HepAD38 cells was used as a matrix for cccDNA detection. Intrahepatic cccDNA measurement by dPCR and qPCR was performed to determine the correlation of the analysis results for the two methods. RESULTS: The limit of detection (LOD) of the cccDNA dPCR was 1.05copy/µl, and the linear range of detection was 1.02×104copies/µl, achieving a dynamic detection range of 104-fold. cccDNA measurement using excess rcDNA as the matrix did not reveal false-positive detection, indicating that dPCR was highly specific. In the HepAD38 cells, the cccDNA levels measured by dPCR were highly correlated with those measured by qPCR but had a higher sensitivity. The CDK inhibitor AZD-5438 was found to block intracellular cccDNA synthesis. CONCLUSIONS: Dpcr greatly improved the sensitivity and specificity of cccDNA detection. Host CDK activities are likely required for cccDNA synthesis. dPCR can potentially be applied for drug screening for effective cccDNA inhibitors.

Insights for clinical diagnostic indicators of virus and host in chronic hepatitis B infection.

Covalently closed circular DNA (cccDNA), which is stably present in the nucleus of hepatocytes, is an important indicator for evaluating antiviral efficacy. Since cccDNA quantification requires an invasive procedure, serum biological markers that can effectively reflect the transcriptional activity of intrahepatic virus and the efficacy of treatment are required. Here, from the aspects of virus and host, we outline the focus of clinical research of HBV in recent years, including HBV RNA, empty virus, hepatitis B core-related antigen and changes in the immune response. We briefly discuss their significance in predicting disease activity and monitoring treatment response in chronic hepatitis B. On this basis, some issues worthy of attention in laboratory diagnosis are proposed.

Assembly and annotation of the mitochondrial minicircle genome of a differentiation-competent strain of Trypanosoma brucei.

Kinetoplastids are protists defined by one of the most complex mitochondrial genomes in nature, the kinetoplast. In the sleeping sickness parasite Trypanosoma brucei, the kinetoplast is a chain mail-like network of two types of interlocked DNA molecules: a few dozen ∼23-kb maxicircles (homologs of the mitochondrial genome of other eukaryotes) and thousands of ∼1-kb minicircles. Maxicircles encode components of respiratory chain complexes and the mitoribosome. Several maxicircle-encoded mRNAs undergo extensive post-transcriptional RNA editing via addition and deletion of uridines. The process is mediated by hundreds of species of minicircle-encoded guide RNAs (gRNAs), but the precise number of minicircle classes and gRNA genes was unknown. Here we present the first essentially complete assembly and annotation of the kinetoplast genome of T. brucei. We have identified 391 minicircles, encoding not only ∼930 predicted 'canonical' gRNA genes that cover nearly all known editing events (accessible via the web at http://hank.bio.ed.ac.uk), but also ∼370 'non-canonical' gRNA genes of unknown function. Small RNA transcriptome data confirmed expression of the majority of both categories of gRNAs. Finally, we have used our data set to refine definitions for minicircle structure and to explore dynamics of minicircle copy numbers.

[The quantitative determination method of covalently closed circular DNA HBV in puncture biopsy specimens of the liver.]

To analyze the method HBV covalent-closed circular DNA quantitative determination in liver puncture biopsies and evaluate its significance in identifying HBsAg-negative viral hepatitis B. In this work, samples of liver tissue biopsy material were used from 128 patients living in St. Petersburg, in various regions of the Russian Federation, as well as in the Republic of Uzbekistan. For quantitative analysis of HBV covalently closed circular DNA in a biopsy material a method was developed based on real-time PCR using TaqMan probes for the target fragment and for the endogenous reference gene, based on the detecting ccc HBV DNA method of Pollicino T. et al. When quantifying ccc DNA HBV in liver tissue of 18 moderately HBV activity with HBV DNA PCR positive results patients and 16 inactive HBsAg carriers, the ccc DNA HBV content was significantly different between groups (p<0.034) and in terms 1 copy of the ß-globin gene among moderate activity HBV patients amounted to 1.71±1.32 copies/cell, and for inactive HBsAg carriers 0.15±0.14 copies/cell. In the group of patients with severe liver fibrosis and cirrhosis, the amount of ccc DNA HBV in liver tissue in patients with HBV averaged 2.5±0.4 copies/cell, in patients with HBV + D on average 0.7±0.25 copies/cell, in patients with HCV + HBV co-infection 0.45±0.07 copies/cell, in patients with a preliminary diagnosis of chronic hepatitis C hepatitis, on average 0.12±0.04 copies/cell, in patients with cryptogenic hepatitis 0.2± 0.05 copies/cell. A significant difference was shown between the group of patients with chronic hepatitis B with marked fibrosis and cirrhosis of the liver with other patients groups, except for the group of 18 moderate activity chronic hepatitis B patients. The values of Student's t-test when compared with other groups were respectively: for patients with a HCV preliminary diagnosis t=5,92 p<0,05 f = 19, patients with cryptogenic hepatitis t=5,71 p<0,05 f = 18, with «inactive HBsAg carriage¼ t=5,55 p<0,05 f = 29, with HCV + HBV co-infection t=5,05 p<0,05 f = 15 and HBV + D co-infection t=3,82 p<0,05 f = 17. The covalently closed circular DNA HBV quantitative assessment method in liver puncture biopsies allows identifying HBsAgnegative chronic viral hepatitis B forms and also reflects the virus replication activity, which, in turn, makes it possible to assume further disease progression and evaluate the antiviral therapy effectiveness.

Hepatitis B Virus cccDNA in Hepatocellular Carcinoma Tissue Increases the Risk of Recurrence After Liver Transplantation.

BACKGROUND: High hepatitis B virus (HBV) DNA level is strongly associated with hepatocellular carcinoma (HCC) development in chronic HBV infection. The aim of this study was to investigate the association between intrahepatic HBV DNA titer and post-liver transplantation (LT) prognosis for HBV-related HCC (HBV-HCC) patients. METHODS: A total of 60 patients with HBV-HCC who underwent LT were retrospectively studied. Using quantitative TaqMan fluorescent real-time polymerase chain reaction assay, HBV total DNA (tDNA) and covalently closed circular DNA (cccDNA) were both quantified in tumor tissue (TT) and adjacent non-tumor tissue (ANTT) from the explanted liver. RESULTS: The loads of tDNA and cccDNA in ANTT were associated with serum HBV DNA levels. Multivariate analysis showed that the presence of vascular invasion and cccDNA in TT were independent risk factors for tumor recurrence. The group of patients with cccDNA titers ≥31ogl0 copies/µg in TT had significantly higher cumulative recurrence rates than those with <31ogl0 copies/µg group. The cccDNA titers predicted the tumor recurrence with an area under the receiver operating characteristic curve of 0.664. CONCLUSIONS: Our findings would assist the clinical implementation of a more personalized therapy for tumor recurrence control and improve the prognosis of HBV-HCC patients.

A quantitative method for hepatitis B virus covalently closed circular DNA enables distinguishing direct acting antivirals from cytotoxic agents.

Studying the biogenesis of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) and developing anti-HBV agents require analytical methods to quantify viral DNA levels inside host cells. The well-accepted Southern blotting method is only semi-quantitative, while the other widely used methods (based on qPCR) have been questioned as to their fidelity for cccDNA quantification. In addition, Southern blotting and qPCR results barely reflect the number of host cells present in an analytical sample. We here developed new techniques that substantially improve cccDNA detection and quantification, including a sample pretreatment method that i) exploits high temperature and exonuclease V (an ATP-dependent, bidirectional exonuclease) digestion to effectively increase the amplification efficiency of HBV cccDNA by removing rcDNA and denaturing the cccDNA template, and ii) a method that splits cell samples and uses separate extraction technologies to facilitate "host normalization" based on host genomic DNA signals. Our study introduces new analytical techniques that should be useful for the basic biology and translational studies of HBV.

A rapid bioanalytical tool for detection of sequence-specific circular DNA and mitochondrial DNA point mutations.

Mutations in mitochondrial DNA (mtDNA) have been an essential cause of numerous diseases, making their identification critically important. The majority of mtDNA screening techniques require polymerase chain reaction (PCR) amplification, enzymatic digestion, and denaturation procedures, which are laborious and costly. Herein, we developed a sensitive PCR-free electrokinetic-based sensor combined with a customized bis-peptide nucleic acid (bis-PNA) and gamma-PNA (γ-PNA) probes immobilized on beads, for the detection of mtDNA point mutations and sequence-specific supercoiled plasmid DNA at the picomolar range. The probes are capable of invading the double-stranded circular DNA and forming a stable triplex structure. Thus, this method can significantly reduce the sample preparation and omit the PCR amplification steps prior to sensing. Further, this bioanalytical tool can open up a new paradigm in clinical settings for the screening of double-stranded circular nucleic acids with a single-base mismatch specificity in a rapid and sensitive manner.

Serum HBcrAg is better than HBV RNA and HBsAg in reflecting intrahepatic covalently closed circular DNA.

The correlation between serum HBcrAg and HBV RNA is unclear, and correlations of intrahepatic cccDNA with HBcrAg, HBV RNA and HBsAg are rarely reported in the same cohort. This study aimed to assess the correlation of HBcrAg with HBV RNA and HBsAg, and investigate whether serum HBcrAg is superior to serum HBV RNA and HBsAg in reflecting intrahepatic HBV cccDNA in HBeAg-positive and HBeAg-negative CHB patients. In this study, 85 HBeAg-positive and 25 HBeAg-negative patients who have never received antiviral therapy were included. Among HBeAg-positive patients, HBcrAg was correlated positively with HBsAg (r = 0.564, P < 0.001) and HBV RNA (r = 0.445, P < 0.001), and HBV RNA was also correlated positively with HBsAg (r = 0.323, P = 0.003). Among HBeAg-negative patients, no significant correlation was observed between HBcrAg, HBsAg and HBV RNA. By multivariable linear regression, HBcrAg (ß = -0.563, P < 0.001), HBsAg (ß = -0.328, P < 0.001) and HBV RNA (ß = 0.180, P = 0.003) were all associated with cccDNA levels among HBeAg-positive patients, but only serum HBcrAg was associated with cccDNA level (ß = 0.774, P = 0.000) among HBeAg-negative patients. HBcrAg was better correlated with cccDNA as compared to HBsAg and HBV RNA, irrespective of HBeAg status. Among HBeAg-positive patients, though HBcrAg level was influenced by hepatic inflammatory activity and HBV DNA levels, the good correlations of HBcrAg with cccDNA persisted after stratification by inflammatory activity and HBV DNA levels. In conclusion, correlations of serum HBcrAg, HBV RNA and HBsAg levels differ significantly between HBeAg-positive and HBeAg-negative patients, but serum HbcrAg correlates with cccDNA levels better than HBV RNA and HBsAg, irrespective of HBeAg status.