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1.
World J Gastroenterol ; 27(14): 1369-1391, 2021 Apr 14.
Artigo em Inglês | MEDLINE | ID: mdl-33911462

RESUMO

Infection with the hepatitis B virus (HBV) is still a major global health threat as 250 million people worldwide continue to be chronically infected with the virus. While patients may be treated with nucleoside/nucleotide analogues, this only suppresses HBV titre to sub-detection levels without eliminating the persistent HBV covalently closed circular DNA (cccDNA) genome. As a result, HBV infection cannot be cured, and the virus reactivates when conditions are favorable. Interferons (IFNs) are cytokines known to induce powerful antiviral mechanisms that clear viruses from infected cells. They have been shown to induce cccDNA clearance, but their use in the treatment of HBV infection is limited as HBV-targeting immune cells are exhausted and HBV has evolved multiple mechanisms to evade and suppress IFN signalling. Thus, to fully utilize IFN-mediated intracellular mechanisms to effectively eliminate HBV, instead of direct IFN administration, novel strategies to sustain IFN-mediated anti-cccDNA and antiviral mechanisms need to be developed. This review will consolidate what is known about how IFNs act to achieve its intracellular antiviral effects and highlight the critical interferon-stimulated gene targets and effector mechanisms with potent anti-cccDNA functions. These include cccDNA degradation by APOBECs and cccDNA silencing and transcription repression by epigenetic modifications. In addition, the mechanisms that HBV employs to disrupt IFN signalling will be discussed. Drugs that have been developed or are in the pipeline for components of the IFN signalling pathway and HBV targets that detract IFN signalling mechanisms will also be identified and discussed for utility in the treatment of HBV infections. Together, these will provide useful insights into design strategies that specifically target cccDNA for the eradication of HBV.


Assuntos
Hepatite B Crônica , Hepatite B , Antivirais/farmacologia , Antivirais/uso terapêutico , DNA Circular , DNA Viral/genética , DNA Viral/uso terapêutico , Vírus da Hepatite B/genética , Hepatite B Crônica/tratamento farmacológico , Humanos , Interferons/uso terapêutico , Replicação Viral
2.
Science ; 372(6538)2021 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-33833097

RESUMO

Liquid biopsies that analyze cell-free DNA in blood plasma are used for noninvasive prenatal testing, oncology, and monitoring of organ transplant recipients. DNA molecules are released into the plasma from various bodily tissues. Physical and molecular features of cell-free DNA fragments and their distribution over the genome bear information about their tissues of origin. Moreover, patterns of DNA methylation of these molecules reflect those of their tissue sources. The nucleosomal organization and nuclease content of the tissue of origin affect the fragmentation profile of plasma DNA molecules, such as fragment size and end motifs. Besides double-stranded linear fragments, other topological forms of cell-free DNA also exist-namely circular and single-stranded molecules. Enhanced by these features, liquid biopsies hold promise for the noninvasive detection of tissue-specific pathologies with a range of clinical applications.


Assuntos
Ácidos Nucleicos Livres/sangue , Ácidos Nucleicos Livres/genética , Fragmentação do DNA , Metilação de DNA , DNA/sangue , DNA/genética , Biópsia Líquida , Animais , Biomarcadores/sangue , DNA Circular/sangue , DNA Mitocondrial/sangue , DNA de Neoplasias/sangue , DNA de Neoplasias/genética , Desoxirribonucleases/metabolismo , Epigênese Genética , Feminino , Feto , Humanos , Gravidez , Transplantes
3.
Zhonghua Gan Zang Bing Za Zhi ; 29(2): 126-132, 2021 Feb 20.
Artigo em Chinês | MEDLINE | ID: mdl-33685080

RESUMO

Objective: To study the use of preS1-tp fusion protein as a novel vector to mediate the entry of small interfering RNA (siRNA) targeting the carboxy-terminal nuclear localization signal (NLS) region of hepatitis B virus (HBV) core protein into HBV-infected hepatocytes, and to further explore the HBV replication inhibition and covalently closed circular DNA synthesis. Methods: HepG2.2.15 cells expressing the human sodium taurocholate co-transporting polypeptide were established on the basis of lentivirus infection system. siRNA against HBV NLS region was designed and synthesized. PreS1-tp fusion protein expression and purification was observed to test its ability to cell entry and DNA binding. NLS siRNA were delivered into HepG2.2.15- sodium taurocholate co-transporting polypeptide cells by preS1-tp fusion protein as a vector to observe the effects of NLS siRNA on HBV replication and covalently closed circular DNA levels. Analysis of variance was used for comparison between multiple groups, and the measurement data differences between groups were analyzed by t-test. Results: HepG2.2.15-sodium taurocholate co-transporting polypeptide cell line was successfully constructed. Screened synthetic HBV NLS siRNA had significantly inhibited HBV replication. The preS1-tp fusion protein was expressed and purified on a large-scale. The fusion protein as a vector for HBV NLS siRNA had targeted delivery. The result showed that the fusion protein had effectively targeted siRNA to Hepg2.2.15-sodium taurocholate co-transporting polypeptide cell, which not only had effectively inhibited the expression of HBV mRNA, HBsAg and HBeAg, but also had significantly reduced the levels of HBV DNA and covalently closed circular DNA. Conclusion: The preS1-tp fusion protein constructed in this study uses the dual functional characteristics of preS1 binding to hepatocyte HBV receptor, and tp binding to nucleic acids, and targets HBV NLS siRNA against HBV-infected cells and block rcDNA from being transported to nucleus. siRNA plays a role in inhibiting HBV replication and covalently close circular DNA synthesis, providing a new strategy for the treatment of chronic hepatitis B caused by HBV infection, and a new research perspective for the complete elimination of HBV from the body.


Assuntos
Vírus da Hepatite B , Hepatite B , DNA Circular/genética , DNA Viral/genética , Células Hep G2 , Antígenos de Superfície da Hepatite B/genética , Vírus da Hepatite B/genética , Humanos , RNA Interferente Pequeno/genética , Replicação Viral
4.
Nat Commun ; 12(1): 1591, 2021 03 11.
Artigo em Inglês | MEDLINE | ID: mdl-33707452

RESUMO

Hepatitis B virus (HBV) is a highly contagious pathogen that afflicts over a third of the world's population, resulting in close to a million deaths annually. The formation and persistence of the HBV covalently closed circular DNA (cccDNA) is the root cause of HBV chronicity. However, the detailed molecular mechanism of cccDNA formation from relaxed circular DNA (rcDNA) remains opaque. Here we show that the minus and plus-strand lesions of HBV rcDNA require different sets of human repair factors in biochemical repair systems. We demonstrate that the plus-strand repair resembles DNA lagging strand synthesis, and requires proliferating cell nuclear antigen (PCNA), the replication factor C (RFC) complex, DNA polymerase delta (POLδ), flap endonuclease 1 (FEN-1), and DNA ligase 1 (LIG1). Only FEN-1 and LIG1 are required for the repair of the minus strand. Our findings provide a detailed mechanistic view of how HBV rcDNA is repaired to form cccDNA in biochemical repair systems.


Assuntos
Reparo do DNA/genética , DNA Circular/genética , DNA Viral/genética , Vírus da Hepatite B/genética , Hepatite B Crônica/patologia , Linhagem Celular Tumoral , DNA Ligase Dependente de ATP/metabolismo , DNA Polimerase III/metabolismo , Replicação do DNA/genética , Endonucleases Flap/metabolismo , Células Hep G2 , Humanos , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteína de Replicação C/metabolismo , Replicação Viral/genética
5.
Nat Commun ; 12(1): 1658, 2021 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-33712578

RESUMO

Chronic hepatitis B virus (HBV) infection is a major cause of liver disease and cancer worldwide for which there are no curative therapies. The major challenge in curing infection is eradicating or silencing the covalent closed circular DNA (cccDNA) form of the viral genome. The circadian factors BMAL1/CLOCK and REV-ERB are master regulators of the liver transcriptome and yet their role in HBV replication is unknown. We establish a circadian cycling liver cell-model and demonstrate that REV-ERB directly regulates NTCP-dependent hepatitis B and delta virus particle entry. Importantly, we show that pharmacological activation of REV-ERB inhibits HBV infection in vitro and in human liver chimeric mice. We uncover a role for BMAL1 to bind HBV genomes and increase viral promoter activity. Pharmacological inhibition of BMAL1 through REV-ERB ligands reduces pre-genomic RNA and de novo particle secretion. The presence of conserved E-box motifs among members of the Hepadnaviridae family highlight an evolutionarily conserved role for BMAL1 in regulating this family of small DNA viruses.


Assuntos
Relógios Biológicos/fisiologia , Ritmo Circadiano/fisiologia , Vírus da Hepatite B/fisiologia , Replicação Viral/fisiologia , Animais , Relógios Biológicos/efeitos dos fármacos , Relógios Biológicos/genética , Ritmo Circadiano/genética , DNA Circular , DNA Viral/metabolismo , Regulação da Expressão Gênica , Genoma Viral , Células Hep G2 , Hepatite B/virologia , Vírus da Hepatite B/genética , Hepatite B Crônica/genética , Hepatócitos/metabolismo , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Fígado/metabolismo , Camundongos , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Regiões Promotoras Genéticas , Simportadores/metabolismo , Transcriptoma , Vírion/metabolismo , Internalização do Vírus
6.
Int J Infect Dis ; 105: 418-423, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33676002

RESUMO

OBJECTIVES: To investigate the factors and virological significance of serum hepatitis B virus (HBV) pregenomic RNA (pgRNA) status after long-term antiviral therapy with nucleos(t)ide analogues (NAs) in patients with chronic hepatitis B (CHB). METHODS: In total, 51 treatment-naïve patients with CHB were included in the study. Clinical data were collected at baseline, during 5 years and at year 10 of NA treatment. Serum HBV pgRNA status of 51 patients was determined at year 5. RESULTS: At year 5, 45% of the patients remained positive for HBV pgRNA. There were significant differences in baseline hepatitis B e antigen (HBeAg) status, HBV DNA load and hepatitis B surface antigen (HBsAg) levels between patients testing positive and negative for HBV pgRNA at year 5. Serum HBV pgRNA status and serum HBV DNA load were correlated after 5 years of NA treatment (r = 0.347, P = 0.013). Being HBV pgRNA positive at year 5 was an independent risk factor for sustainedly undetectable HBV DNA after 10 years of NA treatment (odds ratio 13.638, 95% confidence interval 1.32-140.81; P = 0.028). Furthermore, HBV pgRNA status at year 5 was associated with HBV DNA re-appearance at year 10 (P = 0.009). CONCLUSIONS: HBV pgRNA status at year 5 can reveal HBV covalently closed circular DNA (cccDNA) activity, and HBV pgRNA positivity after long-term antiviral therapy may indicate higher transcriptional activity of HBV cccDNA. Long-term dynamic monitoring of HBV pgRNA should be considered.


Assuntos
Antivirais/uso terapêutico , Vírus da Hepatite B/isolamento & purificação , Hepatite B Crônica/tratamento farmacológico , RNA Viral/sangue , Adulto , Idoso , DNA Circular/sangue , Feminino , Vírus da Hepatite B/genética , Hepatite B Crônica/virologia , Humanos , Masculino , Pessoa de Meia-Idade , Fatores de Tempo
7.
Ageing Res Rev ; 67: 101306, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33610814

RESUMO

Extrachromosomal circular DNA (eccDNA) accumulates within the nucleus of eukaryotic cells during physiological aging and in age-related diseases (ARDs) and the accumulation could be caused by the declined exclusion of nuclear eccDNA in these states. This review focuses on the formation of eccDNA and the roles of some main factors, such as nuclear pore complexes (NPCs), nucleoplasmic reticulum (NR), and nuclear actin, in eccDNA exclusion. eccDNAs are mostly formed from non-coding DNA during DNA damage repair. They move to NPCs along nuclear actin and are excluded out of the nucleus through functional NPCs in young and healthy cells. However, it has been demonstrated that defective NPCs, abnormal NPC components and nuclear actin rods are increased in aged cells, various cancers and certain other ARDs such as cardiovascular diseases, premature aging, neurodegenerative diseases and myopathies. Therefore, mainly resulting from the increase of dysfunctional NPCs, the exclusion of nuclear eccDNAs may be reduced and eccDNAs thus accumulate within the nucleus in aging and the aforementioned ARDs. In addition, the protective function of non-coding DNA in tumorigenesis is further discussed.


Assuntos
DNA Circular , DNA , Idoso , Envelhecimento/genética , Núcleo Celular , Humanos
8.
Nucleic Acids Res ; 49(4): 2317-2332, 2021 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-33524154

RESUMO

We recently showed that Saccharomyces cerevisiae telomeric DNA can fold into an unprecedented pseudocircular G-hairpin (PGH) structure. However, the formation of PGHs in the context of extended sequences, which is a prerequisite for their function in vivo and their applications in biotechnology, has not been elucidated. Here, we show that despite its 'circular' nature, PGHs tolerate single-stranded (ss) protrusions. High-resolution NMR structure of a novel member of PGH family reveals the atomistic details on a junction between ssDNA and PGH unit. Identification of new sequences capable of folding into one of the two forms of PGH helped in defining minimal sequence requirements for their formation. Our time-resolved NMR data indicate a possibility that PGHs fold via a complex kinetic partitioning mechanism and suggests the existence of K+ ion-dependent PGH folding intermediates. The data not only provide an explanation of cation-type-dependent formation of PGHs, but also explain the unusually large hysteresis between PGH melting and annealing noted in our previous study. Our findings have important implications for DNA biology and nanotechnology. Overrepresentation of sequences able to form PGHs in the evolutionary-conserved regions of the human genome implies their functionally important biological role(s).


Assuntos
DNA Circular/química , Pareamento de Bases , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Conformação de Ácido Nucleico , Motivos de Nucleotídeos , Saccharomyces cerevisiae/genética , Estereoisomerismo , Telômero/química
9.
Nat Commun ; 12(1): 1053, 2021 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-33594049

RESUMO

In the cell, DNA is arranged into highly-organised and topologically-constrained (supercoiled) structures. It remains unclear how this supercoiling affects the detailed double-helical structure of DNA, largely because of limitations in spatial resolution of the available biophysical tools. Here, we overcome these limitations, by a combination of atomic force microscopy (AFM) and atomistic molecular dynamics (MD) simulations, to resolve structures of negatively-supercoiled DNA minicircles at base-pair resolution. We observe that negative superhelical stress induces local variation in the canonical B-form DNA structure by introducing kinks and defects that affect global minicircle structure and flexibility. We probe how these local and global conformational changes affect DNA interactions through the binding of triplex-forming oligonucleotides to DNA minicircles. We show that the energetics of triplex formation is governed by a delicate balance between electrostatics and bonding interactions. Our results provide mechanistic insight into how DNA supercoiling can affect molecular recognition, that may have broader implications for DNA interactions with other molecular species.


Assuntos
Pareamento de Bases/genética , DNA Super-Helicoidal/química , Conformação de Ácido Nucleico , Oligonucleotídeos/química , DNA Circular/química , Microscopia de Força Atômica , Simulação de Dinâmica Molecular
10.
J Phys Chem B ; 125(9): 2277-2287, 2021 03 11.
Artigo em Inglês | MEDLINE | ID: mdl-33635080

RESUMO

The binding of proteins onto DNA contributes to the shaping and packaging of the genome as well as to the expression of specific genetic messages. With a view to understanding the interplay between the presence of proteins and the deformation of DNA involved in such processes, we developed a new method to minimize the elastic energy of DNA fragments at the mesoscale level. Our method makes it possible to obtain the optimal pathways of protein-decorated DNA molecules for which the terminal base pairs are spatially constrained. We focus in this work on the deformations induced by selected architectural proteins on circular DNA. We report the energy landscapes of DNA minicircles subjected to different levels of torsional stress and containing one or two proteins as functions of the chain length and spacing between the proteins. Our results reveal cooperation between the elasticity of the double helix and the structural distortions of DNA induced by bound proteins. We find that the imposed mechanical stress influences the placement of proteins on DNA and that the proteins, in turn, modulate the mechanical stress and thereby broadcast their presence along DNA.


Assuntos
DNA Circular , DNA , DNA/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Elasticidade , Conformação de Ácido Nucleico
11.
Mol Biol Rep ; 48(1): 677-689, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33442829

RESUMO

The vertebrate mitochondrial genome is typically circular molecules made up of 14,000 to 16,000 bp, including 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), two ribosomal RNA genes (12 s rRNA and 16 s rRNA) and a control region. Compared with nuclear DNA, mitochondrial DNA has a higher mutation rate, so it is one of the most effective and reliable molecular markers in fish phylogeny. Macrotocinclus affinis was the only species in Macrotocinclus (it was classified as Otocinclus in the past) and currently lacks genetic information. Most of the current researches are based on the mitochondrial Cytb gene and RAG1 and RAG2 nuclear genes to study the phylogenetic analysis of Siluriformes. So, the study provides the characteristic features of the Macrotocinclus affinis mitochondrial genome and this is the first time that the phylogenetic relationship of Siluriformes has been reconstructed based on COI. We aimed to sequence the entire mitochondrial genome of Macrotocinclus affinis using conventional PCR techniques and to clarify its phylogenetic status in Siluriformes by using the COI sequence of mitochondria. In this study, we sequenced the whole mitochondrial genome of this species yielding a 16,632 bp circular assembly composed of the typical vertebrate mitochondrial features. It contains 13 protein-coding genes, two rRNA genes, 22 tRNA genes, a putative control region, and one origin of replication on the light-strand. The overall base composition includes A (30.07%), T (24.43%), C (29.43%) and G (16.01%). The genome composition is A + T biased (54.5%), and exhibits AT-skew (0.1036) and GC-skew (-0.2962). Moreover, the 13 PCGs encode 3850 amino acids in total. The result of the phylogenetic tree supports Macrotocinclus affinis has a closest relationship with Otocinclus cf. hoppei far. These results will help to understand the characteristics of the mitochondrial genome of Macrotocinclus affinis and provide molecular basis for the evolutionary relationship of Loricariidae.


Assuntos
Peixes-Gato/genética , DNA Mitocondrial/genética , Genoma Mitocondrial , Mitocôndrias/genética , Fases de Leitura Aberta , Animais , Composição de Bases , Peixes-Gato/classificação , Mapeamento Cromossômico , DNA Circular/genética , Tamanho do Genoma , Filogenia , RNA Ribossômico/classificação , RNA Ribossômico/genética , RNA de Transferência/classificação , RNA de Transferência/genética , Sequenciamento Completo do Genoma
12.
Nature ; 591(7848): 137-141, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33361815

RESUMO

Focal chromosomal amplification contributes to the initiation of cancer by mediating overexpression of oncogenes1-3, and to the development of cancer therapy resistance by increasing the expression of genes whose action diminishes the efficacy of anti-cancer drugs. Here we used whole-genome sequencing of clonal cell isolates that developed chemotherapeutic resistance to show that chromothripsis is a major driver of circular extrachromosomal DNA (ecDNA) amplification (also known as double minutes) through mechanisms that depend on poly(ADP-ribose) polymerases (PARP) and the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs). Longitudinal analyses revealed that a further increase in drug tolerance is achieved by structural evolution of ecDNAs through additional rounds of chromothripsis. In situ Hi-C sequencing showed that ecDNAs preferentially tether near chromosome ends, where they re-integrate when DNA damage is present. Intrachromosomal amplifications that formed initially under low-level drug selection underwent continuing breakage-fusion-bridge cycles, generating amplicons more than 100 megabases in length that became trapped within interphase bridges and then shattered, thereby producing micronuclei whose encapsulated ecDNAs are substrates for chromothripsis. We identified similar genome rearrangement profiles linked to localized gene amplification in human cancers with acquired drug resistance or oncogene amplifications. We propose that chromothripsis is a primary mechanism that accelerates genomic DNA rearrangement and amplification into ecDNA and enables rapid acquisition of tolerance to altered growth conditions.


Assuntos
Cromotripsia , Evolução Molecular , Amplificação de Genes/genética , Neoplasias/genética , Oncogenes/genética , Dano ao DNA , Reparo do DNA por Junção de Extremidades , DNA Circular/química , DNA Circular/metabolismo , DNA de Neoplasias/química , DNA de Neoplasias/metabolismo , Proteína Quinase Ativada por DNA , Resistencia a Medicamentos Antineoplásicos , Células HEK293 , Células HeLa , Humanos , Micronúcleos com Defeito Cromossômico , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Neoplasias/patologia , Poli(ADP-Ribose) Polimerases/metabolismo , Seleção Genética , Sequenciamento Completo do Genoma
13.
Gene ; 765: 145114, 2021 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-32891769

RESUMO

The current study aimed to investigate the role and underlying mechanisms of circ_LARP4 in diabetic nephropathy (DN). Here, mouse mesangial cells (SV40-MES13) were cultured with 30 mM glucose to establish a DN cellular model. The qRT-PCR results indicated that circ_LARP4 expression was downregulated in the DN cellular model compared to that in the control cells. As determined by an MTT assay, circ_LARP4 overexpression via the circ_LARP4 overexpression (OE) plasmids inhibited the cell proliferation rate. As determined by an Annexin V/PI kit and flow cytometry, circ_LARP4 overexpression increased the cell apoptosis rate. As measured by Western blot, circ_LARP4 overexpression enhanced BAX expression but reduced Bcl-2 expression, also suggesting an enhancement of cell apoptosis. Moreover, regarding cell fibrosis, circ_LARP4 overexpression reduced the mRNA levels of fibrosis markers, including fibronectin, collagen I and collagen IV. Interestingly, miR-424 was found to be reduced in the DN cellular model after transfection with the circ_LARP4 OE plasmids. In addition, restoration of miR-424 expression with the miR-424 mimics reversed the negative effects of circ_LARP4 overexpression on cell proliferation and fibrosis. In conclusion, circ_LARP4 was lower in the DN cellular model than in normal cells, and circ_LARP4 overexpression resulted in decreased cell proliferation and cell fibrosis but increased cell apoptosis in the DN cellular model by sponging miR-424.


Assuntos
DNA Circular/genética , Células Mesangiais/metabolismo , Proteínas/genética , Animais , Apoptose/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Nefropatias Diabéticas/genética , Fibrose , Glucose/metabolismo , Células Mesangiais/fisiologia , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , RNA Mensageiro/metabolismo , Transdução de Sinais/genética
14.
Methods Mol Biol ; 2197: 3-12, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32827129

RESUMO

Minicircle DNA (mcDNA) has been considered to be an alternative choice of traditional DNA vaccine due to its much smaller size, resulting in more efficient antigen synthesis, enhanced and long-lasting adaptive immune response, especially cellular immune response. However, the disadvantages such as relative high cost and labor intensiveness severely restrict its direct application in the field of veterinary vaccine. Here, we describe a novel Cre Recombinase-mediated In vivo McDNA platform, named CRIM, in which the parental plasmid could spontaneously transform into mcDNA by itself after transfection or oral administration. This CRIM vaccine platform might serve as a novel oral antigen delivery system for any infectious diseases, especially for veterinary application.


Assuntos
DNA Circular , Engenharia Genética , Recombinação Homóloga , Integrases/metabolismo , Vacinas de DNA/imunologia , Medicina Veterinária , Animais , DNA Circular/genética , DNA Circular/imunologia , Engenharia Genética/métodos , Células HEK293 , Humanos , Plasmídeos/genética , Vacinas de DNA/genética , Medicina Veterinária/métodos
15.
Methods Mol Biol ; 2153: 403-425, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32840795

RESUMO

The ribosomal RNA (rDNA) sequence is the most abundant repetitive element in the budding yeast genome and forms a tandem cluster of ~100-200 copies. Cells frequently change their rDNA copy number, making rDNA the most unstable region in the budding yeast genome. The rDNA region experiences programmed replication fork arrest and subsequent formation of DNA double-strand breaks (DSBs), which are the main drivers of rDNA instability. The rDNA region offers a unique system to understand the mechanisms that respond to replication fork arrest as well as the mechanisms that regulate repeat instability. This chapter describes three methods to assess rDNA instability.


Assuntos
DNA Ribossômico/metabolismo , Eletroforese em Gel de Campo Pulsado/métodos , Saccharomyces cerevisiae/genética , Southern Blotting , Cromossomos Fúngicos/genética , Quebras de DNA de Cadeia Dupla , Replicação do DNA , DNA Circular/química , DNA Circular/metabolismo , DNA Fúngico/química , DNA Fúngico/metabolismo , DNA Ribossômico/química
16.
Methods Mol Biol ; 2197: 193-205, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32827138

RESUMO

Multimodal (MM) chromatography can be described as a chromatographic method that uses more than one mode of interaction between the target molecule and the ligand to achieve a particular separation. Owing to its advantages over traditional chromatography, such as higher selectivity and capacity, its application for the purification of biomolecules with therapeutic interest has been widely studied. The potential of MM chromatography for the purification of plasmid DNA has been demonstrated. In this chapter, a downstream process for the purification of supercoiled plasmid DNA using MM chromatography with two different ligands-Capto™ adhere and PPA HyperCell™-is described. In both the cases, the purification process yields a high purity and highly homogeneous sc plasmid product.


Assuntos
Cromatografia/métodos , DNA Circular/isolamento & purificação , Plasmídeos/isolamento & purificação , DNA Super-Helicoidal/isolamento & purificação , Diálise , Escherichia coli/genética
17.
Methods Mol Biol ; 2197: 207-222, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32827139

RESUMO

Human papillomavirus (HPV ) has been extensively associated with the development of cervical cancer due to the expression of oncoproteins like E7. This protein can interfere with pRB tumor suppressor activity, enabling the uncontrolled proliferation of abnormal cells. DNA vaccines are known as the third-generation vaccines, providing the ability of targeting viral infections such as HPV in a preventive and therapeutic way. Although current strategies make use of plasmid DNA (pDNA) as the vector of choice to be used as a DNA vaccine, minicircle DNA (mcDNA) has been proving its added value as a non-viral DNA vector by demonstrating higher expression efficiency and increased biosafety than the pDNA. However, due to its innovative profile, few methodologies have been explored and implemented for the manufacture of this molecule. This chapter describes the detailed procedures for the production, extraction, and purification of supercoiled E7-mcDNA vaccine, by using size-exclusion chromatography to obtain mcDNA with a purity degree which meets the regulatory agency criteria. Then, the assessment of E7 antigen expression through immunocytochemistry is also described.


Assuntos
DNA Circular/isolamento & purificação , Vacinas contra Papillomavirus/isolamento & purificação , Plasmídeos/isolamento & purificação , Vacinas de DNA/isolamento & purificação , Técnicas de Cultura de Células , Cromatografia em Gel , Escherichia coli/genética , Fermentação , Expressão Gênica , Imuno-Histoquímica , Proteínas E7 de Papillomavirus/genética , Proteínas E7 de Papillomavirus/imunologia , Vacinas contra Papillomavirus/genética , Vacinas contra Papillomavirus/imunologia , Vacinas de DNA/genética , Vacinas de DNA/imunologia
18.
Nat Commun ; 11(1): 5823, 2020 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-33199677

RESUMO

MYCN amplification drives one in six cases of neuroblastoma. The supernumerary gene copies are commonly found on highly rearranged, extrachromosomal circular DNA (ecDNA). The exact amplicon structure has not been described thus far and the functional relevance of its rearrangements is unknown. Here, we analyze the MYCN amplicon structure using short-read and Nanopore sequencing and its chromatin landscape using ChIP-seq, ATAC-seq and Hi-C. This reveals two distinct classes of amplicons which explain the regulatory requirements for MYCN overexpression. The first class always co-amplifies a proximal enhancer driven by the noradrenergic core regulatory circuit (CRC). The second class of MYCN amplicons is characterized by high structural complexity, lacks key local enhancers, and instead contains distal chromosomal fragments harboring CRC-driven enhancers. Thus, ectopic enhancer hijacking can compensate for the loss of local gene regulatory elements and explains a large component of the structural diversity observed in MYCN amplification.


Assuntos
Cromossomos Humanos/genética , Elementos Facilitadores Genéticos/genética , Proteína Proto-Oncogênica N-Myc/genética , Neuroblastoma/genética , Acetilação , Sequência de Bases , Linhagem Celular Tumoral , Metilação de DNA/genética , DNA Circular/genética , Epigênese Genética , Histonas/metabolismo , Humanos , Estimativa de Kaplan-Meier , Lisina/metabolismo , Sequenciamento por Nanoporos
19.
Nucleic Acids Res ; 48(19): 10680-10690, 2020 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-33021630

RESUMO

Circular DNA aptamers are powerful candidates for therapeutic applications given their dramatically enhanced biostability. Herein we report the first effort to evolve circular DNA aptamers that bind a human protein directly in serum, a complex biofluid. Targeting human thrombin, this strategy has led to the discovery of a circular aptamer, named CTBA4T-B1, that exhibits very high binding affinity (with a dissociation constant of 19 pM), excellent anticoagulation activity (with the half maximal inhibitory concentration of 90 pM) and high stability (with a half-life of 8 h) in human serum, highlighting the advantage of performing aptamer selection directly in the environment where the application is intended. CTBA4T-B1 is predicted to adopt a unique structural fold with a central two-tiered guanine quadruplex capped by two long stem-loops. This structural arrangement differs from all known thrombin binding linear DNA aptamers, demonstrating the added advantage of evolving aptamers from circular DNA libraries. The method described here permits the derivation of circular DNA aptamers directly in biological fluids and could potentially be adapted to generate other types of aptamers for therapeutic applications.


Assuntos
Aptâmeros de Nucleotídeos/química , DNA Circular/química , Trombina/metabolismo , Aptâmeros de Nucleotídeos/sangue , Aptâmeros de Nucleotídeos/metabolismo , DNA Circular/sangue , DNA Circular/metabolismo , Quadruplex G , Humanos , Ligação Proteica , Trombina/química
20.
Arch Virol ; 165(12): 2921-2926, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32989573

RESUMO

In this study, we present an analysis of metagenome sequences obtained from a filtrate of a siphon tissue homogenate of otter clams (Lutraria rhynchaena) with swollen-siphon disease. The viral signal was mined from the metagenomic data, and a novel circular ssDNA virus was identified. Genomic features and phylogenetic analysis showed that the virus belongs to the phylum Cressdnaviricota, which consists of viruses with circular, single-stranded DNA (ssDNA) genomes. Members of this phylum have been identified in various species and in environmental samples. The newly found virus is distantly related to the currently known members of the phylum Cressdnaviricota.


Assuntos
Bivalves/genética , Vírus de DNA/classificação , DNA Viral/genética , Genoma Viral , Animais , Vírus de DNA/isolamento & purificação , DNA Circular/genética , DNA de Cadeia Simples/genética , Microbiologia Ambiental , Metagenômica , Filogenia , Análise de Sequência de DNA
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