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Communications Biology Jul 2023Hepatitis B virus (HBV) may integrate into the genome of infected cells and contribute to hepatocarcinogenesis. However, the role of HBV integration in hepatocellular...
Hepatitis B virus (HBV) may integrate into the genome of infected cells and contribute to hepatocarcinogenesis. However, the role of HBV integration in hepatocellular carcinoma (HCC) development remains unclear. In this study, we apply a high-throughput HBV integration sequencing approach that allows sensitive identification of HBV integration sites and enumeration of integration clones. We identify 3339 HBV integration sites in paired tumour and non-tumour tissue samples from 7 patients with HCC. We detect 2107 clonally expanded integrations (1817 in tumour and 290 in non-tumour tissues), and a significant enrichment of clonal HBV integrations in mitochondrial DNA (mtDNA) preferentially occurring in the oxidative phosphorylation genes (OXPHOS) and D-loop region. We also find that HBV RNA sequences are imported into the mitochondria of hepatoma cells with the involvement of polynucleotide phosphorylase (PNPASE), and that HBV RNA might have a role in the process of HBV integration into mtDNA. Our results suggest a potential mechanism by which HBV integration may contribute to HCC development.
Topics: Humans; Hepatitis B virus; Carcinoma, Hepatocellular; Liver Neoplasms; DNA, Mitochondrial; Virus Integration; Mitochondria
PubMed: 37400627
DOI: 10.1038/s42003-023-05017-4 -
Theranostics 2020Liquid biopsy has emerged in the last ten years as an appealing noninvasive strategy to support early cancer diagnosis and follow-up interventions. However, conventional... (Review)
Review
Liquid biopsy has emerged in the last ten years as an appealing noninvasive strategy to support early cancer diagnosis and follow-up interventions. However, conventional liquid biopsy strategies involving specified biomarkers have encountered unexpected inconsistencies stemming from the use of different analytical methodologies. Recent reports have repeatedly demonstrated that integrated detection of multiple liquid biopsy biomarkers can significantly improve diagnostic performance by eliminating the influence of intratumoral heterogeneity. Herein, we review the progress in the field of liquid biopsy and propose a novel integrated liquid biopsy framework consisting of three categories: elementary, intermediate, and advanced integration. We also summarize the merits of the integration strategy and propose a roadmap toward refining cancer diagnosis, metastasis surveillance, and prognostication.
Topics: Biomarkers, Tumor; Circulating Tumor DNA; Early Detection of Cancer; Extracellular Vesicles; Humans; Liquid Biopsy; Neoplasms; Neoplastic Cells, Circulating
PubMed: 32089746
DOI: 10.7150/thno.40677 -
Nucleic Acids Research May 2023Prokaryotic Argonautes (pAgos) are programmable nucleases involved in cell defense against invading DNA. In vitro, pAgos can bind small single-stranded guide DNAs to...
Prokaryotic Argonautes (pAgos) are programmable nucleases involved in cell defense against invading DNA. In vitro, pAgos can bind small single-stranded guide DNAs to recognize and cleave complementary DNA. In vivo, pAgos preferentially target plasmids, phages and multicopy genetic elements. Here, we show that CbAgo nuclease from Clostridium butyricum can be used for genomic DNA engineering in bacteria. We demonstrate that CbAgo loaded with plasmid-derived guide DNAs can recognize and cleave homologous chromosomal loci, and define the minimal length of homology required for this targeting. Cleavage of plasmid DNA at an engineered site of the I-SceI meganuclease increases guide DNA loading into CbAgo and enhances processing of homologous chromosomal loci. Analysis of guide DNA loading into CbAgo also reveals off-target sites of I-SceI in the Escherichia coli genome, demonstrating that pAgos can be used for highly sensitive detection of double-stranded breaks in genomic DNA. Finally, we show that CbAgo-dependent targeting of genomic loci with plasmid-derived guide DNAs promotes homologous recombination between plasmid and chromosomal DNA, depending on the catalytic activity of CbAgo. Specific targeting of plasmids with Argonautes can be used to integrate plasmid-encoded sequences into the chromosome thus enabling genome editing.
Topics: Gene Editing; Plasmids; DNA; Bacteria; DNA, Single-Stranded; Endonucleases
PubMed: 36987855
DOI: 10.1093/nar/gkad191 -
Biopolymers Jan 2017Peptide nucleic acids (PNAs) are extensively studied for the control of genetic expression since their design in the 1990s. However, the application of PNAs in... (Review)
Review
Peptide nucleic acids (PNAs) are extensively studied for the control of genetic expression since their design in the 1990s. However, the application of PNAs in nanotechnology is much more recent. PNAs share the specific base-pair recognition characteristic of DNA together with material-like properties of polyamides, both proteins and synthetic polymers, such as Kevlar and Nylon. The first application of PNA was in the form of PNA-amphiphiles, resulting in the formation of either lipid integrated structures, hydrogels or fibrillary assemblies. Heteroduplex DNA-PNA assemblies allow the formation of hybrid structures with higher stability as compared with pure DNA. A systematic screen for minimal PNA building blocks resulted in the identification of guanine-containing di-PNA assemblies and protected guanine-PNA monomer spheres showing unique optical properties. Finally, the co-assembly of PNA with thymine-like three-faced cyanuric acid allowed the assembly of poly-adenine PNA into fibers. In summary, we believe that PNAs represent a new and important family of building blocks which converges the advantages of both DNA- and peptide-nanotechnologies.
Topics: DNA; Drug Carriers; Molecular Conformation; Nanostructures; Peptide Nucleic Acids
PubMed: 27486924
DOI: 10.1002/bip.22930 -
Experimental & Molecular Medicine Dec 2022PARPs play fundamental roles in multiple DNA damage recognition and repair pathways. Persistent nuclear PARP activation causes cellular NAD depletion and exacerbates...
PARPs play fundamental roles in multiple DNA damage recognition and repair pathways. Persistent nuclear PARP activation causes cellular NAD depletion and exacerbates cellular aging. However, very little is known about mitochondrial PARP (mtPARP) and poly ADP-ribosylation (PARylation). The existence of mtPARP is controversial, and the biological roles of mtPARP-induced mitochondrial PARylation are unclear. Here, we demonstrate the presence of PARP1 and PARylation in purified mitochondria. The addition of the PARP1 substrate NAD to isolated mitochondria induced PARylation, which was suppressed by treatment with the inhibitor olaparib. Mitochondrial PARylation was also evaluated by enzymatic labeling of terminal ADP-ribose (ELTA). To further confirm the presence of mtPARP1, we evaluated mitochondrial nucleoid PARylation by ADP ribose-chromatin affinity purification (ADPr-ChAP) and PARP1 chromatin immunoprecipitation (ChIP). We observed that NAD stimulated PARylation and TFAM occupancy on the mtDNA regulatory region D-loop, inducing mtDNA transcription. These findings suggest that PARP1 is integrally involved in mitochondrial PARylation and that NAD-dependent mtPARP1 activity contributes to mtDNA transcriptional regulation.
Topics: Poly ADP Ribosylation; NAD; Poly(ADP-ribose) Polymerase Inhibitors; Mitochondria; DNA, Mitochondrial
PubMed: 36473936
DOI: 10.1038/s12276-022-00894-x -
Current Opinion in Chemical Biology Feb 2016Methylation and acetylation of DNA and histone proteins are the chemical basis for epigenetics. From bacteria to humans, methylation and acetylation are sensitive to... (Review)
Review
Methylation and acetylation of DNA and histone proteins are the chemical basis for epigenetics. From bacteria to humans, methylation and acetylation are sensitive to cellular metabolic status. Modification rates depend on the availability of one-carbon and two-carbon substrates (S-adenosylmethionine, acetyl-CoA, and in bacteria also acetyl-phosphate). In addition, they are sensitive to demodification enzyme cofactors (α-ketoglutarate, NAD(+)) and structural analog metabolites that function as epigenetic enzyme inhibitors (e.g., S-adenosylhomocysteine, 2-hydroxyglutarate). Methylation and acetylation likely initially evolved to tailor protein activities in microbes to their metabolic milieu. While the extracellular environment of mammals is more tightly controlled, the combined impact of nutrient abundance and metabolic enzyme expression impacts epigenetics in mammals sufficiently to drive important biological outcomes such as stem cell fate and cancer.
Topics: Acetylation; Animals; DNA; Epigenesis, Genetic; Histones; Humans; Methylation; Microbiology
PubMed: 26629854
DOI: 10.1016/j.cbpa.2015.10.030 -
Microbiology Spectrum Apr 2015The site-specific recombinase encoded by bacteriophage λ (Int) is responsible for integrating and excising the viral chromosome into and out of the chromosome of its... (Review)
Review
The site-specific recombinase encoded by bacteriophage λ (Int) is responsible for integrating and excising the viral chromosome into and out of the chromosome of its Escherichia coli host. Int carries out a reaction that is highly directional, tightly regulated, and depends upon an ensemble of accessory DNA bending proteins acting on 240 bp of DNA encoding 16 protein binding sites. This additional complexity enables two pathways, integrative and excisive recombination, whose opposite, and effectively irreversible, directions are dictated by different physiological and environmental signals. Int recombinase is a heterobivalent DNA binding protein and each of the four Int protomers, within a multiprotein 400 kDa recombinogenic complex, is thought to bind and, with the aid of DNA bending proteins, bridge one arm- and one core-type DNA site. In the 12 years since the publication of the last review focused solely on the λ site-specific recombination pathway in Mobile DNA II, there has been a great deal of progress in elucidating the molecular details of this pathway. The most dramatic advances in our understanding of the reaction have been in the area of X-ray crystallography where protein-DNA structures have now been determined for of all of the DNA-protein interfaces driving the Int pathway. Building on this foundation of structures, it has been possible to derive models for the assembly of components that determine the regulatory apparatus in the P-arm, and for the overall architectures that define excisive and integrative recombinogenic complexes. The most fundamental additional mechanistic insights derive from the application of hexapeptide inhibitors and single molecule kinetics.
Topics: Bacteriophage lambda; Crystallography, X-Ray; DNA; Escherichia coli; Integrases; Models, Biological; Models, Molecular; Nucleic Acid Conformation; Protein Binding; Protein Conformation; Recombination, Genetic
PubMed: 26104711
DOI: 10.1128/microbiolspec.MDNA3-0051-2014 -
Bioconjugate Chemistry Jan 2023Over the past 40 years, structural and dynamic DNA nanotechnologies have undoubtedly demonstrated to be effective means for organizing matter at the nanoscale and... (Review)
Review
Over the past 40 years, structural and dynamic DNA nanotechnologies have undoubtedly demonstrated to be effective means for organizing matter at the nanoscale and reconfiguring equilibrium structures, in a predictable fashion and with an accuracy of a few nanometers. Recently, novel concepts and methodologies have been developed to integrate nonequilibrium dynamics into DNA nanostructures, opening the way to the construction of synthetic materials that can adapt to environmental changes and thus acquire new properties. In this Review, we summarize the strategies currently applied for the construction of synthetic DNA filaments and conclude by reporting some recent and most relevant examples of DNA filaments that can emulate typical structural and dynamic features of the cytoskeleton, such as compartmentalization in cell-like vesicles, support for active transport of cargos, sustained or transient growth, and responsiveness to external stimuli.
Topics: Cytoskeleton; Nanotechnology; Nanostructures; Microtubules; DNA
PubMed: 36174970
DOI: 10.1021/acs.bioconjchem.2c00312 -
Topics in Current Chemistry (Cham) Apr 2020Cellular functions rely on a series of organized and regulated multienzyme cascade reactions. The catalytic efficiencies of these cascades depend on the precise spatial... (Review)
Review
Cellular functions rely on a series of organized and regulated multienzyme cascade reactions. The catalytic efficiencies of these cascades depend on the precise spatial organization of the constituent enzymes, which is optimized to facilitate substrate transport and regulate activities. Mimicry of this organization in a non-living, artificial system would be very useful in a broad range of applications-with impacts on both the scientific community and society at large. Self-assembled DNA nanostructures are promising applications to organize biomolecular components into prescribed, multidimensional patterns. In this review, we focus on recent progress in the field of DNA-scaffolded assembly and confinement of multienzyme reactions. DNA self-assembly is exploited to build spatially organized multienzyme cascades with control over their relative distance, substrate diffusion paths, compartmentalization and activity actuation. The combination of addressable DNA assembly and multienzyme cascades can deliver breakthroughs toward the engineering of novel synthetic and biomimetic reactors.
Topics: DNA; Enzymes; Protein Engineering
PubMed: 32248317
DOI: 10.1007/s41061-020-0299-3 -
Sensors (Basel, Switzerland) Nov 2020Contamination by pesticides in the food chain and the environment is a worldwide problem that needs to be actively monitored to ensure safety. Unfortunately, standard... (Review)
Review
Contamination by pesticides in the food chain and the environment is a worldwide problem that needs to be actively monitored to ensure safety. Unfortunately, standard pesticide analysis based on mass spectrometry takes a lot of time, money and effort. Thus, simple, reliable, cost-effective and field applicable methods for pesticide detection have been actively developed. One of the most promising technologies is an aptamer-based biosensor or so-called aptasensor. It utilizes aptamers, short single-stranded DNAs or RNAs, as pesticide recognition elements to integrate with various innovative biosensing technologies for specific and sensitive detection of pesticide residues. Several platforms for aptasensors have been dynamically established, such as colorimetry, fluorometry, electrochemistry, electrochemiluminescence (ECL) and so forth. Each platform has both advantages and disadvantages depending on the purpose of use and readiness of technology. For example, colorimetric-based aptasensors are more affordable than others because of the simplicity of fabrication and resource requirements. Electrochemical-based aptasensors have mainly shown better sensitivity than others with exceedingly low detection limits. This paper critically reviews the progression of pesticide aptasensors throughout the development process, including the selection, characterization and modification of aptamers, the conceptual frameworks of integrating aptamers and biosensors, the ASSURED (affordable, sensitive, specific, user-friendly, rapid and robust, equipment-free and deliverable to end users) criteria of different platforms and the future outlook.
Topics: Aptamers, Nucleotide; Biosensing Techniques; Colorimetry; DNA, Single-Stranded; Pesticides
PubMed: 33260648
DOI: 10.3390/s20236809