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Environmental and Molecular Mutagenesis 2024As final process of every DNA repair pathway, DNA ligation is crucial for maintaining genomic stability and preventing DNA strand breaks to accumulate. Therefore, a...
As final process of every DNA repair pathway, DNA ligation is crucial for maintaining genomic stability and preventing DNA strand breaks to accumulate. Therefore, a method reliably assessing DNA ligation capacity in protein extracts from murine tissues was aimed to establish. To optimize applicability, the use of radioactively labeled substrates was avoided and replaced by fluorescently labeled oligonucleotides. Briefly, tissue extracts were incubated with those complementary oligonucleotides so that in an ensuing gel electrophoresis ligated strands could be separated from unconnected molecules. Originally, the method was intended for use in cerebellum tissue to further elucidate possible mechanisms of neurodegenerative diseases. However, due to its inhomogeneous anatomy, DNA ligation efficiency varied strongly between different cerebellar areas, illuminating the established assay to be suitable only for homogenous organs. Thus, for murine liver tissue sufficient intra- and interday repeatability was shown during validation. In further experiments, the established assay was applied to an animal study comprising young and old (24 and 110 weeks) mice which showed that DNA ligation efficiency was affected by neither sex nor age. Finally, the impact of in vitro addition of the trace elements copper, iron, and zinc on DNA ligation in tissue extracts was investigated. While all three metals inhibited DNA ligation, variations in their potency became evident. In conclusion, the established method can be reliably used for investigation of DNA ligation efficiency in homogenous murine tissues.
Topics: Animals; Mice; DNA; Male; Female; Liver; Cerebellum; Mice, Inbred C57BL; DNA Ligases; DNA Repair
PubMed: 38767089
DOI: 10.1002/em.22602 -
Animal Cells and Systems 2023Circular RNA (circRNA) is a non-coding RNA with a covalently closed loop structure and usually more stable than messenger RNA (mRNA). However, coding sequences (CDSs)...
Circular RNA (circRNA) is a non-coding RNA with a covalently closed loop structure and usually more stable than messenger RNA (mRNA). However, coding sequences (CDSs) following an internal ribosome entry site (IRES) in circRNAs can be translated, and this property has been recently utilized to produce proteins as novel therapeutic tools. However, it is difficult to produce large proteins from circRNAs because of the low circularization efficiency of lengthy RNAs. In this study, we report that we successfully synthesized circRNAs with the splint DNA ligation method using RNA ligase 1 and the splint DNAs, which contain complementary sequences to both ends of precursor linear RNAs. This method results in more efficient circularization than the conventional enzymatic method that does not use the splint DNAs, easily generating circRNAs that express relatively large proteins, including IgG heavy and light chains. Longer splint DNA (42 nucleotide) is more effective in circularization. Also, the use of splint DNAs with an adenine analog, 2,6-diaminopurine (DAP), increase the circularization efficiency presumably by strengthening the interaction between the splint DNAs and the precursor RNAs. The splint DNA ligation method requires 5 times more splint DNA than the precursor RNA to efficiently produce circRNAs, but our modified splint DNA ligation method can produce circRNAs using the amount of splint DNA which is equal to that of the precursor RNA. Our modified splint DNA ligation method will help develop novel therapeutic tools using circRNAs, to treat various diseases and to develop human and veterinary vaccines.
PubMed: 37808549
DOI: 10.1080/19768354.2023.2265165 -
Methods in Molecular Biology (Clifton,... 2024This protocol outlines the construction of a plant transformation plasmid to express both the Cas9 nuclease and individual guide RNA (gRNA), facilitating the induction...
This protocol outlines the construction of a plant transformation plasmid to express both the Cas9 nuclease and individual guide RNA (gRNA), facilitating the induction of double-stranded breaks (DSBs) in DNA and subsequent imprecise repair via the non-homologous end-joining (NHEJ) pathway. The gRNA expression cassettes are assembled from three components. First, the Medicago truncatula U6.6 (MtU6) promoter (352 bp) and scaffold (83 bp) sequences are amplified from a pUC-based plasmid. Additionally, a third fragment, corresponding to the target sequence, is synthesized as an oligonucleotide. The three gRNA expression fragments are then loosely assembled in a ligation-free cloning reaction and used as a template for an additional PCR step to amplify a single gRNA expression construct, ready for assembly into the transformation vector. The benefits of this design include cost efficiency, as subsequent cloning reactions only require 59 oligonucleotides and standard cloning reagents. Researchers engaged in CRISPR/Cas9-mediated genome editing in plants will find this protocol a clear and resource-efficient approach to create transformation plasmids for their experiments.
Topics: CRISPR-Cas Systems; Genetic Vectors; RNA, Guide, CRISPR-Cas Systems; Gene Knockout Techniques; Plasmids; Medicago truncatula; Gene Editing; Plants, Genetically Modified; Cloning, Molecular; Promoter Regions, Genetic; DNA End-Joining Repair; Transformation, Genetic
PubMed: 38656522
DOI: 10.1007/978-1-0716-3782-1_18 -
Theranostics 2023Increasing evidence suggests that hemodynamic disturbed flow induces endothelial dysfunction via a complex biological process so-called endothelial to mesenchymal...
Increasing evidence suggests that hemodynamic disturbed flow induces endothelial dysfunction via a complex biological process so-called endothelial to mesenchymal transition (EndoMT). Recently, DNA methyltransferases (DNMTs) was reported as a key molecular mediator to promote EndoMT. Our understanding of how DNMTs, particularly the maintenance DNMTs, DNMT1, coordinate EndoMT is still lacking. A parallel-plate flow apparatus and perfusion devices were used to apply fluid with endothelial protective pulsatile shear (PS, to mimic the laminar flow) or harmful oscillatory shear (OS, to mimic the disturbed flow) to cultured endothelial cells (ECs). Endothelial lineage tracing mice and conditional EC Dnmt1 knockout mice were subjected to a surgery of carotid partial ligation to generate the flow-accelerated atherogenesis models. Western blotting, quantitative RT-PCR, immunofluorescent staining, methylation-specific PCR, chromatin immunoprecipitation, endothelial functional assays, and assessments for neointimal formation and atherosclerosis were performed. Inhibition of DNMTs with 5-aza-2'-deoxycytidine (5-Aza) suppressed the disturbed flow/OS-induced EndoMT, both in cultured cells and the endothelial lineage tracing mice. 5-Aza also ameliorated the downregulation of aldehyde dehydrogenases (ALDHs) and β-alanine biosynthesis caused by disturbed flow/OS. Knockdown of the ALDH family proteins, ALDH2, ALDH3A1, and ALDH6A1, showed an EndoMT-induction effect as OS. Supplementation of cells with the functional metabolites of β-alanine, carnosine and acetyl-CoA (acetate), reversed EndoMT, likely via inhibiting the phosphorylation of Smad2/3. Endothelial-specific knockout of Dnmt1 protected the vasculature from disturbed flow-induced remodeling and atherosclerosis. Endothelial DNMT1 acts as one of the key epigenetic factors to mediate the hemodynamically regulated EndoMT at least through repressing the expression of ALDH2, ALDH3A1, and ALDH6A1. Supplementation with carnosine and acetate may have a great potential in the prevention and treatment of atherosclerosis.
Topics: Animals; Mice; Aldehyde Dehydrogenase; Aldehyde Dehydrogenase, Mitochondrial; Atherosclerosis; Azacitidine; Carnosine; DNA Modification Methylases; Endothelial Cells; Homeostasis; DNA (Cytosine-5-)-Methyltransferase 1
PubMed: 37649604
DOI: 10.7150/thno.84427 -
Nature Communications Mar 2024Fine-mapping and functional studies implicate rs117701653, a non-coding single nucleotide polymorphism in the CD28/CTLA4/ICOS locus, as a risk variant for rheumatoid...
Fine-mapping and functional studies implicate rs117701653, a non-coding single nucleotide polymorphism in the CD28/CTLA4/ICOS locus, as a risk variant for rheumatoid arthritis and type 1 diabetes. Here, using DNA pulldown, mass spectrometry, genome editing and eQTL analysis, we establish that the disease-associated risk allele is functional, reducing affinity for the inhibitory chromosomal regulator SMCHD1 to enhance expression of inducible T-cell costimulator (ICOS) in memory CD4 T cells from healthy donors. Higher ICOS expression is paralleled by an increase in circulating T peripheral helper (Tph) cells and, in rheumatoid arthritis patients, of blood and joint fluid Tph cells as well as circulating plasmablasts. Correspondingly, ICOS ligation and carriage of the rs117701653 risk allele accelerate T cell differentiation into CXCR5PD-1 Tph cells producing IL-21 and CXCL13. Thus, mechanistic dissection of a functional non-coding variant in human autoimmunity discloses a previously undefined pathway through which ICOS regulates Tph development and abundance.
Topics: Humans; T-Lymphocytes; Arthritis, Rheumatoid; Inducible T-Cell Co-Stimulator Protein; CD28 Antigens; Alleles; T-Lymphocytes, Helper-Inducer; Chromosomal Proteins, Non-Histone
PubMed: 38459032
DOI: 10.1038/s41467-024-46457-8 -
European Journal of Medicinal Chemistry Feb 2024Different metabolic pathways like DNA replication, transcription, and recombination generate topological constrains in the genome. These topological constraints are... (Review)
Review
Different metabolic pathways like DNA replication, transcription, and recombination generate topological constrains in the genome. These topological constraints are resolved by essential molecular machines known as topoisomerases. To bring changes in DNA topology, the topoisomerases create a single or double-stranded nick in the template DNA, hold the nicked ends to let the tangled DNA pass through, and finally re-ligate the breaks. The DNA nicking and re-ligation activities as well as ATPase activities (when present) in topoisomerases are subjected to inhibition by several anticancer and antibacterial drugs, thus establishing these enzymes as successful targets in anticancer and antibacterial therapies. The anti-topoisomerase drugs interfere with the functioning of these enzymes and result in the accumulation of DNA tangles or lethal genomic breaks, thereby promoting host cell (or organism) death. The potential of topoisomerases in the human malarial parasite, Plasmodium falciparum in antimalarial drug development has received little attention so far. Interestingly, the parasite genome encodes orthologs of topoisomerases found in eukaryotes, prokaryotes, and archaea, thus, providing an enormous opportunity for investigating these enzymes for antimalarial therapeutics. This review focuses on the features of Plasmodium falciparum topoisomerases (PfTopos) with respect to their closer counterparts in other organisms. We will discuss overall advances and basic challenges with topoisomerase research in Plasmodium falciparum and our attempts to understand the interaction of PfTopos with classical and new-generation topoisomerase inhibitors using in silico molecular docking approach. The recent episodes of parasite resistance against artemisinin, the only effective antimalarial drug at present, further highlight the significance of investigating new drug targets including topoisomerases in antimalarial therapeutics.
Topics: Humans; Antimalarials; Plasmodium falciparum; Molecular Docking Simulation; Isomerases; DNA; Anti-Bacterial Agents
PubMed: 38171145
DOI: 10.1016/j.ejmech.2023.116056 -
Methods in Molecular Biology (Clifton,... 2024Over the last years, single-molecule force spectroscopy provided insights into the intricate connection between mechanical stimuli and biochemical signaling. The...
Over the last years, single-molecule force spectroscopy provided insights into the intricate connection between mechanical stimuli and biochemical signaling. The underlying molecular mechanisms were uncovered and explored using techniques such as atomic force microscopy and force spectroscopy using optical or magnetic tweezers. These experimental approaches are limited by thermal noise resulting from a physical connection of the studied biological system to the macroscopic world. To overcome this limitation, we recently introduced the DNA origami force clamp (FC) which is a freely diffusing nanodevice that generates piconewton forces on a DNA sequence of interest. Binding of a protein to the DNA under tension can be detected employing fluorescence resonance energy transfer (FRET) as a sensitive readout.This protocol introduces the reader to the working principles of the FC and provides instructions to design and generate a DNA origami FC customized for a protein of interest. Molecular cloning techniques are employed to modify, produce, and purify a custom DNA scaffold. A fluorescently labeled DNA suitable to detect protein binding via FRET is generated via enzymatic ligation of commercial DNA oligonucleotides. After thermal annealing of all components, the DNA origami FC is purified using agarose gel electrophoresis. The final section covers the interrogation of the FC using confocal single-molecule FRET measurements and subsequent data analysis to quantify the binding of a DNA-binding protein to its cognate recognition site under a range of forces. Using this approach, force-dependent DNA-protein interactions can be studied on the single-molecule level on thousands of molecules in a parallelized fashion.
Topics: DNA; Nanotechnology; Mechanical Phenomena; Microscopy, Atomic Force; Spectrum Analysis; Nucleic Acid Conformation; Nanostructures
PubMed: 37824019
DOI: 10.1007/978-1-0716-3377-9_23 -
Analytical Chemistry Apr 2024Oxidative DNA damage is closely associated with the occurrence of numerous human diseases and cancers. 8-Oxo-7,8-dihydroguanine (8-oxoG) is the most prevalent form of...
Oxidative DNA damage is closely associated with the occurrence of numerous human diseases and cancers. 8-Oxo-7,8-dihydroguanine (8-oxoG) is the most prevalent form of DNA damage, and it has become not only an oxidative stress biomarker but also a new epigenetic-like biomarker. However, few approaches are available for the locus-specific detection of 8-oxoG because of the low abundance of 8-oxoG damage in DNA and the limited sensitivity of existing assays. Herein, we demonstrate the elongation and ligation-mediated differential coding for label-free and locus-specific analysis of 8-oxoG in DNA. This assay is very simple without the involvement of any specific labeled probes, complicated steps, and large sample consumption. The utilization of Bsu DNA polymerase can specifically initiate a single-base extension reaction to incorporate dATP into the opposite position of 8-oxoG, endowing this assay with excellent selectivity. The introduction of cascade amplification reaction significantly enhances the sensitivity. The proposed method can monitor 8-oxoG with a limit of detection of 8.21 × 10 M (0.82 aM), and it can identify as low as 0.001% 8-oxoG damage from a complex mixture with excessive undamaged DNAs. This method can be further applied to measure 8-oxoG levels in the genomic DNA of human cells under diverse oxidative stress, holding prospect potential in the dynamic monitoring of critical 8-oxoG sites, early clinical diagnosis, and gene damage-related biomedical research.
Topics: Humans; DNA; DNA-Directed DNA Polymerase; Guanine; DNA Damage; Biomarkers; DNA Repair
PubMed: 38501982
DOI: 10.1021/acs.analchem.4c00387 -
Advanced Science (Weinheim,... Dec 2023Dynamically evolving adhesions between cells and extracellular matrix (ECM) transmit time-varying signals that control cytoskeletal dynamics and cell fate. Dynamic cell...
Dynamically evolving adhesions between cells and extracellular matrix (ECM) transmit time-varying signals that control cytoskeletal dynamics and cell fate. Dynamic cell adhesion and ECM stiffness regulate cellular mechanosensing cooperatively, but it has not previously been possible to characterize their individual effects because of challenges with controlling these factors independently. Therefore, a DNA-driven molecular system is developed wherein the integrin-binding ligand RGD can be reversibly presented and removed to achieve cyclic cell attachment/detachment on substrates of defined stiffness. Using this culture system, it is discovered that cyclic adhesion accelerates F-actin kinetics and nuclear mechanosensing in human mesenchymal stem cells (hMSCs), with the result that hysteresis can completely change how hMSCs transduce ECM stiffness. Results are dramatically different from well-known results for mechanotransduction on static substrates, but are consistent with a mathematical model of F-actin fragments retaining structure following loss of integrin ligation and participating in subsequent repolymerization. These findings suggest that cyclic integrin-mediated adhesion alters the mechanosensing of ECM stiffness by hMSCs through transient, hysteretic memory that is stored in F-actin.
Topics: Humans; Cell Adhesion; Integrins; Actins; Mechanotransduction, Cellular; Extracellular Matrix
PubMed: 37849221
DOI: 10.1002/advs.202302421 -
Science Advances Sep 20235-Methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) are the most abundant DNA modifications that have important roles in gene regulation. Detailed studies of these...
5-Methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) are the most abundant DNA modifications that have important roles in gene regulation. Detailed studies of these different epigenetic marks aimed at understanding their combined effects and dynamic interconversion are, however, hampered by the inability of current methods to simultaneously measure both modifications, particularly in samples with limited quantities. We present DNA analysis by restriction enzyme for simultaneous detection of multiple epigenomic states (DARESOME), an assay based on modification-sensitive restriction digest and sequential tag ligation that can concurrently perform quantitative profiling of unmodified cytosine, 5mC, and 5hmC in CCGG sites genome-wide. DARESOME reveals the opposing roles of 5mC and 5hmC in gene expression regulation as well as their interconversion during aging in mouse brain. Implementation of DARESOME in single cells demonstrates pronounced 5hmC strand bias that reflects the semiconservative replication of DNA. Last, we showed that DARESOME enables integrative genomic, 5mC, and 5hmC profiling of cell-free DNA that uncovered multiomics cancer signatures in liquid biopsy.
Topics: Animals; Mice; Cell-Free Nucleic Acids; Epigenomics; Liquid Biopsy; Genomics; Aging
PubMed: 37713482
DOI: 10.1126/sciadv.adi0197