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Applied Microbiology and Biotechnology Feb 2021It is of great significance to establish sensitive and accurate pathogen detection methods, considering the continuous emergence or re-emergence of infectious diseases... (Review)
Review
It is of great significance to establish sensitive and accurate pathogen detection methods, considering the continuous emergence or re-emergence of infectious diseases seriously influences the safety of human and animals. Proximity ligation assay (PLA) is developed for the sensitive protein detection and also can be used for the detection of pathogens. PLA employs aptamer or monoclonal/polyclonal antibody-nucleic acid complexes as proximity probes. When the paired proximity probes bind to the same target protein or protein complex, they will be adjacent to each other and form an amplifiable DNA sequence through ligation. Combining the specificity of enzyme-linked immunosorbent assay (ELISA) and sensitivity of polymerase chain reaction (PCR), PLA transforms the detection of protein into the detection of DNA nucleic acid sequence. Therefore, as an ultrasensitive protein assay, PLA has great potential for quantification, localization of protein, and clinical diagnostics. In this review, we summarize the basic principles of PLA and its applications in pathogen detection. KEY POINTS: • Different forms of proximity ligation assay are introduced. • Applications of proximity ligation assay in pathogen detection are summarized. • Proximity ligation assay is an ultrasensitive method to quantify protein and pathogen.
Topics: Animals; Biological Assay; DNA; Enzyme-Linked Immunosorbent Assay; Humans; Oligonucleotides; Polymerase Chain Reaction
PubMed: 33427935
DOI: 10.1007/s00253-020-11049-1 -
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 -
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 -
Emerging Topics in Life Sciences Dec 2022Nucleic acids (NAs) in modern biology accomplish a variety of tasks, and the emergence of primitive nucleic acids is broadly recognized as a crucial step for the... (Review)
Review
Nucleic acids (NAs) in modern biology accomplish a variety of tasks, and the emergence of primitive nucleic acids is broadly recognized as a crucial step for the emergence of life. While modern NAs have been optimized by evolution to accomplish various biological functions, such as catalysis or transmission of genetic information, primitive NAs could have emerged and been selected based on more rudimental chemical-physical properties, such as their propensity to self-assemble into supramolecular structures. One such supramolecular structure available to primitive NAs are liquid crystal (LC) phases, which are the outcome of the collective behavior of short DNA or RNA oligomers or monomers that self-assemble into linear aggregates by combinations of pairing and stacking. Formation of NA LCs could have provided many essential advantages for a primitive evolving system, including the selection of potential genetic polymers based on structure, protection by compartmentalization, elongation, and recombination by enhanced abiotic ligation. Here, we review recent studies on NA LC assembly, structure, and functions with potential prebiotic relevance. Finally, we discuss environmental or geological conditions on early Earth that could have promoted (or inhibited) primitive NA LC formation and highlight future investigation axes essential to further understanding of how LCs could have contributed to the emergence of life.
Topics: Liquid Crystals; RNA; Nucleic Acids; DNA; Polymers
PubMed: 36373852
DOI: 10.1042/ETLS20220081 -
Bioconjugate Chemistry Mar 2020We report a chemical DNA-DNA ligation method based on copper-catalyzed azide-alkyne cycloaddition (CuAAC). We demonstrate that ion addition dramatically influences the...
We report a chemical DNA-DNA ligation method based on copper-catalyzed azide-alkyne cycloaddition (CuAAC). We demonstrate that ion addition dramatically influences the efficiency of the so-called click reaction. Even without any further additions, such as typically splint oligonucleotides for preorganization, the "click ligation" yields up to ∼83% product without any byproducts. Additionally, purification of the desired product is straightforward. In comparison to enzymatic ligation methods used to introduce adapters into, e.g., mRNA library preparation, this targeted chemical ligation method exhibits several advantages: increased ligated product and no adapter or cDNA oligomers byproducts. The advantages of the click ligation method were demonstrated by incorporation of azide modified nucleotides by several enzymes as well as broad polymerase acceptance of the obtained triazole linkage in PCR.
Topics: Alkynes; Azides; Catalysis; Click Chemistry; Copper; Cycloaddition Reaction; DNA; Models, Molecular; Nucleic Acid Conformation
PubMed: 31874033
DOI: 10.1021/acs.bioconjchem.9b00805 -
Genes Oct 2019DNA topoisomerase II (TOP2) plays a critical role in many processes such as replication and transcription, where it resolves DNA structures and relieves torsional... (Review)
Review
DNA topoisomerase II (TOP2) plays a critical role in many processes such as replication and transcription, where it resolves DNA structures and relieves torsional stress. Recent evidence demonstrated the association of TOP2 with topologically associated domains (TAD) boundaries and CCCTC-binding factor (CTCF) binding sites. At these sites, TOP2 promotes interactions between enhancers and gene promoters, and relieves torsional stress that accumulates at these physical barriers. Interestingly, in executing its enzymatic function, TOP2 contributes to DNA fragility through re-ligation failure, which results in persistent DNA breaks when unrepaired or illegitimately repaired. Here, we discuss the biological processes for which TOP2 is required and the steps at which it can introduce DNA breaks. We describe the repair processes that follow removal of TOP2 adducts and the resultant broken DNA ends, and present how these processes can contribute to disease-associated mutations. Furthermore, we examine the involvement of TOP2-induced breaks in the formation of oncogenic translocations of leukemia and papillary thyroid cancer, as well as the role of TOP2 and proteins which repair TOP2 adducts in other diseases. The participation of TOP2 in generating persistent DNA breaks and leading to diseases such as cancer, could have an impact on disease treatment and prevention.
Topics: CCCTC-Binding Factor; Chromatin; DNA; DNA Breaks, Double-Stranded; DNA Repair; DNA Topoisomerases, Type II; Humans; Leukemia, Myeloid, Acute; Thyroid Cancer, Papillary; Topoisomerase II Inhibitors; Torsion, Mechanical
PubMed: 31614754
DOI: 10.3390/genes10100791 -
Methods in Molecular Biology (Clifton,... 2021The in vivo footprinting method identifies protein-targeted DNA regions under different conditions such as carbon sources. Dimethyl sulfate (DMS) generates methylated...
The in vivo footprinting method identifies protein-targeted DNA regions under different conditions such as carbon sources. Dimethyl sulfate (DMS) generates methylated purine bases at DNA sites which are not bound by proteins or transcription factors. The DNA is cleaved by HCl, and the resulting DNA fragments are 5'-end [6-FAM]-labeled by a linker-mediated PCR (LM-PCR). Fluorescent fragments are separated and analyzed on a capillary sequencer, followed by automated data analysis using the software tool ivFAST.
Topics: Base Sequence; DNA Footprinting; DNA, Fungal; Electrophoresis, Capillary; Hypocreales; Methylation; Polymerase Chain Reaction; Promoter Regions, Genetic
PubMed: 33165789
DOI: 10.1007/978-1-0716-1048-0_15 -
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 -
Sheng Wu Gong Cheng Xue Bao = Chinese... May 2024To develop an accurate and efficient protocol for multi-fragment assembly and multi-site mutagenesis, we integrated and optimized the common multi-fragment assembly...
To develop an accurate and efficient protocol for multi-fragment assembly and multi-site mutagenesis, we integrated and optimized the common multi-fragment assembly methods and validated the established method by using fructose-1,6-diphosphatase 1 (FBP1) with 4 mutant sites. The fragments containing mutations were assembled by introducing mutant sites and I recognition sequences. After digestion/ligation, the ligated fragment was amplified with the primers containing overlap region to the linearized vector. The amplified fragment was ligated to the linearized vector and the ligation product was transformed into . After screening and sequencing, the recombinant plasmid with 4 mutant sites was obtained. This protocol overcame the major defects of Gibson assembly and Golden Gate assembly, serving as an efficient solution for multi-fragment assembly and multi-site mutagenesis.
Topics: Escherichia coli; Fructose-Bisphosphatase; Homologous Recombination; Plasmids; Genetic Vectors; DNA; Mutation; Mutagenesis, Site-Directed; Cloning, Molecular
PubMed: 38783816
DOI: 10.13345/j.cjb.230793 -
Biochemical Society Transactions Dec 2019Non-homologous end joining (NHEJ) is a major repair pathway for DNA double-strand breaks (DSBs), which is the most toxic DNA damage in cells. Unrepaired DSBs can cause... (Review)
Review
Non-homologous end joining (NHEJ) is a major repair pathway for DNA double-strand breaks (DSBs), which is the most toxic DNA damage in cells. Unrepaired DSBs can cause genome instability, tumorigenesis or cell death. DNA end synapsis is the first and probably the most important step of the NHEJ pathway, aiming to bring two broken DNA ends close together and provide structural stability for end processing and ligation. This process is mediated through a group of NHEJ proteins forming higher-order complexes, to recognise and bridge two DNA ends. Spatial and temporal understanding of the structural mechanism of DNA-end synapsis has been largely advanced through recent structural and single-molecule studies of NHEJ proteins. This review focuses on core NHEJ proteins that mediate DNA end synapsis through their unique structures and interaction properties, as well as how they play roles as anchor and linker proteins during the process of 'bridge over troubled ends'.
Topics: Chromosome Pairing; DNA; DNA Breaks, Double-Stranded; DNA End-Joining Repair; DNA-Binding Proteins; Humans; Nucleic Acid Conformation
PubMed: 31829407
DOI: 10.1042/BST20180518