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The Plant Journal : For Cell and... Jan 2022Agrobacterium tumefaciens-mediated transformation has been for decades the preferred tool to generate transgenic plants. During this process, a T-DNA carrying transgenes...
Agrobacterium tumefaciens-mediated transformation has been for decades the preferred tool to generate transgenic plants. During this process, a T-DNA carrying transgenes is transferred from the bacterium to plant cells, where it randomly integrates into the genome via polymerase theta (Polθ)-mediated end joining (TMEJ). Targeting of the T-DNA to a specific genomic locus via homologous recombination (HR) is also possible, but such gene targeting (GT) events occur at low frequency and are almost invariably accompanied by random integration events. An additional complexity is that the product of recombination between T-DNA and target locus may not only map to the target locus (true GT), but also to random positions in the genome (ectopic GT). In this study, we have investigated how TMEJ functionality affects the biology of GT in plants, by using Arabidopsis thaliana mutated for the TEBICHI gene, which encodes for Polθ. Whereas in TMEJ-proficient plants we predominantly found GT events accompanied by random T-DNA integrations, GT events obtained in the teb mutant background lacked additional T-DNA copies, corroborating the essential role of Polθ in T-DNA integration. Polθ deficiency also prevented ectopic GT events, suggesting that the sequence of events leading up to this outcome requires TMEJ. Our findings provide insights that can be used for the development of strategies to obtain high-quality GT events in crop plants.
Topics: Agrobacterium tumefaciens; Arabidopsis; DNA, Bacterial; DNA, Plant; DNA-Directed DNA Polymerase; Gene Targeting; Homologous Recombination; Plants, Genetically Modified; Transgenes
PubMed: 34713516
DOI: 10.1111/tpj.15557 -
ACS Nano Jan 2023An orthogonal, noncovalent approach to direct the assembly of higher-order DNA origami nanostructures is described. By incorporating perfluorinated tags into the edges...
An orthogonal, noncovalent approach to direct the assembly of higher-order DNA origami nanostructures is described. By incorporating perfluorinated tags into the edges of DNA origami tiles we control their hierarchical assembly via fluorous-directed recognition. When we combine this approach with Watson-Crick base-pairing we form discrete dimeric constructs in significantly higher yield (8x) than when either molecular recognition method is used in isolation. This integrated "catch-and-latch" approach, which combines the strength and mobility of the fluorous effect with the specificity of base-pairing, provides an additional toolset for DNA nanotechnology, one that enables increased assembly efficiency while requiring significantly fewer DNA sequences. As a result, our integration of fluorous-directed assembly into origami systems represents a cheap, atom-efficient means to produce discrete superstructures.
Topics: Nucleic Acid Conformation; Nanostructures; DNA; Nanotechnology; Base Pairing
PubMed: 36537902
DOI: 10.1021/acsnano.2c10727 -
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 -
Wiley Interdisciplinary Reviews. RNA Mar 2023Transcription factors (TFs) are present in all life forms and conserved across great evolutionary distances in eukaryotes. From yeast to complex multicellular organisms,... (Review)
Review
Transcription factors (TFs) are present in all life forms and conserved across great evolutionary distances in eukaryotes. From yeast to complex multicellular organisms, they are pivotal players of cell fate decision by orchestrating gene expression at diverse molecular layers. Notably, TFs fine-tune gene expression by coordinating RNA fate at both the expression and splicing levels. They regulate alternative splicing, an essential mechanism for cell plasticity, allowing the production of many mRNA and protein isoforms in precise cell and tissue contexts. Despite this apparent role in splicing, how TFs integrate transcription and splicing to ultimately orchestrate diverse cell functions and cell fate decisions remains puzzling. We depict substantial studies in various model organisms underlining the key role of TFs in alternative splicing for promoting tissue-specific functions and cell fate. Furthermore, we emphasize recent advances describing the molecular link between the transcriptional and splicing activities of TFs. As TFs can bind both DNA and/or RNA to regulate transcription and splicing, we further discuss their flexibility and compatibility for DNA and RNA substrates. Finally, we propose several models integrating transcription and splicing activities of TFs in the coordination and diversification of cell and tissue identities. This article is categorized under: RNA Processing > Splicing Regulation/Alternative Splicing RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications RNA Processing > Splicing Mechanisms.
Topics: Cell Differentiation; RNA Splicing; Transcription, Genetic; Transcription Factors; Cell Lineage; Spatio-Temporal Analysis; DNA Polymerase II; DNA; RNA; Humans; Animals
PubMed: 35899407
DOI: 10.1002/wrna.1752 -
Topics in Current Chemistry (Cham) Feb 2020Nucleic acids are considered not only extraordinary carriers of genetic information but also are perceived as the perfect elemental materials of molecular recognition... (Review)
Review
Nucleic acids are considered not only extraordinary carriers of genetic information but also are perceived as the perfect elemental materials of molecular recognition and signal transduction/amplification for assembling programmable artificial reaction networks or circuits, which are similar to conventional electronic logic devices. Among these sophisticated DNA-based reaction networks, catalytic hairpin assembly (CHA), hybridization chain reaction (HCR), and DNAzyme represent the typical nonenzymatic amplification methods with high robustness and efficiency. Furthermore, their extensive hierarchically cascade integration into multi-layered autonomous DNA circuits establishes novel paradigms for constructing more different catalytic DNA nanostructures and for regenerating or replicating diverse molecular components with specific functions. Various DNA and inorganic nanoscaffolds have been used to realize the surface-confined DNA reaction networks with significant biomolecular sensing and signal-regulating functions in living cells. Especially, the specific aptamers and metal-ion-bridged duplex DNA nanostructures could extend their paradigms for detecting small molecules and proteins in even living entities. Herein, the varied enzyme-free DNA circuits are introduced in general with an extensive explanation of their underlying molecular reaction mechanisms. Challenges and outlook of the autonomous enzyme-free DNA circuits will also be discussed at the end of this chapter.
Topics: Biosensing Techniques; DNA; DNA, Catalytic; Entropy; Fluorescence Resonance Energy Transfer; Humans; Microscopy, Fluorescence; Nanostructures; Nucleic Acid Hybridization
PubMed: 32016608
DOI: 10.1007/s41061-020-0284-x -
Journal of Translational Medicine Nov 2022Clinical CAR T-cell therapy using integrating vector systems represents a promising approach for the treatment of hematological malignancies. Lentiviral and...
BACKGROUND
Clinical CAR T-cell therapy using integrating vector systems represents a promising approach for the treatment of hematological malignancies. Lentiviral and γ-retroviral vectors are the most commonly used vectors in the manufacturing process. However, the integration pattern of these viral vectors and subsequent effect on CAR T-cell products is still unclear.
METHODS
We used a modified viral integration sites analysis (VISA) pipeline to evaluate viral integration events around the whole genome in pre-infusion CAR T-cell products. We compared the differences of integration pattern between lentiviral and γ-retroviral products. We also explored whether the integration sites correlated with clinical outcomes.
RESULTS
We found that γ-retroviral vectors were more likely to insert than lentiviral vectors into promoter, untranslated, and exon regions, while lentiviral vector integration sites were more likely to occur in intron and intergenic regions. Some integration events affected gene expression at the transcriptional and post-transcriptional level. Moreover, γ-retroviral vectors showed a stronger impact on the host transcriptome. Analysis of individuals with different clinical outcomes revealed genes with differential enrichment of integration events. These genes may affect biological functions by interrupting amino acid sequences and generating abnormal proteins, instead of by affecting mRNA expression. These results suggest that vector integration is associated with CAR T-cell efficacy and clinical responses.
CONCLUSION
We found differences in integration patterns, insertion hotspots and effects on gene expression vary between lentiviral and γ-retroviral vectors used in CAR T-cell products and established a foundation upon which we can conduct further analyses.
Topics: Humans; Lentivirus; Retroviridae; Genetic Vectors; Virus Integration; T-Lymphocytes; DNA
PubMed: 36348415
DOI: 10.1186/s12967-022-03729-5 -
G3 (Bethesda, Md.) Sep 2023In maize, the community-standard transformant line B104 is a useful model for dissecting features of transfer DNA (T-DNA) integration due to its compatibility with...
In maize, the community-standard transformant line B104 is a useful model for dissecting features of transfer DNA (T-DNA) integration due to its compatibility with Agrobacterium-mediated transformation and the availability of its genome sequence. Knowledge of transgene integration sites permits the analysis of the genomic environment that governs the strength of gene expression and phenotypic effects due to the disruption of an endogenous gene or regulatory element. In this study, we optimized a fusion primer and nested integrated PCR (FPNI-PCR) technique for T-DNA detection in maize to characterize the integration sites of 89 T-DNA insertions in 81 transformant lines. T-DNA insertions preferentially occurred in gene-rich regions and regions distant from centromeres. Integration junctions with and without microhomologous sequences as well as junctions with de novo sequences were detected. Sequence analysis of integration junctions indicated that T-DNA was incorporated via the error-prone repair pathways of nonhomologous (predominantly) and microhomology-mediated (minor) end-joining. This report provides a quantitative assessment of Agrobacterium-mediated T-DNA integration in maize with respect to insertion site features, the genomic distribution of T-DNA incorporation, and the mechanisms of integration. It also demonstrates the utility of the FPNI-PCR technique, which can be adapted to any species of interest.
Topics: Agrobacterium; Zea mays; Transformation, Genetic; DNA, Bacterial; DNA, Plant; Plants, Genetically Modified
PubMed: 37523773
DOI: 10.1093/g3journal/jkad166 -
Progress in Biophysics and Molecular... Oct 2019In vertebrates, double-strand breaks in DNA are primarily repaired by Non-Homologous End-Joining (NHEJ). The ring-shaped Ku heterodimer rapidly senses and threads onto... (Review)
Review
In vertebrates, double-strand breaks in DNA are primarily repaired by Non-Homologous End-Joining (NHEJ). The ring-shaped Ku heterodimer rapidly senses and threads onto broken DNA ends forming a recruiting hub. Through protein-protein contacts eventually reinforced by protein-DNA interactions, the Ku-DNA hub attracts a series of specialized proteins with scaffolding and/or enzymatic properties. To shed light on these dynamic interplays, we review here current knowledge on proteins directly interacting with Ku and on the contact points involved, with a particular accent on the different classes of Ku-binding motifs identified in several Ku partners. An integrated structural model of the core NHEJ network at the synapsis step is proposed.
Topics: Amino Acid Motifs; Animals; DNA; DNA End-Joining Repair; Humans; Ku Autoantigen
PubMed: 30851288
DOI: 10.1016/j.pbiomolbio.2019.03.001 -
Methods in Molecular Biology (Clifton,... 2023DNA nanotechnology provides efficient methods for the sequence-programmable construction of mechanical devices with nanoscale dimensions. The resulting nanomachines...
DNA nanotechnology provides efficient methods for the sequence-programmable construction of mechanical devices with nanoscale dimensions. The resulting nanomachines could serve as tools for the manipulation of macromolecules with similar functionalities as mechanical tools and machinery in the macroscopic world. In order to drive and control these machines and to perform specific tasks, a fast, reliable, and repeatable actuation mechanism is required that can work against external loads. Here we describe a highly effective method for actuating DNA structures using externally applied electric fields. To this end, electric fields are generated with controllable direction and amplitude inside a miniature electrophoresis device integrated with an epifluorescence microscope. With this setup, DNA-based nanoelectromechanical devices can be precisely controlled. As an example, we demonstrate how a DNA-based nanorobotic system can be used to dynamically position molecules on a molecular platform with high speeds and accuracy. The microscopy setup also described here allows simultaneous monitoring of a large number of nanorobotic arms in real time and at the single nanomachine level.
Topics: Nanostructures; Nanotechnology; DNA
PubMed: 37166722
DOI: 10.1007/978-1-0716-3028-0_15 -
Chemical Communications (Cambridge,... Mar 2022The diverse surface interactions and functions of a bacterium play an important role in cell signaling, host infection, and colony formation. To understand and... (Review)
Review
The diverse surface interactions and functions of a bacterium play an important role in cell signaling, host infection, and colony formation. To understand and synthetically control the biological functions of individual cells as well as the whole community, there is growing attention on the development of chemical and biological tools that can integrate artificial functional motifs onto the bacterial surface to replace the native interactions, enabling a variety of applications in biosynthesis, environmental protection, and human health. Among all these functional motifs, DNA emerges as a powerful tool that can precisely control bacterial interactions at the bio-interface due to its programmability and biorecognition properties. Compared with conventional chemical and genetic approaches, the sequence-specific Watson-Crick interaction enables almost unlimited programmability in DNA nanostructures, realizing one base-pair spatial control and bio-responsive properties. This highlight aims to provide an overview on this emerging research topic of DNA-engineered bacterial interactions from the aspect of synthetic chemists. We start with the introduction of native bacterial surface ligands and established synthetic approaches to install artificial ligands, including direct modification, metabolic engineering, and genetic engineering. A brief overview of DNA nanotechnology, reported DNA-bacteria conjugation chemistries, and several examples of DNA-engineered bacteria are included in this highlight. The future perspectives and challenges in this field are also discussed, including the development of dynamic bacterial surface chemistry, assembly of programmable multicellular community, and realization of bacteria-based theranostic agents and synthetic microbiota as long-term goals.
Topics: Bacteria; DNA, Bacterial; Genetic Engineering; Ligands; Metabolic Engineering
PubMed: 35077527
DOI: 10.1039/d1cc06138k