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Nanoparticle Protein RNAVersatile Click Chemistry-based Approaches to Illuminate DNA and RNA G-Quadruplexes in Human Cells
The existence and functional relevance of DNA and RNA G-quadruplexes (G4s) in human cells is now beyond debate, but how did we reach such a level of confidence? Thanks to a panoply of molecular tools and techniques that are now routinely implemented in wet labs. Among them, G4 imaging ranks high because of its reliability and practical convenience, which now makes cellular G4 detection quick and easy; also, because this technique is sensitive and responsive to any G4 modulations in cells, which thus allows gaining precious insights into G4 biology. Herein, we briefly explain what a G4 is and how they can be visualized in human cells; then, we present the strategy we have been developing for several years now for in situ click G4 imaging, which relies on the use of biomimetic G4 ligands referred to as TASQs (for template-assembled synthetic G-quartets) and is far more straightforward and modular than classically used immunodetection methods. We thus show why and how to illuminate G4s with TASQs and provide a detailed, step-by-step methodology (including the preparation of the materials, the methodology per se, and a series of notes to address any possible pitfalls that may arise during the experiments) to make G4 imaging ever easier to operate.
Attachment of a 32P-phosphate to the 3′ Terminus of a DNA Oligonucleotide
Biochemical investigations into DNA-binding and DNA-cutting proteins often benefit from the specific attachment of a radioactive label to one of the two DNA termini. In many cases, it is essential to perform two versions of the same experiment: one with the 5′ DNA end labeled and one with the 3′ DNA end labeled. While homogeneous 5′-radiolabeling can be accomplished using a single kinase-catalyzed phosphorylation step, existing procedures for 3′-radiolabeling often result in probe heterogeneity, prohibiting precise DNA fragment identification in downstream experiments. We present here a new protocol to efficiently attach a 32P-phosphate to the 3′ end of a DNA oligonucleotide of arbitrary sequence, relying on inexpensive DNA oligonucleotide modifications (2′-O-methylribonucleotide and ribonucleotide sugar substitutions), two enzymes (T4 polynucleotide kinase and T4 RNA ligase 2), and the differential susceptibility of DNA and RNA to hydroxide treatment. Radioactive probe molecules produced by this protocol are homogeneous and oxidant-compatible, and they can be used for precise cleavage-site mapping in the context of both DNase enzyme characterization and DNA footprinting assays.
Graphic abstract
Graphic abstract
Localizing Genome Segments and Protein Products of a Multipartite Virus in Host Plant Cells
A founding paradigm in virology is that the spatial unit of the viral replication cycle is an individual cell. This concept applied to multipartite viruses–which have a genome composed of two or more nucleic acid segments, each individually encapsulated–implies that all segments constituting a viral genome need to coinfect the same host cell for replication to occur. Would this requirement be verified, it would constitute a major cost for extreme cases of multipartition such as the Faba bean necrotic stunt virus (FBNSV, nanovirus) whose genome is composed of eight complementary segments, each encoding a single gene (Grigoras et al., 2009). To address this question, we followed the distribution of the FBNSV genome segments by fluorescence in situ hybridization combined to immunolocalization of the replication-controlling viral protein within the cells of the host plant: Vicia Faba.
A rapid and efficient protocol to localize viral transcripts in plant and insect hosts has been developed earlier (Ghanim et al., 2009). We here improve this method by using random-primed labeled probes and apply it to the detection and quantification of the individual segments composing the FBNSV genome. Moreover, we combine this technique with immunolocalization so that both viral segments and proteins can be visualized within the same samples.
A rapid and efficient protocol to localize viral transcripts in plant and insect hosts has been developed earlier (Ghanim et al., 2009). We here improve this method by using random-primed labeled probes and apply it to the detection and quantification of the individual segments composing the FBNSV genome. Moreover, we combine this technique with immunolocalization so that both viral segments and proteins can be visualized within the same samples.
In situ Hybridization of Plant-parasitic Nematode Globodera pallida Juveniles to Detect Gene Expression
In this study, we describe a standard whole mount in situ hybridization method which is used to determine the spatial-temporal expression pattern of genes from Globodera spp. Unlike more invasive radioactive labeling approaches, this technique is based on a safe, highly specific enzyme-linked immunoassay where a Digoxigenin (DIG)-tagged anti-sense probe hybridized to a target transcript is detected by anti-DIG antibodies conjugated with alkaline phosphatase enzyme (AP) (anti-DIG-AP). The hybrid molecules are visualized through an AP-catalyzed color reaction using as the substrate 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitro blue tetrazolium chloride (NBT). This method can be applied to both free-living pre-parasitic juveniles and early endoparasitic stages of cyst nematodes.
Analysis of DNA Exchange Using Thymidine Analogs (ADExTA) in Trypanosoma cruzi
Trypanosoma cruzi is a protozoan parasite belonging to the Trypanosomatidae family. Although the trypanosomatids multiply predominantly by clonal generation, the presence of DNA exchange in some of them has been puzzling researchers over the years, mainly because it may represent a novel form that these organisms use to gain variability. Analysis of DNA Exchange using Thymidine Analogs (ADExTA) is a method that allows the in vitro detection and measurement of rates of DNA exchange, particularly in trypanosomatid cells, in a rapid and simple manner by indirect immunofluorescence assay (IFA). The method can be used to detect DNA exchange within one trypanosomatid lineage or among different lineages by paired analysis. The principle of this assay is based on the incorporation of two distinguishable halogenated thymidine analogs called 5′-chloro-2′-deoxyuridine (CldU) and 5′-iodo-2′-deoxyuridine (IdU) during DNA replication. After mixing the two cell cultures that had been previously incorporated with CldU and IdU separately, the presence of these unusual deoxynucleosides in the genome can be detected by specific antibodies. For this, a DNA denaturation step is required to expose the sites of thymidine analogs incorporated. Subsequently, a secondary reaction using fluorochrome-labeled antibodies will generate distinct signals under fluorescence analysis. By using this method, DNA exchange verification (i.e., the presence of both CldU and IdU in the same cell) is possible using a standard fluorescence microscope. It typically takes 2-3 days from the thymidine analogs incorporation to results. Of note, ADExTA is relatively cheap and does not require transfections or harsh genetic manipulation. These features represent an advantage when compared to other time-consuming protocols that demand DNA manipulation to introduce distinct drug-resistance markers in different cells for posterior selection.
Direct Visualization of the Multicopy Chromosomes in Cyanobacterium Synechococcus elongatus PCC 7942
Cyanobacteria are prokaryotic organisms that carry out oxygenic photosynthesis. The fresh water cyanobacterium Synechococcus elongatus PCC 7942 is a model organism for the study of photosynthesis and gene regulation, and for biotechnological applications. Besides several freshwater cyanobacteria, S. elongatus 7942 also contains multiple chromosomal copies per cell at all stages of its cell cycle. Here, we describe a method for the direct visualization of multicopy chromosomes in S. elongatus 7942 by fluorescence in situ hybridization (FISH).
TUNEL Assay to Assess Extent of DNA Fragmentation and Programmed Cell Death in Root Cells under Various Stress Conditions
DNA damage is one of the common consequences of exposure to various stress conditions. Different methods have been developed to accurately assess DNA damage and fragmentation in cells and tissues exposed to different stress agents. However, owing to the presence of firm cellulosic cell wall and phenolics, plant cells and tissues are not easily amenable to be subjected to these assays. Here, we describe an optimized TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling) assay-based protocol to determine the extent of DNA fragmentation and programmed cell death in plant root cells subjected to various stress conditions. The method described here has the advantages of simplicity, reliability and reproducibility.
Modification of 3’ Terminal Ends of DNA and RNA Using DNA Polymerase θ Terminal Transferase Activity
DNA polymerase θ (Polθ) is a promiscuous enzyme that is essential for the error-prone DNA double-strand break (DSB) repair pathway called alternative end-joining (alt-EJ). During this form of DSB repair, Polθ performs terminal transferase activity at the 3’ termini of resected DSBs via templated and non-templated nucleotide addition cycles. Since human Polθ is able to modify the 3’ terminal ends of both DNA and RNA with a wide array of large and diverse ribonucleotide and deoxyribonucleotide analogs, its terminal transferase activity is more useful for biotechnology applications than terminal deoxynucleotidyl transferase (TdT). Here, we present in detail simple methods by which purified human Polθ is utilized to modify the 3’ terminal ends of RNA and DNA for various applications in biotechnology and biomedical research.
Single-molecule Analysis of DNA Replication Dynamics in Budding Yeast and Human Cells by DNA Combing
The DNA combing method allows the analysis of DNA replication at the level of individual DNA molecules stretched along silane-coated glass coverslips. Before DNA extraction, ongoing DNA synthesis is labeled with halogenated analogues of thymidine. Replication tracks are visualized by immunofluorescence using specific antibodies. Unlike biochemical and NGS-based methods, DNA combing provides unique information on cell-to-cell variations in DNA replication profiles, including initiation and elongation. Finally, this assay can be used to monitor the effect of DNA lesions on fork progression, arrest and restart.
Detection of the Cell Proliferation Zone in Leaves by Using EdU
Visualization of nuclei in S-phase cells in tissues is important for not only cell cycle research but also developmental research because morphogenesis is usually achieved by a combination of cell proliferation and cell expansion. Recently, DNA labeling with 5-ethynyl-2′-deoxyuridine (EdU), which is an analog of thymidine, has been used to visualize nuclei in S-phase cells to assess the activity of cell proliferation during development of plants. EdU is efficiently incorporated into newly synthesized DNA, and detection of EdU is based on the covalent reaction between EdU and Alexa Fluor® dye, which is one of useful fluorescent dyes; this allows us to use mild conditions for the assay without any DNA denaturation. This method could be easily applicable, and, indeed, has been used for various model and non-model plant species. Here, we have described a protocol developed for the detection of nuclei in S-phase cells in leaves.
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