G-Quadruplex Visualization in Cells via Antibody and Fluorescence Probe
G-quadruplexes (G4s) are noncanonical nucleic acids structures involved in key regulatory and pathological roles in eukaryotes, prokaryotes, and viruses: the development of specific antibodies and fluorescent probes represent an invaluable tool to underst
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Introduction G-quadruplexes (G4s) are unique, noncanonical nucleic acids structures adopted by guanine-rich sequences. The building block of these structures is the so-called guanine quartet (G-quartet): two or more G-quartets, stacking on each other, form the G-quadruplex. From a structural point of view, G4s are characterized by a high polymorphism: their topology can be classified as parallel, antiparallel, or hybrid basing on strands orientation and the multiple orientations adopted by the nucleotide linkers between guanine tracts (loops) contribute to increase G4 diversity. G4s are involved in key regulatory and pathological roles in eukaryotes [1–5], prokaryotes, and viruses [6–10]: given their biological significance, many efforts have been devoted to the development of specific and selective G4 stabilizing molecules [11–14], as well as of probes able to modify their fluorescence behavior upon G4 binding [15–17]. Both antibodies and fluorescence probes that specifically recognize G4 structures represent invaluable tools to visualize G4s in cells and to understand their biological relevance. Recently, two antibodies recognizing G4s
Danzhou Yang and Clement Lin (eds.), G-Quadruplex Nucleic Acids: Methods and Protocols, Methods in Molecular Biology, vol. 2035, https://doi.org/10.1007/978-1-4939-9666-7_24, © Springer Science+Business Media, LLC, part of Springer Nature 2019
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Matteo Nadai and Sara N. Richter
have been developed: BG4 [18] and 1H6 [19]. BG4 is a singlechain fragment variable antibody generated by phage display employing a library of different single-chain antibody clones and selecting the best G4 binder, while 1H6 is a monoclonal antibody produced immunizing mice with stable G4 DNA structures. Both antibodies were used to detect G4s in cells [20–22], in our studies the monoclonal antibody 1H6 was used. Many G4-specific fluorescent probes have been developed in the last years [23–25], but only a few of them can be used in both fixed and live cells, because of their cellular and subcellular permeability. The core-extended NDI (c-exNDI) is a potent G4 binder with an antiviral and anticancer activity [9]. Given its light-up properties upon G4 binding and its very fast cellular and nuclear entry, c-exNDI was used to visualize G4, in combination with the 1H6 anti-G4 antibody [23], both in uninfected and in HSV-1-infected cells.
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Materials All solutions and materials used for cell culturing must be sterile.
2.1 Cell Culture and Virus
1. Cell line of interest (must be chosen, depending on the experiment purpose: in our case HEK293T and Vero). 2. Cell culture medium, DMEM–Dulbecco’s Modified Eagle Medium: NaCl 110.34 mM, NaHCO3 44.05 mM, D-Glucose 25.00 mM, KCl 5.33 mM, L-Glutamine 3.97 mM, Fe(NO3)3 2.47 mM, CaCl2 1.80 mM, NaH2PO4 0.92 mM, MgSO4 0.81 mM, L-Valine 0.80 mM, L-Isoleucine 0.80 mM, L-Leucine 0.80 mM, L-Lysine 0.80 mM, L-Threonine 0.80 mM, LPhenylalanine 0.40 mM, L-Serine 0.40 mM, Glycine 0.40 mM, L-Tyrosine 0.40 mM, L-Arginine 0.40 mM, L-Cystine 0.20 mM, L-Methionine 0.20 mM
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