Journal: Nucleic Acids Research

Article Title: Development and application of G4-Flame as a visual biosensor for G4-DNA

doi: 10.1093/nar/gkag179

Figure Lengend Snippet: G4-Flame specifically binds and responds to G4-DNA structures. ITC analysis of G4-Flame binding to different DNA topologies: ( A ) G4-DNA, ( B ) ssMUT, and ( C ) dsDNA. Upper panels show the raw ITC thermograms obtained by titrating each DNA into purified G4-Flame in G4 buffer, and lower panels show the corresponding integrated binding isotherms plotted as a function of molar ratio. The data were fitted using a one-site binding model to determine the binding enthalpy (Δ H ). A clear binding signal was observed for G4-DNA, whereas ssMUT and dsDNA showed no detectable binding. EMSA analysis of G4-Flame binding specificity using 10 nM biotinylated DNA substrates: ( D ) G4-DNA, ( E ) ssMUT, and ( F ) dsDNA. Protein–DNA complexes were resolved by native PAGE and detected via streptavidin–HRP chemiluminescence. A pronounced mobility shift was observed for G4-DNA upon incubation with G4-Flame, whereas ssMUT and dsDNA showed no detectable complex formation. ( G ) Binding specificity of G4-Flame assessed by fluorescence shift assay. Two micromolar TAMRA-labeled G4 DNA showed interaction with 2 µM G4-Flame after 5 min incubation at RT. No detectable interaction was observed between 2 µM G4-Flame and 10 µM TAMRA-labeled oligo dT16 single-stranded DNA. Fluorescent signals were captured using a gel documentation system. ( H ) Fluorescence response (R485/405 ratio) of G4-Flame at the indicated concentrations of G4-DNA, ssMUT, and dsDNA ( n = 3). The R485/405 ratio decreased progressively with increasing concentrations of G4-DNA, whereas no comparable change was observed for ssMUT or dsDNA. Data information: (H) are mean ± SD, one-way ANOVA with Dunnett’s multiple comparisons test; ns, not significant; * P < .05, ** P < .01, *** P < .001, **** P < .0001.

Article Snippet: After that, the oligonucleotides were diluted to 100 μM with the same G4 buffer for isothermal titration calorimetry (ITC) experiments, which were performed using a MicroCal PEAQ-ITC instrument.

Techniques: Binding Assay, Purification, Clear Native PAGE, Mobility Shift, Incubation, Fluorescence, Shift Assay, Labeling

Journal: Nucleic Acids Research

Article Title: Development and application of G4-Flame as a visual biosensor for G4-DNA

doi: 10.1093/nar/gkag179

Figure Lengend Snippet: G4-Flame exhibits specific binding activity toward multiple G4-DNA topologies. NMR spectroscopy was used to characterize G4-Flame’s interactions with different G4-DNA topologies: ( A ) parallel, ( B ) hybrid, and ( C ) antiparallel structures. Upon incremental addition of G4-Flame, the imino proton signals of all three G4-DNA conformations progressively decreased in intensity. ITC analysis of G4-Flame binding to different G4-DNA topologies: ( D ) hybrid and ( E ) antiparallel structures. Upper panels show the raw ITC thermograms obtained by titrating each G4-DNA into purified G4-Flame in G4 buffer, and lower panels show the corresponding integrated binding isotherms plotted as a function of molar ratio. The data were fitted using a one-site binding model to determine the binding enthalpy (Δ H ). Clear binding signals were observed for both hybrid and antiparallel G4-DNA structures. EMSA analysis of G4-Flame binding specificity using 500 nM TAMRA-labeled DNA substrates: ( F ) hybrid, ( G ) antiparallel, and ( H ) parallel structures. The complexes were resolved by native PAGE and visualized using a fluorescence imaging system. A pronounced mobility shift was observed for all tested G4-DNA topologies upon incubation with G4-Flame. ( I ) Native PAGE analysis of G4-Flame–G4 interactions. Two micromolar G4-DNA (unlabeled) were incubated with 2 or 4 µM G4-Flame at RT for 5 min. The resulting samples were applied to native PAGE. Four micromolar of Flame were loaded as control. Signal from fluorescent of Flame was monitored. Upon incubation with G4-DNA, a mobility shift of G4-Flame was observed. ( J ) Fluorescence response (R485/405 ratio) of G4-Flame at the indicated concentrations of parallel-G4, hybrid-G4, and antiparallel-G4 ( n = 3). The R485/405 ratio decreased progressively with increasing concentrations of G4-DNA, with the largest decrease observed for parallel G4-DNA. Data information: (J) are mean ± SD, two-way ANOVA with Dunnett’s multiple comparisons test; ns, not significant; * P < .05, ** P < .01, *** P < .001, **** P < .0001.

Article Snippet: After that, the oligonucleotides were diluted to 100 μM with the same G4 buffer for isothermal titration calorimetry (ITC) experiments, which were performed using a MicroCal PEAQ-ITC instrument.

Techniques: Binding Assay, Activity Assay, Structural Proteomics, Purification, Labeling, Clear Native PAGE, Fluorescence, Imaging, Mobility Shift, Incubation, Control