Characterization of G-Quadruplexes in Enterovirus A71 Genome and Their Interaction with G-Quadruplex Ligands

Figures

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  FIG 1. Schematic representation of the position of PQSs in the EV-A71 genome. (A) Schematic diagram of G-quadruplex structure. Guanines forming G-tetrad structure with a potassium ion inside through Hoogsteen-type hydrogen bonds. (B) EV-A71 genome diagram showing the sites of putative G-quadruplex sequences.

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  FIG 2. Conservation analysis of representative PQSs in the EV-A71 genome. (A) The selected G-quadruplex sequences at EV-A71 strain FY23 genome, in which the G-tracts are underlined. The four G-quadruplex sequences used in the study are distributed in the VP2, 2A, 2B, and 3C regions. (B-C) WebLogo images of the four G-quadruplex sequences showing that the conservation of particular nucleotides among the Enterovirus family (B) and different EV-A71 strains (C).

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  FIG 3. Characterization of the G-quadruplex sequences. (A) ThT fluorescent assay of the selected G-quadruplex and G-mut sequences analyzing the extent of formation. The emission and excitation were at 495 nm and 425 nm, respectively. ****P < 0.0001 and ns, no significance based on the Student's t test. Error bars represent the SD. (B) Native gel electrophoretic analysis of PQSs and G-mut formation. 0.32 nmol RNA solution was prepared in 10 mM Tris HCl (pH = 7.4) buffer with 100 mM KCl. (C) CD spectral analysis for G-quadruplexes oligonucleotide showing RNA topology generated by GraphPad Prism software version 8.0. The wavelength range was from 200 to 350 nm. RNA oligonucleotides were diluted to 20 μM final concentration in 10 mM Tris HCl (pH = 7.4) buffer with 100 mM KCl.

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  FIG 4. Binding and stabilization of G-quadruplex structure by G4 ligands. (A) The chemical structures of BRACO-19, PDS, and TMPyP4 generated by KingDraw v2.0.2. (B) Native gel electrophoresis of G-quadruplex structures. The annealed 0.4 nmol G-quadruplex RNA solution with BRACO-19 is at ratios of 1:1 M. (C) Native gel electrophoresis of G-quadruplex structures in the presence of G4 ligands. The 30 μM ligands were added to the annealed 0.24 nmol G-quadruplex RNA solution. (D) G-quadruplex RNA oligonucleotides pre-incubated with three compounds, the fluorescence was measured after the addition of ThT and normalized to the initial fluorescence signal. ****P < 0.0001 based on the Student's t test. Error bars represent the SD. (D) Comparison of CD melting profiles at 265 nm wavelength for G-quadruplex structures in the presence (green) of BRACO-19 generated by GraphPad Prism software version 8.0. Tm values of each G-quadruplex in the absence of ligand are represented in the left bottom corner.

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  FIG 5. Analysis of G-quadruplex-ligands interactions by SPR spectroscopy. The SPR analysis of G-quadruplex structure showed their binding affinities toward BRACO-19 (A) and TMPyP4 (B) generated by GraphPad Prism software version 8.0.

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  FIG 6. Effection of G‐quadruplex structure on gene transcription. (A) Schematic diagram showing plasmids construction and primer extension assay. The pEGFP-C1 vector was engineered with G-quadruplex or PQS‐mut sequences sited after the ATG start codon. (B) Primer extension assay showed that the intensity of PCR products was decreased with the increasing concentration of BRACO‐19, PDS, and TMPyP4. (C) Primer extension assay showed that the intensity of PCR products from PQS-mut remained the same in the presence of BRACO‐19, PDS, and TMPyP4.

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  FIG 7. Inhibition of EV-A71 replication by G-quadruplex ligands. (A) RD cells were infected with EV-A71 strain FY23 (MOI = 3) for 1h and then treated with G-quadruplex ligands BRACO-19 for 5h. The viral RNA was detected with quantitative PCR. Data were presented with average ± SD based on the Student's t test. ****, P < 0.0001. (B) Effects of BRACO-19 on cell viability of RD cells (A) were determined by measuring ATP content using a CellTiter-Glo assay. Data were presented with average ± SD based on the Student's t test. ns, no significance; **, P < 0.01. (C) RD cells were infected with EV-A71 strain FY23 (MOI = 3) for 1h then treated with BRACO-19 overnight. The lysates were immunoblotted for 2C and β-actin. (D) RD cells infected with EV-A71 strain FY23 (MOI = 3) for 24h in the presence of 50 μM BRACO-19 and extracellular virus titer was measured by plaque assay in RD cells. (E) RD cells were infected with EV-A71 strain FY23 (MOI = 3) for 1h and then treated with G-quadruplex ligands PDS for 5h. The viral RNA was detected with quantitative PCR. Data were presented with average 6 SD based on the Student's t test. **, P < 0.01. (F) ATP levels in cells (E) were measured by CellTiter-Glo…