Chemical Targeting of a G-Quadruplex RNA in the Ebola Virus L Gene Shao-Ru Wang, Qiu-Yan Zhang, Jia-Qi Wang, Xing-Yi Ge, Yan-Yan Song, Ya-Fen Wang, Xiao-Dan Li, Bo-Shi Fu, Guo-Hua Xu, Bo Shu, Peng Gong, Bo Zhang, Tian Tian, Xiang Zhou  Cell Chemical Biology  Volume 23, Issue 9, Pages 1113-1122 (September 2016) DOI: 10.1016/j.chembiol.2016.07.019 Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 1 A Graphical Representation of the Consensus G-Rich Sequence in the EBOV L Gene (A) The G-quartet structure is stabilized by Hoogsteen hydrogen bonding, with alkali metal ions coordinated to the O6 guanine atoms. (B) 162 released EBOV complete genomes were retrieved from the NCBI website (http://www.ncbi.nlm.nih.gov) and aligned. The graphical representation of the consensus sequence was created through WebLogo software. Cell Chemical Biology 2016 23, 1113-1122DOI: (10.1016/j.chembiol.2016.07.019) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 2 Demonstration of G4 Structures Using Different Methods (A) 1H-NMR was performed to characterize G4 RNA. (B–D) PAGE analysis was performed under three different conditions, and three gels were demonstrated together. Lanes 1 and 2, 70 mM KCl; lanes 3 and 4, 70 mM LiCl; lanes 5 and 6, denaturing conditions. Lanes 1, 3, 5, FAM-labeled mutant sequence (PGS-LMut-FAM); lanes 2, 4, 6, FAM-labeled wild-type sequence (PGS-L-FAM). Formation of compact G4 was characterized by the species moving faster than the mutant oligonucleotide in the presence of K+ (lanes 1 and 2), while an opposite phenomenon was observed in the presence of Li+ (lanes 3 and 4). The mutant oligonucleotide showed very similar migration to the wild-type one under denaturing conditions (lanes 5 and 6). (E) RNase T1 footprinting was used to identify the G residues involved in the formation of G4 structure. Cell Chemical Biology 2016 23, 1113-1122DOI: (10.1016/j.chembiol.2016.07.019) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 3 The EBOV L Gene Contains a Parallel G4 RNA (A) The influence of different alkali metal ions (as indicated in the figure legends) on the thermal stabilities of folded PGS-L. (B) TMPyP4 stabilizes folded PGS-L, while TMPyP2 does not. Cell Chemical Biology 2016 23, 1113-1122DOI: (10.1016/j.chembiol.2016.07.019) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 4 TMPyP4 Inhibits RNA-Dependent RNA Synthesis through G4 RNA Stabilization An RNA template containing the EBOV G-rich sequence was used. The extended RNA products were analyzed by denaturing PAGE analysis. Arrows indicate the full-length extended RNA product, G4 pausing product, and the free primer. Lane 1 corresponds to RNA markers (marker 17, marker 19, and marker 44); lanes 2 and 3, no enzyme or DMSO control; lanes 4–6, TMPyP4 treatment in the absence of K+; lane 7, TMPyP2 treatment in the absence of K+; lane 8, DMSO treatment; lanes 9–11, TMPyP4-dependent inhibition in the presence of K+; lane 12, TMPyP2 treatment in the presence of K+; lanes 13 and 14, no enzyme or DMSO control; lane 15, TMPyP4 treatment in the absence of K+; lane 16, DMSO treatment; lane 17, TMPyP4 treatment in the presence of K+. Cell Chemical Biology 2016 23, 1113-1122DOI: (10.1016/j.chembiol.2016.07.019) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 5 TMPyP4 Inhibits EGFP Expression through G4 RNA Stabilization (A) Schematic representation of the plasmids used. Plasmid pEGFP-C1 was engineered to harbor 27 nt of the target G4 sequence or the G4-mutant one. (B) HEK293 cells transfected with pEB-G4 or pEB-G4Mut were treated with different compounds, followed by staining the cell nuclei with Hoechst dye 33,258. Confocal images of the same cells with EGFP (left), Hoechst 33258 (middle), or merge (right) were demonstrated. Scale bar, 20 μm. (C) Flow cytometry was used to determine EGFP expression in transfected HEK293 cells that were treated in triplicate with DMSO or different compounds. Inhibition of EGFP expression compared with DMSO-treated control is shown. Error bars reflect the SEM. All the data are presented as means ± SEM from three independent experiments. TMPyP4 group versus TMPyP2 group, *p < 0.05. Cell Chemical Biology 2016 23, 1113-1122DOI: (10.1016/j.chembiol.2016.07.019) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 6 TMPyP4 Inhibits L Gene and Suppresses EBOV Mini-Genome Replication Error bars reflect the SEM. All the data are presented as the means ± SEM from three independent experiments. (A) TMPyP4 suppress the intracellular L RNA level. BSR-T7 cells harboring pCAGGS-L or pCAGGS-L-Mut were treated in triplicate with DMSO or different compounds. L gene inhibition compared with the DMSO-treated control is shown. The values observed were normalized to β-actin. (B) The mini-replicon assay was performed on BSR-T7 cells that were treated in triplicate with TMPyP4 or DMSO. Ribavirin and TMPyP2 were used as controls. Cell Chemical Biology 2016 23, 1113-1122DOI: (10.1016/j.chembiol.2016.07.019) Copyright © 2016 Elsevier Ltd Terms and Conditions

Cell Chemical Biology 2016 23, 1113-1122DOI: (10. 1016/j. chembiol Copyright © 2016 Elsevier Ltd Terms and Conditions