Figure 1.
A) structure of G-quartet with cyclic array of four guanines linked by Hoogsteen H-bonds, B) Anti-paralled G-quadruplex, C) Mixed-hybrid G-quadruplex, D)An ORTEP drawing of [(dmb)2Ru(obip)Ru(dmb)2]4+.
Figure 2.
Synthetic routes for the preparation of the complex [(dmb)2Ru(obip)Ru(dmb)2]4+.
Figure 3.
CD titration of 22AG with complex in 10 mM Tris buffer.
CD titration spectra of 22AG 20 µM in 10 mM Tris-HCl: with (a) 0 µM, (b) 1µM, (c) 2 µM, (d) 4 µM, (e) 6 µM, (f) 8 µM, (g) 10 µM, (h) 12 µM, (i) 14 µM [(dmb)2Ru(obip)Ru(dmb)2]4+.
Figure 4.
Melting of the G-quadruplex assessed by UV absorbance at 295 nm.
Normalized UV melting curves for 10 µM G-quadruplex (■), 10 µM G-quadruplex + 10 µM [(dmb)2Ru(obip)Ru(dmb)2]4+ (●)in a buffer of 100 mM NaCl, 10 mMNaH2PO4/Na2HPO4, 1mM Na2EDTA; 10 µM G-quadruplex (▼) 10 µM G-quadruplex + 10 µM [(dmb)2Ru(obip)Ru(dmb)2]4+ (▲)in a buffer of 100 mM KCl10 mM K2HPO4/K2HPO4, 1mM K2EDTA (pH 7.0).
Figure 5.
Binding modes assessed by Emission quenching studies.
Emission quenching curves of [(dmb)2Ru(obip)Ru(dmb)2]4+ with increasing concentration of quencher [Fe(CN)6]4- in the absence: (■) and presence of G-quadruplex DNA (•), [Ru] = 4μM, [DNA]/[Ru] = 1.
Figure 6.
Binding modes assessed by molecular docking studies.
A) The schematic diagram of the interaction of [(dmb)2Ru(obip)Ru(dmb)2]4+ with the antiparallel G-quadruplex structure, B) the mixed parallel/antiparallel G-quadruplex structure. G in green yellow, A in red, T in cyan and [(dmb)2Ru(obip)Ru(dmb)2]4+ in green and blue, C) the complex, the antiparallel G-quadruplex and D) the mixed parallel/antiparallel G-quadruplexare shown in CPK mode.
Figure 7.
Selectivity for G-quadruplex DNA assessed by competition dialysis.
Results of competition dialysis experiment with the amount of [(dmb)2Ru(obip)Ru(dmb)2]4+ bound to each DNA structure plotted as a bar graph.
Figure 8.
Telomerase inhibition by [(dmb)2Ru(obip)Ru(dmb)2]4+.
TRAP Assay with Hela cells at gradient concentrations of [(dmb)2Ru(obip)Ru(dmb)2]4+ for 24 h shown in (A), quantitative analysis of the telomerase inhibition by ELISA shown in (B). PC means positive control with HEK293 provided by the kit, NC means the negative control obtained by heating Hela cell protein for 10 min at 85 °C. Hela means Hela cells untreated with [(dmb)2Ru(obip)Ru(dmb)2]4+. 24 h, 48 h, 72 h means Hela cells respectively treated with the complex at MTTIC50= 120 nM for 24 h, 48 h and 72 h. Data were represented as mean +/- S.E.M. * means significant difference by GraphPad Prism5 One way ANOVA (Tukey's Multiple Comparison Test).
Figure 9.
Cytotoxic effects of complexes on cells.
MTT assay was performed on human normal fibroblast cells treated with [(dmb)2Ru(obip)Ru(dmb)2]4 A), human cervical cancer Hela cells treated with [(dmb)2Ru(obip)Ru(dmb)2]4+ at gradient concentrations B), Hela cells treated with a clinical chemotherapeutic cisplatin C), K562 cells treated with [(dmb)2Ru(obip)Ru(dmb)2]4 D) and K562 cells treated with cisplatin respectively at gradient concentrations. The inhibition ratio was collected as the average of triplicate wells relative to that of the untreated wells. Result shown here was the representative one among three independent experiments. Data were presented as mean +/- S.E.M.* means P < 0.05, ** means P < 0.005, *** means P < 0.0001 significant difference was observed by GraphPad Prism5 Two-way RM ANOVA (Bonferroni posttests).
Figure 10.
TUNEL assay with Hela cells to detect the oligonucleosomal DNA cleavage during cell apoptosis induced by [(dmb)2Ru(obip)Ru(dmb)2]4+.
A) The representative green fluorescence images taken under microscope after treatment with [(dmb)2Ru(obip)Ru(dmb)2]4+ at MTTIC50 = 120 nM at different time points, B) the percentage of green fluorescence positive cells after [(dmb)2Ru(obip)Ru(dmb)2]4+ treatment in comparison with both the negative and positive controls. NC means the untreated Hela cells used as the negative control, PC means Hela cells treated with DNase I recombinant (3000U/ml-3U/ml in 50 mM Tris-HCl, PH 7.5, 1 mg/ml BSA) for 10 min at 15-25 °C used as the positive control. 24 h, 48 h and 72 h mean Hela cells treated with [(dmb)2Ru(obip)Ru(dmb)2]4+ for 24 h, 48 h and 72 h respectively before labelling. Scale bar 25 um. Data were presented as mean +/- S.E.M. * means significant difference was observed by GraphPad Prism5 One way ANOVA (Tukey's Multiple Comparison Test).