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Sci Rep
2013 Jan 01;3:1811. doi: 10.1038/srep01811.
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Existence of G-quadruplex structures in promoter region of oncogenes confirmed by G-quadruplex DNA cross-linking strategy.
Yuan L
,
Tian T
,
Chen Y
,
Yan S
,
Xing X
,
Zhang Z
,
Zhai Q
,
Xu L
,
Wang S
,
Weng X
,
Yuan B
,
Feng Y
.
Abstract
Existence of G-quadruplex DNA in vivo always attract widespread interest in the field of biology and biological chemistry. We reported our findings for the existence of G-quadruplex structures in promoter region of oncogenes confirmed by G-quadruplex DNA cross-linking strategy. Probes for selective G-quadruplex cross-linking was designed and synthesized that show high selectivity for G-quadruplex cross-linking. Further biological studies demonstrated its good inhibition activity against murine melanoma cells. To further investigate if G-quadruplex DNA was formed in vivo and as the target, a derivative was synthesized and pull-down process toward chromosome DNAs combined with circular dichroism and high throughput deep sequencing were performed. Several simulated intracellular conditions, including X. laevis oocytes, Ficoll 70 and PEG, was used to investigate the compound's pure cross-linking ability upon preformed G-quadruplex. Thus, as a potent G-quadruplex cross-linking agent, our strategy provided both valuable evidence of G-quadruplex structures in vivo and intense potential in anti-cancer therapy.
Figure 1. a) Structures of compound 1 and o-quinone intermediate induced by tyrosinase. b) CD spectra of Pu27 DNA (20 μM) in 10 mM Tris/HCl buffer at pH 7.0 in the absence or presence of compound 1 ([compound 1]/[DNA strand] = 5) without or with incubation with tyrosinase (400 Units) at 37°C for 1 h. c) CD melting of Pu27 DNA curves in the presence of compound 1 ([compound 1]/[DNA strand] = 5) without or with incubation with tyrosinase (400 Units) at 37°C for 1 h, as monitored by the CD intensity at 265 nm. Tm values of the two G-quadruplexes are 30.3°C and 59.9°C, respectively.
Figure 2. a) Concentration dependence of compound 1 for tyrosinase oxidation DNA alkylating were incubated with 5′-end TAMRA labeled Pu27 DNA in 10 mM Tris/HCl buffer at pH 7.0 for 1 h at 37°C. The amounts of labeled DNA and tyrosinase were fixed as 10 pmol and 40 Units, respectively. Lane 1–3 are control lanes, lane 4–11 are increasing concentrations of compound 1 (10 μM, 20 μM, 50 μM, 0.1 mM, 0.2 mM, 0.5 mM, 1 mM, 2 mM) incubation with labeled Pu27 DNA and tyrosinase (40 Units). The alkylated oligo was separated from the nonreacted DNA by 20% denaturing polyacrylamide gel. b) Compound 1 (1 mM) was incubated with labeled Pu27 DNA (10 pmol) and tyrosinase (40 Units) in the presence of increasing molar ratios (0.5, 1, 2, 5, 10) of unlabeled Pu27 DNA(G4) or unlabeled ds-Pu27 DNA(double-stranded, ds) for 1 h at 37°C. The alkylated oligo was separated from the nonreacted DNA by 20% denaturing polyacrylamide gel. “C” refers to labeled Pu27 DNA (10 pmol) treated at 37°C for 1 h, and “R” refers to the Pu27-compound 1 reaction product in the absence of any competitor DNA.
Figure 3. a) Analysis of cross-linking G-quadruplex in the presence of complementary strand of Pu27 DNA (Pu27-c) in 10 mM Tris/HCl buffer at pH 7.0 and 10 mM KCl by 20% polyacrylamide gel. Lane 1: labeled Pu27 DNA (10 pmol) treated at 37°C for 1 h in 10 mM KCl; lane 2: control lane without tyrosinase and compound 1; lane 3: control lane only with tyrosinase and without the compound 1; lane 4: control lane only with compound 1 and without the tyrosinase; lane 5: compound 1 (2 mM) incubation with labeled Pu27 DNA (10 pmol) and tyrosinase (40 Uints) in the presence of excess of Pu27-c (20 pmol). b) CD spectrum for examining the structural features of the cross-linked complex which was extracted from gel. c) CD melting curves of the cross-linked complex as monitored by the CD intensity at 265 nm. d) MALDI-TOF MS spectrum of the cross-linked complex, calcd. [M + Na-2H]− 9649.77, found: 9648.32. e) MALDI-TOF MS spectrum of the cross-linked complex degradation products by DNase I, calcd. [M + H]+ 1037.52, found: 1037.57.
Figure 4. Western Blot was used to determine the expression of c-myc gene in the B16-F1 cells treated with compound 1.The cells were treated with medium (Line 1) and increasing concentrations of compound 1(20, 40 and 50 μM) for 3 days, and the total protein was extracted and subjected to Western Blot for c-Myc, and β-actin (control).
Figure 5. a) Experimental design for detecting formation of G-quadruplexes and sequencing in oncogene promoters in B16-F1 cells. b)1.5% agarose gel. Lane 1: DNA ladder and lane 2: approximately 100 bp sonicated in genomic DNA. c) CD spectrum of genomic DNA short fragments which was extracted from B16-F1 cells in the absence or presence of compound 1a (40 μM) for 3d.
Figure 6. G-quadruplex formation in long dsDNA carrying G-quadruplex-forming sequence from the c-myc gene examined by native gel electrophoresis.a) DNA samples were loaded on 8% polyacrylamide gel containing 150 mM KCl, 40% (w/v) PEG 200 and electrophoresed at in 1× TBE buffer containing 150 mM KCl. Lane 1:c-myc and c-myc-c without PEG; Lane 2: c-myc and c-myc-c with PEG; Lane 3 and 4 are control lanes, c-myc and c-myc-c incubation with compound 1a (2 mM) or tyrosinase (40 Units); lane 5-10 are increasing concentrations of compound 1a (100 μM, 200 μM, 500 μM, 1 mM, 2 mM) incubation with c-myc and c-myc-c in the presence of tyrosinase (40 Units); Lane 10: c-myc-c only. All samples contain 150 mM KCl. b) DNA samples were loaded on 8% polyacrylamide gel containing 40% (w/v) PEG 200 and electrophoresed at in 1× TBE buffer. Lane 1: c-myc-c only; Lane 2: c-myc and c-myc-c without PEG; Lane 3: c-myc and c-myc-c with PEG; Lane 4 and 5 are control lanes, c-myc and c-myc-c incubation with compound 1a (2 mM) or tyrosinase (40 Units); lane 6-8 are increasing concentrations of compound 1a (500 μM, 1 mM, 2 mM) incubation with c-myc and c-myc-c in the presence of tyrosinase (40 Units), All samples were free off KCl.
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