Cytotoxicity of guanine-based degradation products contributes to the antiproliferative activity of guanine-rich oligonucleotides† †Electronic supplementary information (ESI) available: Experimental details and supplementary figures. See DOI: 10.1039/c4sc03949a

Guanine-rich oligonucleotides with lower nuclease resistance exhibited higher antiproliferative activity; guanine-based compounds showed highly concentration-dependent cytotoxicity.


Growth observation of Jurkat E6-1 cells after treated by dG
Although Jurkat E6-1 is a suspension cultured cell line, the cells prefer to form aggregates when they grow to high density. As shown in Figure S4, the cell density and the aggregate size of untreated cells (control) increased with time of culture; similar results was also observed in cells treated with 10 μM dG, indicating that 10 μM dG did not affect cell growth much. However, after treated with 20 μM dG, the cell growth was significantly inhibited, only some small size aggregates were observed after 96 h. After treated with 30 μM dG, the cell growth was totally inhibited, no notable aggregates were observed after 96 h. All cell lines were routinely cultured at 37 o C in humidified atmosphere with 5% CO 2 .

Circular Dichroism (CD) Spectra measurement
Oligonucleotides were diluted to a concentration of 4 μM by PBS，and stabilized at room temperature for 2-4 h. The CD spectra (220-320 nm) were collected at room temperature on Jasco J-815 spectropolarimeter (Japan). The scanning rate was 500 nm/min. Each sample was scaned three times and the mean value was taken. To facilitate analysis, all CD spectra were applied background subtraction and smoothed.
Oligonucleotides degradation in serum-containing medium. For high sensitive detection, oligonucleotides used in this experiment were labeled with fluorescein (FAM) at 5' end. After annealed, 10 μM oligonucleotides were added into RPMI 1640 medium with 10% FBS and incubated in a humidified 37 o C/5% CO 2 incubator.
Aliquots (20 L) of the incubated samples was taken out at 0, 6, 12, 24, 48 and 96 h, added with 25 mM EDTA, and heated at 95 o C for 5 min to quench the nuclease, and then stored at -20 o C before analysis. 20% denaturing-polyacrylamide gel electrophoresis was used to analyze these urea-denaturing samples. The gels were exposed under UV light and photographed. The fluorescence intensities (quantified by Image J (NIH, USA)) of the major bands corresponding to intact DNAs were used to evaluate the stability of the FAM-labeled oligonucleotides.

Cell proliferation Assay.
Cells were seeded in 96-well plates (100 μL per well). Different cell lines were seeded at different densities and pre-incubated for different time due to their intrinsic different growth rate. Jurkat and K562 were seeded at 3000/well and pre-incubated 4 h. A549, PC-3, and DU145 were seeded at 1000/well and pre-incubated overnight. Control samples incubated with PBS instead of oligonucleotides. Cell viability was obtained by comparing the absorbance of treated cells to that of control cells.

Cell apoptosis assay
Jurkat cells were seeded at a density of 1×10 5 /mL. After pre-incubation for 4 h, cells were added with oligonucleotides (10 μM) or dG (10, 20, 30 and 100 μM) and cultured. Aliquots of treated cells were taken for analysis at time points of 6, 12, 24, 48, 72 and 96 h. After washed, cells were stained with Annexin V-FITC for 30 min at room temperature in the dark and then stained by PI on the ice, and then were measured by flow cytometry with FL1 and FL2 channels. Annexin V-FITC Apoptosis Detection Kit was purchased from Dojindo Co. Ltd (Japan).
Quantification of S phase and sub-G1 phase was done by FlowJo (Treestar, San Caros, USA).

Cell cycle profiles analysis
Jurkat cells were seeded at a density of 1×10 5 /mL in 48-well plates. After preincubation for 4 h, cells were added with oligonucleotides (10 μM) or dG (10, 20, 30 and 100 μM) and cultured. Aliquots of treated cells were taken for analysis at time points of 6, 12, 24, 48, 72 and 96 h. Cells were centrifuged and re-suspended in 50 μL ice-cold PBS, fixed by addition of 500 μL of 78% ethanol, and stored at -20 o C at least overnight. After fixed, cells were washed twice with ice cold PBS and re-suspended in 400 μL of PI staining buffer (PBS containing 50 μg/mL propidium iodide (PI)，10 μg/mL RNase A and 0.5% Triton-X-100) for 30 min at room temperature in the dark.
The DNA content was acquired by a Becton Dickinson FACScalibur flow cytometer (Becton Dickinson, USA) with FL2 channel. The data were analyzed by FlowJo software (Treestar, San Caros, USA).