Sybr gold

Transcription reactions were carried out at 37°C in a total volume of 20 μl. T7 RNA polymerase was present at 0.3 μM. The DNA templates used were 5′-cholesteryl-TEG-GGGGTTAGGGGTTAGGGGTTAGGGGTTGTAACTATCGGGTGTGTAGTTCGTGTCATCTCCTATAGTGAGTCGTATTAGTGATC-3′ (containing a G-quadruplex forming region) and 5′-cholesteryl-TEG-GGTGTATTGTAACTATCGAGGCAGAGAGAGCACCGAGCCTAGTTCGTGTCATCTCCTATAGTGAGTCGTATTAGTGATC-3′ (control without G-quadruplex forming region). The T7 promoter regions are underlined. The template concentration in reactions was 1.5 μM. After incubating at 37°C for 10 min, ribonucleoside triphosphate (NTPs) were added to a final concentration of 1 mM each to initiate the reaction. The final reaction buffer contained 30 mM KCl, 40 mM Tris-HCl (pH 8.0 at 37°C), 8 mM MgCl₂, 2 mM spermidine and 5 mM dithiothreitol (DTT). Reactions were quenched after incubation at the time indicated by addition of DNase I. After incubation for 10 min, a 20-fold excess volume of transcription stop solution (80 wt% formamide, 10 mM Na₂EDTA and 0.01% blue dextran) was added. The samples were then heated to 90°C for 5 min, cooled rapidly and loaded onto a 10% polyacrylamide, 7 M urea gel. After electrophoresis at 60°C, the gels were stained by SYBR Gold (PerkinElmer Life Sciences), and levels were quantified with a fluorescent imager (FUJIFILM, FLA-5100).

Native gel electrophoresis was conducted to determine the effect of CK on G4 DNA. In the absence and presence of 100, 200, and 300 μM of CK, G4s and complementary sequences with a ratio of 1:1 were mixed in a buffer containing 40 mM of Tris-HCl (pH 7.6), 8 mM of MgCl₂, 100 mM of KCl, and 20% PEG200. Samples were incubated at 90 °C for 5 min and then cooled down to room temperature. The samples were mixed 1:1 with 2× loading buffer. Samples were conducted in 20% polyacrylamide gels containing 1× TBE buffer with 10 mM of KCl at 90 V for 2 h. After staining with SYBR Gold (Perkin Elmer Life Sciences, Waltham, MA, USA), scanning was performed with an iBright FL1000 Instrument (Thermo, Woodlands, Singapore).

Native gel electrophoresis was carried out on 10% nondenaturing polyacrylamide gels in buffer containing 30 mM KCl, 40 mM Tris-HCl (pH 8.0 at 37°C), 8 mM MgCl₂, and 2 mM spermidine at 37°C. Loading buffer (1 µL of 40% glycerol and 1% blue dextran) was mixed with 2 µL of 2 µM DNA sample. Gels were stained with SYBR® Gold (PerkinElmer Life Sciences) and imaged using a fluorescent imager (FUJIFILM, FLA-5100). Before the measurement, the samples were heated to 95°C, cooled at a rate of −1°C min⁻¹, and incubated at 37°C for 30 min. Unstructured DNAs of 12, 15, 25, and 30 nt were electrophoresed in parallel. When hairpin or G-quadruplex is formed, migration of the DNA should be fast relative to unstructured DNA of the same length. The migration of all template DNAs with hairpins were faster than that of the 12-nt unstructured DNA and migration of templates designed to form G-quadruplexes were faster than that of unstructured DNA of 15 nt indicating the all DNA sequences formed hairpin or G-quadruplexes.