The yield of transcripts for RNA polymerase regulated by hairpin structures in nascent RNA

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1 Supporting Information The yield of transcripts for RNA polymerase regulated by hairpin structures in nascent RNA Satoru Nagatoishi a, Ryoya Ono b, Naoki Sugimoto a,b * a Frontier Institute for Biomolecular Engineering Research (FIBER), b Faculty of Frontiers of Innovative Research in Science and Technology (FIRST), Konan University, Minatojima-minamimachi, Chuo-ku, Kobe ,Japan. Page S1: Page S2: Page S4: Page S5: Page S6: Page S7: Contents. Materials and Methods. Fig. S1 for the predicted secondary structures of RNA using Mfold. Fig. S2 for CD spectra and UV melting curves of template DNAs. Fig. S3 for native PAGE of template DNAs. Fig. S4 for denaturing PAGE for template DNAs in the Hae III reaction. S1

2 Materials and Methods Preparation of Oligonucleotides DNA and RNA strands used in this study were purchased from Japan Bio Services Co., Ltd. (Saitama, JAPAN) and were purified by high-performance liquid chromatography (HPLC). Single-strand concentrations of the DNA and RNA were determined by measurement of the absorbance at 260 nm at a high temperature using a Shimadzu (Kyoto, Japan) 1800 spectrophotometer connected to a thermoprogrammer. Single-strand extinction coefficients were calculated from mononucleotide and dinucleotide data using nearest-neighbor approximations. Transcription reactions The transcription reaction was carried out with 0.6 µm T7 RNA polymerase (T7 RNAP) and 0.4 µm DNA templates at 37 C using a buffer solution consisting 10 mm MgCl 2, 100 mm NaCl, 40 mm Tris-HCl (ph 8.0), 5.0 mm DTT, and 0.4 unit ml 1 RNase inhibitor. Samples were incubated for 30 min, followed by the addition of the T7 RNAP solution to start the reaction. The transcription reaction was quenched by 10-fold excess volume of stop solution containing 80% formamide, 50 mm Na 2 EDTA, and 0.2% (w/v) blue dextran, and the product was immediately stored at 0 C. Hae III digestion The reactions were performed with 10 unit of Hae III. Samples were incubated at 37 C for 30 min. The reactions were terminated by adding the gel loading buffer containing 80% formamide, 50 mm Na 2 EDTA, and 0.2% (w/v) blue dextran followed by cooling on ice. Gel electrophoresis Denaturing gel electrophoresis was carried out using 12% polyacrylamide, 7 M urea. Samples of 5.0 µl were loaded and analyzed by electrophoresis at 200 V for 1.5 hr at 60 C. Gels were stained using SYBR Gold and imaged using an FLA-5100 (Fujifilm Co., Ltd., Tokyo, Japan). Nondenaturing gel electrophoresis was carried out in gels of 12% polyacrylamide. Samples of 5.0 µl were loaded and analyzed by electrophoresis at 200 V for 1.5 hr at 37 C. Gels were stained using SYBR Gold and imaged using an FLA S2

3 CD measurements Spectra were collected on a JASCO J-820 spectropolarimeter (JASCO, Hachioji, Japan) at 37 C in 1.0-mm path length cuvettes. The concentration of RNA was 10 µm in 40 mm Tris- HCl (ph 8.0) and 100 mm NaCl. The CD spectra shown are averages of six scans from 200 to 350 nm. The temperature of the cell holder was regulated by a JASCO PTC-348 temperature controller, and the cuvette-holding chamber was flushed with a constant stream of dry N 2 gas to avoid condensation of water on the cuvette exterior. Before measurement, the sample was heated to 95 C, cooled at a rate of 1 C min 1, and incubated at 4 C. UV measurements UV absorbance was measured with a Shimadzu 1800 spectrophotometer equipped with a temperature controller. Melting curves of 5.0 µm DNA templates and 10 µm RNA oligonucleotides were obtained by measuring the UV absorbance at 260 nm in 40 mm Tris- HCl (ph 8.0) and 100 mm NaCl. The heating rate was 1 C min 1. The thermodynamic stabilities (free energy change G ) were calculated from the fit of the melting curves to a theoretical equation for an intramolecular association as described previously. 1 Before measurement, the sample was heated to 95 C, cooled at a rate of 1 C min 1, and incubated at 0 C for 5 min. S3

4 (A) (B) (C) (D) Fig. S1. Secondary structures of RNAs transcribed from templates (A) DNA7, (B) DNA5, (C) DNA3 and (D) DNA2 predicted using Mfold. S4

5 (A) (B) [ θ ] (10 5 deg cm 2 dmol -1 ) Normalized absorbance at 260 nm Wavelength / nm Temperature / o C Fig. S2. (A) CD spectra of 5 μm DNA7 (circle), DNA5 (square), DNA3 (diamond) and DNA2 (triangle) at 37 C, and (B) normalized UV melting curves of 5 μm DNA7 (circle), DNA5 (square), DNA3 (diamond) and DNA2 (triangle) at 280 nm in a solution containing 40 mm Tris-HCl (ph 8.0), 100 mm NaCl and 10 mm MgCl 2. S5

6 Fig. S3. Native 12% PAGE of DNA7 (lanes 2 and 3), DNA5 (lanes 4 and 5), DNA3 (lanes 6 and 7) and DNA2 (lanes 8 and 9) at 37 C in a buffer containing 40 mm Tris-HCl (ph 8.0), 10 mm MgCl 2, 100 mm NaCl and 5.0 mm DTT. The samples in the absence of T7 RNA polymerase were loaded in lanes 2, 4, 6 and 8. The samples in the presence of T7 RNA polymerase were loaded in lanes 3, 5, 7 and 9. The 10-base-pair ladder was loaded in lane 1. The gel was stained with SYBR Gold. S6

Fig. S4. Denaturing 12% PAGE containing 7 M urea analysis of DNA7 (lanes 2 4), DNA5 (lanes 5 7), DNA3 (lanes 8 10) and DNA2 (lanes 12 14) with and without restriction enzyme.

7 Fig. S4. Denaturing 12% PAGE containing 7 M urea analysis of DNA7 (lanes 2 4), DNA5 (lanes 5 7), DNA3 (lanes 8 10) and DNA2 (lanes 12 14) with and without restriction enzyme. The Hae III reaction was carried out in the presence of Hae III for 60 minutes at 37 C in a buffer containing 40 mm Tris-HCl (ph 8.0), 10 mm MgCl 2, 100 mm NaCl and 5.0 mm DTT. The samples in the absence of Hae III were loaded in lanes 2, 5, 8 and 12. The samples in the presence of Hae III and absence of T7 RNA polymerase were loaded in lanes 3, 6, 9 and 13. The samples in the presence of both Hae III and T7 RNA polymerase were loaded in lanes 4, 7, 10 and 14. The 10-base-pair ladder was loaded in lanes 1 and 11. This gel was stained with SYBR Gold. 1 D. Miyoshi, H. Karimata and N. Sugimoto, J. Am. Chem. Soc., 2006, 128, S7

Supporting Information

Supporting Information Wiley-VCH 2006 69451 Weinheim, Germany Rolling-circle Amplification of a DNA Nanojunction Chenxiang Lin, Mingyi Xie, Julian J.L. Chen, Yan Liu and Hao Yan A. RCA replication of the