Supporting Information Destabilization of DNA G-quadruplexes by chemical environment changes during tumor progression facilitates transcription Hisae Tateishi-Karimata, 1 Keiko Kawauchi, 2 and Naoki Sugimoto 1,3* 1 Frontier Institute for Biomolecular Engineering Research (FIBER), 2 Faculty of Frontiers of Innovative Research in Science and Technology (FIRST) and 3 Graduate School of FIRST, Konan University, 7-1-20 Minatojima minamimachi, Kobe, 650-0047, Japan Page S1: Page S2: Page S3: Page S4: Page S5: Page S6: Page S7: Page S8: Page S9: Page S10: Page S11: Page S12: Page S13: Page S14: Page S15: Page S16: Page S17: Page S18: Page S19: Page S20: Page S21: Page S22: Page S23: Contents. Figure S1. Schematic illustration of effects of structures in template DNA on RNA polymerase elongation Figure S2. Schematic illustration of expected structure of oligonucleotides with complete and incomplete G-quartets. Figure S3. CD spectra for t1, t2, and t3.at 37 o C Figure S4. CD spectra for d1, d2, d3, and d4 at different temperatures. Figure S5. UV melting curves for G-quadruplexes for t1, t2, and t3 Figure S6. G-quadruplex formation evaluated by analysis of ligand binding. Figure S7. Time-course analyses for T7 RNA polymerase-catalyzed multiple turnover transcription Figure S8. Denaturing gel electrophoresis of products of transcription reactions for Linear, D1, D2, D3, and D4 Figure S9. Denaturing gel electrophoresis of products of transcription reactions for T1, T2, and T3 Figure S10. Effect of the G-quadruplex stability on the production of run-off transcripts in vitro. Figure S11. Effect of the dielectric constants on the production of run-off transcripts. Figure S12. Effect of the water activities on the production of run-off transcripts. Figure S13. CD melting curves for G-quadruplexes at different KCl concentrations. Figure S14. Effect of G-quadruplex stability on production of run-off transcripts in vitro. Figure S15. UV melting curves for t1 at different KCl concentrations. ypes. Figure S16. Comparison of TEin cell values in cancer cells for non-aggressive and aggressive phenotypes. Figure S17. Evaluation of reporter gene expression in cells Figure S18. CD spectra for t1, t2, and t3 at different KCl concentrations Table S1. G-quadruplex forming sequences in c-myc Table S2. Sequences of the double stranded DNA templates Table S3. Dielectric constants and logarithms of water activity Table S4. The free energy changes for t1 at various concentrations of KCl S1
Figure S1. Effects of structures in template DNA on RNA polymerase elongation. (A) An unstructured template, (B) a template with a slippage site, (C) a template with a pause site, and (D) a template with an arrest site. S2
Figure S2. Expected structure of oligonucleotides with complete and incomplete G-quartets. (A) Expected structures of complete and incomplete G-quartets. (B) Expected structures of G-quadruplexes for d1 d4. S3
12 8.0 t2 4.0 0 t3 t1-4.0 200 250 300 Wavelength (nm) 350 Figure S3. CD spectra for 20 µm t1, t2, and t3 in a buffer containing 150 mm KCl, 40 mm Tris-HCl (ph 7.2) and 10 wt% PEG200 at 37 o C. S4
Figure S4. CD spectra for 20 µm (A) d1, (B) d2, (C) d3, and (D) d4 in a buffer containing 150 mm KCl, 40 mm Tris-HCl (ph 7.2) and 10 wt% PEG200 at temperatures ranging from 0 to 90 o C in 10 o C increments. S5
Figure S5. UV melting curves for G-quadruplexes for 20 µm t1 (light blue), t2 (orange), and t3 (light green) in a buffer containing 150 mm KCl, 40 mm Tris-HCl (ph 7.2), 8 mm MgCl2, and 10 wt% PEG200. The Tm value for t1 was 58.4 C. t2 and t3 did not show the clear melting curves due to high stability. S6
Fluorescence intensity at 611 nm Figure S6. G-quadruplex formation in the template DNAs evaluated by analysis of NMM binding. Fluorescence intensity of NMM in the presence of indicated template DNA at 37 C in 150 mm KCl, 40 mm Tris-HCl (ph 7.2), 8 mm MgCl 2 and 10 wt% PEG 200. S7
Figure S7. Time-course analyses for T7 RNA polymerase-catalyzed multiple turnover transcription. Production of full-length transcript from the Linear template was measured at 37 o C in a buffer containing 150 mm KCl, 40 mm Tris-HCl (ph 7.2), 8 mm MgCl2 10 wt% PEG 200, 1 mm DTT, and 1 mm each NTP. (A) Gel electrophoretic analysis of the amount of product as a function of time. Lanes 1 to 6 were from reactions incubated 20, 40 60, 90, 120, and 180 min. M and D indicate size maker and template DNA, respectively. After electrophoresis, the gels were stained with SYBR Gold, and levels were quantified with a fluorescent imager. (B) The time course of the transcript production quantified by gel bands in Figure S7a. S8
Figure S8. Denaturing gel electrophoresis of products of transcription reactions carried out for 120 min at 37 C. Reaction mixtures contained 0.3 μm T7 polymerase, 1.5 μm DNA template, 150 mm KCl, 40 mm Tris-HCl (ph 7.2), 8 mm MgCl2, and 10 wt% PEG 200. Lanes R and M, 35-nt RNA and 10-nt size markers; lanes 1 5, transcription products for Linear, D1, D2, D3, and D4 templates, respectively. The transcript from the IC-template is indicated by a red dashed line. S9
R Size 70 nt- M 1 2 3 * * - Longer slipped transcript Run-off - Full-length transcript transcripts 40 nt- 35 nt- - Arrested transcript (35nt) R: 35 nt RNA M: 10 nt DNA size makers Figure S9. Denaturing gel electrophoresis of products of transcription reactions carried out for 120 min at 37 C. Reaction mixtures contained 0.3 μm T7 polymerase, 1.5 μm DNA template, 150 mm KCl, 40 mm Tris-HCl (ph 7.2), 8 mm MgCl2, and 10 wt% PEG 200. Lane M, size markers; lanes 1 to 3, transcription products for T1, T2, and T3 templates, respectively. Blue stars and red arrows indicate the slipped and arrested transcripts, respectively. S10
TE (%) 150 100 50 0 Figure S10. Effect of the G-quadruplex stability on the production of run-off transcripts in vitro. TE (%) from each template DNA from reactions that included 0.3 μm T7 polymerase and 1.5 μm DNA template in a buffer containing 40 mm Tris-HCl (ph 7.2), 8 mm MgCl2, 10 wt% PEG 200, and 150 mm KCl incubated for 120 min at 37 C. S11
Figure S11. Effect of the dielectric constants on the production of run-off transcripts. The TE values for (a) Linear, (b) D1, (c) D2, (d) D3, and (e) D4 as a function of dielectric constant. The dielectric constant was controlled by the addition of cosolute (Table S3). S12
Figure S12. Effect of the water activities on the production of run-off transcripts. The TE values for (a) Linear, (b) D1, (c) D2, (d) D3, and (e) D4 as a function of water activity. The water activity was controlled by the addition of cosolute (Table S3). S13
Figure S13. CD melting curves for G-quadruplexes at different KCl concentrations. Normalized CD intensity at 263 nm as a function of temperature for 20 µm d1 (blue), d2 (red), d3 (green), and d4 (orange) in a buffer containing 40 mm Tris-HCl (ph 7.6), 10 wt% PEG200, and (A) 30, (B) 60 or (C) 100 mm KCl. CD melting curves in 150 mm KCl solution were shown in Figure 3b. S14
Figure S14. Effect of G-quadruplex stability on production of run-off transcripts in vitro. Denaturing gel electrophoresis of transcription reaction products from (A) T1, T2, and T3 template DNAs after 120 min at 37 C. Blue stars indicate the slipped transcript. (B) Correlation between TE and K + concentration for templates T1, T2, and T3. Reaction mixtures were incubated for 120 min at 37 C and contained 0.3 μm T7 polymerase and 1.5 μm DNA template in a buffer containing 40 mm Tris-HCl (ph 7.2), 8 mm MgCl2, 10 wt% PEG 200, and KCl as indicated. S15
Normalized absorbance at 295 nm Temperature ( o C) Figure S15. UV melting curves for t1 at different KCl concentrations. UV melting curves for G-quadruplexes for 20 µm t1 in a buffer containing 40 mm Tris-HCl (ph 7.2), 8 mm MgCl2, 10 wt% PEG 200, and 1 to 150 mm KCl. S16
Figure S16. Comparision of TEin cell values in cancer cells for non-aggressive and aggressive phenotypes. TEin cell values for T1, T2 and T3 in MCF-7 and MDA-MB-231. S17
Figure S17. Evaluation of reporter gene (Firefly luciferase, F-Luc) expression in (A) MCF-7 and (B) MDA-MB-231 cells 24 h after transfection by luciferase assay. We inserted the DNA template sequences of Linear, D1, D2, D3, and D4 upstream of the Firefly luciferase (F-Luc) gene in the pgl3 plasmid. The phrl plasmid encoding Renilla luciferase (R-Luc) was used as a control (Figure 7). The plasmids were co-transfected, and luminescence from F-Luc and R-Luc was quantified. The F-Luc signals relative to the R-Luc signals are defined as the gene expression of F-Luc. Values are the means ± standard deviations of three samples. Values are the means ± standard deviations of three samples. S18
Figure S18. Circular dichroism (CD) spectra of 20 µm (A) t1, (B) t2, and (C) t3 in a buffer containing 40 mm Tris-HCl (ph 7.2), 10 wt% PEG200, and 60 mm (green) or 150 mm (blue) KCl at 37 C. S19
Table S1. G-quadruplex forming sequences in c-myc a,b Sequence position (Start End) Sequence 369 390 CCTAGAGTCCCAACCCCGGCCC 377 393 CCCAACCCCGGCCCTCC 2988 3007 CCCCCACCTGACCCCCGCCC 2978 3007 CCTGCTCCTGCCCCCACCTGACCCCCGCCC 3943 3967 CCCCCTGCCCCTCCCATATTCTCCC 3943 3977 CCCCCTGCCCCTCCCATATTCTCCCGTCTAGCACC 4139 4158 CCTCCCTCTCGCCCTAGCCC 4506 4537 CCCATTAATACCCTTCTTTCCTCCACTCTCCC 4851 4875 CCTCCCCACCTTCCCCACCCTCCCC 4854 4884 CCCCACCTTCCCCACCCTCCCCACCCTCCCC 4877 4899 CCCTCCCCATAAGCGCCCCTCCC 5038 5059 CCCGGCCGTCCCTGGCTCCC 5031 5060 CCGCCGGGCCCCGGCCGTCCCTGGCTCCCC 5039 5067 CCCCGGCCGTCCCTGGCTCCCCTCCTGCC 5301 5323 CCCAGCCCTCCCGCTGATCCCCC 5294 5323 CCTCTGGCCCAGCCCTCCCGCTGATCCCCC 5301 5327 CCCAGCCCTCCCGCTGATCCCCCAGCC 7362 7381 CCCACCCCGCCCCTGTCCCC 7362 7389 CCCACCCCGCCCCTGTCCCCTAGCCGCC 7676 7695 CCCGAACCCCGCCCGCGGCC a C-rich sequences in c-myc. The template DNA strands are complementary to the sequences in the table. Sequences 5038 5059, 5301 5323, 7362 7381, and 7676 7695 were used in this study (underlined). b G-quadruplex-forming sequences in genome were identified using the algorithm described by Huppert and Balasubramanian (Nucleic Acids Res 33, 2908 (2005) with G-quadruplexes identified as d(g3+n1 7G3+N1 7G3+N1 7G3+) [N refers to any base]. We also selected G-quadruplex-forming sequences with one incomplete G-quartet. S20
Table S2. Sequences of the double stranded DNA templates used in this study Abbreviation Sequence a,b Linear 5 GCCGTTTCGTAGTATTTGGGTTGTAACTATCGAGGCAGAGAGAGCACCGAGCCTAG TTCGTGTCATCTCCTATAGTGAGTCGTATTAGTGATC 3 / 5 GATCACTAATACGACTCACTATAGGAGATGACACGAACTAGGCTCGGTGCTCTCTCT GTCTCGATAGTTACAACCCAAATACTACGAAACGGC 3 D1 5 GCCGTTTCGTAGTATGGGAGCCAGGGACGGCCGGGCAGAGAGAGCA CCGAGCCTA GTTCGTGTCATCTCCTATAGTGAGTCGTATTAGTGATC 3 / 5 GATCACTAATACGACTCACTATAGGAGATGACACGAACTAGGCTCGGTGCTCTCTCT GCC CGGCCGTCCC TGGCTCCCATACTACGAAACGGC 3 D 2 5 GCCGTTTCGTAGTATGGGGACAGGGGCGGGGTGGGCAGAGAGAGCACCGAGCCTA GTTCGTGTCATCTCCTATAGTGAGTCGTATTAGTGATC 3 / 5 GCCGTTTCGTAGTATGGGGACAGGGGCGGGGTGGGCAGAGAGAGCACCGAGCCTA GTTCGTGTCATCTCCTATAGTGAGTCGTATTAGTGATC 3 D 3 5 GCCGTTTCGTAGTATGGCCGCGGGCGGGGTTCGGGCAGAGAGAGCACCGAGCCTA GTTCGTGTCATCTCCTATAGTGAGTCGTATTAGTGATC 3 / 5 GATCACTAATACGACTCACTATAGGAGATGACACGAACTAGGCTCGGTGC TCTCTCT GCCCGAACCCCGCCCGCGGCCATACTACGAAACGGC 3 D4 5 GCCGTTTCGTAGGGGGGATCAGCGGGAGGGCTGGGCAGAGAGAGCACCGAGCCTA GTTCGTGTCATCTCCTATAGTGAGTCGTATTAGTGATC 3 / 5 GATCACTAATACGACTCACTATAGGAGATGACACGAACTAGGCTCGGTGC TCTCTCT GCCCAGCCCTCCC GCTGATCCCCCCTACGAAAC GGC 3 T1 5 GCCGTTTCGTAGTATTTCTAGGTTGGTGTGGTTGGTTGTAACTATCGGGTGTGTAGTT CGTGTCATCTCCTATAGTGAGTCGTATTAGTGATC 3 5 GATCACTAAT ACGACTCACT ATAGGAGATG ACACGAACTA CACACCCGATAGTTAC AACCAACCACACCA ACCTAGAAATACTACGAAACGGC 3 T2 5 GCCGTTTCGTAGTATTGGGTTGGGTGTGGGTTGGGTTGTAACTATCGGGTGTGTAGT TCGTGTCATCTCCTATAGTGAGTCGTATTAGTGATC 3 / 5 GATCACTAATACGACTCACTATAGGAGATG ACACGAACTACACACCCGATAGTTAC AACCCAACCCACACCCAACCCAATACTACGAAACGGC 3 T3 5 GCCGTTTCGTAGGGGGTTGGGGTGTGGGGTTGGGGTTGTAACTATCGGGTGTGTAG TTCGTGTCATCTCCTATAGTGAGTCGTATTAGTGATC 3 / 5 GATCACTAAT ACGACTCACT ATAGGAGATG ACACGAACTA CACACCCGATAGTTA CAACCCCAACCCCACACCCCAACCCCCTACGAAACGGC 3 IC-template 5 CTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGGCC GTTTCG TAGTATTTGGGTTGTAACTATCGAGGCAGAGAGAGCACCGAGCCTAGTTCGTGTCATCTC CTATAGTGAGTCGTATTAGTGATC 3 / 5 GATCACTAATACGACTCACTATAGGAGATGACACGAACTAGGCTCGGTGCT CTCTCT GCCTCGATAGTTACAACCCAAATACTACGAAACGGCCAAAAAACCCCTCAAGACCCGTT TAGAGGCCCCAAGGGGTTATGCTAG 3 a Sequences of the G-quadruplex-forming region are indicated with bold, italics and underlining. b Sequences of the T7 promoter are shown in bold. S21
Table S3. Dielectric constants and logarithms of water activity in the presence of 10 wt% cosolute at 25 o C a Cosolute ε ln a w ( 10 2 ) 1,2-dimethoxyethane 73.0 1.97 PEG 200 74.2 1.26 methanol 74.4 1.97 ethanol 73.0 4.09 1,3-propanediol 75.6 2.41 ethylene glycol 76.4 2.77 glycerol 76.5 2.25 acetonitrile 76.6 3.28 a All experiments were performed in a buffer containing 10 wt% cosolute. The data for ethylene glycol, 2-methoxyethanol, 1,2-dimethoxyethane, and 1,3-propandiol were obtained using freezing point depression osmometry and the data for the other cosolutes were obtained using vapor phase osmometry. S22
Table S4. The free energy changes for t1 at variousconcentrations of KCl a KCl concentration ΔG o 37 (kcal mol -1 ) 1 mm 0.2±0.4 20 mm 1.8±0.5 30 mm 2.2±0.2 60 mm 2.5±0.5 100 mm 2.8±0.2 150 mm 2.9±0.2 a All experiments were carried out in a buffer containing 40 mm Tris-HCl (ph 7.2), 8 mm MgCl2, 10 % PEG 200, and 1-150 mm KCl. Thermodynamic parameters were evaluated using the average values obtained from curve fitting at the different DNA concentrations. S23