Recovery of the Formation and Function of Oxidized G-Quadruplexes by a Pyrenemodified

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1 Supporting Information Recovery of the Formation and Function of Oxidized G-Quadruplexes by a Pyrenemodified Guanine Tract Shuntaro Takahashi, Ki Tae Kim, Peter Podbevsek, Janez Plavec, Byeang Hyean Kim, and,,* Naoki Sugimoto FIBER (Frontier Institute for Biomolecular Engineering Research), Konan University, Minatojima-Minamimachi, Chuo-ku, Kobe , Japan Department of Chemistry, Division of Advanced Materials Science, Pohang University of Science and Technology (POSTECH), Pohang 37673, Republic of Korea. Slovenian NMR Center, National Institute of Chemistry, SI-1000 Ljubljana, Slovenia FIRST (Graduate School of Frontiers of Innovative Research in Science and Technology), Konan University, Minatojima-Minamimachi, Chuo-ku, Kobe , Japan Contents: 1. Table S1, sequences used in this study 2. Figure S1, CD spectra of c-kit2 and its oxidative derivatives 3. Figure S2, NMR spectra of c-kit2 and its oxidative derivatives 4. Figure S3, CD melting of VEGF, c-kit2, and their oxidative derivatives at 10 mm KCl. 5. Figure S4, PAGE images of replication products from the c-kit2 template 6. Figure S5, time course of replication of a full-length product 7. Figure S6, time course of replication of a full-length product in the presence of MBP-NCL 8. Figure S7, a binding isotherm of frequency changes of QCM 9. Figure S8, an NMR spectrum of PyG3 10. Figure S9, CD analysis of c-kit2 and its oxidative derivatives with PyG3 11. Figure S10, CD melting profiles of VEGF G4 oxidative derivatives with PyG3 12. Figure S11, PAGE images showing the effect PyG3 on replication stalling by VEGF G1O, VEGF G3O, ckit2 G1O, c-kit2 G2O, or c-kit2 G3O 13. Figure S12, PAGE images showing the effect PyG3 on replication stalling by wild-type VEGF G2O or wild-type c-kit2 G2O 14. Figure S13, PAGE images showing the effect of the scaffold sequence on a replication stall 15. Figure S14, CD melting profiles and replication assays of VEGF GXT with PyG3 S 1

2 Table S1 Sequences used in this study a) Name Sequence (5 3 ) VEGF native VEGF G1O VEGF G2O VEGF G3O VEGF G1T VEGF G2T VEGF G3T VEGF template VEGF G1O template VEGF G2O template VEGF G3O template VEGF G5O template VEGF G8O template VEGF G11O template Wild type VEGF G2O template VEGF G1T template VEGF G2T template VEGF G3T template c-kit2 native c-kit2 G1O c-kit2 G2O c-kit2 G3O c-kit2 template c-kit2 G1O template c-kit2 G2O template c-kit2 G3O template Wild type c-kit2 G2O template Bcl2 G2O template Telomere G2O template F-Primer VEGF G2O template with guide PyG3 PyG3-g Random template F-Primer for Random template a) O indicates 8-oxoG CAGGGCGGGCCTTGGGCGGGAT CAOGGCGGGCCTTGGGCGGGAT CAGOGCGGGCCTTGGGCGGGAT CAGGOCGGGCCTTGGGCGGGAT CATGGCGGGCCTTGGGCGGGAT CAGTGCGGGCCTTGGGCGGGAT CAGGTCGGGCCTTGGGCGGGAT CAGGGCGGGCCTTGGGCGGGATCGGACCTATAGTGAGTCGTATTCCC CAOGGCGGGCCTTGGGCGGGATCGGACCTATAGTGAGTCGTATTCCC CAGOGCGGGCCTTGGGCGGGATCGGACCTATAGTGAGTCGTATTCCC CAGGOCGGGCCTTGGGCGGGATCGGACCTATAGTGAGTCGTATTCCC CAGGGCGOGCCTTGGGCGGGATCGGACCTATAGTGAGTCGTATTCCC CAGGGCGGGCCTTGOGCGGGATCGGACCTATAGTGAGTCGTATTCCC CAGGGCGGGCCTTGGGCGOGATCGGACCTATAGTGAGTCGTATTCCC GOGCGGGCCGGGGGCGGGCGGACCTATAGTGAGTCGTATTCCC CATGGCGGGCCTTGGGCGGGATCGGACCTATAGTGAGTCGTATTCCC CAGTGCGGGCCTTGGGCGGGATCGGACCTATAGTGAGTCGTATTCCC CAGGTCGGGCCTTGGGCGGGATCGGACCTATAGTGAGTCGTATTCCC CGGGCGGGCGCTAGGGAGGGT COGGCGGGCGCTAGGGAGGGT CGOGCGGGCGCTAGGGAGGGT CGGOCGGGCGCTAGGGAGGGT CGGGCGGGCGCTAGGGAGGGTCGGACCTATAGTGAGTCGTATTCCC COGGCGGGCGCTAGGGAGGGTCGGACCTATAGTGAGTCGTATTCCC CGOGCGGGCGCTAGGGAGGGTCGGACCTATAGTGAGTCGTATTCCC CGGOCGGGCGCTAGGGAGGGTCGGACCTATAGTGAGTCGTATTCCC GOGCGGGCGCGAGGGAGGGGCGGACCTATAGTGAGTCGTATTCC GOGCGCGGGAGGAATTGGGCGGGCGGACCTATAGTGAGTCGTATTCCC TTAGOGTTAGGGTTAGGGTTAGGGCGGACCTATAGTGAGTCGTATTCCC [FITC]-GGGAATACGACTCACTATAGG GTCCAGCTAAGCATACGAGACAGOGCGGGCCTTGGGCGGGATCGGACCTA TAGTGAGTCGTATTCCC [Pyrene-dU]-GGGTT [Pyrene-dU]-GGGTTTATGCTTAGCTGGA GATTGATGTGATTGATTTGATTGATGTGATTGACCCTATAGTGAGTCGTATT AAGCCGAAGCACTAGTATCATCCC [FITC]-GGGATGATACTAGTGCTTCGGCTTAATACGACTCACTATAGGG S 2

3 Figure S1. CD spectra of 10 µm (A) c-kit2 native and its oxidized sequences (B) c-kit2 G1O, (C) c-kit2 G2O, and (D) c-kit2 G3O. All the experiments were conducted in the buffer consisting of 10 mm Tris- HCl (ph 7.5), 8 mm MgCl 2, and 50 mm KCl. S 3

4 G4 peaks VEGF G3O VEGF G2O VEGF G1O VEGF native G4 peaks c-kit2 G3O c-kit2 G2O c-kit2 G1O c-kit2 native Figure S2. Imino and aromatic regions of 1D 1 H NMR spectra of VEGF, c-kit2, and their oxidized derivatives recorded in 10 mm K 2HPO 4 buffer (ph 7.5) with the addition of 50 mm KCl, 10% 2 H 2O, and 0.1 mm oligonucleotide per strand on an 800 MHz spectrometer. S 4

5 Figure S3. CD melting profiles of (A) 10 µm VEGF native (blue), VEGF G1O (red), VEGF G2O (green), and VEGF G3O (pink), and (B) 10 µm c-kit2 native (blue), c-kit2 G1O (red), c-kit2 (green), and c-kit2 G3O (pink) measured at 265 nm in a buffer consisting of 10 mm Tris-HCl (ph 7.5), 8 mm MgCl 2, and 10 mm KCl. S 5

6 Figure S4. Denaturing-PAGE images of replication products from the c-kit2 template with or without 8-oxoG. All the assays were performed in the buffer consisting of 10 mm Tris-HCl (ph 7.5), 8 mm MgCl 2, and 10 mm KCl with 1 µm primer, 1 µm template, 250 µm dntps, and 1 µm KF exo- at 37 C. S 6

7 Figure S5. Time course analyses of replication of a full-length product from the template DNA containing (A) VEGF native with 10 mm KCl, (B) VEGF G1O with 10 µm PyG3 and 50 mm KCl, (C) VEGF G2O with PyG3 and 50 mm KCl, (D) VEGF G3O with PyG3 and 50 mm KCl, (E) c-kit2 native with 10 mm KCl, (F) c-kit2 G2O with 10 mm KCl, (G) c-kit2 G2O with 50 mm KCl, (H) c-kit2 G3O with 10 mm KCl, (I) c-kit2 G3O with 50 mm KCl, (J) c-kit2 G1O with 10 µm PyG3 and 50 mm KCl, (K) c-kit2 G2O with 10 µm PyG3 and 50 mm KCl, (L) c-kit2 G3O with 10 µm PyG3 and 50 mm KCl, or (M) Random with 10 mm KCl. All the assays were performed in the buffer consisting of 10 mm Tris-HCl (ph 7.5), 8 mm MgCl 2, and 10 or 50 mm KCl with 1 µm primer, template, 250 µm dntps and 1 µm KF exoat 37 C. S 7

8 VEGF native VEGFG1O VEGFG2O VEGFG3O VEGF native + MBP-NCL VEGFG1O +MBP-NCL VEGFG2O +MBP-NCL VEGFG3O +MBP-NCL Figure S6. Time course analyses of the ratio of a full-length product of replication of VEGF native template (black), VEGF G1O template (purple), VEGF G2O template (blue), VEGF G3O (sky blue), VEGF native in the presence of 5 µm MBP-NCL (light blue), VEGF G1O in the presence of 5 µm MBP- NCL (green), VEGF G2O in the presence of 5 µm MBP-NCL (yellow), or VEGF G3O in the presence of 5 µm MBP-NCL (red). All the assays were performed in 10 mm Tris-HCl (ph 7.5), 8 mm MgCl 2, and 10 mm KCl with 1 µm primer, template, 250 µm dntps, and 1 µm KF exo- at 37 C. S 8

9 Figure S7. The binding isotherm of frequency changes of QCM after the binding of VEGF native (red), VEGF G2O (yellow), or VEGF G2O with PyG3 (blue) to MBP-NCL immobilized on the QCM plate. The frequency changes were normalized to the maximum changes at the infinite concentration of DNA. The fitting curves are shown. S 9

10 ppm Figure S8. A 1D 1 H NMR spectrum of PyG3 recorded at 25 C. Oligonucleotide concentration in the NMR tube was 100 µm in 10 mm potassium phosphate buffer (ph 7.5) containing 50 mm KCl. S 10

(A) Fluorescence spectra of PyG3 excited at 386 nm at 37 C in the absence of target

concentration of c-kit2 native and its oxidized derivatives at 37 C.

2.9 µm, c-kit2 G2O; 2.0 µm, and c-kit2 G3O; 2.

11 (A) (B) (C) (D) Figure S9. Regulation of G4 formation in an oxidized c-kit2 sequence using PyG3. (A) Fluorescence spectra of PyG3 excited at 386 nm at 37 C in the absence of target G4 (black) and in the presence of c- Kit2 (blue), c-kit2 G1O (red), c-kit2 G2O (green), or c-kit2 G3O (pink). (B) Fluorescence changes of 10 µm PyG3 measured at 448 nm with an increasing concentration of c-kit2 native and its oxidized derivatives at 37 C. The line indicates data fitted to obtain the dissociation constant (K d: c-kit2 G1O; 2.9 µm, c-kit2 G2O; 2.0 µm, and c-kit2 G3O; 2.9 µm) (C) CD spectra of 10 µm c-kit2 G1O, c-kit2 G2O, or c-kit2 G3O, and (D) CD melting profiles of each c-kit2 derivative. All the experiments were conducted in the buffer consisting of 10 mm Tris-HCl (ph 7.5), 8 mm MgCl 2, and 50 mm KCl in the presence of 10 µm PyG3. S 11

12 Figure S10. CD melting profiles of 10 µm VEGF G4 oxidative derivatives with 10 µm PyG3 that were acquired at 265 nm in a buffer consisting of 10 mm Tris-HCl (ph 7.5), 8 mm MgCl 2, 140 mm KCl, and 12 mm NaCl. S 12

13 VEGF G1O VEGF G3O c-kit2 G1O c-kit2 G2O c-kit2 G3O Time (min) 0 1 F S P Figure S11. Denaturing-PAGE images of replication of VEGF G1O, VEGF G3O, c-kit2 G1O, c-kit2 G2O, and c-kit2 G3O. The stalled product is indicated with the red box. The letters F, S, and P on the left side of the gel image indicate a Full-length product, Stalled product, and Primer, respectively. S 13

14 Figure S12. Denaturing PAGE images of replication of wild-type VEGF G2O and wild-type c-kit2 G2O in the absence or presence of PyG3. The stalled product is indicated with the red box. The letters F, S, and P on the left side of the gel image denote a Full-length product, Stalled product, and Primer, respectively. S 14

15 Figure S13. The ability of a guide sequence to stall more effectively at the intermolecular G4 on a template DNA. Schematic illustration of (A) the structure of designed sequences, (B) replication along the VEGF G2O template in the presence of PyG3, (C) replication along the VEGF G2O template in the presence of PyG3-g, and (D) a dissociation assay of PyG3-g on QCM. S 15

Figure S14. Recovery of G4 formation by VEGF GXT with PyG3. (A) CD spectra of 10 µm VEGF G1T, VEGF G2T, and VEGF G3T and (B) CD melting profiles of each VEGF derivative with 10 µm PyG3.

All the thermodynamic parameters in the presence of PyG3 were determined (VEGF G1T: T m = 67.9 C, - G 37 = 4.1 kcal mol -1 ; VEGF G2T: T m = 65.3 C, - G 37 = 4.2 kcal mol -1 ; VEGF G3T: T m = 68.

16 Figure S14. Recovery of G4 formation by VEGF GXT with PyG3. (A) CD spectra of 10 µm VEGF G1T, VEGF G2T, and VEGF G3T and (B) CD melting profiles of each VEGF derivative with 10 µm PyG3. All the experiments were conducted in the buffer consisting of 10 mm Tris-HCl (ph 7.5), 8 mm MgCl 2, and 50 mm KCl in the presence of 10 µm PyG3. All the thermodynamic parameters in the presence of PyG3 were determined (VEGF G1T: T m = 67.9 C, - G 37 = 4.1 kcal mol -1 ; VEGF G2T: T m = 65.3 C, - G 37 = 4.2 kcal mol -1 ; VEGF G3T: T m = 68.1 C, - G 37 = 5.0 kcal mol -1 ). (C) Denaturing PAGE images of replication of VEGF GXT in the absence or presence of PyG3. The stalled product is indicated with the red box. The letters F, S, and P on the left side of the gel image indicate the Full-length product, Stalled product, and Primer, respectively. S 16

Destabilization of DNA G-quadruplexes by chemical environment. changes during tumor progression facilitates transcription

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