Supplementary information

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1 Supplementary information ligonucleotide models of telomeric DA and RA form a hybrid G-quadruplex structure as a potential component of telomeres Yan Xu*, Takumi Ishizuka, Jie Yang, Kenichiro Ito, Hitoshi Katada, Makoto Komiyama and Tetsuya Hayashi Division of Chemistry, Department of Medical Sciences, Faculty of Medicine, University of Miyazaki, 5200 Kihara, Kiyotake, Miyazaki, , Japan Research Center for Advanced Science and Technology, The University of Tokyo, Komaba, Meguro-ku, Tokyo , Japan. Supplementary Fig. S1. (a) CD spectrum of DA-1 (10 µm) in the presence of 100 mm KCl, 5 mm Tris-HCl buffer (ph 7.0) at 25 ºC. (b) CD melting curve for DA-1 monitored at 280 nm in the presence of 100 mm KCl, 5 mm Tris-HCl buffer (ph 7.0). Supplementary Fig. S2. CD melting curves for RA-1 and DA-2 complex monitored at 265 nm in the presence of 100 mm KCl (Tm = 65 C).

2 Supplementary Fig. S3. MALDI-TFMS spectrum of DA RA G-quadruplex formed by DA-2 and RA-1 in the presence of 200 mm acl. RA-1/DA-2 Supplementary Fig. S4. ne-dimensional 600 MHz spectrum ( ppm) of the RA-1 and DA-2 complex in 90% H 2 /10% D 2 solution containing 200 mm KCl and 10 mm K-phosphate (upper) or 200 mm acl and 10 mm a-phosphate (bottom), ph 6.8, 25 C.

3 Supplementary Fig. S5. (a) A fluorescence resonance energy transfer (FRET) molecular beacon to investigate telomeric DA RA G-quadruplex formation. Fuorophore reporter FAM and quencher (DABCYL) were incorporated to 5 and 3 ends of DA 5 -FAM-CCTACGTTGTTAGGGTTAGGGTAGG-DABCYL-3, containing telomere DA sequence 5 -TTAGGGTTAGGGT-3. (b) Fluorescence spectra for molecular beacon without and with telomere RA 5 -UAGGGUUAGGGU-3. Supplementary Fig. S6. (a) Schematic for detection of G-quadruplex formation using molecular beacon. (b) Fluorescence signal of molecular beacon in cells. ph: phase-contrast imaging.

4 Synthesis of 7-azidocoumarin-labeled oligonucleotide (D-3). Scheme 1 H 2 H AcEt Cl H 1 2 H H H H 2 S 4 H 3 C 2 H ah H 2 a 2 a 3 3 C 2 H C 2 H H 3 3--(Carbethoxy)aminophenol (2) To a 1 L flask fitted with a stir bar were added 3-aminophenol 1 (15 g, mmol) and ethyl acetate (50 ml). The flask was fitted with a condenser, and the mixture was heated to reflux for 30 min. Ethyl chloroformate (6.54 ml, 7.46 g, 68.7 mmol) was added dropwise over a 1 h period. The reaction mixture was allowed to cool to room temperature at which time a white precipitate formed. The precipitate was removed by filtration and washed with ethyl acetate (3 30 ml) and diethyl ether (3 30 ml). The combined filtrate was concentrated to afford 12.4 g (99%) of 2 as a white solid. 1 H-MR (500 MHz, DMS-d 6 ): δ 1.24 (t, J = 7.1 Hz, 3H), 4.11 (q, J = 7.1 Hz, 2H), (m, 1H), (m, 1H), (m, 2H), 9.31 (br s, 1H), 9.46 (s, 1H). 7--(Carbethoxy)aminocoumarin-4-acetic acid (3) To a 5 L flask fitted with a stir bar were added 2 (12.4 g, 68.4 mmol) and 70% H 2 S 4 (275 ml). The rapidly stirred reaction mixture was cooled in an ice bath, and 1,3-acetonedi- carboxylic acid (11.0 g, 75.3 mmol) was added in portions. The reaction mixture was allowed to warm to room temperature and stirred for 8 h, after which the reaction mixture was poured onto ice (400 g) and stirred for 30 min. The resultant white precipitate was collected by filtration and washed with diethyl ether (3 200 ml). The crude material was then partially dissolved in hot CH 3 C (100 ml). Upon cooling to room temperature, the precipitate was collected to afford 8.2 g (63%) of 3 as a white solid. 1 H-MR (500 MHz, DMS-d 6 ): δ 1.29 (t, J = 7.1 Hz, 3H), 3.87 (s, 2H), 4.19 (q, J = 7.1 Hz, 2H), 6.33 (s, 1H), (m, 1H), (m, 2H), (s, 1H). 7-Aminocoumarin-4-acetic Acid (4). To a 500 ml flask fitted with a condenser were added 3 (8.2 g, 28.2 mmol), ah (11.3 g, mmol), and H 2 (70 ml). The reaction mixture was stirred at reflux for 16 h. After cooling to room temperature, the ph of the reaction mixture was adjusted to 2.0, by the dropwise addition of conc. HCl. The resultant yellow precipitate that formed was collected by filtration to afford 5.1 g (83%) of 4 as a fluffy yellow powder. 1 H MR (500 MHz, DMS-d 6 ): δ 3.73 (s, 2H), 5.98 (s, 1H), 6.15 (br s, 2H), 6.42 (d, J = 1.9 Hz, 1H), 6.54 (dd, J = 1.9, 8.7 Hz, 1H), 7.33 (d, J = 8.7 Hz, 1H), (br s, 1H).

5 7-azidocoumarin-4-acetic acid (5) 7-aminocoumarin-4-acetic acid 4 (2.1 g, 9.58 mmol) was suspended in H 2 (45 ml) and cooled to 0 C. conc. H 2 S 4 (16 ml) was added slowly to maintain the reaction at 0 C and the solution became homogeneous. A solution of sodium nitrite (0.79 g, 11.5 mmol) in H 2 (14 ml) was added drop wise over 30 min and the mixture was stirred 1h at 0 C. A solution of sodium azide (1.03 g, 15.8 mmol) in H 2 (8 ml) was added drop wise to the reaction mixture and it was stirred. overnight while warming up to room temperature. The product precipitated as dark-yellow solid. It was filtered on glass fritted funnel, washed extensively with water and dried in vacuo to remove traces of water thus affording 1.65 g (70%) of pure 5. 1 H MR (DMS-d 6 ): δ 3.91 (s, 2H), 6.44 (s, 1H), 7.14 (d, J = 8.5 Hz, 1H), 7.19 (s, 1H), 7.72 (d, J = 8.5 Hz, 1H). Succinimidyl-7-azidocoumarin (6),'-diisopropylcarbodiimide (DIC) (0.89 g, 4.3 mmol) was added to a suspension of 5 (1.00 g, 4.08 mmol) and -hydroxysuccinimide (0.56 g, 4.89 mmol) in DMF (20 ml) at room temperature, and the reaction was left to stir. After 4 h, the solvent was removed in vacuo. Then saturated aqueous KCl (20 ml) was added, and the organic layer was separated, washed with water, dried over a 2 S 4 and filtered. The solvent was removed in vacuo. Upon purification by column chromatography (1:2, n-hexane/ethyl acetate) the title compound was isolated as a white solid (0.47 g, 34%). 1 H MR (CDCl 3 ): δ 2.86 (s, 4H), 4.05 (s, 2H), 6.46 (s, 1H), (m, 2H), 7.58 (d, J = 8.1 Hz, 1H). ESI-MS: [M+H] + calcd, ; found, As chromatography resulted in partial decomposition and the product was pure by LCMS, the compound was used for coupling with DA oligomers after careful drying without further purification. ESI-MS spectrum of compound 6

6 1 H-MR spectrum of compound 6 3 a b c d 6 e f CHCl 3 H 2 TMS (f) (a), (b) (c) (d) (e)

7 Synthesis of 7-azidocoumarin-labeled oligonucleotide. To incorporate the azido group at the 5 -end of C6-aminoalkyl oligonucleotide, 30 nmol of the oligonucleotide in 300 µl of 0.5 M a 2 C 3 /ahc 3 buffer (ph 9.2) was incubated for 12 h at room temperature with 10 µmol of Succinimidyl-7-azidocoumarin 6 in 80 µl of DMS. The crude oligonucleotide was purified by reversed-phase HPLC. MALDI-TF-MS: calcd, ; found, Scheme 2 Synthesis of 5 -alkyne labeled oligonucleotides (RA-2). ligonucleotide with 5 -alkyne was prepared by phosphoamidite method on controlled pore glass supports (1 µmol). After automated synthesis, the glass supports were treated with a mixture of saturated 30% ammonia aq. at 50 C for 16 h. Detached and deprotected oligomers were evaporated to dryness, then remove the TBDMS-protecting group, and purified by RP-HPLC. The oligomer was characterized by MALDI-TF MS. Scheme 3 5' 3' UAGGGUUAGGGU (i) P C P 5' 3' UAGGGUUAGGGU C (ii) P 5' 3' UAGGGUUAGGGU C RA-2