Supporting Information. Quencher Group Induced High Specificity Detection of. Telomerase in Clear and Bloody Urines by AIEgens

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1 Supporting Information Quencher Group Induced High Specificity Detection of Telomerase in Clear and Bloody Urines by AIEgens Yuan Zhuang, a Mengshi Zhang, a Bin Chen, c Ruixue Duan, a Xuehong Min, a Zhenyu Zhang, a Fuxin Zheng, b Huageng Liang, b Zujin Zhao, c Xiaoding Lou, a * and Fan Xia a a Key Laboratory for Large-Format Battery Materials and System, Ministry of Education, School of Chemistry and Chemical Engineering, Huazhong University of Science and Technology, Wuhan , China b Department of Urology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan , China c State Key Laboratory of Luminescent Materials and Devices, South China University of Technology, Guangzhou , China * Correspondence and requests for materials should be addressed to X. L. ( louxiaoding@hust.edu.cn). S-1

2 Table of contents: 1 Experimental section 1.1 Synthesis of Silole-R 1.2 Fluorescence measurement 1.3 Investigation of telomerase inhibition 1.4 Assay of S1 Nase degradation 1.5 Non-denaturating polyacrylamide gel (PAGE) analysis 2 Figures & Tables Scheme S1. Synthetic route to Silole-R Figure S1. Mass Spectrum of Compound 3 Figure S2. FRET effect certification between Silole-R and Dabcyl Table S1. Oligonucleotides used in this study Figure S3. Fluorescence spectra in the presence of QP or TP with or without telomerase Figure S4. Fluorescence spectra as time changes Figure S5. Fluorescence spectra in the presence of different reagents Figure S6. Fluorescence spectra in the absence and presence of Silole-R Figure S7. Linear relationship in optical density at wavelength of 450 nm against standard telomerase Figure S8. Histogram for telomerase concentration (activity) of E-J cells obtained from commercial telomerase ELISA Kit and (I/I 0 )-1 value of our method Figure S9. Enhancement multiples of fluorescence intensity before and after adding telomerase in the presence of QP, TP and the difference between them Table S2. Specific values between fluorescence enhancement times of active E-J telomerase and other interferents Figure S10. Fluorescence intensity enhancement ratio in response to telomerase extracted from urine specimens of 15 normal people Table S3. Corresponding disease information of bladder cancer urine samples Table S4. Corresponding information of urine samples from normal people Figure S11. Histogram for telomerase concentrations (activity) of HeLa cells and bloody specimen obtained from commercial telomerase ELISA Kit and (I/I 0 )-1 value of our method S-2

3 1 Experimental section 1.1 Synthesis of Silole-R The synthetic route to Silole-R is shown in Scheme S1. Compound 3 was successfully synthesized according to the procedures below: A solution of lithium 1-naphthalenide (LiNaph) was prepared by stirring a mixture of naphthalene (1.28 g, 10 mmol) and lithium granular (0.07 g, 10 mmol) in dry THF (10 ml) for 4 h at room temperature under nitrogen atmosphere. Then, bis(phenylethynyl)dimethylsilane 1 (0.65 g, 2.5 mmol) dissolved in dry THF (10 ml) was added dropwise into the solution of LiNaph, and the resultant mixture was stirred for 30 min at room temperature. After cooling to 10 o C, the solution was added ZnCl 2 -TMEDA (3.2 g, 12.5 mmol), and subsequent additional THF (10 ml). After the formed fine black suspension was stirred for 1 h, Pd(PPh 3 ) 2 Cl 2 (0.1 g) and 1-(4-bromophenyl)-N,N-dimethylmethanamine (0.81 ml, 5 mmol) were added. After reflux for 12 h, the reaction was cooled to room temperature and terminated by addition of 2 M hydrochloric acid. The mixture was poured into water and extracted twice with dichloromethane. The combined organic layers were washed successively with aqueous sodium chloride solution and water and then dried over magnesium sulfate. After filtration, the solvent was evaporated under reduced pressure and the residue was purified by silica-gel column chromatography using CH 2 Cl 2 /CH 3 OH as eluent. Green solid of compound 3 was obtained in 56% yield. 1 H NMR: (500 MHz, CDCl 3 ), (TMS, ppm): 7.07 (d, 4H, J = 8.0 Hz), (m, 6H), 6.90 (d, 4H, J = 8.0 Hz), 6.82 (dd, 4H, J 1 = 7.5 Hz, J 2 = 2.5 Hz), 3.35 (s, 4H), 2.23 (s, 12H), 0.49 (s, 6H). 13 C NMR: (125 MHz, CDCl 3 ), (TMS, ppm): 153.9, 141.4, 139.0, 138.7, 135.9, 130.0, 128.8, 128.7, 127.4, 126.1, 64.1, 45.3, 3.8. Mass Spectrum of Compound 3 is shown in Figure S1. Compound Silole-R was successfully synthesized according to the procedures below: Compound 3 (0.53 g, 1 mmol) and bromoethane (0.15 ml, 2 mmol) dissolved in acetone (20 ml) were stirred for 2 days at room temperature. The salt precipitated from solution, and then the product was obtained through vacuum filtration. Virescent solid of compound Silole-R was obtained in 94% yield. 1 H NMR: (500 MHz, DMSO), (TMS, ppm): 7.35 (d, 4H, J = 8.0 Hz), (m, 6H), 7.01 (d, 4H, J = 8.0 Hz), (m, 4H), 4.45 (s, 4H), 3.30 (m, 4H), 2.89 (s, 12H), 1.31 (t, 6H, J = 7.0 Hz), 0.51 (s, 6H). 13 C NMR: (125 MHz, DMSO), (TMS, ppm): 155.1, 141.4, 138.6, 133.1, 129.8, 129.1, 128.2, 127.2, 125.9, 65.9, 59.3, 49.0, 8.5, Fluorescence measurement The fluorescence detection was carried out on a Cary eclipse fluorescence spectrophotometer (Agilent Technologies). The excitation wavelength was 360 nm and the emission wavelengths were in the range from 400 to 600 nm. S-3

4 1.3 Investigation of telomerase inhibition In the telomerase inhibition experiments, 1 mm 3 -azido-3 -deoxythy-midine (AZT) was added into telomerase extracts from E-J cancer cells to incubated at 37 ºC for 30 min before telomerase used. Then the AZT-treated telomerase was used in a similar way as active telomerase. 1.4 Assay of S1 Nase degradation In the assay of S1 Nase degradation, firstly, telomerase extracted from E-J cancer cells was added into mixture of QP, dntps and recombinant RNase inhibitor. After incubation at 37 ºC for 60 min and 95 ºC for 10 min, Silole-R was added to solution with final concentration of 10 μm and total volume of 200 μl. Subsequently, S1 Nase with activity of 18 U was added into the solution, then the mixture was incubated at 37 ºC for 10 min and 95 ºC for 10 min. Afterwards, S1 Nase with activity of 18 U was added into the solution for the second time before incubation at 37 ºC for 10 min and 95 ºC for 10 min. Fluorescence spectra of this solution were measured every time after incubation. 1.5 Non-denaturating polyacrylamide gel (PAGE) analysis Telomerase extracts from E-J cells, HeLa cells and MCF-7 cells were incubated with 4.8 mm dntps, 9.5 μm QP and 20 U RNase inhibitor in telomerase extension reaction buffer with the total volume of 10.5 μl at 37 ºC for 60 min. For the control experiment, 9.5 μm QP in telomerase extension reaction buffer with the total volume of 10.5 μl was incubated at 37 ºC for 60 min. A 10% non-denaturing PAGE analysis of 5 μl mixture and 1 μl 6X loading buffer was carried out in 1X TEB buffer at 90 V constant voltage for about 1 h. Then the gels were stained with 3X Gel Red in 50 ml 1X TBE buffer for 30 min and imaged with Gel-Pro Transilluminator 2020D (Carestream Health). S-4

5 Scheme S1. Synthetic route to Silole-R. S-5

6 Figure S1. Mass spectrum of compound 3. S-6

7 Figure S2. FRET effect certification between Silole-R and Dabcyl. The emission of Silole-R (λ max = 478 nm, blue line) and the absorption of Dabcyl (λ max = 480 nm, black line) have a high degree of overlap. S-7

8 Table S1. Oligonucleotides used in this study. Name Sequence (5' to 3') Length (nt) Purify method QP (Dabcyl)-AATCCG TCGAGC AGAGTT 18 HPLC TP AATCCG TCGAGC AGAGTT 18 HPLC S-8

9 Figure S3. (a) Fluorescence emission spectra of Silole-R and QP (TP) in the presence and absence of telomerase extracted from E-J cancer cells. Enhancement multiples of fluorescence intensity at 478 nm before and after adding telomerase to the system are marked in the figure. (b) Fluorescence intensity at 478 nm of Silole-R and QP (TP) in the presence and absence of telomerase extracted from E-J cancer cells. The concentration of Silole-R is 10 μm. S-9

10 Figure S4. (a) Fluorescence emission spectra of Silole-R in the presence of telomerase extracted from E-J cancer cells with different telomerase extension reaction time in the range from 0 to 120 min. (b) Relationship between fluorescence intensity (at 478 nm) of complex S-QP and telomerase extension reaction time. The concentration of Silole-R is 10 μm. S-10

11 Figure S5. Fluorescence emission spectra of Silole-R in the absence (black) and presence of QP (red), dntps (blue) or telomerase extracted from E-J cancer cells (pink), respectively or all (green). The concentration of Silole-R is 10 μm. S-11

12 Figure S6. Fluorescence emission spectra of QP, dntps and telomerase extracted from E-J cancer cells in the absence (black) and presence (red) of Silole-R with concentration of 10 μm. S-12

13 Figure S7. Linear relationship in optical density (OD) at wavelength of 450 nm against standard telomerase at a concentration range of 0-40 IU/L. S-13

14 Figure S8. Histogram for telomerase concentration (activity) obtained from commercial telomerase ELISA Kit and (I/I 0 )-1 value of our method in detecting the activity of telomerase extracted from 10, 100, and 1000 E-J cancer cells, respectively. S-14

15 Figure S9. Enhancement multiples of fluorescence intensity at 478 nm before and after adding telomerase to the system in the presence of QP (black square), TP (red circle) and the difference between them (green triangle). Telomerase used in this assay are extracted from different number of E-J cancer cells in the range from 0 to cells. The concentration of Silole-R is 10 μm. S-15

16 Table S2. Specific values between fluorescence enhancement ratios of active E-J telomerase and other interferents. Signal increase specific value Interferent compared to active E-J telomerase QP TP HLF telomerase AZT-treated HeLa telomerase Lysis buffer Bst DNA polymerase Trypsin Thrombin BSA S-16

17 Figure S10. Fluorescence intensity enhancement ratios between specimens and blanks at 478 nm in response to telomerase extracted from urine specimens of 15 normal people. Error bars indicate standard deviation of triplicate tests. S-17

18 Table S3. Corresponding disease information of bladder cancer urine samples. Sample No. Urine appearance I/I 0 at 478 nm Relative standard deviations (RSD) Detection 1 Clear Yes 2 Clear Yes 3 Clear Yes 4 Clear Yes 5 Clear Yes 6 Clear Yes 7 Clear Yes 8 Clear Yes 9 Clear Yes 10 Clear Yes 11 Clear Yes 12 Clear Yes 13 Clear Yes 14 Clear Yes 15 Clear Yes 16 Clear Yes 17 Clear Yes 18 Clear Yes 19 Clear Yes 20 Bloody Yes 21 Bloody Yes 22 Bloody Yes 23 Bloody Yes 24 Bloody Yes 25 Bloody Yes 26 Bloody Yes 27 Bloody Yes 28 Bloody Yes 29 Bloody Yes 30 Bloody Yes 31 Bloody Yes 32 Bloody No 33 Bloody No 34 Bloody Yes 35 Bloody Yes 36 Bloody Yes 37 Bloody Yes 38 Bloody Yes S-18

19 Table S4. Corresponding information of urine specimens from normal people. Sample No. I/I 0 at 478 nm Relative standard deviations (RSD) S-19

20 Figure S11. Histogram for telomerase concentration (activity) obtained from commercial telomerase ELISA Kit and (I/I 0 )-1 value of our method in detecting the activity of telomerase extracted from HeLa cancer cells and bloody urine specimen (No.30), respectively. S-20