Obtaining More Accurate Signals: Spatiotemporal Imaging of Cancer Sites Enabled by a Photoactivatable Aptamer-Based Strategy

Transcription

1 Supporting Information Obtaining More Accurate Signals: Spatiotemporal Imaging of Cancer Sites Enabled by a Photoactivatable Aptamer-Based Strategy Heng Xiao,,, Yuqi Chen,, Erfeng Yuan,, Wei Li, Zhuoran Jiang, Lai Wei, Haomiao Su, Weiwu Zeng, Yunjiu Gan, Zijing Wang, Bifeng Yuan, Shanshan Qin, Xiaohua Leng, Xin Zhou, Songmei Liu*, and Xiang Zhou*, College of Chemistry and Molecular Sciences and Institute of Advanced Studies, Wuhan University, Wuhan, Hubei , P. R. China Zhongnan Hospital, Wuhan University, Wuhan, Hubei , P. R. China Research Center for Tissue Engineering and Regenerative Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan , China These authors contributed equally. * Address correspondence to xzhou@whu.edu.cn or smliu@whu.edu.cn S-1

2 Table of Contents Experimental Methods. Figure S1. Molecular design of ACTG. Figure S2. Oligonucleotide conjugation and photoactivation of caged fluorescent oligonucleotide. Figure S3. The maximum excitation and emission spectra of ACTG. Figure S4. Change of fluorescence intensity of ACTG upon UV irradiation. Figure S5. Photographs of the solution of ACTG took under the UV light irradiation Figure S6. Change of absorption spectra of ACTG upon UV light irradiation Figure S7. The ESI-TOF results of azide-as1411, CTG-AS1411 before and after UV irradiation. Figure S8. Absorption spectra of CTG-AS1411. Figure S9. The native PAGE analysis of CTG-AS1411 upon UV light irradiation. Figure S10. Relative fluorescence intensity of ACTG upon 408 nm or 488 nm light irradiation. Figure S11. Spatiotemporally controlled imaging in intact living cells. Figure S12. Spatiotemporally controlled imaging of MCF-7 cells after CTG-AS1411 or CTG-random sequence incubation. Figure S13. Flow Cytometric Analysis. Figure S14. The native PAGE analysis of the nuclease resistance ability of the aptamer probe. Figure S15. Photoactivatable fluorescent detection in FFPE 4T1 model nude mice tissue specimens using CTG-AS1411 probe. Figure S16. Spatiotemporally controlled imaging of FFPE tissue sections. Figure S17. Spatiotemporally controlled imaging of FFPE breast cancer tumour tissue sections using CTG-AS1411 probe. Figure S18. Spatiotemporally controlled imaging of FFPE breast cancer tumour tissue sections using FAM-AS1411 probe. Scheme S1. Synthesis of ACTG. Synthesis process of ACTG. Figure S19. The 1 H NMR spectra of ACTG. Figure S20. The 13 C NMR spectra of ACTG. Figure S21. The high-resolution mass spectra of ACTG. S-2

3 Experimental Methods Confocal microscopy imaging for adherent cell For confocal microscopy imaging, MCF-7 and CHO cells were plated in a 35-mm confocal dish (Nest, China) for 24 h and grown to around 30% confluence prior to experiments. Then cells were washed three times with PBS and incubated with 300 nm CTG-AS1411 probe or random probe at 37 C in 5% CO 2 for 40 min. Cells were then washed three times with PBS to remove the excess probes and immersed in 1 ml PBS. Then, confocal imaging was acquired on Nikon Confocal Laser Scanning Microscope (TE2000, Japan) with an objective lens ( 60). The fluorescent images were taken with green filter (excitation: 488 nm) at 15.0 % power. Cells were activated with scans from the 488 nm laser at 15.0 % power or 408 nm laser at 1.0 % power for 20 s one time. Images and merges were obtained with EZ-C1 software. Fluorescence intensity was analyzed by imagej software. Confocal microscopy imaging for cells in serum Living MCF-7 and CHO cells were scraped from culture dishes, then CTG-AS1411 probe (300 nm) was incubated with 200 μl unmodified human healthy serum (Zhongnan Hospital of Wuhan University, School of Medicine) containing cells at 37 for 40 min. After centrifugation at 2000 rpm for 2 min, the cells were collected and washed with PBS 3 times. Subsequently, the cells were resuspended in 1 PBS and maken into slide samples for confocal imaging. The fluorescence probes captured by cells were photoirradiated according to the same procedure as that described in the adherent cell confocal imaging section. Confocal microscopy imaging for cells in human whole blood Living MCF-7 and CHO cells were scraped from culture dishes, then CTG-AS1411 probe (300 nm) was incubated with 180 μl PBS mixed with 20 μl human whole blood (Zhongnan Hospital of Wuhan University, School of Medicine) containing cells at 37 for 40 min. The cells were incubated with the probes and treated with or without photoirradiation according to the same procedure as that described in the confocal microscopy imaging for cells in serum section. Photoactivatable fluorescence imaging in frozen tissue sections. Frozen human breast cancer tumor tissue and human benign breast tissue sections (Zhongnan Hospital of Wuhan University) were incubated with the probes and treated with or without photoirradiation according to the same procedure as that described in confocal imaging of FFPE tissue section. S-3

4 Figure S1: Molecular design of ACTG. Figure S2: Oligonucleotide conjugation and photoactivation of caged fluorescent oligonucleotide. S-4

5 Fluorescence intensity (a. u.) Ex Em Wavelength (nm) Figure S3: The maximum excitation and emission spectra of ACTG (Ex: 488 nm, Em: 513 nm) (sample concentration: 1 mm) in 10 mm phosphate buffered saline, ph 7.4, containing 0.1% DMSO as a cosolvent. Figure S4: Change of fluorescence intensity of ACTG upon UV irradiation. (A) Change of fluorescence spectra of ACTG (sample concentration: 1 mm) upon UV irradiation (365 nm) in 10 mm phosphate buffered saline, ph 7.4, containing 0.1% DMSO as a cosolvent. (B) Change of fluorescence intensity of ACTG at the fluorescence maxima wavelength (516 nm) upon irridiation. For fluorescence, λ ex = 488 nm. S-5

6 Figure S5: Photographs of the solution of ACTG took under the UV light irradiation (sample concentration: 100 mm) in 10 mm phosphate buffered saline, ph 7.4, containing 0.1% DMSO as a cosolvent. Figure S6: Change of absorption spectra of ACTG (sample concentration: 1 mm) upon UV light irradiation (303 nm) in 10 mm phosphate buffered saline, ph 7.4, containing 0.1% DMSO as a cosolvent. S-6

7 Absorbance (a. u.) Figure S7: The ESI-TOF results of azide-as1411 (A), CTG-AS1411 before (B) and after (C) 365 nm UV light irradiation wavelength (nm) Figure S8: Absorption spectra of CTG-AS1411 (sample concentration: 1 μm, 10 mm phosphate buffered saline, ph=7.4). S-7

8 Figure S9: The native PAGE analysis of CTG-AS1411 upon UV light irradiation. (a) Fluorescence image of an native PAGE gel after irradiation. Marker was FAM-labeled oligonucleotide, FAM-AS1411 with same sequence. The solutions of CTG-AS1411 were irradiated with UV light (365 nm) for various periods of time (0, 5, 10, 15, 20, 25, 30 min). (b) PAGE-gel (a) upon 30min UV irradiation (365 nm). (c) Fluorescence intensity analysis of gel (a). (d) Fluorescence intensity analysis of gel (b). S-8

9 Figure S10: Relative fluorescence intensity of ACTG after 408 nm (A) or 488 nm (B) light irradiation for various periods of time. (sample concentration: 100 μm, 10 mm PBS, ph=7.4) S-9

10 Figure S11: Spatiotemporally controlled imaging in intact living cells in PBS buffer (A), human serum (B) and whole blood (C) (arrows point to leukocyte in the whole blood) : DIC (panel 1), time-lapsed fluorescence (panel 2-8), and merged DIC/fluorescence (panel 9) images of MCF breast cancer cells (top row) and CHO normal cells (bottom row) upon 488nm or 408nm laser illumination at 15% power. All cells were treated with 300 nm of CTG-AS1411. Scale bar = 20 μm. DIC: bright-field images. (D-F) Fluorescence density analysis of (A-C) by ImageJ software. S-10

11 Figure S12: Spatiotemporally controlled imaging of MCF-7 cells after CTG-AS1411 or CTG-random sequence incubation in each image. Scale bar = 10 μm. DIC: bright-field images. S-11

12 Figure S13: Flow Cytometric Analysis of MCF breast cancer cells (top row) and CHO normal cells (bottom row) treated with CTG-random aptamer and CTG-AS1411 respectively before and after 365nm UV light irradiation. S-12

13 Figure S14: The native PAGE analysis of the nuclease resistance ability of the aptamer probe. (a) Fluorescence image of a native PAGE gel of FAM-AS1411, CTG-AS1411 and CTG-random DNA sequences upon different concentration (0, 0.2 U, 0.5 U, 1 U, 2 U) of S1 nuclease treatment. Lane 1-5: FAM-AS1411 with different concentration (0, 0.2 U, 0.5 U, 1 U, 2 U) of DNase I treatment; Lane 6-10: CTG-AS1411 with different concentration (0, 0.2 U, 0.5 U, 1 U, 2 U) of DNase I treatment; Lane 11-15: CTG-random with different concentration (0, 0.2 U, 0.5 U, 1 U, 2 U) of DNase I treatment. (b) PAGE-gel (a) upon 30 min UV irradiation (365 nm). S-13

14 Figure S15: Photoactivatable fluorescent detection in paraffin-embedded 4T1 model nude mice tissue specimens using CTG-AS1411 probe. Spatiotemporally controlled imaging of formalin-fixed 4T1 nude mice tumour tissue sections: DIC (panel 1), DAPI (panel 2), time-lapsed fluorescence (panels 3-9), and merged DAPI/fluorescence (panel 10) images of 4T1 model nude mice tumor tissue sections (top row) and nude mice normal tissue sections (bottom row) upon 488 nm illumination and subsequently 408 nm. All tumour sections embedded in paraffin were stained by 300 nm CTG-AS1411 probe. A 20 s 488 nm or 408 nm laser was applied to the selected area. Scale bar = 20 μm. DIC: bright-field images. S-14

15 Figure S16: Spatiotemporally controlled imaging of FFPE tissue sections: DIC (panel 1), fluorescence before (panel 2) and after (panel 3) laser light irradiation, and merged DIC/fluorescence (panel 4) images of 2 cases breast cancer tumor sections (A, B) and 3 cases benign hyperplastic breast tumor sections (C, D, and E) upon 488 nm and subsequently 408 nm illumination. All tumor sections embedded in paraffin were stained by 300 nm CTG-AS1411 probe. A 20 s 488nm or 408nm laser was applied to the selected area. Scale bar = 40 μm. DIC: bright-field images. (F) Fluorescence density analysis of (A-E) by ImageJ software. S-15

16 Figure S17: Spatiotemporally controlled imaging of FFPE breast cancer tumour tissue sections using CTG-AS1411 probe: DIC (panel 1), time-lapsed fluorescence (panels 2-8), and merged DIC/fluorescence (panel 9) images of breast cancer tumour sections upon 488 nm illumination and subsequently 408 nm. All FFPE tumour sections were stained by 300 nm CTG-AS1411 probe. A 20 s 488 nm or 408 nm laser was applied to the selected area. Scale bar = 20 μm. DIC: bright-field images. S-16

17 Figure S18: Spatiotemporally controlled imaging of FFPE breast cancer tumour tissue sections using FAM-AS1411 probe: DIC (panel 1), time-lapsed fluorescence (panels 2-8), and merged DIC/fluorescence (panel 9) images of breast cancer tumour sections upon 488 nm illumination and subsequently 408 nm. All FFPE tumour sections were stained by 300 nm FAM-AS1411 probe. A 20 s 488 nm or 408 nm laser was applied to the selected area. Scale bar = 20 μm. DIC: bright-field images. S-17

18 Scheme S1: Synthesis of ACTG Synthesis process of ACTG TG-NPE was synthesized by following a literature procedure. 1 TG-NPE 52.6 mg (0.1 mmol), EDCI 20 mg (0.1 mmol), HOAT 7 mg (0.05 mmol) were suspended in DMF (5mL) and propargylamine 11 mg (0.2 mmol) in DMF (1 ml) was added with stirring. The reaction mixture was stirred for 12 hours at room temperature under an Ar atmosphere. After the vacuum evaporation of the solvent, the residue was purified on a silica gel column (CH 2 Cl 2 /MeOH = 97/3) to afford compound ACTG 39 mg (0.07 mmol, 70%, orange solid). 1 H-NMR (300 MHz, CDCl 3 ) δ: 1.75 (s, 3H, J = 4.2 Hz), 2.02 (d, 3H), 2.29 (s, 1H), 4.19 (d, 2H, J = 2.1 Hz), 4.58 (s, 1H), 6.18 (q, 1H, J = 6 Hz), 6.40 (s, 1H), 6.53 (d, 1H, J = 9.9 Hz), 6.71 (t, 1H, J = 8.1 Hz), (m, 4H), 7.07 (d, 1H, J = 8.4 Hz), 7.26 (s, 1H), 7.48 (t, 1H, J = 7.5 Hz), 7.63 (t, 1H, J = 6 Hz), 7.71 (d, 1H, J = 6.9 Hz), 8.06 (d, 1H, J = 8.1 Hz). 13 C-NMR (75 MHz, CDCl 3 ) δ: 19.9, 23.3, 28.7, 67.2, 71.7, 71.8, 72.3, 78.9, 102.5, 102.7, 105.7, 105.7, 112.2, 113.4, 113.6, 114.8, 114.9, 116.7, 118.8, 125.0, 126.1, 127.0, 128.8, 129.4, 130.3, 134.2, 137.6, 138.4, 147.2, 148.5, 154.0, 154.2, 157.7, 158.6, 161.7, 167.4, HRMS (ESI + ): m/z calcd for M+H; found; (+1.00 mmu). S-18

19 Figure S19: The 1 H NMR spectra of ACTG. Figure S20: The 13 C NMR spectra of ACTG. S-19

20 Figure S21: The high-resolution mass spectra of ACTG. References (1) Kobayashi, T. Urano, Y. Kamiya, M. Ueno, T. Kojima, H. and Nagano, T. Highly Activatable and Rapidly Releasable Caged Fluorescein Derivatives. J. Am. Chem. Soc. 2007, 129, S-20