Fluorogenic âPhotoclickâ Labeling and Imaging of DNA with Coumarin

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Fluorogenic “Photoclick” Labeling and Imaging of DNA with Coumarin-fused Tetrazole in Vivo Yunxia Wu, Guanlun Guo, Judun Zheng, Da Xing, and Tao Zhang ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.8b00565 • Publication Date (Web): 12 Dec 2018 Downloaded from http://pubs.acs.org on December 15, 2018

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Fluorogenic “Photoclick” Labeling and Imaging of DNA with Coumarin-fused Tetrazole in Vivo Yunxia Wu,† Guanlun Guo,‡ Judun Zheng,† Da Xing,*,† and Tao Zhang*,† †

MOE key Laboratory of Laser Life Science & Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou, 510631, (P.R. China) ‡

Hubei Key Laboratory of Advanced Technology for Automotive Components & Hubei Collaborative Innovation Center for Automotive Components Technology, Wuhan University of Technology, Wuhan, 430070 (P.R. China)

Supporting Information Placeholder ABSTRACT: Photoclickable fluorogenic probes will enable visualization of specific biomolecules with precise spatiotemporal control in their native environment. However, the fluorogenic tagging of DNA with current photocontrolled clickable probes is still challenging. Herein, we demonstrated the fast (19.5 ± 2.5 M-1 s-1) fluorogenic labeling and imaging of DNA in vitro and in vivo with rationally designed coumarin-fused tetrazoles under UV LED photoirradiation. With a water-soluble, nuclear-specific coumarin-fused tetrazole (CTz-SO3), the metabolically synthesized DNA in cultured cells was effectively labeled and visualized, without fixation, via “photoclick” reaction. Moreover, the photoaclickable CTz-SO3 enabled the real-time, spatially controlled imaging of DNA in live zebrafish. KEYWORDS：fluorogenic, photoclick, DNA, metabolic labeling, cell imaging

Fluorogenic tagging of nucleic acids and other biomolecules using chemical probes via click reactions can enable selective visualization in their native conditions.1-8 Significant progress has been achieved in the postsynthetic or metabolic labeling of DNA with fluorogenic probes activated by various click reactions, including copper(I)-catalyzed azide-alkyne click cycloaddition,9-13 strain-promoted 1,3-dipolar cycloadditions, inverse electron demand Diels–Alder reactions and nucleophilic additions.14-23 Light-induced click reactions not only effectively combine the classical advantages of click reactions, but also provide spatiotemporal control and promoted reaction rate without metal-catalysts.24-28 Labeling of DNA in their endogenous environment with photoacitivable fluorogenic probes is significant for spatiotemporal-controlled investigation of cellular processes involving nucleic acids, such as DNA replication, transcription, repair and recombination. However, the fast labeling of metabolically synthesized DNA in their live settings with fluorogenic probes via photoactivated click reaction, to the best of our knowledge, has not yet been achieved.Light-induced click reactions not only effectively combine the classical advantages of click reactions, but also provide spatiotemporal control and promoted reaction rates without metal-catalysts,29-31 Tetrazole-based “photoclick” chemistry is a photoactivated inherently fluorogenic reaction and has been established as an efficient tool for site-selective modification of proteins.32-40 Recently, it has been demonstrated that “photoclick” reaction can be activated with visible (405-nm laser)41 or near-infrared (NIR) light (two-photon excitation, λex = 700 nm)42 by regulating the substructures of tetrazoles to minimize the limitation of UV light. Compared with the well-established fluorogenic “photoclick” labeling of proteins, however, the consequent fluorescence of the photoactivated cycloadducts of DNA are

always rather weak, even though phototriggered fluorogenic labeling of RNA with general tetrazoles has been reported.43-46 Fluorescence quenching behaviors during the “photoclick” labeling of DNA are still an imperative issue for further application of “photoclick” chemistry in labeling DNA, particularly in living systems. Therefore, we designed and synthesized a series of coumarin-fused tetrazole probes and demonstrated their fluorescent “switch-on” labeling of DNA under UV light activation. The fluorescence behaviors during the photoactivated modification of DNA were explained. Moreover, we developed a watersoluble nuclear-specific coumarin-fused tetrazole. With this probe and 5-vinyl-2’-deoxyuridine (VdU), an alkene-modified low-genotoxic thymidine analogue developed by Luedtke and coworkers47, we succeeded in fluorogenic “photoclick” labeling and imaging of DNA in proliferating cells and living zebrafish larvae without the need for the removal of excess probes.

EXPERIMENTAL SECTION General synthetic methods of tetrazoles 1-7. A cooled solution of NaNO2 (1 mmol) in 1.5 mL water was added to a solution of coumarin derivatives (1 mmol) in 1 mL of concentrated HCl and 6 mL EtOH/H2O (1:1) kept at a temperature below 5°C. The resulting coumarin diazonium chloride solution was then added dropwise to a stirred solution of (E)-methyl 4-((2-(phenylsulfonyl) hydrazono) methyl) benzoate (1.1 mmol) in 8 mL pyridine at -15°C for 1 h. The reaction mixture was allowed to slowly warm up to room temperature with stirring over a period of 12 hours. The mixture was extracted by DCM (3 × 5 mL), and the organic layer was concentrated under reduced pressure. Then, the

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crude product was purified by silica gel flash chromatography with eluted by CH2Cl2/methyl alcohol (v/v, 5:1) to produce pure product. Monitoring fluorescence and absorption during “photoclick” labeling of DNA in aqueous solution. One microliter of 5 mM tretrazoles 1-8 in DMSO and 5 μL of 100 μM acrylamide-modified DNA1-3 in PBS were mixed in a 200 μL PE tube containing 94 μL PBS,. After irradiation with 350 nm LEDs for various times, the solution was then filtered with a 3K ultrafiltration tube to remove the small molecules. The filtrate was transferred into a 100 μL quartz cuvette for examination of fluorescence and absorption spectra. Or: Five microliters of 100 μM tetrazole-modified DNA4-5 and 1μL of 5 mM alkene analogues in DMSO and in PBS were mixed in 200 μL PE tube containing 94 μL PBS. After irradiation with 350 nm LEDs for various times, the solution was then filtered with a 3K ultrafiltration tube to remove the small molecules. The filtrate was transferred into a 100 μL quartz cuvette for examination of fluorescence and absorption spectra. Cell Viability Assay. One hundred microliters of 5 × 104 A549 cells suspended in DMEM medium were added to the individual wells in a 96 well plate. After incubation at 37°C in a humidified CO2 incubator for 24 hours, the cells were treated with the compounds diluted with a concentration range from 100 nM to 1 mM for 24 hours. Afterwards, 10 μl CCK-8 solution was added to each well, and the plate was returned to the CO2 incubator for an additional 1hour incubation. The absorbance at 450 nm was then recorded using a microplate reader to determine the cell viabilities. Each treatment condition was repeated five times to obtain the average absorbance and the standard deviation. And: One hundred microliters of 5 × 104 A549 cells suspended in DMEM medium were added to the individual wells in a 96 well plate. After incubation at 37°C in a humidified CO2 incubator for 24 hours, the cells were incubated with VdU (30 μM) for 16 h and then washed with PBS (pH 7.4) for three times, followed by incubation with CTz-SO3 (10 μM) for 4 h. The media was replaced with fresh culture media and incubated further for 0.5 h. Then, the cells were illuminated with with 350 nm LEDs for various times (0, 1, 5, 10, 15, 20 min), All group cells were incubated for another 24 h at 37°C in a humidified CO2 incubator, and CCK-8 was added and the wavelength was detected at 450 nm using a microplate reader to determine the cell viabilities. Each treatment condition was repeated five times in order to obtain the average absorbance and the standard deviation. Cell culture and microscope. A549, U87, EMT6 and 4T1 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM). All cell lines were supplemented with 10% fetal bovine serum and 100 U/ml 1% antibiotics penicillin/streptomycin and maintained at 37°C in a 100% humidified atmosphere containing 5% CO2 at 37°C. Cell density was determined using a hemocytometer before experimentation. Images were acquired on a ZEISS LSM 510 META confocal microscope. A 40× objective was used for image capturing. Images were processed and analyzed using the LSM software.

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Live-cell “photoclick” labeling and imaging of synthesized DNA. Cells were seeded onto 35 mm confocal culture dishes (1 × 106 cells/well) and allowed to grow for 24 h. The media was then replaced with fresh culture media containing with variation of the VdU concentration 30 μM VdU (control groups do not without VdU). After incubating for 16 h, the cells were washed with 3 x 300 μL PBS and then incubated with fresh culture media containing with 10 μM tetrazoles. After incubation for 4 h, the media was replaced with fresh culture media. The cells were then photoirradiated directly with 350 nm LEDs for various times and the images were captured with the exctiation at 405 nm. Fixed-cell “photoclick” labeling and imaging of synthesized DNA. Cells were seeded onto 35 mm confocal culture dishes (1 × 106 cells/well) and allowed to grow for 24 h. The media was then replaced with fresh culture media containing with 30 μM VdU. After incubating for 16 h, the cells were washed with 3 x 300 μL PBS and then incubated with fresh culture media containing with 10 μM tetrazoles. After incubation for 4 h, the media was replaced with fresh culture media and incubated further for 0.5 h. Then the cells were fixed with 300 μL 3.7% paraformaldehyde in PBS at room temperature for 10 minutes. The paraformaldehyde was removed and the cells were washed with 3 x 300 μL PBS. The cells were then permeabilized with 300 μL 0.5% Triton-X for 20 minutes. The Triton-X was removed and the cells were washed twice with 300 μL PBS The cells were then photoirradiated with 350 nm LEDs for 10 minutes and the images were captured with the excitation at 405 nm. Dual staining of cellular DNA with tetrazole probe CTzSO3, Tz-SO3 and PI. Cells were seeded onto 35 mm confocal culture dishes (1 × 106 cells/well) and allowed to grow for 24 h. The media was then replaced with fresh culture media containing with 30 μM VdU .After incubating for 16 h, the cells were washed with 3 x 300 μL PBS and then incubated with fresh culture media containing with 10 μM tetrazoles(CTz-SO3 or Tz-SO3). After incubating further for 4 h, the media was replaced with fresh culture media and incubated further for 0.5 h. The cells were then photoirradiated directly with 350 nm LEDs for 10 minutes, the media was removed and the cells were fixed with 300 μL 3.7% paraformaldehyde in PBS at room temperature for 10 minutes. The paraformaldehyde was removed and the cells were washed with 3 x 300 μL PBS. The cells were then permeabilized with 300 μL 0.5% Triton-X for 20 minutes. The Triton-X was removed and the cells were washed twice with 300 μL PBS. After that, the media was removed and the cells were treated with 200 μL 1% RNase in PBS for 30 min. The RNase was removed and the cells were washed with 3 x 300 μL PBS. The cells were then incubated with 10 μg/mL PI (propidium iodide) for 30 min. The cells were washed with 3 x 300 μL PBS and the images were captured with the excitation at 405 nm (tetrazoles) and 535 nm (PI), respectively. Zebrafish culture and microscope. Zebrafish Husbandry: Adult wild-type zebrafish were kept at 28.5°C on a cycle of 14 hours of light and 10 hours of darkness. Embryos were obtained from natural spawning and were maintained in embryo medium (EM; 150 mM NaCl, 0.5 mM KCl, 1.0 mM CaCl2, 0.37 mM KH2PO4, 0.05 mM Na2HPO4, 2.0 mM MgSO4, 0.71 mM NaHCO3 in deionized water).Images were

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Scheme1. Structures of photoclickable tetrazoles and their pyrazoline cycloadducts with DNA1.

Table 1. General features and photophyscial properties of coumarin-fused tetrazoles over photoinduced cycloaddition reaction with DNA. tetrazoles

λmaxa (nm)

εmaxb (M-1 cm-1)

1 2 3 4 5 6 7 8

339 341 335 335 334 334 335 290

6490 5700 5460 6480 5840 6250 5510 6840

ε350c (M-1 cm1) 6200 5310 4900 5570 5010 4980 4910 2282

λemd (nm)

Φfle (%)

450 495 449 445 445 440 445 490

0.37 0.47 0.39 0.34 0.40 0.24 0.13 0.01

a

Measurements were recorded in pH 7.4 PBS (0.5% DMSO). Extinction coefficient at λmax. c Extinction coefficient at 350 nm. d Emission and e Quantum yield of pyrazoline cycloadducts with DNA1. Cycloaddition reactions of tetrazoles (100 μM) with DNA1 (10 μM) were conducted under 350 nm LEDs; fluorescence was measured in pH 7.4 PBS, λex = 390 nm. b

acquired on a Leica DM6B+DFC 550 fluorescence microscope. A 5× objective was used for image capturing. Live-zebrafish “photoclick” labeling and imaging of synthesized DNA. Embryos were injected into the yolk at the 1–4 cell stage with 2 nL of a solution containing variation of VdU concentration in 0.2 M KCl. After developing to 96 hpf, zebrafish were washed three times with embryo medium, and then incubated with either 10 μM CTz-SO3 or Tz-SO3. After incubating further for 20 h, he media was replaced with fresh embryo medium and incubated further for 2 h. The zebrafish were anesthetized with 40 mg/L tricaine and incubated with glycerin for imaging, and they awakened after about 18-20 minutes. The images were captured with the excitation at 405

nm. Localized labeling of zebrafish. The groups of zebrafish treaed with CTz-SO3 were anesthetized with tricaine and then localized irradiation with 350 nm LEDs and a single pin hole diaphragm for 14 min. The images were captured with the exctiation at 405 nm.

RESULTS AND DISCUSSION

Figure 1. Relationship between the calculated EHOMO of the fused coumarin moiety and the fluorescence quantum yield (Φ fl) of the coumarin-fused tetrazoles (1-7) and their pyrazoline cycloadducts with DNA1.

Design, synthesis and characterization of photoclickable tetrazole. Our initial study focused on the investigation of the photophysical properties of a series of newly designed coumarin-fused tetrazoles bearing different substituents 1-7 (scheme 1) and their corresponding pyrazoline cycloadducts with DNA by UV-VIS absorption and emission spectra (Table 1 and Figure S1 and Figure S2). Under normal conditions, all these coumarin-fused tetrazoles are nonfluorescent, except 2. Upon photoirradiation by 350 nm LEDs of the coumarin-fused tetrazoles with DNA1, a random DNA sequence modified with acrylamide by an alkyl linker at the 5’ end (Table S1), the fluorescence of their pyrazoline cycloadducts with DNA1 increased by different degrees in a range from 440 to 450 nm, which seems to be mainly originating from the coumarin moiety. To better understand the optical behaviors of these photoactivatable tetrazoles, density functional theory calculations were performed using the wB97XD/6-311+G (d, p) basis set (Figure S3). As shown in Figure 1, the fluorescence of the coumarin-fused tetrazoles was suppressed efficiently when the calculated highest occupied molecular orbitals (HOMOs) energy values (EHOMO) of the coumarin moiety were below -


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Figure 2. a) “Photoclick” labeling of DNA 1 with tetrazole 1 and 8. b) Emission spectra of the resulted labeling product of the above reaction solution after irradiated with 350 nm LEDs for 10 min. c) Fluorescence intensity changes of the reaction mixture of tetrazole 1 and 8 (100 μM) with DNA1 (10 μM), Em: 450 nm, n=5; d) PAGE analysis of the reaction of tetrazole 1 with DNA1. e) Emission spectra of the “photoclick” labeling product of DNA2 (G-rich), DNA3 (G-free) and DNA1 (general one) with tetrazole 1 and 8 in PBS solution (pH 7.4) after irradiated with 350 nm LEDs, Ex = 390 nm. The concentrations of DNA and tetrazoles were 10 and 100 μM, respectively. Cycloaddition reactions were conducted under 350 nm LEDs (photoirradiation for 10 min unless specifically mentioned).

0.324 Hartree. Along with the decrease of EHOMO and the electron affinity of the substituent on coumarin, the fluorescence quantum yield of the corresponding cycloadducts increased from 0.129 to 0.389. A continuous decrease of EHOMO to -0.337 Hartree would lead to an enhancement of the quantum yield of the cycloadduct, but meanwhile, it would increase the fluorescence background of the tetrazoles dramatically. The result can also be interpreted as the lowest unoccupied molecular orbitals (LUMOs) of tetrazole 2 are mainly delocalized over the coumarin moiety, whereas the LUMOs of 1 are localized on the entire molecular backbone. Fluorogenic “photoclick” labeling of DNA in vitro. To conduct further investigation into coumarin-fused tetrazole performance for tagging DNA in detail, we selected tetrazole 1 as a model probe. Upon photoirradiation of the solution of coumarin-fused tetrazole 1 and general tetrazole 8 with DNA1 (Figure.2a), a time-dependent emission at 450 nm (λmax) was observed and gradually leveled off eventually (Figure 2b, Figure 2c and Figure S4a). Furthermore, accompanied by the “switch-on” emission, a newly appeared absorption band centered at 395 nm indicated the pyrazoline cycloadduct formed (Figure S4b). PAGE analysis further confirmed the pyrazoline cycloadduct as the abeling product 1-DNA1 (Figure 2d). However, unlike the reaction of tetrazole 1 with DNA1, we can only detect very weak emission at 490 nm in the reaction mixtures of tetrazole 8 with DNA1, even though we extended the irradiation time and confirmed the formation of the 8DNA1 product (Figure S5). By contrast, only very weak emission was detected when irradiation of tetrazole 1 alone or in presence of a DNA oligonucleotide with no alkene modification (DNA1*) (Figure S6 and Table S1). By photoirradiating and isolating the pure cycloadduct (1-IsoAc) of tetrazole 1

with isopropyl acrylamide (IsoAc), a basic acrylamide analogue, the UV-VIS absorption extinction coefficient of the pyrazoline 1-IsoAc at 395 nm was determined as 5780 ± 117 M-1 cm-1 (Figure S7). The yield of 1-DNA1 was then calculated as 9.0%, 33.7%, 57.6%, 62.9%, and 69.1%, at irradiation time of 1 min, 5 min, 10 min, 20 min and 30 min, respectively,

Figure 3. Fluorescence “switch-on” with “photoclick” labeling of DNA. a) Emission units of the cycloadducts of coumarin-fused tetrazoles. b) Emission spectra of the reaction mixture of DNA4/DNA5 (5 μM) with IsoAc/NMal (50 μM) under 350 nm LEDs for10 min. c) Emission changes of the reaction mixture of DNA4 (5 μM) with DmapAc (50 μM) at different pH values. Cycloaddition reactions were conducted under 350 nm LEDs (for 10 min).


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Figure 4. No-wash “photoclick” labeling of cellular DNA in A549 cells. a) Photoinitiated labeling scheme of VdU incorporated DNA with the probe CTz-SO3. b) Labeling and imaging of synthesized DNA in live cells and fixed cells. Ex: 405 nm, Em: 420 -500 nm. Scale bar = 5 μm. c) Time courses of fluorescence changes in the photoirradiated live A549 cells treated with CTz-SO3 (10 μM) in the presence and absence of VdU (30 μM). d) PAGE analysis of total genomic DNA extracted from labeled live cells.

which was consistent with the results of PAGE analysis. Given the relatively intense fluorescence quenching effect of Guanine in DNA due to the photo-induced electron transfer (PeT), we then treated tetrazole 1 and 8 with a G-rich DNA sequence (DNA2) and a G-free DNA sequence (DNA3) under 350 nm photoirradiation, respectively (Table S1). It was found that tetrazole 1 could resist the quenching effect well and afforded a robust increase in fluorescence intensity after photoirradiation with DNA2. In contrast, only very weak emission was detected when tetrazole 8 was used for the labeling of the G-free DNA3, not to mention DNA2 (Figure 2e). Because there should exist two latent fluorescence units (coumarin I and pyrazoline II) for the coumarin-fused tetrazoles (Figure. 3a), to further clarify the fluorogenic pattern of the coumrin-fused tetrazoles in labeling of DNA, we modified the DNA sequence as that of DNA1 with tetrazole 1 and 8 to produce DNA4 and DNA5 (Figure S8a), respectively, and investigated their reactivity with different alkene analogues. The two tetrazole-modified DNA were first conducted photoactivated cycloaddition reaction with the general IsoAc. We found that both cycloadducts of DNA4 and DNA5 are remarkably fluorogenic and exhibited intense emission at 490nm and 510 nm, respectively (Figure 3b, Figure S8b and Figure S9). When we treated DNA4 and DNA5 with N-(2-hydroxyethyl)

maleimide (NMal), a randomly selected maleimide, we found that the cycloadduct of DNA4 still exhibited obvious fluorescence enhancement but at 445 nm. On the other hand, we can only detect very weak emission at 510 nm for that of DNA 5 (Figure. 3b), even though the pyrazoline cycloadducts DNA5IsoAc and DNA5-NMal have been formed under bother conditions (Figure S10). Interestingly, the findings indicate that the alkene with electron-rich substitutions possess a fatal quenching effect by the PeT mechanism on the fluorescence of pyrazoline but less impact on that of fused fluorophores. This deduction was further confirmed by the emission changes of the cycloadduct of DNA4 with N,N-dimethylaminopropyl acrylamide (DmapAc) at different pH conditions by regulating the electron density of the N, N-dimethylamino group. As shown in Figure 3c, accompanied by the pH adjusted from 7.5 to 5.5 and protonation of the N,N-dimethylamino group, the emission at 490 nm increased gradually until the initial emission peak at 450 nm overlapped (IsoAc as reference see Figure S11). These results further prove that the emission at approximately 450 nm and 490 nm of the DNA cycloadducts with coumarin-fused tetrazoles originates from the coumarin and pyrazoline moieties, respectively.


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Figure 5. Dual staining of cellular DNA with tetrazole probe

CTz-SO3, Tz-SO3 and PI. After A549 cells were treated with VdU (30 μM) for 16h and photoirradiated with CTz-SO3 and Tz-SO3 for 10 min, PI was added and stained for 30 min. green: Ex: 405 nm, Em: 420 - 500 nm, red: Ex: 543 nm, Em: 560 - 650 nm . Scale bar = 10 μm. Figure 6. Fluorescence microscopy images of cellular DNA

Application of CTz-SO3 to labeling and imaging of DNA in living cells and in vivo.The successful photoactivated fluorogenic labeling of DNA with coumarin-fused tetrazoles in vitro enabled us to investigate the metabolic visualization of DNA in live cells. First, deliver the tetrazoles to the cellular nucleus more smoothly, we modified the coumarin-fused tetrazole 1 to obtain a water-soluble probe CTz-SO3 by conjugation of tetrazole 1 with an anionic sulfonyl group that has been found to enhance the cell nuclear localization efficiency. 48 By comparison, modification of tetrazole 8 was also conducted to produce Tz-SO3. We then investigated their performance in the photoactivated cycloaddition with VdU in PBS solution. Spectral monitoring of the reaction mixture of CTz-SO3 and VdU revealed a typical pyrazoline absorption at 395 nm and a time-dependent emission at 450 nm (Figure S12). The fluorescence of thepyrazoline cylcoadduct of Tz-SO3 with VdU at 490 nm was found, as expected, very weak (Figure S13). The kinetic for the photoligation of CTz-SO3 with VdU was evaluated under pseudofirst-order conditions based on the HPLC analysis at two concentrations of CTz-SO3, upon irradiation of the reaction mixture for different time (Figure S14). The photoligation shows a significantly high rate constant (k) of 19.5 ± 2.5 M- 1 s-1 with reference to the inverse electron demand Diels–Alde VdU-tetrazine ligation (0.02 M-1 s-1).47 Recent studies have showed that tetrazoles react with different kinds of nucleophiles, upon photolysis, in a biological system49-52. Therefore, we performed negative control experiments to prove the importance of the tetrazoleene photoclick reaction in living systerms (Figure S15a). The cell lysates containing CTz-SO3 exhibited very weak fluorescence under normal conditions. After photoirradiation for 10 min with 350 nm LEDs, the cell lysates showed minimal enrichment fluorescence. By contrast, both VdU and CTz-SO3 are all contained in cell lysates, and after photoirradiation, they show a strong emission. Futhermore, ccomparison of the reactivity of CTz-SO3 with VdU, glutathione(GSH) and acetic acid(AcOH) in PBS buffer solution re-

spatially labeled by CTz-SO3 or Tz-SO3 on live zebrafish. a) Photoinitiated labeling scheme of VdU incorporated DNA with tetrazole probes on live zebrafish; b) Zebrafish embryos were injected with 2 nL of 0.3 mM VdU at the 1-4 cell stage and develop to 96 h postfertilization (hpf). Embryos were incubated with either 10 μM of CTz-SO3 or Tz-SO3 for 20 h. The top row is photoirradiation fluorescence images were after photoirradiation for 14 min with 350 nm LED. ROI group: localized irradiation with 350 nm LEDs and a single pin hole diaphragm for 14 min .Ex: 405 nm, Scale bar = 500 μm.

vealed that our probe possessed high selectivity towards the alkene over other competitive nucleophilic species (Figure S15b). Taken together, these results suggested that CTz-SO3 was still efficient enough to reacted with alkene-modified compounds in complex biological systems containing various nucleophilic species. To evaluate CTz-SO3, with relatively low cytotoxicity (Figure S16), as a photoactivated cellular DNA imaging probe, proliferating A549 cells were treated with 30 μM VdU for 16 h and then, without fixation, incubated further with 10 μM CTz- SO3 for 4 h(Figure 4a). Confocal fluorescence microscopy revealed that these cells exhibited very weak fluorescence under current circumstances (Figure 4b and Figure S17). Upon photoirradiation for 10 min with 350 nm LEDs the cell nuclei were all robustly labeled, whereas the control cells, without treated VdU, showed only a slightly increased fluorescence, potentially due to the photogenerated weakly fluorescent nitrile imine intermediate as observed before.53 Similar results were also achieved in live U87, EMT6 and 4T1 cells (Figure S18). As a point of comparison, we also evaluated VdU treated A549 cells under fixed (3.7% paraform-aldehyde) conditions for CTz-SO3 photocontrolled labeling (Figure 4b). In this case, robust labeling of cell nuclei was also observed as that of live cells. Moreover, live-cell DNA labeling with CTz-SO3 was observed in and real-time, which was in agreement with the ob-


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servations in vitro (Figure 2c and Figure S19). And dosedependent nuclear staining was observed in cells treated with 0 to 50 μM of VdU (Figure S20). The significant cell nuclei labeling and imaging over background should be attributed to the well fluorogenic behavior of coumarin-fused tetrazole and the high nuclear specificity of our probe CTz-SO3 (Figure S21 and Figure S22). To further confirm the newly synthesized DNA labeling, the total genomic DNA of these tested A549 cells was then subsequently extracted. Analysis of the extracted mixture by PAGE showed that VdU was selectively incorporated into the DNA backbone and could be robustly labeled with CTz-SO3 (Figure 4d and Figure S23). Then, to assess whether the cell integrity was damaged after photoirradiation, we added PI, which was inefficient to pass through an intact cell membrane, into the irradiated cells and found that the cells only exhibited a slight fluorescence (Figure S24). Furthermore, a second probe BrdU was employed into the cells for DNA synthesis and then labeled with antibody-AlexaFluor-488 conjugate to examine the cell viability after the photoclick reaction (Figure S25). The result indicated that BrdU was inserted into the synthesized DNA and the photolabeled cells still retained the ability to divide. The results offered, for the first time, the photocontrolled detection and visualization of newly synthesized DNA in living cells. To evaluate the compatibility of our probe CTz-SO3 with other color of a well-established probe, we tested photoactivated nuclear staining experiments with red-emitting PI and DRAQ-5 (Figure 5 and Figure S26). Significant intranuclear staining over background was captured after phototriggered with CTz-SO3 and no obvious effect on the postlabeling of PI staining was observed. Bycontrast, the celltreated with Tz-SO3 showed inconspicuous changes after UV photoirradiation. In order to evaluate whether our probe CTz-SO3 could be used to spatially and temporally label the synthesized DNA in vivo, zebrafish embryos were first injected with 2 nL of 0.3 mM VdU at the 1-4 cell stage. At 96 h postfertilization (hpf), embryos were incubated with either 10 μM of CTz-SO3 or TzSO3 for 20 h (Figure 6a). As shown in Figure 6b, upon photoirradiation for 14 min with 350 nm LEDs，the zebrafish incubated with probe CTz-SO3 were robustly labeled via fluorescence microscopy, and showed fast, time-dependent and dose-dependent fluorescence enhancement over background (Figure S27 and Figure S28). In contrast, the zebrafish treated with Tz-SO3 showed inconspicuous changes after UV photoirradiation. Significantly, after localized photoirradiation, within a region of interest (ROI), the fluorescent signal was specifically limited to the irradiated area, revealing the well spatial resolution of this protocol with our photo-clickable probe CTz-SO3. The higher magnification microscopy images and co-staining with DRAQ-5 experiments further demonstrated CTz-SO3 is suitable for labeling the synthesized DNA in vivo (Figure S29). More importantly, after photoirradiation, the zebrfish with both VdU and CTz-SO3 contained still exhibited a survival rate more than 80% in 40 days (Figure S30).

imported latent florogenic chromophore coumarin as a principle for the design of coumarin-fused tetrazoles for photoinitiated fluorogenic labeling of nucleic acids. Additionally, we have clarified that DNA and other electron-rich alkene counterparts are liable to quench the fluorescence of pyrzaline by photoinduced electron transfer but have little effect on that of the introduced coumarin fluorophore. We anticipate that these findings will be useful for the development of other fluorogenic photoclickable an alogues with more specific optical properties, such as photoexcitation and emission with long wavelengths for nucleic acids labeling and imaging. Moreover, CTz-SO3, a newly synthesized water-soluble coumarin-fused tetrazole probe, can sepecifically localize in the cell nucleus to fast label and visualize VdU incorporated cellular DNA via UV-triggered cycloaddition reactions. This sensitive and selective labeling of cellular and zebrefish DNA in real time can be achieved in a spatiotemporally controlled manner without fixation and DNA denaturation. To the best of our knowledge, this is the first example of a photoinitiated fuorogenic labeling and imaging cellular DNA in live systems. Thereby, we believe that this platform should facilitate to realize more potential of “photoclick” chemistry for fast tracking DNA synthesis in living animals with spatiotemporal control.

ASSOCIATED CONTENT Supporting Information Supporting information figures, tables, synthetic procedures, and experimental protocols. The Supporting Information is available free of charge on the ACS Publications website at: Additional text, figures with experimental details for chemical synthesis of all compounds; supplementary photophysical characterization of probes, and imaging methods and data (PDF)

AUTHOR INFORMATION Corresponding Author *[email protected]; *[email protected]

Notes The authors declare no competing financial interests.

ACKNOWLEDGMENT This research was supported by the National Natural Science Foundation of China (2177065, 81630046), the Program for Changjiang Scholars and Innovative Research Team in University (IRT0829), the Key Program of the National Natural Science Foundation of China (613300033), the Natural Science Foundation of Guangdong Province, China (2017A020215088), the Scientific and Technological Planning Project of Guangzhou - Zhujiang Nova Program (201806010189), the Scientific and Technological Planning Project of Guangzhou (201805010002). the Science and Technology Planning Project of Guangdong Province, China (2015B020233016, 2014B020215003).

REFERENCES

CONCLUSIONS In conclusion, we present a “photoclick” labeling of nucleic acid platform that enables fluorescent “switch-on” labeling of synthesized DNA with coumarin-fused tetrazole probes. In this work, we have proposed the relationship between the fluorescence quantum yield and the calculated EHOMO of the

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Table of Contents For TOC only

“Photoclick” chemistry: Rational design of coumarin-fused tetrazoles were reported for fluorescent “switch-on” labeling of DNA via “photoclick” chemistry in vitro and in vivo. A nuclear-specific coumarin-fused tetrazole (CTz-SO3) was demonstrated for fast (17 ± 3 M-1 s-1) fluorogenic labeling and imaging, without fixation, of metabolically synthesized DNA with 5-vinyl-2’-deoxyuridine (VdU) under photoirradiation of UV LEDs in live cells. Moreover, the use of CTz-SO3 for real-time, spatially controlled imaging of DNA in live zebrafish under the photoirradiation was demonstrated.


Fluorogenic âPhotoclickâ Labeling and Imaging of DNA with Coumarin

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Fluorogenic âPhotoclickâ Labeling and Imaging of DNA with Coumarin