Amplified Visualization of Protein-specific Glycosylation in Zebrafish

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Amplified Visualization of Protein-specific Glycosylation in Zebrafish via Proximity-induced Hybridization Chain Reaction Jingying Li, Shuya Liu, Liqin Sun, Wei Li, Su-Yun Zhang, Sheng Yang, Juan Li, and Huang-Hao Yang J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b08442 • Publication Date (Web): 08 Nov 2018 Downloaded from http://pubs.acs.org on November 9, 2018

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Journal of the American Chemical Society

Amplified Visualization of Protein-specific Glycosylation in Zebrafish via Proximity-induced Hybridization Chain Reaction Jingying Li,† Shuya Liu,† Liqin Sun,† Wei Li,† Su-Yun Zhang,ǁ Sheng Yang,ǁ Juan Li*,‡ and Huang-Hao Yang*,†,‡ †College

of Biological Science and Engineering, Fuzhou University, Fuzhou 350116, P. R. China.

MOE Key Laboratory for Analytical Science of Food Safety and Biology, Fujian Provincial Key Laboratory of Analysis and Detection Technology for Food Safety, State Key Laboratory of Photocatalysis on Energy and Environment, College of Chemistry, Fuzhou University, Fuzhou 350116, P. R. China. ‡

ǁDepartment

of Medical Oncology, Fujian Medical University Union Hospital, Fuzhou, 350001, P. R. China.

ABSTRACT: The visualization of glycosylation states of specific proteins in vivo is of great importance for uncovering their roles in disease development. However, the ubiquity of glycosylation makes probing the glycans on a certain protein difficult as looking for a needle in a haystack. Herein, we demonstrate a proximity-induced hybridization chain reaction (HCR) strategy for amplified visualization of protein-specific glycosylation. The strategy relies on designing two kinds of DNA probes, glycan conversion probes and protein recognition probes, which are attached to glycans and target proteins respectively. Upon sequential binding to the targets, the proximity-induced hybridization between two probes occurs, which leads to the structure-switching of protein recognition probes, followed by triggered HCR assembly. This strategy has been used to visualize tyrosine-protein kinase 7 (PTK7)-specific sialic acid in living CEM cells and zebrafish, and monitor its variation during drug treatment. It provides a potential tool for investigating protein-specific glycosylation, and researching the relation between dynamic glycans state with disease process.

INTRODUCTION As a complex and ubiquitous protein posttranslational modification in organisms, protein glycosylation plays a critical role in regulating physiological and pathological states of cells.1-2 A plethora of evidence has suggested that glycosylation on specific proteins, rather than overall cellular glycosylation status, mainly contributes to malignant transformation.3-4 Therefore, precisely monitoring glycosylation of a certain glycoprotein is important for uncovering the molecular mechanism in clinical diagnostics and therapeutics. In view of the increasing interest in proteinspecific glycans analysis, several techniques, such as mass spectrometry, have been developed.5-6 Unfortunately, these methods could not be applied under living conditions owing to requirement of complicated sample pre-preparation processes.7 To meet the needs for in vivo application, fluorescencebased imaging techniques have been explored. Recently, a series of methods based on förster resonance energy transfer (FRET) have been developed for protein-specific glycans investigation.8-9 In general, the key point for analyzing glycosylation in a protein-specific manner is to integrate the identification of both target proteins and glycans. Traditionally, target proteins are identified through fluorescent protein fusion, which has been relying on genetic engineering and usually limit their application in the physiological state.10-12 Besides, aptamer or antibody-based protein recognition has offered an alternative for protein-specific labeling in living cells, which

benefits from strengths in feasible operation and non-genetic modification.13-16 On the other hand, labeling of glycans is more difficult than that of proteins because of the nongenetically encoded nature and lack of antibodies. Usually, fluorescence tags are anchored on glycans through metabolic oligosaccharide engineering (MOE), which is a universal labeling strategy but incompetent to specifically image the glycans on a certain protein.17-19 On that account, selective labeling of protein-specific glycans is still a challenge and urgent demand. In addition to selective labeling, the relatively low sensitivity of FRET-based methods is another challenge to overcome.20-21 The background signals derived from thousands of other proteins carrying the same glycans require an efficient strategy to yield high signal-to-noise.22-23 To this end, several amplification strategies have been reported to enhance the sensitivity, such as proximity ligation assay (PLA),24-25 rollingcircle amplification (RCA)26 and exonuclease III-aided recycling hybridization.27 Nevertheless, all of these methods are highly enzyme-dependent, thereby impeding their application under living conditions. For this reason, it is still highly desired to develop sensitive imaging strategies for visualizing protein-specific glycosylation in vivo. Herein, we develop a proximity-induced hybridization chain reaction (HCR) strategy to amplify the visualization of protein-specific glycosylation in living cells and zebrafish (Figure 1). HCR is an enzyme-free amplification method, which shows great potential for bioanalysis under living

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conditions.28-31 The strategy designs two kinds of DNA probes, glycan conversion probes and protein recognition probes. First, the glycans on cell surface are efficiently modified with Glycan Conversion Probes (named GCP, colored green) via MOE and bioorthogonal reaction. In this way, the information of glycans can be transferred to that of DNA probes. The protein recognition probes (named ASI) are composed of Aptamer domain (colored blue), Spacer domain (colored green) and Initiator domain (colored red). The aptamer domain is responsible for protein recognition. The initiator domain is a trigger of HCR assembly and initially protected by forming a hairpin structure with the spacer domain. Importantly, the GCP probes are complementary to the spacer domain, which promote the conformational changes of ASI probes and open the hairpin structure. Once binding to the same glycoprotein molecule on cell surfaces, their proximity induces the hybridization of GCP with ASI probes, thus triggering structure-switching of ASI probes and releasing the HCR initiator domain. Finally, in the presence of signal output probes (H1 and H2), the HCR reaction is restricted to occur at the protein-specific glycans. Thus, benefiting from simple DNA labeling and proximity-induced HCR assembly, this proposed strategy will provide a powerful tool for sensitive and amplified visualization of protein-specific glycans in living cells and zebrafish.

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FAM fluorophore and dabcyl quencher was designed as a signal probe. Upon the hybridization of GCP and ASI probes, the structure-switching of ASI probes occurred and triggered HCR assembly of H1 and H2 probes, thus generating an intense fluorescence emission after 24 h incubation (Figure 2c, line i). In the absence of GCP or ASI probes, the H1 and H2 probes could coexist stably in solution and merely gave a very low background fluorescence signal (Figure 2c, lines ii-iv). Amplified visualization of protein-specific glycans on cell surfaces. After ensuring a proper operation of the proposed proximity-induced HCR assembly, we explored this strategy in living cells. In order to visualize glycans on cell surface proteins, the glycans were decorated by azido through MOE, then DBCO modified GCP probes (DBCO-GCP) were attached onto the azido-SiaNAz residues via the copper-free bioorthogonal chemical reaction.33 First, we demonstrated that the azido groups were metabolically incorporate onto the target proteins. CEM (PTK7 positive) and Ramos (PTK7 negative) cells were pretreated with tetra-acylated Nazidoacetylmannosamine (Ac4ManNAz), while cells were treated with DMSO as control. After being labeled by DBCObiotin, cells were lysed and the lysates were analyzed by western blotting using streptavidin-linked horseradish peroxidase (stv-HRP) to confirm the successful azido modification of glycans. Besides, western blotting assay with anti-PTK7 antibody was also performed to detect the PTK7 expression of cells. The gel images indicated that azido tags were successfully attached on glycans of the Ac4ManNAztreated cells (Figure S1a), besides the MOE did not influence cellular PTK7 expression (Figure S1b).

Figure 1. Illustration of amplified visualization of protein-specific glycosylation via proximity-induced HCR.

RESULTS AND DISCUSSION In vitro assay of proximity-induced HCR assembly. As a proof-of-concept, glycosylation of tyrosine-protein kinase 7 (PTK7) was selected as a model, which is an overexpressed cancer biomarker in human acute lymphoblastic leukemia cells. Therefore, the aptamer sgc8 was used to construct the ASI probes,32 and other corresponding DNA probes (GCP, H1 and H2) were also designed (see Table S1 in the Supporting Information). First of all, agarose gel electrophoresis and fluorescence profile were used to assess the feasibility of the designed HCR assembly (Figure 2). As shown in Figure 2a, hybridization between GCP and ASI probes (lane 4) could induce the assembly of H1 and H2 probes in a cascading manner through HCR, thus resulting in large smeared DNA bands (lane 1). Otherwise, in the absence of either GCP or ASI probes, H1 and H2 probes showed only a single band indicating the HCR reaction was not triggered (lane 2 and 3). This assembly process was also verified by fluorescence profile. The occurrence of FRET signal between Cy3 and Cy5 confirmed the proximity hybridization between GCP-Cy3 and ASI-Cy5 probes (Figure 2b). Then, hairpin probe H1 with a

Figure 2. (a) Agarose gel electrophoresis image of HCR products. (b) Fluorescence profile of FRET-based structure-switching of ASI-Cy5 probes upon hybridization with GCP-Cy3 probes. Cy3 and FRET: excitation 530 nm/emission 550 - 750 nm. Cy5: excitation 640 nm/emission 650 - 750 nm. (c) Fluorescence profile of HCR-based DNA assembly (i: ASI + GCP + H1 + H2; ii: GCP + H1 + H2; iii: ASI + H1 + H2; iv: H1 + H2); excitation 488 nm/emission 500 - 650 nm.

Next, confocal laser scanning microscopy (CLSM) was used to visualize the labeling of glycans on PTK7. After being


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fed with Ac4ManNAz and followed by DBCO-GCP-Cy3 labeling, both CEM and Ramos cells showed obvious green fluorescence on the cell surfaces, and vice versa (Figure S1c). Meanwhile, the selective ASI-Cy5 labeling on CEM rather than Ramos cells verified the specificity of aptamer recognition (Figure S1c). Furthermore, FRET-induced Cy5 fluorescence signal was only observed on Ac4ManNAz-treated CEM cells (Figure 3a), which was generated by the hybridization between ASI and GCP and represented the PTK7-specific glycosylation in CEM cells. The FRET on CEM cell surfaces was also verified by fluorescent spectra (Figure S2). In order to confirm the FRET process, acceptor Cy5 was completely photo-bleached with high-power laser. As a result, the FRET-induced fluorescence signal disappeared and the donor Cy3 emission was enhanced (Figure 3b), suggesting the glycosylation of PTK7 could be visualized by FRET signal due to the proximity hybridization between GCP and ASI probes. To prove the versatility of our strategy, we used epithelial cell adhesion molecule (EpCAM) as a second glycoprotein model. The aptamer SYL3C was used to design the EpCAM-targeted ASI probes,34,35 which was feasible to hybridize with GCP probes and subsequently induced HCR assembly of H1 and H2 probes (Figure S3a). In this case, HT29 and MCF10A cells were selected as positive and negative cells. As shown in Figure S3b, EpCAM and glycans on the target proteins were labeled by ASI-Cy5 and DBCOGCP-Cy3 probes, respectively. Besides, the FRET signal caused by proximity hybridization between GCP-Cy3 and ASI-Cy5 probes could only be observed on Ac4ManNAztreated HT29 cells (Figure S4), indicating the successful recognition of glycans on EpCAM. Therefore, by using this strategy, we succeeded in visualizing the glycans of particular proteins in living cells.

However, owing to the low proportion of a particular glycoprotein to the total number of membrane proteins on cell surfaces, signal amplification of the protein-specific glycans visualization is required. The proximity-induced HCR amplification strategy was applied to enhance the proteinspecific glycosylation fluorescence signal. After being labeled by GCP and ASI probes, Ac4ManNAz-treated CEM cells were subsequently incubated with FAM-labeled H1 and H2 probes. As shown in Figure 4, very bright fluorescence could be observed at the rim of Ac4ManNAz-treated CEM cells. However, with respect to CEM cells without Ac4ManNAz treatment and Ramos cells, the fluorescence of HCR assembly could not be observed, owing to the lack of glycans conversion or PTK7 expression on cell surfaces. Several control experiments were also conducted to verify the high specificity of the designed HCR amplification on cell surfaces (Figure S5). In addition, the HCR interaction time in living cells was optimized to be 2 hours in advance (Figure S6). The similar amplified visualization of EpCAM-specific glycans could be achieved on Ac4ManNAz-treated HT29 cells, but neither on non-treated HT29 cells nor MCF10A cells (Figure S7). Furthermore, the high efficiency of HCR amplification in living cells was also testified. The result indicated the effect of HCR amplification was nearly 5 times over that of nonamplified method (Figure S8). These observations verified that the proximity-induced HCR assembly could be used to identify and amplify the protein-specific glycans on cell surfaces.

Figure 4. The CLSM images of PTK-7 specific glycosylation on the CEM or Ramos cell surfaces with or without Ac4ManNAz treatment by using proximity-induced HCR amplification strategy. Top panel: merge of fluorescence and bright field images; bottom panel: fluorescence field images, excitation 488 nm/emission 500 - 550 nm. Scale bar: 20 μm.

Figure 3. The CLSM images of PTK7-specific glycans in living cells via FRET signal. (a) CEM and Ramos cells were treated with Ac4ManNAz, followed by sequential labeling with DBCOGCP-Cy3 and ASI-Cy5, scale bar: 20 μm (inset: enlarged image, scale bar: 2 μm). (b) Donor was dequenched after acceptor photobleaching. Green channel: Cy3 excitation 543 nm/emission 560 610 nm. Red channel: FRET excitation 543 nm/emission 650LP filter. Merge channel: overlay of fluorescence image and bright filed image. Ratio channel: Cy5/Cy3. Scale bar: 5 μm.

Verification of intramolecular proximity-induced signals. Although proximity-induced FRET and HCR signals were detected with protein-specific glycans, it remained to be clarified whether the above obtained signals were generated from intramolecular hybridization between GCP and ASI probes. First, to verify the intramolecular proximity effect, we used SKBR3 cell line as a control (Figure S9a). The EpCAM protein in SKBR3 cells was lack of glycosylated isoforms,36 while that in HT29 cells contained both glycosylated isoforms and basic isoforms (Figure S9b). Thus, in SKBR3 cells, the GCP-Cy3 probes could not be modified on target EpCAM protein, but could on neighbor glycoproteins. After incubated with ASI-Cy5 probes, only EpCAM protein on both of these two cells could be selectively labeled. As shown in Figure S9c, obvious FRET signal was observed on HT29 cells due to the proximity-induced hybridization between GCP-Cy3 and ASI-



Cy5 probes on EpCAM protein. However, negligible FRET occurred on SKBR3 cells, indicating the ASI-Cy5 probes on EpCAM protein could not hybridize with GCP-Cy3 probes on other neighbor glycoproteins. Therefore, the proximity effect was proved to be generated from the glycoprotein and the intermolecular glycan rather than the intramolecular glycans. Furthermore, to exclude intermolecular proximity-induced signal, FRET between cell membrane lipid-derived GCP-Cy3 probes and ASI-Cy5 probes on target protein was investigated. For this purpose, CEM cells were pretreated with propargyl choline and the phospholipid molecules were born with terminal alkyne groups, then N3-GCP-Cy3 probes were modified on cell membrane by copper(I)-catalyzed azidealkyne cycloaddition (CuAAC) (Figure S10a). As shown in Figure S10b, slight FRET signal occurred between ASI-Cy5 probes on PTK7 with the GCP-Cy3 probes labeled on phospholipid. Next, to verify whether the hybridization between GCP and ASI probes occurred, H1-FAM and H2 probes were added. However, barely fluorescence signal of HCR assembly was generated on the cell surface, suggesting that surrounding lipid-derived GCP probes did not hybridize with the ASI-Cy5 probes on target protein and could not contribute to the proximity-induced HCR assembly as well. All of these results consistently showed that taking advantages of the small size and precise designability of DNA probes, the proposed strategy achieved visualization of protein-specific glycosylation via intramolecular proximity-induced HCR amplification. Visualization of the glycosylation states on target proteins under drug treatment. Besides the ability to visualize stably modified glycoprotein, the proximity-induced HCR strategy also enabled in situ amplified visualization of glycosylation changes on target proteins. Tunicamycin (TM) is a wellknown drug to induce a concentration-dependent decrease in glycosylation level of cells. Here, we pretreated CEM cells with the mixture of Ac4ManNAz and different doses of TM (0, 12.5, 20, 25 and 30 ng/mL) to remove N-glycan residues in living cells. Then, cells were analyzed with or without the proximity-induced HCR amplification as above mentioned. Compared with the fluorescence imaging without amplification, the proximity-induced HCR amplification led to a higher sensitivity against the changes of PTK7-specific glycosylation (Figure 5a). Inhibition with higher dose of TM produced a lower HCR fluorescence signal, which confirmed that the HCR signal represented PTK7-specific Nglycosylation status. By quantifying and normalizing the HCR fluorescence signal under treatment by different concentration of TM, the HCR signal was found to be weakened as TM concentration increasing. At the highest TM concentration (30 ng/mL), the HCR signal was reduced to nearly 50% of that of the cells without TM inhibition (Figure 5b). Flow cytometry assay also showed a coincident tendency as CLSM imaging (Figure 5c), demonstrating that the HCR amplification could discriminate the slight difference in protein-specific glycosylation level. In contrast, the quantitative analysis of fluorescence signal without amplification showed less sensitivity towards the change of glycosylation level in CEM cells. Besides, no significant distinction could be observed under various TM concentrations (Figure S11). All these results indicated that the proximity-induced HCR assembly could amplify the visualization of protein-specific glycosylation on the target proteins, prompting us to research glycosylation in more complex biological conditions.

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Figure 5. (a) CLSM images of CEM cells pretreated with a mixture of 25μM Ac4ManNAz and TM at different concentrations (0, 12.5, 20, 25, 30 ng/mL) with HCR amplification (top row) or without amplification (bottom row). Scale bar: 20 μm. (b) Quantitative CLSM intensity and (c) flow cytometry assay of HCR fluorescence in top row of Figure 5a (n ≥ 10; **, 0.01 ≥ P > 0.001; ***, P < 0.001 for tests of significant difference).

Visualization of protein-specific glycans in zebrafish larvae. Furthermore, we explored the capability of proposed amplified strategy to visualize protein-specific glycosylation in zebrafish larvae. As shown in Figure 6a, azido tagged CEM cells were harvested after 48 h Ac4ManNAz treatment and non-treated CEM cells were used as control. Then cells were injected into the yolk sac of zebrafish larvae at 48 hours post-fertilization. The bright fluorescence indicated the feasible implantation of CEM cells (Figure S12a) and the successful GCP probes labeling of glycans on Ac4ManNAz-treated CEM cells in zebrafish larvae (Figure S12b). Afterwards, the zebrafish larvae were further incubated with DBCO-GCP, ASI, H1 and H2 probes in sequence, which allowed the proximity-induced HCR reaction on the implanted CEM cell surfaces. The bright green fluorescence in the zebrafish abdomen suggested the effective HCR assembly in zebrafish carrying Ac4ManNAztreated CEM cells (Figure 6b and 6c, panel iii). In comparison, much weaker fluorescence was generated in the zebrafish larvae without HCR amplification (Figure 6b and 6c, panel ii), in which the larvae were injected with Ac4ManNAz-treated CEM cells and then incubated with DNA probes except H2 probes. Besides, barely fluorescence could be observed in zebrafish larvae which were injected with non-treated CEM cells (Figure 6b and 6c, panel i). These results indicated that the proximity-induced HCR assembly could identify the protein-specific glycans in zebrafish larvae.

Figure 6. (a) Scheme of protein-specific glycosylation imaging in zebrafish larvae. (b) 3D reconstruction fluorescence images and (c) merged cross section images of zebrafish larvae were viewed


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under different conditions: (i) injected with non-treated CEM cells and incubated with all DNA probes; (ii) injected with Ac4ManNAz-treated CEM cells and incubated with all DNA probes except H2 probes and (iii) injected with Ac4ManNAztreated CEM cells and incubated with all DNA probes. Green channel: excitation 488 nm/emission 500 - 550 nm. Scale bar:100 μm.

CONCLUSION In summary, we designed a proximity-induced HCR strategy for amplified visualization of the protein-specific glycosylation in living cells and zebrafish larvae. Making use of glycan conversion probes and protein recognition probes, selective labeling of glycans on target proteins and following HCR assembly could be simply achieved. The proposed strategy has been used to realize the amplified visualization of PTK7-specific sialic acid states in living cells and zebrafish. This straightforward and non-genetically engineered strategy could be easily expanded to other proteins of interest by simply changing the protein recognition probes. Thus, it provides a potential tool for investigating protein-specific glycosylation, and the corresponding protein glycosylation states in disease development.

EXPERIMENTAL DETAILS Reagents and materials. All of the used DNA probes were synthesized and HPLC purified by Sangon Biotechnology Co., Ltd (Shanghai, China). The sequences are listed in Table S1. N-azidoacetylmannosamine-tetraacylated (Ac4ManNAz) was purchased from Click Chemistry Tools LLC (USA). Propargyl choline was obtained from Glixx Laboratories Inc (USA). BTTAA was purchased from MedChemExpress (China). Tunicamycin and BCA protein quantitation kit were obtained from Solarbio Life Sciences (Beijing, China). Dulbecco's Phosphate-Buffered Saline (DPBS) was purchased from Gibco (USA). Magnesium chloride (MgCl2), glucose, bovine serum albumin (BSA), yeast tRNA, DBCO-Biotin, streptavidin-HRP, CuSO4 and sodium ascorbate were obtained from Sigma Aldrich (USA). Ultrapure water was obtained through a MilliQ water purification system (Millipore, USA) with an electric resistivity >18.2 MΩ. Cell lines CCRF-CEM (human T-cell ALL), Ramos (human B-cell Burkitt’s lymphoma), HT29 (human colon carcinoma cells) and MCF-10A (human mammary gland epithelia) were purchased from the American Type Culture Collection (Manassas, VA). In vitro DNA assembly. In this system, four DNA probes (ASI, GCP, H1 and H2) were snap-cooled respectively before use (heated at 95 °C for 3 min, incubated on ice for 3 min, then left at room temperature for 1 h). They were mixed at appropriate molar ratio (GCP: ASI: H1: H2= 200 nM: 200 nM: 1μM: 1μM) in HCR reacting buffer (DPBS containing 5 mM MgCl2) and incubated at room temperature for 24 h. Agarose gel electrophoresis. The formation of HCR assembly was validated by 3% agarose gel electrophoresis (100 V, 60 min) in 1× TBE buffer. The bands were stained with ethidium bromide (EB) and imaged using a ChemiDocTM Touch gel imaging system (Bio-Rad, USA). Fluorescence spectrometry. The fluorescence profile was monitored on a Cary Eclipse fluorescence spectrophotometer (Agilent, USA) at room temperature. For FRET between GCPCy3 and ASI-Cy5, the snap-cooled DNA probes were mixed

at the ratio of 1: 1 at room temperature for 30 min, then the solution was excited at 530 nm and the emission spectra were recorded from 540-750 nm. For HCR amplification signal, four snap-cooled DNA probes (GCP, ASI, FAM-H1-Dabcyl, H2) were mixed at the ratio of 200 nM: 200 nM: 1μM: 1μM at room temperature for 24 h. Then, the resultant solution was excited at 488 nm and the fluorescence emission were measured from 500-650 nm. Cell culture and metabolic labeling. CCRF-CEM, Ramos and SKBR3 cells were cultured in RPMI 1640 medium (Hyclone) supplemented with 10% FBS (Hyclone), penicillin (100 IU/mL) and streptomycin (100 IU/mL). HT29 cells were cultured in McCoy's 5a Medium Modified (ATCC) supplemented with 10% FBS (Hyclone), penicillin (100 IU/mL) and streptomycin (100 IU/mL). MCF-10A cells were cultured in MEGM (Mammary Epithelial Cell Growth Medium, Lonza) along with 100 ng/mL cholera toxin (Sigma). All cells were cultured at 37 °C in a humidified incubator containing 5% wt/vol CO2. For cellular metabolic labeling, cells were treated by 25 μM Ac4ManNAz for 48 h. Preparation of total cell lysates. Cells with or without Ac4ManNAz treatment were labeled with DBCO-biotin in labeling buffer (DPBS with 1% FBS) for 30 min at room temperature. Then, cells were harvested and washed twice with PBS. Cell pellets were resuspended with RIPA lysis buffer (Beyotime) containing protease inhibitor cocktail (Roche) and phosphatase inhibitor cocktail (Nacalai Tesque). Western blotting. The prepared cell lysates or protein extraction were separated by 8% SDS-PAGE and transferred onto PVDF membrane (Bio-rad, USA). After blocking with 5% BSA in PBST for 2 h, the membrane was washed three times with PBST. For identifying azido-tag glycans, the membrane was incubated with streptavidin-HRP for 1 h. For identifying target protein expression, the membrane was incubated with the primary antibody for PTK7 or actin (Abcam) overnight at 4 °C. After being washed with PBST for three times, the membrane was incubated with HRP-labeled anti-rabbit secondary antibody (Abcam) for 1 h and probed using SuperSignal West Pico Chemiluminescent Substrate (Thermo). The blotting images were obtained using a ChemiDocTM Touch gel imaging system (Bio-Rad, USA). Labeling of DNA probes and proximity-induced HCR assembly on cell surfaces. Cells were firstly labeled with 200 nM DBCO-GCP probes in labeling buffer (DPBS with 1% FBS) for 30 min at 37 °C and washed twice with DPBS. Then, cells were co-cultured with 200 nM PTK7 (for CEM and Ramos) or EpCAM (for HT29, MCF10A and SKBR3) targeted ASI probes in binding buffer (DPBS containing 5 mM MgCl2, 4.5 g/L glucose, 5 mM MgCl2, 1 mg/mL BSA and 0.1 mg/mL yeast tRNA) for 30 min at 37 °C and washed three times with washing buffer (DPBS containing 5 mM MgCl2, 4.5 g/L glucose and 5 mM MgCl2). Finally, 1 μM H1-FAM and H2 probes in reacting buffer (DBPS containing 5 mM MgCl2) were incubated with the cells at 37 °C for no more than 3 hours. For visualizing without amplification, the cells were labeled by DNA probes except H2 probes. In order to prevent the cellular internalization of DNA probes, 10 μg/mL chlorpromazine was added to the medium during the HCR assembly. For labeling GCP probes on the phospholipid of cell membrane, the CEM cells were pretreated with propargyl choline for 5 h, following by incubation at room temperature with a mixture of BTTAA: CuSO4 (6:1), N3-GCP-Cy3 probes



and sodium ascorbate solution for 20 min. Then, ASI-Cy5, H1-FAM and H2 probes were added for further incubation. Inhibition of glycosylation by drug treatment. CEM cells were seeded on 6-well dishes with the mixture of 25 μM Ac4ManNAz and tunicamycin at different concentration (0, 12.5, 20, 25, 30 ng/mL) in 1640 medium for 48 h. After being washed three times with 1640 medium, cells were labeled by the above-mentioned HCR strategy. Confocal laser scanning microscopic (CLSM) imaging. CEM and Ramos cells (5×106) were incubated with DNA probes with or without HCR amplification as previous mentioned, followed by three washes with washing buffer and resuspension in 200 μL reacting buffer before observation. The cell suspension was dropped on 35 mm glass-bottom dishes (Nest Biotechnology Co.,Ltd, China) and fluorescence images were acquired on a Nikon A1 confocal laser scanning fluorescence microscope (Japan). HT29, MCF10A and SKBR3 cells (4×104) were seeded on the glass-bottom dishes and incubated with or without Ac4ManNAz for 48 h. After being washed with DPBS, the cells were incubated with DNA probes with or without HCR amplification as previous mentioned, followed by three washes with washing buffer and imaged with the CLSM. FAM fluorescence was excited by 488 nm and recorded emission from 500 to 550 nm; Cy3 fluorescence was excited by 543 nm and recorded emission from 560 to 610 nm); FRET-induced fluorescence of Cy5 was excited by 543 nm and recorded emission using 650LP filter. The mean fluorescence intensities on the cell surface were quantified by using ImageJ software. For each case, we utilized more than 50 cells to calculate the average fluorescence level. Flow cytometric analysis. CEM cells were pretreated with mixture of 25 μM Ac4ManNAz and tunicamycin at different concentration in medium for 48 h. Then, the cells were collected and washed with DPBS. 200 μL cell suspension with a density of 5×106 cells/mL was then added to 200 nM DBCOGCP probes in labeling buffer for 30 min, 200 nM ASI probes in binding buffer for 30 min, 1 μM H1-FAM and with or without 1 μM H2 probes in reacting buffer for 2 h in sequence. Cells were washed with washing buffer for three times after each probe labeling. Finally, the cells were collected by centrifugation at 1000 rpm for 3 min, washed three times with reacting buffer and resuspended in 1 mL reacting buffer, and assayed by a Canto II flow cytometry (BD Bioscience, CA) equipped with a 488 nm laser by counting 10000 events. The data were analyzed with FlowJo software. Zebrafish experiments. Wild type zebrafish were purchased from Nanjing Eze-Rinka Biotechnology Co., Ltd. Zebrafish larvae were prepared using a protocol modified from previous report.19,28 Briefly, 48 hours post-fertilization (hpf) zebrafish larvae were fixed in 4% paraformaldehyde (PFA) for 24 hours at 4 °C, then washed with 3×5 min with PBS to stop the fixation. The fixed larvae were dehydrated and permeabilized with methanol (MeOH) wash. Before use, larvae were rehydrated with a series of graded MeOH/PBST washes for 5 min each cycle. Afterwards, Ac4ManNAz-treated or nontreated CEM cells were harvested after 48 hours pretreatment at a concentration of ~ 1×108 cells/mL with 0.05% phenol red. Then, 10 nL cells suspension were implanted into the yolk sac of fixed zebrafish larvae by using a programmable nanoliter Injector (Drummond, USA, Nanoject III). After cell implantation, the larvae were incubated with DBCO-GCP

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probes (200 nM), ASI probes (200 nM), H1-FAM probes (1 μM) and H2 probes (1 μM) in sequence. For visualizing without HCR amplification, the larvae were incubated with all DNA probes except H2 probes. After 2 hours incubation, the larvae were washed with E3 medium (5 mM NaCl, 0.17 KCl, 0.33 CaCl2, 0.33 MgSO4, pH 7.0) for several times to remove the unlabeled DNA probes. Besides, some control experiments were performed. CEM cells were stained with DiO for 30 min and then washed three time by 1640 medium, then the CEM cells with or without DiO labeling were harvest and implanted into zebrafish larvae respectively. CEM cells with or without Ac4ManNAz pretreatment were harvest and injected into zebrafish larvae, then the larvae were incubated with DBCOGCP-Cy3 probes for 30 min and washed by labeling buffer for three times. Finally, the fluorescence imaging of all zebrafish larvae samples was characterized by using a Nikon A1 CLSM (Japan).

ASSOCIATED CONTENT Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org. Supporting Table S1, Supporting Figures S1-S12 (PDF)

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected]; [email protected]

Author Contributions The manuscript was written through contributions of all authors.

ACKNOWLEDGMENT This work is supported by the National Natural Science Foundation of China (No. 21622502, 21475026, 21505021, 21605023), the Natural Science Foundation of Fujian Province of China (No. 2016J05035, 2017J06004), the Program for Changjiang Scholars and Innovative Research Team in University (No. IRT15R11), the outstanding youth scientific research personnel training plan of colleges and universities in Fujian Province, the Health-Education joint research project of Fujian Province (No. KJ2016-2-23).

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Figure 1. Illustration of amplified visualization of protein-specific glycosylation via proximity-induced HCR. 139x84mm (300 x 300 DPI)



Figure 2. (a) Agarose gel electrophoresis image of HCR products. (b) Fluorescence profile of FRET-based structure-switching of ASI-Cy5 probes upon hybridization with GCP-Cy3 probes. Cy3 and FRET: excitation 530 nm/emission 550 - 750 nm. Cy5: excitation 640 nm/emission 650 - 750 nm. (c) Fluorescence profile of HCR-based DNA assembly (i: ASI + GCP + H1 + H2; ii: GCP + H1 + H2; iii: ASI + H1 + H2; iv: H1 + H2); excitation 488 nm/emission 500 - 650 nm. 140x147mm (300 x 300 DPI)


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Figure 3. The CLSM images of PTK7-specific glycans in living cells via FRET signal. (a) CEM and Ramos cells were treated with Ac4ManNAz, followed by sequential labeling with DBCO-GCP-Cy3 and ASI-Cy5, scale bar: 20 μm (inset: enlarged image, scale bar: 2 μm). (b) Donor was dequenched after acceptor photo-bleaching. Green channel: Cy3 excitation 543 nm/emission 560 - 610 nm. Red channel: FRET excitation 543 nm/emission 650LP filter. Merge channel: overlay of fluorescence image and bright filed image. Ratio channel: Cy5/Cy3. Scale bar: 5 μm. 139x141mm (300 x 300 DPI)



Figure 4. The CLSM images of PTK-7 specific glycosylation on the CEM or Ramos cell surfaces with or without Ac4ManNAz treatment by using proximity-induced HCR amplification strategy. Top panel: merge of fluorescence and bright field images; bottom panel: fluorescence field images, excitation 488 nm/emission 500 - 550 nm. Scale bar: 20 μm. 129x75mm (300 x 300 DPI)


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Figure 5. (a) CLSM images of CEM cells pretreated with a mixture of 25μM Ac4ManNAz and TM at different concentrations (0, 12.5, 20, 25, 30 ng/mL) with HCR amplification (top row) or without amplification (bottom row). Scale bar: 20 μm. (b) Quantitative CLSM intensity and (c) flow cytometry assay of HCR fluorescence in top row of Figure 5a (n ≥ 10; **, 0.01 ≥ P > 0.001; ***, P < 0.001 for tests of significant difference). 132x120mm (300 x 300 DPI)



Figure 6. (a) Scheme of protein-specific glycosylation imaging in zebrafish larvae. (b) 3D reconstruction fluorescence images and (c) merged cross section images of zebrafish larvae were viewed under different conditions: (i) injected with non-treated CEM cells and incubated with all DNA probes; (ii) injected with Ac4ManNAz-treated CEM cells and incubated with all DNA probes except H2 probes and (iii) injected with Ac4ManNAz-treated CEM cells and incubated with all DNA probes. Green channel: excitation 488 nm/emission 500 - 550 nm. Scale bar:100 μm. 139x41mm (300 x 300 DPI)


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Amplified Visualization of Protein-specific Glycosylation in Zebrafish

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