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Enhanced Imaging of Specific Cell– Surface Glycosylation Based on Multi–FRET Baoyin Yuan, Yuanyuan Chen, Yuqiong Sun, Qiuping Guo, Jin Huang, Jianbo Liu, Xiangxian Meng, Xiaohai Yang, Xiaohong Wen, Zenghui Li, Lie Li, and Kemin Wang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b00424 • Publication Date (Web): 26 Apr 2018 Downloaded from http://pubs.acs.org on April 26, 2018

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Analytical Chemistry

Enhanced Imaging of Specific Cell–Surface Glycosylation Based on Multi–FRET Baoyin Yuan, Yuanyuan Chen, Yuqiong Sun, Qiuping Guo,* Jin Huang, Jianbo Liu, Xiangxian Meng, Xiaohai Yang, Xiaohong Wen, Zenghui Li, Lie Li and Kemin Wang* State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Biology, College of Chemistry and Chemical Engineering, Hunan University, Key Laboratory for Bio-Nanotechnology and Molecular Engineering of Hunan Province, Changsha 410082, China ABSTRACT: Cell–surface glycosylation contains abundant biological information that reflects cell physiological state, it is of great value to image cell–surface glycosylation to elucidate its functions. Here we present a hybridization chain reaction (HCR)– based multi–fluorescence resonance energy transfer (multi–FRET) method for specific imaging of cell–surface glycosylation. By installing donors through metabolic glycan labeling and acceptors through aptamer–tethered nanoassemblies on the same glycoconjugate, the intramolecular multi–FRET occurs due to near donor–acceptor distance. Benefiting from amplified effect and spatial flexibility of the HCR nanoassemblies, the enhanced multi–FRET imaging of specific cell–surface glycosylation can be obtained. With this HCR–based multi–FRET method, we achieved obvious contrast in imaging of protein–specific GalNAcylation on 7211 cell surfaces. In addition, we demonstrated the general applicability of this method by visualizing the protein–specific sialylation on CEM cell surfaces. Furthermore, the expression changes of CEM cell–surface protein–specific sialylation under drug treatment was accurately monitored. This developed imaging method may provide a powerful tool in researching glycosylation functions, discovering biomarkers and screening drugs.

Glycosylation, which occurs on their protein or lipid scaffolds, is rich with information that reflects cell physiological state.1 Specifically, most of cell membrane proteins are glycosylated though post–translational modification, which plays an important role in regulation of biological functions.2 Cell–surface glycosylation are proved to work in many critical biological processes, including cancer development, inflammation, cardiovascular disease, and so on.3 Aberrant cell–surface glycosylation is often a hallmark of disease occurrence. In particular, altered cell–surface glycosylation patterns are highly related to cancer phenotype.4 Therefore, it is of great value to specifically visualize cell– surface glycosylation to understand glycan metabolism and regulation mechanism, to discover new diagnostic biomarkers and identify new therapeutic targets. However, glycans are difficult to be detected relative to other biomolecules such as oligonucleotides and proteins because of their complex conformation and non–templated synthesis.4 Recently, a metabolic glycan labeling strategy, which is vigorously developed by Bertozzi's group,5, 6 has been proposed to meet this purpose. In this ingenious strategy, azide modified monosaccharide analogues are first incorporated into global glycans of target cells though glycan metabolic pathways. Then the azide groups are covalently coupled with alkyne or cyclooctyne–functionalized molecules such as fluorescence dyes or affinity ligands via click reaction chemistry for subsequent imaging or target identification.7–12 This two–step labeling method has been applied to detect glycosylation on a variety of research objects, for instance, cells,13–18 tissues,10 virus19 and model species1, 2, 6, 20, 21. Due to labeling of all cell–surface glycans that containing the added unnatural monosaccharide, this method could not be alone used in specific imaging of cell–surface glycosylation. Fluorescence resonance energy transfer (FRET) technique is an effective method that has been widely applied to biochemical analysis22–25. For specific imaging of cell–surface glycosylation, FRET is a talented solution. This solution has been successfully used in specific imaging of different cell–

surface glycosylation.7, 13, 15, 17 In this strategy, the target glycoconjugate is dual–labeled with FRET donor and acceptor through metabolic labeling and targeted labeling, owing to near donor–acceptor distance on the same glycoconjugate, the FRET occurs and thus FRET signal. For targeted labeling of glycoconjugate, some methods such as fluorescent protein fusions16, 17, site–specific protein labeling16 and affinity ligand targeted labeling7, 13,18 are usually used. Although fluorescent protein fusions are powerful tools for protein labeling, their relatively large size may perturb the expression, location or function of the target glycoproteins.26 Site–specific protein labeling often requires complicated enzyme system and is not suitable to glycoproteins that lack of specific sites. For affinity ligand targeted labeling, the common targeting ligands rely on fluorescence dye–modified antibodies. As we know, the number of dyes could be conjugated to antibodies is really limited, but the copy number of incorporated monosaccharide analogues is usually higher than that of dye–modified antibodies with several orders of magnitudes, and this probably leads to low FRET signal.7 Moreover, the rigid spherical structure of antibodies may not be beneficial for FRET. To facilitate the occurrence of FRET, amplified labeling of target glycoconjugates with flexible ligands is an effective method. Aptamers, which developed by SELEX (systematic evolution of ligands by exponential enrichment),27, 28 are short single–stranded DNA or RNA and can bind targets with high affinity and specificity.29, 30 Aptamers are excellent substitutes for antibodies for amplified labeling of target glycoconjugates in specific FRET imaging of cell–surface glycosylation. Benefiting from the Watson–Crick base pairing of oligonucleotides, aptamers are easy to design for amplified labeling combined with DNA amplification techniques. Hybridization chain reaction (HCR) amplification, which is triggered by an initiator strand and yields a relatively long and flexible double–strand DNA nanostructure, is a non–enzyme amplification process under room temperature and could be used to general sensing and therapeutic applications.31–33 To

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our knowledge, HCR amplification has not been applied to enhanced imaging of cell–surface glycosylation. Herein, we report a HCR amplification–based multi–FRET strategy for specific imaging of cell–surface glycosylation by employing metabolic glycan labeling. First, the target cells are subjected to the metabolic glycan labeling for donor installation, that is incorporated with azide–modified monosaccharide analogues on cell surfaces, followed by reacting with dibenzocyclooctynol (DIBO)–modified FRET donors through copper–free click chemistry.34 Then, to label target glycoconjugates with FRET acceptors, target cells are incubated with acceptor dye–labeled nanoassemblies that produced by aptamer–triggered HCR. In the previously reported FRET–based methods for cell–surface glycosylation assay, the target glycoconjugates are usually labeled with a single FRET donor or acceptor, considering the huge discrepancy between the incorporated monosaccharide and the binding ligands, this go against the obtaining of high FRET signal. Besides, the commonly used target ligands, antibodies, may not be perfect for FRET because of their rigid spherical structure. However, the amplified labeling of target glycoconjugates and rational intramolecular proximity between donors and acceptors can be induced by the HCR nanoassemblies in our strategy, and the high FRET signal is probably to be obtained, which can be used to enhanced imaging of cell–surface glycosylation.

EXPERIMENTAL SECTION Chemicals and Materials. Ac4GalNAz (N–azidoacetyl galactosamine–tetraacylated) and Ac4ManNAz (N–azidoacetyl mannosamine–tetraacylated) were purchased from Thermo Scientific (USA). Click–iT DIBO–Alexa Fluor 488 (DIBO– AF488) and SYBR Gold nucleic acid dye was purchased from Invitrogen (USA). 1,1'–dioctadecyl–3,3,3',3'–tetramethyl indocarbocyanine perchlorate (DiI) was purchased from Beyotime (Shanghai, China). Tunicamycin was purchased from Solarbio (Beijing, China). PNGase F, O–Glycosidase and α (2–3, 6, 8, 9) neuraminidase were purchased from NEB (USA). 5'–Alexa Fluor 488–TTTTT–Cholesterol–3' (AF488– Cholesterol) probe, 5'–Texas red–TTTTT–Cholesterol–3' (TR– Cholesterol) probe and all other DNA sequences used in the experiment were synthesized by Sangon Biotechnology Co., Ltd. (Shanghai, China). All DNA sequences are listed in Table S1. Water was purified by the Milli–Q ultrapure water system (Millipore System Inc., USA). Hoechst 33342 and Dulbecco's phosphate–buffered saline (D–PBS) was purchased from Sigma–Aldrich (USA). The binding buffer contained 5 mM MgCl2, 4.5 g/L glucose, 1 mg/mL bovine serum albumin (BSA) and 0.1 mg/mL yeast tRNA in D–PBS. Other chemicals were of analytical grade at least. Cells. Hepatocellular carcinoma SMMC–7721 (7721) cells, human acute lymphoblastic leukemia CCRF–CEM (CEM) cells, and hepatocyte L02 cells used in the experiment were obtained from the cell bank of the Committee on Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Hepatocellular carcinoma HepG2 cells used in the experiment were purchased from American Type Culture Collection. All cells were cultured in basic RPMI 1640 medium supplemented with 12% fetal bovine serum (FBS) and 0.02% penicillin–gentamicin (200 µg/mL) at 37 °C in a humidified incubator containing 5% CO2 by volume. Both cell subculture and pretreatment were operated at the clean bench.

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Preparation of the Aptamer–Triggered Nanoassemblies. The probe H1, H2 and aptamer–trigger were dissolved in D– PBS supplemented with 5 mM Mg2+ respectively, and then heated at 95 °C for 5 min and cooled at room temperature for 30 min. The probe H1 (5 µM), H2 (5 µM) and aptamer–trigger (0.5 µM) were mixed together and then incubated at room temperature for 2 hours for the formation of the nanoassemblies. Agarose gel electrophoresis. The obtained nanoassemblies were characterized by 2.5% agarose gel electrophoresis (100 V, 45 min), after stained with SYBR Gold, the gel was imaged by a C600 multifunctional molecular imaging system (Azure Biosystems, USA). For UV imaging, the gel was excited using a 302–nm light and collected with a 595/55–nm filter; for TR fluorescent imaging, the gel was excited using a 628–nm light and collected with a 676/28–nm filter. Metabolic Glycan Labeling. 7721 cells were seeded on glass bottom dishes (In Vitro Scientific, USA) and cultured in 1640 medium containing 40 µM Ac4GalNAz for 24 hours. CEM cells were seed on a 12–well plate and cultured in RPMI 1640 medium containing 40 µM Ac4ManNAz for 48 hours.8 After washed with D–PBS containing 1% FBS for three times, the cells were incubated with 10 µM DIBO–AF488 at room temperature for 60 min for AF488 labeling. Then the cells were washed with D–PBS containing 1% FBS for three times for subsequent experiments. Flow Cytometry Assays. To obtain FRET–induced signal, the 7721 cells that subjected to AF488 metabolic glycan labeling were incubated with Texas Red (TR)–labeled ZYsls (25 nM) or as–prepared ZYsls–tethered nanoassemblies (equivalent 25 nM) in 200 µL of binding buffer at 4 °C for 30 min. After washed with binding buffer for twice, the cells were analyzed by a flow cytometer (Gallios, Beckman Coulter, USA) by counting 10000 events. The AF488 fluorescence signal was collected in FL1 with a 488–nm laser and a 505– to 525–nm band–pass filter. The FRET fluorescence signal was collected in FL3 with a 488–nm laser and a 620–nm long–pass filter. To test the recognition ability of aptamer–trigger and nanoassemblies, the 7721 cells were incubated with 25 nM TR–labeled aptamer, aptamer–trigger and nanoassemblies in 200 µL of binding buffer at 4 °C for 30 min. After washed with binding buffer for twice, the cells were analyzed by the flow cytometer by counting 10000 events. The TR fluorescence signal was collected in FL6 with a 633–nm laser and a 660–nm long–pass filter. Confocal Imaging. For FRET–induced specific imaging of cell–surface glycosylation, the 7721 cells that subjected to AF488 metabolic glycan labeling were incubated with ZYsls (25 nM) or as–prepared ZYsls–tethered nanoassemblies (equivalent 25 nM) in binding buffer at 4 °C for 30 min, the CEM cells that subjected to AF488 metabolic glycan labeling were incubated with sgc8–tethered nanoassemblies (equivalent 100 nM) in binding buffer at 4 °C for 60 min. After washed with binding buffer for twice, the cells were fixed with 4% paraformaldehyde for 15 min followed by washing with D– PBS for three times, and imaged by the FV500 (Olympus, Japan) laser scanning confocal microscope (LSCM). To verify intramolecular FRET, 7721 cells were seeded on glass bottom dishes, and incubated with Ac4GalNAz for 24 hours. After washed three times with D–PBS, the cells were incubated with 200 µl of bundle of 2000 unit/mL O– glycosidase and 100 unit/mL α (2–3, 6, 8, 9) neuraminidase at 37 °C for 2 hours, and then followed by the above metabolic

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glycan labeling, incubated with the nanoassemblies and imaged by LSCM. In the drug treatment experiments, the SiaNAz labeled CEM cells were treated with 10 µg/mL of tunicamycin in RPMI 1640 culture medium at 37 °C for 24 hours or 100 unit/mL α (2–3, 6, 8, 9) neuraminidase in binding buffer at 37 °C for 2 hours, after washing three times with D–PBS, were subjected to the above AF488 and TR dual–labeling and imaged by LSCM. The FRET signal quantification was analyzed using the ImageJ software. First, the integrated optical density of each image was calculated by the ImageJ. Then, the average optical density of a single cell was calculated. The obtained average value was used as the statistical FRET signal intensity of each sample. The fluorescence signal was collected by a 100× oil immersion objective. Hoechst 33342 fluorescence was excited using a 405–nm laser and collected with a 430– to 460–nm band–pass filter. AF488 fluorescence was excited using a 488– nm laser and collected with a 505– to 525–nm band–pass filter. TR and DiI fluorescence were excited using a 543–nm laser and collected with a 560–nm long–pass filter. FRET–induced fluorescence was excited using a 488–nm laser and collected with a 610–nm long–pass filter.

RESULTS AND DISCUSSION Design of a HCR–Based Multi–FRET Experiment. As a proof of concept, two TR–labeled hairpin probes (H1, H2, sequences in Table S1) were designed for HCR polymerization in the presence of a trigger probe. To obtain the aptamer– triggered HCR nanoassemblies, ZYsls aptamer that derived from intact ZY11 aptamer (sequences in Table S1), which was developed by our lab using cell–SELEX and can bind to target 7721 cells with high affinity and specificity,35, 36 was chosen to construct the nanoassemblies. The target of ZYsls locates on cell membrane, and was preliminarily identified as a N– glycoprotein (Figure S1, Figure S2). To initiate HCR, a trigger DNA was fused into the 5'–end of ZYsls. The obtained chimeric aptamer–trigger (ZYsls–trigger, sequences in Table S1) retained high affinity to 7721 cells (Figure S3). When ZYsls–trigger was added into the mixture of H1 and H2, the self–assembly of these building blocks occurred and thus ZYsls–tethered DNA nanoassemblies (Scheme 1). Then the Scheme 1. Schematic Illustration of the HCR–Based Multi– FRET Method for Imaging of Protein–Specific GalNAcylation on 7721 Cell Surfaces. Aptamer-trigger

Nanoassemblies

n

HCR TR

Binding H1

Ac4GalNAz N3

H2

N3

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Analytical Chemistry

AF488-DIBO

N3

N3

Membrane Target glycoprotein

Cytosol

TR–labeled nanoassemblies were used to stain target cells that subjected to AF488 metabolic glycan labeling (Scheme 1). AF488 and TR are a pair of FRET dyes, proved by the suitable overlap of AF488 emission spectrum and TR excitation spectrum (Figure S4). Due to intramolecular proximity of multiple AF488 and TR, which the distance of AF488 and TR was probably within 1–10 nm for FRET,37 the enhanced FRET signal was obtained. Construction and Characterization of Nanoassemblies. For construction of nanoassemblies, the molar ratio of ZYsls–

A

B Nanoassemblies 500bp 200bp 100bp

ZYsls

Random

+ -

+ -

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+ + -

+ + +

Marker H1 H2 ZYsls-trigger

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TR

Figure 1. Characterization of the formation, and enhanced target cell binding of the nanoassemblies. (A) Agarose gel electrophoresis demonstrating the HCR polymerization of nanoassemblies initiated by ZYsls–trigger. The used marker is 20 bp DNA Ladder. (B) Flow cytometric assays of recognition abilities of TR–labeled random sequence, ZYsls and nanoassemblies to target 7721 cells.

trigger to hairpins was investigated by agarose gel electrophoresis, the results demonstrated that a 1:5 ratio was the best, and it was used in subsequent experiments (Figure S5). The formation of the nanoassemblies in the presence of ZYsls–trigger was verified by agarose gel electrophoresis (Figure 1A, Figure S6). After successful construction of nanoassemblies, the binding ability of nanoassemblies to target 7721 cells was investigated through flow cytometric assays, the results significantly showed enhanced fluorescence intensity of 7721 cells stained with nanoassemblies compared to that of 7721 cells stained with ZYsls, suggested amplified labeling of target glycoproteins by the nanoassemblies (Figure 1B). To further improve the binding ability of nanoassemblies to 7721 cells, the probe concentration and incubation temperature were optimized respectively, the results demonstrated that the combination of 4 °C with 25 nM probes was the desired experimental condition (Figure S7). Imaging of Protein–Specific GalNAcylation on 7721 Cell Surfaces. Cell–surface GalNAc (N–acetylgalactosamine) is a key monosaccharide unit as a recognition element, which functions not only in normal physiological progression but also in cancer development.38 It is of great value to specifically image of cell–surface GalNAcylation to elucidate its functions in protein regulation and cancer development. Because the binding target of ZYsls is a cell–surface N–glycoprotein, the so–called GalNAcylation is target N–glycoprotein–specific. For global labeling of cell–surface GalNAc of 7721 cells, Ac4GalNAz, which is modified with a azide group for coupling with DIBO–AF488 through click reaction39, 40, was used as a substitute for GalNAc in cellular glycometabolism. Chen et al recently reported that increasing artificial S– glycosylation of cysteine residues in various proteins could be induced with increasing number of per–O–acetylated unnatural monosaccharides.41 To confirm whether the used Ac4GalNAz would induce lots of artificial S–glycosylation on 7721 cell

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Analytical Chemistry

TR

FRET signal

AF488

TR

B

1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0

a

Figure 2. LSCM imaging of protein–specific GalNAcylation on 7721 cell surfaces with the multi–FRET method. 7721 cells were treated with Ac4GalNAz for AF488 installation, and then incubated with the nanoassemblies for TR labeling (the lower). Negative controls in which the nanoassemblies (the upper) or GalNAz (the middle) was absent. The fluorescence signal was collected by a 100× objective.

GalNAc and red TR staining for target glycoproteins on surface of 7721 cells that treated with Ac4GalNAz and nanoassemblies, a strong gray FRET signal was observed because of near distance between donor and acceptor on the same target glycoprotein, while negative controls without the

FRET signal

No treat

A

Normalized FRET signal intensity

AF488

nanoassemblies or Ac4GalNAz treatment showed no FRET signals except for corresponding AF488 or TR fluorescence. The FRET signal was further verified by flow cytometric assays, the results demonstrated that the FRET signal intensity of dual–labeled 7721 cells was obviously higher than negative controls (Figure S12). Furthermore, the specificity of this multi–FRET imaging method for visualizing of 7721 cell– surface protein–specific GalNAcylation was investigated. As displayed in Figure S13, strong TR fluorescence signal and protein–specific GalNAcylation FRET signal were observed on target 7721 cells because of specific recognition of ZYsls– tethered nanoassemblies to 7721 cells, but no signals on control liver cancer HepG2 cells and hepatocyte L02 cells. These results showed excellent specificity of the proposed method to image protein–specific GalNAcylation on 7721 cell surfaces. Verification of Intramolecular FRET. Because all cell– surface GalNAc of target cells were labeled with AF488, whether the above obtained signals were produced from intramolecular FRET needs to be verified. Generally, N–linked and O–linked glycosylation are the most common forms on cell surfaces. If the FRET signals come from N–glycoproteins, inhibition of O–glycans will not affect the FRET signals, and vice versa. A bundle of O–Glycosidase and neuraminidase was used to inhibit O–glycosylation since it could catalyze the removal of O–glycans from glycoproteins.42 As shown in Figure 3A, after treated with the enzyme bundle, green AF488

＋ O-Glycosidase ＋ Neuraminidase

surfaces, the fixed cells were subjected to AF488 labeling and DiI cell membrane staining for co–staining. The results showed no obvious co–staining (Figure S8), indicated that the cell–surface S–glycosylation induced by Ac4GalNAz could be ignored. To obtain the strongest AF488 fluorescence with the least amount of Ac4GalNAz in metabolic glycan labeling, the incubation concentration of Ac4GalNAz to 7721 cells was optimized, as shown in Figure S9, the fluorescence intensity reached a plateau at 40 µM of Ac4GalNAz, which was used in subsequent studies. Moreover, incubation time of 7721 cells to Ac4GalNAz was investigated, the results demonstrated that the AF488 fluorescence reached the highest when the incubation time was 24 hours, which was applied in subsequent experiments (Figure S10). Although 7721 cell surfaces were expectedly labeled with AF488 by metabolic glycan labeling, some intracellular regions were bright with AF488 fluorescence. To explore this phenomena, Hoechst 33342 and TR–Cholesterol were respectively used to stain cell nucleus and membrane system for colocalization experiment. TR– Cholesterol is a membrane probe can stain cell outer and internal membrane due to excellent membrane–anchoring capability of cholesterol. As shown in Figure S11, AF488 co– stained with TR rather than Hoechst 33342, indicated that these bright regions belonged to internal membrane. According to the facts that endoplasmic reticulum (ER) and Golgi apparatus (GA) are close to cell nucleus and function in glycan synthesis and processing, these bright regions mostly located on ER and GA. After metabolic labeling of cell–surface GalNAc with AF488 as donor, 7721 cells were incubated with TR–labeled ZYsls–tethered nanoassemblies for acceptor installation. As shown in Figure 2, in addition to green AF488 staining for all ＋ Ac4GalNAz －Ac4GalNAz ＋ Ac4GalNAz ＋ Nanoassemblies ＋ Nanoassemblies － Nanoassemblies

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b

Figure 3. (A) LSCM imaging verifying intramolecular FRET of target glycoproteins. The upper cells were subjected to AF488 metabolic labeling and TR staining. The cells below were treated with O– Glycosidase and neuraminidase for O–glycan inhibition after Ac4GalNAz incorporation, followed by the above AF488 and TR dual–labeling. The fluorescence signals were collected by a 100× objective. (B) Corresponding quantitative analysis of the FRET signal intensity of 7721 cells without (a) and with (b) drug treatment. Error bars indicate the standard deviations of three experiments.

fluorescence of 7721 cells became weaker than that of untreated cells, while the gray FRET signals did not change significantly, indicating that the cleavage of O–glycans by the

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enzyme bundle showed negligible influence on FRET imaging of 7721 cell–surface protein–specific GalNAcylation. Corresponding statistical results demonstrated that the FRET signal intensity of cells with enzyme treatment was 92% of that of cells without enzyme treatment (Figure 3B). These results suggested that AF488 donors located on surrounding O–glycoproteins could not contribute to the FRET signals obviously, thus verified that the signals probably derived from intramolecular FRET of target N–glycoproteins. In addition, to further exclude nonspecific FRET signal, the FRET between cell membrane lipid–derived AF488 and TR was investigated by using a AF488–Cholesterol cell membrane probe. From the results, we observed no obvious FRET signal of 7721 cells stained with AF488–Cholesterol compared with that of stained with Ac4GalNAz for AF488 glycan labeling (Figure S14), suggested that surrounding lipid–derived AF488 could not contribute to the FRET signals significantly, further verified that the obtained signals were from intramolecular FRET of target N–glycoproteins. Enhanced Effect of the Multi–FRET. As we know, one affinity ligand usually binds to one glycoprotein, while the copy number of monosaccharide units of the glycoprotein is much higher than that of the binding ligand. This would lead to enormous quantity variance between donors and acceptors labeled on target glycoproteins after metabolic glycan labeling and ligand staining, and would affect FRET efficiency. Besides, uncertain spatial distribution of the glycosylation sites and binding ligand may be unfavorable for FRET. As a result, the HCR–based multi–FRET method was designed to improve FRET efficiency in protein–specific imaging of cell–surface glycosylation. Because of multiple acceptor labelling and more rational acceptor distribution induced by the relatively long and flexible structures of the aptamer–tethered nanoassemblies, the FRET was easy to occur effectively. As displayed in Figure 4A, the FRET signal of 7721 cells incubated with TR

Normalized FRET signal intensity

B

FRET signal

1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0

nanoassemblies was obviously stronger than that of 7721 cells incubated with ZYsls, and corresponding stronger TR fluorescence was also observed because of multiple TR installed on nanoassemblies. According to the statistical average value of FRET fluorescence intensity, the enhanced HCR–based multi–FRET fluorescence intensity was approximately two times higher than that of the FRET fluorescence intensity with no HCR (Figure 4B). These results suggested that the HCR–based multi–FRET method could effectively image protein–specific cell–surface GalNAcylation with an enhanced FRET signal. Visualizing of Protein–Specific Sialylation on CEM Cell Surfaces. To further investigate the general applicability of this method, we applied it to visualize cell–surface sialylation. Cell–surface sialylation plays a vital role in most physiological and pathological processes, and abnormal sialylation usually appears in cancer development, especially cancer metastasis.43, 44 As a proof of concept, cell–surface protein tyrosine kinase–7 (PTK7), which was closely related to a variety of cancers and overexpressed on human acute lymphoblastic leukemia CEM cells,45, 46 was chosen as the target glycoprotein. Ac4ManNAz was used to label sialic acid with AF488 as donor on the CEM cell surfaces. To evaluate the Ac4ManNAz induced S– glycosylation on CEM cell surfaces, the fixed cells were labeled with AF488 and DiI membrane dyes for co–staining. As shown in Figure S15, no obvious co–staining contrast was observed between the Ac4ManNAz treated cells and control cells, indicated that the number of Ac4ManNAz induced S– glycosylation was really limited. Meanwhile, the sgc8 aptamer, which selectively binds to PTK7,45, 47 was selected to design sgc8–trigger for HCR polymerization. The formation of sgc8– tethered nanoassemblies in the presence of sgc8–trigger was verified by agarose gel electrophoresis (Figure S16). The obtained sgc8–tethered nanoassemblies retained great binding ability to CEM cells (Figure S17). As shown in Figure 5, after ＋ Ac4ManNAz － Ac4ManNAz ＋ Ac4ManNAz ＋ Nanoassemblies ＋ Nanoassemblies － Nanoassemblies

AF488

ZYsls

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Analytical Chemistry

a

b

Figure 4. LSCM imaging of protein–specific GalNAcylation on 7721 cells incubated with TR–labeled ZYsls (A, the upper) and TR–labeled nanoassemblies (A, the below) after AF488 metabolic labeling. The fluorescence signals were collected by a 100× objective. (B) Quantitative analysis of the FRET signal intensity of 7721 cells incubated with TR–labeled ZYsls (a) and TR–labeled nanoassemblies (b). Error bars indicate the standard deviations of three experiments.

AF488

TR

FRET signal

Figure 5. Visualizing of PTK7–specific sialylation on CEM cell surfaces with the multi–FRET method by LSCM. CEM cells were treated with Ac4ManNAz for AF488 installation, and then were incubated with sgc8–tethered nanoassemblies for TR labeling (the lower). Negative controls in which the sgc8–tethered nanoassemblies (the upper) or Ac4ManNAz (the middle) was absent. The fluorescence signal was collected by a 100× objective.

the CEM cells were dual–labeled with AF488 donor and TR acceptor through metabolic glycan labeling and nanoassembly

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Analytical Chemistry staining respectively, in addition to green AF488 staining for all sialic acids and red TR staining for PTK7 on CEM cell surfaces, a strong gray FRET fluorescence was observed, while negative controls without the nanoassemblies or Ac4ManNAz treatment showed no FRET fluorescence. This result indicated that the information of CEM cell–surface PTK7–specific sialylation could be visualized by the multi– FRET method. Monitoring Cell–Surface Protein–Specific Sialylation under Drug Treatment. To evaluate the expression changes of protein–specific sialylation under drug treatment by the multi–FRET method, the target CEM cells were treated with tunicamycin for N–glycan inhibition and neuraminidase for sialic acid hydrolysis, respectively. As shown in Figure 6A,

A

TR

FRET signal

+ Tunicamycin

No treat

AF488

CONCLUSION In summary, we present a HCR–based multi–FRET method for specific imaging of cell–surface glycosylation by installing donors through metabolic glycan labeling and acceptors through aptamer–tethered nanoassembly staining on the same target glycoprotein. Because of amplified effect and spatial flexibility induced by HCR nanoassemblies, the enhanced FRET imaging of 7721 cell–surface protein–specific GalNAcylation can be successfully achieved. Furthermore, this method can enable visualizing of protein–specific sialylation on CEM cell surfaces, and monitoring of expression changes of the protein–specific sialylation under drug treatment. In theory, we can detect different glycosylation types on cell surfaces with this method as long as the aptamers of target glycoconjugates are available. This developed method may provide a new insight into researching biological functions of cell–surface glycosylation and pathogenetic mechanism of aberrant glycosylation.

Supporting Information

Normalized FRET signal intensity

B

tunicamycin and neuraminidase were 40% and 16% of that of cells without drug treatment, respectively. These results suggested that this multi–FRET method could be used to monitor the cell–surface protein–specific sialylation expression under drug treatment, and possess great potential in drug screening.

ASSOCIATED CONTENT

+ Neuraminidase

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1.2 1.0 0.8 0.6 0.4 0.2 0.0

a

b

c

Figure 6. (A) LSCM imaging of expression changes of PTK7– specific sialylation on CEM cell surfaces with drug treatment by the multi–FRET method. The CEM cells were treated with Ac4ManNAz, and then subjected to drug treatment, followed by AF488 and TR dual–labeling. The upper CEM cells were treated without drug, the middle CEM cells were treated with tunicamycin, and the CEM cells below were treated with neuraminidase. The fluorescence signal was collected by a 100× objective. (B) Corresponding quantitative analysis of the FRET signal intensity of 7721 cells treated with no drug (a), tunicamycin (b) and neuraminidase (c). Error bars indicate the standard deviations of three experiments.

after the CEM cells were treated with drugs and labeled with AF488 donors and TR acceptors through the above dual– labeling strategy, the green AF488 fluorescence and gray PTK7–specific FRET fluorescence observed on CEM cells with drug treatment was significantly weaker than that of CEM cells without drug treatment, which was contributed to the decreased number of sialic acids on CEM cell surfaces induced by drug inhibition. From the statistical average value (Figure 6B), the FRET signal intensity of cells treated with

The Supporting Information is available free of charge on the ACS Publications website. The table listing detailed sequences of all oligonucleotides used in this work; 17 figures showing target type identification of ZYsls, recognition ability of ZYsls–trigger, fluorescence spectrum of the FRET pair, optimization of ZYsls–trigger concentration for HCR, nanoassembly concentration for 7721 cell binding, Ac4GalNAz concentration for glycan labeling, incubation time of Ac4GalNAz to 7721 cells and incubation temperature of nanoassembly to 7721 cells, S–glycosylation induced by Ac4GalNAz and Ac4ManNAz, colocalization analysis of AF488 to cell nucleus and membrane system in 7721 cells, verification of intramolecular FRET on 7721 cells, agarose gel electrophoresis of the HCR initiated by ZYsls–trigger and sgc8–trigger, the specificity of protein–specific GalNAcylation on 7211 cell surfaces, confocal images of CEM cells stained with nanoassemblies.

AUTHOR INFORMATION Corresponding Authors * E–mail: [email protected]; Tel/Fax: +86–731–88821566. * E–mail: [email protected]; Tel/Fax: +86–731–88821566.

Notes The authors declare no competing financial interests.

ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (Grants 21735002, 21575037, 21778016) and the Foundation for Innovative Research Groups of NSFC (Grant 21521063).

REFERENCES

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Analytical Chemistry

Table of Contents (TOC)

Aptamer-trigger

Nanoassemblies

n

HCR TR

Binding H1

Ac4GalNAz N3

H2

N3

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

AF488-DIBO

N3

N3

Membrane Target glycoprotein

Cytosol

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