A nongenetic approach for imaging protein dimerization by aptamer

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A nongenetic approach for imaging protein dimerization by aptamer recognition and proximity-induced DNA assembly Hong Liang, Shan Chen, Peipei Li, Liping Wang, Jingying Li, Juan Li, Huang-Hao Yang, and Weihong Tan J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b11311 • Publication Date (Web): 09 Mar 2018 Downloaded from http://pubs.acs.org on March 9, 2018

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Journal of the American Chemical Society

A nongenetic approach for imaging protein dimerization by aptamer recognition and proximity-induced DNA assembly Hong Liang,† Shan Chen,† Peipei Li,† Liping Wang,† Jingying Li,‡ Juan Li,†,||,* Huang-Hao Yang†,‡,* and Weihong Tan||,

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MOE Key Laboratory for Analytical Science of Food Safety and Biology, College of Chemistry, Fuzhou University, Fuzhou 350116, P. R. China ‡ College of Biological Science and Engineering, Fuzhou University, Fuzhou 350116, P. R. China || Institute of Molecular Medicine, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai Jiao Tong University, Shanghai, 200240, China ⊥

Department of Chemistry and Department of Physiology and Functional Genomics, Center for Research at the Bio/Nano Interface, UF Health Cancer Center, University of Florida, Gainesville, FL 32611-7200, USA Fax: (+1) 352-846-2410 Supporting Information Placeholder ABSTRACT: Herein, we report a nongenetic and real-time approach for imaging protein dimerization on living cell surfaces by aptamer recognition and proximity-induced DNA assembly. We use the aptamer specific for the receptor monomer as a recognition probe. When receptor dimerization occurs, the dimeric receptors bring two aptamer probes into close proximity, thereby triggering dynamic DNA assembly. The proposed approach was successfully applied to visualize dimerization of Met receptor and transforming growth factor-β type II receptor. This approach allows us to image the two states (monomer/dimer) of a receptor protein on living cell surfaces in real time, opening a universal method for further investigation of protein dimerization and the corresponding activation processes in signal transduction.

Protein-protein interactions play pivotal roles in a wide variety of biological processes. In particular, receptor protein dimerization on the cell surface is often believed to be the first step in intracellular signal transduction, which is crucial for normal biological processes and cancer development.1 For instance, receptor tyrosine kinases (RTKs) are widely expressed transmembrane proteins. They act as receptors for growth factors, neurotrophic factors, and other extracellular signaling molecules, which regulate fundamental cellular activities.2 The canonical oligomerization model for RTK activation proposes that ligand binding shifts the equilibrium from monomeric inactive receptors towards active receptor dimers.3 The dimerization of RTKs and the subsequent phosphorylation of the intracellular domain are key steps for triggering a series of intracellular downstream signaling cascades.4 Therefore, validation of the expression levels and oligomerization states of RTKs is critical for an in-depth understanding of signal transduction networks. Fluorescence techniques are frequently used for tracking and analyzing receptor dimerization or protein modifications on cell surfaces due to their speed, simplicity, and sensitivity.5-7 These imaging methods have been successfully applied in imaging the dimerization of several RTKs, including the fibroblast growth

factor receptor (FGFR),8 Met receptor,9 and tropomyosin-related kinase receptor.10 However, to date, most of these methods have relied on genetic engineering of receptors with fluorescent proteins. Genetic modification has been reported to affect the receptor dimerization efficiency or perturb other biological processes in cells.11, 12 Thus, development of a nongenetic and real-time approach for imaging of protein dimerization on living cell surfaces remains a desirable goal. Herein, we propose a nongenetic approach for real-time imaging of receptor monomers and their dimerization based on aptamer recognition and proximity-induced DNA assembly. Aptamers are functional single-stranded nucleic acids with high specificity and affinity toward specific molecular targets.13-15 The principle of our approach is illustrated in Scheme 1. The approach is designed to have receptor recognition (abc and ab*d) and signal output probes (c*d* and d). Region a is the aptamer sequence (colored pink), which recognizes the receptor monomer. Regions b and b* (colored blue) are designed to have only 8 complementary bases, so that they cannot form a stable duplex at room temperature. Regions c and d are complementary to the signal output probes c*d* (colored purple and yellow, respectively). Upon ligand stimulation, the receptor monomers are activated to form dimers. The dimeric receptors bring two aptamer probes into close proximity, greatly increasing their local effective concentrations. Consequently, the two probes hybridize to each other to form a stable abc:ab*d DNA duplex, which accelerates the strand displacement reaction between c*d* and d and leads to the fluorescence recovery of Cy5 for the dimers on the cell surface. Moreover, receptor recognition probes labeled with FAM can be used to detect the amount of receptor monomer. In addition, the concept of proximity-induced DNA assembly or enzymatic proximity ligation assay has been widely used for protein detection, as well as protein-protein interactions.16-22 Thus, the proposed approach combining the advantages of aptamer recognition and proximity-induced DNA assembly would provide an efficient avenue for visualizing both the amount of dimers and monomers on living cell surfaces.

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Scheme 1. Schematic illustration of the principle of imaging protein dimerization on the cell membrane by aptamer recognition and proximity-induced DNA assembly. As a proof-of-principle, we selected the mesenchymal epithelial transition (Met) receptor as a model, which is a cognate receptor for the hepatocyte growth factor (HGF).23, 24 HGF activates the Met receptor by binding and promoting receptor dimerization.25 A 40-mer Met-binding DNA aptamer was used for targeting the Met receptor.26 Tables S1 and S2 show the sequences of the DNA probes used in this work. Of note, one of the key points for proximity-induced DNA assembly is the design of the complementary sequences of receptor recognition probes, abc and ab*d. We carefully examined complementary sequences of abc and ab*d (Figure S1). Indeed, the background fluorescence intensity scarcely increased when increasing lengths of complementary sequences of abc and ab*d from 6 to 8 nt. However, further increasing the complementary lengths of DNA probes from 9 to15 nt, resulted in significant high background in the blank. Therefore, 8 nt complementary sequences were selected for probe design in this work, unless otherwise specified. Then, we explored this approach for analyzing receptor dimerization on living cell surfaces. The cell line MKN-45 was used as a model due to its high Met expression level.27 Overexpression of Met in MKN-45 cells can lead to HGF-independent dimerization of receptors and subsequent autophosphorylation of Met.28 The Met and phosphorylated Met (p-Met) expression levels in MKN-45 cells were determined by western blot analysis (Figure 1A). As shown in Figure 1B, after incubating all DNA probes with MKN-45 cells for 10 minutes, intense fluorescence signals in the red and green channels were observed on the cell surfaces by confocal laser scanning microscopy (CLSM) (Figure 1B, line i). To illustrate the function of a pair of receptor recognition probes, we performed control experiments in the absence of ab*d (Figure 1B, line ii) or abc (Figure 1B, line iii). Negligible fluorescence of Cy5 in the red channel and bright fluorescence of FAM in the green channel on the MKN-45 cell surface were found in both controls. In addition, there were no fluorescence signals of Met receptor monomers and dimers in LNCaP cells, which is Met receptor negative cell line (Figure S2).29 The absence of Met and p-Met expression was also confirmed by western blot analysis (Figure 1A). The results of flow cytometry analysis were consistent with those of CLSM images (Figure 1C). The fluorescence emission ratio (Cy5/FAM) upon incubation with all DNA probes was distinguishable from incomplete controls (no abc or ab*d). Therefore, these results demonstrated that the proposed approach can be applied for imaging ligand-independent Met dimerization.

Figure 1. (A) Western blot analysis of Met and p-Met expression in MKN-45 cells. (B) CLSM images and (C) Flow cytometry analysis of Met protein dimerization in the MKN-45 cells with different DNA probes. (i) abc, ab*d, and c*d*:d; (ii) abc and c*d*:d; (iii) ab*d and c*d*:d. Ratio images generated from the emission ratio of the Cy5 to the FAM (Cy5/FAM). Scale bars represent 20 µm. We further evaluated this approach for ligand-dependent Met dimerization. The DU145 cell line, which expresses moderate levels of endogenous Met protein, was chosen as a model.30 Ratiometric imaging with receptor recognition and signal output DNA probes was applied. At first, the fluorescence signal from the isotype probes (abc and c*d*:d) served as the negative control. Negligible fluorescence was observed in the red channel, suggesting low background noise (Figure S3). Next, the DU145 cells were incubated with all DNA probes (abc, ab*d, and c*d*:d). As shown in Figure 2A, a slight red emission and strong green emission were observed without HGF stimulation, suggesting that Met receptors mainly existed as monomers (Figure 2A, line i). Upon incubation with HGF for 30 minutes and then adding DNA probes, the DU145 cells showed a marked increase in the red channel (Figure 2A, line ii), suggesting that the ligand HGF is indeed able to induce the dimerization of Met receptor monomers on living cells. Quantitative analysis of the cell fluorescence imaging data revealed that the emission ratio (Cy5/FAM) displayed about 2.3-fold enhancement than that of without stimulation (Figure S4). Furthermore, based on the calculations, ~89.3% and ~10.7% of Met receptors were monomers and dimers in the DU145 cells, respectively (Figure S5). Upon HGF stimulation, the dimer fraction increased to ~39.2 %, which was comparable with those obtained by single molecule total internal reflection fluorescence microscopy (Figures S5 and S6). In addition, a significant reduction of red fluorescence signal was observed in cells pretreated with HGF inhibitors (Figure 2A, line iii). We next examined the real-time imaging capability of our approach. Time-lapse fluorescence imaging was carried out with a one-minute interval. Only a small increase in the fluorescence signal was observed in the control group without HGF treatment (Figure 2B line i, Figure S7 line i, Videos S1 and S2). However, after HGF stimulation, a detectable fluorescence signal was detected as early as one minute and increased over the 30 minutes measurement window (Figure 2B line ii, Figure S7, line ii, Videos S3 and S4). Further quantitative measurement of the cell imaging data revealed that new Met dimers rapidly formed on the cell surface in initial 10 minutes, and followed by a slow and sustained increase within 30 minutes (Figure S8). This real-time imaging capability would potentially contribute to rapid signal transduction by proximity-induced DNA assembly.


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Journal of the American Chemical Society We also used flow cytometry to analyze Met protein dimerization in DU145 cells under different conditions. The results from flow cytometry were consistent with those of CLSM images (Figure 2C). Then, we conducted western blot analysis to check the Met phosphorylation level with different types of stimulation. A significant increase in the Met phosphorylation level was observed in cells that were treated with HGF (Figure 2D lane 2) compared to untreated cells (Figure 2D lane 1). However, the Met phosphorylation expression was decreased when cells were pretreated with HGF inhibitors (Figure 2D lane 3). This result was consistent with a widely accepted hypothesis for the mechanism of Met activation, which states that the formation of dimeric Met receptors induces autophosphorylation of Met proteins.23

Figure 2. (A) CLSM images of Met protein dimerization under different conditions. Scale bars represent 20 µm. (B) Time-lapse images of Met dimerization under different conditions. Scale bars represent 10 µm. (C) Flow cytometry analysis of Met protein dimerization under different conditions. (D) Western blot analysis of p-Met and Met expression in DU145 cells. (i) without treatment, (ii) addition of HGF and (iii) addition of HGF and inhibitor. Ratio images generated from Cy5/FAM. To evaluate the generality of the approach, we used transforming growth factor-β (TGF-β) type II receptor (TβRII), which belongs to the serine/threonine kinase family, as another model for imaging protein dimerization.31,32 It has been reported that TβRII exists in a dynamic equilibrium between monomeric and dimeric forms on the cell surface.33 After ligand stimulation, dimeric receptor population increases, which can be used as an indicator for TGF-β signaling activation.34 We tested the potential of the proposed approach for TβRII imaging on living cell surfaces. An aptamer sequence which binds to TβRII was used as a targeting unit.35 Compared with the negative control (abc and c*d*:d, Figure 3 line i), a low red fluorescence signal and obvious green fluorescence signal in the untreated DU145 cells were observed (line ii), suggesting that TβRII partly existed as dimers. Interestingly, the red fluorescence signal increased after cells were treated with TGF-β, indicating that dimeric receptor population increased after ligand stimulation (line iii). Quantitative analysis of fluorescence imaging data revealed that the emission ratio (Cy5/FAM) displayed about 1.7-fold enhancement (Figure S9). To further prove that the fluorescence signal was triggered by the TβRII dimers, we pretreated cells with TGF-β inhibitors. A significant reduction of red fluorescence signal was observed in cells treated with inhibitors (line iv). Further, the real-time imaging of TβRII dimerization was achieved successfully in living DU145 cells. Time-lapse

images of TβRII dimerization in response to TGF-β stimulation were taken with a one-minute interval (Figure S10 and Videos S5-S8). Quantitative analysis of the cell imaging data showed that TβRII dimers gradually increased within 30 minutes (Figure S11).

Figure 3. CLSM images of TβRII protein dimerization in the DU145 cells under different conditions. (i) negative control, (ii) without treatment, (iii) addition of TGF-β and (iv) addition of TGF-β and inhibitor. Ratio images generated from Cy5/FAM. Scale bars represent 20 µm. Next, to confirm TGF-β signaling activation upon TβRII dimerization, we evaluated downstream signaling stimulation through phosphorylation of the intracellular mediator Smad2 protein.36 Comparing the untreated and inhibitor-treated cells, the cells with TGF-β treatment showed phosphorylation of Smad2 protein, while the TβRII protein expression level stayed the same (Figure S12). All these results indicated that this approach also can be used for TβRII dimerization imaging and can be expanded easily to other receptors by simply changing aptamers. In summary, we have designed a nongenetic approach for real-time imaging of receptor dimerization on living cell surfaces by aptamer recognition and proximity-induced DNA assembly. This approach is simple, convenient and highly versatile, thus can potentially be applied to any target protein of interest by simply changing aptamers. Furthermore, this method can be performed in various types of samples, thus holding great potential in practical applications. Therefore, the proposed real-time imaging approach provides a powerful tool for in-depth study of protein dimerization and the corresponding activation processes in signal transduction. ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Experimental details, supporting figures and table (PDF). AUTHOR INFORMATION Corresponding Author *[email protected] *[email protected] Notes The authors declare no competing financial interests. ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (Nos. 21622502, 21475026, 21505021, 21605023), Natural Science Foundation of Fujian Province of



China (Nos. 2015H6011, 2016J05035, 2017J06004), the Program for Changjiang Scholars and Innovative Research Team in University (No. IRT15R11), and the Health-Education joint research project of Fujian Province (No. WKJ2016-2-23). REFERENCES (1) Heldin, C. H. Cell 1995, 80, 213-223. (2) Lemmon, M. A.; Schlessinger, J. Cell 2010, 141, 1117. (3) Maruyama, I. N. Cells 2014, 3, 304. (4) Trusolino, L.; Bertotti, A.; Comoglio, P. M. Nat. Rev. Mol. Cell Biol. 2010, 11, 834-848. (5) Sako, Y.; Minoghchi, S.; Yanagida, T. Nat. Cell Biol. 2000, 2, 168-72. (6) Persani, L.; Calebiro, D.; Bonomi, M. Nat. Clin. Pract. Endocrinol. Metab. 2007, 3, 180-90. (7) Chen, Y.; Liu, H.; Ding, L.; Ju, H. Anal Chem. 2018, Doi: 10.1021/acs.analchem.7b03587. (8) Sarabipour, S.; Hristova, K. Nat. Commun. 2016, 7, 10262. (9) Koschut, D.; Richert, L.; Pace, G.; Niemann, H. H.; Mely, Y.; Orian-Rousseau, V. Biochim. Biophys. Acta. 2016, 1863, 1552-8. (10) Shen, J.; Maruyama, I. N. J. Mol. Signal. 2012, 7, 2. (11) Michelini, E.; Cevenini, L.; Mezzanotte, L.; Coppa, A.; Roda, A. Anal. Bioanal. Chem. 2010, 398, 227. (12) Ueki, R.; Atsuta, S.; Ueki, A.; Sando, S. J. Am. Chem. Soc. 2017, 139, 6554-6557. (13) Liang, H.; Zhang, X. B.; Lv, Y.; Gong, L.; Wang, R.; Zhu, X.; Yang, R.; Tan, W. Acc. Chem. Res. 2014, 47, 1891-901. (14) Li, J.; Mo, L.; Lu, C. H.; Fu, T.; Yang, H. H.; Tan, W. Chem. Soc. Rev. 2016, 45, 1410-1431. (15) Zhou, J.; Rossi, J. Nat. Rev. Drug Discov. 2017, 16, 440. (16) Zong, C.; Wu, J.; Liu, M.; Yang, L.; Liu, L.; Yan, F.; Ju, H. Anal Chem. 2014, 86, 5573-5578. (17) Guo, Y.; Wu. J.; Ju, H. Chem Sci. 2015, 6, 4318-4323. (18) Zong, C.; Wu, J.; Liu, M.; Yan, F.; Ju, H. Chem. Sci. 2015, 6, 2602-2607. (19) Li. F.; Lin, Y.; Le, X. C. Anal Chem. 2013, 85, 10835-10841. (20) Tang, Y.; Wang, Z.; Yang, X.; Chen, J.; Liu, L.; Zhao, W.; Le, X. C.; Li, F. Chem. Sci. 2015, 6, 5729-5733.

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Scheme 1. Schematic illustration of the principle of imaging protein dimerization on the cell membrane by aptamer recognition and proximity-induced DNA assembly. 199x146mm (300 x 300 DPI)


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Figure 1. (A) Western blot analysis of Met and p-Met expression in MKN-45 cells. (B) Confocal fluorescence imaging and (C) Flow cytometry analysis of Met protein dimerization in the MKN-45 cells with different DNA probes. (i) abc, ab*d, and c*d*:d; (ii) abc and c*d*:d; (iii) ab*d and c*d*:d. Ratio images generated from the emission ratio of the Cy5 to the FAM (Cy5/FAM). Scale bars represent 20 µm. 199x196mm (300 x 300 DPI)



Figure 2. (A) Confocal fluorescence imaging of Met protein dimerization in DU145 cells under different conditions. Scale bars represent 20 µm. (B) Time-lapse images of Met dimerization under different conditions. Scale bars represent 10 µm. (C) Flow cytometry analysis of Met protein dimerization in DU145 cells under different conditions. (D) Western blot analysis of p-Met and Met expression in DU145 cells. (i) without treatment, (ii) addition of HGF and (iii) addition of HGF and inhibitor. Ratio images generated from Cy5/FAM. 267x308mm (300 x 300 DPI)


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Figure 3. Confocal fluorescence imaging of TβRII protein dimerization in the DU145 cells under different conditions. (i) negative control, (ii) without treatment, (iii) addition of TGF-β and (iv) addition of TGF-β and inhibitor. Ratio images generated from Cy5/FAM. Scale bars represent 20 µm. 477x359mm (300 x 300 DPI)


A nongenetic approach for imaging protein dimerization by aptamer

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