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Photoregulation of protein plasmid expression in vitro and in vivo using BHQ caging group
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Chinese Chemical Letters 22 (2011) 338–341 www.elsevier.com/locate/cclet
Photoregulation of protein plasmid expression in vitro and in vivo using BHQ caging group Zhi Ping Zhang a, Yi Ming Li a,b,*, Xiao Yun Chen a, Qing Xiang Guo a,** a
Department of Chemistry, University of Science and Technology of China, Hefei 230026, China b Department of Chemistry, Tsinghua University, Beijing 100084, China Received 25 June 2010 Available online 22 December 2010
Abstract Green fluorescent protein (GFP) plasmid was caged by 8-bromo-7-hydroxyquinolinyl chromophore (BHQ) for controlling its expression with exact spatiotemporal resolution. In vitro and in vivo experiments clearly verified that, comparing with Bhc caging, the expression level of caged GFP plasmid was dramatically decreased and then efficiently restored after subsequent photolysis. # 2010 Yi Ming Li. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved. Keywords: Photoregulation; Caging (uncaging); BHQ; Green fluorescent protein plasmid; Photolysis
Photolabile protecting groups offer an ideal approach for regulating cellular functions in a time-dependent and space-focused manner [1]. ‘‘Caging’’ is a specific term meaning that a bioactive molecule was encapsulated in an inactive form and can be subsequently restored by photolysis [2]. Recently, a number of novel photocaging groups, such as 1-(4,5-dimethoxy-2-nitrophenyl)ethyl (DMNPE) [3–5] and 6-bromo-7-hydroxycoumarin-4-ylmethyl (Bhc) [6,7] have been successfully used to cage nucleic acids in vivo. However, insufficient uncaging efficiency, which is a primary obstacle to in vivo studies, still exists because of several negative properties of the aforementioned caging groups. In comparison with them, the 8-bromo-7-hydroxyquinolinyl (BHQ) has a very improved property. The BHQ group was first developed by Dore et al. for photoreleasing the acetate, phosphate, and diol groups commonly found in bioactive molecules including neurotransmitters, nucleic acids, and drugs [8–10]. Compared with DMNPE and Bhc, many photochemical and photophysical properties of BHQ, such as two-photon cross section, photolysis sensitivity, water solubility are substantially improved. These features were believed to be highly important for the study on living cells [11]. In the present work, we examine, for the first time, the use of the BHQ group to photoregulate the plasmid expression both in vitro and in vivo. The diazomethane form of BHQ (BHQ-diazo) was used in our study, and green fluorescent protein (GFP) was performed as a model protein. Our results show that protein expression can be efficiently photoregulated by the proposed approach, which may provide an important and useful tool for future studies on the photo-control of protein activities in cellular systems.
* Corresponding author at: Department of Chemistry, University of Science and Technology of China, Hefei 230026; Department of Chemistry, Tsinghua University, Beijing 100084, China. ** Corresponding author. E-mail addresses: [email protected] (Y.M. Li), [email protected] (Q.X. Guo). 1001-8417/$ – see front matter # 2010 Yi Ming Li. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved. doi:10.1016/j.cclet.2010.10.007
[()TD$FIG]
Z.P. Zhang et al. / Chinese Chemical Letters 22 (2011) 338–341
339
Scheme 1. The synthesis procedures of BHQ-diazo.
1. Results and discussion BHQ-diazo was synthesized in five steps as previously described (Scheme 1) and stored at 20 8C under dark conditions [12]. The caging group was co-incubated with the GFP plasmid within 6–8 h and then the resulting photocaged plasmid was purified using Microcon YM (10,000 MW cutoff) centrifugal filters. The spectrophotometric analysis of the caged plasmid showed two characteristic absorbance peaks of BHQ-diazo at 320 nm and 370 nm in addition to the main peak at 260 nm. This observation was similar to that previous reported about BHQ caging nucleic acid [8]. The appearance of these additional peaks was attributed to the attachment of BHQ-diazo indicating the success of the photo-caging process. Subsequent photolysis of the caged plasmid with 365 5 nm ultraviolet light and purification, the plasmid was de-caged and the photocleaved product resembles the control GFP plasmid (Fig. 1). Likewise, the result of gel electrophoresis further confirmed that the caged GFP plasmids reduced the mobility and intensity of the plasmid band compared to that of the control, suggesting an alteration of the GFP plasmid that [()TD$FIG]interferes with its staining (Fig. 2). A light-induced change was observed between the caged and light-activated 2.5
Absorbance Units (AU)
2.0
GFP plasmid Caged GFP plasmid
1.5
Caged - light
1.0
0.5
0.0 250
300
350
400
450
[()TD$FIG]
Wavelengh(nm) Fig. 1. Spectrophotometric analysis of caged and light-activated GFP plasmid. All samples were processed in parallel.
Fig. 2. Gel electrophoresis analysis of caged and light-activated GFP plasmids. All samples were processed in parallel.
[()TD$FIG] 340
Z.P. Zhang et al. / Chinese Chemical Letters 22 (2011) 338–341 5' O
HO
N2 + nucleic acid
N Br
H
Base
nucleic acid
uncaging
caging HO
N Br
O O P O O
+
H Base
HO
N
OH
Br
BHQ-diazo O '3
H
Scheme 2. Reaction of GFP plasmid caged by BHQ-diazo and photo-removal (ucaging) of the BHQ compounds.
[()TD$FIG]
Fig. 3. Fluorescent images of native GFP (A), Bhc caged (B) and light-activated (C), BHQ caged (D) and light-activated (E) plasmid expression in HeLa cell.
samples, with the photolysis band having increased mobility and intensity than the process control GFP plasmid [13,14]. All the above results clearly demonstrated that BHQ-diazo could cage with GFP plasmid through forming a covalent bond with the phosphate moiety of nucleic acid during simply co-incubation and compounds were subsequently released upon light-activation (Scheme 2). After confirmed that BHQ could link and cage with the GFP plasmid in vitro, we further compared the expression level of BHQ and Bhc caged GFP plasmid in vivo. HeLa cells were liposome-transfected with caged and native plasmids coding for GFP. As shown in Fig. 3, the fluorescence intensity which shown by BHQ and Bhc caged protein plasmid expressed decreased to about 15% of native GFP protein and partly restored with subsequent photolysis. Compared with Bhc caged plasmid, BHQ caged sample showed a much higher expression level for higher fluorescence intensity with similar photolysis period. We considered that the higher photolysis sensitivity and improved water solubility of BHQ may contribute to its better uncaging efficiency in vivo. 2. Conclusion In summary, the diazomethane form of BHQ compound was successfully used to cage with GFP plasmid both in vitro and in vivo. This work is the first description of the application of BHQ to photoregulate nucleic acid expression in vivo with higher caging and uncaging efficiency than the previously reported Bhc caging method. Because
Z.P. Zhang et al. / Chinese Chemical Letters 22 (2011) 338–341
341
insufficient uncaging efficiency is the primary obstacle to in vitro and in vivo studies [2,15], we anticipate that the positive results presented here will provide a wide application and convenient strategy of BHQ-diazo to photoregulate plasmid as well as mRNA expressions in vivo. The applications of this new approach to the study of protein manipulation under photo-controlled conditions are undergoing in our laboratory and will be reported elsewhere. Acknowledgment This study was supported by the National Natural Science Foundation of China (No. 90713009). References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15]
G. Mayer, A. Heckel, Angew. Chem. Int. Ed. 45 (2006) 4900. X.J. Tang, I.J. Dmochowski, Mol. Biosyst. 3 (2007) 100. W.T. Monroe, M.M. McQuain, M.S. Chang, et al. J. Biol. Chem. 274 (1999) 20895. S.B. Cambridge, D. Geissler, F. Calegari, et al. Nat. Methods 6 (2009) 527. R.A. Blidner, K.R. Svoboda, R.P. Hammer, et al. Mol. Biosyst. 4 (2008) 431. H. Ando, T. Furuta, R.Y. Tsien, et al. Nat. Genet. 28 (2001) 317. H. Ando, M. Kobayashi, T. Tsubokawa, et al. Dev. Biol. 287 (2005) 456. O.D. Fedoryak, T.M. Dore, Org. Lett. 4 (2002) 3419. Y. Zhu, C.M. Pavlos, J.P. Toscano, et al. J. Am. Chem. Soc. 128 (2006) 4267. M.J. Davis, C.H. Kragor, K.G. Reddie, et al. J. Org. Chem. 174 (2009) 1721. Y. Li, J. Shi, R. Cai, et al. Tetrahedron Lett. 51 (2010) 1609. Y. Li, J. Shi, R. Cai, et al. J. Photochem. Photobiol. A: Chem. 211 (2010) 129. B. Ghosn, F.R. Haselton, K.R. Gee, et al. Photochem. Photobiol. 81 (2005) 953. Y. Li, J. Shi, Z.F. Luo, et al. Bioorg. Med. Chem. Lett. 19 (2009) 5368. J.P. Casey, R.A. Blidner, W.T. Monroe, Mol. Pharm. 6 (2009) 669.
Chinese Chemical Letters 22 (2011) 338–341 www.elsevier.com/locate/cclet
Photoregulation of protein plasmid expression in vitro and in vivo using BHQ caging group Zhi Ping Zhang a, Yi Ming Li a,b,*, Xiao Yun Chen a, Qing Xiang Guo a,** a
Department of Chemistry, University of Science and Technology of China, Hefei 230026, China b Department of Chemistry, Tsinghua University, Beijing 100084, China Received 25 June 2010 Available online 22 December 2010
Abstract Green fluorescent protein (GFP) plasmid was caged by 8-bromo-7-hydroxyquinolinyl chromophore (BHQ) for controlling its expression with exact spatiotemporal resolution. In vitro and in vivo experiments clearly verified that, comparing with Bhc caging, the expression level of caged GFP plasmid was dramatically decreased and then efficiently restored after subsequent photolysis. # 2010 Yi Ming Li. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved. Keywords: Photoregulation; Caging (uncaging); BHQ; Green fluorescent protein plasmid; Photolysis
Photolabile protecting groups offer an ideal approach for regulating cellular functions in a time-dependent and space-focused manner [1]. ‘‘Caging’’ is a specific term meaning that a bioactive molecule was encapsulated in an inactive form and can be subsequently restored by photolysis [2]. Recently, a number of novel photocaging groups, such as 1-(4,5-dimethoxy-2-nitrophenyl)ethyl (DMNPE) [3–5] and 6-bromo-7-hydroxycoumarin-4-ylmethyl (Bhc) [6,7] have been successfully used to cage nucleic acids in vivo. However, insufficient uncaging efficiency, which is a primary obstacle to in vivo studies, still exists because of several negative properties of the aforementioned caging groups. In comparison with them, the 8-bromo-7-hydroxyquinolinyl (BHQ) has a very improved property. The BHQ group was first developed by Dore et al. for photoreleasing the acetate, phosphate, and diol groups commonly found in bioactive molecules including neurotransmitters, nucleic acids, and drugs [8–10]. Compared with DMNPE and Bhc, many photochemical and photophysical properties of BHQ, such as two-photon cross section, photolysis sensitivity, water solubility are substantially improved. These features were believed to be highly important for the study on living cells [11]. In the present work, we examine, for the first time, the use of the BHQ group to photoregulate the plasmid expression both in vitro and in vivo. The diazomethane form of BHQ (BHQ-diazo) was used in our study, and green fluorescent protein (GFP) was performed as a model protein. Our results show that protein expression can be efficiently photoregulated by the proposed approach, which may provide an important and useful tool for future studies on the photo-control of protein activities in cellular systems.
* Corresponding author at: Department of Chemistry, University of Science and Technology of China, Hefei 230026; Department of Chemistry, Tsinghua University, Beijing 100084, China. ** Corresponding author. E-mail addresses: [email protected] (Y.M. Li), [email protected] (Q.X. Guo). 1001-8417/$ – see front matter # 2010 Yi Ming Li. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved. doi:10.1016/j.cclet.2010.10.007
[()TD$FIG]
Z.P. Zhang et al. / Chinese Chemical Letters 22 (2011) 338–341
339
Scheme 1. The synthesis procedures of BHQ-diazo.
1. Results and discussion BHQ-diazo was synthesized in five steps as previously described (Scheme 1) and stored at 20 8C under dark conditions [12]. The caging group was co-incubated with the GFP plasmid within 6–8 h and then the resulting photocaged plasmid was purified using Microcon YM (10,000 MW cutoff) centrifugal filters. The spectrophotometric analysis of the caged plasmid showed two characteristic absorbance peaks of BHQ-diazo at 320 nm and 370 nm in addition to the main peak at 260 nm. This observation was similar to that previous reported about BHQ caging nucleic acid [8]. The appearance of these additional peaks was attributed to the attachment of BHQ-diazo indicating the success of the photo-caging process. Subsequent photolysis of the caged plasmid with 365 5 nm ultraviolet light and purification, the plasmid was de-caged and the photocleaved product resembles the control GFP plasmid (Fig. 1). Likewise, the result of gel electrophoresis further confirmed that the caged GFP plasmids reduced the mobility and intensity of the plasmid band compared to that of the control, suggesting an alteration of the GFP plasmid that [()TD$FIG]interferes with its staining (Fig. 2). A light-induced change was observed between the caged and light-activated 2.5
Absorbance Units (AU)
2.0
GFP plasmid Caged GFP plasmid
1.5
Caged - light
1.0
0.5
0.0 250
300
350
400
450
[()TD$FIG]
Wavelengh(nm) Fig. 1. Spectrophotometric analysis of caged and light-activated GFP plasmid. All samples were processed in parallel.
Fig. 2. Gel electrophoresis analysis of caged and light-activated GFP plasmids. All samples were processed in parallel.
[()TD$FIG] 340
Z.P. Zhang et al. / Chinese Chemical Letters 22 (2011) 338–341 5' O
HO
N2 + nucleic acid
N Br
H
Base
nucleic acid
uncaging
caging HO
N Br
O O P O O
+
H Base
HO
N
OH
Br
BHQ-diazo O '3
H
Scheme 2. Reaction of GFP plasmid caged by BHQ-diazo and photo-removal (ucaging) of the BHQ compounds.
[()TD$FIG]
Fig. 3. Fluorescent images of native GFP (A), Bhc caged (B) and light-activated (C), BHQ caged (D) and light-activated (E) plasmid expression in HeLa cell.
samples, with the photolysis band having increased mobility and intensity than the process control GFP plasmid [13,14]. All the above results clearly demonstrated that BHQ-diazo could cage with GFP plasmid through forming a covalent bond with the phosphate moiety of nucleic acid during simply co-incubation and compounds were subsequently released upon light-activation (Scheme 2). After confirmed that BHQ could link and cage with the GFP plasmid in vitro, we further compared the expression level of BHQ and Bhc caged GFP plasmid in vivo. HeLa cells were liposome-transfected with caged and native plasmids coding for GFP. As shown in Fig. 3, the fluorescence intensity which shown by BHQ and Bhc caged protein plasmid expressed decreased to about 15% of native GFP protein and partly restored with subsequent photolysis. Compared with Bhc caged plasmid, BHQ caged sample showed a much higher expression level for higher fluorescence intensity with similar photolysis period. We considered that the higher photolysis sensitivity and improved water solubility of BHQ may contribute to its better uncaging efficiency in vivo. 2. Conclusion In summary, the diazomethane form of BHQ compound was successfully used to cage with GFP plasmid both in vitro and in vivo. This work is the first description of the application of BHQ to photoregulate nucleic acid expression in vivo with higher caging and uncaging efficiency than the previously reported Bhc caging method. Because
Z.P. Zhang et al. / Chinese Chemical Letters 22 (2011) 338–341
341
insufficient uncaging efficiency is the primary obstacle to in vitro and in vivo studies [2,15], we anticipate that the positive results presented here will provide a wide application and convenient strategy of BHQ-diazo to photoregulate plasmid as well as mRNA expressions in vivo. The applications of this new approach to the study of protein manipulation under photo-controlled conditions are undergoing in our laboratory and will be reported elsewhere. Acknowledgment This study was supported by the National Natural Science Foundation of China (No. 90713009). References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15]
G. Mayer, A. Heckel, Angew. Chem. Int. Ed. 45 (2006) 4900. X.J. Tang, I.J. Dmochowski, Mol. Biosyst. 3 (2007) 100. W.T. Monroe, M.M. McQuain, M.S. Chang, et al. J. Biol. Chem. 274 (1999) 20895. S.B. Cambridge, D. Geissler, F. Calegari, et al. Nat. Methods 6 (2009) 527. R.A. Blidner, K.R. Svoboda, R.P. Hammer, et al. Mol. Biosyst. 4 (2008) 431. H. Ando, T. Furuta, R.Y. Tsien, et al. Nat. Genet. 28 (2001) 317. H. Ando, M. Kobayashi, T. Tsubokawa, et al. Dev. Biol. 287 (2005) 456. O.D. Fedoryak, T.M. Dore, Org. Lett. 4 (2002) 3419. Y. Zhu, C.M. Pavlos, J.P. Toscano, et al. J. Am. Chem. Soc. 128 (2006) 4267. M.J. Davis, C.H. Kragor, K.G. Reddie, et al. J. Org. Chem. 174 (2009) 1721. Y. Li, J. Shi, R. Cai, et al. Tetrahedron Lett. 51 (2010) 1609. Y. Li, J. Shi, R. Cai, et al. J. Photochem. Photobiol. A: Chem. 211 (2010) 129. B. Ghosn, F.R. Haselton, K.R. Gee, et al. Photochem. Photobiol. 81 (2005) 953. Y. Li, J. Shi, Z.F. Luo, et al. Bioorg. Med. Chem. Lett. 19 (2009) 5368. J.P. Casey, R.A. Blidner, W.T. Monroe, Mol. Pharm. 6 (2009) 669.