brief communication published online: 2 November 2014 | doi: 10.1038/nchembio.1656
Diels-Alder reaction–triggered bioorthogonal protein decaging in living cells
© 2014 Nature America, Inc. All rights reserved.
Jie Li1,3, Shang Jia1,3 & Peng R Chen1,2* Small molecules that specifically activate an intracellular protein of interest are highly desirable. A generally applicable strategy, however, remains elusive. Herein we describe a small molecule–triggered bioorthogonal protein decaging technique that relies on the inverse electron-demand DielsAlder reaction for eliminating a chemically caged protein side chain within living cells. This method permits the efficient activation of a given protein (for example, an enzyme) in its native cellular context within minutes. Using small molecules to specifically modulate the activity of a given protein inside living cells, particularly in a gain-of-function fashion, holds major application potential1–3. We recently developed a biocompatible palladium-mediated deprotection reaction that can liberate the ε-amine from a chemically caged lysine site-specifically incorporated into a target protein, enabling the use of a simple palladium catalyst to activate the lysine-dependent activity of a specific intracellular protein4. Although this strategy is sufficient for probing the signaling roles of enzymes, the low elimination efficiency (15–30%) and long reaction time (~3 h) may hinder its usage when rapid activation is needed. As part of our efforts to exploit diverse elimination reactions for noninvasive activation of intracellular proteins, we turned to the inverse electron-demand Diels-Alder (invDA) reaction, which has seen success as a highly bioorthogonal and efficient reaction for biomolecular conjugations in living systems5,6. Although invDA is a [4+2] cycloaddition reaction that has been extensively used for various bioconjugation purposes, previous studies of an invDA reaction pair consisting of a 1,2,4,5-tetrazine and cyclic alkenes showed that the primary bicyclic adduct, a dihydropyridazine, could be converted into a pyridazine by eliminating a leaving group (for example, alcohol or amine) from the vinyl or allylic position7,8 (Supplementary Results, Supplementary Fig. 1). This unique mechanism makes it a useful prodrug release methodology8. Nevertheless, the compatibility and efficiency of this reaction had not been examined on biomolecules, particularly on intracellular proteins. We thus decided to exploit the invDAmediated elimination reaction as a bioorthogonal decaging strategy for fast protein activation in living cells. We began by examining the invDA reaction between the TCO-caged Fmoc-lysine analog N-Fmoc-N-(((E)-cyclooct-2-en1-yl)-oxy)carbonyl-L-lysine (Fmoc-TCOK, 1; Fig. 1a) and different tetrazine derivatives. We synthesized Fmoc-protected TCOK with a carbamate group at either the axial (isomer 1a; Fig. 1) or the equatorial position (isomer 1e; Fig. 1) from cycloctene according to a photolysis strategy9 (Supplementary Note 1). We also prepared or purchased four tetrazine derivatives bearing substitution groups with different electronic and steric properties (Online Methods and Supplementary Note 1). When treating 1a or 1e with each of these tetrazines, we found that the yields of eliminated products
(8) varied in the presence of different tetrazine derivatives (Supplementary Table 1). This is most likely due to the different electronic and steric properties of these tetrazines8, which may largely affect the equilibrium ratio between the two tautomers 7 and 9 (Fig. 1a). Notably, 3,6-dimethyl-1,2,4,5-tetrazine (Me2Tet) demonstrated the highest elimination efficiency (Supplementary Table 1 and Supplementary Fig. 2) for both 1a and 1e among all the tetrazines we tested. We therefore used Me2Tet for the remainder of our study. Next, we conducted a time-course examination of the final product 8 from reactions between Me2Tet and 1a or 1e under identical conditions (Fig. 1b). Interestingly, the elimination reaction between Me2Tet and 1a reached maximum efficiency (>95%) within only 5 min, whereas 1e reacted with Me2Tet at a much slower rate, reaching maximum efficiency after 3 h. The higher reactivity of the axial isomer (TCO-a) relative to the equatorial isomer (TCO-e) is consistent with previous reports8. Taken together, we identified TCO-a and Me2Tet as a highly efficient decaging pair. We next aimed to apply the TCO-a–containing lysine analog (TCOK-a; 10a) and Me2Tet for invDA-mediated decaging on intact proteins in vitro and in living cells (Fig. 1a). TCOK-a and its equatorial isomer (TCOK-e; 10e) are analogs of pyrrolysine (Pyl), the twenty-second naturally occurring amino acid in certain archaea species that can be site-specifically and genetically incorporated into proteins via the Pyl-tRNA synthetase (PylRS)-tRNAPylCUA pair in response to an in-frame amber codon (TAG)10. This Pyl-based system has emerged in recent years as a ‘one-stop shop’ for encoding unnatural amino acids (UAAs) in diverse living systems, including prokaryotic and eukaryotic cells, and even in multicellular organisms11–14. Recently, a Y306A Y384F double mutant derived from Methanosarcina mazei PylRS (MmPylRS) has been shown to recognize a broad range of aromatic and/or cyclic Pyl analogs including the cyclooctene-containing TCOK (TCOK-a and TCOK-e mixture)15–17. We used this mutant MmPylRS to site-specifically incorporate isomers TCOK-a and TCOK-e into the model protein GFP at residue N149 (a previously used residue site with high incorporation efficiency) to generate GFP-N149-TCOK-a and GFPN149-TCOK-e, respectively (Supplementary Fig. 3). The yields of these TCOK isomer–containing proteins were essentially the same, indicating that this mutant MmPylRS recognizes the isomers with a similar efficiency. MS of the purified GFP-N149-TCOK-a and GFP-N149-TCOK-e (Fig. 2a and Supplementary Fig. 4) confirmed the site specificity of this incorporation. The purified proteins before and after Me2Tet-mediated decaging were also analyzed by MS (Fig. 2a and Supplementary Fig. 4). A −152 Da shift of the main peak, corresponding to the removal of the TCO caging group, was clearly detected (Fig. 2a and Supplementary Fig. 4). In addition to the main elimination products, a small amount of both GFP-N149-TCOK-a and GFP-N149-TCOK-e proteins remained unreacted regardless of the incubation time (Supplementary
Synthetic and Functional Biomolecules Center, Beijing National Laboratory for Molecular Sciences, Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, College of Chemistry and Molecular Engineering, Peking University, Beijing, China. 2Peking-Tsinghua Center for Life Sciences, Beijing, China. 3These authors contributed equally to this work. *e-mail: [email protected] 1
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1
brief communication 1a and 1e:R = FMOC 10a and 10e:R = H, then a protein chain
R' R' H
N
H
N
H N
–N2
+ N
N
N
N
R'
R'
2:R' = H 3:R' = CH3 4:R' = Ph 5:R' = 2-pyridyl
O O
H shift
H N
O
O
COOH
Byproduct (9)
NH
–CO2
Axial (a)
RHN
O
H2N
H
6a and 6e
COOH
RHN
7a and 7e
COOH
75 50
NH
0
Equatorial (e)
RHN
1a + 3 1e + 3
25
O
COOH RHN
100
H H N O
O
O
+
NH
R' O
R'
H R'
O O
COOH
b
N H
NH
N
RHN
H N
R'
Yield of 8 (%)
a
Nature chemical biology doi: 10.1038/nchembio.1656
0
50
Target product (8)
100 Time (min)
150
200
2
enzymatic activity, which could be subsequently reactivated by Me2Tet. Indeed, HEK293T cells bearing the TCOK-a caged fLuc (C-fLuc; fLuc-K529-TCOK-a cells) showed negligible luminescence activity (Supplementary Fig. 12). As expected, largely increased bioluminescence was detected 60 min after the addition of 100 μM Me2Tet into the cells. Alternatively, the ‘rescue ratio’ of C-fLuc (see Online Methods for definition) was calculated by quenching and transferring the cell lysate to luciferin solution for measuring bioluminescence signal. The mean rescue ratio of C-fLuc from three independent experiments reached 89% (Fig. 3a). a
b
40 20
X = TCOK
100 27,889 27,971
0 27,000
27,500
(m/z 851.91 Da, 2+) (m/z 734.85 Da, 2+) (m/z 642.29 Da, 2+)
27,889
28,000 Mass
80 60 40
20 28,500 0 min
X′ = TCOK+Me2Tet-N2
P4
P1
eT 2 mi et n
60
(m/z 810.89 Da, 2+)
P2: P3: P4:
15
80
27,737 Before decaging After decaging -Met 27,758 -Met 27,606
+ Me2Tet P1:
+M
100
Intensity (%)
Fig. 5). Some GFP-N149-TCOK-a underwent cycloaddition without further elimination to give the cycloaddition product with a MS increase of 82 Da (Fig. 2a). The unreacted GFPN149-TCOK-a and GFP-N149-TCOK-e might have experienced a cis-trans isomerization of the carbon-carbon double bond mediated by thiol radicals in the cellular environment, which converted the TCO moiety into the nonreactive cis form18. The overall yield of Me2Tet-decaged GFP-N149-TCOK-a reached 75% within 10 min, which is much faster than that of Me2Tet-decaged GFP-N149-TCOK-e (80% at ~4 h; Fig. 2a and Supplementary Fig. 6). To further demonstrate that our invDA-mediated elimination strategy is effective in cells, we used the Me2Tet-triggered decaging reaction on intracellular GFP bearing a site-specifically incorporated TCOK residue. We expressed GFP carrying TCOK-a or TCOK-e at residue Tyr40 (a previously used residue site with high incorporation efficiency, producing GFP-Y40-TCOK-a or GFPY40-TCOK-e) in HEK293T cells (Supplementary Fig. 7 and Online Methods) and applied Me2Tet-mediated invDA elimination on these intracellular proteins. At varying time points, the GFP variants were purified for LC/MS/MS analysis (Fig. 2b and Supplementary Figs. 8 and 9). Time-course LC/MS/MS analysis indicated that the initial GFP-Y40-TCOK-a protein was converted to the elimination product GFP-Y40-K within 15 min (~80% yield; Fig. 2b), and the reaction yield can reach 90% after 60 min (Supplementary Fig. 8). In contrast, the GFP-Y40-TCOK-e protein only gave a moderate reaction yield (~55%; Supplementary Fig. 8) after 60 min. We also treated HEK293T cells with up to 2 mM Me2Tet for 2 h, and results of an MTS assay showed that the tetrazine molecules had negligible toxicity (>95% viability) with a concentration equal or below 0.5 mM, which is fivefold higher than the concentration used for intracellular protein decaging (Supplementary Fig. 10). Notably, even 2 mM Me2Tet still gave ~70% cell viability after 2 h, demonstrating excellent biocompatibility. Taken together, our in vitro and cellular results confirmed TCOK-a and Me2Tet as an excellent invDA-mediated decaging pair for rapid and efficient protein activation. Finally, we applied this strategy for the activation of an intracellular enzyme. For proof of concept, we chose firefly luciferase (fLuc), which has a catalytic lysine (K529) that is necessary to convert the nonluminescent luciferin to its highly luminescent product, oxyluciferin (Supplementary Fig. 11). We have previously shown that substitution of K529 with a photocaged lysine disrupts substrate binding and thus blocks fLuc’s catalytic activity19. Similarly, we envisioned that replacing K529 by TCOK-a might inhibit fLuc’s
t e Te +M 2 in 15 m
© 2014 Nature America, Inc. All rights reserved.
Figure 1 | InvDA-mediated elimination reaction for protein activation in living cells. (a) The model reaction scheme on with TCOK is shown. The target product is the decaged lysine 8, whereas the adduct 9 is the unwanted byproduct. The cascade electron shift direction (starting from the electron lone pair of NH into the ring) is marked with arrows. 2, 1,2,4,5-tetrazine; 3, 3,6-dimethyl-1,2,4,5-tetrazine (Me2Tet); 4, 3,6-diphenyl-1,2,4,5tetrazine; 5, 3,6-di(pyridin-2-yl)-1,2,4,5-tetrazine. In living cells, TCOK-a is first site-specifically incorporated into a protein of interest through genetic code expansion to block its lysine-dependent activity. The protein is then decaged and activated by the same process. (b) Axial (a) and equatorial (e) isomers of (E)-cyclooct-2-en-1-yl carbamates are shown, together with the yield of elimination product 8 (%) released from 1a (red) or 1e (black) following the addition of tetrazine 3 (5 equiv.) at indicated time points as measured by ultra-performance LC (UPLC) analysis (photo diode array detector at 210 nm). Error bars represent ± s.d. from three independent experiments.
15 min
P3 P2
20
P1
30 40 Time (min)
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Figure 2 | Demonstration of the invDA-mediated elimination reaction on model GFP protein in aqueous buffer and in living cells. (a) TCOK-a was site-specifically incorporated at N149 on GFP to generate GFP-N149TCOK-a. The deconvoluted mass spectra of GFP-N149-TCOKs (black) and their reaction products (red) upon addition of 3 (Me2Tet) are shown. For GFP-N149-TCOK: expected mass 27,889 Da, found mass 27,889 Da (major) and 27,758 Da (minor). For GFP-N149-K (the elimination product): expected mass 27,737 Da, found mass 27,737 Da (major), 27,760 Da (27,737 Da with Na+) and 27,606 Da (minor). The minor peak corresponds to the same protein (the major peak) with the N-terminal Met (−132 Da) post-translationally cleaved. The mass peak of 27,971 Da found in the products of GFP-N149-TCOK-a refers to the addition product between GFP-N149-TCOK and Me2Tet. (b) The invDA-mediated elimination reaction inside living HEK 293T cells was monitored by LC/MS/MS analysis of the four target peptides after trypsin digestion. The target peptides sequences are listed above the LC spectra, and the relative intensity, retention time and molecular weight are shown. Peptide-1 (P1, black) corresponds to the reaction substrate GFP-Y40TCOK, whereas peptide-2 (P2, blue) is related to the addition product between GFP-Y40-TCOK and Me2Tet. Peptide-3 and peptide-4 (P3 and P4, red) refer to the elimination product GFP-Y40-K. The reaction site on target peptides are labeled in purple and are located in the box.
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Nature chemical biology doi: 10.1038/nchembio.1656 a
Luc-WT Luc-K529-TAG TCOK-a – – + + Me2Tet – + – + Anti-His Relative 100 ± 7 N.D. N.D. 80% in about 15 min, consistent with the yield from intracellularly decaged GFPY40-TCOK-a (Fig. 3b). Similarly, a dose-dependent assay showed that Me2Tet as low as 100 μM was sufficient for full activation of intracellular C-fLuc (Fig. 3c). Moreover, this titration indicated a correlation between the Me2Tet concentration and the rescue ratio, which could be used for controlled rescue of the enzyme activity by varying the Me2Tet concentration. Finally, cell lysates were subjected to bioluminescence measurement, which showed similar activation efficiency (Supplementary Fig. 14). In summary, we have developed an invDA-mediated bioorthogonal protein decaging strategy to mask and liberate a lysine’s ε-amine for efficient and rapid protein activation in living cells. Compared to previous protein activation strategies, this method has unique advantages. First, the TCOK-a and Me2Tet reagents provide a generally applicable caging-decaging pair for chemical control of a lysine side chain. This strategy may also be further extended to modulate the activation of additional amino acids beyond lysine. Second, the time scale for efficient decaging is comparable to that of the widely adopted UV-mediated protein photodecaging method (for example, efficient intracellular activation of photocaged fLuc or photocaged MAP kinase kinase, MEK1, with a 10-min time scale19,20), thus making it suitable for rapid protein activation in living cells. Third, a small-molecule Me2Tet was used to trigger the decaging event, which could potentially be used in deep tissues or even in intact animals that are hardly accessible
brief communication
by UV irradiation. Finally, Me2Tet is highly biocompatible, and its fluorescent derivatives have been widely used for intracellular labeling of diverse biomolecules. Therefore, other laboratories may easily adopt our Me2Tet-triggered elimination strategy. Such a rationally designed, small molecule–triggered bioorthogonal protein activation strategy may become a generally applicable tool in turning on intracellular enzymes, and thus their downstream cellular responses or signaling events, with value in cell engineering and therapeutics21. The bioorthogonal protein manipulation toolkit could be further expanded from conjugation to elimination chemistry22–25, with potential applications in an in vivo setting. Received 26 June 2014; accepted 26 August 2014; published online 2 November 2014
Methods
Methods and any associated references are available in the online version of the paper.
References
1. Zorn, J.A. & Wells, J.A. Nat. Chem. Biol. 6, 179–188 (2010). 2. Banaszynski, L.A., Chen, L.-c., Maynard-Smith, L.A., Ooi, A.G.L. & Wandless, T.J.A. Cell 126, 995–1004 (2006). 3. Qiao, Y., Molina, H., Pandey, A., Zhang, J. & Cole, P.A. Science 311, 1293–1297 (2006). 4. Li, J. et al. Nat. Chem. 6, 352–361 (2014). 5. Knall, A.-C. & Slugovc, C. Chem. Soc. Rev. 42, 5131–5142 (2013). 6. Selvaraj, R. & Fox, J.M. Curr. Opin. Chem. Biol. 17, 753–760 (2013). 7. Sauer, J. et al. Eur. J. Org. Chem. 1998, 2885–2896 (1998). 8. Versteegen, R.M., Rossin, R., tenHoeve, W., Janssen, H.M. & Robillard, M.S. Angew. Chem. Int. Ed. Engl. 52, 14112–14116 (2013). 9. Royzen, M., Yap, G.P.A. & Fox, J.M. J. Am. Chem. Soc. 130, 3760–3761 (2008). 10. Hao, B. et al. Science 296, 1462–1466 (2002). 11. Liu, C.C. & Schultz, P.G. Annu. Rev. Biochem. 79, 413–444 (2010). 12. Chin, J.W. Annu. Rev. Biochem. 83, 379–408 (2014). 13. Lang, K. & Chin, J.W. Chem. Rev. 114, 4764–4806 (2014). 14. Wan, W., Tharp, J.M. & Liu, W.R. Biochim. Biophys. Acta 1844, 1059–1070 (2014). 15. Yanagisawa, T. et al. Chem. Biol. 15, 1187–1197 (2008). 16. Yanagisawa, T. et al. Mol. Biosyst. 8, 1131–1135 (2012). 17. Nikić, I. et al. Angew. Chem. Int. Ed. Engl. 53, 2245–2249 (2014). 18. Chatgilialoglu, C. & Ferreri, C. Acc. Chem. Res. 38, 441–448 (2005). 19. Zhao, J., Lin, S., Huang, Y., Zhao, J. & Chen, P.R. J. Am. Chem. Soc. 135, 7410–7413 (2013). 20. Gautier, A., Deiters, A. & Chin, J.W. J. Am. Chem. Soc. 133, 2124–2127 (2011). 21. Wei, P. et al. Nature 488, 384–388 (2012). 22. Sasmal, P.K., Streu, C.N. & Meggers, E. Chem. Commun. (Camb.) 49, 1581–1587 (2013). 23. Bielski, R. & Witczak, Z. Chem. Rev. 113, 2205–2243 (2013). 24. Li, L. et al. Nat. Commun. 5, 3276 (2014). 25. Chan, J., Dodani, S.C. & Chang, C.J. Nat. Chem. 4, 973–984 (2012).
Acknowledgments
We acknowledge support from R. Meng and the Proteomic Mass Spectrometry Core of the National Facilities for Protein Sciences (the Phoenix Project) at Peking University. This work was supported by the National Basic Research Program of China (2010CB912300 and 2012CB917301) and the National Natural Science Foundation of China (21225206 and 91313301). J.L. acknowledges support from the Peking University Principal Foundation. S.F. Reichard edited the manuscript.
Author contributions
P.R.C. and J.L. designed the experimental strategy and wrote the manuscript. J.L. and S.J. performed the experiments. All authors prepared the figures and edited the manuscript.
Competing financial interests
The authors declare no competing financial interests.
Additional information
Supplementary information and chemical compound information is available in the online version of the paper. Reprints and permissions information is available online at http://www.nature.com/reprints/index.html. Correspondence and requests for materials should be addressed to P.R.C.
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ONLINE METHODS
© 2014 Nature America, Inc. All rights reserved.
Plasmid construction. The plasmids pSupAR-MmPylRS and pCMVMmPylRS encoding mutant MmPylRS and the cognitive tRNAPylCUA for TCOK have been described in previous literature4. The plasmids pBAD-GFPN149-TAG-His6, pCDNA3.1-GFP-Y40TAG-His6 and pCDNA3.1-fLuc/fLucK529TAG-His6 have been reported in our previous work4,19. Reagents and equipment. All of the compounds used in the synthesis were purchased from J&K Scientific. Synthesis information is included in Supplementary Note 1. Compounds 4 and 5 were purchased from SigmaAldrich (cat. nos. 403555 and 403547). All of the other chemicals were analytical grade or better. 1H NMR and 13C NMR spectra were recorded on a Bruker-500 MHz NMR (AVANCE III) or Varian-300 MHz NMR instrument. High-resolution mass spectra (HRMS) were recorded on a Fourier Transform Ion Cyclotron Resonance Mass Spectrometer (APEX IV). Protein purifications were all performed on an AKTA UPC 900 system (GE Healthcare). Protein MS was performed with LC/MS, using an ACQUITY UPLC I-Class SQD-2 (Waters) system with electrospray ionization (ESI). LC/MS/MS analysis of tryptic peptides was performed on an Easy nLC 1000 system coupled to an LTQ-orbitrap-XL mass spectrometer equipped with a nano-ESI source. MS survey scans were performed in FT mode at a resolution of 30,000, and MS2 scans were performed in an ion trap. Images of protein gels, including Coomassie blue–stained SDS-PAGE gel and western blotting membranes, were taken on a ChemiDoc XRS+ (Bio-Rad). The luminescence and fluorescence intensity were monitored in a Multi-Mode Microplate Reader (Synergy 4, Bio-Tek, USA) or ChemiDoc XRS+ (Bio-Rad). Cell culture. HEK293T cells were purchased from the American Type Culture Collection (ATCC). Cells were grown in DMEM (Dulbecco’s modified Eagle’s medium, Gibco) supplemented with 10% FBS (FBS) and cultured at 37 °C under 5% CO2. Reactions between 1a/1e and tetrazines. These Fmoc-protected UAAs (1a and 1e) were diluted in the reaction solvent (CH3CN/H2O = 1/4) to a final concentration of 100 μM in a 1.5-ml Eppendorf tube from a 10 mM stock in CH3CN. The solutions were warmed to 37 °C before the tetrazines (10 mM stock in CH3CN) were added to a final concentration of 500 μM. The reactions were conducted at 37 °C for varying times (400 r.p.m. in Eppendorf Thermomixer C) and were then diluted twofold with CH3CN before immediate UPLC-MS/PDA analysis. The UPLC-yields for the conversion of 1a and 1e to 8 were determined by an external standard method (making a calibration line of 1a, 1e and 8 for quantification with different concentrations). The UPLC analysis was performed on an ACQUITY UPLC I-Class SQD 2 (Waters) system with a BEH C18 Acquity column (1.7 μm, 2.1× 50 mm). The mobile phase consisted of acetonitrile (MeCN) and water. The sample was eluted with a 4.8-min linear gradient (5–95% acetonitrile, 0.1% HCOOH, 0.4 ml/min) directed to the mass detector. Retention times: 1a: 2.460–2.522 min; 1e: 2.472–2.549 min; 8: 1.573–1.672 min; 9 (tetrazine=3): 1.995–2.035 min. Expression and purification of UAA-incorporated proteins in E. coli. Expression of green fluorescent protein was carried out in DH10B cells cotransformed with plasmids carrying pMmPylRSY306A Y384F-tRNACUAPyl and pBAD-GFP-N149-TAG-His6, respectively. Cells were recovered in 1 ml LB medium for 1 h at 37 °C before further growth in LB medium containing ampicillin (50 mg/ml) and chloramphenicol (34 mg/ml) overnight at 37 °C. After 1:100 dilution in 500 ml LB medium containing ampicillin (50 mg/ml) and chloramphenicol (34 mg/ml), the cell culture was grown at 37 °C to an OD600 ~0.6. TCOK-a and TCOK-e (100 mM) were added at a 1:100 dilution to a final concentration of 1 mM. After 30 min of incubation, protein expression was induced by the addition of 5 ml 20% arabinose to give a final concentration of 0.2%, and cells were harvested after 12 h expression at 30 °C by centrifugation (6,000 r.p.m., 10 min) followed by resuspension in lysis buffer (Tris-HCl, pH 7.8, 300 mM NaCl). Lysate after sonication was loaded onto a Ni-NTA column (Histrap 5 ml, GE Healthcare), which was washed with 30 ml washing buffer (20 mM Tris-HCl, pH 7.8, 300 mM NaCl, 40 mM imidazole) and then eluted with elution buffer (20 mM Tris-HCl, pH 7.8, 300 mM NaCl and 250 mM imidazole) to yield ~5 mg/ml full-length GFP containing TCOK. Protein was then desalted to 1× DPBS buffer (pH 7.4) and concentrated to 20 μM. nature chemical biology
ESI-MS analysis. LC/MS analysis of GFP-N149-TCOK and its products after the decaging reaction were performed using a Waters ACQUITY UPLC I-Class SQD 2 MS spectrometer with electrospray ionization (ESI). 0.1% formic acid in H2O as buffer A and 0.1% formic acid in acetonitrile as buffer B were used as the solvent system. LC separation for GFP and its variants was carried out with a BEH300 C4 Acquity column (1.7 μm, 2.1 × 100 mm), and positive mode was chosen for ESI-MS to analyze all samples. The total mass of proteins was calculated using MassLynx V4.1 software (Waters). Theoretical mass of the wild-type protein was calculated using PROTEIN CALCULATOR v3.3 (http://protcalc.sourceforge.net/), and the theoretical mass for all modified proteins was adjusted manually. InvDA-mediated elimination reaction on purified proteins. For in vitro cleavage reactions on purified proteins, recombinant GFP proteins bearing the site-specifically incorporated TCOK-a and TCOK-e (all at the 10 μM final concentration in 1× DPBS buffer, pH 7.4) were incubated with 20 equiv. of Me2Tet (200 μM final concentration). The solution was incubated at 37 °C for varying times and was then subjected to LC/MS analysis. Expression and decaging of TCOK-a and TCOK-e containing GFP in mammalian cells. For GFP-40-TCOK-a and GFP-40-TCOK-e expression in HEK293T cells, plasmids of pCDNA3.1-GFP-Y40TAG-His 6 and pCMVmutant MmPylRS were co-transfected via X-tremeGENE HP (Roche) in DMEM (10% FBS) supplemented with 1 mM TCOK-a and TCOK-e. Cells were allowed to grow for additional 24 h to express the desired protein bearing a site-specifically incorporated TCOK-a and TCOK-e residue. For elimination reactions on proteins, cells harboring the TCOK-a and TCOK-e incorporated proteins were first cultured in DMEM without UAA for 180 min to exocytose free UAA inside cells, followed by treatment with Me2Tet reagents (100 μM) in fresh DMEM with 10% FBS to enable the invDA-mediated cleavage reaction to proceed inside mammalian cells. The reactions were subsequently quenched by TCOK-a (2 mM in 1× DPBS) at varying times. Quantifying invDA-mediated elimination efficiency on intracellular proteins. After the decaging reaction, HEK293T cells were lysed, and the target GFP proteins were purified through the C-terminus His6 tag and digested in SDS-PAGE gel by trypsin for the following LC/MS/MS analysis. After SDS-PAGE separation, the corresponding protein bands were excised and digested in-gel with sequencing grade trypsin (5 ng/μl trypsin, 50 mM ammonium bicarbonate, pH 8.0) overnight at 37 °C. Prior to the addition of the enzyme, gel pieces were dehydrated in acetonitrile, incubated in 10 mM DTT in 50 mM ammonium bicarbonate at 56 °C for 30 min, incubated in 55 mM iodoacetamide in 50 mM ammonium bicarbonate at ambient temperature for 1 h in the dark, and then dehydrated again. The resulting peptides were extracted twice with 5% formic acid/50% acetonitrile in acetonitrile and then vacuum-centrifuged to dryness. For LC/MS/MS analysis, the extracted peptides were reconstituted in 0.2% formic acid, loaded onto a 100 μm × 2 cm precolumn and separated on a 75 μm × 20 cm capillary column. Both columns were packed in-house with 4 μm C18 bulk materials (InnosepBio, China). An Easy nLC 1000 system (Thermo Scientific, USA) was used to generate the following HPLC gradient: 7–35% B in 50 min, 35–75% B in 1 min, then holding at 75% B for 14 min (A = 0.1% formic acid in water, B = 0.1% formic acid in acetonitrile). The eluted peptides were sprayed into a LTQ Orbitrap XL mass spectrometer (Thermo Scientific, USA) equipped with a nano-ESI source. The mass spectrometer was operated in data-dependent mode with one MS scan in FT mode at a resolution of 30,000 followed by 5 CID (collision-induced dissociation) MS/MS scans in the ion trap for each cycle. Cytotoxicity study of the tetrazine Me2Tet. HEK 293T cells were cultured at a density of ~2,000–5,000 cells per well, depending on the cell type, in flat-bottomed 96-well plates and left to grow for 24 h. Cells were then treated with different concentrations of tetrazine molecule Me2Tet for 120 min, and each condition was carried out in quintuplicate. After incubation, cells were washed with fresh DMEM, and CellTiter 96 Aqueous One Solution Reagent (Promega, Madison, WI) was added to each well according to the manufacturer’s instructions. After 4 h incubation, the cell viability was determined by measuring the absorbance at 490 nm using a microplate reader. InvDA-mediated activation of caged fLuc in living cells. For fLuc-529-TCOK-a and fLuc-529-TCOK-e expression in HEK293T cells, plasmids of pCDNA3.1fLuc-K529TAG-His6 and pCMV-mutant MmPylRS were cotransfected via doi:10.1038/nchembio.1656
Determination of rescue ratio of C-fLuc. Rescue ratio was calculated as: Rescue ratio =
r.l. of C - fLuc /r.l. of fLuc - WT b.i. of C - fLuc/ b.i. of fLuc - WT
× 100%
where r.l. represents relative luminescence measured from samples containing C-fLuc or fLuc-WT under Synergy 4 Hybrid Microplate Reader or ChemiDoc, and b.i. represents band intensity, as measured from western blotting analysis on samples containing C-fLuc or fLuc-WT. Statistical analysis. Error bars are presented as mean ± s.d. from three independent experiments. The statistical analysis was performed using an unpaired Student’s t-test when the two groups were compared. P < 0.05 was considered to be significant.
© 2014 Nature America, Inc. All rights reserved.
X-tremeGENE HP (Roche) in DMEM (10% FBS) supplemented with 1 mM TCOK-a and TCOK-e. Cells bearing the chemical-caged C-fLuc protein were treated with Me2Tet to undergo the invDA-mediated elimination reaction after 24 h transfection and expression. After reaction, cells were washed and incubated with DPBS supplemented with 2 mM TCOK-a to quench the reaction. For live cell imaging using ChemiDoc instrument (Bio-Rad), luciferin solution (20 mM Tricine, pH 7.8, 1 mM MgSO4, 0.1 mM EDTA, 1 mM dithiothreitol, 270 μM coenzyme A, 500 μM D-luciferin, 530 μM ATP and 2 mM TCOK-a) was added and incubated for another 1 min before cells were imaged on a ChemiDoc XRS+ (Bio-Rad) using the chemiluminescence channel. Alternatively, cells were lysed using universal lysis buffer (containing 2 mM TCOK-a) after reaction, and cell lysates were then mixed with luciferin solution. Relative bioluminescence was measured with a Synergy 4 Hybrid Microplate Reader. The same samples were used for SDS-PAGE analysis to quantity the amount of the protein.
doi:10.1038/nchembio.1656
nature CHEMICAL BIOLOGY
Diels-Alder reaction–triggered bioorthogonal protein decaging in living cells
© 2014 Nature America, Inc. All rights reserved.
Jie Li1,3, Shang Jia1,3 & Peng R Chen1,2* Small molecules that specifically activate an intracellular protein of interest are highly desirable. A generally applicable strategy, however, remains elusive. Herein we describe a small molecule–triggered bioorthogonal protein decaging technique that relies on the inverse electron-demand DielsAlder reaction for eliminating a chemically caged protein side chain within living cells. This method permits the efficient activation of a given protein (for example, an enzyme) in its native cellular context within minutes. Using small molecules to specifically modulate the activity of a given protein inside living cells, particularly in a gain-of-function fashion, holds major application potential1–3. We recently developed a biocompatible palladium-mediated deprotection reaction that can liberate the ε-amine from a chemically caged lysine site-specifically incorporated into a target protein, enabling the use of a simple palladium catalyst to activate the lysine-dependent activity of a specific intracellular protein4. Although this strategy is sufficient for probing the signaling roles of enzymes, the low elimination efficiency (15–30%) and long reaction time (~3 h) may hinder its usage when rapid activation is needed. As part of our efforts to exploit diverse elimination reactions for noninvasive activation of intracellular proteins, we turned to the inverse electron-demand Diels-Alder (invDA) reaction, which has seen success as a highly bioorthogonal and efficient reaction for biomolecular conjugations in living systems5,6. Although invDA is a [4+2] cycloaddition reaction that has been extensively used for various bioconjugation purposes, previous studies of an invDA reaction pair consisting of a 1,2,4,5-tetrazine and cyclic alkenes showed that the primary bicyclic adduct, a dihydropyridazine, could be converted into a pyridazine by eliminating a leaving group (for example, alcohol or amine) from the vinyl or allylic position7,8 (Supplementary Results, Supplementary Fig. 1). This unique mechanism makes it a useful prodrug release methodology8. Nevertheless, the compatibility and efficiency of this reaction had not been examined on biomolecules, particularly on intracellular proteins. We thus decided to exploit the invDAmediated elimination reaction as a bioorthogonal decaging strategy for fast protein activation in living cells. We began by examining the invDA reaction between the TCO-caged Fmoc-lysine analog N-Fmoc-N-(((E)-cyclooct-2-en1-yl)-oxy)carbonyl-L-lysine (Fmoc-TCOK, 1; Fig. 1a) and different tetrazine derivatives. We synthesized Fmoc-protected TCOK with a carbamate group at either the axial (isomer 1a; Fig. 1) or the equatorial position (isomer 1e; Fig. 1) from cycloctene according to a photolysis strategy9 (Supplementary Note 1). We also prepared or purchased four tetrazine derivatives bearing substitution groups with different electronic and steric properties (Online Methods and Supplementary Note 1). When treating 1a or 1e with each of these tetrazines, we found that the yields of eliminated products
(8) varied in the presence of different tetrazine derivatives (Supplementary Table 1). This is most likely due to the different electronic and steric properties of these tetrazines8, which may largely affect the equilibrium ratio between the two tautomers 7 and 9 (Fig. 1a). Notably, 3,6-dimethyl-1,2,4,5-tetrazine (Me2Tet) demonstrated the highest elimination efficiency (Supplementary Table 1 and Supplementary Fig. 2) for both 1a and 1e among all the tetrazines we tested. We therefore used Me2Tet for the remainder of our study. Next, we conducted a time-course examination of the final product 8 from reactions between Me2Tet and 1a or 1e under identical conditions (Fig. 1b). Interestingly, the elimination reaction between Me2Tet and 1a reached maximum efficiency (>95%) within only 5 min, whereas 1e reacted with Me2Tet at a much slower rate, reaching maximum efficiency after 3 h. The higher reactivity of the axial isomer (TCO-a) relative to the equatorial isomer (TCO-e) is consistent with previous reports8. Taken together, we identified TCO-a and Me2Tet as a highly efficient decaging pair. We next aimed to apply the TCO-a–containing lysine analog (TCOK-a; 10a) and Me2Tet for invDA-mediated decaging on intact proteins in vitro and in living cells (Fig. 1a). TCOK-a and its equatorial isomer (TCOK-e; 10e) are analogs of pyrrolysine (Pyl), the twenty-second naturally occurring amino acid in certain archaea species that can be site-specifically and genetically incorporated into proteins via the Pyl-tRNA synthetase (PylRS)-tRNAPylCUA pair in response to an in-frame amber codon (TAG)10. This Pyl-based system has emerged in recent years as a ‘one-stop shop’ for encoding unnatural amino acids (UAAs) in diverse living systems, including prokaryotic and eukaryotic cells, and even in multicellular organisms11–14. Recently, a Y306A Y384F double mutant derived from Methanosarcina mazei PylRS (MmPylRS) has been shown to recognize a broad range of aromatic and/or cyclic Pyl analogs including the cyclooctene-containing TCOK (TCOK-a and TCOK-e mixture)15–17. We used this mutant MmPylRS to site-specifically incorporate isomers TCOK-a and TCOK-e into the model protein GFP at residue N149 (a previously used residue site with high incorporation efficiency) to generate GFP-N149-TCOK-a and GFPN149-TCOK-e, respectively (Supplementary Fig. 3). The yields of these TCOK isomer–containing proteins were essentially the same, indicating that this mutant MmPylRS recognizes the isomers with a similar efficiency. MS of the purified GFP-N149-TCOK-a and GFP-N149-TCOK-e (Fig. 2a and Supplementary Fig. 4) confirmed the site specificity of this incorporation. The purified proteins before and after Me2Tet-mediated decaging were also analyzed by MS (Fig. 2a and Supplementary Fig. 4). A −152 Da shift of the main peak, corresponding to the removal of the TCO caging group, was clearly detected (Fig. 2a and Supplementary Fig. 4). In addition to the main elimination products, a small amount of both GFP-N149-TCOK-a and GFP-N149-TCOK-e proteins remained unreacted regardless of the incubation time (Supplementary
Synthetic and Functional Biomolecules Center, Beijing National Laboratory for Molecular Sciences, Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, College of Chemistry and Molecular Engineering, Peking University, Beijing, China. 2Peking-Tsinghua Center for Life Sciences, Beijing, China. 3These authors contributed equally to this work. *e-mail: [email protected] 1
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1
brief communication 1a and 1e:R = FMOC 10a and 10e:R = H, then a protein chain
R' R' H
N
H
N
H N
–N2
+ N
N
N
N
R'
R'
2:R' = H 3:R' = CH3 4:R' = Ph 5:R' = 2-pyridyl
O O
H shift
H N
O
O
COOH
Byproduct (9)
NH
–CO2
Axial (a)
RHN
O
H2N
H
6a and 6e
COOH
RHN
7a and 7e
COOH
75 50
NH
0
Equatorial (e)
RHN
1a + 3 1e + 3
25
O
COOH RHN
100
H H N O
O
O
+
NH
R' O
R'
H R'
O O
COOH
b
N H
NH
N
RHN
H N
R'
Yield of 8 (%)
a
Nature chemical biology doi: 10.1038/nchembio.1656
0
50
Target product (8)
100 Time (min)
150
200
2
enzymatic activity, which could be subsequently reactivated by Me2Tet. Indeed, HEK293T cells bearing the TCOK-a caged fLuc (C-fLuc; fLuc-K529-TCOK-a cells) showed negligible luminescence activity (Supplementary Fig. 12). As expected, largely increased bioluminescence was detected 60 min after the addition of 100 μM Me2Tet into the cells. Alternatively, the ‘rescue ratio’ of C-fLuc (see Online Methods for definition) was calculated by quenching and transferring the cell lysate to luciferin solution for measuring bioluminescence signal. The mean rescue ratio of C-fLuc from three independent experiments reached 89% (Fig. 3a). a
b
40 20
X = TCOK
100 27,889 27,971
0 27,000
27,500
(m/z 851.91 Da, 2+) (m/z 734.85 Da, 2+) (m/z 642.29 Da, 2+)
27,889
28,000 Mass
80 60 40
20 28,500 0 min
X′ = TCOK+Me2Tet-N2
P4
P1
eT 2 mi et n
60
(m/z 810.89 Da, 2+)
P2: P3: P4:
15
80
27,737 Before decaging After decaging -Met 27,758 -Met 27,606
+ Me2Tet P1:
+M
100
Intensity (%)
Fig. 5). Some GFP-N149-TCOK-a underwent cycloaddition without further elimination to give the cycloaddition product with a MS increase of 82 Da (Fig. 2a). The unreacted GFPN149-TCOK-a and GFP-N149-TCOK-e might have experienced a cis-trans isomerization of the carbon-carbon double bond mediated by thiol radicals in the cellular environment, which converted the TCO moiety into the nonreactive cis form18. The overall yield of Me2Tet-decaged GFP-N149-TCOK-a reached 75% within 10 min, which is much faster than that of Me2Tet-decaged GFP-N149-TCOK-e (80% at ~4 h; Fig. 2a and Supplementary Fig. 6). To further demonstrate that our invDA-mediated elimination strategy is effective in cells, we used the Me2Tet-triggered decaging reaction on intracellular GFP bearing a site-specifically incorporated TCOK residue. We expressed GFP carrying TCOK-a or TCOK-e at residue Tyr40 (a previously used residue site with high incorporation efficiency, producing GFP-Y40-TCOK-a or GFPY40-TCOK-e) in HEK293T cells (Supplementary Fig. 7 and Online Methods) and applied Me2Tet-mediated invDA elimination on these intracellular proteins. At varying time points, the GFP variants were purified for LC/MS/MS analysis (Fig. 2b and Supplementary Figs. 8 and 9). Time-course LC/MS/MS analysis indicated that the initial GFP-Y40-TCOK-a protein was converted to the elimination product GFP-Y40-K within 15 min (~80% yield; Fig. 2b), and the reaction yield can reach 90% after 60 min (Supplementary Fig. 8). In contrast, the GFP-Y40-TCOK-e protein only gave a moderate reaction yield (~55%; Supplementary Fig. 8) after 60 min. We also treated HEK293T cells with up to 2 mM Me2Tet for 2 h, and results of an MTS assay showed that the tetrazine molecules had negligible toxicity (>95% viability) with a concentration equal or below 0.5 mM, which is fivefold higher than the concentration used for intracellular protein decaging (Supplementary Fig. 10). Notably, even 2 mM Me2Tet still gave ~70% cell viability after 2 h, demonstrating excellent biocompatibility. Taken together, our in vitro and cellular results confirmed TCOK-a and Me2Tet as an excellent invDA-mediated decaging pair for rapid and efficient protein activation. Finally, we applied this strategy for the activation of an intracellular enzyme. For proof of concept, we chose firefly luciferase (fLuc), which has a catalytic lysine (K529) that is necessary to convert the nonluminescent luciferin to its highly luminescent product, oxyluciferin (Supplementary Fig. 11). We have previously shown that substitution of K529 with a photocaged lysine disrupts substrate binding and thus blocks fLuc’s catalytic activity19. Similarly, we envisioned that replacing K529 by TCOK-a might inhibit fLuc’s
t e Te +M 2 in 15 m
© 2014 Nature America, Inc. All rights reserved.
Figure 1 | InvDA-mediated elimination reaction for protein activation in living cells. (a) The model reaction scheme on with TCOK is shown. The target product is the decaged lysine 8, whereas the adduct 9 is the unwanted byproduct. The cascade electron shift direction (starting from the electron lone pair of NH into the ring) is marked with arrows. 2, 1,2,4,5-tetrazine; 3, 3,6-dimethyl-1,2,4,5-tetrazine (Me2Tet); 4, 3,6-diphenyl-1,2,4,5tetrazine; 5, 3,6-di(pyridin-2-yl)-1,2,4,5-tetrazine. In living cells, TCOK-a is first site-specifically incorporated into a protein of interest through genetic code expansion to block its lysine-dependent activity. The protein is then decaged and activated by the same process. (b) Axial (a) and equatorial (e) isomers of (E)-cyclooct-2-en-1-yl carbamates are shown, together with the yield of elimination product 8 (%) released from 1a (red) or 1e (black) following the addition of tetrazine 3 (5 equiv.) at indicated time points as measured by ultra-performance LC (UPLC) analysis (photo diode array detector at 210 nm). Error bars represent ± s.d. from three independent experiments.
15 min
P3 P2
20
P1
30 40 Time (min)
50
Figure 2 | Demonstration of the invDA-mediated elimination reaction on model GFP protein in aqueous buffer and in living cells. (a) TCOK-a was site-specifically incorporated at N149 on GFP to generate GFP-N149TCOK-a. The deconvoluted mass spectra of GFP-N149-TCOKs (black) and their reaction products (red) upon addition of 3 (Me2Tet) are shown. For GFP-N149-TCOK: expected mass 27,889 Da, found mass 27,889 Da (major) and 27,758 Da (minor). For GFP-N149-K (the elimination product): expected mass 27,737 Da, found mass 27,737 Da (major), 27,760 Da (27,737 Da with Na+) and 27,606 Da (minor). The minor peak corresponds to the same protein (the major peak) with the N-terminal Met (−132 Da) post-translationally cleaved. The mass peak of 27,971 Da found in the products of GFP-N149-TCOK-a refers to the addition product between GFP-N149-TCOK and Me2Tet. (b) The invDA-mediated elimination reaction inside living HEK 293T cells was monitored by LC/MS/MS analysis of the four target peptides after trypsin digestion. The target peptides sequences are listed above the LC spectra, and the relative intensity, retention time and molecular weight are shown. Peptide-1 (P1, black) corresponds to the reaction substrate GFP-Y40TCOK, whereas peptide-2 (P2, blue) is related to the addition product between GFP-Y40-TCOK and Me2Tet. Peptide-3 and peptide-4 (P3 and P4, red) refer to the elimination product GFP-Y40-K. The reaction site on target peptides are labeled in purple and are located in the box.
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Nature chemical biology doi: 10.1038/nchembio.1656 a
Luc-WT Luc-K529-TAG TCOK-a – – + + Me2Tet – + – + Anti-His Relative 100 ± 7 N.D. N.D. 80% in about 15 min, consistent with the yield from intracellularly decaged GFPY40-TCOK-a (Fig. 3b). Similarly, a dose-dependent assay showed that Me2Tet as low as 100 μM was sufficient for full activation of intracellular C-fLuc (Fig. 3c). Moreover, this titration indicated a correlation between the Me2Tet concentration and the rescue ratio, which could be used for controlled rescue of the enzyme activity by varying the Me2Tet concentration. Finally, cell lysates were subjected to bioluminescence measurement, which showed similar activation efficiency (Supplementary Fig. 14). In summary, we have developed an invDA-mediated bioorthogonal protein decaging strategy to mask and liberate a lysine’s ε-amine for efficient and rapid protein activation in living cells. Compared to previous protein activation strategies, this method has unique advantages. First, the TCOK-a and Me2Tet reagents provide a generally applicable caging-decaging pair for chemical control of a lysine side chain. This strategy may also be further extended to modulate the activation of additional amino acids beyond lysine. Second, the time scale for efficient decaging is comparable to that of the widely adopted UV-mediated protein photodecaging method (for example, efficient intracellular activation of photocaged fLuc or photocaged MAP kinase kinase, MEK1, with a 10-min time scale19,20), thus making it suitable for rapid protein activation in living cells. Third, a small-molecule Me2Tet was used to trigger the decaging event, which could potentially be used in deep tissues or even in intact animals that are hardly accessible
brief communication
by UV irradiation. Finally, Me2Tet is highly biocompatible, and its fluorescent derivatives have been widely used for intracellular labeling of diverse biomolecules. Therefore, other laboratories may easily adopt our Me2Tet-triggered elimination strategy. Such a rationally designed, small molecule–triggered bioorthogonal protein activation strategy may become a generally applicable tool in turning on intracellular enzymes, and thus their downstream cellular responses or signaling events, with value in cell engineering and therapeutics21. The bioorthogonal protein manipulation toolkit could be further expanded from conjugation to elimination chemistry22–25, with potential applications in an in vivo setting. Received 26 June 2014; accepted 26 August 2014; published online 2 November 2014
Methods
Methods and any associated references are available in the online version of the paper.
References
1. Zorn, J.A. & Wells, J.A. Nat. Chem. Biol. 6, 179–188 (2010). 2. Banaszynski, L.A., Chen, L.-c., Maynard-Smith, L.A., Ooi, A.G.L. & Wandless, T.J.A. Cell 126, 995–1004 (2006). 3. Qiao, Y., Molina, H., Pandey, A., Zhang, J. & Cole, P.A. Science 311, 1293–1297 (2006). 4. Li, J. et al. Nat. Chem. 6, 352–361 (2014). 5. Knall, A.-C. & Slugovc, C. Chem. Soc. Rev. 42, 5131–5142 (2013). 6. Selvaraj, R. & Fox, J.M. Curr. Opin. Chem. Biol. 17, 753–760 (2013). 7. Sauer, J. et al. Eur. J. Org. Chem. 1998, 2885–2896 (1998). 8. Versteegen, R.M., Rossin, R., tenHoeve, W., Janssen, H.M. & Robillard, M.S. Angew. Chem. Int. Ed. Engl. 52, 14112–14116 (2013). 9. Royzen, M., Yap, G.P.A. & Fox, J.M. J. Am. Chem. Soc. 130, 3760–3761 (2008). 10. Hao, B. et al. Science 296, 1462–1466 (2002). 11. Liu, C.C. & Schultz, P.G. Annu. Rev. Biochem. 79, 413–444 (2010). 12. Chin, J.W. Annu. Rev. Biochem. 83, 379–408 (2014). 13. Lang, K. & Chin, J.W. Chem. Rev. 114, 4764–4806 (2014). 14. Wan, W., Tharp, J.M. & Liu, W.R. Biochim. Biophys. Acta 1844, 1059–1070 (2014). 15. Yanagisawa, T. et al. Chem. Biol. 15, 1187–1197 (2008). 16. Yanagisawa, T. et al. Mol. Biosyst. 8, 1131–1135 (2012). 17. Nikić, I. et al. Angew. Chem. Int. Ed. Engl. 53, 2245–2249 (2014). 18. Chatgilialoglu, C. & Ferreri, C. Acc. Chem. Res. 38, 441–448 (2005). 19. Zhao, J., Lin, S., Huang, Y., Zhao, J. & Chen, P.R. J. Am. Chem. Soc. 135, 7410–7413 (2013). 20. Gautier, A., Deiters, A. & Chin, J.W. J. Am. Chem. Soc. 133, 2124–2127 (2011). 21. Wei, P. et al. Nature 488, 384–388 (2012). 22. Sasmal, P.K., Streu, C.N. & Meggers, E. Chem. Commun. (Camb.) 49, 1581–1587 (2013). 23. Bielski, R. & Witczak, Z. Chem. Rev. 113, 2205–2243 (2013). 24. Li, L. et al. Nat. Commun. 5, 3276 (2014). 25. Chan, J., Dodani, S.C. & Chang, C.J. Nat. Chem. 4, 973–984 (2012).
Acknowledgments
We acknowledge support from R. Meng and the Proteomic Mass Spectrometry Core of the National Facilities for Protein Sciences (the Phoenix Project) at Peking University. This work was supported by the National Basic Research Program of China (2010CB912300 and 2012CB917301) and the National Natural Science Foundation of China (21225206 and 91313301). J.L. acknowledges support from the Peking University Principal Foundation. S.F. Reichard edited the manuscript.
Author contributions
P.R.C. and J.L. designed the experimental strategy and wrote the manuscript. J.L. and S.J. performed the experiments. All authors prepared the figures and edited the manuscript.
Competing financial interests
The authors declare no competing financial interests.
Additional information
Supplementary information and chemical compound information is available in the online version of the paper. Reprints and permissions information is available online at http://www.nature.com/reprints/index.html. Correspondence and requests for materials should be addressed to P.R.C.
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3
ONLINE METHODS
© 2014 Nature America, Inc. All rights reserved.
Plasmid construction. The plasmids pSupAR-MmPylRS and pCMVMmPylRS encoding mutant MmPylRS and the cognitive tRNAPylCUA for TCOK have been described in previous literature4. The plasmids pBAD-GFPN149-TAG-His6, pCDNA3.1-GFP-Y40TAG-His6 and pCDNA3.1-fLuc/fLucK529TAG-His6 have been reported in our previous work4,19. Reagents and equipment. All of the compounds used in the synthesis were purchased from J&K Scientific. Synthesis information is included in Supplementary Note 1. Compounds 4 and 5 were purchased from SigmaAldrich (cat. nos. 403555 and 403547). All of the other chemicals were analytical grade or better. 1H NMR and 13C NMR spectra were recorded on a Bruker-500 MHz NMR (AVANCE III) or Varian-300 MHz NMR instrument. High-resolution mass spectra (HRMS) were recorded on a Fourier Transform Ion Cyclotron Resonance Mass Spectrometer (APEX IV). Protein purifications were all performed on an AKTA UPC 900 system (GE Healthcare). Protein MS was performed with LC/MS, using an ACQUITY UPLC I-Class SQD-2 (Waters) system with electrospray ionization (ESI). LC/MS/MS analysis of tryptic peptides was performed on an Easy nLC 1000 system coupled to an LTQ-orbitrap-XL mass spectrometer equipped with a nano-ESI source. MS survey scans were performed in FT mode at a resolution of 30,000, and MS2 scans were performed in an ion trap. Images of protein gels, including Coomassie blue–stained SDS-PAGE gel and western blotting membranes, were taken on a ChemiDoc XRS+ (Bio-Rad). The luminescence and fluorescence intensity were monitored in a Multi-Mode Microplate Reader (Synergy 4, Bio-Tek, USA) or ChemiDoc XRS+ (Bio-Rad). Cell culture. HEK293T cells were purchased from the American Type Culture Collection (ATCC). Cells were grown in DMEM (Dulbecco’s modified Eagle’s medium, Gibco) supplemented with 10% FBS (FBS) and cultured at 37 °C under 5% CO2. Reactions between 1a/1e and tetrazines. These Fmoc-protected UAAs (1a and 1e) were diluted in the reaction solvent (CH3CN/H2O = 1/4) to a final concentration of 100 μM in a 1.5-ml Eppendorf tube from a 10 mM stock in CH3CN. The solutions were warmed to 37 °C before the tetrazines (10 mM stock in CH3CN) were added to a final concentration of 500 μM. The reactions were conducted at 37 °C for varying times (400 r.p.m. in Eppendorf Thermomixer C) and were then diluted twofold with CH3CN before immediate UPLC-MS/PDA analysis. The UPLC-yields for the conversion of 1a and 1e to 8 were determined by an external standard method (making a calibration line of 1a, 1e and 8 for quantification with different concentrations). The UPLC analysis was performed on an ACQUITY UPLC I-Class SQD 2 (Waters) system with a BEH C18 Acquity column (1.7 μm, 2.1× 50 mm). The mobile phase consisted of acetonitrile (MeCN) and water. The sample was eluted with a 4.8-min linear gradient (5–95% acetonitrile, 0.1% HCOOH, 0.4 ml/min) directed to the mass detector. Retention times: 1a: 2.460–2.522 min; 1e: 2.472–2.549 min; 8: 1.573–1.672 min; 9 (tetrazine=3): 1.995–2.035 min. Expression and purification of UAA-incorporated proteins in E. coli. Expression of green fluorescent protein was carried out in DH10B cells cotransformed with plasmids carrying pMmPylRSY306A Y384F-tRNACUAPyl and pBAD-GFP-N149-TAG-His6, respectively. Cells were recovered in 1 ml LB medium for 1 h at 37 °C before further growth in LB medium containing ampicillin (50 mg/ml) and chloramphenicol (34 mg/ml) overnight at 37 °C. After 1:100 dilution in 500 ml LB medium containing ampicillin (50 mg/ml) and chloramphenicol (34 mg/ml), the cell culture was grown at 37 °C to an OD600 ~0.6. TCOK-a and TCOK-e (100 mM) were added at a 1:100 dilution to a final concentration of 1 mM. After 30 min of incubation, protein expression was induced by the addition of 5 ml 20% arabinose to give a final concentration of 0.2%, and cells were harvested after 12 h expression at 30 °C by centrifugation (6,000 r.p.m., 10 min) followed by resuspension in lysis buffer (Tris-HCl, pH 7.8, 300 mM NaCl). Lysate after sonication was loaded onto a Ni-NTA column (Histrap 5 ml, GE Healthcare), which was washed with 30 ml washing buffer (20 mM Tris-HCl, pH 7.8, 300 mM NaCl, 40 mM imidazole) and then eluted with elution buffer (20 mM Tris-HCl, pH 7.8, 300 mM NaCl and 250 mM imidazole) to yield ~5 mg/ml full-length GFP containing TCOK. Protein was then desalted to 1× DPBS buffer (pH 7.4) and concentrated to 20 μM. nature chemical biology
ESI-MS analysis. LC/MS analysis of GFP-N149-TCOK and its products after the decaging reaction were performed using a Waters ACQUITY UPLC I-Class SQD 2 MS spectrometer with electrospray ionization (ESI). 0.1% formic acid in H2O as buffer A and 0.1% formic acid in acetonitrile as buffer B were used as the solvent system. LC separation for GFP and its variants was carried out with a BEH300 C4 Acquity column (1.7 μm, 2.1 × 100 mm), and positive mode was chosen for ESI-MS to analyze all samples. The total mass of proteins was calculated using MassLynx V4.1 software (Waters). Theoretical mass of the wild-type protein was calculated using PROTEIN CALCULATOR v3.3 (http://protcalc.sourceforge.net/), and the theoretical mass for all modified proteins was adjusted manually. InvDA-mediated elimination reaction on purified proteins. For in vitro cleavage reactions on purified proteins, recombinant GFP proteins bearing the site-specifically incorporated TCOK-a and TCOK-e (all at the 10 μM final concentration in 1× DPBS buffer, pH 7.4) were incubated with 20 equiv. of Me2Tet (200 μM final concentration). The solution was incubated at 37 °C for varying times and was then subjected to LC/MS analysis. Expression and decaging of TCOK-a and TCOK-e containing GFP in mammalian cells. For GFP-40-TCOK-a and GFP-40-TCOK-e expression in HEK293T cells, plasmids of pCDNA3.1-GFP-Y40TAG-His 6 and pCMVmutant MmPylRS were co-transfected via X-tremeGENE HP (Roche) in DMEM (10% FBS) supplemented with 1 mM TCOK-a and TCOK-e. Cells were allowed to grow for additional 24 h to express the desired protein bearing a site-specifically incorporated TCOK-a and TCOK-e residue. For elimination reactions on proteins, cells harboring the TCOK-a and TCOK-e incorporated proteins were first cultured in DMEM without UAA for 180 min to exocytose free UAA inside cells, followed by treatment with Me2Tet reagents (100 μM) in fresh DMEM with 10% FBS to enable the invDA-mediated cleavage reaction to proceed inside mammalian cells. The reactions were subsequently quenched by TCOK-a (2 mM in 1× DPBS) at varying times. Quantifying invDA-mediated elimination efficiency on intracellular proteins. After the decaging reaction, HEK293T cells were lysed, and the target GFP proteins were purified through the C-terminus His6 tag and digested in SDS-PAGE gel by trypsin for the following LC/MS/MS analysis. After SDS-PAGE separation, the corresponding protein bands were excised and digested in-gel with sequencing grade trypsin (5 ng/μl trypsin, 50 mM ammonium bicarbonate, pH 8.0) overnight at 37 °C. Prior to the addition of the enzyme, gel pieces were dehydrated in acetonitrile, incubated in 10 mM DTT in 50 mM ammonium bicarbonate at 56 °C for 30 min, incubated in 55 mM iodoacetamide in 50 mM ammonium bicarbonate at ambient temperature for 1 h in the dark, and then dehydrated again. The resulting peptides were extracted twice with 5% formic acid/50% acetonitrile in acetonitrile and then vacuum-centrifuged to dryness. For LC/MS/MS analysis, the extracted peptides were reconstituted in 0.2% formic acid, loaded onto a 100 μm × 2 cm precolumn and separated on a 75 μm × 20 cm capillary column. Both columns were packed in-house with 4 μm C18 bulk materials (InnosepBio, China). An Easy nLC 1000 system (Thermo Scientific, USA) was used to generate the following HPLC gradient: 7–35% B in 50 min, 35–75% B in 1 min, then holding at 75% B for 14 min (A = 0.1% formic acid in water, B = 0.1% formic acid in acetonitrile). The eluted peptides were sprayed into a LTQ Orbitrap XL mass spectrometer (Thermo Scientific, USA) equipped with a nano-ESI source. The mass spectrometer was operated in data-dependent mode with one MS scan in FT mode at a resolution of 30,000 followed by 5 CID (collision-induced dissociation) MS/MS scans in the ion trap for each cycle. Cytotoxicity study of the tetrazine Me2Tet. HEK 293T cells were cultured at a density of ~2,000–5,000 cells per well, depending on the cell type, in flat-bottomed 96-well plates and left to grow for 24 h. Cells were then treated with different concentrations of tetrazine molecule Me2Tet for 120 min, and each condition was carried out in quintuplicate. After incubation, cells were washed with fresh DMEM, and CellTiter 96 Aqueous One Solution Reagent (Promega, Madison, WI) was added to each well according to the manufacturer’s instructions. After 4 h incubation, the cell viability was determined by measuring the absorbance at 490 nm using a microplate reader. InvDA-mediated activation of caged fLuc in living cells. For fLuc-529-TCOK-a and fLuc-529-TCOK-e expression in HEK293T cells, plasmids of pCDNA3.1fLuc-K529TAG-His6 and pCMV-mutant MmPylRS were cotransfected via doi:10.1038/nchembio.1656
Determination of rescue ratio of C-fLuc. Rescue ratio was calculated as: Rescue ratio =
r.l. of C - fLuc /r.l. of fLuc - WT b.i. of C - fLuc/ b.i. of fLuc - WT
× 100%
where r.l. represents relative luminescence measured from samples containing C-fLuc or fLuc-WT under Synergy 4 Hybrid Microplate Reader or ChemiDoc, and b.i. represents band intensity, as measured from western blotting analysis on samples containing C-fLuc or fLuc-WT. Statistical analysis. Error bars are presented as mean ± s.d. from three independent experiments. The statistical analysis was performed using an unpaired Student’s t-test when the two groups were compared. P < 0.05 was considered to be significant.
© 2014 Nature America, Inc. All rights reserved.
X-tremeGENE HP (Roche) in DMEM (10% FBS) supplemented with 1 mM TCOK-a and TCOK-e. Cells bearing the chemical-caged C-fLuc protein were treated with Me2Tet to undergo the invDA-mediated elimination reaction after 24 h transfection and expression. After reaction, cells were washed and incubated with DPBS supplemented with 2 mM TCOK-a to quench the reaction. For live cell imaging using ChemiDoc instrument (Bio-Rad), luciferin solution (20 mM Tricine, pH 7.8, 1 mM MgSO4, 0.1 mM EDTA, 1 mM dithiothreitol, 270 μM coenzyme A, 500 μM D-luciferin, 530 μM ATP and 2 mM TCOK-a) was added and incubated for another 1 min before cells were imaged on a ChemiDoc XRS+ (Bio-Rad) using the chemiluminescence channel. Alternatively, cells were lysed using universal lysis buffer (containing 2 mM TCOK-a) after reaction, and cell lysates were then mixed with luciferin solution. Relative bioluminescence was measured with a Synergy 4 Hybrid Microplate Reader. The same samples were used for SDS-PAGE analysis to quantity the amount of the protein.
doi:10.1038/nchembio.1656
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