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A Localizable and Photoactivatable Fluorophore for Spatiotemporal Two-Photon Bioimaging Liyi Zhou, Xiaobing Zhang, Yifan Lv, Chao Yang, Danqing Lu, Yuan Wu, Zhuo Chen, Qiaoling Liu, and Weihong Tan Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b00691 • Publication Date (Web): 23 Apr 2015 Downloaded from http://pubs.acs.org on May 10, 2015

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Analytical Chemistry

A

Localizable

and

Photoactivatable

Fluorophore

for

Spatiotemporal Two-Photon Bioimaging

Liyi Zhou,a Xiaobing Zhang,a* Yifan Lv,a Chao Yang,a Danqing Lu,a Yuan Wu,a,b Zhuo Chen,a Qiaoling Liu,a and Weihong Tana,b* a

Molecular Science and Biomedicine Laboratory, State Key Laboratory for

Chemo/Bio-Sensing and Chemometrics, College of Chemistry and Chemical Engineering, College of Biology, and Collaborative Research Center of Molecular Engineering for Theranostics, Hunan University, Changsha 410082, China b

Departments of Chemistry, Department of Physiology and Functional Genomics, Center

for Research at Bio/Nano Interface, Shands Cancer Center, University of Florida Genetics Institute and McKnight Brain Institute, University of Florida, Gainesville, FL 32611-7200, USA

* To whom correspondence should be addressed. Email: [email protected], [email protected]

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ABSTRACT Photoactivatable probe-based fluorescent imaging has become an efficient and attractive technique for spatiotemporal microscopic studies of biological events. However, almost all previously reported photoactivatable organic probes have been based on hydrosoluble precursors, which have produced water-soluble active fluorophores able to readily diffuse away from the photocleavage site, thereby dramatically reducing spatial resolution. Hydroxyphenylquinazolinone (HPQ), a small organic dye known for its classic luminescence mechanism through excited-state intramolecular proton transfer (ESIPT), shows strong light-emission in the solid-state, but no emission in solution. In this work, HPQ was employed as a precursor to develop a localizable, photoactivatable two-photon probe (PHPQ) for spatiotemporal bioimaging applications. After photocleavage, PHPQ releases a precipitating HPQ fluorophore which shows both one-photon and two-photon excited yellow-green fluorescence, thereby producing a localizable fluorescence signal that affords high spatial resolution for bioimaging, with more than 200-fold one-photon and 150-fold two-photon fluorescence enhancement. INTRODUCTION Photoactivatable probe-based fluorescence imaging has become an efficient and attractive technique for spatiotemporal microscopic imaging of tissues and animals.1-2 However, the development of high-performance photoactivatable probes has encountered some challenges.3-6

In

order to

achieve high-quality fluorescence images,

several

photoactivatable probes generated from fluorescent proteins (FPs), 7-12 quantum dots,13-14 or organic fluorophores15-17 have been widely developed for cellular bioimaging with temporal and spatial resolution. Among them, organic fluorophore-based photoactivatable probes have attracted particular attention based on their high membrane permeability, excellent photophysical features, and the chemical flexibility of their well-known precursors like coumarin and fluorescein. For a traditional organic molecular photoactivatable probe, the fluorophore is usually protected with a caging group, and the fluorescence is quenched.17 Under experimental conditions, illumination with ultraviolet light (UV) triggers a break in the photosensitive bond, resulting in release of the caging group and recovery of fluorescence. Thus, it is theoretically possible to endow these 2


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probes with high spatiotemporal resolution by controlling both the illumination time and site. Unfortunately, upon UV stimulation, almost all such probes produce water-soluble active fluorophores that readily diffuse away from the site of the photocleavage reaction, hence reducing spatial resolution. This property limits intracellular applications requiring accurate spatial localization. To address the effects of diffusion, an alternative probe is needed which uses photoactivation to release a precipitating active fluorophore after light stimulation, producing a localizable fluorescence signal and, consequently, yielding high spatial resolution. Moreover, most previous probes have been one-photon excited, requiring a rather short excitation wavelength (usually 130nm) and significant photostability.30 By replacing the phenolic hydrogen of HPQ with a hydrophilic group, the insoluble and fluorescent HPQ becomes water soluble and lightless by blocking ESIPT, because the molecule cannot form the fluorescent structure (enol and keto forms). Moreover, similar to the classic naphthalene-based two-photon fluorophores,21-23, 25-27 HPQ possesses a Donor-π-Acceptor structure, which is expected to exhibit good two-photon fluorescent activity. These unique features make HPQ different from common water-soluble and diffusible fluorescent dyes and thus favorable for the design of activatable probes that release a precipitating fluorophore with localizable fluorescence signal and high spatial resolution. Accordingly, quite a few HPQ-based enzyme-activatable fluorogenic probes have been developed over the past two decades and commercialized to detect the activity of phosphatase, esterases, lipases, glycosidases, and beta-lactamase.33-36 More recently, by employing smart cyclizing spacer design, the Hasserodt group has adopted HPQ to develop fluorescent probes able to detect peptidase activity.37 Upon enzymatic removal of the hydrosoluble residue, a bright fluorescent precipitating product was released from these probes at the site of enzymatic reaction. This effectively solved the diffusion problem faced by common probes and enabled good spatial resolution of enzymatic activities in cells. Inspired by these successful designs, we, for the first time, report herein the use of HPQ as a precusor to develop a localizable photoactivatable two-photon probe (PHPQ) for spatiotemporal bioimaging applications (Figure 1 and Figure 2). PHPQ contains a quaternary ammonium group that serves as both water-soluble moiety and mitochondria-targeting unit and a 2-nitrobenzyl group that serves as a photolabile caging group. When triggered with ultraviolet light, the probe releases a precipitating HPQ fluorophore that shows both one-photon- and two-photon-excited bright solid-state yellow-green fluorescence in aqueous media with more than 200-fold one-photon and 150-fold TP fluorescence enhancement, thereby producing a localizable fluorescence signal that affords high spatial resolution for bioimaging. The HPQ with larger two-photon action cross section was measured to be 95 GM using 720 nm excitation, which is comparable to that of the classic Donor-π-Acceptor-structured naphthalene derivative, indicating that HPQ is an efficient TP fluorophore. These 4


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excellent features all favor bioimaging applications. This probe was then applied in phototriggered one-photon and/or TP spatiotemporal imaging in live cells, and both showed satisfactory results. EXPERIMENTAL SECTION Reagents and Apparatus. All chemicals were obtained from commercial suppliers and used without further purification. Water used in all experiments was doubly distilled and purified by a Milli-Q system (Millipore, USA). LC-MS analyses were performed using an Agilent 1100 HPLC/MSD spectrometer. 1H-NMR spectra were recorded on a BrukerDRX-400 spectrometer operating at 400 MHz. All chemical shifts are reported in the standard notation of parts per million. All fluorescence measurements were carried out on a Hitachi-F4500 fluorescence spectrometer with both excitation and emission bandwidths set at 5.0 nm. Two-photon fluorescence data were measured by exciting with a mode-locked Ti: sapphire pulsed laser (Chameleon Ultra II, Coherent Inc.) and then recording with a DCS200PC single-photon counter (Beijing Zolix Instruments Co., Ltd.). One- or two-photon fluorescence images of HeLa cells were obtained using an Olympus FV1000-MPE multiphoton laser scanning confocal microscope (made in Japan). PHPQ compounds were efficiently synthesized following the synthetic methodology shown in the synthetic routes. Spectroscopic Materials and Methods. Experiments to measure the fluorescence of PHPQ were conducted in pure water or in 99% glycerin. The fluorescence emission spectra were recorded using an excitation wavelength of 365 nm with emission wavelength ranging from 460 to 600 nm. A 5×10-4 M stock solution of PHPQ was prepared by dissolving PHPQ in pure water or in 99 % glycerin. Because glycerin has a sufficiently larger viscosity than water, thus preventing HPQ from sedimentation, HPQ was uniformly dispersed in glycerin. To recount specific operations, HPQ was first dissolved in tetrahydrofuran. The HPQ solution was then added to an appropriate volume of glycerol, thus making a homogeneous solution. Finally, tetrahydrofuran was evaporated to obtain the required test solution.

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Preparation and Staining of Cell Cultures. Live HeLa cells were obtained from the Biomedical Engineering Center of Hunan University (Changsha, China). Immediately prior to the imaging experiments, the cells were washed with phosphate-buffered saline (PBS), incubated with 1 µM PHPQ for 0.5 h at 37 oC, washed with PBS three times, and then imaged. Confocal fluorescence imaging was observed under an Olympus FV 1000 laser confocal microscope. The laser excitation wavelength was 405 nm or 720 nm (two-photon), and emissions were centered at 460-550 nm. For the colocalization reagent FMD, the excitation wavelength was 635 nm, and the emission wavelength was 640-680 nm. Cytotoxicity test. Cell viability was evaluated by the reduction of MTT3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyltetrazolium bromide to formazan crystals by mitochondrial dehydrogenases (Mosmanm, 1983). A sample of 1×105 HeLa cells in 50 µL of washing buffer was seeded into each test well on a 96-well plate. After overnight culture, PHPQ (0~6 mM/L) in 50 µL of washing buffer was added to respective test wells. After 2 h treatment, 10 µL of MTT solution (5 mg/mL in phosphate buffer solution) was added to the each well. After 4 h incubation at 37 °C, 100 µL of a solution containing 10% SDS and 0.01 M HCl was added to dissolve the purple crystals. After 12 h incubation, the optical density readings at 595 nm were recoded using a plate reader. Each of the experiments was performed at least 3 times. Probe synthesis. Detailed description of the synthesis of each probe can be found in the Supplementary Methods. Each step was characterized by thin-layer chromatography, high-resolution mass spectra, and 1H-NMR. RESULTS AND DISCUSSION Diffusion-resistance experiments in vitro. The diffusion-resistance of the HPQ fluorophore was first tested with a water-soluble 7-hydroxycoumarin chosen as a control fluorophore. HPQ and 7-hydroxycoumarin were spotted on silica-gel plates, and the plates were immersed in water. As shown in Fig. 1, HPQ showed strong emission and precisely localized fluorescence, even after immersion in water for 6 h. In contrast, 7-hydroxycoumarin diffused quickly on the water-soaked silica-gel plate with fluorescence rapidly fading within only 30 min. These results demonstrated that HPQ 6


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could solve the problem of dye diffusion, making it a promising precursor for the development of spatiotemporal imaging probes for bioimaging applications. a

b

Figure 1. Diffusion-resistance experiments for HPQ (A) and 7- hydroxycoumarin (B). The compounds were spotted on silica-gel plates, immersed in water and irradiated under UV light at 365 nm. Design and synthesis of PHPQ. Having confirmed the diffusion resistance of HPQ, a photocaged-HPQ (PHPQ) was then designed and synthesized. PHPQ contains a 2-nitrobenzyl group as a photolabile caging unit, as shown in Figure 2. It was also modified with a quaternary ammonium moiety, which could localize in the mitochondria and afford good water solubility for the entire probe molecule. Upon irradiation with ultraviolet light, the quaternary ammonium moiety, together with the 2-nitrobenzyl group of PHPQ, are cleaved from the probe molecule to release a precipitating HPQ, affording a localizable fluorescence signal for imaging mitochondria in vivo. A photocaged 7-hydroxycoumarin (PCM) containing a 2-nitrobenzyl group and a quaternary ammonium moiety was also synthesized as a control probe (Figure 2). See supporting materials for synthesis details and spectral information (Figure S1).

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NH

NH

NH

NH

N

N

O keto

OH

NO2 O

enol Excitation at 365nm or 405nm

N

O

O

O

Cl

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Localizes in the mitochondria and emits strong green fluorescence

O PHPQ NO2 O

O

O

O

Excitation at 365nm or 405nm Cl

N

O

O

Cannot localize in the mitochondria and emits strong blue fluorescence

O PCM

Figure 2. Structures and response mechanisms of the localizable, photoactivable probe (PHPQ) and the control photoactivable probe (PCM). Photoactivatable response property of PHPQ. As shown in Figure 3, under ultraviolet light irradiation, the fluorescent intensity of PHPQ increased gradually. After 12 min, a clear green color could be observed by the naked eye under a UV lamp (Figure 3C and D). Measurement of the intensity showed a 200-fold enhancement, indicating that PHPQ had good photoactivatable response within 12 min. At the same time, the absorption spectra before and after the UV radiation were also recorded, with obvious difference observed (See Figure 3e). The quantum yield for the caged dye PHPQ was calculated to be 0.00095, while the quantum yield for the photocleaved product HPQ to be as high as 0.22, corresponding to a 231.6- fold enhancement after photoconversion, which is comparable to that of previously reported 2-nitrobenzyl derivatives.16

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400 Fluorescent Intensity / a.u.

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Time / s

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Figure 3. (a), (b) Time-dependent fluorescent spectral changes of PHPQ under UV irradiation (0-12min) at RT, PHPQ dissolved in glycerol to form 1 µM testing solution; (c), (d) visual fluorescence color change of PHPQ before and after irradiation by UV light at 365 nm for 12 min, λex=365 nm; (e) Normalized absorption spectra PHPQ before and after UV irradiation (10 min) at RT. Cell viability and co-staining imaging for mitochondria. Previous results demonstrated that PHPQ had good chemical and spectral properties. The cytotoxicity of PHPQ, HPQ and UV irradiation at 365 nm (0~30 min) were then evaluated using a 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyltetrazolium bromide (MTT) assay (Figure S7).38 Experimental results showed that negligible cytotoxicity was observed when up to 6.0 µM PHPQ or HPQ was added to the culture medium. Moreover, the cytotoxicity of UV irradiation less than 10 min is also negligible, with UV irradiation time more than 10 min, increased cytotoxicity was observed. The effect of metal ions on the response of the photoactivatable fluorescent probe was also investigated. As shown in Figure S2, 10 µM of common bio-related metal ions such as Na+, K+, Mg2+, Ca2+ exhibited negligible effect on the response of PHPQ. In addition, the effect of pH on the fluorescence response of PHPQ was also studied, with results added in Figure S2. One can find that the probe could achieve the best performance at neutral pH, which is favorable for its applications. The ability of PHPQ to localize and stain mitochondria in living cells was then assessed by fluorescence microscopy. HeLa cells were incubated with PHPQ (1 µM) and then irradiated with a 405 nm laser line. As shown in Figure 4, the irradiated cell grew bright, and the fluorescent signal intensity increased as a function of time. 9



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a

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b

c

d

f

g

h

10 µm e

Figure 4. Confocal fluorescent images. HeLa cells stained with (1µM) PHPQ or (5µM) PCM and (50nM) MitoTracker Deep Red FM (FMD). (A) green fluorescence of PHPQ in cells after 30min of irradiation at 405nm; (B) and (F) red fluorescence of FMD in cells; (C) overlay of (A), (B) and bright field image; (E) blue fluorescence of PCM in cells after 30min of irradiation at 405nm; (G) overlay of (E), (F) and bright field image; (D) and (H) Pearson’s coefficient 0.946 and 0.565, respectively. Conditions: blue fluorescence channel: λEx=405nm, λEm=415-500nm; green fluorescence channel: λEx= 405 nm, λEm=460-550nm; red fluorescence channel: λEx=635nm, λEm= 640-680nm. Scale bar: 10µm. Co-staining experiments with 1 µM PHPQ and 50 nM MitoTracker® Deep Red FM (FMD), a commercially available mitochondrial indicator (Figure S8, for FMD structure), were then conducted in order to illustrate that HPQ has the ability to self-localize. After photocleaving the localization unit, the fluorescenct moiety remained firmly adhered to the mitochondria. More importantly, HPQ was strongly attracted to the mitochondria, and no diffusion was observed. Thus, after 30 min of irradiation, HeLa cells loaded with HPQ displayed a marked increase in localized fluorescence compared to control cells. The mitochondrial imaging results of HPQ and FMD overlapped well. Additionally, the colocalization photobleaching experiment showed that intracellular fluorescence intensity was almost unchanged with increased irradiation time, whereas the fluorescence intensity of FMD became increasingly weaker (Figure S4 and S6).18 Because HPQ is a solid-fluorophore and has a larger stokes shift than FMD, these results indicate outstanding photostability and self-localization of HPQ.

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To further illustrate the performance of HPQ for localizable fluorescence bioimaging with high spatial resolution, we used 7-hydroxycoumarin to synthesize a control molecule, photocaged coumarin (PCM, Figure 2), with an analogous photoresponsive unit. Based on the previous experimental results, the 7-hydroxycoumarin easily diffused in the medium. By using FMD co-staining, we also investigated the diffusion properties of conditional dyes in vivo. After UV irradiation, the real-time imaging results showed that 7-hydroxycoumarin did not localize in the mitochondria, with a small Pearson’s coefficient (0.565) estimated; thus, the mitochondrial morphology could not be visualized, as shown in Figure 4 and S5. In contrast, PHPQ staining firmly adhered to mitochondria after photocleaving the localization unit, indicating the superior localization effect with a large Pearson’s coefficient (0.946). From Figure 4 and Figure S5, we can clearly see the shape of the mitochondria, and the real-time results can be seen in Figure S5. These results further showed that the water-soluble dyes easily diffused in the media, whereas the solid HPQ dye did not, thereby achieving spatiotemporal imaging. Two-photon activity absorption cross-section and two-photon confocal microscopy images. Similar to the classic naphthalene-based two-photon fluorophores, HPQ also possesses a Donor-π-Acceptor structure, which is expected to exhibit good two-photon fluorescence activity. To confirm this, the two-photon absorption properties of HPQ and PHPQ were investigated. The two-photon active absorption cross-section was calculated by using the following formula: δ=δr (Ss Φr ϕr cr)/(Sr Φsϕs cs), where the subscripts s and r denote the sample and reference molecule, respectively. HPQ was calculated to have a two-photon active absorption cross-section of 95 GM (1GM=10-50 (cm

4

s)/photon) at

495 nm upon excitation at 720 nm (Figure 5a) using Rhodamine B as the reference.39-40 This value is comparable to that of the classic Donor-π-Acceptor-structured naphthalene derivative. As shown in Figure 5, PHPQ had a two-photon action cross-section of only 5 GM; however, after 12 min of ultraviolet light irradiation, the fluorescent signal intensity increased by nearly 150-fold, indicating the excellent two-photon absorption property of PHPQ after UV triggering. Two-photon properties of HPQ and PHPQ. Finally, intracellular imaging performance was tested using a two-photon laser scanning confocal microscope. As shown in Figure 6, 11



HeLa cells were incubated with PHPQ (1 µM) for 30 min at 37 oC, and the cells showed weak fluorescence in the green channel under 720 nm laser excitation. However, after 365 nm UV excitation for 30 min, strong fluorescence in the green channel was observed under the same conditions. Unfortunately, the probe PHPQ is activated by UV, which severely limits the depth of penetration. Herein, the depth fluorescence images of HPQ in onion tissues were obtained with spectral confocal multiphoton microscopy, HPQ was capable of tissue imaging at depths of over 80-220 µm by 2PFM (Figure S9).

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Figure 5. Two-photon properties of HPQ and PHPQ. (A) The two-photon activity absorption cross-section (TPA) for HPQ and PHPQ with Rhodamine B as the reference; (B) TP signal intensity of PHPQ before and after UV-radiation; Two-photon excitation at 720 nm in glycerin. a

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Figure 6. Two-photon confocal microscopy images. HeLa cells stained with 1 µM PHPQ 12


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before and after UV irradiation. (A) bright field image of the HeLa cells before UV irradiation; (B) fluorescence image of (A) from green fluorescence channel; (C) overlay of (A) and (B); (D) bright field image of the HeLa cells after UV irradiation; (E) fluorescence image of (D) from green channel; (F) overlay of (D) and (E). λEx = 720 nm. Scale bar: 100 µm CONCLUSIONS The development of innovative fluorescence imaging probes has offered powerful tools for biological studies at a subcellular level, such as high spatiotemporal resolution imaging and real-time monitoring of subcellular structures such as metabolites and inorganic ion pools.41-44 Traditional techniques in this field have, however, encountered some obstacles. Limited imaging resolution and imprecise signal localization need to be addressed in order to make improvements in the imaging quality of organelles. It can be reasonably speculated that such limitations result from the undesired diffusion of water-soluble probes typically employed for signal reporting. To solve this problem, we employed the solid-state fluorophore HPQ as a precusor and developed a localizable photoactivatable two-photon probe (PHPQ) for spatiotemporal bioimaging applications. A photocaged and co-staining strategy was successfully adopted for mitochondrial imaging, and the innovative probe was demonstrated to possess outstanding properties of nondiffusion and self-localization. In our experiments, the water-soluble PHPQ probe self-located on mitochondria, and the insoluble HPQ molecule was precipitated in situ without diffusing into the cytoplasm after the caging group was cleaved by UV irradiation. The released HPQ molecule served as a reporter and gave a high-resolution two-photon fluorescence signal. Compared with traditional water-soluble probes, the HPQ-based two-photon probe could realize co-staining and self-localization imaging of mitochondria with higher quality and resolution. In addition, we, for the first time, studied the two-photon activity of HPQ. The TPA of HPQ was calculated to be 95 GM, which is comparable to that of the classic Donor-π-Acceptor-structured naphthalene derivative. Unfortunately, PHPQ is activated by UV light, which severely limits the depth of penetration. Such a problem might be solved by design a caged probe which employs NIR or two-photon irradiation for photocleavage.

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SUPPORTING INFORMATION AVAILABLE

This material is available free of charge via the Internet at http://pubs.acs.org.

ACKNOWLEDGEMENTS This work was supported by the National Key Scientific Program of China (2011CB911000), NSFC Grants (21325520, 21327009, 21375076, J1210040, 21177036, 21135001), the Foundation for Innovative Research Groups of NSFC (Grant 21221003), the National Instrumentation Program (2011YQ030124), and Hunan Provincial Natural Science Foundation (Grant 11JJ1002). REFERENCES (1) Fernández-Suárez, M.; Ting, A. Nat. Rev. Mol. Cell. Biol. 2008, 9: 929-943. (2) Lim, R. K. V.; Lin, Q. Acc. Chem. Res. 2011, 44, 828-839. (3) Vaughan, J. C.; Jia, S.; Zhuang, X. W. Nat. Meth. 2012, 9: 1181–1184. (4) Li, W. H.; Zheng, G. Photochem. Photobiol. Sci. 2012, 11, 460–471. (5) Shao, Q.; Xing, B. G. Chem. Soc. Rev. 2010, 39, 2835-2846. (6) Zhao, J.; Lin, S.; Huang, Y.; Zhao, J.; Chen, P. R. J. Am. Chem. Soc. 2013, 135, 7410-7413. (7) Vaillancourt, R. R.; Dhanasekaran, N.; Johnson, G. L.; Ruoho, A. E. Proc. Natl. Acad. Sci. USA, 1990, 87, 3645-3649. (8) Lee, S. H.; Shin, J. Y.; Lee, A.; Bustamante, C. Proc. Natl. Acad. Sci. USA, 2012, 109, 17436–17441. (9) Magliery, T. Med. Chem. 2005, 2, 303-323. (10) Koh, J. Chem. Biol. 2005, 12, 613-614. (11) Manley, S.; Gillette, J. M.; Lippincott-Schwartz, J. Methods Enzymol 2010, 475, 14


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