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Monitoring Nitric Oxide in Subcellular Compartments by Hybrid Probe Based on Rhodamine Spirolactam and SNAP-tag Chao Wang, Xinbo Song, Zhuo Han, Xiaoyu Li, Yongping Xu, and Yi Xiao ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.5b01032 • Publication Date (Web): 16 May 2016 Downloaded from http://pubs.acs.org on May 16, 2016
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ACS Chemical Biology
Monitoring Nitric Oxide in Subcellular Compartments by Hybrid Probe Based on Rhodamine Spirolactam and SNAP-tag Chao Wang,†,# Xinbo Song,†,# Zhuo Han,‡ Xiaoyu Li,‡ Yongping Xu,‡ and Yi Xiao*,† †
‡
State Key Laboratory of Fine Chemicals and School of Life Science and Technology, Dalian University of Technology, Dalian 116024, China. ABSTRACT: By connection of O6-benzylguanine (BG) to an “o-phenylenediamine-locked” rhodamine spirolactam responsive to nitric oxide (NO), a novel substrate (TMR-NO-BG) of genetically encoded SNAP-tag has been constructed. In living cells, labeling SNAP-tag fused proteins with TMR-NO-BG will in situ generate corresponding probe-protein conjugates (TMR-NO-SNAP) that not only inherit high NO sensitivity from the small-molecule parent, but also guarantee the site-specificity to the designated subcellular compartments such as mitochondrial inner membrane, nucleus and cytoplasm. In two representative cellular processes, TMR-NO-BG demonstrates its applicability to monitor endogenous subcellular NO in the activated RAW264.7 cells stimulated by lipopolysaccharide and in the apoptotic COS-7 cells induced by etoposide.
Nitric oxide (NO) is a critical modulator of diverse physiological and pathological processes.1-3 Because this gaseous free radical is highly reactive toward variety of biomacromolecules,4-6 it is consumed fast near its generating sites in various subcellular compartments.7, 8 Thus, accurately monitoring NO’s local distribution in different organelles is crucial for the understanding of its complex biological effects.
stability and specificity of genetically encoded techniques with the diversity and versatility of small-molecule chemical labels for protein-targetable labeling.18-24 Hence, it is very meaningful and practical to develop the genetically encoded probes for specifically sensing and imaging subcellular NO.25
Sensing and imaging NO in cells by fluorescent probes have attracted much attention.9-14 To this date, a number of molecular probes have been successfully developed and applied to analyze the overall or average levels of NO in living cells. However, most of these known probes without targeting capability can only be randomly/unevenly distributed in uncertain intracellular areas. This limitation prevents them from tracing the local generation of NO, which makes it difficult to clarify NO’s roles in terms of organelles, not to mention proteins. To solve above-mentioned problem, a new trend has emerged toward molecular probes with organelletargeting ability.15-17 We previously introduced lysosomes15 and mitochondria16 targeting NO probes. Purposely attachments of functional groups as targeting moieties enable these new NO probes to fast localize in corresponding organelles. However, such targeting capabilities based on weak attractions (e.g. mutual affinity between positive and negative charges) are susceptible to changes of local microenvironment under physiological or pathological conditions, as we have demonstrated in a mitochondrial NO probe.16 In contrast to unreliability of above-mentioned targeting small-molecule probes, the genetically encoded protein-tag methodologies have been known to integrate the
Figure 1. Mechanism of protein-targetable NO sensing. As a part of our continuous works on NO molecular probes15, 16 and fluorescent SNAP-tag,26 we have attempted to integrate both them into a hybrid NO probe, for the sake to take good use of individual advantages. On the one hand, the widely used SNAP-tag (20 kDa) evolved from human O6-alkylguanine-DNA alkyltransferase
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detection limit) to the reactive substrate of SNAP-tag (O6benzylguanine, BG). As illustrated in Figure 1, small molecule TMR-NO-BG is supposed to freely diffuse into cells and then react with the cysteine at the active site of SNAP-tag protein to form covalent attachment specifically and efficiently, which in situ generates a protein-probe conjugate TMR-NO-SNAP; the latter can sense NO by ring-opening reaction to produce strongly emissive TMRSNAP. The targeting ability of TMR-NO-SNAP in specific locations is guaranteed by the fusion of SNAP-tag to any protein of interest. The SNAP-tag-rhodamine spirolactam hybrid NO probe is of inventiveness for its unique molecular design that improves biological applicability in terms of small tag protein size, high NO sensitivity and excellent quality of subcellular NO imaging.
(hAGT) is a high specific and stable protein labeling method. On the other hand, ring-opening transformation of rhodamine spirocyclic structures represents an important mechanism to develop a very large class of ultrasensitive fluorescence ‘turn-on’ probes.27, 28 The spirocyclic forms are completely nonfluorescent, which is helpful to eliminate background noise. But, once an analyte-induced ring-opening process takes place to generate a rhodamine fluorophore, very strong fluorescence will be switched on. To our knowledge, no sensing derivative from rhodamine spirocyclic scaffold has yet been introduced to any protein-tag technique. Based on above considerations, we construct the ‘hybrid’ probe TMR-NO-BG, by connecting a rhodamine spirolactam TMR-NO highly sensitive to NO (nM scale Scheme 1. Synthesis of TMR-NO-BG.
O Cl N
+ H2N
N N H
N H
O
N
N NH2
N
NH2
N
PYBG N
N
O
N
N H
Br
N O
N
O NH2
O
NH2 COOH
O
O
OH N
O N
N N
NH2
N
N
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TMR-NO-BG
N O NH2
Br Br-TMR-NO
RESULTS AND DISCUSSION Spectra of TMR-NO-BG and TMR-NO-SNAP. As demonstrated in Scheme 1, to synthesize TMR-NO-BG, terminal alkyne substituted BG (PYBG26) is conjugated with the Br-TMR-NO through Sonogashira coupling. TMR-NO-BG is well characterized by NMR and HRMS, and purified TMR-NO-BG (confirmed by HPLC) is suitable for spectral investigation and cell imaging experiments. We first attempt to evaluate the in vitro NO responsiveness of TMR-NO-BG and TMR-NO-SNAP (i.e. TMRNO-BG being attached to purified SNAP-tag protein) by adding a NO solution (the concentration was quantitated by the Griess method29) into aqueous buffer. As shown in Figure 2a and Figure S2a, TMR-NO-BG and TMR-NOSNAP showed no absorption in visible region in the absence of NO; Upon the addition of NO, an intensive absorption band peaked at 560 nm (characteristic for rhodamine chormophore) appeared, as the result of the NOinduced ring-opening process to generate TMR-BG (Figure S3) and TMR-SNAP. Correspondingly, a remarkable fluorescence enhancement (19-fold at 564 nm for TMRNO-SNAP and 8.5-fold at 566 nm for TMR-NO-BG) was also observed just a few seconds after the addition of NO, as shown in Figure 2b, c and Figure S2b. The fluorescence intensity of TMR-NO-SNAP was increased with a good linear correlation (R2 = 0.9895) in the range of low NO concentration from 1eq to 25 eq (Figure 2b). And the de-
tection limit of TMR-NO-BG labeled to SNAP-tag protein was calculated by the concentration corresponding to triple signal-to-noise ratio to be 67.3 nM. While, the detection limit of TMR-NO-BG was calculated to be 29.4 nM (Figure 2b and Figure S2b). The reaction efficiency is high as shown in Figure 2c. The Photophysical properties of TMR-NO-BG and TMR-NO-SNAP with excess NO were described in Table 1. The covalent labeling of SNAPtag protein with TMR-NO-BG is confirmed by SDS-PAGE analysis (Figure 2d). The labeled protein stimulated with NO shows remarkable fluorescence band at 23 kD in gel but no fluorescence without NO, which demonstrated the specificity of covalent labeling and NO induced fluorescence enhancement of protein labeled probe. The probe also shows high selectivity for NO over various species (Figure 2e). Hence, the probe TMR-NO-BG attached to SNAP-tag protein maintained the NO sensitivity, which implied the potential application in living cells. SNAP-tag protein labelling and sensing of NO in cells. We next examined the usability of TMR-NO-BG for the subcellular labeling and sensing of NO in living cells. The nucleus and cytoplasm diffusely expressed pSNAPf Vector (SNAP-tag without any signaling sequence), nucleus histone H2B proteins targeted pSNAPf-H2B
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ACS Chemical Biology with TMR-NO-BG, subsequently. Although TMR-NO-BG molecules can freely diffuse anywhere in cells, they are specifically fastened and retained in corresponding compartments where they react with above SNAP-tag fusion proteins to form covalent TMR-NO-SNAP. The “ophenylenediamine-locked” TMR-NO-BG labeled to all the transfected cells show almost no fluorescence in the absence of NO (Figure 3a, c, e). But the fluorescence show conspicuous 40- to 50-fold enhancement immediately after NO solution (20 eq) is added, respectively. (Figure 3b, d, f, g). This indicates that, even in living cells, the NO sensitivity of TMR-NO-SNAP remains as high as the small-molecule probe parent, despite the conjugation of the NO sensing unit to protein tags. In control experiments (Figure S4), although non-transfected cells stained with TMR-NO-BG also show NO-induced fluorescence enhancement, washing the cells will result in the complete disappearance of fluorescence signals, further confirming fusion protein technique’s advantage of stable labeling.
Figure 2. Spectra of TMR-NO-SNAP. (a) Absorption spectra of TMR-NO-SNAP (10 μM in PBS at PH 7.4) before and after the addition of NO (200 eq). (b) Fluorescence spectra of TMR-NO-SNAP (0.5 μM in 20 mM HEPES at PH 7.2) excited at 535 nm upon the addition of NO. Inset: the response of fluorescent intensity at 564 nm upon the addition of NO from 0 to 320 eq. (c) Fluorescence spectra of TMR-NO-SNAP (0.5 μM) upon the addition of NO from 0 to 25 eq. Inset: the linear response of fluorescent intensity at 564 nm upon the addition of NO. (d) SDS-PAGE analysis of TMR-NO-BG labeled SNAP-tag protein. Coomassie blue stained (left) and fluorescence (right) imaging of the same gel before (-NO) and after (+NO) the labeled probe incubated with NO. (e) Selectivity of TMR-NO-SNAP for NO over various species. Fluorescence intensity (at 564 nm) of TMR-NO-SNAP (0.5 μM in PBS) in the presence of various species (200 eq) after incubate at r.t. for 2 h. Table 1. Photophysical properties of probes.a Compound
λex (nm)
λem (nm)
ε (M−1 cm−1)
Φf b
6-Br-TMR
557
570
48000
0.58
TMR-NO-BG + NO
557
571
19000
0.48
TMR-NOSNAP + NO
556
567
34000
0.59
a
TMR-NO-BG and TMR-NO-SNAP show very weak absorbance and fluorescence at the corresponding region of TMR-BG and TMRSNAP. b Quantum yields are measured in PBS (pH 7.2) using Rhodamine B as a standard (ΦF = 0.69 in methanol).
and the mitochondria cytochrome c oxidase submit 8 (COX8A) targeted pSNAPf-COX8A were chosen. These plasmid SNAP-tag fusion proteins were specifically expressed to the corresponding subcellular compartments in COS-7 cells by transfecting with the three plasmids, respectively. All the stable transfected cells were labeled
The site-specificity of TMR-NO-SNAP is also notable. As shown in Figure 3b and d, in the cells transfected with pSNAPf vector labeled with TMR-NO-BG, NO addition results in an almost homogeneous fluorescence distribution in nucleus and cytoplasm of entire cells; in the cells transfected with pSNAPf-H2B plasmid, fluorescence signals are exclusively emitted in nucleus. And in the cells transfected with pSNAPf-COX8A, the mitochondrial labeling is confirmed by colocalization with a commercial mitochondrial dye MitoTracker Deep Red FM (Figure S5). The Pearson’s colocalization coefficient 0.87 and overlap coefficient 0.88 indicate the excellent labeling of TMRNO-BG to SNAP-tag expressed in mitochondria. To further evaluate the specificity of the labeling with TMRNO-BG, the signal-to-background ratio (SBR) of different SNAP-tag transfected cells in Figure 3b, d and f are calculated to be 14, 10 and 19 (SBR determination detail recorded in Figure S6) respectively, reflecting the high specificity of labeling with TMR-NO-BG. Fluorescent imaging of endogenous NO. Further, we apply TMR-NO-BG in the detection of induced endogenous NO in subcellular compartments. Inducible nitric oxide synthase (iNOS) in murine macrophages RAW264.7 cells can be activated by lipopolysaccharide (LPS) and interferon-γ (IFN-γ) to produce NO. The RAW264.7 cells are stably transfected with diffusely expressed (pSNAPf vector) and mitochondrial targeted (SNAP-COX8A) plasmids, respectively. Then, the cells are labeled with TMR-NO-BG by the same way for COS-7 cells labeling. These labeled macrophage cells are incubated with LPS (20 μg mL-1) and IFN-γ (400 U mL-1) for 12 h. In the case of diffusely expressed SNAP-tag labeled with TMR-NO-BG, the total intensity of intracellular fluorescence shows an obvious 3-fold increase (Figure 4a, b, g) after stimulation. Interestingly, the fluorescence in subcellular regions shows inhomogeneous distribution (Figure 4b), which is not consistent with the diffusely homogeneous expressed
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Figure 3. Fluorescent imaging of exogenous NO in different subcellular compartments of COS-7 cells labeled with TMRNO-BG. Cells stable transfected with diffusely expressed (a, b), nucleus targeted(c, d) and mitochondrial targeted (e, f) plasmids encoding SNAP-tag are stained with TMR-NO-BG (5 μM). Fluorescent imagings were collected before (a, c, e) and after NO solution (20 μM) were added (b, d, f). (λex: 559 nm, λem: 575-675 nm). (g) The relative fluorescence intensity of a-f. Thirty cells were calculated for three independent imagings. Error bars represent SD. Significance was determined using the T test (***, P
