Multifunctional stimuli-responsive chemogenetic platform for conditional multicolor cell-selective labeling

Multicolor conditional labeling is a powerful tool that can simultaneously and selectively visualize multiple targets for bioimaging analysis of complex biological processes and cellular features. We herein report a multifunctional stimuli-responsive Fluorescence-Activating and absorption-Shifting Tag (srFAST) chemogenetic platform for multicolor cell-selective labeling. This platform comprises stimuli-responsive fluorogenic ligands and the organelle-localizable FAST. The physicochemical properties of the srFAST ligands can be tailored by modifying the optical-tunable hydroxyl group with diverse reactive groups, and their chemical decaging process caused by cell-specific stimuli induces a conditionally activatable fluorescent labeling upon binding with the FAST. Thus, the resulting switch-on srFASTs were designed for on-demand labeling of cells of interest by spatiotemporally precise photo-stimulation or unique cellular feature-dependent activation, including specific endogenous metabolites or enzyme profiles. Furthermore, diverse enzyme-activatable srFAST ligands with distinct colors were constructed and simultaneously exploited for multicolor cell-selective labeling, which allow discriminating and orthogonal labeling of three different cell types with the same protein tag. Our method provides a promising strategy for designing a stimuli-responsive chemogenetic labeling platform via facile molecular engineering of the synthetic ligands, which has great potential for conditional multicolor cell-selective labeling and cellular heterogeneity evaluation.


Introduction
Elucidating the localization and the activities of intracellular biomolecules for a deep understanding of biological processes in cells and organisms calls for effective imaging tools. [1][2][3][4] Chemogenetic strategy, as a powerful toolkit, provides many options and great potential for visualizing biological events in real-time and in situ. In this strategy, genetically encoded activators (consisting of peptides or proteins) can specically activate small synthetic ligands (uorogens) to a uorescent state by transient or covalent binding. [5][6][7] Such hybrid approaches combine the excellent targeting-specicity of the genetically encoded protein tags with superior photophysical properties as well as tunability of the small synthetic ligands, which ensures a great deal of experimental versatility and designability. 6 Accordingly, SNAP-tags, 8,9 HaloTags, 10 PYP-tags, 11,12 uorogen-activating proteins (FAPs) 13 and uorescence-activating and absorption-shiing Tags (FASTs) [14][15][16] have been developed.
As an emerging member of chemogenetic strategy, the FAST system contains a 14 kDa protein tag, which can non-covalently bind with a series of cell-permeant and nontoxic 4-hydroxybenzylidene rhodanine (HBR) derivatives and activate their uorescence. [14][15][16] The small size of the tag and its instantaneous and reversible binding with the ligands endow the FAST system with additional excellent properties such as (a) minimal functional perturbation to the target proteins, (b) excellent photophysical properties such as brightness and antiphotobleaching, and (c) much shorter maturation time than GFP-like uorescent proteins. 14 By changing the substituents on the 4-hydroxybenzyl moiety of the ligands, colours of the FAST:uorogen complex have spanned from green-yellow to farred, signicantly improving its spectral tunability. 15 Although the uorogen promiscuity of the FAST system facilitates spectral exibility, it is still challenging for FASTs to simultaneously label multiple targets with different colors because of indiscriminate recognition of structurally similar ligands by its protein tag. To address this problem, directed evolution of the FAST presented a feasible strategy to develop orthogonal labeling systems. Accordingly, two FAST variants that can selectively bind with different colored ligands (HMBR or HBR- Cite this: Chem. Sci., 2022, 13, 12187 All publication charges for this article have been paid for by the Royal Society of Chemistry 3,5DOM) were recently developed, allowing for selective labeling of two targets with dual colors. 17 However, further expanding orthogonal FAST tagging proteins requires laborious procedures to implement protein evolution and sophisticated multiple gene expressions. Hence, these challenges may present an opportunity for seeking additional solutions toward this problem.
Besides manipulation of FASTs, ligand engineering is an alternative approach with the potential to achieve multicolor selective labeling based on the identical FAST, which is yet to be demonstrated. Rationally designed ligands capable of responding to particular targets independently 18-25 may offer an opportunity to realize not only conditional selective labeling, but also multicolor selective labeling. Given that complex biological samples include many diverse cell types, cellular heterogeneity in phenotypes and genotypes, a fundamental property of cellular ecosystems, 26-28 might function as a key factor to enable conditional multicolor labeling of specic cell types of interest. The unique spatiotemporal distribution, metabolite prole, and endogenous enzymes of a cell of interest not only play a central relevance to its biological function, but are also highly useful as identication markers for discriminating diverse cell types. [28][29][30][31] It is enlightening that these cellular characteristics may act as specic triggers for stimuli-responsive ligands with different colors to achieve heterogeneity-dependent cell-selective labeling, providing an additional approach for on-demand multicolor labeling of specic cell types without the requirement of diverse protein tags.
Herein, taking advantage of the cellular heterogeneity features and rational tunability of the ligands, we introduce a FAST-derived strategy to construct a multifunctional stimuliresponsive labeling platform, namely stimuli-responsive FAST (srFAST), for conditional multicolor cell-selective labeling. The srFAST consists of two parts: one is the FAST scaffold, which can activate the uorescence of ligands exquisitely and provide an excellent subcellular location through genetic fusion with a localizable peptide or protein; the other is the rationally designed HBR-derived "caged" ligands, which are endowed with responsiveness to diverse stimuli such as light-inducible spatiotemporal activation, unique small-molecule metabolites, or the enzymatic expressions of specic cell types. These stimuli-activatable ligands can be conditionally activated by cell-specic physical or biochemical characteristics to enable on-demand uorescent labeling of FAST-fused proteins, only in target cells. Meanwhile, using multicolor enzyme-responsive srFASTs, different cell types were selectively labeled with orthogonal multicolor uorescence dependent on their independent endogenous enzymatic signatures. We envision that this proposed platform would enrich the applications of the FAST system in cell-specic multicolor labeling and provide a meaningful tool for cellular heterogeneous studies.

Results and discussion
Design principle of the srFAST platform As an emerging uorogenic chemogenetic protein tag, the FAST system features signicant uorescent enhancement and large absorption redshi of its HBR-derived ligands upon binding with the FAST, the protein ( Fig. 1a and S1 †). 14,15 These noticeable spectroscopic changes depend on the inhibition of the intramolecular rotation of ligands by FAST binding and the protein microenvironment-caused deprotonation of the ligands' phenolic hydroxyl group. In addition to the key role in the spectroscopic variation, its characteristic hydroxyl group is likely to function as an optical tunable group ready for modication-mediated regulation, which has been widely exploited in various uorescent dyes 32-35 but still unexplored in the FAST system. Accordingly, we sought to investigate whether caging the hydroxyl group of the ligands can modulate the optical properties of the FAST system.
Red-emissive ligand HBR-3,5DOM and its methoxy derivative HBR-3,4,5TOM were prepared for comparison and synthesized in one step by condensation of rhodanine to the corresponding substituted 4-hydroxy-benzaldehyde. We rst investigated the photophysical properties of the compounds ( Fig. 1b and c). The maximal absorption peak of HBR-3,4,5TOM is 393 nm, a slight blue shi to that of HBR-3,5DOM in the phenolic form (409 nm), and both compounds have little uorescence in PBS buffer, with relative uorescence quantum yields of 0.05% and 0.11%, respectively. In the presence of the FAST, deprotonated HBR-3,5DOM was stabilized and showed a large absorption redshi (409 nm to 518 nm) and a signicant uorescence increase at 600 nm. The FAST:HBR-3,5DOM complex (the original FAST system, O-FAST) exhibits a relative uorescence quantum yield of 32% (Table S1 †) comparable to that reported (31%) in the literature. 15 However, HBR-3,4,5TOM exhibited a negligible absorption change, and only a weak uorescence upon FAST binding with a relative uorescence quantum yield of 4.4%, which was about 6.3% of that of the O-FAST at 600 nm. Time-dependent density functional theory (TDDFT) calculations revealed that the protonation and methylation of the oxygen atom increase the excitation energy gaps from 2.53 (HBR-3,5DOM in phenolate form) to 2.82 (HBR-3,5DOM in phenolic form) and 2.92 eV (HBR-3,4,5TOM), respectively, supporting the similarly blue-shied absorption of the FAST:HBR-3,4,5TOM complex and free HBR-3,5DOM in comparison with the O-FAST. A close examination of the S 1 excited state (Fig. S2 †) showed that the electrons on the LUMO of HBR-3,4,5TOM and HBR-3,5DOM in the phenolic form are mainly located on the rhodanine framework, plausibly reasoning the decrease in the uorescence efficiency of the FAST:HBR-3,4,5TOM complex due to intramolecular charge transfer (ICT). 32,35 Further uorescence competition assay (Fig. S3 †) showed that HBR-3,4,5TOM could moderately interfere with the association between HBR-3,5DOM and the FAST when its concentration is signicantly higher. The proposed K d (28.2 mM) of HBR-3,4,5TOM upon FAST binding was much greater than that of HBR-3,5DOM (0.97 mM), 15 indicating a decrease in affinity. Molecular docking studies (Fig. 1d) demonstrated that HBR-3,4,5TOM could enter the binding cavity of the FAST and be stabilized by the hydrogen bond with T98 as well as apolar interactions with I31, F62, V66, A67, T70, I96, P97 and V122. However, the hydrogen-bond network formed by the conserved residues Y42 and E46 to stabilize the phenolate anion 36,37 in the O-FAST disappeared in the FAST:HBR-3,4,5TOM complex, potentially resulting in a decrease in affinity. Taken together, modifying the hydroxyl group of the ligand could diminish its binding with the FAST and efficiently reduce the complex uorescence. Hence, the feature of the optically tunable hydroxyl group provides a good entrance to designing activatable ligands for a versatile srFAST platform.
Photo-srFAST for spatiotemporal cell-selective labeling of the nucleus Light activation as an external regulation and control means has been widely used in spatiotemporal controlled imaging due to its quick, straightforward, and precisely controllable properties. 38,39 Here, an optically controlled tagging system is explored as the preliminary attempt to achieve spatiotemporally selective labeling of cells. Here, the photoactivatable ligand photo-DOM containing a 2-nitrobenzyl group 39,40 was successfully synthesized through a one-step nucleophilic reaction of HBR-3,5DOM and 1-(bromomethyl)-2-nitrobenzene (Fig. 2a). In comparison with HBR-3,5DOM, photo-DOM has a greater absorption enhancement with a large redshi from 445 nm to 567 nm aer binding with the FAST (Fig. 2b), but shows weak uorescence at 600 nm (Fig. 2c). Further competition assay indicated that 2-nitrobenzyl group modication of photo-DOM partially diminished the binding affinity with the FAST (K d ¼ 7.4 mM, Fig. S4 †). Under UV irradiation (l ¼ 365 nm, 3 mW cm −2 ), the uorescence intensity of the FAST:photo-DOM complex at 600 nm increased rapidly, and tended to be stable aer 12 min with a signicant enhancement about 15 times (Fig. 2c, d, and S5 †), which are consistent with the uncaging dynamics of the same caging group reported in other literatures. 40,41 These results proved the feasibility of our photo-responsive FAST system, named photo-srFAST. To demonstrate the ability of the photo-srFAST for spatiotemporally selective labeling, we performed patterned imaging in a 96-well plate. As shown in  , the photo-srFAST could pattern a specic "X" area with red uorescence aer UV irradiation. However for the O-FAST, red uorescence was detected in all areas without selectivity. Thus, photo-decaging of the ligand's spectrum-tunable hydroxyl group is an efficient approach to develop srFASTs, and the constructed photo-srFAST can efficiently respond to photostimulation with an excellent S/N ratio.
Then, we evaluated the feasibility of photo-srFAST in spatiotemporally selective labeling of target cells (Fig. 3a). The nucleus-localized FAST fusion protein (NLS-sfGFP-FAST) was transiently expressed in HEK293T cells, where the nuclear localization signal (NLS) and super-folded green uorescent protein (sfGFP) were fused at FAST's N-terminal sequentially. As can be seen from Fig. 3b, the nucleus of the cells emitted the typical green uorescence of sfGFP under the excitation of 488 nm, which conrmed the successful expression and localization of the FAST fusion protein. Being incubated with photo-DOM did not induce any detectable signal. Aer being illuminated with UV (l ¼ 365 nm, 3 mW cm −2 ) for 4 min, the cells appeared with red uorescence, which was colocalized well with the green uorescence, and the uorescence intensity was signicantly enhanced about 12 times aer irradiation of 10 min (Fig. 3b, c and S6 †), indicating the gradual formation of the photo-decaged uorogenic complex. Flow cytometry analysis (Fig. S7 †) also conrmed that photo-DOM could light up the target cell populations aer UV illumination. Thus, photo-DOM is cell-permeant with a negligible uorescence background, and the photo-srFAST is suitable for selective labeling of cells with a rapid and signicant uorescence response under UV irradiation. Comparatively, in the cells incubated with HBR-3,5DOM (the original FAST ligand) (Fig. 3b and c), both red and green uorescence remained bright before and aer UV irradiation, and the red uorescence from the O-FAST was bleached by 15% aer 10 min UV irradiation (Fig. S8 †). In contrast, only the cells located at the selected area exposed to UV irradiation showed red uorescence that merged as yellow in the presence of photo-DOM, while other cells out of the irradiated area remained green ( Fig. 3d and S9 †), suggesting spatially selective cell labeling by the photo-srFAST. At the same time, sequential labeling of the selected cells (Fig. 3e) was performed on a Leica DMi8 inverted microscope with an optical manipulative module, where the time and area of illumination could be nely controlled by its laser (l ¼ 405 nm, 40 mW cm −2 ). When the cells at specic locations were exposed to the laser in sequence, red uorescence appeared instantaneously (5 s) and sequentially with 8 or 11-fold uorescence enhancement, respectively, allowing for the near real-time implementation of the cell-specic labeling processes. Notably, even the adjacent cells in 50 mm can be manipulated precisely. CCK8 assay (Fig. S10 †) showed that neither photo-DOM nor illumination had an obvious inuence on the cell survival rate under the experimental conditions. Taken together, the photo-srFAST enables us to selectively and quickly label cells with high spatiotemporal imaging resolution through controlling UV irradiation, which has a potential application in spatiotemporal labeling and analysis. To demonstrate the versatility and expansibility of our srFAST platform, we sought to fabricate another stimuli-responsive ligand that can be specically activated by a particular metabolite for cell-selective labeling. Generally, different cell types respond distinctively to the uctuating environment and external stimuli and produce different metabolites under certain stress, leading to heterogeneous cellular responses. 26,27 For example, respiratory burst is a rapid release process of reactive oxygen species (ROS) usually observed in immune cells, such as macrophages, as a pathogen-defending mechanism. Phorbol 12-myristate 13-acetate (PMA) is a potent stimulant of respiratory burst of macrophages, enabling activation of NADH oxidase and consequently massive generation of hydrogen peroxide and other ROS. Recently, mounting evidence supported that H 2 O 2 produced by respiratory burst has a profound effect on macrophage signaling, 42 (Fig. S11 †), and the resulting FAST:H 2 O 2 -DOM complex exhibited relative weak uorescence at 600 nm in comparison with the O-FAST at an equimolecular concentration (Fig. S12 †). Furthermore, the competition assay between H 2 O 2 -DOM and HBR-3,5DOM revealed a moderate affinity between H 2 O 2 -DOM and the FAST (K d ¼ 5.0 mM, Fig. S13 †). Addition of H 2 O 2 triggered a remarkable enhancement in uorescence intensity due to H 2 O 2 -mediated boronate decaging of H 2 O 2 -DOM (Fig. 4b, c and S14 †). A titration experiment presented that the emission intensity of the H 2 O 2 -srFAST at 600 nm exhibited a linear response toward H 2 O 2 in the range of 5-250 mM with the detection limit of 1.5 mM (Fig. 4c and S14a †), which ts the endogenous H 2 O 2 concentration in macrophages under PMA stimulation. 42 Other compounds except for H 2 O 2 generated negligible uorescence in PBS buffer, illustrating the high selectivity of the H 2 O 2 -srFAST (Fig. S14b †).
Next, we investigated whether the H 2 O 2 -srFAST can be used for H 2 O 2 -responsive labeling of cells. HEK293T cells expressing the mitochondrial-targeted FAST protein (mito-FAST, the mitochondrial targeting sequence from subunit VIII of human cytochrome c oxidase was fused at FAST's N-terminal) were treated with H 2 O 2 -DOM and a commercialized mito-tracker (a specic dye for the localization of mitochondria in living cells). Aer the addition of 200 mM H 2 O 2 , bright red uorescence was observed in cells, which overlapped with the green uorescence from the mito-tracker (Fig. S15 †) Fig. 4d, in the absence of PMA, cells incubated with H 2 O 2 -DOM showed negligible red uorescence. Aer PMA stimulation, bright red uorescence was detected, which was perfectly colocalized with the green uorescence from the mito-tracker. Flow cytometry analysis (Fig. S16 †) also depicted that the red uorescence intensity from the cell populations stimulated by PMA is about 4 times of that without PMA treatment. By contrast, the cells incubated with HBR-3,5DOM maintained red uorescence before and aer PMA stimulation. In addition, CCK8 assay suggested that H 2 O 2 -DOM had no obvious toxic or adverse effects on cells under experimental conditions (Fig. S17 †) (Fig. S18 †). Both cells expressing mito-FAST were co-cultured and stimulated with PMA, and then incubated with H 2 O 2 -DOM or HBR-3,5-DOM. For the O-FAST, it is impossible to distinguish the signals between the two different cell lines, since they were both labeled with similar red uorescence ( Fig. 4e-g). The merged images suggested the much higher transfection efficiency of HEK293T cells than RAW 264.7 cells. Even then we can easily distinguish the two co-cultured cell lines with the H 2 O 2 -srFAST. Only RAW 264.7 cells were labeled with strong red uorescence due to the high level of respiratory burst-caused endogenous H 2 O 2 , with the signal intensity more than 2 times that from HEK293T cells (Fig. 4e-g and S19 †). Therefore, the H 2 O 2 -srFAST can distinguish the cellular response of different cell types and selectively label the cell line of interest with the featured metabolite, demonstrating that our proposed srFAST platform has extensive potential in studying specic cell populations with metabolic heterogeneity.
Multicolor enzyme-srFASTs for protein-dependent orthogonal labeling of multiple cells Encouraged by cell-selective labeling based on light-triggered spatiotemporal activation and specic metabolite-dependent chemical decaging, we further adapted the srFAST platform relying on cellular heterogeneity at the expression level of endogenous enzymes. Unique cell types have cell-specic endogenous enzyme expression and activity proles to maintain diverse cellular functions, presenting a fundamental mechanism of cellular differentiation and heterogeneity. Moreover, aberrant expression of crucial enzymes in growth and metastasis regulation is highly associated with cancer development. 28 For instance, alkaline phosphatase (ALP), a hydrolase in phosphate metabolism and associated with cell differentiation and viability, is overexpressed in several tumor cell lines including HeLa cells (a cell line of cervical cancer). 44-46 b-Galactosidase (b-gal) removes galactose residues from substrates such as gangliosides, glycoproteins, and sphingolipids. It is known as an important biomarker for cell senescence and primary ovarian cancers, and is overexpressed in SKOV-3 cells (a cell line of ovarian cancer). [47][48][49] Comparatively, both enzymes are expressed at a low level in normal cells, such as HEK293T cells. 50,51 Thus, these cell-specic endogenous enzymes can be exploited to discriminate distinct cell types. We attempted to design two enzyme-activatable ligands with different colors, termed enzyme-srFASTs, for orthogonal multicolor selective labeling of cells with a distinct enzyme expression.
For responding to ALP, a ligand ALP-HMBR was designed by blocking the hydroxyl group of the green-emissive ligand HMBR with a phosphate group (Fig. 5a). The FAST:ALP-HMBR complex (ALP-srFAST) displayed very weak uorescence at 540 nm ( Fig. S20 and S21 †) and their interaction affinity was moderate (K d ¼ 5.4 mM, Fig. S22 †). As expected, the phosphate group of ALP-HMBR could be specically hydrolyzed to generate HMBR when ALP was presented (Fig. S23 †), 45,46,50 resulting in the recovery of green uorescence of the decaged FAST:HMBR complex (Fig. S24 †). The emission intensity of the ALP-srFAST at 540 nm is linearly proportional to the ALP concentration in the range of 2.0-50.0 U L −1 (Fig. S24 †). Similarly, we developed the ligand b-gal-DOM by modifying HBR-3,5DOM with a galactose group and obtained a b-gal-srFAST (Fig. 5b). Unlike other activatable HBR-3,5DOM derivatives, b-gal-DOM has no detectable change in the absorption and uorescence spectra before and aer FAST addition (Fig. S26 †). Ligand competition experiments revealed that the FAST was able to bind b-gal-DOM with low affinity (K d ¼ 16.3 mM, Fig. S27 †). Addition of b-gal caused a remarkable uorescence recovery of the FAST:b-gal-DOM complex at 600 nm owing to the hydrolyzation of the galactose group from b-gal-DOM, and there was an obvious linear relationship with the b-gal concentration from 0.01 to 0.12 U mL −1 (Fig. S28 and S29a †). [47][48][49]51 Thanks to the excellent substrate specicity, both enzyme-srFASTs exhibited good selectivity toward their targets, including the modied ligands, respectively (Fig. S24d, S25 and S29b †). Collectively, srFASTs responding to two enzymes with distinct colors were developed in vitro.
For live-cell labeling, good biocompatibility of ALP-HMBR and b-gal-DOM was rstly conrmed for their negligible inuence on cell viability in CCK8 assays ( Fig. S30 and S31 †). Next, the feasibility of the ALP-srFAST for efficient uorogenic labeling of the cells with highly expressed endogenous ALP was conrmed. Both cell imaging and ow cytometry experiments exhibited that HeLa cells expressing the membrane-localized FAST (mem-FAST, the membrane localization signal GAP43 was fused at FAST's N-terminal) appeared with typical green uorescence of the FAST:HMBR complex on their cell membrane when treated with ALP-HMBR, while there was no obvious uorescence in the cells pre-treated with Na 3 VO 4 , a typical ALP inhibitor (Fig. S32 †). Likewise, the cytomembrane of SKOV-3 cells overexpressing b-gal was labeled by the b-gal-srFAST with red uorescence, conrmed by cell imaging and ow cytometric analysis (Fig. S33 †). Together, these results proved that endogenous enzymes are potent markers for responsive labeling of target cells.
Further, the labeling selectivity of the enzyme-srFASTs was evaluated. Here, both HeLa and SKOV-3 cells transfected with the pcDNA3.1-mem-fast were separately incubated with the mixed ligands, where the uncaged ligands were devoted to verifying the successful expression and localization of mem-FAST. For the ALP-srFAST group (Fig. 5c and d), green uorescence was only observed in ALP-positive HeLa cells, and the average uorescence intensity was 6 times that of SKOV-3 cells, suggesting that the ALP-srFAST can optionally label HeLa cells. For the b-gal-srFAST group (Fig. 5e and f), only b-gal-positive SKOV-3 cells were stained by red uorescence, with the average uorescence intensity 8 times that of HeLa cells. However, for the corresponding O-FAST group, both cell lines incubated with the mixed original ligands were indiscriminately labeled with the same green or red uorescence (Fig. 5c-f). In general, the two-color enzyme-srFASTs could respond to multiplex enzymes and label different cancer cells with distinct colors, opening novel prospects for multiple cell line discrimination and analysis.
Finally, we sought to study the potential of multicolor enzyme-srFASTs for simultaneous orthogonal labeling of distinct cell lines in a complex multi-cell co-culture system (Fig. 6a). According to the different expression levels of the two enzymes, two cancer cell lines, HeLa (ALP + , b-gal − ) and SKOV-3 (ALP − , b-gal + ), and one normal cell line HEK293T (ALP − , b-gal − ) were chosen. The three types of cells expressing the same mem-FAST were co-cultured, and then incubated with the mixture of ALP-HMBR and b-gal-DOM (srFAST) or HBR-3,5DOM and HMBR (O-FAST). For the srFAST group, it is quite easy to distinguish the three cell types, as HeLa cells were labeled with green uorescence and SKOV-3 cells were labeled with red uorescence, whereas no obvious uorescence appeared in HEK293T cells (Fig. 6b-d and S34 †). For the O-FAST group, comparatively, all cells were labeled with green and red uorescence, merging as yellow equally (Fig. 6b, e, f and S34 †). Therefore, multicolor enzyme-srFASTs are suitable for selectively labeling cells with orthogonal colors via the identical protein tag, and distinguishing different cell types. The ability to respond to multiple targets by using the same protein tag but different target-responding ligands with various spectral properties provides unprecedented experimental versatility and exibility for multicolor imaging, without the need for reengineering. It is reasonable to envision that if there are cell lines co-overexpressing the two enzymes, they could be observed in yellow by employing multicolor enzyme-srFASTs. Specic labeling and distinguishing three or four cell lines with only one protein tag is a promising step toward overcoming the uorogen promiscuity of the original FAST system, which also opens up exciting prospects for conditional multicolor cell-selective labeling and cellular heterogeneity monitoring at multiplex levels.

Conclusions
In summary, we developed a multifunctional stimuli-responsive chemogenetic platform (srFAST platform) for conditional cellselective labeling via facile ligand engineering of the modular FAST system. The srFAST platform combines the subcellular localization of the genetically encoded FAST and the color and target tunability of synthetic ligands, providing excellent experimental versatility. Based on the srFAST platform, cells of interest were labeled on demand by spatiotemporally precise photo-activation or specic endogenous metabolites or enzyme proles, demonstrated by proof-of-concept models, including photo-srFAST, H 2 O 2 -srFAST or enzyme-srFASTs, respectively. Further, multicolor enzyme-srFASTs were constructed for concurrent orthogonal labeling of three cell types with different colors, proving feasibility to achieve multiplex bioimaging with the same protein tag. The generality and modularity of this platform make it facile to enrich the srFAST platform via endowing an increasing number of spectrally tunable FAST uorogens with additional responsiveness to diverse molecular features of cell heterogeneity. Moreover, the fast binding and reversible dynamics of the FAST system might enable further development of phase-specic enzyme-dependent srFAST systems to track diverse phases and progression of the cell cycle by simply changing the corresponding ligands. As a promising alternative approach for orthogonal multicolor labeling, we believe that the srFAST platform could expand the FAST toolbox and be of great potential in sophisticated imaging applications in multicellular systems.

Data availability
Experimental procedures, compound characterization, NMR spectra, and additional data are located within the ESI. †