Rapid labeling of intracellular His-tagged proteins inliving cellsYau-Tsz Laia,1, Yuen-Yan Changa,1, Ligang Hua,1, Ya Yanga, Ailun Chaoa, Zhi-Yan Dub, Julian A. Tannerc, Mee-Len Chyeb,Chengmin Qianc, Kwan-Ming Nga, Hongyan Lia, and Hongzhe Suna,2

Departments of aChemistry and cBiochemistry, bSchool of Biological Sciences, University of Hong Kong, Pokfulam, Hong Kong, People’s Republic of China

Edited by Kenneth N. Raymond, University of California, Berkeley, CA, and approved February 3, 2015 (received for review October 12, 2014)

Small molecule-based fluorescent probes have been used for real-time visualization of live cells and tracking of various cellularevents with minimal perturbation on the cells being investigated.Given the wide utility of the (histidine)6-Ni

2+-nitrilotriacetate(Ni-NTA) system in protein purification, there is significant interestin fluorescent Ni2+-NTA–based probes. Unfortunately, previous Ni-NTA–based probes suffer from poor membrane permeability andcannot label intracellular proteins. Here, we report the design andsynthesis of, to our knowledge, the first membrane-permeablefluorescent probe Ni-NTA-AC via conjugation of NTA with fluoro-phore and arylazide followed by coordination with Ni2+ ions. Theprobe, driven by Ni2+-NTA, binds specifically to His-tags geneti-cally fused to proteins and subsequently forms a covalent bondupon photoactivation of the arylazide, leading to a 13-fold fluo-rescence enhancement. The arylazide is indispensable not only forfluorescence enhancement, but also for strengthening the bindingbetween the probe and proteins. Significantly, the Ni-NTA-ACprobe can rapidly enter different types of cells, even plant tissues,to target His-tagged proteins. Using this probe, we visualized thesubcellular localization of a DNA repair protein, Xeroderma pigmen-tosum group A (XPA122), which is known to bemainly enriched in thenucleus.We also demonstrated that the probe can image a geneticallyengineered His-tagged protein in plant tissues. This study thus offersa new opportunity for in situ visualization of large libraries of His-tagged proteins in various prokaryotic and eukaryotic cells.

His-tagged protein | Ni-NTA | fluorescent probe | live cell | photoactivation

Chemical and biochemical labeling of proteins can elucidateprotein function, localization, and dynamics as well as otherbiological events in live cells (1–3). Small molecule-based fluo-rescent labeling of recombinant proteins holds particular prom-ise as an alternative to fluorescent protein fusion technology (4, 5)without deleterious perturbation of protein functions. A repre-sentative technique is small tag-based fluorescent imaging, in whicha protein of interest is genetically fused with a short peptide thatbinds site-specifically to a designed synthetic fluorescent probe.Over recent decades, tremendous progress has been made in usingsmall molecule-based probes to monitor cellular events (6, 7). Inparticular, metal-chelation labeling of a protein is attractive owingto its simplicity and high specificity. Among these probes, FlAsHand its derivatives, including ReAsH and SplAsH, are successfulsmall molecule-based metal-containing probes that can light upintracellular proteins fused with a tetracysteine motif (1, 8, 9).However, this system suffers from drawbacks, including a highbackground that requires extensive washing (10), and unsuitabilityin a cellular oxidizing environment (11). Nevertheless, the pioneeringdevelopment of biarsenical-based fluorescent probes inspiredresearchers to design various probes that target other tag-ging systems.Given the wide utility of the (histidine)6-Ni

2+-nitrilotriacetatesystem (Ni-NTA) in molecular biology and biotechnology foraffinity chromatography-based protein purification, this systemhas also been exploited to site-selectively label large libraries ofexisting hexahistidine-tagged (His-tagged) proteins via conjuga-tion with fluorophores (12–16). Various NTA-based fluorescent

probes have been developed via conjugation of fluorophoreswith mono-NTA (12, 17) or di-, tri-, and tetra-NTA derivativesto either mimic the concept of FlAsH or overcome the weakbinding nature of His-tag with Ni2+-NTA (Kd = 13 μM) (11, 18–21). Despite significant increases in stability of the multiplechelator heads/His-tag adducts compared with mono-NTA,negative charges of these moieties might prevent them fromentering cells. Indeed, all reported Ni-NTA–based fluorescentprobes that target His-tagged proteins are exclusively limited tolabel membrane proteins because none can cross cell membranesto label intracellular proteins in live cells (15, 17). A previousclaim that a di-NTA dibromobimane conjugate entered cells totarget polyhistidine-containing proteins is not convincing be-cause the fluorescence measurement was carried out using wholecells and results were not corroborated with in vivo imaging data(22). It is thus possible that the observed fluorescence upon treat-ment of whole cells with the probe could be derived from in-teraction with polyhistidine-containing proteins on cell membranes.Recently a di-NTA–based fluorescent probe containing an

α-chloroacetamide that targets the Cys-appended His-tag (Cys-His6-tag) was reported to be able to label intracellular Cys-His6–tagged proteins in live cells (16). However, the di-NTA fluorophoreconjugate itself could hardly enter cells, unless the conjugate wasattached to a cargo consisting of a cell-penetrating peptide andfluorescent quencher (a dabsyl-appended icosapeptide with a dabsylgroup, a tetra-His, and an octa-Arg), enforcing the probe to entercells to label Cys-His6–tagged instead of His6-tagged proteins.

Significance

The hexahistidine-Ni2+-NTA system is used extensively in pro-tein purification, and large numbers of His-tagged protein li-braries exist worldwide. The application of this tagging systemto image proteins in live cells would offer significant oppor-tunities to track cellular events with minimal spatial andfunctional perturbation on a protein of interest. However,previously reported Ni-NTA–based probes suffer from poormembrane permeability and have been limited to label mem-brane proteins only. Here we report, to our knowledge, thefirst small fluorescent probe, Ni-NTA-AC, that can rapidly crosscell membranes to specifically target His-tagged proteins invarious types of live cells, even in plant tissues. The probe willprovide new opportunities for in situ analysis of variouscellular events.

Author contributions: Y.-T.L., Y.-Y.C., L.H., Y.Y., A.C., Z.-Y.D., M.-L.C., C.Q., H.L., and H.S.designed research; Y.-T.L., Y.-Y.C., L.H., Y.Y., A.C., Z.-Y.D., and K.-M.N. performed re-search; Y.-T.L., Y.Y., and A.C. performed syntheses; Y.-T.L., Y.-Y.C., L.H., Z.-Y.D., J.A.T.,M.-L.C., C.Q., H.L., and H.S. analyzed data; and Y.-T.L., Y.-Y.C., L.H., M.-L.C., H.L., andH.S. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.1Y.-T.L., Y.-Y.C., and L.H. contributed equally to this work.2To whom correspondence should be addressed. Email: hsun@hku.hk.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1419598112/-/DCSupplemental.

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Furthermore, the probe is relatively large and complicated to make.The consequential high cost of application precludes its use in la-beling many existing His-tag libraries of proteins. Moreover, therelatively slow labeling (80% yield within an hour) also hinders itsuse for real-time imaging. Therefore, it is preferable to design smalland simple fluorescent probes with good membrane permeability torapidly label intracellular His-tagged proteins.Herein, we present the design, synthesis, and application of

a fluorescent probe Ni-NTA-AC that exhibits excellent mem-brane permeability and can rapidly enter cells to image intracellularHis-tagged proteins (Fig. 1A). The probe targets a His-taggedprotein specifically through Ni2+-NTA with ∼13-fold fluorescence“turn-on” responses. An arylazide was incorporated into the probeinitially with the purpose of overcoming weak binding betweenNi2+ and histidines. Unexpectedly, the arylazide was also in-dispensable for fluorescence enhancement. We have demon-strated that our probe could image His-tagged proteins in differenttypes of cells, even in plant tissues. The ability to rapidly visualizeintracellular proteins genetically fused with a His-tag offers greatpotential for spatial and functional analysis of libraries of existingHis-tagged proteins in different types of live cells.

Results and DiscussionDesign and Synthesis of a Fluorescent Probe Ni-NTA-AC. Previouslymany fluorescent probes used the Ni-NTA system, includingdi-, tri-, or tetra-NTA derivatives conjugated with fluorophores,but could only label membrane proteins due to poor mem-brane permeability (11, 18). We reasoned that negative chargesmight prohibit crossing of cell membranes, although introductionof multi-NTA to the probes did overcome the weak binding of

Ni2+-NTA to the His-tag. We therefore designed a probe (Fig.1A) consisting of a mono-Ni-NTA moiety, a small membrane-permeable fluorophore (a coumarin derivative) (23), and anarylazide moiety. Ni2+-NTA targets the His-tag to achieve specificlabeling of a protein of interest, and the arylazide group providesan additional covalent bond between the probe and its targetprotein upon photoactivation (24) to resolve the intrinsic weakbinding of Ni2+-NTA to His-tag. A linker between mono-Ni-NTAand the fluorophore was designed to allow flexibility in facilitatingefficient protein labeling.We first synthesized a coumarin-based ligand NTA-AC via

a three-step synthesis with an overall yield of 64% (Fig. 1A andSI Appendix, Figs. S1 and S2) by conjugating the NTA moietywith a coumarin fluorophore and arylazide, and the purity ofNTA-ACwas confirmed by both NMR and electrospray ionization massspectrometry. The ligand exhibited amaximumabsorption at∼342nm (e = 11,100 M−1·cm−1) and emitted at 448 nm (Φ = 0.056; SIAppendix, Fig. S3). The lowquantumyield ofNTA-AC is attributedto the presence of an azide at the seventh position of the coumarinmoiety, which quenches the fluorescence of coumarin as reportedpreviously (25, 26). The probeNi-NTA-ACwas then generated bysubsequent incubation of NTA-AC with Ni2+ (as NiSO4) inbuffered aqueous solution. As shown in Fig. 1B, upon addition ofequimolar amounts of Ni2+ to NTA-AC, the fluorescence wassignificantly quenched by ∼70%, in sharp contrast with the 5%reduction observed in previously reported NTA-DCF conjugate(17), thus Ni-NTA-AC only has very weak emission at 448 nm. Thetitration data were nonlinearly fitted using the Ryan–Weberequation (27), which gave rise toKd of 38± 13 nM. To evaluate thebinding stoichiometry, a Job’s plot was constructed by monitoringfluorescence changes upon complexation ofNTA-AC with Ni2+ at448 nm excited at 342. Maximum fluorescence changes wereobserved at amolar ratio of Ni2+:NTA-AC of 0.5, indicative of theformation of Ni-NTA-AC complex with a ratio ofNTA-AC:Ni2+ of1:1 (Fig. 1C); this was further verified in electrospray ionizationmass spectrum (a peak at m/z 558.6), in agreement with the cal-culated value of 558.9.

Evaluation of Ni-NTA-AC Probe in Labeling His-Tagged Proteins inVitro. To examine the feasibility that Ni-NTA-AC can labela His-tagged protein in vitro, we applied the functional domainof Xeroderma pigmentosum group A (XPA122) as a showcasestudy. Xeroderma pigmentosum group A serves as a classic formof XP proteins, which is important for repairing DNA damagecaused by UV radiation (λ ≤ 310 nm); and the functional do-main, XPA122, serves as the site of damaged-DNA bindingto initiate DNA repair (28). Proteins with (denoted as His-XPA122) or without (XPA122) genetically fused His-tag at itsN terminus were overexpressed and purified as described pre-viously (SI Appendix) (29). The interaction of Ni-NTA-AC withthe protein was first investigated by fluorescence spectroscopy.Incubation of 10-M equivalents of His-XPA122 with Ni-NTA-ACled to an increase in fluorescence intensity with time, reachinga plateau at ∼9 min, where an approximate 13-fold increase influorescence intensity was observed (Fig. 2A). In contrast, noobvious fluorescence changes (less than 50% increases) werenoted upon mixing of Ni-NTA-AC with XPA122 under identicalconditions (SI Appendix, Fig. S4). Similarly, premixing of the li-gand NTA-AC (without coordination of Ni2+) with His-XPA122under identical conditions resulted in no fluorescence enhance-ment at all (SI Appendix, Fig. S5). These combined results in-dicate that Ni-NTA-AC selectively targets the His-tag of theprotein through Ni2+, resulting in fluorescence turn-on responses.Nonspecific binding is negligible under the conditions used. Al-though the underlying mechanism of fluorescence turn-on responsesof the probe toward His-tagged proteins is not fully understood, itis possible that the sandwich-like structure of the probe withNi2+-NTAmoiety weakly bound to the fluorophore is abolished upon

Fig. 1. (A) Schematic diagram demonstrating intracellular labeling of His-tagged proteins using Ni-NTA-AC (Top) and the synthetic scheme of NTA-AC(Bottom). The probe enters the cells rapidly and targets His-tagged proteinswith significant fluorescence turn-on. (B) Normalized fluorescence changesof NTA-AC (5 μM) upon addition of Ni2+ (as NiSO4). Approximately 70%decrease in fluorescence is noted upon Ni2+ chelation. (C) Job’s plot of thefluorescence changes (λex = 342 nm, λem = 448 nm) upon complexation ofNTA-AC with Ni2+ in Tris buffer. Maximum fluorescence changes were ob-served at a molar ratio of Ni2+:NTA-AC of 0.5, indicative of the formation ofNi-NTA-AC complex with a ratio of NTA-AC: Ni2+ of 1:1.

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binding to His-tagged proteins (30), then subsequent interactionof the fluorophore with protein targets might lead to fluores-cence enhancement. Moreover, the arylazide is also likely to playan important role in fluorescence turn-on because it was reportedpreviously that attachment of arylazide to coumarin at the seventhposition led to fluorophore quenching. However, such an effect isabolished upon the reduction of arylazide (25, 31).The binding properties of Ni-NTA-AC toward His-XPA122

were also studied by isothermal titration calorimetry (ITC),which gave rise to a dissociation constant of 7.1 ± 0.6 μM andbinding capacity of 1.4 (SI Appendix, Fig. S6A), consistent withthe weak binding strength of Ni2+-NTA to histidine residues.The nonintegral stoichiometry of Ni-NTA-AC binding to the His-tagged protein is attributable to the fact that one His-tag possiblybinds 1–2 Ni2+ ions (32, 33), verified by our MS data (Fig. 3C).The weak binding might lead to dissociation of the probe fromlabeled proteins in the complex environments of live cells. Totackle this problem, we incorporated an arylazide with a ratio-nale of providing additional binding between the probe andtargeted proteins upon photoactivation. The role of arylazidewas examined subsequently. First, we examined the time re-quired for arylazide activation. A mixture of equimolar amountsof His-XPA122 and Ni-NTA-AC was irradiated with 365-nm UVlight using a 4-W long-wave compact UV lamp (720 μW/cm2) atdifferent time intervals. Samples were analyzed by SDS/PAGEgel electrophoresis and fluorescence signals were quantified. Asshown in SI Appendix, Fig. S7, a fluorescent band could obviouslybe seen after 1 min of UV exposure and the fluorescence in-creased with exposure time and maximized at 10 min, suggestingthat the efficiency of photoactivation of arylazide increasescorrespondingly. A mixture of Ni-NTA-AC and 10-M equivalentsof His-XPA122 was thus subjected to irradiation under UV light(365 nm) for 10 min to ensure complete photoactivation ofarylazide. As expected, over 10-fold fluorescence enhancementcompared with the fluorescence of Ni-NTA-AC was observableowing to the probe binding to His-XPA122 (Fig. 2B). Uponaddition of 40-M equivalents of EDTA to the mixture to strip offNi2+ from the probe–protein complex, the observed fluorescenceintensities, instead of being decreased, were slightly increased(∼30%), attributable to recovery of the fluorescence being quenchedby Ni2+ (Fig. 2B). Subsequently, we carried out a similar experimentexcept under darkness to prevent arylazide from photoactivation.As shown in Fig. 2B, ∼60% decrease in fluorescence were noted,indicating that without photoactivation of arylazide, removal ofNi2+ from the probe–His-XPA122 complex abolishes the probe’sability to image His-tagged proteins.The capability of arylazide to strengthen binding between the

probe and His-tagged proteins upon photoactivation was alsoinvestigated by observation of the probe–protein complex under

denatured conditions. The Ni-NTA-AC–labeled His-XPA122was irradiated with UV light (365 nm) for 10 min, then subjectedto SDS/PAGE gel electrophoresis. An intense blue fluorescentband corresponding to His-XPA122 was observable in the SDS/PAGE, corroborating with strong binding of the probe to His-XPA122 even under denatured conditions (Fig. 2C). In contrast,no corresponding blue fluorescent band was detected whenNi-NTA-AC (12 μM) was mixed with equimolar amounts ofXPA122 or His-XPA122 in the presence of 40-M equivalentsof EDTA, which removes Ni2+ from the probe (Fig. 2C), inline with the above observations that the probe targets His-tagof XPA122 through Ni2+ and subsequent photoactivation ofthe arylazide group strengthens binding between the probeand the protein.We further examined the role of arylazide within the probe by

synthesizing a coumarin-based ligand without arylazide attached—namely, NTA-C, for comparison via a three-step synthesis (SIAppendix, Figs. S8–S10). Addition of equimolar amounts of Ni2+

led to ∼50% fluorescence reduction for NTA-C (SI Appendix, Fig.S11A). Unexpectedly, incubation of Ni-NTA-C with His-XPA122under similar conditions resulted in no fluorescence enhancement

Fig. 2. (A) Fluorescence spectra of Ni-NTA-AC (1 μM) at different time intervals after addition of His-XPA122 (10 μM). (Inset) Time-dependent fluorescencechanges (λem = 448 nm) of Ni-NTA-AC upon binding to His-XPA122. (B) Normalized fluorescence of Ni-NTA-AC incubated with His-XPA122 under variousconditions. Without photoactivation of arylazide (under dark), addition of excess EDTA (40-M equivalents) to the mixture of Ni-NTA-AC and His-XPA122 leadsto the fluorescence decrease by ∼60% owing to chelation of Ni2+ by EDTA from Ni-NTA-AC, dissociating the probe from His-XPA122. After binding of Ni-NTA-AC toHis-XPA122 and upon photoactivation of arylazide, addition of excess EDTA showed a slight increase (∼30%) in fluorescence, corresponding to fluorescence re-covered from Ni2+ quenching. (C) SDS/PAGE of protein labeling by equimolar amounts of Ni-NTA-AC (or Ni-NTA-C) under different conditions. Lane 1: His-XPA122;lane 2: His-XPA122 in the presence of excess EDTA (50 μM); lane 3: His-XPA122 and Ni-NTA-C (without arylazide); lane 4: XPA122 (without His-tag).

Fig. 3. Labeling efficiency of Ni-NTA-AC to His-XPA122. (A) SDS/PAGE ofprotein labeling upon incubation with different amounts (0–10 M equiv-alents) of Ni-NTA-AC monitored by Coomassie Blue and fluorescence stain-ing. (B) Labeling yield of Ni-NTA-AC to His-XPA122 determined by SDS/PAGEin Fig. 3A. (C) MALDI-TOF MS spectra of His-XPA122 (10 μM) in the absenceand presence of 1- and 2-M equivalents of Ni-NTA-AC. The peak at m/z of14,981 is assignable to the intact protein (calculated 14,979), and peaks atm/z of 15,549 and 16,100 correspond to the protein bound to one and twoprobes, respectively. The labeling efficiency was evaluated by comparing thepeak areas of intact His-XPA122 and the probe bound His-XPA122, to be46% and 70%, respectively, upon the addition of 1- and 2-M equivalents ofthe probes to the protein solutions.

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(SI Appendix, Fig. S11B), although Ni-NTA-C still bound toHis-XPA122 with an affinity similar to the probe Ni-NTA-ACbased on ITC titration data (SI Appendix, Fig. S6B). Similarly,no evident blue fluorescence was detected on SDS/PAGE whenNi-NTA-C (12 μM) was mixed with equimolar amounts of His-XPA122 (Fig. 2C). These results demonstrate that arylazide iscrucial not only for strengthening binding between the probeand its targets, but also for significant fluorescence turn-onresponses toward His-tagged proteins.We further evaluated the labeling efficiency of Ni-NTA-AC

to His-tagged proteins by monitoring reactivity toward His-XPA122. His-XPA122 was incubated with 0, 0.2, 0.5, 1.0, 2.0, 5.0,and 10 M equivalents of Ni-NTA-AC, and subsequently UV-irradi-ated and subjected to SDS/PAGE (Fig. 3A). By comparing thefluorescent intensities of the protein bands with respect to thoseof Coomassie blue staining, we determined that 1-M equivalentof Ni-NTA-AC could label ∼50% of His-XPA122 (Fig. 3B). Wealso examined labeling efficacy using MALDI-TOF mass spec-trometry. Equimolar amounts of Ni-NTA-AC and His-XPA122were incubated and then subjected to UV irradiation, followedby MALDI-MS analysis. As shown in Fig. 3C, two peaks at m/z14,981 and 15,549 were observed, corresponding to the intactprotein and the protein with one probe bound (calculated 14,979and 15,541, respectively). The intensity of the peak at m/z 15,549further increased upon incubation with 2-M equivalents of theprobe to the protein solution. A weak peak at m/z 16,100 wasassignable to the protein with two probes bound. By comparingthe peak areas, we found that ∼46% and 70% of His-XPA122were labeled upon addition of 1- and 2-M equivalents ofNi-NTA-AC, respectively (Fig. 3C), consistent with the resultsfrom SDS/PAGE (Fig. 3B).

Evaluation of Ni-NTA-AC Probe Membrane Permeability and Toxicity.We then investigated the cell permeability of Ni-NTA-AC in livemammalian cells. We genetically fused a His-tag to the N ter-minus of red fluorescent protein (RFP; His-RFP) and transientlyexpressed this fusion protein in HeLa cells. The fluorescence

responses to the His-RFP transfected cells upon administrationof Ni-NTA-AC were monitored by confocal microscopy using redfluorescence as a reference. As shown in Fig. 4 A and B and SIAppendix, Fig. S12, blue fluorescence appeared upon treatmentof cells with Ni-NTA-AC and reached saturation within 2 min,indicating that the probe can rapidly cross cell membranes, andfluorescence turn-on upon binding to His-RFP can be initiatedquickly; this also indicates that the fluorescent probe Ni-NTA-ACcan readily label cytosolic proteins such as His-RFP in HeLa aslong as the proteins are fused with a His-tag. In contrast, no bluefluorescence was observed after treatment of cells with NTA-AC,i.e., without Ni2+ coordination, indicating that NTA-AC itselffailed to enter cells (SI Appendix, Fig. S13). This observation is inline with our rational design of the probe considering chargemight be important in determining probe membrane perme-ability. In the absence of Ni2+, the charged hydrophilic NTAmoiety remains exposed, thereby prohibiting NTA-AC fromcrossing cell membranes. Upon coordination of Ni2+ to the NTAmoiety, the overall charge reduces dramatically and, moreover,a sandwich-like structure is probably formed owing to the weakinteractions between Ni2+-NTA and the fluorophore as well asflexible linker (30), leading to Ni2+-NTA moiety to be “buried,”which enables Ni-NTA-AC to cross the hydrophobic cell mem-branes. It should also be noted that neither interaction betweenNTA-AC and histidines nor the fluorescence turn-on effectwas possible without Ni2+-binding to NTA-AC.The potential toxicity of the probe in bacterial and mammalian

cells was also examined. The viability of E. coli reached ∼99 ±1% even when 100 μM of Ni-NTA-AC was incubated with thecells (SI Appendix, Fig. S14). The viability of HeLa cells in-vestigated by MTT assay showed that over 90% cells remainedalive upon incubation with 25 and 50 μM Ni-NTA-AC, againconfirming that the probe exhibits no toxicity toward the cellsunder the conditions used (SI Appendix, Fig. S15). We also ex-amined the potential toxicity of UV irradiation to the cells bylight microscope. No morphological changes were observed un-der illumination conditions over 40 min, indicating that the

Fig. 4. Ni-NTA-AC labeling of protein in prokaryoticand eukaryotic cells. (A) Images of Ni-NTA-AC–labeledHis-RFP–transfected cells at different times. Ni-NTA-ACenters the cells rapidly and labels intracellular His-RFPprotein in 2 min. (B) Relative fluorescent intensityplotted against incubation time. (C) Images of E. colicells with or without His-XPA122 overexpression aftertreatment with Ni-NTA-AC (10 μM) for 30 min (n = 5).Only cells expressing His-tagged proteins show bluefluorescence. (Scale bars: 5 μm.) (D) Images of His-XPA122-transfected HeLa cells after incubation withNi-NTA-AC (25 μM) for 30min. The signals are enrichedin the nucleus, where XPA protein is located (29). HeLacells without transfection served as a control, showingno fluorescence under identical treatments (n = 5).(Scale bar: 10 μm.) (E) SDS/PAGE and Western blottinganalysis of cells used for confocal imaging in D. Lane 1:purified His-XPA122 and Ni-NTA-AC; lane 2: cell lysateof HeLa cells without His-XPA122 transfection; lane 3:nuclear extract of His-XPA122 transfected HeLa cells. Ablue band appeared in the nuclear extract sample ofHis-XPA122 transfected cells matches with the band ofpurified His-XPA122, confirming the occurrence of la-beling of His-tagged protein by Ni-NTA-AC in trans-fected cells but not in untransfected cells. (F) Images offluorescent labeling of His-RFP-His-XPA122–transfectedHeLa cells after treatment with Ni-NTA-AC (25 μM;n = 5). The protein was expressed all over the cells, incontrast to fluorescent labeling using Ni-NTA-AC. Theblue and red fluorescence are colocalized and shownin purple in the overlay image. (Scale bar: 10 μm.)

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imaging conditions are apparently nontoxic to live cells, and thusapplicable for live-cell imaging.

Evaluation of Ni-NTA-AC Probe for Labeling of His-Tagged Proteins inCells. We first investigated the photoactivation conditions ofarylazide for imaging experiments because this is crucial forfluorescence enhancement. We illuminated His-RFP-transfectedHeLa cells with different light sources, including 405-nm laser,a DAPI filter cube equipped in the illumination system of theconfocal microscope, and the UV lamp using 4-W long-wavecompact UV lamp (720 μW/cm2) at different time intervals be-fore confocal imaging. As shown in SI Appendix, Fig. S16, HeLacells with His-RFP transfection exhibited the largest blue fluo-rescence increase upon 1- to 2-min irradiation by a DAPI filtercube, whereas irradiation for 2 min by UV lamp gave rise tolower photoactivation efficiency, similar to that activated by 405-nmlaser. Thus, in all cell-imaging experiments, it is not necessary toapply an external light source for arylazide photoactivation. In-stead, an illumination system equipped with a DAPI filter cubeof the confocal microscope was used for optimal photoactivationwithin 1 min before image capturing. Cellular autofluorescencewas minimized through optimization of imaging parameters us-ing the cells without treatment of Ni-NTA-AC.To examine the feasibility of the probe in imaging His-tagged

proteins in live cells, we investigated the applicability of Ni-NTA-ACfor labeling of His-XPA122 in E. coli cells. The probe was in-cubated with E. coli cells overexpressing His-XPA122 for 30 minat 37 °C, then subjected to confocal imaging (n = 5) afterwashing with Hepes buffer. As shown in Fig. 4C, E. coli cellsoverexpressing His-XPA122 were stained with blue fluorescence,and subsequent SDS/PAGE analysis of the cell lysates showedthat only one protein band with molecular weight of ∼15 kDaexhibited blue fluorescence, verifying that indeed His-XPA122was the only protein labeled (SI Appendix, Fig. S17). In contrast,cells without His-XPA122 expression exhibited no blue fluores-cence, revealing the feasibility of the probe in labeling intracellularHis-tagged proteins in live bacterial cells. The viability and mem-brane integrity of these cells were also examined by propidiumiodide staining. As shown in Fig. 4C, no cells were stained in red bypropidium iodide, suggesting that they are live cells.

Imaging His-Tagged Proteins in Live Mammalian Cells Using Ni-NTA-ACProbe. We further investigated the capability of the probe to labelHis-tagged proteins inside mammalian cells. HeLa cells with orwithout His-XPA122 transfection were supplemented with 25 μMNi-NTA-AC in HBSS buffer for 30 min at 37 °C, washed, andsubjected to confocal imaging. As shown in Fig. 4D, an intenseblue fluorescence located mainly at the nuclei was observed onlyin the cells transfected with His-XPA122, but not in those cellswithout transfection. To further confirm the identity of the labeledprotein, cells with or without His-XPA122 transfection weretreated with the probe and irradiated at 365 nm by UV lamp for10 min to ensure complete photoactivation of arylazide, then thenuclei of transfected and untransfected HeLa cells were extrac-ted and concentrated, and subsequently subjected to SDS/PAGEfor fluorescence imaging and Western blotting (Fig. 4E). Thepurified His-XPA122 labeled with Ni-NTA-AC (5 μM) was alsoused for comparison. The blue fluorescent and Western blottingbands from the nuclei of His-XPA122–transfected cells were similarto those of purified His-XPA122, confirming that the labeled pro-tein was indeed His-XPA122 expressed in HeLa cells. In contrast,no corresponding bands were observed for the nuclei of untrans-fected cells (Fig. 4E).Fluorescent proteins have been widely used to study protein

function and localization as well as other biological events in thephysiological context of living cells when genetically fused to theprotein of interest (34). However, the use of fluorescent proteinsmight potentially interfere with the proper localization or function

of the protein of interest due to its large size (1), in particular forrelatively small proteins. Therefore, small molecule-based fluores-cent probes have significant advantages; to demonstrate this, weincorporated His-tagged RFP into the N terminus of His-taggedXPA122 to generate a His-RFP-His-XPA122 plasmid, then trans-fected HeLa cells to investigate the cellular localization of theprotein under identical conditions. Both blue fluorescence (due toNi-NTA-AC labeling) and red fluorescence (due to the expressionof RFP) were observed with colocalization (Fig. 4F). Interestingly,the fluorescence signals, instead of being enriched in the nucleus asfound for His-XPA122 (Fig. 4D), were distributed evenly through-out the cells, suggesting that RFP disrupts XPA122 localization.The perturbation of protein localization may be attributed to therelatively large size of RFP (27.5 kDa) (5) compared with XPA122(15 kDa). By contrast, using the Ni-NTA-AC probe, His-XPA122was found to be enriched in the nucleus, in agreement with thepreviously reported intracellular localization of XPA, a protein in-volved in the recognition of DNA damage during nucleotide exci-sion repair processes (16, 29). Taken together, we demonstrate thatthe new fluorescent probe Ni-NTA-AC can be preferentially appliedto track the abundances and localization of His-tagged proteins inlive mammalian cells, in particular for small proteins.

Application of Ni-NTA-AC Probe for Imaging of His-Tagged Protein inPlant Tissues.We finally show that Ni-NTA-AC can label proteinsin other eukaryotic systems. Transplastomic Nicotiana tabacumvar. Xanthi (tobacco) plants expressing His-tagged Brassicajuncea chitinase BjCHI1 (His-BjCHI1) generated as describedpreviously were tested (35). Protoplasts were extracted fromleaves of 4-wk-old wild-type and His-BjCHI1 transplastomic to-bacco (SI Appendix, Fig. S18) according to previously reportedprocedures (36). Upon incubation of protoplasts with Ni-NTA-AC (10 μM) for 30 min, blue fluorescence was detected in thechloroplasts (SI Appendix, Fig. S19), where His-BjCHI1 wasexpressed and subsequently accumulated (35). Colocalization ofblue fluorescence with red autofluorescence of chloroplasts byconfocal microscopy was observed only in cells expressing His-BjCHI1, but not in wild-type cells, indicating labeling of His-BjCHI1 in the chloroplasts by Ni-NTA-AC (SI Appendix, Fig.S19). Furthermore, we added Ni-NTA-AC (10 μM) into PBSbuffer for root immersion of 7-d-old transplastomic seedlingsovernight, which were then washed and blotted dry before im-aging. Confocal microscopy of the abaxial surface of trans-plastomic tobacco leaves revealed blue fluorescence in tobaccoleaves expressing His-BjCHI1 (Fig. 5), suggesting that the probewas taken up through the roots of seedlings and the His-taggedprotein inside living leaves was subsequently detected. Theseresults clearly demonstrate that the probe, Ni-NTA-AC, can

Fig. 5. Confocal imaging of guard cells from the abaxial surface of atransplastomic tobacco leaf expressing His-BjCHI1 (Lower) in comparisonwith the wild-type (Upper) leaf (n = 5). Seedlings (Left) were incubated inNi-NTA-AC–containing buffer (10 μM) overnight. Wild-type tobacco servedas a negative control. (Scale bars: 10 μm.)

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readily be extended to label His-tagged proteins in variouseukaryotic cells, including plant tissues.

ConclusionsThe wide prevalence of a (histidine)6-Ni

2+-nitrilotriacetate sys-tem in protein purification has led to significant interest in thedevelopment of small Ni-NTA–based fluorescent probes to im-age His-tagged proteins. Given the existing large libraries of His-tagged proteins, such probes would have significant impact infunctional investigation of intracellular proteins under physio-logically relevant conditions. Here, we have to our knowledgedeveloped the first small cell-permeable fluorescent probeNi-NTA-AC that enables rapid labeling (2 min) and observation ofintracellular His-tagged proteins in different types of live cellsand even in plant tissues with a plethora of potential applica-tions. The probe exhibits high specificity and labeling efficiencytoward intracellular His-tagged proteins. Background stainingresulted from binding of the probe to endogenously expressedhistidine-rich proteins in certain type of cells might lead toa lower sensitivity compared with fluorescent protein labeling,a similar problem that was also encountered by FlAsH. Never-theless, it will not prohibit the use of the Ni-NTA-AC probe whenthe protein of interest is overexpressed. Moreover, the probeposed less perturbation than RFP on protein function and locali-zation in live cells owing to its small size. Further efforts to

develop differently colored NTA-AC analogs are underway inour laboratory. A recently developed green probe also exhibitsexcellent membrane permeability and turn-on fluorescence uponbinding to His-RFP in COS-7 cells (SI Appendix, Fig. S20). Thestrategy we reported here should find many possible applica-tions, such as in situ visualization of subcellular localization ofproteins, tracking protein trafficking, and monitoring proteindynamics in live cells.

Materials and MethodsNTA-AC was synthesized from 2-(7-azido-4-methyl-2-oxo-2H-chromen-3-yl)acetic acid by the conversion of aromatic amine to arylazide, and subsequentlyamidation was performed to connect the metal-chelating NTA to the fluo-rophore, which yielded NTA-AC. NTA-C was synthesized similarly. Ni-NTA-AC(and Ni-NTA-C) was prepared by the addition of 1 M equivalent of NiSO4 toNTA-AC (or NTA-C) in buffered aqueous solution at pH 7.2. Details of thesynthesis of NTA-AC and NTA-C, preparation of the probe (Ni-NTA-AC),characterization, and cell experiments are described in SI Appendix.

ACKNOWLEDGMENTS. We thank Prof. Peter J. Sadler for helpful commentsand acknowledge the assistance of Li Ka Shing Faculty of Medicine FacultyCore Facility, The University of Hong Kong. Funding was provided by TheResearch Grants Council of Hong Kong Grants 704612P and 703913P, theUniversity of Hong Kong emerging Strategic Research Theme (e-SRT) on In-tegrative Biology, the Wilson and Amelia Wong Endowment Fund, anda university scholarship (to Y.Y.).

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