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Title:
METAL CHELATION-BASED FLUORESCENT PROBES FOR PROTEIN OR OTHER BIOMOLECULE LABELING IN CELLS
Document Type and Number:
WIPO Patent Application WO/2015/097313
Kind Code:
A1
Abstract:
The present invention provides metal chelation-based fluorescent probes for imaging intracellular proteins or other biomolecules in living cells to monitorbiological events. The probes can label poly-Histidine-tagged proteins or biomolecules while also being able to covalently bind to labeled proteins for further protein analysis.

Inventors:
SUN, Hongzhe (Flat H, 30th Floor Block 11,,Yee Fung Court, South Horizons,Apleichau, Hong Kong, CN)
LAI, Yau Tsz (Flat 914, 9th Floor Wah Yiu House,Lai Yiu Estate, Kwai Chung,New Territories, Hong Kong, CN)
YANG, Ya (Room A13-12, Lung WahResidential College,9 Lung Wah Street,Kennedy Town, Hong Kong, CN)
Application Number:
EP2015/050063
Publication Date:
July 02, 2015
Filing Date:
January 05, 2015
Export Citation:
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Assignee:
THE UNIVERSITY OF HONG KONG (Pokfulam Road, Hong Kong, CN)
HGF LIMITED (140 London Wall, London EC2Y 5DN, GB)
International Classes:
G01N33/533; G01N33/566; G01N33/58; G01N33/68; G03C1/695
Domestic Patent References:
WO2008145609A12008-12-04
WO2001072458A12001-10-04
WO2007070479A22007-06-21
Other References:
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Attorney, Agent or Firm:
HGF LIMITED (140 London Wall, London Greater London EC2Y 5DN, GB)
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Claims:
Claims

1. A fluorescent probe for targeting biomolecules in biological samples, comprising a fluorophore reporting moiety which generates a fluorescent signal, a metal-chelating moiety to chelate metal ions for coordinating to poly-Histidine-tag encoded to a targeted protein, a linker connecting the fluorophore reporting moiety and metal-chelating moieties, and a photoreactive crosslinker serving as an anchor point onto the targeted protein.

2. The fluorescent probe according to claim 1, wherein the fluorescent signal of the reporting moiety has a wavelength of about 400 to about 800 nm after absorption of optical energy.

3. The fluorescent probe according to any of claims 1 - 2, wherein the reporting moiety comprises a coumarin-derivative, fluorescein-derivative or Rhodamine-derivative.

4. The fluorescent probe according to any of claims 1 - 3, wherein the metal-chelating moiety comprises a polydentate ligand.

5. The fluorescent probe according to claim 4, wherein the polydentate ligand comprises nitrilotriacetic acid ( TA) or iminodiacetic acid (IDA).

6. The fluorescent probe according to any of claims 1 - 5, wherein the metal-chelating moiety comprises a chelating metal ion selected from nickel(II), cobalt(II), and copper(II) metal ions.

7. The fluorescent probe according to any of claims 1 - 6, wherein the linker between the fluorophore reporting moiety and the metal-chelating moiety is a hydrocarbon chain or a peptide sequence.

8. The fluorescent probe according to any of claims 1 - 7, wherein the photoreactive crosslinker comprises arylazide, diazirine, or benzophenone.

9. The fluorescent probe according to any of claim 8, wherein the photo reactive crosslinker exhibits photo-activation when subjected to ultraviolet radiation for about 5 to about 15 minutes after the fluorescent probe coordinates to a poly-Histi dine -tagged protein in a biological sample.

10. The fluorescent probe according to claim 9, wherein the ultraviolet radiation is in a range of about 340 nm to about 380 nm.

11. The fluorescent probe according to any of claims 1 - 10, wherein the chelation of metal ion selected from nickel(II), cobalt(II), and copper(II) ions generates a fluorescent quenching of the metal-chelated probe.

12. The fluorescent probe according to any of claims 1 - 11 , wherein the probe coordinates to metal ions in 1 : 1 molar ratio.

13. The fluorescent probe according to any of claims 1 - 12, wherein labeling a targeted biomolecule is achieved through coordinating to poly-Histidine-tag encoded to the biomolecule by metal-chelation of the probe.

14. The fluorescent probe according to claim 13, wherein the metal-chelating fluorescent probe exhibits an elevation of fluorescent signals (a "turn-on" response) and is achieved when the metal-chelating fluorescent probe labels a poly-Histidine -tagged protein in buffer at pH 6-8.

15. The fluorescent probe according to claim 14, wherein the metal-chelating fluorescent probe exhibits an elevation of fluorescent signals when the metal-chelating fluorescent probe labels a poly-Histidine -tagged protein at a temperature from about 4°C to about 40°C.

16. The fluorescent probe according to claim 15, wherein the labeling of a poly- Histidine -tagged protein is stable for overnight incubation.

17. The fluorescent probe according to any of claims 1 - 16, wherein the labeling of a poly-Histidine -tagged protein is retained after the protein is denatured with a temperature of about 90°C to about 110°C.

18. The fluorescent probe according to claim 16, wherein the fluorescent labeling of a poly-Histidine -tagged protein is able to be visualized on gel after native or denaturing gel electrophoresis.

19. A method of labeling of poly-Histidine -tagged protein, comprising:

coordinating poly-Histidine -tag encoded proteins to target biomolecules by the metal- chelation of fluorescent probes, the fluorescent probes each comprising a fluorophore reporting moiety which generates a fluorescent signal after absorption of optical energy, a metal-chelating moiety to chelate metal ions for coordinating to a poly-Histidine -tag encoded to a targeted protein, a linker connecting the fluorophore and metal-chelating moieties, and a photoreactive crosslinker serving an anchor point onto the targeted protein.

20. The method according to claim 19, wherein the fluorescent probes generate a fluorescent signal after absorption of optical energy having a wavelength of about 400 to about 800 nm.

21. The method according to any of claims 19-20, wherein the labeling of poly-Histidine - tagged proteins is achieved in a biological sample comprising bacterial cells, mammalian cells, mammalian tissues, plant cells, or plant tissues.

22. The method according to claim 21 , wherein the labeling is carried out at a temperature of about 4°C to about 40°C.

23. The method according to claim 21 , further comprising undergoing washing by a buffer.

24. The method according to any of claims 19-23, further comprising undergoing confocal imaging.

25. The method according to claim 21 , wherein the introduction of the fluorescent probes does not damage the biological sample.

Description:
METAL CHELATION-BASED FLUORESCENT PROBES FOR PROTEIN OR OTHER

BIOMOLECULE LABELING IN CELLS

Background of Invention

The identification and molecular level understanding of the interacting chemical reactions during the whole life process are of great value in basic biological research and medical science. Fluorescent imaging has long been used for this purpose because it allows us to spy on events in living cells and organisms, including humans, in real time and with high spatial resolution. Site- specific chemical labeling using small fluorescent probes is a powerful and attractive technique to study biological events in cells and tissues, and thus for probing mechanisms of diseases. In particular, metal chelation based fluorescent labeling has the advantage of high selectivity, small size and covalent labeling.

In modern biochemistry, the genetic encoding of poly-Histi dine -tag to the POI has been a robust technique in the protein chemistry. His-tag basically refers to a short peptide motif with oligohistidines, with hexahistidine sequence as the most common His-tag. The imidazole ring of histidines of the His-tag can interact with various kinds of transition metals, such as Ni 2+ , Cu 2+ , Zn 2+ , etc. Accordingly, His-tag has been ordinarily found to interact with transition metal complexes such as Ni 2+ -nitrilotriacetic acid (NTA) complex, thereby aids the purification of overexpressed proteins using immobilized metal affinity chromatography (IMAC). Such an interacting combination has also been widely applied in site-specific protein labeling accounted to the compatibility to the extensive library of existing oligohistidine -tagged proteins as reported, and the genetic encoding of the His-tag is flexible, in which it could be genetically engineered to the termini or internal sites of the protein targets for the labeling (Soh, N. Sensors 8, 1004-1024 (2008); Kapanidis, A. N., Ebright, Y. W. & Ebright, R. H. J. Am. Chem. Soc. 123, 12123-12125 (2001)).

The most matured and widely used metal-based (or metalloid-based) small molecule fluorescent probe is FlAsH or its analogues such as ReAsH and SplAsH (Griffin, B. A., Adams, S. R. & Tsien, R. Y. Science 281, 269-272 (1998); Hoffmann, C. et al. Nat. Protocols 5, 1666- 1677 (2010)). Another two typical small fluorescent sensors for poly-Histidine -tagged proteins were published by Vogel in 2004 with selective, rapid and reversible metal chelating NTA probes while by Auer in 2008 who synthesized the irreversible ones (Guignet, E. G., Hovius, R. & Vogel, H. Nat. Biotechnol. 22, 440-444 (2004); Hintersteiner, M. et al. ChemBioChem 9, 1391-1395 (2008)). Most other histidine labeling probes were discovered in the same way but have only been reported to label proteins on the cell surface. Extensive study has been carried out to image CysHis 6 -tagged (C¾-tagged) proteins inside living cells using small molecule fluorescent probes only in the presence of a penetrating peptide with an incubation time of 30 minutes or more (Uchinomiya, S., Nonaka, H., Wakayama, S., Ojida, A. & Hamachi, I. Chem. Commun. 49, 5022-5024 (2013)), and unfortunately, no such probe can directly enter into cells and label intracellular proteins.

As mentioned above, small molecule-based fluorescent probes for protein labeling has long been used in biomedical and clinical science, however poor membrane permeability of these probes prevents their application. A few such probes, particularly based on chelation, have been marketed (e.g. FlAsH, ReAsH, and SplAsH by Invitrogen®). All of these are biarsenical fluorescent probes which can cross cell membrane to label genetically tetracysteine fused proteins of interest. However a small organic compound, 1, 2-ethanedithiol (EDT) must be synergistically used with the probe to increase labeling rate and to reduce the background. Arsenic has been known to be toxic to humans, and is also an environmental contaminant. The synthesis of mentioned biarsenical probes involves the exploitation of highly toxic mercury and arsenic in large quantity, causing serious environmental issues. Moreover, in oxidizing environments, a specific labeling is difficult since the reduced form of tetracysteine motif can be easily converted into the oxidized form. In contrast, histidine was chosen to be abundant focused in particular and important proteins and poly-Histidine tag was used for protein purification. Tetracysteine motif is not as common as poly-Histidine -tag to be genetically fused to proteins in cell biology and biomedical research (Rowinska-Zyrek, M., Witkowska, D., Potocki, S., Remelli, M. & Kozlowski, H. New J. Chem. 37. 58-70 (2013)).

Brief Summary

Presently, there are no metal tunable probes which achieve high throughput for the visualization and subsequent identification of labeled proteins. Described herein are metal chelating-based fluorescent probes for imaging intracellular proteins or other biomolecules in living cells and tissues thus to monitor their biological events and study the disease mechanisms. A series of probes are provided with at least three different emissions of blue, red and green by conjugating various fluorophores to metal-chelating agents and a photoreactive crosslinker. By coordinating to different metal ions, the metal-chelated probes serve as potential probes to label poly-Histidine -tagged proteins/ biomolecules. Moreover, the probes are also able to covalently bind to labeled proteins, enabling further protein identification.

Compared with the most extensively used current probe (FlAsH) which use the toxic arsenic and require introduction of tetracysteines, our probes use non-toxic metals such as nickel(II) chelated by nitrogen containing ligand(s) for imaging poly-Histidine -tagged proteins, which are more rapidly across cell membrane and less toxic. More importantly, the probes are used more conveniently considering the wide spread usage of poly-Histidine-tag in biochemical and biomedical research, in view of the large library of existing poly-Histidine -tagged proteins. The present invention relates to detection and visualization of biomolecules in cells. It is applicable for tracking or following a biochemical event including proteins in cells readily and rapidly. The preparation is simple with low cost accordingly, and the probes could avoid the use of arsenic and tetracysteines (which may affect the redox environment in cells). Instead, other non-toxic metal ions such as nickel(II) are incorporated into the current probe, which targets poly-Histidine-tag encoded genetically to proteins or other biomolecules. Such probes thus have the potential to replace or at least complement the current most-used class of probes such as FlAsH (ReAsH).

This description relates to the development of novel metal chelation-based fluorescent probes for imaging intracellular proteins or other biomolecules in living cells to monitor their biological events. Described further herein are the design and synthesis of probes.

In one aspect, fluorescent probes for targeting biomolecules in biological samples, particularly living samples, are provided. The fluorescent probes comprise a fluorophore reporting moiety which generates a fluorescent signal, a metal-chelating moiety to chelate metal ions for coordinating to poly-Histidine-tag encoded to the targeted protein, a linker connecting the fluorophore reporting moiety and metal-chelating moieties, and a photoreactive crosslinker serving as an anchor point onto the targeted protein to proliferate labeling affinity and stability.

In some embodiments, the fluorescent signal of the reporting moiety has a wavelength of about 400 to about 800 nm after absorption of optical energy. The reporting moiety comprises coumarin-derivatives, fluorescein-derivatives and Rhodamine-derivatives.

In some embodiments, the metal-chelating moiety comprises polydentate ligands. The polydentate ligands comprise nitrilotriacetic acid ( TA) and iminodiacetic acid (IDA). The metal-chelating moiety also comprises chelating metal ions comprising nickel(II), cobalt(II) and copper(II) metal ions.

In some embodiments, the linker between the fluorophore and the metal-chelating moiety is designed as a hydrocarbon chain or a peptide sequence.

In some embodiments, the photoreactive crosslinker comprises arylazide, diazirine and benzophenone. The photoreactive crosslinker embraces or, alternatively, does not embrace as part of the conjugated system of the fluorophore. The photoreactive crosslinker may exhibit photo-activation being accomplished by ultraviolet radiation for about 5 to about 15 minutes after the fluorescent probes coordinate to poly-Histidine -tagged proteins in the biological samples. The ultraviolet radiation is typically in a range of about 340nm to about 380 nm.

In some embodiments, the chelation of metal ions including nickel(II), cobalt(II) and copper(II) ions generate a fluorescent quenching of the metal-chelated probes.

In some embodiments, the probes coordinate to metal ions in 1 : 1 molar ratio.

In some embodiments, labeling targeted biomolecules is achieved through coordinating to poly-Histidine-tag encoded to the biomolecules by the metal-chelation of the probes. The metal-chelating fluorescent probes exhibit an elevation of fluorescent signals (a "turn-on" response) and is achieved when the metal-chelating fluorescent probes label poly-Histidine - tagged proteins in buffers at pH 6-8, which may be at a temperature from about 4°C to about 40°C. In one embodiment, the labeling process takes about 5-30 minutes. In another embodiment, the label is stable for overnight incubation.

In one embodiment, the labeling of poly-Histidine -tagged proteins is retained after the proteins are denatured with a temperature of about 90°C to about 1 10°C.

In additional embodiments, the fluorescent labeling of poly-Histidine -tagged proteins is able to be visualized on gels after native or denaturing gel electrophoresis.

In another aspect, methods of labeling of poly-Histidine -tagged proteins are provided.

The methods comprise: coordinating poly-Histidine-tag encoded proteins to target biomolecules by the metal-chelation of a fluorescent probe, the fluorescent probe comprising a fluorophore reporting moiety which generates a fluorescent signal after absorption of optical energy, a metal- chelating moiety to chelate metal ions for coordinating to poly-Histidine-tag encoded to the targeted protein, a linker connecting the fluorophore and metal-chelating moieties, and a photoreactive crosslinker serving an anchor point onto the targeted protein to proliferate labeling affinity and stability. In some embodiments, the fluorescent probe generates a fluorescent signal after absorption of optical energy having a wavelength of about 400 to about 800 nm.

In some embodiments, labeling of poly-Histidine -tagged proteins is achieved in biological samples comprising bacterial cells, mammalian cells, mammalian tissues, plant cells, and plant tissues. In one embodiment, the introduction of the fluorescent probes does not damage the biological samples.

In some embodiments, the method of labeling is carried out at a temperature of about 4°C to about 40°C. The labeling can take from about 5 to about 60 minutes.

In further embodiments, the method includes washing by a buffer and/or undergoing confocal imaging.

In some embodiments, fluorescent agents having the chemical structure represented in FIG.l contain:

(a) a cyclic moiety with established conjugated system as the fluorophore with an emission ranged from 400 to 800 nm;

(b) a metal-chelating moiety comprising poly dentate ligands, including but is not limited to, carboxylic acids-containing ligands such as nitrilotriacetic acids (NTA) and iminodiacetic acid (IDA) to chelate metal ions;

(c) metal ions which partially coordinate to the metal-chelating moiety, including but not limited to, one or more of nickel(II), cobalt(II), and copper(II) ions;

(d) a linker between the fluorophore and the metal-chelating moiety, which can include a short hydrocarbon chain or a short peptide sequence; and/or

(e) a photoreactive crosslinker including, but not limited to, arylazide, diazirine, and benzophenone, which may or may not be part of the conjugated system of the fluorophore.

The present invention provides effective labeling of intracellular (and extracellular) poly- histidine -tagged proteins/biomolecules, while the probes are also able to covalently bind to labeled proteins for further protein identification. Upon addition to the biological samples, the partially-coordinated metal ions can direct the fluorescent agent to label the poly-Histidine- tagged proteins, while in proximity the photoreactive crosslinker can be photo-activated by UV irradiation at 340-380 nm to generate covalent linkage to the targeted proteins.

In some embodiments, advantages of the present invention include the rapid labeling of the poly-histidine -tagged proteins in 10 minutes or less (such as 9 minutes) as compared to more than 30 minutes in conventional methods. Advantages can also include the labeling of the targeted poly-Histidine -tagged proteins that can generate a "turn-on" response which elevates the fluorescence intensity of the fluorescent agent more than 5 times, more than 10 times, and even up to 13 times. Also, in some embodiments, the disclosed labeling method does not damage the biological sample or its components.

Brief Description of Drawings

Figure 1 illustrates a schematic diagram of the fluorescent agent design.

Figure 2 illustrates the structure of metal-chelated N7¾ - ^ 4C and NTA-AF.

Figure A shows a schematic diagram demonstrating intracellular labeling of I Iis6- tagged proteins using ΉΊ-ΝΤΑ-AC {top) and synthetic scheme of NT A- AC {bottom). The probe enters the cells rapidly and targets His6-tagged proteins with significant fluorescence "turn-on". Figure 3B shows the normalized fluorescence changes of NT A- AC (5 μΜ) upon addition of Ni 2+ (as NiS0 4 ). About 70% decrease in fluorescence was noted upon Ni 2+ chelation with NT A- AC. Figure 3C shows a Job ' s plot of the fluorescence changes (λ εχ = 342 nm, em = 448 nm) upon compicxation of NTA-AC with Ni 2+ in 20 niM Tris-HCl buffer, pi I 7.2. The total concentrations of NTA-AC and Ni 2+ were kept constant (10 μΜ). Maximum fluorescence changes were observed at a molar ratio of Ni 2+ to NTA-AC of 0.5, indicative of the formation of Ni-A .-f-.-i complex with a ratio of NTA-A C: Ni 2+ of 1 : 1.

Figure 4A illustrates 1H (4-A) and Figure 4B illustrates 13 C (4-B) NMR spectra of NTA- AC.

Figure 5 illustrates ESI-MS spectra of NTA-AC.

Figure 6 illustrates the excitation (solid line) and emission (dashed line) spectra of NTA- AC with λ εχ = 342 nm and X em = 448 nm.

Figure 7 A shows the fluorescence spectra of Ni- V7>i-.-iC (1 μΜ) at different time intervals after addition of His-XPA 122 (10 μΜ). Inset: Time-dependent fluorescence changes (λ εηι = 448 nm) of NI-NT4-AC upon binding to His-XPA 122. A 13-fold increase in fluorescence was observed and the signals reached a plateau at 9 minutes for Ni-.V7 -.iC Figure 7B shows the normalized fluorescence of Ni-NTA-AC incubated w ith His-XPA 1 22 under various conditions. Without photoactivation of arylazide (under dark), addition of excess amounts of EDTA (40-foid) to the mixture of Ni-NTA-AC and His-XPA 122 led to the

.2 +

fluorescence decreased by ca. 60% owing to chelation of by EDTA from Ni-NTA-AC, which resulted in dissociation of the probe from His-XPA122. In contrast, after binding of ~ Ni-NTA-AC to His-XPA122 and upon photoactivation of arylazide, addition of excess EDTA would not disturb the covalent linkage between the probe and protein, and a slight increase (30%) in fluorescence was attributed to fluorescence recov ered from that quenched by Ni . Figure 7C shows SDS-PAGE analysis of protein labeling (12 μΜ) by equimolar amounts of Ni-NTA-AC (or ~ Ni-NTA-C) under di fferent conditions. The probe binds the His-tag of the protein through Nr , whereas the arylazide moiety further enhances the binding via covalent linkage upon photo-activ ation, thus label ing is retained even under denaturing electrophoresis. Lane I : His-X PA 122; Lane 2: His- PA 122 in the presence of excess EDTA (50 μΜ); Lane 3: His-XPA122 and Ni-A -i-C (w ithout arylazide); Lane 4: X PA I 22 (without His-tag). A ll samples were incubated at 4 °C overnight and subjected to photo-activation of arylazide by UV radiation (λ= 365 nm ) for 1 0 minutes prior to gel electrophoresis.

Figure 8A shows the labeling efficiency of Ni-NTA-AC to His-XPA 1 22 by SDS- PAGE analysis of protein labeling (10 μ M ) upon incubation w ith di fferent amounts (0- 10 molar equiv alents) of Ni-NTA-AC monitored by Coomassie Blue and fluorescence staining. Figure SB shows the labeling yield of Ni-NTA-AC to His- XPA I 22 determined by SDS- PAGE in Figure 8A. Figure 8C shows MALDI-TOF MS spectra of His-XPA 1 22 (10 μΜ) in the absence and presence of 1 and 2 molar equivalents of ~ Ni-NTA-AC. The peak at m/z of 14981 Da is assignable to the intact protein (calcd. 14979 Da) and peaks at m/z of 15549 and 1 6 1 00 Da appeared after incubation of His-XPA 1 22 w ith Ni-NTA-AC, corresponding to the protein bound to one and two probes respectively. Assuming the incorporation of ~ Ni-NTA- AC to His-X PA 1 22 exhibited negligible effect on the ionization efficiency of the native protein His-XPA 1 22 as the size of the protein is much bigger than that of the probe, the labeling efficiency was ev aluated by compari ng the peak areas of intact His-XPA 1 22 and the probe bound His-XPA 1 22. The labeling efficiency using I and 2 molar equivalents were calculated to be 38% and 62%, respectivel y.

Figure 9 sh o w s Ni-NTA-AC labeling of protein in prokaryotic and eukaryotic cells. Figure 9 A I mages of N i -Λ ' 7 ' .-ί -A C- la be led H i s - R F P - 1 ra n s fe c t ed cells at di fferent times. Ni- NTA-AC enters the cel ls rapidly and labels intracel lular His-RFP protein in 2 minutes. Figure 9B shows a g r a h 0 f the relati ve fluorescence intensity plotted against incubation time. Figure 9C shows images of E. coli cells with or without His-XPA 1 22 overexprcssion after treatment with ΉΊ-ΝΤΑ-AC (10 μΜ) for 30 minutes (n= 5). Only cel ls expressing His- tagged proteins showed blue fluorescence. Scale bars: 5 μιτι. Figure 9D shows images of His- XPA 122-transfeeted HeLa cells after incubation with Ni-NTA-AC (25 μΜ) for 30 minutes. The signals arc enriched in the nucleus, where XPA protein is located ( Kuraoka 1. et al. (1996) Mutat Res 362(l):87-95). HeLa cells without transfection served as a control showed no fluorescence under identical treatments ( n = 5). Scale bar: 10 μΜ. Figure 9E shows SDS-PAGE and western blotting analysis of cells used for con focal imaging in Figure 9D. Lane 1: Purified His-XPA l 22 and Ni- NT A- AC; Lane 2: Cell lysate of HeLa cells without His-XPA l 22 transfection; Lane 3: Nuclear extract of His-XPA l 22 transfected HeLa cells. A blue band in the nuclear extract of His- PA 1 22 transfected cells matched the band of purified His-XPA 122, confirming the occurrence of labeling of His-tagged protein by ~ Ni-NTA-AC in cells, but not in untransfected cells. Figure 9F shows images of fluorescent labeling of His-RFP-His-XPA 122-transfected HeLa cells after treatment with Ni-NTA-AC (25 uM ) (n= 5). The protein was expressed all over the cells, in contrast to fluorescent label ing using Ni-NTA- AC. The blue and red fluorescence were co-localized and was show n in purple in the overlay image. Scale bar: 10 um.

Figure 10 shows confocal imaging of guard cells from the abaxial surface of a transplastomic tobacco leaf expressing His-BjCHIl (Bottom) in comparison to the w ild-type (Top) leaf (n= 5). Seedlings (Left) were incubated in N i - V TA -A C ' -c o n t ai n i n g buffer (10 μΜ) overnight. Wild-type tobacco served as a negative control. Scale bars: Ι Ομηι.

Figure 11 shows wild-type ( VVT ) and His-BjCHl 1 -expressing 4-week-old tobacco plants for protoplast extraction and confocal images of protoplasts from WT and His-BjCHI - ex pressing 4 -week-old to bac co plants (n= 5) after 30-minute incubation with (10 μΜ). Scale bars, 10 μηι.

Figure 12 illustrates the synthesis of NTA-AF.

Figure 13 illustrates the fluorescence spectra of NTA-AF, demonstrating the excitation maxima at 496 nm while the emission maxima at 518 nm.

Figure 14 illustrates the fluorescent labeling of poly-Histidine -tagged protein His- XPA122 with Ni 2+ -NTA-AF in SDS-PAGE denaturing gel in comparison to Coomassie blue staining. Figure 15 shows a graph of the fluorescence response of ΉΊ-ΝΤΑ-AC (1 μΜ) to concentrations of XPA122 ranging from 0 to 10 μΜ. ΉΊ-ΝΤΑ-AC did not show obvious fluorescence response upon the addition of XPA under identical conditions in the absence of the His 6 -tag.

Figure 16 shows a graph of the fluorescence response of NTA-AC (1 μΜ) to concentrations of His-XPA ! 22 ranging from 0 to 10 μΜ. NTA-AC did not exhibit any fluorescence response upon the addition of His-XPA 122 under identical conditions in the absence of Ni 2 \

Figure 17 shows the binding affinities of (A) Ni-NTA-AC and (B) Ni-NTA-C to I lis- XPA122 (in 20 niM HEPES, 100 niM NaCl, pH 7.4) by isothermal titration calorimetry. ~ Ni-NTA-AC and Νί-Λ .-ί- (500 μΜ each ) was injected stepwise into the cell containing apo-His-XPA 1 22 (35 μΜ) and the heat of binding was recorded for every injection.

Figure 18 shows a schematic of the synthetic scheme of NTA-C.

Figure 19 shows the 13 C NMR (125 MHz) spectrum of NTA-C.

Figure 20 shows the ESI-MS spectrum of NTA-C. The ion at m/z 514.5 corresponding to [M-2H + +K + ] (calcd. 514.5).

Figure 21 shows a graphical evaluation of the role of arylazide of NTA-AC through comparing with its deriv ativ e NTA-C. (A) Normalized fluorescence changes of NTA-C (5 μΜ)

·2~

upon addition of Ni " ' . Note that the incorporation of Ni ' to NTA-C resulted in fluorescence quenched in a less extent (50%) compared with binding of Ni 2+ to NTA-AC (70%). (B) Time- dependent fluorescence changes of Ni-NZ i-C at X em = 448 nm upon incubation with His- XPA 22. No obvious fluorescent turn-on responses were observed despite that Ni-NTA- C binds to His- XPA 22.

Figure 22 shows con focal images o HeLa cells w ith His-RFP-expression after supplementation of Ni- V7 ' -i- AC (25 μΜ) for 2 minutes (n= 5). Note that the blue fluorescence was co-localized with the red fluorescence from RFP, confirming that the probe entered the ceils and labeled H is-tagged proteins. Scale bar: 10 μιτι.

Figure 23 shows microscopic images of fluorescence labeling of His-RFP -His- XPA 1 22-transfected HeLa cells after treatment w ith NTA-AC (25 μΜ) or M-NTA-AC (25 μΜ) for 30 minutes (n= 5). No blue fluorescence was observed inside the cells as NTA-AC could not enter cells in the absence of Ni ' . Scale bar: 10 μηι. Figure 24 is a graph showing the viability of His-XPA 1 22-exprcsscd E. coli incubated with different concentration of Ni-NTA-AC (0, 10, 25 50, 1 00 μΜ) determined by microscopic imaging (n = 5). The viability of E. coli reached 99% +/- 1% even when 100 μΜ of Ni-NTA-AC was incubated with the cells.

Figure 25 shows a graph of the toxicity determination of Ni-NTA-AC in HeLa c e l ls .

Ni-NTA-AC (25 and 50 μΜ) was incubated with HeLa cells and cytotoxicity was determined by MTT assay (n= 5). The probe exhibited negligible effect on cell viability to HeLa cells.

Figure 26 shows an SDS-PAGE gel with Coomassie blue stain and fluorescence. E. coli cells with (Lane 1) or without (Lane 2) His-XPA 1 22 overexpression were incubated with -NTA-AC ( 10 μΜ) for 30 minutes at 37°C, washed with HE PES buffer, lysed and subjected to electrophoresis. Note that only the ceils with His-XPA 122 overexpressed exhibited an intense fluorescence band (corresponding to a molecular mass of - 1 5 kDa, i.e. His-XPA 122), in contrast to E. coli cells without overexpression. M: protein marker.

Figure 27 illustrates ESI-MS spectra of Ni-NTA-AF.

Figure 28 show s t h e c o n f o c a 1 i mag i ng o f ' Ni-NTA-AF labeling of protein in COS-7 ceils. His- FP was transfected and expressed in COS-7 ceils, and samples were treated w ith with ϋ-ΝΤΑ-AF (25 μΜ) (n= 3). The blue and red fluorescence were co-localized and was shown in purple in the overlay image. Scale bar: 1 0 μιη.

Brief Description of Sequences

SEQ ID NO: 1 is a primer utilized for plasmid construction containing a BamHI restriction site.

SEQ ID NO: 2 is a primer utilized for plasmid construction containing a Xhol restriction site.

SEQ ID NO: 3 is a primer utilized for plasmid construction containing a BamHI restriction site. SEQ ID NO: 4 is a primer utilized for plasmid construction containing a Xhol restriction site.

SEQ ID NO: 5 is a primer utilized for plasmid construction containing a Nhel restriction site.

SEQ ID NO: 6 is a primer utilized for plasmid construction containing a BamHI restriction site.

Detailed Disclosure

Definitions

The term "biomolecule" refers to molecules produced by living organisms. Examples used herein include, but are not limited to, polypeptides, proteins, nucleic acids and lipids. The term "target biomolecule" as used herein refers to a biomolecule that is: (1) able to actively direct the entity to which it is attached (e.g., a fiuorogenic moiety) to a target region, e.g., a cell; or (2) is preferentially passively absorbed by or entrained within a target region. The targeting biomolecule can be a small molecule, which is intended to include both non-peptides and peptides. The targeting group can also be a macromolecule, which includes, but is not limited to, saccharides, lectins, receptors, ligand for receptors, proteins such as BSA, antibodies, poly( ethers), dendrimers, poly(amino acids) and so forth.

The term "photoreactive crosslinker" refers to photo-activable reactive groups for labeling proteins, nucleic acids and other biomolecules in an irreversible manner after activated by ultraviolet or visible light. Examples of photoreactive crosslinkers include, but are not limited to, arylazides, azido-methyl-coumarins, benzophenones, anthraquinones, certain diazo compounds, diazirines, and psoralen derivatives. In certain embodiments, the probes have arylazides in their backbone as crosslinkers. The term "arylazides" also called phenylazides, refers to compounds containing an aryl group directly linked with an azide group.

The term "fluorophore" refers to fluorescent compounds which absorb light of a specific wavelength and re-emit light at a different and typically longer wavelength. Most fluorophores are small molecular fluorophores, which means, typical macrocyclic compounds with established conjugated system containing combined aromatic groups or π systems. Typical small fluorophores include xanthenes derivatives, coumarin derivatives, fluorescein derivatives and BODIPY derivatives and so on. This tag-label pair strategy certainly could benefit from the diverse choices of chemical fluorophores, and the response time of these fiuorogenic compounds is relatively short and stable in comparison to fluorescent proteins. Coumarin and fluorescein are chosen at present for the purpose but a variety of fluorophores can be readily obtained commercially or through simple synthesis for future plan.

The term "linker" refers to a carbon chain (C) n for different purpose, "C" herein refers to carbon atom. It can be a hydrocarbon chain to increase lipophilicity of the agent or a peptide sequence for elevating hydrophilicity. The appropriate length of the linker is important for the purpose. An exemplary link group -(C4H8)- used in some embodiments is to minimize interference caused by the macrocyclic fluorophore and chelator when permeating cell membrane and approaching the biomolecular targets. Link groups are not limited to alkyl groups. A "cleavable linker" is a linker that has one or more cleavable groups that may be broken by the result of a reaction or condition.

The term "metal chelation" refers to the way for a metal-chelating moiety binds metal ions with increasing stability. In some embodiments, metal chelation happens between metal ions and metal chelating moiety, and between metal ions and poly-Histidine -tagged proteins with a relatively stronger affinity.

The term "metal-chelating moiety" refers to the part for metal ions attached to, which is usually a polydentate ligand in a cyclic or ring structure, which coordinates to metal ions according to the size, charge, coordination geometry and Lewis acid character. Examples of metal-chelating moieties include, but are not limited to, nitrilotriacetic acid (NT A), ethylenediamine, Ethylenediaminetetraacetic acid (EDTA) and iminodiacetic acid (IDA). They are frequently used for binding metals to enable the availability of metals to coordinate to proteins. In some embodiments, NTA moiety chelates the nickel(II) ion to form a Ni 2+ -NTA chelating compartment for site-specific labeling of his-tagged proteins. Other chelating ligands were considered in some embodiments for better results.

The term "irradiation" refers to a process by which an object is exposed to radiation, such as ultraviolet, visible light, microwave and infra-red. The term "ultraviolet irradiation" refers to the irradiation process under particular wavelength of ultraviolet light. It has different applications in the field of sterilization, agriculture, medicine and industry. Photoreactive crosslinkers label with proteins, nucleic acids and other biomolecules in an irreversible manner after activated by ultraviolet light. In some embodiments, fluorescent probes are excited with optical energy or UV light (generally 200 to 400 nm wavelength) or typically UV light with wavelength between 340 to 380 nm. The term "poly-Histidine-tag" refers to amino acid motif contains at least six histidine ((His) n ,n>6) residues often at the N- or C- terminus of proteins. Examples of poly-histidine proteins include, but are not limited to, hexahistidine ((His) 6 ) and decahistidine (His)io, which are also well known and widely used in biochemistry.

The term "covalent" refers to the stable balance of force between atoms when they share electron pairs to form a bond. The crosslinker employed can be generated through photo- activation to provide covalent linkage of targets. Self- modifying protein fusion tags were employed to generate irreversible binding with the respective ligands through bioorthogonal reactions, thereby providing prominent specificity with minimized background and assuring further analysis in denatured conditions. In some embodiments, the metal coordination to targeted protein provided non-covalent binding of the probe to the target and self-assembling of the photoreactive crosslinker to the respective protein, and subsequently the covalent linking would be achieved through photo-activation of the sample for further separation and protein identification processes. Such a covalent bonding to protein targets is for the identification of proteins, preserving fluorescent labeling throughout the denaturing separation process and allowing excision or respective proteins for detection in proteome-wide.

The term "turn-on" herein refers to the nondestructive and prompt detection of fluorescence enhancement. In some embodiments, fluorescence of metal-chelating probes was quenched upon chelating metal ions, for example nickel(II) ion. The term "quench" herein refers to the rapid vanishing of fluorescence, which is also known as fluorescence turn-off. Yet when the Ni 2+ -chelated probe was mixed with Ni 2+ -chelating poly-Histidine -tagged proteins, a dramatic increase in fluorescence intensity is resulted. The recorded increasing fluorescence assures a turn-on system.

The term "physiological conditions" herein refers to laboratory conditions monitoring external or internal milieu that may occur in living organisms with suitable buffer solutions, for example, pH 7.2-7.4, temperature 20-40°C and atmospheric oxygen concentration. Some common buffering systems are used herein, including 20 mM HEPES (4-(2-hydroxyethyl)-l- piperazineethanesulfonic acid) with 100 mM sodium chloride in pH 7.2, PBS in pH 7.4 (phosphate buffer saline) or 20 mM Tris-HCl (tris(hydroxymethyl)aminomethane) in pH 7.2, which are applied in some embodiments.

The term "biological sample" refers to a biological specimen used in the laboratory as an experiment system for research. Examples used herein include, but are not limited to, bacterial cells, plant cells and tissues, mammalian cell lines and tissues, to detect, localize and analyze his- tagged proteins in cells.

The term "confocal imaging" refers to fluorescent imaging performed with confocal dish on. For example, a Carl Zeiss LSM700 Inverted Confocal Microscope with a Plan-Apochromat 63x 1.40NA oil-immersion objective may be utilized.

The term "SDS-PAGE" refers to the proteins separation process with denaturing gel electrophoresis. In some embodiments, SDS-PAGE was performed using 15% resolving gel. Fluorescence gel imaging may be captured by ImageQuant 350 and Typhoon 9410 system from GE Healthcare. The denaturing gel can be stained by Coomassie blue (and/or Western blotting) for comparison.

The term "carrier molecule" as used herein refers to a fluorogenic or fluorescent compound that is covalently bonded to a biological or a non-biological component. Such components include, but are not limited to, an amino acid, a peptide, a protein, a polysaccharide, a nucleoside, a nucleotide, an oligonucleotide, a nucleic acid, a hapten, a psoralen, a drug, a hormone, a lipid, a lipid assembly, a synthetic polymer, a polymeric microparticle, a biological cell, a virus and combinations thereof.

The term "detectable response" as used herein refers to a change in or an occurrence of, a signal that is directly or indirectly detectable either by observation or by instrumentation. Typically, the detectable response is an optical response resulting in a change in the wavelength distribution patterns or intensity of absorbance or fluorescence or a change in light scatter, fluorescence lifetime, fluorescence polarization, or a combination of the above parameters.

The hexahistidine -Ni-NTA system has been exhaustively utilized in protein purification and large amounts of His-tagged protein libraries exist worldwide. Exploration of such a system for imaging of proteins in live cells offers enormous opportunities for tracking of various cellular events with minimal spatial and functional perturbation on a protein of interest. However, previously reported Ni-NTA based probes suffer from poor membrane permeability and were limited to label membrane proteins only. The present invention provides the first small fluorescent probe Ήι-ΝΤΑ-AC, which can rapidly across cell membranes to specifically target His 6 -tagged proteins in various types of live cells even in plant tissues. The probe provides new opportunities for in-situ analysis of various cellular events.

In an embodiment, the fluorescent probe is comprised of a reporting moiety (a fluorophore), a metal-chelating moiety linked to the fluorophore by a linker, and a photoreactive crosslinker (Figure 1). Metal ions-chelated to the fluorescent probe directs the agent to label poly-Histidine -tagged proteins, while in proximity the photoreactive crosslinker is photo- activated by UV irradiation to serve as the second anchor point on the targeted proteins and thus to provide extra stability to the fluorescent labeling. An advantage of such an anchoring is that the binding is covalent in nature, thus proliferating binding affinity of the probes, and the labeling could be retained even after targeted protein is being denatured (which could destruct the metal-coordination of the metal-chelating moiety to the poly-Histidine -tag).

In an embodiment, the probe includes a blue coumarin derivative (7-amino-4- methylcoumarin-3-acetic acid, AMCA) as the fluorophore accounted to its excellent sensitivity and small size. Metal-chelating moiety nitrilotriacetic acid (NT A) is employed to partially coordinate to metal ions, allowing the tracking of poly-Histidine -tagged proteins based on metal coordination to the tag, which is classified as a universal metal-chelating agent and potentially coordinates to various hard to borderline metal ions (Lewis acids) such as Ni 2+ , Cu 2+ and Co 2+ (Haas, K. L. & Franz, K. J. Chem. Rev. 109, 4921-4960 (2009)), making it relatively suitable for tracing poly-Histidine -tagged proteins bound to transition metals. The fluorescent agent also possesses a photoreactive crosslinker arylazide to anchor onto the target of interest in an irreversible manner, provided that the trigger of crosslinking only requires simple photo- activation by ultraviolet radiation at 365 nm (Hintersteiner, M. et al. ChemBioChem 9, 1391- 1395 (2008)). Such a covalent bonding to protein targets helps preserving fluorescent labeling throughout the denaturing protein separation process (e.g. gel electrophoresis) and allowing excision of respective proteins for detection in proteome-wide (Figure 2).

An advantage of the fluorescent labeling is that the chelation of metal ions by the probe initiates a quenching of fluorescence while the labeling of poly-Histidine -tagged protein elevates the fluorescent signals to generate a "turn-on" response. However, the presence of poly- Histidine -tagged proteins initiates a significant increase in fluorescence accounted to the launching of small-molecule-fluorescent labeling to the protein of interest. In another embodiment, introduction of poly-Histidine -tagged XPA122 protein (His-XPA122) could initiate

• 2+

an elevation of fluorescence of Ni -NTA-AC by 13 times while the complete labeling could be achieved in 9 minutes according to the time-course plot (Figure 7A).

In some embodiments, the probes label poly-Histidine -tagged protein with a covalent linkage and thus maintain the staining after proteins undergo denaturing protein separation processes (e.g. SDS-PAGE). In some embodiments, the fluorescent agents could apply to living biological samples to target intracellular (and membrane -bound) poly-Histidine -tagged proteins accordingly. For example, biological samples include, but are not limited to, bacterial samples, mammalian cell lines and tissues, in addition to plant cells and tissues.

Another probe NTA-AF (Figure 2) with the same structural design in Figure 1 is provided herein, which includes a fluorescein-derived fluorophore, a metal-chelating nitrilotriacetic acid ( TA) moiety linker to the fluorophore, and an arylazide photoreactive crosslinker (Figure 12).

Thus, the following non-limiting embodiments are provided:

1. A fluorescent probe for targeting biomolecules in biological samples, comprising a fluorophore reporting moiety which generates a fluorescent signal, a metal-chelating moiety to chelate metal ions for coordinating to poly-Histidine-tag encoded to the targeted protein, a linker connecting the fluorophore reporting moiety and metal-chelating moieties, and a photoreactive crosslinker serving as an anchor point onto the targeted protein to proliferate labeling affinity and stability.

2. The fluorescent probe according to embodiment 1 , wherein the fluorescent signal of the reporting moiety has a wavelength of about 400 to about 800 nm after absorption of optical energy. 3. The fluorescent probe according to any of embodiments 1 - 2, wherein the reporting moiety comprises coumarin-derivatives, fluorescein-derivatives and Rhodamine-derivatives.

4. The fluorescent probe according to any of embodiments 1 - 3, wherein the metal- chelating moiety comprises poly dentate ligands.

5. The fluorescent probe according to embodiment 4, wherein the poly dentate ligands comprise nitrilotriacetic acid (NTA) and iminodiacetic acid (IDA).

6. The fluorescent probe according to any of embodiments 1 - 5, wherein the metal- chelating moiety comprises chelating metal ions comprising at least one of nickel(II), cobalt(II), and copper(II) metal ions. 7. The fluorescent probe according to any of embodiments 1 - 6, wherein the linker between the fluorophore and the metal-chelating moiety is a hydrocarbon chain or a peptide sequence. 8. The fluorescent probe according to any of embodiments 1 - 7, wherein the photoreactive crosslinker comprises arylazide, diazirine, and benzophenone,

9. The fluorescent probe according to embodiment 8, wherein the photoreactive crosslinker embraces or, alternatively, does not embrace as part of the conjugated system of the fluorophore.

10. The fluorescent probe according to any of embodiments 8 - 9, wherein the photoreactive crosslinker exhibits photo-activation being accomplished by ultraviolet radiation for about 5 to about 15 minutes after the fluorescent probes coordinate to poly-Histidine -tagged proteins in the biological samples.

11. The fluorescent probe according to embodiment 10, wherein the ultraviolet radiation is in a range of about 340nm to about 380 nm. 12. The fluorescent probe according to any of embodiments 1 - 11 , wherein the chelation of metal ions including nickel(II), cobalt(II), and copper(II) ions generates a fluorescent quenching of the metal-chelated probes.

13. The fluorescent probe according to any of embodiments 1 - 12, wherein the probes coordinate to metal ions in 1 : 1 molar ratio.

14. The fluorescent probe according to any of embodiments 1 - 13, wherein labeling targeted biomolecules is achieved through coordinating to poly-Histidine-tag encoded to the biomolecules by the metal-chelation of the probes.

15. The fluorescent probe according to embodiment 14, wherein the metal-chelating fluorescent probes exhibit an elevation of fluorescent signals (a "turn-on" response) and is achieved when the metal-chelating fluorescent probes label poly-Histidine -tagged proteins in buffers at pH 6-8.

16. The fluorescent probe according to embodiment 15, wherein the metal-chelating fluorescent probes exhibit an elevation of fluorescent signals when the metal-chelating fluorescent probes label poly-Histidine -tagged proteins at a temperature from about 4°C to about 40°C.

17. The fluorescent probe according to embodiment 16, wherein the labeling of poly- Histi dine -tagged proteins is stable for overnight incubation.

18. The fluorescent probe according to any of embodiments 1 - 17, wherein the labeling of poly-Histidine -tagged proteins is retained after the proteins are denatured with a temperature of about 90°C to about 1 10°C.

19. The fluorescent probe according to embodiment 17, wherein the fluorescent labeling of poly-Histidine -tagged proteins is able to be visualized on gels after native or denaturing gel electrophoresis. 20. A method of labeling of poly-Histidine -tagged proteins, comprising:

coordinating poly-Histidine -tag encoded proteins to target biomolecules by the metal- chelation of a fluorescent probe, the fluorescent probe comprising a fluorophore reporting moiety which generates a fluorescent signal after absorption of optical energy, a metal-chelating moiety to chelate metal ions for coordinating to poly-Histidine-tag encoded to the targeted protein, a linker connecting the fluorophore and metal-chelating moieties, and a photoreactive crosslinker serving as an anchor point onto the targeted protein to proliferate labeling affinity and stability.

21. The method according to embodiment 20, wherein the fluorescent probe generates a fluorescent signal after absorption of optical energy having a wavelength of about 400 to about 800 nm. 22. The method according to any of embodiments 20 - 21 , wherein the labeling of poly- Histidine -tagged proteins is achieved in a biological sample comprising bacterial cells, mammalian cells, mammalian tissues, plant cells, or plant tissues. 23. The method according to embodiment 22, wherein the labeling is carried out at a temperature of about 4°C to about 40°C.

24. The method according to embodiment 22, further comprising undergoing washing by a buffer.

25. The method according to any of embodiments 20 - 24, further comprising undergoing confocal imaging.

26. The method according to embodiment 22, wherein the introduction of the fluorescent probes does not damage the biological samples.

MATERIALS AND METHODS

Synthesis of NTA-AC. Synthesis of NTA-AC involves three steps with an overall yield of 64% (Figure 3). Analytical thin layer chromatography (TLC) was performed using Macherey-Nagel pre-coated 0.25 mm thick TLC-plates (silica gel 60 with fluorescent indicator UV254). Silica gel 60 from Merck was used for flash column chromatography (230-400 mesh ASTM). HPLC-grade water for electrospray ionization mass spectrometry (ESI-MS) was received from Labscan. Deuterated solvents for NMR were purchased from Cambridge Isotope Laboratories (for acetone-d6) and Sigma-Aldrich (for D 2 0). Proton and Carbon magnetic resonance spectra ( ! H and 13 C NMR) experiments were carried out on Bruker Avance-300 and Avance-500 spectrometers at 298 K. ESI-MS spectra were collected using a Finnigan LCQ spectrometer.

The probe NTA-AC was synthesized through a three step synthesis as shown in Figure 3A. All reactions were performed avoiding light exposure.

In an embodiment, the fluorescence spectra of NTA-AC were determined in water at 25°C on a Hitachi F-7000 fluorescence spectrophotometer using 1000 W xenon lamp source, with the excitation and emission slit width set at 5.0 nm while the photomultiplier voltage set at 700 V. A 1 cm x 1 cm quartz cuvette with a sample volume of 1.5 mL applied for experiments. Sample with 5 μΜ of NTA-AC dissolved in water at 25°C was excited with near-UV wavelength and emitted in the blue light region, exhibiting an excitation maxima at λ εχ = 342 nm and emission centered at X em = 448 nm (Figure 6).

In an embodiment, the binding stoichiometry of NTA-AC to Ni ions was determined by

Job's plot of the fluorescence changes of NTA-AC and Ni 2+ ions in Tris-HCl buffer. The

• 2+

concentrations of NTA-AC and Ni ions were kept constantly in a total of 10 μΜ, with 11 solutions with varied concentrations of NTA-AC and Ni 2+ ions and incubated for 30 minutes

• 2+

before detection. The stoichiometry of Ni -NTA-AC complexation was measured by monitoring the fluorescence changes (λ εχ = 342 nm and X em = 448 nm) and the plotting of points intersected at [Ni 2+ ]/{[Ni 2+ ]+[NTA-AC] equaled to 0.5, indicating that the complexation of Ni 2+ -NTA-AC was originated with a ratio of Ni 2+ : NTA-AC of 1 : 1 (Figure 3C). The coordination of Ni 2+ ions to NTA-AC was monitored by mass spectrometry, which exhibited a peak at 560.0 m z (calc. 560.0 m/z), confirming the formation of Ni 2+ -NTA-AC with a ratio of Ni 2+ : NTA-AC equaled to 1 : 1.

2-(7-Azido-4-methyl-2-oxo-2H-chromen-3-yl)acetic acid (1)

(1) was synthesized according to the literature. (Thcvenin. BJ, et al. (1992) Eur J Biochem 206(2):471-477). Briefly. 7-amino-4-methylcoumarin-3-acetic acid (0.143 g, 0.61 mmol) was mixed with sodium nitrite (0.045 g, 0.65 niiiiol) in water (6 mL) containing concentrated sulfuric acid (1 mL) in ice bath. Subsequently, sodium azide (0.051 g, 0.79 mmol) was added to the reaction mixture in ice bath and was continuously stirred for 45 minutes, and precipitate was formed. The precipitate was filtered, washed with ice-cold water, then dried by lyophilization and obtained as a light brown powder (0.135 g, 0.52 mmol, 85% yield).

2,5-Dioxopyrrolidin-l-yl 2-(7-azido-4-methyl-2-oxo-2H-chromen-3-yl)acetate (2)

(2). Compound ( 1) (0.129 g, 0.50 mmol) was dissolved in 25 ml. of acetonitrile and stirred at room temperature. V- ydroxysuecinimide (0.058 g, 0.51 mmol) and Ν,Ν'- dicyci o h e x y I c a r bo d i i m i d e (0.105 g, 0.51 mmol) were added subsequently into the reaction flask and the reaction mixture was stirred overnight at room temperature. The solution was filtered, and the filtrate was rotary-evaporated to yield crude yellow product, which was then dissolved in chloroform for simple solvent extraction. A crude yellow solid (2) was obtained after rotary-evaporation.

(S)-2,2'-(5-(2-(7-azido-4-methyl-2-oxo-2H-chromen-S-yl)ac etamido)-l-carboxy

pentylazanediyl)diacetic acid (NTA-AC)

Compound (2) was dissolved in acetonitrile (50 ml. ) with stirring at room temperature. N ( i ,.V ( < -bis(carboxymethyl )-L-lysine hydrate (0.180 g, 0.69 mmol ) was dissolved in water (10 ml.) supplemented with triethylamine (0.5 ml.). The solution was then added dropvvisc to the solution of (2), and the reaction mixture was stirred continuously overnight at room temperature. After rotary-evaporation and lyophilization, the crude product was purified by column chromatography to give pale yellow powder NTA-AC (0.198 g, 0.39 mmol, 79% yield compared to reactant (1) in the second reaction) (overall yield of 64%). IR (Nujol, c m " 1 ): 3413.8 (br), 2725.2 (m), 21 19.6 (m), 1645.2 (s), 1461.9 (s), 1307.6 (m), 1 1 59. 1 (w), 1097.4 (w), 1035.7 (w), 964.3 (w), 866.0 (w), 72 1 .3 (m ). Ή NMR (500 MHz, D20) (Figure 4A): δ 7.70 (d, J = 8.69 Hz, 1H), δ 7.03 (d, J = 8.62 Hz, 1H), δ 6.94 (s, 1H), δ 3.80 (br s, 5H), δ 3.63 (s, 2H), δ 3.24 (s, 2H), δ 2.39 (s, 3H), δ 1.98 (s, 2H), δ 1.59 (br s, 4H). 13 C NMR (500 MHz, D 2 0) (Figure 4B): δ 180.794, δ 172.698, δ 171.057, δ 163.884, δ 152.817, δ 152.765, δ 144.057, δ 127.385, δ 1 17.926, δ 1 17.378, δ 1 16.407, δ 106.835, δ 68.977, δ 55.999, δ 55.696, δ 39.606, δ 34.383, δ 28.616, δ 27.1 19, δ 24.320, δ 23.042, δ 15.298. ESI-MS (m/z) (Figure 5): [M+Na] ' calcd. 526. 1 , obsd. 526. 1 . Ni-NTA-AC ESI-MS (m/z): [ -3H] " calcd. 558.9, obsd. 558.6.

Synthesis of NTA-C

(S)-dimethyl 2,2 , -((6-aniino-l -niethoxy-l-oxohexan-2-yl)azanediyl)diacetate (3).

(S)-2,2'-((5-amino-l - earboxypenty! )azanediyl)diacctic acid ( 1 00 mg, 0.43 mmol ) was dissolved in 30 ml . methanol and the solution was cooled to 0°C, then SOCl 2 (623 μΐ, 8.58 mmol) was added in a drop wise manner. The mixture was stirred for 48 hours under reflux at 55°C, then the solvent was rotary-evaporated to afford a colorless oily product. The protection step was assumed 100% yield and (3) was used in the next step without further purification. Ή NMR. (300 MHz, CD30D): δ 4.53-4.35 (m, 5H), 3.90 (s, 3H), 3.88 (s, 6H), 3.08-2.96 (m, 2H), 2.15-2.02 (m, 2H), 1 .87- 1 .73 (m, 2H), 1.72-1.60 (m, 2H). ESI-MS

(m/z): [M+H] + calcd. 305.2, obsd. 305.2.

(S)-dimethyl 2,2'-((6-(2-(7-amino-4-methyl-2 )xo-2H-chromen-3-yl)acetamido)-l- methoxy-l-oxohexan-2-yl)azanedi l)diacetate (4). 2-(7-amino-4-methyl-2-oxo-2H- chromen-3-yl )acctic acid (30 mg, 0. 13 mmol) was dissolved in I ml . DMF, followed by the addition of HATU (GL Biochem) (98 mg, 0.26 mmol ). After 5 minutes, a solution of DIE A (90 PL, 0.516 mmol) and ( 3) (51mg, 0. 167 mmol) in 0.5 ml, DMF and 2 ml . DC was added, and the reaction mixture was stirred for 2 hours before dilution into 50 ml. of IX M. The organic phase was washed with 5% acetic acid, water and brine, dried over anhydrous magnesium sulfate. After rotary-evaporation, the residue was purified by flash chromatography to afford ( 4) (12 mg, 18% yield). ESI-MS (m/z): [M+Na] + calcd. 542.2, obsd. 542.3.

(S)-6^2-(7-amino-4-methyl-2-oxo-2II-chromen-3-yl)acetamido)- 2- ((carboxymethyl)(hydroperoxymethyl)amino)hexanoic acid ( 'TA-C). LiOH-H 2 0 (8 mg,

0. 19 mmol) was dissolved in 4.5 m 1. of solution mixture (1 :4: 1 of H 2 0:THF:MeOH), then subsequently ( 4) (10 mg, 0.019 mmol ) was added with the solution further stirred for 48 hours. Resin (Dowex® 50Wx8 hydrogen form) was used to remove Li ions by adjusting p!l to 6, then the solvent was rotary-evaporated and water was removed by lyophilization to yield NTA-C. (3.5 mg, 39% yield)Ή NMR. (300 MHz, D20): δ 7.49 (d, J = 8.3 Hz, Hi ), 6.76 (d, J = 9.3 Hz, H I ). 6.64 (s, H I ), 3.78-3.65 (m , 5H), 3.45 (s, 2H), 3. 1 7-3.04 (m, 2H), 2,24 (s, 3H), 1.88-1.63 ( m, 2H), 1.53-1.27 (m, 4H). 13 C NMR (400 Hz, D20): ι 172.94, 172.63, 170.36, 164.38, 153.38, 148.02, 126.90, 1 14.40. 1 14.36, 113.29, 102.77, 68.23, 55.39,

39.03. 33.90, 28.12, 26.59, 23.63, 14.77. ESI-MS (m/z): [M-2H+K]- calcd 514.5, obsd. 514.5. Synthesis of NTA-AF

Synthesis of azido fluorescein - Fluorescein-amine (0.1340 g, 0.38 mmol) was dissolved in 10 mL of methanol with 0.1506 g NaN0 2 being dissolved in 4 mL of water and added to the solution, followed by 4 mL of 5 M hydrochloric acid. Sodium azide (0.1827 g) was dissolved in 4 mL of water and added dropwise to the reaction mixture. After stirring for 140 minutes at room temperature and checking with TLC, the solution was concentrated under vacuum then filtered and washed with cold water. All steps were performed in the dark or foil package. Yield: 92.06%. 1H NMR (400 MHz, CO(CD 3 ) 2 ): δ= 7.57 (s, 1H, ArH), 7.47 (d, 1H, ArH), 7.29 (d, 1H, ArH), 6.71 -6.58 (m, 6H, ArH). ESI-MS (m/z): [M+H] + calcd. 374.1 , obsd. 374.1.

Protection NTA-amine - In an ice bath, NTA-amine (0.1006 g, 0.38 mmol) was dissolved in 30 mL methanol with SOCl 2 (332 uL, 4.57 mmol) being added dropwise to the solution. The mixture was left to reflux and stirred for 48 hours in oil bath, with an oily product being obtained after evaporation. 1H NMR (400 MHz, MeOD): δ= 4.52-4.38 (m, 5H), 3.81 (d, 3H+6H), 2.95 (t, 2H), 2.10-1.59 (m, 6H, 2H+2H+2H). ESI-MS (m/z): [M+H] + calcd. 304.2, obsd. 305.3.

Synthesis of pro-probe - At 0°C azidofluorescein (0.1 141 g, 0.3 mmol), l-ethyl-3-(3- dimethylaminopropyl)carbodiimide (EDCI, 0.0544 g, 0.35 mmol), 1 -hydro xybenzotriazole hydrate (HOBT, 0.1210 g, 0.79 mmol) and N-methylmorpholine (NMM, 0.15 mL, 1.2 mmol) were dissolved in dry DMF (5 mL) and stirred for 1 hour in nitrogen. Then, pro-NTA synthesized before was dissolved in 2 mL dry DMF and added dropwise. The solution was stirred overnight at room temperature under nitrogen. After checking with TLC, the solvent was removed under pressure, the residue was dissolved in ethyl acetate and washed with saturated sodium chloride, then the organic phase was dried over MgSO After removing the ethyl acetate, the crude product was purified by silica gel column chromatography (solvent: EA Hexane=2.5: l), the clean product was obtained with a yield of 25.07%. 1H NMR (400 MHz, CDCI 3 ): δ= 7.58 (s, 1H, ArH), 7.10 (d, 1H, ArH), 7.04 (d, 1H, ArH), 6.68-6.42 (m, 6H, ArH), 3.66-3.51 (d, 6H+3H), 3.46 (m, 4H), 3.17 (t, 1H), 2.98 (t, 2H), 1.98-0.91 (m, 6H, 2H+2H+2H). 13 C MR (400MHz, CDC1 3 ): δ 23.341 , 27.696, 30.000, 40.422, 51.606, 51.932, 52.521 , 64.812, 64.983, 77.287, 98.916, 103.289, 1 10.007, 1 10.1 15, 1 12.622, 1 13.051 , 123.951 , 125.233, 129.136, 132.738, 141.208, 149.376, 152.720, 152.761 , 154.138, 157.955, 158.023, 167.526, 172.41 1 , 173.300. dept MR: δ 23.263, 27.617, 29.914, 40.349, 52.432; 51.527, 51.853, 64.723, 103.201 , 103.245, 1 12.543, 1 12.970, 123.877, 125.177, 129.058. ESI-MS (m/z): [M+Na] + calcd. 682.2, obsd. 682.2.

De-protection - 30.6 mg pro-probe (0.046 mmol), Lithium hydroxide (LiOH ' H 2 0, 0.0201 g, 0.48 mmol) was dissolved in 6 mL 4: 1 : 1 THF: methanol: water at 0°C. The reaction mixture was stirred at room temperature for 60 hours, then evaporated in vacuo. Added 5 mL methanol to dissolve and evaporated twice to get rid of the solvent. ! H NMR (400 MHz, CD 3 OD): δ= 7.55 (s, 1H, ArH), 7.24 (d, 1H, ArH), 7.06 (d, 1H, ArH), 6.64-6.39 (m, 6H, ArH), 3.68-3.58 (m, 4H+1H), 3.09 (t, 2H), 1.60-0.99 (m, 6H, 2H+2H+2H). 13 C NMR (400MHz, CDC1 3 ): δ 23.387, 27.417, 28,757, 39.504, 53.898, 65.008, 65.442, 102.284, 109.253, 109.326, 1 12.324, 123.721 , 125.120, 128.699, 128.777, 132.636, 141.275, 149.645, 152.839, 152.925, 158.979, 167.599, 173.924. dept NMR: δ: 23.290, 27.328, 28.661 , 39.404, 53.809; 65.346, 102.192, 1 12.026, 1 12.236, 123.635, 125.032, 128.61 1 , 128.689. ESI-MS (m/z): [M+H] + calcd. 618.2, obsd. 618.5.

In some embodiments, the fluorescein-based fluorescent probe NTA-AF exhibits its green fluorescence at X em = 518 nm with the excitation maxima located at λ εχ = 496 nm (Figure 13). Poly-Histidine -tagged protein His-XPA122 (12 μΜ) was also pre-incubated with equimolar concentration of Ni 2+ -NTA-AF for 1 hours at 4°C and subsequently the photoreactive crosslinker arylazide was photo-activated by ultraviolet radiation at 365 nm for 10 minutes at room temperature. SDS-PAGE analysis was performed using 15% resolving gel, and fluorescence gel images were captured by Typhoon 9410 system from GE Healthcare (λ εχ = 488 nm, em = -520 nm) and with Coomassie blue staining for comparison. The probe facilitated a green fluorescent labeling of poly-Histidine -tagged protein His-XPA122 (lane 1) on the gel and thus demonstrating its applicability similar to NTA-AC but in a different color to for providing spectral varieties (Figure 14).

Construction of Mammalian Expression Plasmids. pRSETB-mRFP ! containing the mrfp gene was purchased from Clontech Laboratories, Inc. Consecutive histidine residues were added before the N-termini of mrfp and xpal22. A linker (Gly-Gly-Scr-Gly- Gly-Ser) was inserted between the poiyhistidine tag and mrfp or xpal22 gene to enhance the flexibi lity between poiyhistidine tag and target proteins.

Full length mrfp gene was amplified by polymerase chain reaction (PGR) with the primer-pair I I is-m RFP-For/His-m R FP-Rev (Table 1). The xpal22 gene was amplified by PGR with the primer pair His-XPA 122- For His6-XPA 122-Rev (Table 1). Restriction sites BamW 1 and Xhol were introduced at the 5'- and 3'- ends of the PGR products, respectively. The PGR products and the vector pcDNA3.1 -(+) ( Life Technologies Corporation) were digested by restrictive endonue leases (New England Biolabs, Inc.). After digestion, the PGR products were ligated into the pcDNA3.1 -(+) vector by T4 ligase ( Life Technologies Corporation) to obtain pcDNA3. 1 -His-mRFP and pcDNA3. 1 -I lis-XPA 1 22.

Table 1. Primers used for plasmid construction.

Alternatively, for constructing pcDN A3. 1 -His-mRFP-Uis-XPA 122, ful l length mrfp gene was amplified with primer pair I l is-mRFPtag-For I lis-mRFPtag-Rev, introducing

Nhel and BamHl sites at the 5' and 3 '-end respectively. PGR product and vector pcDNA3. 1 -His-XPA 1 22 were digested by restrictive endonucleases (New England Biolabs, Inc.). After digestion, the PGR product was ligated into the vector pcDNA3. 1 -His- XPA 122 by T4 ligase (Life Technologies Corporation) to obtain pcDN A3. 1 -I iis-mRFP-I iis- XPA122. The sequence of the constructed plasmids was confirmed by DNA sequencing.

Mammalian Cell Culture and Transient Transfection. Del. a cell line was purchased from American Type Culture Collection (ATCC). All of the chemicals regarding ceil culturing were purchased from Gibco® by Life Technologies or otherwise stated. HeLa cells were grown in Dulbecco ' s Modified Eagle Medium (DMEM) with the supplementation of 10% fetal bovine serum ( FBS) and 1% antibiotics (Pen Strep), and cultured in 5% CO 2 incubator at 37°C. Transient transfcctions of HeLa cells with prepared plasmids were performed with Lipofcctamine 2000 (Invitrogen). HeLa cells were seeded on 6-weil plates (for iysates collection) or confocal dishes (for confocal imaging) in the presence of DMEM and 10% FBS without antibiotics. When the cell density reached 90% confluency, Lipofcctamine and prepared plasmid. were supplemented to the ceil samples at the ratio of 2 PL/Pg. After 24 hours of transfection, the medium was replaced by Hank's Balanced Salt Solution (HBSS) for subsequent experiments.

Overexpression and Purification of l lis-XPA122 and XPA 122. Plasmid pET-His6-

XPA I 22 was transformed into Escherichia coli BL2 1 (DE3). The overnight culture inoculated into fresh Luria-Bertani ( LB) broth supplemented with 34 iig mL kanamycin was grown at 37°C until an optical density at 600 nm (OD 600 ) of about 0.6. Protein expression was induced at 16°C overnight with 0.2 mM isopropyl β-D- thiogalactopyranoside (IPTG). Bacteria were then harv ested by centrifugation at 4000 rpm at 4°C for 15 minutes and were suspended in Tris buffer A (20 mM Tris-HCl, pH 7.6, 500 mM NaCl and 20 mM imidazole).

Cells were lysed by sonication in the presence of I mM phen y 1 me t h y Isu 1 fon y 1 fluoride (PMSF). The cell lysate was centrifuged at 10.000 g at 4°C for 30 minutes to separate the inclusion body. The supernatant was filtered through a 0.45 μηι filter unit and was applied to HisTrap Ni-NTA column (GE Healthcare), equilibrated with the same buffer. The protein was eluted with Tris buffer containing 300 mM imidazole. The fractions were analyzed by 12% sodium dodecyl su I phate-po 1 yacry I am i de gel electrophoresis (SDS- PAGE). The fraction w ith the His-XPA 1 22 was concentrated by Am icon Ultra- 15 centrifugal filter unit (Miiiipore).

To obtain XPA122 without the His-tag, His-XPA 122 was concentrated in Tris buffer

B (20 mM Tris-HCl, pH 7.2, 130 mM Nad) by Amicon Ultra- 15 centrifugal filter unit (Millipore). Removal of His-tag was done by thrombin cleavage at 20°C overnight with gentle shaking and the cleavage product was purified by HisTrap Ni-ΝΤΆ column (GE Healthcare).

His-XPA 122 and XPA122 were subjected to further purification by Superdex 75 size- exclusion column (GE Healthcare) in Tris buffer C (20 mM Tris-HCl, pH 7.4, 300 mM NaCl). The peak fractions were col lected and concentrated with Am icon Ultra- 15 centrifugal filter unit (Millipore). The purity of protein was confirmed by 15% SDS-PAGE and the protein concentration was determined by BCA Protein Assay Kit (Novagen).

Fluorescent Spectroscopic Measurements. Binding of the fluorescent probe to proteins under di fferent conditions were carried out on a Hitachi F-7000 fluorescence spectrophotometer with 1000 W xenon lamp using a 1 cm x 1 cm quartz cuvette (1.5 mi . sample volume). The binding stoichiometry of NT A- AC to Ni 2+ ions was determined by Job's plot of the fluorescence changes of NT A- AC and Ni ' ions. The concentrations of NT A- AC and Ni 2+ ions were kept constant in a total of 10 μΜ, with various concentrations of NT A- AC and Ni 2 ions and incubation for 30 minutes before each measurement. The changes in fluorescence of Νί-ΛΤ.-ί-.-l or Ni-NTA-C (1 uM) upon the addition of His-XPA I 22 (10 molar equivalents) were measured in every minute interval at 25 °C.

To demonstrate the significance of the presence of His 6 -tag and Ni 2† for specific interaction, the changes in fluorescence of Ni-NTA-AC upon incubation with proteins were determined. Apo-protein XPA 1 22 was prepared in 20 mM HE PES, 100 mM NaCl, pH 7.4 and was titrated into Ni-AT.-i-.-iC (1 μΜ) in 1 μΜ increment. Apo-protein His- XPA 1 22 was also prepared in 20 mM HEPES, 100 mM NaCl, pi I 7.4 and was titrated into NT A -AC (1 μΜ) in 1 μΜ increment.

Isothermal Titration Calorimetry (ITC). Apo-protein His-XPA 122 was prepared freshly in 20 mM HEPES, 100 mM NaCl, pH 7.4, while Ni-NTA-AC or Ni-NTA-C was pre- incubated in the same buffer overnight at 4°C before use. For probe-protein interaction, Ni- NTA-AC or Ni-NTA-C (500 μΜ) was titrated into His- XPA 122 (35 μΜ). All ITC experiments were performed at 25 °C on an ITC200 isothermal titration calorimeter (Microcal).

In vitro Imaging of Proteins on SDS-PAGE. Solutions containing Ni-NTA-AC were first incubated with 1 0 molar equivalents of ethyienediaminetetraacetic acid (EDTA) overnight at 4°C to prepare NT A- AC solutions. Subsequently, proteins ( 12 μΜ each) were pre-incubated with equimolar concentration of the probe or NTA-C for 2 hours at 4°C, the covalent linkage was then photo-activated by ultraviolet radiation at 365 run using a UVP UVGL-25 Mineralight UV lamp for 10 minutes at room temperature. SDS-PAGE analysis was performed using 15% resolving gel. Fluorescence gel images were captured by ImageQuant 350 (GE Healthcare) (λ εχ =365 mil, - 460-500 nm ). The denaturing gel was stained by Coomassie blue afterwards for comparison and the presence of His-tag in protein was validated by immunoblotting.

Labeling Yield of His-XPA 122 by M-NTA-AC on SDS-PAGE and ALI) I -MS. His-XPA 122 protein (10 μ M each) was pre-incubated with 0, 0.2, 0.5. 1 , 2, 5, 10 molar equivalents of Ni-A .-i- -/C overnight at 4°C. The covalent linkage was achiev ed v ia photo- activ ation under ultrav iolet radiation at 365 nm using a UVP UVGL-25 Mineralight UV lamp for 1 0 minutes at room temperature. SDS-PAGE analysis was performed using 15% resolving gel. Fluorescence gel images were captured by ImageQuant 50 (GE Healthcare) (λ εχ =365 nm, >. tn 460-500 nm). The denaturing gel was stained by Coomassie blue afterwards for comparison. The labeling yield was obtained by quantifying the areas of protein bands after fluorescence and Coomassie blue staining using Image J and was normalized against the maximum intensity.

His-XPA 1 22 protein (10 μΜ each) was incubated with or without various ratios of -NTA-AC in 20 mM HEPES, 100 mM NaCl, pl l 7.4. then were analyzed by Ultraflex I I TOF/TOF MALDI-TOF MS (Bruker). The labeling efficiencies were evaluated using the peak areas via ImageJ .

Con focal Imaging and in vivo study of E. oli. pET-His-XP A 122 or pET32a (as a control) transformed BL21 (DE3) cells were cultured in Luria-Bertani ( LB) overnight containing 34 n ml. kanamycin (for pET- His-XPA 1 22) or 100 ng mL ampicil lin (for pET32a) at 37°C, and then subcultured by 1 : 100 dilution. When the OD 600 reached 0.6, isopropyl β-D-thiogalactopyranoside (IPTG) (0.2 mM) was added to induce the protein expression at 16°C ov ernight. ~ Ni-NTA-AC (10 M ) was added and further incubated in the dark. The E. coli cells with or without His-XPA 1 22 overexpression were then washed w ith 50 mM HEPES, 1 00 mM NaCl, pH 7.3 at 4°C. The OD 600 were adjusted to 0.3 with HEPES buffer and propidium iodide (PI) (1 [ig ' mL) was added to the samples prior to imaging to examine the viability of the ceils. Images were captured on a Carl Zeiss LSM700 Inverted Confocal Microscope with the use of a 405 nm laser (for excitation) and a 555 nm laser (for excitation of propidium iodide ( PI)). Cells were visualized using a Plan-Apochromat 63x 1.40NA oil-immersion objective for fluorescent and phase contrast imaging respectively. Cell v iability was determined by the measurement of the percentage of no n- PI -stained dead cells among His-XPA 122-expressed E. coli captured by confocal imaging (n 5) upon incubation with different concentrations of Ni-,V7M-.-iC (0-100 μΜ).

Confocal Imagi g of Transfected HeLa Cells. After transient transfection of I lei. a cells on confocal dishes, cells were washed once with HBSS, and the solution was replaced by HBSS preloaded with 25 uM of Ni- NT A- AC to further incubate for 30 minutes. The buffer solution was then discarded and cells were washed ith HBSS for three times and subjected to confocal imaging. Fluorescent and phase contrast images were captured on a Carl Zeiss LSM700 Inverted Confocal Microscope using 405 nm and 555 lasers under a Plan- Apochromat 63 x 1.40NA oil-immersion objective, while the measuring ranges of emission (430 - 500 nm) were fixed ( for emission of the probe) and 575 - 700 nm ( for mRFP emission). To quantify the time required to label intracellular targets by the probe, His-m FP transfected HeLa cel ls were washed once w ith HBSS, subjected to confocal microscope and imaging was initiated every 30 seconds after 25 μΜ of Ni-NTA-AC was supplemented to the cells. The probe fluorescence intensity from each transfected cell was quantified by Zen software (Carl Zeiss) and plotted.

Measurement of Cell Viability. HeLa cells were seeded into 96-well plates ( 10,000 cells/well) and incubated with respective medium overnight at 37°C w ith 5% C0 2 . The medium was replaced and the ceils were incubated with different concentrations (0, 25 and 50 uM) of Ni-,Y7>i-.-iC in medium for 30 minutes at 37°C and protected from l ight. The medium was then removed and replaced w ith fresh medium, then 10 L of (3-(4,5- dimethylthiazoi-2-yl)-2,5-diphenyl-tetrazoiium bromide ( M IT, 5 mg/mL in sterile PBS) were added and further incubated for 4 hours. After removing all but 25 L medium, 50 u L of DM SO were applied to incubate for 10 minutes at 37°C. Absorbance of each well was recorded at λ = 540 nm using a microplate reader ( BIO-RAD, iMark 1 ), and cell v iability was reported relativ e to those of untreated cells. Confocal Imagi g of Tobacco Plant Cells. Transplastomic Nicotiana tabacum var. Xanthi (tobacco) His-BjCHIl plants expressing His-tagged Brass tea juncea chitinase BjCHIl were used (Guan Y, amalingam S, Nagegowda D, Taylor PWJ, Chye M-L (2008) J Exp Bot 59(12):3475-3484). Tobacco seeds were surface sterilized, sown on Murasliige and Skoog (MS) medium supplemented with 2% sucrose and grown as previously reported (Guan Y, Ramalingam S, Nagegowda D, Taylor PWJ, Chye M-L (2008) J Exp Bot 59(12):3475-3484). Protoplasts were extracted from leaves of 4-week-old wild-type and His-BjCHIl tobacco plants according to a previous protocol (Papadakis AK, Siminis CI, Roubelakis-Angelakis KA (2001) Plant Physiol 126(l):434-444). Lower epidermis of leaves was pealed and incubated 3 hours with the extraction solution. The isolated cells were incubated with ΉΊ-ΝΤΑ-AC (10 μΜ) for 30 minutes and applied on microscopic slides for imaging. For applications in living whole plants, seven-day-old seedlings grown on MS medium were transferred to PBS buffer (pH 7.4) supplemented with Ήι-ΝΤΑ-AC (10 μΜ) to immerse roots for 24 hours. The seedlings were then washed with PBS buffer and blotted dry before imaging. Tobacco plant images were captured on a Carl Zeiss LSM7 10 NLO Inverted Confocal Microscope with a Spectra Physics MaiTai HP tunable 2-photon laser (λ £Χ =780 nm and λ 6ΐη =435-470 n , 572-674 nm). Protoplasts were imaged using a 40x1.30 oil-immersion objectiv e lens while images on leaves were captured using a 63x1.40 oil- immersion objective lens.

ImiTiunoblotting. Cells were lysed by sonication in sonication buffer (50 niM

HEPES, pH 7.3, 100 mM NaCl). The cell lysates were separated by 12% SDS-PAGE and were transferred onto a PVDF membrane ( Hybond-P, GE Healthcare). The membrane was blocked with 5% BSA in I B ST buffer (10 mM Tris-HCi, pH 7.6, 1 30 mM NaCl, 0.1% (v/v) Tween-20) for an hour and was incubated with Anti-6X His tag® antibody (Abeam) for an hour at room temperature. The membrane was then incubated w ith horseradish peroxidase- conjugated goat anti-mouse IgG secondary antibody (Abeam) for detection by chemi luminescence with LumiGLO® reagent (Cell Signaling Technology).

EXAMPLES

An advantage of the fluorescent labeling is that the chelation of metal ions by the probe initiates a quenching of fluorescence while the labeling of poly-Histidine -tagged protein elevates the fluorescent signals to generate a "turn-on" response. Measurements on the fluorescence changes of NTA-AC upon the addition of Ni 2+ ions (λ εχ = 342 nm and em = 448 nm) were performed by the titration of Ni 2+ ions with concentration of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15 and 20 μΜ to 5 μΜ of NTA-AC at 25°C, and upon the addition of Ni 2+ , fluorescence intensity of NTA-AC was gradually reduced by 72% (Figure 3B). However, the presence of poly-Histidine- tagged proteins initiates a significant increase in fluorescence accounted to the launching of small-molecule-fluorescent labeling to the protein of interest. In another embodiment, introduction of poly-Histidine -tagged XPA122 protein (His-XPA122) could initiate an elevation

• 2+

of fluorescence of Ni -NTA-AC by 13 times while the complete labeling could be achieved in 9 minutes according to the time-course plot (Figure 7A).

Poly-Histidine-tagged protein His-XPA122 and non-Histidine-tagged XPA122 (12 μΜ)

• 2+

were pre-incubated with equimolar concentration of Ni -NTA-AC for 1 hours at 4°C and subsequently the photoreactive crosslinker was photo-activated by ultraviolet radiation at 365 nm for 10 minutes at room temperature. SDS-PAGE analysis was performed using 15% resolving gel, and fluorescence gel images were captured by ImageQuant 350 from GE Healthcare (λ εχ = 365 nm, em = 460-500 nm). The denaturing gel was stained by Coomassie blue afterwards for comparison, while the presence of poly-Histidine -tag in proteins was assured by Western blotting. Only protein with poly-Histidine -tagged (lane 1) could be fluorescently labeled in comparison to protein with poly-Histidine -tag deleted (lane 4), thus demonstrating the specificity of fluorescent labeling towards poly-Histidine -tag-encoded proteins (Figure 7C).

To test the fluorescent agents' application to living biological samples to target intracellular (and membrane-bound) poly-Histidine -tagged proteins, nickel(II) preloaded probe Ni 2+ -NTA-AC (10 u ) was added directly to the Escherichia coli cultures and further incubated in the dark for an hour in 37°C, and subsequently washed with 20 mM Tris-HCl, 100 mM NaCl, pH 7.2 at 4°C. The densities of the cells were adjusted to 0.3 (OD600) with buffer and propidium iodide (PI) (1 μg/mL) was added to the samples prior to imaging to examine the viability of the cells. Under confocal imaging, only E. coli cells overexpressing poly-Histidine -tagged protein His-XPA122 could be lighted-up in comparison to the pET 32a sample without poly-Histidine - tagged protein overexpression, and the cells were maintained alive throughout the labeling process (Figure 9C). In another experiment, protoplasts of transplastomic Nicotiana tabacum var. Xanthi (tobacco) plants expressing poly-Histidine -tagged Brassica juncea chitinase BjCHIl (termed as His-BjCHIl) were extracted from leaves of 4-week-old wild-type and His-BjCHIl tobacco plants (Figure 11), subsequently the isolated protoplasts were treated with 10 μΜ Ni 2+ - NTA-AC for 30 minutes in the dark under room temperature prior to confocal imaging analysis. An intense blue fluorescence was only received under confocal microscope for the cells expressing His-BjCHIl in comparison to the wild-type (WT) sample, while in addition accounted to the fact that His-BjCHIl was expressed and subsequently accumulated in tobacco chloroplasts (Guan, Y., Ramalingam, S., Nagegowda, D., Taylor, P. W. J. & Chye, M.-L. J. Exp. Bot. 59, 3475-3484 (2008)), co-localization of the blue fluorescence in tobacco protoplasts could be observed with the autofluorescence of chlorophyll, indicating the occurrence of the fluorescent labeling of His-BjCHIl (which only expressed in chloroplasts) by the probe (Figure 11). Labeled plant tissues were directly investigated under confocal microscope in another embodiment, with 7-day-old transplastomic tobacco plant seedlings transferred to PBS buffer (pH 7.4) supplemented with 10 μΜ Ni 2+ -NTA-AC to immerse roots for 24 hours, then washed and blotted dry prior to imaging. The abaxial surface of transplastomic tobacco leaves was analyzed directly under confocal microscope, and again blue fluorescence was observed only in the chloroplasts of guard cells on the tobacco leaf transplastomic for His-BjCHIl , indicating that the probe was up-taken through the roots and the Ni 2+ -binding poly-Histidine-tagged proteins inside living plants could be detected (Figure 10).

Design and Synthesis of Fluorescent Probe Ήι-ΝΤΑ-AC. Previously many fluorescent probes utilized Nr ' -nitri lotriacetate system including <//-, tri- or feira-NTA derivatives conjugated with fluorophores could only be used in label membrane proteins in cells as these probes suffer from poor membrane permeability and could hardly enter cells (Soh N (2008) Sensors 8(2): 1004-1024; Jing C & Cornish VW (201 1 ) Acc Chem Res 44(9):784- 792). 1 is reasoned feat that highly negative charges might prohibit these probes from crossing cell membranes although introduction of m lti-NTA to the probes could overcome the weak binding feature of Ni-NTA with His6-tag. Therefore, a probe was desi gned ( Figure A), consisting of a mono-Ni-ΝΊΑ moiety, a small membrane-permeable fluorophore (a coumarin derivative) (Uttamapinant C, et al. ( 2010) Proc Natl Acad Sci USA 107(24): 10914- 1 091 9) and an arylazide moiety. The Ni- nitrilotriacetate (NTA) will target the His6-tag to achieve specific labeling of a protein of interest, and arylazide group was incorporated into the probe to provide an additional covalent bond between the probe and its target proteins upon photo-activation (Melcher K (2004) Curr Protein Pept Sci 5(4):287-296.), thus to resolve the intrinsic weak binding nature of Ni-NTA with His6-tag. A linker between mono-Ni-NTA and the fluorophore was designed to allow flexibility of the Ni-NTA to facilitate efficient protein labeling, in addition, such a linker could also enhance membrane permeability of Ύϋ-ΝΤΑ-AC during liv e cell labeling, which would be further elaborated (vide infra).

A coumarin-based ligand NTA-AC was first synthesized via a three-step reaction with an overall yield of 64% ( Figure A, Figures B and 5) by conjugating the nitrilotriacetate moiety with a coumarin fluorophore and arylazide. The purity of the compound was confirmed by both ' I I NMR. and ESI- MS. The ligand exhibited a maximum absorption at ca. 342 nm (ε = 1 1 ,100 M ~ 'cm ~ ' ) and emitted at 448 nm ( Φ = 0.056) ( Figure 6). The probe Ni-NTA- AC was then generated by subsequent reaction of NTA-AC with Ni 2+ (as NiS04) in buffered aqueous solution. As shown in Figure 3B, upon addition of equimolar amounts of Ni 2+ to NTA-AC in 20 mM Tris buffer at pH 7.2, the fluorescence was significantly quenched by ca. 70%; in sharp contrast with the 5% reduction observed in previously reported NTA-DCF conjugate (Goldsmith CR, Jaworski J, Sheng M, & Lippard SJ (2006) J Am. Chem Soc 128(2):418-419.), thus ~ Ni-NTA-AC has only very weak emission at 448 nm.. The titration data were nonlincarly fitted using Ryan Weber equation ( Bai YC, et al. (2008) Anal Chim Acta 616(1): 1 15-121.), w hich gave rise to a dissociation constant (A ' d) of 38± 13 iiM. To evaluate the binding stoichiometry, a Job's plot was constructed by monitoring fluorescence changes upon complexation of NTA-AC with Ni 2+ at 448 nm excited at 342 nm in 20 mM Tris buffer, pH 7.2. Maximum fluorescence changes were observed at a molar ratio of Ni 2+ to NTA-AC of 0.5, indicative of the formation of Ni-NTA-AC complex with a ratio of NTA-AC: Ni 2+ of 1 : 1 (Figure 3C). This was further verified by observation of a peak at m/z of 558.6 from ESI-MS, in agreement w ith the calculated value (m/z) of 558.9.

Evaluation of the ΉΙ-ΝΤΑ-AC Probe in Labeling His6-tagged Proteins in vitro.

To examine the feasibility of I i-NTA-AC in labeling a His 6 -tagged protein in vitro, the functional domain of a DNA repairing protein. Xeroderma pigmentosum group A

(XPA.122) was used as a showcase study. Xeroderma pigmentosum, group A serves as the class form of XP proteins, which is important for repairing DNA. damage caused by ultraviolet radiation; the functional domain, X A 122, serves as the site of damaged-DNA binding and thus initiates repairing (Cleaver JE (2005) Nat Rev Cancer 5(7):564-573). The protein with (denoted as His- XPA122) or without (XPA122) genetically fused His6-tag to its N- term in us were overexpressed and purified as described previously ( Supporting Information) ( Kuraoka I, et al. (1996) Mutat Res 362(l):87-95). The interaction of M-NTA-AC probe with the protein was first investigated by fluorescence spectroscopy, incubation of 10 molar equivalents of His-XPA122 with I μΜ of ϋ-ΝΤΑ-AC led to a quick increase in fluorescence intensity with time, reaching a plateau at about 9 minutes, where about 13-fold increase in fluorescence was observed (Figure 7A). In contrast, no obvious fluorescence changes (less than 50% increases) were noted upon mixing of Ni-NTA-AC with XPA122 under identical conditions (Figure 15). Similarly, premixing of the ligand NTA-AC (without coordination of Ni 2+ ) with His-XPA 1 22 under identical conditions resulted in no fluorescence enhancement at all ( Figure 16). These combined results indicate that Ni-NTA- AC selectively targets the His ( ,-tag of the protein through Ni 2† , resulting in fluorescence "turn-on " responses. Nonspecific binding is negligible under the condition used. Although the underlying mechanism of fluorescence turn-on responses of the probe towards His 6 -tagged proteins is not ful ly understood, it is likely that weak interaction between Ni 2+ -NTA and the fluorophore led to a "sandwich-like" structure owing to the presence of a flexible linker as reported previously ( Kamoto , Umezawa N, Kato N, & H iguchi T (2008) Chem Eur J 14(26):8004-8012), which quenched fluorescence of the fluorophore. Such weak interaction might be abolished upon binding of " Ni-NTA-AC to intracellular His 6 -tagged protein, subsequent interaction of the fluorophore with protein targets resulted in significant increases in fluorescence.

The binding properties of Ni-NTA-AC towards His-XPA 1 22 were also studied by isothermal titration calorimetry (ITC), which gave rise to a dissociation constant of 7.1 ± 0.6 μΜ and binding capacity of 1 .4 ± 0.1 ( Figure 17A), in consistence with the weak binding nature of Ni-NTA with histidine residues. The non-integral stoichiometry of Ni- NTA-AC binding to His<,-tag was attributed to the fact that one His6-tag could possibly bind one to two Ni 2+ ions (Valenti LE, De Pauli CP, & Giacomelli CE (2006) J Inorg Biochem 100(2): 192-200; Knecht S, Rickiin D, Eberle AN, & Ernst B (2009) J Mol Recognit 22(4):270- 279). The weak binding might lead to dissociation of the probe from labeled proteins in the complex environment of live cells. To tackle this problem, aryiazide was incorporated with the probe for the purpose of providing additional binding between the probe and its target proteins upon photoactivation. The role of arylazide was examined subsequently. Firstly, the mixture of 1 μΜ of Ni-A -i-.-iC and 10 molar equivalents of His-XPA 1 22 was subjected to irradiation under UV light (365 nm) for 15 min to ensure photoactivation of arylazide. A significant fluorescent enhancement (over 10-fold) compared to the fluorescence of Ni-NT.-i-AC was observable owing to the probe binding to His- XPA 1 22 (Figure 7B). Upon addition of 40 molar equivalents of ethylenediaminetetraacctate (EDTA) to the mixture to strip off Ni 2" from the probe-protein complex, the observed fluorescent intensities, instead of being decreased, were sl ightly increased (ca. 30%), attributable to recovery of the fluorescent being quenched by Ni 2" ( Figure 7B). Subsequently, a similar experiment was carried out in darkness to prevent photoactivation of arylaizde, and the results showed a signi ficant decrease in fluorescence (by -60%) ( Figure 7B), indicating that without photoactivation of arylazide, removal of Ni 2† from the probe-His-XPA122 complex abolished the capability of the probe in imaging His 6 - tagged proteins. Based on these data, we concluded that incorporation of arylazide to the probe could overcome the weak binding nature of Ni-NTA towards histidine residues.

The capability of arylazide for strengthening the binding between the probe and His-tag proteins upon photoactiv ation was also manifested by observation of the probe - protein complex under denatured conditions. The ϋ-ΝΤΑ-AC labeled His-XPA 1 22 was irradiated with 11 V light (365 nm) for 10 minutes by 4W Longwave Compact UV lamp (720 PW cm ), then subjected to SDS-PAGE electrophoresis. An intense blue fluorescent band corresponding to His-XPA 1 22 was observable in the SDS-PAGE gel, corroborated with strong binding of the probe to His-XPA 1 22 even under denatured conditions. In contrast, no corresponding blue fluorescent band was detected when Ni- NT A- AC ( 12 μΜ) was mixed with equi molar amounts of XPA 122 or His-XPA 1 22 in the presence of 0 μΜ of EDTA, which removed Ni ! from the probe ( Figure 7C), in line w ith the above observation that the probe targets to the Hisg-tag of XPA 1 22 through Ni 2+ and subsequent photoactivation of the arylazide group strengthens binding between the probe and the protein.

To further ev aluate the role of arylazide of the probe, a coumarin-based ligand w ithout arylazide attached was synthesized, namely NTA-C, for comparison via a three-step reaction (Figures 18-20). Addition of equimolar amounts of Ni 2" led to ca. 50% fluorescence reduction for NTA-C ( Figure 21 A ). Unexpectedly, incubation I μΜ of Ni-NZ¾-C with His-XPA122 under similar conditions as the probe resulted in no fluorescence increase (Figure 21AB) though Ni- VXi-C still binds to His-XPA122 with an affinity similar to the probe ~ Ni-NTA-AC as evidenced from ITC titration data (Figure 17B). Similarly, no evident blue fluorescence was detected on SDS-PAGE when Ni-NTA-C ( 12 μΜ) was mixed with equi molar amounts of His-XPA I 22 ( Figure 7C). These results demonstrated that arylazide is benefic ial not only for strengthening binding between the probe and its targets, but also for significant fluorescence "turn-on" responses towards His 6 - tagged proteins.

The labeling efficiency of ~ Ni-NTA-AC to His 6 -tagged proteins wa.s further ev aluated by monitoring its reactivity towards His-XPA I 22. His-XPA I 22 ( 10 μΜ) was incubated with different molar equivalents of ~ Ni-NTA-AC, photoactiv ated and subjected to SDS-PAGE electrophoresis ( Figure 8A). By comparing the fluorescence intensities of the protein bands to that of Coomassic staining, it was determined that one molar equiv alent of ~ Ni-NTA-AC could label -50% of His- XPA 122 ( Figure 8B) The labeling efficacy using the MALDI- TOF mass spectrometry was also examined. Equimolar of ~ Ni-NTA-AC and His-XPA I 22 was incubated and then photoactivated before subjecting to MALDI-MS. In the spectra ( Figure 8C), two ion peaks at m/z 14981 and 15549 were observed, corresponding to the intact protein and the protein with probe bound (calcd. 14979 and 15541). The intensity of the peak at m/z 15549 Da relativ e to that of the peak at m/z 14981 further increased upon addition of two molar equivalent of the probe to the protein solution. A weak peak at m/z 1 1 00 is assignable to the protein w ith two probes bound. Through comparing the peak area, it was found that 38% and 62% of His-XPA 1 22 were labeled upon addition of 1 and 2 molar equiv alents of Ni-NTA-AC respectively ( Figure 8C), in consistent with the results determined from SDS-PAGE ( Figures 8A-8B).

valuation of the Ni-NTA-AC Probe Membrane Permeability and Toxicity. The cell permeability of N\-\ TA-AC in live mammal ian cells was investigated. A His ( ,-tag w a s g e n e t i c a 1 1 y fus e d to the N-terminus of red fluorescent protein ( RFP) ( His-RFP) and transiently expressed this fusion protein in He La ceils. The fluorescence responses to the His-R P transfected cells upon administration of Ni-NTA-AC (25 μΜ) were monitored under confocal microscope using the red fluorescence as a reference. Given that arylazide can readily be photoactivated under 405 11m laser utilized by confocal microscopy for capturing image, no additional UV irradiation was used in all cell imaging experiments. Cellular autofluorescence was minimized through optimizing imaging parameters ith the cells in the absence of ~ Ni-NTA-AC. As shown in Figure 9 A, B and Figure 22, blue fluorescence appeared immediately upon treatment of Ni-NTA-AC and reached saturation within 2 minutes, indicating that the probe can rapidly cross cell membrane and exhibit turn-on fluorescence upon binding to I lis-RFP. In contrast, no blue fluorescence was observed after treatment of NTA-AC, i.e. without Ni 2† coordination, indicating that NTA-AC itself failed to enter the cells ( Figure 23). This is consistent w ith our probe design rationale that the charge might be an important factor determining the membrane permeability of a probe. In the absence of Ni , the charged hydrophilic NTA moiety remained exposed, and thus prohibiting NTA-AC from crossing cell membranes. Upon coordination of Ni 2† to the NTA moiety, the overal l charge reduced dramatically and moreover, a "sandwich-like " structure was probably formed owing to the w eak interaction between Ni-NTA and the fluorophorc as well as flexible linker ( Kamoto M, Umezawa N, Kato N, & Higuchi T (2008) Chem Eur .1 14(26): 8004-8012 ), leading to Ni-NTA moiety to be "buried " , which enabled ϋ-ΝΤΑ-AC to cross the hydrophobic cell membranes.

The toxicity of the probe in bacterial and mammalian cells was also examined. The viability of E. coli reached ca. 99%+/- 1% even w hen 100 μΜ of Ni-.Y7M-.-iC was incubated with the cells ( Figure 24). The v iability of HeLa cells investigated by MTT assay showed that over 90% cells are live upon incubation w ith 25 and 50 μΜ ϋ-ΝΤΑ-AC, again confirming that the probe exhibits no toxicity towards the cells ( Figure 25).

Evaluation of the Ni-NTA-AC Probe for Labeling of His-tagged Proteins in E. coli.

To examine the feasibility of the probe in imaging His-tagged proteins in live cells, the applicability of Ήι-ΝΤΑ-AC for labeling of His-XPA122 in E. coli cells was investigated. The probe (10 μΜ) was incubated with E. coli cells overexpressing His-XPA122 for 30 minutes at 37°C, then subjected to confocal imaging (n= 5) after washing with HEPES buffer at pH 7.4. As shown in Figure 9C, E. coli cells overexpressing His-XPA122 were stained with blue fluorescence, and subsequent SDS-PAGE analysis of the cell lysates showed that only one protein band with molecular weight of about 15 kDa exhibited blue fluorescence, verifying that indeed His-XPA 122 is the only protein being labeled ( Figure 26). In contrast, cells without His- XPA122 expression exhibited no blue fluorescence, revealing the feasibility of the probe in labeling of intracellular His-tagged proteins in live bacterial cells. The viability and membrane integrity of these cells were also examined by propidium iodide (PI) staining. As shown in Figure 9C, no cells were stained in red by PI, suggesting that they are live cells.

Imaging His-tagged Proteins in Live Mammalian Cells using the Ni-NTA-AC Probe. The capability of the probe to label His-tagged proteins inside mammalian cells was investigated. The HeLa cells with or without His-XPA122 transfection were supplemented with 25 μΜ of Ni-NTA-AC in HBSS buffer for 30 minutes at 37°C, washed and subjected to confocal imaging. As shown in Figure 9D, an intense blue fluorescence located mainly at the nuclei was observed only in the cells transfected with His -XP A 122, but not in those cells without transfection. To further confirm the identity of the labeled protein, cells with or without His-XPA122 transfection were treated with the probe (25 μΜ) and were radiated at 365 nm for 10 minutes, enabling photo-activation of arylazide, the nuclei of transfected and un- transfected HeLa cells were extracted and concentrated, and subsequently subjected to fluorescence imaging and western blotting (Figure 9E). For comparison, the purified His- XPA122 labeled with Ni-NTA-AC (5 μΜ) was also studied. The blue fluorescent and western blotting bands from the nucleus of His-XPA122 transfected cells are similar to those of purified His-XPA122, confirming that the labeled protein was indeed the His-XPA122 expressed in HeLa cells. In contrast, no corresponding bands were observed for the nucleus of un -transfected cells (Figure 9E).

Fluorescent proteins have been widely used to study protein function, localization as well as other biological events in the physiological context of living cells when genetically fused to the protein of interest (Lam AJ, et al. (2012) Nat Meth 9: 1005-1012.). However, the use of fluorescent proteins might potentially interfere with the proper localization or function of the protein of interest due to its large size (Giepmans BNG, Adams SR, Ellisman MH, & Tsien RY (2006) Science 312(5771):217-224), in particular, for relatively small proteins. On the contrary, small molecule-based fluorescent probes might have advantages on this issue. To demonstrate this, His 6 -tagged RFP was incorporated into the N-terminus of His 6 -tagged XPA122 to generate His 6 -RFP-His 6 -XPA122 plasmid, subsequently transfected into HeLa cells to investigate the cellular localization of the protein under identical conditions. Both blue fluorescence (due to ~ Ni-NTA-AC labeling) and red fluorescence (due to the expression of RFP) were observed with co-localization (Figure 9F). Interestingly, the fluorescence signals, instead of being enriched in the nucleus as found for His-XPA122 (Figure 9D), were distributed evenly throughout the cells. The perturbation of the protein localization may be attributed to the relatively large size of RFP (27.5 kDa) (Shaner NC, Steinbach PA, & Tsien RY (2005) Nat Meth 2(12):905-909) compared to XPA122 (15 kDa). By contrast, using the Ni-NTA-AC probe, His-XPA122 was found to be enriched in the nucleus, which is in agreement with the previously reported intracellular localization of XPA, a protein involved in the recognition of DNA damage during nucleotide excision repair processes (Uchinomiya S, Nonaka H, Wakayama S, Ojida A, & Hamachi I (2013) Chem Commun 49(44):5022-5024; Kuraoka I, et al. (1996) Mutat Res 362(l):87-95). Taken together, it is demonstrated that the new fluorescent probe ΉΊ-ΝΤΑ-AC could be preferentially applied to track the abundance and localization of His-tagged proteins in live mammalian cells, and in particular for small proteins.

Application of Ni-NTA-AC Probe for Imaging of His-tagged Protein in Plant Tissues. It is shown that Ni-NTA-AC could label proteins in other eukaryotic systems. Transplastomic Nicotiana tabacum var. Xanthi (tobacco) plants expressing His-tagged Brassica juncea chitinase BjCHIl (His- BjCHIl) were generated as described previously (Guan Y, Ramalingam S, Nagegowda D, Taylor PWJ, & Chye M-L (2008) J Exp Bot 59(12):3475-3484). Protoplasts were extracted from leaves of 4-week-old wild-type and His-BjCHIl transplastomic tobacco (Figure 11) according to a previous procedure (Papadakis AK, Siminis CI, & Roubelakis-Angelakis KA (2001) Plant Physiol. 126(l):434-444). Upon incubation of protoplasts with ~ Ni-NTA-AC (10 μΜ) for 30 minutes, blue fluorescence was detected in the chloroplasts (Figure 11), where His-BjCHIl was expressed and subsequently accumulated (Guan Y, Ramalingam S, Nagegowda D, Taylor PWJ, & Chye M-L (2008) J Exp Bot 59(12):3475-3484). Co-localization of blue fluorescence with red autofluorescence of chloroplasts under confocal microscope was only seen in cells expressing His-BjCHIl , but not in the wild-type (WT), indicative of labeling of His-BjCHIl in the chloroplasts by Ni-NTA-AC (Figure 11). Furthermore, Ni-NTA-AC (10 μΜ) was added into PBS buffer (pH 7.4) for root immersion of 7-day- old transplastomic seedlings overnight, which were washed and blotted dry prior to imaging. The abaxial surface of transplastomic tobacco leaves was subjected to confocal microscopy and blue fluorescence was again observed in tobacco leaves expressing His-BjCHIl (Figure 10), suggesting that the probe were taken up through the roots of seedlings and the His-tagged proteins inside living leaves were subsequently be detected. These results clearly demonstrated that the probe, Ni-NTA-AC, could readily be extended to label His-tagged proteins in various eukaryotic cells including plant tissues.

The previous examples illustrate embodiments of the subject invention. Unless otherwise indicated in the examples and elsewhere in the specification and claims, all parts and percentages are by weight, all temperatures are in degrees Centigrade, and pressure is at or near atmospheric pressure.

With respect to any figure or numerical range for a given characteristic, a figure or a parameter from one range may be combined with another figure or a parameter from a different range for the same characteristic to generate a numerical range.

Other than in the operating examples, or where otherwise indicated, all numbers, values and/or expressions referring to quantities of ingredients, reaction conditions, etc., used in the specification and claims are to be understood as modified in all instances by the term "about."

DISCUSSION

Chemical and biochemical labeling of proteins serves as a powerful tool for elucidation of protein functions, localization, dynamics as well as other biological events in liv e cells (Giepmans BNG, Adams SR, Ellisman MH, & Tsien RY (2006) Science 3 12( 577 1 ):2 1 7-224; Sletten EM & Bertozzi CR (2009) Angew Chem Int Ed 48(38):6974~ 6998; Uttamapinant C, Sanchez MI, Liu DS, Yao JZ, & Ting AY (2013) Nat Protoc 8(8): 1620- 1634). Small molecule -based fluorescent labeling of recombinant proteins holds particular promise as an alternative to fluorescent protein (FP)-flxsion technology (Tsien RY (1998) Ann Rev Biochem 67:509-544; Shaner NC, Steinbach PA, & Tsien RY (2005) Nat Meth 2( 12):905-909), without deleterious perturbation of protein functions. The introduction of a short peptide to a protein of interest that is able to site-specifically bind with a designed synthetic fluorescent probe is representative techniques, allowing in vivo functional protein to be analyzed Over the last several decades, there has been tremendous progress in using small molecule -based probes to monitor cellular events (Ueno T & Nagano T (201 1 ) Nat Meth 8(8):642-645; Marks KM & Nolan G (2006) Nat Meth 3(8):591 -596), in particular, metal-che!ation labeling of a protein appears to be one of attractive approaches owing to its simplicity and high specificity. Amongst large quantities of small molecule-based probes, FlAsH and its derivatives including RcAsH and SplAsH appears to be ones of the most successful small-molecules based metal-containing probes, which have been frequently used to light-up intracellular proteins fused with a tetracysteine motif (Giepmans BNG, Adams SR, Eilisman MH, & Tsien RY (2006) Science 1 2(5771 ):2 1 7-224; Hoffmann C, et al . (2010) Nat Protoc 5( 10) : 1666- 1677; Adams SR & Tsien RY (2008) Nat Protoc 3( 9): 1 527- 1 534). Despite that this system has been pointed out as h a v i n g several limitations, such as the requirement of extensive washing for reducing background (Stroffekova K, Proenza C, & Beam K (2001 ) Pflugers Archiv 442(6):859-866) or its inability to apply to cellular oxidizing environment (Soh N (2008) Sensors 8( 2): 1 04- 1024), the development of biarsenical-based fluorescent probes serves as an important work that has inspired researchers to design various probes that target other tagging system.

Given the wide utility of (histidine)rt-Nr ' -nitrilotriacetate system (Ni 2† -NTA.) in molecular biology and biotechnology for affinity-chromatography-based protein purification, this system has also been extensively exploited previously to site-selectively label large library of existing hexahistidine -tagged (His-tagged) proteins via conjugation with a fluorophore (Guignet EG, Hovius R, & Vogel I I (2004) Nat Biotcchnol 22(4):44()-444; Meredith GD, Wu HY, & Al lbritton NL (2004) Bioconiugate Chem 15(5):969-982; Mauser CT & Tsien RY (2007) Proc Natl Acad Sci USA 104(10):3693-3697; Hintcrstcincr M, et al. (2008) ChemBioChem 9(9): 1391 -1395; Uchinomiya S, Nonaka H, Wakayama S, Ojida A, & Hamachi I (2013 ) Chem Com num. 49(44): 5022-5024). Various NTA- based fluorescent probes have been synthesized via conjugation of fluorophores with mono-ΝΊ A (Guignet EG, Hovius R, & Vogel I I (2004) Nat Biotcchnol 22(4):440-444; Goldsmith CR, Jaworski J, Sheng M, & Lippard SJ (2006) J Am Chem Soc 1 28(2 ):4 18-419) or di-, tri- and t tra-NW derivatives to either mimic the concept of FlAsH or overcome the weak binding nature of His-tag with

Ni -NTA (Kd= 13 μΜ) (Soh N (2008) Sensors 8(2): 1004-1024; Jing C & Cornish VW (2011) Acc Chem Res 44(9):784-792; Uchinomiya S, Ojida A, & Hamachi I (2013) Inorg Chem 53(4): 1816- 1823; Lata S, Gawtis M, Tampe R, & Piehler J (2006) J Am. Chem Soc 128(7):2365-2372; Kapamdis AN, Ebright YW, & Ebright Rf l (2001) J Am Chem Soc 123(48): 12123- 12125). Despite signi ticant increase in stability of the multiple chelator heads His-tag complex compared with mono- NT A, highly negative charges of these moieties might prev ent them from entering cells. Indeed, all Ni-NTA-based fluorescent probes reported so far that target His6-tagged proteins are exclusively limited to label membrane proteins, as none of them could cross cell membranes to label intracellular proteins in live cel ls (Hintersteiner M, et al. (2008) ChemBioChem 9(9): 1391 -1395; Goldsmith CR, Jaworski J, Sheng M, & Lippard SJ (2006) J Am. Chem Soc 128(2):418-419). A prev ious claim that a J/-NTA dibromobimane conjugate entered cells to target polyhistidine containing protei s is not convincing as the fluorescence measurement was carried out using whole cells and their results were not corroborated with in vivo cel l imaging data ( rishnan B, Szymanska A, & Gierasch LM ( 2007 ) Chem Biol Drug Pes 69( 1 ):3 1 -40). Therefore it is highly possible that the observed fluorescence upon treatment of whole cells with the probe would most likely be derived from bindi ng of polyhistidinc-containing proteins from cel l membranes.

Recently a J/- TA-based fluorescent probe containing an a-chloroacetamide, which targets the Cys-appended His-tag (Cys-His 6 -tag), was reported to be able to label intracel lular Cys-His 6 - tagged proteins in liv e cells. ( Uchinomiya S, Nonaka H, Wakayama S, Oj ida A, & Hamachi I ( 201 3 ) Chem Commun 49(44):5022-5024). However, the <//-NTA. fluorophore conjugate itself could hardly entered ceils, unless the conjugate was attached with a cargo consisting o a cell-penetrating peptide and fluorescent quencher (a dabsyl- appended icosapcptide with a dabsyl group, a tetra-His and an octa-Arg), enforcing the probe to enter cells to label Cys-His 6 -tagged instead of His6-tagged proteins. Apart from that the probe is relatively large and much more compl icated to make, the method precludes its use in labeling many existing His6-tag libraries without re-subcloning every single gene. Moreover, the relativ e slow kinetics on the labeling (with 80% yield within an hour) also prevents its use for real-time imaging of proteins. Therefore, it is highly preferable to design a smal l and si mple fluorescent probe with good membrane permeabi lity to rapidly label any intracellular proteins as long as fused with a His 6 -tag.

This disclosure presents the design, synthesis, and applications of a new fluorescent probe Ni-N7¾- AC which exhibits excellent membrane permeability, can rapidly enter cel ls to image intracellular His 6 -tagged proteins (Figure 3A). The probe targets a His 6 -tagged protein specifically through Ni 2† - NTA with ca. 1 3 -fold fluorescence turn -on responses. An aryiazide was incorporated into the probe initially with the purpose of overcoming weak binding nature between Ni 2+ and histidines, and unexpectedly, it is also indispensable for fluorescent enhancement. Our probe can be used to image His-tag proteins in different types of cells, ev en in plant tissues. The abi l ity to rapidly visualize intracel lular proteins genetically fused with His<,-tag offers great potential for spatial and functional analysis of vast amounts of existing His 6 -tagged proteins in different types of live cells.

Conclusions. Enormous success of the (histidine)rt-Nr ' -nitrilotriacetate system ( Ni- NTA) in protein purification aroused great interest in the development of small Ni-NTA based fluorescent probes to image His-tagged proteins. Given the existing large libraries of His-tagged proteins, such probes offer a wide utility and versatility in functional investigation of various intracellular proteins under physiologically relevant conditions. The present invention discloses the first smal l cel l permeable fl uorescent probe Ni-NTA-AC, which enables rapid labeling (approximately 2 minutes) and observation of intracellular His-tagged proteins in many different types of l ive cells, and even in plant tissues, with a plethora of potential applications. The probe exhibits high specificity and labeling efficiency towards intracellular His-tag proteins. Moreover, the probe poses less perturbation on protein function and localization in live ceils owing to its small size, an d it has significant advantages over large fl uorescent proteins when label ing smal l proteins. This invention opens up a highly informative approach for the in situ analysis of spatial distribution and function of all proteins in different types of cells.

While embodiments of the invention have been explained, it is to be understood that various modifications thereof will become apparent to those skilled in the art upon reading the specification. Therefore, it is to be understood that the invention disclosed herein is intended to cover such modifications as fall within the scope of the appended claims.

All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification.

It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application.