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WO2015097313A1 - Sondes fluorescentes basées sur la chélation de métaux pour le marquage de protéines ou d'autres biomolécules dans des cellules - Google Patents

Sondes fluorescentes basées sur la chélation de métaux pour le marquage de protéines ou d'autres biomolécules dans des cellules Download PDF

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WO2015097313A1
WO2015097313A1 PCT/EP2015/050063 EP2015050063W WO2015097313A1 WO 2015097313 A1 WO2015097313 A1 WO 2015097313A1 EP 2015050063 W EP2015050063 W EP 2015050063W WO 2015097313 A1 WO2015097313 A1 WO 2015097313A1
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fluorescent
metal
nta
protein
poly
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Hongzhe SUN
Yau Tsz LAI
Ya YANG
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HGF Ltd
University of Hong Kong HKU
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HGF Ltd
University of Hong Kong HKU
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    • G—PHYSICS
    • G03—PHOTOGRAPHY; CINEMATOGRAPHY; ANALOGOUS TECHNIQUES USING WAVES OTHER THAN OPTICAL WAVES; ELECTROGRAPHY; HOLOGRAPHY
    • G03C—PHOTOSENSITIVE MATERIALS FOR PHOTOGRAPHIC PURPOSES; PHOTOGRAPHIC PROCESSES, e.g. CINE, X-RAY, COLOUR, STEREO-PHOTOGRAPHIC PROCESSES; AUXILIARY PROCESSES IN PHOTOGRAPHY
    • G03C1/00—Photosensitive materials
    • G03C1/695—Compositions containing azides as the photosensitive substances
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • 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.
  • metal chelation based fluorescent labeling has the advantage of high selectivity, small size and covalent labeling.
  • 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).
  • NTA Ni 2+ -nitrilotriacetic acid
  • 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)).
  • 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
  • biarsenical probes involve 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)).
  • metal tunable probes which achieve high throughput for the visualization and subsequent identification of labeled proteins.
  • 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.
  • the metal-chelated probes serve as potential probes to label poly-Histidine -tagged proteins/ biomolecules.
  • the probes are also able to covalently bind to labeled proteins, enabling further protein identification.
  • 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.
  • 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.
  • fluorescent probes for targeting biomolecules in biological samples, particularly living samples 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.
  • 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.
  • 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.
  • the linker between the fluorophore and the metal-chelating moiety is designed as a hydrocarbon chain or a peptide sequence.
  • 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.
  • the chelation of metal ions including nickel(II), cobalt(II) and copper(II) ions generate a fluorescent quenching of the metal-chelated probes.
  • the probes coordinate to metal ions in 1 : 1 molar ratio.
  • 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.
  • the labeling process takes about 5-30 minutes.
  • the label is stable for overnight incubation.
  • 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.
  • the fluorescent labeling of poly-Histidine -tagged proteins is able to be visualized on gels after native or denaturing gel electrophoresis.
  • 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.
  • the fluorescent probe generates a fluorescent signal after absorption of optical energy having a wavelength of about 400 to about 800 nm.
  • labeling of poly-Histidine -tagged proteins is achieved in biological samples comprising bacterial cells, mammalian cells, mammalian tissues, plant cells, and plant tissues.
  • the introduction of the fluorescent probes does not damage the biological samples.
  • 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.
  • the method includes washing by a buffer and/or undergoing confocal imaging.
  • fluorescent agents having the chemical structure represented in FIG.l contain:
  • 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;
  • NTA nitrilotriacetic acids
  • IDA iminodiacetic acid
  • 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;
  • a linker between the fluorophore and the metal-chelating moiety which can include a short hydrocarbon chain or a short peptide sequence;
  • 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.
  • 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.
  • 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.
  • Figure 1 illustrates a schematic diagram of the fluorescent agent design.
  • Figure 2 illustrates the structure of metal-chelated N73 ⁇ 4 - ⁇ 4C and NTA-AF.
  • Figure A shows a schematic diagram demonstrating intracellular labeling of I Iis6- tagged proteins using ⁇ - ⁇ -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 ⁇ ) upon addition of Ni 2+ (as NiS0 4 ). About 70% decrease in fluorescence was noted upon Ni 2+ chelation with NT A- AC.
  • the total concentrations of NTA-AC and Ni 2+ were kept constant (10 ⁇ ).
  • 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 7 A shows the fluorescence spectra of Ni- V7>i-.-iC (1 ⁇ ) at different time intervals after addition of His-XPA 122 (10 ⁇ ).
  • Time-dependent fluorescence changes ( ⁇ ⁇ 448 nm) of NI-NT4-AC upon binding to His-XPA 122.
  • ⁇ ⁇ 448 nm
  • 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
  • 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 ⁇ ); Lane 3: His-XPA122 and Ni-A -i-C (w ithout arylazide); Lane 4: X PA I 22 (without His-tag).
  • Figure 8A shows the labeling efficiency of Ni-NTA-AC to His-XPA 1 22 by SDS- PAGE analysis of protein labeling (10 ⁇ 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 ⁇ ) 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.
  • the labeling efficiency was ev supered 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 A I mages of N i - ⁇ ' 7 ' .- ⁇ -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.
  • 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 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 ⁇ - ⁇ -AC (1 ⁇ ) to concentrations of XPA122 ranging from 0 to 10 ⁇ . ⁇ - ⁇ -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 ⁇ ) to concentrations of His-XPA ! 22 ranging from 0 to 10 ⁇ . 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 ⁇ - ⁇ .- ⁇ - (500 ⁇ each ) was injected stepwise into the cell containing apo-His-XPA 1 22 (35 ⁇ ) 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.
  • 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 ⁇ )
  • Figure 25 shows a graph of the toxicity determination of Ni-NTA-AC in HeLa c e l ls .
  • 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 ⁇ ) 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.
  • 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.
  • biomolecule refers to molecules produced by living organisms. Examples used herein include, but are not limited to, polypeptides, proteins, nucleic acids and lipids.
  • target biomolecule 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.
  • 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.
  • photoreactive crosslinkers include, but are not limited to, arylazides, azido-methyl-coumarins, benzophenones, anthraquinones, certain diazo compounds, diazirines, and psoralen derivatives.
  • the probes have arylazides in their backbone as crosslinkers.
  • arylazides also called phenylazides, refers to compounds containing an aryl group directly linked with an azide group.
  • 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 ⁇ 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • UV ultraviolet
  • visible light visible light
  • microwave and infra-red ultraviolet
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • turn-on refers to the nondestructive and prompt detection of fluorescence enhancement.
  • fluorescence of metal-chelating probes was quenched upon chelating metal ions, for example nickel(II) ion.
  • quench herein refers to the rapid vanishing of fluorescence, which is also known as fluorescence turn-off.
  • 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.
  • physiological conditions 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.
  • suitable buffer solutions for example, pH 7.2-7.4, temperature 20-40°C and atmospheric oxygen concentration.
  • 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.
  • 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.
  • confocal imaging refers to fluorescent imaging performed with confocal dish on.
  • a Carl Zeiss LSM700 Inverted Confocal Microscope with a Plan-Apochromat 63x 1.40NA oil-immersion objective may be utilized.
  • 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.
  • carrier molecule 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.
  • detectable response refers to a change in or an occurrence of, a signal that is directly or indirectly detectable either by observation or by instrumentation.
  • 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.
  • 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 ⁇ - ⁇ -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.
  • 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).
  • a reporting moiety a fluorophore
  • metal-chelating moiety linked to the fluorophore by a linker
  • 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).
  • 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.
  • 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)).
  • 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.
  • 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.
  • introduction of poly-Histidine -tagged XPA122 protein could initiate
  • 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).
  • the fluorescent agents could apply to living biological samples to target intracellular (and membrane -bound) poly-Histidine -tagged proteins accordingly.
  • biological samples include, but are not limited to, bacterial samples, mammalian cell lines and tissues, in addition to plant cells and tissues.
  • 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).
  • TA metal-chelating nitrilotriacetic acid
  • 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.
  • 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.
  • poly dentate ligands comprise nitrilotriacetic acid (NTA) and iminodiacetic acid (IDA).
  • the linker between the fluorophore and the metal-chelating moiety is a hydrocarbon chain or a peptide sequence.
  • the photoreactive crosslinker comprises arylazide, diazirine, and benzophenone
  • 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.
  • NTA-AC 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.
  • 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.
  • the binding stoichiometry of NTA-AC to Ni ions was determined by
  • concentrations of NTA-AC and Ni ions were kept constantly in a total of 10 ⁇ , with 11 solutions with varied concentrations of NTA-AC and Ni 2+ ions and incubated for 30 minutes
  • 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.
  • NTA-AC pentylazanediyl
  • 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): ⁇ 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.
  • 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.
  • Poly-Histidine -tagged protein His-XPA122 (12 ⁇ ) 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.
  • 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.).
  • the PGR products were ligated into the pcDNA3.1 -(+) vector by T4 ligase ( Life Technologies Corporation) to obtain pcDNA3.
  • 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).
  • DMEM Dulbecco ' s Modified Eagle Medium
  • FBS fetal bovine serum
  • Pen Strep 1% antibiotics
  • 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.
  • HBSS Hank's Balanced Salt Solution
  • XPA I 22 was transformed into Escherichia coli BL2 1 (DE3).
  • Protein expression was induced at 16°C overnight with 0.2 mM isopropyl ⁇ -D- thiogalactopyranoside (IPTG).
  • IPTG isopropyl ⁇ -D- thiogalactopyranoside
  • 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).
  • 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 ⁇ , with various concentrations of NT A- AC and Ni 2 ions and incubation for 30 minutes before each measurement. The changes in fluorescence of ⁇ - ⁇ .- ⁇ -.-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.
  • ITC Isothermal Titration Calorimetry
  • His-XPA 122 Labeling Yield of His-XPA 122 by M-NTA-AC on SDS-PAGE and ALI) I -MS.
  • His-XPA 122 protein (10 ⁇ 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.
  • 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 ⁇ 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 .
  • 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.
  • PI propidium iodide
  • 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).
  • 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 ⁇ 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.
  • 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.
  • 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 ⁇ - ⁇ -AC (10 ⁇ ) 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 ⁇ - ⁇ -AC (10 ⁇ ) to immerse roots for 24 hours.
  • PBS buffer pH 7.4
  • 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 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 ⁇ to 5 ⁇ 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).
  • poly-Histidine-tagged proteins initiates a significant increase in fluorescence accounted to the launching of small-molecule-fluorescent labeling to the protein of interest.
  • introduction of poly-Histidine -tagged XPA122 protein could initiate an elevation
  • Ni 2+ -NTA-AC 10 u
  • PI propidium iodide
  • 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 ⁇ Ni 2+ - NTA-AC for 30 minutes in the dark under room temperature prior to confocal imaging analysis.
  • a probe was desi gned ( Figure A), consisting of a mono-Ni- ⁇ 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.
  • Ni- nitrilotriacetate 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 ⁇ - ⁇ -AC during liv e cell labeling, which would be further elaborated (vide infra).
  • Ni-NTA- AC was then generated by subsequent reaction of NTA-AC with Ni 2+ (as NiS04) in buffered aqueous solution.
  • Ni 2+ as NiS04
  • 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..
  • XPA.122 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).
  • 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.
  • 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.
  • Ni-NTA-AC 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 ⁇ and binding capacity of 1 .4 ⁇ 0.1 ( Figure 17A), in consistence with the weak binding nature of Ni-NTA with histidine residues.
  • ITC isothermal titration calorimetry
  • 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 ⁇ - ⁇ -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.
  • the cell permeability of N ⁇ - ⁇ TA-AC in live mammal ian cells was investigated.
  • RFP red fluorescent protein
  • His-RFP red fluorescent protein
  • 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 ⁇ 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 ⁇ ⁇ - ⁇ -AC, again confirming that the probe exhibits no toxicity towards the cells ( Figure 25).
  • 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.
  • 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).
  • 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 ⁇ ) 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).
  • 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.
  • 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).
  • NTA-based fluorescent probes have been synthesized via conjugation of fluorophores with mono- ⁇ 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
  • 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.
  • the method precludes its use in labeling many existing His6-tag libraries without re-subcloning every single gene.
  • 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-N73 ⁇ 4- 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.
  • 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.

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Abstract

La présente invention concerne des sondes fluorescentes basées sur la chélation de métaux pour l'imagerie de protéines ou d'autres biomolécules intracellulaires dans des cellules vivantes pour surveiller des événements biologiques. Ces sondes peuvent marquer des protéines ou des biomolécules portant une étiquette polyhistidine tout en étant capables de se lier de manière covalente à des protéines marquées pour l'analyse ultérieure de protéines.
PCT/EP2015/050063 2013-12-27 2015-01-05 Sondes fluorescentes basées sur la chélation de métaux pour le marquage de protéines ou d'autres biomolécules dans des cellules Ceased WO2015097313A1 (fr)

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