EP1000172A4 - Detection de cibles au moyen d'une proteine fluorescente verte et de ses variantes fluorescentes - Google Patents

Detection de cibles au moyen d'une proteine fluorescente verte et de ses variantes fluorescentes

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Publication number
EP1000172A4
EP1000172A4 EP19980908588 EP98908588A EP1000172A4 EP 1000172 A4 EP1000172 A4 EP 1000172A4 EP 19980908588 EP19980908588 EP 19980908588 EP 98908588 A EP98908588 A EP 98908588A EP 1000172 A4 EP1000172 A4 EP 1000172A4
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EP
European Patent Office
Prior art keywords
ligand
target
labeled
bind
antibody
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP19980908588
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German (de)
English (en)
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EP1000172A1 (fr
Inventor
Todd W Plaia
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Oncor Inc
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Oncor Inc
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Publication of EP1000172A1 publication Critical patent/EP1000172A1/fr
Publication of EP1000172A4 publication Critical patent/EP1000172A4/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
    • C07D405/12—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings linked by a chain containing hetero atoms as chain links
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N47/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid
    • A01N47/08—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid the carbon atom having one or more single bonds to nitrogen atoms
    • A01N47/28—Ureas or thioureas containing the groups >N—CO—N< or >N—CS—N<
    • A01N47/38—Ureas or thioureas containing the groups >N—CO—N< or >N—CS—N< containing the group >N—CO—N< where at least one nitrogen atom is part of a heterocyclic ring; Thio analogues thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
    • C07D307/02—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings
    • C07D307/04—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having no double bonds between ring members or between ring members and non-ring members
    • C07D307/10—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having no double bonds between ring members or between ring members and non-ring members with substituted hydrocarbon radicals attached to ring carbon atoms
    • C07D307/16—Radicals substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D309/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings
    • C07D309/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings having no double bonds between ring members or between ring members and non-ring members
    • C07D309/08—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings having no double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D309/14—Nitrogen atoms not forming part of a nitro radical
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D309/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings
    • C07D309/16—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member
    • C07D309/28—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/14—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
    • C07D409/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D413/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing three or more hetero rings

Definitions

  • the present invention relates to detection and/or isolation from or in living or non-living organisms of target compounds such as proteins, peptides, DNA, RNA, carbohydrates and the like. More particularly, such compounds are detected and/or isolated by using fluorescent markers incorporating green fluorescent protein or fluorescent variants thereof.
  • Detection, localization and isolation of compounds from or in living organisms is often necessary for a better understanding of such organisms.
  • various procedures have been developed to locate target molecules in biological systems.
  • antibodies which are proteins produced by vertebrates as a defense against infection, recognize specific molecules, i.e., antigens.
  • Antibodies which are labeled with a detectable label such as a radioactive compound or a fluorescent compound are powerful tools for locating particular molecules due to the affinity of such antibodies for- a specific antigen. Once the labeled antibody binds to the antigen, the resulting complex can be detected by observing the site of radioactivity or fluorescence.
  • Nucleic acid probes are used to detect, localize and/or isolate genetic material.
  • Radioactive or fluorescent labels are placed on oligonucleotides to create a detectable labeled probe.
  • Specific genes can be located in cDNA and genomic libraries by use of such probes. More specifically, double stranded nucleic acid is denatured by high temperatures or high pH to dissociate complementary base pairs and divide the double stranded helix into two separate strands. The single strands can be reassociated with themselves or other complementary nucleic acid in a process known as hybridization.
  • gene sequences may be detected by denaturing a quantity of nucleic acid, creating a complementary nucleic sequence to a target portion of the denatured nucleic acid, labeling the complementary nucleic acid sequence with a radioactive substance such as 32 P and allowing the complementary nucleic acid to hybridize with the denatured nucleic acid, thus incorporating the detectable radioactive probe into the double stranded nucleic acid.
  • the labeled nucleic acid known as the probe, allows the target nucleic acid to be localized by observing the site of radioactivity. Labeled nucleic acid probes are also used to detect pathogens such as viruses and bacteria in fluids and tissues .
  • the labels used in the above detection procedures may be radioactive or non-radioactive.
  • Common non-radioactive labels include detectable enzymes and fluorescent molecules. Fluorescent molecules absorb light at one wavelength and emit it at another, thus allowing visualization with, e.g., a fluorescent microscope. Spectrophotometers, fluorescence microscopes, fluorescent plate readers and flow sorters are well-known and are often used to detect specific molecules which have been made fluorescent by coupling them covalently to a fluorescent dye.
  • Fluorochro es such as amino coumarin acetic acid (AMCA) , fluorescein isothiocyanate (FITC) , tetramethylchodamine isothiocyanate (TRITC) , Texas Red, Cy3.0 and Cy5.0 are covalently coupled to antibody molecules and nucleic acid probes in connection with detection using fluorescence microscopy.
  • AMCA amino coumarin acetic acid
  • FITC fluorescein isothiocyanate
  • TRITC tetramethylchodamine isothiocyanate
  • Texas Red Cy3.0 and Cy5.0
  • Green fluorescent protein has recently attracted interest since cloning of the gene encoding GFP.
  • GFP is a bioluminescent protein produced in coelenterates, e.g., the jelly fish Aecruorea victoria and the sea pansy Renilla reniformis .
  • the emission spectra of Renilla GFP is almost identical to Aecruorea GFP, the excitation maxima of Aequorea GFP is 393 nm versus 498nm in Renilla. See Delagrave et al . , "Red-shifted Excitation Mutants of the Green Fluorescent Protein," Bio/Technology, Vol. 13, pp. 151-154 (1995) .
  • Aequorea and Renilla GFPs have structurally identical chromophores consisting of a hexapeptide and including a cyclic tripeptide sequence covalently linked through the protein's backbone.
  • the fluorescent properties of the chromophore in Aecruorea and Renilla GFP are influenced substantially by the surrounding protein matrix. See Ehrig et al . , "Green- fluorescent protein mutants with altered fluorescence excitation spectra," FEBS Letters, 367, pp 163-166 (1995). Neither the isolated hexapeptide nor the denatured protein is fluorescent. Id.
  • GFP fusion proteins have been used as markers of gene expression and protein localization in living and fixed tissues. See, for example, U.S. Patent No. 5,491,084. See also, Wang et al .
  • the search for new fluorescent molecules and procedures that can be used to detect targets of interest is ongoing.
  • the search is essential for increasing the level of detection sensitivity and resolution in procedures utilizing spectrophotometry, fluorescent microscopy, fluorescent plate readers and flow sorters.
  • a labeled marker for detection of a target which includes a label selected from the group consisting of green fluorescent protein and a fluorescent variant thereof, and a ligand configured to bind to the target.
  • the ligand includes any molecule or combination of molecules which have an affinity for another substance.
  • the ligand can be selected from the group consisting of nucleic acid probe, antibody, hapten conjugate, biotin, avidin and streptavidin.
  • Also provided is a method for detecting a target which includes providing a labeled ligand which includes a label selected from the group consisting of green fluorescent protein and a fluorescent variant thereof, and a ligand for binding the target.
  • the target is contacted with the labeled ligand and the labeled ligand is allowed to bind to the target.
  • the labeled ligand and target are subjected to light having a wavelength which excites the label and the locus of fluorescence is observed.
  • a method for detecting a target includes providing a primary ligand configured to bind to the target and providing a secondary ligand configured to bind to the primary ligand.
  • the secondary ligand includes a label selected from the group consisting of green fluorescent protein and a fluorescent variant thereof.
  • the target is contacted with the primary ligand and the primary ligand is allowed to bind to the target.
  • the labeled secondary ligand is contacted with the primary ligand and the secondary ligand is allowed to bind to the primary ligand.
  • the bound labeled secondary ligand is subjected to light having a wavelength which excites the label and the locus of fluorescence is observed.
  • a method for detecting a target includes providing a primary ligand configured to bind to the target and providing a secondary ligand configured to bind to the primary ligand.
  • a ternary ligand incorporating a label and configured to bind to the secondary ligand is also provided.
  • the label is selected from the group consisting of green fluorescent protein and a fluorescent variant thereof.
  • the target is contacted with the primary ligand and the primary ligand is allowed to bind to the target.
  • the primary ligand is contacted with the secondary ligand and the secondary ligand is allowed to bind to the primary ligand.
  • the secondary ligand is contacted with the labeled ternary ligand and the labeled ligand is allowed to bind to the secondary ligand.
  • the bound labeled ternary ligand is subjected to light having a wavelength which excites the label and the locus of fluorescence is observed.
  • a labeled marker/target complex including a target complexed with a marker having a label selected from the group consisting of green fluorescent protein and a fluorescent variant thereof.
  • the marker further incorporates at least one ligand configured to bind to the target, where the ligand is selected from the group consisting of antibody, nucleic acid probe, biotin, streptavidin, avidin and hapten conjugate.
  • the marker may optionally incorporate other ligands which are capable of binding to the at least one ligand configured to bind to the target .
  • a method for manufacturing a labeled marker includes providing a label selected from the group consisting of green fluorescent protein and a fluorescent variant thereof and linking the label to a ligand.
  • kits which comprises suitable packaging material and a ligand which includes a label selected from the group consisting of green fluorescent protein and a fluorescent variant thereof.
  • FIG. 1A is a photograph depicting a green fluorescent signal from a labeled marker complexed to a target near the centromere of metaphase human chromosome 17 homologues .
  • FIG. IB is a photograph depicting two green fluorescent signals from labeled markers complexed to targets near the centromere of each interphase chromosome 17 homologue.
  • FIG. 2 is a photograph depicting three green fluorescent signals from labeled markers complexed to targets, i.e., two near the respective centromere of two chromosome 16 homologues, and one near the centromere on the Yql2 region of the single chromosome Y in metaphase nuclei .
  • FIG. 3A is a photograph depicting a green fluorescent signal from a labeled marker complexed to targets at the location of each copy of the HER-2/neu gene in normal metaphase nuclei.
  • FIG. 3B is a photograph depicting a green fluorescent signal from a labeled marker complexed to targets at the location of each copy of the HER-2/neu gene in normal interphase nuclei .
  • Labeled markers according to the present invention include a label portion and a ligand portion.
  • the label provides a signal which allows the location of the labeled marker to be discerned from the surroundings.
  • the ligand binds to a desired target and thus provides a vehicle for transporting the label to the target .
  • the label is green fluorescent protein (GFP) or a fluorescent variant thereof.
  • GFP is a well-characterized protein having 238 amino acids. Fluorescent variants thereof have also been described. See, e.g., Heim et al . , "Improved Green Fluorescence", Nature, Vol 373, pp. 663-664 (1995) (ser 65 —» ala, leu, cys or thr) or Delgrave et al . ,
  • Variant EGFP is commercially available from Clontech Laboratories, Inc., California. Thus, fluorescent variants of GFP and methods of obtaining them are well- known to those skilled in the art. Although it has been established that denaturation or misfolding of GFP causes loss of fluorescence, the labels used in accordance with the present invention are not detrimentally altered to the point of losing fluorescence by cross-linking to ligands as described below.
  • the ligand herein is any molecule or combination of molecules which demonstrates an affinity for a target.
  • ligands include nucleic acid probes, antibodies, hapten conjugates, biotin, streptavidin and avidin. The mechanisms involved in obtaining and using such ligands are well-known.
  • nucleic acid probes are constructed with one or more nucleotides that incorporate GFP or fluorescent variants thereof. Suitable cross-linkers for connecting a label to a nucleotide are discussed below.
  • the present invention is especially well-adapted for use in in situ hybridization, i.e., the use of nucleic acid probes to locate specific nucleic acid sequences in situ.
  • Nucleic acid probes labeled directly or indirectly with GFP or a fluorescent variant thereof are hybridized to chromosomes that have been denatured by, e.g., high pH. The chromosomal regions that bind the labeled probe during the hybridization step are visualized by fluorescence microscopy in a process known in the art by the acronym FISH.
  • Antibodies that specifically recognize antigens are useful in accordance with another embodiment of the present invention.
  • Antibodies which are labeled with GFP or fluorescent variants thereof are used to locate specific target molecules by fluorescence detecting techniques such as fluorescence microscopy. Monoclonal or polyclonal antibodies are raised and purified using conventional techniques. After purification, the antibodies are labeled with GFP or a fluorescent variant thereof. Cross-linkers suitable for use in coupling a label to an antibody are discussed below.
  • GFP labeled antibodies or fluorescent GFP variant labeled antibodies are used to detect and quantify target molecules in cell extracts or, if the target is on a cell or viral surface, to pick out specific types of living cells or virus from a heterogeneous population. In this manner, detection markers according to the present invention can be used to detect the presence of pathogens, cancer cells or other disease states characterized by the presence of unique target molecules such as peptides, proteins, nucleic acids and the like.
  • GFP or a fluorescent variant thereof are linked to hapten conjugates such as digoxigenin and dinitrophenyl .
  • hapten conjugates such as digoxigenin and dinitrophenyl .
  • hapten conjugates such as digoxigenin and dinitrophenyl .
  • hapten conjugates such as digoxigenin and dinitrophenyl .
  • a detectable marker is made by linking GFP or a fluorescent variant thereof to known haptens or to antibodies which recognize such haptens.
  • Antibody/antigen complexes which form in response to hapten conjugates are easily detected by linking GFP or a fluorescent variant thereof to the hapten or carrier for the hapten or to antibodies which recognize such hapten or carrier for the hapten, and then observing the site of fluorescence using fluorescence detecting techniques such as fluorescence microscopy.
  • the sensitivity of nucleic acid probes or of antibodies is enhanced by signal amplification techniques.
  • GFP or a fluorescent variant thereof can be linked directly to the ligand which binds the target.
  • a stronger signal is possible when a first or primary ligand is bound to the target and then the primary ligand is detected with a group of secondary ligands labeled with GFP or a fluorescent variant thereof that bind to it.
  • such amplification techniques incorporate indirect immunocytochemistry when the ligands are antibodies.
  • the primary antibody is bound by a plurality of secondary antibodies incorporating GFP or a fluorescent variant thereof as a label . Since a plurality of labels are attracted to the target site, the signal strength is multiplied.
  • the high binding affinity of biotin for streptavidin or avidin provides a well-suited detection marker which can provide an amplified signal in accordance with the present invention. It is also possible to use labeled anti-biotin, anti-streptavidin or anti- avidin antibodies in accordance with the present invention. In one aspect, GFP or a fluorescent variant thereof is linked to biotin to create a detectable marker.
  • biotin is linked to nucleic acid probes using conventional techniques.
  • the biotinylated probes are then allowed to hybridize with denatured target nucleic acid.
  • Streptavidin or avidin which has been labeled with GFP or a fluorescent variant thereof is then contacted with the hybridized nucleic acid which leads to binding of the labeled streptavidin or avidin to the biotin portion of the biotinylated probe.
  • the streptavidin or avidin thus functions as a ligand. Fluorescence microscopy affords visualization of the fluorescent signal which allows the location of the target to be pinpointed.
  • streptavidin or avidin may be linked to the nucleic acid probe while GFP or a fluorescent variant thereof are linked to biotin.
  • labeled biotin acts as a ligand in binding to the streptavidin or avidin linked nucleic acid probe. Since each streptavidin or avidin molecule is capable of binding four biotin molecules, as many as four fluorescent signals may be concentrated at the target site.
  • a nucleic acid probe is constructed with one or more nucleotides having biotin linked thereto. The probe is then allowed to hybridize with a target nucleic acid sequence, thus acting as a primary ligand. After hybridization, the hybridized nucleic acid is then contacted with streptavidin or avidin, which acts as a secondary ligand in binding to the biotinylated probe. Additional biotin, which is labeled with GFP or a fluorescent variant thereof, is then contacted with the streptavidin or avidin.
  • the labeled biotin acts as a ligand and binds to the streptavidin or avidin. Since each streptavidin or avidin molecule is capable of binding four labeled biotin molecules, a relatively large three- dimensional network is created which includes numerous fluorescent protein molecules.
  • the amplified signal is then detected by fluorescence detecting techniques such as fluorescence microscopy.
  • biotin, streptavidin or avidin can be linked to antibody molecules to provide detection and amplified detection.
  • biotin is linked by conventional cross-linking techniques to an antibody specific for a defined target antigen.
  • the biotinylated antibody is then allowed to contact and bind the target.
  • Streptavidin or avidin which has been labeled with GFP or a fluorescent variant thereof is then contacted with the antibody/target antigen complex which leads to binding of the labeled streptavidin or avidin to the biotin portion of the biotinylated antibody.
  • the streptavidin or avidin thus functions as a ligand for binding to biotin. Fluorescence microscopy affords visualization of the fluorescent signal and allows the target to be pinpointed.
  • streptavidin or avidin may be linked to the antibody specific for a defined target antigen.
  • the streptavidin or avidin linked antibody is allowed to contact and bind the target .
  • Biotin which has been labeled with GFP or a fluorescent variant thereof is then contacted with the antibody/target antigen complex which leads to binding of the labeled biotin to the streptavidin or avidin portion of the antibody.
  • labeled biotin acts as a ligand in binding to the streptavidin or avidin linked antibody.
  • as many as four fluorescent molecules are concentrated at the target site.
  • streptavidin or avidin to bind four biotin molecules provides a further technique for amplifying the signal at a target antigen.
  • An antibody is linked with biotin using conventional techniques.
  • the antibody acting as a primary ligand, is then allowed to complex with the target antigen.
  • the antibody/antigen complex is then contacted with streptavidin or avidin which acts as a secondary ligand in binding to the biotinylated antibody.
  • Additional biotin which is labeled with GFP or a fluorescent variant thereof, is then contacted with the streptavidin or avidin to act as a labeled ligand and bind to the streptavidin or avidin. Since each streptavidin or avidin molecule binds four labeled biotin molecules, a relatively large three dimensional network is created which includes numerous fluorescent protein molecules.
  • Antibodies which are raised to complex with biotin, streptavidin or avidin and which incorporate GFP or a fluorescent variant thereof are also useful as labeled ligands for target detection.
  • a nucleic acid probe containing nucleotides linked to biotin hybridizes to a complementary sequence of denatured target nucleic acid.
  • the hybridized nucleic acid is then contacted with anti- biotin antibody having GFP or a fluorescent variant thereof linked thereto, thus forming a complex containing a detectable fluorescent marker.
  • the location of the complex is visualized using fluorescence detecting techniques such as fluorescence microscopy.
  • streptavidin or avidin may be linked to a nucleic acid probe.
  • the hybridized nucleic acid After hybridization to a complementary target nucleic acid, the hybridized nucleic acid is contacted with anti- streptavidin antibody or anti-avidin antibody depending on whether streptavidin or avidin linked probe have been used to form a labeled complex. The location of the complex is visualized using fluorescence microscopy.
  • GFP or any fluorescent variant thereof are linked to ligands by cross-linking procedures which, in accordance with the present invention, do not cause denaturing or misfolding of GFP or fluorescent variants thereof.
  • the terms "linked” or “conjugated” as used herein are intended to include any or all of the mechanisms known in the art for coupling the label to the ligand. For example, any chemical or enzymatic linkage known to those with skill in the art is contemplated including those which result from photoactivation and the like. Homofunctional and heterobifunctional cross linkers are all suitable. Reactive groups which can be cross-linked with a cross- linker include primary amines, sulfhydryls, carbonyls, carbohydrates and carboxylic acids.
  • Cross-linkers are available with varying lengths of spacer arms or bridges.
  • Cross-linkers suitable for reacting with primary amines include homobifunctional cross-linkers such as imidoesters and N-hydroxysuccinimidyl (NHS) esters.
  • imidoester cross-linkers include dimethyladipimidate, dimethylpimelimidate, and dimethylsuberimidate .
  • NHS-ester cross-linkers include disuccinimidyl glutamate, disucciniminidyl suberate and bis (sulfosuccinimidyl) suberate.
  • Accessible ⁇ amine groups present on the N-termini of peptides react with NHS- esters to form amides.
  • NHS-ester cross-linking reactions can be conducted in phosphate, bicarbonate/carbonate, HEPES and borate buffers . Other buffers can be used if they do not contain primary amines.
  • the reaction of NHS-esters with primary amines should be conducted at a pH of between about 7 and about 9 and a temperature between about 4°C and room temperature for about 30 minutes to about 2 hours.
  • the concentration of NHS-ester cross-linker can vary from about 0.1 to about lOmM.
  • NHS-esters are either hydrophilic or hydrophobic. Hydrophilic NHS-esters are reacted in aqueous solutions although DMSO may be included to achieve greater solubility. Hydrophobic NHS-esters are dissolved in a water miscible organic solvent and then added to the aqueous reaction mixture.
  • Sulfhydryl reactive cross-linkers include maleimides, alkyl halides, aryl halides and -haloacyls which react with sulfhydryls to form thiol ether bonds and pyridyl disulfides which react with sulfhydryls to produce mixed disulfides.
  • Sulfhydryl groups on peptides and proteins can be generated by techniques known to those with skill in the art, e.g., by reduction of disulfide bonds or addition by reaction with primary amines using 2-iminothiolane.
  • maleimide cross-linkers include succinimidyl 4- (N-maleimido-methyl) cyclohexane-1-carboxylate and m- maleimidobenzoyl-N-hydroxysuccinimide ester.
  • haloacetal cross-linkers include N-succinimidyl (4- iodoacetal) aminobenzoate and sulfosuccinimidyl (4- iodoacetal) aminobenzoate.
  • pyridyl disulfide cross-linkers examples include 1, 4-Di- [3 ' -2 ' - pyridyldithio (propionamido) butane] and N-succinimidyl-3- (2-pyridyldithio) -propionate.
  • Carboxyl groups are cross-linked to primary amines or hydrazides by using carbodimides which result in formation of amide or hydrazone bonds. In this manner, carboxy termini of peptides or proteins can be linked.
  • carbodiimide cross-linkers include l-ethyl-3- (3- dimethylaminopropyl) -carbodiimide hydrochloride and N, N'- dicyclohexylcarbodiimide .
  • Arylazide cross-linkers become reactive when exposed to ultraviolet radiation and form aryl nitrene.
  • arylazide cross-linkers examples include azidobenzoyl hydrazide and N-5-azido-2-nitrobenzoyl- oxysuccinimide.
  • Glyoxal cross linkers target the guanidyl portion of arginine.
  • An example of a glyoxal cross-linker is p-azidophenyl glyoxal monohydrate.
  • Heterobifunctional cross-linkers which possess two or more different reactive groups are suitable for use herein.
  • cross-linkers which are amine-reactive at one end and sulfhydryl-reactive at the other end such as 4- succinimidyl-oxycarbonyl- ⁇ - (2-pyridyldithio) -toluene, N- succinimidyl-3- (2-pyridyldithio) -propionate and the maleimide cross-linkers discussed above.
  • the kit of the present invention comprises suitable packaging material and a ligand (as described above) which includes a label selected from the group consisting of green fluorescent protein and a fluorescent variant thereof.
  • a ligand is an antibody, most preferably an anti-digoxigenin antibody.
  • the kit may include other materials such as a counterstain for use in FISH applications, such as DAPI or propidium iodide. The following examples are included for purposes of illustration and should not be construed as limiting the present invention.
  • Biotin (Long Arm) N-hydroxy succinimide ester (BNHS) (commercially available from Vector Laboratories, CA) powder was dissolved in DMSO to a concentration of 25 mg/ml. 100 ⁇ g GFP Protein (in 100 ⁇ l) (commercially available from Clontech Laboratories, CA) was supplied in Tris buffer, which interferes with labeling of protein amino groups. To remove Tris buffer, GFP was dialyzed overnight against 3 changes of untitrated 100 mM sodium bicarbonate.
  • the resulting 250 ⁇ l of protein was concentrated to a volume of 40 ul using a Nanosep spin filter (Filtron, OMEGA membrane and molecular weight cutoff of 10,000) by loading the GFP on top of the filter and centrifuging the spin column at 6000 rpm in the microfuge for 1 hr, 4°C. 50 ⁇ g of dissolved BNHS was added to the GFP solution and the mixture was incubated in the dark at room temperature on a rotator for 2 hr. The reaction was stopped by the addition of 10 mg glycine. The resulting biotin-conjugated GFP was dialyzed overnight against 3 changes of untitrated 100 mM sodium bicarbonate.
  • a Nanosep spin filter Frtron, OMEGA membrane and molecular weight cutoff of 10,000
  • the nucleic acid probes were labeled with biotin-14-deoxycytosine triphosphate (Oncor, Inc.) by standard nick translation. Incorporation of the biotin nucleotide into the labeled DNA was verified by dot- blot procedures. DNA fragment size was determined by gel electrophoresis (3% NuSieve agarose) such that the majority of the nucleic acid smear was less than 600 base pairs.
  • Ong of a labeled repetitive sequence probe 65% formamide/2XSSC/10% dextran sulfate, was denatured at 72°C for five minutes and applied to the sample on the slides under a coverslip. Following hybridization the samples were washed in a solution of SSC (IX SSC for unique sequence probes, 0.25XSSC for repetitive sequence probes) at 72°C for five minutes and then immediately placed in a coplin jar containing 1XPBD (phosphate buffered detergent) at room temperature for at least two minutes prior to fluorescence detection procedures .
  • IX SSC unique sequence probes
  • 0.25XSSC for repetitive sequence probes
  • All samples were stained with sixty microliters of detection reagent, consisting of avidin (lOug/ml) (Vector Laboratories), IXPBS, 5% powered dry milk, and 0.02% sodium azide, which was applied to the slide under a plastic coverslip and incubated at 37°C for fifteen minutes in a humidified chamber. The slides were washed in 1XPBD several times at room temperature. Next, the samples were stained with sixty microliters of the GFP-biotinconjugate (lOO ⁇ g/ml or lO ⁇ g/ml) in lOOmM sodium bicarbonate buffer and incubated for 30 minutes at 37°C in a humidified chamber.
  • detection reagent consisting of avidin (lOug/ml) (Vector Laboratories), IXPBS, 5% powered dry milk, and 0.02% sodium azide, which was applied to the slide under a plastic coverslip and incubated at 37°C for fifteen minutes in a humidified chamber. The slides were washe
  • microphotographs were made on Kodak Ektachrome color slide film (EL 400), using a Zeiss Axioskop epi- fluorescence microscope (Zeiss, West Germany) equipped with a 100-Watt mercury-arc lamp, a 100X Plan-Neofluor oil immersion objective (Zeiss, West Germany) , a Zeiss MC-100 camera (Zeiss, West Germany), and the appropriate filter sets for FITC/Pl fluorescence (Chroma, VT) having an excitation wavelength of 490nm and an emission wavelength of 525m.
  • FITC/Pl fluorescence Chroma, VT
  • the satellite chromosome 17-specific probe hybridizes to the highly repeated alphoid DNA located at the centromere of human chromosome 17.
  • a green fluorescent signal is visible near the centromere of both metaphase chromosome 17 homologues (See Fig. 1A) , while two fluorescent signals are also visible in each interphase nucleus (See Fig. IB) .
  • Hybridization of the alpha satellite chromosome 16-specific probe in combination with the chromosome Y cocktail probe was successful.
  • a fluorescent signal is present near the centromere of both chromosome 16 homologues, and a single signal is visible near the centromere and on the Yql2 region of the single chromosome Y in metaphase nuclei. Accordingly, interphase nuclei display three signals (See Fig. 2) .
  • the expected results for visualization of the HER- 2 /neu gene were obtained.
  • a single green fluorescent signal is visible at the location of each copy of the HER- 2 /neu gene.
  • the number of signals in normal metaphase chromosomes is two; one on each- chromatid (See Fig. 3A) , and the number of signals in normal interphase nuclei is also two, or two pairs if the cell is in G2 phase (See Fig. 3B) .
  • the fluorescent signals were photostable and remained visible with no noticeable loss of signal intensity under the conditions tested.
  • sulfo-succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo- SMCC) as follows: GFP is dissolved in lxPBS (phosphate buffered saline, Biofluids Inc.), pH 7.2, at lOmg/ml. lmg sulfo-SMCC (Pierce Cat. #22322) is added per milliliter of GFP with mixing until dissolved. The mixture is allowed to react for 30-60 minutes at room temperature in a darkened room.
  • the resulting maleimide-activated GFP is separated from excess cross-linker and reaction products by gel filtration (Sephadex G-25) against lxPBS, pH 7.2. After elution, the concentration is adjusted to lOmg/ml for the conjugation reaction. The resulting solution is used immediately to conjugate with the thiolated antibody as described below.
  • Modified sheep anti-digoxigenin, Fab fragments (Boehringer Mannheim) are prepared as follows: The Fab fragments are dissolved in lxPBS, pH 7.2, at lOmg/ml. N-Succinimidyl-S-acetylthioacetate (SATA) (Pierce Cat.
  • stock solution is prepared by dissolving SATA in DMSO at 13 mg/ml under a fume hood. 25 ⁇ l of SATA stock solution is added to each milliliter of lOmg/ml Fab fragment solution (not to exceed 10% DMSO) . The resulting solution is left to react for 30 minutes in a darkened room. Resulting SATA conjugated anti-digoxigenin Fab fragments are purified by gel filtration (Sephadex G-25) against lxPBS, pH 7.2 containing lOmM EDTA.
  • the acetylated sulfhydryl groups on the SATA-anti-digoxigenin are deprotected as follows: 100 ⁇ l hydroxylamine stock solution containing 0.5M hydroxylamine solution in 0.1M sodium phosphate, ph 7.2, lOmM EDTA, is added to each milliliter of SATA-anti-digoxigenin such that the final concentration of hydroxylamine is 50mM. The resulting mixture is allowed to react in a darkened room at room temperature for 2 hours. Resulting thiolated anti-digoxigenin is purified by gel filtration (Sephadex G-25) against lxPBS containing lOmM EDTA.
  • the GFP/anti-digoxigenin complex is produced by immediately mixing thiolated anti-digoxigenin with maleimide-activated GFP in a 4:1 (antibody: GFP) molar ratio. The resulting mixture is allowed to react at 37°C for two hours to yield the conjugate. In the alternative, the resulting mixture may be allowed to react for 2 hours at room temperature in a darkened room or at 4°C overnight in a darkened room. Dialysis may be substituted for gel filtration in the above example.
  • ligands or binding partners other than those described herein may be linked to GFP or fluorescent variants thereof. It is also contemplated that GFP or fluorescent variants thereof may be linked to the same or other fluorescent molecules prior to linkage to a ligand for increased signal amplification.
  • GFP can be bound to fluorescein and linked in combination to another molecule.
  • GFP can also be linked to additional GFP to create an oligomer chain of GFP. Such fluorescent combinations are then linked to ligands and used as described above .
  • each and every ligand can be separately labeled with GFP or fluorescent variants thereof to provide increased signal amplification when procedures incorporating at least primary ligands and secondary ligands are utilized.
  • the order of any method steps may be varied to facilitate binding of ligands to targets or other ligands. Therefore, the above description should not be construed as limiting, but as exemplifications of the preferred embodiments. Those skilled in the art can envision other modifications within the scope of the following claims.

Abstract

On combine une protéine fluorescente verte ou ses variantes fluorescentes avec des ligands afin de les utiliser en tant que marqueurs fluorescents dans la détection de cibles. Les ligands appropriés comprennent des sondes d'acide nucléique, des anticorps, des conjugués d'haptènes, biotine, avidine et streptavidine. On met en application des techniques, telles que la microscopie fluorescente, afin visualiser le marqueur.
EP19980908588 1997-02-18 1998-02-18 Detection de cibles au moyen d'une proteine fluorescente verte et de ses variantes fluorescentes Withdrawn EP1000172A4 (fr)

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PCT/US1998/003147 WO1998036099A1 (fr) 1997-02-18 1998-02-18 Detection de cibles au moyen d'une proteine fluorescente verte et de ses variantes fluorescentes

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US6495664B1 (en) 1998-07-24 2002-12-17 Aurora Biosciences Corporation Fluorescent protein sensors of post-translational modifications
US6410255B1 (en) 1999-05-05 2002-06-25 Aurora Biosciences Corporation Optical probes and assays
ATE487943T1 (de) 2004-04-08 2010-11-15 Hoffmann La Roche Fluoreszenzpolypeptidkomplex einer grösse von 40 bis 500 nm
US20130085095A1 (en) * 2011-09-29 2013-04-04 Trustees Of Boston University Protein complementation regulators
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EP1000172A1 (fr) 2000-05-17
AU6658898A (en) 1998-09-08

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