WO1999051986A1 - Procedes et reactifs pour le ciblage de composes organiques a des emplacements cellulaires selectionnes - Google Patents

Procedes et reactifs pour le ciblage de composes organiques a des emplacements cellulaires selectionnes Download PDF

Info

Publication number
WO1999051986A1
WO1999051986A1 PCT/US1999/007847 US9907847W WO9951986A1 WO 1999051986 A1 WO1999051986 A1 WO 1999051986A1 US 9907847 W US9907847 W US 9907847W WO 9951986 A1 WO9951986 A1 WO 9951986A1
Authority
WO
WIPO (PCT)
Prior art keywords
cell
probe
specific binding
binding partner
ligand
Prior art date
Application number
PCT/US1999/007847
Other languages
English (en)
Inventor
Javier Farinas
Original Assignee
The Regents Of The University Of California
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by The Regents Of The University Of California filed Critical The Regents Of The University Of California
Priority to AU34874/99A priority Critical patent/AU3487499A/en
Priority to US09/403,882 priority patent/US7045305B1/en
Publication of WO1999051986A1 publication Critical patent/WO1999051986A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals 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
    • G01N33/6854—Immunoglobulins
    • G01N33/6857—Antibody fragments
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • the present invention is directed to membrane permeant compounds that have an organic compound coupled to a ligand and methods of localizing and using such compounds as spectroscopic probes, or reagents.
  • reagents or spectroscopic probes are typically improved in both selectivity and sensitivity if the reagents or spectroscopic probes can be targeted to specific locations within a cell or organism.
  • the selective targeting of spectroscopic probes within a cell enables defined measurements of subcellular microenvironments such as within subcellular organelles to be precisely probed.
  • the use of spectroscopic probes tagged to macromolecules enables spatio-temporal aspects of a tagged macromolecule to be monitored in real time in vivo.
  • Such methods can be used to develop a variety of specific assays for cellular activation, or to create functional assays of enzymatic function.
  • the location of a nuclear receptor within a cell can be determined by creating a fusion protein of the receptor to a specific binding partner and then observing its movement, after addition of a fluorescent ligand for the specific binding partner, in response to a test stimulus, such as a chemical.
  • a test stimulus such as a chemical.
  • activation of nuclear receptor results in their translocation into the nucleus, which can be used as an assay to determine the relative activity of a series of different chemicals.
  • the targeting of NMR contrast agents or positron emission probes enables whole organism imaging of specific tissues or cell types in the intact organism.
  • the present invention provides a targeting method that combines the ability to genetically encoded a protein sequence, and specifically express that sequence within living cells, with the use of well characterized spectroscopic probes or other probes.
  • the invention thus enables the use of a wide range of fluorophores, as well as other spectroscopic probes, including nuclear magnetic resonance (NMR), positron emission tomography (PET), relaxation reagents, chromophores, and other reagents such as chemical cross linkers, caged compounds, enzymatic substrates, activators and substrates to be selectively targeted within intact cells or organisms.
  • GFP green fluorescent protein
  • FLASH an arseno-fiuorescein derivative
  • FLASH involves the addition of a membrane permeant arseno-fiuorescein derivative to the cells that binds to a short alpha helix containing 4 cysteines that can be added to a protein of interest (Griffin, et al, (1998) Science 281 269-72).
  • the FLASH approach is limited to a single fluorophore, however in combination with the present invention both methods may be used to specifically label two proteins with two different fluorophores.
  • the invention provides for the intracellular expression of a high affinity specific binding partner that specifically interacts with a fluorophore- coupled ligand.
  • the specific binding partner comprises a single chain antibody (sFv) as the specific binding partner, and a hapten (phOx) as the ligand.
  • the present invention is directed towards methods and uses of targeted probes in living organisms or cells.
  • the probe is a spectroscopic probe moiety coupled to a ligand to create a spectroscopic probe/ligand conjugate that can be localized to a specific binding partner within an organism.
  • the specific binding partner is a single chain antibody, which specifically binds to the spectroscopic probe/ligand conjugate.
  • the compound is membrane permeant and is useful for localizing the spectroscopic probe/ligand conjugate to intracellular structures comprising a high affinity specific binding partner for the fluorescent ligand within a living cell.
  • the organic molecule is substantially non-membrane permeable, and the specific binding partner is attached to the extracellular face of the plasma membrane.
  • the spectroscopic probes may be detected using a variety of techniques including fluorescence, nuclear magnetic resonance and positron emission tomography.
  • One embodiment of the method further comprises the step of adding a stimulus to the cell and comparing the intracellular spatial distribution of the specific binding partner fusion protein, before, and after addition of the stimulus. In one aspect this method further comprises the step of adding a test chemical to determine if it can modulate the effect of the stimulus.
  • the method involves detecting post-translational activity using a specific binding partner fusion protein /spectroscopic probe/ ligand conjugate expressed within a cell. In one aspect, the method involves detecting said spectroscopic probe/ ligand conjugate before and after activation of a post-translational activity within the cell. In one aspect, this method involves visualizing the localization, or detecting a fluorescent property, of said spectroscopic probe/ ligand conjugate, such as fluorescence emission, polarization or fluorescence lifetime. In another aspect, detecting involves NMR imaging or positron emission tomography. In one aspect of this method, the method further comprises the step of adding a test chemical to determine if it can modulate the effect of post-translational activation.
  • the invention is an expression vector comprising expression control sequences operatively linked to a nucleic acid sequence encoding a specific binding partner for a spectroscopic probe/ligand conjugate fused to a protein of interest and a fluorescent protein.
  • the spectroscopic probe moiety and the fluorescent protein change position relative to each upon post-translational modification of the protein of interest.
  • the invention is an expression vector comprising expression control sequences operatively linked to a nucleic acid sequence encoding a specific binding partner for a spectroscopic probe/ligand conjugate fused to a protein of interest and a protein comprising a second specific binding domain for a second fluorescent moiety.
  • the spectroscopic probe moiety and the second fluorescent moiety change position relative to each upon post-translational modification of the protein of interest.
  • the invention includes a host cell transfected with a specific binding partner fused to another protein.
  • the invention features a transgenic non-human animal having a phenotype characterized by expression of the nucleic acid sequence coding for the expression of the specific binding partner.
  • the phenotype is conferred by a transgene contained in the somatic and germ cells of the animal.
  • the animal can be a mouse.
  • a further aspect of the present invention is a method of screening a test chemical for an activity using a cell comprising a nucleic acid encoding a specific binding partner having a binding region that binds a spectroscopic probe/ligand conjugate.
  • the nucleic acid molecule encoding the specific binding partner may be randomly integrated into the genome or operably linked to a response element or promoter element.
  • the specific binding partner is a protein or polypeptide that is adapted to bind to the fluorescent ligand with high affinity within the cell.
  • the protein is selected from a random pool of proteins expressed in a plurality of cells containing a diverse population of randomly arranged sequences.
  • the polypeptide comprises a single chain antibody.
  • the single chain antibody is adapted for functional expression within the reducing environment of one or more subcellular compartments of a mammalian cell.
  • the invention provides a method for localizing a probe, comprising contacting a sample comprising a cell expressing a specific binding partner with a probe/ligand conjugate, said probe/ligand conjugate, said probe/ligand conjugate comprising a probe moiety, a ligand that can bind with said specific binding partner, and a linker moiety coupling said probe to said ligand, wherein said ligand and said specific binding partner bind non-covalently, wherein said probe/ligand conjugate is membrane permeant, and wherein the specific binding partner is expressed from a recombinant nucleic acid.
  • the invention provides a method for detecting a post- translational activity in a cell, comprising contacting a cell expressing a specific binding partner fused to a protein of interest with a spectroscopic probe/ligand conjugate, said spectroscopic probe/ligand conjugate comprising a spectroscopic probe moiety, a ligand that can bind with said specific binding partner, and a linker moiety coupling said spectroscopic probe to said ligand, activating said post-translational activity in said cell, and detecting said spectroscopic probe/ligand conjugate, before and at least one time after activation of said post-translational activity, wherein said ligand and said specific binding partner bind non-covalently, wherein said probe/ligand conjugate is membrane permeant, and wherein the specific binding partner is expressed from a recombinant nucleic acid.
  • the invention provides a method of screening a test chemical for activity, comprising contacting a cell with a test chemical, said cell comprising, a nucleic acid encoding a specific binding partner, said specific binding partner comprising, a binding region which binds a spectroscopic probe/ligand conjugate, contacting said cell with a spectroscopic probe/ligand conjugate, said spectroscopic probe/ligand conjugate comprising (1) a spectroscopic probe moiety, (2) a ligand that can bind with said specific binding partner, and a linker moiety coupling said spectroscopic probe to said ligand, and detecting said spectroscopic probe/ligand conjugate, wherein said ligand and said specific binding partner bind non-covalently, wherein said probe/ligand conjugate is membrane permeant, and wherein the specific binding partner is expressed from a recombinant nucleic acid.
  • FIG. 1 represents a schematic representation of several embodiments of the specific binding partners of the present invention.
  • the specific binding partner (10) is fused in frame with a protein of interest (20).
  • the protein of interest typically comprises cellular location motifs that can direct the fusion protein to specific subcellular locations as described herein.
  • the fusion of interest (20) is fused to a fluorescent protein or homologue thereof (30).
  • the protein of interest is fused to a second specific binding partner (40) that recognizes a distinct ligand.
  • FIG. 2A represents one strategy for fluorophore targeting.
  • a single-chain antibody, specific binding partner is targeted to an intracellular site by cDNA transfection.
  • Ligand -fluorophore conjugate added to the media is trapped by the specific binding partner. Fluorescence in the cell increases upon binding to the specific binding partner.
  • FIG. 2B chemical structures of preferred conjugates are shown.
  • FIG. 2C and FIG. 2D represent fluorescence and biding affinity of phOx-fluorescein.
  • FIG. 2C shows the excitation and emission spectra of phOx-fluorescein (1 nM) in PBS (solid line).
  • FIG. 2D shows the phOx-fluorescein fluorescence at indicated concentrations in regions of an image without cells (filled squares), regions in non-sFv expressing cells (triangles), and regions in cells expressing sFv at the plasma membrane (circles).
  • the fluorescence of sFv bound phOx-fluorescein was fitted to a single site binding model (dashed line) with I 6.8 nM.
  • FIG. 3 represents site-specific labeling of CHO cells.
  • FIG. 3A is a confocal image of CHO cells transfected with the plasma membrane-targeted sFv vector and shows plasma membrane staining by phOx-rhodamine.
  • FIG. 3B is a confocal fluorescence image of the cells in FIG. 3 A stained with a fluorescein-labeled anti-c-myc antibody.
  • FIG. 3C shows the brightfield image of the cells in FIG.3A.
  • FIG. 3D shows a fluorescence image of CHO cells with Golgi-targeted sFv in the presence of 10 nM phOx-BodipyFl.
  • FIG. 3E shows the same cells as in FIG.
  • FIG. 3D in the presence of 500 nM phOx-ethanolamine and 10 nM phOx-BodipyFL.
  • FIG. 3F is a fluorescence image of a cell with ER-targeted sFv in the presence of 10 nM phOx-Bodipy.
  • FIG. 3G shows immunostaining of Golgi-sFv transfected cells with fluorescein-labeled, anti-c-myc antibody.
  • FIG. 3H shows immunostaining of Golgi-sFv transfected cells with rhodamine-labeled anti -mouse antibody directed against a mouse 58k protein antibody.
  • FIG. 31 shows immunostaining of ER-sFv transfected cells with rhodamine-labeled anti- mouse antibody directed against an anti-c-myc antibody.
  • FIG. 3J shows immunostaining of ER-sFv transfected cells with fluorescein-labeled concavalin A. The scale bar represents 10 micrometers.
  • FIG. 4 represents the measurement of Golgi pH.
  • FIG. 4A shows the average ratio generated by pixel-by-pixel division of 490 nm images by 440 nm images after background subtraction. The ratio was converted to pH using a calibration relating pH to fluorescence signal ratios for Golgi (circles) and plasma membrane (squares) targeted phOx-fluorescein.
  • Plasma membrane data was acquired in the presence of 10 nM phOx- fluorescein. The ratios of absorbance at 490-to-440 nm are shown for unbound phOx- fluorescein (triangles) in solution. Calibration data were fitted to a single site titration model (fluorescence solid line, absorbance dashed line).
  • FIG. 4B represents the fluorescence ratio (490/440) time course in Golgi. At indicated times, bafilomycin Ai (100 nM) and high K + buffers (120 mM) containing 5 ⁇ M monensin at indicated pH were added. Calibration solutions were used to convert ratios to pH values (scale at right).
  • FIG. 4C represents the calculated pH time course in response to a 20 mM sodium acetate pre-pulse in the absence and presence of 100 nM bafilomycin Aj. Lines (fitted from 120 to 300 seconds after pre-pulse) indicate the initial rate of pH change.
  • FIG. 4D represents the calculated proton pump rates (circles) and leak rates (filled squares) as a function of Golgi pH. Dashed lines are linear fits to the data.
  • polypeptide refers to a polymer in which the monomers are amino acids and are joined together through amide bonds, alternatively referred to as a peptide.
  • unnatural amino acids for example, beta-alanine, phenylglycine and homoarginine are also meant to be included.
  • Commonly encountered amino acids which are not gene-encoded, may also be used in the present invention.
  • All of the amino acids used in the present invention may be either the D- or L-isomer.
  • the L-isomers are preferred.
  • Chemically modified amino acids for example including phosphorylated, sulfated, methylated, or prenylated residues may also be used to create polypeptides for specific applications.
  • naturally-occurring refers to the fact that an object can be found in nature.
  • a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally-occurring.
  • probe refers to a compound useful as marker or environmental indicator, or modifying reagent for use with the present invention. Probes may comprises fluorescent, spectroscopic or modifying moieties as described herein.
  • spectroscopic probe or moiety refers to a compound that can be used as a marker or indicator, or contrast agent for nuclear magnetic resonance imaging, positron emission tomography, absorption spectroscopy, luminescence spectroscopy and fluorescence spectroscopy.
  • fluorescent probe or moiety refers to a compound that can absorb electromagnetic energy and is capable of at least partially remitting some fraction of that energy as electromagnetic radiation over some time period.
  • Suitable fluorescent moieties include, but are not limited to, coumarins and related dyes, xanthene dyes such as fluoresceins, rhodols, and rhodamines, resorufms, cyanine dyes, bimanes, acridines, isoindoles, dansyl dyes, aminophthalic hydrazides such as luminol, and isoluminol derivatives, aminophthalimides, aminonaphthalimides, aminobenzofurans, aminoquinolines, dicyanohydroquinones, semiconductor fluorescent nanocrystals, fluorescent proteins and fluorescent europium and terbium complexes and related compounds. Fluorescent moieties may also be environmental sensitive or be indicators of various ions such as calcium, magnesium or pH, as is known in the art.
  • modify moiety refers to compounds that can modify the function of a protein or macromolecule within the cell.
  • modify moieties include but are not limited to chemical cross linkers, caged compounds, enzymatic substrates, activators and inhibitors.
  • optical property refers to the molar extinction coefficient at an appropriate excitation wavelength, the fluorescent quantum efficiency, the shape of the excitation spectrum or emission spectrum, the excitation wavelength maximum or emission wavelength maximum, the ratio of excitation amplitudes at two different wavelengths, the ratio of emission amplitudes at two different wavelengths, the excited state lifetime, the fluorescent anisotropy or any other measurable property of a compound.
  • post-translational activity refers to activities that mediate a modification of a protein such that its activity, function or cellular localization is changed. Such modifications include phosphorylation, glycosylation, methylation, sulfation, ubiquitination, proteolysis, prenylation and ADP-ribsoylation.
  • the term includes non-covalent modifications including protein-protein interactions, and the binding of allosteric or other modulators or second messengers such as calcium, cAMP or inositol phosphates to a protein of interest.
  • Specific binding partner refers to a member of a specific binding pair as that term is known in the art.
  • Specific binding pairs include, for example, antigen- antibody, hapten-antibody, receptor-ligand, nucleic acid-nucleic acid, nucleic acid- protein, enzyme-inhibitor and enzyme-substrate pairs.
  • the interaction between specific binding pairs can be covalent or non-covalent in nature.
  • Specific binding members can be
  • 10 be of any chemical composition, such as protein, carbohydrate, lipid, chemical, nucleic acid or any combination thereof.
  • acceptor refers to a quencher that operates via energy transfer. Acceptors may re-emit the transferred energy as fluorescence and are "acceptor fluorescent moieties". Examples of acceptors include coumarins and related fluorophores, xanthenes such as fluoresceins, rhodols, and rhodamines, resorufins, cyanines, difluoroboradiazaindacenes, and phthalocyanines. Other chemical classes of acceptors generally do not re-emit the transferred energy. Examples include indigos, benzoquinones, anthraquinones, azo compounds, nitro compounds, indoanilines, and di- and tripheny lmethanes .
  • homologue refers to two sequences or parts thereof, that are greater than, or equal to 75% identical when optimally aligned using the ALIGN program.
  • Homology or sequence identity refers to the following. Two amino acid sequences are homologous if there is a partial or complete identity between their sequences. For example, 85% homology means that 85% of the amino acids are identical when the two sequences are aligned for maximum matching. Gaps (in either of the two sequences being matched) are allowed in maximizing matching; gap lengths of 5 or less are preferred with 2 or less being more preferred.
  • two protein sequences are homologous, as this term is used herein, if they have an alignment score of more than 5 (in standard deviation units) using the program ALIGN with the mutation data matrix and a gap penalty of 6 or greater. See Dayhoff, M.O., in Atlas of Protein Sequence and Structure, 1972, volume 5, National Biomedical Research Foundation, pp. 101-110, and Supplement 2 to this volume, pp. 1-10.
  • reference sequence is a defined sequence used as a basis for a sequence comparison; a reference sequence may be a subset of a larger sequence, for example, as a segment of a full-length cDNA or gene sequence given in a sequence listing such as a SEQ. ID. NO.: 1, or may comprise a complete cDNA or gene sequence. Generally, a reference sequence is at least
  • two polynucleotides may each (1) comprise a sequence (i.e., a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides, and (2) may further comprise a sequence that is divergent between the two polynucleotides, sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a "comparison window" to identify and compare local regions of sequence similarity.
  • a “comparison window”, as used herein, refers to a conceptual segment of at least 20 contiguous nucleotide positions wherein a polynucleotide sequence may be compared to a reference sequence of at least 20 contiguous nucleotides and wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20 percent or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • Optimal alignment of sequences for aligning a comparison window may be conducted by the local homology algorithm of Smith and Waterman (1981) Adv. Appl. Math. 2: 482, by the homology alignment algorithm of Needleman and Wunsch (1970) J.
  • sequence identity means that two polynucleotide sequences are identical (i.e., on a nucleotide-by-nucleotide basis) over the window of comparison.
  • percentage of sequence identity is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or I) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
  • substantially identity denotes a characteristic of a polynucleotide sequence, wherein the polynucleotide
  • sequence 12 comprises a sequence that has at least 30 percent sequence identity, preferably at least 50 to 60 percent sequence identity, more usually at least 60 percent sequence identity as compared to a reference sequence over a comparison window of at least 20 nucleotide positions, frequently over a window of at least 25-50 nucleotides, wherein the percentage of sequence identity is calculated by comparing the reference sequence to the polynucleotide sequence which may include deletions or additions which total 20 percent or less of the reference sequence over the window of comparison.
  • the term "substantial identity” means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 30 percent sequence identity, preferably at least 40 percent sequence identity, more preferably at least 50 percent sequence identity, and most preferably at least 60 percent sequence identity.
  • residue positions, which are not identical differ by conservative amino acid substitutions.
  • Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine. A group of amino acids having aliphatic-hydroxyl side chains is serine and threonine.
  • a group of amino acids having amide-containing side chains is asparagine and glutamine.
  • a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan.
  • a group of amino acids having basic side chains is lysine, arginine, and histidine.
  • a group of amino acids having sulfur-containing side chains is cysteine and methionine.
  • Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine- valine, glutamic-aspartic, and asparagine-glutamine.
  • test chemical refers to a chemical to be tested by one or more screening method(s) of the invention as a putative modulator.
  • a test chemical can be any chemical, such as an inorganic chemical, an organic chemical, a protein, a peptide, a carbohydrate, a lipid, or a combination thereof.
  • various predetermined concentrations of test chemicals are used for screening, such as 0.01 micromolar, 1 micromolar and 10 micromolar.
  • Test chemical controls can include the measurement of a signal in the absence of the test compound or comparison to a compound known to modulate the target.
  • Membrane permeant means that a compound can pass through a membrane, such as a cell membrane, such as a eukaryotic cell membrane, preferably a mammalian cell membrane.
  • a membrane such as a cell membrane, such as a eukaryotic cell membrane, preferably a mammalian cell membrane.
  • membrane permeant compounds pass through a membrane without the assistance of transport molecules within a membrane, such as porins, but the present invention recognizes such transport molecules as a mechanism of membrane permeability of compounds.
  • Substantially membrane permeant means that a compound can pass through a membrane and accumulate within a cell to a concentration useful in the methods of the present invention, such as the detection of intracellular specific binding members using fluorescent detection methods.
  • the compound is at lest as membrane permeable as CCF2/AM (as described in U.S. patent No. 5,741,657 issued 4/21/98 to Tsien et al).
  • Membrane-permeant derivative refers a chemical derivative of a compound of that increases membrane permeability of the compound. These derivatives are made better able to cross cell membranes, i.e. membrane permeant, because hydrophilic groups are masked to provide more hydrophobic derivatives. Also, the masking groups are designed to be cleaved from the fluorogenic substrate within the cell to generate the derived substrate intracellularly. Because the substrate is more hydrophilic than the membrane permeant derivative it becomes trapped within the cell.
  • Operaably linked refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner.
  • a control sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.
  • Control sequence refers to polynucleotide sequences which are necessary to effect the expression of coding and non-coding sequences to which they are ligated. The nature of such control sequences differs depending upon the host organism; in prokaryotes, such control sequences generally include promoter, ribosomal binding site, and transcription termination sequence; in eukaryotes, generally, such control sequences include promoters and transcription termination sequence.
  • control sequences is intended to include, at a minimum, components whose presence can influence
  • nucleic acid refers to a polymeric form of nucleotides of at least ten bases in length, either ribonucleotides or deoxynucleotides or a modified form of either type of nucleotide.
  • the term includes single and double stranded forms of DNA.
  • Modulation refers to the capacity to either enhance or inhibit a functional property of a biological activity or process (e.g., enzyme activity or receptor binding). Such enhancement or inhibition may be contingent on the occurrence of a specific event, such as activation of a signal transduction pathway, and/or may be manifest only in particular cell types.
  • modulator refers to a chemical (naturally occurring or non-naturally occurring), such as a biological macromolecule (e.g. nucleic acid, protein, non-peptide, or organic molecule), or an extract made from biological materials such as bacteria, plants, fungi, or animal (particularly mammalian) cells or tissues. Modulators are typically evaluated for potential activity as inhibitors or activators (directly or indirectly) of a biological process or processes (e.g., agonist, partial antagonist, partial agonist, antagonist, antineoplastic agents, cytotoxic agents, inhibitors of neoplastic transformation or cell proliferation, cell proliferation-promoting agents, and the like) by inclusion in assays described herein. The activity of a modulator may be known, unknown or partial known. Other chemistry terms herein are used according to conventional usage in the art, as exemplified by The McGraw-Hill Dictionary of Chemical Terms (ed. Parker, S., 1985), McGraw-Hill, San Francisco, incorporated herein by reference).
  • a biological macromolecule e.
  • the present invention recognizes that a probe can be coupled to a ligand, to create a probe/ligand conjugate that is useful for a range of cell based assays.
  • the probe/ligand conjugate of the present invention is used in conjunction with an intracellular specific binding member or receptor, that specifically binds to the probe/ligand conjugate.
  • the specific binding partner is typically a protein or polypeptide, and in one preferred embodiment is a single chain antibody.
  • the specific binding partner is typically a protein or polypeptide, and in one preferred embodiment is a single chain antibody.
  • binding partner can be part of a fusion protein that comprises a known or unknown protein of interest, so that the intracellular specific binding member localizes with the protein of interest.
  • the intracellular localization of the specific binding partner and fusion protein can be located and monitored in a living cell. Additionally a variety of detection methods can be used to provide information on the post-translational modification state and interactions of a fusion protein comprising a protein of interest, coupled to a specific binding partner.
  • the choice of the spectroscopic probe moiety is governed by a number of factors including, the type of measurements being made, the availability of specific instrumentation and the ease of coupling of the probe moiety to the hapten. Additionally, other factors that are specific to a particular application are also relevant and include, the effect of labeling on the solubility of the ligand, permeability of the probe/ ligand conjugate across the plasma membrane, the stability of the conjugate within the cell and the required detection sensitivity of the assay.
  • preferred fluorophores typically exhibit good quantum yields, lifetimes, and extinction coefficients, are resistant to collisional quenching and bleaching, and should preferably be easily conjugated to the ligand.
  • fluorophores that show absorbance and emission in the red and near infrared range, which are useful in whole animal studies, because of reduced scattering background fluorescence, and greater transmission through tissues.
  • moieties include cyanines, oxazines, thiazines, porphyrins, phthalocyanines, fluorescent infrared-emitting polynuclear aromatic hydrocarbons such as violanthrones, and near LR squaraine dyes.
  • fluoroscein isothicyanate especially fluorescein-5-
  • 16 isothiocyanate), dichlorotriazinylaminofluorescein, tetramethylrhodamine-5 (and -6)- isothiocyanate, 1,3-bis- (2-dialkylamino-5-thienyl)-substituted squarines, and the succinimidyl esters of: 5 (and 6) carboxyfluoroscein; 5 (and 6)- carboxytetramethylrhodamine; and 7-amino-4-methylcoumarin-3-acetic acid.
  • preferred spectroscopic probes include lipophilic cationic complexes of radioactive metal ions such as gallium-68 (III) with metal chelating ligands, as described in U.S. patent No. 5,324,502, issued June 28, 1994 to Green et al, and carbon -11 containing compounds as described in J. Nucl. Med. 38 1305-10 (1997).
  • Preferred spectroscopic probes for use as NMR contrast agents include chelates of paramagnetic, ferromagnetic or diamagnetic metal ions complexed to lipophilic complexes as described in U.S. patents 5,628,982, issued May 13, 1997 to Lauffer et al. and U.S.
  • the spectroscopic probe is generally attached by a linker that provides a spacer between the spectroscopic probe and the ligand thereby preventing sterric interference of the spectroscopic probe on the ligand specific binding partner interaction.
  • Preferred spacers are substantially stable under cellular conditions, non ionic and easily coupled to the ligand and spectroscopic probe.
  • Preferred examples include flexible aliphatic linkers such as diaminopentane, and aminohexanoyl as well as rigid aromatic linkers such as trans cyclohexane. Such linkers are know in the art and described for example in the
  • the specific binding partner or receptor for use in the present invention is a polypeptide or protein that can bind to high affinity to the spectroscopic probe/ligand conjugate.
  • the specific binding partner comprises a single chain antibody that exhibits high specificity and selectivity for the probe/ligand conjugate.
  • the single chain antibody is essentially as described in Griffith et al. (1984) Nature 312 271-275 (SEQ ID. No 1).
  • the single chain antibody may also comprise one or more antibody recognition motifs fused in frame with the coding sequence for immunological confirmation of specific binding partner expression. For example the hemagglutinin, Poly His, V5 or myc epitopes are well characterized and monoclonal antibodies in each case are commercially available.
  • single chain antibody includes single chain antibodies with separate light and heavy chains co-expressed in cells, and ioslated VH domains such as used for camelid antibodies as described in EMBO J. 9 101-102 (1990) and Prot. Eng. 9 531-7 (1996).
  • the specific binding partner can also include a localization sequence to direct the specific binding partner to particular cellular sites by fusion to appropriate organellar targeting signals or localized host proteins.
  • a polynucleotide encoding a localization sequence, or signal sequence can be ligated or fused at the 5' terminus of a polynucleotide encoding the specific binding partner such that the signal peptide is located at the amino terminal end of the resulting fusion polynucleotide/polypeptide.
  • the signal peptide is believed to function to transport the fusion polypeptide across the endoplasmic reticulum.
  • the secretory protein is then transported through the Golgi apparatus, into secretory vesicles and into the extracellular space or, preferably, the external environment.
  • Signal peptides which can be utilized according to the invention, include pre-pro peptides that contain a proteolytic enzyme recognition site. Other signal peptides with similar properties to pro-calcitonin described herein are known to those skilled in the art, or can be readily ascertained without undue experimentation.
  • the localization sequence can be a nuclear localization sequence, an endoplasmic reticulum localization sequence, a peroxisome localization sequence, a mitochondrial localization sequence, or a localized protein. Localization sequences can be targeting sequences which are described, for example, in "Protein Targeting", chapter 35 of Stryer, L., Biochemistry (4th ed.). W.H. Freeman, 1995.
  • the localization sequence can also be a localized protein.
  • Some important localization sequences include those targeting the nucleus (KKKRK), mitochondrion (amino terminal MLRTSSLFTRRVQPSLFRNILRLQST-), endoplasmic reticulum (KDEL at C-terminus, assuming a signal sequence present at N-terminus), peroxisome (SKF at C-terminus), prenylation or insertion into plasma membrane (CaaX, CC, CXC, or CCXX at C- terminus), cytoplasmic side of plasma membrane (fusion to SNAP-25), or the Golgi apparatus (fusion to furin).
  • specific binding partners can be created by producing libraries of peptides containing a diverse population of amino acid sequences, and then screening the library to identify peptides that bind to the probe/ligand conjugate with high affinity.
  • a genetically engineered library of putative specific binding partners having a randomized sequence can be used to define a sequence that binds to a probe/ligand conjugate with high affinity.
  • a "library" refers to a collection containing at least 5 different members, preferably at least 100 different members and more preferably at least 200 different members.
  • amino acid sequences for the peptide will typically be in the range or 10 to 20 amino acids in length and may be completely random or biased towards a particular sequence based on a particular structural motif, for example based on a known structure that has some binding affinity with the target ligand.
  • the library will created genetically and the individual members expressed in bacterial or a mammalian cells.
  • the library can contain peptides with a diverse collection of amino acids in which most or all of the amino acid positions are randomized.
  • the library can contain variable peptide in which only a few, e.g., one to ten, amino acid positions are varied, but in which the probability of substitution is very high.
  • libraries of peptides are created by expressing of recombinant nucleic acid molecules having expression control sequences operatively linked to nucleic acid sequences that code for the expression of different candidate specific binding partners.
  • Methods of making nucleic acid molecules encoding a diverse collection of peptides are described in, for example, U.S. patent 5,432,018 (Dower et al), U.S. patent 5,223,409 (Ladner et al.) and International patent publication WO 92/06176 (Huse et al).
  • recombinant nucleic acid molecules are used to transfect cells, such that a cell contains on average one member of the library. This produces, in turn, a library of host cells capable of expressing a library of different candidate peptides. The library of host cells can then be used to screen for peptides that can bind to the spectroscopic probe/ligand conjugate.
  • a diverse collection of oligonucleotides having random codon sequences are combined to create polynucleotides encoding the candidate peptides.
  • the oligonucleotides preferably are prepared by chemical synthesis.
  • the polynucleotides encoding the peptides of variable composition can then be ligated to the 5' or 3' end of a nucleic acid encoding a defined gene, if required. For example, in one embodiment, they could be fused to a functional engineered green fluorescent protein. This creates a recombinant nucleic acid molecule coding for the expression of a variable peptide moiety fused to the amino or carboxy- terminus of a fluorescent protein.
  • This recombinant nucleic acid molecule is then inserted into an expression vector to create a recombinant nucleic acid molecule comprising expression control sequences operatively linked to the sequences encoding the fluorescent protein fused to the variable peptide region.
  • a codon motif is used, such as (NNK) X , where N may be A, C, G, or T (nominally equimolar), K is G or T (nominally equimolar), and x is the desired number of amino acids in the peptide moiety, e.g., 15 to produce a library of 15-mer peptides.
  • the third position may also be G or C, designated "S”.
  • NNK or NNS (i) code for all the amino acids, (ii) code for only one stop codon, and (iii) reduce the range of codon bias from 6:1 to 3:1.
  • NNK 6 An exemplified codon motif (NNK) 6 produces 32 codons, one for each of 12 amino acids, two for each of five amino acids, three for each of three amino acids and one (amber) stop codon. Although this motif produces a codon distribution as equitable as available with standard methods of oligonucleotide synthesis, it results in a bias against peptides containing one-codon residues.
  • An alternative approach to minimize the bias against one-codon residues involves the synthesis of 20 activated tri -nucleotides, each representing the codon for one of the 20 genetically encoded amino acids. These are synthesized by conventional means, removed from the support but maintaining the base and 5-HO-protecting groups, and activating by the addition of 3'0-phosphoramidite (and phosphate protection with beta- cyanoethyl groups) by the method used for the activation of mononucleosides, as generally described in McBride and Caruthers, Tetrahedron Letters 22:245 (1983). Degenerate "oligocodons" are prepared using these trimers as building blocks.
  • the trimers are mixed at the desired molar ratios and installed in the synthesizer.
  • the ratios will usually be approximately equimolar, but may be a controlled unequal ratio to obtain the over- to under-representation of certain amino acids coded for by the degenerate oligonucleotide collection.
  • the condensation of the trimers to form the oligocodons is done essentially as described for conventional synthesis employing activated mononucleosides as building blocks. See generally, Atkinson and Smith, Oligonucleotide Synthesis, M.J. Gait, ed. p35-82 (1984).
  • this procedure generates a population of oligonucleotides for cloning that is capable of encoding an equal distribution (or a controlled unequal distribution) of the possible peptide sequences.
  • Libraries of host cells expressing candidate peptides described above are then used in identifying peptide sequences that can bind to high affinity to the spectroscopic probe/ligand conjugate.
  • one begins with a library of recombinant host cells, each of which expresses a different peptide candidate specific binding partner. Each cell is expanded into a clonal population that is genetically homogeneous.
  • the method consists of measuring the fluorescence of the cell after addition and incubation of the cell with the probe/ligand conjugate at a limiting concentration. Binding of the probe/ligand
  • conjugate to the specific binding partner results in a net uptake of probe/ligand conjugate into the cell that can be readily measured over background non-specific uptake.
  • specific binding can also be determined by measuring the degree of fluorescence energy transfer (FRET) between the fluorescent ligand conjugate and a fluorescent protein - peptide fusion protein.
  • FRET fluorescence energy transfer
  • binding of the fluorescent ligand conjugate to the peptide fusion would result in an increase in FRET between the ligand and GFP.
  • FRET fluorescence Activated Cell Sorting
  • nucleic acids from cells exhibiting a change in FRET or enhancement in fluorescence as a result of fluorescent ligand binding can be isolated for example by PCR amplification, and the peptide sequences identified by sequencing. The results from these studies could used as the basis for the generation of more targeted libraries to identify optimal peptides through repeated rounds of analysis and selection of clones exhibiting the largest and most rapid changes in FRET in the presence, but not the absence of the fluorescent ligand conjugate. Evpression of the specific binding partner within cells
  • the method would involve the expression of the specific binding partner into living eukaryotic or prokaryotic cells as a fusion protein to a protein of interest to enable the subcellular localization of the protein to be determined.
  • a variety of host-expression vector systems may be utilized to express the specific binding partner coding sequence.
  • microorganisms such as bacteria transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing a specific binding partner coding sequence; yeast transformed with recombinant yeast expression vectors containing the specific binding partner coding sequence; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing a specific binding partner coding sequence; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing a specific binding partner coding sequence; or animal cell systems infected with recombinant virus expression vectors
  • any of a number of suitable transcription and translation elements including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, etc. may be used in the expression vector (see, e.g., Bitter, et al, Methods in Enzymology 153:516-544, 1987).
  • inducible promoters such as pL of bacteriophage ⁇ , plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like may be used.
  • promoters derived from the genome of mammalian cells e.g., metallothionein promoter
  • mammalian viruses e.g., the retrovirus long terminal repeat; the adenovirus late promoter; the vaccinia virus 7.5K promoter
  • Promoters produced by recombinant DNA or synthetic techniques may also be used to provide for transcription of the inserted specific binding partner coding sequence.
  • a number of expression vectors may be advantageously selected depending upon the use intended for the specific binding partner expressed.
  • yeast a number of vectors containing constitutive or inducible promoters may be used.
  • Current Protocols in Molecular Biology Vol. 2, Ed. Ausubel, et al, Greene Publish. Assoc. & Wiley Interscience, Ch. 13, 1988; Grant, et al, Expression and Secretion Vectors for Yeast, in Methods in Enzymology, Eds. Wu &
  • a constitutive yeast promoter such as ADH or LEU2 or an inducible promoter such as GAL may be used (Cloning in Yeast, Ch. 3, R. Rothstein In: DNA Cloning Vol.l 1, A Practical Approach, Ed. DM Glover, LRL Press, Wash., D.C., 1986).
  • vectors may be used which promote integration of foreign DNA sequences into the yeast chromosome.
  • the expression of a specific binding partner coding sequence may be driven by any of a number of promoters.
  • viral promoters such as the 35S RNA and 19S RNA promoters of CaMV (Brisson, et al, Nature 310:511-514, 1984), or the coat protein promoter to TMV (Takamatsu, et al, EMBOJ. 3:1671-1680; Broglie, et al, Science 224:838-843, 1984); or heat shock promoters, e.g., soybean hspl7.5-E or hspl7.3-B (Gurley, et al, Mol. Cell.
  • Biol 6:559-565, 1986 may be used. These constructs can be introduced into plant cells using Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, microinjection, electroporation, etc.
  • Ti plasmids Ri plasmids
  • plant virus vectors direct DNA transformation, microinjection, electroporation, etc.
  • Grierson & Corey Plant Molecular Biology, 2d Ed., Blackie, London, Ch. 7-9, 1988.
  • An alternative expression system that could be used to express specific binding partner is an insect system.
  • Autographa calif ornica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes.
  • the virus grows in Spodoptera frugiperda cells.
  • the specific binding partner coding sequence may be cloned into non-essential regions (for example, the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter).
  • Successful insertion of the specific binding partner coding sequence will result in inactivation of the polyhedrin gene and production of non-occluded recombinant virus (i.e., virus lacking the proteinaceous coat coded for by the polyhedrin gene).
  • Mammalian cell systems that utilize recombinant viruses or viral elements to direct expression may be engineered.
  • the specific binding partner coding sequence may be ligated to an adenovirus transcription translation control complex, e.g. , the late promoter and tripartite leader sequence.
  • This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region El or E3) will result in a recombinant virus that is viable and capable of expressing the specific binding partner in infected hosts (e.g., see Logan & Shenk, Proc.
  • the vaccinia virus 7.5K promoter may be used, (e.g., see, Mackett, et al, Proc. Natl. Acad. Sci. USA ,79: 7415- 7419, 1982; Mackett, et al, J. Virol. 49: 857-864, 1984; Panicali, et al, Proc. Natl. Acad. Sci. USA 79: 4927-4931, 1982).
  • vectors based on bovine papilloma virus which have the ability to replicate as extrachromosomal elements
  • the plasmid replicates to about 100 to 200 copies per cell. Transcription of the inserted cDNA does not require integration of the plasmid into the host's chromosome, thereby yielding a high level of expression.
  • These vectors can be used for stable expression by including a selectable marker in the plasmid, such as the neo gene.
  • the retroviral genome can be modified for use as a vector capable of introducing and directing the expression of the specific binding partner gene in host cells (Cone & Mulligan, Proc. Natl. Acad. Sci. USA, 81:6349-6353, 1984). High level expression may also be achieved using inducible promoters, including, but not limited to, the metallothionine IIA promoter and heat shock promoters.
  • host cells can be transformed with the specific binding partner cDNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker.
  • expression control elements e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.
  • the selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines.
  • engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media.
  • a number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler, et al, Cell, 11: 223, 1977), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA, 48:2026, 1962), and adenine phosphoribosyltransferase (Lowy, et al, Cell, 22: 817, 1980) genes can be employed in tk " , hgprt " or aprt " cells respectively.
  • antimetabolite resistance can be used as the basis of selection for dhfr, which confers resistance to methotrexate (Wigler, et al, Proc. Natl. Acad. Sci. USA, 77: 3567, 1980; O'Hare, et al, Proc. Natl.
  • trpB which allows cells to utilize indole in place of tryptophan
  • hisD which allows cells to utilize histinol in place of histidine
  • ODC ornithine decarboxylase
  • DFMO 2-(difluoromethyl)-DL-ornithine
  • DNA sequences encoding the specific binding partner polypeptide of the invention can be expressed in vitro by DNA transfer into a suitable host cell.
  • "Host cells” are cells in which a vector can be propagated and its DNA expressed.
  • the term also includes any progeny of the subject host cell. It is understood that all progeny may not be identical to the parental cell since there may be mutations that occur during replication. However, such progeny are included when the term "host cell” is used. Methods of stable transfer, in other words when the foreign DNA is continuously maintained in the host, are known in the art.
  • the invention provides a transgenic non-human animal that expresses a nucleic acid sequence that encodes the specific binding partner.
  • non-human animals comprise any non-human animal having nucleic acid sequence which encodes a suitable specific binding partner.
  • Such non-human animals include vertebrates such as rodents, non-human primates, sheep, dog, cow, pig, amphibians, and reptiles.
  • Preferred non-human animals are selected from the rodent family including rat and mouse, most preferably mouse.
  • Introducing "transgenes" into the germline of the non-human animal produces the "transgenic non-human animals” of the invention.
  • Embryonic target cells at various developmental stages can be used to introduce transgenes. Different methods are used depending on the stage of development of the embryonic target cell. The zygote is the best target for micro-injection. In the mouse, the male pronucleus reaches the size of approximately 20 micrometers in
  • zygotes as a target for gene transfer has a major advantage in that in most cases the injected DNA will be incorporated into the host gene before the first cleavage (Brinster et al, Proc. Natl. Acad. Sci. USA 82:4438-4442, 1985). As a consequence, all cells of the transgenic non-human animal will carry the incorporated transgene. This will in general also be reflected in the efficient transmission of the transgene to offspring of the founder since 50% of the germ cells will harbor the transgene. Microinjection of zygotes is the preferred method for incorporating transgenes in practicing the invention.
  • transgenic is used to describe an animal that includes exogenous genetic material within all of its cells.
  • a “transgenic” animal can be produced by crossbreeding two chimeric animals which include exogenous genetic material within cells used in reproduction. Twenty-five percent of the resulting offspring will be transgenic i.e., animals that include the exogenous genetic material within all of their cells in both alleles. 50% of the resulting animals will include the exogenous genetic material within one allele and 25% will include no exogenous genetic material.
  • Retroviral infection can also be used to introduce transgene into a non-human animal. The developing non-human embryo can be cultured in vitro to the blastocyst stage.
  • the blastomeres can be targets for retro viral infection (Jaenich, R., Proc. Natl. Acad. Sci USA 73:1260-1264, 1976). Efficient infection of the blastomeres is obtained by enzymatic treatment to remove the zona pellucida (Hogan, et al. (1986) in Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).
  • the viral vector system used to introduce the transgene is typically a replication-defective retro virus carrying the transgene (Jahner, et al, Proc. Natl. Acad. Sci. USA 82:6927-6931, 1985; Van der Putten, et al, Proc. Natl.
  • ES cells are obtained from pre-implantation embryos cultured in vitro and fused with embryos (M. J. Evans et al. Nature 292:154-156, 1981; M.O. Bradley et al, Nature 309: 255-258, 1984; Gossler, et al, Proc. Natl. Acad.
  • Transgenes can be efficiently introduced into the ES cells by DNA transfection or by retro virus-mediated transduction. Such transformed ES cells can thereafter be combined with blastocysts from a nonhuman animal. The ES cells thereafter colonize the embryo and contribute to the germ line of the resulting chimeric animal. (For review see Jaenisch, R., Science 240: 1468-1474, 1988).
  • Transformed means a cell into which (or into an ancestor of which) has been introduced, by means of recombinant nucleic acid techniques, a heterologous nucleic acid molecule.
  • Heterologous refers to a nucleic acid sequence that either originates from another species or is modified from either its original form or the form primarily expressed in the cell.
  • Transgene means any piece of DNA which is inserted by artifice into a cell, and becomes part of the genome of the organism (i.e., either stably integrated or as a stable extrachromosomal element) which develops from that cell.
  • a transgene may include a gene which is partly or entirely heterologous (i.e., foreign) to the transgenic organism, or may represent a gene homologous to an endogenous gene of the organism. Included within this definition is a transgene created by the providing of an RNA sequence that is transcribed into DNA and then incorporated into the genome.
  • the transgenes of the invention include DNA sequences that encode the specific binding partner that may be expressed in a transgenic non-human animal.
  • transgenic as used herein additionally includes any organism whose genome has been altered by in vitro manipulation of the early embryo or fertilized egg or by any transgenic technology to induce a specific gene knockout.
  • gene knockout refers to the targeted disruption of a gene in vivo with complete loss of function that has been
  • transgenic animals having gene knockouts are those in which the target gene has been rendered nonfunctional by an insertion targeted to the gene to be rendered non-functional by homologous recombination.
  • transgenic includes any transgenic technology familiar to those in the art which can produce an organism carrying an introduced transgene or one in which an endogenous gene has been rendered nonfunctional or "knocked out.”
  • Fluorescent methods that are preferred with the present invention include, time resolved fluorescence spectroscopy, fluorescence correlation spectroscopy, fluorescence polarization spectroscopy, and resonance energy based fluorescence spectroscopy.
  • Methods of performing such assays on fluorescent materials are well known in the art and are described in, e.g., Lakowicz, J.R., Topics in Fluorescence Spectroscopy, volumes 1 to 3, New York: Plenum Press (1991); Herman, B., Resonance energy transfer microscopy, in: Fluorescence Microscopy of Living Cells in Culture, Part B, Methods in Cell Biology, vol. 30, ed. Taylor, D.L.
  • the present invention can be used to determine the expression and localization of the specific binding partner-fusion protein over time or after the cell has received a stimulus, such as being contacted with a test chemical.
  • the method involves the creation of a fusion protein between the protein of interest and the specific binding partner, and
  • the specific binding partners are randomly integrated into the host cells genome in order to tag unknown genes.
  • the nucleic acid molecule can integrate into the genome of the host cell and expression can be modulated by expression control elements or promoters contained within the genome using gene trapping or promoter trapping techniques (see, PCT/US97/17395) or as described above.
  • the specific binding partner can be used to monitor the induction, repression and spatial localization of a particular protein.
  • the specific binding partners may be expressed on the extracellular surface of the cell, within a transgenic organism in order to enable the spatial distribution of a particular cell type or tissue to be analyzed.
  • the present invention can be used to develop assays for post- translational modifications such as proteolysis, phosphorylation and protein-protein interactions of the protein of interest fused to specific binding partner.
  • the method used to determine the degree of post-translational modification of the protein of interest is dependent on the assay format used.
  • the method may be based on the difference in fluorescence anisotropy of the fluorescent ligand/specific binding partner- usion protein complex before and after exposure to a stimulus.
  • modification of the protein of interest by the post translational activity in response to stimulation of the cell, results in a change in the rotational flexibility of the complex that results in a measurable change in fluorescence polarization of the fluorescent specific binding par ⁇ ner/ligand complex.
  • the method involves the creation of a fusion protein between the specific binding partner and the protein of interest that contains a site of post-translational modification.
  • the protein of interest would contain
  • proteolysis would result in the cleavage of the protein of interest resulting an increase in rotational movement of the fluorophore.
  • protein binding to the protein of interest, as a result of phosphorylation, or otherwise would result in a decrease in rotational flexibility. In either case, the resulting change in rotational flexibility would influence the florescence anisotropy of the fluorescent ligand/specific binding partner- fusion protein complex.
  • Polarization measurements are based on the relative rotational movement of the fluorophore compared to the excited state life-time of that fluorophore.
  • polarization p
  • degree of rotational movement can be readily derived (see Weber, Polarization of the fluorescence of solutions, in Fluorescence and Phosphorescence Analysis, Don Hercules (ed.), Interscience Publishers New York. Chapter 8, pages 217-240 (1966)).
  • Rotational movement can be related to the rotational diffusion constant of the molecule, and hence to the molecular volume. In practice there is a close correlation between the molecular size and relative polarization of emitted light from a fluorophore.
  • Polarization based measurements are relatively easy to set up, and can be obtained over a wide concentration, temperature, and ionic strength range.
  • the present invention takes advantage of fluorescence resonance energy transfer (FRET) between two fluorescent moieties to provide a ratiometric fluorescent readout.
  • the specific binding partner typically comprises a second fluorescent moiety, for example by the creation of a fusion protein between a fluorescent protein or second fluorescent moiety and the protein of interest and the specific binding partner.
  • cleavage of the protein of interest by a protease results in an alteration in energy transfer between the specific binding partner bound fluorescent ligand and the fluorescent protein or second fluorescent moiety that may be used to detect protease activity.
  • the fluorescent moieties are typically chosen such that the excitation spectrum of one of the moieties (the acceptor fluorescent moiety) overlaps with the emission spectrum of the donor fluorescent moiety. The selection and
  • the donor fluorescent moiety then transfers the absorbed energy by non radiative means to the acceptor, which subsequently re-emits some of the absorbed energy as fluorescence emission, at a characteristic wavelength.
  • FRET can be manifested as a reduction in the intensity of the fluorescent signal from the donor, reduction in the lifetime of its excited state, and an increase in emission of fluorescence from the acceptor fluorescent moiety.
  • the peptide substrate that connects the donor fluorescent moiety and acceptor fluorescent moiety is cleaved, the donor fluorescent moiety and the acceptor fluorescent moiety physically separate, and FRET is diminished or eliminated. Under these circumstances fluorescence emission from the donor increases and fluorescence emission from the acceptor decreases.
  • E is the efficiency of FRET
  • F and F° are the fluorescence intensities of the donor in the presence and absence of the acceptor, respectively
  • R is the distance between the donor and the acceptor.
  • R Q the distance at which the energy transfer efficiency is 50%, is given (in A) by
  • K 2 is an orientation factor having an average value close to 0.67 for freely mobile donors and acceptors
  • Q is the quantum yield of the unquenched fluorescent donor
  • n is the refractive index of the intervening medium
  • J is the overlap integral, which expresses in quantitative terms the degree of spectral overlap
  • changes in the degree of FRET are determined as a function of the change in the ratio of the amount of fluorescence from the donor and acceptor moieties, a process referred to as "ratioing.”
  • ratioing a process referred to as "ratioing.”
  • the method would involve a fusion protein comprising a peptide containing one or more binding sites for a second fluorescent moiety (FIG. 1).
  • the binding site could comprises a sequence that recognizes a fluorescent moiety as described in the pending US Patent applications, identified by serial number 08/955,050, filed October 21, 1997, entitled Methods of using synthetic molecules and target sequences; 08/955,859, filed October 21, 1997, entitled Synthetic molecules that specifically react with target sequences, and 08/955,206, filed October 21, 1997, entitled Target sequences for synthetic molecules.
  • expression of the specific binding partner fusion protein and binding site could be accomplished recombinantly as described above.
  • the binding site and specific binding partner would enable the creation, in situ of a fluorescent substrate capable of detecting proteolysis.
  • the binding sites for the second fluorescent moiety could be created through the fusion to a second specific binding partner that recognized a distinct ligand.
  • the second fluorescent moiety is a fluorescent protein (FIG. 1).
  • Such fluorescent proteins include endogenously fluorescent proteins, functional engineered fluorescent proteins, and homologues thereof, because the entire fluorophore and specific binding partner can be synthesized within intact living cells without the addition of other co-factors or fluorophores (for review see Cubitt et al, TIBS 20, pp. 448-455 1995).
  • Endogenously fluorescent proteins have been isolated and cloned from a number of marine species including the sea pansies Renilla reniformis, R. kollikeri andR. mullerei and from the sea pens Ptilosarcus, Stylatula and Acanthoptilum, as well as from the Pacific Northwest jellyfish, Aequorea victoria.; Szent-Gyorgyi et al.
  • fluorescent proteins have also been observed in other organisms, although in most cases these require the addition of some exogenous factor to enable fluorescence development.
  • yellow fluorescent protein from Vibrio fischeri strain Y-l has been described by T.O. Baldwin et al, Biochemistry (1990) 29:5509-15. This protein requires flavins as fluorescent co-factors.
  • the cloning of Peridinin-chlorophyll a binding protein from the dinoflagellate Symbiodinium sp. was described by B.J. Morris et al, Plant Molecular Biology, (1994) 24:673:77.
  • One useful aspect of this protein is that it fluoresces in red.
  • phycobiliproteins from marine cyanobacteria such as Synechococcus, e.g., phycoerythrin and phycocyanin, is described in S.M. Wilbanks et al, J. Biol. Chem. (1993) 268:1226- 35. These proteins require phycobilins as fluorescent co-factors, whose insertion into the proteins involves auxiliary enzymes. The proteins fluoresce at yellow to red wavelengths.
  • the fluorescent moieties are typically chosen such that the excitation spectrum of one of the moieties (the acceptor fluorescent moiety) overlaps with the emission spectrum of the donor fluorescent moiety.
  • a contemplated version of the method is to use inducible controlling nucleotide sequences to produce a sudden increase in the expression of either the specific binding partner-fusion protein or the post-translational activity being assayed, e.g., by inducing expression of the construct.
  • a post-translational activity could be expressed within the cell, or induced, or introduced using a membrane translocating sequence (Rojas et al., (1998) Nature Biotech 16 370-375) if did not naturally occur in that cell.
  • the efficiency of FRET is monitored at one or more time intervals after the onset of increased expression of the post-translational activity.
  • the post-translational activity could be monitored one or more times after the addition of a stimulus, such as an agonist, antagonist or test chemical.
  • a stimulus such as an agonist, antagonist or test chemical.
  • the ability of a test chemical to alter a post- translational activity, in a cell-based assay may be determined.
  • cells transfected with an expression vector encoding a specific binding partner fused to a protein of interest, as described above are exposed to different amounts of the test chemical, and the effect on FRET or fluorescence polarization in each cell can be determined before or after exposure of a stimulus.
  • the difference in FRET or polarization of treated cells is compared to that of untreated controls.
  • the methods of the present invention can also take place in vivo, such that compounds and test chemicals are administered to an animal or transgenic organism rather than the procedure taking place in culture or using samples from an organism.
  • the fluorescent ligand/specific binding partner systems of the present invention can be used with a system for spectroscopic measurement, comprising: a reagent for an assay, and a device comprising at least one plate or container, preferably a multi-well platform, and a second platform to hold said plate or container for detecting a signal from a sample.
  • the system can further comprise a detector, such as a detector appropriate for detecting a signal from a sample or a plate on in a container as such detectors are known in the art or are later developed.
  • the system can comprise multiple plates or containers or multi-well platforms.
  • a reagent for an assay includes any reagent useful to perform biochemical or biological in vitro or in vivo testing procedures, such as, for example, buffers, co-factors, proteins such as enzymes or proteases, carbohydrates, lipids, nucleic acids, active fragments thereof, organic solvents such as DMSO, chemicals, analytes, therapeutics, compositions, cells, antibodies, ligands, and the like.
  • an active fragment is a portion of a reagent that has substantially the activity of the parent reagent.
  • the choice of spectroscopic probe/ligand conjugate - specific binding partner pair depends on the type of assay to be performed.
  • FRET based assays would typically comprise a specific binding partner with fused to a fluorescent protein, or containing a binding site for a second fluorophore.
  • Fluorescent polarization based assays would typically be completed with specific binding partners comprising one a fusion protein to a protein of interest. In both cases assays would typically be run with cells.
  • the fluorescent substrates of the present invention are suited for use with systems and methods that utilize automated and integratable workstations for identifying modulators, and chemicals having useful activity. Such systems are described generally in the art (see, U.S. Patent Nos: 4,000,976 to Kramer et al. (issued January 4, 1977),
  • such a system includes: A) a storage and retrieval module comprising storage locations for storing a plurality of chemicals in solution in addressable chemical wells, a chemical well retriever and having programmable selection and retrieval of the addressable chemical wells and having a storage capacity for at least 100,000 addressable wells, B) a sample distribution module comprising a liquid handler to aspirate or dispense solutions from selected addressable chemical wells, the chemical distribution module having programmable selection of, and aspiration from, the selected addressable chemical wells and programmable dispensation into selected addressable sample wells (including dispensation into arrays of addressable wells with different densities of addressable wells per centimeter squared) or at locations, preferably pre-selected, on a plate, C) a sample transporter to transport the selected addressable chemical wells to the sample distribution module and optionally having programmable control of transport of the selected addressable chemical wells or locations on a plate (including adaptive routing and parallel processing), D) a reaction module comprising either
  • the storage and retrieval module, the sample distribution module, and the reaction module are integrated and programmably controlled by the data processing and integration module.
  • the storage and retrieval module, the sample distribution module, the sample transporter, the reaction module and the data processing and integration module are operably linked to facilitate rapid processing of the addressable sample wells or locations on a plate.
  • devices of the invention can process at least 100,000 addressable wells or locations on a plate in 24 hours. This type of system is described in
  • each separate module is integrated and programmably controlled to facilitate the rapid processing of liquid samples, as well as being operably linked to facilitate the rapid processing of liquid samples.
  • the invention provides for a reaction module that is a fluorescence detector to monitor fluorescence.
  • the fluorescence detector is integrated to other workstations with the data processing and integration module and operably linked with the sample transporter.
  • the fluorescence detector is of the type described herein and can be used for epi-fluorescence.
  • Other fluorescence detectors that are compatible with the data processing and integration module and the sample transporter, if operable linkage to the sample transporter is desired, can be used as known in the art or developed in the future.
  • detectors are available for integration into the system. Such detectors are described in U.S. Patent 5,589,351 (Harootunian), U.S. Patent 5,355,215 (Schroeder), U.S. Patent Application (serial number pending), entitled “Detector and Screening Device for Ion Channels” filed 7/17/98, and PCT patent application WO 93/13423 (Akong).
  • an entire plate may be "read” using an imager, such as a Molecular Dynamics Fluor-Imager 595 (Sunnyvale, CA).
  • Multi-well platforms having greater than 864 wells, including 3,456 wells, can also be used in the present invention (see, for example, the PCT Application PCT/US98/11061, filed 6/2/98. These higher density well plates require miniaturized assay volumes that necessitate the use of highly sensitivity assays that do not require washing.
  • the present invention provides such assays as described herein.
  • Example I plasmids and cell transfection.
  • the cDNA encoding the sFv (25kDa protein) with two c-myc epitopes was amplified by polymerase chain reaction using the plasmid pHook-1 (Chestnut et al., J.
  • ER and trans-Golgi targeting the cDNA was subcloned at EcoR I and Xbal restriction sites in the expression plasmid pCDNA3.1 containing the specific targeting sequences (ER: preprolactin signal sequence and C- terminal SEKDEL, trans-Golgi: amino acids 1 to 47 of the human beta- 1,4- galactosyltransferase).
  • Plasma membrane targeting was achieved with the pHook plasmid.
  • CHO cells ATCC CRL 9618
  • PhOx-Bodipy FI was prepared by reaction of excess Bodipy FL succinimidyl ester (Molecular Probes) with 1 mM of phOx- aminopentane in borate buffer for 2 h. The product was obtained as a precipitate.
  • PhOx-tetramethylrhodamine was prepared by reaction of excess tetramethylrhodamine succinimidyl ester (Molecular Probes) with 5 mM phOx-aminocyclohexane in borate buffer for 6 h. The product was obtained as a precipitate.
  • PhOx-fluorescein was prepared by reaction of equimolar amounts of phOx (0.5 mg in 25 ⁇ l acetone) and fluorescein cadaverine (Molecular Probes) (1 mg in 50 ⁇ l dimethylformamide) for 1 h. PBS was added, unreacted phOx was removed by hexane extraction, and the product was extracted with butanol. PhOx-ethanolamine was prepared by reaction of 1 mg phOx with 0.3 ⁇ l ethanolamine in 10 ml ethanol for 1 h. All
  • Example III Fluorescence measurements.
  • Example IV Cell labeling using phOx in CHO cells
  • a cell labeling method was developed that combines the site specificity conferred by genetically encoded protein targeting sequences with the spectral and indicator properties of fluorophores.
  • the strategy is to express a high affinity specific binding partner at a specified intracellular location to trap a conjugate of an indicator linked to a specific ligand (FIG. 2A).
  • Fv single-chain antibody
  • phOx high affinity hapten
  • sFv targeting cells are transfected with cDNAs encoding sFv in fusion with Golgi, ER or plasma membrane targeting sequences. Fluorophore-hapten conjugates were added to the extracellular solution at low concentrations, diffused to sites of sFv expression, and bound to the sFv. Conjugates of different indicator, spectral and linker properties were synthesized (FIG. 2B), including phOx-Bodipy FL (green fluorescent, flexible linker), phOx-fluorescein (green fluorescent, pH-sensitive, flexible linker), and phOx-tetramethylrhodamine (red fluorescent, rigid linker). The flexible linkers were designed to permit stacking of the unbound hapten with its covalently attached fluorophore to form a dark (non-fluorescent) complex.
  • the sFv/hapten-fluorophore system preferably include: bright fluorescence of the bound hapten- fluorophore conjugate, high affinity biding of the conjugate to sFv, strong cellular expression of functional sFv, membrane permeability of the conjugate, and minimal cellular toxicity. These characteristics were fulfilled for cellular sFv expression (using the Golgi, ER and plasma membrane vectors) and binding of the hapten- fluorophore conjugates in FIG 2B. Fluorescence spectra of phOx-fluorescein bound to sFv and of an equal concentration of unbound phOx-fluorescein in solution had similar spectral shape (FIG. 2C).
  • the toxicity and stability of the conjugates were investigated. There were no differences in cell growth as assessed by cell counting and viability as assessed by Trypan Blue exclusion between control cells and cells incubated for 24 hours with 200 nM of each conjugate. The stability of the imine bond in the conjugates was determined in cells. In freshly prepared phOx-fluorescein and phOx-fluorescein incubated with cell
  • FIG. 3A shows a fluorescence image of living cells expressing the sFv at the plasma membrane and stained with phOx-rhodamine. A plasma membrane staining pattern was found.
  • FIG. 3B shows staining of sFv in the same cells with a fluorescein- labeled anti-c-myc antibody. Comparison with FIG. 3A demonstrates that only sites of sFv expression were significantly labeled with phOx-rhodamine. There was no significant staining of adjacent cells that did not express sFv (FIG. 3C).
  • FIG. 3D shows specific phOx-Bodipy staining of Golgi. Staining was reversed by addition of 1 ⁇ M phOx-ethanolamine (FIG. 3E).
  • FIG. 3F shows phOx-Bodipy staining of ER, seen as a characteristic reticular pattern.
  • the high expression level of the sFv, the relatively high affinity of the hapten/sFv, and the low fluorescence of the unbound conjugate allowed images to be obtained in the presence of ⁇ 10 nM concentrations of unbound conjugate with little contribution from free conjugate. For quantitative measurement of organelle pH, the free dye was washed out of the bathing solutions.
  • the membrane permeability of the conjugates was high enough to load cells by incubation at 37 °C for 4 hours for phOx-fluorescein, 2 hours for phOx-rhodamine, or 10 minutes for the less polar phOx-Bodipy. Cells could be loaded at 4 °C, indicating that the conjugate entered the cells primarily by transmembrane diffusion and not by endocytosis.
  • FIG. 4B shows that the fluorescence ratio increases upon addition of the vacuolar H + pump inhibitor bafilomycin A ⁇ .
  • the ratios measured using calibration buffers were used to convert fluorescence ratios to pH (scale at right). Golgi pH initially at ⁇ 6.3 promptly alkalinized after the addition of bafilomycin Ai.
  • the buffer capacity, ⁇ was measured by the NH C1 pulse method (Roos and Boron Physiol. Rev. 61, 296-421 (1981)) to be constant (38 ⁇ 3 mM pH units) in the pH range 6-7 (not shown).
  • the H + leak rate, dH + /dt ⁇ ea k was measured from the pH change (solid line, FIG. 4C) after an identical 20 mM Na acetate pre-pulse with the H + pump inhibited by bafilomycin A ⁇ . Similar pre-pulse measurements were done at different Golgi pH by varying Na acetate and NH 4 C1 concentrations.
  • FIG. 4D shows that computed H + pump
  • H + pump rate In the steady-state H + pump rate must equal H + leak rate.
  • the dependence of the leak rate on Golgi pH was measured from the kinetics of pH change after bafilomycin Ai addition as shown in FIG. 4B.
  • the data for different pH are summarized in FIG. 4D, showing decreased H + leak as Golgi pH increases.
  • the intersection of the H + pump and leak curves predicts correctly the observed steady- state Golgi pH, supporting a balanced pump/leak mechanism for setting Golgi pH. .
  • the resting pH is determined by the kinetics of proton leak versus pump. Shifts in the leak or pump curves could provide a simple mechanism for the observed differences in resting pH in organelles of the secretory pathway.
  • the specific binding partner-mediated probe targeting strategy should have numerous applications for the labeling of specified cellular structures with fluorescent or other indicator molecules.
  • Specific binding partner-mediated targeting of chemical probes permits many types of measurements that cannot be made using GFP.
  • Multiple fluorophores with excitation and emission wavelengths from the ultraviolet to the infrared can be targeted, permitting well-resolved multicolor fluorescence detection.
  • red and infrared probes are especially useful to minimize background autofluorescence, photodamage and photobleaching.
  • bright fluorophores can be chosen with substantially improved molar absorbance and quantum yield over GFP.
  • a unique application of specific binding partner-mediated probe targeting is the labeling of intracellular compartments with fluorescent indicators that are sensitive to pH (FIG.
  • Conjugates can also be designed to deliver magnetic resonance probes, caged compounds or chemical cross-linkers.
  • cell-specific promoters and gene transfer should allow the in vivo targeting of hapten-probe complexes to specific cell types in multicellular organisms.

Landscapes

  • Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Biochemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • General Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Cell Biology (AREA)
  • Microbiology (AREA)
  • Food Science & Technology (AREA)
  • Pathology (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • Peptides Or Proteins (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)

Abstract

La présente invention concerne des procédés et des réactifs permettant de cibler des sondes dans des zones cellulaires sélectionnées grâce à l'expression dans une cellule de partenaires de liaison par rapport à ces sondes. Dans une réalisation, les sondes peuvent être du type spectroscopique et servir à localiser un partenaire de liaison dans une cellule et à créer des essais pour des activités post-translationnelles. L'invention permet de surveiller l'emplacement de partenaires de liaison intracellulaires spécifiques de ce type, dans le temps et en réponse à certains stimuli, par exemple à des substances chimiques test. Les sondes spectroscopiques peuvent être utilisées pour contrôler l'activité d'une substance chimique test. La présente invention concerne également des cellules et des organismes transgéniques renfermant le partenaire de liaison spécifique intracellulaire, lequel peut se lier avec la sonde spectroscopique/ le conjugué ligand.
PCT/US1999/007847 1998-04-08 1999-04-08 Procedes et reactifs pour le ciblage de composes organiques a des emplacements cellulaires selectionnes WO1999051986A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
AU34874/99A AU3487499A (en) 1998-04-08 1999-04-08 Methods and reagents for targeting organic compounds to selected cellular locations
US09/403,882 US7045305B1 (en) 1998-04-08 1999-04-08 Methods and reagents for targeting organic compounds to selected cellular locations

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US8111898P 1998-04-08 1998-04-08
US60/081,118 1998-04-08
US8134098P 1998-04-09 1998-04-09
US60/081,340 1998-04-09

Publications (1)

Publication Number Publication Date
WO1999051986A1 true WO1999051986A1 (fr) 1999-10-14

Family

ID=26765219

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1999/007847 WO1999051986A1 (fr) 1998-04-08 1999-04-08 Procedes et reactifs pour le ciblage de composes organiques a des emplacements cellulaires selectionnes

Country Status (2)

Country Link
AU (1) AU3487499A (fr)
WO (1) WO1999051986A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002041001A1 (fr) * 2000-11-16 2002-05-23 Roche Diagnostics Gmbh Couple de colorants pour mesures de transfert d'energie de fluorescence-resonance

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4618827A (en) * 1984-09-10 1986-10-21 General Electric Company Method for high-spatial-resolution spectroscopic NMR imaging of chemically-shifted nuclei
US5328984A (en) * 1991-03-04 1994-07-12 The United States As Represented By The Department Of Health & Human Services Recombinant chimeric proteins deliverable across cellular membranes into cytosol of target cells
US5561049A (en) * 1994-09-21 1996-10-01 Behringwerke Ag Method for detecting antibodies
US5602095A (en) * 1992-06-18 1997-02-11 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Recombinant pseudomonas exotoxin with increased activity
US5703369A (en) * 1995-10-20 1997-12-30 Hamamatsu Photonics K.K. Positron emission computed tomography apparatus and image reconstruction method

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4618827A (en) * 1984-09-10 1986-10-21 General Electric Company Method for high-spatial-resolution spectroscopic NMR imaging of chemically-shifted nuclei
US5328984A (en) * 1991-03-04 1994-07-12 The United States As Represented By The Department Of Health & Human Services Recombinant chimeric proteins deliverable across cellular membranes into cytosol of target cells
US5602095A (en) * 1992-06-18 1997-02-11 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Recombinant pseudomonas exotoxin with increased activity
US5561049A (en) * 1994-09-21 1996-10-01 Behringwerke Ag Method for detecting antibodies
US5703369A (en) * 1995-10-20 1997-12-30 Hamamatsu Photonics K.K. Positron emission computed tomography apparatus and image reconstruction method

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
J. RICHARDSON ET AL.: "Phenotypic knockout of the high-affinity human interleukin 2 receptor by intracellular single-chain antibodies against the alpha subunit of the receptor.", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, NATIONAL ACADEMY OF SCIENCES, US, vol. 92., 11 April 1995 (1995-04-11), US, pages 3137 - 3141., XP002097645, ISSN: 0027-8424, DOI: 10.1073/pnas.92.8.3137 *
YUAN Q., ET AL.: "INTRACELLULAR SINGLE-CHAIN INHIBITS VLA-4 MATURATION AN FUNCTION.", BIOCHEMICAL JOURNAL, PORTLAND PRESS LTD., GB, vol. 318., 1 January 1996 (1996-01-01), GB, pages 591 - 596., XP002921472, ISSN: 0264-6021 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002041001A1 (fr) * 2000-11-16 2002-05-23 Roche Diagnostics Gmbh Couple de colorants pour mesures de transfert d'energie de fluorescence-resonance
US6908769B2 (en) 2000-11-16 2005-06-21 Roche Diagnostics Gmbh Dye pair for fluorescence resonance energy transfer (FRET) measurements

Also Published As

Publication number Publication date
AU3487499A (en) 1999-10-25

Similar Documents

Publication Publication Date Title
CA2283629C (fr) Detecteurs de proteines fluorescentes permettant de detecter des substances a analyser
EP1088233B1 (fr) Systeme de transfert d'energie de resonance par bioluminescence et utilisation dudit systeme
US7060869B2 (en) Fluorescent protein sensors for detection of analytes
US8101364B2 (en) Fragments of fluorescent proteins for protein fragment complementation assays
US6627449B1 (en) Fluorescent protein sensors for measuring the pH of a biological sample
US6900304B2 (en) Emission ratiometric indicators of phosphorylation
US9103790B2 (en) Linker for unimolecular FRET biosensor based on principle of fluorescence resonance energy transfer
US7060793B2 (en) Circularly permuted fluorescent protein indicators
US8669074B2 (en) Chimeric phosphorylation indicator
WO2001046694A2 (fr) Molecule de fusion de transfert d'energie de resonance par bioluminescence et procede d'utilisation
AU2002312149A1 (en) Emission ratiometric indicators of phosphorylation
US6852906B2 (en) Assay for measuring enzyme activity in vivo
WO2002033102A1 (fr) Activation induite par un recepteur de proteines g heterotrimeriques
JP2003513271A (ja) 汎用蛍光センサー
CA2432782A1 (fr) Polypeptides fluorescents dimeres
US7045305B1 (en) Methods and reagents for targeting organic compounds to selected cellular locations
WO2008094316A2 (fr) Nouvelles protéines fluorescentes dérivées du genre branchiostoma
US20030143634A1 (en) Method to detect interactions between cellular components in intact living cells, and to extract quantitative information relating to those interactions by fluorescence redistribution
WO1999051986A1 (fr) Procedes et reactifs pour le ciblage de composes organiques a des emplacements cellulaires selectionnes
EP2802600B1 (fr) Procédés pour identifier les ligands de récepteurs de sigma-1
WO2001075142A2 (fr) Dosage de proteine kinase
RU2338785C2 (ru) Флуоресцирующие белки и хромопротеины из видов hydrozoa, не относящихся к aequorea, и способы их получения
WO2006020550A2 (fr) Capteurs d'analytes et procede de construction de motifs de liaison d'analyte
WO2013087922A1 (fr) Procédés de production de protéines fluorescentes génétiquement modifiées à des fins d'amélioration du fret, produits et leurs utilisations
CA2541920A1 (fr) Systeme de transfert d'energie de resonance par bioluminescence et utilisation dudit systeme

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AL AM AT AU AZ BA BB BG BR BY CA CH CN CU CZ DE DK EE ES FI GB GD GE GH GM HR HU ID IL IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT UA UG US US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW SD SL SZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
WWE Wipo information: entry into national phase

Ref document number: 09403882

Country of ref document: US

NENP Non-entry into the national phase

Ref country code: KR

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase