WO2002085667A2 - Methode de fret par trois au cube (fret-33) destinee a la detection d'un transfert d'energie de fluorescence - Google Patents

Methode de fret par trois au cube (fret-33) destinee a la detection d'un transfert d'energie de fluorescence Download PDF

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WO2002085667A2
WO2002085667A2 PCT/US2002/004563 US0204563W WO02085667A2 WO 2002085667 A2 WO2002085667 A2 WO 2002085667A2 US 0204563 W US0204563 W US 0204563W WO 02085667 A2 WO02085667 A2 WO 02085667A2
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fret
donor
acceptor
molecule
filter
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PCT/US2002/004563
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WO2002085667A3 (fr
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David T. Yue
Michael G. Erickson
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The Johns Hopkins University School Of Medicine
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Priority to US10/467,964 priority patent/US20040191786A1/en
Publication of WO2002085667A2 publication Critical patent/WO2002085667A2/fr
Publication of WO2002085667A3 publication Critical patent/WO2002085667A3/fr

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    • 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
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching
    • 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
    • G01N2500/02—Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)

Definitions

  • the invention relates to methods for detecting and quantifying Fluorescent Resonance Energy Transfer (FRET).
  • FRET Fluorescent Resonance Energy Transfer
  • the invention termed the 3 3 -FRET method, furnishes the means to perform quantitative, FRET-based assays for measuring inter- or intramolecular interactions, in a manner that is especially suited to the conditions encountered in living cells.
  • High throughput screening (HTS) assays for compounds that alter either inter- or intra-molecular interactions are widely used to screen large numbers of test compounds for potential therapeutic activity.
  • Methods for monitoring cellular responses of a drug target (e.g., such as an extracellular receptor) to a test compound using optically detectable labels can provide a sensitive and quantitative measure of the target's activity.
  • cell-based assays also can be used to characterize the physiological function of a target biomolecule, for example, by identifying changes in a target biomolecule's function in response to physiological stimuli.
  • Functional assays can range from binding assays (e.g., library-based screening methods) to genetic assays (e.g., screens for extragenic suppressors or activators) (see, e.g., Phizicky and Fields, 1995, Microbiol. Rev. 59: 94-123).
  • One technique for assessing intermolecular interactions is based on fluorescence resonance energy transfer (FRET) (see Selvin, 1995, Methods Enzymol. 246: 300- 334). In this process, a "donor" fluorophore transfers its excited-state energy to an "acceptor” fluorophore which typically emits fluorescence of a different color.
  • FRET fluorescence resonance energy transfer
  • Suitable donor and acceptor fluorophore pairs are those that exhibit substantial overlap between respective emission and excitation spectra (Selvin, 1995, Methods Enzymol. 246: 300-334).
  • FRET has been used in both in vitro and in vivo assays to monitor protein-protein interactions by chemically attaching appropriate fluorophores to pairs of purified proteins and measuring fluorescence spectra of protein mixtures or cells microinjected with the labeled proteins (see, e.g., Adams, et al., 1991, Nature 349: 694-697).
  • spontaneously fluorescent proteins has facilitated genetic labeling of proteins with fluorophores.
  • GFP green fluorescent protein
  • the cDNA encoding GFP can be fused with coding sequences from a number of other proteins, thus enabling such proteins to fluoresce without interfering with their biological activity or cellular localization.
  • mutant variants of spontaneously fluorescent proteins with different emission wavelengths across the visible spectrum provide a variety of suitable dono ⁇ acceptor pairs for FRET (see, e.g., Heim, et al., 1994, Proc. Nat. Acad. Sci. USA. 97: 12501-12504).
  • enhanced cyan fluorescent protein ECFP
  • enhanced yellow fluorescent protein EYFP
  • ECFP enhanced cyan fluorescent protein
  • EYFP enhanced yellow fluorescent protein
  • the invention (3 3 -FRET) provides a fast, simple, and nondestructive method for detecting and quantifying FRET, despite the aforementioned challenges.
  • One advantage of the 3 -FRET method is that it provides a way to nondestructively determine a quantitative index of the strength of FRET interactions, despite variable expression levels and variable bound fractions of acceptor- and donor-tagged molecules.
  • the specific index of FRET is termed "the FRET ratio," or FR.
  • a second advantage of the 3 -FRET method is that it provides a way to nondestructively determine: the fraction of acceptor-tagged molecules that are bound by donor-tagged molecules; the relative affinity of a binding reaction; and the strength of FRET interactions when all acceptor-tagged molecules are bound by donor-tagged molecules. The latter determination enables estimates of the physical distance and/or orientation between interacting acceptor and donor fluorophore molecules to be obtained.
  • This second advantage may be conveniently applied to determinations of FR, but may also be applied to many other quantitative FRET indices.
  • the invention provides a method for detecting interactions between two molecules or between different portions of a single molecule.
  • the method comprises processing measurements made from a specimen containing donor and acceptor fluorophores, which are atttached to either separate molecules or different parts of the same molecule.
  • the specimen is exposed to a wavelength of light suitable for exciting donor molecules and the light emitted by the specimen is detected and decomposed to determine whether acceptor molecules have received energy from donor molecules, i.e., indicating the relative proximity of the donor and acceptor molecules.
  • the individual filter sets each comprise a filter between the light source and the specimen and a filter between the specimen and the detector.
  • Each filter set transmits and/or reflects specific wavelengths of light.
  • the filter between the light source and specimen maximally transmits a wavelength of light that excites the donor (and possibly the acceptor), and the filter between the specimen and the detector maximally transmits wavelengths of light where only the donor emits photons.
  • the filter between the light source and specimen maximally transmits a wavelength of light that preferentially excites the acceptor, and the filter between the specimen and the detector maximally transmits wavelengths of light where mainly the acceptor emits photons (and possibly the donor emits photons).
  • the filter between the light source and specimen maximally transmits a wavelength of light that excites the donor (and possibly the acceptor)
  • the filter between the specimen and the detector maximally transmits wavelengths of light where mainly the acceptor emits photons (and possibly the donor emits photons).
  • the 3 3 -FRET method processes these three light intensity readings, each obtained with a different filter set engaged, and yields a quantitative readout of the strength of FRET interaction, termed "the FRET ratio" or FR.
  • FR furnishes the fractional increase in acceptor fluorescence due to FRET.
  • each filter cube comprises the first, second, and third filter sets.
  • each filter cube contains an excitation filter, a dichroic mirror, and an emission filter.
  • the donor molecule is a polypeptide such as ECFP and the acceptor molecule is a polypeptide such as EYFP.
  • the FRET ratio is produced by processsing sequential filter set measurements according to:
  • S F RET(DA) is a measure of light intensity transmitted to the detector from the FRET filter set
  • SD(DA) is a measure of light intensity transmitted to the detector from donor filter set
  • S A (DA) is a measure of light intensity transmitted to the detector from the acceptor filter set.
  • R DI , R A I, and R D2 are predetermined constants determined from measurements of light emissions from specimens expressing only donor (D) or acceptor (A) molecules (see Equations A6-A8 in Detailed Description, below). In practice, no two optical systems are identical; for example, small aberrations in optical components comprising the filter sets are common. Because FR is unitless, this index of FRET has the special advantage of being independent of these small aberrations; all errors of this sort are "normalized out” in producing this ratio.
  • the method comprises processing like measurements from multiple specimens, and furnishing an estimate of the relative affinity of the binding of donor-tagged molecules to acceptor-tagged molecules, the fractional binding of acceptor-tagged molecules by donor-tagged molecules in any individual specimen, and the maximum FRET efficiency when every acceptor-tagged molecule is associated with a donor-tagged molecule.
  • the method comprises providing an estimate of the relative affinity of the binding of acceptor-tagged molecules to donor- tagged molecules, the fractional binding of donor-tagged molecules by acceptor-tagged molecules in any individual specimen, and the maximum FRET efficiency when every donor-tagged molecule is associated with a acceptor-tagged molecule.
  • the maximum FRET efficiency can be used to determine the physical distance and/or orientation between donor and acceptor molecules. In one aspect, the maximum FRET efficiency can be gauged by FR mm , the maximum FRET ratio when every acceptor-tagged molecule is associated with a donor-tagged molecule.
  • the classic index of FRET efficiency, termed E, can then be produced by processing ERmax according to:
  • bracketed term is the ratio of acceptor and donor molar extinction coefficients at the preferred wavelength of the filter between the light source and specimen in the FRET filter set.
  • the specimen is a cell and the method further comprises the step of introducing the donor and acceptor molecule into the cell.
  • the donor and acceptor molecule can be introduced by transfection (e.g., cDNA transfection), transformation, electroporation, microinjection, or a combination thereof.
  • the donor and acceptor molecule can each be linked to different biomolecules, using standard molecular biological techniques.
  • the different biomolecules are binding partners, e.g., interacting polypeptides, nucleic acids, or nucleic acids and nucleic acid binding proteins.
  • one of the polypeptides is selected from the group consisting of calmodulin (CaM), cGMP-dependent protein kinase, a steroid hormone receptor or a ligand binding domain thereof, protein kinase C, inositol- 1,4,5- triphosphate receptor, alphachymotrypsin, or recoverin.
  • CaM calmodulin
  • cGMP-dependent protein kinase a steroid hormone receptor or a ligand binding domain thereof
  • protein kinase C inositol- 1,4,5- triphosphate receptor
  • alphachymotrypsin alphachymotrypsin
  • recoverin recoverin.
  • One or both of the polypeptides can contain an intracellular localization signal for specific targeting of one or both of the polypeptides within a cell. Detection of FRET can be used to assay for intermolecular interactions in this system.
  • the cell is exposed to a sample suspected of comprising a modulator of the binding partners and the measure of FRET provides an indication of whether or not the sample comprises the modulator.
  • one of the binding partners is an intracellular signaling molecule.
  • Suitable binding partners include, but are not limited to: a ligand and receptor; antibodies and antigens; calmodulin and ion channels; G-proteins and ion channels; and GTP and G-protein coupled receptors.
  • the method is used to identify interacting molecules (e.g., such as those involved in intracellular signaling processes).
  • the donor molecule is linked to a "bait" polypeptide (e.g., encoding a polypeptide being evaluated such as an orphan receptor), while the acceptor molecule is linked to a "prey" polypeptide (e.g., an unknown polypeptide sequence taken from a library or expressed sequences such as a cDNA library).
  • the measure of FRET provides a measure of whether the bait polypeptide and prey polypeptide specifically bind to each other.
  • Single-cell purification of plasmid DNA (“single-cell miniprep") can be used to specify the sequence identity of nucleic acids encoding the interacting prey polypeptide. In this manner, discovery of unknown interaction partners with a specified bait polypeptide can be determined.
  • the assay can be used to identify ligands for orphan receptors. Application of this approach to many cells in parallel, such as using plate-reader technology, permits high-throughput identification of interacting molecules.
  • the assay also can be used to identify interacting molecules in living mammalian cells.
  • the method can be used to identify mutations or compounds that inhibit and/or promote binding between two molecules known to interact.
  • mutations can be introduced into polypeptides fused to either donor or acceptor fluorophores. Loss or enhancement of FRET interaction between binding partners indicates a critical site for interaction was mutated.
  • cells expressing interacting FRET partners can be exposed to a library of compounds. Loss and/or enhancement of FRET indicate a compound that may modulate the interaction between specific FRET-pair molecules. Because the 3 3 -FRET method is nondestructive, time-dependent aspects of compound modulation may be examined. Application of this approach to many cells in parallel, such as using plate-reader, technology, permits high-throughput identification of important mutations or modulatory molecules.
  • the donor and acceptor molecule also can be linked to a single molecule (e.g., a nucleic acid or polypeptide) for detecting an analyte.
  • a single molecule e.g., a nucleic acid or polypeptide
  • the molecule for detecting an analyte specifically binds to the analyte.
  • the molecule for detecting an analyte is cleavable by the analyte.
  • the molecule for detecting an analyte may comprise a polypeptide comprising a protease cleavage site or may comprise a nucleic acid comprising a nuclease digestion site.
  • the molecule for detecting an analyte is immobilized on a solid phase, thereby forming a FRET sensor.
  • the FRET sensor can be exposed to a sample suspected of comprising the analyte, and the measure of FRET obtained can be correlated with the presence or level of the analyte.
  • the donor molecule and acceptor molecule linked to a single molecule for detecting an analyte also can be introduced into a cell and the measure of FRET can be correlated with the presence or level of analyte in the cell.
  • the method further comprises the step of sorting cells comprising donor and acceptor molecules from those which do not comprise both donor and acceptor molecules.
  • the method comprises the further step of sorting cells in which FRET occurs from cells in which FRET does not occur.
  • the optical system comprises a light source for providing excitation light to the specimen; the detector; a specimen holder for positioning the specimen in a suitable position to receive light from the light source sufficient to excite the donor; and to transmit light emitted by the cell to the detector; and a holder for sequentially receiving the first, second, and third filter sets, and for positioning each of the filters.
  • the optical system is selected from the group consisting of an epifluorescence microscope, a confocal microscope, a flow cytometer, and a plate reader.
  • the 3 3 -FRET invention provides a fast, simple, and nondestructive method for detecting and quantifying FRET.
  • One main part of the 3 - FRET method provides means to sensitively and selectively produce a quantitative index of the strength of FRET interaction. The process controls for variability in expression levels and fractional binding of acceptor- and donor-tagged molecules; for inevitable small aberrations in optical components used to perform FRET measurements; and for optical crosstalk between acceptor and donor fluorophores.
  • a advantage of the 3 -FRET method provides a means to determine: the fraction of acceptor-tagged molecules that are bound by donor-tagged molecules; the relative affinity of that binding reaction; and the strength of FRET interaction when all acceptor-tagged molecules are bound by donor-tagged molecules. The latter determination enables estimates of the physical distance and/or orientation between interacting acceptor and donor fluorophore molecules.
  • Figures 1 A-F show that CaMwr-ECFP and ⁇ ic-EYFP preserve Ca 2+ - dependent inactivation.
  • Figure 1 A shows the ⁇ 2a subunit and CI region (Peterson, et al., 1999, Neuron 22: 549-558) of ⁇ ic-EYFP.
  • Figure IB shows a confocal image and intensity profile for a cell expressing ⁇ c-EYFP/ ⁇ 2a / ⁇ 2 ⁇ . Peaks indicate membrane targeting.
  • Figure 1 C shows HEK293 lysates probed with anti-CaM or anti-GFP (labelled).
  • Upper left comparison of control (mock transfected) cells with cells overexpressing CaMw ⁇ -ECFP or CaM M ur-ECFP; arrowhead indicates endogenous CaM at -20 kD.
  • Lower left same lysates as above, optimized for visualization of endogenous CaM, showing that endogenous CaM expression is unchanged.
  • Lower right calibration ladder for purified recombinant CaM ⁇ v ⁇ and CaM ⁇ viu ⁇ > conditions same as at left.
  • Upper right immunoblot probed with anti-GFP antibody comparing CMV and SV40 promoter systems.
  • Figure ID shows whole-cell currents from cells co-expressing ⁇ c-EYFP/ ⁇ 2a / ⁇ 2 ⁇ and CaMwr- ECFP.
  • the upper graph shows Ba (black) and scaled Ca (gray) currents during steps to -10 mV.
  • the lower graph shows the fraction of current remaining at the end of 300 ms depolarizations (r 30 o)-
  • Figure IE shows results from cells co-expressing ⁇ ic- EYFP/ ⁇ 2a / ⁇ 2 ⁇ and CaM M u ⁇ -ECFP using a format identical to Figure ID.
  • Figure IF shows confocal images and intensity profiles for cells expressing CaMwr-EYFP alone (left) or together with ⁇ c ⁇ 2a ⁇ 2 b ⁇ (right) showing some perimembrane enrichment of CaMwr-EYFP (peaks in intensity profile) when coexpressed with unlabeled channels.
  • Figure 2 illustrates FRET detection by 3 -FRET.
  • Figure 2A shows dissection of 535 nm emission with 440 nm excitation.
  • the graph shows the overall emission spectrum from a single cell expressing ECFP- and EYFP-tagged proteins (black line), reflecting underlying ECFP (thick gray) and EYFP (thin gray) spectra. Portions of the EYFP emission are due to direct excitation (gray dashed spectra).
  • Points (1 - 5) are: SF RE r(DA); ⁇ D1 S CF p(DA); S FREr (DA) -R D ⁇ S CF p(DA); R M S ⁇ F p(DA); and, Sc F p(DA); where and are pre-computed constants from cells expressing only ECFP- or EYFP-tagged proteins, respectively, and are described further in the text below.
  • Figure 2B shows 3 3 -FRET control experiments on single live cells expressing indicated constructs. Horizontal axes correspond to the FRET Ratio (FR) and FRET percent efficiency (E). For yellow cameleon-2 constructs, cells were incubated in 10 ⁇ M ionomycin for 15 minutes before application of either 5 mM EGTA or 20 mM CaCl in buffered Tyrode's.
  • Figures 3A-B show preassociation of CaM with L-type Ca + channel complexes. Horizontal axes correspond to the FRET Ratio (FR) and FRET percent efficiency (E); ⁇ 2 b ⁇ subunits also are transfected. As shown in Figure 3A, 3 3 - FRET reveals that and CAM M U T preassociate with L-type channels in resting cells. Asterisk,/? ⁇ 0.01 vs. free ECFP; dagger, * ⁇ 0.05. Figure 3B shows that preassociation with L-type channel complexes requires the ⁇ ic pore-forming subunit. dagger, p ⁇ 0.05
  • Figure 4 shows preassociation of CaM with R-Type and P/Q Type Ca 2+ channel complexes. Format Identical to Figure 3; ⁇ 2 ⁇ subunits also are transfected. Asterisk, /? ⁇ 0.01 vs. free ECFP
  • Figure 5 shows a model of CaM preassociation.
  • Figure 5 A shows analysis of FR data for cells coexpressing CaMw ⁇ -ECFP and ⁇ c-EYFP/ ⁇ 2a / ⁇ 2b ⁇ .
  • the upper panels show a comparison of measured (filled circles) and predicted (black line) FR values for cells coexpressing FRET between pairings plotted versus calculated fraction bound, A b .
  • Arrowhead indicates the maximal FR, FR max .
  • FRET also was measured by swapping ECFP and EYFP and quantitating ECFP dequenching following complete acceptor photodestraction (open circles).
  • N_s Black and N A (gray) are relative numbers of ECFP-and EYFP-tagged molecules, respectively, as determined using ⁇ 2b ⁇ .
  • FIG. 5B shows FR data for cells coexpressing ECFP and ⁇ c-EYFP/ ⁇ 2a / ⁇ 2b ⁇ using a format analogous to Figure 5A.
  • FR-A_ data is plotted as mean +/- SD for visual clarity.
  • Figure 5C shows FR data for cells expressing yellow cameleon-2 (YC2) in the Ca 2+ free state. The format is analogous to that of Figure 5A.
  • FR-A_ data is plotted as mean +/-SD for visual clarity.
  • Figure 5D shows a table of K_£_ ⁇ and FR max values from fits of measured FR.
  • Figure 5E show FR data for cells coexpressing CaMwr- ECFP and ⁇ 2a -EYFP (left) or ⁇ 2a -ECFP and ⁇ c-EYFP/ ⁇ 2b ⁇ .
  • the format is identical to the upper panel of Figure 5 A.
  • Figure 5F shows course triangulation of key channel landmarks using 3 3 -FRET analysis. ECFP and EYFP are not represented.
  • Figure 6 shows fluorescence behavior of donor (ECFP) and acceptor (EYFP) molecules in a microscope field of view, represented quantitatively as three sequential subsystems: an excitation subsystem, afluorophore-rate-constant subsystem, and emission-detection subsystem.
  • the three output signals on the right are those that comprise aggregate fluorescence output obtained with any of the filter filter sets or cubes used an optical system according to the invention.
  • Figures 7A-C show application of 3 -FRET to two-hybrid screening of Ca channel/CaM interactions.
  • Figure 7A shows examplar "prey” segments from the ctic CI region, and the relevant "bait.”
  • EF, PrelQ and IQ are ⁇ 33-residue domains.
  • Figure 7B, left, shows screen results for the labelled prey-bait pair, showing that PrelQ, IQ and PrelQ-IQ each interact with CaM MUT - Right, preliminary fits using 1 : 1 binding model as in Figure 5.
  • FIG. 7C shows Ca 2+ -dependent movements in CaM binding to segments of the Ca + channel (same format as in Figure 7B). Cells were clamped to either high (10 mM, gray bars) or low (5 mM EGTA, gray bars) Ca 2+ following 15 minute incubation in ionomycin, a potent Ca + -ionophore.
  • Figure 8 A shows a flow-chart depicting the major steps of the 3 3 -FRET method for producing FR, the quantitative index of FRET according to one aspect of the invention.
  • Figure 8B shows a flow-chart depicting the major steps of the 3 3 -FRET method for producing K_ ⁇ FF and FR mSL .
  • the invention (3 3 -FRET) provides a fast, simple, and nondestructive method for detecting and quantifying FRET.
  • the 3 -FRET method can be used to sensitively and selectively determine a quantitative index of the strength of FRET interaction, based on a series of fluorescent intensity readings from a specimen, such as a cell, using three filter sets.
  • the specific index of FRET is termed "the FRET ratio," or FR.
  • the 3 -FRET method also can be used to determine one or more of the following: the fraction of acceptor-tagged molecules that are bound by donor-tagged molecules; the relative affinity of that binding reaction; and the strength of FRET interaction when all acceptor-tagged molecules are bound by donor-tagged molecules.
  • the latter determination enables estimates of the physical distance and/or orientation between interacting acceptor and donor fluorophore molecules.
  • the method can be applied to determinations of FR, but may also be applied to many other quantitative FRET indices.
  • the method can be used to monitor inter- or intra-molecular interactions, detect analytes, identify polypeptide-binding partners from a library of expressed sequences (e.g., such as a cDNA library) and probe compounds for their ability to inhibit or enhance polypeptide binding.
  • a library of expressed sequences e.g., such as a cDNA library
  • probe compounds for their ability to inhibit or enhance polypeptide binding.
  • the method is incorporated into an HTS assay for parallel screening of molelecular interactions across many samples.
  • a "polypeptide” refers to a polymer in which the monomers are amino acid residues which are joined together through amide bonds.
  • the amino acids are alpha-amino acids, either the L-optical isomer or the D-optical isomer can be used; however, the term also includes the polymers comprising unnatural amino acids such as beta-alanine, phenylglycine, and homo-arginine.
  • a fluorescent protein refers to any protein capable of emitting light when excited with appropriate electromagnetic radiation. Fluorescent proteins include proteins having amino acid sequences that are either natural or engineered, such as the fluorescent proteins derived from fluorescent proteins.
  • GFP is the green fluorescent protein from the jellyfish, Aequorea victoria.
  • CFP CFP
  • ECFP E-YFP
  • GFP variant enhanced yellow fluorescent protein as described by Miyawaki, et al. (supra).
  • nucleic acid refers to DNA, RNA, DNA:RNA hybrids, single stranded or double stranded forms thereof, and includes modified or variant forms thereof.
  • a "heterologous" region of a DNA construct is an identifiable segment of DNA within a larger DNA molecule that is not found in association with the larger molecule in nature.
  • the heterologous region encodes a mammalian gene
  • the gene will usually be flanked by DNA that does not flank the mammalian genomic DNA in the genome of the source organism (e.g., such as viral promoter sequences).
  • a "donor molecule” refers to a fluorophore which when in the excited state can transfer energy to an acceptor molecule, provided that the donor fluorescence emission spectrum overlaps significantly with the acceptor absorption spectrum.
  • An "acceptor molecule” refers to a fluorophore which, upon receiving energy from a donor molecule, can enter the excited state and emit a photon.
  • suitable donor:acceptor pair refers to a pairing of donor and acceptor fluorophores that satisfies the definitions of donor molecule and acceptor molecule.
  • a FRET signal refers to the emission produced when an acceptor molecule receives energy from a donor molecule.
  • energy transfer can only occur when two conditions are met: the donor and acceptor are separated by less than approximately 100 A; and, the donor emission transition dipole and acceptor absorption transition dipole are not perpendicular (i.e., the orientation factor, K , does not equal zero).
  • a donor and acceptor molecule in "close proximity” refer to donor and acceptor molecules in sufficient proximity and at appropriate orientations to cause a FRET signal.
  • a "light path” refers the geometrical distance between a light source and a light detector or photodetector.
  • a "ratio of acceptor and donor molar extinction coefficients scaled for the third (FRET) filter” refers to the ratio of acceptor and donor molar extinction coefficients at the preferred wavelength of the filter between the light source and specimen in the FRET filter set.
  • a molecule which is "linked” to another molecule refers to a molecule which is stably coupled to another molecule, for example, by a covalent linkage.
  • a "linked molecule” can be chemically conjugated to another molecule using methods routine in the art, or, if a polypeptide, can be engineered so as to be fused in frame with the other molecule (e.g., the covalent linkage may be an amide bond).
  • a "modulator" of a molecular interaction refers to a compound which produces a statistically significant change in the interaction relative to the interaction as measured in the absence of compound.
  • a molecular interaction refers to an intermolecular or an intramolecular interaction.
  • FRET coupling between donor and acceptor fluorophores provides one of the most promising approaches for detecting polypeptide interactions in living samples, such as single cells.
  • Donor and acceptor fluorophores can be chemically attached to two polypeptides (or, to different parts of the same polypeptide) within the sample, and FRET between the donor and acceptor then becomes an optical means of detecting whether the "tagged" polypeptides associate (i.e., are within close proximity).
  • Detection and quantification of FRET signals generally relies on measurements of light in the visible or near-visible wavelengths, which is inherently non-invasive (i.e., does not require destruction of the sample).
  • FRET can monitor polypeptide interactions in the setting of ultimate biological relevance — the living cell.
  • ECFP enhanced cyan fluorescent protein
  • EYFP enhanced yellow fluorescent protein
  • EYFP and EYFP are held together at a distance of less than about 100 , energetically excited ECFP can return to its ground state by transferring its energy to EYFP (i.e., via FRET) without emitting a fluorescent photon. An excited EYFP molecule can then relax and emit a yellow photon ( ⁇ 535 nm), a phenomenon called sensitized EYFP emission (see, e.g., as described in Clegg, 1992, Methods Enzymol. 277: 353-358).
  • a shift from cyan to yellow fluorescence in a sample comprising a mixture of ECFP and EYFP thus indicates that ECFP and EYFP are within about 100 of each other.
  • separations of less than 100 generally imply that the two fluorophores, and by inference the polypeptides to which they are fused, are closely associated with one another.
  • a sample, such as a cell, comprising molecules tagged with ECFP and EYFP can thus be evaluated using an appropriate optical system to determine whether intermolecular interactions are taking place and/or whether compounds added to the sample are capable of modifying such interactions.
  • filter sets The individual filters which comprise a filter set transmit and/or reflect specific wavelengths of light. In the case of epifluorescent microscopes, these filter sets are usually combined as filter cubes. A wide spectrum of filter sets and/or cubes is available from most major manufacturers. Filter sets comprise one or more of the following: excitation filter, emission filter, and dichroic mirror (or, dichroic beamsplitter). Excitation filters permit only selected wavelengths from a light source to pass to a specimen, such as a cell.
  • Emission filters are filters that block or absorb the excitation wavelengths and permit only selected emission wavelengths to pass to a photodetector, such as the eye, photomultiplier tube, or CCD camera. Emission filters generally suppress shorter wavelengths and have high transmission for longer wavelengths.
  • Dichromatic mirrors are filters designed to reflect excitation wavelengths and transmit emission wavelengths. They are used in reflected light fluorescence illuminators and are positioned in the light path after the exciter filter but before the emission filter and are generally at a 45° angle with respect to light passing through the excitation filter and light passing through the emission filter.
  • a filter set generally combines these elements to provide appropriate wavelengths of light to enable detection of a fluorophore.
  • 3 -FRET processes the signals obtained from a combination of filter sets (or filter cubes) to produce an index of the strength of energy transfer between donor and acceptor molecules.
  • acceptor emission To determine FRET from measurements of sensitized acceptor emission, fluorescence emission from the acceptor must be dissected from contaminating donor emission (i.e., donor "crosstalk”). (4) Fluorescence emission from the acceptor must be dissected from contaminating donor emission (i.e., donor "crosstalk”). (5) Some FRET assays require photo-destruction (e.g., photobleaching over many minutes) of the donor or acceptor fluorophores, which precludes measurements of FRET at different time points from the same sample.
  • photo-destruction e.g., photobleaching over many minutes
  • FRETN multi-filter method
  • the 3 -FRET method provides a fast, simple, and nondestructive method for detecting and quantifying FRET, despite the challenges described above.
  • One advantage of the 3 -FRET method is that it provides a way to nondestructively produce a quantitative index of the strength of FRET signal.
  • the specific index of FRET is termed "the FRET ratio," or FR.
  • a second advantage of the 3 3 -FRET method is that it provides a way to nondestructively determine: the fraction of acceptor-tagged molecules that are bound by donor-tagged molecules; the relative affinity of a binding reaction; and the strength of FRET interactions when all acceptor-tagged molecules are bound by donor-tagged molecules. The latter determination enables estimates of the physical distance and/or orientation between interacting acceptor and donor fluorophore molecules to be obtained.
  • This second advantage may be conveniently applied to determinations of FR, but may also be applied to many other quantitative FRET indices.
  • the EBFP/EGFP FRET pair is not as favorable due to the relatively poor quantum yield of EBFP (Miyawaki et al., 1997, supra).
  • FRET pairs involving red-shifted fluorescent proteins, such as DsRed often suffer from slow fluorophore maturation and intracellular aggregation (see Lauf, et al., 2001, FEBS Letters 498A 1-15).
  • the 3 3 -FRET method generalizes to many other suitable FRET pairs.
  • the 3 3 -FRET method will likely be of considerable advantage for quantifying FRET from these pairs.
  • the invention provides a method for detecting a FRET signal from a specimen containing suitable donor-tagged and acceptor-tagged molecules utilizing 3 3 -FRET.
  • 3 3 -FRET involves "optical dissection” by obtaining sequential intensity readings from a single specimen (e.g., such as a cell) at a time, using measurements made with the three filter sets. Simple equations manipulate readings from each of the filter sets to specify a unitless index of FRET called the FRET ratio (FR). FR bears a linear relation to FRET efficiency E, described further below.
  • sequential light intensity readings are obtained from the specimen using an optical system that can sequentially engage three filter sets or cubes (Figure 8A).
  • One filter set preferentially detects donor emission, one filter set preferentially detects acceptor emission, and one filter set detects emissions from both donor and acceptor fluorophores.
  • An exemplary optical system for use in the method comprises a light source for providing excitation light to the specimen; a detector; a specimen holder for positioning the specimen in a suitable position to receive light from the light source and to transmit light emitted by the specimen to the detector; and a filter set holder for sequentially receiving first, second, and third filter sets and for positioning each of the filters.
  • the optical system is selected from the group consisting of an epifluorescence microscope, a confocal microscope, a flow cytometer, and a plate reader.
  • Figure 2A shows a fluorescence emission spectrum produced by illuminating a cell expressing both ECFP and EYFP with light at 440 nm.
  • the double-humped shape results from superposition of individual ECFP (thick line) and EYFP (thin line) spectra.
  • FRET alters this spectrum by decreasing the ECFP (energy donor) peak near 480 nm and enhancing the EYFP (energy acceptor) peak near 535 nm.
  • FRET can therefore be nondestructively dissected from the enhanced EYFP emission at 535 nm by eliminating signal from secondary EYFP emissions due to direct excitation (dashed line) from total EYFP emission (thin line) due to both FRET and direct excitation.
  • Emission at 535 nm ( Figure 2 A, number 1) is the sum of CFP emission (number 2) and YFP emission (number 3), a portion of which is due to direct excitation (number 4).
  • 3 3 -FRET employs filter sets that isolate CFP and YFP signals from a cell expressing both fluorophores.
  • the CFP filter set excites both fluorophores but measures fluorescence where only CFP emits (number 5). Multiplying this measurement by a predetermined constant provides CFP emission at 535 nm (number 2), which is subtracted from number (1) to determine total YFP emission ( F A ; number 3).
  • FRET fluorophores
  • D donor
  • A acceptor
  • D_ represents the fraction of donor molecules bound by an acceptor
  • A_ is the fraction of acceptor molecules bound by a donor. It is assumed that no FRET occurs between unassociated donor and acceptor molecules.
  • the excitation subsystem models the effects of properties of components of an optical detection system used to perform FRET measurements on the excitation rate of a fluorophore.
  • the subsystem accounts for the effects of properties of an excitation light source, excitation filter, and dichroic mirror (e.g., such as are found in an epifluorescence microscope) on excitation rates.
  • the excitation rate (in units of transitions per second) of a single ground-state fluorophore may be represented by IoG ⁇ (y, ⁇ e X , x ), where I_ is the overall intensity of the xenon lamp (over all wavelengths), x specifies which of three filter sets is being used (D, A, or FRET), y specifies a donor or acceptor molecule (D or A) is being evaluated, and ⁇ eX , x is the predominant wavelength of excitation light (determined mainly by the excitation filter of filter set or cube x).
  • G x (y, ⁇ ex , x ) is thus a constant that incorporates spectral properties of a light source used in an optical detector, such as a epifluorescence microscope, optical properties of the excitation filter and dichroic mirror of filter set or cube x, and wavelength-dependent absorption properties of the fluorophore in question as given by a molar extinction coefficient ( ⁇ y ( ⁇ )).
  • the fluorophore-rate-constant subsystem models behavior of fluorophores as a state diagram with interstate transitions governed by various rate constants.
  • the rate constants relating to emission of fluorescent photons are functions of intrinsic properties of donor and acceptor molecules, and are independent of the wavelengths used for excitation or detection of emission.
  • the rate constant pertaining to resonance energy transfer (k ) is a function of many factors, including distance and orientation between bound donor and acceptor molecules, as well as overlap between emission and excitation spectra of donor and acceptor molecules, respectively.
  • k ⁇ is also independent of the wavelengths used for excitation or detection of emission.
  • the rate-constant model shown in Figure 4, describes the probabilities of occupying A, A*, D, and D* states, and the probability flux of transitions among the various states.
  • the steady- state behavior of the system is considered because measurement times are far larger than the characteristic relaxation times.
  • a standard assumption for derivation of FRET equations is that the system is in the "low-excitation limit," where excitation power is low enough that the steady-state probabilities of being in D or A PQ or PA) are essentially unity and this has been experimentally verified for this system.
  • k- ⁇ is the rate constant for FRET between donor and acceptor molecules (all rate constants in units of s "1 )
  • k D is the rate constant for the emission of non-FRET relaxation from D* to D.
  • the rate constants k_- and k_, are independent of wavelength, but k- ⁇ is a function of donor-acceptor distance according to the F ⁇ rster equation
  • G X (A, ⁇ eX ⁇ X ) is a measure of direct excitation of the acceptor by the light source of an optical system (e.g., such as a xenon lamp), which occurs regardless of whether a donor is bound
  • G X (A, ⁇ eX ⁇ X ) is a measure of FRET excitation of the acceptor (which only can occur if donor is bound).
  • the emission-detection subsystem accounts for properties of the emission filter, dichroic mirror, photomultiplier electronics, as well as fluorophore emission spectrum and quantum yield.
  • the three output signals on the right of Figure 6 are those that comprise aggregate fluorescence output obtained with any of the filter sets or cubes.
  • the rate of excited donor relaxations which can possibly give rise to fluorescence emissions by acceptor molecules is represented as k_ ⁇ P_ * .
  • the fluorescence output from the photodetector of an optical system e.g., such as a photomultiplier tube or PMT
  • mV output per second arising from excited donor relaxations
  • F x (D, ⁇ em , x ) is an "output transfer function," corresponding to the signal actually produced by the optical detector, with units of mV per non-FRET donor relaxation.
  • F x (D, ⁇ em, x ) is a constant that incorporates the emission spectrum and quantum yield of the donor, the dichroic mirror and emission filter optical properties of filter set or cube x, and frequency-dependent sensitivity of the photodetector.
  • Equation Al for fluorescence output resulting from donor fluorescence, as measured with a filter set or cube x:
  • Equation A2 provide the full equation for fluorescence output resulting from acceptor fluorescence, as measured with filter set or cube x. This entity is given by two terms relating to direct and FRET excitation (A4 and A5, respectively).
  • the actual fluorescence signal output obtained from a given sample using a particular optical filter set or cube can be denoted by the descriptor S x (specimen), where x is the name of the filter set or cube (e.g., ECFP, EYFP, FRET) and the specimen is either the donor (D), acceptor (A), or both (DA).
  • x is the name of the filter set or cube (e.g., ECFP, EYFP, FRET) and the specimen is either the donor (D), acceptor (A), or both (DA).
  • the longer specifier of the fluorescence signal output S x (specimen, ⁇ eX , X) ⁇ cm , x ) (which is equivalent to S x (specimen)) can be used.
  • excitation wavelength is denoted herein as ⁇ eXlX
  • emission wavelength is denoted as ⁇ emjX as detected by a photo detection device (e.g., a CCD camera).
  • a photo detection device e.g., a CCD camera
  • the signal output obtained from ECFP with the ECFP filter set or cube is S C FP (D, 440,480).
  • the added information in the longer specifier serves as a reminder of dominant operating features of the filter set employed.
  • R _ S FRET (A,440,535) G FRET (A,440) - E FRET (A,535) Al S YFP (A,500,530LP) G YFP (A,500) E YFP (A,530LP)
  • these ratios are independent of the excitation intensity I 0 and the number of donor or acceptor molecules in the field of view N__ or N A . Thus, they can be determined in cells in which acceptor or donor alone are expressed, and then applied to calculations of equations representing the signals obtained from cells in which mixtures of acceptor and donor are expressed.
  • ECFP filter set or cube is used to obtain a fluorescence measurement from a cell expressing both a donor and acceptor. Because EYFP does not detectably emit photons in the 480 nm range, the ECFP filter set or cube measurement SCF P (DA,440,480) is equivalent to the contribution due to ECFP fluorescence alone, or CFP CF p(440,480,direct), as calculated from Equation A3.
  • determining the ratio 7? DI provides a way to transform the optically isolated ECFP signal, Sc F p(DA,440,480), into the ECFP contribution to fluorescence at 535 nm, where both EYFP and ECFP fluorescence are appreciable.
  • the FRET ratio (FR) to be produced by the 3 3 -FRET method is defined as FR ⁇ Y " FP- F - R t E E T . ( v 44 -0,.5-3-5-,. F- R"E • "T) / + ⁇ Y -FP ⁇ FRET ( ⁇ 440-,'535, direct) ' [A10]
  • Equations A4 and A5 where the terms refer to EYFP fluorescence due to direct and FRET excitation, as defined in Equations A4 and A5.
  • the numerator of the expression is easily determined from the experimental measurement S FRET (DA,440,535) by considering its constituent components (see, e.g., as shown in Figure 1 A) and Equations A3-A5:
  • Equation A10 the numerator of the FR expression in Equation A10 is experimentally determined as
  • Equation Al [A12] To solve for YFP FRET (440,535,direct), the denominator of the FR expression in Equation A10, the EYFP filter set or cube measurement S YFP (DA,500,530LP) can be expressed in terms of its three constituent components (see, Figure Al). With reference to Equations A3-A5, an expression strictly analogous to Equation Al 1 can be represented as follows:
  • the second term dominates the expression, consistent with the near selective excitation of EYFP with the EYFP filter set or cube.
  • the third term is considerably smaller, while the first term is even smaller, and negligible from a practical standpoint. In practice, the first term can be ignored.
  • Equation A3 Equation A8
  • Equation A14 yields:
  • Equation A5 allows us to relate YFP YFP (500,530LP,FRET) to YFP FR E T (440,535,FRET) by the relation G YFP (D,500) G FRET (A,440)
  • Equation A17 Substituting Equation A17 into Equation A16 yields
  • Equations A12 and A18 can be solved simultaneously to give the denominator term for FR (in Equation A 10), in terms of experimentally measurable entities, as given by
  • Equation A10 Substituting Equations A12 and A19 into the FR expression in Equation A10 provide 3Ss tthhee FFRREETT rratio expressed in terms of 3 -FRET experimental measures. The complete relation is:
  • the magnitude of Y turns out to be exceedingly small, and can be estimated from the ratio of molar extinction coefficients for ECFP and EYFP ( ⁇ c F p( ⁇ ) or ⁇ F p( ⁇ )), as given by
  • SPECIMEN Scu BE
  • CUBE denotes a particular filter set or cube (ECFP, EYFP or FRET)
  • SPECIMEN indicates the nature of a sample being evaluated using the filter set or cube, for example, a sample (e.g., a cell) expressing donor only (D; ECFP), acceptor only (A; EYFP) or both (DA, FRET).
  • S FR ⁇ (DA) is the sum of both ECFP emission (number 2) and EYFP emission (number 3), a portion of which is due to direct excitation (number 4).
  • the key to dissecting these components requires obtaining measurements from both ECFP and EYFP filter sets or cubes (Sc F p(DA) and S YF p(DA)), to optically isolate ECFP and EYFP signals received from a sample (e.g., a cell) expressing both fluorophores.
  • Sc F p(DA) (number 5) represents a filter set or cube which transmits light to a sample (e.g., such as a cell) which excites ECFP and EYFP but which transmits fluorescence to a detector only in the range that ECFP emits.
  • the term can be multiplied by predetermined constant, 7?D I to determine what the contribution of ECFP emission is at 535 nm (number 2). Subtracting this value from S FRE ⁇ (DA) leaves F AD .
  • FRET efficiency (E) is determined from ER by
  • bracketed term is the ratio of ⁇ YFP and ⁇ CFP molar extinction coefficients, scaled for the FRET filter set or cube excitation filter (Selvin, 1995, Methods Enzymol. 246: 300-334). This transformation can be derived from standard results in the field.
  • This transformation presumes that each acceptor-tagged molecule is bound by a donor-tagged molecule, and that donor-acceptor orientations are randomized. The binding assumption will be further expanded in a section below.
  • E enabled another specific validation test of the 3 3 -FR ⁇ T method.
  • E also can be determined by measuring dequenching of donor emission following near complete acceptor photodestruction (while sparing the donor) by several minutes of strong illumination through an excitation filter that excites the acceptor but not the donor (see, e.g., as described in Miyawaki, and Tsien, 2000, Methods Enzymol. 327: 472-500). This approach complements 3 3 -FRET but is slower, destructive, and entails unavoidable collateral photobleaching (e.g., of cells in addition to one being analyzed). By comparing this value of E with ET?
  • ⁇ FP (440)/ ⁇ c F p(440) was experimentally found to be 0.096, which is within 3% of the predicted value based on published extinction coefficients for ⁇ CFP and ⁇ YFP (Patterson, et al., 2001, J Cell Sci 774: 837-838).
  • the 3 3 -FRET process can be used to quantify FRET, and therefore the presence or absence of interaction between donor and acceptor molecules.
  • the process can also be used to provide information about the properties of binding between donor and acceptor molecules, under the presumption that donor- acceptor interaction follows a 1 :1 stoichiometry.
  • the steps involved are summarized in Figure 8B.
  • a key underlying principle is that ECFP and EYFP filter-set or cube measurements, as processed according to the invention, provide a method for estimating the relative concentrations of ECFP- and EYFP-tagged molecules in single cells.
  • the fraction of EYFP-tagged molecules associated with ECFP-tagged partners can be calculated.
  • E kj I (kj + k__) is defined as the FRET efficiency of a donor-acceptor pair, and the ratio of GFR ET (D,440)/G FRET (A,440) is essentially equal to the ratio of molar extinction coefficients 8 C F P (440) / ⁇ F p(440).
  • This equation can be used to address the scenario where low expression levels of donor and acceptor result in unpaired donor and/or acceptor molecules and highlights three important features of incomplete labeling of acceptor molecules.
  • the measured ER varies linearly with an increasing fraction of acceptor bound to donor, according to slope ⁇ ET? max .
  • ⁇ ER max must be estimated from some type of regression analysis based upon measured ET? as a function of A b . E would be required to constrain actual distances between donor and acceptor moieties according to the Forster equation. The last point underscores the need for an experimental estimate of A b .
  • a b can be represented by the classic binding equation
  • a b l / ( ⁇ + 2 - K_ / [Dr ree ] ) [A24] assuming acceptor molecules which are membrane associated (e.g., such as ion channels), free donor molecules which are soluble cytoplasmic moieties (like tagged CaM), and a stoichiometry of donor-acceptor binding of 1 : 1.
  • K_ is the dissociation constant (in M units)
  • [D fr ee] is the concentration of free (unbound) donor molecules (in M units)
  • the factor of 2 relates to the fact that donor molecules can only bind to acceptor from the cytoplasmic side of the membrane. This can be restated in terms of the total number of donor and acceptor molecules in a cell (which is always within a field of view) as
  • a b 1 / [ 1 + 2 ⁇ K d ⁇ V • Navogadro / (ND - A. ⁇ NO ] [A25]
  • N av0 gadro is Avogadro's number
  • No and N A are number of donor and acceptor molecules in the cell
  • V is the volume of the cell (in liters).
  • Equation A27 can be recast into the very useful form below:
  • CFP FRE ⁇ (440,535,direct) [N D - E el ⁇ N A ] • I 0 ⁇ G FRET (D,440) ⁇ E FRET (D,535)
  • C is a constant
  • each function Prior to averaging and/ ⁇ , each function is scaled such that the total area under each spectrum is equal to the quantum yield of EYFP or ECFP, respectively.
  • Equations A30 and A31 into Equations A28 and A29 then yields the following expressions for N A and No
  • Equation A26 Substituting Equations A32 and A33 into Equation A26 yields an experimentally- based estimate of A_, according to
  • Equation A34 will translate the 3 3" FRET measurements into a prediction of A b
  • Equation A23 will in turn translate the predicted A into a predicted ET? (ER pre dicted)- Parameters ⁇ ET? max and d
  • ⁇ FF can be adjusted until the squared error (ER ex - ERp r edicted) is minimized. Minimizing the error for a single cell, in itself, would not be a very stringent constraint on the parameters. However, the same ⁇ ET?
  • 3 3 -FRET determination of ⁇ .E FF and ET? max is conveniently applied to measurements of ET?; however, it may also be applied to many other quantitative FRET indices.
  • measurements of FRET can be based on the enhancement of acceptor fluorescence emission (as with ET?) or on the quenching of donor fluorescence emission.
  • 3 3 -FR ⁇ T can be applied exactly as illustrated above to any method based on acceptor emission, provided that the method generates an index that can be linearly related to E.
  • donor emission that generate an index related to E e.g., acceptor photobleaching method
  • two simple alterations make the method compatible with 3 3 -FRET.
  • ER equ i v 1 + E [6 CFP (440)/ ⁇ Fp(440)], and ERequiv can be substituted for ER in the description of 3 -FRET above.
  • terms describing the number and/or concentration of donor and acceptor molecules in the field of view e.g., No and N A ) must be swapped. These changes reflect the fact that a FRET method based on donor fluorescence will generate binding affinities with respect to the donor-tagged molecule, whereas a FRET method based on acceptor fluorescence will generate binding affinities with respect to the acceptor- tagged molecule.
  • 3 3 -FRET requires donor and acceptor filter set readings that can be related, as above, to the number of donors and acceptors in the field of view.
  • the system can be generalized for other dono ⁇ acceptor pairs which meet the following criteria: there is significant overlap between the donor emission spectrum and the acceptor absorption spectrum; the donor: acceptor spectral properties permit use of a filter set that can detect emission predominantly from the donor while predominantly excluding emission from the acceptor; and, donor: acceptor spectral properties permit use of a filter set that can predominantly excite the acceptor while predominantly excluding excitation of the donor.
  • all the formulations throughout can be applied directly by substituting a suitable donor for ECFP and a suitable acceptor for EYFP.
  • FRET assays have been applied to measure FRET for the pairs EGFP/DsRed and ECFP/DsRed (Moon, et al. Biophys. J. 80: 362a.).
  • Other exemplary fluorophores which can be used in FRET assays as donor or acceptor include, but are not limited to, those listed in Table 1 , below.
  • the fluorophores are peptides or polypeptides (e.g., such as GFP- related proteins) which can be fused to a polypeptide(s) of interest.
  • GFP-related proteins are described in U.S. Patent No. 5,625,048; U.S. Patent No. 5,77,079; U.S. Patent No 6,306,600; U.S. Patent No 6,251,384; U.S. Patent No. 6,235,968; U.S. Patent No 6,232,523; 6,130,313; U.S. Patent No 6,090,919; U.S. Patent No. 6,020,192; U.S. Patent No. 6,054,387; and U.S. Patent No 5,804,387; for example, the entireties of which are incorporated herein by reference.
  • a number of optical systems can be used to detect FRET between a donor and acceptor molecule such as ECFP and EYFP.
  • the invention provides a system optimized for performing 3 -FRET. To accomplish this 3 -FRET process, three filter sets are sequentially placed between the light source and the specimen, and between the specimen and the detector. The individual filter sets each comprise a filter between the light source and the specimen and a filter between the specimen and the detector. Each filter set transmits and/or reflects specific wavelengths of light.
  • the filter between the light source and specimen maximally transmits a wavelength of light that excites the donor (and possibly the acceptor), and the filter between the specimen and the detector maximally transmits wavelengths of light where only the donor emits photons.
  • the filter between the light source and specimen maximally transmits a wavelength of light that preferentially excites the acceptor
  • the filter between the specimen and the detector maximally transmits wavelengths of light where mainly the acceptor emits photons (and possibly the donor emits photons).
  • the filter between the light source and specimen maximally transmits a wavelength of light that excites the donor (and possibly the acceptor), and the filter between the specimen and the detector maximally transmits wavelengths of light where mainly the acceptor emits photons (and possibly the donor emits photons).
  • the 3 3 -FRET method processes these three light intensity readings, each obtained with a different filter set engaged, and yields a quantitative readout of the strength of FRET interaction, termed "the FRET ratio" or ER.
  • ER furnishes the fractional increase in acceptor fluorescence due to FRET.
  • each filter cube contains an excitation filter, a dichroic mirror, and an emission filter.
  • the excitation filter is a band pass or high pass filter that allows only short wavelength light from a light source to pass through.
  • the emission filter is a band pass or low pass filter that passes only long wavelength light emitted by the object in response to illumination by the shorter wavelength exciting light.
  • the dichroic mirror is a beam splitter that reflects the excitation light onto a specimen, e.g., such as a cell, and then allows emitted light from the specimen to pass through.
  • the "cut on" wavelength of the dichroic mirror generally lies between the transmission bands of the excitation filter and the emission filter.
  • 3 3 -FRET filter-cubes used comprise a ECFP cube comprising an excitation filter of D440/20M, a dichroic mirror of 455DCLP, and an emission filter of D480/30M (available commercially from Chroma, Inc., Brattleboro, VT; a EYFP cube comprising an excitation filter of 500DF25, a dichroic mirror of 535DRLP, and an emission filter of 530EFLP; and a FRET filter cube comprising an excitation filter of 440DF20, a dichroic mirror of 455DRLP, and an emission filter of 535DF25 (EYFP cubes and FRET cubes are obtainable from Omega Optical).
  • the numerical designators in each case refers to the peak wavelength of light transmitted by each filter.
  • an excitation filter of D440/20M transmits light maximally at 440 nm, with a 20 nm bandwidth.
  • LP signifies a longpass filter.
  • filter cubes are precisely machined as described in PCT/US98/113909855026 to minimize overlap in the emission spectra between donor and acceptor molecules.
  • the system further comprises a light source and also can comprise one or more optical fibers for transmitting light to a specimen (e.g., such as a cell).
  • a specimen e.g., such as a cell
  • a powerful light source generally is preferred, such as a mercury or xenon arc (burner) lamp, for producing high-intensity illumination powerful enough to image faintly visible fluorescence specimens.
  • a laser light source e.g., a gas laser such as a nitrogen, helium, neon, or argon laser; uv laser; semiconductor laser; pulsed laser, solid-state diode laser, and the like
  • a scanning mechanism is provided for moving the light source relative to the sample so that light can be scanned across the specimen (e.g., for obtaining three-dimensional images).
  • the cubes are connectable to an appropriate detection device which can comprise, but is not limited to: one or more photodetectors, a filter, a CCD camera, a streak tube, an endoscopic imaging system, an endoscopic fluorescence imaging microscope, a fiber optic fluorescence imaging microscope, a computer used in the fluorescence analysis, and the like.
  • the system also comprises a holder for holding at least one of the cubes in position relative to a specimen, the light source, and the detector, so that light from the light source can be received by the specimen and light emitted by the specimen can be received by the detector.
  • each filter cube can be sequentially positioned (e.g., via a holder slideable in a horizontal plane), relative to a light source and light detector to obtain sequential light intensity readings.
  • the cube when a filter cube is so positioned, the cube is rotatable about a vertical axis for selectively aligning an optical path with a light source and one or more focusing lens.
  • the system also can include a wavelength divider such as a filter, prism, diffraction grating, or image-subtracting double monochromator.
  • the system further can comprise a sample support, e.g., such as a stage, and a scanning mechanism for scanning the support relative to both the light source and the detection device. Scanning can be mechanical or automated. Preferably, at least a portion of the sample support is optically transmissive.
  • the system also can include an image processor and/or an image display device.
  • the image processor may be a suitably programmed personal computer, while the image display device may be a computer monitor (e.g., CRT or LCD display) or a printer.
  • an image of an illuminated sample can be obtained by the detector device and input into the processor as a digitized pixel image.
  • a set of three such images from each channel (donor, acceptor, and FRET) can be processed as three spatially coregistered images or can be treated as single images in which each pixel has three color space coordinates corresponding to the monochrome wavelengths.
  • the processor comprises software for implementing 3 3 -FRET analysis. A flowchart of the process that such software would implement is shown in Figure 8.
  • the optical detection system can include, but is not limited to: an epifluorescent microscope, a 3D imaging system, such as a confocal microscope (single-photon confocal microscope) or a two-photon microscope. In addition to permitting subcellular monitoring, such the latter two systems would facilitate the identification of molecular interactions (e.g., such as protein-protein interactions) deep in living tissue samples.
  • an epifluorescent microscope such as a confocal microscope (single-photon confocal microscope) or a two-photon microscope.
  • confocal microscope single-photon confocal microscope
  • two-photon microscope two-photon microscope
  • the optical system is a flow cytometer comprising a dropping nozzle through which individual cells can be passed in a single small droplet of suspending media (e.g., a buffer or cell culture media).
  • At least one coherent light source e.g., a laser
  • At least one coherent light source is placed in optical proximity to the droplet to excite fluorescence in the cell.
  • Light emitted by the cell is channeled into a light path using a least one focusing element which is separated into various wavelengths using at least three dichroic mirrors which divert light into each of at least three filters: a donor filter, an acceptor filter, and a FRET filter.
  • Light transmitted through the filters are detected using separate detector devices (e.g., such as photomultipliers).
  • Signals from the photomultipliers are sent to a processor which performs 3 -FRET computations to calculate FRET.
  • droplets with particular fluorescent characteristics e.g., reflecting interacting dono ⁇ acceptor pairs
  • Charged and uncharged droplets are separated as they fall between charged plates.
  • the system can be used to both evaluate molecular interactions as well as to identify and sort cell populations in which donor acceptor interactions have or have not occurred.
  • the optical system is a plate reader.
  • a plate reader can be coupled to a robotic fluid transfer system to maximize assay throughput.
  • the 3 3 -FRET assays described herein are generally nondestructive of cells, as compared, for example, to the acceptor bleaching method of Miyawaki and Tsien, 2000, supra.
  • the assays are also rapid, facilitating high throughput screening (HTS) of specimens.
  • HTS high throughput screening
  • a cell-based assay according to the invention can be performed in 3-5 minutes using an epifluorescence microscope.
  • HTS screens also can be performed using FACs sorting machines, making it possible to evaluate responses of single cells in under 5 minutes, and even within the time-of-flight requirements of these machines (i.e., within seconds). This contrasts with the bleaching method of Miyawaki and Tsien, 2000, supra, which take minutes, precluding its use in a FACS sorting machine.
  • 3 3 -FRET can be used to monitor the responses of a FRET-based sensor in analyte detection assays.
  • a donor tagged molecule and an acceptor tagged molecule can be bound to a binding protein that changes its conformation upon binding to an analyte (see, e.g., as described in U.S. Patent No. 6,197,928).
  • the change in conformation leads to a change in the relative position and orientation of the donor and acceptor molecules and FRET.
  • the binding protein can be in solution or immobilized on a solid phase (e.g., a particle, microparticle, bead, microbead, sphere, magnetized particle, capillary, slide, wafer, cube, membrane, filter, and the like), creating a FRET-based sensor for the analyte.
  • the degree of FRET can be correlated with the concentration of analyte in the sample. In one aspect, the degree of FRET is determined over different time periods to determine changes in the concentration of an analyte in the sample.
  • the donor molecule is ECFP while the acceptor molecule is EYFP.
  • Suitable binding proteins which change conformation upon binding to an analyte include, but are not limited to, calmodulin (CaM), cGMP-dependent protein kinase, steroid hormone receptors (or ligand binding domains thereof), protein kinase C, inositol-l,4,5-rriphosphate receptor, alphachymotrypsin, or recoverin (see, e.g., as described in Katzenellenbogen and Katzenellenbogen, 1996, Chemistry & Biology 3: 529-536; Ames, et al., Curr. Opin. Struct. Biol. 6: 432-438; U.S. Patent No. 5,254,477).
  • CaM calmodulin
  • cGMP-dependent protein kinase e.g., steroid hormone receptors (or ligand binding domains thereof)
  • protein kinase C inositol-l,4,5-rriphosphate receptor
  • the binding protein is also responsive to an intracellular signaling molecule (e.g., such as Ca 2+ ) (see, e.g., Falke, et al., 1994, Quart. Rev. Biophys. 27;. 219-290).
  • an intracellular signaling molecule e.g., such as Ca 2+
  • signaling molecules include, but are not limited to, the calmodulin-binding domain of Ml 3, smMLCKp, CaMKII, Caldesmon, Calspermin, Calcineurin, PhK5, PhK13, C28W, 59-kDa PDE, 60-kDa PDE, NO-30, AC-28, Bordetella pertussis AC, Neuro-modulin, Spectrin, MARCKS, F52, [beta] - Adducin, HSP9Oa, HIV-1 gpl60, BBMHBI, Dilute MHC, Mastoparan, Melittin, Glucagon, Secretin, VIP, GIP, or Model Peptide CBP2.
  • the binding of these signaling molecules also can be monitored by monitoring changes in the interactions between the binding protein and the analyte.
  • the binding protein is an enzyme and FRET is an indication of substrate catalysis as well as binding (see, e.g., as described in U.S. Patent No. 5,254,477).
  • a donor and acceptor molecule are held together by a cleavable linker, e.g., such as a peptide linker comprising a cleavage site for cleaving molecule. While in their linked state, FRET occurs between the donor and acceptor molecule; however, upon cleavage by a cleaving molecule (e.g., such as an enzyme), the donor and acceptor molecule are separated resulting in a decrease in FRET.
  • a cleaving molecule e.g., such as an enzyme
  • a decrease in FRET is used as a measure of an analyte in a sample.
  • the assay is used to detect an intracellular protease. Suitable linkers comprising cleavage sites are described in U.S. Patent 5,981,200, for example.
  • the donor/acceptor tagged binding protein can be generated using methods routine in the art and as described in U.S. Patent 6,197,928, for example, the entirety of which is incorporated by reference herein. Sequences for both ECFP and EYFP are known in the art, as are sequences for the coding regions of the binding proteins exemplified above. It is contemplated that additional coding sequences for binding proteins will become known and the examples provided herein are non-limiting.
  • the donor molecule and acceptor molecule are linked to the binding protein using a suitable linker for maintaining the donor and acceptor molecule greater than lOOA away from each other when the tagged binding protein is in a solution or immobilized on a substrate, and less than 100 A when the tagged binding protein is bound to an analyte.
  • the average distance between the donor and acceptor molecules is between about 1 nm and about 10 nm, preferably between about 1 nm and about 6 nm, and more preferably between about 1 nm and about 4 nm, when the analyte is bound (or released).
  • the linker comprises between about one and 30 amino acid residues in length, preferably between about two and 15 amino acid residues.
  • One preferred linker moiety is a -Gly-Gly- linker.
  • One preferred linker moiety is a linker comprising a plurality of serines and glycines. Preferably, such a linker is about 50% serine.
  • Flexible linker molecules and constraints on the design of linker molecules are known in the art and are described in U.S. Patent No. 6,197,928; U.S. Patent No. 5,254,477; Huston, et al., 1988, Proc. Natl. Acad. Sci.
  • donor and acceptor molecules are not peptides or polypeptides, they can be conjugated to the binding protein using chemical conjugation methods as are well known in the art.
  • the sensor also can be used to sense molecules in an intracellular environment.
  • the tagged binding protein can be introduced into a cell and changes in the proximity of donor and acceptor molecules upon binding of an intracellular molecule binding to the binding protein can be detected using 3 -FRET and the optical system as described above.
  • the tagged binding protein comprises a localization signal to facilitate introduction of the sensor into the cell and/or to target the sensor to a particular intracellular compartment.
  • Suitable localization sequences include, but are not limited to: a nuclear localization sequence, an endoplasmic reticulum localization sequence, a peroxisome localization sequence, a mitochondrial localization sequence, and a peroxisome localization sequence. Additional localization sequences are described in U.S. Patent No. 6,197,928 and in Stryer, 1995, Biochemistry (4th ed.). W. H. Freeman, Ch. 35, for example.
  • cells are electroporated to transiently introduce pores into the cells to facilitate uptake of the tagged binding protein.
  • donor and acceptor pair interactions are used to detect and or quantitate a nucleic acid analyte.
  • a first and second oligonucleotide probe can be labeled with a donor and acceptor molecule, respectively, for example, by chemical conjugation.
  • the sequence of the first probe is selected to be complementary to a first portion of a target sequence while the sequence of a second probe is selected to be complementary to a second portion of the target sequence, such that hybridization of the first and second probe to the hybridization sequence brings the donor and acceptor molecule in sufficient proximity to each other to cause FRET (see, e.g., as described in Wittwer, et al., 1997, Biotechniques 22;.
  • the first and second oligonucleotides are introduced into a cell using methods routine in the art (e.g., transfection, transformation, electroporation, microinjection) and FRET is detected using 3 3 -FRET and the optical system as described above.
  • a nucleic acid substrate is provided for measuring DNA-polypeptide interactions using FRET.
  • the substrate is linked to a donor and acceptor (e.g., by chemical conjugation) in such a way that the donor and acceptor are less than lOOA apart.
  • the nucleic acid substrate is incubated with a sample and binding of a polypeptide increases the distance between the donor and acceptor molecule, i.e., decreasing FRET.
  • the polypeptide is a nucleic acid cleaving enzyme, such as a nuclease.
  • the nucleic acid substrate is immobilized on a substrate (e.g., such as a glass slide) and FRET is detected using 3 3 -FRET and the optical system as described above.
  • Some donor acceptor pair interactions are susceptible to pH, such that FRET changes upon a change in the pH of a solution surrounding the donor acceptor molecules. For example, the absorption of the basic form of phenol red rises with increased pH and overlaps the emission spectrum of eosin, resulting in increased FRET as pH is raised from 6 to 10. A change in pH can thus be monitored by monitoring changes in FRET.
  • FRET sensors are generated by immobilizing appropriate donor acceptor pairs on a substrate (e.g., a polymer) at suitable distances using linker molecules.
  • a substrate e.g., a polymer
  • linker molecules e.g., linker molecules
  • Suitable fluorophore pairs that can be used and their excitation and emission wavelength(s) are described in U.S. Patent No. 5,254,477, the entirety of which is incorporated by reference herein. Changes in FRET can be detected readily using the optical system and 3 3 -FRET methods described above.
  • Stable cells lines expressing a known interacting pair of donor and acceptor - tagged molecules can be used in HTS assays to screen for modulators of these molecules, such as drugs.
  • a screen for compounds that disrupt the protein-protein interactions is performed.
  • Such a screen can be made high throughput by robotic application of different compounds to cultures of cells in multi- well plates.
  • a custom plate reader designed to perform 3 3 -FRET on each of the wells can be used to rapidly identify candidate compounds that inhibit the protein-protein interaction of interest. Plate readers need only be modified to allow engagement of three filter sets, as described above under optical systems.
  • first and second molecules are tagged with donor and acceptor molecules (e.g., by chemical conjugation or by genetic engineering). Interaction between the first and second molecules brings the donor and acceptor molecules sufficiently close together to cause FRET.
  • the first and second molecules are introduced into a cell using methods known in the art (e.g., transfection, transformation, electroporation, microinjection) and the cell is contacted with a sample suspected of comprising a modulator of the interaction.
  • Suitable interacting molecules include, but are not limited to, ligands and receptors; antibodies and antigens; calmodulin and calcium; G proteins, GTP and G-Protein Coupled Receptors; and the like.
  • FRET in the cell is detected using 3 3 -FRET, for example, with the optical system described above.
  • the strength of FRET is compared to a baseline, e.g., the amount of FRET in the cell prior to exposure to the sample, or the strength of FRET in a substantially identical cell into which the first and second molecules have been introduced but which has not been exposed to sample.
  • a modulator is identified as a compound which produces a significant change with respect to the baseline ER, using routine statistical methods.
  • a "substantially identical cell” refers to a genetically identical cell.
  • the method further includes the step of contacting the cell with a compound at a first time and a second time, and measuring a change in FRET at the first time and the second time.
  • the method further includes the step of contacting a cell with a first concentration of a compound, and a substantially identical cell with a second, different concentration and determining FRET after each contacting to determine a dose-response curve for the compound.
  • a donor and acceptor tagged molecules are provided as part of a two-hybrid system to identify molecules which interact with a polypeptide of interest (see, e.g., Fields and Song, 1989, Nature 340: 245-246; WO 94/10300; U.S. Patent No. 5,283,173).
  • a bait protein can be generated by fusing the polypeptide of interest to a donor polypeptidepeptide (e.g., such as ECFP), while prey proteins can be generated from random sequences fused to an acceptor peptide (e.g., such as EYFP).
  • Interactions between the polypeptide of interest and the bait protein can be identified by the FRET which occurs as donor and acceptor polypeptides are brought in sufficient proximity.
  • 3 3 -FRET analysis of bait and prey interactions would provide for a high-throughput discovery strategy, since protein-protein interactions are almost instantaneously detected by 3 3 -FRET (e.g., as compared to systems such as yeast two-hybrid systems).
  • Single-cell rescue of nucleic acid sequences encoding an interacting prey polypeptide can be used to specify the identity of the interacting prey polypeptide. In this manner, discovery of unknown interaction partners with a specified bait polypeptide can be determined.
  • the assay can be used to identify ligands for orphan receptors.
  • the assay can be used to identify interacting molecules in living mammalian cells.
  • the assays above can be used to provide clinical tests (e.g., diagnostic and prognostic assays), as well as screening assays.
  • a cellular process or condition can be diagnosed by performing the analyte detection assays described above to detect a marker of a disease (e.g., such as a tumor-specific antigen).
  • 3 3 -FRET can be used to screen for altered molecular interactions that are known to be perturbed during the cellular process or condition.
  • a specimen can be obtained from a patient suspected of having, or at risk for developing a disease and can be evaluated for the presence of an analyte or altered interaction by using 3 3 FRET, after introduction of a suitable FRET-based biosensor into the patient specimen.
  • the measure of FRET obtained from the specimen can then be compared to a measure obtained from a control, such as a normal patient.
  • the assays can be performed in living cells, the effect of a test compound, such as a drug on the expression of the analyte/molecular interaction can be evaluated over time to examine the effect of the drug on the normalization of a physiological response.
  • the localization of FRET also can be monitored.
  • the specimen can be place on a sample holder comprising a culture medium.
  • Various parameters of the culture medium can be regulated, such as pH and temperature, using automated controls (e.g., sensors and tubing systems which can deliver appropriate reagents to the culture medium in response to conditions sensed by the sensors).
  • automated controls e.g., sensors and tubing systems which can deliver appropriate reagents to the culture medium in response to conditions sensed by the sensors).
  • a FACs sorting system as described above, cells comprising analytes, or in which molecular interactions have occurred, can be identified and sorted because of their unique spectral properties.
  • Voltage-gated Ca + channels trigger essential physiological processes, including contraction, secretion and expression.
  • Ca 2+ channel modulation are the feedback regulation of L-type ( ⁇ .c) and P/Q-type ( ⁇ X IA ) channels by intracellular Ca fluctuations, acting in an unconventional channel- calmodulin (CaM) interaction.
  • CaM M ur Ca 2+ -insensitive mutant CaM eliminates Ca dependent modulaUon in both channel types, hinting that CaM may be "preassociated" with these channel complexes even before channel opening, so as to enhance detection of local Ca 2+ .
  • 3 -FRET was used to probe constitutive associations between Ca channel subunits and CaM in single living cells, using variants of the green fluorescent protein (GFP) as fluorophore tags.
  • GFP green fluorescent protein
  • This rapid, non-destructive assay detects steady-state associations between CaM (or CaMiviu ⁇ ) and the pore- for ing ⁇ i subunit of L-type, P/Q-type, and surprisingly, R-type ( ⁇ i ⁇ ) Ca channels.
  • the assay was used to map a triangle formed by three key channel landmarks: the ⁇ i subunit, the auxiliary ⁇ 2a subunit, and CaM.
  • apoCaM sometimes preassociates with a target molecule, whose activity is subsequently modulated as Ca 2+ -CaM shifts to a different site on the target.
  • This arrangement is a potent means of ensuring selective responsiveness to local Ca 2+ and, in the case of Ca 2+ channels, of permitting accelerated modulation initiated by local Ca 2+ influx.
  • traditional in vitro biochemistry confirms such preassociation, the actual prevalence of this mechanism may be far greater, especially for ion channels whose potential apoCaM interaction might be disrupted by detergents required to solubilize channels for in vitro biochemistry.
  • ECFP/EYFP tagged proteins were generated as described below and assayed to verify that resulting fusion proteins preserved the functions and interactions of the tagged proteins. Focusing on L-type (otic) channels, the functional modulation produced by CaM-channel interaction is feedback inhibition of channel opening by elevated intracellular Ca 2+ (Ca 2+ -dependent inactivation). Two CaM-channel interactions are believed to underlie such inactivation: (1) Ca 2+ -CaM binding to an "IQ-like" domain on the proximal otic carboxyl tail ( Figure 1 A), which initiates Ca 2+ - dependent inactivation; and (2) inferred preassociation of the Ca -free form of this CaM with the channel complex, at a presently uncertain site.
  • HEK293 cells were thinly plated into 3.5-cm culture dishes with No. 0 glass cover slip bottoms (MatTek Corp.) optimized for inverted microscopes. Cells were transiently transfected with FuGene 6 as a means of optimizing transfection using the manufacturer's standard protocol (Roche Molecular Biochemicals) and three days later assessed optically.
  • the sideport optical train includes an adjustable aperture in the image plane to clip spurious light from neighboring cells or other background sources, a selectable eyepiece for precise adjustment of image position and focus, an optional beam- splitter, and two 30-mm EMI 9124B (Electron Tubes Limited, England) ambient temperature photon-counting photomultiplier tubes (PMTs).
  • EMI 9124B Electro Tubes Limited, England
  • PMTs ambient temperature photon-counting photomultiplier tubes
  • PMT signals were conditioned by pre-amplifiers, integrated and filtered (at 10 kHz) in the dual -channel fluorometer, and digitized with an ITC-18 programmable data acquisition board (Instrutech Corp.). Shutter control, data acquisition, and automatic dark-current subtraction were managed by custom software combining
  • the level of CaM expression was qualitatively evaluated by immunostaining to determine by the level of expression of CaM-ECFP, CaM M u ⁇ and HEK293 cell endogenous CaM.
  • HEK293 cells were scraped from a 10-cm plate, washed with PBS, pelleted and lysed in a small volume of lysis buffer (1% NP40, 20 mM Tris [pH 7.4], 150 mM NaCl) with protease inhibitor cocktail (Complete; Roche).
  • Proteins in the lysates were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transfe ⁇ ed to a hydrophobic membrane (Immobilon-PSQ; Millipore).
  • SDS-PAGE SDS-polyacrylamide gel electrophoresis
  • Immobilon-PSQ Millipore
  • Mouse anti-CaM (Research Diagnostics, Inc.) and secondary anti-mouse with conjugated horseradish peroxidase (Amersham) were used in immunoblotting assays and bands were visualized with enhanced chemiluminescence (ECL; Amersham) to determine the presence and relative amounts of the approximately 45 kD CaM fusion proteins and the approximately 20 kD CaM.
  • the fusion constructs preserved the functional properties of Ca -dependent inactivation, as well its underlying CaM-channel interactions.
  • HEK293 cells expressing labelled L-type channels ( ⁇ ic EYFP/ ⁇ 2a / ⁇ 2 ⁇ displayed a distinct fluorescent ring at the cell perimeter ( Figure IB) and had substantial recombinant currents (not shown), confirming that labelled channels are functional and properly target to the plasma membrane.
  • Western blots Figure IC
  • taken from HEK293 cells transfected with CaMwr-ECFP or CaM M u ⁇ -ECFP showed strong expression of labelled CaMs and no cleavage of linked ECFP.
  • FRET could therefore be nondestructively quantified from the enhanced EYFP emission at 535 nm, but only if it was possible to dissect out EYFP emission secondary to direct excitation (dashed line) from total EYFP emission (thin line) due to both FRET and direct excitation.
  • P/Q-type ( ⁇ A ) and R-type ( ⁇ i ⁇ ) channel subunits possess homologous IQ-like domains that bind Ca 2+ -CaM in vitro.
  • ⁇ i ⁇ -EYFP and ⁇ i A -EYFP constructs were generated, with carboxyl terminus truncations and EYFP fusions produced as described above.
  • FRET not only provides a qualitative indication of whether two tagged protein interact, but in the best cases, it can be used to estimate physical distances between donor and acceptor molecules.
  • this estimation requires that each EYFP- tagged molecule be associated with a ECFP-tagged molecule. Since ECFP-tagged moieties (like CaMw - ECFP) were intentionally limited to avoid trivial concentration-dependent FRET, this condition may not have been satisfied. Using the strategies as discussed above for calculating Equation A23, this limitation was actually turned to advantage.
  • the ECFP and EYFP cube measurements provided the means to estimate the relative concentrations of ECFP- and EYFP-tagged molecules in single cells. When combined with estimation of a single Langmuir binding function, the fraction of EYFP-tagged molecules associated with ECFP-tagged partners can be calculated and the calculated fraction can be used to predict ET? according to Equation A23.
  • Figure 5 A shows the application of such analysis to the pairing of ctic-YFP and CaMwr-CFP.
  • 3 3 -FRET is uniquely equipped to answer these questions, in particular because of its ability to compare ER max and ⁇ ⁇ FF among different FRET pairs. 3 3 -FRET was therefore applied for single cell, two-hybrid screening of channel/CaM interactions, with ECFP-tagged CaM serving as "bait” and EYFP-tagged segments of the Ca 2+ channel as "prey.”
  • the first objective was to identify which Ca + channel segments coordinate binding of apoCaM.
  • a library of short (-100 basepair, or -33 residue) and long (-200 basepair, or -66 residue) segments from the L-type Ca 2+ channel carboxyl tail was generated, and each segment was fused in frame to EYFP.
  • Four such segments are illustrated in Figure 7A (EF, PrelQ, IQ and PrelQ-IQ).
  • the EYFP-tagged segments were then cotransfected in cells with CaM M ur-CFP, which inco ⁇ orates the Ca 2+ insensitive mutant CaM, and the cells where probed with 3 3 -FRET.
  • the 3 3 -FRET method was also applied to investigate how Ca 2+ -activation of preassociated CaM could trigger channel modulation.
  • the cells were clamped to either high (10 mM) or low (5 mM EGTA) internal Ca 2+ before application of 3 3 - FRET.
  • Both PrelQ/CaM and IQ/CaM exhibited marked conformational changes
  • 3 -FRET generally exhibits a very low false- positive rate, as FRET signals are only detected when the donor and acceptor fluorophores are within 100 A.
  • the false-negative rate can be high, since the orientation and/or distance of the fluorophores tagging two polypeptides might not be conducive to FRET, even when the two polypeptides are tightly bound to one another.
  • 3 3 -FRET based two-hybrid screening compliments existing hybridization assays that exhibit high false-positive rates, such as yeast two-hybrid screening.
  • 3 3 -FRET provides the unique ability to determine the binding affinity for polypeptides that do interact, which enables discrimination between strong and weak interactions.

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Abstract

La présente invention concerne une méthode permettant de déterminer une mesure du FRET, consistant à obtenir des valeurs d'intensité de fluorescence séquentielles à partir d'un échantillon, tel qu'une cellule, à l'aide de trois cubes filtrants. Une équation simple permet de manipuler les valeurs de chacun des ensembles filtrantes ou cubes pour spécifier un indice sans unités de FRET dit rapport de FRET (FR). FR a une relation linéaire par rapport à l'efficacité E de FRET. Cette méthode permet également de déterminer la fraction des molécules marquées avec un accepteur liées à des molécules marquées par un donneur ; l'affinité relative de la liaison ; et la force des interactions de FRET lorsque toutes les molécules marquées avec un accepteur sont liées par un donneur. Cette dernière détermination permet d'estimer la distance physique et/ou l'orientation entre les molécules fluorophores interagissant. Cette méthode peut être utilisée pour détecter des analytes ou des interactions inter- ou intramoléculaires. Dans un aspect préféré de l'invention, cette méthode est utilisée dans un criblage à haut rendement pour identifier des modulateurs de ces interactions.
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Cited By (5)

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Publication number Priority date Publication date Assignee Title
WO2008104638A2 (fr) * 2007-02-27 2008-09-04 Hidex Oy Dosage biologique homogène par luminescence amélioré
WO2008104638A3 (fr) * 2007-02-27 2008-11-06 Hidex Oy Dosage biologique homogène par luminescence amélioré
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CN113899722B (zh) * 2021-09-14 2022-11-01 华南师范大学 基于单一标准fret质粒测量fret系统矫正参数的方法及其应用

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