Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
  • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
  • G01N21/64—Fluorescence; Phosphorescence
  • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
  • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
  • G01N21/64—Fluorescence; Phosphorescence
  • G01N21/6445—Measuring fluorescence polarisation

Definitions

• the invention relates to the use of the fluorescence polarization phenomenon, with a view to improving the detection of fluorescence signals during non-radiative energy transfer (FRET).
• the invention relates to a method for improving the signal-to-noise ratio in a FRET measurement.
• Fluorescence non-radiative energy transfer (FRET) is a spectroscopic tool widely used in the detection of biological events and in particular molecular interactions.
• FRET Fluorescence Activated FRET
• a spatial approximation between donor and acceptor fluorescent molecules that will be involved in energy transfer proves to be a powerful tool in the demonstration of biological interactions. It can be used in fields as varied as molecular biology, in-vitro or in-cellulo detection of enzymatic phenomena (peptide cleavage, phosphorylation) or interactions between proteins (1, 2, 3). Detection of the FRET phenomenon can be done by measuring different parameters of the fluorescence signal emitted either by the donor, by the acceptor or by the two molecules. Among the most used techniques, we can mention:
• the FRET phenomenon proves to be complex to detect in many applications based on fluorescence intensities measurements.
• the need for substantial energy compatibility between the donor and the acceptor often leads to the use of molecules having relatively close fluorescence emission spectra.
• the overlap of the donor and acceptor spectra that results makes it very difficult to accurately measure the variations of signals recorded on the donor or on the acceptor (9).
• FRET experiments such as the most widely used Cyan Fluorescent Protein (CFP) / Yellow Fluorescent Protein (YFP) donor / acceptor pair.
• CFP Cyan Fluorescent Protein
• YFP Yellow Fluorescent Protein
• FRET experiments capable of being expressed in fluorescent form in many cell types, allow the demonstration of numerous intracellular events.
• an important overlap of fluorescence spectra exists between them, resulting in the direct parasitic excitation of the acceptor by the excitation beam of the donor molecule.
• the signal-to-background ratio of the FRET experiments performed with this donor / acceptor pair is low, often less than 1.5 (1). Therefore, it is necessary to implement complex experimental protocols including many experimental controls to be able to interpret the results obtained.
• the technical problem to be solved therefore consists in providing a simple and reproducible method for correcting the FRET measurement, in particular by improving the signal-to-noise ratio.
• a homoFRET between two GFP molecules was detected by measuring their depolarization (12).
• the depolarization measurement of lectin-coupled rhodamine was used to detect a FRET occurring between fluorescein and rhodamine (5).
• the measurement of the increase in the polarization of a Concanaviline A-fluorescein donor has made it possible to demonstrate a FRET indicating the formation of a cluster (in English "cluster”) molecular in the lymphocyte membranes (10).
• GFP-type fluorescent proteins for example, because of their structure and their molecular weight, are highly polarized molecules. Their degree of polarization varies when they are involved in a transfer of energy: as a donor molecule, their polarization increases a little, whereas as an acceptor molecule, they undergo a strong depolarization through the FRET phenomenon.
• the signal measured at the emission wavelength of the acceptor will comprise:
• a depolarized signal from the FRET (the specific signal that one wants to measure)
• the method according to the invention is therefore based on the use of this important polarization variation between the donor and the acceptor to improve the spectral selectivity of the FRET measurement.
• the measurement of the emitted fluorescence signal is carried out either in the plane of polarization parallel to that of the polarized excitation light, or in the plane of polarization orthogonal to that of the polarized excitation light, according to the state of polarization of the donor molecule and that of the acceptor molecule.
• the invention therefore relates to a method for detecting an energy transfer between a donor fluorescent compound and an acceptor fluorescent compound present in a measurement medium, wherein the selectivity of the measurement of the energy transfer is improved by using the polarization properties of said fluorescent donor and acceptor compounds.
• the energy transfer is detected by measuring the signal resulting from the florescence emitted by the acceptor fluorescent compound at a wavelength ⁇ 3. This emission results from the transfer of energy between a fluorescent donor compound, excited in the measuring medium at a wavelength ⁇ 1 and said acceptor fluorescent compound.
• measuring medium a solution comprising the fluorescent donor and acceptor compounds; this solution may be a biological sample, or may contain the elements necessary for the study of a biological phenomenon.
• the measuring medium can also be a tissue sample living cells or live cells placed in an appropriate culture medium.
• the fluorescent donor and acceptor compounds are present either in the culture medium of said sample of tissue or said cells, or in the tissue itself or in the cells.
• the measuring medium may finally consist of a living organism, an animal, in particular a mammal to which the fluorescent donor and acceptor compounds have been administered.
• the administration of the fluorescent donor and acceptor compounds to a living animal can be carried out topically, by simply bringing said compounds into contact with the animal; donor and acceptor compounds may also be injected into the animal; fluorescent donor and acceptor compounds may also be directly produced in the body of the animal by genetic engineering.
• the general method according to the invention solves the problems related to the use of fluorescent compounds donors and acceptors of low spectral selectivity, and in particular to limit the background noise related d part of the emission of light by the donor at the emission wavelength of the acceptor, and secondly at the emission of light by the acceptor not involved in the transfer of energy the acceptor being in this case excited directly by the excitatory light.
• the invention relates to a method for detecting an energy transfer between a donor fluorescent compound and an acceptor fluorescent compound present in a measurement medium, comprising the following steps: (i) excitation of the measuring medium by a light beam polarized at the wavelength ⁇ 1, ⁇ 1 being the wavelength at which said fluorescent donor compound is excited, and
• the measurement of the signal emitted at the emission wavelength of the acceptor fluorescent compound in a different (ie non-parallel) plane of the polarization plane of the exciting light will make it possible to promote the measurement of the signal emitted by the strongly depolarized species, and in particular the signal of the acceptor engaged in the transfer of energy, thereby reducing the measured signal portion not resulting from the energy transfer.
• the plane in which the measurement is made is preferably the plane orthogonal to the plane of polarization of the exciting light. Measures in other plans may also be appropriate.
• the correction of step (iv) above may, for example, consist of a calculation of the ratio of the intensity of the fluorescence measured at the wavelength ⁇ 3 by that measured at the wavelength ⁇ 2.
• the measurement of the signal resulting from the fluorescence emitted can be carried out in a parallel or different plane, preferably orthogonal to the plane of the excitatory light.
• the polarization properties of the donor and acceptor fluorescent compounds are used to improve the selectivity of the energy transfer measurement, in a method for determining the polarization variation due to energy transfer. Like the first method described above, this method will make it possible to improve the selectivity of the measurement, which will thus be better correlated with the energy transfer phenomenon that it is desired to detect.
• This second embodiment comprises the following steps: (i) excitation of the measurement medium by a polarized light beam at the wavelength ⁇ 1, where ⁇ 1 is the wavelength at which said fluorescent donor compound is excited, (ii) measuring the total fluorescence intensity (lt //) ⁇ 2 emitted at the wavelength ⁇ 2 in the plane parallel to the plane of the excitatory light, ⁇ 2 being the wavelength at which the light of the fluorescent donor compound is emitted (iii) measuring the total fluorescence intensity (It 1 K 2 emitted at the wavelength ⁇ 2 in a plane different from the polarization plane of the exciting light,
• A represents the proportionality factor between the signals resulting from the fluorescence emitted at the wavelengths ⁇ 2 and ⁇ 3 by the donor alone, in a plane parallel to the plane of the excitatory light,
• G is a factor that makes it possible to correct the sensitivity difference of the detection in the parallel and orthogonal planes. This factor is either provided by the manufacturer, or easily determined by those skilled in the art by measuring the polarization of known polarization substances.
• a and B are calculated as follows:
• the measurements made in a plane different from the plane of polarization of the exciting light are preferably carried out in the plane orthogonal to the plane of polarization of the exciting light. Measurements in other planes could also be appropriate, as long as the chosen plane is not the plane parallel to the plane of polarization of the exciting light.
• the method according to the invention thus makes it possible to improve the selectivity of the measurement of a phenomenon of energy transfer between a donor compound and an acceptor compound.
• This is particularly advantageous in the case where the spectral selectivity between the donor and the acceptor is not optimal, ie in the following cases: - where the emission spectra of the donor and the acceptor overlap.
• the methods according to the invention are particularly effective in the case where 5nm ⁇ 3- ⁇ 2 ⁇ 100 nm, ⁇ 3- ⁇ 2 representing the difference between the wavelengths ⁇ 3 and ⁇ 2. - case where a direct parasitic excitation of the acceptor is possible at the excitation wavelength of the donor ( ⁇ 1).
• the process according to the invention can be carried out with numerous fluorescent donor and acceptor compounds: these compounds can be chosen from fluorescent proteins or organic fluorophores.
• the donor and the acceptor may be fluorescent proteins chosen from: GFP (Green fluorescent protein), CFP (Cyan fluorescent protein), YFP (Yellow fluorescent protein) and, in general, GFP derivatives ( BFP.eGFP), as well as among the family of Reef Coral Fluorescent Proteins (RCFP) such as DsRed HcRed.
• the donor and the acceptor may also be organic fluorophores, for example: rhodamines, cyanines, squaraines, fluoresceins, bodipies, compounds of the AlexaFluor family and their derivatives, or the fluorescent compounds described in US Pat. WO2003104685.
• the donor and the acceptor may be fluorescent microspheres or nano-crystals of Quantom-dots type.
• the fluorescent donor and acceptor compounds have a high polarization, in particular greater than 50 mP, preferably greater than 10OmP.
• Donor and acceptor compounds whose intrinsic polarization is less than 50 mP can be coupled or adsorbed to carrier molecules (organic molecules, proteins, peptides, antibodies, or other molecules as described below), which will have the effect of increase the apparent polarization of the fluorophore and allow it to be used in according to the invention.
• the fluorescent donor and acceptor compounds are chosen such that, following excitation at the donor excitation length ⁇ 1, no emission of the acceptor is detected at the wavelength of donor emission ⁇ 2.
• the method according to the invention therefore makes it possible to substantially improve the detection of energy transfer phenomena, which makes it possible to study in a more precise manner in particular the biological interactions.
• the method according to the invention can thus be used in a biological system in which the distance between the donor and acceptor fluorescent compounds varies according to biochemical events occurring in the measuring medium.
• the fluorescent donor and acceptor compounds are linked to molecules chosen from the group comprising: a peptide, a protein, an antibody, an antigen, an intercellular messenger, an intracellular messenger, a hapten, a lectin, biotin, avidin, streptavidin, a toxin, a carbohydrate, an oligosaccharide, a polysaccharide, a nucleic acid.
• the fluorescent donor and / or acceptor compounds are fluorescent proteins, they may be linked to other proteins in the form of fusion proteins, produced by recombinant DNA techniques, well known to those skilled in the art.
• the fluorescent donor and acceptor compounds are directly or indirectly bound to a hydrolyzable substrate.
• the measuring medium contains, for example, an enzyme capable of cleaving said substrate, the detection of the evolution of the FRET may be correlated with the enzymatic activity.
• direct or indirect bonding of the fluorescent compounds to the substrate is meant a covalent bond, optionally via spacer arms, or non-covalent bonds via couples. molecules that can bind to each other.
• Such indirect linkages include, for example, the case where a fluorescent compound is covalently bound to biotin, and the substrate has a streptavidin group, or the case where the fluorescent compound is bound to a specific antibody of a label present on the substrate. the substrate, such as groups 6his, flag etc.
• the method according to the invention can also be used to study the variation of a FRET in the case of an interaction between two compounds.
• the fluorescent donor and acceptor compounds are covalently bound to two molecules capable of recognizing each other.
• the donor compound may be linked to an antibody or antibody fragment and the acceptor compound may be bound to the antigen recognized by that antibody.
• the donor and acceptor compounds are each bound to a member of a ligand-receptor pair, or to two interacting proteins, or the donor is bound to a compound that regulates the activity of a protein and the acceptor compound is bound to said protein.
• the method according to the invention can also be used to study the binding of two compounds X and Y to a third compound Z. This may be of interest for studying phenomena of complex recognition between different proteins.
• compound X is covalently bound to a donor fluorescent compound
• compound Y is bound to an acceptor fluorescent compound, and energy transfer will occur if X and Y bind to molecule Z.
• the fluorescent donor and acceptor compounds are different.
• the invention finally relates to a measuring apparatus adapted to the implementation of the method according to the invention.
• Such an apparatus comprises the following elements:
• means for illuminating a measuring medium with a polarized exciter light for example, lasers, flash or continuous lamps associated with a polarizer; means for collecting the fluorescence emitted by the measuring medium at lengths of different waves and in different polarization planes, in particular parallel or non-parallel, preferentially orthogonal to that of the exciting light, the detection means being photomultiplier tubes, CDD cameras or intensified cameras in front of which will be placed the appropriate polarizers, and - computer means for correcting the signal measured at the wavelength of emission of the acceptor fluorescent compound by that measured at the emission wavelength of the donor fluorescent compound, in particular computer programs capable of calculating the ratio of the intensity of the signal collected at the wavelength d emission of the acceptor by the intensity of the signal collected at the emission wavelength of the donor.
• Another apparatus for implementing the method according to the invention comprises:
• a polarized excitation light for example, lasers, flash or continuous lamps associated with a polarizer
• the detection means being able to detect be photomultiplier tubes, CDD cameras or intensified cameras in front of which the appropriate polarisers will be placed, and
• These devices can be for example microscopes for measuring the fluorescence intensity emitted by a sample.
• the method according to the invention makes it possible to study in a precise manner the FRET phenomena occurring in complex measuring media and in particular biological media containing mixtures of proteins, animal or plant cells, membranes derived from animal or plant cells or artificial membranes. Since the methods according to the invention fundamentally make it possible to optimize energy transfer measurements (FRET), they are perfectly suitable for all techniques based on FRET measurements.
• FRET energy transfer measurements
• the methods according to the invention can also be used to refine the data obtained by the type techniques
• FLIM Fluorescence Lifetime Imaging Microscopy
• Time-resolved fluorescence imaging allows for quantitative monitoring of FRET with high sensitivity, via changes induced in the fluorescence lifetime of the donor and / or acceptor.
• A647 Alexa Fluor 647 from Molecular Probes.
• VIa-YFP Yellow Fluorescent Protein
• Receptor fusion protein CXCR4-CFP (Cyan Fluorescent Protein) expressed in HEK293 cells.
• CAM fusion protein whose structure is as follows: CFP-peptide linker-YFP (this construct is described by Zhou et al in The Journal of Pharmacology and Experimental Therapeutics, 305: 460-466, 2003). Direct Interaction Between the Heterotrimeric G Protein subunit G ⁇ 35 and the G G protein signaling 11: Gain of function of fluorescent cyan protein-tagged G ⁇ 3). This fusion protein has also been expressed in HEK 293 cells.
• the peptide linker can be any peptide of about 9 amino acids, regardless of these amino acids.
• HEK293 cells are transiently transfected by electroporation with plasmids encoding different fusion proteins. The cells are then placed at 37 ° C. in a controlled medium. After 24 hours, the cells are recovered, washed in PBS buffer, counted and fixed in a solution based on paraformaldehyde.
• HEK293 containing the different molecules were distributed in the different wells of the microplate.
• the same amount of control cells (not containing any fluorescent protein) was distributed in "control" wells.
• the level of polarization was determined using a microplate fluorescence reader, the Analyst (Molecular Devices). According to the fluorescent molecule to be detected, the Analyst was equipped with the following filters (all from Omega Optical):
• P is expressed in mP units.
• Table 1 below reports the polarization levels obtained for the various molecules observed.
• Example 2 Determination of the signal / noise ratio (S / B) of an intracellular FRET experiment.
• Receptor fusion protein VIa-YFP Yellow Fluorescent Protein expressed in HEK293 cells.
• CXCR4-CFP Receptor Fusion Protein CXCR4-CFP Receptor Fusion Protein
• CAM fusion protein whose structure is as follows: CFP-peptide linker-YFP. This fusion protein was also expressed in HEK 293 cells. The expression of these different proteins was carried out as described in Example 1.
• the cells containing the CAM are those which allow the measurement of a FRET between the CFP (donor molecule) and the YFP (acceptor molecule).
• the ratios are then calculated for positive or negative wells for total fluorescence measurements (without polarizers) or for orthogonal fluorescence measurement (with polarizers).
• the signal / noise (S / B) of the experiment is then calculated as follows for measurements made with or without the polarizers.
• the graph shown in FIG. 1 gives the signal / noise values obtained in the absence or in the presence of polarizers.
• the so-called “orthogonal" fluorescence measurement makes it possible to promote the detection of the signal emitted by the acceptor after energy transfer (depolarized fluorescence) with respect to the fluorescence signals emitted by donors or fluorescence acceptors not involved in a FRET phenomenon (highly polarized fluorescence).
• Example 3 Measurement of the degree of polarization of the acceptor for the detection of a FRET. Correction of donor contamination in measured fluorescence signals and quantification of FRET.
• the fusion proteins used in this example are identical to those described in Example 2. Their expression was carried out as described in Example 1.
• P is expressed in mP.
• I ⁇ 35 nm // is the fluorescence intensity obtained during measurement 3 either on the positive wells or on the negative wells or on the contaminated wells.
• l 53 5 ⁇ m i is the fluorescence intensity obtained during measurement 4 either on the positive wells or on the negative wells or on the contaminated wells.
• the overall degree of polarization measured at 535 nm represents the sum of the degree of polarization of the YFP acceptor involved in the FRET and the degree of polarization of the CFP donor measured at 535 nm due to the high contamination of CFP signal at this wavelength.
• the determination of the degree of polarization of the YFP acceptor involved in the FRET can be obtained by subtracting the signal from the CFP donor from the signals obtained during the fluorescence measurements at 535 nm (measurements 3 and 4).
• IW n mx is the average of the fluorescence intensities obtained during measurement 2 on the control wells.
• It 535 nm // is the average of the fluorescence intensities obtained when measuring on three control wells.
• K 535 nm x is the average of the fluorescence intensities obtained during measurement 4 on the control wells.
• U ⁇ o n m // is the fluorescence intensity obtained during measurement 1 either on the positive wells or on the negative wells or on the contaminated wells.
• U ⁇ o nm x is the fluorescence intensity obtained during measurement 2 either on the positive wells or on the negative wells or on the contaminated wells.
• l535 ⁇ m // is the fluorescence intensity obtained during measurement 3 either on the positive wells or on the negative wells or on the contaminated wells.
• I535 nm 1 is the fluorescence intensity obtained during measurement 4 either on the positive wells or on the negative wells or on the contaminated wells.
• Pf is expressed in mP.
• Table 2 shows the degree of overall polarization (ie Pgiob a) and the degree of polarization of the acceptor YFP involved in the FRET (Pf) for the different samples:

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• 2004
  • 2004-06-28 FR FR0407087A patent/FR2872287B1/fr not_active Expired - Lifetime
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