EP1929294A2 - Method for detecting intracellular interaction between biomolecules - Google Patents

Abstract

The invention concerns a quantitative non-microscopic method for detecting intracellular interaction between biomolecules, on living cells, responsive to a biological or pharmacological stimulation, by time-resolved proximate energy transfer effect between a fluorescence donor/acceptor pair. The invention is useful for high throughput screening of molecules and intracellular signalling pathway detection.

Description

Method for detecting intracellular interaction between bio-molecules

The present invention relates to a non-microscopic, quantitative method for detecting intracellular interactions between biomolecules, on living cells, in response to biological or pharmacological stimulation, by the effect of proximity energy transfer in time resolved between two members. a fluorescence donor / acceptor pair and its applications such as in particular the high-throughput screening of molecules and the detection of intracellular signaling pathways.

Technical field and prior art

The molecules with pharmacological activity (drugs for example) act on molecular targets located either at the plasma membrane or inside living cells. The selection and optimization of candidate molecules in research processes within the pharmaceutical industry requires the determination of their molecular actions and, as far as possible, in a native environment in order to integrate the modulations possibly brought about by interactions between different partners. From a more fundamental point of view, in physiological or pathological conditions, it is important to have cell models where the direct interactions between bio-molecules involved in intracellular signaling pathways can be easily studied, characterized and quantified [see for example Takesono et al. Journal of CeII Sciences, 115, 3039-3048, (2002)]. This desire to understand mechanisms of action in native conditions explains why more and more cell assays are used in primary screening of molecules. But currently, high throughput screening techniques can primarily detect the end products of biological events, such as the production of second messengers. In this case, the cells must be lysed to allow access to the intracellular target molecule to be assayed. Very few kinetic observations are currently possible and rare events are easily undetected. New approaches using live cell microscopy or imaging techniques provide access to dynamic and kinetic parameters of one or more bio-molecules of interest in their native environment, in response to stimulation. These techniques are based on movement studies of these biomolecules in response to a stimulus. The analysis of these displacements makes use of a treatment of the images which give access, a posteriori and by indirect methods, with arbitrary values which make it possible to quantify the biological events considered.

These microscopy techniques consist of determining the location of a molecular target and monitoring any localization changes. For these approaches, the molecular target is generally fused to a fluorescent protein that may be of the GFP family. Such direct visualization techniques inside a cell are carried out using a conventional fluorescence microscope, cofocal microscopes or new platforms integrating microscopy and image acquisition. Such platforms are, for example, used in TRANSFLUOR technology (Molecular Devices) or on instruments such as OPERA (Evotech Technologies) or In CeII analyze (GE Healthcare) (see John Comley review, DDW, summer 2005, pp 31-53).

Many disadvantages, however, limit the use of such technologies:

- Microscopic studies are delicate, difficult to automate and they require complex tools of image analysis, specific software and very experienced users and they require a lot of time.

- The analysis of the images is complicated, often source of errors and these technologies are difficult to reproduce and unreliable (low parameter value ZQ.

They allow the monitoring of biological events in living cells with an average rate of screening of molecules limiting their use in the primary screening steps.

- The visualized effects are generally downstream of the signaling cascade. It is for these main reasons that these techniques are not compatible with high throughput screening of molecules.

In addition to these aspects, these technologies are used to qualify one or more cellular events, which most often result from a cascade of past events, giving indirect access to the event that one wishes to measure. Moreover, these techniques do not make it possible to directly give information on the interactions between biomolecules or on conformational changes within the same molecule, both of which are direct indicators of the biological event that is desired. detect and quantify. Currently intracellular technologies to characterize interactions between biomolecules or conformational changes within the same bio-molecule, both witnesses of biological events, are based on energy transfer technology (FRET) between different fluorescent proteins such as green fluorescent protein (GFP) and its different mutants. For example, two protein variants of GFP (CFP and YFP) form a compatible pair to establish FRET. They can be fused with the target proteins of interest and expressed in living cells. Many interaction events or conformational changes between proteins thus labeled were detected using a FRET method (see Janetopoulos et al., SCIENCE (2001) 291: 2408-2411, Vilardaga et al., NATURE BIOTECH (2003). 21: 807-812 and WO 2004 057333 A1).

Both variants of GFP (CFP and YFP) can also be expressed in living cells in fusion with a single target protein. This biomolecule, doubly fused with the two variants of GFP, is defined as a biro-sensor. A bio-sensor is able to adopt different conformations depending on changes in the biological environment, these conformational changes being able to be followed by a FRET process. Such biomolecules can be used in the monitoring of numerous cellular events such as the production of cAMP, IP3 or cGMP (see Nikolaev et al., JBC, vol 279, No. 36, pp. 37215-37218, 2004). , Tanimura et al., JBC, vol 279, No. 37, pp. 38095-38098, 2004 and US Patent 6,924,191 B2).

However, the use of these fluorescent proteins has certain major disadvantages;

- The overlapping spectra of the two fluorescent molecules (CFP and YFP) are broad and they cause a strong parasitic excitation of the acceptor (YFP) in the wavelengths used to excite the donor (CFP). This parasitic contamination induces a strong background that reduces the signal-to-noise ratio to a value less than 1.5. Such small modulation differences preclude comfortable use of these techniques in high throughput molecule screening.

At the wavelengths used for the detection of FRET with these fluorescent proteins, a self-fluorescence intrinsic to the cells disturbs the reading of FRET. - Microscopic analyzes may be necessary to reveal subtle changes in FRET signals. Such analyzes then have the same disadvantages as those described above for the first detection approach.

- The limitation of the number of GFP variants restricts the use of such proteins to applications where the proximity changes are in agreement with the Fôrster (Ro) radius of the CFP / YFP pair.

- These proteins are fused with the target proteins. The fluorescence of these proteins is detected as soon as the cell expresses it and can not be triggered or controlled by the experimenter without setting up much more complex expression systems.

An alternative to FRET using GFP or its variants, which can be controlled by the experimenter, is the use of a molecule that generates bioluminescence, so-called BRET. A BRET process can take place in living cells between luciferase and GFP labeled proteins respectively. Several intracellular protein interactions could be measured by fusing luciferase to one partner and GFP to another (see Milligan G Eur J Pharm Sci 2004 Mar; 21 (4): 397-405, Boute N et al. Trends Pharmacol Sci., 2002 Aug; 23 (8) 351-4 and Trugnan G. et al., Sci Med (Paris), 2004 Nov; 20 (11); 1027-34.). The energy transfer process is initiated when the substrate of the luciferase enzyme is provided in the cell preparation to be tested. Luciferase produces a light which, in the case of a protein interaction allowing close proximity between the two partners, excites the GFP. The fluorescence signal emitted by the GFP is then measured.

As for the CFP-YFP couple, many disadvantages limit the use of the Luciferase-GFP couple in high-throughput screening (HTS):

- The signal-to-noise ratio of the BRET tests is quite low.

The fluorescence signal can be easily masked by an autofluorescence of the tested products. The BRET technology is not robust enough for screening molecules because of the low stability of the signal over time, which leaves a very short reading window, which is not very compatible with the high-throughput screening of molecules.

Another set of technologies for studying interactions between biomolecules is to use a functional complementation process. Biological molecules of interest are fused with two inactive fragments of a ^3rd protein. When the direct interaction between the biomolecules of interest occurs, the 2 active fragments of the 3 ^rd protein then reconstitute, by direct interaction or by a more complex process of protein splicing, a 3 ^rd functional protein whose activity can be measured. This protein, the activity of which has been restored by the interaction between the biomolecules of interest, may be a luciferase; in this case, a luminescence signal is measured, a fluorescent protein such as GFP (see Ozawa T, Current Opinion in Chemical Biology, 2001, No. 5, pp578-583 for review) or an enzyme such as β-galactosidase which enzymatic activity is measured by colorimetric method (see Eglen R, ASSAY and Drug Develop Technologies, 2002, vol 1, pp 97-104) and DISCOVER's Path Hunter ^® method.

The main disadvantage of these approaches using functional complementation lies in the fact that the measurement of interaction between biomolecules is dependent on perfect reconstitution of the 3 ^rd protein in its active form, such as β-galactosidase and requires sometimes a displacement of the interaction complex in a cell compartment other than that where the biomolecules normally interact. Finally, it is an indirect method of characterizing an interaction between biomolecules of interest where the measurement of an enzymatic activity accounts for the interaction. The use of the functional complementation is more complex to implement in the case of a single bio-molecule of the biosensor type.

Depending on the intrinsic properties of the proteins used in these energy transfer processes, technologies using fluorescent proteins such as GFP (or its other variants) or bio-luminescent proteins such as luciferase, do not allow have great flexibility in the choice of the donor / acceptor pair involved in the energy transfer process.

The use of particular fluorescent molecules, called "QuantumDots", offers a much wider possible number of possible donor / acceptor couples, makes it possible to work on a range of longer excitation wavelengths and, by combining different couples, to measure concomitantly different interactions between bio-molecules (multiplex approach). However, it is difficult to specifically label the biomolecules by these compounds in the cell.

The use of fluorescent organic molecules for intracellular FRET applications has, for example, been described for visualizing messenger RNAs in living cells. The interaction involved in this case is based on an apartment between the target messenger RNA and anti-sense oligonucleotide sequences labeled with fluorescent organic molecules (see Dirks et al., 2003, METHODS, Jan. 29, No. 1). pp. 51-57 and Tsuji et al., Biophys J. 2001 JuI 81 (1): 501-15). The information obtained in this case is not quantitative because it uses microscopic detection. Furthermore, it does not qualify an interaction between two biomolecules of interest and serves only to reveal the presence of a messenger RNA of interest.

There is a real need in the scientific community for new tools and processes to study biological phenomena in living cells, while avoiding the drawbacks associated with the use of known techniques.

DESCRIPTION OF THE INVENTION

The present method of quantitative and direct analysis of live cell intracellular interactions without the aid of microscopy exploits the properties of long-lived fluorescent compounds for time-resolved FRET (TR-FRET) detection.

This technique uses a first long-lived donor fluorescent compound and one or more acceptor fluorescent compounds having spectral characteristics compatible with the first fluorescent compound.

This measurement of FRET in time resolved makes it possible to overcome the problems of background noise, auto-fluorescence of the cells and possibly molecules to be tested by two ways:

the long life of the fluorescent compound, preferably greater than 100 nanoseconds, makes it possible to define a window of time to achieve the measurement in which the emissions of short-lived fluorescent compounds are not present;

the measurement of two tracers, one of which as a reference, makes it possible to compensate the residual auto-fluorescence effects by using as a reading the fluorescence emission ratio between the two tracers.

Due to its sensitivity, its specificity, its robustness and its molecular resolution, the TR-FRET allows in the method of the invention to eliminate all the disadvantages of FRET applications between CFP / YFP or BRET with luciferase and GFP observed in living cells, in particular;

- the wide choice of fluorescent compounds and time-resolved reading allows to eliminate the strong parasitic excitation of the acceptor in the wavelengths used to excite the donor;

the time-resolved reading makes it possible to suppress the effects of the auto-fluorescence of the cells and products to be tested;

the amplitude of the signals in TR-FRET is higher than that measured with FRET techniques using dyes / fluorescent proteins.

The TR-FRET technique has never been used in living cells until now. This is due to many technical obstacles that the Applicant has been able to overcome.

One of the major technical problems in implementing this technique in a living cell is that most fluorescent compounds are often very hydrophilic and do not cross the plasma membrane. Another problem is related to the fact that these compounds are sensitive to their environment and in particular their fluorescence yield can be very affected in a biological medium. Finally, the Applicant had to overcome another obstacle in order to label biomolecules present in the cell by fluorescent compounds introduced outside the cell, without affecting the biological integrity of the cells.

The method of the invention makes it possible to circumvent these obstacles and thus provides effective means for studying biological phenomena within living cells.

The method of the invention allows the detection and quantification of conformational interactions or modifications between living cell biomolecules in response to specific stimulation.

The invention relates to a method for detecting interactions between bio-molecules, translocation or conformational change of biomolecules in living cells, comprising the following steps:

1) labeling a first bio-molecule in the living cell with a first fluorescent compound having a long fluorescence lifetime;

2) marking, in the living cell, at least one second bio-molecule with a second fluorescent compound;

3) subject the living cells to a specific stimulation adapted to the biological response to be studied, in the presence or absence of a library of molecules with pharmacological activity (potential candidates for future drugs) to be tested; 4) subjecting living cells to a light source having a wavelength for exciting the first long-lived fluorescent compound;

5) measuring the intensity of the fluorescence emitted by said first and second fluorescent compounds, and calculating the ratio between the fluorescence intensities of said first fluorescent compound and said second fluorescent compound, or measuring the lifetime of the first or second fluorescent compound ;

6) compare the signals measured with those obtained before stimulation of the cells. The light excitation of living cells can be achieved by methods well known to those skilled in the art, such as by a laser source, flash lamps or Xenon.

The emission, intensity and lifetime measurements of the various fluorescent compounds used in the method of the invention are carried out by means of the FRET signals by conventional methods well known to those skilled in the art. The appropriate readers for detecting the time resolved FRET signal according to the method of the invention are of the RUBYstar or PHERAstar type from BMG Labtech or other readers compatible with time resolved such as ULTRA, ULTRA Evolution or the GENios Pro from TECAN or PAnalyst from Molecular Devices. The calculation of the ratio between the fluorescence of the first fluorescent compound and the fluorescence of the second fluorescent compound or others (in the context of a multiplexing analysis) is carried out by a method known to those skilled in the art and described in particular in EP 569 496 Bl.

The fluorophores used to label biomolecules are fluorescent compounds that partner with TR-FRET. According to the invention, the term "TR-FRET partner fluorescent compounds" means pairs of fluorescent compounds whose fluorescence spectra partially overlap, and whose donor is a long-lived fluorescent compound and the acceptor is a fluorescent compound with a shorter lifetime than the donor. Those skilled in the art are able to select couples of fluorescent compounds partners of TR-FRET to implement the method according to the invention.

In an alternative implementation of the method according to the invention, and in the case where it is desired to study conformational changes of a bio-molecule in the cell, the two fluorescent compounds are used to mark one and the same biomolecule.

According to an implementation variant, the method according to the invention comprises additional steps for labeling biomolecules with other fluorescent compounds, which makes it possible to study more complex biological phenomena. In this case, the system will always include a first long-lived donor fluorescent compound, as well as several other fluorescent acceptor partners partners of TR-FRET, which will make it possible to measure several interactions, translocations or conformational changes in the same cell.

Bio-molecules

By bio-molecule, we mean a molecule present in a living organism, and in particular the molecules constituting the structure of an organism, those involved in the production and transformation of energy or in the transmission of biological signals. This definition includes nucleic acids, proteins, sugars, lipids, peptides, oligonucleotides, metabolic intermediates, enzymes, hormones and neurotransmitters.

The biomolecules that can be studied using the process according to the invention are all types of molecules expressed, thanks to heterologous expression techniques known to those skilled in the art, in living cells in culture. These bio-molecules may be, for example, any proteins, all lipids, sugars or oligonucleotides artificially produced by living cells, such as for example membrane receptors, such as tyrosine kinase receptors, 7-transmembrane domain-coupled receptors. G-proteins and subunits of hetero-trimeric G-proteins, non-membrane receptors, such as hormone receptors, ion channels, transporters present at the membrane (aquaria, sodium transporters, bicarbonate carriers (HCO ₃ ), ionic pumps (sodium / potassium pump), signal transduction proteins, such as monomeric G proteins, enzymes, such as kinases, phosphatases, transglutaminases, hydrolases, halogenases, lipases, transferases, metabolic enzymes, scaffolding proteins, such as SOS, GRB, 1RS, HSP, x lipids, proteins containing PH domains, or FYVE, associating with the membrane by lipid anchoring of N-myristoylation type, S-palmitoylation, S-prenylation geranylgéranylation, proteins associated with G-protein-coupled receptors (monomeric or trimeric) such as GEFs, GAPs and RGS, heterotrimeric G-protein subunits and G-protein-coupled transmembrane domain receptors, adapter proteins such as proteins containing SH2, SH3 domains, transcription factors such as c-fos, c-jun, CREB, mitochondrial proteins (such as respiratory chain proteins, cytochromes C, caspases, PBR ...), phospholipids , nuclear proteins such as DNA polymerases, helicases, topoisomerases, proteins of the family of so-called "death" receptors, such as FAS, apoptosis proteins, such as those of the Bcl family; 2, cell cycle proteins, such as cdk and cyclin.

Living cells

The living cells used in the present invention are all types of living cells cultured according to techniques known to those skilled in the art, such as, for example, prokaryotic cells (bacteria), yeasts, immortalized eukaryotic cell lines, insect cells, primary cultures, such as cultures from mammalian blood, tissue or organs. It is important to emphasize that the method of the invention makes it possible to work on living cells and to preserve the integrity of intracellular biochemical mechanisms: the cell membranes are not permeabilized as is the case in other processes of the invention. state of the art and the cells do not need to be fixed.

The interactions between molecules that can be demonstrated by the process according to the invention are numerous and varied and are dependent on the nature of the bio-molecules used in said process. These interactions may be, for example, the interactions between the androgen receptor and androgen which induce a translocation of the cytoplasmic receptor to the cell nucleus, PKC-γ which undergoes a translocation after stimulation of the cytoplasm to the lipids composing the membrane. cell plasma, the calcineurin complex which requires a calmodulin binding to be active, p65 / p50, etc.

The conformational changes of biomolecules within the living cell can take place in certain stimulation circumstances, such modifications can be, for example, beta-arrestin, EPAC (binding proteins), cAMP), exchange factors of small monomeric G proteins (GEFs), ion channels, such as K + channels dependent on fast inactivation potential, etc.

Fluorescent compounds

Several fluorescent compounds, also known as fluorophores, are used in the process of the invention, the first of these compounds has a long fluorescence lifetime and the others have generally short fluorescence lifetimes. In all cases, the fluorescent compounds are partners of TR-FRET.

The long-lived fluorescent compounds particularly suitable for the purposes of the invention preferably have a lifetime equal to or greater than 100 nanoseconds.

More specifically, these suitable fluorescent compounds are rare earth complexes, such as cryptates and chelates, in particular cryptates containing one or more pyridine units. Such rare earth cryptates are described, for example, in EP 180 492, EP 321 353, EP 601 113 and WO 01/96 877. Terbium (Tb3 +) and Europium (Eu3 +) cryptates are particularly suitable for the purposes of the invention. of the present invention. The rare earth chelates are described in particular in patents US 4,761,481, US 5,032,677, US 5,055,578, US 5,106,957, US 5,116,989, US 4,761,481, US 4,801,722 and US 4,794,191. , US 4,637,988, US 4,670,572, US 4,837,169, US 4,859,777. Other chelates are composed of a nonadentant ligand such as terpyridine (EP 403,593, US 5,324,825, US 5,202,423). , US 5,316,909).

A particular example of a rare earth chelate which is suitable for the purposes of the invention is the chelate having the formula:

The synthesis of this compound which can be used as donor member of a pair of FRET partners, is described by Poole R. et al. Org. In Biomol. Chem, 2005, 3, 1013-1024 "Synthesis and characterization of highly emissive and kinetically stable lanthanide complexes, suitable for use in cellulo". Example 6 below shows that this type of compound can be used to quantify time-resolved signals in the cell.

Fluorescent compounds having a short lifetime can be selected from fluorescent proteins, such as green fluorescent protein (GFP) and its derivatives (especially CFP, YFP), or fluorescent compounds having a lifetime of less than 100 nanoseconds, such as cyanines, rhodamines, fluoresceins, squarenes and fluorescent molecules known as BODIPY's (difluoroboradiazaindacenes), the compounds known under the name AlexaFluor, fluorescent proteins extracted from corals, phycobiliproteins, such as B- phycoerythrin, R-phycoerythrin, C-phycocyanin, allophycocyanins, in particular those known under the name XL665, the "Quantum Dots". Other suitable fluorescent compounds are described in patent application FR 2,840,611 and comprise a fluorophore coupled to an oligonucleotide.

Marking of biological molecules by your fluorescent compounds

The different methods for labeling biomolecules with fluorescent compounds are detailed below.

1) Generation of a fusion protein between the bio-molecules of interest and a protein having intrinsic fluorescence properties, such as the GFP family proteins. Expression of such fusion proteins is based on molecular biology techniques well known to those skilled in the art, such as transiently transfecting into the living cell, stably or transiently, expression vectors, such as plasmids, whose DNA encodes the fusion protein.

2) Generation of a fusion protein between the biomolecules of interest and a protein having a suicide enzyme activity, such as, for example, SnapTag (Covalys) or HaloTag (Promega) proteins that transfer covalently and irreversibly the fluorescent compound on the bio-molecules of interest. The compound In this case, the fluorescent is covalently bound to the substrate of the suicide enzyme and is introduced into the extracellular medium. The techniques described below make it possible to cross the cell membrane conjugates fluorescent compound - substrate that would not be naturally lipophilic.

Suicide enzymes are proteins that have enzymatic activity modified by specific mutations that give them the ability to bind a substrate quickly and covalently. These enzymes are called suicides because each can bind only one fluorescent molecule, the activity of the enzyme being blocked by the attachment of the substrate. Currently two families of known suicide enzymes allow this type of labeling:

the mutant of an alkylguanine-DNA alkyltransferase (or SnapTag marketed by Covalys) (see Klepper A. et al (2003) in Nature Biotechnology, Vol 21, P. 86 and WO 02/083937 A2), one of the substrates is benzylguanine.

- The mutant of a dehalogenase (HaloTag marketed by Promega) which also generates a suicide type enzymatic reaction (see WO 04/072232 A2), some of the substrates are compounds of the chloroalkane family.

In both cases, the substrate that will be incorporated by these suicide enzymes must first be labeled with a fluorescent organic compound.

3) Generation of a fusion protein between the bio-molecules of interest and a sequence recovering a protein splicing activity after binding with a complementary sequence previously labeled with the fluorescent molecule of interest thus transferring covalente and irreversible by a peptide bond the fluorescent compound on the target bio-molecules, such as the sequence encoding the intein domain of Ssp DnaE.

This approach exploits a biological process of post-translational processing, called protein splicing. This process catalyzes a series of chemical reactions whose ultimate goal is to remove a domain named intein present in a precursor protein and to bind by a peptide bond the two domains lying on either side of the intein domain. Protein trans-splicing requires two halves of complementary intein domain. The first half is fused to the target protein and the second half to the fluorescent molecule. This method is described by Muir TW et al. (2003) J. Am. Chem. Soc. 2003, 125, 7180-7181. The techniques described below make it possible to cross the cell membrane with conjugated "fluorescent compound - intein" which would not be naturally epophilic.

4) Generation of a fusion protein between the bio-molecule (s) to be studied and a peptide sequence or a member of a pair of binding partners having specific recognition and high affinity binding characteristics with a previously labeled substrate with the fluorescent molecule (s) of interest such as, for example:

The cysteine-cysteine-X-X-cysteine-cysteine (CCXXCC) sequences that have the property of binding to bi-arsenic compounds. These bi-arsenic compounds can be easily labeled with an organic molecule of the fluorescein or rhodamine type (see BAGriffin et al (1998) Science, 1998, 281, 269-271 and SA Adams et al (2002) J. Am. Soc., 2002, 124, 6063-6076 for details on the technology).

The PoIy histidine repeats bind to metal ions that can be coupled to "quencher" molecules of fluorescent compounds (see E.G. Guignet et al (2004), Nature Biotech., 2004, 22, 440-444).

The BTX tag (bungarotoxin), composed of a peptide of 13 amino acids that is recognized by bungarotoxin (BTX), can be previously coupled to a fluorescent molecule (see CM McCann et al (2005), Biotechnique (2005)). 38, 945-952).

The streptavidin binding sequence (SBP-Tag) is a 38 amino acid sequence that has a high affinity for biotin that can be previously labeled with a fluorophore (see CM McCann et al (2005), Biotechnology (2005)). 38, 945-952).

5) Generation of a fusion protein between the bio-molecules to be studied and a peptide sequence for the recognition of certain enzymes, for example transglutaminases or ligases which transfer to a specific amino acid of the Tag sequence the compound fluorescent previously fixed on the substrate of the enzyme.

The peptide sequence PKPQQFM (Proline-Lysine-Proline-Glutamine-Glutamine-Phenylalanine-Methionine) is for example recognized by an enzyme called transglutaminase. Transglutaminase transfers the coupled organic fluorescent compound to its substrate, cadaverine, directly to the first glutamine residue of the PKPQQFM sequence (see Taki et al (2004) Protein Engeneering, Design Selection, 2004, 17, 119-12). The techniques described below make it possible to cross the cell membrane with fluorescent compound-substrate conjugates that would not be naturally lipophilic. The sequence of the dihydrofolate reductase enzyme of E. coli (eDHFR) which binds specifically and with high affinity ligands such as trimethoprim onto which fluorescent compounds can be grafted according to the technology called "Ligand link Universal labeling technology" of the Active Motif Company,

6) Nucleic sequences can also be specifically recognized by enzymes having transferase activities allowing them to covalently link a fluorescent organic molecule present on a cofactor or a substrate directly to the specific sequence. A te! The method can be illustrated by the specific 5'-TCGA-3 'nucleic sequence on which the TaqI methyltransferase transfers the previously grafted fluorescent compound to the aziridine cofactor (see Pljevaljcic G. et al., (2004) ChemBiochem, 5: 265-269). .

In the process according to the invention, the fluorescent compound / biomolecule labeling is thus carried out by coupling the fluorescent compound and the bio-molecule, respectively with the members of the pairs chosen from: a SNAP-Tag substrate / the SNAP-Tag enzyme , a HALO-Tag substrate / the HALO-Tag enzyme, an intein part such as the intein of Ssp DnaE / the complementary intein part for reconstituting a functional intein, a bi-arsenic unit / the Cys-Cys-X-X-Cys-Cys sequence, X representing any amino acid, metal ion / poly-histidine sequence, biotin / streptavidin, streptavidin / biotin, bungarotoxin / BTX tag, cadaverine / the PKPQQFM protein sequence, the aziridine / the TCGA nucleic sequence.

The labeling techniques mentioned above sometimes involve passing the cell membrane to compounds that are not naturally lipophilic and do not naturally enter the cell. These compounds or their conjugates with substrates, tags, or members of a pair of binding partners as described above can be introduced into the cell using, for example, one of the following techniques: :

1). Use of esters which mask the charged groups during the passage of the lipid bilayer; these esters fall into the category of compounds named pro-drugs and refer to compounds that are rapidly transformed in vivo to give the "parent" compound following hydrolysis under conditions physiological. [T. Higuchi and V. Stella in "Pro-drugs as Novel Deliver Systems", Vol. 14 of the ACS Symposium Series, American Chemical Society (1975)]. Examples include mainly pivaloyloxymethyl, acetoxymethyl and glycol esters [Nielsen and Bundgaard, Int. J. of Pharmacy. 39 (1984) 75-85]. When this technique is used, one of these groups is grafted covalently on the fluorophore.

2). Use of viral peptides grafted covalently to the fluorophore: some of these peptides make it possible to convey the fluorophore through the cell membrane. Examples of such viral peptides include the analogues of "penetratin" and "transportan" [Langel et al. Bioconj. Chem. (2000), 11, 619-626], poly-Arginines [Wender et al. Arginine-based molecular transporters. Org. Lett. (2003), 5 (19), 3459-3462], or peptoids, peptides analogs carrying guanidine groups [The design, synthesis, and evaluation of molecules that enable or enhance cellular uptake: peptoid molecular transporters. Wender et al. Proc Natl Acad Sci U S A. (2000), 13003-8], or the non-hydrolyzable derivatives tetraguanidinium pattern [Fernandez-Carneado J. et al. J. Am. Chem. Soc. 2005, 127, 869-874]

3). Modification of the lipophilicity and polarity of fluorescent compounds by the addition of side chains containing cholesterol molecules, vitamin E or aliphatic chains that interact with cell membranes using the exemplified approach on oligonucleotides using undecyl chains or 1,2-di-O-hexadecyl-glycerol. [3'-end conjugates of minimally phosphorothioate-protected oligonucleotides with 1-O-hexadecylglycerol: synthesis and anti-ras activity in radiation-resistant cells. RaIt et al. Bioconj. Chem. (2000), 11, 153-160].

These modifications are more effective than cationic Tipids, which form supramolecular complexes with nucleic acids and increase endocytosis but are toxic and damage cell membranes.

Advantageously, in the method according to the invention, at least one fluorescent compound has a pattern enabling it to cross the plasma membrane, this pattern being selected from the following groups; esters such as valvaloyloxymethyl ester, acetoxymethyl ester, or glycol esters; viral peptides supported by membrane transporters, such as penetratin and its like, transport it! and its analogs, polyarginine groups, peptoids bearing guanidine groups, such as oligoguanidinium groups; cholesterol groups, vitamin E or aliphatic chains, such as undecyl or 1,2-di-O-hexadecylglycerol chains.

In an alternative implementation, at least two TR-FRET partner fluorescent compounds include such motifs allowing them to cross the plasma membrane.

Advantageously, the fluorescent compound can be bonded to the substrate of the suicide enzyme and at the same time generate on said fluorescent compound a reactive chemical function, such as, in particular, an amine or acid function which subsequently makes it possible to couple onto said fluorescent compound a motif allowing him to cross the plasma membrane.

By way of example, reference may be made to FIGS. 7 and 8 which illustrate the synthesis of a conjugate between a fluorescent compound, the compound DY647 (from the DYOMICS Company) and the substrate of a suicide enzyme (substrate of the "SnapTag" enzyme), namely benzylguanine on which an amine functional group has been added and the introduction of a reactive chemical function on said fluorescent compound (this conjugate is hereinafter referred to as "tripod").

By virtue of this reactive chemical function, it is possible subsequently to introduce, by covalent bonding, said pattern making it possible to cross the plasma membrane.

The tripode with an NH ₂ function will integrate the vector systems having a COOH function and that with a COOH function will integrate the vector systems having an NH ₂ function.

The invention also provides a kit of components (commonly called "kit of parts" in English) comprising the reagents for implementing the method for detecting interactions between bio-molecules, translocation or change of conformation of bio -molecules in living cells according to the invention.

This kit (or kit) includes:

a first fluorescent compound and a second fluorescent compound, these compounds being partners of TR-FRET and at least one of these compounds having a motif enabling it to cross the plasma membrane;

live cells comprising said bio-molecules; means for labeling the biomolecules with the fluorescent compounds;

instructions for studying phenomena of interactions between biomolecules, translocation or conformational change of bio-molecules in living cells.

The reagents present in this kit allow the labeling of the biomolecules to be studied present in the living cell by the fluorescent compounds partnering with TR-FRET; in practice, this means that the fluorescent compound and the bio-molecule are coupled, respectively, with the members of the pairs chosen from: a SNAP-Tag substrate such as benzylguanine / SNAP-Tag enzyme, a HALO-substrate; Tag such as a chloroalkane / HALO-Tag enzyme, a part of intein such as the internal of Ssp DnaE / the complementary intein part allowing to reconstitute a functional intein, a bi-arsenic motif / the Cys sequence -Cys-XX-Cys-Cys, X representing any amino acid, metal ion / poly-histidine sequence, biotin / streptavidin, streptavidin / biotin, bungarotoxin / BTX tag, cadaverine / sequence PKPQQFM protein, aziridine / TCGA nucleic acid sequence.

At least one of the fluorescent compounds furthermore comprises a motif enabling it to cross the plasma membrane, and this unit is chosen from the following compounds: esters such as pivaloyloxymethyl ester, acetoxymethyl ester, or glycol esters; viral peptides supported by membrane transporters such as penetratin and analogues thereof, transanan and analogues thereof, polyarginine groups, peptoids bearing guanidine groups; cholesterol groups, vitamin E or aliphatic chains such as undecyl or 1,2-di-O-hexadecyl-glycerol chains.

The living cells included in this kit may be genetically modified so as to express bio-molecules whose interaction we wish to study, these modifications make use of conventional molecular biology techniques well known to those skilled in the art, such as transfectomy. stable or transient cells by plasmids expressing the bio-molecules, or fusion proteins comprising the bio-molecules of interest.

The fluorophore partners of TR-FRET present in the kit according to the invention are a long-lived fluorescent compound and at least one fluorescent compound chosen from the following: fluorescent proteins such as the protein green fluorescent (GFP) and its derivatives (in particular CFP, YFP), or fluorescent compounds having a lifetime of less than 100 nanoseconds, such as cyanines, rhodamines, fluoresceins, squarenes and fluorescent molecules known under the name of BODIPY's (difluorobora-diaza indacenes), the compounds known under the name AlexaFluor, the fluorescent proteins extracted from corals, the phycobiliproteins, such as B-phycoerythrin, R-phycoerythrin, C-phycocyanin, allophycocyanins, in particular those known as XL665.

Preferably, the long-lived fluorescent compound has a lifetime greater than 100 ns and even more preferably is a rare earth chelate or cryptate. In a particular formulation of the kit according to the invention, the rare earth is terbium or europium.

The present invention may now be described in greater detail by the following illustrative examples, which should in no way limit the applications of the present invention.

EXAMPLE NO. 1 Measurement of the Translocation of the Protein Kinase C -Gamma (PKC-γ) (See Diagram 11)

Protein kinases C are proteins belonging to a group of serine / threonine kinases dependent on phospholipids. These proteins play a large role in many cell signaling pathways. Physiologically PKC-γ is activated in a calcium-dependent manner by phosphodiesterines (PS) and binds diacylglycerol (DAG), but PKC-γ can be activated independently of the presence of DAG by phorbol esters inducing tumors (PMA).

After stimulation, PKC-γ undergoes translation from the cytoplasm to the plasma membrane, this translocation can be measured using a fusion protein with GFP after stimulation with PMA (Sakai NJ CeII Bio (1997) 139 , 1465-1476).

A fusion protein between PKC-γ and a suicide enzyme, named HaloTag, was created to label PKC-γ with an acceptor fluorescent organic compound and a tool was developed to specifically label the plasma membrane. with a donor fluorescent organic compound that can enter FRET with the labeled PKC-γ.

The plasma membrane is specifically labeled with a suicide enzyme, named SnapTag fused to a Cys-Ala-Ala-X sequence (X is any amino acid) which is a recognition sequence for post-translational modification enzymes. This sequence will cause the grafting of a C-terminal farnesyl group of the Snaptag enzyme, which results in the addressing and anchoring of this enzyme on the inner sheet of the plasma membrane. COS-7 cells were transiently transfected with both plasmid constructs with pofectamine 2000 or by electroporation.

24 h or 48 h after the transient transfection, the cells are incubated for 1 hour with cell medium containing 5 μM of each specific substrate of the enzymes HaloTag and SnapTag, each of the substrates carrying the fluorescent organic molecules forming FRET.

After 3 washes with medium, the translocation is measured by TR-FRET after induction by different stimulations, for example after addition of 12-myristate 13-acetate phorbol ester (PIWA). An increase in the TR-FRET signal is correlated with an increase in the translocation process.

Potentially inhibitory molecules of the PKC-γ translocation pathway can be added to the culture medium to test their effects on stimulation of transitization.

Example 2: Demonstration of Androgen Receptor-Coactivator or Orroqene-Correpressor Receptor Interactions. (see block diagram figure 2)

Androgens and their functional receptor (AR) are responsible for the normal differentiation of the external male phenotype. The cytosolic androgen receptors, in the inactive state, are associated with chaperone proteins (known by the English name "heat shock proteins"). After binding with the ligand, the receptors dimerize and are translocated to the nucleus. The coregulators (coactivators or corepressors) of the AR will then interact or not with the receptor and the result of these interactions will eventually cause the binding of the complex to the DNA at the level of a specific sequence (called ARE for "androgen receptor responsive element ") and thus regulate transcription.

The method according to the invention is used to highlight the interactions between a corepressor, a coactivator, and an AR. For this purpose, expression vectors are introduced into the cell in order to express in the intracellular medium:

an AR-enzyme SnapTag fusion protein. The suicide enzyme Snaptag allows coupling with a fluorescent compound labeled with a snaptag substrate.

an HDAC1-Halotag fusion protein. HDAC1 (histone acetyltransferase 1) is a co -pressor capable of binding to RA. The suicide halotag enzyme allows coupling with a fluorescent compound labeled with a halotag substrate.

a pl60-fragment intein fusion protein. P160 is a coactivator of likely to fix on AR. The intein fragment allows coupling with a fluorescent compound comprising the complementary intein fragment by protein trans-splicing.

When the AR is localized in the nucleus after stimulation (binding of its ligand), one can assay its final effect by measuring the TR-FRET signal; the AR is labeled with the donor fluorescent compound and the coactivator and the corepressor with two acceptor fluorescent compounds which are quite distinct in their spectral properties, and whose emission wavelength following a transfer of energy is respectively

665 and 780 nm.

The measurement of a TR-FREET signal at 665 nm will therefore be representative of the binding of the AR with its coactivator, whereas the measurement of a TR-FREET at 780 nm will be significant of an AR-corepressor interaction. This test format thus allows the detection of two types of interaction in a single cellular test. This multi-detection assay can be further enhanced by the addition of other fluorescent acceptor compounds to simultaneously reveal other interactions.

Example 3: Bio-sensor cAMP: Detection of conformational changes of a fusion protein comprising a TAMPc binding domain (See Schematic diagram, Fig. 31)

In this example, the variations of the TR-FREET signal of a pair of fluorescent donor / acceptor compounds are measured by coupling these fluorophores to a cAMP binding domain (for example that of the regulatory subunit β11 of the PKA , that of the exchange proteins activated by cAMP) known by the term of CAMPS. These molecular constructs can be used to quantify in living cells the levels of cAMP after pharmacological stimulation of a G protein-coupled receptor (GPCR) (see (Nikolaev et al., JBC, vol 279, No. 36, pp. 37215-37218, 2004).

The plasmid encoding a SNAP-tag / CAMPS / Halo-tag fusion protein is transfected into HEK cells. 24 hours or 48 hours after transfection, cells are incubated (1 hour at 37 ° C) with SNAP-Tag and HALO-Tag substrates, each coupled to a member of a TR-FREH donor / acceptor pair ^"

At the end of the incubation step, the fluorophores are covalently coupled via the suicide reactions of the SNAP-Tag and HALO-Tag enzymes, and the CAMPS protein is labeled by the pair of fluorophores involved in TR-FRET. .

In the basal state, a TR-FRET signal is measured because of the proximity of the fluorophores. After pharmacological stimulation (eg, agonist stimulation of a Gs protein-coupled receptor expressed in HEK cells), intracellular cAMP concentration will increase and cAMP binding to CAMPS will cause a conformational change in this latter protein. . A decrease in the measured TR-FRET signal is then observed.

This example shows that the amplitude of the TR-FRET signal is directly correlated to the binding of the cAMP to the CAMPS protein.

The same experiment can be conducted with a GPCR antagonist, which causes a decrease in cAMP concentration in the cell and a conformational change in CAMPS protein that can be detected by measuring an increase in the TR-FRET signal.

Example 4: Measurement of the calcineurin / calmodulin interaction (see block diagram FIG. 4)

Calcineurin possesses phosphatase activity and plays a fundamental role in the cellular signaling pathway involving intracellular calcium levels. Ca / cineurine is involved in the development and adaptation to stress in mammals, through two classes of transcription factors: NFAT and MEF2.

Calcineurin is a heterodimer with two subunits, calcineurin A (CnA) and calcineurin B (CnB). The enzyme is active in its heterodimeric form and the phosphatase activity is stimulated by the formation of a calcineurin-calmodulin complex upon increasing the intracellular calcium concentration.

A plasmid encoding a fusion protein comprising the CnA subunit and the HALO-Tag enzyme and a plasmid encoding a fusion protein comprising calmodulin and the SNAP-Tag enzyme are co-transfected into cells using the lipofectamine 2000 or by electroporation. After 24 or 48 hours, the cells are incubated for 1 hour at 37 ° C. in a medium comprising the SNAP-Tag and HALO-Tag enzyme substrates, each coupled to a member of a pair of fluorophores compatible for TR-FRET.

After washing, the cells are stimulated pharmacologically or by stress (for example by cyclosporin A, or acidification of the medium). The amplitude of the measured TR-FRET signal increases and is correlated with the association of the CnA-CnB heterodimer with calmodulin.

It is possible to add to the measuring medium before stimulation molecules whose effect on the activation of the calcineurin-dependent signaling pathway is to be tested. This example shows that the method according to the invention makes it possible to study the dependent calcineurin signaling pathway in living cells, optionally in the presence of compounds to be tested. This type of test allows the high-throughput screening of candidate compounds capable of allowing the treatment of pathologies involving this signaling pathway.

Example 5 Screening of Compounds Before Anti-inflammatory Activity

TNF alpha is a factor of inflammation that causes several cellular responses when bound to its membrane receptor. One of the responses caused by TNF alpha in B cells is the activation of the transcription factor NFkB, which controls the production of antibody light chains - a critical step in the immune response. Inhibitors of TNF alpha are therefore useful for regulating the immune response, and may for example be used in the prevention of septic shock, or as anti-inflammatory drugs. Such inhibitors of TNFalpha activity can be discovered by measuring the activation level of NFkB, which the present invention allows. FIG. 5 illustrates the cytosolic retention of NFkB by the cytosolic protein IkB: the NFkB complex (p65, p50) -IkB is retained in the cytosol. Appropriate stimulation (mitogens of T and B cells, lipopolysaccharide, and TNFalpha) causes IkB phosphoryiation, causes degradation and releases NFkB which can then penetrate the cell nucleus and activate the production of antibody light chains.

The method according to the invention is applied by introducing into B lymphocytes by conventional molecular biology techniques the following constructions:

a vector expressing an IkB-Snaptag fusion protein, which may be labeled with a fluorescent donor compound conjugated to the Snaptag substrate;

a vector expressing a p50-Halotag fusion protein, which may be labeled with a fluorescent acceptor compound conjugated to a Halotag substrate.

Under resting conditions, the NFkB complex (ρ65 / p50) forms a complex with IκB and a TR-FRFJT signal can be measured. After stimulation of the cells by TNFalpha, the phosphoryiation of IκB and its dissociation of the complex will cause a decrease of the TR-FREET signal. The magnitude of this decrease is representative of the degree of activation of NFkB.

These cells are cultured in a microwell plate, and one of the following treatments is applied to different wells:

addition of fluorescent donor and acceptor compounds coupled to Snaptag and Halotag substrates, measurement of the TR-FRET signal (high TR-FRET control well);

addition of fluorescent compounds coupled to Snaptag and Halotag substrates, stimulation of the cells by addition of TNFafpha, measurement of the TR-FRET signal (low TR-FRET control well);

addition of the fluorescent compounds coupled to the Snaptag and Halotag substrates, addition of a test compound, stimulation of the cells by addition of TNFalpha, measurement of the TR-FRET signal (test wells).

When the signal from the test wells is similar to that measured in the high TR-FRET control wells, it can be concluded that the compounds to be tested have an inhibitory effect on the TIMF alpha activity. When this value is close to the value measured in the low TR-FRET control wells, it can be concluded that the compounds to be tested have no effect on the action of TNF alpha.

This example illustrates the application of the method according to the invention to the screening of drugs and is particularly suitable for so-called high throughput screening.

Example 6: Time-resolved microscopy of a europium chelate

This example illustrates the use of a fluorescent compound capable of naturally crossing the plasma membrane and its ability to emit a time-resolved signal in a cell, a) Protocol

Cells are seeded in LABTEK culture chambers [density 80 000c / ml], cultured for 24 hours and then washed with culture medium.

The europium chelate of formula below is added to the culture medium (concentration 20 .mu.m) and the cells are incubated for 24 hours at 37.degree.

After washing with culture medium, Hoechst stain 33342 [concentration 2 μg / ml; supplier reference: Sigma Aldrich B2261] is added to the culture chambers, which are then incubated for 15 min. protected from light at room temperature. This dye has the property of specifically labeling the cell nuclei.

The culture chambers are washed again with culture medium before proceeding with the acquisition of images by microscopy, in "time resolved" mode, with the following equipment: an Axiovert 200M microscope (Zeiss), a source of UV excitation: a pulsed nitrogen laser (Spectra Physics) and a Pi-Max intensified CCD (charge coupled device) camera (Roper Scientific), with the following parameters:

• Playback time 2 milliseconds: The camera detects for 2 milliseconds to recover all the chelate signal;

• Delay 100 microseconds: delay imposed to eliminate parasitic fluorescence before capturing the signal in time resolved;

Number of cycles per reading: the signal is acquired during 30 exposures during 2 milliseconds and compiled.

b) Results;

The resulting images are shown in Figure 6.

The image in transmission (image 3) makes it possible to ensure the integrity of the cells and the density of the cellular mat. The image Hoechst (Image 2) allows to locate the cellular nuclei. The negative control (Figure 5) shows the signal obtained in time-resolved detection mode in the absence of fluorescent compound.

The image obtained with the donor fluorescent compound tested at a concentration of 20 μM (FIG. 1) makes it possible to demonstrate an intracellular localization of the FRET donor compound. The superposition of the images (2) and (1) shows that the fluorescence is localized in the cell.

This type of time-resolved measurement can be quantified by designating regions of interest on signal and noise areas, and subtracting the average value of the noise from the measured signal value.

This example shows that a rare earth chelate capable of crossing the plasma membrane can be used to implement the invention, and that the signal emitted by such a compound can be measured by time-resolved microscopy.

Example 7 Synthesis of a Fluorescent Conjugate Containing a Polyglycine Unit and a Benzylquanine Group, Microscopic Data a) Synthesis of BG-DY647 and Dy647-R9-BG

The synthetic scheme of a conjugate comprising benzylguanine BG (substrate of the enzyme SNAPTAG) covalently bound to the fluorescent compound DY647 is described in FIG. 9.

10 represents the synthesis scheme of a conjugate comprising a sequence of 9 arginines (R9), on which are grafted on the one hand, fluorophore DY647, and on the other hand benzylguanine BG. b) Microscopic analysis:

CHO cells are cultured at 37 ^° C. under a controlled atmosphere at 5% CO ₂ in F-12 HAM medium (Invitrogen) + 10% fetal calf serum (FCS) previously inactivated for 20 min at 60 ^° C.

The day before the experiment, the cells are dissociated and seeded in LABTECK culture chambers (Nunc) at a density of 75,000 cells / well. On the day of the experiment, the test compounds (BG-DY647 or Dy647-R9-BG) are prepared in culture medium at a concentration of 5 μM and placed in contact for one hour with the cells.

After the incubation, 3 washes are carried out with culture medium. Prior to microscopic analysis, nuclear staining is performed with Hoescht stain.

The microscopic analysis is carried out using an Axiovert 200M (Zeiss) epifluorescence microscope, objective 40 X, a source of UV excitation; a pulsed nitrogen laser (Spectra Physics) and a CooISNAP CCD camera (Photometrics).

Figure 11 shows the images obtained with either the BG-DY647 conjugate or the DY647-R9-BG conjugate. It is found that the DY647-R9-BG conjugate is located in the cells while the BG-DY647 conjugate remains in solution in the culture medium. A compound such as DY647-R9-BG can therefore be used in the method according to the invention, especially as an acceptor conjugate capable of crossing the plasma membrane to label an intracellular protein comprising the SnapTag enzyme,

EXAMPLE 8 Comparative Example of the Effect of a Modification PolyAraInin or Oliquoquanidinium on the Entry of a Fluorophore into Cells in Culture, Data on Fluorescence Reader a) Protocol

CHO cells are cultured at 37 ⁰ C, under a controlled atmosphere of 5% COi medium HAM F-12 (Invitrogen) + 10% fetal calf serum (FCS) previously inactivated 20 min at 60 ⁰ C. The day before experiment, CHO-M1 cells are seeded in a plate 96 well at the density of 10,000 cells per well.

On the day of the experiment, the culture medium is aspirated and replaced by the test compounds at a concentration of 2.5 μM or 5 μM in KREBS buffer (Sigma). After

1 hour of incubation, the wells are washed with KREBS buffer + 0.05% Tween-20.

The cells are then lysed in PBS buffer + 0.1% Triton X100. The fluorescence is measured on a reader of the Analyst AD (UL, Molecular Devices) type with the filters and the dichroic suitable for the spectral specificities of the compound to be detected.

Example for a compound bearing a fluorescein:

Excitation filter: 485/22 nm bandwidth

Dichroic: high pass 505 nm

Emission filter: 535/35 nm bandwidth

Example for a compound carrying a rare earth complex: Excitation filter: 330/80 nm bandwidth Dichroic: BBUV Emission filter: 620/10 nm bandwidth

b) Results Under these conditions, 4 compounds were tested:

Fluorescein capable of naturally crossing the plasma membrane and carboxyfluorescein which is not able to naturally cross the plasma membrane as controls; the R7-fluorescein derivative of formula below:

obtained by grafting a poly arginine sequence comprising 7 arginine units on the fluorescein structure according to the synthesis scheme depicted in FIG.

12;

The tetraguanidinium-fluorescein derivative: a tetraguanidinium sequence (as described by Femandez-Cameado J. et al, X Am Chem Soc 2005, 127, 869-874) was grafted onto the carboxyfluorescein structure

The fluorescence intensity measured for each of the compounds (FIG. 13) according to the above protocol shows that the poly arginine and tetraguanidinium sequences can be used to introduce into the cell one of the FRET partners, and therefore as a pattern allowing one of FRET's partners to cross the cell membrane.

Example 9: Example of Constitutive Intracellular TR-FRlT (Chameleon HaloTaq-SπapTaq)

The method according to the invention is used to highlight a FRET signal in a living cell. For this purpose, an expression vector is introduced into the cell in order to express in the intracellular medium a fusion protein composed of SnapTag and HaloTag. The suicide enzyme SnapTag allows coupling with a fluorescent compound labeled with a SnapTag substrate (benzylguanine). The suicide enzyme Halo-tag allows coupling with a fluorescent compound labeled with a substrate of the Halo-tag (a chloroalkane).

Obtaining the plasmid construct for cellular expression of a SnapTag-HaloTag fusion protein:

The pHT2 plasmid carrying the HafoTag sequence is from Promega. The plasmid pSem-Sl-ST26m carrying the SnapTag sequence comes from the company

Covalys.

The cassette corresponding to the coding sequence of HaloTag is isolated from the plasmid pHT2 by enzymatic digestion EcoRV-Not I (Biolabs) and transferred into the plasmid pCDNA3.1 (Invitrogen) previously digested with the same enzymes.

The ligation is done by T4 ligase (Invitrogen). The ligating product is transformed into chemocompetent Turbo celis bacteria (GenetherapySystem).

The transformed bacteria are spread on LB-Agar medium (Sigma) + 0.1 mg / ml of ampicillin (Eurogentec) allowing the selection of bacteria possessing the plasmid pCDNA3.1Halo-Tag.

The plasmid DNA is obtained by column purification (QIAGEN). The integrity of the sequence is verified by sequencing.

The cassette corresponding to the coding sequence of SnapTag is isolated from pSEM-Sl-

ST26m by Clal-Xhol enzymatic digestion (Biolabs) and transferred into the plasmid pBIuescript KS (Stratagene) previously digested with the same enzymes.

Ligation is by T4 Ligase (Invitrogen). The ligation product is transformed into chem / ocompetent bacteria Turbo cells (GenetherapySystem).

The transformed bacteria are spread on one of LB-Agar medium (Sigma) + 0.1 mg / ml of ampicillin (Eurogentec) allowing the selection of bacteria possessing the plasmid pBfescriptKS-ST26m. Plasmid DNA is obtained by column purification

(QIAGEN). The integrity of the sequence is verified by sequencing.

The plasmid making it possible to obtain the coding sequence of the SnapTag-HaloTag fusion protein is obtained by transferring the cassette corresponding to the sequence of SnapTag, isolated by digestion of the plasmid pBlescrIptKS-ST26m with XbaI-Kpnel (Biolabs), into the plasmid pCDNA3 .1Haio-Tag previously digested by Nhel-Kpnel. The plasmid DNA corresponding to plasmid pB) escriptKS-ST26m-HaloTag is obtained by column purification (QIAGEN). ). The integrity of the sequence is verified by sequencing.

Cos-7 cells are transiently transfected with the plasmid construct pBlescriptKS-ST26m-HaloTag with lipofectamine 2000 or by 96 well plate electroporation. 24 h or 48 h after the transient transfection, the COS-7 cells are incubated for 1 hour with cell medium containing 5 μM of each specific substrate of the enzymes HaloTag and SnapTag, each of the substrates being covalently bound to a member of a couple of FREET partners and a pattern allowing it to cross the plasma membrane (manufactured for example according to the diagram of Figures 7 or 8).

After 3 washes in KREBS + 0.05% Tween-20, a FREET signal is measured on a RubyStar (BMG) reader, which shows that the method according to the invention can be used to measure a FRET signal generated at the same time. inside of living celMes.

Example 10: Example of Induced Intracellular TR-FRET (FRB-FKBP)

Model used:

The method according to the invention is here applied to the demonstration of the interaction of the intracellular proteins FKBP12 and the FRB domain of the FRAP protein when rapamycin is added to the culture medium.

FKBP12 is a 12 kDa protein that belongs to the family of immunophilins. Rapamycin binding to FKBP12 renders the protein cytosoluble by dissociating FKBP from these partners.

The FRAP (or mTOR) protein contains the FRB domain: this domain is composed of 99 amino acids (corresponding to the mTOR sequence E2015 - Q 2114),

Rapamycin is a molecule isolated from a bacterium Streptomyces hygroscopicus and commercially available. Rapamycin occupies two distinct hydrophobic pockets, one on FKBP and one on FRB, it binds both proteins at the same time.

rapamycin

FKBP _{j |} JTg C ooommpplieexxe Ternary

FRB Construction for obtaining a Halotag-FRB fusion protein in a cell expression plasmid

A PCR on the plasmid pHT2 (Promega) is carried out with the following primers:

ERV-Nco-HT-s primer; 5 'gcggatatcgccaccatgggatcc 3 ¹

HT-Pstl-HT-as primer: 5 'acttaattaactgcaggccggcccc 3'

The PCR is made with Phosponase polymerase (Finnzyme) at a hybridization temperature of 72 ^° C.

The PCR product is digested with the enzymes NcoI-PstI, 1 h at 37 ° C, the enzymes are then inactivated at 80 ⁰ C for 20 min.

The plasmid pBAD-ST26-FRB supplied by Covaiys is digested with Ncol and

PstI to remove the cassette corresponding to SnapTag (ST26). The linear plasmid pBAD-FRB obtained is ligated with the HaloTag PCR product described above.

The ligation is made by T4 ligase (Invitrogen). The ligation product is transformed into chemiocompetent bacteria Turbo cells

(GenetherapySystem). The transformed bacteria are plated on LB-medium.

Agar (Sigma) + 0.1 mg / ml ampicillin (Eurogentec) allowing the selection of bacteria with plasmid.

The plasmid obtained pBAD-HT-FRB is purified on a column (QIAGEN) and controlled by sequencing.

The cassette corresponding to the coding sequence of the HaloTag fusion protein

(HT) -FRB is amplified by PCR from the pBAD-HT-FRB template with the following primers:

ERV-Nco-HT-s primer: 5 'gcggatatcgccaccatgggatcc 3'

Primer pBAD rev: 5 'gttctgatttaatctgtatca 3'

The PCR is made with the Phusion polymerase (Finnzyme) at a hybridization temperature of 72 ^° C.

The PCR product is digested with the enzymes EcoRV-AscI, 1 h at 37 ⁰ C, the enzymes are then inactivated at 65 ⁰ C for 20 min.

The digested PCR product is transferred into a pSEM-XT cell expression plasmid (cova (ys) digested with EcoRV and AscI.

The ligation is made by T4 Legase (Invitrogen). The ligation product is transformed into chemiocompetent bacteria Turbo cells (GenetherapySystem). The transformed bacteria are plated on LB-Agar medium (Sigma) + 0.1 mg / ml of ampicillin (Eurogentec) allowing the selection of the bacteria possessing the plasmid.

L ¹ plasmid DNA is obtained by purification on column (QIAGEN). ). The integrity of the sequence is verified by sequencing.

- Construction for obtaining a SNAPTAG-FKBP fusion protein in a cell expression plasmid.

The cassette corresponding to the coding sequence of the FKBP protein is isolated from the plasmid pBAD-ST-FKBP (Covalys) by PstI-AscI digestion, and transferred into a cell expression plasmid pSEMXT-26 (covalys).

The ligation is made by T4 ligase (Invitrogen). The ligation product is transformed into chemiocompetent bacteria Turbo cells (GenetherapySystem). The transformed bacteria are plated on LB-Agar medium (Sigma) + 0.1 mg / ml of ampicillin (Eurogentec) allowing the selection of the bacteria possessing the plasmid.

The plasmid DNA is obtained by column purification (QIAGEN). ). The integrity of the sequence is verified by sequencing.

The Cos-7 cells are transiently transfected with the two plasmid constructs (Halo-Tag-FRB and SNAP-Tag-FKBP) with lipofectamine 2000 or by 96-well plate electroporation at a cell concentration of 10,000 cells per well. .

24 h or 48 h after the transient transfection the COS-7 cells are incubated for 1 hour with cell medium containing 5 μM of each specific substrate of the enzymes HaloTag and SnapTag, each of the substrates being covalently bound to a member of a couple FRET partners and a pattern allowing it to cross the plasma membrane (manufactured for example according to the diagram of Figures 7 and 8).

After 3 washes in KREBS + 0.05% tween-20, the fluorescence is measured on a RubyStar type reader (BMG) before and after induction of the protein interaction with 100 nM of rapamycin (Calbiochem): the TR-FRET signal is significantly more elevated after incubation with rapamycin.

This example shows that the method according to the invention makes it possible to demonstrate a biological interaction in a living cell by means of the TR-FRET technique.

Claims

A method for detecting interactions between bio-molecules, translocation or conformational change of bio-molecules in living cells, comprising the steps of:

1) to mark, in a living cell, a first bio-molecule with a first fluorescent compound having a long fluorescence lifetime;

2) marking, in said living cell, at least one second biomolecule with a second fluorescent compound;

3) subjecting the living cells to a specific stimulation adapted to the biological response to be studied, in the presence or absence of a compound belonging to a library of molecules to be tested;

4) subjecting living cells to a light source having a wavelength for exciting said first long-lived fluorescent compound;

5) measuring the intensity of the fluorescence emitted by said first and second fluorescent compounds, calculating the ratio between the fluorescence intensities of said first fluorescent compound and said second fluorescent compound, or measuring the lifetime of the first or second fluorescent compound; and

6) compare the measured signals with those obtained before stimulation of the cells; the first and at least second fluorescent compounds being partners of TR-FRCT.

2. Method according to claim 1, characterized in that a change in the conformation of a bio-molecule is studied and in that the fluorescent compounds are coupled to the same bio-molecule.

3. Method according to claims 1 or 2, characterized in that the labeling of the biomolecules by the fluorescent compounds is carried out using a technique chosen from the following: covalent labeling by the action of a suicide enzyme, labeling covalent by protein splicing, expression labeling of a fusion protein comprising the bio-molecule and a fluorescent protein as a compound fluorescent, high affinity non-covalent labeling via binding partners.

4. Method according to claim 1 or 2, characterized in that (e) fluorescent / bio-moiécuie compound labeling is carried out by coupling the fluorescent compound and the bio-molecule, respectively with the members of the pairs chosen from: a SNAP- Tag / the enzyme SNAP-Tag, a HALO-Tag substrate / the HALO-Tag enzyme, an intein part such as the intein of Ssp DnaE / the complementary intein part allowing to reconstitute a functional intein , a bi-arsenic unit / the Cys-Cys-XX-Cys-Cys sequence, X representing any amino acid, a metal ion / a poly-histidine sequence, biotin / Ja streptavidin, streptavidin / biotin, bungarotoxin BTX tag, cadaverine / PKPQQFM protein sequence, aziridin / TCGA nucleic acid sequence

5. Method according to any one of the preceding claims, characterized in that at least one of the fluorescent compounds comprises a pattern enabling it to cross the plasma membrane.

6. Method according to claim 5 characterized in that the pattern allowing the fluorescent compound to cross the plasma membrane is selected from the following units: ester such as pivaloyl-oxymethyl ester, acetoxymethyl ester, or glycol esters; viral peptides supported by membrane transporters such as penetratin and its analogs, transanan and its analogues, polyarginine groups, peptoids bearing guanidine groups; nonhydrolyzable derivatives with tetraguanidinium units; cholesterol groups, vitamin E or aliphatic chains, such as undecyl or 1,2-di-O-hexadecyl-glycerol chains.

7. Method according to any one of the preceding claims, characterized in that the lifetime of the first fluorescent compound is greater than 100 nanoseconds.

8. Method according to claim 7, characterized in that the first fluorescent compound is selected from chelates or cryptates of rare earths.

9. Process according to claim 7, characterized in that the first fluorescent compound is a rare earth cryptate comprising a pyridine unit.

10. Process according to claims 7 to 9, characterized in that the rare earth is terbium or europium.

11. Process according to any one of Claims 1 to 10, characterized in that that the second fluorescent compound is chosen from the following compounds: fluorescent proteins such as green fluorescent protein (GFP) and its derivatives (in particular CFP, YFP), fluorescent proteins extracted from corals, phycobiliproteins, such as B-phycoerythrin , R-phycoerythrin, C-phycocyanin, allophycocyanines, in particular those known under the name XL665 or organic fluorescent compounds having a lifetime of less than 100 nanoseconds, such as cyanines, rhodamines, fluoresceins, squarenes and the fluorescent molecules known under the name BODIPY's (difluorobora-diaza indacenes), the compounds known under the name AlexaFluor.

12. The method of claim 11, characterized in that the second fluorescent compound is an organic fluorescent compound having a pattern allowing it to cross the plasma membrane.

13. Necessary of components (kit of parts), including;

a first fluorescent compound and at least one second fluorescent compound, these compounds being partners of TR-FRET and at least one of these compounds having a motif enabling it to cross the plasma membrane;

live cells comprising said bio-molecules;

means for labeling the biomolecules with said fluorescent compounds;

instructions for studying phenomena of interactions between bio-molecules, translocation or conformational change of bio-molecules in living cells.

14. Required components according to claim 13, characterized in that the means for labeling the biomolecules by the fluorescent compounds consist in coupling the fluorescent compound and the bio-molecule respectively with the members of the pairs chosen from: a substrate of SNAP-Tag / the enzyme SNAP-Tag, a substrate of HALO-Tag / the HALO-Tag enzyme, a part of intein such as the intein of Ssp DnaE / the part of complementary ntaine allowing to reconstitute a functional intein, a bi-arsenic unit / the Cys-Cys-XX-Cys-Cys sequence, X representing any amino acid, a metal ion / a poly-histidine sequence, biotin / streptavidin, streptavidin / biotin, bungarotoxin / BTX tag, cadaverine / PKPQQFM protein sequence, aziridine / TCGA nucleotide sequence, enzyme dihydrofolate reductase / trimethoprim.

15. A kit of components according to claim 13, characterized in that the pattern allowing at least one of the fluorescent compounds to cross the plasma membrane is chosen from the following: ester such as pivaloyi-oxymethyl ester, acetoxymethyl ester, or esters glycolic; viral peptides supported by membrane transporters such as penetratin and its analogues, transanan and its analogues, polyarginine groups, peptoids bearing guanidine groups; nonhydrolyzable derivatives with tetraguanidintum units of cholesterol groups, vitamin E or aliphatic chains such as undecyl or 1,2-di-O-hexadecylglycerol chains.

16. A kit of components according to claim 13, characterized in that the cells are genetically modified so as to express the bio-molecules to be studied, or fusion proteins comprising the molecules to be studied.

17. Component kit according to claim 13, characterized in that it comprises a long-lived fluorescent compound and at least one other fluorescent compound selected from the following: fluorescent proteins, such as green fluorescent protein (GFP) and its derivatives (especially CFP, YFP), or fluorescent compounds having a lifetime of less than 100 nanoseconds, such as cyanines, rhodamines, fluoresceins, squarenes and fluorescent molecules known as BODIPY's (difluoroboran). diaza indacenes), the compounds known under the name AlexaFluor, the fluorescent proteins extracted from corals, phycobiliproteins, such as β-phycoerythrin, R-phycoerythrin, C-phycocyanin, allophycocyanines, in particular those known under the name XL665. .

18. Kit according to claim 17, characterized in that the long-lived fluorescent compound has a lifetime greater than 100 ns.

19. Kit according to claim 18, characterized in that the long-lived fluorescent compound is a rare earth chelate or a rare earth cryptate.

20. Kit according to claim 18, characterized in that the long-lived fluorescent compound is a rare earth chelate or a terbium or europium cryptate.