SYSTEM AND USES THEREOF

FIELD OF INVENTION The present invention pertains to the field of protein detection, notably detection of protein-protein interactions through fluorescent labeling. In particular, the present invention relates to a complementation system comprising two fragments of photoactive yellow protein (PYP), and its use with a fluorogen for detecting interactions between biological molecules of interest, in particular proteins of interest.

BACKGROUND OF INVENTION

Interactions between proteins play an essential role in metabolic and signaling pathways, cellular processes, and organismal systems. Because of their central role in biological function, protein interactions control mechanisms leading to healthy and diseased states in organisms. Diseases often result from mutations affecting the binding interface or leading to dysfunctional allosteric changes in proteins. Deciphering protein interaction networks can thus allow understanding of the molecular basis of diseases, which in turn can lead to methods for prevention, diagnosis, and treatment.

For deciphering protein interaction networks, not only is it essential to monitor protein- protein interactions in living cells, but, in addition, the subcellular localization and timing of interactions are important parameters. Fluorescent reporters able to report on protein- protein interactions in space and time are thus of utmost importance for (i) the monitoring of protein-protein interactions in living cells, (ii) the dissection of complex interaction networks, and (iii) the development of high-throughput screening of inhibitors or stabilizers of protein-protein interactions for drug development and biological studies.

The visualization of protein-protein interactions in living cells is in general achieved by Forster resonance energy transfer (FRET) also referred to as fluorescence resonance energy transfer (Piston DW & Kremers GJ, Trends Biochem Sci. 2007 Sep;32(9):407-14), or by bimolecular fluorescence complementation (BiFC) (Kerppola TK, Annu Rev Biophys. 2008;37:465-87).

Visualizing protein-protein interactions by FRET necessitates the fusion of two interacting proteins (often called the bait and prey proteins) to two fluorescent proteins (also referred to as fluorophores), acting respectively as FRET donor and FRET acceptor. The FRET donor is able to transfer its excitation energy to the FRET acceptor if the latter is close enough. FRET thus allows the real-time monitoring of complex association and dissociation through the monitoring of how the FRET efficiency changes over time. However, FRET is difficult to implement. It often requires high expression level of the bait and prey proteins to detect energy transfer between the FRET donor and the FRET acceptor. Moreover, structural information is necessary to place the two fluorophores within a distance where energy transfer is efficient. FRET requires also that a large fraction of bait and prey proteins interact to produce a sufficient change in the donor and acceptor fluorescence intensities. In addition, precise quantification of FRET-based interaction is ideally achieved by dedicated systems. Finally, numerous controls and high quantitative accuracy are required to exclude alternative interpretations.

Bimolecular fluorescence complementation (BiFC)-based assays are often preferred to FRET because they are easy to implement, straightforward to interpret and less sensitive to the relative levels of the two interacting proteins. In BiFC assays, the bait and prey proteins are fused to two complementary fragments of a fluorescent protein (FP), which assemble into a functional reporter (so-called split-FP) if the bait and prey proteins do interact. As the two complementary fragments are not fluorescent when taken separately, high contrast is obtained no matter the relative proportion of the two interacting proteins. However, monitoring protein-protein interactions with BiFC has posed its own challenges. First, spontaneous self-assembly of the two complementary fragments can generate unspecific fluorescence background. Furthermore, for BiFC based on proteins of the GFP family, complementation is followed by chromophore maturation, which results in irreversible complex formation (Magliery TJ et al., J Am Chem Soc. 2005 Jan 12; 127(1): 146-57). With BiFC based on phytochrome-based infrared proteins, the attachment of the biliverdin chromophore is slow and often also results in irreversibility (Filonov GS & Verkhusha VV, Chem Biol. 2013 Aug 22;20(8): 1078-86). The slow formation of fluorescent complexes prevents the monitoring of transient protein-protein interactions and the performance of dynamic studies involving active and inactive states, and may induce dominant negative or positive effects. There is thus a need for an improved fluorescence-based complementation system providing a dynamic system to detect protein-protein interactions. In particular, there is a need for an improved fluorescence-based complementation system characterized by a low self-assembly, to enhance contrast, and by a reversible complex formation, to detect the dissociation of the studied proteins and prevent adverse effect in the studied cells. Patent application W02016001437 and Plamont, et al. (Plamont et al., P Natl Acad Sci Usa 2016, 113 (3), 497-502) disclose new peptide tags derived from the photoactive yellow protein (PYP). In particular, W02016001437 and Plamont, et al. (Plamont et al., P Natl Acad Sci Usa 2016, 113 (3), 497-502) disclose the yellow fluorescence-activating and absorption-shifting tag (Y-FAST, hereafter“FAST”), which is a fluorogen-based fluorescent reporter developed by the Applicants. FAST is a 14-kDa protein tag derived from the photoactive yellow protein (PYP) which can form complexes with various fluorogens. Detection with FAST relies on two spectroscopic changes for fluorogen activation: increase of fluorescence quantum yield and absorption red-shift. The absorption red-shift undergone by the fluorogen upon binding with FAST ensures higher imaging selectivity and contrast, as unbound or unspecifically bound fluorogen can be discriminated via the choice of the excitation wavelength. In particular, FAST binds fluorogenic hydroxybenzylidene rhodanine (HBR) analogs displaying various spectral properties, such as for example, HMBR (which provides green-yellow fluorescence) and HBR-3,5-DOM (which provides orange-red fluorescence). Fluorogenic HBR analogs are weakly fluorescent in solution but strongly fluoresce when immobilized in the binding cavity of FAST.

Here, the Applicants developed a PYP -based fluorescence complementation system, wherein two PYP fragments, or truncated fragments thereof, assemble reversibly into a functional reporter, or a functional truncated fragment thereof, that binds a fluorogen and thus turns on its fluorescence if the protein(s) of interest to which the PYP fragments are bound do interact. In particular, the Applicants showed that a PYP -based fluorescence complementation system (split-FAST) comprising two fragments of the FAST protein tag, or truncated fragments thereof, displays a low unspecific fluorescence background due to a low self-assembly of the two FAST fragments. The Applicants also demonstrated that split-FAST enables the detection of both the association and dissociation of proteins in cells with high resolution in space and time. The Applicants further demonstrated that split-FAST enables the development of protein and cell-based sensors. The Applicants also developed PYP-based fluorescence complementation systems comprising PYP fragments derived from orthologs of FAST with properties comparable to that of the split-FAST complementation system.

The present invention thus relates to a complementation system comprising a first photoactive yellow protein (PYP) fragment, or a truncated fragment thereof, and a second photoactive yellow protein (PYP) fragment, or a truncated fragment thereof, wherein the first and second PYP fragments are able to reconstitute a functional PYP, or a functional truncated fragment thereof, that binds a fluorogenic chromophore, in particular a fluorogenic hy droxyb enzy li dene rhodanine (HBR) analog, reversibly. The present invention also relates to methods for detecting an interaction between biological molecules of interest, in particular proteins of interest, and assays relying on the detection of the interaction between two proteins in a sample.

SUMMARY

The present invention relates to a complementation system comprising a first photoactive yellow protein (PYP) fragment and a second photoactive yellow protein (PYP) fragment, wherein:

- the first PYP fragment comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 23, or a truncated fragment thereof, or an amino acid sequence having at least about 70% identity with the amino acid sequence as set forth in SEQ ID NO: 23, or a truncated fragment thereof, wherein said truncated fragment comprises at least 89 consecutive amino acids from the C -terminal end of the amino acid sequence as set forth in SEQ ID NO: 23 or of an amino acid sequence having at least about 70% identity with SEQ ID NO: 23; and the second PYP fragment comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 34, or a truncated fragment thereof, or an amino acid sequence having at least about 70% identity with the amino acid sequence as set forth in SEQ ID NO: 34, or a truncated fragment thereof, wherein said truncated fragment comprises at least 8 consecutive amino acids, preferably from the N-terminal end, of the amino acid sequence as set forth in SEQ ID NO: 34 or of an amino acid sequence having at least about 70% identity with SEQ ID NO: 34. In one embodiment, the first PYP fragment comprises or consists of an amino acid sequence selected from the group comprising or consisting of the amino acid sequences as set forth in SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29, and truncated fragments thereof comprising at least 89 consecutive amino acids from the C -terminal end of said amino acid sequences. In one embodiment, the second PYP fragment comprises or consists of an amino acid sequence selected from the group comprising or consisting of the amino acid sequences as set forth in SEQ ID NO:34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39 and SEQ ID NO: 40, and truncated fragments thereof comprising at least 8 consecutive amino acids, preferably from the N-terminal end, of said amino acid sequences.

In one embodiment,

the first PYP fragment comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 or SEQ ID NO: 29, or a truncated fragment thereof comprising at least 89 consecutive amino acids from the C -terminal end of said amino acid sequences; and

the second PYP fragment comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39 or SEQ ID NO: 40, respectively, or a truncated fragment thereof comprising at least 8 consecutive amino acids, preferably from the N-terminal end, of said amino acid sequences.

Thus, in one embodiment,

the first PYP fragment comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 23, or a truncated fragment thereof comprising at least

89 consecutive amino acids from the C -terminal end of said amino acid sequence and the second PYP fragment comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 34, or a truncated fragment thereof comprising at least 8 consecutive amino acids, preferably from the N-terminal end, of said amino acid sequence; or

the first PYP fragment comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 24, or a truncated fragment thereof comprising at least 89 consecutive amino acids from the C -terminal end of said amino acid sequence and the second PYP fragment comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 35, or a truncated fragment thereof comprising at least

8 consecutive amino acids, preferably from the N-terminal end, of said amino acid sequence; or

the first PYP fragment comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 25, or a truncated fragment thereof comprising at least 89 consecutive amino acids from the C -terminal end of said amino acid sequence and the second PYP fragment comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 36, or a truncated fragment thereof comprising at least 8 consecutive amino acids, preferably from the N-terminal end, of said amino acid sequence; or

- the first PYP fragment comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 26, or a truncated fragment thereof comprising at least 89 consecutive amino acids from the C -terminal end of said amino acid sequence and the second PYP fragment comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 37, or a truncated fragment thereof comprising at least 8 consecutive amino acids, preferably from the N-terminal end, of said amino acid sequence; or the first PYP fragment comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 27, or a truncated fragment thereof comprising at least 89 consecutive amino acids from the C -terminal end of said amino acid sequence and the second PYP fragment comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 38, or a truncated fragment thereof comprising at least

8 consecutive amino acids, preferably from the N-terminal end, of said amino acid sequence; or

the first PYP fragment comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 28, or a truncated fragment thereof comprising at least 89 consecutive amino acids from the C -terminal end of said amino acid sequence and the second PYP fragment comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 39, or a truncated fragment thereof comprising at least 8 consecutive amino acids, preferably from the N-terminal end, of said amino acid sequence; or

the first PYP fragment comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 29, or a truncated fragment thereof comprising at least 89 consecutive amino acids from the C -terminal end of said amino acid sequence and the second PYP fragment comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 40, or a truncated fragment thereof comprising at least 8 consecutive amino acids, preferably from the N-terminal end, of said amino acid sequence.

In one embodiment, the first PYP fragment comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 23, or a truncated fragment thereof comprising at least 89 consecutive amino acids from the C -terminal end of said amino acid sequence and the second PYP fragment comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 34, SEQ ID NO: 42, SEQ ID NO: 43, or SEQ ID NO: 44.

In one embodiment, the amino acid sequence of the first PYP fragment, or a truncated fragment thereof comprising at least 89 consecutive amino acids from the C -terminal end of said amino acid sequence, further comprises at least one of the following amino acid substitutions with reference to SEQ ID NO: 23: an asparagine at position 19, a leucine at position 62, a cysteine or a glutamic acid at position 68, an arginine at position 71, a serine at position 73, and/or an isoleucine a position 107; and/or wherein the amino acid sequence of the second PYP fragment, or a truncated fragment thereof comprising at least 8 consecutive amino acids of said amino acid sequence, further comprises the following amino acid substitution with reference to SEQ ID NO: 34: an isoleucine at position 8.

The present invention also relates to a kit comprising at least one vector comprising: a first nucleic acid sequence encoding the first photoactive yellow protein (PYP) fragment, or a truncated fragment thereof, as defined hereinabove; and a second nucleic acid sequence encoding the second photoactive yellow protein (PYP) fragment, or a truncated fragment thereof, as defined hereinabove.

In one embodiment, the kit of the invention comprises:

a first vector comprising the nucleic acid sequence encoding the first photoactive yellow protein (PYP) fragment, or a truncated fragment thereof, as defined hereinabove; and

a second vector comprising the nucleic acid sequence encoding the second photoactive yellow protein (PYP) fragment, or a truncated fragment thereof, as defined hereinabove.

According to one embodiment, the complementation system of the invention and the kit of the invention further comprise a fluorogenic hydroxybenzylidene rhodanine (HBR) analog of formula (I):

f ormula (I) wherein

Rl, R2, R5 and R6 may be identical or different and each represents H, halo, hydroxyl, aryl, alkyl, cycloalkyl, heteroalkyl or heterocycloalkyl group, saturated or unsaturated, linear or branched, optionally substituted by at least one group selected from halo, hydroxyl, oxo, nitro, ami do, carboxy, amino, cyano, haloalkoxy, haloalkyl;

R3 represents a non-binding doublet ( i.e ., a free pair of electrons) or H, halo, hydroxyl, aryl, alkyl, cycloalkyl, heteroalkyl or heterocycloalkyl group, saturated or unsaturated, linear or branched, optionally substituted by at least one group selected from halo, hydroxyl, oxo, nitro, ami do, carboxy, amino, cyano, haloalkoxy, haloalkyl;

R4 is a single or a double bound, interrupted or terminated by S, O or N atom, optionally substituted by at least one group selected from H, hydroxyl, aryl, alkyl, cycloalkyl, heteroalkyl or heterocycloalkyl group, saturated or unsaturated, linear or branched, optionally substituted by at least one group selected from halo, hydroxyl, oxo, nitro, ami do, carboxy, amino, cyano, haloalkoxy, haloalkyl;

X is OH, SH, NHR7, or N(R7)2, wherein R7 is H, halo, hydroxyl, aryl, alkyl, cycloalkyl, heteroalkyl or heterocycloalkyl group, saturated or unsaturated, linear or branched, optionally substituted by at least one group selected from halo, hydroxyl, oxo, nitro, ami do, carboxy, amino, cyano, haloalkoxy, haloalkyl; and

Y is O, NH or S.

In one embodiment, the fluorogenic HBR analog is selected from the group comprising or consisting of 4-hydroxy-3-methylbenzylidene rhodanine (HMBR), (Z)-2-(5-(4- hydroxy-3-methoxybenzylidene)-4-oxo-2-thioxothiazolidin-3-yl )acetic acid (HBR- 30M), (Z)-2-(5-(4-hydroxy-3, 5-dimethylbenzylidene)-4-oxo-2-thioxothiazolidin-3-yl) acetic acid (HBR-3,5DM) and (Z)-2-(5-(4-hydroxy-3, 5-dimethoxybenzylidene)-4-oxo- 2-thioxothiazolidin-3-yl) acetic acid (HBR-3,5DOM).

The present invention also relates to a method for detecting an interaction between two biological molecules of interest in a sample, preferably two proteins of interest, comprising the steps of: fusing a first photoactive yellow protein (PYP) fragment, or a truncated fragment thereof, as defined hereinabove, to a first biological molecule of interest, thereby tagging the first biological molecule of interest with said first PYP fragment; fusing a second photoactive yellow protein (PYP) fragment, or a truncated fragment thereof, as defined hereinabove, to a second biological molecule of interest, thereby tagging the second biological molecule of interest with said second PYP fragment;

contacting the sample with a fluorogenic hydroxybenzylidene rhodanine (HBR) analog; and

- detecting a fluorescence resulting from the binding of the fluorogenic HBR analog to the functional PYP, or a functional truncated fragment thereof, reconstituted upon interaction of the two biological molecules of interest;

thereby detecting the interaction of the two biological molecules of interest present in the sample, through the binding of the fluorogenic HBR analog to the functional PYP, or a functional truncated fragment thereof, reconstituted upon interaction of the two biological molecules of interest.

In one embodiment, the method of the invention is for monitoring over time and/or space the association and dissociation of the two biological molecules of interest, preferably of the two proteins of interest, through the detection of the interaction between said biological molecules of interest.

The present invention also relates to a screening method for identifying a new protein-protein interaction between two protein candidates of interest in a sample, comprising the steps of:

fusing a first photoactive yellow protein (PYP) fragment, or a truncated fragment thereof, as defined hereinabove, to a first protein candidate of interest, thereby tagging the first protein candidate of interest with said first PYP fragment;

fusing a second photoactive yellow protein (PYP) fragment, or a truncated fragment thereof, as defined hereinabove, to a second protein candidate of interest, thereby tagging the second protein candidate of interest with said second PYP fragment; contacting the sample with a fluorogenic hydroxybenzylidene rhodanine (HBR) analog; and

detecting a fluorescence resulting from the binding of the fluorogenic HBR analog to the functional PYP, or a functional truncated fragment thereof, reconstituted upon interaction of the two protein candidates of interest;

thereby identifying a new protein-protein interaction between the two protein candidates of interest present in the sample, through the binding of the fluorogenic HBR analog to the functional PYP, or a functional truncated fragment thereof, reconstituted upon interaction of the two protein candidates of interest. The present invention also relates to an assay relying on the detection of the interaction between two proteins in a sample, said assay comprising the steps of:

obtaining a first tagged protein, wherein the protein is tagged with a first photoactive yellow protein (PYP) fragment, or a truncated fragment thereof, as defined hereinabove;

- obtaining a second tagged protein, wherein the protein is tagged with a second photoactive yellow protein (PYP) fragment, or a truncated fragment thereof, as defined hereinabove;

contacting the sample with a fluorogenic hydroxybenzylidene rhodanine (HBR) analog; and

- detecting a fluorescence resulting from the binding of the fluorogenic HBR analog to the functional PYP, or a functional truncated fragment thereof, reconstituted upon interaction of the two proteins;

thereby detecting the interaction of the two proteins present in the sample, through the binding of the fluorogenic HBR analog to the functional PYP, or a functional truncated fragment thereof, reconstituted upon interaction of the two proteins.

In one embodiment, the assay of the invention is for assessing the capacity of a molecule of interest to stabilize or to inhibit protein-protein interactions. In one embodiment, the assay of the invention is for assessing a signaling pathway of interest, with the interaction of the two proteins depending on the activation of the signaling pathway of interest; or is for assessing the capacity of a molecule of interest to modulate said signaling pathway of interest.

DEFINITIONS

As used herein, the term“about” preceding a figure means plus or minus 10%, or less, of the value of said figure. It is to be understood that the value to which the term“about” refers is itself also specifically, and preferably, disclosed.

As used herein, the term“alkoxy” refers to any O-alkyl group. Suitable alkoxy groups include ethoxy and m ethoxy.

As used herein, the term“alkyl” refers to a hydrocarbyl radical of formula Cnkhn+i wherein n is a number greater than or equal to 1. Generally, alkyl groups of this invention comprise from 1 to 12 carbon atoms, preferably from 1 to 6 carbon atoms. Alkyl groups may be linear or branched and may be substituted as indicated herein. Suitable alkyl groups include methyl, ethyl, propyl (n-propyl, i-propyl), butyl (n-butyl, i-butyl, s-butyl and t-butyl), pentyl and its isomers (e.g., n-pentyl, iso-pentyl), and hexyl and its isomers (e.g., n-hexyl, iso-hexyl).

As used herein, the term“amido” refers to the -NR-CO- function wherein R may be -H or an alkyl group.

As used herein, the term“amino” refers to a -NH2 group or any group derived thereof by substitution of one or two hydrogen atom(s) by an organic aliphatic or aromatic group. Preferably, groups derived from - NH2 are alkylamino groups, i.e., N-alkyl groups, comprising monoalkylamino and dialkylamino. According to a specific embodiment, the term "amino" refers to NH2, NHMe or NMe2.

As used herein, the term“amino acid” refers to both natural and synthetic amino acids, and both D- and L-amino acids. They are represented by their full name, their three-letter code or their one-letter code as well-known in the art. Amino acid residues in peptides are thus abbreviated as follows: phenylalanine is Phe or F; leucine is Leu or L; isoleucine is He or I; methionine is Met or M; valine is Val or V; serine is Ser or S; proline is Pro or P; threonine is Thr or T; alanine is Ala or A; tyrosine is Tyr or Y; histidine is His or H; glutamine is Gin or Q; asparagine is Asn or N; lysine is Lys or K; aspartic acid is Asp or D; glutamic acid is Glu or E; cysteine is Cys or C; tryptophan is Trp or W; arginine is Arg or R; and glycine is Gly or G.“Standard amino acid” or“naturally occurring amino acid” means any of the twenty standard L-amino acids commonly found in naturally occurring peptides.“Non-standard amino acid” means any amino acid, other than the standard amino acids, regardless of whether it is prepared synthetically or derived from a natural source. For example, naphtlylalanine can be substituted for tryptophan to facilitate synthesis. Other synthetic amino acids that can be substituted include, but are not limited to, L-hydroxypropyl, L-3,4-dihydroxyphenylalanyl, alpha-amino acids such as L-alpha- hydroxylysyl and D-alpha-methylalanyl, L-alpha-methylalanyl, beta-amino acids, and isoquinolyl. The PYP fragments of the invention may comprise standard amino acids or non-standard amino acids. The term“amino acid” also encompasses chemically modified amino acids, including, but not limited to, salts, amino acid derivatives (such as amides), and substitutions. Amino acids contained within the polypeptides of the present invention ( i.e ., PYP fragments and fusion proteins of the present invention), and particularly at the carboxy- or amino-terminus, can thus be modified by methylation, amidation, acetylation or substitution with other chemical groups which can change the circulating half-life of the polypeptides without adversely affecting their activity. Additionally, a disulfide linkage may be present or absent in the polypeptides of the invention. Other polypeptide mimetics encompassed herein include polypeptides of the present invention (i.e., PYP fragments and fusion proteins of the present invention) having the following modifications: i) polypeptides wherein one or more of the peptidyl -C(0)NR- linkages (bonds) have been replaced by a non-peptidyl linkage such as a -CFE-carbamate linkage (-CH20C(0)NR-), a phosphonate linkage, a -CFE-sulfonamide (-CH2- S (0)2NR-) linkage, a urea (-NHC(O)NH-) linkage, a -CEE-secondary amine linkage, or with an alkylated peptidyl linkage (-C(O)NR-) wherein R is C1-C4 alkyl; ii) polypeptides wherein the N-terminus is derivatized to a -NRR ¹ group, to a -NRC(0)R group, to a -NRC(0)OR group, to a -NRS(0)2R group, to a -NHC(0)NHR group, where R and R ¹ are hydrogen or C1-C4 alkyl with the proviso that R and R ¹ are not both hydrogen; iii) polypeptides wherein the C terminus is derivatized to -C(0)R ² , where R ² is selected from the group consisting of C1-C4 alkoxy, and -NR ³ R ⁴ , where R ³ and R ⁴ are independently selected from the group consisting of hydrogen and C1-C4 alkyl. As used herein, the term“aryl” refers to a polyunsaturated, aromatic hydrocarbyl group having a single ring ( i.e ., phenyl) or multiple aromatic rings fused together (e.g., naphtyl) or linked covalently, typically containing 5 to 12 atoms, preferably 6 to 10, wherein at least one ring is aromatic. The aromatic ring may optionally include one to two additional ring(s) (either cycloalkyl, heterocyclyl or heteroaryl) fused thereto. Aryl is also intended to include the partially hydrogenated derivatives of the carbocyclic systems enumerated herein. Non-limiting examples of aryl comprise phenyl, biphenylyl, biphenylenyl, 5- or 6- tetralinyl, naphthalen-1- or -2-yl, 4-, 5-, 6 or 7-indenyl, 1- 2-, 3-, 4- or 5- acenaphtylenyl, 3-, 4- or 5-acenaphtenyl, 1- or 2-pentalenyl, 4- or 5-indanyl, 5-, 6- , 7- or 8 -tetrahy dronaphthy 1 , 1,2,3,4-tetrahydronaphthyl,

1 ,4-dihydronaphthyl, 1-, 2-, 3-, 4- or 5-pyrenyl.

As used herein, the term“carboxy” refers to the -COOH function, including -COO- and salts thereof.

As used herein, the term“cyano” refers to the -CºN function.

As used herein, the term“cycloalkyl” refers to a cyclic alkyl group, that is to say, a monovalent, saturated, or unsaturated hydrocarbyl group having 1 or 2 cyclic structure(s). Cycloalkyl includes monocyclic or bicyclic hydrocarbyl groups. Cycloalkyl groups may comprise 3 or more carbon atoms in the ring and generally, according to this invention, comprise from 3 to 10 carbon atoms, preferably from 3 to 8 carbon atoms, more preferably from 3 to 6 carbon atoms. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl.

As used herein the term“complementation system” refers to a system comprising at least two components, e.g., two fragments of a polypeptide or protein, that together form a structure having a property of interest, such as for instance the capacity to bind a fluorogenic chromophore, that each component taken separately, e.g., each polypeptide fragment, does not have. Accordingly, the term“complementation” refers to the reconstitution of the structure having the property of interest, such as for instance the capacity to bind a fluorogenic chromophore. For example, the term“complementation” refers to the reconstitution of a polypeptide (or protein) scaffold when the two fragments of said polypeptide (or protein) are in close proximity, the reconstituted polypeptide (or protein) scaffold then having the capacity to bind a fluorogenic chromophore.

As used herein, the term“functional PYP” refers to a photoactive yellow protein (PYP) able to bind to a fluorogenic HBR analog, preferably reversibly. Accordingly, as used herein, the term“functional truncated fragment of PYP” refers to a truncated fragment of photoactive yellow protein (PYP) still able to bind to a fluorogenic HBR analog, preferably reversibly.

As used herein, the terms“fluorogenic chromophore” or“fluorogen” refer to a chromophore, the brightness of which can be significantly enhanced by an environmental change. A fluorogenic chromophore is substantially non-fluorescent in solution under its free form, but brightens up when placed into an environment constraining its conformation and excluding deexcitation of its excited state by non-radiative pathways. In one embodiment of the invention, the fluorogenic chromophore, e.g., the fluorogenic 4-hydroxybenzylidene-rhodanine (HBR) analog, is almost invisible in solution and becomes fluorescent upon binding of a protein scaffold, such as the one formed by the complementation of the PYP fragments of the invention.

As used herein, the term “fluorogenic HBR analog” refers to a fluorogenic 4-hydroxybenzylidene-rhodanine (HBR) analog (also referred to as fluorogenic hydroxybenzylidene-rhodanine (HBR) analog) which absorbs light at a specific frequency and is thus colored, and has the properties of a fluorogenic chromophore. In one embodiment, as described hereinafter, the fluorogenic HBR analog is a compound of formula (I).

As used herein, the term“halo” means fluoro, chloro, bromo or iodo. Preferred halo groups are fluoro and chloro.

As used herein, the term“haloalkyl” refers to any alkyl group substituted by one or more halo group(s). Examples of preferred haloalkyl groups are CF3, CHF2 and CH2F.

As used herein, the term“haloalkoxy” refers to any alkoxy group substituted by one or more halo group(s). As used herein, the term“heteroalkyl” refers to an alkyl group wherein at least one carbon atom is replaced by a heteroatom; preferably, said heteroatom is selected from N, S, P or O. In heteroalkyl groups, the heteroatoms are bound along the alkyl chain only to carbon atoms, i.e., each heteroatom is separated from any other heteroatom by at least one carbon atom. However, the nitrogen, sulfur and phosphorus heteroatoms may optionally be oxidized, and the nitrogen heteroatoms may optionally be quatemized. Heteroalkyl groups especially include alkoxy groups.

As used herein, the term“heterocycloalkyl” refers to a cycloalkyl group wherein at least one carbon atom is replaced by a heteroatom; preferably, said heteroatom is selected from N, S, P or O. In heterocycloalkyl groups, the heteroatoms are bound along the alkyl chain only to carbon atoms, i.e., each heteroatom is separated from any other heteroatom by at least one carbon atom. However, the nitrogen, sulfur and phosphorus heteroatoms may optionally be oxidized, and the nitrogen heteroatoms may optionally be quaternized.

As used herein, the term“hydroxyl” refers to the -OH function.

As used herein, the term“identity”, when used in a relationship between the sequences of two or more polypeptides or of two or more nucleic acid, refers to the degree of sequence relatedness between polypeptides or nucleic acids (respectively), as determined by the number of matches between strings of two or more amino acid residues or of two or more nucleotides, respectively.“Identity” measures the percent of identical matches between the smaller of two or more sequences with gap alignments (if any) addressed by a particular mathematical model or computer program (i.e.,“algorithms”). Identity of related polypeptides or nucleic acid sequences can be readily calculated by known methods. Such methods include, but are not limited to, those described in Arthur M. Lesk, Computational Molecular Biology: Sources and Methods for Sequence Analysis (New- York: Oxford University Press, 1988); Douglas W. Smith,

Biocomputing: Informatics and Genome Projects (New- York: Academic Press, 1993); Hugh G. Griffin and Annette M. Griffin, Computer Analysis of Sequence Data, Part 1 (New Jersey: Humana Press, 1994); Gunnar von Heinje, Sequence Analysis in Molecular Biology: Treasure Trove or Trivial Pursuit (Academic Press, 1987); Michael Gribskov and John Devereux, Sequence Analysis Primer (New York: M. Stockton Press, 1991); and Carillo et al., 1988. SIAM J. Appl. Math. 48(5): 1073-1082. Preferred methods for determining identity are designed to give the largest match between the sequences tested. Methods of determining identity are described in publicly available computer programs. Preferred computer program methods for determining identity between two sequences include the GCG program package, including GAP (Devereux et al., 1984. Nucl. Acid. Res. 12(1 Pt l):387-395; Genetics Computer Group, University of Wisconsin Biotechnology Center, Madison, WI), BLASTP, BLASTN, TBLASTN and FASTA (Altschul et al., 1990. J. Mol. Biol. 215(3):403-410). The BLASTX program is publicly available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul et al. NCB/NLM/NIH Bethesda, Md. 20894; Altschul et al., 1990. J. Mol. Biol. 215(3):403-410). The well-known Smith Waterman algorithm may also be used to determine identity.

As used herein, the term“nitro” refers to the -NO2 function.

As used herein the term“nucleic acid” refers to a polymer of nucleotides covalently linked by phosphodiester bonds, such as deoxyribonucleic acids (DNA) or ribonucleic acids (RNA), in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, single nucleotide polymorphisms (SNPs), and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al.,

Mol. Cell. Probes 8:91-98 (1994)). Unless otherwise specified, a nucleotide sequence encoding an amino acid sequence includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence that encodes a protein or a RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron(s). As used herein, the term“oxo” refers to the -C=0 function.

As used herein, the term“PYP fragment” refers to a peptide or polypeptide originating from a functional photoactive yellow protein PYP, but not comprising itself a functional photoactive yellow protein (PYP), the latter only being able to bind a fluorogenic HBR analog. In other words, as used herein, the term“PYP fragment” refers to a peptide or polypeptide originating from a functional photoactive yellow protein PYP but not able on its own to bind a fluorogenic HBR analog. Accordingly, not being able to bind a fluorogenic HBR analog on its own, a PYP fragment as described herein (e.g., a first PYP fragment or a second PYP fragment) cannot induce or generate fluorescence on its own. As used herein, the term“reporter protein” refers to a protein the interactions of which may be detected, localized or quantified as a way to indirectly assess a target of interest or a mechanism of interest.

As used herein, the term“sample” refers to a specimen or small quantity of material, in particular of biological material, generally solid or liquid.“Sample” may thus also refer to cells or tissues or organisms of interest.

DETAILED DESCRIPTION

The present invention relates to a complementation system comprising two photoactive yellow protein (PYP) fragments as described herein, a first PYP fragment and a second PYP fragment, or truncated fragments thereof.

According to the present invention, the complementation system comprises a first PYP fragment and a second PYP fragment, or truncated fragments thereof, wherein the first and second PYP fragments are able to associate with each other and thus to reconstitute a functional PYP, or a functional truncated fragment thereof, that binds a fluorogenic hydroxybenzylidene rhodanine (HBR) analog, preferably reversibly. As mentioned hereinabove, only a functional PYP or a functional truncated fragment thereof, such as a reconstituted functional PYP or a reconstituted functional fragment thereof, is able to bind a fluorogenic HBR analog. The first PYP fragment of the invention and the second PYP fragment of the invention, or truncated fragments thereof, are not able on their own to bind a fluorogenic HBR analog, and therefore are not able on their own to induce or generate fluorescence, which is emitted by a fluorogenic HBR analog upon binding to a functional PYP, or a functional truncated fragment thereof.

“Photoactive yellow protein” or“PYP” is a photoreceptor protein isolated, for instance, from purple photosynthetic bacteria Ectothiorhodospira halophila (Halorhodospira halophila). The wild-type PYP is a relatively small protein (14 kDa), which can bind p- coumaric acid, a chromophore, through a thioester covalent bond with the 69 ^th cysteine residue.

According to the present invention, the two PYP fragments or truncated fragments thereof, of the complementation system reconstitute the structure of a functional PYP, or a functional truncated fragment thereof, when they are in close proximity, with the functional PYP being capable of binding a fluorogenic hydroxybenzylidene-rhodanine (HBR) analog. As mentioned hereinabove, the two PYP fragments or truncated fragments thereof, of the complementation system of the invention are able to associate with each other when they are in close proximity.

In one embodiment, by close proximity it is meant a distance shorter than about 20 nm, preferably shorter than about 10 nm, and more preferably shorter than about 5 nm.

Thus, according to the present invention, the reconstituted functional PYP, or the reconstituted functional truncated fragment thereof, is able to bind a fluorogenic HBR analog. Preferably, in one embodiment, the reconstituted functional PYP, or the reconstituted functional truncated fragment thereof, is able to bind a fluorogenic HBR analog reversibly.

In the present invention, by reconstitution of a functional PYP (or in short reconstitution of a PYP), it is meant reconstitution of the structure of a functional PYP, or a functional truncated fragment thereof, allowing the binding of a fluorogenic hydroxybenzylidene-rhodanine (HBR) analog to the reconstituted functional PYP, or to the reconstituted functional truncated fragment thereof. Thus, according to the present invention, the reconstitution of a functional PYP, or a functional truncated fragment thereof, can be detected through the detection of the fluorescence emitted by a fluorogenic hydroxybenzylidene-rhodanine (HBR) analog upon binding to the reconstituted functional PYP or to the reconstituted functional truncated fragment thereof.

In one embodiment, the functional PYP derives from an amino acid sequence as set forth in SEQ ID NO: 1 or from an amino acid sequence having at least about 30%, 35%, 40%, 45%, 50%, 55%, 60% or 65% identity, preferably at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more identity with the amino acid sequence as set forth in SEQ ID NO: 1.

In one embodiment, the functional PYP derives from the PYP of bacteria species selected from the group comprising or consisting of Halorhodospira halophila (SEQ ID NO: 1), Halomonas boliviensis LC1 (SEQ ID NO: 2), Halomonas sp. GFAJ-1 (SEQ ID NO: 3), Rheinheimera sp. A13L (SEQ ID NO: 4), Idiomarina loihiensis (SEQ ID NO: 5), Thiorhodospira sibirica ATCC 700588 (SEQ ID NO: 6) and Rhodothalassium salexigens

(SEQ ID NO: 7).

According to the present invention, the functional PYP comprises a cysteine at position 69 by reference to the sequence as set forth in SEQ ID NO: 1 or at the corresponding position in the sequences as set forth in SEQ ID NO: 2-7, and one or more amino acid substitutions in the amino acid region from position 94 to position 101 with reference to SEQ ID NO: 1 or in the corresponding amino acid region in the sequences as set forth in SEQ ID NO: 2-7, one of said substitutions being a proline at position 97 with reference to SEQ ID NO: 1, or at the corresponding position in the sequences as set forth in SEQ ID NO: 2-7. In one embodiment, the functional PYP comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 8, or a functional truncated fragment thereof, or an amino acid sequence having at least about 30%, 35%, 40%, 45%, 50%, 55%, 60% or 65% identity, preferably at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more identity with the amino acid sequence as set forth in SEQ ID NO: 8 or a functional truncated fragment thereof, said amino acid sequence further comprising one or more amino acid substitutions in the amino acid region from position 94 to position 101 with reference to SEQ ID NO: 8, one of said substitution being a proline at position 97 with reference to SEQ ID NO: 8. In one embodiment, said functional PYP further comprises at least one, at least two or at least three amino acid substitution(s) in the amino acid region from position 94 to position 101 with reference to SEQ ID NO: 8, said amino acid substitution(s) being selected from the group comprising or consisting of a tryptophan at position 94; an isoleucine, valine or leucine at position 96; and a threonine at position 98. In one embodiment, said functional PYP further comprises the following amino acid substitutions in the amino acid region from position 94 to position 101 with reference to SEQ ID NO: 8: a tryptophan at position 94, a methionine at position 95, an isoleucine at position 96, a threonine at position 98, a serine at position 99, an arginine at position 100, and a glycine at position 101. In one embodiment, the functional PYP comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 9, or a functional truncated fragment thereof, or an amino acid sequence having at least about 30%, 35%, 40%, 45%, 50%, 55%, 60% or 65% identity, preferably at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more identity with the amino acid sequence as set forth in SEQ ID NO: 9 or a functional truncated fragment thereof.

In one embodiment, the functional PYP comprises or consists of an amino acid sequence as set forth in any one of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15, or a functional truncated fragment thereof. The amino acid sequences as set forth in any one of SEQ ID NO: 10-15 have at least 70% identity with the amino acid sequence as set forth in SEQ ID NO: 9.

In one embodiment, the functional PYP as described hereinabove further comprises at least one of the following amino acid substitutions at the positions defined with reference to SEQ ID NO: 9: an asparagine at position 19, a leucine at position 62, a cysteine or a glutamic acid at position 68, an arginine at position 71, a serine at position 73, an isoleucine at position 107, and/or an isoleucine at position 122.

In one embodiment, the functional PYP comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 8, or a functional truncated fragment thereof, further comprising one of the following combination of substitutions:

- Y94W, T95M, F96I, D97P, Y98T, Q99S, M100R and T101G (SEQ ID NO: 9);

- Y94W, T95M, F96I, D97P, Y98T, Q99S, M100R, T101G and V107I

(SEQ ID NO: 16);

- Y94W, T95M, F96I, D97P, Y98T, Q99S, M100R, T101G and V122I

(SEQ ID NO: 17);

- Y94W, T95M, F96I, D97P, Y98T, Q99S, M100R, T101G, V107I and V122I (SEQ ID NO: 18);

- F62L, P68C, D71R, P73S, Y94W, T95M, F96I, D97P, Y98T, Q99S, M100R and T101G (SEQ ID NO: 19);

- D19N, F62L, P68C, D71R, P73S, Y94W, F96I, D97P, Y98T, Q99K, M100R and T101G (SEQ ID NO: 20); or

- F62L, P68E, C69G, D71R, P73S, Y94W, F96I, D97P, Y98T, Q99K, M100R and T101G (SEQ ID NO: 21),

wherein the substitutions are defined using SEQ ID NO: 8 as a reference.

In one embodiment, the functional PYP comprises or consists of an amino acid sequence as set forth in any one of SEQ ID NO: 9, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21, or a functional truncated fragment thereof, or an amino acid sequence having at least about 30%, 35%, 40%, 45%, 50%, 55%, 60% or 65% identity, preferably at least about 70%, 75%, 80%,

85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more identity with an amino acid sequence selected from the group comprising or consisting of SEQ ID NO: 9; SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, or a functional truncated fragment thereof. In one embodiment, the functional PYP comprises or consists of an amino acid sequence as set forth in any one of SEQ ID NO: 9-21, or a functional truncated fragment thereof.

In one embodiment, the first PYP fragment of the invention comprises or consists of a N-terminal fragment of a functional PYP as described hereinabove ending at position 114 with reference to SEQ ID NO: 8, or a truncated fragment thereof.

In one embodiment, the first PYP fragment of the invention comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 22 or a truncated fragment thereof , or an amino acid sequence having at least about 30%, 35%, 40%, 45%, 50%, 55%, 60% or 65% identity, preferably at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more identity with the amino acid sequence as set forth in SEQ ID NO: 22, or a truncated fragment thereof, said amino acid sequence further comprising one or more amino acid substitutions in the amino acid region from position 94 to position 101 with reference to SEQ ID NO: 22, one of said substitution being a proline at position 97 with reference to SEQ ID NO: 22. In one embodiment, said first PYP fragment of the invention, or a truncated fragment thereof, further comprises at least one, at least two or at least three amino acid substitution(s) in the amino acid region from position 94 to position 101 with reference to SEQ ID NO: 22, said amino acid substitution(s) being selected from the group comprising or consisting of a tryptophan at position 94; an isoleucine, valine or leucine at position 96; and a threonine at position 98.

In one embodiment, the first PYP fragment of the invention, or a truncated fragment thereof, as described hereinabove comprises the following amino acid substitutions in the amino acid region from position 94 to position 101 with reference to SEQ ID NO: 22: a tryptophan at position 94, a methionine at position 95, an isoleucine at position 96, a threonine at position 98, a serine at position 99, an arginine at position 100, and a glycine at position 101.

In one embodiment, the first PYP fragment of the invention comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 23

(MEHVAF GSEDIENTLAKMDDGQLDGLAF GAIQLDGDGNILQYNAAEGDITGR DPKQVIGKNFFKDVAPGTDSPEFYGKFKEGVASGNLNTMFEWMIPTSRGPTKV KVHMKKAL S) or a truncated fragment thereof, or an amino acid sequence having at least about 30%, 35%, 40%, 45%, 50%, 55%, 60% or 65% identity, preferably at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more identity with the amino acid sequence as set forth in SEQ ID NO: 23, or a truncated fragment thereof.

In one embodiment, the first PYP fragment of the invention thus comprises, or consists of, an amino acid sequence as set forth in any one of SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 or SEQ ID NO: 29, or a truncated fragment thereof.

The amino acid sequences as set forth in any one of SEQ ID NO: 24-29 have at least 70% identity with the amino acid sequence as set forth in SEQ ID NO: 23.

In one embodiment, the first PYP fragment of the invention, or a truncated fragment thereof, as described hereinabove further comprises at least one of the following amino acid substitutions at the positions defined with reference to SEQ ID NO: 23: an asparagine at position 19, a leucine at position 62, a cysteine or a glutamic acid at position 68, an arginine at position 71, a serine at position 73, and/or an isoleucine at position 107.

In one embodiment, the first PYP fragment of the invention, or a truncated fragment thereof, as described hereinabove further comprises the following amino acid substitution at the position defined with reference to SEQ ID NO: 23: an isoleucine at position 107.

In one embodiment, the first PYP fragment of the invention, or a truncated fragment thereof, as described hereinabove further comprises at least one of the following amino acid substitutions at the positions defined with reference to SEQ ID NO: 23: an asparagine at position 19, a leucine at position 62, a cysteine or a glutamic acid at position 68, an arginine at position 71, and/or a serine at position 73.

In one embodiment, the first PYP fragment of the invention, or a truncated fragment thereof, as described hereinabove further comprises at least one of the following amino acid substitutions at the positions defined with reference to SEQ ID NO: 23: a leucine at position 62, an arginine at position 71, and/or a serine at position 73.

In one embodiment, the first PYP fragment of the invention comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 22, or a truncated fragment thereof, further comprising at least one of the following combination of substitutions:

- Y94W, T95M, F96I, D97P, Y98T, Q99S, M100R and T101G (SEQ ID NO: 23);

- Y94W, T95M, F96I, D97P, Y98T, Q99S, M100R, T101G and V107I (SEQ ID NO: 30);

- F62L, P68C, D71R, P73S, Y94W, T95M, F96I, D97P, Y98T, Q99S, M100R and T101G (SEQ ID NO: 31);

- D19N, F62L, P68C, D71R, P73S, Y94W, F96I, D97P, Y98T, Q99K, M100R and T101G (SEQ ID NO: 32); or

- F62L, P68E, C69G, D71R, P73S, Y94W, F96I, D97P, Y98T, Q99K, M100R and T101G (SEQ ID NO: 33),

wherein the substitutions are defined using SEQ ID NO: 22 as a reference.

In one embodiment, the first PYP fragment of the invention comprises or consists of an amino acid sequence as set forth in any one of SEQ ID NO: 23, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, or SEQ ID NO: 33, or a truncated fragment thereof, or an amino acid sequence having at least about 30%, 35%, 40%, 45%, 50%, 55%, 60% or 65% identity, preferably at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,

94%, 95%, 96%, 97%, 98% or 99% or more identity with an amino acid sequence selected from the group comprising or consisting of SEQ ID NO: 23, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO: 33, or a truncated fragment thereof.

In one embodiment, the first PYP fragment of the invention comprises or consists of an amino acid sequence as set forth in any one of SEQ ID NO: 23-33, or a truncated fragment thereof.

In one embodiment, the truncated fragment of the first PYP fragment of the invention comprises the deletion of amino acids starting from the N-terminal end of the first PYP fragment as described hereinabove, preferably the deletion of a number of amino acids ranging from 1 amino acid to 50 amino acids, more preferably ranging from 1 amino acid to 40 amino acids, from 1 amino acid to 35 amino acids, from 1 amino acid to 30 amino acids, even more preferably from 1 amino acid to 25 amino acids.

In one embodiment, the truncated fragment of the first PYP fragment of the invention is truncated of the amino acids from position 1 to at most position 25 with reference to SEQ ID NO: 22 (or with reference to any other amino acid sequence of the same length, such as any one of SEQ ID NO: 23-33). Thus, in one embodiment, the truncated fragment of the first PYP fragment of the invention comprises at least 89 consecutive amino acids, from position 26 to position 114, of the first PYP fragment as described hereinabove. In other words, the truncated fragment of the first PYP fragment of the invention comprises at least 89 consecutive amino acids from the C -terminal end of the first PYP fragment as described hereinabove.

In one embodiment, the truncated fragment of the first PYP fragment of the invention is truncated of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acids from the N-terminal end (also referred to as N-ter) of the first PYP fragment as described hereinabove.

In one embodiment, the truncated fragment of the first PYP fragment of the invention comprises 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, or 113 consecutive amino acids from the C-terminal end of the first PYP fragment as described hereinabove.

In one embodiment, the second PYP fragment of the invention comprises or consist of the C-terminal fragment of a functional PYP as described hereinabove starting at position 115 with reference to SEQ ID NO: 8, or a truncated fragment thereof.

In one embodiment, the second PYP fragment of the invention thus comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 34 (GDSYWVFVKRV), or a truncated fragment thereof, or an amino acid sequence having at least about 30%, 35%, 40%, 45%, 50%, 55%, 60% or 65% identity, preferably at least about 70%, 75%, 80%, 85% or 90% or more identity with the amino acid sequence as set forth in SEQ ID NO: 34, or a truncated fragment thereof.

In one embodiment, the second PYP fragment of the invention comprises or consists of an amino acid sequence as set forth in any one of SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, or SEQ ID NO: 40, or a truncated fragment thereof.

The amino acid sequences as set forth in any one of SEQ ID NO: 35-40 have at least about 70% identity with the amino acid sequence as set forth in SEQ ID NO: 34.

In one embodiment, the second PYP fragment of the invention, or a truncated fragment thereof, as described hereinabove further comprises the following amino acid substitution with reference to SEQ ID NO: 34: an isoleucine at position 8.

In one embodiment, the second PYP fragment of the invention comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 41, or a truncated fragment thereof, or an amino acid sequence having at least about 30%, 35%, 40%, 45%, 50%, 55%, 60% or 65% identity, preferably at least about 70%, 75%, 80%, 85% or 90% or more identity with the amino acid sequence as set forth in SEQ ID NO:41, or a truncated fragment thereof.

In one embodiment, the second PYP fragment of the invention comprises or consists of an amino acid sequence as set forth in any one SEQ ID NO: 34-41, or a truncated fragment thereof.

In one embodiment, the truncated fragment of the second PYP fragment of the invention comprises at least 8 consecutive amino acids of the second PYP fragment as described hereinabove.

In one embodiment, the truncated fragment of the second PYP fragment of the invention comprises 8, 9 or 10 consecutive amino acids of the second PYP fragment as described hereinabove. In one embodiment, the truncated fragment of the second PYP fragment of the invention comprises the deletion of amino acids starting from the N-terminal end and/or the C -terminal end of the second PYP fragment as described hereinabove, preferably the deletion of 1, 2 or 3 amino acids. In one embodiment, the truncated fragment of the second PYP fragment of the invention comprises the deletion of amino acids starting from the N-terminal end of the second PYP fragment as described hereinabove, preferably the deletion of 1, 2 or 3 amino acids.

In one embodiment, the truncated fragment of the second PYP fragment of the invention comprises the deletion of amino acids starting from the C -terminal end of the second PYP fragment as described hereinabove, preferably the deletion of 1, 2 or 3 amino acids.

In one embodiment, the truncated fragment of the second PYP fragment of the invention comprises 8, 9 or 10 consecutive amino acids from the N-terminal end of the second PYP fragment as described hereinabove.

In one embodiment, the truncated fragment of the second PYP fragment of the invention comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 42 (GDSYWVFVKR), SEQ ID NO: 43 (GDSYWVFVK) or SEQ ID NO: 44 (GDSYWVFV), or an amino acid sequence having at least about 30%, 35%, 40%, 45%, 50%, 55%, 60% or 65% identity, preferably at least about 70%, 75%, 80%, 85% or 90% or more identity with the amino acid sequence as set forth in SEQ ID NO: 42, SEQ ID NO: 43 or SEQ ID NO: 44.

According to one embodiment, the complementation system of the invention comprises a first PYP fragment as described hereinabove, or a truncated fragment thereof as described hereinabove, and a second PYP fragment as described hereinabove, or a truncated fragment thereof as described hereinabove. In one embodiment, the complementation system of the invention comprises:

a first PYP fragment of the invention comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 23, or a truncated fragment thereof as described hereinabove, or an amino acid sequence having at least about 30%, 35%, 40%, 45%, 50%, 55%, 60% or 65% identity, preferably at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more identity with the amino acid sequence as set forth in SEQ ID NO: 23, or a truncated fragment thereof as described hereinabove; and

- a second PYP fragment of the invention comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 34, or a truncated fragment thereof, or an amino acid sequence having at least about 30%, 35%, 40%, 45%, 50%, 55%, 60% or 65% identity, preferably at least about 70%, 75%, 80%, 85% or 90% or more identity with the amino acid sequence as set forth in SEQ ID NO: 34, or a truncated fragment thereof as described hereinabove.

In said embodiment, the first and second PYP fragments, or truncated fragments thereof, are thus able to reconstitute a functional PYP comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 9, or a functional truncated fragment thereof, or an amino acid sequence having at least about 30%, 35%, 40%, 45%, 50%, 55%, 60% or 65% identity, preferably at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more identity with the amino acid sequence SEQ ID NO: 9, or a functional truncated fragment thereof.

In one embodiment, the complementation system of the invention comprises:

a first PYP fragment of the invention comprising or consisting of an amino acid sequence as set forth in any one of SEQ ID NO: 23, SEQ ID NO: 24,

SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 or SEQ ID NO: 29, or a truncated fragment thereof as described hereinabove; and a second PYP fragment of the invention comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 34, or a truncated fragment thereof, or an amino acid sequence having at least about 30%, 35%, 40%, 45%, 50%, 55%, 60% or 65% identity, preferably at least about 70%, 75%, 80%, 85% or 90% or more identity with the amino acid sequence as set forth in SEQ ID NO: 34, or a truncated fragment thereof as described hereinabove. In one embodiment, the complementation system of the invention comprises:

a first PYP fragment of the invention comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 23, or a truncated fragment thereof as described hereinabove, or an amino acid sequence having at least about 30%, 35%, 40%, 45%, 50%, 55%, 60% or 65% identity, preferably at least about 70%,

75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more identity with the amino acid sequence as set forth in SEQ ID NO: 23, or a truncated fragment thereof as described hereinabove; and

a second PYP fragment of the invention comprising or consisting of an amino acid sequence as set forth in any one of SEQ ID NO:34, SEQ ID NO: 35,

SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39 or SEQ ID NO: 40, or a truncated fragment thereof as described hereinabove.

In one embodiment, the complementation system of the invention comprises:

a first PYP fragment of the invention comprising or consisting of an amino acid sequence as set forth in any one of SEQ ID NO: 23, SEQ ID NO: 24,

SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 or SEQ ID NO: 29, or a truncated fragment thereof as described hereinabove; and a second PYP fragment of the invention comprising or consisting of an amino acid sequence as set forth in any one of SEQ ID NO:34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39 or

SEQ ID NO: 40, or a truncated fragment thereof as described hereinabove.

In one embodiment, the complementation system of the invention comprises:

a first PYP fragment of the invention comprising or consisting of an amino acid sequence as set forth in any one of SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 or

SEQ ID NO: 29, or a truncated fragment thereof as described hereinabove; and a second PYP fragment of the invention comprising or consisting of an amino acid sequence as set forth in any one of SEQ ID NO:34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39 or SEQ ID NO: 40, respectively, or a truncated fragment thereof as described hereinabove.

Thus, in one embodiment, the complementation system of the invention comprises: a first PYP fragment comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 23, or a truncated fragment thereof as described hereinabove and a second PYP fragment comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 34, or a truncated fragment thereof as described hereinabove;

a first PYP fragment comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 24, or a truncated fragment thereof as described hereinabove and a second PYP fragment comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 35, or a truncated fragment thereof as described hereinabove;

a first PYP fragment comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 25, or a truncated fragment thereof as described hereinabove and a second PYP fragment comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 36, or a truncated fragment thereof as described hereinabove;

a first PYP fragment comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 26, or a truncated fragment thereof as described hereinabove and a second PYP fragment comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 37, or a truncated fragment thereof as described hereinabove;

a first PYP fragment comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 27, or a truncated fragment thereof as described hereinabove and a second PYP fragment comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 38, or a truncated fragment thereof as described hereinabove;

a first PYP fragment comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 28, or a truncated fragment thereof as described hereinabove and a second PYP fragment comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 39, or a truncated fragment thereof as described hereinabove; or

a first PYP fragment comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 29, or a truncated fragment thereof as described hereinabove and a second PYP fragment comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 40, or a truncated fragment thereof as described hereinabove.

In one embodiment, the complementation system of the invention comprises:

a first PYP fragment of the invention comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 24 or a truncated fragment thereof as described hereinabove; and

a second PYP fragment of the invention comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 35, or a truncated fragment thereof as described hereinabove. In said embodiment, the first and second PYP fragments, or truncated fragments thereof, are thus able to reconstitute a functional PYP comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 10, or a functional truncated fragment thereof.

In one embodiment, the complementation system of the invention comprises:

a first PYP fragment of the invention comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 25 or a truncated fragment thereof as described hereinabove; and

a second PYP fragment of the invention comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 36, or a truncated fragment thereof as described hereinabove. In said embodiment, the first and second PYP fragments, or truncated fragments thereof, are thus able to reconstitute a functional PYP comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 11, or a functional truncated fragment thereof. In one embodiment, the complementation system of the invention comprises:

a first PYP fragment of the invention comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 26 or a truncated fragment thereof as described hereinabove; and

- a second PYP fragment of the invention comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 37, or a truncated fragment thereof as described hereinabove.

In said embodiment, the first and second PYP fragments, or truncated fragments thereof, are thus able to reconstitute a functional PYP comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 12, or a functional truncated fragment thereof.

In one embodiment, the complementation system of the invention comprises:

a first PYP fragment of the invention comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 27 or a truncated fragment thereof as described hereinabove; and

a second PYP fragment of the invention comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 38, or a truncated fragment thereof as described hereinabove.

In said embodiment, the first and second PYP fragments, or truncated fragments thereof, are thus able to reconstitute a functional PYP comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 13, or a functional truncated fragment thereof.

In one embodiment, the complementation system of the invention comprises:

a first PYP fragment of the invention comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 28 or a truncated fragment thereof as described hereinabove; and

- a second PYP fragment of the invention comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 39, or a truncated fragment thereof as described hereinabove. In said embodiment, the first and second PYP fragments, or truncated fragments thereof, are thus able to reconstitute a functional PYP comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 14, or a functional truncated fragment thereof.

In one embodiment, the complementation system of the invention comprises:

- a first PYP fragment of the invention comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 29 or a truncated fragment thereof as described hereinabove; and

a second PYP fragment of the invention comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 40, or a truncated fragment thereof as described hereinabove.

In said embodiment, the first and second PYP fragments, or truncated fragments thereof, are thus able to reconstitute a functional PYP comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 15, or a functional truncated fragment thereof.

In one embodiment, the complementation system of the invention comprises:

a first PYP fragment of the invention comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 23, or a truncated fragment thereof as described hereinabove, or an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more identity with the amino acid sequence as set forth in SEQ ID NO: 23, or a truncated fragment thereof as described hereinabove; and

a second PYP fragment of the invention comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 34, SEQ ID: 42, SEQ ID NO: 43 or SEQ ID NO: 44, or an amino acid sequence having at least about 70%, 75%, 80%, 85% or 90% or more identity with any one of the amino acid sequence as set forth in SEQ ID NO: 34, SEQ ID: 42, SEQ ID NO: 43 or SEQ ID NO: 44.

In one embodiment, the first PYP fragment, or a truncated fragment thereof, of the complementation assay of the invention as described hereinabove further comprises at least one of the following amino acid substitutions at the positions defined with reference to SEQ ID NO: 23: an asparagine at position 19, a leucine at position 62, a cysteine or a glutamic acid at position 68, an arginine at position 71, a serine at position 73, and/or an isoleucine at position 107; and/or the second PYP fragment, or a truncated fragment thereof, of the complementation assay of the invention as described hereinabove further comprises the following amino acid substitution with reference to SEQ ID NO: 34: an isoleucine at position 8.

In one embodiment, the first PYP fragment, or a truncated fragment thereof, of the complementation assay of the invention as described hereinabove further comprises at least one of the following amino acid substitutions at the positions defined with reference to SEQ ID NO: 23: an asparagine at position 19, a leucine at position 62, a cysteine or a glutamic acid at position 68, an arginine at position 71, a serine at position 73, and/or an isoleucine at position 107.

In one embodiment, the first PYP fragment, or a truncated fragment thereof, of the complementation assay of the invention as described hereinabove further comprises the following amino acid substitution at the position defined with reference to SEQ ID NO: 23: an isoleucine at position 107.

In one embodiment, the first PYP fragment, or a truncated fragment thereof, of the complementation assay of the invention as described hereinabove further comprises at least one of the following amino acid substitutions at the positions defined with reference to SEQ ID NO: 23: an asparagine at position 19, a leucine at position 62, a cysteine or a glutamic acid at position 68, an arginine at position 71, and/or a serine at position 73.

In one embodiment, the first PYP fragment, or a truncated fragment thereof, of the complementation assay of the invention as described hereinabove further comprises at least one of the following amino acid substitutions at the positions defined with reference to SEQ ID NO: 23: a leucine at position 62, an arginine at position 71, and/or a serine at position 73.

In one embodiment, the second PYP fragment, or a truncated fragment thereof, of the complementation assay of the invention as described hereinabove further comprises the following amino acid substitution with reference to SEQ ID NO: 34: an isoleucine at position 8. In one embodiment, the complementation system of the invention comprises:

a first PYP fragment of the invention comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 30 or a truncated fragment thereof as described hereinabove; and

- a second PYP fragment of the invention comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 34, or a truncated fragment thereof as described hereinabove.

In said embodiment, the first and second PYP fragments, or truncated fragments thereof, are thus able to reconstitute a functional PYP comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 16, or a functional truncated fragment thereof.

According to one embodiment, the truncated fragment of the first PYP fragment of the complementation assay of the invention as described hereinabove comprises at least 89 consecutive amino acids from the C -terminal end of said first PYP fragment.

According to one embodiment, the truncated fragment of the second PYP fragment of the complementation assay of the invention as described hereinabove comprises at least 8 consecutive amino acids of said second PYP fragment, preferably at least 8 consecutive amino acids from the N-terminal end of said second PYP fragment.

As mentioned hereinabove, according to the present invention, the complementation system comprises a first PYP fragment and a second PYP fragment, or truncated fragments thereof, wherein the first and second PYP fragments, or truncated fragments thereof, are able to reconstitute a functional PYP, or a functional truncated fragment thereof, that binds a fluorogenic hydroxybenzylidene rhodanine (HBR) analog, preferably reversibly.

According to the present invention, the reconstitution of a functional PYP, or a functional truncated fragment thereof, can be obtained by coupling or fusing the two PYP fragments, or truncated fragments thereof, of the complementation system of the invention to molecules interacting together, wherein the first PYP fragment, or a truncated fragment thereof, is coupled or fused to a first molecule and the second PYP fragment, or a truncated fragment thereof, is coupled or fused to a second molecule. Through their interaction, the molecules bring the two PYP fragments, or truncated fragments thereof, of the complementation system of the invention sufficiently close to each other so that a functional PYP, or a functional truncated fragment thereof, is reconstituted.

As mentioned above, the reconstitution of a functional PYP, or a functional truncated fragment thereof, can be detected through the detection of the fluorescence emitted by a fluorogenic hydroxybenzylidene-rhodanine (HBR) analog upon binding to the reconstituted functional PYP, or to the reconstituted functional truncated fragment thereof.

Indeed, the complex subsequently formed by the functional PYP, or functional truncated fragment thereof, reconstituted from the two PYP fragments, or truncated fragments thereof, of the complementation system of the invention, and a fluorogenic HBR analog is fluorescent, i.e. can emit light upon light excitation.

According to one embodiment, the functional PYP, or functional truncated fragment thereof, reconstituted from the two PYP fragments, or truncated fragments thereof, of the complementation system of the invention binds a fluorogenic HBR analog reversibly, i.e., through non-covalent interactions. Thus, according to one embodiment, the binding of the fluorogenic HBR analog to the functional PYP, or functional truncated fragment thereof, reconstituted from the two PYP fragments, or truncated fragments thereof, of the complementation system of the invention is reversible. Methods for assessing the binding of a fluorogenic chromophore to a polypeptide are well-known in the art. Such methods may notably rely on the assessment of the fluorescence emitted by the fluorogenic chromophore upon binding to the polypeptide and include spectrofluorimetry.

According to one embodiment, the functional PYP, or functional truncated fragment thereof, reconstituted from the two PYP fragments, or truncated fragments thereof, of the complementation system of the invention binds reversibly a fluorogenic HBR analog with: a KD ranging from about 0.01 to about 20 mM when measured at a temperature of about 25°C, preferably ranging from about 0.05 to about 10 mM, more preferably ranging from about 0.1 to about 5 mM; and/or

a koff ranging from about 0.5 to about 50 s ¹ when measured at a temperature of about 25°C, preferably from about 1 to about 25 s ¹ , more preferably from about 5 to about 20 s ¹ ; and/or

a kon ranging from about 0.05 x 10 ⁷ to about 50 x 10 ⁷ M V ¹ when measured at a temperature of about 25°C, preferably from about 0.1 x 10 ⁷ to about 25 x 10 ⁷ M V ¹ , more preferably from about 1 x 10 ⁷ to about 15 x 10 ⁷ M V ¹ . Methods for measuring the KD, k _on and k _0ff constants are well-known in the art, and include, for example, those described by Scatchard et al. (Ann. N.Y. Acad. Sci. USA 51 :660 (1949)). In particular, methods for measuring the thermodynamic dissociation constant KD are well-known in the art, and include, for example, those described by Plamont, et al. (Plamont et al., P Natl Acad Sci Usa 2016, 113 (3), 497-502). According to one embodiment, the molar absorption coefficient (e) of a fluorogenic HBR analog bound to the functional PYP, or functional truncated fragment thereof, reconstituted from the two PYP fragments, or truncated fragments thereof, of the complementation system of the invention ranges from about 1 to about 100 mM km ¹ when measured at its wavelength of maximal absorption (kabs). In one embodiment, the molar absorption coefficient (e) ranges from about 10 to about 200 mM km ¹ at kabs. In a particular embodiment, the molar absorption coefficient (e) ranges from about 20 to about 100 mM km ¹ at kabs.

Methods for measuring the molar absorption coefficient (e) are known in the art, and include, for example, those described by Plamont, M.-A. et al. (Plamont, M.-A. et al. , P Natl Acad Sci Usa 2016, 113 (3), 497-502).

According to one embodiment, the fluorescence quantum yield (f) of a fluorogenic HBR analog bound to the functional PYP, or functional truncated fragment thereof, reconstituted from the two PYP fragments, or truncated fragments thereof, of the complementation system of the invention is greater than about 0.1% when measured at its wavelength of maximal absorption (kabs). In an embodiment, f is greater than about 0.25%, about 0.75%, or about 1% at ka _bs. In a particular embodiment, f is greater than about 2% at kabs.

Methods for measuring the fluorescence quantum yield (f) are known in the art, and include, for example, those described by Plamont, M.-A. et al. (Plamont, M.-A. et al., P Natl Acad Sci Usa 2016, 113 (3), 497-502). In one embodiment, the fluorescence quantum yields after one-photon excitation f are calculated from the relation: where the subscript ref stands for standard samples. A(kexc) is the absorbance at the excitation wavelength kexc, D is the integrated emission spectrum, and n is the refractive index for the solvent.

According to one embodiment, upon binding to a fluorogenic HBR analog, the functional PYP, or functional truncated fragment thereof, reconstituted from the two PYP fragments, or truncated fragments thereof, of the complementation system of the invention enhances the brightness of said fluorogenic HBR analog.

According to one embodiment, the brightness of a fluorogenic HBR analog bound to the functional PYP, or functional truncated fragment thereof, reconstituted from the two PYP fragments, or truncated fragments thereof, of the complementation system of the invention is greater than about 50. In an embodiment, the brightness is greater than about 200. In a particular embodiment, the brightness is greater than about 1000.

Methods for measuring the brightness are well-known in the art. Brightness corresponds to the fluorescence output per emitter and is the product of the molar absorption coefficient (at the excitation wavelength) and the fluorescence quantum yield.

According to one embodiment, the first and second PYP fragments as described hereinabove, or truncated fragments thereof as described hereinabove, are able to reconstitute a functional PYP, or a functional truncated fragment thereof, with low affinity.

Thus, in one embodiment, the first and second PYP fragments as described hereinabove, or truncated fragments thereof as described hereinabove, display a low unspecific fluorescence background in presence of a fluorogenic HBR analog due to a low self-assembly of said first and second PYP fragments, or truncated fragments thereof.

In one embodiment, the first and second PYP fragments of the invention, or truncated fragments thereof, bind each other with a dissociation constant (KD) higher than about 0.1 mM, preferably higher than about 0.2 pM, more preferably higher than about 0.5 pM, even more preferably higher than 1 pM, wherein said KD is measured at a temperature of about 25°C in presence of a fluorogenic HBR analog, preferably in presence of 10 pM of a fluorogenic HBR analog.

In one embodiment, the first and second PYP fragments of the invention, or truncated fragments thereof, bind each other with a dissociation constant (KD) higher than about 0.2 pM, preferably higher than about 0.5 pM, more preferably higher than about 1 pM, even more preferably higher than 5 pM, wherein said KD is measured at a temperature of about 25°C in presence of a fluorogenic HBR analog, preferably in presence of 10 pM of a fluorogenic HBR analog.

In one embodiment, the first and second PYP fragments of the invention, or truncated fragments thereof, bind each other with a dissociation constant (KD) higher than about 0.2 pM, preferably higher than about 0.75 pM, more preferably higher than about 1 pM, even more preferably higher than about 5 pM, wherein said KD is measured at a temperature of about 25°C in presence of HMBR, preferably in the presence of 10 pM HMBR. In one embodiment, the first and second PYP fragments of the invention, or truncated fragments thereof, bind each other with a dissociation constant (KD) higher than about 1 pM, preferably higher than about 2 pM, more preferably higher than about 5 pM, even more preferably higher than about 10 pM, wherein said KD is measured at a temperature of about 25°C in presence of HBR-3,5-DOM, preferably in presence of 10 mM HBR-3,5-DOM.

As indicated above, methods for measuring the thermodynamic dissociation constant KD are well-known in the art, and include, for example, those described by Plamont, M.-A. et al., (Plamont, M.-A. et al., P Natl Acad Sci Usa 2016, 113 (3), 497-502).

In one embodiment, the thermodynamic dissociation constant KD is determined by spectrofluorometric titration as described in the Examples.

The complementation system of the invention may thus be used to study the interaction between two molecules, and in particular to study either or both of the association and the dissociation of said molecules.

The present invention also relates to a biological molecule of interest comprising the first or second PYP fragment as described hereinabove, or a truncated fragment thereof as described hereinabove. According to one embodiment, a biological molecule of interest comprising the first or second PYP fragment, or a truncated fragment thereof, is a biological molecule of interest to which the first or second PYP fragment, or a truncated fragment thereof, is attached, either covalently or non-covalently.

As used herein, the term“biological molecule of interest” encompasses any molecules present inside a living organism. In particular, the term includes, but is not limited to, amino acids, monosaccharides, nucleotides, polypeptides, proteins, polysaccharides, nucleic acids, lipids, fatty acids, glycolipids, sterols, vitamins, hormones, neurotransmitters, and metabolites.

Methods for non-covalently attaching a peptide or polypeptide to a biological molecule of interest are well-known. Methods for covalently attaching a peptide or polypeptide to a biological molecule of interest are well-known. The present invention also relates to a pair of biological molecules of interest comprising the first and second PYP fragments as described hereinabove, or truncated fragments thereof as described hereinabove, wherein one biological molecule of interest comprises the first PYP fragment as described hereinabove, or a truncated fragment thereof as described hereinabove, and the other biological molecule of interest comprises the second PYP fragment as described hereinabove, or a truncated fragment thereof as described hereinabove.

According to one embodiment, the biological molecule of interest is a protein. The present invention thus also relates to a fusion protein comprising the first or second PYP fragment as described hereinabove, or a truncated fragment thereof as described hereinabove.

Indeed, the PYP fragments as described hereinabove, or truncated fragments thereof as described hereinabove, can be expressed in fusion with any protein of interest within a host cell by inserting (for example via transformation or transfection) a nucleic acid sequence which encodes the resulting fusion protein.

Methods for fusing a peptide or polypeptide to a protein of interest are well-known. Briefly, such methods comprise inserting the nucleic acid sequence encoding a protein of interest in frame with the nucleic sequence encoding the peptide(s) or polypeptide(s). The nucleic sequence encoding the protein of interest can be inserted so that the encoded peptide is situated at the N-terminal end of the protein of interest or at the C-terminal end of the protein of interest, or internally, as desired. Additionally, a short nucleic sequence encoding a linker or spacer may be present between the sequences coding for the peptide and the protein of interest. In one embodiment, the fusion protein of the invention comprises the first PYP fragment as described hereinabove, or a truncated fragment thereof as described hereinabove, and a protein of interest. In one embodiment, the fusion protein of the invention comprises the second PYP fragment as described hereinabove, or a truncated fragment thereof as described hereinabove, and a protein of interest. In one embodiment, the fusion protein of the invention comprises at least one additional element other than the PYP fragments of the invention, or truncated fragments thereof, and the protein of interest. Example of additional elements that may be considered for the fusion proteins of the invention include, but are not limited to, linkers, targeting signals, protease target sites, antibody crystallizable region (Fc), enzymes (such as the alkaline phosphatase or horseradish peroxidase), hemagglutinin tag, poly arginine tag, poly histidine tag, myc tag, strep tag, S-tag, HAT tag, 3x flag tag, calmodulin-binding peptide tag, SBP tag, chi tin binding domain tag, GST tag, maltose-binding protein tag, fluorescent protein tag, preferably the fluorescence of which may be spectrally separated from the fluorescence associated with the complementation system of the invention, T7 tag, V5 tag, and X-press tag. In one embodiment, the fusion protein as described hereinabove comprises a linker.

Methods for designing or selecting a linker are well-known to one skilled in the art.

Examples of linkers include, without being limited to, linkers comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 45.

The present invention also relates to a pair of fusion proteins comprising the first and second PYP fragments as described hereinabove, or truncated fragments thereof as described hereinabove, wherein one fusion protein comprises the first PYP fragment as described hereinabove, or a truncated fragment thereof as described hereinabove, and the other fusion protein comprises the second PYP fragment as described hereinabove, or a truncated fragment thereof as described hereinabove. In one embodiment, both fusion proteins of the pair comprise the same protein of interest. In other words, in one embodiment, one fusion protein comprises the first PYP fragment as described hereinabove, or a truncated fragment thereof as described hereinabove, and a protein of interest, and the other fusion protein comprises the second PYP fragment as described hereinabove, or a truncated fragment thereof as described hereinabove, and the same protein of interest.

In one embodiment, each fusion protein of the pair comprises a distinct protein of interest. In other words, in one embodiment, one fusion protein comprises the first PYP fragment as described hereinabove, or a truncated fragment thereof as described hereinabove, and a first protein of interest, and the other fusion protein comprises the second PYP fragment as described hereinabove, or a truncated fragment thereof as described hereinabove, and a second, different, protein of interest.

In one embodiment, the first and the second proteins of interest are two reporter proteins able to interact with each other.

The present invention also relates to a nucleic acid sequence encoding the first PYP fragment and/or the second PYP fragment as described hereinabove, or truncated fragments thereof as described hereinabove, and to a nucleic acid sequence encoding the fusion protein of the invention as described hereinabove. According to the present invention, the nucleic acid sequence of the invention encoding the first PYP fragment and/or the second PYP fragment as described hereinabove, or truncated fragments thereof as described hereinabove, includes all nucleic acid sequences that are degenerate versions of each other and that encode the same first PYP fragment and/or second PYP fragment, or truncated fragments thereof. One skilled in the art is familiar with methods for adapting a coding sequence on the basis of the genetic code, for instance and without limitation, methods making use of codon degeneracy to introduce silent mutations and methods taking into account codon usage bias and variation of the standard genetic code relevant to the host cell considered.

According to one embodiment, the nucleic acid sequence of the invention encodes a first PYP fragment comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 23 or a truncated fragment thereof as described hereinabove, or encodes an amino acid sequence having at least about 30%, 35%, 40%, 45%, 50%, 55%, 60% or 65% identity, preferably at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more identity with the amino acid sequence as set forth in SEQ ID NO: 23, or a truncated fragment thereof as described hereinabove.

Examples of nucleic acid sequences encoding a first PYP fragment comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 23 or an amino acid sequence having at least about 70% identity with the amino acid sequence as set forth in SEQ ID NO: 23 include, without being limited to, the nucleic acid sequences as set forth in SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51 and SEQ ID NO: 52.

An example of a nucleic acid sequence encoding a first PYP fragment comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 23 includes, without being limited to, the nucleic acid sequence as set forth in SEQ ID NO: 46.

Examples of nucleic acid sequences encoding an amino acid sequence having at least about 70% identity with the amino acid sequence as set forth in SEQ ID NO: 23 include, without being limited to, the nucleic acid sequences as set forth in SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51 and SEQ ID NO: 52.

According to one embodiment, the nucleic acid sequence of the invention encodes a second PYP fragment comprising or consisting an amino acid sequence as set forth in SEQ ID NO: 34, or encodes a truncated fragment thereof as described hereinabove, or an amino acid sequence having at least about 30%, 35%, 40%, 45%, 50%, 55%, 60% or 65% identity, preferably at least about 70%, 75%, 80%, 85% or 90% or more identity with the amino acid sequence as set forth in SEQ ID NO: 34, or a truncated fragment thereof as described hereinabove.

Examples of nucleic acid sequences encoding a second PYP fragment comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 34 or an amino acid sequence having at least about 70% identity with the amino acid sequence as set forth in SEQ ID NO: 34 include, without being limited to, the nucleic acid sequences as set forth in SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58 and SEQ ID NO: 59.

An example of a nucleic acid sequence encoding a second PYP fragment comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 34 includes, without being limited to, the nucleic acid sequence as set forth in SEQ ID NO: 53.

Examples of nucleic acid sequences encoding an amino acid sequence having at least about 70% identity with the amino acid sequence as set forth in SEQ ID NO: 34 include, without being limited to, the nucleic acid sequences as set forth in SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58 and SEQ ID NO: 59.

In one embodiment, the nucleic acid sequence of the invention encodes a second PYP fragment comprising or consisting an amino acid sequence as set forth in SEQ ID NO: 34, SEQ ID NO: 42, SEQ ID NO: 43 or SEQ ID NO: 44, or encodes an amino acid sequence having at least about 30%, 35%, 40%, 45%, 50%, 55%, 60% or 65% identity, preferably at least about 70%, 75%, 80%, 85% or 90% or more identity with the amino acid sequence as set forth in SEQ ID NO: 34, SEQ ID NO: 42, SEQ ID NO: 43 or SEQ ID NO: 44.

Examples of nucleic acid sequences encoding a second PYP fragment comprising or consisting an amino acid sequence as set forth in SEQ ID NO: 34, SEQ ID NO: 42, SEQ ID NO: 43 or SEQ ID NO: 44, include, without being limited to, the nucleic acid sequences as set forth in SEQ ID NO: 53, SEQ ID NO: 60, SEQ ID NO: 61 and SEQ ID NO: 62, respectively.

The present invention also relates to a pair of nucleic acid sequences encoding the first PYP fragment and the second PYP fragment as described hereinabove, or truncated fragments thereof as described hereinabove, wherein one nucleic acid sequence encodes the first PYP fragment as described hereinabove, or a truncated fragment thereof as described hereinabove, and the other nucleic acid sequence encodes the second PYP fragment as described hereinabove, or a truncated fragment thereof as described hereinabove.

The present invention also relates to a pair of nucleic acid sequences encoding a first fusion protein and a second fusion protein as described hereinabove, wherein one fusion protein comprises the first PYP fragment as described hereinabove, or a truncated fragment thereof as described hereinabove, and the second fusion protein comprises the second PYP fragment as described hereinabove, or a truncated fragment thereof as described hereinabove.

The present invention also relates to a vector comprising at least one nucleic acid sequence as described hereinabove. According to one embodiment, the vector of the invention comprises:

a first nucleic acid sequence encoding the first PYP fragment, or a truncated fragment thereof, as described hereinabove; and

a second nucleic acid sequence encoding the second PYP fragment, or a truncated fragment thereof, as described hereinabove.

In one embodiment, the vector of the invention comprises:

a first nucleic acid sequence encoding a first PYP fragment comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 23 or a truncated fragment thereof as described hereinabove, or an amino acid sequence having at least about 30%, 35%, 40%, 45%, 50%, 55%, 60% or 65% identity, preferably at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,

98% or 99% or more identity with the amino acid sequence as set forth in SEQ ID NO: 23, or a truncated fragment thereof as described hereinabove; and a second nucleic acid sequence encoding a second PYP fragment comprising or consisting an amino acid sequence as set forth in SEQ ID NO: 34, or a truncated fragment thereof as described hereinabove, or an amino acid sequence having at least about 30%, 35%, 40%, 45%, 50%, 55%, 60% or 65% identity, preferably at least about 70%, 75%, 80%, 85% or 90% or more identity with the amino acid sequence as set forth in SEQ ID NO: 34, or a truncated fragment thereof as described hereinabove.

In one embodiment, the vector of the invention comprises:

a first nucleic acid sequence encoding a first PYP fragment comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 23 or a truncated fragment thereof as described hereinabove, or an amino acid sequence having at least about 30%, 35%, 40%, 45%, 50%, 55%, 60% or 65% identity, preferably at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more identity with the amino acid sequence as set forth in SEQ ID NO: 23, or a truncated fragment thereof as described hereinabove; and a second nucleic acid sequence encoding a second PYP fragment comprising or consisting an amino acid sequence as set forth in SEQ ID NO: 34, SEQ ID NO: 42, SEQ ID NO: 43 or SEQ ID NO: 44, or an amino acid sequence having at least about 30%, 35%, 40%, 45%, 50%, 55%, 60% or 65% identity, preferably at least about 70%, 75%, 80%, 85% or 90% or more identity with the amino acid sequence as set forth in SEQ ID NO: 34, SEQ ID NO: 42, SEQ ID NO: 43 or SEQ ID NO: 44.

The present invention also relates to a pair of vectors comprising the nucleic acid sequences encoding the first PYP fragment and the second PYP fragment as described hereinabove, or truncated fragments thereof as described hereinabove, wherein one vector comprises a nucleic acid sequence encoding the first PYP fragment as described hereinabove, or a truncated fragment thereof as described hereinabove, and the other vector comprises a nucleic acid sequence encoding the second PYP fragment as described hereinabove, or a truncated fragment thereof as described hereinabove.

In one embodiment, the first vector comprises a nucleic acid sequence encoding a first PYP fragment comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 23 or a truncated fragment thereof as described hereinabove, or an amino acid sequence having at least about 30%, 35%, 40%, 45%, 50%, 55%, 60% or 65% identity, preferably at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more identity with the amino acid sequence as set forth in SEQ ID NO: 23, or a truncated fragment thereof as described hereinabove. In one embodiment, the second vector comprises a nucleic acid sequence encoding a second PYP fragment comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 34, or a truncated fragment thereof as described hereinabove, or an amino acid sequence having at least about 30%, 35%, 40%, 45%, 50%, 55%, 60% or 65% identity, preferably at least about 70%, 75%, 80%, 85% or 90% or more identity with the amino acid sequence as set forth in SEQ ID NO: 34, or a truncated fragment thereof as described hereinabove. In one embodiment, the second vector comprises a nucleic acid sequence encoding a second PYP fragment comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 34, SEQ ID NO: 42, SEQ ID NO: 43 or SEQ ID NO: 44, or an amino acid sequence having at least about 30%, 35%, 40%, 45%, 50%, 55%, 60% or 65% identity, preferably at least about 70%, 75%, 80%, 85% or 90% or more identity with the amino acid sequence as set forth in SEQ ID NO: 34, SEQ ID NO: 42, SEQ ID NO: 43 or SEQ ID NO: 44.

As used herein the term“vector” refers to nucleic acid molecules used in molecular cloning as a vehicle to artificially carry foreign genetic material into a cell where it can be replicated and/or expressed. Examples of vectors include, without being limited to, plasmids, viral vectors and artificial chromosomes.

In particular, in the context of the present invention, the vector may comprise the required nucleic acid sequences for the fusion of the sequence encoding a protein of interest in frame with a sequence encoding a PYP fragment as described hereinabove, or a truncated fragment thereof as described hereinabove.

The present invention also relates to a cell expressing at least one of the PYP fragment as described hereinabove, or a truncated fragment thereof as described hereinabove, or a fusion protein as described hereinabove.

According to one embodiment, the cell of the invention expresses both the first and second PYP fragments as described hereinabove, or truncated fragments thereof as described hereinabove.

In one embodiment, the cell of the invention expresses a first biological molecule as described hereinabove comprising the first PYP fragment as described hereinabove, or a truncated fragment thereof as described hereinabove, and a second biological molecule as described hereinabove comprising the second PYP fragment as described hereinabove, or a truncated fragment thereof as described hereinabove.

In one embodiment, one of the biological molecules of interest is a protein. In one embodiment, both of the biological molecules of interest are a protein.

Thus, in one embodiment, the cell of the invention expresses a first fusion protein comprising the first PYP fragment as described hereinabove, or a truncated fragment thereof as described hereinabove, and a second fusion protein comprising the second PYP fragment as described hereinabove, or a truncated fragment thereof as described hereinabove. In one embodiment, the fusion protein comprises a reporter protein.

Thus, in one embodiment, the cell of the invention expresses:

a first fusion protein comprising the first PYP fragment as described hereinabove, or a truncated fragment thereof as described hereinabove, and a reporter protein; and

a second fusion protein comprising the second PYP fragment as described hereinabove, or a truncated fragment thereof as described hereinabove, and a reporter protein;

wherein the reporter proteins are identical or different. In one embodiment, the cell of the invention expresses:

a first fusion protein comprising the first PYP fragment as described hereinabove, or a truncated fragment thereof as described hereinabove, and a reporter protein; and

a second fusion protein comprising the second PYP fragment as described hereinabove, or a truncated fragment thereof as described hereinabove, and a reporter protein;

wherein the reporter proteins are identical or different, and wherein their interaction may be detected, localized or quantified as a way to indirectly assess a target of interest or a mechanism of interest. Thus, in one embodiment, the cell of the invention may be used as a cellular sensor to assess a target of interest or a physiological mechanism of interest.

Examples of targets or mechanisms of interest include, without being limited to, assessment of the presence in the cell of an analyte such as calcium, and assessment of a physiological mechanism such as apoptosis. Thus, the cell of the invention may be used as a cellular sensor to assess the presence in the cell of an analyte of interest such as calcium. The cell of the invention may be used as a cellular sensor to assess a physiological mechanism in the cell such as apoptosis. The cell of the invention may also be used as a cellular sensor to screen chemical libraries for identifying drugs regulating the target or mechanism of interest. According to one embodiment, the complementation system as described hereinabove further comprises a fluorogenic HBR analog.

In one embodiment, the complementation system of the invention further comprises a fluorogenic HBR analog of formula (I):

wherein:

Rl, R2, R5 and R6 may be identical or different and each represents H, halo, hydroxyl, aryl, alkyl, cycloalkyl, heteroalkyl (e.g., alkoxy) or heterocycloalkyl group, saturated or unsaturated, linear or branched, optionally substituted by at least one group selected from halo, hydroxyl, oxo, nitro, ami do, carboxy, amino, cyano, haloalkoxy, haloalkyl;

R3 represents a non-binding doublet ( i.e ., a free pair of electrons) or H, halo, hydroxyl, aryl, alkyl, cycloalkyl, heteroalkyl or heterocycloalkyl group, saturated or unsaturated, linear or branched, optionally substituted by at least one group selected from halo, hydroxyl, oxo, nitro, ami do, carboxy, amino, cyano, haloalkoxy, haloalkyl; R4 is a single or a double bound, interrupted or terminated by S, O orN atom, optionally substituted by at least one group selected from H, hydroxyl, aryl, alkyl, cycloalkyl, heteroalkyl or heterocycloalkyl group, saturated or unsaturated, linear or branched, optionally substituted by at least one group selected from halo, hydroxyl, oxo, nitro, ami do, carboxy, amino, cyano, haloalkoxy, haloalkyl; X is OH, SH, NHR7, or N(R7)2, wherein R7 is H, halo, hydroxyl, aryl, alkyl, cycloalkyl, heteroalkyl or heterocycloalkyl group, saturated or unsaturated, linear or branched, optionally substituted by at least one group selected from halo, hydroxyl, oxo, nitro, ami do, carboxy, amino, cyano, haloalkoxy, haloalkyl; and

Y is O, NH or S.

In one embodiment, the fluorogenic HBR analog is selected from the group comprising or consisting of: (Z)-5-(4-hydroxybenzylidene)-2-thioxo-l,3-thiazolidin-4-one (HBR);

(Z)-5-(4-hydroxy-3-methylbenzylidene)-2-thi oxo-1, 3-thiazolidin-4-one (HMBR); (Z)-5- (4-Hydroxy-3,5-dimethoxybenzylidene)-2-thioxothiazolidin-4-o ne (HBR-3,5DOM); (Z)-5-(4-Hydroxy-3-methoxybenzylidene)-2-thi oxo-1, 3-thiazolidin-4-one (HBR-30M); (Z)-5-(4-Hydroxy-3,5-dimethylbenzylidene)-2-thioxothiazolidi n-4-one (HBR-3,5DM); (Z)-5-(4-Hydroxy-2,5-dimethylbenzylidene)-2-thioxo-l,3-thiaz olidin-4-one (HBR-

2,5DM); (Z)-5-(3-Ethyl-4-hydroxybenzylidene)-2-thioxothiazolidin-4-o ne (HBR-3E); (Z)-5-(3-Ethoxy-4-hydroxybenzylidene)-2-thioxothiazolidin-4- one (HBR-30E); (Z)-5- (4-hydroxybenzylidene)-3-methyl-2-thioxothiazolidin-4-one (HBMR); (Z)-5-(2,4- Dihydroxybenzylidene)-2-thioxo-l,3-thiazolidin-4-one (DHBR) (Z)-5-(4-hydroxy-3- methoxybenzylidene)-2-thioxo-l,3-thiazolidin-4-one (HMOBR); (Z)-2-(5-(3-ethyl-4- hydroxybenzylidene)-4-oxo-2-thioxothiazolidin-3-yl)acetic acid (HBRAA-3E); (Z)-2- (5-(4-hydroxy-3-ethoxybenzylidene)-4-oxo-2-thioxothiazolidin -3-yl)acetic acid

(HBRAA-30E); (Z)-2-(5-(4-hydroxy-2-methoxybenzylidene)-4-oxo-2- thioxothiazolidin-3-yl)acetic acid (HBRAA-20M); (Z)-2-(5-(4-hydroxybenzylidene)-4- oxo-2-thioxothiazolidin-3-yl)acetic acid (HBRAA); (Z)-2-(5-(4-hydroxy-3- methylbenzylidene)-4-oxo-2-thioxothiazolidin-3-yl)acetic acid (HBRAA-3M); (Z)-2-(5- (4-hydroxy-3-methoxybenzylidene)-4-oxo-2-thioxothiazolidin-3 -yl)acetic acid

(HBRAA-3 OM); (Z)-2-(5-(4-hydroxy-2-methoxybenzylidene)-4-oxo-2- thi oxothi azoli din-3 -yl)aceti c acid (HBRAA-20M); (Z)-2-(5-(4-hydroxy-2, 5- dimethylbenzylidene)-4-oxo-2 -thi oxothi azolidin-3-yl) acetic acid (HBRAA-2,5DM);

(Z)-2-(5-(4-hydroxy-3, 5-dimethylbenzylidene)-4-oxo-2 -thi oxothi azolidin-3-yl)acetic acid (HBRAA-3 , 5DM) and (Z)-2-(5-(4-hydroxy-3, 5-dimethoxybenzylidene)-4-oxo-2- thi oxothi azolidin-3-yl) acetic acid (HBRAA-3, 5DOM).

In one embodiment, the fluorogenic HBR analog is selected from the group comprising or consisting of: (Z)-5-(4-hydroxy-3-methylbenzylidene)-2-thi oxo-1, 3-thiazolidin-4-one (HMBR); (Z)-5-(4-Hydroxy-3,5-dimethoxybenzylidene)-2-thioxothiazolid in-4-one (HBR-3,5DOM); (Z)-5-(4-Hydroxy-3-methoxybenzylidene)-2-thioxo-l,3-thiazoli din-4- one (HBR-30M) and (Z)-5-(4-Hydroxy-3,5-dimethylbenzylidene)-2-thioxothiazolidi n- 4-one (HBR-3,5DM).

In one embodiment the fluorogenic HBR analog is membrane-permeant.

As used herein, the term“membrane-permeant” refers to a property of a compound which is able to cross a biological membrane ( i.e ., a membrane consisting of a polar lipid layer, preferably a polar lipid bilayer). The term“membrane-impermeant” refers, in opposition, to a compound which is not able to cross a biological membrane.

In one embodiment, the fluorogenic HBR analog is membrane-permeant and is selected from the group comprising or consisting of: (Z)-5-(4-hydroxybenzylidene)-2-thioxo-l,3- thiazolidin-4-one (HBR); (Z)-5-(4-hydroxy-3-methylbenzylidene)-2-thioxo-l,3- thiazolidin-4-one (HMBR); (Z)-5-(4-Hydroxy-3,5-dimethoxybenzylidene)-2- thioxothiazolidin-4-one (HBR-3,5DOM); (Z)-5-(4-Hydroxy-3-methoxybenzylidene)-2- thioxo- 1 ,3 -thiazolidin-4-one (HBR-30M); (Z)-5-(4-Hydroxy-3,5-dimethylbenzylidene)- 2-thioxothiazolidin-4-one (HBR-3,5DM); (Z)-5-(4-Hydroxy-2,5-dimethylbenzylidene)- 2-thi oxo-1, 3-thiazolidin-4-one (HBR-2,5DM); (Z)-5-(3-Ethyl-4-hydroxybenzylidene)-2- thioxothiazolidin-4-one (HBR-3E); (Z)-5-(3-Ethoxy-4-hydroxybenzylidene)-2- thioxothiazolidin-4-one (HBR-30E); (Z)-5-(4-hydroxybenzylidene)-3-methyl-2- thioxothiazolidin-4-one (HBMR); (Z)-2-(5-(4-hydroxybenzylidene)-4-oxo-2- thioxothiazolidin-3-yl)acetic acid (HBAAR); (Z)-5-(2,4-Dihydroxybenzylidene)-2- thioxo- 1 ,3 -thiazolidin-4-one (DHBR) and (Z)-5-(4-hydroxy-3-methoxybenzylidene)-2- thioxo- 1 ,3 -thiazolidin-4-one (HMOBR).

In one embodiment, the fluorogenic HBR analog is membrane-permeant and selected from the group comprising or consisting of: (Z)-5-(4-hydroxy-3-methylbenzylidene)-2- thioxo- 1 ,3 -thiazolidin-4-one (HMBR); (Z)-5-(4-Hydroxy-3,5-dimethoxybenzylidene)-2- thioxothiazolidin-4-one (HBR-3 , 5DOM); (Z)-5-(4-Hydroxy-3-methoxybenzylidene)-2- thioxo- 1 ,3 -thiazolidin-4-one (HBR-3 OM) and (Z)-5-(4-Hydroxy-3,5- dimethylbenzylidene)-2-thioxothiazolidin-4-one (HBR-3, 5DM). In one embodiment the fluorogenic HBR analog is membrane-impermeant.

In one embodiment, the fluorogenic HBR analog is membrane-impermeant and selected from the group comprising or consisting of: (Z)-2-(5-(3-ethyl-4-hydroxybenzylidene)-4- oxo-2-thioxothiazolidin-3-yl)acetic acid (HBRAA-3E); (Z)-2-(5-(4-hydroxy-3- ethoxybenzylidene)-4-oxo-2-thioxothiazolidin-3-yl)acetic acid (HBRAA-30E); (Z)-2-

(5-(4-hydroxy-2-methoxybenzylidene)-4-oxo-2-thioxothiazol idin-3-yl)acetic acid (HBRAA-20M); (Z)-2-(5-(4-hydroxybenzylidene)-4-oxo-2-thioxothiazolidin-3- yl)acetic acid (HBR A A); (Z)-2-(5-(4-hydroxy-3-methylbenzylidene)-4-oxo-2- thioxothiazolidin-3-yl)acetic acid (HBRAA-3M); (Z)-2-(5-(4-hydroxy-3- methoxybenzylidene)-4-oxo-2-thioxothiazolidin-3-yl)acetic acid (HBRAA-3 OM); (Z)-

2-(5-(4-hydroxy-2-methoxybenzylidene)-4-oxo-2-thioxothiaz olidin-3-yl)acetic acid (HBRAA-20M); (Z)-2-(5-(4-hydroxy-2, 5-dimethylbenzylidene)-4-oxo-2- thioxothiazolidin-3-yl) acetic acid (HBRAA-2,5DM); (Z)-2-(5-(4-hydroxy-3, 5- dimethylbenzylidene)-4-oxo-2-thioxothiazolidin-3-yl)acetic acid (HBRAA-3, 5DM) and (Z)-2-(5-(4-hydroxy-3, 5-dimethoxybenzylidene)-4-oxo-2-thioxothiazolidin-3-yl) acetic acid (HBRAA-3, 5DOM).

According to one embodiment, the complementation system of the invention comprises: a first PYP fragment as described hereinabove, or a truncated fragment thereof as described hereinabove;

- a second PYP fragment of the invention comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 34, or an amino acid sequence having at least about 30%, 35%, 40%, 45%, 50%, 55%, 60% or 65% identity, preferably at least about 70%, 75%, 80%, 85% or 90% or more identity with the amino acid sequence as set forth in SEQ ID NO: 34; and

- a fluorogenic HBR analog selected form the list comprising or consisting of

HMBR, HBR-3,5DOM, HBR-30M and HBR-3,5DM.

In one embodiment, the complementation system of the invention comprises a first PYP fragment as described hereinabove, or a truncated fragment thereof as described hereinabove; a second PYP fragment of the invention comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 34, or an amino acid sequence having at least about 30%, 35%, 40%, 45%, 50%, 55%, 60% or 65% identity, preferably at least about 70%, 75%, 80%, 85% or 90% or more identity with the amino acid sequence as set forth in SEQ ID NO: 34; and HMBR or HBR-3,5DOM.

In one embodiment, the complementation system of the invention comprises a first PYP fragment as described hereinabove, or a truncated fragment thereof as described hereinabove; a second PYP fragment of the invention comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 34, or an amino acid sequence having at least about 30%, 35%, 40%, 45%, 50%, 55%, 60% or 65% identity, preferably at least about 70%, 75%, 80%, 85% or 90% or more identity with the amino acid sequence as set forth in SEQ ID NO: 34; and HMBR.

In one embodiment, the complementation system of the invention comprises a first PYP fragment as described hereinabove, or a truncated fragment thereof as described hereinabove; a second PYP fragment of the invention comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 34, or an amino acid sequence having at least about 30%, 35%, 40%, 45%, 50%, 55%, 60% or 65% identity, preferably at least about 70%, 75%, 80%, 85% or 90% or more identity with the amino acid sequence as set forth in SEQ ID NO: 34; and HBR-3,5DOM.

In one embodiment, the complementation system of the invention comprises a first PYP fragment as described hereinabove, or a truncated fragment thereof as described hereinabove; a second PYP fragment of the invention comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 34, or an amino acid sequence having at least about 30%, 35%, 40%, 45%, 50%, 55%, 60% or 65% identity, preferably at least about 70%, 75%, 80%, 85% or 90% or more identity with the amino acid sequence as set forth in SEQ ID NO: 34; and HBR-30M. In one embodiment, the complementation system of the invention comprises a first PYP fragment as described hereinabove, or a truncated fragment thereof as described hereinabove; a second PYP fragment of the invention comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 34, or an amino acid sequence having at least about 30%, 35%, 40%, 45%, 50%, 55%, 60% or 65% identity, preferably at least about 70%, 75%, 80%, 85% or 90% or more identity with the amino acid sequence as set forth in SEQ ID NO: 34; and HBR-3,5DM.

According to one embodiment, the complementation system of the invention comprises: a first PYP fragment comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 23 or SEQ ID NO: 30, or a truncated fragment thereof, or an amino acid sequence having at least about 30%, 35%, 40%, 45%, 50%, 55%, 60% or 65% identity, preferably at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more identity with the amino acid sequence as set forth in SEQ ID NO: 23 or SEQ ID NO: 30, or a truncated fragment thereof;

a second PYP fragment of the invention comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 34, or an amino acid sequence having at least about 30%, 35%, 40%, 45%, 50%, 55%, 60% or 65% identity, preferably at least about 70%, 75%, 80%, 85% or 90% or more identity with the amino acid sequence as set forth in SEQ ID NO: 34; and

a fluorogenic HBR analog selected form the list comprising or consisting of HMBR, HBR-3,5DOM, HBR-30M and HBR-3,5DM, preferably the fluorogenic HBR analog is HMBR or HBR-3,5DOM.

According to one embodiment, the complementation system of the invention comprises: a first PYP fragment as described hereinabove, or a truncated fragment thereof as described hereinabove;

a second PYP fragment of the invention comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 42, or an amino acid sequence having at least about 30%, 35%, 40%, 45%, 50%, 55%, 60% or 65% identity, preferably at least about 70%, 75%, 80%, 85% or 90% or more identity with the amino acid sequence as set forth in SEQ ID NO: 42; and

a fluorogenic HBR analog selected form the list comprising or consisting of HMBR, HBR-3,5DOM, HBR-30M and HBR-3,5DM.

In one embodiment, the complementation system of the invention comprises a first PYP fragment as described hereinabove, or a truncated fragment thereof as described hereinabove; a second PYP fragment of the invention comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 42, or an amino acid sequence having at least about 30%, 35%, 40%, 45%, 50%, 55%, 60% or 65% identity, preferably at least about 70%, 75%, 80%, 85% or 90% or more identity with the amino acid sequence as set forth in SEQ ID NO: 42; and HMBR or HBR-3,5DOM.

In one embodiment, the complementation system of the invention comprises a first PYP fragment as described hereinabove, or a truncated fragment thereof as described hereinabove; a second PYP fragment of the invention comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 42, or an amino acid sequence having at least about 30%, 35%, 40%, 45%, 50%, 55%, 60% or 65% identity, preferably at least about 70%, 75%, 80%, 85% or 90% or more identity with the amino acid sequence as set forth in SEQ ID NO: 42; and HMBR.

In one embodiment, the complementation system of the invention comprises a first PYP fragment as described hereinabove, or a truncated fragment thereof as described hereinabove; a second PYP fragment of the invention comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 42, or an amino acid sequence having at least about 30%, 35%, 40%, 45%, 50%, 55%, 60% or 65% identity, preferably at least about 70%, 75%, 80%, 85% or 90% or more identity with the amino acid sequence as set forth in SEQ ID NO: 42; and HBR-3,5DOM. In one embodiment, the complementation system of the invention comprises a first PYP fragment as described hereinabove, or a truncated fragment thereof as described hereinabove; a second PYP fragment of the invention comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 42, or an amino acid sequence having at least about 30%, 35%, 40%, 45%, 50%, 55%, 60% or 65% identity, preferably at least about 70%, 75%, 80%, 85% or 90% or more identity with the amino acid sequence as set forth in SEQ ID NO: 42; and HBR-30M.

In one embodiment, the complementation system of the invention comprises a first PYP fragment as described hereinabove, or a truncated fragment thereof as described hereinabove; a second PYP fragment of the invention comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 42, or an amino acid sequence having at least about 30%, 35%, 40%, 45%, 50%, 55%, 60% or 65% identity, preferably at least about 70%, 75%, 80%, 85% or 90% or more identity with the amino acid sequence as set forth in SEQ ID NO: 42; and HBR-3,5DM.

In one embodiment, the complementation system of the invention comprises:

a first PYP fragment comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 23 or SEQ ID NO: 30, or a truncated fragment thereof, or an amino acid sequence having at least about 30%, 35%, 40%, 45%, 50%, 55%, 60% or 65% identity, preferably at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more identity with the amino acid sequence as set forth in SEQ ID NO: 23 or SEQ ID NO: 30, or a truncated fragment thereof;

a second PYP fragment of the invention comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 42, or an amino acid sequence having at least about 30%, 35%, 40%, 45%, 50%, 55%, 60% or 65% identity, preferably at least about 70%, 75%, 80%, 85% or 90% or more identity with the amino acid sequence as set forth in SEQ ID NO: 42; and

a fluorogenic HBR analog selected form the list comprising or consisting of HMBR, HBR-3,5DOM, HBR-30M and HBR-3,5DM, preferably the fluorogenic HBR analog is HMBR or HBR-3,5DOM.

According to one embodiment, the complementation system of the invention comprises: a first PYP fragment as described hereinabove, or a truncated fragment thereof as described hereinabove;

a second PYP fragment of the invention comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 43, or an amino acid sequence having at least about 30%, 35%, 40%, 45%, 50%, 55%, 60% or 65% identity, preferably at least about 70%, 75%, 80%, 85% or 90% or more identity with the amino acid sequence as set forth in SEQ ID NO: 43; and

a fluorogenic HBR analog selected form the list comprising or consisting of HMBR, HBR-3,5DOM, HBR-30M and HBR-3,5DM. In one embodiment, the complementation system of the invention comprises a first PYP fragment as described hereinabove, or a truncated fragment thereof as described hereinabove; a second PYP fragment of the invention comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 43, or an amino acid sequence having at least about 30%, 35%, 40%, 45%, 50%, 55%, 60% or 65% identity, preferably at least about 70%, 75%, 80%, 85% or 90% or more identity with the amino acid sequence as set forth in SEQ ID NO: 43; and HMBR or HBR-3,5DOM.

In one embodiment, the complementation system of the invention comprises a first PYP fragment as described hereinabove, or a truncated fragment thereof as described hereinabove; a second PYP fragment of the invention comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 43, or an amino acid sequence having at least about 30%, 35%, 40%, 45%, 50%, 55%, 60% or 65% identity, preferably at least about 70%, 75%, 80%, 85% or 90% or more identity with the amino acid sequence as set forth in SEQ ID NO: 43; and HMBR. In one embodiment, the complementation system of the invention comprises a first PYP fragment as described hereinabove, or a truncated fragment thereof as described hereinabove; a second PYP fragment of the invention comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 43, or an amino acid sequence having at least about 30%, 35%, 40%, 45%, 50%, 55%, 60% or 65% identity, preferably at least about 70%, 75%, 80%, 85% or 90% or more identity with the amino acid sequence as set forth in SEQ ID NO: 43; and HBR-3,5DOM.

In one embodiment, the complementation system of the invention comprises a first PYP fragment as described hereinabove, or a truncated fragment thereof as described hereinabove; a second PYP fragment of the invention comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 43, or an amino acid sequence having at least about 30%, 35%, 40%, 45%, 50%, 55%, 60% or 65% identity, preferably at least about 70%, 75%, 80%, 85% or 90% or more identity with the amino acid sequence as set forth in SEQ ID NO: 43; and HBR-30M. In one embodiment, the complementation system of the invention comprises a first PYP fragment as described hereinabove, or a truncated fragment thereof as described hereinabove; a second PYP fragment of the invention comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 43, or an amino acid sequence having at least about 30%, 35%, 40%, 45%, 50%, 55%, 60% or 65% identity, preferably at least about 70%, 75%, 80%, 85% or 90% or more identity with the amino acid sequence as set forth in SEQ ID NO: 43; and HBR-3,5DM.

In one embodiment, the complementation system of the invention comprises:

a first PYP fragment comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 23 or SEQ ID NO: 30, or a truncated fragment thereof, or an amino acid sequence having at least about 30%, 35%, 40%, 45%, 50%, 55%, 60% or 65% identity, preferably at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more identity with the amino acid sequence as set forth in SEQ ID NO: 23 or SEQ ID NO: 30, or a truncated fragment thereof;

a second PYP fragment of the invention comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 43, or an amino acid sequence having at least about 30%, 35%, 40%, 45%, 50%, 55%, 60% or 65% identity, preferably at least about 70%, 75%, 80%, 85% or 90% or more identity with the amino acid sequence as set forth in SEQ ID NO: 43; and

a fluorogenic HBR analog selected form the list comprising or consisting of HMBR, HBR-3,5DOM, HBR-30M and HBR-3,5DM, preferably the fluorogenic HBR analog is HMBR or HBR-3,5DOM.

According to one embodiment, the complementation system of the invention comprises: a first PYP fragment as described hereinabove, or a truncated fragment thereof as described hereinabove;

a second PYP fragment of the invention comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 44, or an amino acid sequence having at least about 30%, 35%, 40%, 45%, 50%, 55%, 60% or 65% identity, preferably at least about 70%, 75%, 80%, 85% or 90% or more identity with the amino acid sequence as set forth in SEQ ID NO: 44; and

a fluorogenic HBR analog selected form the list comprising or consisting of HMBR, HBR-3,5DOM, HBR-30M and HBR-3,5DM.

In one embodiment, the complementation system of the invention comprises a first PYP fragment as described hereinabove, or a truncated fragment thereof as described hereinabove; a second PYP fragment of the invention comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 44, or an amino acid sequence having at least about 30%, 35%, 40%, 45%, 50%, 55%, 60% or 65% identity, preferably at least about 70%, 75%, 80%, 85% or 90% or more identity with the amino acid sequence as set forth in SEQ ID NO: 44; and HMBR or HBR-3,5DOM.

In one embodiment, the complementation system of the invention comprises a first PYP fragment as described hereinabove, or a truncated fragment thereof as described hereinabove; a second PYP fragment of the invention comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 44, or an amino acid sequence having at least about 30%, 35%, 40%, 45%, 50%, 55%, 60% or 65% identity, preferably at least about 70%, 75%, 80%, 85% or 90% or more identity with the amino acid sequence as set forth in SEQ ID NO: 44; and HMBR.

In one embodiment, the complementation system of the invention comprises a first PYP fragment as described hereinabove, or a truncated fragment thereof as described hereinabove; a second PYP fragment of the invention comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 44, or an amino acid sequence having at least about 30%, 35%, 40%, 45%, 50%, 55%, 60% or 65% identity, preferably at least about 70%, 75%, 80%, 85% or 90% or more identity with the amino acid sequence as set forth in SEQ ID NO: 44; and HBR-3,5DOM.

In one embodiment, the complementation system of the invention comprises a first PYP fragment as described hereinabove, or a truncated fragment thereof as described hereinabove; a second PYP fragment of the invention comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 44, or an amino acid sequence having at least about 30%, 35%, 40%, 45%, 50%, 55%, 60% or 65% identity, preferably at least about 70%, 75%, 80%, 85% or 90% or more identity with the amino acid sequence as set forth in SEQ ID NO: 44; and HBR-30M.

In one embodiment, the complementation system of the invention comprises a first PYP fragment as described hereinabove, or a truncated fragment thereof as described hereinabove; a second PYP fragment of the invention comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 44, or an amino acid sequence having at least about 30%, 35%, 40%, 45%, 50%, 55%, 60% or 65% identity, preferably at least about 70%, 75%, 80%, 85% or 90% or more identity with the amino acid sequence as set forth in SEQ ID NO: 44; and HBR-3,5DM.

In one embodiment, the complementation system of the invention comprises:

a first PYP fragment comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 23 or SEQ ID NO: 30, or a truncated fragment thereof, or an amino acid sequence having at least about 30%, 35%, 40%, 45%, 50%, 55%, 60% or 65% identity, preferably at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more identity with the amino acid sequence as set forth in SEQ ID NO: 23 or SEQ ID NO: 30, or a truncated fragment thereof;

a second PYP fragment of the invention comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 44, or an amino acid sequence having at least about 50%, 55%, 60% or 65% identity, preferably at least about 70%, 75%, 80%, 85% or 90% or more identity with the amino acid sequence as set forth in SEQ ID NO: 44; and

a fluorogenic HBR analog selected form the list comprising or consisting of HMBR, HBR-3,5DOM, HBR-30M and HBR-3,5DM, preferably the fluorogenic HBR analog is HMBR or HBR-3,5DOM.

According to one embodiment, the complementation system of the invention comprises: a first PYP fragment comprising or consisting of an amino acid sequence as set forth in any one of SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 or SEQ ID NO: 29, or a truncated fragment thereof as described hereinabove;

a second PYP fragment comprising or consisting of an amino acid sequence as set forth in any one of SEQ ID NO:34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39 or SEQ ID NO: 40, or a truncated fragment thereof as described hereinabove; and

a fluorogenic HBR analog selected form the list comprising or consisting of HMBR, HBR-3,5DOM, HBR-30M and HBR-3,5DM, preferably the fluorogenic HBR analog is HMBR or HBR-3,5DOM. In one embodiment, the complementation system of the invention comprises:

a first PYP fragment comprising or consisting of an amino acid sequence as set forth in any one of SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25,

SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 or SEQ ID NO: 29, or a truncated fragment thereof as described hereinabove;

a second PYP fragment comprising or consisting of an amino acid sequence as set forth in any one of SEQ ID NO:34, SEQ ID NO: 35, SEQ ID NO: 36,

SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39 or SEQ ID NO: 40, respectively, or a truncated fragment thereof as described hereinabove; and a fluorogenic HBR analog selected form the list comprising or consisting of HMBR, HBR-3,5DOM, HBR-30M and HBR-3,5DM, preferably the fluorogenic

HBR analog is HMBR or HBR-3,5DOM.

The present invention also relates to a kit comprising at least one vector comprising: a first nucleic acid sequence encoding the first PYP fragment, or a truncated fragment thereof, as described hereinabove; and

- a second nucleic acid sequence encoding the second PYP fragment, or a truncated fragment thereof, as described hereinabove.

In one embodiment, the kit of the invention comprises at least one vector comprising: a first nucleic acid sequence encoding a first PYP fragment comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 23 or a truncated fragment thereof as described hereinabove, or an amino acid sequence having at least about 30%, 35%, 40%, 45%, 50%, 55%, 60% or 65% identity, preferably at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more identity with the amino acid sequence as set forth in SEQ ID NO: 23, or a truncated fragment thereof as described hereinabove; and - a second nucleic acid sequence encoding a second PYP fragment comprising or consisting an amino acid sequence as set forth in SEQ ID NO: 34, or a truncated fragment thereof as described hereinabove, or an amino acid sequence having at least about 30%, 35%, 40%, 45%, 50%, 55%, 60% or 65% identity, preferably at least about 70%, 75%, 80%, 85% or 90% or more identity with the amino acid sequence as set forth in SEQ ID NO: 34, or a truncated fragment thereof as described hereinabove.

In one embodiment, the kit of the invention comprises at least one vector comprising: a first nucleic acid sequence encoding a first PYP fragment comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 23 or a truncated fragment thereof as described hereinabove, or an amino acid sequence having at least about 30%, 35%, 40%, 45%, 50%, 55%, 60% or 65% identity, preferably at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more identity with the amino acid sequence as set forth in SEQ ID NO: 23, or a truncated fragment thereof as described hereinabove; and - a second nucleic acid sequence encoding a second PYP fragment comprising or consisting an amino acid sequence as set forth in SEQ ID NO: 34, SEQ ID NO: 42, SEQ ID NO: 43 or SEQ ID NO: 44, or an amino acid sequence having at least about 30%, 35%, 40%, 45%, 50%, 55%, 60% or 65% identity, preferably at least about 70%, 75%, 80%, 85% or 90% or more identity with the amino acid sequence as set forth in SEQ ID NO: 34, SEQ ID NO: 42,

SEQ ID NO: 43 or SEQ ID NO: 44.

The present invention also relates to a kit comprising:

a first vector comprising a nucleic acid sequence encoding the first PYP fragment, or a truncated fragment thereof, as described hereinabove; and a second vector comprising a nucleic acid sequence encoding the second PYP fragment, or a truncated fragment thereof, as described hereinabove.

In one embodiment, the kit of the invention comprises:

a first vector comprising a nucleic acid sequence encoding a first PYP fragment comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 23 or a truncated fragment thereof as described hereinabove, or an amino acid sequence having at least about 30%, 35%, 40%, 45%, 50%, 55%, 60% or 65% identity, preferably at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more identity with the amino acid sequence as set forth in SEQ ID NO: 23, or a truncated fragment thereof as described hereinabove; and

a second vector comprising a nucleic acid sequence encoding a second PYP fragment comprising or consisting an amino acid sequence as set forth in SEQ ID NO: 34, or a truncated fragment thereof as described hereinabove, or an amino acid sequence having at least about 30%, 35%, 40%, 45%, 50%, 55%, 60% or 65% identity, preferably at least about 70%, 75%, 80%, 85% or 90% or more identity with the amino acid sequence as set forth in SEQ ID NO: 34, or a truncated fragment thereof as described hereinabove.

In one embodiment, the kit of the invention comprises:

- a first vector comprising a nucleic acid sequence encoding a first PYP fragment comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 23 or a truncated fragment thereof as described hereinabove, or an amino acid sequence having at least about 30%, 35%, 40%, 45%, 50%, 55%, 60% or 65% identity, preferably at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more identity with the amino acid sequence as set forth in SEQ ID NO: 23, or a truncated fragment thereof as described hereinabove; and

a second vector comprising a nucleic acid sequence encoding a second PYP fragment comprising or consisting an amino acid sequence as set forth in SEQ ID NO: 34, SEQ ID NO: 42, SEQ ID NO: 43 or SEQ ID NO: 44, or an amino acid sequence having at least about 30%, 35%, 40%, 45%, 50%, 55%, 60% or 65% identity, preferably at least about 70%, 75%, 80%, 85% or 90% or more identity with the amino acid sequence as set forth in SEQ ID NO: 34, SEQ ID NO: 42, SEQ ID NO: 43 or SEQ ID NO: 44. The present invention also relates to a kit comprising:

a first biological molecule of interest as described hereinabove comprising the first PYP fragment, or a truncated fragment thereof, as described hereinabove; and a second biological molecule of interest as described hereinabove comprising the second PYP fragment, or a truncated fragment thereof, as described hereinabove. The present invention also relates to a kit comprising:

a first fusion protein comprising the first PYP fragment, or a truncated fragment thereof, as described hereinabove; and

a second fusion protein comprising the second PYP fragment, or a truncated fragment thereof, as described hereinabove. The present invention also relates to a kit comprising:

a first fusion protein comprising the first PYP fragment, or a truncated fragment thereof, as described hereinabove and a reporter protein; and

a second fusion protein comprising the second PYP fragment, or a truncated fragment thereof, as described hereinabove and a reporter protein,

wherein the reporter proteins are identical or different.

The present invention also relates to a kit comprising a cell or a population of cells as described hereinabove.

In one embodiment, the kit of the invention comprises a cell or a population of cells expressing a first fusion protein comprising the first PYP fragment as described hereinabove, or a truncated fragment thereof as described hereinabove, and a reporter protein; and a second fusion protein comprising the second PYP fragment as described hereinabove, or a truncated fragment thereof as described hereinabove, and a reporter protein. According to one embodiment, the kit as described hereinabove further comprises at least one fluorogenic HBR analog as described hereinabove.

The present invention also relates to a method for detecting an interaction between two biological molecules of interest in a sample, preferably two proteins of interest, comprising the steps of:

attaching a first fragment of PYP as described hereinabove, or a truncated fragment thereof as described hereinabove, to a first biological molecule of interest;

attaching a second fragment of PYP as described hereinabove, or a truncated fragment thereof as described hereinabove, to a second biological molecule of interest;

contacting the sample with a fluorogenic HBR analog as described hereinabove; and

detecting a fluorescence resulting from the binding of the fluorogenic HBR analog to the functional PYP, or a functional truncated fragment thereof, reconstituted upon interaction of the two biological molecules of interest;

thereby detecting the interaction of the two biological molecules of interest present in the sample, through the binding of the fluorogenic HBR analog to the functional PYP, or a functional truncated fragment thereof, reconstituted upon interaction of the two biological molecules of interest.

In one embodiment, the first biological molecule of interest is attached to the first PYP fragment as described hereinabove, or a truncated fragment thereof as described hereinabove, and the second biological molecule of interest is attached to the second PYP fragment as described hereinabove, or a truncated fragment thereof as described hereinabove.

Thus, in one embodiment, the method of the invention is for detecting an interaction in a sample between a first biological molecule of interest attached to the first PYP fragment, or a truncated fragment thereof, as described hereinabove and a second biological molecule of interest attached to the second PYP fragment, or a truncated fragment thereof as described hereinabove, and comprises the steps of: contacting the sample with a fluorogenic HBR analog as described hereinabove; and

detecting a fluorescence resulting from the binding of the fluorogenic HBR analog to the functional PYP, or a functional truncated fragment thereof, reconstituted upon interaction of the two biological molecules of interest;

thereby detecting the interaction of the two biological molecules of interest present in the sample, through the binding of the fluorogenic HBR analog to the functional PYP, or a functional truncated fragment thereof, reconstituted upon interaction of the two biological molecules of interest In one embodiment the first and second biological molecules of interest are identical. In one embodiment the first and second biological molecules of interest are different.

In a preferred embodiment, at least one of the biological molecules of interest is an amino acid chain such as a peptide, a polypeptide or a protein, preferably a protein. In such embodiment, at least one of the PYP fragments of the invention, or fragments thereof, may form with said at least one biological molecule a fusion or tagged protein.

In one embodiment, both the first and the second biological molecules of interest are amino acid chains such as a peptide, a polypeptide or a protein, preferably a protein.

The protein of interest may be a natural protein, a chimeric protein resulting from the fusion of various protein domains or a synthetic protein. In one embodiment, the protein of interest is an intracellular protein, a membrane protein, a cell surface protein present at least in part at the extra membranous surface, or a secreted protein.

In one embodiment, the two biological molecules of interest are reporter proteins.

As used herein, the term reporter proteins refer to two proteins, the interaction of which reflects a variation in the sample. Examples of variation that can be followed using reporter proteins include, but are not limited to, cell signaling, gene expression, calcium concentration, cell-cell interaction, cell movement, cell death, intracellular transport, secretion, cell-cycle phase, and circadian rhythm. According to one embodiment, the sample is a biological sample. In one embodiment the sample is an organism. In one embodiment, the organism is not a human organism. In one embodiment, the sample is a cell culture. Example of cells include yeast cells, bacteria cells, plant cells, animal cells. In one embodiment, the cells in the sample are alive. In one embodiment, the cells in the sample are fixed for microscopy and imaging.

Organisms considered in the context of the present invention are for instance, and without limitation, model organisms used in biomedical research such as for example, bacterium, yeast, fruit fly, nematode, zebrafish, mouse, rat, guinea pig, rabbit, and dog.

In one embodiment the previously produced, and optionally purified, PYP fragments of the invention, or truncated fragments thereof, attached to the biological molecules of interest, preferably proteins of interest, are injected inside the organism or cells.

In a preferred embodiment, the model organism or the cells in the sample, or a subset thereof, are modified to express the PYP fragments of the invention, or truncated fragments thereof, attached to the biological molecules of interest, preferably proteins of interest.

The skilled artisan is familiar with methods allowing to modify a model organism or cells in a culture in order for them to express a protein of interest. Examples of such methods include, without being limited to, transfection, electroporation, injection and transgenesis of nucleic acid sequences, such as a nucleic acid sequence as described hereinabove. Said methods also include, but are not limited to, transplantation, injection and co-culture of cells modified to express proteins of interest.

In one embodiment, the sample is an extract from cells modified to express the PYP fragments of the invention, or truncated fragments thereof, attached to biological molecules of interest, preferably proteins of interest. In one embodiment, the sample is a cell extract complemented with the PYP fragments of the invention, or truncated fragments thereof, attached to biological molecules of interest, preferably proteins of interest. In one embodiment, the sample is acellular or does not comprise cells producing a PYP fragment of the invention, or a truncated fragment thereof, attached to a biological molecule of interest. In such an embodiment, the PYP fragments of the invention, or truncated fragments thereof, attached to biological molecules of interest, preferably proteins of interest, previously produced, optionally purified, are added in the sample.

In one embodiment, the method for detecting an interaction between two biological molecules of interest in a sample, preferably two proteins of interest, comprises the steps of:

fusing a first fragment of PYP as described hereinabove, or a truncated fragment thereof as described hereinabove, to a first biological molecule of interest, preferably a first protein of interest;

fusing a second fragment of PYP as described hereinabove, or a truncated fragment thereof as described hereinabove, to a second biological molecule of interest, preferably a second protein of interest;

contacting the sample with a fluorogenic HBR analog as described hereinabove; and

detecting a fluorescence resulting from the binding of the fluorogenic HBR analog to the functional PYP, or a functional truncated fragment thereof, reconstituted upon interaction of the two biological molecules of interest, preferably two proteins of interest;

thereby detecting the interaction of the two biological molecules of interest, preferably two proteins of interest, present in the sample, through the binding of the fluorogenic HBR analog to the functional PYP, or a functional truncated fragment thereof, reconstituted upon interaction of the two biological molecules of interest, preferably two proteins of interest.

As mentioned above, methods for fusing a peptide or a polypeptide, i.e., a PYP fragment according to the invention, or a truncated fragment thereof, to a protein of interest are well-known. In one embodiment, the fluorogenic HBR analog is added in the sample at a concentration ranging from about 0.1 mM to about 50 mM, preferably ranging from about 1 mM to about 25 mM, more preferably ranging from about 1 mM to about 15 mM.

As used herein the term“interaction” means that the two biological molecules of interest, preferably the two proteins of interest, are sufficiently close for the two PYP fragments of the complementation system of the invention, or truncated fragments thereof, to bind each other and consequently form a functional PYP, or a functional truncated fragment thereof, able to bind a fluorogenic HBR analog.

According to one embodiment, the interaction is mediated by the direct binding of the two biological molecules of interest, preferably of the two proteins of interest, to each other.

According to one embodiment the interaction is indirect, i.e., is mediated by the binding of the two biological molecules of interest, preferably the two proteins of interest, to at least one other biological molecule. Examples of biological molecules that may induce the interaction of the two biological molecules of interest, preferably of the two proteins of interest, include, without being limited to, cations, anions, peptides, metabolites, secondary messengers, RNAs, DNAs, and proteins.

Thus, in one embodiment, the method of the invention comprises the steps of:

- attaching a first fragment of PYP as described hereinabove, or a truncated fragment thereof as described hereinabove, to a first biological molecule of interest;

attaching a second fragment of PYP as described hereinabove, or a truncated fragment thereof as described hereinabove, to a second biological molecule of interest;

contacting the sample with a fluorogenic HBR analog as described hereinabove; and

detecting a fluorescence resulting from the binding of the fluorogenic HBR analog to the functional PYP, or a functional truncated fragment thereof, reconstituted upon induction by a biological molecule of the interaction of the two biological molecules of interest;

thereby detecting the interaction of the two biological molecules of interest present in the sample, through the binding of the fluorogenic HBR analog to the functional PYP, or a functional truncated fragment thereof, reconstituted upon induction by a biological molecule of the interaction of the two biological molecules of interest.

In one embodiment, the interaction is transient.

In one embodiment, the method of the invention comprises a step of detecting a variation of the fluorescence resulting from the binding of the fluorogenic HBR analog to the functional PYP, or a functional truncated fragment thereof, reconstituted upon interaction of the two biological molecules of interest.

In one embodiment, the method of the invention comprises a step of detecting an increase of the fluorescence resulting from the binding of the fluorogenic HBR analog to the functional PYP, or a functional truncated fragment thereof, reconstituted upon interaction of the two biological molecules of interest.

In one embodiment, the method of the invention comprises a step of detecting a decrease of the fluorescence resulting from the unbinding of the fluorogenic HBR analog upon disassembly of a functional PYP, or a functional truncated fragment thereof, resulting from the destruction of the interaction between two biological molecules of interest. The person of the art is familiar with the techniques available to detect fluorescence in a sample and is able to choose among those depending on the sample, the excitation and emission spectra of the fluorophore(s) and the desired readout.

Such techniques include, but are not limited to, direct observation, fluorescence spectroscopy, flow cytometry, fluorescence microscopy, and fluorescence tomography. In one embodiment, the method of the invention is for monitoring in space the interaction between the biological molecules of interest, preferably the proteins of interest, in a sample. For example, the localization in the sample of the interaction between the biological molecules of interest, preferably proteins of interest, may be determined by:

(i) using a fluorescence detection technique allowing to determine the localization of the source of a fluorescence emission in a sample such as fluorescence tomography or fluorescence microscopy (including laser scanning- and spinning-disk based confocal microscopy, multiphoton-microscopy, super-resolution microscopy);

(ii) targeting the PYP fragments of the invention attached to the biological molecules of interest, preferably proteins of interest, to a particular cell population and/or subcellular compartment; and/or

(iii) controlling the diffusion of the HBR analog in the sample, notably by using either membrane-permeant or membrane-impermeant HBR analogs as described hereinabove.

In one embodiment, the method of the invention further allows to measure the amount of the interacting biological molecules of interest relative to a reference value by measuring fluorescence intensity. In one embodiment, said reference value corresponds to the fluorescence intensity value in a control sample. In one embodiment, said reference value corresponds to the fluorescence intensity value(s) at a different location(s) and/or time point(s) in the same sample. In one embodiment, the method of the invention is for monitoring over time the interaction between the biological molecules of interest, preferably proteins of interest, in a sample as described hereinabove.

In one embodiment, the method of the invention is for monitoring over time and/or space the interaction between the biological molecules of interest, preferably proteins of interest, in a sample as described hereinabove.

The present invention also relates to a screening method for identifying new a protein-protein interaction in a sample, comprising the steps of: fusing a first fragment of PYP as described hereinabove, or a truncated fragment thereof as described hereinabove, to a first protein, thereby tagging the first protein with the first PYP fragment;

fusing a second fragment of PYP as described hereinabove, or a truncated fragment thereof as described hereinabove, to a second protein, thereby tagging the second protein with the second PYP fragment;

contacting the sample with a fluorogenic HBR analog as described hereinabove; and

detecting a fluorescence resulting from the binding of the fluorogenic HBR analog to the functional PYP, or a functional truncated fragment thereof, reconstituted upon interaction of the two proteins;

thereby identifying a new protein-protein interaction between the two proteins present in the sample, through the binding of the fluorogenic HBR analog to the functional PYP, or a functional truncated fragment thereof, reconstituted upon interaction of the two proteins.

In one embodiment, the method of the invention is for screening new protein-protein interactions between a protein of interest and protein candidates which may interact with said protein of interest.

In one embodiment, the method of the invention is for screening protein-protein interactions induced by a biological molecule as described hereinabove, with examples of said biological molecule including, without being limited to, cations, anions, peptides, metabolites, secondary messengers, RNAs, DNAs, and proteins.

The present invention also relates to an assay relying on the detection of the interaction between two proteins in a sample, said assay comprising the steps of:

- obtaining a first tagged protein, wherein the protein is tagged with a first PYP fragment as described hereinabove, or a truncated fragment thereof as described hereinabove;

obtaining a second tagged protein, wherein the protein is tagged with a second PYP fragment as described hereinabove, or a truncated fragment thereof as described hereinabove; contacting the sample with a fluorogenic HBR analog; and detecting a fluorescence resulting from the binding of the fluorogenic HBR analog to the functional PYP, or a functional truncated fragment thereof, reconstituted upon interaction of the two tagged proteins;

thereby detecting the interaction of the two tagged proteins present in the sample, through the binding of the fluorogenic HBR analog to the functional PYP, or a functional truncated fragment thereof, reconstituted upon interaction of the two tagged proteins.

In one embodiment, the two proteins are reporter proteins.

In one embodiment, the assay of the invention is for assessing the capacity of a molecule of interest to stabilize or to inhibit protein-protein interactions.

Thus, according to one embodiment, the assay of the invention comprises the steps of: contacting the sample with a molecule of interest;

detecting the fluorescence resulting from the binding of the fluorogenic HBR analog to the functional PYP, or a functional truncated fragment thereof, reconstituted upon interaction of the two proteins in the presence of said molecule of interest;

comparing the fluorescence resulting from the binding of the fluorogenic HBR analog to the functional PYP, or a functional truncated fragment thereof, reconstituted upon interaction of the two proteins detected in the absence and in the presence of the molecule of interest;

thereby assessing the effect of said molecule of interest on the interaction of the two proteins.

In one embodiment, a decrease of the fluorescence detected in the presence of the molecule of interest, as compared to the fluorescence detected in the absence of the molecule of interest, is indicative of an inhibition or destabilization of the interaction of the two proteins by the molecule of interest.

In one embodiment, an increase of the fluorescence detected in the presence of the molecule of interest, as compared to the fluorescence detected in the absence of the molecule of interest, is indicative of an induction or stabilization of the interaction of the two proteins by the molecule of interest.

In one embodiment, by“decrease or increase of the fluorescence” it is meant decrease or increase of the fluorescence intensity. In one embodiment, the assay of the invention is for assessing a signaling pathway of interest, with the interaction of the two proteins depending on the activation or on the inactivation of the signaling pathway of interest.

In one embodiment, the assay of the invention is for detecting the activation of a signaling pathway. Thus, in one embodiment, the detection of a variation in the fluorescence resulting from the binding of the fluorogenic HBR analog to the functional PYP, or a functional truncated fragment thereof, reconstituted upon interaction of the two proteins, is indicative of an activation of the signaling pathway of interest.

In one embodiment, the assay of the invention is for detecting the inactivation of a signaling pathway. Thus, in one embodiment, the detection a variation of the fluorescence resulting from the binding of the fluorogenic HBR analog to the functional PYP reconstituted upon interaction of the two proteins, is indicative of an inactivation of the signaling pathway of interest.

In one embodiment, by“variation of the fluorescence” it is meant a decrease or an increase of the fluorescence intensity. In one embodiment, the assay of the invention is for detecting the modulation of a signaling pathway. In one embodiment, the assay of the invention is for assessing the capacity of a molecule of interest to modulate a signaling pathway of interest. In one embodiment, the assay of the invention is for assessing the capacity of a molecule of interest to activate or to inactivate a signaling pathway of interest Thus, according to one embodiment, the assay of the invention comprises the steps of: contacting the sample with a molecule of interest;

detecting the fluorescence resulting from the binding of the fluorogenic HBR analog to the functional PYP, or a functional truncated fragment thereof, reconstituted upon interaction of the two proteins in the presence of said molecule of interest;

comparing the fluorescence resulting from the binding of the fluorogenic HBR analog to the functional PYP, or a functional truncated fragment thereof, reconstituted upon interaction of the two proteins detected in the absence and in the presence of the molecule of interest;

thereby assessing the effect of said molecule of interest on the signaling pathway of interest on which depends the interaction of the two proteins.

The present invention also relates to an assay relying on the detection of the interaction between two proteins in a cell or cell population, said assay comprising the steps of: expressing a first tagged protein in the cell or cell population, wherein the protein is tagged with a first PYP fragment as described hereinabove, or a truncated fragment thereof as described hereinabove;

expressing a second tagged protein in the cell or cell population, wherein the protein is tagged with a second PYP fragment as described hereinabove, or a truncated fragment thereof as described hereinabove;

contacting the cell or cell population with a fluorogenic HBR analog; and detecting a fluorescence resulting from the binding of the fluorogenic HBR analog to the functional PYP, or a functional truncated fragment thereof, reconstituted upon interaction of the two tagged proteins in the cell or cell population;

thereby detecting the interaction of the two tagged proteins present in the cell or cell population, through the binding of the fluorogenic HBR analog to the functional PYP, or a functional truncated fragment thereof, reconstituted upon interaction of the two tagged proteins. According to one embodiment, the assay of the invention is for assessing the presence of an analyte of interest in the cell or cell population, with the interaction of the two proteins depending on the presence in the cell or cell population of the analyte of interest.

Thus, in one embodiment, detecting a fluorescence resulting from the binding of the fluorogenic HBR analog to the functional PYP, or a functional truncated fragment thereof, reconstituted upon interaction of the two proteins in the cell or cell population is indicative of the presence of the analyte of interest in the cell or cell population.

In one embodiment, the assay of the invention is for detecting a variation of calcium concentration in a cell or cell population. According to one embodiment, the assay of the invention is for assessing a signaling pathway of interest in the cell or cell population, with the interaction of the two proteins depending on the activation or on the inactivation of said signaling pathway in the cell or cell population.

Thus, in one embodiment, detecting a fluorescence resulting from the binding of the fluorogenic HBR analog to the functional PYP, or a functional truncated fragment thereof, reconstituted upon interaction of the two proteins in the cell or cell population is indicative of the activation or inactivation of the signaling pathway of interest in the cell or cell population.

In one embodiment, the assay of the invention is for detecting a modulation of the mitogen-activated protein kinase pathway in a cell or cell population.

According to one embodiment, the assay of the invention is for assessing a physiological mechanism of interest of the cell or cell population, with the interaction of the two proteins depending on said physiological mechanism of the cell or cell population.

Thus, in one embodiment, detecting a fluorescence resulting from the binding of the fluorogenic HBR analog to the functional PYP, or a functional truncated fragment thereof, reconstituted upon interaction of the two proteins in the cell or cell population is indicative of the activation or inactivation of the physiological mechanism of interest of the cell or cell population.

In one embodiment, the assay of the invention is for detecting apoptosis in a cell or cell population, for example through the detection of caspase-3 activation in the cell or cell population. BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1A-B are a schematic representation of the working principle (Fig. 1A) and design (Fig. IB) of the fluorescence complementation system of the invention. N and C represent the first and second PYP fragments according to the invention, respectively, able to interact and thus to reconstitute a functional PYP that can reversibly bind a fluorogen and turn on its fluorescence; X and Y represent the potentially interacting protein(s) of interest, to which the PYP fragments are fused. FAST corresponds to the PYP consisting of the amino acid sequence as set forth in SEQ ID NO: 9. NFAST corresponds to the first PYP fragment according to the invention consisting of the amino acid sequence as set forth in SEQ ID NO: 23. CFAST11 corresponds to the second PYP fragment according to the invention consisting of the amino acid sequence as set forth in SEQ ID NO: 34. CFAST10 corresponds to the second PYP fragment according to the invention consisting of the amino acid sequence as set forth in SEQ ID NO: 42. Split-FAST corresponds to the reconstituted functional PYP, i.e., FAST, resulting from the complementation of the two FAST fragments, NFAST and either CFAST11 or CFAST10.

Figure 2A-C are a set of graphs showing the normalized excitation and emission spectra of complexes formed of either FAST or split-FAST and the fluorogenic HBR analog HMBR: FAST:HMBR (Fig. 2A), splitFASTl 1 :HMBR (Fig. 2B), and splitFAST10:HMBR (Fig. 2C). split-FASTl 1 results from the complementation of NFAST and CFAST11; split-FAST 10 results from the complementation of NFAST and CFAST10.

Figure 3A-D are a set of graphs showing the flow cytometry analysis of E. coli cells expressing the indicated fusion proteins in absence or presence of the fluorogenic HBR analog HMBR: E3-CFAST(65-125) + NFAST(l-64)-K3 (Fig.3A), E3-CFASTQ 15-125) + NFAST(1-114)-K3 (Fig. 3B), K3-NFASTQ-64) + CFAST(65-125)-E3 (Fig. 3C), and K3-NFAST(1-114) + CFASTQ 15-125)-E3 (Fig. 3D) K3 and E3 are two proteins interacting with high affinity. Figure 4A-D are a set of histograms showing the relative in-cell brightness of various split-FAST with different fluorogenic HBR analogs as indicated (Fig. 4A: HMBR, Fig. 4B: HBR-3,5DM, Fig. 4C: HBR-30M, and Fig. 4D: HBR-3,5DOM) in function of the C -terminal fragment CFASTn (n = 11, 10 or 9) and the N-terminal fragment used. The iFAST label indicates when NFAST from iFAST ( i.e ., the first PYP fragment according to the invention consisting of the amino acid sequence NFAST with an isoleucine at position 107 instead of a valine as set forth in SEQ ID NO: 30) was used. If absent, NFAST from FAST was used (i.e., the first PYP fragment according to the invention consisting of the amino acid sequence as set forth in SEQ ID NO: 23). CFAST11, CFAST10 and CFAST9 correspond to the second PYP fragment according to the invention consisting of the amino acid sequence as set forth in SEQ ID NO: 34, SEQ ID NO: 42 and SEQ ID NO: 43, respectively. NFAST fragments were fused to FRB and CFAST fragments were fused to FKBP. Complementation was induced by addition of rapamycin. Split-FAST fluorescence was normalized by expression level using the fluorescence of co-expressed fluorescent proteins.

Figure 5A-G illustrate the detection of rapamycin-induced FRB -FKBP dimerization with the complementation system of the invention. Fig. 5A-B show HEK293T cells co-expressing FKBP-NFAST and FRB-CFASTn (n = 10 or 11) which were labeled with 5 mM HMBR (Fig. 5A) or 10 pM HBR-3,5DOM (Fig. 5B) and imaged before and after addition of 100 nM rapamycin. Scale bars 10 pm. Fig. 5C shows the fluorescence fold increase upon FKBP-FRB association (mean ± sem, n = 84, 83, 107, 112 cells respectively from 3-4 experiments). Fig. 5D shows the temporal evolution of the fluorescence intensity after rapamycin addition in HMBR-treated cells co-expressing FKBP-NFAST and FRB-CFASTl 1 (n = 11 cells). Fig. 5E shows the evolution of the cellular fluorescence of HMBR-labeled HEK293 cells expressing FAST, split-FASTl l (resulting from the complementation of NFAST and CFAST11), and split-FAST 10 (resulting from the complementation of NFAST and CFAST 10) upon imaging by confocal microscopy. Cells expressing FRB-NFAST and FKBP-CFASTn (n = 10 or 11) were treated with rapamycin to form split-FASTl l and split-FASTlO. Cells expressing FAST were used as control. HMBR concentration was 10 pM. Excitation at 488 nm at 6.3 kW/cm2. Six cells were analyzed per condition. Fig. 5F shows selected time lapse frames of representative HEK293 cells co-expressing FKBP-NFAST and FRB-CFASTn (n = 10 or 11) (labeled with 5 mM HMBR) upon addition of 100 nM rapamycin. Scale bars 20 pm. Fig. 5G shows selected frames of representative HEK293 cells co-expressing FKBP-NFAST and FRB-CFAST11 (labeled with 10 pM HBR-3,5DOM) upon addition of 100 nM rapamycin. Scale bars 20 pm.

Figure 6A-F illustrate the detection of rapamycin-induced dissociation of AP1510-induced FKBP homodimers with the complementation system of the invention. Fig. 6A-B show AP1510-treated HEK293T cells co-expressing FKBP-NFAST and FKBP-CFASTn (n = 10 or 11) which were labeled with 5 pM HMBR (Fig. 6A) or 10 pM HBR-3,5DOM (Fig. 6B) and imaged before and after addition of 1 pM rapamycin.

Scale bars 10 pm. Fig. 6C shows the fluorescence fold decrease upon FKBP-FKBP dissociation (mean ± sem, n = 142, 175, 219, 125 cells respectively from 3-4 experiments). Fig. 6D shows the temporal evolution of the fluorescence intensity after rapamycin addition in HMBR-labeled, AP1510-treated cells co-expressing FKBP-NFAST and FKBP-CFAST11 (n = 8 cells). Fig. 6E shows selected time lapse frames of representative AP 1510-treated HEK293 cells co-expressing FKBP-NFAST and FKBP-CFASTn (n = 10 or 11) (labeled with 5 pM HMBR) upon addition of 1 pM rapamycin. Scale bars 20 pm. Fig. 6F shows selected time lapse frames of representative AP1510-treated HEK293 cells co-expressing FKBP-NFAST and FKBP-CFAST11 (labeled with 10 pM HBR-3,5DOM) upon addition of 1 pM rapamycin. Scale bars 20 pm.

Figure 7A-D illustrate the detection of the dimerization of FKBP-FKBP homodimer and the dissociation of said FKBP-FKBP homodimer with the complementation system of the invention. Fig. 7A shows HMBR-labeled cells co-expressing FKBP-NFAST and FKBP-CFASTn (n = 11 or 10) which were first treated with 100 nM AP1510 for 160 min, then AP1510 was removed, and 1 pM rapamycin was added. Selected frames are shown, scale bars: 30 pm. Fig. 7B shows the temporal evolution of the fluorescence intensity upon sequential treatment of HMBR-labeled cells co-expressing FKBP-NFAST and FKBP-CFAST11 with AP1510 and then rapamycin (n = 8 cells). Fig. 7C-D show HMBR-labeled cells co-expressing FKBP-NFAST and FKBP-CFASTn (n = 11 (Fig. 1C) or n = 10 (Fig. 7D)) which were first treated with 100 nM AP1510 for 160 min (association phase), then AP1510 was removed, and 1 mM rapamycin was added (dissociation phase). Selected frames are shown, scale bars 30 pm.

Figure 8A-B illustrate the detection of the interaction between a membrane protein and a cytosolic protein with the complementation system of the invention. Fig. 8A shows HEK293T cells co-expressing Lynl 1-FRB-NFAST and FKBP-CFASTn (n = 10 or 11) which were labeled with 5 pM HMBR and imaged before and after addition of 100 nM rapamycin. Scale bars 10 pm. Fig. 8B shows the temporal evolution of the fluorescence intensity after rapamycin addition in HMBR-treated cells co-expressing Lynl 1-FRB-NFAST and FKBP-CFAST11.

Figure 9A-D are a set of images illustrating the use of split-FAST for imaging K-Ras/Rafl (Fig. 9A), MEK1/ERK2 (Fig. 9B) and ERK2/MKP1 (Fig. 9C) interactions. Representative images of cells co-expressing the indicated constructs were imaged in presence of 10 pM HMBR. Scale bar 20 pm. Fig. 9D shows controls relating to the use of split-FAST for imaging K-Ras/Rafl, MEK1/ERK2 and ERK2/MKP1 interactions. Representative cells co-expressing the indicated constructs were imaged in presence of 10 pM HMBR. Positive and negative controls are shown. Scale bars 10 pm. Split-FAST corresponds to the reconstituted functional PYP, i.e., FAST, resulting from the complementation of the two FAST fragments, NFAST and CFAST10. Figure 10A-B illustrate the use of split-FAST for imaging the evolution of MEK 1 /ERK2 interaction upon EGF stimulation. HMBR-labeled Hela cells co-expressing MEK1-NFAST and mCherry-ERK2-CFAST10 were imaged after stimulation with EGF. Fig. 10A depicts the experimental design. N and C represent the first and second PYP fragments according to the invention, respectively, able to interact and thus to reconstitute a functional PYP that can reversibly bind a fluorogen and turn on its fluorescence. Fig. 10B shows the temporal evolution of split-FAST fluorescence, cytoplasmic mCherry fluorescence and nuclear mCherry fluorescence intensities (mean ± sem, n = 6 cells, 5 experiments). Data were synchronized using the beginning of the nuclear import of mCherry-ERK2-CFAST10 as reference. Figure 11A-B illustrate the use of split-FAST for imaging of the Ca ²⁺ -dependent interaction of calmodulin (CaM) and the Ca ²⁺ -CaM-interacting peptide Ml 3. Fig. 11A depicts the experimental design. N and C represent the first and second PYP fragments according to the invention (NFAST and CFAST10, respectively) able to interact and thus to reconstitute a functional PYP that can reversibly bind a fluorogen and turn on its fluorescence. The sensor is composed of M13-NFAST and CFASTlO-CaM. Fig. 11B shows the temporal evolution of the intracellular fluorescence intensity for a representative HMBR-treated HeLa cell (n = 14 cells from 2 experiments) treated with histamine (histamine addition is shown by the arrow). Figure 12A-B illustrate the use of split-FAST for detecting caspase-3 activity. Fig. 12A depicts the experimental design. N and C represent the first and second PYP fragments according to the invention (NFAST and CFAST11, respectively) able to interact and thus to reconstitute a functional PYP that can reversibly bind a fluorogen and turn on its fluorescence. The sensor consists ofbFos-CFASTl l and bJun-NFAST-NLS3- DEVDG-mCherry-NES. Fig. 12B shows the temporal evolution of the nuclear split-FAST fluorescence intensity after treatment with staurosporine in HMBR-treated cells (n = 9 cells).

Figures 13A-I are a combination of graphs illustrating the use of complementation systems comprising PYP fragments of FAST or orthologs of FAST for detecting the rapamycin-induced dimerization of FRB and FKBP. Human embryonic kidney (HEK) 293T cells expressing FRB fused to a N-terminal PYP fragment ( i.e first PYP fragment of the invention) and FKBP fused to a C -terminal PYP fragment (i.e., second PYP fragment of the invention) of different complementation systems according to the present invention were incubated with the fluorogen HMBR at the following concentrations: 0, 1, 5, 10, 25, 50 mM. Cell fluorescence was analyzed in absence and in presence of 500 nM rapamycin by flow cytometry. The graphs show the fluorescence mean of split-FAST (with CFAST11) (Fig. 13A), split-FAST (with CFAST10) (Fig. 13B), split-iFAST (Fig. 13C), Oi-splitFAST derived from Halomonas boliviensis LC1 PYP (Fig. 13D), Ck-splitFAST derived from Halomonas sp. GFAJ-1 PYP (Fig. 13E), Ch-splitFAST derived from Rheinheimera sp. A13L PYP (Fig. 13F), Ck-splitFAST derived from Idiomarina loihiensis PYP (Fig. 13G), Os-splitFAST derived from Thiorhodospira sibirica ATCC 700588 PYP (Fig. 13H), and 0 ₆ -splitFAST derived from Rhodothalassium salexigens PYP (Fig. 131) at the indicated HMBR concentrations in presence and absence of rapamycin.

EXAMPLES

The present invention is further illustrated by the following examples.

Materials and Methods

Synthetic oligonucleotides used for cloning were purchased from Sigma Aldrich or Integrated DNA Technology. The sequences of the oligonucleotides used in this study are provided in Table 1. Polymerase chain reactions (PCRs) were performed with Q5 polymerase (New England Biolabs) in the buffer provided. PCR products were purified using QIAquick PCR purification kit (Qiagen). The products of restriction enzyme digestions were purified by preparative gel electrophoresis followed by QIAquick gel extraction kit (Qiagen). Restriction endonucleases, T4 ligase, Phusion polymerase, Taq ligase, and Taq exonuclease were purchased from New England Biolabs and used with their accompanying buffers according to manufacturer’ s protocols. Isothermal assemblies (Gibson assembly) were performed using a homemade mix prepared according to Gibson et al, Nat. Meth. 6, 343-345 (2009). Small-scale isolation of plasmid DNA was done using QIAprep miniprep kit (Qiagen) from 2 mL of overnight culture. Large-scale isolation of plasmid DNA was done using the QIAprep maxiprep kit (Qiagen) from 150 mL of overnight culture. All plasmid sequences were confirmed by Sanger sequencing with appropriate sequencing primers (GATC -Biotech). Table 2 lists the plasmids used in this study. Peptides corresponding to CFASTl l-8 were purchased from Clini sciences at 98% purity and are acetylated and ami dated at the N and C termini. Rapamycin was purchased from Sigma Aldrich and dissolved in DMSO to a concentration of 3 mM. AP1510 was purchased from Clontech and dissolved in ethanol to a concentration of 0.5 mM. Human recombinant EGF was purchased from Sigma Aldrich and dissolved in 0.1% BSA to a concentration of 100 pg/mL. Staurosporine was purchased from Cell Signaling Technologies and dissolved in ethanol to a concentration of 1 mM. HMBR (4-hydroxy-3-methylbenzylidene rhodanine) and HBR-3,5DOM (4-hydroxy-3,5-dimethoxybenzylidene rhodanine) were provided by The Twinkle Factory under the references ^Lime and ^TF Coral (thetwinklefactory . com) . Table 1: list of oligonucleotides.

Table 2: list of plasmids.

Molecular cloning

Bacterial expression plasmids

The plasmid pAG209 was obtained by inserting the gene encoding for NFAST (amplified using primers agl26 and ag216) into plasmid pET28a using restriction enzymes Nhe I and Xho I.

FRB-FKBP fusion plasmids for mammalian expression

In general, fusion proteins were constructed by PCR assembly and contain an 11 amino acid linker, SGGGGSGGGGS (SEQ ID NO: 45), between the two proteins. The plasmid pAG148 was obtained by inserting the gene encoding FRB-NFAST (the sequence coding for FRB-NFAST was assembled by PCR from the sequences coding for FKBP-rapamycin binding domain of mTOR (FRB) and NFAST amplified with the primers agl81/agl82 and agl75/agl76) into plasmid pAG1042 using the restriction enzymes, Bgl II and Not I. The plasmid pAG149 was generated by inserting the gene encoding FKBP-NFAST (the sequence coding for FKBP-NFAST was assembled by PCR from the sequences coding for FK506 binding protein (FKBP) and NFAST amplified with the primers agl83/agl84 and agl75/agl76) into plasmid pAG104 using the restriction enzymes Bgl II and Not I. The plasmid pAG152 was obtained by inserting the gene encoding FRB-CFAST11 (synthesized by Eurofms Genomics) into pAG104 using restriction enzymes Bgl II and Not I. The plasmid pAG153 was generated by inserting the gene coding for FKBP (amplified using primers agl83 and agl84) into pAG152 using restriction enzymes Bgl II and BspE I. The plasmid pAG241 was constructed by Gibson assembly of two fragments obtained by amplification of the plasmid pAG153 with the primers ag345/ag313 and ag347/ag314. The plasmid pAG336 was cloned by Gibson assembly of two fragments obtained by amplification of the plasmid pAG148 with the primers ag468/313 and ag469/314. To determine the photostability of split-FAST in cells, the plasmid pAG439 encoding FRB -NF AST-P2 A-mCherry was generated by Gibson assembly of the sequences of

FRB-NFAST (amplified from pAG148 with primers ag308 and ag313) and mCherry (amplified from pAG962 with ag412 and ag550) assembled with the plasmid backbone of pAG104 (amplified using primers and ag347 and ag314). The plasmid pAG496 encoding FKBP-CF AST 10-IRES-mCherry was cloned from the plasmid pAG241 (amplified using ag700 and ag313), mCherry (amplified using ag701 and ag314), and the IRES sequence amplified from the plasmid pIRES (using primers ag694 and ag695) via Gibson assembly. The plasmid pAG490 encoding FRB-NFAST-IRES-mTurquoise2 was cloned from the plasmid pAG148 (amplified using ag697 and ag313), the IRES sequence amplified from the plasmid pIRES (using ag694 and ag695), and a g-block encoding mTurquoise2 (IDT). The plasmid pAG491 encoding lynl 1-FRB-NFAST-IRES- mTurquoise2 was cloned from the plasmid pAG336 (amplified using ag697 and ag313), the IRES sequence amplified from the plasmid pIRES (using ag694 and ag695), and a g-block encoding mTurquoise2 (IDT). Signaling pathway plasmids

The genes coding for NFAST-MKP1 (MKP1 GenBank accession number: NM_004417.3), NFAST-KRas (K-Ras GenBank accession number: NM_004985.4), and CF AST 10-Raf 1 -mCherry (Rafl GenBank accession number: NM_001354689.1) were synthesized by Eurofms genomics. The plasmids pAG301, pAG341 and pAG340 were generated via Gibson assembly of the sequences of NFAST-MKP1 (amplified using ag357/ag412), NFAST-KRas (amplified using ag357/ag475), and CFASTIO-Rafl- mCherry (amplified using ag474/ag412), and the backbone of pAG104 amplified in two fragments using primers ag311/ag314 and ag358/ag313. The genes coding for MEK1, ERK2, and mCherry were amplified using primers ag418/419, ag416/417, and ag414/415, respectively, and assembled via Gibson assembly with the corresponding fragments of NFAST (amplified with primers 374/313) and CFAST10 (amplified with primers 413/314) to generate the plasmids pAG298 and pAG296 encoding NFAST-MEK1 and mCherry-ERK2-CFAST10, respectively. The plasmids pAG492, pAG493 and pAG494 were constructed by Gibson assembly from the initial plasmid encoding the signaling pathway partner pAG298, pAG301, pAG341 (amplified with primers 697/313, 696/314, 699/313, 698/313), the IRES sequence (amplified from the plasmid pIRES using ag694 and ag695), and a g-block encoding mTurquoise2 (IDT).

Sensor construction The plasmid pAG334 was generated from the plasmid pAG148 by Gibson assembly by amplifying NFAST using primers ag455/ag456 with M13 encoded, and the backbone of pAG148 with primers ag313/ag314. The plasmid pAG335 was generated by Gibson assembly by amplification of calmodulin using primers ag465 and ag466, and inserted into the plasmid pAG144 by amplifying CFAST10 with primers ag467 and ag313 and assembling with plasmid amplified with ag311 and ag314. The genes encoding bJun-NFAST (bJun GenBank accession number: NM_021835.3) and bFos-CFASTl l (bFos GenBank accession number: M34001.1) were synthesized by Eurofms genomics. The plasmid pAG384 was cloned by Gibson assembly by amplification of the gene encoding bFos-CFASTl l (using primers ag541/542), and the backbone of pAG104 (using primers ag358/ag313 and ag347/314). The plasmid pAG385 was generated by amplifying the gene coding for bJun-NFAST using primers ag543/544, followed by a Gibson assembly with the sequence of mCherry amplified with ag545/546, the primer ag535 for NLSx3 and the backbone of pAG104 amplified using the primers ag358/313 and ag539/ag314. Protein expression and purification

Expression vectors were transformed in Rosetta (DE3) pLysS E. coli (New England Biolabs). Cells were grown at 37°C in LB medium complemented with 50 pg/mL kanamycin and 34 pg/mL chloramphenicol to OD6oonm 0.6. Expression was induced for 4 hours by adding isopropyl b-D-l-thiogalactopyranoside (IPTG) to a final concentration of 1 mM. Cells were harvested by centrifugation (4,000 x g for 20 min at 4°C) and frozen. The cell pellet was resuspended in lysis buffer (phosphate buffer 50 mM, NaCl 150 mM, MgC12 2.5 mM, protease inhibitor, DNase, pH 7.4) and sonicated (5 min at 20 % of amplitude, 3 sec on, 1 sec off). The lysate was incubated for 2 hours at 4 °C to allow DNA digestion by DNase. Cellular fragments were removed by centrifugation (9200 x g for 1 hour at 4°C). The supernatant was incubated overnight at 4°C under gentle agitation with Ni-NTA agarose beads in phosphate buffered saline (PBS) (sodium phosphate 50 mM, NaCl 150 mM, pH 7.4) complemented with 10 mM imidazole. Beads were washed with 20 volumes of PBS containing 20 mM imidazole, and with 5 volumes of PBS complemented with 40 mM imidazole. His-tagged proteins were eluted with 5 volumes of PBS complemented with 0.5 M imidazole. The buffer was exchanged to

PBS (50 mM phosphate, 150 mM NaCl, pH 7.4) using PD- 10 desalting columns.

Physico-chemical measurements

Steady state UV-Vis absorption spectra were recorded using a Cary 300 El V- Vis spectrometer (Agilent Technologies), equipped with a Versa20 Peltier-based temperature-controlled cuvette chamber (Quantum Northwest) and fluorescence data were recorded using a LPS 220 spectrofluorometer (PTI, Monmouth Junction, NJ), equipped with a TLC50TM Legacy /PTI Peltier-based temperature-controlled cuvette chamber (Quantum Northwest). Thermodynamic dissociation constants for NFAST :CFASTn (n = 8 to 11) couples were determined using peptides synthesized for CFASTn (n = 8 to 11) and recombinantly purified NFAST. The affinity for NFAST:CFAST11 in the presence of 10 mM HMBR was determined independently from a minimum of three different purifications of NFAST. NFAST:CFAST11 was then run in parallel as an internal control for the determination of the other NFAST-CFAST combinations, which were all performed on the same day with the same preparation of NFAST. Thermodynamic dissociation constants were determined with a Spark 10M plate reader (Tecan) and fit in Prism 6 to a one- site specific binding model. Mammalian cell culture

HEK 293T cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with phenol red, Glutamax I, and 10% (vol/vol) fetal calf serum (FCS), at 37 °C in a 5% C02 atmosphere. HeLa cells were cultured in Modified Eagle Medium (MEM) supplemented with phenol red, IX non-essential amino acids, IX sodium pyruvate, and 10% (vol/vol) fetal calf serum at 37 °C in a 5% C02 atmosphere. For imaging, cells were seeded in pDish IBIDI (Biovalley) coated with poly-L-lysine. Cells were transiently transfected using Genejuice (Merck) or Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol for 24 hours prior to imaging.

Fluorescence microscopy Confocal micrographs were acquired on a Zeiss LSM 710 Laser Scanning Microscope equipped with a Plan Apochromat 63x/1.4 NA oil DIC M27 immersion objective, heated stage, and XL-LSM 710 SI incubation chamber for temperature and C02 control. Images were acquired using ZEN software and processed in Fiji (ImageJ).

Photobleaching measurements for HMBR were carried out at 10 mM fluorogen at 488 nm excitation (4.6 kW / cm2, 1.27 psec pixel dwell); FAST was used as a control. Samples were imaged continuously for 1000 images at a 1 frame per second frequency.

To image the rapamycin-mediated interaction between FRB and FKBP, the cells were imaged in DMEM without phenol red supplemented with 5 pM HMBR or 10 mM HBR-3,5DOM. Tile images were taken before rapamycin addition. A solution of rapamycin, prepared in fluorogen-containing DMEM in order to maintain fluorogen concentration constant, was added to obtain a final rapamycin concentration of 100 nM and images were taken every minute. A final tile image was taken after fluorescence saturation.

To image the AP1510-mediated interaction, AP1510 was added to a final concentration of 100 nM to the cells for 2 hours before imaging. The cells were rinsed and the media was replaced with DMEM without phenol red, supplemented with 5 pM HMBR or 10 mM HBR-3,5DOM. Tile images were taken before rapamycin addition. A solution of rapamycin (prepared in DMEM supplemented with fluorogen in order to maintain fluorogen concentration constant) was added to obtain a final rapamycin concentration of 1 pM and images were taken every 30 seconds. A final tile image was taken after fluorescence ceased changing.

To image the association and dissociation of FKBP-FKBP in the same sample, optiMEM (Gibco) was supplemented with 5 pM HMBR and AP1510 was added to a final concentration of 100 nMjust before the beginning of imaging. The cells were maintained at 37 °C and 5% C02 over the duration of the experiment. Images were taken every 5 minutes until the fluorescence signal saturated. The acquisition was then paused and the sample was washed with IX DPBS (supplemented with HMBR in order to maintain fluorogen concentration constant) and the imaging solution was replaced. The acquisition frequency was reduced to 2 images per minute and 7-10 images were acquired before a solution of rapamycin, prepared in optiMEM (supplemented with HMBR in order to maintain fluorogen concentration constant), was added to obtain a final rapamycin concentration of 1 pM. To image interactions in the Raf-MEK-ERK pathway, the cells were serum-starved for 24 hours before imaging after transfection. The cells were imaged in DMEM without phenol red supplemented with 10 pM HMBR. For time course experiments, the pathway was activated using purified EGF added to a final concentration of 200 ng/mL. Calcium imaging was performed in HHBSS (HEPES-Buffered Hanks Balanced Salt Solution) supplemented with 5 mM HMBR. Calcium oscillations were triggered using 50 mM histamine in HHBSS (supplemented with HMBR in order to maintain fluorogen concentration constant) and images were acquired every 500 ms. To image caspase activity, the cells were imaged at 37 °C in 5% C02 in optiMEM supplemented with 5 pM HMBR. Just before the start of acquisition, staurosporine was added to a final concentration of 2 pM. Images were acquired every 5 minutes over 3 hours.

Influence of the length of CFAST on in-cell brightness of complexes HEK 293T cells were seeded in ibidi pDish microscopy dishes and 24 hours prior to imaging were co-transfected with plasmids encoding CMV-FRB-NFAST (or NF AST(V 107I))-P2 A-mCherry (or EGFP)) and CMV-FKBP-CFASTn (n = 11, 10 or 9). The cells were imaged before and after addition of 500 nM rapamycin.

Evaluation of split site efficiency in E. coli The de novo designed peptides IAAL-E3 and IAAL-K3 form antiparallel alpha helical coiled coils that interact constitutively with an affinity of 70 nM. The interacting system was expressed in a bis-cistronic vector with two T7 promoters for simultaneous expression in E. coli. The genes encoding IAAL-E3-CFAST(65-125)-T7p- NF AST( 1 -64)-I AAL-K3 , IAAL-K3_NFAST(l-64)-T7p-CFAST(65-125)-IAAL-E3, IAAL-E3 -CF AST(115- 125)-T7p-NF AST( 1-114)-IAAL-K3 , IAAL-K3_NFAST(1-114)-

T7p-CFAST(115-125)-IAAL-E3 were purchased (Eurofms genomics) and inserted using the restriction enzymes Nco I and Xho I into the plasmid, pET28a.

The resulting plasmids were transformed into E. coli BL21 Rosetta cells. Overnight pre cultures were used into inoculate 5 mL cultures, which were then grown to OD -0.6 and induced with 1 mM IPTG for two hours. The cytometry samples were prepared using 1.5 mL of culture that was pelleted and then washed in IX PBS + BSA (1 g/L). The samples were then resuspended in 1.5 mL PBS + BSA with HMBR and 50,000 events were analyzed on a BD Accuri c6 cytometer. Example 1: generation of the split-FAST fluorescence complementation system

The complementation system split-FAST (Fig. 1A) was engineered from the PYP-derived Fluorescence- Activating and absorption Shifting Tag (FAST - amino acid sequence SEQ ID NO: 9), a small protein of 14 kDa that specifically and reversibly binds fluorogenic hy droxyb enzy li dene rhodanine (HBR) analogs displaying various spectral properties. Fluorogenic HBR analogs are weakly fluorescent in solution but strongly fluoresce when immobilized in the binding cavity of FAST. The design is based on the splitting of FAST in two fragments between amino acids 114 and 115 (Fig. IB). The two fragments 1-114 (hereafter called NFAST - SEQ ID NO: 23) and 115-125 (hereafter called CFAST11 - SEQ ID NO: 34) showed modest affinity in presence ofHMBR (which provides green-yellow fluorescence) or HBR-3,5DOM (which provides orange-red fluorescence). The apparent affinity of the two fragments could be further decreased by the successive removal of residues at the C terminus of CFAST11, resulting in CFAST10 - SEQ ID NO: 42; CFAST9 - SEQ ID NO: 43 and CFAST8 - SEQ ID NO: 44 (Table 3). Table 3: affinities of the split fragments in the presence of fluorogens

As shown in Fig. 2, the excitation and emission spectra of the complemented split- FAST:fluorogen assembly (resulting from the complementation of NFAST and CFAST11 (Fig. 2B), and of NFAST and CFAST10 (Fig. 2C)) were identical to those of the regular FAST:fluorogen complex (Fig. 2A). The results of Fig. 2 thus demonstrate that, once complemented, the complementation system of the invention behaves as the full-length FAST in terms of binding of fluorogenic HBR analogs, induction of fluorescence following said binding and photophysical properties. The complementation system of the invention thus allows the reconstitution of a functional PYP (e.g., split-FASTl 1), or a functional truncated fragment thereof (e.g., split-FAST 10). The design of the fluorescence complementation system of the invention was validated by comparing the fluorescence intensity obtained in E. coli cells when a pair of proteins interacting with high affinity (i.e., E3 and K3) were fused to different fragments of the PYP-derived FAST (Fig. 3). A first PYP fragment, i.e., NFAST, was fused to K3 and a second PYP fragment, i.e., CFAST, was fused to E3. FAST was split in two fragments between amino acids 114 and 115 and the resulting fragments FASTI-114 (i.e., NFAST) and FAST115-125 (i.e., CFAST11) were fused to K3 and E3, respectively (either NFAST-K3 or K3 -NFAST, and E3 -CFAST or CFAST-E3). The combinations of either NFAST-K3 and E3 -CFAST 11 (Fig. 3B) or K3 -NFAST and CFAST11-E3 (Fig. 3D) led to an increase in fluorescence intensity in the presence of HMBR compared to the fluorescence intensity obtained without HMBR. By contrast, when FAST was split in two fragments between amino acids 64 and 65 and the resulting fragments FASTI-64 and FAST65-125 were fused to K3 or E3, no such increase was observed for any of the combinations tested (Fig. 3A & 3C). An alternative N-terminal fragment was also developed using a variant ofFAST (referred to as improved FAST or iFAST - amino acid sequence SEQ ID NO: 16), wherein the valine at position 107 is replaced by an isoleucine. Using different fluorogenic HBR analogs, the relative in-cell brightness of various split-FAST was assessed (Fig. 4).

As shown in Fig. 4, with any of the fluorogenic HBR analogs tested: HMBR (Fig. 4A), HBR-3,5DM (Fig. 4B), HBR-30M (Fig. 4C) and HBR-3,5DOM (Fig. 4D), the fluorescence observed was highest with CFAST11, lower with CFAST10 and lowest with CFAST9. The lower affinity between the two fragments (see Table 3 above) coincides with a lower in-cell brightness.

As shown in Fig. 4, the fluorescence observed with a system comprising NFAST (SEQ ID NO: 23) and CFAST is similar to that observed with a system comprising the corresponding Nter fragment of iFAST (SEQ ID NO: 30) and CFAST. These data thus demonstrate the suitability of iFAST in a complementation system according to the invention. Example 2: split-FAST allows the characterization of dynamic and reversible protein interactions

To test the ability of split-FAST to detect protein interactions in mammalian cells (pretreated with fluorogenic HBR analogs), NFAST and CFASTn (n = 10 or 11) were fused to the FK506 binding protein (FKBP) and to the FKBP-rapamycin binding domain of mTOR (FRB), respectively. FKBP and FRB interact together in the presence of rapamycin.

As shown in Fig. 5, rapamycin-induced FRB -FKBP dimerization led to a large fluorescence increase, in accordance with interaction-dependent complementation of split-FAST. The use of either HMBR or HBR-3,5DOM gave similar results (Fig. 5A-C), demonstrating that the color of split-FAST can be tuned by changing the nature of the fluorogen added.

Time lapse imaging after rapamycin addition showed fluorescence saturation within a few minutes, in agreement with the rapid formation of the FRB-FKBP-rapamycin complex (Fig. 5D, F and G). These results thus demonstrate that split-FAST can monitor protein complex formation in real-time. In cells, split-FAST :fluorogen assemblies (with either CFAST11 or CFAST10) were furthermore shown to be as photostable as the regular F AST:fluorogen complex (Fig. 5E).

The ability of rapamycin to dissociate AP1510-induced FKBP homodimers was used to test the reversibility of split-FAST. Cells co-expressing FKBP-NFAST and FKBP-CFASTn (n = 10 or 11) were incubated with AP1510 for two hours to pre-form FKBP homodimers and treated with HMBR. Addition of rapamycin led to a significant loss of split-F AST:fluorogen fluorescence in agreement with FKBP homodimer dissociation (Fig. 6A-C). These results thus demonstrate that the split-FAST :fluorogen assembly is reversible. Rapid loss of fluorescence within a few minutes was observed after rapamycin addition, demonstrating the rapid disassembly of split-FAST when two proteins dissociate (Fig. 6D, E and F). The ability of split-FAST to image dynamic and reversible protein interactions was further demonstrated by monitoring in a single experiment, first, the association of FKBP-NFAST and FKBP-CFASTn (n = 11 or 10) upon addition of AP1510, and then, the dissociation of the FKBP-FKBP homodimer by removal of AP1510 and addition of rapamycin (Fig. 7A-D).

In conclusion, the data presented hereinabove demonstrate that split-FAST is a reversible complementation system that allows the real-time monitoring of both the association and the dissociation of proteins of interest. Moreover, the NFAST and CFAST fragments are characterized by a low affinity, resulting in a limited self-assembly and thus in a low unspecific fluorescence background.

Example 3: split-FAST allows the detection of the interaction between a membrane protein and a cytosolic protein. As many protein interactions occur at the plasma membrane, the use of split-FAST to detect the interaction between a membrane protein and a cytosolic protein was next tested. FKBP-CFASTn (n = 11 or 10) was expressed in the cytosol and FRB-NFAST at the plasma membrane using a Lynl 1 membrane-anchoring sequence (Lynl 1-FRB -NFAST). Addition of rapamycin led to the rapid formation of fluorescent split-F AST:fluorogen assemblies at the plasma membrane in HMBR-treated cells (Fig. 8A-B), demonstrating the ability of split-FAST to detect proteins interactions at the plasma membrane in real-time.

Example 4: split-FAST enables the observation of dynamic protein interactions in a signaling pathway in real time. Split-FAST was then benchmarked with known, physiologically relevant protein interactions from the mitogen-activated protein kinase (MARK) signaling pathway. NFAST was fused to K-Ras, a small GTPase downstream of growth factor receptors, and CFAST10 was fused to an mCherry fusion of Rafl, known to be recruited to the membrane by interaction with K-Ras. The fluorescence of split-FAST :HMBR was colocalized with that of mCherry and concentrated at the membrane, in agreement with a specific recruitment of Rafl at the plasma membrane by K-Ras (Fig. 9A). Next, the interaction between the MAP kinase kinase MEK1, and the downstream extracellular signal-regulated protein kinase ERK2, one of the central interactions in the Raf/MEK/ERK signaling pathway was assessed using split-FAST. When MEKl was fused to NFAST (MEK1-NFAST) and a mCherry fusion of ERK2 was fused to CFAST10 (mCherry-ERK2-CF AST 10), a specific, cytosolic split-FAST :HMBR fluorescence was observed in accordance with MEK1 anchoring ERK2 in the cytosol (Fig. 9B). Finally, split-FAST allowed to detect the nuclear interaction between ERK2 and MKP1 (DEiSPl), which is a phosphatase localized in the nucleus responsible for deactivating ERK2 after its activation and subsequent translocation to the nucleus (Fig. 9C). Controls relating to the use of split-FAST for imaging the K-Ras/Rafl, MEK1/ERK2 and ERK2/MKP1 interactions are shown in Fig. 9D.

To explore the applicability of split-FAST to study dynamic protein interactions, interaction between MEK1 and ERK2 upon activation of the MAPK signaling pathway was followed. Upon cell stimulation, MEK1 phosphorylates ERK2, which detaches from MEK1 and translocates to the nucleus, where it regulates the activity of transcription factors. Dephosphorylation by nuclear phosphatases deactivates ERK2, returning it to the cytoplasm. In resting HMBR-treated cells expressing MEK1 -NFAST and mCherry-ERK2-CFAST10, mCherry and split-FAST :HMBR fluorescence were cytoplasmic, in agreement with MEK1 anchoring ERK2 in the cytoplasm. Upon cell stimulation with epidermal growth factor (EGF), mCherry -ERK2-CF AST 10 dissociated from MEK1-NFAST and translocated to the nucleus, as shown by the simultaneous loss of split-FAST :HMBR fluorescence and the nuclear accumulation of mCherry fluorescence. The nuclear accumulation of mCherry -ERK2-CFAST10 was transitory: desensitized mCherry -ERK2-CFAST10 returned to the cytoplasm and re assembled with MEK1 -NFAST, as revealed by the simultaneous increase of split-FAST :HMBR fluorescence and cytosolic mCherry fluorescence (Fig. 10A-B). This experiment illustrates how split-FAST can be used to observe dynamic protein interactions in signaling pathways in real-time.

Example 5: split-FAST enables the observation of transient and short-lived interactions.

To further demonstrate the use of split-FAST for the detection of rapid and transient interactions, the Ca ²⁺ -dependent interaction between calmodulin (CaM) and the Ca ²⁺ -CaM-interacting peptide M13 was monitored. In HMBR-treated HeLa cells expressing CFASTlO-CaM and M13-NFAST, addition of histamine led to a large increase of split-F AST -HMBR fluorescence followed by rapid oscillations of the fluorescence signals and eventually desensitization (Fig. 11A-B). This response was in agreement with the known change in Ca ²⁺ concentration in mammalian cells upon histamine stimulation, and demonstrated the ability of split-F AST to image transient, short-lived interactions.

Example 6: split-FAST enables the generation of cellular sensors.

To examine the utility of split-FAST for imaging other signaling processes, a caspase biosensor was created. The transcriptional regulator bFos was fused to CFAST (bFos-CFASTl l), and a gene encoding bJun-NFAST-NLS3-DEVDG-mCherry-NES was constructed, where NES is a genetically fused nuclear export signal, DEVD is the caspase-3 substrate sequence Asp-Glu-Val-Asp, NLS is a nuclear localization signal, and bJun is a peptide known to form a heterodimer with bFos (Fig. 12A). Induction of apoptosis (and thus caspase-3 activity) by treatment with staurosporine released bJun- NFAST from mCherry-NES, resulting in the translocation ofbJun-NFAST to the nucleus, and the subsequent complementation of split-FAST by interaction of bJun and bFos. Approximately 1-2 hour(s) after the induction of apoptosis, the red fluorescence of mCherry was segregated in the cytoplasm and the bright green fluorescence of complemented split-FAST :HMBR appeared in the nucleus (Fig. 12B). Beyond further demonstrating the potential of split-FAST to monitor protein interactions formation in real-time, this experiment showed the great potential of split-FAST for the design of cellular sensors.

To conclude, the data presented hereinabove demonstrate that split-FAST, a split reporter displaying rapid and reversible complementation, allows one to observe transient protein interactions in real-time. Split-FAST :fluorogen fluoresces green-yellow or orange-red light depending on the fluorogen used, thus providing a system adaptable to multi-color imaging. Split-FAST allows the observation of protein interactions in various cellular compartments (cytosol, nucleus, plasma membrane) and, in contrast to traditional BiFC systems, allows the monitoring of both the formation and dissociation of protein assembly in real-time. This unprecedented behavior can be exploited to study the role and function of protein interactions in various cellular processes and dissect complex interaction networks.

Example 7: complementation systems with orthologs of FAST. FAST, iFAST and 6 functional PYP deriving from the orthologs On (n = 1-6) were split into two complementary fragments in their last loop between residues 114 and 115 as indicated above. The 6 functional PYP deriving from the orthologs Oi- ₆ have an amino acid sequence with at least 70% identity with the amino acid sequence of FAST (SEQ ID NO: 9). As used hereinafter:

Oi refers to a functional PYP deriving from Halomonas boliviensis LC 1 PYP and having the sequence set forth in SEQ ID NO: 10;

O2 refers to a functional PYP deriving from Halomonas sp. GFAJ-1 PYP and having the amino acid sequence set forth in SEQ ID NO: 11;

- O3 refers to a functional PYP deriving from Rheinheimera sp. A13L PYP and having the amino acid sequence set forth in SEQ ID NO: 12;

O4 refers to a functional PYP deriving from Idiomarina loihiensis PYP and having the amino acid sequence set forth in SEQ ID NO: 13;

Os refers to a functional PYP deriving from Thiorhodospira sibirica ATCC 700588 PYP and having the amino acid sequence set forth in SEQ ID NO: 14; and

Oό refers to a functional PYP deriving from Rhodothalassium salexigens PYP and having the amino acid sequence set forth in SEQ ID NO: 15.

The N-terminal fragments (corresponding to residues 1-114) obtained from FAST, iFAST and the 6 orthologs O1-6 were called NFAST, N-iFAST and O1-6NFAST, respectively. As previously indicated, the NFAST and N-iFAST fragments have the amino acid sequences as set forth in SEQ ID NO: 23 and SEQ ID NO: 30, respectively. O1-6NFAST have the amino acid sequences as set forth in SEQ ID NO:24 (OiNFAST), SEQ ID NO: 25 (O2NFAST), SEQ ID NO: 26 (O3NFAST), SEQ ID NO: 27 (O4NFAST), SEQ ID NO: 28 (OsNFAST) and SEQ ID NO: 29 (OeNFAST).

The C -terminal fragments (corresponding to residues 115- 125) obtained from FAST and iFAST were identical and called CFAST11, the C -terminal fragments (corresponding to residues 115- 125) obtained from the 6 orthologs O1-6 were called O1-6CFAST. For split-FAST, a truncated C -terminal fragment (residues 115-124, named CFAST10) was also tested. As previously indicated, the CFAST11 and CFAST10 fragments have the amino acid sequences as set forth in SEQ ID NO: 34 and SEQ ID NO: 42, respectively. O1-6CFAST have the amino acid sequences as set forth in SEQ ID NO:35 (OiCFAST), SEQ ID NO: 36 (O2CFAST), SEQ ID NO: 37 (O3CFAST), SEQ ID NO: 38 (O4CFAST), SEQ ID NO: 39 (OsCFAST) and SEQ ID NO: 40 (OeCFAST).

To test the ability of the split-FAST, split-iFAST and split-Oi-6FAST complementation systems to detect protein-protein interactions in mammalian cells, their N-terminal fragment ( i.e ., first PYP fragment according to the present invention) was fused to the C -terminus of the FKBP-rapamycin-binding domain of mammalian target of rapamycin (FRB) and their C -terminal fragment (i.e., second PYP fragment according to the present invention) was fused at the C -terminus of the FK506-binding protein (FKBP). FKBP and FRB are known to interact together upon the addition of rapamycin. Addition of rapamycin is thus expected to induce the complementation of the split-FAST, split-iFAST and split-Oi-eFAST systems.

The following transfection reporters were used to easily detect doubly transfected cells: mTurquoise2 (which provides cyan fluorescence) and iRFP670 (which provides far-red fluorescence). Internal ribosome entry site (IRES)-containing bi-cistronic vectors were generated, allowing the simultaneous expression of FKBP fusions and iRFP670 separately from a single RNA transcript. Internal ribosome entry site (IRES)-containing bi-cistronic vectors were generated, allowing the simultaneous expression of FRB fusions and mTurquoise2 separately from a single RNA transcript.

Human embryonic kidney (HEK) 293T cells were transfected with the two bi-cistronic vectors. The transfected cells were incubated with the fluorogen HMBR at the following concentrations: 0, 1, 5, 10, 25, and 50 mM. Cell fluorescence was analyzed in absence and in presence of 500 nM rapamycin by flow cytometry. The mean fluorescence of doubly transfected cells was extracted for the different conditions.

As shown on Fig. 13, at a given HMBR concentration, with each of the complementation system tested, an increase of the mean cell fluorescence was observed upon addition of rapamycin, in accordance with an interaction-dependent complementation of the PYP fragments. The data thus demonstrate that the split-FAST (Fig. 13A-B), split-iFAST (Fig. 13C) and split-Oi- ₆ FAST (Fig. 13D-I) complementation systems can be successfully used to detect protein-protein interactions. Fig. 13 also shows that increasing HMBR concentration increased the self-assembly of the PYP fragments (, i.e ., fluorescence detected in the absence of rapamycin). However, the self-assembly of the PYP fragments remained low and little fluorescence was detected in the absence of rapamycin at lower concentration of HMBR, notably at HMBR concentrations of 25 mM or less, and in particular at HMBR concentrations of 10 mM or less.