Proximity guidance system and its use

Abstract

The present invention relates to a method for inducing proximity between two biomolecules in a sample, an assay that relies on inducing proximity between two biomolecules in a sample, and a proximity induction system comprising two polypeptides and a molecular proximity inducer.

Description

[Technical Field]

[0001] The present invention relates to the localization and, optionally, detection of biomolecules. In particular, the present invention relates to a proximity induction system that allows for induction and, optionally, detection of proximity between two biomolecules. The present invention also relates to the use of said proximity induction system. [Background technology]

[0002] Many essential biological processes, such as gene regulation, protein transport, signal transduction, or metabolism, are governed and regulated by the physical proximity / interaction, or proximity, between biomolecules such as proteins and / or nucleic acids. In particular, protein-protein proximity plays an essential and universal role in numerous biological processes.

[0003] Molecular tools that allow for the control and observation of protein proximity are essential for studying the functional role of the physical distance between two proteins. Therefore, various molecular tools have been developed to understand the role of proximity in cellular and physiological mechanisms. Most of these tools allow for either the control or observation of protein proximity.

[0004] In particular, chemically induced proximity (CIP) technology has been developed to enable precise temporal control of biological processes such as transcription, cell signaling, or protein localization. CIP technologies or systems generally rely on the genetic fusion of proteins to dimerization domains that interact in a specific manner in the presence of small molecules that function as chemical proximity inducers. An example of a CIP system is one based on the interaction of FK506-binding protein (FKBP) with the FKBP rapamycin-binding domain (FRB), which can be induced by rapamycin or synthetic analogs of rapamycin called rapalogs.

[0005] Most CIP systems aim to control protein proximity and do not allow for the detection of protein interactions induced by chemical proximity inducers and / or visualization of protein localization after proximity induction. Therefore, such detection and / or visualization usually require the use of an additional reporter, separate from the interacting dimerization domain and the chemical proximity inducer. Furthermore, CIP systems typically rely on two dimerization domains, each larger than 10 kDa, which can result in dysfunctional fusion proteins. For example, in rapamycin- or rapalog-based CIPs, FKBP is approximately 12 kDa and FRB is approximately 11 kDa. Finally, most CIP systems rely on chemical proximity inducers to induce the formation of very tightly bound ternary assemblies. These ternary assemblies are usually too stable to dissociate by washing out the inducer, preventing reversal of the recruitment process.

[0006] Thus, there is a need for improved proximity induction systems, particularly those that are robust, rapid, and fully reversible, allowing for the control and advantageous visualization of protein proximity with high temporal resolution.

[0007] The present inventors have surprisingly demonstrated that a first polypeptide consisting of 114 amino acid residues having the sequence set forth in SEQ ID NO:1 and a second polypeptide consisting of 11 amino acid residues having the sequence set forth in SEQ ID NO:2 can complement each other and form a ternary assembly with a small synthetic compound that functions as a molecular proximity inducer. In fact, the ternary assembly between these two polypeptides and the small synthetic compound is induced by the small synthetic compound regardless of the initial proximity between the two polypeptides of SEQ ID NO:1 and SEQ ID NO:2. Thus, in the absence of the molecular proximity inducer, the two polypeptides of SEQ ID NO:1 and SEQ ID NO:2 cannot complement each other. Thus, the two polypeptides of SEQ ID NO:1 and SEQ ID NO:2 and the molecular proximity inducer form a proximity inducer system that can be used to control the localization of biomolecules such as proteins.

[0008] The molecular proximity inducer may be a fluorescent chromophore that emits significant fluorescence only upon interaction with the two polypeptides of SEQ ID NO:1 and SEQ ID NO:2. Thus, the present proximity-inducing system advantageously allows for detection and visualization of ternary assembly without the need for an additional reporter. Furthermore, the present inventors have shown that ternary assembly between these two polypeptides and the molecular proximity inducer is extremely rapid and remains stable for extended periods of time unless the molecular proximity inducer is removed. Indeed, the present proximity-inducing system is fully reversible and can therefore be used to control the proximity between two proteins with high temporal resolution by adding and removing the molecular proximity inducer. As shown in the Experimental Section, its ability to induce and optionally detect proximity between two biomolecules makes the system described herein a suitable tool for biological and biomedical applications requiring control of the localization, transport, stability, and / or function of biomolecules. Summary of the Invention

[0009] The present invention provides a method for inducing proximity between two biomolecules in a sample, comprising the steps of: obtaining a first biological molecule bound to a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or an amino acid sequence having at least about 70% identity to SEQ ID NO: 1, or a truncated fragment thereof comprising at least 89 consecutive amino acid residues from the C-terminus of SEQ ID NO: 1 or a sequence having at least about 70% identity to SEQ ID NO: 1; obtaining a second biological molecule bound to a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO:2 or an amino acid sequence having at least about 70% identity to SEQ ID NO:2, or a truncated fragment thereof comprising at least 8 consecutive amino acid residues from the N-terminus of SEQ ID NO:2 or a sequence having at least about 70% identity to SEQ ID NO:2; A step of adding a molecular proximity inducer to the sample. Including, wherein upon addition of a molecular proximity inducer, the first polypeptide, the second polypeptide, and the molecular proximity inducer form a ternary assembly; This method induces proximity between a first and a second biomolecule present in the sample.

[0010] The present invention provides an assay that relies on inducing proximity between two biomolecules in a sample, comprising: obtaining a first biological molecule bound to a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or an amino acid sequence having at least about 70% identity to SEQ ID NO: 1, or a truncated fragment thereof comprising at least 89 consecutive amino acid residues from the C-terminus of SEQ ID NO: 1 or a sequence having at least about 70% identity to SEQ ID NO: 1; obtaining a second biological molecule bound to a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO:2 or an amino acid sequence having at least about 70% identity to SEQ ID NO:2, or a truncated fragment thereof comprising at least 8 consecutive amino acid residues from the N-terminus of SEQ ID NO:2 or a sequence having at least about 70% identity to SEQ ID NO:2; A step of adding a molecular proximity inducer to the sample. Including, wherein upon addition of a molecular proximity inducer, the first polypeptide, the second polypeptide, and the molecular proximity inducer form a ternary assembly; The present invention also relates to an assay whereby proximity is induced between a first and a second biomolecule present in the sample.

[0011] In some embodiments, the concentration of molecular proximity inducer required to reach half-maximal assembly in the sample is less than about 5 μM.

[0012] In some embodiments, the molecular proximity inducer is fluorescent, and the method or assay further comprises detecting fluorescence resulting from the formation of a ternary assembly between the first polypeptide, the second polypeptide, and the fluorescent molecular proximity inducer.

[0013] In some embodiments, the first polypeptide consists of the amino acid sequence set forth in SEQ ID NO:1 and the second polypeptide consists of the amino acid sequence set forth in SEQ ID NO:2.

[0014] In some embodiments, the molecular proximity inducer is a compound of formula (I): [ka] or a salt and / or solvate thereof, During the ceremony, R 1 , R 2 , R 5 and R 6 each independently represents H, halo, hydroxyl, aryl, alkyl, cycloalkyl, heteroalkyl (e.g., alkoxy), or heterocycloalkyl; wherein said aryl, alkyl, cycloalkyl, heteroalkyl, or heterocycloalkyl is optionally substituted with at least one group selected from halo, hydroxyl, oxo, nitro, amido, carboxy, amino, cyano, haloalkoxy, and haloalkyl; R 3 represents a non-bonded pair (i.e., a free electron pair), H, halo, hydroxyl, aryl, alkyl, cycloalkyl, heteroalkyl, or heterocycloalkyl; wherein said aryl, alkyl, cycloalkyl, heteroalkyl, or heterocycloalkyl is optionally substituted with at least one group selected from halo, hydroxyl, oxo, nitro, amido, carboxy, amino, cyano, haloalkoxy, and haloalkyl; R 4 represents a single or double bond interrupted or terminated by one S, O or N heteroatom optionally substituted with at least one group selected from H, hydroxyl, aryl, alkyl, cycloalkyl, heteroalkyl and heterocycloalkyl; wherein said aryl, alkyl, cycloalkyl, heteroalkyl, or heterocycloalkyl is optionally substituted with at least one group selected from halo, hydroxyl, oxo, nitro, amido, carboxy, amino, cyano, haloalkoxy, and haloalkyl; W is OH, SH, NHR 7 or NR 7 R 8 wherein R 7 and R 8 each independently represents H, halo, hydroxyl, aryl, alkyl, cycloalkyl, heteroalkyl, or heterocycloalkyl; wherein said aryl, alkyl, cycloalkyl, heteroalkyl, or heterocycloalkyl is optionally substituted with at least one group selected from halo, hydroxyl, oxo, nitro, amido, carboxy, amino, cyano, haloalkoxy, and haloalkyl; X represents O, S or NH, and Z represents O, S or NH.

[0015] In some embodiments, the assay is for controlling the localization, trafficking, and / or function of a biomolecule. In some embodiments, the assay is for inducing degradation of a biomolecule. In some embodiments, the assay is for detecting the co-occurrence of two biomolecules in the sample.

[0016] In some embodiments, the first and second biomolecules are proteins.

[0017] The present invention provides a proximity induction system comprising two polypeptides and a molecular proximity inducer, the first polypeptide comprises the amino acid sequence set forth in SEQ ID NO:1 or an amino acid sequence having at least about 70% identity to SEQ ID NO:1, or a truncated fragment thereof comprising at least 89 contiguous amino acid residues from the C-terminus of SEQ ID NO:1 or a sequence having at least about 70% identity to SEQ ID NO:1; and the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO:2 or an amino acid sequence having at least about 70% identity to SEQ ID NO:2, or a truncated fragment thereof comprising at least 8 consecutive amino acid residues from the N-terminus of SEQ ID NO:2 or a sequence having at least about 70% identity to SEQ ID NO:2; The first polypeptide, the second polypeptide and the molecular proximity inducer also refer to a proximity induction system, which forms a ternary assembly.

[0018] In some embodiments, the concentration of molecular proximity inducer required to reach half-maximal assembly is less than about 5 μM. In some embodiments, the molecular proximity inducer is fluorescent. In some embodiments, the molecular proximity inducer is a compound of formula (I) described above, or a salt and / or solvate thereof.

[0019] In some embodiments, the first polypeptide consists of the amino acid sequence set forth in SEQ ID NO:1 and the second polypeptide consists of the amino acid sequence set forth in SEQ ID NO:2.

[0020] The present invention provides a cell or cell line comprising two biomolecules, the first biological molecule is bound to a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1 or an amino acid sequence having at least about 70% identity to SEQ ID NO:1, or a truncated fragment thereof comprising at least 89 consecutive amino acid residues from the C-terminus of SEQ ID NO:1 or a sequence having at least about 70% identity to SEQ ID NO:1; the second biological molecule is linked to a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO:2 or an amino acid sequence having at least about 70% identity to SEQ ID NO:2, or a truncated fragment thereof comprising at least 8 consecutive amino acid residues from the N-terminus of SEQ ID NO:2 or a sequence having at least about 70% identity to SEQ ID NO:2; Also contemplated is a cell or cell line in which, in the presence of a molecular proximity inducer, the first polypeptide, the second polypeptide and the molecular proximity inducer are capable of forming a ternary assembly.

[0021] In some embodiments, the concentration of molecular proximity inducer required to reach half-maximal assembly is less than about 5 μM. In some embodiments, the first and second biological molecules are proteins.

[0022] definition For the purposes of the present invention, the following terms have the following meanings:

[0023] chemical definition "Alkyl" alone or as part of another group is a group of formula C n H 2n+1 (where n is a number equal to or greater than 1) refers to a hydrocarbyl radical of the formula: (where n is a number equal to or greater than 1). Generally, alkyl groups of the present invention contain 1 to 12 carbon atoms, preferably 1 to 6 carbon atoms. Alkyl groups can be straight or branched chain and can be substituted as provided herein. Non-limiting examples of 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, isopentyl), and hexyl and its isomers (e.g., n-hexyl, isohexyl).

[0024] "Alkoxy" refers to any -O-alkyl or -O-aryl group. Generally, the alkoxy groups of this invention are -O-alkyl groups.

[0025] "Amido" refers to the -CONH2 group.

[0026] "Amino" refers to the group -NH2.

[0027] "Aryl" refers to a polyunsaturated aromatic hydrocarbyl group having a single ring (i.e., phenyl) or multiple aromatic rings fused together (e.g., naphthyl) or covalently bonded together, typically containing 5 to 12 carbon atoms, preferably 6 to 10 carbon atoms, in which at least one ring is aromatic. The aromatic ring may optionally have one to two additional rings (either cycloalkyl, heterocycloalkyl, or heteroaryl) fused thereto. Aryl is also intended to include partially hydrogenated derivatives of the carbocyclic ring systems enumerated herein, so long as at least one ring is aromatic. Non-limiting examples of aryl include phenyl, biphenyl, biphenylenyl, 5- or 6-tetralinyl, naphthalen-1- or -2-yl, 4-, 5-, 6- or 7-indenyl, 1,2-, 3-, 4- or 5-acenaphthylenyl, 3-, 4- or 5-acenaphthenyl, 1- or 2-pentalenyl, 4- or 5-indanyl, 5-, 6-, 7- or 8-tetrahydronaphthyl, 1,2,3,4-tetrahydronaphthyl, 1,4-dihydronaphthyl and 1-, 2-, 3-, 4- or 5-pyrenyl.

[0028] "Carboxy" refers to the group --COOH.

[0029] "Cyano" refers to the group -CN.

[0030] "Cycloalkyl" refers to a cyclic alkyl group, i.e., a monovalent saturated or unsaturated hydrocarbyl group having one or two ring structures. Cycloalkyl includes monocyclic and bicyclic hydrocarbyl groups. A cycloalkyl group can contain three or more carbon atoms in its ring; generally, cycloalkyl groups of this invention contain 3 to 10, preferably 3 to 8, and more preferably 3 to 6 carbon atoms. This definition of "cycloalkyl" encompasses polycyclic cycloalkyls (e.g., bicyclic cycloalkyls) and bridged cycloalkyl structures, including cycloalkyls linked together through one atom ("spiro") or through two atoms. Non-limiting examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopropyl, cyclohexyl, cycloheptyl, cyclooctanyl, cyclononanyl, cyclodecanyl, norbornyl, adamantyl, bicyclo[2.2.2]octanyl, bicyclo[4.4.0]decanyl, bicyclo[3.2.1]octanyl, bicyclo[3.3.1]nonanyl, bicyclo[2.1.1]hexane, 2,3-dihydro-1H-indenyl, 1,2,3,4-tetrahydronaphthalenyl, decahydronaphthalenyl, 1,2,3,4-tetrahydronaphthalenyl, and octahydropentalenyl.

[0031] "Halogen" or "halo" means fluoro, chloro, bromo, or iodo. Typically, halo groups of this invention are fluoro, chloro, or bromo.

[0032] "Haloalkyl" refers to an alkyl group in which one or more hydrogen atoms have been replaced with a halogen atom. Non-limiting examples of haloalkyl groups include chloromethyl, 1-bromoethyl, fluoromethyl, difluoromethyl, trifluoromethyl, and 1,1,1-trifluoroethyl.

[0033] "Haloalkoxy" refers to an alkoxy group in which one or more hydrogen atoms have been replaced with a halogen atom. Non-limiting examples of haloalkoxy groups include chloromethoxy, 1-bromoethoxy, fluoromethoxy, difluoromethoxy, trifluoromethoxy, and 1,1,1-trifluoroethoxy.

[0034] "Heteroalkyl" refers to an alkyl group in which one or more carbon atoms are replaced with a heteroatom, such as oxygen, nitrogen, or sulfur, and the resulting heteroalkyl group contains at least one carbon atom. In the heteroalkyl group, the heteroatoms are bonded only to carbon atoms along the alkyl chain; i.e., each heteroatom is separated from every other heteroatom by at least one carbon atom, typically at least two carbon atoms. The nitrogen and sulfur heteroatoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quaternized (e.g., sulfur may be oxidized as SO or SO). The heteroalkyl group may further contain one or more =0 and / or =S groups. In one embodiment, at least two carbon atoms are replaced with heteroatoms. In one embodiment, a heteroalkyl is bonded to another group or molecule through a carbon atom, i.e., the bond atom is not selected from among the heteroatoms contained therein. In one embodiment, a heteroalkyl is bonded to another group or molecule through one of the heteroatoms contained therein. When substituted by one or more other groups, a heteroalkyl may be substituted through either a carbon atom or a heteroatom (e.g., nitrogen), unless otherwise specified. Non-limiting examples of heteroalkyls include alkoxy, ethers, polyethers (e.g., polyethylene glycol), secondary and tertiary amines, polyamines, thioethers, polythioethers, and combinations thereof.

[0035] "Heterocycloalkyl" refers to a cyclic heteroalkyl group typically containing 2 to 15 carbon atoms, preferably 2 to 11 carbon atoms, more preferably 2 to 7 carbon atoms, and even more preferably 2 to 6 carbon atoms. Heterocycloalkyl groups are typically 3 to 7-membered, preferably 5- or 6-membered rings. Heterocycloalkyls are typically monocyclic or bicyclic, preferably monocyclic. This definition encompasses polycyclic heterocycloalkyls (e.g., bicyclic heterocycloalkyls) and bridged heterocycloalkyl structures, including rings connected to each other through one atom ("spiro") or two atoms. In one embodiment, a heterocycloalkyl is connected to another group or molecule through a carbon atom, i.e., the connecting atom is not selected from among the heteroatoms contained therein. In one embodiment, a heterocycloalkyl is connected to another group or molecule through one of the heteroatoms contained therein. When substituted with one or more other groups, a heterocycloalkyl may be substituted through either a carbon atom or a heteroatom (e.g., nitrogen), unless otherwise specified. Non-limiting examples of heterocycloalkyls include aziridine, pyrrolidine, piperidine, piperazine, morpholine, thiomorpholine, azepane, azocane, octahydro-1H-isoindole, decahydroisoquinoline, tetrahydrofuran, tetrahydropyran, tetrahydroisoquinoline (e.g., 1,2,3,4-tetrahydroisoquinoline), hexahydropyridazine, hexahydropyrazine, hexahydropyrimidine, decahydroquinoline, octahydropyrrolo[3,4-c]pyrrole, isoindoline, 1,2,3,4-tetrahydroquinoline, and oxetane.

[0036] "Hydroxyl" refers to the -OH group.

[0037] "Nitro" refers to the -NO2 group.

[0038] "Oxo" refers to the group =O.

[0039] A "solvate" refers to a molecular complex comprising a compound of the invention and one or more pharmaceutically acceptable solvent molecules, such as ethanol, in stoichiometric or substoichiometric amounts. The term "hydrate" refers to when the solvent is water.

[0040] General definition The terms "a" and "an" refer to one or to more than one (i.e., at least one) element of the grammatical object of the article. By way of example, "an element" means one or more elements.

[0041] The term "about" before a number means ±10% or less of the value of said number. It should be understood that the value to which the term "about" refers is itself specifically and preferably disclosed.

[0042] The term "amino acid" refers to both natural and synthetic amino acids and both D and L amino acids. Amino acids are represented by their full name or their three-letter or one-letter codes as known in the art. "Standard amino acid" or "naturally occurring amino acid" refers to any of the 20 standard L-amino acids generally present in naturally occurring polypeptides. "Non-standard amino acid" refers to any amino acid other than the standard amino acids, whether synthetically prepared or derived from natural sources. For example, naphthylalanine can be used in place of tryptophan to facilitate synthesis. Other synthetic amino acids that can be substituted include, but are not limited to, L-hydroxypropyl, L-3,4-dihydroxyphenylalanyl, L-α-hydroxylysyl, and D-α-methylalanyl, α-amino acids such as L-α-methylalanyl, β-amino acids, and isoquinolyl. The polypeptides described herein may contain standard or non-standard amino acids. The term "amino acid" also encompasses chemically modified amino acids, including, but not limited to, salts, amino acid derivatives (e.g., amides), and substitutions. Thus, amino acids contained in the polypeptides and fusion proteins described herein, particularly at the carboxy or amino termini, can be modified by methylation, amidation, acetylation, or substitution with other chemical groups. Additionally, disulfide bonds may or may not be present in the polypeptides described herein. Other polypeptide mimetics encompassed herein include polypeptides or fusion proteins described herein having the following modifications: i) polypeptides in which one or more peptidyl -C(O)NR- linkages are replaced by a non-peptidyl linkage, such as a -CH2-carbamate linkage (-CH2OC(O)NR-), a phosphonate linkage, a -CH2-sulfonamide (-CH2-S(O)NR-), a urea (-NHC(O)NH-), a -CH2-secondary amine linkage, or by an alkylated peptidyl linkage (-C(O)NR-), where R is C1-C4 alkyl; ii) polypeptides in which the N-terminus is -NR-R 1group, —NRC(O)R group, —NRC(O)OR group, —NRS(O)R group, —NHC(O)NHR group (wherein R and R 1 R and R are not hydrogen atoms. 1 is hydrogen or C1-C4 alkyl), iii) a polypeptide derivatized to a C-terminus of -C(O)R 2 (In the formula, R 2 is C1-C4 alkoxy and -NR 3 R 4 wherein R is selected from the group consisting of 3 and R 4 are independently selected from the group consisting of hydrogen and C1-C4 alkyl).

[0043] "Cell line" refers to cells derived from the propagation of a single cell, thus forming a homogeneous population of cells containing identical genetic material.

[0044] "Complementary" or "complementary" with respect to a first and second polypeptide described herein refers to the ability of the two polypeptides to assemble together to reconstitute a scaffold to which a molecular proximity inducer described herein can bind.

[0045] The term "CIP system" refers to a chemically induced proximity system.

[0046] The term "em" refers to emission as in emission wavelength.

[0047] The term "ex" refers to excitation as in the excitation wavelength.

[0048] "Fluorescent chromophore" or "fluorogen" refers to a chromophore whose brightness can be significantly enhanced by a change in the environment. A fluorescent chromophore or fluorogen is substantially non-fluorescent in its free form in solution, but emits light when placed in an environment that constrains its conformation and precludes de-excitation of its excited state by non-radiative pathways. In some embodiments, the fluorescent chromophore is nearly invisible in solution but becomes fluorescent when bound to a protein scaffold, such as the scaffold formed by two polypeptides described herein.

[0049] "Identity" or "identical," when used in the context of two or more polypeptide sequences or two or more nucleic acid sequences, refers to the degree of sequence relatedness between the polypeptide or nucleic acid sequences (respectively) as determined by the number of matches between sequences of two or more amino acid residues or two or more nucleotide residues, respectively. "Identity" is a measure of the percent exact match between the shorter sequence of two or more sequences, with gap alignments (if any) handled by a particular mathematical model or computer program (i.e., "algorithm"). The identity of related polypeptide or nucleic acid sequences can be readily calculated by known methods.Such methods include, for example, 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 to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity are described in publicly available computer programs.Examples of methods for determining identity between two sequences using computer programs include GAP (Devereux et al., 1984. Nucl. Acid. Res. 12(1 Pt 1):387-395; Genetics Computer Group, University of Wisconsin Biotechnology Center, Madison, Wisconsin), BLASTP, BLASTN, TBLASTN, and the GCG program package, which includes 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). Identity may also be determined using the well-known Smith Waterman algorithm.

[0050] "Nucleic acid" or "nucleic acid molecule" refers to a polymer of nucleotides covalently linked by phosphodiester bonds, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), in either single- or double-stranded form. Thus, as used herein, the term "nucleic acid" encompasses single-stranded, partially double-stranded, and fully double-stranded nucleic acids. Unless otherwise 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.

[0051] A "nucleic acid sequence" or "nucleotide sequence" refers to the consecutive sequence of nucleotides in a single nucleic acid. Unless otherwise specified, a particular nucleic acid sequence implicitly encompasses its conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs (single nucleotide polymorphisms), and complementary sequences, as well as the explicitly indicated sequence. Specifically, degenerate codon substitutions may be achieved by creating 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, nucleotide sequences encoding amino acid sequences include all nucleotide sequences that are degenerate versions of each other and encode the same amino acid sequence. As long as the nucleotide sequence encoding protein or RNA may contain at least one intron in some versions, the nucleotide sequence encoding protein or RNA may also contain intron.In particular, the specific nucleic acid sequence described herein implicitly includes its corresponding complementary sequence.It should be noted that the specific nucleic acid sequence described herein implicitly includes DNA sequence and corresponding RNA sequence.

[0052] A "pharmaceutically acceptable excipient" or "pharmaceutically acceptable carrier" refers to an excipient or carrier that does not produce adverse, allergic, or other untoward reactions when administered to a subject, such as an animal, particularly a mammal, preferably a human. It includes any and all solvents, such as dispersion media, coatings, antibacterial agents, antifungal agents, isotonic and absorption delaying agents. A pharmaceutically acceptable excipient or carrier refers to any type of non-toxic solid, semisolid, or liquid filler, diluent, encapsulating agent, or formulation auxiliary. For human administration, formulations must meet sterility, pyrogenicity, general safety, and purity standards required by regulatory authorities, such as the FDA (U.S. Food and Drug Administration) or EMA (European Medicines Agency).

[0053] "Reporter protein" refers to a protein that can be detected, localized or quantified as a way to indirectly assess a target of interest or a mechanism of interest.

[0054] A "sample" generally refers to a solid or liquid specimen or small amount of material, particularly biological material. Thus, a "sample" can also refer to a cell or tissue or organism of interest.

[0055] A "vector" refers to a vehicle by which a nucleotide sequence (e.g., a DNA sequence or an RNA sequence), such as a nucleotide sequence encoding a polypeptide or protein, can be introduced into a host cell in order to transform, transfect, or transduce the host cell and promote the replication and / or expression (e.g., transcription and / or translation) of the introduced nucleotide sequence.

[0056] An "expression vector" refers to a vector containing regulatory elements (or control sequences) operably linked to a nucleotide sequence to be expressed. Thus, an expression vector contains sufficient cis-acting regulatory elements to control expression of the nucleotide sequence; other elements that may be required to control expression of the nucleotide sequence of interest may be supplied by the host cell. DETAILED DESCRIPTION OF THE INVENTION

[0057] One object of the present invention is a proximity induction system comprising two polypeptides as described herein and a molecular proximity inducer, the first polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO:1 as described herein or an amino acid sequence having at least about 30% identity to SEQ ID NO:1, or a truncated fragment thereof as described herein; and the second polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO:2 as described herein or an amino acid sequence having at least about 30% identity to SEQ ID NO:2, or a truncated fragment thereof as described herein; It is a close-in guidance system.

[0058] In some embodiments, the invention provides a proximity induction system comprising two polypeptides described herein and a molecular proximity inducer, the first polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO:1 as described herein or an amino acid sequence having at least about 30% identity to SEQ ID NO:1, or a truncated fragment thereof as described herein; and the second polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO:2 as described herein or an amino acid sequence having at least about 30% identity to SEQ ID NO:2, or a truncated fragment thereof as described herein; The first polypeptide (or truncated fragment thereof), the second polypeptide (or truncated fragment thereof) and the molecular proximity inducer are involved in a proximity induction system that forms a ternary assembly.

[0059] In some embodiments, the invention provides a proximity induction system comprising two polypeptides described herein and a molecular proximity inducer, the first polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO:1 as described herein or an amino acid sequence having at least about 30% identity to SEQ ID NO:1, or a truncated fragment thereof as described herein; and the second polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO:2 as described herein or an amino acid sequence having at least about 30% identity to SEQ ID NO:2, or a truncated fragment thereof as described herein; The first polypeptide (or truncated fragment thereof), the second polypeptide (or truncated fragment thereof), and the molecular proximity inducer are related to a proximity induction system that forms a ternary assembly at a concentration of the molecular proximity inducer required to reach half-maximal assembly that is less than about 5 μM.

[0060] As used herein, the phrase "a first polypeptide, a second polypeptide, and a molecular proximity inducer form a ternary assembly" encompasses the formation of a ternary assembly between a cleaved fragment of a first polypeptide described herein, a cleaved fragment of a second polypeptide described herein, and a molecular proximity inducer described herein.

[0061] According to the present invention, in the presence of a molecular proximity inducer, a first polypeptide described herein and a second polypeptide described herein (or truncated fragments thereof) can complement and bind to the molecular proximity inducer described herein to form a ternary assembly. Surprisingly, as shown in the examples below, the presence of a molecular proximity inducer induces the first and second polypeptides (or truncated fragments thereof) to complement and bind to the molecular proximity inducer, regardless of the initial proximity between the first and second polypeptides. In other words, pre-existing proximity between the first and second polypeptides (or truncated fragments thereof) is not required for the molecular proximity inducer to be able to induce the first and second polypeptides (or truncated fragments thereof) to complement and bind to the molecular proximity inducer.

[0062] Thus, according to the present invention, a first polypeptide (or a truncated fragment thereof), a second polypeptide (or a truncated fragment thereof), and a molecular proximity inducer form a ternary assembly regardless of any initial proximity between the first and second polypeptides (or their truncated fragments). Thus, in some embodiments, a first polypeptide (or a truncated fragment thereof), a second polypeptide (or a truncated fragment thereof), and a molecular proximity inducer form a ternary assembly in the absence of pre-existing proximity between the first and second polypeptides (or their truncated fragments).

[0063] In some embodiments, "proximity" means a distance of less than about 20 nm, preferably less than about 10 nm, and more preferably less than about 5 nm.

[0064] Thus, a first polypeptide described herein, or a truncated fragment thereof, cannot bind by itself to a molecular proximity inducer described herein. Similarly, a second polypeptide described herein, or a truncated fragment thereof, cannot bind by itself to a molecular proximity inducer described herein. Furthermore, a first polypeptide described herein and a second polypeptide described herein (or a truncated fragment thereof) cannot complement each other in the absence of a molecular proximity inducer described herein.

[0065] In some embodiments, the formation of a ternary assembly between a first polypeptide described herein (or a cleaved fragment thereof), a second polypeptide described herein (or a cleaved fragment thereof), and a molecular proximity inducer described herein is rapid. In some embodiments, "rapid" means that the ternary assembly between a first polypeptide described herein (or a cleaved fragment thereof), a second polypeptide described herein (or a cleaved fragment thereof), and a molecular proximity inducer described herein is formed in less than about 90 seconds. In some embodiments, "rapid" means that half-maximal assembly between a first polypeptide described herein (or a cleaved fragment thereof), a second polypeptide described herein (or a cleaved fragment thereof), and a molecular proximity inducer described herein is reached in less than about 90 seconds, preferably less than about 60 seconds, more preferably less than about 45 seconds, and even more preferably less than about 30 seconds.

[0066] In some embodiments, the formation of a ternary assembly between a first polypeptide described herein (or a truncated fragment thereof), a second polypeptide described herein (or a truncated fragment thereof), and a molecular proximity inducer described herein is stable.

[0067] In some embodiments, the formation of a ternary assembly between a first polypeptide described herein (or a truncated fragment thereof), a second polypeptide described herein (or a truncated fragment thereof), and a molecular proximity inducer described herein is reversible.

[0068] As used herein, the concentration of molecular proximity inducer required to reach half-maximal assembly (sometimes referred to as half-maximal ternary assembly) refers to the concentration of molecular proximity inducer required to reach 50% of the ternary assembly formed between the first polypeptide, the second polypeptide, and the molecular proximity inducer.

[0069] Methods for assessing or quantifying the ternary assembly formed between a first polypeptide, a second polypeptide, and a molecular proximity inducer are well known in the art. Such methods may rely, inter alia, on assessing or quantifying the fluorescence emitted by a fluorescent molecular proximity inducer upon binding to the first and second polypeptides, and include fluorescence spectroscopy, flow cytometry, and fluorescence microscopy (including laser scanning and spinning disk confocal microscopy, multiphoton microscopy, and super-resolution microscopy).

[0070] In some embodiments, the concentration of molecular proximity inducer required to reach half-maximal assembly is less than about 5, 4.5, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.5, or 0.25 μM. In some embodiments, the concentration of molecular proximity inducer required to reach half-maximal assembly is in the range of about 0.05 to about 5 μM, preferably about 0.1 to about 2.5 μM, and more preferably about 0.1 to about 1 μM.

[0071] In some embodiments, a first polypeptide described herein comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1 or an amino acid sequence having at least about 30%, 35%, 40%, 45%, 50%, or 55%, preferably at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 1. In some embodiments, a first polypeptide described herein comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1 or an amino acid sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 1.

[0072] Examples of sequences having at least about 60% identity to SEQ ID NO:1 include SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14. In some embodiments, the sequence having at least about 60% identity to SEQ ID NO:1 is selected from the group comprising or consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14.

[0073] Examples of sequences having at least about 65% identity to SEQ ID NO:1 include SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14. In some embodiments, the sequence having at least about 65% identity to SEQ ID NO:1 is selected from the group comprising or consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14.

[0074] Examples of sequences having at least about 70% identity to SEQ ID NO:1 include SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12. In some embodiments, the sequence having at least about 70% identity to SEQ ID NO:1 is selected from the group comprising or consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12.

[0075] In some embodiments, the first polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1. In the experimental section below, a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 is referred to as " FIRE It's called "Mate".

[0076] In some embodiments, an amino acid sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:1 described herein contains at least one, two, three, four or five of the following amino acid residues at the positions shown with respect to SEQ ID NO:1: valine (V) at position 30, leucine (L) at position 41, threonine (T) at position 72, alanine (A) at position 83, threonine (T) at position 95, and / or leucine (L) at position 109.

[0077] In some embodiments, an amino acid sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:1 described herein contains two, three, four, five or all of the following amino acid residues at the positions shown with respect to SEQ ID NO:1: valine (V) at position 30, leucine (L) at position 41, threonine (T) at position 72, alanine (A) at position 83, threonine (T) at position 95, and / or leucine (L) at position 109.

[0078] In some embodiments, an amino acid sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:1 described herein contains the following amino acid residues at the positions shown with respect to SEQ ID NO:1: valine (V) at position 30, leucine (L) at position 41, threonine (T) at position 72, alanine (A) at position 83, threonine (T) at position 95, and leucine (L) at position 109.

[0079] In some embodiments, an amino acid sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:1 described herein contains at least one, two, three, four, five, six, seven or eight of the following amino acid residues at the positions shown with respect to SEQ ID NO:1: asparagine (N) at position 17, glutamic acid (E) at position 21, arginine (R) at position 25, valine (V) at position 30, leucine (L) at position 41, threonine (T) at position 72, alanine (A) at position 83, threonine (T) at position 95, and / or leucine (L) at position 109.

[0080] In some embodiments, an amino acid sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:1 described herein contains two, three, four, five, six, seven, eight or all of the following amino acid residues at the positions shown with respect to SEQ ID NO:1: asparagine (N) at position 17, glutamic acid (E) at position 21, arginine (R) at position 25, valine (V) at position 30, leucine (L) at position 41, threonine (T) at position 72, alanine (A) at position 83, threonine (T) at position 95, and / or leucine (L) at position 109.

[0081] In some embodiments, an amino acid sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:1 described herein contains the following amino acid residues at the positions shown with respect to SEQ ID NO:1: asparagine (N) at position 17, glutamic acid (E) at position 21, arginine (R) at position 25, valine (V) at position 30, leucine (L) at position 41, threonine (T) at position 72, alanine (A) at position 83, threonine (T) at position 95, and leucine (L) at position 109.

[0082] In some embodiments, an amino acid sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:1 described herein further comprises a glycine (G) at position 69 relative to SEQ ID NO:1.

[0083] In some embodiments, an amino acid sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:1 described herein further comprises at least one, two, three, four, five or six of the following amino acid residues at the positions shown with respect to SEQ ID NO:1: tryptophan (W) at position 94, isoleucine (I) at position 96, proline (P) at position 97, threonine (T) at position 98, serine (S) at position 99, arginine (R) at position 100, and / or glycine (G) at position 101.

[0084] In some embodiments, an amino acid sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:1 described herein further comprises the following amino acid residues at the positions shown with respect to SEQ ID NO:1: tryptophan (W) at position 94, isoleucine (I) at position 96, proline (P) at position 97, threonine (T) at position 98, serine (S) at position 99, arginine (R) at position 100, and glycine (G) at position 101.

[0085] In some embodiments, an amino acid sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:1 described herein is the following amino acid residues at the positions shown with respect to SEQ ID NO: 1: glycine (G) at position 69, tryptophan (W) at position 94, isoleucine (I) at position 96, proline (P) at position 97, threonine (T) at position 98, serine (S) at position 99, arginine (R) at position 100 and glycine (G) at position 101, and at least one, two, three, four or five of the following amino acid residues at the positions indicated with respect to SEQ ID NO: 1: valine (V) at position 30, leucine (L) at position 41, threonine (T) at position 72, alanine (A) at position 83, threonine (T) at position 95 and / or leucine (L) at position 109 Includes.

[0086] In some embodiments, an amino acid sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:1 described herein is the following amino acid residues at the positions shown with respect to SEQ ID NO: 1: glycine (G) at position 69, tryptophan (W) at position 94, isoleucine (I) at position 96, proline (P) at position 97, threonine (T) at position 98, serine (S) at position 99, arginine (R) at position 100 and glycine (G) at position 101, and two, three, four, five or all of the following amino acid residues at the positions shown with respect to SEQ ID NO: 1: valine (V) at position 30, leucine (L) at position 41, threonine (T) at position 72, alanine (A) at position 83, threonine (T) at position 95 and / or leucine (L) at position 109 Includes.

[0087] In some embodiments, an amino acid sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:1 described herein is the following amino acid residues at the positions shown with respect to SEQ ID NO: 1: glycine (G) at position 69, tryptophan (W) at position 94, isoleucine (I) at position 96, proline (P) at position 97, threonine (T) at position 98, serine (S) at position 99, arginine (R) at position 100 and glycine (G) at position 101, and at least 1, 2, 3, 4, 5, 6, 7 or 8 of the following amino acid residues at the positions shown with respect to SEQ ID NO: 1: asparagine (N) at position 17, glutamic acid (E) at position 21, arginine (R) at position 25, valine (V) at position 30, leucine (L) at position 41, threonine (T) at position 72, alanine (A) at position 83, threonine (T) at position 95 and / or leucine (L) at position 109 Includes.

[0088] In some embodiments, an amino acid sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:1 described herein is the following amino acid residues at the positions shown with respect to SEQ ID NO: 1: glycine (G) at position 69, tryptophan (W) at position 94, isoleucine (I) at position 96, proline (P) at position 97, threonine (T) at position 98, serine (S) at position 99, arginine (R) at position 100 and glycine (G) at position 101, and two, three, four, five, six, seven, eight or all of the following amino acid residues at the positions shown with respect to SEQ ID NO: 1: asparagine (N) at position 17, glutamic acid (E) at position 21, arginine (R) at position 25, valine (V) at position 30, leucine (L) at position 41, threonine (T) at position 72, alanine (A) at position 83, threonine (T) at position 95 and / or leucine (L) at position 109 Includes.

[0089] In some embodiments, an amino acid sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:1 described herein contains the following amino acid residues at the indicated positions with respect to SEQ ID NO:1: valine (V) at position 30, leucine (L) at position 41, glycine (G) at position 69, threonine (T) at position 72, and ribonucleotides (RIs) at positions 100, 102, 104, 106, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, ), alanine (A) at position 83, tryptophan (W) at position 94, threonine (T) at position 95, isoleucine (I) at position 96, proline (P) at position 97, threonine (T) at position 98, serine (S) at position 99, arginine (R) at position 100, glycine (G) at position 101, and / or leucine (L) at position 109.

[0090] In some embodiments, an amino acid sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:1 described herein contains the following amino acid residues at the positions shown with respect to SEQ ID NO:1: valine (V) at position 30, leucine (L) at position 41, glycine (G) at position 69, threonine (T) at position 72, alanine (A) at position 83, tryptophan (W) at position 94, threonine (T) at position 95, isoleucine (I) at position 96, proline (P) at position 97, threonine (T) at position 98, serine (S) at position 99, arginine (R) at position 100, glycine (G) at position 101, and leucine (L) at position 109.

[0091] In some embodiments, an amino acid sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:1 described herein contains the following amino acid residues at the positions shown with respect to SEQ ID NO:1: asparagine (N) at position 17, glutamic acid (E) at position 21, arginine (R) at position 25, valine (V) at position 30, leucine (L) at position 41, glycine (G) at position 69, and ribonucleotides (RI) at position 11. at position 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 169, 169, 170, 171, 172, 173, 174, 175

[0092] In some embodiments, an amino acid sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:1 described herein contains the following amino acid residues at the positions shown with respect to SEQ ID NO:1: asparagine (N) at position 17, glutamic acid (E) at position 21, arginine (R) at position 25, arginine (R) at position 30, and ribonucleotides (RI) at position 31. It contains valine (V) at position 41, leucine (L) at position 42, glycine (G) at position 69, threonine (T) at position 72, alanine (A) at position 83, tryptophan (W) at position 94, threonine (T) at position 95, isoleucine (I) at position 96, proline (P) at position 97, threonine (T) at position 98, serine (S) at position 99, arginine (R) at position 100, glycine (G) at position 101 and leucine (L) at position 109.

[0093] In some embodiments, a truncated fragment of a first polypeptide described herein results from the deletion of consecutive amino acid residues starting from the N-terminus of the first polypeptide described herein, preferably a deletion of a number of consecutive amino acid residues ranging from 1 amino acid residue to 40, 35, 30 or 25 amino acid residues from the N-terminus of the first polypeptide described herein.

[0094] In some embodiments, the truncated fragment of a first polypeptide described herein is a truncated fragment consisting of amino acid residues 1 through 25 of SEQ ID NO:1 or a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:1 described herein.

[0095] In some embodiments, the truncated fragment of a first polypeptide described herein is a truncated fragment consisting 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 consecutive amino acid residues from the N-terminus (also referred to as N-ter) of SEQ ID NO:1 or a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:1 described herein.

[0096] In some embodiments, a truncated fragment of a first polypeptide described herein comprises at least 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, or 112 contiguous amino acid residues from the C-terminus of SEQ ID NO:1 or a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:1 described herein.

[0097] In some embodiments, a truncated fragment of a first polypeptide described herein 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 contiguous amino acid residues from the C-terminus of SEQ ID NO:1 or a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:1 as described herein.

[0098] In some embodiments, the second polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO:2 or an amino acid sequence having at least about 30%, 35%, 40%, 45%, 50%, or 55%, preferably at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:2.

[0099] In some embodiments, the second polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO:2 or an amino acid sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:2.

[0100] Examples of sequences having at least about 60% identity to SEQ ID NO:2 include SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20. In some embodiments, the sequence having at least about 60% identity to SEQ ID NO:2 is selected from the group comprising or consisting of SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20.

[0101] Examples of sequences having at least about 70% identity to SEQ ID NO:2 include SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20. In some embodiments, the amino acid sequence having at least about 70% identity to SEQ ID NO:2 is selected from the group comprising or consisting of SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20.

[0102] Examples of sequences having at least about 80% identity to SEQ ID NO:2 include SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20. In some embodiments, the amino acid sequence having at least about 80% identity to SEQ ID NO:2 is selected from the group comprising or consisting of SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20.

[0103] In some embodiments, the second polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 2. In the experimental section below, a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2 is referred to as " FIRE It's called a "tag."

[0104] In some embodiments, an amino acid sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:2 described herein contains the following amino acid residues at the positions shown with respect to SEQ ID NO:2: arginine (R) at position 3;

[0105] In some embodiments, an amino acid sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:2 described herein further comprises the following amino acid residues at the positions shown with respect to SEQ ID NO:2: isoleucine (I) at position 8;

[0106] In some embodiments, a cleaved fragment of a second polypeptide described herein comprises at least 8 consecutive amino acid residues of a second polypeptide described herein, hi some embodiments, a cleaved fragment of a second polypeptide described herein comprises 8, 9, or 10 consecutive amino acid residues of SEQ ID NO:2 described herein or a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:2.

[0107] In some embodiments, the truncated fragment of a second polypeptide described herein results from the deletion of consecutive amino acid residues, preferably 1, 2 or 3 consecutive amino acid residues, starting from the N-terminus and / or C-terminus of a second polypeptide described herein.

[0108] In some embodiments, a truncated fragment of a second polypeptide described herein results from the deletion of consecutive amino acid residues, preferably 1, 2, or 3 consecutive amino acid residues, starting from the N-terminus of a second polypeptide described herein. In some embodiments, a truncated fragment of a second polypeptide described herein results from the deletion of consecutive amino acid residues, preferably 1, 2, or 3 consecutive amino acid residues, starting from the C-terminus of a second polypeptide described herein.

[0109] In some embodiments, a cleaved fragment of a second polypeptide described herein comprises at least 8, 9, or 10 contiguous amino acid residues from the N-terminus of SEQ ID NO:2 described herein or a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 2. In some embodiments, a cleaved fragment of a second polypeptide described herein comprises 8, 9, or 10 contiguous amino acid residues from the N-terminus of SEQ ID NO:2 described herein or a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:2.

[0110] In some embodiments, a second polypeptide described herein or a truncated fragment thereof described herein is fused to a 113 amino acid fragment of a photoactive yellow protein (PYP) polypeptide having the amino acid sequence set forth in SEQ ID NO:21 or an amino acid sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to the amino acid sequence set forth in SEQ ID NO:21, or a truncated fragment thereof. The truncated fragment may comprise at least 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111 or 112 consecutive amino acid residues from the C-terminus of SEQ ID NO:21 or a sequence having at least about 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99 or more identity to SEQ ID NO:21 as set forth herein. Thus, the truncated fragment may comprise 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111 or 112 consecutive amino acid residues from the C-terminus of SEQ ID NO:21 or a sequence having at least about 60, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:21 as set forth herein.

[0111] In some embodiments, the molecular proximity inducer is a compound of formula (I): [ka] or a salt and / or solvate thereof, During the ceremony, R 1 , R 2 , R 5 and R 6each independently represents H, halo, hydroxyl, aryl, alkyl, cycloalkyl, heteroalkyl (e.g., alkoxy), or heterocycloalkyl; wherein said aryl, alkyl, cycloalkyl, heteroalkyl, or heterocycloalkyl is optionally substituted with at least one group selected from halo, hydroxyl, oxo, nitro, amido, carboxy, amino, cyano, haloalkoxy, and haloalkyl; R 3 represents a non-bonded pair (i.e., a free electron pair), H, halo, hydroxyl, aryl, alkyl, cycloalkyl, heteroalkyl, or heterocycloalkyl; wherein said aryl, alkyl, cycloalkyl, heteroalkyl, or heterocycloalkyl is optionally substituted with at least one group selected from halo, hydroxyl, oxo, nitro, amido, carboxy, amino, cyano, haloalkoxy, and haloalkyl; R 4 represents a single or double bond interrupted or terminated by one S, O or N heteroatom optionally substituted with at least one group selected from H, hydroxyl, aryl, alkyl, cycloalkyl, heteroalkyl and heterocycloalkyl; wherein said aryl, alkyl, cycloalkyl, heteroalkyl, or heterocycloalkyl is optionally substituted with at least one group selected from halo, hydroxyl, oxo, nitro, amido, carboxy, amino, cyano, haloalkoxy, and haloalkyl; W is OH, SH, NHR 7 or NR 7 R 8 wherein R 7 and R 8 each independently represents H, halo, hydroxyl, aryl, alkyl, cycloalkyl, heteroalkyl, or heterocycloalkyl; wherein said aryl, alkyl, cycloalkyl, heteroalkyl, or heterocycloalkyl is optionally substituted with at least one group selected from halo, hydroxyl, oxo, nitro, amido, carboxy, amino, cyano, haloalkoxy, and haloalkyl; X represents O, S or NH, and Z represents O, S or NH.

[0112] According to the present invention, the alkyl, cycloalkyl, heteroalkyl, or heterocycloalkyl group can be saturated or unsaturated, hi some embodiments, the alkyl, cycloalkyl, heteroalkyl, or heterocycloalkyl group is saturated.

[0113] In some embodiments, R 1 represents H, alkyl, or alkoxy. In some embodiments, R 2 represents H, alkyl, or alkoxy. In some embodiments, R 3 represents H. In some embodiments, R 4 represents a double bond terminated with S, O, or NH. In some embodiments, R 5 represents H. In some embodiments, R 6 represents H, alkyl, or alkoxy. In some embodiments, W represents OH. In some embodiments, Z represents O.

[0114] In some embodiments, the molecular proximity inducer is (Z)-5-(4-hydroxybenzylidene)-2-thioxo-1,3-thiazolidin-4-one (HBR), (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-3OM), (Z)-5-(4-hydroxy-3,5-dimethylbenzylidene)-2 ... Oxothiazolidin-4-one (HBR-3,5DM), (Z)-5-(4-hydroxy-2,5-dimethylbenzylidene)-2-thioxo-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-3OE), (Z)-5-(4-hydroxybenzylidene)-3-methyl-2-thioxothiazolidin-4-one (HBMR), (Z)-5-(2,4-dihydroxybenzylidene)-2-thioxo-1,3-thiazolidin-4-one (DHBR), (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-3OE), (Z)-2-(5-(4-hydroxy-2-methoxybenzylidene)-4-oxo-2-thioxothiazolidin-3-yl)acetic acid ( HBRAA-2OM), (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-3OM), (Z)-2-(5- (4-Hydroxy-2-methoxybenzylidene)-4-oxo-2-thioxothiazolidin-3-yl)acetic acid (HBRAA-2OM), (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), (Z)-2-(5 (Z)-5-(4-hydroxy-3,5-dimethoxybenzylidene)-4-oxo-2-thioxothiazolidin-3-yl)acetic acid (HBRAA-3,5DOM), (Z)-5-(4-hydroxy-3-methylbenzylidene)-4-thioxo-1,3-thiazolidin-2-one (HBIR-3M), and (Z)-5-(4-hydroxy-2,5-dimethoxybenzylidene)-2-thioxo-1,3-thiazolidin-4-one (HBR-2,5DOM).

[0115] In some embodiments, the molecular proximity inducer is a compound of formula (II): [ka] or a salt and / or solvate thereof, During the ceremony, R 1 , R 2 and R 6 each independently represents H, alkyl, or alkoxy; and X, Y, and Y each independently represent O, S, or NH.

[0116] In some embodiments, R 1 represents H, methyl (Me), or methoxy (OMe). In some embodiments, R 2 represents H, methyl, or methoxy. In some embodiments, R 6 represents H, methyl or methoxy.

[0117] In some embodiments, R 1 , R 2 and R 6 represent "Me, H and H", "H, Me and Me", "Me, H and Me", "OMe, H and OMe", or "H, OMe and OMe", respectively.

[0118] In some embodiments, X, Y, and Y represent "S, S, and O," "S, O, and S," "S, NH, and O," "S, O, and O," or "O, O, and O," respectively.

[0119] In some embodiments, R 1 , R 2 , R 6 , X, Y, and Y represent "Me, H, H, S, S, and O," "H, Me, Me, S, O, and S," "Me, H, Me, S, NH, and O," "OMe, H, OMe, S, O, and O," or "H, OMe, OMe, O, O, and O," respectively.

[0120] In some embodiments, the molecular proximity inducer is (Z)-5-(4-hydroxy-2,5-dimethylbenzylidene)-2-thioxo-1,3-thiazolidin-4-one (HBR-2,5DM), (Z)-5-(4-hydroxy-3-methylbenzylidene)-2-thioxo-1,3-thiazolidin-4-one (HMBR), (Z)-5-(4-hydroxy-3-methylbenzylidene)-4-thioxo-1,3-thiazolidin-2-one (HBIR-3M), (Z)-5-(4-hydroxy-3,5-dimethylbenzylidene)-2-thioxo-1,3-thiazolidin-2-one (HBIR-3M), (Z)-5-(4-hydroxy-3,5-dimethylbenzylidene)-2-thioxo-1,3-thiazolidin-4 ... oxothiazolidin-4-one (HBR-3,5DM), (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-3OM), and (Z)-5-(4-hydroxy-2,5-dimethoxybenzylidene)-2-thioxo-1,3-thiazolidin-4-one (HBR-2,5DOM).

[0121] In some embodiments, the molecular proximity inducer comprises or is selected from the group consisting of (Z)-5-(4-hydroxy-2,5-dimethylbenzylidene)-2-thioxo-1,3-thiazolidin-4-one (HBR-2,5DM), (Z)-5-(4-hydroxy-3-methylbenzylidene)-2-thioxo-1,3-thiazolidin-4-one (HMBR), and (Z)-5-(4-hydroxy-3-methylbenzylidene)-4-thioxo-1,3-thiazolidin-2-one (HBIR-3M).

[0122] In some embodiments, the molecular proximity inducer is (Z)-5-(4-hydroxy-2,5-dimethylbenzylidene)-2-thioxo-1,3-thiazolidin-4-one (HBR-2,5DM): [ka] is.

[0123] In some embodiments, the molecular proximity inducer is (Z)-5-(4-hydroxy-3-methylbenzylidene)-2-thioxo-1,3-thiazolidin-4-one (HMBR): [ka] is.

[0124] In some embodiments, the molecular proximity inducer is (Z)-5-(4-hydroxy-3-methylbenzylidene)-4-thioxo-1,3-thiazolidin-2-one (HBIR-3M): [ka] is.

[0125] In some embodiments, the molecular proximity inducer is a chromophore.

[0126] In some embodiments, the molecular proximity inducer is non-fluorescent. In some embodiments, the molecular proximity inducer is a non-fluorescent chromophore. An example of a non-fluorescent molecular proximity inducer is (Z)-5-(4-hydroxy-3-methylbenzylidene)-4-thioxo-1,3-thiazolidin-2-one (HBIR-3M).

[0127] In some embodiments, the molecular proximity inducer is fluorescent. In some embodiments, the molecular proximity inducer is a fluorescent chromophore. Examples of fluorescent molecular proximity inducers include (Z)-5-(4-hydroxy-2,5-dimethylbenzylidene)-2-thioxo-1,3-thiazolidin-4-one (HBR-2,5DM), (Z)-5-(4-hydroxy-3-methylbenzylidene)-2-thioxo-1,3-thiazolidin-4-one (HMBR), and (Z)-5-(4-hydroxy-3,5-dimethylbenzylidene)-2-thioxothiazolidin-4-one (HBR-3,5DM). , (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-3OM), and (Z)-5-(4-hydroxy-2,5-dimethoxybenzylidene)-2-thioxo-1,3-thiazolidin-4-one (HBR-2,5DOM). In some embodiments, the fluorescent chromophore is (Z)-5-(4-hydroxy-2,5-dimethylbenzylidene)-2-thioxo-1,3-thiazolidin-4-one (HBR-2,5DM), (Z)-5-(4-hydroxy-3-methylbenzylidene)-2-thioxo-1,3-thiazolidin-4-one (HMBR), (Z)-5-(4-hydroxy-3,5-dimethylbenzylidene)-2-thioxothiazolidin-4-one (HBR-3,5DM), (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-3OM) and (Z)-5-(4-hydroxy-2,5-dimethoxybenzylidene)-2-thioxo-1,3-thiazolidin-4-one (HBR-2,5DOM).In some embodiments, the fluorescent chromophores are (Z)-5-(4-hydroxy-2,5-dimethylbenzylidene)-2-thioxo-1,3-thiazolidin-4-one (HBR-2,5DM) and (Z)-5-(4-hydroxy-3-methylbenzylidene)-2-thioxo-1,3-thiazolidin-4-one (HMBR).

[0128] Another object of the present invention is a biomolecule comprising a first polypeptide described herein or a truncated fragment thereof described herein and / or a second polypeptide described herein or a truncated fragment thereof described herein.

[0129] In some embodiments, a biomolecule comprising a first and / or second polypeptide or a cleaved fragment thereof is a biomolecule to which the first and / or second polypeptide or a cleaved fragment thereof is covalently or non-covalently bound.

[0130] Methods for non-covalently linking a peptide or polypeptide to a biomolecule described herein are well known and include, for example, chemical conjugation. Methods for covalently linking a peptide or polypeptide to a biomolecule described herein are well known and include, for example, chemical conjugation or genetic fusion as described herein.

[0131] As used herein, the term "biomolecule" encompasses any molecule present in a living organism. Examples of biomolecules include amino acids, polypeptides, proteins, monosaccharides, polysaccharides, nucleotides, nucleic acids, lipids, fatty acids, glycolipids, sterols, vitamins, hormones, neurotransmitters, and metabolites. In particular, biomolecules may be nucleic acids or proteins. Examples of nucleic acids include DNA and RNAs such as messenger RNA (mRNA) and transfer RNA (tRNA). Examples of proteins include transcription factors, enzymes, receptors, immunoreceptors, immunoglobulins, and signaling proteins.

[0132] In some embodiments, the biomolecule is a protein.

[0133] Accordingly, another object of the present invention is a fusion protein comprising a protein of interest fused to a first polypeptide described herein or a truncated fragment thereof described herein and / or a second polypeptide described herein or a truncated fragment thereof described herein. As shown in the experimental section below, the first and second polypeptides or truncated fragments thereof may each be expressed in fusion with any protein of interest in a host cell by inserting (e.g., by transformation or transfection) nucleotide sequences encoding both the polypeptide and the protein of interest.

[0134] Methods for fusing a peptide or polypeptide to a protein of interest are well known. Briefly, such methods involve inserting a nucleotide sequence encoding the protein of interest in frame with a nucleic acid sequence encoding the peptide or polypeptide. The nucleic acid sequence encoding the protein of interest can be inserted so that the encoded peptide or polypeptide is located at the N-terminus of the protein of interest, at the C-terminus of the protein of interest, or internally, as desired. Additionally, a short nucleic acid sequence encoding a linker or spacer may be present between the sequence encoding the peptide or polypeptide and the sequence encoding the protein of interest.

[0135] The protein of interest may be a naturally occurring protein, a chimeric protein resulting from the fusion of various protein domains, or a synthetic protein. The protein of interest may be an intracellular protein, a membrane protein, a cell surface protein that is at least partially present on the outer membrane surface, or a secreted protein. The protein of interest may be a transcription factor, an enzyme, a receptor, an immunoreceptor, an immunoglobulin, or a signal transduction protein.

[0136] In some embodiments, the protein of interest is a reporter protein.Reporter protein can detect, evaluate and / or monitor the change in sample.Examples of changes that can be detected, evaluated and / or monitored using reporter protein include cell signaling, gene expression, metabolite or analyte concentration, cell-cell interaction, cell motility, cell death, intracellular transport, secretion, cell cycle stage and circadian rhythm.

[0137] In some embodiments, the fusion proteins described herein comprise at least one additional element other than the first and / or second polypeptides described herein or truncated fragments thereof described herein and the protein of interest, such as a linker, a targeting signal, a localization signal, a protease target site, a fluorescent protein (such as a fluorescent protein tag), an antibody crystallizable fragment (Fc), or an enzyme.

[0138] In some embodiments, the fusion proteins described herein comprise a linker. Methods for designing or selecting linkers are well known to those of skill in the art.

[0139] Another object of the present invention is a kit comprising a biomolecule described herein, such as a fusion protein described herein, and a molecular proximity inducer described herein.

[0140] Another object of the present invention is a pair of biomolecules as described herein, wherein one biomolecule comprises a first polypeptide as described herein or a truncated fragment thereof as described herein, and the other biomolecule comprises a second polypeptide as described herein or a truncated fragment thereof as described herein.

[0141] In some embodiments, the present invention relates to a pair of biomolecules described herein, wherein one biomolecule comprises a first polypeptide described herein or a cleaved fragment thereof described herein, and the other biomolecule comprises a second polypeptide described herein or a cleaved fragment thereof described herein, wherein in the presence of a molecular proximity inducer described herein, the first polypeptide, the second polypeptide, and the molecular proximity inducer can form a ternary assembly at a concentration of molecular proximity inducer required to reach half-maximal assembly that is less than about 5 μM.

[0142] In some embodiments, a pair of biomolecules consists of one biomolecule to which a first polypeptide, or a truncated fragment thereof, is covalently or non-covalently bound, and another biomolecule to which a second polypeptide, or a truncated fragment thereof, is covalently or non-covalently bound.

[0143] A pair of biomolecules may consist of two different biomolecules. A pair of biomolecules may consist of two biomolecules of different structures, such as a nucleic acid and a protein. A pair of biomolecules may consist of two different biomolecules of the same structure, such as two nucleic acids or two proteins. A pair of biomolecules may consist of two copies of the same biomolecule (such as two copies of the same protein), where one copy contains a first polypeptide or a truncated fragment thereof and the other copy contains a second polypeptide or a truncated fragment thereof.

[0144] In some embodiments, the pair of biomolecules is a pair of fusion proteins described herein, where one fusion protein comprises a protein of interest fused to a first polypeptide described herein or a truncated fragment thereof described herein, and the other fusion protein comprises a protein of interest fused to a second polypeptide described herein or a truncated fragment thereof described herein.

[0145] In some embodiments, both fusion proteins of a pair comprise the same protein of interest. In other words, in some embodiments, one fusion protein comprises a first polypeptide or a cleaved fragment thereof and a protein of interest, and the other fusion protein comprises a second polypeptide or a cleaved fragment thereof and the same protein of interest. In some embodiments, each fusion protein of a pair comprises a different protein of interest. In other words, in some embodiments, one fusion protein comprises a first polypeptide or a cleaved fragment thereof and a first protein of interest, and the other fusion protein comprises a second polypeptide or a cleaved fragment thereof and a second, different protein of interest.

[0146] In some embodiments, at least one of the fusion proteins of the pair comprises a protein of interest that is a reporter protein, hi some embodiments, both fusion proteins of the pair comprise proteins of interest that are reporter proteins (either the same reporter protein or different reporter proteins).

[0147] In some embodiments, at least one of the pair of fusion proteins comprises an additional element described herein, such as a linker, a targeting signal, a localization signal, a protease target site, an antibody crystallizable fragment (Fc), a fluorescent protein (such as a fluorescent protein tag), or an enzyme.

[0148] Another object of the present invention is a kit comprising a pair of biomolecules as described herein, such as a pair of fusion proteins as described herein, and a molecular proximity inducer as described herein.

[0149] Another object of the present invention is a nucleic acid molecule comprising or consisting of a nucleotide sequence encoding a first polypeptide described herein or a truncated fragment thereof described herein and / or a second polypeptide described herein or a truncated fragment thereof described herein. Examples of nucleic acid molecules include DNA molecules and RNA molecules. Examples of DNA molecules include cDNA molecules (complementary DNA). Examples of RNA molecules include mRNA molecules (messenger RNA).

[0150] In some embodiments, "a nucleotide sequence encoding a first and / or second polypeptide described herein or a truncated fragment thereof" refers to all nucleotide sequences that are degenerate versions of each other and that encode the same first and / or second polypeptide or a truncated fragment thereof. Those skilled in the art are familiar with methods for adapting coding sequences based on the genetic code, such as methods that take advantage of codon degeneracy to introduce silent mutations and methods that take into account codon usage biases and variations in the standard genetic code associated with the host cell under consideration.

[0151] In some embodiments, the nucleic acid molecule comprises or consists of a nucleotide sequence that encodes a first polypeptide or a truncated fragment thereof, comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 1 as described herein. An example of a nucleotide sequence that encodes a first polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 1 is the nucleotide sequence that comprises or consists of the sequence set forth in SEQ ID NO: 22. Thus, in some embodiments, the nucleic acid molecule comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 22 or a nucleotide sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 22.

[0152] In some embodiments, the nucleic acid molecule comprises or consists of a nucleotide sequence that encodes a second polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO:2 as described herein, or a truncated fragment thereof. An example of a nucleotide sequence that encodes a second polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO:2 is the nucleotide sequence comprising or consisting of the amino acid sequence set forth in SEQ ID NO:23. Thus, in some embodiments, the nucleic acid molecule comprises or consists of the nucleotide sequence set forth in SEQ ID NO:23 or a nucleotide sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:23.

[0153] In some embodiments, the nucleic acid molecule comprises or consists of a nucleotide sequence encoding a fusion protein described herein.

[0154] Another object of the present invention is a kit comprising a nucleic acid molecule as described herein and a molecular proximity inducer as described herein.

[0155] Another object of the present invention is a pair of nucleic acid molecules as described herein, wherein one nucleic acid molecule comprises or consists of a nucleotide sequence encoding a first polypeptide as described herein or a truncated fragment thereof as described herein, and the other biological molecule comprises or consists of a nucleotide sequence encoding a second polypeptide as described herein or a truncated fragment thereof as described herein.

[0156] In some embodiments, a pair of nucleic acid molecules consists of one nucleic acid molecule that comprises or consists of a nucleotide sequence encoding a first polypeptide or a truncated fragment thereof that comprises or consists of the amino acid sequence set forth in SEQ ID NO:1 as described herein, and another nucleic acid molecule that comprises or consists of a nucleotide sequence encoding a second polypeptide or a truncated fragment thereof that comprises or consists of the amino acid sequence set forth in SEQ ID NO:2 as described herein.

[0157] In some embodiments, a pair of nucleic acid molecules consists of one nucleic acid molecule that comprises or consists of the nucleotide sequence set forth in SEQ ID NO:22 or a nucleotide sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:22, and another nucleic acid molecule that comprises or consists of the nucleotide sequence set forth in SEQ ID NO:23 or a nucleotide sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:23.

[0158] In some embodiments, a pair of nucleic acid molecules consists of two nucleic acid molecules that comprise or consist of a nucleotide sequence encoding a fusion protein as described herein, where one nucleic acid molecule comprises or consists of a nucleotide sequence encoding a fusion protein comprising a first polypeptide as described herein or a truncated fragment thereof and a protein of interest as described herein, and the other biological molecule comprises or consists of a nucleotide sequence encoding a second fusion protein comprising a second polypeptide as described herein or a truncated fragment thereof and a protein of interest as described herein.

[0159] Another object of the present invention is a kit comprising a pair of nucleic acid molecules as described herein and a molecular proximity inducer as described herein.

[0160] Another object of the present invention is a vector, particularly an expression vector, comprising a nucleotide sequence encoding a first polypeptide described herein or a truncated fragment thereof described herein and / or a second polypeptide described herein or a truncated fragment thereof described herein. Examples of vectors include, but are not limited to, plasmids, viral vectors, artificial chromosomes, liposomes, and lipid nanoparticles.

[0161] In some embodiments, the vector is a nucleic acid molecule described herein. Examples of such vectors include plasmids and artificial chromosomes.

[0162] In some embodiments, the vectors described herein include: a first nucleotide sequence encoding a first polypeptide described herein or a truncated fragment thereof described herein, and a second nucleotide sequence encoding a second polypeptide described herein or a truncated fragment thereof described herein; Includes.

[0163] In some embodiments, the vectors described herein include: a first nucleotide sequence encoding a first polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO:1 as described herein, or a truncated fragment thereof; and a second nucleotide sequence encoding a second polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO:2, as described herein, or a truncated fragment thereof; Includes.

[0164] In some embodiments, the vectors described herein include: a first nucleotide sequence comprising or consisting of the sequence set forth in SEQ ID NO:22 or a nucleotide sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:22, and a second nucleotide sequence comprising or consisting of the sequence set forth in SEQ ID NO:23 or a nucleotide sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:23; Includes.

[0165] The vector may contain the nucleotide sequences required for the fusion of a nucleic acid sequence encoding a protein of interest in frame with a nucleic acid sequence encoding a first polypeptide described herein and / or a second polypeptide described herein or a truncated fragment thereof described herein.

[0166] In some embodiments, the vectors described herein comprise a nucleotide sequence encoding at least one fusion protein described herein.

[0167] Another object of the present invention is a kit comprising a vector as described herein and a molecular proximity inducer as described herein.

[0168] Another object of the present invention is a pair of vectors as described herein, wherein one vector comprises a nucleotide sequence encoding a first polypeptide as described herein or a truncated fragment thereof as described herein, and the other vector comprises a nucleotide sequence encoding a second polypeptide as described herein or a truncated fragment thereof as described herein.

[0169] In some embodiments, the pair of vectors consists of one vector containing a nucleotide sequence encoding a first polypeptide or truncated fragment thereof that comprises or consists of the amino acid sequence set forth in SEQ ID NO:1 as described herein, and another vector containing a nucleotide sequence encoding a second polypeptide or truncated fragment thereof that comprises or consists of the amino acid sequence set forth in SEQ ID NO:2 as described herein.

[0170] In some embodiments, a pair of vectors consists of one vector comprising the nucleotide sequence set forth in SEQ ID NO:22 or a nucleotide sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:22, and another vector comprising the nucleotide sequence set forth in SEQ ID NO:23 or a nucleotide sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:23.

[0171] In some embodiments, a pair of vectors consists of two vectors, each containing a nucleotide sequence encoding a fusion protein described herein, where one vector contains a nucleotide sequence encoding a fusion protein comprising a first polypeptide described herein or a truncated fragment thereof and a protein of interest described herein, and the other vector contains a nucleotide sequence encoding a second fusion protein comprising a second polypeptide described herein or a truncated fragment thereof and a protein of interest described herein.

[0172] Another object of the present invention is a kit comprising a pair of vectors as described herein and a molecular proximity inducer as described herein.

[0173] Another object of the present invention is a cell or cell line comprising or expressing a first polypeptide described herein or a truncated fragment thereof described herein and / or a second polypeptide described herein or a truncated fragment thereof described herein. In some embodiments, the cell or cell line described herein comprises or expresses both a first polypeptide described herein or a truncated fragment thereof described herein and a second polypeptide described herein or a truncated fragment thereof described herein.

[0174] Another object of the present invention is a cell or cell line comprising or expressing a nucleic acid molecule comprising or consisting of a nucleotide sequence encoding a first polypeptide described herein or a truncated fragment thereof described herein and / or a second polypeptide described herein or a truncated fragment thereof described herein. In some embodiments, the cell or cell line comprises or expresses a pair of nucleic acid molecules described herein, where one nucleic acid molecule comprises or consists of a nucleotide sequence encoding a first polypeptide described herein or a truncated fragment thereof described herein, and the other biomolecule comprises or consists of a nucleotide sequence encoding a second polypeptide described herein or a truncated fragment thereof described herein.

[0175] The cells or cell lines described herein may be genetically modified cells or cell lines, i.e., cells or cell lines genetically modified to express a first polypeptide described herein or a truncated fragment thereof described herein and / or a second polypeptide described herein or a truncated fragment thereof described herein.

[0176] The cell or cell line may be a prokaryotic cell or cell line or a eukaryotic cell or cell line. The cell or cell line may be a bacterial cell or cell line, an archaeal cell or cell line, a yeast cell or cell line, a plant cell or cell line, or an animal cell or cell line. The cell or cell line may be a mammalian cell or cell line. The cell or cell line may be a human cell or cell line. The cell or cell line may be a primary cell or cell line, particularly a human primary cell or cell line. The cell or cell line may be an immortalized cell or cell line, particularly an immortalized human cell or cell line. The cell or cell line may be an immune cell or cell line, particularly a human immune cell or cell line.

[0177] In some embodiments, a cell or cell line described herein comprises a vector described herein. In some embodiments, a cell or cell line described herein comprises a pair of vectors described herein.

[0178] Within the cells or cell lines described herein, a first polypeptide described herein or a truncated fragment thereof described herein and / or a second polypeptide described herein or a truncated fragment thereof described herein may be comprised in a biomolecule described herein. Thus, within the cells or cell lines described herein, the first and / or second polypeptide or truncated fragment thereof may be covalently or non-covalently bound to a biomolecule.

[0179] In some embodiments, the cells or cell lines described herein comprise or express at least one biomolecule comprising a first polypeptide described herein or a truncated fragment thereof described herein and / or a second polypeptide described herein or a truncated fragment thereof described herein.

[0180] In some embodiments, the cells or cell lines described herein comprise or express at least one fusion protein comprising a first polypeptide described herein or a truncated fragment thereof described herein and / or a second polypeptide described herein or a truncated fragment thereof described herein.

[0181] In some embodiments, the cells or cell lines described herein comprise or express a pair of biomolecules described herein, such that in some embodiments, the cells or cell lines described herein comprise or express a first biomolecule comprising a first polypeptide described herein or a truncated fragment thereof described herein, and a second biomolecule comprising a second polypeptide described herein or a truncated fragment thereof described herein.

[0182] In some embodiments, the cells or cell lines described herein comprise or express two biomolecules: the first biomolecule is attached to a first polypeptide described herein or a truncated fragment thereof described herein; the second biomolecule is linked to a second polypeptide described herein or a truncated fragment thereof described herein; In the presence of a molecular proximity inducer described herein, the first polypeptide, the second polypeptide, and the molecular proximity inducer can form a ternary assembly at a concentration of molecular proximity inducer required to reach half-maximal assembly that is less than about 5 μM.

[0183] In some embodiments, the cells or cell lines described herein comprise or express a pair of fusion proteins described herein, such that in some embodiments, the cells or cell lines described herein comprise or express a first fusion protein comprising a first polypeptide described herein or a truncated fragment thereof described herein, and a second fusion protein comprising a second polypeptide described herein or a truncated fragment thereof described herein.

[0184] In some embodiments, a cell or cell line described herein comprises or expresses two fusion proteins described herein, the first fusion protein comprises a first polypeptide described herein or a truncated fragment thereof described herein and a protein of interest described herein; the second fusion protein comprises a second polypeptide described herein or a truncated fragment thereof described herein and a protein of interest described herein; In the presence of a molecular proximity inducer described herein, the first polypeptide, the second polypeptide, and the molecular proximity inducer can form a ternary assembly at a concentration of molecular proximity inducer required to reach half-maximal assembly that is less than about 5 μM.

[0185] Another object of the present invention is a kit comprising a cell or cell line as described herein and a molecular proximity inducer as described herein.

[0186] In some embodiments, at least one of the fusion proteins contained in or expressed by a cell or cell line described herein comprises a reporter protein, hi some embodiments, both of the fusion proteins contained in or expressed by a cell or cell line described herein comprise a reporter protein.

[0187] In some embodiments, the cells or cell lines described herein are biosensors. By "biosensor," it is meant that the cells or cell lines described herein can be used to assess and / or monitor a target of interest, a physiological mechanism, or a biological process of interest.

[0188] Examples of targets of interest include analytes or metabolites, and thus, in some embodiments, the cells described herein may be used to assess and / or monitor the intracellular presence of an analyte or metabolite.

[0189] Examples of physiological mechanisms or biological processes of interest include apoptosis, cell cycle, viral infection, and intracellular protein trafficking.

[0190] In some embodiments, the cells or cell lines described herein are proximity biosensors. By "proximity biosensor," we mean that the cells or cell lines described herein can be used to assess and / or monitor the intracellular localization and / or trafficking of biomolecules, particularly proteins such as cargo proteins.

[0191] In some embodiments, at least one of the biomolecules contained in or expressed by the cells or cell lines described herein is a therapeutic effector. In some embodiments, at least one of the biomolecules contained in or expressed by the cells or cell lines described herein is a fusion protein comprising a protein of interest that is a therapeutic effector. By "therapeutic effector" is meant a biomolecule, particularly a protein, that can produce a therapeutic effect when administered to a subject.

[0192] The therapeutic effector may be a protein selected from the group including or consisting of transcription factors, enzymes, receptors, immunoreceptors, immunoglobulins, and signaling proteins. The therapeutic effector may be a signaling protein, such as a chimeric antigen receptor (CAR). The therapeutic effector may be a transcription factor.

[0193] In some embodiments, the activity and / or function of the therapeutic effector is controlled by chemically induced proximity, chemically induced localization, chemically induced transport, chemically induced dimerization, and / or chemically induced degradation.

[0194] In some embodiments, at least one of the biomolecules contained in or expressed by the cells or cell lines described herein is a safety switch. In some embodiments, at least one of the biomolecules contained in or expressed by the cells or cell lines described herein is a fusion protein comprising a protein of interest that is a safety switch. By "safety switch" is meant a protein that can control the activity and / or viability of a biomolecule, particularly a cell, for example, by controlling gene expression and / or inducing cell death (apoptosis).

[0195] The safety switch may be a protein selected from the group including or consisting of transcription factors and enzymes. The safety switch may be an enzyme, such as a caspase. The safety switch may be a transcription factor.

[0196] In some embodiments, the activity and / or function of the safety switch is controlled by chemically induced proximity, chemically induced localization, chemically induced transport, chemically induced dimerization, and / or chemically induced degradation.

[0197] Another object of the present invention is to provide a biomolecule described herein, a pair of biomolecules as described herein, a nucleic acid molecule described herein, a pair of nucleic acid molecules as described herein; a vector as described herein, a pair of vectors as described herein, or a cell or cell line as described herein, and optionally at least one pharmaceutically acceptable excipient or pharmaceutically acceptable carrier and a pharmaceutical composition comprising or consisting of any one of:

[0198] In some embodiments, the pharmaceutical compositions described herein further comprise a molecular proximity inducer described herein.

[0199] Another object of the present invention is to provide a biomolecule described herein, a pair of biomolecules as described herein, a nucleic acid molecule described herein, a pair of nucleic acid molecules as described herein; a vector as described herein, a pair of vectors as described herein, or A cell or cell line described herein A drug containing or consisting of any one of the following:

[0200] In some embodiments, the medicament described herein further comprises a molecular proximity inducer described herein.

[0201] Another object of the present invention is to provide a biomolecule described herein, a pair of biomolecules as described herein, a nucleic acid molecule described herein, a pair of nucleic acid molecules as described herein; a vector as described herein, a pair of vectors as described herein, or A cell or cell line described herein a first part including any one of a second portion comprising a molecular proximity inducer as described herein; It is a kit of parts that includes or consists of:

[0202] Another object of the present invention is to provide a method for the preparation of a pharmaceutical composition comprising: a biomolecule described herein, a pair of biomolecules as described herein, a nucleic acid molecule described herein, a pair of nucleic acid molecules as described herein; a vector as described herein, a pair of vectors as described herein; a cell or cell line described herein, a kit comprising any one of the above and a molecular proximity inducer as described herein; a pharmaceutical composition described herein, or Kit-of-parts as described herein Any one of the following:

[0203] In some embodiments, a cell or cell line described herein, a pharmaceutical composition as described herein comprising said cell or cell line, a drug described herein comprising said cell or cell line, or a kit-of-parts as described herein, comprising the cells or cell lines; Any one of the following is for use as a cell therapy:

[0204] In some embodiments, a cell or cell line described herein, a pharmaceutical composition as described herein comprising said cell or cell line, a drug described herein comprising said cell or cell line, or a kit-of-parts as described herein, comprising the cells or cell lines; Any one of is for use for in vivo implantation in a subject in need thereof.

[0205] Another object of the present invention is a method for inducing proximity between two biomolecules in a sample, said method comprising: obtaining a first biological molecule bound to a first polypeptide described herein or a truncated fragment thereof described herein; obtaining a second biomolecule bound to a second polypeptide described herein or a truncated fragment thereof described herein; adding to the sample a molecular proximity inducer as described herein. Including, Here, upon addition of a molecular proximity inducer, the first polypeptide, the second polypeptide and the molecular proximity inducer form a ternary assembly, thereby inducing proximity between the first and second biological molecules present in the sample.

[0206] In some embodiments, the concentration of molecular proximity inducer required to reach half-maximal assembly is less than about 5, 4.5, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.5, or 0.25 μM. In some embodiments, the concentration of molecular proximity inducer required to reach half-maximal assembly is in the range of about 0.05 to about 5 μM, preferably about 0.1 to about 2.5 μM, and more preferably about 0.1 to about 1 μM.

[0207] Thus, in some embodiments, the present invention provides a method for inducing proximity between two biomolecules in a sample, comprising: obtaining a first biological molecule bound to a first polypeptide described herein or a truncated fragment thereof described herein; obtaining a second biomolecule bound to a second polypeptide described herein or a truncated fragment thereof described herein; adding to the sample a molecular proximity inducer as described herein. Including, wherein upon addition of a molecular proximity inducer, the first polypeptide, the second polypeptide, and the molecular proximity inducer form a ternary assembly at a concentration of molecular proximity inducer required to reach half-maximal assembly in the sample that is less than about 5 μM; This method induces proximity between a first and a second biomolecule present in the sample.

[0208] In some embodiments, the method is for inducing proximity between two biomolecules in a sample, the method comprising: conjugating a first biological molecule to a first polypeptide described herein or a truncated fragment thereof described herein; conjugating a second biomolecule to a second polypeptide described herein or a truncated fragment thereof described herein; adding to the sample a molecular proximity inducer as described herein. Including, wherein upon addition of a molecular proximity inducer, the first polypeptide, the second polypeptide, and the molecular proximity inducer form a ternary assembly at a concentration of molecular proximity inducer required to reach half-maximal assembly in the sample that is less than about 5 μM; This induces proximity between the first and second biomolecules present in the sample.

[0209] In some embodiments, the method is for inducing proximity between two biomolecules in a sample, the method comprising: expressing in the sample a first biological molecule linked to a first polypeptide described herein or a truncated fragment thereof described herein; expressing in the sample a second biological molecule linked to a second polypeptide described herein or a truncated fragment thereof described herein; adding to the sample a molecular proximity inducer as described herein. Including, wherein upon addition of a molecular proximity inducer, the first polypeptide, the second polypeptide, and the molecular proximity inducer form a ternary assembly at a concentration of molecular proximity inducer required to reach half-maximal assembly in the sample that is less than about 5 μM; This induces proximity between the first and second biomolecules present in the sample.

[0210] The two biomolecules may be two different biomolecules. The two biomolecules may be two biomolecules of different structures, such as a nucleic acid and a protein. The two biomolecules may be two different biomolecules of the same structure, such as two nucleic acids or two proteins. The two biomolecules may be two copies of the same biomolecule (such as two copies of the same protein), where one copy is bound to a first polypeptide described herein or a cleaved fragment thereof described herein, and the other copy is bound to a second polypeptide described herein or a cleaved fragment thereof described herein.

[0211] The biomolecule may be selected from the group including or consisting of amino acids, polypeptides, proteins, monosaccharides, polysaccharides, nucleotides, nucleic acids, lipids, fatty acids, glycolipids, sterols, vitamins, hormones, neurotransmitters, and metabolites. In some embodiments, the biomolecule is a protein or a nucleic acid. In some embodiments, the biomolecule is a protein.

[0212] In some embodiments, the biomolecule attached to a first or second polypeptide described herein, or a truncated fragment thereof described herein, is a fusion protein comprising said first or second polypeptide, or a truncated fragment thereof, and a protein of interest described herein. The two fusion proteins may comprise the same protein of interest or different proteins of interest.

[0213] In some embodiments, the biomolecule is non-covalently linked to a first or second polypeptide described herein or a truncated fragment thereof described herein. In some embodiments, the biomolecule is covalently linked to a first or second polypeptide described herein or a truncated fragment thereof described herein. In some embodiments, the biomolecule is a protein, and the protein is fused to a first or second polypeptide described herein or a truncated fragment thereof described herein.

[0214] In some embodiments, the method is for inducing proximity in a sample between a first biological molecule bound to a first polypeptide described herein or a cleaved fragment thereof described herein and a second biological molecule bound to a second polypeptide described herein or a cleaved fragment thereof described herein, the method comprising: adding to the sample a molecular proximity inducer as described herein. Including, wherein upon addition of a molecular proximity inducer, the first polypeptide, the second polypeptide, and the molecular proximity inducer form a ternary assembly at a concentration of molecular proximity inducer required to reach half-maximal assembly in the sample that is less than about 5 μM; This induces proximity between the first and second biomolecules present in the sample.

[0215] The sample may be a biological sample. The sample may be an organism. Organisms considered herein are, for example, model organisms used in biomedical research, such as bacteria, yeast, fruit flies, nematodes, zebrafish, mice, rats, guinea pigs, rabbits, and dogs. Other organisms considered herein are plant organisms, such as Arabidopsis thaliana, tobacco (e.g., Nicotiana tabacum), wheat (e.g., bread wheat (Triticum aestivum), durum wheat (Triticum durum), club wheat (Triticum compactum)), and rice (e.g., Oryza sativa). Thus, the organism is not a human organism. The sample may be a cell, a cell line, or a cell culture. Examples of cells include bacterial cells, archaeal cells, yeast cells, plant cells, and animal cells. Examples of animal cells include mammalian cells, particularly human cells.

[0216] The cells may be primary cells. The cells may be immortalized cells. The cells in the sample may be live. The cells in the sample may be fixed for microscopy and imaging.

[0217] In some embodiments, the organisms or cells in the sample, or a subset thereof, are genetically modified to express a biomolecule described herein, preferably a first and second polypeptide described herein or a truncated fragment thereof described herein, linked to a protein of interest.

[0218] In some embodiments, the method is for inducing proximity between two biomolecules in a cell sample, the method comprising: expressing in cells of the sample a first biological molecule linked to a first polypeptide described herein or a truncated fragment thereof described herein; expressing in cells of the sample a second biological molecule linked to a second polypeptide described herein or a truncated fragment thereof described herein; adding a molecular proximity inducer described herein to the cell sample. Including, wherein upon addition of a molecular proximity inducer, the first polypeptide, the second polypeptide, and the molecular proximity inducer form ternary assemblies in a cell sample at a concentration of molecular proximity inducer required to reach half-maximal assembly in the sample that is less than about 5 μM; This induces proximity between the first and second biomolecules expressed in the cells of the sample.

[0219] By "in a cell sample" is meant that the ternary assembly formed between the first polypeptide, the second polypeptide, and the molecular proximity inducer may be intracellular, extracellular, or localized in the membrane of the cell. Indeed, the first and / or second biomolecule expressed in the cells of the sample may be secreted by the cells of the sample or may be localized in the membrane of the cells of the sample.

[0220] In some embodiments, the method is for inducing proximity between two biomolecules in an organism, such as a plant organism or a non-human model organism, the method comprising: expressing in the organism a first biological molecule linked to a first polypeptide described herein or a truncated fragment thereof described herein; expressing in the organism a second biological molecule linked to a second polypeptide described herein or a truncated fragment thereof described herein; administering to the organism a molecular proximity inducer as described herein. Including, wherein upon administration of a molecular proximity inducer, the first polypeptide, the second polypeptide, and the molecular proximity inducer form a ternary assembly at a concentration of molecular proximity inducer required to reach half-maximal assembly in the sample that is less than about 5 μM; This induces proximity between the first and second biological molecules expressed in the organism.

[0221] Those skilled in the art are familiar with the methods that allow organisms or cells in culture to be genetically modified to express target biomolecules.Examples of such methods include transfection, electroporation, injection and gene transfer of nucleic acid molecules such as the nucleic acid molecules described herein.Methods also include transplantation, injection and co-culture of cells that are modified to express target protein.

[0222] In some embodiments, the method is for inducing proximity between two biomolecules in a sample, the method comprising: fusing a first biological molecule, preferably a protein, to a first polypeptide described herein or a truncated fragment thereof described herein; fusing a second biological molecule, preferably a protein, to a second polypeptide described herein or a truncated fragment thereof described herein; expressing both biomolecules in the sample; adding to the sample a molecular proximity inducer as described herein. Including, wherein upon addition of a molecular proximity inducer, the first polypeptide, the second polypeptide, and the molecular proximity inducer form a ternary assembly at a concentration of molecular proximity inducer required to reach half-maximal assembly in the sample that is less than about 5 μM; This induces proximity between the first and second biomolecules present in the sample.

[0223] In some embodiments, the methods described herein involve adding a molecular proximity inducer described herein to the sample at a concentration of less than about 5, 4.5, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.5, or 0.25 μM. In some embodiments, the methods described herein involve adding a molecular proximity inducer described herein to the sample at a concentration ranging from about 0.05 to about 5 μM, preferably from about 0.1 to about 2.5 μM, and more preferably from about 0.1 to about 1 μM.

[0224] The molecular proximity inducer may be non-fluorescent. The molecular proximity inducer may be fluorescent.

[0225] Thus, the method may further comprise detecting and / or visualizing fluorescence resulting from the formation of a ternary assembly between the first polypeptide, the second polypeptide, and the fluorescent molecular proximity inducer. Thus, the method may allow for the detection and / or visualization of the localization of the ternary assembly formed between the first polypeptide, the second polypeptide, and the fluorescent molecular proximity inducer.

[0226] The method may further comprise monitoring the fluorescence resulting from the formation of a ternary assembly between the first polypeptide, the second polypeptide, and the fluorescent molecular proximity inducer over time. Thus, the method may allow the formation of a ternary assembly between the first polypeptide, the second polypeptide, and the fluorescent molecular proximity inducer to be monitored over time.

[0227] Those skilled in the art are familiar with available techniques for detecting fluorescence in a sample and can select from among them depending on the sample, the excitation and emission spectra of the fluorescent compound, and the desired readout. Examples of such techniques include direct observation, fluorescence spectroscopy, flow cytometry, fluorescence microscopy (including laser scanning and spinning disk confocal microscopy, multiphoton microscopy, super-resolution microscopy), and fluorescence tomography.

[0228] The method may further comprise assessing and / or quantifying the fluorescence resulting from the formation of a ternary assembly between the first polypeptide, the second polypeptide, and the fluorescent molecular proximity inducer. Thus, the method may allow for assessing and / or quantifying the formation of a ternary assembly between the first polypeptide, the second polypeptide, and the fluorescent molecular proximity inducer.

[0229] In some embodiments, the method comprises detecting a change in fluorescence resulting from the formation of a ternary assembly between the first polypeptide, the second polypeptide, and the fluorescent molecular proximity inducer. Thus, the method may comprise detecting an increase or decrease in fluorescence resulting from the formation of a ternary assembly between the first polypeptide, the second polypeptide, and the fluorescent molecular proximity inducer.

[0230] In some embodiments, the method further allows for measuring the amount of ternary assembly formed between the first polypeptide, the second polypeptide, and the fluorescent molecular proximity inducer relative to a reference value by measuring fluorescence intensity. In some embodiments, the reference value corresponds to a fluorescence intensity value in a control sample. In some embodiments, the reference value corresponds to a fluorescence intensity value at a different location and / or time point in the same sample.

[0231] Another object of the present invention is an assay that relies on inducing proximity between two biomolecules in a sample, said assay comprising: obtaining a first biological molecule bound to a first polypeptide described herein or a truncated fragment thereof described herein; obtaining a second biomolecule bound to a second polypeptide described herein or a truncated fragment thereof described herein; adding to the sample a molecular proximity inducer as described herein. Including, Here, upon addition of a molecular proximity inducer, the first polypeptide, the second polypeptide and the molecular proximity inducer form a ternary assembly, thereby inducing proximity between the first and second biological molecules present in the sample.

[0232] In some embodiments, the concentration of molecular proximity inducer required to reach half-maximal assembly is less than about 5, 4.5, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.5, or 0.25 μM. In some embodiments, the concentration of molecular proximity inducer required to reach half-maximal assembly is in the range of about 0.05 to about 5 μM, preferably about 0.1 to about 2.5 μM, and more preferably about 0.1 to about 1 μM.

[0233] Thus, in some embodiments, the present invention provides an assay that relies on inducing proximity between two biomolecules in a sample, comprising: obtaining a first biological molecule bound to a first polypeptide described herein or a truncated fragment thereof described herein; obtaining a second biomolecule bound to a second polypeptide described herein or a truncated fragment thereof described herein; adding to the sample a molecular proximity inducer as described herein. Including, wherein upon addition of a molecular proximity inducer, the first polypeptide, the second polypeptide, and the molecular proximity inducer form a ternary assembly at a concentration of molecular proximity inducer required to reach half-maximal assembly in the sample that is less than about 5 μM; The present invention relates to an assay that induces proximity between a first and a second biomolecule present in the sample.

[0234] In some embodiments, the assay comprises: conjugating a first biological molecule to a first polypeptide described herein or a truncated fragment thereof described herein; conjugating a second biomolecule to a second polypeptide described herein or a truncated fragment thereof described herein; adding to the sample a molecular proximity inducer as described herein. Including, wherein upon addition of a molecular proximity inducer, the first polypeptide, the second polypeptide, and the molecular proximity inducer form a ternary assembly at a concentration of molecular proximity inducer required to reach half-maximal assembly in the sample that is less than about 5 μM; This induces proximity between the first and second biomolecules present in the sample.

[0235] In some embodiments, the assay comprises: expressing in the sample a first biological molecule linked to a first polypeptide described herein or a truncated fragment thereof described herein; expressing in the sample a second biological molecule linked to a second polypeptide described herein or a truncated fragment thereof described herein; adding to the sample a molecular proximity inducer as described herein. Including, wherein upon addition of a molecular proximity inducer, the first polypeptide, the second polypeptide, and the molecular proximity inducer form a ternary assembly at a concentration of molecular proximity inducer required to reach half-maximal assembly in the sample that is less than about 5 μM; This induces proximity between the first and second biomolecules present in the sample.

[0236] In some embodiments, the assay relies on inducing proximity between a first biomolecule bound to a first polypeptide described herein or a cleaved fragment thereof described herein, and a second biomolecule bound to a second polypeptide described herein or a cleaved fragment thereof described herein in a sample, and the assay comprises: adding to the sample a molecular proximity inducer as described herein. Including, wherein upon addition of a molecular proximity inducer, the first polypeptide, the second polypeptide, and the molecular proximity inducer form a ternary assembly at a concentration of molecular proximity inducer required to reach half-maximal assembly in the sample that is less than about 5 μM; This induces proximity between the first and second biomolecules present in the sample.

[0237] The assays described herein may be used to control the localization, trafficking, stability, degradation, and / or function of a biomolecule or pair of biomolecules described herein. In particular, the assays described herein may be used to control the localization, trafficking, stability, degradation, and / or function of a protein or pair of proteins described herein in a cell. As referred to herein, the protein may be a fusion protein comprising a first or second polypeptide described herein or a truncated fragment described herein and a protein of interest described herein.

[0238] In some embodiments, the assays described herein are for controlling the localization, trafficking and / or function of a biomolecule or pair of biomolecules.

[0239] In some embodiments, the assays described herein are for controlling the localization of a single biomolecule, particularly a single protein, or a pair of biomolecules, particularly a pair of proteins. For example, as shown in the experimental section below, the assays may be used to control the localization of a cytoplasmic protein, particularly a cytoplasmic fusion protein, in a cell by inducing proximity between the protein and a protein, particularly a fusion protein, that is localized in a cellular compartment, such as an organelle.

[0240] In some embodiments, the assays described herein are for controlling the transport of a single biomolecule, particularly a single protein, or a pair of biomolecules, particularly a pair of proteins. For example, as shown in the Experimental Section below, the assays may be used to control the nucleocytoplasmic transport of proteins in cells by inducing proximity between a protein containing a nuclear localization signal (NLS), particularly a fusion protein, and a cytoplasmic protein, particularly a cytoplasmic fusion protein, such as a protein containing a nuclear export signal (NES).

[0241] In some embodiments, the assays described herein are for controlling the function of a single biomolecule, particularly a single protein, or a pair of biomolecules, particularly a pair of proteins.

[0242] In some embodiments, the assays described herein are for controlling the stability of a biomolecule, particularly a protein, or a pair of biomolecules, particularly a pair of proteins. In some embodiments, the assays described herein are for inducing the degradation of a biomolecule, particularly a protein, or a pair of biomolecules, particularly a pair of proteins.

[0243] In some embodiments, the assays described herein are for detecting the co-occurrence of two biomolecules, particularly two proteins, in a sample of interest. For example, the assays may be used in a cell to detect the co-occurrence of two biomolecules, particularly two proteins, in a cellular compartment, such as an organelle.

[0244] In some embodiments, the assays described herein involve adding a molecular proximity inducer described herein to the sample at a concentration of less than about 5, 4.5, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.5, or 0.25 μM. In some embodiments, the assays described herein involve adding a molecular proximity inducer described herein to the sample at a concentration ranging from about 0.05 to about 5 μM, preferably from about 0.1 to about 2.5 μM, and more preferably from about 0.1 to about 1 μM.

[0245] The molecular proximity inducer may be non-fluorescent. The molecular proximity inducer may be fluorescent.

[0246] The assay may further comprise detecting and / or visualizing fluorescence resulting from the formation of a ternary assembly between the first polypeptide, the second polypeptide, and the fluorescent molecular proximity inducer. Thus, the assay may allow for the detection and / or visualization of the localization of the ternary assembly formed between the first polypeptide, the second polypeptide, and the fluorescent molecular proximity inducer.

[0247] The assay may further comprise monitoring the fluorescence resulting from the formation of a ternary assembly between the first polypeptide, the second polypeptide, and the fluorescent molecular proximity inducer over time. Thus, the assay may allow for the formation of a ternary assembly between the first polypeptide, the second polypeptide, and the fluorescent molecular proximity inducer to be monitored over time.

[0248] The assay may further comprise assessing and / or quantifying the fluorescence resulting from the formation of a ternary assembly between the first polypeptide, the second polypeptide, and the fluorescent molecular proximity inducer. Thus, the assay may allow for assessing and / or quantifying the formation of a ternary assembly between the first polypeptide, the second polypeptide, and the fluorescent molecular proximity inducer.

[0249] In some embodiments, the assay comprises detecting a change in fluorescence resulting from the formation of a ternary assembly between the first polypeptide, the second polypeptide, and the fluorescent molecular proximity inducer. Thus, the assay may comprise detecting an increase or decrease in fluorescence resulting from the formation of a ternary assembly between the first polypeptide, the second polypeptide, and the fluorescent molecular proximity inducer.

[0250] In some embodiments, the assay further comprises measuring the amount of ternary assembly formed between the first polypeptide, the second polypeptide, and the fluorescent molecular proximity inducer relative to a reference value by measuring fluorescence intensity. In some embodiments, the reference value corresponds to a fluorescence intensity value in a control sample. In some embodiments, the reference value corresponds to a fluorescence intensity value at a different location and / or time point in the same sample. [Table 1] TIFF2025529306000008.tif234159TIFF2025529306000009.tif237159TIFF2025529306000010.tif100159 [Brief explanation of the drawings]

[0251] [Figure 1]Schematic diagram of the operating principle of the proximity induction system described herein. The system relies on the binding (e.g., by genetic fusion) of two proteins to two distinct polypeptides, termed the FIRE mate (i.e., the polypeptide of SEQ ID NO: 1) and the FIRE tag (i.e., the polypeptide of SEQ ID NO: 2). These two polypeptides can only specifically interact in the presence of a small synthetic molecule (i.e., a molecular proximity inducer termed a "match") that stabilizes the interaction between these two polypeptides. Advantageously, the match may be a fluorescent chromophore, such as HBR-2,5DM or HMBR, that becomes fluorescent (i.e., a "bright match," also known as "FIRE fluorescence" or "FIRE signal") upon specific formation of a ternary assembly consisting of these two polypeptides and the match (a so-called "FIRE" ternary assembly). Thus, the system described herein can induce proximity between any two proteins and, optionally, visualize the recruitment process by fluorescence imaging. [Figure 2] Figures 2A and 2B are graphs showing the fluorogen-dependent interaction between two polypeptides, FIRE-mate and FIRE-tag. These graphs show the normalized mean fluorescence detected from approximately 50,000 HEK293T cells co-expressing FK506-binding protein (FKBP) fused to the FIRE-tag and the FKBP rapamycin-binding domain (FRB) of the mammalian target of rapamycin fused to the FIRE-mate, with or without treatment (triangles) or 500 nM rapamycin (squares), and with 1, 5, 10, 25, or 50 μM HBR-2,5DM (Figure 2A) or HMBR (Figure 2B). Data represent the mean ± SD of three independent experiments. [Figure 3]Figures 3A-3E show the recruitment of cytosolic proteins to mitochondria by the proximity-guided system described herein. Figure 3A is a schematic of the experimental design, in which HeLa cells co-expressing mCherry-FIRE tag and Tom20-ECFP-FIRE mate were treated with 10 µM match (either HBR-2,5DM or HMBR) and imaged by time-lapse confocal microscopy. Figures 3B-3C are graphs showing the time course of the FIRE signal detected at 0 min upon addition of HBR-2,5DM (match 1 - Figure 3B) or HMBR (match 2 - Figure 3C). Data represent the mean ± SD of 20 cells from n = 3 independent experiments (Figure 3B, Figure 3D) or the mean ± SD of 10 cells from n = 3 independent experiments (Figure 3C, Figure 3E). Figure 3D-Figure 3E are a set of representative confocal micrographs of cells before (0 min) and after (5 min) the addition of HBR-2,5DM (match 1 - Figure 3D) or HMBR (match 2 - Figure 3E) (mCherry: ex / em = 561 / 606-675 nm, FIRE: ex / em = 488 / 508-570 nm, ECFP: ex / em = 445 / 455-499 nm). The experiment was repeated three times with similar results. The scale bar is 20 µm. [Figure 4]Figures 4A-4D show the time-lapse recruitment of cytosolic proteins to mitochondria using the proximity-guided system described herein. Figures 4A and 4C show HeLa cells co-expressing mCherry-FIRE tag and Tom20-ECFP-FIRE mate treated with 10 μM HBR-2,5DM and imaged by time-lapse confocal microscopy with one image every 5 seconds. Figures 4B and 4D show U2OS cells co-expressing mCherry-FIRE tag and Tom20-ECFP-FIRE mate treated with 10 μM HBR-2,5DM and imaged by time-lapse confocal microscopy with one image every 2 minutes. Figure 4A shows representative time-lapse images (ex / em = 488 / 508-570 nm) of HeLa cells obtained by adding HBR-2,5DM at 0 seconds. The experiment was repeated three times with similar results. The scale bar is 20 μm. Figure 4B shows representative time-lapse images of U2OS cells after addition of HBR-2,5DM at 0 min (mCherry: ex / em = 561 / 606-675 nm, FIRE: ex / em = 488 / 508-570 nm, ECFP: ex / em = 445 / 455-499 nm). The experiment was repeated three times with similar results. The scale bar is 20 μm. Figure 4C shows the time course of the FIRE signal detected in HeLa cells after addition of HBR-2,5DM at 0 s. Data represent the mean ± SD of 15 cells from three independent experiments. Figure 4D shows the time course of the FIRE signal detected in U2OS cells after addition of HBR-2,5DM at 0 min. Data represent the mean ± SD of 10 cells from three independent experiments. [Figure 5]Figures 5A-5D demonstrate that the positioning of the FIRE tag does not affect the proximity-guided system described herein. HeLa cells co-expressing either FIRE tag-mCherry (Figure 5A, 5C) or mCherry-FIRE tag-mCherry (Figure 5B, 5D) and Tom20-ECFP-FIRE Mate were treated with 10 µM HBR-2,5DM (match 1) and imaged by time-lapse confocal microscopy. Figures 5A-5B are a set of representative confocal micrographs of cells before (0 min) and after (5 min) the addition of HBR-2,5DM (match 1) (mCherry: ex / em = 561 / 606-675 nm, FIRE: ex / em = 488 / 508-570 nm, ECFP: ex / em = 445 / 455-499 nm). The experiment was repeated three times with similar results. The scale bar is 20 µm. Figures 5C-5D are graphs showing the time course of the FIRE signal detected upon addition of HBR-2,5DM at 0 min. Data represent the mean ± SD of 17 cells from n = 3 independent experiments (Figure 5A, Figure 5C) or 13 cells from n = 3 independent experiments (Figure 5B, Figure 5D). [Figure 6]Figures 6A-6E show the recruitment of cytosolic proteins to the Golgi apparatus by the proximity-guided system described herein. Figure 6A is a diagram of the experimental design, in which HeLa cells co-expressing mCherry-FIRE tag and FIRE mate-ECFP-Giantin were treated with 10 µM match (either HBR-2,5DM or HMBR) and imaged by time-lapse confocal microscopy. Figures 6B-6C are graphs showing the time course of the FIRE signal detected upon addition of HBR-2,5DM (match 1 - Figure 6B) or HMBR (match 2 - Figure 6C) at 0 min. Data represent the mean ± SD of 10 cells from n = 3 independent experiments (Figure 6B, Figure 6D) or 12 cells from n = 3 independent experiments (Figure 6C, Figure 6E). Figure 6D-Figure 6E are a set of representative confocal micrographs of cells before (0 min) and after (5 min) the addition of HBR-2,5DM (match 1 - Figure 6D) or HMBR (match 2 - Figure 6E) (mCherry: ex / em = 561 / 606-675 nm, FIRE: ex / em = 488 / 508-570 nm, ECFP: ex / em = 445 / 455-499 nm). The experiment was repeated three times with similar results. The scale bar is 20 µm. [Figure 7]Figures 7A-7E show the recruitment of cytosolic proteins to the endoplasmic reticulum by the proximity-guided system described herein. Figure 7A is a diagram of the experimental design, in which HeLa cells co-expressing mCherry-FIRE tag and FIRE mate-ECFP-Cb5 were treated with 10 µM match (either HBR-2,5DM or HMBR) and imaged by time-lapse confocal microscopy. Figures 7B-7C are graphs showing the time course of the FIRE signal detected upon addition of HBR-2,5DM (match 1 - Figure 7C) or HMBR (match 2 - Figure 7D) at 0 min. Data represent the mean ± SD of 17 cells from n = 3 independent experiments (Figure 7B, Figure 7D) or 18 cells from n = 3 independent experiments (Figure 7C, Figure 7E). Figure 7D-Figure 7E are a set of representative confocal micrographs of cells before (0 min) and after (5 min) the addition of HBR-2,5DM (Match 1 - 7D) or HMBR (Match 2 - Figure 7E) (mCherry: ex / em = 561 / 606-675 nm, FIRE: ex / em = 488 / 508-570 nm, ECFP: ex / em = 445 / 455-499 nm). The experiment was repeated three times with similar results. The scale bar is 20 µm. [Figure 8]Figures 8A-8E show the recruitment of cytoplasmic proteins to the plasma membrane by the proximity-guided system described herein. Figure 8A is a diagram of the experimental design, in which HEK293 cells co-expressing mCherry-FIRE tag and Lyn11-ECFP-FIRE mate were treated with 10 µM match (either HBR-2,5DM or HMBR) and imaged by time-lapse confocal microscopy. Figures 8B-8C are graphs showing the time course of the FIRE signal detected upon addition of HBR-2,5DM (match 1 - Figure 8B) or HMBR (match 2 - Figure 8C) at 0 min. Data represent the mean ± SD of 22 cells from n = 3 independent experiments (Figure 8B, Figure 8D) or 30 cells from n = 3 independent experiments (Figure 8C, Figure 8E). Figure 8D-Figure 8E are a set of representative confocal micrographs of cells before (0 min) and after (5 min) the addition of HBR-2,5DM (match 1 - Figure 8D) or HMBR (match 2 - Figure 8E) (mCherry: ex / em = 561 / 606-675 nm, FIRE: ex / em = 488 / 508-570 nm, ECFP: ex / em = 445 / 455-499 nm). The experiment was repeated three times with similar results. The scale bar is 20 µm. [Figure 9] Figures 9A-9D demonstrate the reversible and non-fluorescent recruitment potential of the proximity-guided system described herein. HeLa cells co-expressing mCherry-FIRE tag and FIRE mate-ECFP-Giantin were treated with 10 μM HBR-2,5DM (match 1), washed with medium without HBR-2,5DM, and then treated with 10 μM HBIR-3M. Cells were imaged by time-lapse spinning disk confocal microscopy. Figures 9A-9D are a set of representative confocal micrographs of untreated cells (Figure 9A), cells after HBR-2,5DM (match 1) addition (Figure 9B), cells after HBR-2,5DM (match 1) washing (Figure 9C), and cells after HBIR-3M addition (Figure 9D) (mCherry: ex / em = 561 / 606-675 nm, FIRE: ex / em = 488 / 508-570 nm). The scale bar is 10 μm. [Figure 10]Graphs depicting the fluorogen-dependent interaction of caged FIRE tags with FIRE mate. These graphs show the normalized mean fluorescence detected from approximately 50,000 HEK293T cells co-expressing FK506 binding protein (FKBP) fused to a caged FIRE tag and the FKBP rapamycin-binding domain of mammalian target of rapamycin (FRB) fused to FIRE mate, with or without treatment with 500 nM rapamycin, but with treatment with 1, 5, 10, 25, or 50 μM HBR-2,5DM. Data represent the mean ± SD of three independent experiments. [Figure 11] Figures 11A-11C illustrate the use of the caged proximity guidance system described herein. Figure 11A is a diagram of the experimental design, in which HeLa cells co-expressing mCherry-caged FIRE tag and FIRE mate-ECFP-Giantin were treated with 10 μM of match (i.e., HBR-2,5DM) and imaged by time-lapse confocal microscopy. Figure 11B is a graph showing the time course of FIRE signal detected with either FIRE tag or caged FIRE tag upon addition of HBR-2,5DM at 0 min. Data represent the mean ± SD of seven cells from n=3 independent experiments. Figure 11C is a set of representative confocal micrographs of cells before (0 min) and after (9 min) the addition of HBR-2,5DM (match 1) (mCherry: ex / em = 561 / 606-675 nm, FIRE: ex / em = 488 / 508-570 nm, ECFP: ex / em = 445 / 455-499 nm). The experiment was repeated three times with similar results. The scale bar is 20 µm. [Figure 12]Figure 12A-12B demonstrate that the proximity-guided system described herein is orthogonal to the FRB / FKBP / rapamycin CIP system. Figure 12A is a diagram of the experimental design, in which HeLa cells co-expressing mCherry-FKBP-FIRE tag, TOM20-FIRE mate, and FRB-ECFP-Giantin were treated with 10 µM match (i.e., HBR-2,5DM) and 500 nM rapamycin and imaged by time-lapse confocal microscopy. Figure 12B is a set of representative confocal micrographs of cells before and after the addition of HBR-2,5DM (match 1) and rapamycin (rap) (mCherry: ex / em = 561 / 606-675 nm, FIRE: ex / em = 488 / 508-570 nm, ECFP: ex / em = 445 / 455-499 nm). The scale bar is 20 µm. [Figure 13] Figures 13A-13D show protein nuclear export mediated by the proximity-guided system described herein. Figure 13A shows the experimental design, in which HeLa cells co-expressing NLS-mCherry-FIRE mate and NES-ECFP-FIRE tag were treated with 10 μM of match (i.e., HBR-2,5DM) and imaged by time-lapse confocal microscopy. Figure 13B shows the time course of the nuclear:cytoplasmic mCherry fluorescence intensity ratio for n = 5 cells from two independent experiments. Figure 13C shows the time course of the FIRE signal detected upon addition of HBR-2,5DM at 0 min for n = 5 cells from two independent experiments. Figure 13D is a set of representative confocal micrographs before (0 min) and after (4 min) treatment with HBR-2,5DM (mCherry: ex / em = 561 / 606-675 nm, FIRE: ex / em = 488 / 508-570 nm, ECFP: ex / em = 445 / 455-499 nm). Scale bar is 20 µm. [Example]

[0252] The present invention is further illustrated by the following examples.

[0253] example material and method Molecular cloning Synthetic oligonucleotides used for cloning were purchased from Integrated DNA Technologies. PCR reactions were performed using Q5 polymerase (New England Biolabs) in the provided buffer. PCR products were purified using a QIAquick PCR Purification Kit (Qiagen). Deoxyribonuclease I, T4 ligase, Fusion polymerase, Taq ligase, and Taq exonuclease were purchased from New England Biolabs and used with the provided buffer according to the manufacturer's protocol. Isothermal assembly (Gibson assembly) was performed using a homemade mix prepared according to a previously described protocol (Gibson et al., "Enzymatic assembly of DNA molecules up to several hundred kilobases." Nat Methods. 2009 May, 6(5):343-5). Small-scale isolation of plasmid DNA was performed from 2 mL overnight bacterial cultures supplemented with the appropriate antibiotic using a QIAprep miniprep kit (Qiagen). Large-scale isolation of plasmid DNA was performed from 150 mL overnight bacterial cultures supplemented with the appropriate antibiotic using a QIAprep maxiprep kit (Qiagen). All plasmid sequences were confirmed by Sanger sequencing using appropriate sequencing primers (GATC Biotech).

[0254] Chromophore synthesis The synthesis of HMBR and HBR-2,5DM has been previously reported (Plamont et al., "Small fluorescence-activating and absorption-shifting tag for tunable protein imaging in vivo." Proc Natl Acad Sci U S A. 2016 Jan. 19, 113(3):497-502; and Li et al., "Dynamic multicolor protein labeling in living cells." Chem Sci. 2017 Aug. 1, 8(8):5598-5605).

[0255] cell culture HeLa cells (ATCC CRM-CCL2) were cultured in minimum essential medium (MEM) supplemented with phenol red, Glutamax I, 1 mM sodium pyruvate, 1% (vol / vol) non-essential amino acids, and 10% (vol / vol) fetal calf serum (FCS) at 37°C in a 5% CO atmosphere. HEK293T cells (ATCC CRL-3216) were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with phenol red and 10% (vol / vol) FCS at 37°C in a 5% CO atmosphere. U2OS cells (ATCC HTB-96) were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with phenol red, 10% (vol / vol) FCS, and 1% (vol / vol) penicillin-streptomycin at 37°C in a 5% CO atmosphere. For imaging, cells were seeded on poly-L-lysine-coated μDish IBIDI (Biovalley). 24 hours before imaging, cells were transiently transfected using Genejuice (Merck) according to the manufacturer's protocol. After washing with DPBS (Dulbecco's phosphate-buffered saline), cells were treated with DMEM medium (serum- and phenol red-free) supplemented with the compound of interest (e.g., molecular proximity inducer) at the indicated concentrations.

[0256] Flow cytometry analysis Flow cytometry of HEK293T cells was performed using a MACSQuant® Analyzer equipped with 405 nm, 488 nm, and 635 nm lasers and seven channels. To prepare samples, cells were first grown in cell culture flasks and then transiently transfected for 24 hours using Genejuice (Merck) according to the manufacturer's protocol. After 24 hours, cells were centrifuged in PBS-BSA (phosphate-buffered saline containing 1 mg / mL bovine serum albumin) and resuspended in PBS-BSA supplemented with an appropriate amount of compound (e.g., molecular proximity inducer). For each experiment, 20,000 cells positively expressing mTurquoise2 (ex: 434 nm / em: 473 nm) and iRFP670 (ex: 638 nm / em: 660 ± 10 nm) were analyzed using the following parameters: excitation (i.e., ex): 488 nm, emission (i.e., em): 525 ± 20 nm. mTurquoise2 and iRFP670 are transfection markers used to select cells expressing the construct of interest. Data were analyzed using FlowJo v10.7.1.

[0257] Fluorescence microscopy Confocal micrographs were acquired using a Zeiss LSM 980 laser scanning microscope equipped with a Plan-Apochromat 63x / 1.4NA oil immersion objective. Data were collected using ZEN software. These images were analyzed using Icy (2.4.0.0) and Fiji (Image J). Briefly, to track fluorescent signals in specific organelles, regions of interest (ROIs) were defined by masking the signal in the enhanced cyan fluorescent protein (ECFP) channel using the plugin HK-instruments with the following parameters: intensity class = 100, minimum object size (pixels, i.e., px) = 500, and maximum object size (px) = 1500–3000. Signal intensity in the ROIs was tracked over time for each channel using the active contour plugin. Background signals were subtracted. Data were processed using GraphPad Prism v9.3.0.

[0258] result Proximity Guidance System Bisection of the mutant photoactive yellow protein (PYP) yielded two fragments: a first polypeptide ( FIRE mate) and a second polypeptide consisting of 11 amino acid residues having the sequence set forth in SEQ ID NO:2 ( FIRE As shown in Figure 1, these two fragments, i.e. SEQ ID NO: 1 (i.e. FIRE mate) and SEQ ID NO:2 (i.e. FIRE It has been surprisingly found that the two polypeptides, i.e., the nucleotide sequence of SEQ ID NO: 1 (i.e., the tag), are complementary and can form a fluorescent ternary assembly (also referred to as "FIRE") with a fluorogen (also referred to as a "match"), such as HBR-2,5DM or HMBR, which is represented by SEQ ID NO: 1 (i.e., the tag). FIRE mate) and SEQ ID NO:2 (i.e. FIRESurprisingly, the first polypeptide of SEQ ID NO: 1 (i.e., the tag) emits significant fluorescence only when in an environment that constrains their conformation and precludes de-excitation of their excited state, such as the assembly formed with the two polypeptides of SEQ ID NO: 1 (i.e., the tag). FIRE mate), a second polypeptide of SEQ ID NO:2 (i.e. FIRE It was found that ternary assembly between the nucleotide sequence (tag) and the fluorogen occurs upon addition of the fluorogen regardless of the initial proximity between the two polypeptides of SEQ ID NO:1 and SEQ ID NO:2. FIRE Mate and FIRE When a tag is used, a fluorogen such as HBR-2,5DM or HMBR functions as a molecular proximity inducer.

[0259] fused to the C-terminus of FKBP (FK506-binding protein) and FRB (FKBP rapamycin-binding domain of mammalian target of rapamycin), respectively FIRE Mate and FIRE The formation of fluorescent ternary assemblies was assessed by flow cytometry in HEK293 cells expressing the tag. FKBP and FRB can dimerize upon the addition of rapamycin. Thus, treatment of HEK293 cells with rapamycin induces the dimerization of FKBP and FRB, followed by FIRE Tags and FIRE As shown in Figure 2, treatment of HEK293 cells with the fluorogen HBR-2,5DM (Figure 2A) or HMBR (Figure 2B) induced the formation of ternary fluorescent assemblies at all tested fluorogen concentrations (1, 5, 10, 25, or 50 μM, as indicated). Note that addition of fluorogen resulted in the formation of ternary fluorescent assemblies regardless of the presence or absence of rapamycin, which is consistent with the previous study. FIRE tag, FIRE To induce the formation of ternary fluorescent assemblies between Mate and HBR-2,5DM or HMBR FIRE Tags and FIRE This indicates that initial proximity between the mate is not required, even in the absence of rapamycin. FIRE tag, FIREMate and HBR-2,5DM or HMBR were able to induce dimerization of FKBP and FRB, i.e., induce proximity between FKBP and FRB.

[0260] Proximity does not occur in the absence of fluorogen FIRE Mate and FIRE To test whether the tag is self-complementary in the absence of a fluorogen, we spatially separated these two fragments and analyzed their mutual affinity. As shown in Figure 3A, for diffuse cytoplasmic expression, FIRE The tag was fused to the C-terminus of the red fluorescent protein mCherry, so that pFAST1-114 faces the cytosol. FIRE Mate was fused to the C-terminus of the N-terminal domain of the mitochondrial outer membrane protein TOM20 (TOM20_1-34). Enhanced cyan fluorescent protein (ECFP) was used. FIRE The fusion protein was inserted between Mate and TOM20_1-34 to monitor the localization of the resulting fusion protein (blue fluorescence). Fluorescence imaging of HeLa cells co-expressing the two fusion proteins in the absence of a fluorogen showed that the localization of the mCherry- FIRE Diffuse cytoplasmic localization of tagged fusion proteins (red fluorescence) and TOM20-ECFP- FIRE This shows mitochondrial localization of the Mate fusion protein (blue fluorescence). FIRE Mate and FIRE This suggests that the tags are not naturally complementary (Figures 3B-3E).

[0261] Addition of HBR-2,5DM resulted in the uptake of mCherry-1 from the cytosol to mitochondria, as observed by the transfer of red fluorescence (i.e., mCherry fluorescence) from the cytosol to mitochondria. FIRE Very rapid translocation of the tagged protein was induced, resulting in the simultaneous appearance of a strong green fluorescent signal (i.e., the FIRE signal corresponding to HBR-2,5DM fluorescence) in the mitochondria (Figure 3B, Figure 3D). FIRE Mate and FIREThese results suggest that fluorogen-induced complementation of the tags mediates the interaction of mCherry and TOM20-ECFP in a sustainable manner, allowing the formed complex to be simultaneously imaged. Similar results were observed when using the fluorogen HMBR (Figure 3C, Figure 3E).

[0262] Therefore, these experiments were performed using fluorogens such as HBR-2,5DM or HMBR, respectively. FIRE Mate and FIRE Induce rapid and sustainable proximity / interaction of two proteins fused to the tag, and FIRE mate, FIRE We demonstrate that the recruitment process can be illuminated by the formation of a sustainable ternary fluorescent assembly between the tag and a fluorogen such as HBR-2,5DM or HMBR.

[0263] Inductive proximity is extremely fast and stable Two fusion proteins (i.e., mCherry- FIRE Tag and TOM20-ECFP- FIRE Further time-lapse confocal microscopy experiments were performed on HeLa cells treated with HBR-2,5DM co-expressing the chromosome mate (Mate). As shown in Figures 4A and 4C, these experiments demonstrated that the formation of the ternary fluorescent assembly occurred in a half-time of approximately 20 seconds, suggesting that the process is extremely rapid and limited almost exclusively by cellular uptake and diffusion of the fluorogen. Furthermore, time-lapse confocal microscopy experiments demonstrated that the ternary fluorescent assembly was stable over long periods, as observed in Figures 4B and 4D, with HBR-2,5DM fluorescence detected for over 25 minutes.

[0264] Induced proximity is independent of positioning within the fusion protein for the efficiency of formation of fluorescent ternary assemblies FIRE To investigate the effect of tag positioning, FIRE The tag is fused to the N-terminus of mCherry ( FIRE tag-mCherry) or inserted between two mCherry (mCherry- FIRETOM20-ECFP- (Tag-mCherry) FIRE Mate fusion proteins were expressed in HeLa cells. FIRE Tag-mCherry fusion protein or mCherry- FIRE The GFP-mCherry fusion protein was co-expressed with either the GFP-mCherry tag or the GFP-mCherry tag. FIRE Tag-mCherry fusion protein (Figure 5A, Figure 5C) and mCherry- FIRE Similar rapid and stable translocation of the tag-mCherry fusion protein (Figure 5B, Figure 5D) into both mitochondria occurred, indicating that the fusion protein FIRE We demonstrate that the positioning of the tag does not affect the efficiency of formation of the fluorescent ternary assembly.

[0265] Use of the present proximity-guided system to control protein localization within cells To further appreciate the scope and demonstrate the versatility of the proximity guidance systems described herein, FIRE Mate facing the cytosol FIRE Mate was anchored to the membranes of various organelles or cellular structures. FIRE Mate, The N-terminus of the Giantin transmembrane domain (amino acid residues 3131–3259) for positioning at the cis- to medial edge of the Golgi apparatus (Figure 6A), the N-terminus (amino acid residues 100–134) of the transmembrane domain of cytochrome b5 (Cb5) for placement in the endoplasmic reticulum membrane (Figure 7A), and The C-terminus of the Lyn11 inner membrane targeting sequence for targeting to the plasma membrane (Figure 8A) was fused into For each construct, ECFP FIRE The fusion proteins were inserted between the mCherry- and other protein domains / sequences, and the resulting fusion proteins (blue fluorescence) were monitored for localization. Each fusion protein was expressed in mammalian cells (HeLa or HEK293 cells, as indicated). FIREThe addition of either HBR-2,5DM (Figures 6B and 6D, 7B and 7D, 8B and 8D) or HMBR (Figures 6C and 6E, 7C and 7E, 8C and 8E) increased the expression of mCherry-. FIRE Rapid translocation of the tag to organelles occurs, as observed by the movement of red fluorescence from the cytosol to the organelle and the simultaneous appearance of organelle-localized green fluorescence (i.e., FIRE signal corresponding to HBR-2,5DM or HMBR fluorescence). FIRE The mate fusion protein is expressed, which FIRE tag, FIRE We demonstrate the efficient formation of fluorescent ternary assemblies between Mate and either HBR-2,5DM or HMBR. mCherry-, regardless of organelle or cellular structure (i.e., Golgi apparatus, endoplasmic reticulum membrane, or plasma membrane), is expressed in the ternary assemblies. FIRE Translocation of the tags occurred at very similar rates.

[0266] Induced proximity is reversible To test whether the formation of the fluorescent ternary assembly could be reversed by removal of the fluorogen, we used the fusion protein mCherry- FIRE tag (red fluorescence) and FIRE Mate-ECFP-Giantin (blue fluorescence) was co-expressed in HeLa cells (Figure 9A). FIRE tag, FIRE We induced the formation of a fluorescent ternary assembly between Mate and HBR-2,5DM (Figure 9B). Washing the cells with HBR-2,5DM-free medium resulted in rapid dissociation of the fluorescent ternary assembly and the simultaneous release of mCherry into the cytosol (Figure 9C), demonstrating the complete reversibility of the fluorescent ternary assembly. The ability to reverse the fluorescent ternary assembly at will allowed us to induce repeated catch-and-release cycles by sequentially adding and removing HBR-2,5DM. The ability to control protein proximity by adding and removing a molecular proximity inducer is unprecedented and opens up great possibilities for controlling the proximity of two proteins with high temporal resolution.

[0267] Proximity can be induced by non-fluorescent chromophores Next, we conducted experiments to determine whether other chromophores could function as molecular proximity inducers and provide new properties. In particular, we investigated non-fluorescent chromophores such as HBIR-3M. HBIR-3M: FIRE Tags and FIRE It can form very tight, non-fluorescent, "invisible" assemblies with its mate. FIRE tag (red fluorescence) and FIRE Mate-ECFP-Giantin (blue fluorescence) was co-expressed in HeLa cells as described above. By successive addition and removal cycles, HBIR-3M was as efficient as HBR-2,5DM. FIRE Mate (i.e., ECFP-Giantin) and FIRE It was found to induce proximity of proteins fused to a tag (i.e., mCherry) (Figure 9D).

[0268] Caged Proximity Guidance System As shown above, FIRE Tags and FIRE Self-complementation between mates cannot be observed in the absence of fluorogen (see Figure 3), and even at high expression levels, FIRE Caged to prevent any interaction with mates FIRE Tag designed. FIRE To cage the tag, N-terminal domains 2-114 of Halorhodospira halophila photoactive yellow protein (PYP) C69G (corresponding to SEQ ID NO: 21) were used. FIRE Merged with tags. FIRE Intermolecular interaction between the tag and the N-terminal domain 2-114 of Halorhodospira halophila PYPC69G caged FIRE Induce the folding of the tagged protein, thus caged FIRE Tags and FIRE Prevents any intermolecular interactions with the mate. FKBP-caged fusion protein FIRETags and Fed- FIRE Mate was co-expressed in HEK293T cells. The cells were then treated with various concentrations (1, 5, 10, 25, or 50 μM, as indicated) of the fluorogen HBR-2,5DM in the presence or absence of rapamycin. As shown in Figure 10, flow cytometric analysis revealed that even in the absence of rapamycin, i.e., caged β-glucan was expressed. FIRE Tags and FIRE Caging occurs regardless of initial proximity between the mate. FIRE Tag FIRE It was found that fluorescent ternary assemblies could still be formed with Mate and HBR-2,5DM. FIRE Following tag caging, higher concentrations of HBR-2,5DM were required to reach a complete ternary assembly in the absence of rapamycin. These results support the formation of highly stable ternary assemblies, which are driven by mutually exclusive folding mechanisms. FIRE For efficient complementation with mates FIRE This suggests that the driving force required to uncage the tag can be obtained.

[0269] Caging FIRE Tags and FIRE To further verify that the mates do not interact with each other, we used the fusion protein mCherry-caged FIRE Tags and FIRE Mate-ECFP-Giantin were co-expressed in HeLa cells (Fig. 11A). The cells were then imaged in the absence or presence of HBR-2,5DM (Fig. 11C). In the absence of HBR-2,5DM, the two fusion proteins did not co-localize, demonstrating the presence of caged fusion proteins. FIRE Tags and FIRE However, treatment with HBR-2,5DM inhibits the binding of mCherry-caged mAbs to the mAbs. FIRE This results in efficient recruitment of TAG to the Golgi, which is caged. FIRE Tags and FIRE Efficient complementation of the mate has been demonstrated. FIREBecause it is necessary to expand the tag, FIRE tag, FIRE The rate was reduced when compared to the fluorescent ternary assembly between Mate and HBR-2,5DM (Figure 11B).

[0270] This proximity guidance system can be used simultaneously with the CIP system. Next, to control interactions between two or more proteins, we investigated the combination of the "FIRE" proximity induction system described herein with the FRB-FKBP-rapamycin CIP system (Figure 12). FIRE The tags were FRB-EGFP-Giantin and TOM20- FIRE The mCherry-FKBP- and mate fusion proteins were expressed in HeLa cells (Figure 12A). FIRE Translocation of the tag to the Golgi apparatus and mitochondria was induced (Figure 12B), demonstrating that the two systems are orthogonal and can be used to simultaneously control two interactions.

[0271] Use of the proximity induction system to control protein nucleocytoplasmic transport To further demonstrate the potential of the proximity-guided system described herein, we developed an assay to control protein nucleocytoplasmic transport (Figure 13). Nucleocytoplasmic transport was tightly controlled by a mechanism involving nuclear localization signals (NLS) and nuclear export signals (NES). First, we used NLS-mCherry as the NLS-containing cargo molecule. FIRE Mate is used, and NES-ECFP- is used as the outgoing NES-containing cargo partner. FIRE The NLS-mCherry tag was used to examine the regulation of nuclear protein export (Figure 13A). FIRE Mate was distributed approximately equally between the nucleus and the cytosol. FIREThe tag was able to freely enter the nuclear compartment by diffusion. As shown in Figure 13D (4 min), addition of HBR-2,5DM resulted in the NLS-mCherry- FIRE Efficient cytoplasmic localization of the formed protein complex occurred, as judged by the translocation of mate fluorescence from the nucleus to the cytoplasm (Figure 13B) and the appearance of strong cytoplasmic green fluorescence (Figure 13C). These results confirm that in protein assemblies containing both an NLS and an NES, export activity predominates over import activity (Busch et al., "Quantification of nuclear protein transport using induced heterodimerization." Traffic. 2009 Sep;10(9):1221-7).

Claims

1. A method for inducing proximity between two biomolecules in a sample, - A step of obtaining a first biomolecule bound to a first polypeptide comprising the amino acid sequence described in SEQ ID NO: 1 or an amino acid sequence having at least about 70% identity with SEQ ID NO: 1, or a cleaved fragment thereof containing at least 89 consecutive amino acid residues from the C-terminus of the sequence having at least about 70% identity with SEQ ID NO: 1 or SEQ ID NO: 1, - A step of obtaining a second biomolecule bound to a second polypeptide containing the amino acid sequence described in SEQ ID NO: 2 or an amino acid sequence having at least about 70% identity with SEQ ID NO: 2, or a cleaved fragment thereof containing at least eight consecutive amino acid residues from the N-terminus of the sequence having at least about 70% identity with SEQ ID NO: 2, - Step of adding a molecular proximity inducing substance to the sample. Includes, Here, when the molecular proximity inducing substance is added, the first polypeptide, the second polypeptide, and the molecular proximity inducing substance form a ternary assembly at a concentration of the molecular proximity inducing substance required to reach the maximum half volume of the assembly in the sample, which is less than approximately 5 μM. A method for inducing proximity between the first and second biomolecules present in the sample.

2. An assay that relies on inducing proximity between two biomolecules in a sample, - A step of obtaining a first biomolecule bound to a first polypeptide comprising the amino acid sequence described in SEQ ID NO: 1 or an amino acid sequence having at least about 70% identity with SEQ ID NO: 1, or a cleaved fragment thereof containing at least 89 consecutive amino acid residues from the C-terminus of the sequence having at least about 70% identity with SEQ ID NO: 1 or SEQ ID NO: 1, - A step of obtaining a second biomolecule bound to a second polypeptide containing the amino acid sequence described in SEQ ID NO: 2 or an amino acid sequence having at least about 70% identity with SEQ ID NO: 2, or a cleaved fragment thereof containing at least eight consecutive amino acid residues from the N-terminus of the sequence having at least about 70% identity with SEQ ID NO: 2, - Step of adding a molecular proximity inducing substance to the sample. Includes, Here, when the molecular proximity inducing substance is added, the first polypeptide, the second polypeptide, and the molecular proximity inducing substance form a ternary assembly at a concentration of the molecular proximity inducing substance required to reach the maximum half volume of the assembly in the sample, which is less than approximately 5 μM. An assay that thereby induces proximity between the first and second biomolecules present in the sample.

3. The method according to claim 1 or the assay according to claim 2, wherein the molecular proximity inducer is fluorescent, and the method or assay further comprises detecting fluorescence resulting from the formation of a ternary assembly between the first polypeptide, the second polypeptide and the fluorescent molecular proximity inducer.

4. The method according to claim 1 or the assay according to claim 2, wherein the first polypeptide consists of the amino acid sequence described in SEQ ID NO: 1, and the second polypeptide consists of the amino acid sequence described in SEQ ID NO:

2.

5. The aforementioned molecular proximity inducing substance is a compound of formula (I): 【Chemistry 1】 or its salt and / or solvate, During the ceremony, R １ , R ２ , R ５ and R ６ Each of these independently represents H, halo, hydroxyl, aryl, alkyl, cycloalkyl, heteroalkyl, or heterocycloalkyl. Here, the aryl, alkyl, cycloalkyl, heteroalkyl, or heterocycloalkyl group is optionally substituted with at least one group selected from halo, hydroxyl, oxo, nitro, amide, carboxy, amino, cyano, haloalkoxy, and haloalkyl groups. R ３ represents an unbonded pair, H, halo, hydroxyl, aryl, alkyl, cycloalkyl, heteroalkyl, or heterocycloalkyl. Here, the aryl, alkyl, cycloalkyl, heteroalkyl, or heterocycloalkyl group is optionally substituted with at least one group selected from halo, hydroxyl, oxo, nitro, amide, carboxy, amino, cyano, haloalkoxy, and haloalkyl groups. R ４ This represents a single or double bond interrupted or terminated by a single S, O, or N heteroatom which is optionally substituted by at least one group selected from H, hydroxyl, aryl, alkyl, cycloalkyl, heteroalkyl, and heterocycloalkyl. Here, the aryl, alkyl, cycloalkyl, heteroalkyl, or heterocycloalkyl group is optionally substituted with at least one group selected from halo, hydroxyl, oxo, nitro, amide, carboxy, amino, cyano, haloalkoxy, and haloalkyl groups. W represents OH, SH, NHR ７ or NR ７ R ８ , wherein R ７ and R ８ each independently represent H, halo, hydroxyl, aryl, alkyl, cycloalkyl, heteroalkyl or heterocycloalkyl, Here, the aryl, alkyl, cycloalkyl, heteroalkyl, or heterocycloalkyl group is optionally substituted with at least one group selected from halo, hydroxyl, oxo, nitro, amide, carboxy, amino, cyano, haloalkoxy, and haloalkyl groups. X represents O, S, or NH, and Z represents O, S, or NH. The method according to claim 1 or the assay according to claim 2.

6. The assay according to claim 2, wherein the assay is for controlling the localization, transport, and / or function of biomolecules.

7. The assay according to claim 2, wherein the assay is for inducing the degradation of biomolecules.

8. The assay according to claim 2, wherein the assay is for detecting the simultaneous generation of two biomolecules in the sample.

9. The method according to claim 1 or the assay according to claim 2, wherein the first and second biomolecules are proteins.

10. A proximity induction system comprising two polypeptides and a molecular proximity induction material, The first polypeptide comprises the amino acid sequence described in SEQ ID NO: 1 or an amino acid sequence having at least about 70% identity with SEQ ID NO: 1, or a cleaved fragment thereof containing at least 89 consecutive amino acid residues from the C-terminus of the sequence having at least about 70% identity with SEQ ID NO: 1, and The second polypeptide comprises the amino acid sequence described in SEQ ID NO: 2 or an amino acid sequence having at least about 70% identity with SEQ ID NO: 2, or a cleaved fragment thereof containing at least eight consecutive amino acid residues from the N-terminus of the sequence having at least about 70% identity with SEQ ID NO: 2 or SEQ ID NO:

2. Herein, the first polypeptide, the second polypeptide, and the molecular proximity inducting substance form a ternary assembly at a concentration of the molecular proximity inducting substance required to reach a maximum half-assembly of less than approximately 5 μM.

11. The proximity induction system according to claim 10, wherein the molecular proximity induction material is fluorescent.

12. The aforementioned molecular proximity inducing substance is a compound of formula (I): 【Chemistry 2】 or its salt and / or solvate, During the ceremony, R １ , R ２ , R ５ and R ６ Each of these independently represents H, halo, hydroxyl, aryl, alkyl, cycloalkyl, heteroalkyl, or heterocycloalkyl. Here, the aryl, alkyl, cycloalkyl, heteroalkyl, or heterocycloalkyl group is optionally substituted with at least one group selected from halo, hydroxyl, oxo, nitro, amide, carboxy, amino, cyano, haloalkoxy, and haloalkyl groups. R ３ represents an unbonded pair, H, halo, hydroxyl, aryl, alkyl, cycloalkyl, heteroalkyl, or heterocycloalkyl. Here, the aryl, alkyl, cycloalkyl, heteroalkyl, or heterocycloalkyl group is optionally substituted with at least one group selected from halo, hydroxyl, oxo, nitro, amide, carboxy, amino, cyano, haloalkoxy, and haloalkyl groups. R ４ This represents a single or double bond interrupted or terminated by a single S, O, or N heteroatom which is optionally substituted by at least one group selected from H, hydroxyl, aryl, alkyl, cycloalkyl, heteroalkyl, and heterocycloalkyl. Here, the aryl, alkyl, cycloalkyl, heteroalkyl, or heterocycloalkyl group is optionally substituted with at least one group selected from halo, hydroxyl, oxo, nitro, amide, carboxy, amino, cyano, haloalkoxy, and haloalkyl groups. W stands for OH, SH, NHR ７ or NR ７ R ８ This represents, and in the formula, R ７ and R ８ Each of these independently represents H, halo, hydroxyl, aryl, alkyl, cycloalkyl, heteroalkyl, or heterocycloalkyl. Here, the aryl, alkyl, cycloalkyl, heteroalkyl, or heterocycloalkyl group is optionally substituted with at least one group selected from halo, hydroxyl, oxo, nitro, amide, carboxy, amino, cyano, haloalkoxy, and haloalkyl groups. X represents O, S, or NH, and Z represents O, S, or NH. The proximity guidance system according to claim 10.

13. The proximity induction system according to claim 10, wherein the first polypeptide consists of the amino acid sequence described in SEQ ID NO: 1, and the second polypeptide consists of the amino acid sequence described in SEQ ID NO:

2.

14. A cell or cell line containing two biomolecules, The first biomolecule is bound to a first polypeptide which includes the amino acid sequence described in SEQ ID NO: 1, or an amino acid sequence having at least about 70% identity with SEQ ID NO: 1, or a cleaved fragment thereof containing at least 89 consecutive amino acid residues from the C-terminus of the sequence having at least about 70% identity with SEQ ID NO: 1, The second biomolecule is bound to a second polypeptide which includes the amino acid sequence described in SEQ ID NO: 2, or an amino acid sequence having at least about 70% identity with SEQ ID NO: 2, or a cleaved fragment thereof containing at least eight consecutive amino acid residues from the N-terminus of the sequence having at least about 70% identity with SEQ ID NO: 2, A cell or cell line in which, in the presence of a molecular proximity inducing substance, the first polypeptide, the second polypeptide, and the molecular proximity inducing substance can form a ternary assembly at a concentration of the molecular proximity inducing substance required to reach a maximum half-assembly of less than about 5 μM.

15. The cell or cell line according to claim 14, wherein the first and second biomolecules are proteins.

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