Proximity-based labeling of biomolecular condensate microenvironments

Abstract

Described herein are composites, systems, and methods that enable profiling of local microenvironments across diverse biomolecular condensates through proximity labeling. In some embodiments, the composites include one or more biomolecular condensates that include nucleic acids and / or functionally diverse proteins, and a transition metal catalyst that couples to the biomolecular condensates through interactions with at least one of the nucleic acids or functionally diverse proteins. In some embodiments, the composites described herein are located within an intracellular environment.

Description

[Technical Field]

[0001] Related application data This application claims priority pursuant to Article 8 of the Patent Cooperation Treaty to U.S. Provisional Patent Application No. 63 / 424,581, filed November 11, 2022, which is incorporated herein by reference in its entirety.

[0002] Field The present invention relates to compositions, systems, and methods for proximity-based labeling, particularly proximity-based labeling for profiling the local microenvironment of diverse biomolecular condensates. [Background technology]

[0003] Protein proximity labeling has emerged as a powerful approach for profiling protein interaction networks. The ability to label associated or nearby proteins through proximity labeling may have important implications for further understanding the cellular environment and biological role of a protein of interest. Current proximity labeling methods all involve the use of enzyme-generated reactive intermediates that label nearby proteins on several selected amino acid residues through diffusion or physical contact. Despite the novel impact of this technology, the conversion of these reactive intermediates, such as phenoxy radicals (t), through peroxidase activation, has not been well documented. 1 / 2 >100 μs), or biotin-AMP via biotin ligase (t 1 / 2 The inherent stability of enzymes (>60 s) can facilitate diffusion far away from their point of origin. As a result, these enzyme-generated reactive intermediates pose challenges to profiling within close microenvironments. Furthermore, the large size of enzymes, their dependence on specific amino acids for labeling, and the inability to temporally control these labeling systems pose further challenges to profiling within confined spatial regions. Given these limitations, new approaches for proximity-based labeling are needed. Summary of the Invention

[0004] In consideration of the above-mentioned disadvantages, this specification describes a composite, system, and method that can profile the local microenvironment of various biomolecular condensates through proximity labeling.Biomolecular condensates are compartments in eukaryotic cells that concentrate biomolecular species, including nucleic acids and proteins.In contrast to intracellular organelles, biomolecular condensates lack surrounding membranes.Liquid-liquid phase separation can also drive the formation of various biomolecular condensates, where various biomolecules aggregate through intramolecular and intermolecular interactions.

[0005] In some embodiments, the complex includes one or more biomolecular condensates containing nucleic acids and / or functionally diverse proteins and a transition metal catalyst coupled to the biomolecular condensates through interaction with at least one of the nucleic acids or functionally diverse proteins. In some embodiments, the complexes described herein are located in an intracellular environment. In some embodiments, for example, the complex is located in the nucleus of a cell. Alternatively, the complex may be located in the cytoplasm or membrane of a cell. The transition metal catalyst may interact with the nucleic acid, e.g., RNA, or protein of the biomolecular condensate via a covalent bond. In other embodiments, the transition metal catalyst may interact with the nucleic acid or protein of the condensate via electrostatic and / or van der Waals interactions.

[0006] The complexes described herein can perform a variety of cellular functions depending on the composition and location of the complex. In some embodiments, the biomolecular condensates of the complexes described herein are selected from the group consisting of P-bodies, U-bodies, stress granules, centrosomes, signaling clusters, membrane clusters, synaptic densities, RNA transport granules, Balbiani bodies, germ granules, nuclear speckles, OPT domains, gems, PcG bodies, Cajal bodies, perinuclear compartments, cleavage bodies, and PML bodies. The type and location of the complex can be specific to a particular cell type. For example, RNA transport granules and synaptic densities are found in neurons, while Balbiani bodies and germ granules are found in germ cells.

[0007] The transition metal catalyst for coupling with the biomolecular condensate may, in some embodiments, comprise a platinum group metal center. Further, in some embodiments, the transition metal catalyst has Formula I: [ka] wherein M is a transition metal; A, D, E, G, Y and Z are independently selected from C and N; R 3 ~R 7 each represent 1 to 4 optional ring substituents, and the 1 to 4 optional ring substituents are each independently selected from alkyl, heteroalkyl, haloalkyl, haloalkenyl, halo, hydroxy, alkoxy, amine, amide, ether, —C(O)O - , -C(O)OR 8 , and -R 9 OH; R 8 is selected from the group consisting of hydrogen and alkyl, and R 9 is alkyl; R 1 is selected from the group consisting of a direct bond, alkylene, alkenylene, cycloaklylene, cycloalkenylene, arylene, heteroalkylene, heteroalkenylene, heterocyclene, and heteroarylene; L is an optional linking moiety selected from the group consisting of amide, ester, sulfonamide, sulfonate, carbamate, and urea; R 2 is an alkyne, amine, protected amine, azide, hydrazide, aryl, heteroaryl, cycloalkyl, cycloalkenyl, cycloalkylnyl, heterocyclyl, hydroxy, carboxyl, halo, alkoxy, maleimide, -C(O)H, -C(O)OR 8 , -OS(O2)R 9 , thiol, biotin, oxyamine, and haloalkyl, and R 8 and R 9is independently selected from the group consisting of alkyl, haloalkyl, aryl, haloaryl, N-succinimidyl, and N-succinimidyl esters; X - is a counter ion, and n is an integer from 0 to 20. As provided in Formula I, the linking moiety L is optional and, therefore, may be absent in some embodiments of the transition metal catalyst.

[0008] The polarity of the transition metal complex is R 3 ~R 7 It is also possible to tailor the R to a particular cellular environment through the selection of R. 3 ~R 7 One or more of R are selected to exhibit hydrophobic, lipophilic, or non-polar properties. 3 ~R 7 One or more of may be alkyl, fluoro, or fluoroalkyl. Transition metal complexes described herein that exhibit hydrophobic, lipophilic, or non-polar properties may also be suitable for placement within or passage into intracellular environments, including intracellular biomolecular condensate environments. Transition metal complexes may cross cell membranes to map local intracellular environments according to the principles described herein. Thus, such transition metal complexes are cell-permeable. Alternatively, R 3 ~R 7 is selected to exhibit hydrophilic properties through charged and / or polar chemical moieties. In such embodiments, the transition metal complex may exhibit hydrophilic properties suitable for placement in an intracellular / extracellular environment.

[0009] As further described herein, the transition metal catalyst may have an electronic structure that allows energy transfer to the protein labeling agent to produce a reactive intermediate. In some embodiments, the energy transfer is Dexter energy transfer or electron transfer, including single-electron transfer. In some embodiments, energy transfer to the protein labeling agent may occur from an excited state of the transition metal catalyst electronic structure. The excited state of the catalyst may be, for example, a singlet excited state or a triplet excited state. The excited state of the catalyst may occur through one or more mechanisms, including energy absorption by the catalyst. In some embodiments, the catalyst is a photocatalyst, in which the excited state is induced by absorption of one or more photons. In other embodiments, the catalyst may be placed in the excited state by interaction with one or more chemical species in the surrounding environment. Alternatively, energy transfer to the protein labeling agent, including electron transfer, may occur from the ground state of the catalyst electronic structure. As described herein, energy transfer from the protein labeling agent to produce a reactive intermediate enables profiling of the microenvironment near biomolecular condensates.

[0010] In another aspect, a system for profiling a microenvironment near a biomolecular condensate is provided. In some embodiments, the system includes a protein labeling agent, a complex comprising one or more biomolecular condensates comprising nucleic acids and / or functionally diverse proteins, and a transition metal catalyst coupled to the biomolecular condensate through at least one of the nucleic acids or functionally diverse proteins, wherein the transition metal catalyst has an electronic structure that allows energy transfer to the protein labeling agent to provide a reactive intermediate. As described herein, the biomolecular condensate can be located in the nucleus, cytoplasm, or membrane(s) of a cell. The reactive intermediate can be manipulated to label proteins or other biomolecules within a predetermined range of the complex. The predetermined range can be the diffusion range of the reactive intermediate.

[0011] The diffusion range of the reactive intermediate may be tailored to specific microenvironment mapping (proximity-based labeling) considerations and may be limited to the nanometer scale. In some embodiments, for example, the diffusion range of the reactive intermediate may be less than 100 nm, less than 50 nm, less than 10 nm, less than 5 nm, less than 4 nm, less than 3 nm, or less than 2 nm before quenching in the surrounding environment. In some embodiments, the diffusion range may be 0.5 nm to 10 nm or 0.5 nm to 100 nm. Thus, the reactive intermediate will react with or crosslink with proteins or other biomolecules within its diffusion range, or, if no proteins or biomolecules are present, will be quenched by the surrounding environment. In this manner, the cooperative forces between the transition metal catalyst and the protein labeling agent may allow the environment near the biomolecular condensate to be mapped with high resolution. Furthermore, in some embodiments, the reactive intermediate may react with or crosslink with proteins or other biomolecules within its diffusion range before quenching. 1 / 2 The reactive intermediate may exhibit, for example, a t of less than 1 to 5 ns. 1 / 2 In further embodiments, the diffusion range can be extended to 5-500 nm through an increase in the reactive intermediate half-life. For example, in some embodiments, the reactive intermediate can have a half-life of 1-100 μs or longer.

[0012] In another aspect, methods for profiling the microenvironment near a biomolecular condensate are described herein. The methods include forming a complex containing a transition metal catalyst coupled to the biomolecular condensate through interaction with a biomolecular species of the condensate, and activating a protein labeling agent with the transition metal catalyst to a reactive intermediate. The reactive intermediate couples to a protein or other biomolecule within a predetermined range of the complex. The biomolecular condensate, transition metal catalyst, protein labeling agent, and reactive intermediate may have any composition and / or properties described herein. Furthermore, the transition metal catalyst may be coupled to the biomolecular condensate through covalent bonds, electrostatic interactions, or van der Waals interactions. In some embodiments, the protein labeling agent is activated at different points in the biomolecular condensate life cycle, including early, intermediate, and late degradation. Furthermore, the methods described herein further include detecting or analyzing the protein or other biomolecule coupled to the reactive intermediate.

[0013] These and other embodiments are further described in the detailed description that follows. [Brief explanation of the drawings]

[0014] [Figure 1] 1 illustrates the transition metal catalysts described herein, according to some embodiments. [Figure 2] 1 illustrates the binding of a transition metal complex described herein to a protein through a haloalkane dehalogenase, according to some embodiments. [Figure 3] 1 illustrates activation of protein labeling agents to map important proteins of interest across the biomolecular condensate life cycle, including early, middle, and late degradation, according to some embodiments. [Figure 4] 1 is a schematic detailing stress granule interactome mapping with compounds and protein labeling agents described herein, according to some embodiments. [Figure 5]Illustrates ubiquitination of stress granule proteins during granule formation. [Figure 6] We illustrate that protein ubiquitination is impaired by the presence of TAK243 during granule formation and degradation. [Figure 7] 1 is a volcano plot identifying HECT ubiquitin ligases and autography adaptors identified by stress granule interactome mapping with the compounds and protein labeling agents described herein, according to some embodiments. [Figure 8] According to some embodiments, time-resolved recruitment of HECT E3 ligase to stress granules is provided as determined by interactome mapping with the complex and protein labeling agents described herein. [Figure 9] FIG. 1 is a schematic diagram for using Heclin to determine the effect on HECT E3 ligase function in the context of stress granule disassembly. [Figure 10] 1 illustrates that inhibition of HECT E3 ligase with heclin delays stress granule disassembly. [Figure 11] We demonstrate that HECT E3 ligase siRNA knockdown delays stress granule disassembly. DETAILED DESCRIPTION OF THE INVENTION

[0015] The embodiments described herein may be more readily understood by reference to the following detailed description and examples, as well as the preceding and following descriptions. However, the elements, devices, and methods described herein are not limited to the specific embodiments presented in the detailed description and examples. It should be recognized that these embodiments are merely illustrative of the principles of the present invention. Numerous modifications and adaptations will be readily apparent to those skilled in the art without departing from the spirit and scope of the invention.

[0016] definition The term "alkyl," as used herein, alone or in combination, refers to a straight-chain or branched-chain saturated hydrocarbon group, optionally substituted with one or more substituents. For example, alkyl refers to C1 to C6 30 or C1~C 18 may be.

[0017] The term "alkenyl," as used herein, alone or in combination, refers to a straight-chain or branched-chain hydrocarbon group having at least one carbon-carbon double bond and optionally substituted with one or more substituents.

[0018] The term "alkynyl," as used herein, alone or in combination, refers to a straight-chain or branched-chain hydrocarbon group having at least one carbon-carbon triple bond and optionally substituted with one or more substituents.

[0019] The term "aryl," as used herein, alone or in combination, refers to an aromatic monocyclic or polycyclic ring system optionally substituted with one or more ring substituents.

[0020] The term "heteroaryl," as used herein, alone or in combination, refers to an aromatic monocyclic or polycyclic ring system in which one or more of the ring atoms is an element other than carbon, for example, nitrogen, boron, oxygen, and / or sulfur.

[0021] The term "heterocycle," as used herein, alone or in combination, refers to a monocyclic or polycyclic ring system in which one or more atoms of the ring system is an element other than carbon, such as boron, nitrogen, oxygen, and / or sulfur or phosphorus, and the ring system is optionally substituted with one or more ring substituents. Heterocyclic ring systems may include aromatic and / or non-aromatic rings, including rings with one or more points of unsaturation.

[0022] The term "cycloalkyl," as used herein, alone or in combination, refers to a non-aromatic mono- or multi-cyclic ring system, optionally substituted with one or more ring substituents.

[0023] The term "heterocycloalkyl," as used herein, alone or in combination, refers to a non-aromatic monocyclic or polycyclic ring system in which one or more of the atoms in the ring system is an element other than carbon, for example, boron, nitrogen, oxygen, sulfur, or phosphorus, alone or in combination, and the ring system is optionally substituted with one or more ring substituents.

[0024] The term "alkoxy," as used herein, alone or in combination, refers to the moiety RO--, where R is alkyl, alkenyl, or aryl as defined above.

[0025] The term "halo," as used herein, alone or in combination, refers to an element of Group VIIA of the periodic table (a halogen). Depending on the chemical environment, a halo can be in a neutral or anionic state.

[0026] Terms not specifically defined herein have their ordinary meaning in the art.

[0027] I. compound In one aspect, described herein are complexes that enable profiling of the local microenvironment across biomolecular condensates through proximity labeling. In some embodiments, the complexes include one or more biomolecular condensates containing nucleic acids and / or functionally diverse proteins, and a transition metal catalyst that couples to the biomolecular condensates through interactions with at least one of the nucleic acids or functionally diverse proteins. In some embodiments, the complexes described herein are localized in the intracellular environment. In some embodiments, for example, the complex is located in the nucleus of a cell. Alternatively, the complex may be localized in the cytoplasm or membrane of a cell. The transition metal catalyst may interact with the nucleic acid, e.g., RNA, or protein of the biomolecular condensate through a covalent bond. In other embodiments, the transition metal catalyst may interact with the nucleic acid or protein of the condensate through electrostatic and / or van der Waals interactions.

[0028] In some embodiments, the transition metal complex couples to a protein of a biomolecular condensate through interaction with a haloalkane dehalogenase (HaloTag). The haloalkane dehalogenase may be co-expressed with the protein using standard cloning methods. The transition metal complex may be functionalized with a haloalkane moiety for covalent attachment to the haloalkane dehalogenase, resulting in coupling of the protein to the biomolecular condensate. Figure 2 illustrates the attachment of a transition metal complex described herein to a protein via a haloalkane dehalogenase, according to some embodiments. Alternatively, the transition metal complex may be attached to a derivatized amino acid of a biomolecular condensate protein. Suitable click chemistry moieties of the transition metal complex and / or the derivatized amino acid may be selected from the group consisting of DBCO, BCN, TCO, tetrazine, alkyne, and azide. The transition metal complex may be of formula (I) herein. In other embodiments, the transition metal complex is coupled to a protein of the biomolecular condensate through protein trans-splicing, and the transition metal complex is first coupled to a split intein. The split intein carrying the transition metal complex can be an N-intein or a C-intein.

[0029] In some embodiments, the transition metal catalyst has Formula I: [ka] wherein M is a transition metal; A, D, E, G, Y and Z are independently selected from C and N; R 3 ~R 7 each represent 1 to 4 optional ring substituents, and the 1 to 4 optional ring substituents are each independently selected from alkyl, heteroalkyl, haloalkyl, haloalkenyl, halo, hydroxy, alkoxy, amine, amide, ether, —C(O)O - , -C(O)OR 8 , and -R 9 OH; R 8is selected from the group consisting of hydrogen and alkyl, and R 9 is alkyl; R 1 is selected from the group consisting of a direct bond, alkylene, alkenylene, cycloalkylene, cycloalkenylene, arylene, heteroalkylene, heteroalkenylene, heterocyclene, and heteroarylene; L is an optional linking moiety selected from the group consisting of amide, ester, sulfonamide, sulfonate, carbamate, and urea; R 2 is an alkyne, amine, protected amine, azide, hydrazide, aryl, heteroaryl, cycloalkyl, cycloalkenyl, cycloalkylnyl, heterocyclyl, hydroxy, carboxyl, halo, alkoxy, maleimide, -C(O)H, -C(O)OR 8 , -OS(O2)R 9 , thiol, biotin, oxyamine, and haloalkyl, and R 8 and R 9 is independently selected from the group consisting of alkyl, haloalkyl, aryl, haloaryl, N-succinimidyl, and N-succinimidyl esters; X - is a counter ion, and n is an integer from 0 to 20. As provided in Formula I, the linking moiety L is optional and, therefore, may be absent in some embodiments of the transition metal catalyst.

[0030] Optional Substituent R 3 ~R 7 It is understood that in the absence of a counterion (X), hydrogen occupies the positions on the aryl ring of formula I. Additionally, in some embodiments, - ) is tetraalkylborate, tetrafluoroborate, tetraphenylborate, PF6 - and chlorine.

[0031] The polarity of the transition metal complex is R 3 ~R 7It is also possible to tailor the R to a particular cellular environment through the selection of R. 3 ~R 7 One or more of R are selected to exhibit hydrophobic, lipophilic, or non-polar properties. 3 ~R 7 One or more of may be alkyl, fluoro, or fluoroalkyl. Transition metal complexes described herein that exhibit hydrophobic, lipophilic, or non-polar properties may also be suitable for placement or passage into intracellular environments. Transition metal complexes may cross cell membranes to map biomolecular condensates in intracellular environments according to the principles described herein. Thus, such transition metal complexes are cell-permeable. Figure 1 illustrates various transition metal complexes described herein. In some embodiments, L is selected from R 2 amide combined with a polyethylene glycol (PEG) moiety for linkage to the PEG moiety. As provided in Figure 1, the PEG moiety may be substituted with an alkylene moiety. In some embodiments, for example, R 2 R comprises a haloalkane or click chemistry moiety including, but not limited to, BCN, DBCO, TCO, tetrazine, alkyne, and azide. As exemplified in Figure 1, R 2 These click chemistries may be coupled directly to the linker (L) or through a heteroatom, aryl, or carbonyl.

[0032] The transition metal catalyst may have an electronic structure that allows it to transfer energy to the protein labeling agent to generate a reactive intermediate. In some embodiments, the energy transfer is Dexter energy transfer or electron transfer. In some embodiments, the energy transfer to the protein labeling agent may occur from an excited state of the transition metal catalyst electronic structure. The excited state of the catalyst may be, for example, a singlet excited state or a triplet excited state. In some embodiments, the transition metal complex of Formula I may exhibit a long-lived triplet excited state (T1) that facilitates energy transfer to the protein labeling agent. The T1 state may have a t of, for example, 0.2 to 2 μs. 1 / 2 The transition metal complexes described herein may be photocatalysts and, in some embodiments, absorb light in the visible or infrared region of the electromagnetic spectrum. Absorption of electromagnetic radiation excites the transition metal complex to the S1 state, which can then undergo quantitative intersystem crossing to the T1 state. The transition metal catalyst can then return to the ground state, S0, via short-range Dexter energy transfer to the protein labeling agent. Energy transfer to the labeling agent activates the labeling agent for reaction with proteins or other biomolecules in the local biomolecular condensate environment. The T1 state of the transition metal complex may, in some embodiments, be greater than 60 kcal / mol. The metal center may be selected, for example, from transition metals of the platinum group. In some embodiments, the metal center may be iridium.

[0033] In other embodiments, the catalyst may be placed in an excited state by interaction with one or more chemical species in the surrounding environment. Alternatively, energy transfer to the protein labeling agent, including electron transfer, may occur from the ground state of the catalyst electronic structure. Energy transfer from the protein labeling agent to produce reactive intermediates allows for profiling of the microenvironment near the biomolecular condensate, as described herein.

[0034] II. A system for profiling the interactome of biomolecular condensates In another aspect, a system for profiling a microenvironment near a biomolecular condensate is provided. In some embodiments, the system includes a complex comprising a protein labeling agent, one or more biomolecular condensates comprising nucleic acids and / or functionally diverse proteins, and a transition metal catalyst coupled to the biomolecular condensate through interaction with at least one of the nucleic acids or functionally diverse proteins, the transition metal catalyst having an electronic structure that allows energy transfer to the protein labeling agent to provide a reactive intermediate. The transition metal catalyst may be of Formula I and may have any of the compositions, structures, and / or properties described in Section I above. The biomolecular condensate may be located within the nucleus, cytoplasm, or membrane(s) of a cell. The reactive intermediate is operable to label proteins or other biomolecules within a predetermined range of the complex. The predetermined range may be the diffusion range of the reactive intermediate.

[0035] The diffusion range of the reactive intermediate may be tailored to specific microenvironment mapping (proximity-based labeling) considerations and may be limited to the nanometer scale. In some embodiments, for example, the diffusion range of the reactive intermediate may be less than 100 nm, less than 50 nm, less than 10 nm, less than 5 nm, less than 4 nm, less than 3 nm, or less than 2 nm before quenching in the surrounding environment. In some embodiments, the diffusion range may be between 0.5 nm and 10 nm. Thus, the reactive intermediate will react with or crosslink with proteins or other biomolecules within its diffusion range, or, if no proteins or biomolecules are present, will be quenched by the surrounding environment. In this manner, the cooperative forces between the catalyst and the protein labeling agent allow the environment near the biomolecular condensate to be mapped with high resolution. Furthermore, in some embodiments, the reactive intermediate will react with or crosslink with proteins or other biomolecules within its diffusion range before quenching. 1 / 2 The reactive intermediate may exhibit, for example, a t of less than 1 to 5 ns. 1 / 2In further embodiments, the diffusion range can be extended to 5-500 nm through an increase in the half-life of the reactive intermediate. For example, in some embodiments, the reactive intermediate can have a half-life of 1-100 μs or longer. In some embodiments, more than one species of protein labeling agent type can be used in the systems described herein. For example, two or more species with different diffusion ranges can be used in the system. In such embodiments, the different diffusion ranges can allow for labeling at different distances from the transition metal complex, thereby further mapping the interactome of biomolecular condensates.

[0036] In some embodiments, the protein labeling agent may be diazirine. Triplet energy transfer from the excited-state photocatalyst may promote the diazirine to its triplet (T1) state. The diazirine triplet undergoes elimination of N2 to release a free triplet carbene, which undergoes spin equilibration on the picosecond time scale to its reactive singlet state (t 1 / 2 <1 ns), which crosslinks with nearby proteins or is quenched in an aqueous environment. In some embodiments, the extinction coefficient of the transition metal complex is 3-5 orders of magnitude greater than that of the diazirine.

[0037] Any diazirine consistent with the technical principles discussed herein can be used. Diazirine sensitization can be extended to, for example, a variety of p- and m-substituted aryltrifluoromethyldiazirines, which carry valuable payloads for microscopy and proteomics applications, including free carboxylic acids, phenols, amines, alkynes, carbohydrates, and biotin groups. Diazirines may be functionalized with markers such as biotin. In some embodiments, the marker is desthiobiotin. Markers can aid in the identification of proteins labeled with protein labeling agents. Markers can be useful, for example, in assay results via Western blots and / or other analytical techniques. In addition to biotin and desthiobiotin, markers can include alkyne, azide, FLAG tags, fluorophores, and chloroalkane functionalities.

[0038] In further embodiments where the transition metal catalyst is a photocatalyst, the protein labeling agent may be an azide. Triplet energy transfer from the excited-state photocatalyst may promote nitrene formation from the azide. The reactive nitrene either crosslinks with nearby proteins or is quenched in an aqueous environment. Any azide operable to undergo energy transfer with the transition metal photocatalyst to form a nitrene may be used. In some embodiments, the azide is an aryl azide.

[0039] III. A method for profiling biomolecular condensate microenvironments In another aspect, methods for profiling the microenvironment near a biomolecular condensate are described herein. The methods include forming a complex containing a transition metal catalyst that couples to the biomolecular condensate through an interaction with a biomolecular species in the condensate, and activating a protein labeling agent with the transition metal catalyst to a reactive intermediate. The reactive intermediate couples to a protein or other biomolecule within a predetermined range of the complex. The biomolecular condensate, transition metal catalyst, protein labeling agent, and reactive intermediate may have any of the compositions and / or properties described herein in Sections I and II above. Furthermore, the transition metal catalyst may be coupled to the biomolecular condensate through covalent bonds, electrostatic interactions, or van der Waals interactions, as described in Section I above. In some embodiments, the protein labeling agent is activated at different points in the biomolecular condensate life cycle, including early, intermediate, and late degradation. In some embodiments, the same protein labeling agent may be used throughout the condensate life cycle. Alternatively, multiple different protein labeling agents may be used throughout the condensate life cycle. Different protein labeling agents may have similar or different diffusion ranges to provide a complete mapping of the biomolecular condensate interactome.

[0040] Additionally, the methods described herein further include detecting or analyzing proteins or other biomolecules coupled to the reactive intermediate.

[0041] Figure 3 illustrates the activation of protein labeling agents over the biomolecular condensate life cycle, including early, middle, and late degradation. Staged activation of protein labeling agents with complex transition metal complexes can determine key proteins of interest in condensate degradation control.

[0042] These and other embodiments are further illustrated in the following non-limiting examples. [Example]

[0043] Example 1 - Stress granule interactome mapping The protein-transition metal complex conjugate in Figure 2 was prepared as follows: Cells (HEK243T stably expressing HaloTag-G3BP1) were seeded and grown to 95% confluence before labeling. The cells were incubated for 1 hour in DMEM medium containing 5 μM of the iridium catalyst in Figure 2. During this period, the iridium catalyst crosses the cell membrane and conjugates with the HaloTag-G3BP1 protein. The cells were then incubated for 1 hour in fresh DMEM medium to eliminate or minimize noise from any unbound iridium catalyst.

[0044] The first portion of the Ir catalyst-loaded cells was then transferred to DMEM medium pre-treated with 2 μM TAK243 and incubated for an additional hour. The remaining second portion (control) was treated with the same volume of DMSO. The cells were stressed with 500 mM NaAsO2 in DMEM for 30 minutes. After stress treatment, the medium was replaced with fresh DMEM for the indicated time period for cell recovery. During the stress treatment period, 30 minutes prior to each photolabeling time point, the cells were treated with 500 μM biotin-diazirine protein labeling agent in the medium. During photolabeling, the cells were exposed to 450 nm blue light irradiation for 10 minutes. Figure 4 illustrates the aforementioned experimental protocol.

[0045] A typical proteomics preparation protocol was followed: 600 μg of total protein was loaded for streptavidin enrichment. After on-bead denaturation, reduction, alkylation, and washing, the biotin-labeled proteins were digested with trypsin to obtain a peptide solution for TIMSTOF LCMS analysis.

[0046] Figure 5 illustrates the ubiquitination of stress granule proteins during granule formation. Furthermore, Figure 6 illustrates that when protein ubiquitination is reduced or inhibited by the presence of TAK243 during granule formation, granule disassembly is delayed. Figure 7 is a volcano plot identifying HECT ubiquitin ligases and autograft adaptors identified in stress granule interactome mapping with this example's complex and protein labeling agents. Figure 8 provides a time-resolved view of HECT E3 ligases recruited to stress granules.

[0047] Armed with this information provided by interactome mapping, the E3 ligase inhibitor heclin was used to determine any effect on HECT E3 ligase function in relation to stress granule disassembly, as illustrated in Figure 9. Figure 10 illustrates that HECT E3 ligase inhibition with heclin delays stress granule disassembly. Furthermore, Figure 11 shows that HECT E3 ligase siRNA knockdown delays stress granule disassembly.

[0048] Various embodiments of the present invention have been described in fulfillment of various objectives of the present invention. It should be recognized that these embodiments are merely illustrative of the principles of the present invention. Numerous modifications and adaptations thereof will be readily apparent to those skilled in the art without departing from the spirit and scope of the present invention.

Claims

1. one or more biomolecular condensates comprising nucleic acids and / or functionally diverse proteins; a transition metal catalyst that couples to the biomolecular condensate through an interaction with at least one of the nucleic acid or functionally diverse protein; A compound containing

2. 2. The composite of claim 1, wherein the interaction is selected from the group consisting of a covalent bond, an electrostatic interaction(s), and a van der Waals interaction.

3. The transition metal complex has the formula: 【Chemistry 1】 wherein M is a transition metal; A, D, E, G, Y and Z are independently selected from C and N; R 3 ~R 7 each represent 1 to 4 optional ring substituents, wherein the 1 to 4 optional ring substituents are each independently selected from alkyl, heteroalkyl, haloalkyl, haloalkenyl, halo, hydroxy, alkoxy, amine, amide, ether, —C(O)O - , -C(O)OR 8 , and -R 9 OH; R 8 is selected from the group consisting of hydrogen and alkyl, and R 9 is alkyl; R 1 is selected from the group consisting of a direct bond, alkylene, alkenylene, cycloalkylene, cycloalkenylene, arylene, heteroalkylene, heteroalkenylene, heterocyclene, and heteroarylene; L is an optional linking moiety selected from the group consisting of amide, ester, sulfonamide, sulfonate, carbamate, and urea; R 2 is an alkyne, amine, protected amine, azide, hydrazide, aryl, heteroaryl, cycloalkyl, cycloalkenyl, cycloalkylnyl, heterocyclyl, hydroxy, carboxyl, halo, alkoxy, maleimide, -C(O)H, -C(O)OR 8 , -OS(O 2 )R 9 , thiol, biotin, oxyamine, and haloalkyl, and R 8 and R 9 is independently selected from the group consisting of alkyl, haloalkyl, aryl, haloaryl, N-succinimidyl, and N-succinimidyl esters; X - is a counterion, and n is an integer from 0 to 20.

2. The composite of claim 1,

4. 4. The composite of claim 3, wherein the transition metal complex couples to a protein of the biomolecular condensate through binding with a haloalkane dehalogenase.

5. 4. The compound of claim 3, wherein M is iridium.

6. The composite of claim 1 , wherein the transition metal catalyst is a photocatalyst.

7. The composite of claim 1 , wherein the transition metal catalyst has an electronic structure for energy transfer to the protein labeling agent.

8. 8. The composite of claim 7, wherein the energy transfer is Dexter energy transfer.

9. 2. The compound of claim 1, wherein the biomolecular condensate is selected from the group consisting of P bodies, U bodies, stress granules, centrosomes, signaling clusters, membrane clusters, synaptic densities, RNA transport granules, Balbiani bodies, germ granules, nuclear speckles, OPT domains, gems, PcG bodies, Cajal bodies, perinuclear compartments, cleavage bodies, and PML bodies.

10. 1. A system for profiling the interactome of a biomolecular condensate, comprising: a protein labeling agent; a composite comprising one or more biomolecular condensates comprising nucleic acids and / or functionally diverse proteins; and a transition metal catalyst coupled to the biomolecular condensates through an interaction with at least one of the nucleic acids or functionally diverse proteins, the transition metal catalyst having an electronic structure that allows energy transfer to the protein labeling agent to provide a reactive intermediate; The system comprising:

11. 11. The system of claim 10, wherein the reactive intermediate is operable to label proteins or other biomolecules within a predetermined range of the complex.

12. The transition metal complex has the formula: 【Chemistry 2】 wherein M is a transition metal; A, D, E, G, Y and Z are independently selected from C and N; R 3 ~R 7 each represent 1 to 4 optional ring substituents, wherein the 1 to 4 optional ring substituents are each independently selected from alkyl, heteroalkyl, haloalkyl, haloalkenyl, halo, hydroxy, alkoxy, amine, amide, ether, —C(O)O - , -C(O)OR 8 , and -R 9 OH; R 8 is selected from the group consisting of hydrogen and alkyl, and R 9 is alkyl; R 1 is selected from the group consisting of a direct bond, alkylene, alkenylene, cycloalkylene, cycloalkenylene, arylene, heteroalkylene, heteroalkenylene, heterocyclene, and heteroarylene; L is an optional linking moiety selected from the group consisting of amide, ester, sulfonamide, sulfonate, carbamate, and urea; R 2 is an alkyne, amine, protected amine, azide, hydrazide, aryl, heteroaryl, cycloalkyl, cycloalkenyl, cycloalkylnyl, heterocyclyl, hydroxy, carboxyl, halo, alkoxy, maleimide, -C(O)H, -C(O)OR 8 , -OS(O 2 )R 9 , thiol, biotin, oxyamine, and haloalkyl, and R 8 and R 9 is independently selected from the group consisting of alkyl, haloalkyl, aryl, haloaryl, N-succinimidyl, and N-succinimidyl esters; X - is a counterion, and n is an integer from 0 to 20. The system of claim 10.

13. 13. The system of claim 12, wherein the transition metal complex couples to a protein of the biomolecular condensate through binding with a haloalkane dehalogenase.

14. 13. The system of claim 12, wherein M is iridium.

15. The system of claim 10 , wherein the transition metal catalyst is a photocatalyst.

16. The system of claim 10 , wherein the energy transfer is Dexter energy transfer.

17. 11. The system of claim 10, wherein the protein labeling agent is diazirine.

18. 18. The system of claim 17, wherein the diazirine comprises a molecular marker.

19. The system of claim 10, wherein the biomolecular condensates are selected from the group consisting of P bodies, U bodies, stress granules, centrosomes, signaling clusters, membrane clusters, synaptic densities, RNA transport granules, Balbiani bodies, germ granules, nuclear speckles, OPT domains, GEMs, PcG bodies, Cajal bodies, perinuclear compartments, cleavage bodies, and PML bodies.

20. 1. A method for profiling a microenvironment near a biomolecular condensate, comprising: forming a complex comprising a transition metal catalyst coupled to the biomolecular condensate through interaction with the biomolecular species of the biomolecular condensate; activating a protein labeling agent with said transition metal catalyst to a reactive intermediate; coupling said reactive intermediate to a protein or other biomolecule within a predetermined range of said conjugate; The method comprising:

21. 21. The method of claim 20, wherein activating the protein labeling agent to the reactive intermediate comprises transferring energy from the transition metal catalyst to the protein labeling agent.

22. 21. The method of claim 20, wherein the protein labeling agent is diazirine.

23. 23. The method of claim 22, wherein the diazirine is functionalized with a marker.

24. 21. The method of claim 20, wherein the predetermined range is 2 to 100 nm.

25. 21. The method of claim 20, wherein the reactive intermediate is quenched outside the predetermined range to eliminate binding to the biomolecule outside the predetermined range.

26. 21. The method of claim 20, further comprising detecting or analyzing the protein coupled to the reactive intermediate.

27. 21. The method of claim 20, wherein the protein labeling agents are activated at different times during the biomolecular condensate life cycle.

28. 28. The method of claim 27, wherein the biomolecular condensate life cycle comprises early degradation, middle degradation, and late degradation.

29. The transition metal complex has the formula: 【Transformation 3】 wherein M is a transition metal; A, D, E, G, Y and Z are independently selected from C and N; R 3 ~R 7 each represent 1 to 4 optional ring substituents, wherein the 1 to 4 optional ring substituents are each independently selected from alkyl, heteroalkyl, haloalkyl, haloalkenyl, halo, hydroxy, alkoxy, amine, amide, ether, —C(O)O - , -C(O)OR 8 , and -R 9 OH; R 8 is selected from the group consisting of hydrogen and alkyl, and R 9 is alkyl; R 1 is selected from the group consisting of a direct bond, alkylene, alkenylene, cycloalkylene, cycloalkenylene, arylene, heteroalkylene, heteroalkenylene, heterocyclene, and heteroarylene; L is an optional linking moiety selected from the group consisting of amide, ester, sulfonamide, sulfonate, carbamate, and urea; R 2 is an alkyne, amine, protected amine, azide, hydrazide, aryl, heteroaryl, cycloalkyl, cycloalkenyl, cycloalkylnyl, heterocyclyl, hydroxy, carboxyl, halo, alkoxy, maleimide, -C(O)H, -C(O)OR 8 , -OS(O 2 )R 9 , thiol, biotin, oxyamine, and haloalkyl, and R 8 and R 9 is independently selected from the group consisting of alkyl, haloalkyl, aryl, haloaryl, N-succinimidyl, and N-succinimidyl esters; X - is a counterion, and n is an integer from 0 to 20.

21. The method of claim 20,

30. 30. The method of claim 29, wherein the transition metal complex is coupled to a protein of the biomolecular condensate through binding with a haloalkane dehalogenase.

31. 30. The method of claim 29, wherein M is iridium.

32. 21. The method of claim 20, wherein the transition metal catalyst is a photocatalyst.

33. 21. The method of claim 20, wherein the biomolecular condensates are selected from the group consisting of P bodies, U bodies, stress granules, centrosomes, signaling clusters, membrane clusters, synaptic densities, RNA transport granules, Balbiani bodies, germ granules, nuclear speckles, OPT domains, GEMs, PcG bodies, Cajal bodies, perinuclear compartments, cleavage bodies, and PML bodies.

34. 21. The method of claim 20, wherein the biomolecular condensate is located in the cytoplasm or membrane of a cell.

35. 21. The method of claim 20, wherein the biomolecular condensate is located in the nucleus of a cell.

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