Proximity-based labeling of biomolecular aggregate microenvironments
By coupling the transition metal catalyst with biomolecular agglomerates, reactive intermediates are generated for labeling, the problem of uncontrolled diffusion of enzyme-generated intermediates is solved, and high-resolution analysis of the local microenvironment of biomolecular agglomerates is achieved.
Patent Information
- Application Number
- CN202380086169.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Priority Date
- 2022-11-11
- Filing Date
- 2023-11-13
- Publication Date
- 2025-08-01
AI Technical Summary
When the existing proximity labeling method analyzes protein interaction networks, the diffusion of reactive intermediates generated by enzymes is uncontrolled, making it difficult to analyze in a tight microenvironment and is highly dependent on certain amino acids, making it impossible to perform effective analysis in a limited space.
Complexes containing transition metal catalysts are used to couple with biomolecular agglomerates through covalent bonding, electrostatic or van der Waals interactions, and reactive intermediates are generated by energy transfer for labeling. The diffusion radius can be adjusted within the nanoscale, with a short quenching time and adapted to different cellular environments.
High-resolution analysis of the local microenvironment of biomolecular agglomerates is achieved. Reactive intermediates react or cross-link with proteins within a specific radius, and are quickly quenched, which is suitable for localization analysis of the environment inside and outside the cell.
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Figure CN120418006A_ABST
Abstract
Description
[0001] Related application data
[0002] This application claims priority to U.S. Provisional Patent Application Serial No. 63 / 424,581, filed on November 11, 2022, under Article 8 of the Patent Cooperation Treaty, which is hereby incorporated by reference in its entirety. Technical field
[0003] The present invention relates to proximity-based labeling compositions, systems, and methods, particularly for analyzing the local microenvironment of various biomolecular condensates based on proximity-based labeling. Background art
[0004] Protein proximity labeling has emerged as a powerful method for analyzing protein interaction networks. The ability to label associated or bystander proteins through proximity labeling is important for further understanding the cellular environment and biological functions of target proteins. Current proximity labeling methods all involve the use of enzyme-generated reactive intermediates that label adjacent proteins at a few selected amino acid residues through diffusion or physical contact. Although this technology has had a transformative impact, the inherent stability of these reactive intermediates (such as phenoxyl radicals activated by peroxidase (t 1 / 2 >100 μs) or biotin-AMP by biotin ligase (t 1 / 2 >60 s)) can promote diffusion away from their origin. Thus, these enzyme-generated reactive intermediates pose challenges for analysis in a tight microenvironment. In addition, the large enzyme size, the dependence of labeling on certain amino acids, and the inability to temporally control these labeling systems pose additional challenges for analysis in a limited spatial region. Given these limitations, new proximity-based labeling methods are needed. Summary of the invention
[0005] In view of the foregoing drawbacks, complexes, systems, and methods are described herein that are capable of analyzing the local microenvironment across various biomolecular condensates through proximity labeling. Biomolecular condensates are compartments in eukaryotic cells where biomolecular substances (including nucleic acids and proteins) are concentrated. Distinct from organelles, biomolecular condensates lack a surrounding membrane. Liquid-liquid phase separation can drive the formation of various biomolecular condensates, in which various biomolecules aggregate through intramolecular and intermolecular interactions.
[0006] In some embodiments, the complex comprises one or more biomolecular condensates and a transition metal catalyst, the biomolecular condensate contains nucleic acids and / or functionally diverse proteins, and the transition metal catalyst is coupled to the biomolecular condensate by interacting with at least one of the nucleic acid or the functionally diverse protein. In some embodiments, the complex described herein is in an intracellular environment. In some embodiments, for example, the complex is in the nucleus of a cell. Optionally, the complex can be in the cytoplasm or cell membrane of the cell. The transition metal catalyst can interact with the nucleic acid (such as RNA) or protein of the biomolecular condensate by covalent bonding. In other embodiments, the transition metal catalyst can interact with the nucleic acid or protein of the condensate by electrostatic interactions and / or van der Waals interactions.
[0007] The complex described herein can perform various cellular functions depending on the composition and location of the complex. In some embodiments, the biomolecular condensate of the complex described herein 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. 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 neuronal cells, while Balbiani bodies and germ granules are found in germ cells.
[0008] In some embodiments, the transition metal catalyst coupled to the biomolecular condensate can contain a platinum group metal center. Additionally, in some embodiments, the transition metal catalyst has the following formula I:
[0009]
[0010] where M is a transition metal;
[0011] where A, D, E, G, Y, and Z are independently selected from C and N;
[0012] where R 3 to R 7 each represents one to four optional ring substituents, and each of the one to four optional ring substituents is independently selected from the group consisting of alkyl, heteroalkyl, haloalkyl, haloalkenyl, halogen, hydroxy, alkoxy, amine, amide, ether, -C(O)O - 、-C(O)OR 8 and -R 9 OH, where R 8 is selected from the group consisting of hydrogen and alkyl, and R9 is an alkyl group;
[0013] wherein R 1 is selected from the group consisting of a direct bond, an alkylene group, an alkenylene group, a cycloalkylene group, a cycloalkenylene group, an arylene group, a heteroalkylene group, a heteroalkenylene group, a heterocyclo group, and a heteroarylene group;
[0014] wherein L is an optional linking moiety selected from the group consisting of an amide, an ester, a sulfonamide, a sulfonate, a carbamate, and a urea; and
[0015] R 2 is selected from the group consisting of an alkynyl group, an amine, a protected amine, an azide, a hydrazide, an aryl group, a heteroaryl group, a cycloalkyl group, a cycloalkenyl group, a cycloalkynyl group, a heterocyclic group, a hydroxyl group, a carboxyl group, a halogen, an alkoxy group, a maleimide, -C(O)H, -C(O)OR 8 , -OS(O2)R 9 , a thiol, biotin, a hydroxylamine, and a haloalkyl group, wherein R 8 and R 9 are independently selected from the group consisting of an alkyl group, a haloalkyl group, an aryl group, a haloaryl group, an N-succinimidyl group, and an N-succinimide ester; wherein X - is a counterion and n is an integer from 0 to 20. As provided in Formula I, the linking moiety L is optional and thus may be absent in some embodiments of the transition metal catalyst.
[0016] The polarity of the transition metal complex can be adjusted by the selection of R 3 to R 7 to suit a particular cellular environment. In some embodiments, for example, one or more of R 3 to R 7 are selected to exhibit hydrophobic, lipophilic, or nonpolar characteristics. In some embodiments, for example, one or more of R 3 to R 7 can be an alkyl group, fluorine, or a fluoroalkyl group. The transition metal complexes described herein that exhibit hydrophobic, lipophilic, or nonpolar characteristics may be suitable for placement or entry into the intracellular environment, including the environment of intracellular biomolecular condensates. According to the principles described herein, the transition metal complexes can cross cell membranes for mapping the local intracellular environment. Thus, such transition metal complexes are cell-permeable. Optionally, R 3 to R 7 are selected to exhibit hydrophilic characteristics through charged and / or polar chemical moieties. In such embodiments, the transition metal complexes can exhibit hydrophilic characteristics suitable for placement in the intercellular / extracellular environment.
[0017] As further described herein, a transition metal catalyst can have an electronic structure for transferring energy to a protein label to generate a reactive intermediate. In some embodiments, the energy transfer is Dexter energy transfer or electron transfer, including single electron transfer. In some embodiments, the energy transfer to the protein label can originate from an excited state of the electronic structure of the transition metal catalyst. For example, the excited state of the catalyst can be a singlet excited state or a triplet excited state. The excited state of the catalyst can be generated by one or more mechanisms, including energy absorption by the catalyst. In some embodiments, the catalyst is a photocatalyst, where the excited state is induced by absorption of one or more photons. In other embodiments, the catalyst can be in an excited state by interacting with one or more chemical species in the surrounding environment. Optionally, the energy transfer to the protein label (including electron transfer) can originate from the ground state of the electronic structure of the catalyst. As described herein, the energy transfer to the protein label to generate a reactive intermediate allows for the analysis of the microenvironment local to the biomolecular condensate.
[0018] In another aspect, a system for analyzing the microenvironment local to a biomolecular condensate is provided. In some embodiments, the system comprises a protein label and a complex comprising one or more biomolecular condensates and a transition metal catalyst, the biomolecular condensate comprising nucleic acids and / or functionally diverse proteins, the transition metal catalyst being coupled to the biomolecular condensate by interacting with at least one of the nucleic acids or the functionally diverse proteins, wherein the transition metal catalyst has an electronic structure that allows energy transfer to the protein label to provide a reactive intermediate. As described herein, the biomolecular condensate can be located within the nucleus, cytoplasm, or cell membrane of a cell. The reactive intermediate can be used to label proteins or other biomolecules within a predetermined radius of the complex. The predetermined radius can be the diffusion radius of the reactive intermediate.
[0019] The diffusion radius of the reactive intermediate can be adjusted to accommodate specific microenvironment localization analysis (proximity labeling) considerations and can be restricted to the nanoscale. In some embodiments, for example, before quenching in the surrounding environment, the diffusion radius of the reactive intermediate can 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. In some embodiments, the diffusion radius can be from 0.5 nm to 10 nm or from 0.5 nm to 100 nm. Thus, the reactive intermediate will react or crosslink with proteins or other biomolecules within the diffusion radius or be quenched by the surrounding environment if no proteins or biomolecules are present. In this way, high-resolution localization analysis of the local environment of biomolecular condensates can be achieved through the synergistic action between the transition metal catalyst and the protein label. Additionally, in some embodiments, the reactive intermediate can exhibit a t of less than 5 ns, less than 4 ns, or less than 2 ns before quenching. 1 / 2 . For example, the reactive intermediate can exhibit a t of less than 1 - 5 ns 1 / 2 . In additional embodiments, by extending the half-life of the reactive intermediate, the diffusion radius can be extended to be between 5 - 500 nm. For example, in some embodiments, the reactive intermediate can have a half-life of 1 - 100 μs or longer.
[0020] In another aspect, methods for analyzing the local microenvironment of biomolecular condensates are provided. The methods include forming a complex comprising a transition metal catalyst and activating a protein label into a reactive intermediate with the transition metal catalyst, which is coupled to the biomolecular condensate by interacting with the biomolecular material of the biomolecular condensate. The reactive intermediate couples to proteins or other biomolecules within a predetermined radius of the complex. The biomolecular condensate, transition metal catalyst, protein label, and reactive intermediate can have any composition and / or properties described herein. Additionally, the transition metal catalyst can be coupled to the biomolecular condensate by covalent bonding, electrostatic interaction, or van der Waals interaction. In some embodiments, the protein label is activated at different times during the life cycle of the biomolecular condensate, including early disassembly, mid-disassembly, and late disassembly. Additionally, the methods described herein further include detecting or analyzing the proteins or other biomolecules coupled to the reactive intermediate.
[0021] These and other embodiments are further described in the detailed description below. BRIEF DESCRIPTION OF THE DRAWINGS
[0022] Figure 1 Shows a transition metal catalyst described herein according to some embodiments.
[0023] Figure 2Shows the binding of the transition metal complexes described herein to proteins via haloalkane dehalogenase according to some embodiments.
[0024] Figure 3 Shows that, according to some embodiments, a protein labeler is activated for the localization analysis of key target proteins during the life cycle of biomolecular condensates (including early disassembly, mid - disassembly, and late disassembly).
[0025] Figure 4 Is a schematic diagram showing the stress granule interactome localization analysis using the complexes and protein labelers described herein according to some embodiments.
[0026] Figure 5 Shows the ubiquitination of stress granule proteins during granule formation.
[0027] Figure 6 Shows that protein ubiquitination is disrupted by the presence of TAK243 during granule formation and disassembly.
[0028] Figure 7 Is a volcano plot for identifying HECT ubiquitin ligases and autophagy adaptors, which are identified by performing stress granule interactome localization analysis using the complexes and protein labelers described herein according to some embodiments.
[0029] Figure 8 Provides the time resolution of the HECT E3 ligases recruited to stress granules, as determined by interactome localization analysis using the complexes and protein labelers described herein according to some embodiments.
[0030] Figure 9 Is a schematic diagram showing the use of Heclin to determine the effect on the function of HECT E3 ligases regarding stress granule disassembly.
[0031] Figure 10 Shows that inhibition of HECT E3 ligases with Heclin delays stress granule disassembly.
[0032] Figure 11 Demonstrates that siRNA knockdown of HECT E3 ligases delays stress granule disassembly. Detailed Description
[0033] The embodiments described herein can be more readily understood by reference to the following specific embodiments and examples and the descriptions before and after them. However, the elements, devices, and methods described herein are not limited to the specific embodiments presented in the specific embodiments and examples. It should be recognized that these embodiments are merely illustrative of the principles of the present invention. Many modifications and adaptations will be apparent to those skilled in the art without departing from the spirit and scope of the present invention.
[0034] Definition
[0035] The term "alkyl", used alone or in combination herein, refers to a straight-chain or branched-chain saturated hydrocarbon-based group optionally substituted with one or more substituents. For example, alkyl can be C1-C 30 or C1-C 18 .
[0036] The term "alkenyl", used alone or in combination herein, refers to a straight-chain or branched-chain hydrocarbon-based group having at least one carbon-carbon double bond and optionally substituted with one or more substituents.
[0037] The term "alkynyl", used alone or in combination herein, refers to a straight-chain or branched-chain hydrocarbon-based group having at least one carbon-carbon triple bond and optionally substituted with one or more substituents.
[0038] The term "aryl", used alone or in combination herein, refers to an aromatic monocyclic or polycyclic system optionally substituted with one or more ring substituents.
[0039] The term "heteroaryl", used alone or in combination herein, refers to an aromatic monocyclic or polycyclic system in which one or more of the ring atoms are elements other than carbon (such as nitrogen, boron, oxygen, and / or sulfur).
[0040] The term "heterocycle", used alone or in combination herein, refers to a monocyclic or polycyclic system in which one or more of the atoms in the ring system are elements other than carbon (such as boron, nitrogen, oxygen, and / or sulfur or phosphorus), and in which the ring system is optionally substituted with one or more ring substituents. The heterocyclic system can include aromatic and / or non-aromatic rings, including rings having one or more unsaturation points.
[0041] The term "cycloalkyl", used alone or in combination herein, refers to a non-aromatic monocyclic or polycyclic system optionally substituted with one or more ring substituents.
[0042] The term "heterocycloalkyl", used alone or in combination herein, refers to a non-aromatic monocyclic or polycyclic system in which one or more of the atoms in the ring system are elements other than carbon (such as boron, nitrogen, oxygen, sulfur, or phosphorus), and in which the ring system is optionally substituted with one or more ring substituents.
[0043] As used herein, the term "alkoxy", alone or in combination, refers to the group RO—, where R is an alkyl, alkenyl, or aryl group as defined above.
[0044] As used herein, the term "halogen", alone or in combination, refers to the elements of Group VIIA of the Periodic Table of the Elements (halogens). Depending on the chemical environment, the halogen can be in a neutral or anionic state.
[0045] Terms not specifically defined herein are given their ordinary meanings in the art.
[0046] I. Complex
[0047] In one aspect, the present disclosure describes complexes capable of analyzing the local microenvironment across biomolecular condensates by proximity labeling. In some embodiments, the complex comprises one or more biomolecular condensates and a transition metal catalyst, the biomolecular condensate comprising nucleic acids and / or functionally diverse proteins, and the transition metal catalyst being coupled to the biomolecular condensate by interacting with at least one of the nucleic acid or the functionally diverse protein. In some embodiments, the complexes described herein are in an intracellular environment. In some embodiments, for example, the complex is in the nucleus of a cell. Optionally, the complex can be in the cytoplasm or cell membrane of the cell. The transition metal catalyst can interact with the nucleic acid (such as RNA) or protein of the biomolecular condensate by covalent bonding. In other embodiments, the transition metal catalyst can interact with the nucleic acid or protein of the condensate by electrostatic interactions and / or van der Waals interactions.
[0048] In some embodiments, the transition metal complex is coupled to the protein of the biomolecular condensate by interaction with a haloalkane dehalogenase (HaloTag). The haloalkane dehalogenase can be co-expressed with the protein using standard cloning procedures. The transition metal complex can be functionalized with a haloalkane moiety for covalent binding to the haloalkane dehalogenase, resulting in the coupling of the protein to the biomolecular condensate. Figure 2 Shown is the binding of the transition metal complex described herein to a protein via a haloalkane dehalogenase according to some embodiments. Optionally, the transition metal complex can be bonded to a derivatized amino acid of the biomolecular condensate protein. Suitable click chemistry moieties for the transition metal complex and / or the derivatized amino acid can be selected from the group consisting of DBCO, BCN, TCO, tetrazine, alkyne, and azide. The transition metal complex can have Formula (I) herein. In other embodiments, the transition metal complex is coupled to the protein of the biomolecular condensate by protein trans-splicing, and the transition metal complex is initially coupled to a split intein. The split intein carrying the transition metal complex can be an N-intein or a C-intein.
[0049] In some embodiments, the transition metal catalyst has the following formula I:
[0050]
[0051] wherein M is a transition metal;
[0052] wherein A, D, E, G, Y, and Z are independently selected from C and N;
[0053] wherein R 3 to R 7 each represents one to four optional ring substituents, and each of the one to four optional ring substituents is independently selected from the group consisting of alkyl, heteroalkyl, haloalkyl, haloalkenyl, halogen, hydroxy, alkoxy, amine, amide, ether, -C(O)O - , -C(O)OR 8 and -R 9 OH, wherein R 8 is selected from the group consisting of hydrogen and alkyl, and R 9 is alkyl;
[0054] wherein R 1 is selected from the group consisting of a direct bond, alkylene, alkenylene, cycloalkylene, cycloalkenylene, arylene, heteroalkylene, heteroalkenylene, heterocyclo, and heteroarylene;
[0055] wherein L is an optional linking moiety selected from the group consisting of amide, ester, sulfonamide, sulfonate, carbamate, and urea; and
[0056] R 2 is selected from the group consisting of alkynyl, amine, protected amine, azide, hydrazide, aryl, heteroaryl, cycloalkyl, cycloalkenyl, cycloalkynyl, heterocyclic, hydroxy, carboxy, halogen, alkoxy, maleimide, -C(O)H, -C(O)OR 8 , -OS(O2)R 9 , thiol, biotin, hydroxylamine, and haloalkyl, wherein R 8 and R 9 are independently selected from the group consisting of alkyl, haloalkyl, aryl, haloaryl, N-succinimidyl, and N-succinimide ester; wherein X - is a counterion, and n is an integer from 0 to 20. As provided in formula I, the linking moiety L is optional and thus may be absent in some embodiments of the transition metal catalyst.
[0057] It should be understood that in the absence of the optional substituents R 3 to R 7 , hydrogen occupies the positions on the aryl ring of formula I. Additionally, in some embodiments, the counterion (X -) may be selected from tetraalkyl borates, tetrafluoroborates, tetraphenyl borates, PF6 - and chlorine.
[0058] The polarity of the transition metal complex can be adjusted to suit a particular cellular environment by the selection of R 3 to R 7 . In some embodiments, for example, one or more of R 3 to R 7 are selected to exhibit hydrophobic, lipophilic or non-polar characteristics. In some embodiments, for example, one or more of R 3 to R 7 may be alkyl, fluorine or fluoroalkyl. The transition metal complexes described herein that exhibit hydrophobic, lipophilic or non-polar characteristics may be suitable for placement or entry into the intracellular environment. According to the principles described herein, the transition metal complexes may cross cell membranes for localizing and analyzing biomolecular condensates in the local intracellular environment. Thus, such transition metal complexes have cell permeability. Figure 1 Illustrates various transition metal complexes described herein. In some embodiments, L is an amide bound to a polyethylene glycol (PEG) moiety for linking R 2 . As provided in Figure 1 , the PEG moiety may be replaced with an alkylene moiety. In some embodiments, for example, R 2 comprises a haloalkane or a click chemistry moiety, including but not limited to BCN, DBCO, TCO, tetrazine, alkyne and azide. As shown in Figure 1 , these click chemistries of R 2 can be directly coupled to the linking moiety (L) or through a heteroatom, aryl or carbonyl.
[0059] The transition metal catalyst may have an electronic structure for transferring energy to a protein label 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 label may originate from an excited state of the transition metal catalyst electronic structure. For example, the excited state of the catalyst may be 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 label. For example, the T1 state may have a t of 0.2 - 2 μs 1 / 2。The transition metal complexes described herein can be photocatalytic and, in some embodiments, absorb light in the visible or infrared regions of the electromagnetic spectrum. Absorption of electromagnetic radiation can excite the transition metal complex to the S1 state, followed by quantitative intersystem crossing to the T1 state. The transition metal catalyst can then undergo short-range Dexter energy transfer to the protein tag and return to the ground state S0. Energy transfer to the tag activates the tag to react with proteins or other biomolecules in the local biomolecular condensate environment. In some embodiments, the T1 state of the transition metal complex can be greater than 60 kcal / mol. For example, the metal center can be a transition metal selected from the platinum group. In some embodiments, the metal center can be iridium.
[0060] In other embodiments, the catalyst can be in an excited state by interacting with one or more chemical species in the surrounding environment. Optionally, the energy transfer (including electron transfer) to the protein tag can originate from the ground state of the catalyst's electronic structure. As described herein, energy transfer to the protein tag to generate a reactive intermediate allows for the analysis of the microenvironment local to the biomolecular condensate.
[0061] II. System for analyzing the interactome of biomolecular condensates
[0062] In another aspect, a system for analyzing the local microenvironment of a biomolecular condensate is provided. In some embodiments, the system comprises a protein tag and a complex comprising one or more biomolecular condensates and a transition metal catalyst, the biomolecular condensate comprising nucleic acids and / or functionally diverse proteins, the transition metal catalyst being coupled to the biomolecular condensate by interacting with at least one of the nucleic acids or functionally diverse proteins, wherein the transition metal catalyst has an electronic structure that permits energy transfer to the protein tag to provide a reactive intermediate. The transition metal catalyst can have Formula I and can have any composition, structure, and / or properties described in Section I above. The biomolecular condensate can be located within the nucleus, cytoplasm, or cell membrane of a cell. The reactive intermediate can be used to label proteins or other biomolecules within a predetermined radius of the complex. The predetermined radius can be the diffusion radius of the reactive intermediate.
[0063] The diffusion radius of the reactive intermediate can be adjusted to accommodate specific microenvironment localization analysis (proximity labeling) considerations and can be restricted to the nanoscale. In some embodiments, for example, prior to quenching in the surrounding environment, the diffusion radius of the reactive intermediate can 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. In some embodiments, the diffusion radius can be from 0.5 nm to 10 nm. Thus, the reactive intermediate will react or crosslink with proteins or other biomolecules within the diffusion radius or, if no proteins or biomolecules are present, be quenched by the surrounding environment. In this way, high-resolution localization analysis of the local environment of biomolecular condensates can be achieved through the synergistic action between the catalyst and the protein label. Additionally, in some embodiments, the reactive intermediate can exhibit a t less than 5 ns, less than 4 ns, or less than 2 ns before quenching. 1 / 2 . For example, the reactive intermediate can exhibit a t less than 1 - 5 ns. 1 / 2 . In additional embodiments, by extending the half-life of the reactive intermediate, the diffusion radius can be extended to be between 5 - 500 nm. 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 type of protein label can be employed in the systems described herein. For example, two or more substances with different diffusion radii can be employed in the system. In such embodiments, the different diffusion radii can allow labeling at different distances from the transition metal complex, thereby further localizing the interactome analysis of biomolecular condensates.
[0064] In some embodiments, the protein label can be diazomethane. Triplet energy transfer from the excited-state photocatalyst can promote diazomethane into its triplet state (T1). The diazomethane 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 either crosslinks with nearby proteins or is quenched in an aqueous environment. In some embodiments, the extinction coefficient of the transition metal complex is 3 to 5 orders of magnitude greater than the extinction coefficient of diazomethane.
[0065] Any diazomethane conforms to the technical principles discussed herein. For example, the sensitization effect of diazomethane can be extended to a variety of p- and m-substituted aryltrifluoromethyldiazomethanes that carry valuable payloads for microscopy and proteomics applications, including free carboxylic acids, phenols, amines, alkynes, carbohydrates, and biotin groups. Diazomethane can be functionalized with markers such as biotin. In some embodiments, the marker is desthiobiotin. The marker can help identify proteins labeled by the protein labeling agent. For example, the marker can be used to determine the results by Western blotting and / or other analytical techniques. In addition to biotin and desthiobiotin, the marker can include alkyne, azide, FLAG tag, fluorophore, and chloroalkane functional groups.
[0066] In additional embodiments where the transition metal catalyst is a photocatalyst, the protein labeling agent can be an azide. Triplet energy transfer from the excited state photocatalyst can promote the formation of nitrene from the azide. The reactive nitrene either crosslinks with nearby proteins or quenches in an aqueous environment. Any azide capable of undergoing energy transfer with the transition metal photocatalyst and forming nitrene can be employed. In some embodiments, the azide is an aryl azide.
[0067] III. Method for analyzing the microenvironment of biomolecular condensates
[0068] On the other hand, methods for analyzing the local microenvironment of biomolecular condensates are provided. The methods include forming a complex comprising a transition metal catalyst and activating a protein labeling agent with the transition metal catalyst into a reactive intermediate, the transition metal catalyst being coupled to the biomolecular condensate by interacting with the biomolecular substance of the biomolecular condensate. The reactive intermediate couples with a protein or other biomolecule within a predetermined radius of the complex. The biomolecular condensate, transition metal catalyst, protein labeling agent, and reactive intermediate can have any composition and / or properties described in Sections I and II above herein. Additionally, as described in Sections I and II above, the transition metal catalyst can be coupled to the biomolecular condensate by covalent bonding, electrostatic interaction, or van der Waals interaction. In some embodiments, the protein labeling agent is activated at different times during the lifecycle of the biomolecular condensate, including early disassembly, mid-disassembly, and late disassembly. In some embodiments, the same protein labeling agent can be used during the condensate lifecycle. Optionally, multiple different protein labeling agents can be used during the condensate lifecycle. The different protein labeling agents can have similar or different diffusion radii for providing a complete localization analysis of the biomolecular condensate interactome.
[0069] Additionally, the methods described herein further include detecting or analyzing the proteins or other biomolecules coupled to the reactive intermediate.
[0070] Figure 3 Shows the activation of protein tags during the life cycle of biomolecular condensates, including early disassembly, mid - disassembly, and late disassembly. Staged activation of protein tags using transition metal complexes of the complex can identify key target proteins in condensate disassembly regulation.
[0071] These and other embodiments are further described in the non - limiting examples below.
[0072] Example 1 - Stress Granule Interactome Localization Analysis
[0073] Prepare the Figure 2 protein - transition metal complex composite as follows. Seed cells (HEK293T stably expressing HaloTag - G3BP1) and grow to 95% confluence before labeling. Incubate the cells in DMEM medium for one hour, which contains 5 μM Figure 2 of the catalyst with iridium. During this period, the iridium catalyst crosses the cell membrane and binds to the HaloTag - G3BP1 protein. Then incubate the cells in fresh DMEM medium for one hour to remove or minimize background signals from any unbound iridium catalyst.
[0074] Subsequently, transfer the first part of the cells loaded with the Ir catalyst to DMEM medium pre - supplemented with 2 μM TAK243 and incubate for another hour. The remaining second part (control) is given the same volume of DMSO. Stress the cells for 30 minutes with 500 mM NaAsO2 in DMEM. After stress, replace the medium with fresh DMEM at the designated time to recover the cells. During stress, 30 minutes before each time point of photo - labeling, give the cells 500 μM of the protein tag biotin - diazomethane in the medium. During photo - labeling, irradiate the cells with 450 nm blue light for 10 minutes. Figure 4 Shows the aforementioned test protocol.
[0075] Follow the general proteomics preparation protocol. Load 600 μg of total protein for streptavidin enrichment. After denaturation, reduction, alkylation, and washing on beads, digest the biotin - labeled proteins with trypsin to produce a peptide solution for TIMSTOF LCMS analysis.
[0076] Figure 5 Shows the ubiquitination of stress granule proteins during granule formation. In addition, Figure 6 Shows that when protein ubiquitination during granule formation is disrupted or inhibited by the presence of TAK243, granule disassembly is delayed. Figure 7A volcano plot identifying HECT ubiquitin ligases and autophagy adaptors identified in a stress granule interactome localization analysis using the complex and protein labeler of this example. Figure 8 Provides temporal resolution of HECT E3 ligases recruited to stress granules.
[0077] Using this information provided by interactome localization analysis, the E3 ligase inhibitor Heclin was used to determine any effect on the function of HECT E3 ligases regarding stress granule disassembly, as Figure 9 shown. Figure 10 Shows that inhibition of HECT E3 ligases with Heclin delays stress granule disassembly. Additionally, in Figure 11 , siRNA knockdown of HECT E3 ligases delays stress granule disassembly.
[0078] To achieve the various objects of the present invention, various embodiments of the present invention have been described. It should be recognized that these embodiments are merely illustrative of the principles of the present invention. Many modifications and adaptations thereof will be apparent to those skilled in the art without departing from the spirit and scope of the present invention.
Claims
1. 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 by interacting with at least one of the nucleic acid or the functionally diverse proteins.
2. The complex according to claim 1, wherein the interaction is selected from the group consisting of covalent bonds, electrostatic interactions, and van der Waals interactions.
3. The complex according to claim 1, wherein the transition metal complex has the following formula: Where M is a transition metal; Where A, D, E, G, Y, and Z are independently selected from C and N; wherein R 3 to R 7 each independently represents one to four optional ring substituents, each of the one to four optional ring substituents being independently selected from the group consisting of alkyl, heteroalkyl, haloalkyl, haloalkenyl, halogen, hydroxy, alkoxy, amine, amide, ether, -C(O)O - , -C(O)OR 8 and -R 9 OH, wherein R 8 is selected from the group consisting of hydrogen and alkyl, and R 9 is alkyl; wherein R 1 is selected from the group consisting of a direct bond, an alkylene, an alkenylene, a cycloalkylene, a cycloalkenylene, an arylene, a heteroalkylene, a heteroalkenylene, a heterocyclo and a heteroarylene; Where L is an optional linking moiety selected from the group consisting of amides, esters, sulfonamides, sulfonates, carbamates, and ureas; and R 2 selected from the group consisting of alkynyl, amine, protected amine, azide, hydrazide, aryl, heteroaryl, cycloalkyl, cycloalkenyl, cycloalkynyl, heterocyclic group, hydroxyl, carboxyl, halogen, alkoxy, maleimide, -C(O)H, -C(O)OR 8 、-OS(O2)R 9 、thiol, biotin, hydroxylamine and haloalkyl, wherein R 8 and R 9 are independently selected from the group consisting of alkyl, haloalkyl, aryl, haloaryl, N-succinimidyl and N-succinimide ester; wherein X - is a counterion and n is an integer from 0 to 20.
4. The complex according to claim 3, wherein the transition metal complex is coupled to the protein of the biomolecular condensate by binding to a haloalkane dehalogenase.
5. The complex according to claim 3, wherein M is iridium.
6. The complex according to claim 1, wherein the transition metal catalyst is a photocatalyst.
7. The complex according to claim 1, wherein the transition metal catalyst has an electronic structure for transferring energy to a protein label.
8. The complex according to claim 7, wherein the energy transfer is Dexter energy transfer.
9. The complex according to claim 1, wherein the biomolecular condensate is selected from the group consisting of P-bodies, U-bodies, stress granules, centrosomes, signal transduction 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. A system for analyzing the interactome of a biomolecular condensate, comprising: A protein label; and A complex, the complex comprising one or more biomolecular condensates and a transition metal catalyst, the biomolecular condensate comprising nucleic acids and / or functionally diverse proteins, the transition metal catalyst being coupled to the biomolecular condensate by interacting with at least one of the nucleic acid or the functionally diverse proteins, wherein the transition metal catalyst has an electronic structure that allows energy transfer to the protein label to provide a reactive intermediate.
11. The system according to claim 10, wherein the reactive intermediate can be used to label proteins or other biomolecules within a predetermined radius of the complex.
12. The system according to claim 10, wherein the transition metal complex has the following formula: Where M is a transition metal; Where A, D, E, G, Y, and Z are independently selected from C and N; wherein R 3 to R 7 each independently represents one to four optional ring substituents, each of the one to four optional ring substituents being independently selected from the group consisting of alkyl, heteroalkyl, haloalkyl, haloalkenyl, halogen, hydroxy, alkoxy, amine, amide, ether, -C(O)O - , -C(O)OR 8 and -R 9 OH, wherein R 8 is selected from the group consisting of hydrogen and alkyl, and R 9 is alkyl; wherein R 1 is selected from the group consisting of a direct bond, an alkylene, an alkenylene, a cycloalkylene, a cycloalkenylene, an arylene, a heteroalkylene, a heteroalkenylene, a heterocyclo and a heteroarylene; Where L is an optional linking moiety selected from the group consisting of amides, esters, sulfonamides, sulfonates, carbamates, and ureas; and R 2 selected from the group consisting of alkynyl, amine, protected amine, azide, hydrazide, aryl, heteroaryl, cycloalkyl, cycloalkenyl, cycloalkynyl, heterocyclic group, hydroxy, carboxy, halogen, alkoxy, maleimide, -C(O)H, -C(O)OR 8 、-OS(O2)R 9 、thiol, biotin, hydroxylamine and haloalkyl, wherein R 8 and R 9 are independently selected from the group consisting of alkyl, haloalkyl, aryl, haloaryl, N-succinimidyl and N-succinimide ester; wherein X - is a counterion and n is an integer from 0 to 20.
13. The system according to claim 12, wherein the transition metal complex is coupled to the protein of the biomolecular condensate by binding to a haloalkane dehalogenase.
14. The system according to claim 12, wherein M is iridium.
15. The system according to claim 10, wherein the transition metal catalyst is a photocatalyst.
16. The system according to claim 10, wherein the energy transfer is Dexter energy transfer.
17. The system according to claim 10, wherein the protein label is diazomethane.
18. The system according to claim 17, wherein the diazomethane comprises a molecular marker.
19. The system according to claim 10, wherein the biomolecular condensate is selected from the group consisting of P bodies, U bodies, stress granules, centrosomes, signal transduction 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. A method for analyzing the local microenvironment of a biomolecular condensate, comprising: forming a complex comprising a transition metal catalyst, the transition metal catalyst being coupled to the biomolecular condensate by interacting with the biomolecular material of the biomolecular condensate; activating a protein label with the transition metal catalyst into a reactive intermediate; and coupling the reactive intermediate to a protein or other biomolecule within a predetermined radius of the complex.
21. The method according to claim 20, wherein activating the protein label into the reactive intermediate comprises energy transfer from the transition metal catalyst to the protein label.
22. The method according to claim 20, wherein the protein label is diazomethane.
23. The method according to claim 22, wherein the diazomethane is functionalized with a marker.
24. The method according to claim 20, wherein the predetermined radius is 2 - 100 nm.
25. The method according to claim 20, wherein the reactive intermediate is quenched outside the predetermined radius, precluding binding to biomolecules outside the predetermined radius.
26. The method according to claim 20, the method further comprising detecting or analyzing the protein coupled to the reactive intermediate.
27. The method according to claim 20, wherein the protein label is activated at different times during the life cycle of the biomolecular condensate.
28. The method according to claim 27, wherein the life cycle of the biomolecular condensate comprises early disassembly, mid - disassembly, and late disassembly.
29. The method according to claim 20, wherein the transition metal complex has the following formula: wherein M is a transition metal; wherein A, D, E, G, Y, and Z are independently selected from C and N; wherein R 3 to R 7 each independently represents one to four optional ring substituents, each of the one to four optional ring substituents being independently selected from the group consisting of alkyl, heteroalkyl, haloalkyl, haloalkenyl, halogen, hydroxy, alkoxy, amine, amide, ether, -C(O)O - , -C(O)OR 8 and -R 9 OH, wherein R 8 is selected from the group consisting of hydrogen and alkyl, and R 9 is alkyl; wherein R 1 is selected from the group consisting of a direct bond, an alkylene, an alkenylene, a cycloalkylene, a cycloalkenylene, an arylene, a heteroalkylene, a heteroalkenylene, a heterocyclo and a heteroarylene; wherein L is an optional linking moiety selected from the group consisting of amides, esters, sulfonamides, sulfonates, carbamates, and ureas; and R 2 selected from the group consisting of alkynyl, amine, protected amine, azide, hydrazide, aryl, heteroaryl, cycloalkyl, cycloalkenyl, cycloalkynyl, heterocyclic group, hydroxy, carboxy, halogen, alkoxy, maleimide, -C(O)H, -C(O)OR 8 , -OS(O2)R 9 , thiol, biotin, hydroxylamine and haloalkyl, wherein R 8 and R 9 are independently selected from the group consisting of alkyl, haloalkyl, aryl, haloaryl, N-succinimidyl and N-succinimide ester; wherein X - is a counterion and n is an integer from 0 to 20.
30. The method according to claim 29, wherein the transition metal complex is coupled to the protein of the biomolecular condensate by binding to a haloalkane dehalogenase.
31. The method according to claim 29, wherein M is iridium.
32. The method according to claim 20, wherein the transition metal catalyst is a photocatalyst.
33. The method according to claim 20, wherein the biomolecular condensate is selected from the group consisting of P bodies, U bodies, stress granules, centrosomes, signal transduction 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. The method according to claim 20, wherein the biomolecular condensate is located in the cytoplasm or cell membrane of the cell.
35. The method according to claim 20, wherein the biomolecular condensate is located in the nucleus of the cell.