Hi-APEX, a non-cytotoxic in vivo compatible proximity labeling method for peroxidases, and its applications.

Abstract

This invention provides a non-cytotoxic, in vivo compatible proximity labeling method for peroxidases, Hi-APEX, and its applications, belonging to the field of biotechnology. This invention provides a method for labeling interacting proteins, neighboring proteins, and / or neighboring RNA. By introducing TP probes, it completely overcomes the core limitation of peroxidase dependence on H2O2 without sacrificing the original high spatiotemporal resolution. The method provided by this invention exhibits significantly superior technical effects compared to existing technologies in terms of reduced toxicity, enabling in vivo application, precise analysis of redox-sensitive processes, dynamic tracking of protein transport, and simultaneous spatial multi-omics analysis. It provides a powerful tool for life science research and drug development, possessing significant scientific value and broad application prospects.

Description

Technical Field

[0001] This invention relates to a non-cytotoxic in vivo compatible proximity labeling method for peroxidase, Hi-APEX, and its applications, belonging to the field of biotechnology. Background Technology

[0002] Proximity labeling is a powerful tool for identifying and studying the function of biomolecules (such as proteins, RNA, and DNA) within a specific spatial range surrounding a target protein in living cells or in natural environments. Its core idea is to use genetic methods to anchor a "labeling enzyme" to the target protein or organelle. This enzyme can covalently link a reporter group (usually biotin) to neighboring molecules at close range (typically on the nanoscale), thereby "capturing" potential interacting molecules or spatially adjacent molecules of the target protein.

[0003] Currently, the main proximity labeling techniques include the following:

[0004] 1. Peroxidase-based technology

[0005] This mainly includes APEX (Ascorbate Peroxidase) or HRP (Horseradish Peroxidase). 1-3 wait

[0006] This type of technology utilizes the property of peroxidase to catalyze the conversion of phenol substrates into phenoxy radicals in the presence of hydrogen peroxide (H2O2), and uses biotin-phenol as a probe molecule to label neighboring protein or RNA molecules. Among them, APEX is an engineered product of soybean peroxidase (APX), which has a smaller molecular weight and higher catalytic activity.

[0007] The marking process mainly includes:

[0008] 1) Express APEX by fusing it with the target protein.

[0009] 2) When live cells are co-incubated with biotin-phenol, biotin-phenol will diffuse freely into the cells.

[0010] 3) Add H2O2 to start the reaction (usually lasts for 1 minute).

[0011] 4) APEX uses H2O2 to oxidize biotin-phenol into highly reactive biotin-phenoxy radicals. These radicals have an extremely short lifetime (<1 ms) and a limited diffusion distance (<20 nm), thus they can only label proteins near the APEX enzyme.

[0012] 5) Lyse the cells, enrich the biotinylated proteins using streptavidin beads, and then perform mass spectrometry analysis.

[0013] 2. Biotin ligase-based technology

[0014] Mainly includes BioID 4 TurboID, miniTurbo 5 wait

[0015] These technologies utilize the property of biotin ligases to attach biotin to lysine residues of neighboring proteins, using biotin as a probe molecule to label neighboring protein molecules. BioID is a mutant E. coli biotin ligase (BirA). This mutation causes it to lose its specific binding ability to substrate peptides. TurboID is a further engineered version of BioID obtained through directed evolution, resulting in significantly improved activity and faster labeling speed. miniTurbo is a truncated version of TurboID, with slightly lower activity but a cleaner background.

[0016] The marking process mainly includes:

[0017] 1) Fuse the labeled enzyme with the target protein for expression.

[0018] 2) Provide excess biotin in the cell culture medium.

[0019] 3) BirA* continuously produces active biotin-AMP within the cell.

[0020] 4) If there are reactive lysine residues on the surface of a neighboring protein, biotin-AMP will covalently bind to them to complete the biotinylation labeling.

[0021] 5) Labeling usually lasts for 18-24 hours, followed by cell lysis for streptavidin enrichment and mass spectrometry analysis.

[0022] 3. Techniques based on tyrosinase or laccase

[0023] Mainly includes TyroID 6 LaccID 7 wait

[0024] TyroID utilizes an engineered tyrosinase to catalyze the generation of highly active quinone intermediates from a biotin-phenol probe in the presence of oxygen. These intermediates rapidly undergo Michael addition reactions with nucleophilic groups on neighboring proteins (such as the ε-amino group of lysine and the thiol group of cysteine), thereby achieving covalent labeling. LaccID utilizes an engineered laccase to catalyze the generation of free radicals from a biotin-phenol probe in the presence of oxygen, thereby labeling neighboring proteins. TyroID is a tyrosinase derived from Agaricus bisporus, while LaccID is an engineered copper-containing polyphenol oxidase—laccase.

[0025] The marking process mainly includes:

[0026] 1) Fuse TyroID or LaccID with the target protein for expression.

[0027] 2) Co-incubate live cells with biotinylated phenolic substrates.

[0028] 3) TyroID or LaccID utilizes oxygen in the environment to oxidize the substrate into highly reactive quinones or free radicals. This active intermediate has a short lifespan and limited diffusion distance, therefore it can only label proteins near the enzyme.

[0029] 4) The labeling time is usually 30 minutes to several hours. Then the cells are lysed, and the biotinylated proteins are enriched using streptavidin beads for mass spectrometry analysis.

[0030] 4. Technologies based on photosensitive proteins or photosensitive small molecules

[0031] Mainly includes μMAP 8 PDPL 9 RinID 10 SeeID 11 CAP-seq 12 Halo-seq 13 wait

[0032] These technologies utilize light (usually visible light) to activate photosensitizers, causing them to transition from a ground state to an excited state. The excited photosensitizer can then transfer energy to surrounding molecules through various mechanisms, generating active intermediates that covalently label neighboring biomolecules. For example, μMAP uses blue light to excite an iridium catalyst, activating a diaziridinium probe to generate a carbene intermediate that labels neighboring proteins or RNA molecules. PDPL and RinID use blue light to excite the photosensitizing protein miniSOG, generating singlet oxygen to oxidize neighboring histidine residues, and then use an amino probe to label the protein. SeeID uses red light to excite the photosensitizing small molecule silyl rhodamine (SiR), generating singlet oxygen to oxidize neighboring histidine residues, and then use an amino probe to label the protein. CAP-seq uses blue light to excite the photosensitizing protein miniSOG, generating singlet oxygen to oxidize neighboring guanosine, and then use an amino probe to label the RNA. Halo-seq uses green light to excite the photosensitizing small molecule dibromofluorescein (DBF), generating singlet oxygen to oxidize neighboring guanosine, and then use an amino probe to label the RNA.

[0033] Tagging process:

[0034] 1) Target the photosensitizer to the target location (e.g., through antibody-photosensitizer conjugates, HaloTag ligands, etc.).

[0035] 2) Add biotinylated probe molecules (in some designs, the reporter molecule itself may not be biotinylated and biotin needs to be introduced using a "click" reaction after cell lysis).

[0036] 3) Irradiate the area with light of a specific wavelength to initiate the labeling reaction (usually within seconds to minutes).

[0037] 4) Lyse cells for streptavidin enrichment and omics analysis.

[0038] 5. Technologies based on other enzymes

[0039] For example, PUP-IT 14

[0040] This technology uses the PafA enzyme derived from Mycobacterium tuberculosis to catalyze the covalent attachment of a small protein tag—a prokaryotic ubiquitin-like protein (Pup)—to a lysine residue of a target protein, thereby enabling the labeling of neighboring proteins. Specifically, this technology utilizes an endogenously biotinylated Pup protein tag (bio-PupE) for subsequent enrichment and analysis.

[0041] Tagging process:

[0042] 1) Fuse PafA enzyme with target protein for expression.

[0043] 2) Provide PupE protein with a reporter tag in cell culture medium or lysis buffer.

[0044] 3) PafA enzyme uses ATP to activate PupE and covalently transfers it to a protein lysine residue in the vicinity of the fusion protein.

[0045] 4) After labeling, lyse the cells and enrich and analyze them using an affinity medium corresponding to the tag on PupE (such as streptavidin, if PupE is biotinylated).

[0046] Existing peroxidase-based technologies suffer from a fundamental, yet unresolved, limitation: their catalytic activity is entirely dependent on the addition of exogenous hydrogen peroxide. This inherent characteristic leads to a series of serious problems:

[0047] 1. Severely limited application scope: The cytotoxicity of H2O2 hinders the direct application of this technology in live animal models. Even if transgenic animals are constructed, tissue dissection and immersion in H2O2 buffer are necessary, which is essentially an ex vivo operation and cannot achieve true in vivo in situ labeling.

[0048] 2. Interference with normal cellular physiological state: The introduction of exogenous H2O2 will severely disrupt the redox homeostasis within cells, leading to deviations or even complete distortions in research data on redox-sensitive signaling pathways and cellular processes.

[0049] 3. Existing alternatives have inherent flaws: Attempts to circumvent the H2O2 problem have all been unsuccessful: Endogenous H2O2 generation strategy: fusing APEX2 with an enzyme that generates H2O2 avoids direct addition, but still alters the intracellular redox balance and may introduce new interferences such as phototoxicity.

[0050] Other proximity labeling systems also have their own shortcomings:

[0051] 1. Biotin ligases (such as TurboID) have high background and usually lack RNA labeling capabilities.

[0052] 2. Methods based on tyrosinase and laccase are limited by the toxicity of copper ions and the challenge of spatial localization.

[0053] 3. Although photocatalyst-dependent systems can achieve simultaneous protein / RNA labeling, their poor visible light penetration severely limits their application in thick tissues or in vivo.

[0054] References

[0055] 1.Lam, SS et al. Directed evolution of APEX2 for electronmicroscopy and proximity labeling. Nat. Methods 12, 51–54 (2015).

[0056] 2.Fazal, FM et al. Atlas of Subcellular RNA Localization Revealed by APEX-Seq. Cell 178, 473-490.e26 (2019).

[0057] 3.Zhou, Y. et al. Expanding APEX2 Substrates for Proximity-DependentLabeling of Nucleic Acids and Proteins in Living Cells. Angew. Chem. Int. Ed.58, 11763–11767 (2019).

[0058] 4.Roux, K. J., Kim, D. I., Raida, M. & Burke, B. A promiscuous biotinligase fusion protein identifies proximal and interacting proteins inmammalian cells. J. Cell Biol. 196, 801–810 (2012).

[0059] 5.Branon, T. C. et al. Efficient proximity labeling in living cellsand organisms with TurboID. Nat. Biotechnol. 36, 880–887 (2018).

[0060] 6.Zhang, Z. et al. Spatiotemporally resolved mapping of extracellularproteomes via in vivo-compatible TyroID. Nat. Commun. 16, 2553 (2025).

[0061] 7.Lee, S.-Y. et al. Directed evolution of LaccID for cell surfaceproximity labeling and electron microscopy. Nat. Chem. Biol. 1–11 (2025) doi:10.1038 / s41589-025-01973-6.

[0062] 8.Geri, J. B. et al. Microenvironment mapping via Dexter energytransfer on immune cells. Science 367, 1091–1097 (2020).

[0063] 9.Zhai, Y. et al. Spatiotemporal-resolved protein networks profilingwith photoactivation dependent proximity labeling. Nat. Commun. 13, 4906(2022).

[0064] 10.Zheng, F., Yu, C., Zhou, X. & Zou, P. Genetically encodedphotocatalytic protein labeling enables spatially-resolved profiling ofintracellular proteome. Nat. Commun. 14, 2978 (2023).

[0065] 11.Wang, W. et al. Silicon-rhodamine-enabled identification for near-infrared light controlled proximity labeling in vitro and in vivo. Nat.Commun. 16, 8134 (2025).

[0066] 12.Wang, P. et al. Mapping spatial transcriptome with light-activatedproximity-dependent RNA labeling. Nat. Chem. Biol. 15, 1110–1119 (2019).

[0067] 13.Engel, K. L. et al. Analysis of subcellular transcriptomes by RNAproximity labeling with Halo-seq. Nucleic Acids Res. 50, e24 (2022).

[0068] 14. Liu, Q. et al. A proximity-tagging system to identify membraneprotein–protein interactions. Nat. Methods 15, 715–722 (2018). Summary of the Invention

[0069] The problem the invention aims to solve

[0070] To address the problems of cytotoxicity, physiological interference, and limitations in in vivo application caused by existing peroxidase technologies (such as APEX2 and HRP) due to their reliance on exogenous hydrogen peroxide, this invention provides a novel proximity labeling platform, Hi-APEX.

[0071] Specifically, the present invention aims to solve the following technical problems:

[0072] 1. Eliminate the dependence of existing peroxidase technology on exogenous hydrogen peroxide, and avoid cytotoxicity problems caused by the addition of hydrogen peroxide;

[0073] 2. Maintaining normal redox homeostasis in cells makes it possible to study redox-sensitive biological processes;

[0074] 3. Achieve in-situ protein labeling in living animals, overcoming the limitations of existing technologies that are limited to cell culture or ex vivo tissues;

[0075] 4. Maintaining labeling efficiency comparable to traditional peroxidase technology without the need for exogenous hydrogen peroxide;

[0076] 5. Provide a technical solution that can be seamlessly integrated with existing HRP or APEX2 workflows without requiring modification of existing HRP or APEX2 research systems.

[0077] Solution for solving the problem

[0078] This invention provides a novel proximity labeling method called Hi-APEX (H2O2-independent APEX). The core of this method lies in the discovery and use of tetrazine-Phenol (TP) as a small molecule probe. This probe can be directly activated by APEX2 enzymes or HRP enzymes without the need for exogenous hydrogen peroxide (H2O2), thereby achieving efficient and specific labeling of nanoscale biomolecules (including proteins and RNA) surrounding the target protein.

[0079] This invention provides the following solution:

[0080] This invention provides a method for labeling interacting proteins, neighboring proteins, and / or neighboring RNA, the method comprising the following steps:

[0081] S1) The peroxidase is linked to protein A, which participates in the interaction, or to element A, which expresses protein A, to obtain element A' that expresses peroxidase;

[0082] S2) The peroxidase element A' is contacted with tetrazine-phenol, thereby catalyzing the attachment of tetrazine-phenol to other neighboring proteins and / or other neighboring RNAs adjacent to protein A;

[0083] The peroxidases include horseradish peroxidase (HRP), ascorbate peroxidase (APEX), and / or variants thereof.

[0084] In some embodiments, the APEX variant comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity with the sequence shown in SEQ ID NO:1;

[0085] The APEX variant retains the catalytically active site His42 corresponding to the sequence shown in SEQ ID NO:1, and is capable of catalyzing the conversion of tetrazine-phenol to tetrazine phenoxy without the dependence on exogenous hydrogen peroxide.

[0086] In some optional embodiments, the APEX variant retains sites Arg38, Phe145, Trp179, and Tyr235 corresponding to the sequence shown in SEQ ID NO:1.

[0087] In some embodiments, element A is selected from organelles, cells, or combinations thereof.

[0088] In some embodiments, step S1 includes at least one of the following:

[0089] a) Peroxidase is expressed on element A, thereby attaching the peroxidase to element A;

[0090] b) Fusing the peroxidase to protein A, thereby attaching the peroxidase to element A;

[0091] c) Contacting the protein A or element A with an antibody crosslinked with the peroxidase, wherein the antibody specifically binds to the protein A, thereby linking the peroxidase to the protein A or element A;

[0092] d) Contacting the protein A or element A, a first antibody that specifically binds to the protein A, with a second antibody that is cross-linked with the peroxidase, thereby linking the peroxidase to the protein A or element A, wherein the second antibody specifically recognizes the first antibody.

[0093] This invention provides a method for analyzing protein-protein interactions or protein-RNA interactions, the method comprising:

[0094] Interacting proteins, neighboring proteins, and / or neighboring RNAs were labeled using the methods described above;

[0095] Biotin is linked to a protein or RNA labeled with tetrazine-phenol using trans-cyclooctene-biotin (TCO-Biotin).

[0096] Enrichment of biotin-labeled proteins or RNA using streptavidin magnetic beads;

[0097] Identification and analysis of the enriched biotin-labeled proteins or RNA.

[0098] In some implementations, the enriched biotin-labeled proteins or RNAs are analyzed and identified using LC-MS / MS, Western blot, DNA-seq, RNA-seq, or RT-qPCR methods.

[0099] The present invention provides a composition for labeling interacting proteins, neighboring proteins, and / or neighboring RNA, comprising i) and optionally ii), iii), and / or iv):

[0100] i) Tetraazine-phenol;

[0101] ii) Ascorbate peroxidase (APEX), its localization fusion protein, its antibody cross-linked product, nucleic acid molecule encoding it, or expression vector containing said nucleic acid molecule;

[0102] iii) APEX variants, their localization fusion proteins, their antibody cross-links, nucleic acid molecules encoding them, or expression vectors containing said nucleic acid molecules;

[0103] iv) Horseradish peroxidase (HRP), its localization fusion protein, its antibody cross-linked form, nucleic acid molecule encoding it, or expression vector containing said nucleic acid molecule.

[0104] In some embodiments, the APEX variant comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity with the sequence shown in SEQ ID NO:1.

[0105] The APEX variant retains the catalytic active site corresponding to His42 as shown in SEQ ID NO:1, and is capable of catalyzing the conversion of tetrazine-phenol to tetrazine phenoxy without relying on exogenous hydrogen peroxide.

[0106] This invention provides the use of tetrazine-phenol in the following i) and / or ii):

[0107] i) Prepare kits for labeling interacting proteins, neighboring proteins, and / or neighboring RNA;

[0108] ii) Prepare kits for analyzing protein-protein interactions or protein-RNA interactions.

[0109] In some embodiments, the kit also contains horseradish peroxidase (HRP), ascorbate peroxidase (APEX), and / or variants thereof;

[0110] In some preferred embodiments, the APEX variant comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity with the sequence shown in SEQ ID NO:1.

[0111] The APEX variant retains the catalytic active site corresponding to His42 as shown in SEQ ID NO:1, and is capable of catalyzing the conversion of tetrazine-phenol to tetrazine phenoxy without relying on exogenous hydrogen peroxide.

[0112] The effects of the invention

[0113] This invention provides a hydrogen peroxide (H2O2)-independent peroxidase labeling method (Hi-APEX) based on a tetrazine-phenol (TP) probe, successfully overcoming a series of long-standing technical bottlenecks of traditional peroxidase technologies (such as APEX2 and HRP), including cytotoxicity, oxidative stress interference, and inability to be applied in live animals due to their dependence on exogenous H2O2. The Hi-APEX technology utilizes a novel TP probe, overcoming the H2O2 dependence of traditional APEX2, achieving significant breakthroughs in reducing toxicity, enabling in vivo application, and dynamically analyzing cellular processes. It is a powerful new tool for life science and drug research.

[0114] Specifically, compared with existing technologies, Hi-APEX achieves the following significant technical effects:

[0115] 1. Achieved true in vivo compatible spatial multi-omics analysis

[0116] The most significant technical achievement of this invention is that it is the first time that APEX2-mediated proximity labeling has been performed directly in a living animal.

[0117] Because of the H2O2-independent activation property of the TP probe, it is possible to avoid the direct injection of toxic H2O2 into living tissues, making it possible to perform spatial proteomics and transcriptomics mapping with nanoscale precision in complete living systems, and expanding the application of proximity labeling (PL) technology from cell culture systems to living organisms.

[0118] 2. Significantly reduced cytotoxicity, enabling true capture of redox-sensitive processes.

[0119] Compared to traditional BP / H2O2 labeling, TP labeling (Hi-APEX) showed no significant effect on cell viability and normal cell proliferation during a 48-hour observation period. Hi-APEX removes the interference of exogenous H2O2, eliminating H2O2-induced oxidative stress, and can be used to study biological processes sensitive to H2O2 without introducing interference from the labeling process itself.

[0120] 3. Provides high spatiotemporal resolution and controllable labeling capabilities.

[0121] Hi-APEX inherits the high temporal resolution (labeling can be completed within 1-5 minutes) and high spatial specificity of APEX2. Furthermore, TP labeling is stopable, enabling pulse-chase experiments.

[0122] The inherent rapid catalytic activity of the APEX2 enzyme and the membrane permeability and fast reaction kinetics of the TP probe together ensure the efficiency and speed of labeling.

[0123] 4. Compatible with multi-omics analysis and requires no enzyme modification.

[0124] Hi-APEX can be used simultaneously for spatial proteomics (Hi-APEX-proteomics) and spatial transcriptomics (Hi-APEX-seq) analysis. High-specificity and highly reproducible enrichment of proteins and RNA was achieved in both mitochondrial matrix and stress granule models.

[0125] The free radicals generated by TP probes can label proteins and RNA, giving them the potential for multi-omics analysis.

[0126] The subsequent IEDDA reaction (combined with TCO-biotin) is characterized by rapid reaction (10-30 minutes), bioorthogonality, and no need for metal catalysts, which makes the enrichment process more efficient and has a lower background.

[0127] Any existing APEX2 or HRP system can be seamlessly switched to the Hi-APEX platform immediately, greatly reducing the technical barrier to entry and making it widely applicable and accessible.

[0128] 5. A unique H2O2-independent catalytic mechanism was revealed.

[0129] This invention demonstrates that APEX2 can directly catalyze the generation of free radicals from TP in the absence of exogenous H2O2, and identifies His42 residues as crucial for H2O2-independent catalysis, while Pro132 and Phe175 residues specifically mediate H2O2-dependent TP activation.

[0130] The unique tetrazine group in the TP molecule is key to achieving H2O2-independent labeling. Molecular docking and mutagenesis experiments suggest that this group may interact specifically with the APEX2 active site, potentially forming an "H2O2-like" intermediate. Figure 11 This process, involving His42, directly generates phenoxy radicals. Elucidating this mechanism lays the theoretical foundation for designing more efficient and specific next-generation probes. Attached Figure Description

[0131] Figure 1 This is a schematic diagram of Hi-APEX technology.

[0132] Figure 2 The molecular structure is related to Hi-APEX technology.

[0133] Figure 3 This study compares the labeling capabilities of TP with the tetrazine-free control molecule "4-EP".

[0134] Figure 4 This is a schematic diagram of Hi-APEX allogeneic tumor markers.

[0135] Figure 5 This is a schematic diagram of the labeling of hippocampal neurons in Hi-APEX live mice.

[0136] Figure 6 For Hi-APEX-labeled cytotoxicity assays.

[0137] Figure 7 SG proteomic analysis for Hi-APEX.

[0138] Figure 8 SG transcriptome analysis for Hi-APEX.

[0139] Figure 9 Ferraphobia-related proteomic analysis for Hi-APEX.

[0140] Figure 10 Functional verification for TRMT61B.

[0141] Figure 11 This is a hypothesis about the mechanism of Hi-APEX.

[0142] Figure 12 To analyze the specificity and accuracy of the Hi-APEX SG proteome.

[0143] Figure 13 IGV map of important genes in the Hi-APEX SG transcriptome.

[0144] Figure 14 Western blotting validation of Hi-APEX live markers.

[0145] Figure 15 GO analysis of the neighboring proteome of Hi-APEX in vivo marker mGPx4.

[0146] Figure 16 STRING network analysis of mGPx4 interacting proteins.

[0147] Figure 17 Co-IP validation of TRMT61B protein.

[0148] Figure 18 The experimental procedure and results of Hi-APEX for labeling and enriching secreted proteins are presented.

[0149] Figure 19 The label strength of APEX2 and its variants in the conventional APEX and Hi-APEX methods.

[0150] Figure 20 For traditional tags and Hi-APEX tags when using HRP. Detailed Implementation

[0151] Various exemplary embodiments, features, and aspects of the present invention will be described in detail below. The term "exemplary" as used herein means "serving as an example, embodiment, or illustration." Any embodiment described herein as "exemplary" is not necessarily to be construed as superior to or better than other embodiments.

[0152] Furthermore, to better illustrate the present invention, numerous specific details are set forth in the following detailed embodiments. Those skilled in the art should understand that the present invention can be practiced without certain specific details. In other instances, methods, means, apparatus, and steps well known to those skilled in the art have not been described in detail in order to highlight the spirit of the present invention.

[0153] Unless otherwise stated, all units used in this specification are international standard units, and all numerical values ​​and ranges appearing in this invention should be understood to include systematic errors that are unavoidable in industrial production.

[0154] In this specification, the word "may" has two meanings: to perform a certain process and not to perform a certain process.

[0155] In this specification, references to "some specific / preferred embodiments," "other specific / preferred embodiments," "implementation," etc., refer to specific elements (e.g., features, structures, properties, and / or characteristics) related to that embodiment, which are included in at least one of the embodiments described herein and may or may not be present in other embodiments. Furthermore, it should be understood that these elements may be combined in any suitable manner in various embodiments.

[0156] In this specification, "optional" and "optionally" mean that the events or circumstances described below may or may not occur, and the description includes both cases where the events or circumstances occur and cases where the events or circumstances do not occur.

[0157] In this specification, the range of values ​​referred to as "value A to value B" refers to the range including the endpoint values ​​A and B.

[0158] As used herein, the term “and / or” covers all combinations of items connected by the term and should be regarded as if each combination had been listed separately herein. For example, “A and / or B” covers “A,” “A and B,” and “B.” For example, “A, B, and / or C” covers “A,” “B,” “C,” “A and B,” “A and C,” “B and C,” and “A and B and C.”

[0159] As used in this article, “containing,” “having,” or “including” includes “containing,” “mainly composed of,” “substantially composed of,” and “composed of”; “mainly composed of,” “substantially composed of,” and “composed of” are subordinate concepts of “containing,” “having,” or “including.”

[0160] When the term "comprising" is used herein to describe a protein or nucleic acid sequence, the protein or nucleic acid may consist of the stated sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, while still possessing the activities described in this invention. Furthermore, those skilled in the art will understand that the methionine encoded by the start codon at the N-terminus of a polypeptide may be retained in certain practical situations (e.g., when expressed in a specific expression system) without substantially affecting the polypeptide's function. Therefore, when describing a specific polypeptide amino acid sequence in this specification and claims, although it may not contain the methionine encoded by the start codon at the N-terminus, the sequence containing that methionine is still included, and correspondingly, its encoding nucleotide sequence may also contain the start codon; and vice versa.

[0161] In this invention, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are all widely used terms and routine procedures in their respective fields. For example, the standard recombinant DNA and molecular cloning techniques used in this invention are well known to those skilled in the art and are described more fully in the following literature: Sambrook, Joseph Frank et al. “Molecular Cloning: A Laboratory Manual.” (2001). (Hereinafter referred to as “Sambrook”). Meanwhile, to better understand this invention, definitions and explanations of relevant terms are provided below.

[0162] In this invention, the term "peroxidase" generally refers to a class of biological enzymes that can catalyze the biotin-phenol reaction under the activation of hydrogen peroxide, thereby enabling biotin to attach to a target protein. A representative peroxidase is ascorbate peroxidase (APEX). APEX is an engineered derivative of plant-derived ascorbate peroxidase. In some embodiments of this invention, the "peroxidase" can catalyze the single-electron oxidation of tetrazine-phenol to tetrazine phenoxy groups without the influence of hydrogen peroxide.

[0163] In this invention, the term "Hi-APEX" is an abbreviation for "H2O2-independent APEX", also known as "hydrogen peroxide-independent APEX".

[0164] In this invention, the term "TP" or "tetraazine-phenol" refers to methyltetraazine phenol, a functional organic compound fusing a tetraazine and a phenolic hydroxyl group, with CAS number 58884-35-8, molecular formula C9H8N4O, and molecular weight 188.2. It is the key small molecule probe used in this invention. Tetraazine is a six-membered heterocycle containing four nitrogen atoms and is the key functional group for achieving H2O2-independent catalysis; phenol is the traditional substrate moiety of APEX2.

[0165] In this invention, the term "IEDDA" refers to the inverse electron demand Diels-Alder reaction, a highly efficient bioorthogonal click chemistry reaction. Specifically, it refers to the reaction between a tetrazine (located on the labeled molecule) and a TCO (trans-cyclooctene, located on the biotin reporter molecule). This reaction is fast, highly selective, and requires no metal catalyst.

[0166] In this invention, the term "TCO-Biotin" refers to trans-cyclooctene-biotin, which is a reporter molecule in the IEDDA reaction used to introduce a biotin tag onto a TP-tagged molecule.

[0167] In this invention, the term "spatial multi-omics" refers to a technique for simultaneously analyzing multiple types of biomolecules (such as proteins and RNA) at a specific spatial location (such as organelles or protein complexes).

[0168] In this invention, the term "protein interaction" or "interaction" refers to the interaction between proteins that lead to changes in protein active conformation and signal transduction, also known as protein-protein interaction (PPI). Common protein interactions in the art include the interaction between polypeptide ligands and proteases, and between polypeptide hormones and receptors.

[0169] In this invention, the term "interacting protein" refers to a protein that participates in the interaction.

[0170] In this invention, the term "neighboring protein" refers to a protein located near the interaction region that may interact with a known protein. The two or more proteins involved in the interaction may be known or unknown. The method of this invention can be used to label known interacting proteins or to label unknown proteins that may interact with known proteins.

[0171] In this invention, the term "neighboring RNA" refers to RNA located near the interaction region that may interact with a known protein. The interacting protein, RNA, or other proteins and RNA may be known or unknown. The method of this invention can be used to label known interacting protein-RNA, or to label unknown RNA that may interact with a known protein.

[0172] In this specification, the term "conserved amino acid substitution" refers to the substitution of an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, and include those with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), non-polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan), β-branched chains (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine).

[0173] In this specification, the terms "sequence identity" or "percentage of identity" in comparisons of two nucleic acids or peptides refer to the percentage of identical sequences or identical sequences when compared and aligned using nucleotide or amino acid residue sequence comparison algorithms or by visual inspection to achieve the highest possible correspondence. In other words, the identity of a nucleotide or amino acid sequence can be defined using a ratio that represents the proportion of identical nucleotides or amino acids in the total number of nucleotides or amino acids in the aligned portion, assuming the maximum number of identical nucleotides or amino acids and omitting gaps as needed.

[0174] The technical solution of the present invention will be described in detail below:

[0175] Existing technologies (such as APEX2 and HRP) all rely on exogenous H2O2 to activate phenolic substrates. However, the presence of H2O2 can lead to cytotoxicity and affect in vivo applications. To address these issues, the inventors, through extensive research, discovered and demonstrated that TP can be used as a probe in APEX2-based proximity labeling methods, directly catalyzed by APEX2 in the absence of exogenous H2O2, to label neighboring proteins or RNA (e.g., ...). Figure 1 (As shown). This represents a fundamental breakthrough from "H2O2 dependence" to "H2O2 independence". Furthermore, it was discovered that using TP as a probe and HRP, proximity labeling can also be achieved without relying on H2O2.

[0176] Furthermore, by eliminating exogenous H2O2, this invention achieves for the first time APEX2-mediated proximity labeling directly within a living animal (see this invention). Figure 4 and Figure 5 This is something that no existing peroxidase-based PL technology can achieve, greatly expanding the application scope of the technology.

[0177] Meanwhile, this technical solution avoids the oxidative stress introduced by H2O2, and therefore can be used to study biological processes that are sensitive to redox balance, such as ferroptosis and stress particle formation, without the labeling method itself interfering with the biological results, thus obtaining more realistic and reliable data.

[0178] Furthermore, this technique utilizes the property of TP radicals to label both proteins and RNA, combined with efficient IEDDA bioorthogonal chemistry, to achieve simultaneous spatial proteomics and transcriptomics (spatial multi-omics) analysis. Simultaneously, due to its low toxicity and termination capability, it can be used for "pulse-chase" experiments to dynamically study protein transport processes (such as secretomics), which is difficult to achieve with traditional APEX2.

[0179] Meanwhile, this invention, through in vitro biochemical experiments and point mutagenesis studies, revealed the molecular mechanism by which TP achieves H2O2-independent labeling. The results show that the His42 residue in the APEX2 active site is crucial for the H2O2-independent catalysis of TP, and the tetrazine group in the TP molecule is the key structure for generating this unique activity (see this invention). Figure 3 This provides ideas for a completely new probe design.

[0180] First aspect

[0181] This invention provides a method for labeling interacting proteins, neighboring proteins, and / or neighboring RNA, the method comprising the following steps:

[0182] S1) The peroxidase is linked to protein A, which participates in the interaction, or to element A, which expresses protein A, to obtain element A' that expresses peroxidase;

[0183] S2) The peroxidase element A' is contacted with tetrazine-phenol, thereby catalyzing the attachment of tetrazine-phenol (TP) to other neighboring proteins and / or other neighboring RNAs adjacent to protein A.

[0184] In some embodiments, the peroxidase includes horseradish peroxidase (HRP) and ascorbate peroxidase (APEX). In some exemplary embodiments, the HRP comprises the sequence shown in SEQ ID NO:6, or comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity with the sequence shown in SEQ ID NO:6. In some exemplary embodiments, the APEX comprises the sequence shown in SEQ ID NO:1.

[0185] Building upon the existing APEX, the inventors have further discovered that the unique tetrazine group in the TP molecule is key to achieving H2O2-independent labeling. This group may interact specifically with the APEX2 active site, potentially forming an "H2O2-like" intermediate, thereby directly generating phenoxy radicals with the participation of His42. Therefore, those skilled in the art will recognize that one or more nucleotide or amino acid mutations can be introduced without altering the functional properties of a given nucleic acid or protein.

[0186] Mutations can be introduced using standard molecular biology techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis, to produce variants. For example, one or more amino acid substitutions, additions, or deletions can be made without altering the functional properties of the reference protein. When comparing a reference protein with a putative variant, amino acid similarity can be considered in addition to amino acid identity at corresponding positions in the amino acid sequence. "Amino acid similarity" refers to the amino acid identity and conserved amino acid substitutions in the putative variant compared to the corresponding amino acid positions in the reference protein.

[0187] Based on the above research, in some optional embodiments of the present invention, the peroxidase further includes an APEX variant comprising a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity with the sequence shown in SEQ ID NO:1.

[0188] In some specific embodiments, the APEX variant retains the catalytically active site His42 corresponding to the sequence shown in SEQ ID NO:1, and is capable of catalyzing the conversion of tetrazine-phenol to tetrazine-phenoxy without relying on exogenous hydrogen peroxide. "Exogenous" refers to the addition of hydrogen peroxide to the cells, organelles, or organisms that are already present, or the generation of additional hydrogen peroxide by modification.

[0189] Furthermore, the APEX variant retains the sites Arg38 (R38), Phe145 (F145), Trp179 (W179), and Tyr235 (Y235) corresponding to the sequence shown in SEQ ID NO:1, in order to retain the catalytic performance of the APEX2 variant.

[0190] In some exemplary embodiments, the APEX variant may be a sequence corresponding to one or more mutations as shown in SEQ ID NO:1, namely F41A, H169A, and P34A. Through these mutations, the APEX variant can still catalyze the conversion of tetrazine-phenol to tetrazine-phenoxy without relying on exogenous hydrogen peroxide.

[0191] In some specific embodiments, element A is selected from organelles, cells, or combinations thereof. In some embodiments, element A is derived from animals, plants, microorganisms, etc.; animals include humans, mice, rats, monkeys, dogs, pigs, sheep, cattle, cats, chickens, ducks, geese, etc.; microorganisms include *Escherichia coli*, *Bacillus subtilis*, mycobacteria, etc.; plants include *Arabidopsis thaliana*, tobacco, rice, corn, etc. In some embodiments, the organelles include mitochondria, the nucleus, the Golgi apparatus, the endoplasmic reticulum, mitochondria, or combinations thereof.

[0192] In some specific implementations, step S1 includes at least one of the following:

[0193] a) Peroxidase is expressed on element A, thereby attaching the peroxidase to element A;

[0194] b) Fusing the peroxidase to protein A, thereby attaching the peroxidase to element A;

[0195] c) Contacting the protein A or element A with an antibody crosslinked with the peroxidase, wherein the antibody specifically binds to the protein A, thereby linking the peroxidase to the protein A or element A;

[0196] d) Contacting the protein A or element A, a first antibody that specifically binds to the protein A, with a second antibody that is cross-linked with the peroxidase, thereby linking the peroxidase to the protein A or element A, wherein the second antibody specifically recognizes the first antibody.

[0197] In some exemplary embodiments, protein A includes proteins such as mGPx4 and G3BP1 that can interact with other proteins or RNA.

[0198] In some optional implementations, the method of detecting antibody-antigen interactions using APEX or its variants as markers can be a method known in the art, for example, see Li X, Zhou J, et al. Defining ProximityProteome of Histone Modifications by Antibody-mediated Protein A-APEX2Labeling. Genomics Proteomics Bioinformatics. 2022 Feb;20(1):87-100. doi:10.1016 / j.gpb.2021.09.003. Epub 2021 Sep 30. etc.

[0199] Using the above method, the substrate tetrazine-phenol can be oxidized to form phenoxy groups without relying on exogenous H2O2. Therefore, the present invention can perform efficient, low-toxicity, and high spatiotemporal resolution proximity labeling without relying on exogenous H2O2, and realize multi-dimensional spatial multi-omics analysis from in vitro cells to living animals.

[0200] Second aspect

[0201] This invention provides a method for analyzing protein-protein interactions or protein-RNA interactions, the method comprising:

[0202] The methods described above are used to label interacting proteins, neighboring proteins, and / or neighboring RNAs;

[0203] Biotin is linked to a protein or RNA labeled with tetrazine-phenol using trans-cyclooctene-biotin (TCO-Biotin).

[0204] Enrichment of biotin-labeled proteins or RNA using streptavidin magnetic beads;

[0205] Identification and analysis of the enriched biotin-labeled proteins or RNA.

[0206] In some implementations, the enriched biotin-labeled proteins or RNAs are analyzed and identified using methods such as LC-MS / MS, Western blot, DNA-seq, RNA-seq, or RT-qPCR.

[0207] In some specific embodiments, the step of linking biotin to a tetrazine-phenol-labeled protein or RNA in the method provided in this invention does not require a cytotoxic copper catalyst.

[0208] Third aspect

[0209] The present invention provides a composition for labeling interacting proteins, neighboring proteins, and / or neighboring RNA, comprising i) and optionally ii), iii), and / or iv).

[0210] i) Tetraazine-phenol;

[0211] ii) Ascorbate peroxidase (APEX), its localization fusion protein, its antibody cross-linked product, nucleic acid molecule encoding it, or expression vector containing said nucleic acid molecule;

[0212] iii) APEX variants, their localization fusion proteins, their antibody cross-links, nucleic acid molecules encoding them, or expression vectors containing said nucleic acid molecules;

[0213] iv) Horseradish peroxidase (HRP), its localization fusion protein, its antibody cross-linked form, nucleic acid molecule encoding it, or expression vector containing said nucleic acid molecule.

[0214] In some optional embodiments of the invention, the peroxidase further includes an APEX variant comprising a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity with the sequence shown in SEQ ID NO:1.

[0215] In some specific embodiments, the APEX variant retains the catalytically active site His42 corresponding to SEQ ID NO:1, and is capable of catalyzing the conversion of tetrazine-phenol to tetrazine-phenoxy without the dependence on exogenous hydrogen peroxide.

[0216] Furthermore, the APEX variant retains the sites corresponding to Arg34, Phe145, Trp179, and Tyr235 in the sequence shown in SEQ ID NO:1 to retain the catalytic performance of the APEX2 variant.

[0217] In some exemplary embodiments, the APEX variant may be a sequence corresponding to one or more mutations as shown in SEQ ID NO:1, namely F41A, H169A, and P34A. Through these mutations, the APEX variant can still catalyze the conversion of tetrazine-phenol to tetrazine-phenoxy without relying on exogenous hydrogen peroxide.

[0218] In some exemplary embodiments, the localization fusion protein comprises the sequence shown in SEQ ID NO:2.

[0219] In some exemplary embodiments, the expression vector includes the pLX304 plasmid. Further, based on the intracellular localization of the protein, a corresponding signal peptide is selected to target a specific location. In some optional embodiments, the expression vector also includes the mitochondrial targeting signal MTS, the nuclear export signal NES, the endoplasmic reticulum retention signal KDEL, and the stress granule protein G3BP1, etc.

[0220] Fourth aspect

[0221] This invention provides the use of tetrazine-phenol in the following i) and / or ii):

[0222] i) Prepare kits for labeling interacting proteins, neighboring proteins, and / or neighboring RNA;

[0223] ii) Prepare kits for analyzing protein-protein interactions or protein-RNA interactions.

[0224] In some embodiments, horseradish peroxidase (HRP), ascorbate peroxidase (APEX), and / or variants thereof. In some alternative embodiments, the APEX variant comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or higher identity with the sequence shown in SEQ ID NO:1.

[0225] In some specific embodiments, the APEX variant retains the catalytically active site His42 corresponding to the sequence shown in SEQ ID NO:1, and is capable of catalyzing the conversion of tetrazine-phenol to tetrazine-phenoxy without the dependence on exogenous hydrogen peroxide.

[0226] Furthermore, the APEX variant retains sites Arg34, Phe145, Trp179, and Tyr235 corresponding to the sequence shown in SEQ ID NO:1, in order to retain the catalytic performance of the APEX2 variant.

[0227] In some exemplary embodiments, the APEX variant may be a sequence corresponding to one or more mutations as shown in SEQ ID NO:1, namely F41A, H169A, and P34A. Through these mutations, the APEX variant can still catalyze the conversion of tetrazine-phenol to tetrazine-phenoxy without relying on exogenous hydrogen peroxide.

[0228] In some optional embodiments, the kit may also contain trans-cyclooctene-biotin (TCO-Biotin), streptavidin magnetic beads, etc.

[0229] Example

[0230] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0231] Composition and steps of the technical solution

[0232] 1. Core components:

[0233] a. Labeling enzymes: HRP, APEX2, or variants thereof. This enzyme is expressed at a specific location within the cell or in a complex with a specific protein by fusing it with a target protein or localization signal peptide (e.g., mitochondrial targeting signal MTS, nuclear export signal NES, endoplasmic reticulum retention signal KDEL, stress granule protein G3BP1, etc.) through genetic engineering techniques.

[0234] b. Probe molecule: Tetraazine-phenol (TP).

[0235] c. Reporter molecule and click chemistry reagent: trans-Cyclooctene-Biotin (TCO-Biotin). Used for biotinylation of TP-labeled molecules via the IEDDA reaction.

[0236] d. Enrichment and analysis reagents: Streptavidin magnetic beads for enriching biotinylated molecules; and reagents required for subsequent proteomics (e.g., LC-MS / MS) or transcriptomics (e.g., RNA-seq) analyses.

[0237] 2. Experimental Procedure:

[0238] a. Preparation of cell or in vivo models:

[0239] A fusion construct that stably or transiently expresses APEX2 and the target protein in cells.

[0240] For in vivo applications, APEX2 constructs can be delivered to target tissues (such as the mouse hippocampus) via adeno-associated virus (AAV), or mouse xenograft tumor models or related APEX2 transgenic mice can be constructed using tumor cells that stably express APEX2 fusion protein.

[0241] b. Hi-APEX marking:

[0242] Add the TP probe directly to the cell culture medium or the target tissue in vivo. This step involves no addition of exogenous H2O2. Incubate at 37°C (cells) or in vivo physiological temperature for a period of time (usually 1-30 minutes) to allow the labeling reaction to occur.

[0243] c. Reaction termination and sample collection:

[0244] Remove the TP-containing culture medium and add quenching buffer containing sodium ascorbate (10 mM) and Trolox (5 mM) to terminate the reaction. Collect cells or tissues and lyse them.

[0245] d. IEDDA Click Chemical Reaction:

[0246] Add TCO-Biotin to cell or tissue lysates and incubate at room temperature for 10–30 minutes. This step replaces the copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction required in the traditional APEX2 method based on alkyne-phenol (AP). The IEDDA reaction is faster, more efficient, and does not require a cytotoxic copper catalyst.

[0247] e. Enrichment and Detection:

[0248] Biotinylated proteins or RNA were enriched by co-incubating streptavidin magnetic beads with lysis buffer that had undergone the IEDDA reaction. For proteins, detection was performed by Western blotting (using streptavidin-HRP) or proteomics analysis by liquid chromatography-tandem mass spectrometry (LC-MS / MS). For RNA, specific transcripts were validated by qRT-PCR or transcriptomics analysis was performed by high-throughput sequencing (RNA-seq) (Hi-APEX-seq).

[0249] Example 1: Using Hi-APEX to perform non-interfering spatial protein analysis on stress granules (SG). Plasmic and Transcriptome Mapping

[0250] 1. Experimental Objective

[0251] This embodiment aims to demonstrate the application of Hi-APEX technology in stress particle (SG) research and to verify its technical effects:

[0252] This study achieves specific labeling of stress granules, a dynamic organelle sensitive to oxidative stress, without the use of exogenous H2O2. It demonstrates that the Hi-APEX labeling process itself does not induce stress granule formation, thus enabling precise spatial multi-omics analysis under their natural stress state. Simultaneous acquisition of proteomic and transcriptomic information of stress granules is demonstrated, and comparisons with existing techniques prove Hi-APEX's advantages in identifying authentic SG components.

[0253] 2. Experimental Materials

[0254] Cell line: HEK293T cells (purchased from ATCC).

[0255] plasmid:

[0256] APEX2-G3BP1: APEX2 is fused with the SG core protein G3BP1 (the fusion protein sequence is shown in SEQ ID NO:2) for targeting SG.

[0257] APEX2-NES: APEX2 was fused with the nuclear output signal (fusion protein sequence as shown in SEQ ID NO:3) as a control for cytoplasmic background.

[0258] Key reagents:

[0259] TP probe: Prepare a 500 mM stock solution with DMSO before use and store at -80°C. The working concentration is 500 μM.

[0260] TCO-Biotin (trans-cyclooctene-biotin): used in IEDDA click chemistry, working concentration 20 μM.

[0261] Sodium arsenite (NaAsO2): Used to induce oxidative stress and SG formation, with a working concentration of 500 μM.

[0262] Streptavidin magnetic beads: used to enrich biotinylated molecules.

[0263] 3. Experimental Procedure

[0264] a. Cell culture and construction of stable cell lines

[0265] HEK293T cells were cultured in 10 cm cell culture dishes using DMEM complete medium (containing 10% FBS and 1% Penicillin-Streptomycin) in an incubator at 37°C and 5% CO2.

[0266] Using the pMD2G-psPAX2 lentiviral packaging system, APEX2-G3BP1 and APEX2-NES plasmids were transfected into HEK293T cells, respectively, to obtain HEK293T cells containing APEX2-G3BP1 and APEX2-NES plasmids. The cells were then screened for at least one week using DMEM complete medium containing 2 μg / mL of blastcinin S to obtain stable expression cell lines.

[0267] b. Stress granule induction and Hi-APEX labeling

[0268] Once the cells containing APEX2-G3BP1 and APEX2-NES have grown to 80% confluence and become stable, replace the cell culture medium with fresh DMEM complete medium.

[0269] ① Experimental group: Sodium arsenate was added to the culture medium to a final concentration of 500 μM and incubated in an incubator for 55 minutes to induce SG formation.

[0270] Note: During the last 5 minutes of sodium arsenate treatment, TP probe was added to the culture medium to a final concentration of 500 μM (no exogenous H2O2 was added throughout the process).

[0271] ② Control group 1 (no stress): APEX2-G3BP1 cells, without sodium arsenate treatment, were directly incubated with 500 μMTP for 5 minutes.

[0272] ③ Control group 2 (no enzyme): Wild-type HEK293T cells (not expressing APEX2) were treated with the same sodium arsenate and TP as the experimental group.

[0273] c. Reaction termination and sample collection

[0274] Five minutes after labeling, the culture medium was quickly removed, and the cells were washed twice with pre-cooled quenching buffer (10 mM sodium ascorbate, 5 mM MTrolox in PBS) to terminate the free radical reaction. After washing once with PBS, the cells were collected with a cell scraper and centrifuged at 1000 × g for 3 minutes at 4°C to collect the cell pellet.

[0275] d. IEDDA click chemistry and sample lysis

[0276] Resuspend the cell pellet in RIPA lysis buffer and lyse on ice for 30 minutes. Centrifuge at 20,000 × g for 10 minutes at 4°C, collect the supernatant, and determine the protein concentration using the BCA method, adjusting the concentration to 2 mg / mL. Add TCO-Biotin to each lysis buffer to a final concentration of 20 μM and incubate at room temperature on a rotary mixer for 30 minutes to complete the IEDDA reaction, linking the biotin tag to the TP-labeled molecule.

[0277] e. Streptavidin enrichment and omics analysis

[0278] Proteomics sample preparation: A portion of the lysate from the click reaction was incubated overnight at 4°C with streptavidin beads. Subsequently, the samples were washed with stringent, digested with membrane proteases, and the peptides were collected for LC-MS / MS (liquid chromatography-tandem mass spectrometry) analysis.

[0279] Transcriptomics (Hi-APEX-seq) sample preparation: A portion of the cell pellet was used to extract total RNA using TRIzol reagent. The purified RNA was reacted with 20 μM TCO-Biotin at room temperature for 10 minutes. After the reaction, the RNA was purified again, and 100 μg of RNA was incubated with streptavidin beads to enrich the biotin-labeled RNA. After elution, INPUT and ENRICH libraries were prepared separately for high-throughput sequencing.

[0280] 4. Experimental Results and Analysis

[0281] 4.1 Assessment of SG formation status: By comparing the number and size of SGs in cells treated with sodium arsenate only and those treated with sodium arsenate + Hi-APEX, no significant difference was found. This demonstrates that the TP labeling process of Hi-APEX itself does not additionally promote SG assembly, thus meeting the prerequisite for "native state" analysis of SGs.

[0282] 4.2 Hi-APEX for precise identification of the SG proteome

[0283] Mass spectrometry data quality: By comparing the proteomic data of the experimental group (APEX2-G3BP1 + sodium arsenite + TP) with two control groups (APEX2-NES + sodium arsenite + TP; wild-type cells + sodium arsenite + TP), the inventors identified 186 high-confidence SG-enriched proteins (p < 0.05).

[0284] Specificity and accuracy, such as Figure 12 As shown:

[0285] Gene ontology analysis revealed that these proteins were significantly enriched in pathways such as stress granule assembly and RNA binding. Compared to existing high-resolution techniques (such as µMAP and BRET-ID), Hi-APEX identified a comparable or higher proportion of known SG proteins and RNA-binding proteins. These proteins exhibited a significantly higher phase-separation tendency (P-score), consistent with the biological characteristics of SG.

[0286] 4.3 Hi-APEX-seq reveals the SG core transcriptome

[0287] Hi-APEX successfully mapped the proteome and transcriptome of SG simultaneously under sodium arsenate-induced stress conditions without H2O2 interference. The transcriptome enrichment effect was as follows: Figure 7 and Figure 8 As shown: Hi-APEX-seq analysis identified 155 transcripts significantly enriched in SG, and identified new SG components such as SPDL1 and CEP170, obtaining a more realistic and accurate molecular map of SG. G3BP1 mRNA itself was one of the most enriched transcripts, demonstrating the consistency of the method. Compared with the CAP-seq method, which requires additional optogenetic stimulation, Hi-APEX-seq captured a higher proportion of known SG core transcripts. The sequencing map (IGV) clearly shows ( Figure 13 Key mRNAs such as DCP2 and G3BP1 showed significantly higher read coverage in the ENRICH group than in the INPUT group, while the housekeeping gene GAPDH showed no enrichment in mRNA.

[0288] 5. Conclusion

[0289] This embodiment demonstrates that Hi-APEX successfully achieves low-interference, high-specificity spatial multi-omics analysis of stress granules, a sensitive organelle, by using TP probes and an H2O2-independent labeling mechanism. It not only avoids the toxicity and interference introduced by H2O2 in traditional methods, enabling the capture of more accurate molecular maps, but also possesses the unique ability to simultaneously analyze proteins and RNA. This provides a powerful and reliable new tool for studying other redox-sensitive biological processes.

[0290] Example 2: Using Hi-APEX to map the mGPx4 interactome in a living tumor model and reveal its role in ferroptosis New regulatory factors in death

[0291] 1. Experimental Objective

[0292] This embodiment aims to demonstrate the application of Hi-APEX technology at the in vivo level and verify its following technical effects:

[0293] This study demonstrates that Hi-APEX can achieve APEX2-mediated proximity labeling in tumor tissues of living animals via simple probe injection. In ferroptosis, a physiological and pathological process highly sensitive to H2O2, the dynamic interaction network of the target protein (mGPx4) was successfully mapped, a task that traditional APEX2 could not accomplish. Through in vivo interoperomics, novel mGPx4 interacting proteins and their biological functions in ferroptosis were discovered and validated.

[0294] 2. Experimental Materials and Animals

[0295] Cell line: HT1080 human fibrosarcoma cells (purchased from ATCC).

[0296] Plasmid: pLX304-mGPx4-APEX2 (used to express the mitochondrial-targeted GPx4 and APEX2 fusion protein, the sequence of which is shown in SEQ ID NO:4).

[0297] Key reagents:

[0298] TP probe: Prepare a 5 mM stock solution using PBS containing 5% Tween-80.

[0299] RSL3: A GPx4 inhibitor used to induce ferroptosis; prepared with DMSO before use.

[0300] Laboratory animals: Male BALB / c nude mice, 3-4 weeks old, housed in an SPF-grade environment.

[0301] 3. Experimental procedures (e.g.) Figure 4 and Figure 5 (As shown)

[0302] 3.1 Construction of stable cell lines and tumor models

[0303] A pool of HT1080 cells stably expressing mGPx4-APEX2 was obtained by infecting the cells with the pMD2G-psPAX2 lentiviral system and selecting with Blastidin S. Wild-type HT1080 cells were used as a control.

[0304] Mice were randomly divided into groups. 1×10^6 cells stably expressing mGPx4-APEX2 or wild-type cells were injected subcutaneously into the left and right hind limbs of each mouse to construct a xenograft tumor model.

[0305] 3.2 In vivo Hi-APEX labeling

[0306] When the tumor grows to approximately 100 mm 3 Live marking will be performed approximately 2 weeks later.

[0307] ① Experimental group: Tumor-bearing mice were lightly anesthetized and injected with insulin via multiple sites into the mGPx4-APEX2 tumor with 5 mM TP solution. The total injection volume was 100 μL (TP was injected directly in vivo, and exogenous H2O2 was neither required nor possible).

[0308] ② Control group: An equal volume of TP solution was injected into the wild-type tumor.

[0309] Twenty-four hours later, an intratumoral TP injection was repeated to enhance the labeling signal.

[0310] One hour after the second injection, the mice were euthanized, the tumor tissue was quickly removed, and immediately flash-frozen in liquid nitrogen before being transferred to -80°C for storage.

[0311] 3.3 Hi-APEX markers under in vitro ferroptosis conditions (as a comparison)

[0312] HT1080 cells stably expressing mGPx4-APEX2 were plated.

[0313] Two groups were set up: the Basal group (basic conditions) and the Ferroptosis group (ferroptosis conditions). The Ferroptosis group was treated with RSL3 at a working concentration of 150 nM for 3 hours.

[0314] At the end of the treatment, 500 μM TP was added to both groups of cells and incubated for 5 minutes. Then, quenching, collection, lysis and IEDDA reaction were performed according to the steps in Example 1.

[0315] 3.4 Sample processing and proteomics analysis

[0316] Frozen tumor tissue or cell samples were ground and lysed in RIPA lysis buffer. The lysis buffer was reacted with 20 μM TCO-Biotin at room temperature for 30 minutes. The lysate was enriched by streptavidin magnetic beads, washed with stringent and digested with membrane proteases, and then analyzed by LC-MS / MS in DIA (data-independent acquisition) mode.

[0317] 4. Experimental Results and Analysis

[0318] 4.1 Hi-APEX enables efficient labeling in living tumor tissues

[0319] Western Blot Validation: Streptavidin blotting analysis was performed on the tumor tissue lysate. Results are as follows... Figure 14 The results showed that abundant and widespread biotinylated protein signals were detected only in the mGPx4-APEX2 tumor group injected with TP. In contrast, almost no background signal was observed in the wild-type tumor group. This directly demonstrates that Hi-APEX can still achieve APEX2-dependent specific labeling in the complex in vivo tumor microenvironment.

[0320] 4.2 Plotting the cell-to-live mGPx4 interactionome

[0321] Since ferroptosis itself is driven by lipid peroxidation, conventional APEX2 H2O2 treatment can directly interfere with or even induce this process.

[0322] Intracellular dynamic interactionome such as Figure 9 As shown, under basal conditions, 120 interacting proteins were identified, of which over 97% were mitochondrial proteins. The Hi-APEX method of this invention successfully revealed a dynamic interaction network between mGPx4 and a large number of mitochondrial and cytoplasmic proteins under RSL3-induced ferroptosis conditions: the number of interacting proteins significantly increased to 675, including a large number of cytoplasmic proteins enriched in pathways such as protein phosphorylation and ubiquitination, suggesting that mitochondrial-cytoplasmic communication intensifies during ferroptosis, and membrane permeability may be altered.

[0323] In vivo interactome: From proteomics data of in vivo tumor samples, the inventors identified 228 high-confidence mGPx4 neighboring proteins. GO cellular component analysis showed that these proteins were significantly enriched in locations such as "mitochondria" and "mitochondrial matrix," demonstrating the spatial specificity of the labeling. Figure 15 As shown, the list includes interacting proteins validated in cell models, such as ETHE1, as well as novel interacting proteins specific to the tumor microenvironment, such as the reactive oxygen species regulator ROMO1.

[0324] 4.3 Functional validation of the novel interacting protein TRMT61B

[0325] Network analysis and screening, results as follows Figure 16 As shown: STRING network analysis of top interacting proteins identified under ferroptosis conditions revealed a module closely related to mitochondrial translation and RNA modification, which contains m 1 A RNA methyltransferase TRMT61B.

[0326] Co-IP verification results are as follows: Figure 17 As shown: mGPx4, tagged with the Flag, was overexpressed in HT1080 cells, and immunoprecipitation was performed using an anti-Flag antibody. Western blot results showed that, under RSL3 treatment, endogenous TRMT61B could specifically bind to mGPx4, validating the reliability of the Hi-APEX screening results at the protein level.

[0327] Results of gain-of-function experiments, such as Figure 10 As shown, functional validation confirmed that the interacting protein TRMT61B promotes ferroptosis: TRMT61B was overexpressed in HT1080 cells and treated with gradient concentrations of RSL3. MTS cell viability assays showed that cells overexpressing TRMT61B were more sensitive to RSL3-induced ferroptosis (IC50) compared to wild-type cells. 50 (Significantly reduced). This indicates that TRMT61B is not only an interacting protein of mGPx4, but also functionally promotes the ferroptosis process, revealing a new link between mitochondrial RNA epigenetic regulation and ferroptosis.

[0328] 5. Conclusion

[0329] This embodiment demonstrates the unique and irreplaceable value of Hi-APEX technology in in vivo applications and the study of redox-sensitive processes. Through simple intratumoral injection of TP, the inventors successfully mapped the interaction network of mGPx4 in living tumors, and based on this, discovered and validated TRMT61B as a novel ferroptosis promoter. The entire research paradigm clearly shows that Hi-APEX expands the scope of APEX proximity marker research from cultured cells to living animals, while also enabling the study of dynamic biological processes previously inaccessible due to sensitivity to H2O2.

[0330] This invention, through the two progressive embodiments described above, systematically verifies the core advantages and disruptive applications of Hi-APEX technology. Example 1, at the cellular level, using stress granules sensitive to oxidative stress as a model, demonstrates that Hi-APEX can achieve high-fidelity spatial multi-omics analysis of the "native state" of dynamic organelles without introducing exogenous H2O2 interference. Example 2 further expands the application scenario to the in vivo level, mapping the interactionome of the key ferroptosis regulator mGPx4 in a tumor xenograft model and revealing its novel function, strongly confirming that Hi-APEX is the only technology capable of achieving efficient and specific proximity labeling within living animals and in H2O2-sensitive biological processes. These two examples together constitute a complete chain of evidence from in vitro to in vivo, from principle verification to functional discovery, highlighting the superior performance and broad application prospects of this invention compared to existing technologies.

[0331] Example 3

[0332] 1. Experimental Objective

[0333] This embodiment aims to demonstrate the exploration of reaction mechanisms and active centers in Hi-APEX technology research.

[0334] 2. Experimental Materials

[0335] Purified proteins of APEX2 and its variants

[0336] BP (Biotin-phenol) probe: Prepared as a 500 mM stock solution using DMSO.

[0337] TP probe: Prepared as a 500 mM stock solution using DMSO.

[0338] BSA: Bovine serum albumin, used as the labeled protein, prepared with PBS before use.

[0339] 3. Experimental procedures (e.g.) Figure 4 and Figure 5 (As shown)

[0340] 3.1 Construction and in vitro purification of APEX2 and its variant proteins

[0341] Based on the preliminary research, we selected key residues that could potentially exert enzyme activity around the active site of APEX2. Specifically, we used PCR site-directed mutagenesis to introduce ten mutations into APEX2, namely P34A, R38A, F41A, H42A, P132A, F145A, H169A, F175A, W179A, and Y235A.

[0342] The proteins of 11 enzymes, including WT-APEX2, were purified in vitro using the Ni-NTA affinity purification method.

[0343] 3.2 In vitro Hi-APEX labeling

[0344] ① Experimental group: Add TP probe to a final concentration of 500 μM, 1 mM H2O2 and 1 μM APEX2 or its variant protein to a 4 mg / mL BSA solution.

[0345] ② Control group: The added H2O2 was replaced with PBS.

[0346] ③ Traditional APEX2 control group: The added TP probe was replaced with a BP probe.

[0347] After reacting at 37 degrees Celsius for 5 minutes, eight times the volume of ice-cold methanol was added to precipitate the protein and the reaction was stopped. The protein was then reconstituted according to the steps in Example 1 and the IEDDA reaction was carried out.

[0348] 3.3 Western blot was used to detect the label intensity.

[0349] The labeled protein samples were subjected to SDS-PAGE, transferred to a membrane, blocked, and then subjected to Western blot using SA-HRP (streptavidin-horseradish peroxidase conjugate). The labeling intensity was detected by the band strength and analyzed using ImageJ software.

[0350] 4. Experimental Results and Analysis

[0351] 4.1 H42 is a key residue for APEX2 to undergo Hi-APEX labeling.

[0352] The results of this experiment are as follows: Figure 19 The results showed that among the 10 mutated residues, mutations in R38, F145, W179, and Y235 resulted in the loss of APEX2's catalytic activity; mutations in P132 and F175 resulted in the loss of APEX2's ability to be catalyzed and promoted by H2O2; and the H42A mutation restored APEX2's dependence on hydrogen peroxide during Hi-APEX catalysis. Therefore, H42 is a key residue and active site for APEX2 to perform Hi-APEX labeling.

[0353] 5. Conclusion

[0354] This embodiment, through site-directed mutagenesis screening, revealed that histidine 42 (H42) is a key residue in the active site region of APEX2 that determines its ability to undergo Hi-APEX labeling. This discovery provides an important structural basis for a deeper understanding of the reaction mechanism of Hi-APEX technology and for optimizing its application.

[0355] Example 4

[0356] 1. Experimental Objective

[0357] This embodiment aims to evaluate the cytotoxicity of the TP probe and the positive control BP probe used in the Hi-APEX technology.

[0358] 2. Experimental Materials

[0359] Cell line: HEK293T cells.

[0360] Key reagents:

[0361] TP / BP probes: Probes related to the Hi-APEX technology to be tested.

[0362] H2O2: Positive control treatment.

[0363] MTS Cell Proliferation Detection Kit: Purchased from Promega, used for quantifying cell viability.

[0364] 3. Experimental Procedure

[0365] a. Cell plating

[0366] The cells to be tested were seeded at a density of 5000 cells per well in a 96-well plate and cultured overnight to allow them to adhere to the plate.

[0367] b. Cytotoxicity assay

[0368] Probe and H2O2 treatment: HEK293T cells were treated for 3 minutes with normal culture medium (negative control group), TP probe (experimental group 1), and H2O2 (experimental group 2), or BP probe and H2O2 together (experimental group 3).

[0369] The culture medium was then immediately replaced with fresh medium and cultured for 24 or 48 hours, and cell viability was detected using an MTS kit.

[0370] 4. Experimental Results and Analysis

[0371] The short-term cytotoxicity of the TP probe was assessed by comparing cell viability in different treatment groups (TP / BP / H2O2) with the control group. Results showed that compared to the negative control group (NC, 100%), the TP probe treatment group did not exhibit a significant decrease in cell viability at 24 and 48 hours, indicating that the TP probe had no significant cytotoxicity. Both the positive control H2O2 treatment group and the H2O2 / BP co-treatment group showed some degree of cell damage, demonstrating the oxidative damage to cells caused by traditional methods.

[0372] 5. Conclusion

[0373] The results of this embodiment are as follows: Figure 6 As shown, the TP probe did not significantly affect cell viability at either the 24-hour or 48-hour time points, demonstrating good biocompatibility. However, H2O2 treatment led to a significant decrease in cell viability. This indicates that the TP probe meets the safety requirements for subsequent long-term experiments.

[0374] Example 5

[0375] 1. Experimental Objective

[0376] This embodiment aims to demonstrate that Hi-APEX technology, due to its low toxicity and termination capability, can be used in "pulse-chase" experiments to dynamically study protein transport processes.

[0377] 2. Experimental Materials

[0378] Cell line: HEK293T cells (purchased from ATCC).

[0379] plasmid:

[0380] APEX2-KDEL: APEX2 is fused with the secretory signal peptide Igk and the endoplasmic reticulum resident sequence KDEL (the fusion protein sequence is shown in SEQ ID NO:5) for targeting the endoplasmic reticulum lumen.

[0381] Key reagents:

[0382] TP probe: Prepare a 500 mM stock solution with DMSO before use and store at -80°C. The working concentration is 500 μM.

[0383] TCO-Biotin (trans-cyclooctene-biotin): used in IEDDA click chemistry, working concentration 20 μM.

[0384] Streptavidin magnetic beads: used to enrich biotinylated molecules.

[0385] 3. Experimental Procedure

[0386] a. Cell culture and construction of stable cell lines

[0387] Following the steps in Example 1, a HEK293T cell line stably expressing APEX2-KDEL was obtained.

[0388] b. Hi-APEX's "pulse-chase" label

[0389] Once the cells containing APEX2-KDEL have grown to a stable state with 80% confluence, the cell culture medium is replaced with fresh serum-free DMEM medium containing 500 μM TP probe.

[0390] ① Experimental group: Performed according to the above method

[0391] ② Control group 2 (no enzyme): Wild-type HEK293T cells (not expressing APEX2) were treated with the same TP as the experimental group.

[0392] Five minutes after labeling, the culture medium was quickly removed and the cells were washed twice with pre-cooled quenching buffer (10 mM sodium ascorbate, 5 mM MTrolox in PBS) to terminate the free radical reaction.

[0393] The cell culture medium was replaced with fresh serum-free DMEM medium, and the cells were cultured for a period of time until the labeled protein was secreted into the medium.

[0394] c. Sample collection and IEDDA click chemistry

[0395] After culturing for another 18 hours, the culture medium was collected and concentrated to 2 mg / mL by ultrafiltration using a 10 kD ultrafiltration tube at 4°C. The IEDDA reaction was then performed as described in Example 1 to attach the biotin tag to the TP-labeled molecule.

[0396] d. Streptavidin enrichment and omics analysis

[0397] Proteomics sample preparation: A portion of the lysate from the click reaction was incubated overnight at 4°C with streptavidin beads. Subsequently, the samples were washed with stringent, digested with membrane proteases, and the peptides were collected for LC-MS / MS (liquid chromatography-tandem mass spectrometry) analysis.

[0398] 4. Experimental Results and Analysis

[0399] Mass spectrometry data quality: By comparing the proteomic data of the experimental and control groups, the inventors identified 728 high-confidence SG-enriched proteins (p < 0.05). Nearly 80% of these proteins are known secretory proteins, such as… Figure 18 As shown, by using Hi-APEX to pulse-label the ER cavity, followed by probe removal and tracking, 728 proteins secreted from the ER to the extracellular space were successfully identified, dynamically elucidating the protein secretion and transport process. This is extremely challenging for conventional APEX2 because the toxicity of H2O2 can disrupt normal cellular physiological activities.

[0400] 5. Conclusion

[0401] This embodiment successfully applied Hi-APEX technology to dynamically trace the intracellular protein transport process. It fully validated the high specificity and reliability of Hi-APEX technology combined with a pulse-chase experimental strategy in studying protein secretion pathways and dynamic transport processes. This technology provides a powerful new tool for elucidating the spatiotemporal dynamic mechanisms of secreted proteins.

[0402] Example 6

[0403] 1. Experimental Objective

[0404] This embodiment aims to demonstrate that Hi-APEX technology can also be compatible with HRP for proximity tagging.

[0405] 2. Experimental Materials

[0406] Commercially available HRP purified protein

[0407] BP (Biotin-phenol) probe: Prepared as a 500 mM stock solution using DMSO.

[0408] TP probe: Prepared as a 500 mM stock solution using DMSO.

[0409] Hydrogen peroxide: Prepare a 100 mM stock solution using PBS.

[0410] BSA: Bovine serum albumin, used as the labeled protein, prepared with PBS before use.

[0411] 3. Experimental Procedure

[0412] 3.1 In vitro Hi-APEX labeling

[0413] ① Experimental group: Add TP probe with a final concentration of 500 μM and HRP of 1 μM to a 4 mg / mL BSA solution.

[0414] ② Control group: HRP was replaced with PBS.

[0415] ③ Traditional HRP control group: The added TP probe was replaced with a BP probe, and 1mM H2O2 was added.

[0416] After reacting at 37 degrees Celsius for 5 minutes, eight times the volume of ice-cold methanol was added to precipitate the protein and the reaction was stopped. The protein was then reconstituted according to the steps in Example 1 and the IEDDA reaction was carried out.

[0417] 3.2 Western blot was used to detect the label intensity.

[0418] The labeled protein samples were subjected to SDS-PAGE, transferred to a membrane, blocked, and then subjected to Western blot using SA-HRP (streptavidin-horseradish peroxidase conjugate). The labeling intensity was detected by the intensity of the bands.

[0419] 4. Experimental Results and Analysis

[0420] 4.1 HRP can also replace APEX2 enzyme for Hi-APEX labeling.

[0421] The results of this experiment are as follows: Figure 20 The results show that replacing APEX2 with HRP still allows for traditional peroxidase-based proximity labeling, and HRP can also generate a clear labeling signal using TP probes without the addition of H2O2.

[0422] 5. Conclusion

[0423] This embodiment successfully verified the compatibility of Hi-APEX technology with horseradish peroxidase (HRP) through in vitro labeling experiments. This result expands the application scope of Hi-APEX technology, indicating that it is not limited to APEX2 enzymes but is also compatible with other peroxidases (such as HRP), providing more options for proximity labeling in different experimental systems.

[0424] The sequence involved in this invention:

[0425] SEQ ID NO:1APEX2 adjacent labeling enzyme amino acid sequence:

[0426] GKSYPTVSADYQDAVEKAKKKLRGFIAEKRCAPLMLRLAFHSAGTFDKGTKTGGPFGTIKHPAELAHSANNGLDIAVRLLEPLKAEFPILSYADFYQLAGVVAVEVTGGPKVPFHPGREDKPEP PPEGRLPDPTKGSDHLRDVFGKAMGLTDQDIVALSGGHTIGAAHKERSGFEGPWTSNPLIFDNSYFTELLSGEKEGLLQLPSDKALLSDPVFRPLVDKYAADEDAFFADYAEAHQKLSELGFADA

[0427] SEQ ID NO:2APEX2-G3BP1 fusion protein sequence:

[0428] G3BP1-APEX2- V5 :

[0429] MEKPSPLLVGREFVRQYYTLLNQAPDMLHRFYGKNSSYVHGGLDSNGKPADAVYGQKEIHRKVMSQNFTNCHTKIRHVDAHATLNDGVVVQVMGLLSNNNQALRRFMQTFVLAPEGSVANKFYVHNDIFRYQDEVFGGFVTEPQEESEEEVEEPEERQQTPEVVPDDSGTFYDQAVVSNDMEEHLEEPVAEPEPDPEPEPEQEPVSEIQEEKPEPVLEETAPEDAQKSSSPAPADIAQTVQEDLRTFSWASVTSKNLPPSGAVPVTGIPPHVVKVPASQPRPESKPESQIPPQRPQRDQRVREQRINIPPQRGPRPIREAGEQGDIEPRRMVRHPDSHQLFIGNLPHEVDKSELKDFFQSYGNVVELRINSGGKLPNFGFVVFDDSEPVQKVLSNRPIMFRGEVRLNVEEKKTRAAREGDRRDNRLRGPGGPRGGLGGGMRGPPRGGMVQKPGFGVGRGLAPRQGSGGGKSYPTVSADYQDAVEKAKKKLRGFIAEKRCAPLMLRLAFHSAGTFDKGTKTGGPFGTIKHPAELAHSANNGLDIAVRLLEPLKAEFPILSYADFYQLAGVVAVEVTGGPKVPFHPGREDKPEPPPEGRLPDPTKGSDHLRDVFGKAMGLTDQDIVALSGGHTIGAAHKERSGFEGPWTSNPLIFDNSYFTELLSGEKEGLLQLPSDKALLSDPVFRPLVDKYAADEDAFFADYAEAHQKLSELGFADA GKPIPNPLLGLDST

[0430] SEQ ID NO:3 APEX2-NES fusion protein sequence:

[0431] V5 -APEX2-NES:

[0432] M GKPIPNPLLGLDSTTGGKSYPTVSADYQDAVEKAKKKLRGFIAEKRCAPLMLRLAFHSAGTFDKGTKTGGPFGTIKHPAELAHSANNGLDIAVRLLEPLKAEFPILSYADFYQLAGVVAVEVTGGPKVPFHPGREDKPEPPPEGRLPDPTKGSDHLRDVFGKAMGLTDQDIVALSGGHTIGAAHKERSGFEGPWTSNPLIFDNSYFTELLSGEKEGLLQLPSDKALLSDPVFRPLVDKYAADEDAFFADYAEAHQKLSELGFADALQLPPLERLTLD

[0433] SEQ ID NO:4 mGPx4-APEX2 fusion protein sequence:

[0434] mito - V5 -APEX2-GPX4:

[0435] M SLGRLCRLLKPALLCGALAAPGLAGTGKPIPNPLLGLDST TGGKSYPTVSADYQDAVEKAKKKLRGFIAEKRCAPLMLRLAFHSAGTFDKGTKTGGPFGTIKHPAELAHSANNGLDIAVRLLEPLKAEFPILSYADFYQLAGVVAVEVTGGPKVPFHPGREDKPEPPPEGRLPDPTKGSDHLRDVFGKAMGLTDQDIVALSGGHTIGAAHKERSGFEGPWTSNPLIFDNSYFTELLSGEKEGLLQLPSDKALLSDPVFRPLVDKYAADEDAFFADYAEAHQKLSELGFADAAEFSRADMCASRDDWRCARSMHEFSAKDIDGHMVNLDKYRGFVCIVTNVASQUGKTEVNYTQLVDLHARYAECGLRILAFPCNQFGKQEPGSNEEIKEFAAGYNVKFDMFSKICVNGDDAHPLWKWMKIQPKGKGILGNAIKWNFTKFLIDKNGCVVKRYGPMEEPLVIEKDLPHYF

[0436] SEQ ID NO:5 APEX2-KDEL fusion protein sequence:

[0437] Igk - V5 -APEX2-KDEL:

[0438] METDTLLLWVLLLWVPGSTGDGKPIPNPLLGLDST TGGKSYPTVSADYQDAVEKAKKKLRGFIAEKRCAPLMLRLAFHSAGTFDKGTKTGGPFGTIKHPAELAHSANNGLDIAVRLLEPLKAEFPILSYADFYQLAGVVAVEVTGGPKVPFHPGREDKPEPP PEGRLPDPTKGSDHLRDVFGKAMGLTDQDIVALSGGHTIGAAHKERSGFEGPWTSNPLIFDNSYFTELLSGEKEGLLQLPSDKALLSDPVFRPLVDKYAADEDAFFADYAEAHQKLSELGFADAKDEL

[0439] SEQ ID NO:6 Horseradish peroxidase HRP amino acid sequence:

[0440] QLTPTFYDNSCPNVSNIVRDTIVNELRSDPRIAASILRLHFHDCFVNGCDASILLDNTTSFRTEKDAFGNANSARGFPVIDRMKAAVESACPRTVSCADLLTIAAQQSVTLAGGPSWRVPLGRRDSLQAFLDLANANLPAPFFTLPQLKDSFRN VGLNRSSDLVALSGGHTFGKNQCRFIMDRLYNFSNTGLPDPTLNTTYLQTLRGLCPLNGNLSALVDFDLRTPTIFDNKYYVNLEEQKGLIQSDQELFSSPNATDTIPLVRSFANSTQTFFNAFVEAMDRMGNITPLTGTQGQIRLNCRVVNSNS

[0441] It should be noted that although the technical solution of the present invention has been described with specific examples, those skilled in the art will understand that the present invention should not be limited thereto.

[0442] The various embodiments of the present invention have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is chosen to best explain the principles, practical application, or technical improvements to the embodiments in the market, or to enable others skilled in the art to understand the embodiments disclosed herein.

Claims

1. A method of labeling an interacting protein, a proximal protein and / or a proximal RNA, characterized in that, The method includes the following steps: S1) The peroxidase is linked to protein A, which participates in the interaction, or to element A, which expresses protein A, to obtain element A' that expresses peroxidase; S2) The peroxidase element A' is contacted with tetrazine-phenol, thereby catalyzing the attachment of tetrazine-phenol to other neighboring proteins and / or other neighboring RNAs adjacent to protein A; The peroxidases include horseradish peroxidase (HRP), ascorbate peroxidase (APEX), and / or variants thereof.

2. The method of claim 1, wherein, The APEX variant comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity with the sequence shown in SEQ ID NO:

1. The APEX variant retains the catalytically active site His42 corresponding to the sequence shown in SEQ ID NO:1, and is capable of catalyzing the conversion of tetrazine-phenol to tetrazine-phenoxy without the dependence on exogenous hydrogen peroxide. Optionally, the APEX variant retains sites Arg38, Phe145, Trp179, and Tyr235 corresponding to the sequence shown in SEQ ID NO:

1.

3. The method according to claim 1 or 2, characterized in that, Element A is selected from organelles, cells, or combinations thereof.

4. The method according to any one of claims 1 to 3, characterized in that, Step S1 includes at least one of the following: a) Peroxidase is expressed on element A, thereby attaching the peroxidase to element A; b) Fusing the peroxidase to protein A, thereby attaching the peroxidase to element A; c) Contacting the protein A or element A with an antibody crosslinked with the peroxidase, wherein the antibody specifically binds to the protein A, thereby linking the peroxidase to the protein A or element A; d) Contacting the protein A or element A, a first antibody that specifically binds to the protein A, with a second antibody that is cross-linked with the peroxidase, thereby linking the peroxidase to the protein A or element A, wherein the second antibody specifically recognizes the first antibody.

5. A method for analyzing protein-protein interactions or protein-RNA interactions, characterized in that, The method includes: The method described in any one of claims 1 to 4 is used to label interacting proteins, neighboring proteins, and / or neighboring RNAs; Biotin is linked to a protein or RNA labeled with tetrazine-phenol using trans-cyclooctene-biotin (TCO-Biotin). Enrichment of biotin-labeled proteins or RNA using streptavidin magnetic beads; Identification and analysis of the enriched biotin-labeled proteins or RNA.

6. The method according to claim 5, characterized in that, The enriched biotin-labeled proteins or RNAs were analyzed and identified using LC-MS / MS, Western blot, DNA-seq, RNA-seq, or RT-qPCR methods.

7. A composition for labeling interacting proteins, neighboring proteins, and / or neighboring RNA, comprising i) and optionally ii), iii), and / or iv): i) Tetraazine-phenol; ii) Ascorbate peroxidase (APEX), its localization fusion protein, its antibody cross-linked product, nucleic acid molecule encoding it, or expression vector containing said nucleic acid molecule; iii) APEX variants, their localization fusion proteins, their antibody cross-links, nucleic acid molecules encoding them, or expression vectors containing said nucleic acid molecules; iv) Horseradish peroxidase (HRP), its localization fusion protein, its antibody cross-linked form, nucleic acid molecule encoding it, or expression vector containing said nucleic acid molecule.

8. The composition according to claim 7, characterized in that, The APEX variant comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity with the sequence shown in SEQ ID NO:

1. The APEX variant retains the catalytic active site corresponding to His42 as shown in SEQ ID NO:1, and is capable of catalyzing the conversion of tetrazine-phenol to tetrazine phenoxy without relying on exogenous hydrogen peroxide.

9. Uses of tetrazine-phenol in the following i) and / or ii): i) Prepare kits for labeling interacting proteins, neighboring proteins, and / or neighboring RNA; ii) Prepare kits for analyzing protein-protein interactions or protein-RNA interactions.

10. The use according to claim 9, characterized in that, The kit also contains horseradish peroxidase (HRP), ascorbate peroxidase (APEX) and / or variants thereof; Preferably, the APEX variant comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity with the sequence shown in SEQ ID NO:

1. The APEX variant retains the catalytic active site corresponding to His42 as shown in SEQ ID NO:1, and is capable of catalyzing the conversion of tetrazine-phenol to tetrazine phenoxy without relying on exogenous hydrogen peroxide.

Images