Nanobody-proximity labeling enzyme fusion protein and use thereof

Abstract

The present invention relates to a nanobody-proximity labeling enzyme fusion protein and the use thereof. Specifically, provided is a fusion protein used for proximity labeling; the fusion protein comprises (a) a nanobody and (b) a proximity labeling enzyme which are operably linked; optionally, there is a peptide linker between the nanobody and the proximity labeling enzyme. The nanobody-proximity labeling enzyme fusion protein is obtained by means of in vitro expression and purification; and said fusion protein uses an antibody to target a target protein, and therefore is not dependent on an overexpression system and is compatible with various types of cells, tissue and clinically immobilized specimens. The fusion protein can target to proteins having post-translational modifications and various organelles, such as membraneless organelles, and can reach a 100% accuracy of localization.

Description

Nanobody-proximity labeling enzyme fusion protein and its applications Technical Field

[0001] This application belongs to the field of biotechnology, specifically relating to a nanobody-neighbor labeling enzyme fusion protein and its application. Background Technology

[0002] Protein-protein interactions are fundamental to various intracellular biological processes, including cell signaling, metabolic regulation, gene transcription regulation, protein synthesis, and cell assembly. They hold significant importance in biology, involving the interactions and influences between proteins. Studying protein-protein interactions helps to reveal the complex molecular networks and signaling pathways within cells, and to understand the regulatory mechanisms of cellular function and biological processes.

[0003] Proximity labeling technology offers a complementary alternative to traditional methods for studying protein-protein interaction networks, organelle proteomes and membrane contacts, protein-nucleic acid interactions, and subcellular transcriptome analysis. This technology relies on enzymes with proximity labeling capabilities, enabling biotinylation of neighboring biomolecules at the live-cell level. It is currently widely used for identifying components of macromolecular complexes, analyzing protein types within organelles, and constructing protein-protein interaction networks. The strategy of proximity labeling typically involves fusing the target protein with an enzyme carrying a biotinylated proximity label, thereby localizing the target protein to a specific intracellular location. By adding small molecule substrates such as biotin and its derivatives, proximity labeling enzymes can catalyze biotinylation covalently linked to spatially adjacent proteins. Currently widely used enzymes include: biotin ligase A (BirA) and its mutants BioID / BioID2 and TurboID; peroxidases APEX (engineered ascorbate peroxidase) and APEX2, etc.

[0004] However, current proximity labeling methods rely on the expression of exogenous fusion proteins and cannot be used to identify post-translational modified proteins. While antibody-mediated protein A-APEX2 has been reported to perform in-situ biotinylation labeling, thus better preserving the spatial relationships of protein-protein interactions, and mass spectrometry analysis can identify proteins reacting to histone modifications, further understanding the function and mechanism of these modifications in cells is possible. However, because protein A can interact with various proteins such as endogenous IgG, the orthogonality and reproducibility of the protein A-APEX2 fusion protein with mammalian cells are poor. Although protein A-APEX2 can label post-histone modifications in the cell nucleus, its large labeling radius prevents it from labeling fine intracellular structures such as microtubules and mitochondria.

[0005] In traditional methods for studying protein-protein interactions, such as immunoprecipitation, antibodies derived from mice and rabbits are indispensable tools in many basic research techniques and medical diagnostic assays. Typically, these primary antibodies are detected or immobilized indirectly using polyclonal anti-IgG secondary antibodies. However, meeting the demand for a continuous supply of anti-IgG serum requires large-scale breeding, immunization, and bloodletting, ultimately leading to the slaughter of large numbers of goats, sheep, rabbits, and donkeys. This is not only costly but also raises significant animal welfare and ethical concerns. Researchers have now developed recombinantly expressible nano-secondary antibodies—single-domain antibodies derived from camel heavy chain antibodies—as an alternative to polyclonal anti-IgG secondary antibodies. These nano-secondary antibodies can bind to different regions of rabbit or mouse IgG, partially reducing the need for animal-derived polyclonal secondary antibodies. Due to their small size, high specificity, the potential for regenerable recombinant fusion proteins, and excellent biophysical properties, nano-secondary antibodies are considered powerful tools in fields such as cell biology and structural biology.

[0006] However, in the field of antibody-drug conjugate technology, nanobodies have not been widely used due to their poor efficacy.

[0007] Furthermore, there are currently insurmountable bottlenecks in the identification of protein interactions in protein condensates. For example, (1) traditional biochemical methods cannot effectively separate the aforementioned condensates; (2) the formation of protein condensates is highly dependent on the concentration of the condensing protein, which means that the overexpression system of biotin ligase fused with the target protein cannot represent the true concentration of endogenous phase transition proteins; and (3) some overexpressed proteins may exhibit abnormal localization, resulting in false positives. These factors directly or indirectly affect the detection and analysis of protein interactions.

[0008] To date, no fusion protein or conjugate integrating nanobodies with proximity-labeled enzymes has been publicly disclosed or developed in this field. Furthermore, no method has been developed to leverage the spatiotemporal labeling properties of enzymes and the biological characteristics of nanobodies to overcome or improve the limitations or problems of traditional immunoprecipitation methods and proximity labeling techniques in protein-protein interaction network research. However, there has been a persistent need in life science research to combine the advantages of both enzymes and nanobodies for more efficient and sensitive exploration of protein-protein interaction networks within and outside cells or tissues. Summary of the Invention

[0009] To address the problems existing in the prior art, this disclosure integrates the advantages of both nanobodies and proximity labeling enzymes, providing a fusion protein or conjugate of nanobodies and proximity labeling enzymes. It aims to leverage the spatiotemporal labeling characteristics of the enzymes and the biophysical properties of the nanobodies to break through or improve the application of traditional immunoprecipitation methods and proximity labeling techniques in protein interaction network research.

[0010] On one hand, this disclosure provides a fusion protein for proximity labeling, wherein the fusion protein is operatively linked together by (a) a nanobody and (b) a proximity labeling enzyme; a peptide linker is also included between the nanobody and the proximity labeling enzyme.

[0011] On the other hand, this disclosure provides a nucleic acid molecule encoding the aforementioned fusion protein.

[0012] On the other hand, this disclosure provides a vector containing the aforementioned nucleic acid molecules.

[0013] On the other hand, this disclosure provides a kit comprising the aforementioned fusion protein: preferably, the kit further comprises a primary antibody targeting the target protein;

[0014] Preferably, the kit further comprises a biotinylation reaction solution;

[0015] Preferably, the kit further comprises streptavidin conjugated with a fluorescent agent;

[0016] Preferably, the kit further comprises a fluorescently conjugated secondary antibody;

[0017] Preferably, the biotinylation reaction solution comprises PBS, MgCl2, ATP, and biotin;

[0018] Preferably, the biotinylation reaction solution contains biotin, phenol, and hydrogen peroxide.

[0019] On the other hand, this disclosure provides a proximity labeling method, characterized in that the interacting protein with the target protein is biotin-labeled using the aforementioned fusion protein or the aforementioned kit, comprising the following steps:

[0020] Add a primary antibody derived from rabbit or mouse species that targets the target protein to the cells and incubate them to allow the primary antibody to bind to the target protein;

[0021] According to the species of the primary antibody, the corresponding fusion protein is added to the cell and incubated to form a neighboring labeling enzyme-nano secondary antibody-primary antibody-target protein complex in the cell;

[0022] Add biotinylation reaction solution to cells, incubate, and biotinylate the interacting protein molecules with the target protein;

[0023] After washing, streptavidin and secondary antibody conjugated with fluorescent dye were added separately, incubated, washed, and detected.

[0024] On the other hand, this disclosure provides a method for analyzing intermolecular interactions, the method comprising the following steps:

[0025] Biotin labeling of the interacting protein with the target protein was performed using the aforementioned fusion protein or the aforementioned kit.

[0026] Biotin-labeled protein molecules can be enriched or fluorescently localized using magnetic beads or fluorescent agents coupled with streptavidin.

[0027] The enriched biotin-labeled protein molecules were analyzed and identified by LC-MS / MS.

[0028] On the other hand, this disclosure provides applications of the aforementioned fusion protein, the aforementioned kit, and the aforementioned method, wherein the applications include:

[0029] (a) Biotinylated proximity markers and component analysis of the cytoskeleton and organelles;

[0030] (b) Biotinylated proximity markers and component analysis of post-modified histones;

[0031] (c) Biotinylated proximity markers and component analysis of different nucleolar substructures;

[0032] (d) Biotinylated proximity markers and interaction protein analysis of proteins in FFPE and OCT slices;

[0033] (e) Double biotinylated neighbor markers in the same sample; or

[0034] (f) Biotinylated proximity labeling and interaction protein analysis of proteins in model organisms.

[0035] The beneficial effects of this disclosure are at least as follows:

[0036] The above-described technical solution disclosed herein does not rely on an overexpression system; by recognizing histone-modified antibodies, biotinylate is used to label interacting proteins of post-modified histones, thereby effectively resolving the protein interaction network of post-modified histones; it can also accurately resolve protein components in phase transition particles; it does not rely on the construction of cloning vectors, and can use targeted antibodies to label proteins with large molecular weights and identify their related interacting proteins; it can resolve protein interactions of multilocalized proteins at specific locations at high resolution, such as distinguishing different protein interaction networks of the same protein in the cell nucleus and cytoplasm.

[0037] The nanobody-neighbor-labeled enzyme fusion protein disclosed herein can be used for:

[0038] (a) Biotinylated proximity markers and component analysis of the cytoskeleton and organelles;

[0039] (b) Biotinylated proximity markers and component analysis of post-modified histones;

[0040] (c) Biotinylated proximity markers and component analysis of different nucleolar substructures;

[0041] (d) Biotinylated proximity markers and interaction protein analysis of proteins in FFPE and OCT slices;

[0042] (e) Double biotinylated neighbor markers in the same sample;

[0043] (f) Biotinylated proximity labeling and interaction protein analysis of proteins in model organisms.

[0044] In particular, the designed and provided nanobody-photocontrolled proximity labeling enzyme fusion protein enables flexible and controllable high-efficiency biotin labeling. Furthermore, future integration with a fully automated AI-based microscopy imaging system allows the AI ​​algorithm to quickly and accurately identify structures of interest, enabling the localization and analysis of target regions. Moreover, the combination of AI and microscopy promises to achieve high-throughput, automated image acquisition and analysis, as well as site-specific biotinylation labeling, allowing for the labeling of hundreds of thousands of cells at specific locations, thereby meeting the sample volume requirements of mass spectrometry. Attached Figure Description

[0045] Figure 1 is a schematic diagram illustrating the working principle of the nanobody-neighborhood marker enzyme fusion protein disclosed in this invention in actual sample detection.

[0046] Figure 2 illustrates the working principle of the light-controlled nanobody-neighbor labeling enzyme fusion protein in actual sample detection.

[0047] Figure 3 shows the SDS-PAGE images of the purified anti-mouse or anti-rabbit nanobody-TurboID fusion protein. Lanes 1-8 represent the molecular map lanes of the constructed anti-mouse nanobody-TurboID fusion protein before and after expression and purification in host cells; lane 9 is the protein marker; lanes 10-16 represent the molecular map lanes of the constructed anti-rabbit nanobody-TurboID fusion protein before and after expression and purification in host cells.

[0048] Figure 4 shows the labeling radii of Nano-ID, Nano-APX, Pro-ID, and Pro-APX in the mouse cytoskeleton. Figure 4a shows the neighboring biotinylate ligases expressed by six different types of nanobody fusions: anti-rabbit nanobody-TurboID / APEX2, anti-mouse nanobody-TurboID / APEX2, and Protein A-TurboID / APEX2. Figure 4b shows the in vitro biotinylation experimental procedure: cells are first fixed, blocked, and permeabilized; then, a primary antibody targeting the region of interest is added; followed by the addition of Protein A. A-TurboID / APEX2 or, depending on the primary antibody species, anti-rabbit nanobody-TurboID / APEX2 or anti-mouse nanobody-TurboID / APEX2 were added; Figures 4c-4n show the fluorescence localization results of different anti-mouse fusion proteins in U-2OS cells. Anti-β-actin primary antibody was used to label actin filaments (4c, 4f, 4i, 4l), anti-vimentin primary antibody was used to label intermediate filaments (4d, 4g, 4j, 4m), and anti-TubA4A primary antibody was used to label tubulin (4e, 4h, 4k, 4n); Figures 4c-4e show the addition of anti-mouse nanobody-TurboID, Figures 4f-4h show the addition of anti-mouse nanobody-APEX2, and Figures 4i-4k or 4l-4n show the addition of Protein A-TurboID / APEX2. Green indicates Alexa Fluor. TM 488-conjugated streptavidin, showing the distribution of biotinylated proteins; red markings indicate Alexa Fluor. TM The secondary antibody conjugated to 560 shows the localization of the primary antibody. Scale bar, 5μm; magnified view, 2μm.

[0049] Figure 5 shows the distribution of fluorescence signals in the white-lined areas of Figure 4 (4c, 4f, 4i, 4l) (top row) and the fluorescence signal index fitting (bottom row).

[0050] Figure 6 compares the labeling radii of Nano-ID, Nano-APX, Pro-ID, and Pro-APX using different mouse organelle antibodies. Anti-LAMP1 primary antibody was used for lysosomal labeling (6a, 6d, 6g, 6j), anti-LaminB1 primary antibody for nuclear membrane labeling (6b, 6e, 6h, 6k), and anti-ATP5A1 primary antibody for mitochondrial labeling (6c, 6f, 6i, 6l). Figures 6a-6c show the addition of anti-mouse nanobody-TurboID, Figures 6d-6f show the addition of anti-mouse nanobody-APEX2, and Figures 6g-6i or Figures 6j-6l show the addition of Protein A-TurboID / APEX2. The green label indicates Alexa Fluor. TM488-conjugated streptavidin, showing the distribution of biotinylated proteins; red markings indicate Alexa Fluor. TM The secondary antibody conjugated to 560 shows the localization of the primary antibody. Scale bar, 5 μm; magnified view, 2 μm.

[0051] Figure 7 compares the labeling radii of Nano-ID, Nano-APX, Pro-ID, and Pro-APX using different rabbit organelle antibodies. Anti-Paxillin primary antibody was used for focal adhesion labeling (7a-7d), anti-PEX14 primary antibody for peroxisome labeling (7e-7h), and anti-EDC4 primary antibody for P-body labeling (7i-7l). Figures 7a, 7e, and 7i show the addition of anti-rabbit nanobody-TurboID; Figures 7b, 7f, and 7j show anti-rabbit nanobody-APEX2; and Figures 7c, 7g, and 7k, or Figures 7d, 7h, and 7l show the addition of Protein A-TurboID / APEX2. The green label indicates Alexa Fluor. TM 488-conjugated streptavidin, showing the distribution of biotinylated proteins; red markings indicate Alexa Fluor. TM The secondary antibody conjugated to 560 shows the localization of the primary antibody. Scale bar, 5 μm; magnified view, 2 μm.

[0052] Figure 8 shows the Nano-ID used for labeling organelles or histones and subsequent modifications. The primary antibodies used are: anti-GOLG2 (mouse antibody, Golgi apparatus labeling) (8a), anti-CD98 (rabbit antibody, cell membrane labeling) (8b), anti-SC35 (mouse antibody, nuclear plaque labeling) (8c), anti-PDI (rabbit antibody, endoplasmic reticulum labeling) (8d), anti-CEP250 (rabbit antibody, centriole labeling) (8e), anti-FUS (mouse antibody) (8f), anti-H3K4me3 (mouse antibody) (8g), anti-H3K27Ac (rabbit antibody) (8h), anti-HAK119ub (rabbit antibody) (8i), and anti-L-Lactyl. Lysine (rabbit antibody) (8j); anti-rabbit nanobody-TurboID was added in Figures 8b, 8d, 8e, 8h, 8i, and 8j; anti-mouse nanobody-TurboID was added in Figures 8a, 8c, 8f, and 8g; green markings indicate Alexa Fluor. TM 488-conjugated streptavidin, showing the distribution of biotinylated proteins; red markings indicate Alexa Fluor. TM The secondary antibody conjugated with 488 shows the localization of the primary antibody. Scale bar, 5 μm.

[0053] Figure 9 shows the layered analysis of the nucleolus using Nano-ID. Figure 9a is a schematic diagram of the nucleolus structure, showing the Fibrillar Centers (FC), Dense Fibrillar Component (DFC), and Granular Component (GC). Figures 9b-9d show the in-situ biotin labeling results of the three substructures of the nucleolus. Figure 9e shows the immunoblotting detection of biotinylated proteins in different substructures. Figure 9f shows the signal heatmaps of TCOF1, FBL, and NPM1 after mass spectrometry detection of different nucleolar substructures. Figure 9g shows the mass spectrometry signal heatmaps of nucleolar proteins in different layers. Figures 9h-9j show representative protein interaction networks enriched in the FC (9h), DFC (9i), and GC (9j) structures. The protein interaction networks are illustrated using StringDb.

[0054] Figure 10 shows the application of Nano-ID in FFPE and OCT sections. Figure 10a shows representative immunofluorescence images of in vitro biotinylation on FFPE sections of lung adenocarcinoma tissue using Nano-ID. Figure 10b shows the detection results of biotinylated total protein content in the experimental group (+, with PECAM1 primary antibody) and the control group (-, without PECAM1 primary antibody). Figure 10c shows the mass spectrometry signal intensity of PECAM1 in FFPE samples from the above three patients after enrichment with streptavidin-conjugated magnetic beads in the experimental and control groups; ND, not detected. Figure 10d is a Venn diagram showing the overlap of PECAM1 protein interactions in the three patients. Figure 10e shows the interaction network analysis results of interacting proteins that were detected with PECAM1 in all three samples. The interaction network information is sourced from the Stringdb database, with different colors representing different signaling pathways involved by the proteins.

[0055] Figure 11 shows the application of Nano-ID in mouse brain OCT sections. Figure 11a shows representative immunofluorescence images of in vitro biotinylation of Nano-ID on mouse brain OCT sections. After fixation and permeabilization, GFAP antibody and Nano-ID were added sequentially to the mouse brain OCT sections for in vitro biotinylation. After the reaction, the samples were incubated with Alexa Fluor. TM 560-conjugated secondary antibody marker GFAP, Alexa Fluor TM 488 streptavidin-conjugated biotinylated proteins. Figure 11b shows the total biotinylated protein content in the experimental group (+, with GFAP primary antibody) and the control group (-, without GFAP primary antibody). Figure 11c is a Venn diagram of the 689 endogenous wild-type biotinylated proteins and 795 D4-labeled proteins identified after enrichment. Figure 11d shows the GO analysis results of the proteins identified in Figure 11c. Figure 11e is a schematic diagram of the experimental procedure.

[0056] Figure 12 shows the application of Nano-ID in the brains of adult mice with the HTT disease model. Figure 12a shows representative immunofluorescence images of HTT-adjacent proteins labeled in HTT disease model mice. Figure 12b shows the results of total biotinylated protein content detection in the experimental group (+, with HTT primary antibody) and the control group (-, without HTT primary antibody).

[0057] Figure 13 shows the application of Nano-ID in FFPE and OCT sections, as well as fertilized egg samples. Figures 10a and 10b are representative immunofluorescence images of Nano-ID in vitro biotinylated on FFPE sections of lung adenocarcinoma tissue. Figure 13a shows the addition of PDL1 antibody, and Figure 13b shows the addition of H3K27Ac antibody. Alexa Fluor TM 560 conjugated secondary antibody markers PDL1(a) or H3K27Ac(b), Alexa Fluor TM 488 was conjugated with streptavidin to label biotinylated proteins. Figure 13c shows biotinylated transcription factor PAX6 neighboring proteins on an OCT slice of the brain of a 12.5-day-old mouse. Figure 13d is a schematic diagram of apical domain labeling using Nano-ID in a mouse 8-cell embryo.

[0058] Figure 14 illustrates the dual labeling of a sample using Nano-ID. Figure 14a is a schematic diagram of the dual labeling experimental process. Intracellular ROI1 and ROI2 were labeled with rabbit and mouse antibodies, respectively. For example, in the first round of reaction, Nano-ID(Rb) recognized the rabbit primary antibody, labeling the rabbit-labeled ROI1 with wild-type biotin. After the first round of reaction, TEV enzyme was added for cleavage, cutting Nano-ID-(Rb), and the free Turbo-ID was washed away. In the second round of reaction, Nano-ID(Ms) was added to recognize the mouse primary antibody, and then the biotin in the reaction solution was labeled with D4. The second round of reaction was then performed to label the mouse-labeled ROI2 with D4. After cell lysis, the resulting protein precipitate was digested into peptides, and biotinylation enrichment was performed at the peptide level to obtain wild-type and D4-labeled peptides, thus distinguishing the proteins labeled in the first and second rounds. Figure 14b shows the biotinylate labeling process as described in Figure 14a: first round using rabbit-derived primary antibody anti-TFAM wild-type biotin, and second round using mouse-derived primary antibody anti-NPM1 isotope D4 biotin. The biotinylate labeled in the first and second rounds was labeled using Alexa Fluor. TM 488 and Alexa Fluor TM 560-conjugated streptavidin was observed. Immunoblotting results for single TFAM and NPM1 labeling and double TFAM and NPM1 labeling in Figure 14b are shown in Figure 14c.

[0059] Figure 15 shows the labeling results of Nb2-ID in cell lines and model organisms stably expressing GFP fusion proteins. Figure 15a shows a cell line overexpressing mEmerald-SRSF3, Figure 15b shows a cell line with GFP knocked into the SC35 gene locus using CRISPR, Figure 15c shows a cell line overexpressing mEmerald-ensconsin, and Figure 15d shows a nematode stably expressing ajm-1-GFP with a developmental stage of 1.8 fold. After incubation with Nb2-ID, the above samples underwent in vitro biotinylation. TM 560-conjugated streptavidin localizes biotinylated proteins.

[0060] Figure 16 shows the labeling results of nanobody-photocontrolled proximity labeling enzyme in U-2OS cells. Figure 16a shows the incubation of rabbit anti-PEX14 antibody, and Figure 16b shows the incubation of mouse anti-Tubulin antibody. The corresponding species of nanobody-photocontrolled proximity labeling enzyme was then added to perform biotinylation reactions. Purple labels represent Alexa Fluor. TM The secondary antibody conjugated to 647 shows the localization of the primary antibody; the green marker indicates Alexa Fluor. TM 488-conjugated streptavidin, showing the distribution of biotinylated proteins. Scale bar, 5 μm. Detailed Implementation

[0061] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments other than those of this application. For those skilled in the art, other implementation methods can be obtained based on these drawings.

[0062] definition

[0063] The following definitions are provided to assist the reader. Unless otherwise defined, all technical terms, symbols, and other scientific or medical terms or nouns used herein are intended to have the meaning commonly understood by one of ordinary skill in the art of chemistry and medicine. In some cases, for clarity and / or ease of reference, terms with generally understood meanings are defined herein, and such definitions included herein should not be construed as representing a material difference from the definitions of terms commonly understood in the art. In cases of ambiguity, chemical, pharmaceutical, and medical dictionaries may be used as further sources of information to the extent consistent with the invention.

[0064] Unless the context otherwise indicates, the terms “an” or “a” denote a substance or component, but do not limit its number / quantity, and can be singular or plural, unless otherwise specified herein or the context clearly contradicts it. For example, reference to “a combination of drugs” should be understood as a combination of two or more components.

[0065] In this document, unless otherwise stated, the terms “comprising,” “including,” and “containing,” or equivalents, are open-ended expressions and mean that they may cover other unspecified elements, components, and steps in addition to those listed.

[0066] Unless the context clearly indicates otherwise, singular terms encompass the plural referents, and vice versa. Similarly, unless the context clearly indicates otherwise, the word "or" is intended to include "and," and vice versa.

[0067] As used herein, the terms “about” and “substantially” refer to a deviation from the stated value within the range of -10% to +10%. When the word “about” is used herein to refer to a number, it should be understood that another embodiment of the invention includes that number not modified by the presence of the word “about”.

[0068] As used herein, the term "antibody" includes any immunoglobulin that binds to a specific antigen, including monoclonal antibodies, polyclonal antibodies, multivalent antibodies, bivalent antibodies, monovalent antibodies, multispecific antibodies, or bispecific antibodies. A natural, complete antibody consists of two heavy (H) chains and two light (L) chains. The mammalian heavy chains are divided into α, δ, ε, γ, and μ chains, each including a variable region (VH) and a first, second, third, and optionally fourth constant region (CH1, CH2, CH3, CH4, respectively); the mammalian light chains are divided into λ or κ chains, each including a variable region (VL) and a constant region. Antibodies are Y-shaped, where the stem of the Y-structure includes the second and third constant regions of the two heavy chains linked together by disulfide bonds. Each arm of the Y includes the variable region and the first constant region of a single heavy chain, which bind to the variable and constant regions of a single light chain. The variable regions of the light and heavy chains are responsible for antigen binding. Each chain's variable region typically contains three hypervariable regions called complementarity-determining regions (CDRs). The light chain CDRs include LCDR1, LCDR2, and LCDR3, while the heavy chain CDRs include HCDR1, HCDR2, and HCDR3. These three CDRs are separated by flanking regions called framework regions (FRs). The light chain FRs include LFR1, LFR2, LFR3, and LFR4, while the heavy chain FRs include HFR1, HFR2, HFR3, and HFR4. The framework regions are more conserved than the CDRs and form a scaffold to support the highly variable loops. The constant regions of the heavy and light chains are not involved in antigen binding but exhibit various effector functions. Antibodies can be classified into several classes based on the amino acid sequence of their heavy chain constant regions. The five main classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, characterized by the presence of α, δ, ε, γ, and μ heavy chains, respectively. Several major antibody classes are divided into subclasses, such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain), or IgA2 (α2 heavy chain).

[0069] As used herein, the term "monoclonal antibody" refers to a highly homogeneous antibody produced by cloning a single B cell (whose genes encode only one type of antibody) that targets only a specific antigenic epitope. It is typically prepared using hybridoma technology, which, based on cell fusion techniques, fuses sensitized B cells capable of secreting specific antibodies with myeloma cells capable of unlimited proliferation to create a B-cell hybridoma. A cell population formed from a single hybridoma cell possessing these characteristics can then be used to prepare a specific antibody against a single antigenic epitope, i.e., a monoclonal antibody.

[0070] As used in this article, the term "polyclonal antibody" refers to a group of immunoglobulins secreted by plasma cells of an organism in response to an immune response stimulated by a heterologous antigen (macromolecule antigen, hapten conjugate). They typically contain at least two or more different antibodies, which can recognize multiple antigenic epitopes and induce precipitation reactions. They are inexpensive and relatively quick to prepare, and the preparation process is simpler than that of monoclonal antibodies, making them widely used in research and diagnostics.

[0071] As used herein, the term "primary antibody" or "first antibody" refers to a protein that specifically binds to a non-antibody antigen (specific antigen). Types include monoclonal antibodies and polyclonal antibodies. In other words, it is the antibody that specifically binds to an antigen, as commonly understood.

[0072] As used in this article, the term "secondary antibody" refers to an antibody that can bind to an antibody, i.e., an antibody against an antibody. Its main function is to detect the presence of the primary antibody and amplify its signal. Secondary antibodies utilize the antigenic property of antibodies, which are large protein molecules, to immunize a foreign animal. The immune system of the foreign animal produces immunoglobulins against this antibody. Secondary antibodies are reactive to all antibodies (such as IgG, IgM, or IgA) against a specific species (e.g., mice).

[0073] As used herein, the term "antigen-binding fragment" refers to an antibody fragment comprising one or more CDRs, or any other antibody fragment that binds to an antigen but does not contain the complete structure of a native antibody. Examples of antigen-binding fragments include, but are not limited to, bifunctional antibodies, Fab, Fab', F(ab')2, Fd, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized bifunctional antibodies (ds bifunctional antibodies), single-chain antibody molecules (scFv), scFv dimers (bivalent bifunctional antibodies), multispecific antibody fragments, camelified single-domain antibodies, nanobodies, domain antibodies, and bivalent domain antibodies. An antigen-binding fragment is capable of binding to the same antigen that the parent antibody binds to. In some embodiments, the antigen-binding fragment may comprise one or more CDRs from a particular antibody.

[0074] As used herein, the term "Fab" refers to a monovalent antigen-binding fragment of an antibody, which consists of a single light chain (variable region and constant region) connected by disulfide bonds to a single heavy chain with a variable region and a first constant region. Fab can be obtained by digesting residues near the N-terminus of the disulfide bonds between the heavy chains in the hinge region of the antibody using papain.

[0075] As used herein, the term "Fab" refers to a Fab fragment containing a portion of the hinge region, which can be obtained by digesting residues near the C-terminus of the disulfide bonds between the heavy chains of the antibody hinge region with pepsin, and thus a small number of residues (containing one or more cysteine ​​residues) in the hinge region are different from those in Fab.

[0076] As used in this article, the term “F(ab’)2” refers to the dimer of Fab’, which comprises two light chains and a portion of two heavy chains.

[0077] As used herein, the term "Fc" refers to the antibody moiety, which consists of the second and third constant regions of the first heavy chain bound to the second and third constant regions of the second heavy chain via disulfide bonds. The Fc region of IgG and IgM contains three heavy chain constant regions (the second, third, and fourth constant regions of each chain). It can be obtained by digesting the antibody with papain. The Fc moiety of the antibody is responsible for various effector functions, such as ADCC, ADCP, and CDC, but does not play a role in antigen binding.

[0078] As used herein, the term "Fv" refers to the smallest antibody fragment with an intact antigen-binding site. An Fv fragment consists of a variable region of a single light chain that binds to a variable region of a single heavy chain. "dsFv" refers to a disulfide-stabilized Fv fragment, wherein the bond between the variable region of the single light chain and the variable region of the single heavy chain is a disulfide bond.

[0079] As used herein, the term "single-chain Fv antibody" or "scFv" refers to an engineered antibody composed of light chain variable regions and heavy chain variable regions linked together directly or via peptide linker sequences. "scFv dimer" refers to a single-chain antibody containing two heavy chain variable regions and two light chain variable regions with linkers. In some embodiments, "scFv dimer" is a bivalent bifunctional antibody or bivalent scFv (BsFv) comprising a VH-VL (linked by peptide linkers) dimerized with another VH-VL portion, such that one VH portion coordinates with the other VL portion, forming two binding sites that can target the same antigen (or epitope) or different antigens (or epitopes). In other embodiments, the “scFv dimer” is a bispecific bifunctional antibody comprising VH1-VL2 (linked by peptide linkers) that bind to VL1-VH2 (also linked by peptide linkers), such that VH1 coordinates to VL1 and VH2 coordinates to VL2 and each coordination pair has a different antigen specificity.

[0080] As used herein, the terms “single-chain Fv-Fc antibody” or “scFv-Fc” refer to engineered antibodies that consist of scFvs linked to the Fc region of the antibody.

[0081] As used herein, the term "nanobody" or "camelized single-domain antibody," "single-domain antibody (sdAb)," or "HCAb" has the same meaning and is used interchangeably. It refers to the construction of a nanobody consisting of only one variable region of the heavy chain by cloning the variable region of the antibody's heavy chain. Typically, antibodies lacking both the light chain and the heavy chain constant region 1 (CH1) are first obtained, and then the variable region of the antibody's heavy chain is cloned to construct a nanobody consisting of only one variable region of the heavy chain. Heavy chain antibodies were originally obtained from the Camelidae family (camel, dromedary camel, and llama). Although they do not contain light chains, camelized antibodies possess a reliable antigen-binding library. The variable domain (VHH domain) of a heavy chain antibody represents the smallest known antigen-binding unit generated by an acquired immune response.

[0082] As used herein, the term "bifunctional antibody" comprises a small antibody fragment having two antigen-binding sites, wherein the fragment contains a VH domain (VH-VL or VL-VH) linked to a VL domain on a single polypeptide chain. Because the linker is too short, the two domains on the same chain cannot pair, thus forcing the domain to pair with a complementary domain of another chain, thereby creating two antigen-binding sites. These antigen-binding sites may target the same or different antigens (or epitopes).

[0083] As used herein, the term "enzyme" refers to a protein or RNA produced by living cells that exhibits high specificity and catalytic efficiency towards its substrate. The catalytic activity of an enzyme depends on the integrity of its primary and spatial structures. Denaturation or subunit depolymerization of the enzyme molecule can lead to loss of enzyme activity. Enzymes are biological macromolecules with molecular weights ranging from at least 10,000 to over 1 million. Enzymes are extremely important biocatalysts. Due to the action of enzymes, chemical reactions within organisms can proceed efficiently and specifically even under extremely mild conditions. Based on their chemical composition, enzymes can be divided into two categories: simple enzymes and conjugated enzymes. Simple enzyme molecules are those that, after hydrolysis, consist only of amino acids. Conjugated enzyme molecules are composed of both protein and non-protein components, such as metal ions, iron porphyrins, or small organic molecules containing B vitamins.

[0084] As used in this article, the term "protein interaction" or "protein-protein interaction" refers to the process by which two or more protein molecules form a protein complex through non-covalent bonds. Examples include replication, transcription, translation, cell cycle regulation, and metabolism.

[0085] As used in this article, the term "protein-protein interaction network (PPI)" refers to the network of proteins that interact with each other to participate in various stages of life processes, including biological signal transduction, gene expression regulation, energy and metabolism, and cell cycle regulation. Systematic analysis of the interactions between a large number of proteins in biological systems is crucial for understanding the working principles of proteins, the response mechanisms of biological signals and energy metabolism under specific physiological states such as disease, and the functional connections between proteins. Currently, commonly used methods for identifying protein interactions include yeast two-hybrid (Y2H) screening and immunoprecipitation combined with mass spectrometry (IP-MS) analysis.

[0086] As used herein, the term "proximity labeling (PL)" refers to a technique that uses gene fusion to fuse certain enzymes with a target protein, resulting in a specific reaction catalyzed by the enzyme, thereby labeling proteins spatially adjacent to the target protein. Proximity labeling uses genetically modified enzymes, such as peroxidases or biotin ligases, which catalyze the conversion of inert substrates into highly reactive, short-lived active substances. The active substance diffuses from the enzyme's active site to the surrounding area, covalently labeling nearby biomolecules (proteins, nucleic acids). The labeling extent depends on the half-life of the active substance and the concentration of the quencher. The covalently labeled biomolecules are enriched using streptavidin magnetic beads and identified by mass spectrometry or nucleic acid sequencing. Proximity labeling is widely used to identify protein-protein interaction networks and to study protein-RNA and protein-DNA interactions in living cells. It is also suitable for constructing interaction networks of poorly soluble proteins and for detecting transient or dynamic protein interactions. Furthermore, it is applicable to analyzing protein components localized to subcellular organelles or for studying interaction networks in living organisms. Over the past decade, proximity labeling technology has developed rapidly. Currently, it has reached nanometer-level spatial resolution and minute-level temporal resolution, and has been used to construct molecular interaction maps in living organisms.

[0087] As used herein, the term "proximity labeling enzyme" or "proximity labeling tool enzyme" refers to tool enzymes with proximity labeling function, including peroxidases (e.g., APEX, HRP) and biotin ligases (e.g., BioID, TurboID), which biotinylate neighboring biomolecules at the live-cell level. Because the highly reactive small molecules catalyzed by these enzymes have very short lifetimes, proximity labeling technology exhibits high spatial specificity. Combined with mass spectrometry-based proteomics and high-throughput sequencing technologies, large-scale analysis of neighboring biomolecules can be achieved.

[0088] The ligation-active enzymes used can be mainly divided into two categories structurally: intact and dissociated. Intact proximity labeling enzymes are primarily used to study potential interacting proteins of a single target protein, while dissociated proximity labeling enzymes are used to study proteins associated with known protein complexes or interacting proteins. Proximity labeling assays based on intact proximity labeling enzymes involve fusing a biotin ligase or ascorbate peroxidase (APEX enzyme) with the target protein and expressing it in live cells. Substrate, such as biotin or biotin-phenol and hydrogen peroxide (H2O2), is then added to the culture medium, allowing proteins or RNA near the target protein to be biotin-labeled. Cell lysis and incubation with streptavidin beads enrich the biotin-labeled proteins or RNA for subsequent LC-MS / MS or high-throughput sequencing analysis. Dissociated proximity labeling enzyme systems, used to identify protein complex compositions, involve fusing the N-terminal and C-terminal portions of the proximity labeling enzyme with a pair of known interacting proteins. When these two proteins interact in the cell, the two halves of the adjacent labeling enzyme are pulled close together and reassembled into a complete adjacent labeling enzyme, which labels the protein complex. By lysing the cell and incubating it with streptavidin magnetic beads, the biotin-labeled protein can be enriched for subsequent LC-MS / MS sequencing analysis.

[0089] There are many proximity labeling enzymes used for protein-protein interaction identification, among which commonly used proximity labeling tools include the mutant of E. coli biotin ligase BirA (BioID) and ascorbate peroxidase (APEX). Based on these, many other proximity labeling enzymes have been optimized and developed, such as the optimized APEX series enzyme APEX2; the optimized BioID series enzymes BioID2, AirID, BASU, etc.; and some tools used in smaller areas such as HRP, EXCELL, PUP-IT, and NEDDylation. The development of these tools has continuously broadened the application scope of proximity labeling technology.

[0090] As used herein, the term "fusion protein" has the common and conventional meaning as understood by one of ordinary skill in the art based on reference books in the field and the specification of this application. In this application, a "fusion protein" is the expression product of two recombinant genes obtained through DNA recombination technology, where two different proteins can be linked into a large molecule through gene fusion. In addition to the nanobodies listed in this application, the fusion protein in this application may also include optional tag sequences that assist in expression and / or purification (e.g., 6xHis tag, GGGS sequence, FLAG tag); or optional polypeptide molecules or fragments with therapeutic functions; or optional protein functional domains that assist in physicochemical or pharmaceutical applications (e.g., molecules that can prolong the in vivo half-life of nanobodies, such as Fc fragments, HLE, ABD).

[0091] In this application, the nanobody-neighbor-labeled enzyme fusion protein is formed by coupling a nanobody (Nano) and a neighboring labeling enzyme. When the neighboring labeling enzyme is TurboID, it can be called "Nano-ID (Ms / Rb)", "Nanobody-ID (Ms / Rb)", or "Anti-rabbit / anti-mouse nanobody-TurboID". When the neighboring labeling enzyme is APEX2, it can be called "Nano-APX (Ms / Rb)", "Nanobody-APX (Ms / Rb)", or "Anti-rabbit / anti-mouse nanobody-APEX2". The fusion protein formed by coupling protein A and the neighboring labeling enzyme can be called "ProteinA-TurboID", "Pro-ID", "ProteinA-APEX2", or "Pro-APX". In some embodiments, "Nb2-ID" and "nanobody-photocontrolled neighboring labeling enzyme" are also types of nanobody-neighboring labeling enzyme fusion proteins.

[0092] As used in this article, the term "post-translational modifications" (PTMs) refers to the chemical modifications that occur to proteins after translation. These modifications include acetylation, methylation, phosphorylation, ubiquitination, and ADP-ribosylation, which can occur on the amino acid residues of histones, thereby altering the structure and function of chromatin and regulating gene expression. "Post-modified histones" refers to histones that have undergone post-translational modifications.

[0093] Detailed description of the implementation plan

[0094] On one hand, this disclosure provides a fusion protein for proximity labeling, wherein the fusion protein is operatively linked together by (a) a nanobody and (b) a proximity labeling enzyme; a peptide linker is also included between the nanobody and the proximity labeling enzyme.

[0095] In some embodiments, a peptide linker is also included between the nanobody and the adjacent labeling enzyme.

[0096] In some embodiments, the adjacent labeling enzyme comprises a peroxidase and / or a biotin ligase. In some embodiments, the peroxidase is horseradish peroxidase (HRP) or ascorbate peroxidase. In some embodiments, the ascorbate peroxidase is APEX or APEX2. In some embodiments, the biotin ligase is selected from Mini TurboID, TurboID, AirID, BioID, BASU, or BirA. In some embodiments, the biotin ligase is TurboID. In some embodiments, the biotin ligase is an engineered light-controlled TurboID. In some embodiments, the amino acid sequence of the light-controlled TurboID is as shown in any one of SEQ ID NO. 25-29.

[0097] In some embodiments, the nanobody is a primary antibody targeting a target protein or a secondary antibody targeting an immunoglobulin. In some embodiments, the immunoglobulin is rabbit immunoglobulin, human immunoglobulin, or mouse immunoglobulin. In some embodiments, the immunoglobulin is selected from IgG, IgM, IgD, IgE, IgA, or IgY. In some embodiments, the nanobody is a secondary antibody targeting IgG, which specifically binds to the primary antibody targeting the target protein. In some embodiments, the nanobody is a secondary antibody with an amino acid sequence as shown in SEQ ID NO.1, SEQ ID NO.11, or SEQ ID NO.18.

[0098] In some embodiments, the nanobody is a nanobody that recognizes GFP. In some embodiments, the amino acid sequence of the GFP-recognizing nanobody is shown in SEQ ID NO.46.

[0099] In some embodiments, the peptide linker is a flexible linker or a rigid linker. In some embodiments, the peptide linker is a flexible linker. In some embodiments, the amino acid sequence of the flexible linker is as shown in SEQ ID NO.4.

[0100] In some implementations, the nanobody and the adjacent labeling enzyme are linked via a click chemistry reaction.

[0101] In some embodiments, the nanobody and the adjacent labeling enzyme are fused together in any of the following ways:

[0102] (a) The C-terminus of the nanobody is linked to the N-terminus of the adjacent labeled enzyme; or

[0103] (b) The N-terminus of the nanobody is linked to the C-terminus of the adjacent labeled enzyme.

[0104] In some embodiments, the C-terminus of the nanobody is fused to the N-terminus of a neighboring labeled enzyme via a linker.

[0105] In some embodiments, the amino acid sequence of the fusion protein is selected from:

[0106] (a) A polypeptide having an amino acid sequence as shown in any one of SEQ ID NO. 5, 6, 12, 13, 19, 20, 30-39, 47; or

[0107] (b) A polypeptide that is homologous to or has at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity with an amino acid sequence of any one of SEQ ID NO. 5, 6, 12, 13, 19, 20, 30-39, or 47, and is capable of labeling a neighboring protein molecule that interacts with the target protein; or

[0108] (c) A protein or polypeptide derived from the insertion, substitution or deletion of one or more amino acids in the amino acid sequence of (a) or (b), which can be used to label neighboring protein molecules that interact with the target protein.

[0109] In some embodiments, the fusion protein further comprises a protein tag selected from any one of GST, 6x-His, MBP, Flag, HA, cMyc, GFP, eGFP, eYFP, mCherry, AviTag, or SUMO tags. In some preferred embodiments, the protein tag is a 6xHis and Flag tag.

[0110] In some embodiments, the target protein is any intracellular or extracellular protein that can be recognized by nanobodies or immunoglobulins.

[0111] In some embodiments, the fusion protein is an anti-rabbit nanobody-TurboID, whose amino acid sequence is shown in SEQ ID NO. 5. In some embodiments, the fusion protein is an anti-rabbit nanobody-APEX2, whose amino acid sequence is shown in SEQ ID NO. 6. In some embodiments, the fusion protein is an anti-mouse nanobody-TurboID, whose amino acid sequence is shown in SEQ ID NO. 12. In some embodiments, the fusion protein is an anti-rabbit nanobody-APEX2, whose amino acid sequence is shown in SEQ ID NO. 13. In some embodiments, the fusion protein is Protein A-TurboID, whose amino acid sequence is shown in SEQ ID NO. 19. In some embodiments, the fusion protein is Protein A-APEX2, whose amino acid sequence is shown in SEQ ID NO. 20. In some embodiments, the fusion protein is a nanobody-photocontrolled proximity labeling enzyme fusion protein, which can precisely activate or deactivate the enzymatic activity of TurboID by controlling light irradiation, achieving spatial control of the biotinylation process, with an amino acid sequence shown in any one of SEQ ID NO. 30-39. In some embodiments, the fusion protein is Nb2-ID, whose amino acid sequence is shown in SRQ ID NO.47.

[0112] On the other hand, this disclosure provides a nucleic acid molecule encoding the aforementioned fusion protein.

[0113] On the other hand, this disclosure provides a vector containing the aforementioned nucleic acid molecules.

[0114] On the other hand, this disclosure provides a kit comprising the aforementioned fusion protein: preferably, the kit further comprises a primary antibody targeting the target protein;

[0115] Preferably, the kit further comprises a biotinylation reaction solution;

[0116] Preferably, the kit further comprises streptavidin conjugated with a fluorescent agent;

[0117] Preferably, the kit further comprises a fluorescently conjugated secondary antibody;

[0118] Preferably, the biotinylation reaction solution comprises PBS, MgCl2, ATP, and biotin;

[0119] Preferably, the biotinylation reaction solution contains biotin, phenol, and hydrogen peroxide.

[0120] On the other hand, this disclosure provides a proximity labeling method, characterized in that the protein interacting with the target protein is biotin-labeled using the aforementioned fusion protein or the aforementioned kit, comprising the following steps:

[0121] Add a primary antibody of any species that targets the target protein to the cell and incubate it to allow the primary antibody to bind to the target protein;

[0122] According to the species of the primary antibody, the corresponding fusion protein is added to the cell and incubated to form a neighboring labeling enzyme-nano secondary antibody-primary antibody-target protein complex in the cell;

[0123] Add biotinylation reaction solution to cells, incubate, and biotinylate proteins that interact with the target protein;

[0124] After washing, streptavidin and secondary antibody conjugated with fluorescent dye were added separately, incubated, washed, and detected.

[0125] On the other hand, this disclosure provides a method for analyzing intermolecular interactions, the method comprising the following steps:

[0126] Biotin labeling of proteins that interact with the target protein is performed using the aforementioned fusion protein or the aforementioned kit.

[0127] Biotin-labeled protein molecules can be enriched or fluorescently localized using magnetic beads or fluorescent agents coupled with streptavidin.

[0128] The enriched biotin-labeled proteins were analyzed and identified by LC-MS / MS.

[0129] On the other hand, this disclosure provides applications of the aforementioned fusion protein, the aforementioned kit, and the aforementioned method, wherein the applications include:

[0130] (a) Biotinylated proximity markers and component analysis of the cytoskeleton and organelles;

[0131] (b) Biotinylated proximity markers and component analysis of post-modified histones;

[0132] (c) Biotinylated proximity markers and component analysis of different nucleolar substructures;

[0133] (d) Biotinylated proximity markers and interaction protein analysis of proteins in FFPE and OCT slices;

[0134] (e) Double biotinylated neighbor markers in the same sample;

[0135] (f) Biotinylated proximity labeling and interaction protein analysis of proteins in model organisms.

[0136] A further understanding of this disclosure can be obtained by referring to some specific embodiments given herein, which are for illustrative purposes only and are not intended to limit the scope of this disclosure in any way. It will be apparent that various modifications and variations can be made to this disclosure without departing from its essence, and therefore such modifications and variations are also within the scope of protection claimed in this application.

[0137] Example 1: Preparation of nanobody-neighbor-labeled enzyme fusion protein

[0138] The nanobodies used in this disclosure are all specific secondary antibodies. However, the technical solutions of this disclosure are also applicable to primary antibody nanobodies. A schematic diagram of the working principle of the nanobody-neighbor-labeled enzyme fusion protein is shown in Figure 1.

[0139] 1. Anti-rabbit nanobody-TurboID / APEX2 fusion protein

[0140] The amino acid sequences of the anti-rabbit nanobody are shown in SEQ ID NO.1, TurboID in SEQ ID NO.2, and APEX2 in SEQ ID NO.3. The C-terminus of the nanobody is fused to the N-terminus of a neighboring labeled enzyme via a linker; the amino acid sequence of the linker is shown in SEQ ID NO.4. The amino acid sequence of the fused anti-rabbit nanobody-TurboID is shown in SEQ ID NO.5, with a 3xFlag tag attached to the N-terminus and a 6xHis tag attached to the C-terminus. The amino acid sequence of the anti-rabbit nanobody-APEX2 is shown in SEQ ID NO.6, with both a 6xHis tag and a 3xFlag tag attached to the N-terminus of the fusion protein.

[0141] Table 1. Encoding information of the anti-rabbit nanobody-TurboID / APEX2 fusion protein

[0142] Choose an expression vector suitable for the *E. coli* protein expression system, including but not limited to any one of pET15b, pET28a, pGEX4T1, or pGEX-6p-1, and construct the anti-rabbit nanobody-TurboID fusion protein particle as shown in SEQ ID NO.9, or the anti-rabbit nanobody-APEX2 fusion protein particle as shown in SEQ ID NO.10. The specific process is as follows:

[0143] Vectors are constructed through homologous recombination. The main method of homologous recombination is to recombine the target fragment and the vector in the overlapping region to form a new plasmid.

[0144] The synthesized anti-rabbit nanobody-TurboID fusion protein gene and anti-rabbit nanobody-APEX2 fusion protein gene were constructed into an expression vector through homologous recombination.

[0145] (1) PCR of target fragment and vector

[0146] The PCR primers used are shown in Table 2 below:

[0147] Table 2. PCR Primers

[0148] The PCR system and procedures used are shown in Table 3 below:

[0149] Table 3. PCR reaction system

[0150] The PCR program settings are shown in Table 4 below:

[0151] Table 4. PCR Program Settings

[0152] Perform agarose gel electrophoresis on the PCR products and recover the bands of the correct size for later use.

[0153] (2) The system and reaction conditions for homologous recombination are shown in Table 5 below.

[0154] Table 5. Reaction components and reaction conditions

[0155] The ligation products were transformed into clonal competent cells, plated onto LB agar plates containing antibiotics, and single colonies were picked the following day for sequencing. Fusion protein plasmids were extracted from correctly sequenced strains, transformed into expression-competent BL21(DE3) cells, plated onto LB agar plates containing antibiotics, and single colonies were picked the following day for protein expression.

[0156] 2. Anti-mouse nanobody-TurboID / APEX2 fusion protein

[0157] The amino acid sequences of the anti-mouse nanobody are shown in SEQ ID NO.11, TurboID in SEQ ID NO.2, and APEX2 in SEQ ID NO.3. The C-terminus of the nanobody is fused to the N-terminus of a neighboring labeled enzyme via a linker; the amino acid sequence of the linker is shown in SEQ ID NO.4. The amino acid sequence of the fused anti-mouse nanobody-TurboID is shown in SEQ ID NO.12, with a 3xFlag tag attached to the N-terminus and a 6xHis tag attached to the C-terminus. The amino acid sequence of the anti-mouse nanobody-APEX2 is shown in SEQ ID NO.13, with both a 6xHis tag and a 3xFlag tag attached to the N-terminus of the fusion protein.

[0158] Table 6. Encoding information of the anti-mouse nanobody-TurboID / APEX2 fusion protein.

[0159] Choose an expression vector suitable for the *E. coli* protein expression system, including but not limited to any one of pET15b, pET28a, pGEX4T1, or pGEX-6p-1, and construct the anti-mouse nanobody-TurboID fusion protein particle as shown in SEQ ID NO.16, or the anti-mouse nanobody-APEX2 fusion protein particle as shown in SEQ ID NO.17. The specific process is as follows:

[0160] For PCR primer sequences, procedures, ligation methods, screening, etc., please refer to the relevant content on anti-rabbit nanobodies.

[0161] 3. Protein A-TurboID / APEX2 fusion protein

[0162] The amino acid sequences of Protein A are shown in SEQ ID NO.18, TurboID in SEQ ID NO.2, and APEX2 in SEQ ID NO.3. The C-terminus of Protein A is fused to the N-terminus of a neighboring labeled enzyme via a linker; the amino acid sequence of the linker is shown in SEQ ID NO.4. The amino acid sequences of the fused Protein A-TurboID are shown in SEQ ID NO.19, and those of Protein A-APEX2 are shown in SEQ ID NO.20. The N-terminus of the fusion protein is linked to a 6xHis tag and a 3xFlag tag.

[0163] Table 7. Encoding information of the anti-mouse nanobody-TurboID / APEX2 fusion protein.

[0164] Choose an expression vector suitable for the *E. coli* protein expression system, including but not limited to any one of pET15b, pET28a, pGEX4T1, or pGEX-6p-1, and construct the Protein A-TurboID fusion protein particle as shown in SEQ ID NO.23, or the Protein A-APEX2 fusion protein particle as shown in SEQ ID NO.24. The specific process is as follows:

[0165] The synthesized proteinA-TurboID fusion protein gene and proteinA-APEX2 fusion protein gene were constructed into an expression vector through homologous recombination.

[0166] The PCR primer sequences used are shown in Table 8 below:

[0167] Table 8. PCR Primers

[0168] For PCR procedures, ligation methods, screening, etc., please refer to the relevant content on anti-rabbit nanobodies.

[0169] 4. Anti-rabbit nanobody-photocontrolled proximity labeling enzyme fusion protein

[0170] Photocontrolled proximity labeling enzymes (PLAs) utilize genetic codon expansion technology to introduce photocontrolled non-natural amino acids into the active pocket of TurboID, thereby inactivating TurboID. Under UV irradiation, these non-natural amino acids are converted into their corresponding natural amino acids, thus releasing the enzymatic activity of TurboID. Specifically, PLAs replace lysine or tyrosine at specific sites with corresponding lysine or tyrosine derivatives, etc., non-natural amino acids. This site-specific insertion of non-natural amino acids eliminates the activity of proximity biotin ligases. Under 365 nm UV irradiation, the non-natural amino acids, such as lysine or tyrosine derivatives, at specific sites in the PLA are converted back to lysine or tyrosine, thereby restoring the activity of proximity biotin ligases and specifically biotinylating neighboring proteins.

[0171] Therefore, the fusion expression protein of the nano-secondary antibody and the photo-controlled proximity labeling enzyme (a photoswitch mutant of TurboID) can precisely activate the enzymatic activity of TurboID in a specific region by controlling the illumination area, thus achieving spatial control of the biotinylation process. The introduction of this photoswitch improves the functional flexibility and controllability of TurboID, enabling efficient biotin labeling at specific spatial locations. A schematic diagram of the working principle of the fusion expression protein of the nano-secondary antibody and the TurboID photoswitch mutant is shown in Figure 2.

[0172] The photosensitive proximity labeling enzyme TurboID is a mutant of the biotin ligase BirA derived from *E. coli*, with its amino acid sequence shown in SEQ ID NO. 65 and SEQ ID NO. 66. One or more of the lysine at positions 183, 132, and 172 in the amino acid sequence of the photosensitive proximity labeling enzyme are mutated to non-natural amino acids MNPY-lysine (MNPYK), ONB-lysine (ONBK), or ONB-tyrosine (ONBK). Photosensitive proximity labeling enzymes TurboID-183-MNPYK, TurboID-183-ONBK, TurboID-132-ONBY, TurboID-172-MNPYK, and TurboID-172-ONBK were obtained, and their corresponding amino acid sequences are shown in Table 9. The construction method of the photosensitive proximity labeling enzyme, the specific tRNA and tRNA synthetase used, are described in patent CN113481173B.

[0173] The construction method of the anti-rabbit nanobody-photocontrolled proximity labeling enzyme fusion protein is described in Example 2. The nanobody is fused with the photocontrolled proximity labeling enzyme via a linker. The amino acid sequence of the linker is shown in SEQ ID NO.4, and the amino acid sequence of the fusion protein is shown in Table 9 below.

[0174] Similarly, an anti-mouse nanobody-photosensitive proximity labeling enzyme fusion protein can be constructed. Alternatively, an Nb2-ID photosensitive proximity labeling enzyme fusion protein suitable for recognizing GFP can be developed.

[0175] The photocontrolled biotin ligase can be activated by irradiation with 365nm wavelength ultraviolet light for 1 minute.

[0176] Table 9. Amino acid sequences corresponding to the anti-rabbit nanobody-photosensitive neighbor-labeled enzyme fusion protein

[0177] Example 2: Induction and purification of nanobody-neighbor-labeled enzyme fusion protein

[0178] 2.1 Induced expression of nanobody-neighbor-labeled enzyme fusion protein in prokaryotic system

[0179] The prokaryotic recombinant plasmid containing the target protein was transformed into competent expression cells BL21(DE3). The following day, single colonies were picked and inoculated into 2 mL of LB medium. After shaking, the culture was preserved and amplified to 1 L of LB medium, and cultured at 37°C and 180 rpm in a shaker. When the OD600 of the bacterial culture reached 0.8–1.0, the culture was cooled to 16°C. For the expression of the TurboID fusion protein, IPTG (YEASEN, Cat#10902ES08) at a final concentration of 300 mM was added directly for induction; for the expression of the APEX2 fusion protein, IPTG (Sigma, Cat#1.24802) at a final concentration of 300 mM and 5-Aminolevulinic acid hydrochloride (Sigma, Cat#1.24802) at a final concentration of 1 mM were added. After 16 h of induction, the bacterial cells were collected by centrifugation at 4000 rpm for 20 min.

[0180] 2.2 Expression of nanobody-photocontrolled proximity labeling enzyme fusion protein in eukaryotic systems

[0181] (1) According to 1.4x10 6 HEK293-F cells were inoculated into 200 ml of culture medium in a 1 L shake flask at a seeding rate of 1 cell / mL and cultured in a shaker incubator at 37°C, 120 rpm, and 8% CO2. The next day, when the cell concentration reached 2 x 10⁻⁶ cells / mL... 6 Cells / mL, transfect plasmid using PEI (Polysciences, Cat#24765-100), as follows:

[0182] (2) Place 600 μL of PEI in a metal bath and incubate at 55°C for 20 min;

[0183] Add 100 μg of nanobody-neighbor labeling enzyme fusion protein expression plasmid and 100 μg of plasmid expressing tRNA and tRNA synthetase to 10 mL of Opti-MEM, and vortex to mix.

[0184] Add 600 μL of PEI to 10 mL of Opti-MEM (Gibco, Cat#31985070) and shake to mix.

[0185] Combine the two Opti-MEM tubes, mix them thoroughly by shaking, and let them stand at 37°C for 20 minutes.

[0186] (3) Under light-protected conditions, the above Opti-MEM system was added dropwise to the prepared HEK293-F cells; then, 200 μL of 500 mM light-controlled non-natural amino acids (synthesized by Beijing Oukenas Technology Co., Ltd.) was added, with a final concentration of 500 μL.

[0187] (4) Wrap the cells in aluminum foil to protect them from light and incubate them in a shaker at 37°C, 120 rpm, and 8% CO2 for 72 hours. After expression is complete, centrifuge at 2500 rpm for 10 min and collect the cells.

[0188] 2.3 Fusion Protein Purification

[0189] (1) Suspending bacteria: Add 30 mL of PBS to suspend the bacterial culture, and add protease inhibitor (TargetMOI, Cat#C0001) and PMSF (Beyotime, Cat#ST506).

[0190] (2) Ultrasonic fragmentation: 40% power, turn on for 3 seconds, turn off for 20 seconds, ultrasound for 30 minutes.

[0191] (3) Centrifuge to obtain protein supernatant: Centrifuge at 14000 rpm and 4℃ for 1 h and obtain supernatant.

[0192] (4) Affinity chromatography: Add 1 mL of nickel column packing material (Qiagen, Cat#30210) to the affinity chromatography column and equilibrate the column with 10 mL of PBS before use. Pour the collected protein supernatant into the column and allow it to flow out by gravity (repeat this operation twice).

[0193] (5) Eluting off impurities: Add 10 mL of elution solution (PBS, 30 mM imidazole) to the nickel column and allow it to flow out by gravity.

[0194] (6) Elution of target protein: Add 10 mL of elution solution (PBS, 300 mM imidazole) to the nickel column above, allow it to flow out by gravity, and collect the elution solution.

[0195] (7) Concentration: Using a 50 mL concentration tube with a molecular weight cutoff of 10 kD, centrifuge at 3500 rpm and 4 °C until the eluent is concentrated to 500 μL.

[0196] (8) Dialysis: In order to remove McAc from the protein solution, 10 mL of PBS was added to 500 μL of concentrate, and the concentration was increased to 500 μL. This was repeated once, and finally 500 μL of protein was obtained.

[0197] Figure 3 shows the SDS-PAGE images of the purified anti-rabbit and anti-mouse nanobody-TurboID fusion proteins.

[0198] Example 3: Preparation of biotinylated cell samples

[0199] (1) Prepare cells: One day in advance, seed the required amount of cells (e.g., human osteosarcoma cells U-2OS) in a 6cm cell culture dish. The next day, fix the cells. Different fixation methods can be selected according to different antibodies: 4% PFA / -20℃ methanol / -20℃ ethanol.

[0200] (2) Blocking and permeabilization: Treat cells with PBST + 5% BSA solution to block and permeabilize them for 1 hour (for methanol and ethanol fixation, PBST is not required for permeabilization; simply replace it with PBS).

[0201] (3) Incubation with primary antibody: Dilute the primary antibody with PBS + 5% BSA solution at a ratio of 1:200, add it to the cells, and incubate at room temperature for 2 hours or at 4°C overnight. Wash three times with PBS.

[0202] (4) Incubation of nanobody-neighbor labeling enzyme fusion protein: Dilute the fusion protein with PBS solution at a ratio of 1:200, add it to the cells, and incubate at room temperature for 1 hour. Wash three times with PBS.

[0203] (5) Biotinylation reaction: Add biotinylation reaction solution (1 mL PBS + 20 mM MgCl2 + 100 mM ATP + 500 μM biotin) to the cells. Incubate at 37 °C for 16 h. After the reaction, stain to detect the biotinylation effect, or lyse the cells for the preparation of mass spectrometry samples.

[0204] (6) Cell staining: After the biotinylation reaction, wash three times with PBS, and add Alexa Fluor at a ratio of 1:1000. TM 488 or 560 coupled with streptavidin, and simultaneously added to Alexa Fluor at a ratio of 1:1000. TM The secondary antibody conjugated with 560 or 488 was incubated at room temperature for 1 hour, washed three times with PBS, and the biotinylation reaction was detected by fluorescence microscopy.

[0205] (6) Cell lysis: After the biotinylation reaction, wash three times with PBS, add 500 μL of lysis buffer 1 (300 mM Tris-HCl, 2% SDS, 0.2 M glycine, pH 9.0), scrape the cells into a low-adsorption tube, and lyse the sample by heating in a metal bath: 98℃ for 20 min, 80℃ for 2 h. After lysis, centrifuge at 14000 rpm and 4℃ for 20 min, and collect the supernatant.

[0206] Example 4: Enrichment of Biotinylated Proteins and Preparation of Mass Spectrometry Samples

[0207] (1) Add an equal volume of water to the sample to dilute it by half. Dilute lysis buffer 1 with water by half to make lysis buffer 2.

[0208] (2) Take 50 μL of streptavidin C1 magnetic beads (Invitrogen, Cat#65002), add 200 μL of lysis buffer 2, gently pipette and resuspend thoroughly, place on a magnetic rack for separation for 10 s, remove the supernatant, and repeat this step three times. Add the sample from step 1 to the magnetic beads and incubate at 4°C with shaking for 1 h.

[0209] (3) After incubation, place the beads on a magnetic rack and discard the supernatant; add 200 μL of lysis buffer 2 to wash the magnetic beads, repeating three times. Then, wash once each with 200 μL of buffer A (1M KCl), buffer B (0.1M Na2CO3), and buffer C (50mM Tris-HCl, 2M urea, pH=7.5), and once with 200 μL of 100mM Tris-HCl (pH=8.0).

[0210] (4) Add 40 μL of 100 mM Tris-HCl (pH 8.0) to the magnetic beads, add 1 mM DTT and 0.4 μL of trypsin to a final concentration, and incubate at 25 °C with shaking for 1 h.

[0211] (5) After incubation, place the beads on a magnetic rack, transfer the supernatant to a new tube, and wash the magnetic beads twice with 30 μL of 100 mM Tris (pH 8.0). Combine the washing solutions, and the volume of the elution solution at this time is 100 μL.

[0212] (6) Add 4 mM dithiothreitol to a final concentration and incubate at 25°C with shaking for 30 min.

[0213] (7) Add 10mM iodoacetamide to a final concentration and incubate at 25°C with shaking for 45 minutes.

[0214] (8) Add 0.4 μg of trypsin and incubate overnight at 25°C with shaking.

[0215] (9) After digestion overnight, the pH of the sample was adjusted to less than 3 using 10% trifluoroacetic acid to desalt it.

[0216] Example 5: Enrichment of biotinylated peptides and preparation of mass spectrometry samples

[0217] (1) Reductive alkylation: Add 30 μL of 200 mM TCEP to the cell lysate and incubate at 55 °C for 1 h.

[0218] Add 30 μL of 375 mM iodoacetamide and incubate at 25°C in the dark for 30 min.

[0219] (2) Methanol-chloroform precipitation of proteins: Add 500 μL of methanol and 125 μL of chloroform to the reduced alkylated sample, vortex for 30 s to mix thoroughly. Centrifuge at 14000 rpm at room temperature for 10 min, discard the supernatant. Add 500 μL of methanol, vortex for 30 s to mix thoroughly. Centrifuge at 14000 rpm at room temperature for 10 min, discard the supernatant, and air dry the sample.

[0220] (3) Trypsin digestion: Add 100 μL of PTS buffer (100 mM Tris-HCl, pH 8.0, 12 mM SDC, 12 mM SLS) to the dried protein precipitate and heat in a metal bath at 90 °C for 10 minutes. Dissolve 20 μg of trypsin in 400 μL of 100 mM Tris-HCl (pH 8.0) and add it to the above sample. Incubate overnight at 25 °C with shaking.

[0221] (4) Antibody conjugation of protein G beads: 50 μL of protein G beads (NEB, Cat#S1430S) were washed twice with 100 μL of 300 mM NaAc (pH=3.0). Then, 10 μL of anti-biotin antibody and 400 μL of 300 mM NaAc were added, and the mixture was incubated at 4°C with shaking for 2 hours. The beads were then washed twice with 200 μL of 300 mM NaAc.

[0222] (5) Biotinylated peptide enrichment: The overnight digested peptides were heated in a 90°C metal bath for 10 min to inactivate trypsin. 15 μL of NaAc was added to adjust the sample pH to approximately 6.5. The acidified peptides were then added to protein G beads coupled with antibody and incubated overnight at 4°C with shaking.

[0223] (6) Elution: After discarding the supernatant of the above sample, add 200 μL H2O and wash 3 times, discarding the supernatant each time. Add 25 μL 0.2% TFA and wash 2 times, 10 min each time. Take the supernatant and combine the eluents.

[0224] (7) Desalination.

[0225] Example 6: Labeling radius of nanobody-proximity labeling enzyme fusion protein in mouse cytoskeleton and different organelles

[0226] The experimental procedure for in vitro biotinylation is shown in Figure 4b. Cells are first fixed, blocked, and permeabilized; then, a primary antibody targeting the target region is added; Protein A-TurboID / APEX2 is added, or anti-rabbit nanobody-TurboID / APEX2 or anti-mouse nanobody-TurboID / APEX2 is added depending on the primary antibody species. U-2OS cells are incubated with anti-β-actin primary antibody (labeled actin filaments) (c, f, i, l), anti-vimentin primary antibody (labeled intermediate filaments) (d, g, j, m), or anti-TubA4A primary antibody (labeled tubulin) (e, h, k, n) as described in Figure 4b; then, anti-mouse nanobody-TurboID (ce), anti-mouse nanobody-APEX2 (fh), or Protein A-TurboID (ik) / APEX2 (ln) are added; finally, biotinylation reaction solution is added. The specific experimental procedure is described in Example 3.

[0227] The test results are shown in Figures 4c-4n, where the green markers represent Alexa Fluor. TM The secondary antibody conjugated to 488 shows the localization of the primary antibody; the red marker indicates Alexa Fluor. TM 560-conjugated streptavidin shows the distribution of biotinylated proteins.

[0228] The distribution of fluorescence signals was measured and characterized in the dashed areas c, f, i, l in Figure 4, and the fluorescence signal index was fitted. The results are shown in Figure 5.

[0229] Similarly, the labeling radii of Nano-ID, Nano-APX, Pro-ID, and Pro-APX were compared using different murine organelle antibodies in U-2OS cells. The specific experimental procedure followed Example 3: first, incubation was performed with anti-LAMP1 primary antibody (lysosomal labeling) (a, d, g, j), anti-LaminB1 primary antibody (nuclear membrane labeling) (b, e, h, k), or anti-ATP5A1 primary antibody (mitochondrial labeling) (c, f, i, l); then, anti-mouse nanobody-TurboID (ac), anti-mouse nanobody-APEX2 (df), or Protein A-TurboID (gi) / APEX2 (jl) were added; finally, biotinylated in vitro reaction solution was added for reaction. The detection results are shown in Figure 6.

[0230] As shown in Figures 4-6, when using a mouse-derived primary antibody, Nano-ID can effectively label the target region, forming a label with a high signal-to-noise ratio. Although Pro-ID, Pro-APX, or Nano-APX can also label the target region, the label radius is too large and cannot be effectively labeled.

[0231] Example 7: Labeling radius of nanobody-proximity labeling enzyme fusion protein in rabbit scaffold and different organelles

[0232] U2OS cells were incubated with anti-Paxillin primary antibody (focal adhesion label) (ad), anti-PEX14 primary antibody (peroxisome label) (eh), or anti-EDC4 primary antibody (P-body label) (il) according to the procedure described in Example 3. Then, anti-rabbit nanobody-TurboID (a, e, i), anti-rabbit nanobody-APEX2 (b, f, j), or Protein A-TurboID (c, g, k) / APEX2 (d, h, l) were added. Finally, biotinylated in vitro reaction solution was added for reaction.

[0233] The test results are shown in Figure 7, where the green marker indicates Alexa Fluor. TM 488-conjugated streptavidin, showing the distribution of biotinylated proteins; red markings indicate Alexa Fluor. TM The secondary antibody conjugated to 560 shows the localization of the primary antibody. As shown in Figure 7, when using rabbit-derived primary antibodies, Nano-ID and Pro-ID can effectively label the target region, forming high signal-to-noise ratio markers. However, the marker radius of Pro-APX or Nano-APX is too large, and they cannot effectively label the target region.

[0234] Example 8: Nano-ID for labeling more types of organelles or post-histone modifications

[0235] U-2OS cells were incubated with anti-GOLG2 primary antibody (mouse antibody, Golgi apparatus labeled) (a), anti-CD98 primary antibody (rabbit antibody, cell membrane labeled) (b), anti-SC35 primary antibody (mouse antibody, nuclear plaque labeled) (c), anti-PDI primary antibody (rabbit antibody, endoplasmic reticulum labeled) (d), anti-CEP250 primary antibody (rabbit antibody, centriole labeled), anti-FUS primary antibody (mouse antibody, f), anti-H3K4me3 (mouse antibody, g), anti-H3K27Ac (rabbit antibody, h), anti-HAK119ub (rabbit antibody, i), and anti-L-Lactyl Lysine (j) according to the procedure described in Example 3. Then, anti-rabbit (b, d, e, h, i, j) or anti-mouse (a, c, f, g) nanobody-TurboID was added. Finally, biotinylated in vitro reaction solution was added for reaction.

[0236] The test results are shown in Figure 8. The green marker indicates Alexa Fluor. TM488-conjugated streptavidin, showing the distribution of biotinylated proteins; red markings indicate Alexa Fluor. TM The secondary antibody conjugated to 560 shows the localization of the primary antibody. As shown in Figure 8, the Nano-ID prepared in this disclosure can recognize more types of rabbit or mouse organelles or histone post-modifications, expanding the range of antibodies that can be effectively recognized compared to the Pro-ID in the prior art.

[0237] Example 9: Nano-ID Layered Analysis of Nucleoli

[0238] The nucleolus can be morphologically divided into three layers from the inside out: the fibrillar center (FC, highly correlated with POLI transcription), the dense fibrillar component (DFC, the main region for ribosomal RNA cleavage), and the granular component (GC, involved in ribosome biosynthesis). See Figure 9a for a schematic diagram. U-2OS cells were incubated with anti-TCOF1 (rabbit antibody, labeling the FC region), anti-FBL (rabbit antibody, labeling the DFC region), and anti-NPM1 (mouse antibody, labeling the GC region) according to the procedure described in Example 3. Then, anti-rabbit or anti-mouse nanobody-TurboID was added, followed by a biotinylation reaction solution. The biotinylation reaction was then monitored by adding Alexa Fluor to the cells. TM 488-conjugated streptavidin shows biotinylated protein localization, added to Alexa Fluor TM The secondary antibody conjugated with 560 was used to visualize the localization of the primary antibody. The detection results are shown in Figures 9b-9d. The nucleolar layered structure can be clearly distinguished, and Nano-ID has high localization accuracy. Subsequently, U-2OS cells were treated in the same way. After the biotinylation reaction was completed, the cells were lysed, and the content of biotinylated proteins was detected by immunoblotting, as shown in Figure 9e. Compared with the control group, the experimental group showed a significant enrichment of biotinylated proteins. The total proteins of the experimental and control groups were enriched by conjugating streptavidin and then detected by mass spectrometry. Figure 9f shows the signal heatmap of TCOF1, FBL, and NPM1, and Figure 9g shows the mass spectrometry signal heatmap of nucleolar proteins in different layers. Figures 9h-9j select representative protein interaction networks enriched in the FC structure (9h), DFC structure (9i), and GC structure (9j), which are highly correlated with the functions of the nucleolar layered regions. From the above in-situ biotin labeling of the three substructures of the nucleolar, Nano-ID was used to achieve layered analysis of the nucleolar structure.

[0239] Example 10: Nano-ID is applicable to various types of slicing such as FFPE and OCT.

[0240] FFPE sections of lung adenocarcinoma tissue were selected for in vitro biotinylation labeling. Paraffin-embedded lung adenocarcinoma sections were dewaxed, rehydrated, and then treated with PECAM1 antibody and Nano-ID before in vitro biotinylation. TM 560 conjugated antibody labeled PECAM1, Alexa Fluor TM Biotinylated proteins were labeled with streptavidin using 488 and subjected to immunofluorescence detection. Images were acquired using a PANNORAMIC 1000 scanner. Immunofluorescence results are shown in Figure 10a. The total biotinylated protein content in the experimental group (+, with PECAM1 primary antibody) and the control group (-, without PECAM1 primary antibody) is shown in Figure 10b. In FFPE samples from three patients, the mass spectrometry signal intensity of PECAM1 after enrichment with streptavidin in both the experimental and control groups is shown in Figure 10c (ND indicates undetected). A Venn diagram showing the overlap of PECAM1 protein interactions in the three patients is shown in Figure 10d. Interacting proteins of PECAM1 detected in all three samples were input into Stringdb for analysis, as shown in Figure 10e. Different colors represent different signaling pathways involved by the proteins.

[0241] Similarly, mouse brain OCT sections were subjected to in vitro biotinylation labeling. After fixation and permeabilization, GFAP antibody and Nano-ID were added sequentially to the mouse brain OCT sections for in vitro biotinylation reaction. After the reaction, the samples were incubated with Alexa Fluor. TM 560 conjugated secondary antibody labeled GFAP, Alexa Fluor TM 488 streptavidin-conjugated biotinylated proteins were used, and the immunofluorescence detection results are shown in Figure 11a. Figure 11e illustrates the in vitro biotinylation reaction solution of the sample described in Figure 11a, where biotin was replaced with D4 isotope labeling. After the reaction, the protein was precipitated and digested. The resulting peptides were enriched with anti-biotin antibodies, and the biotinylation sites and biotin tag type were identified by mass spectrometry, i.e., whether they were wild-type biotin (endogenous biotinylated protein) or D4 isotope biotinylated (Nano-ID with D4 isotope biotinylated tag), thus distinguishing between endogenous biotinylated proteins and target biotinylated proteins. The detection results of total biotinylated protein content in the experimental group (+, with GFAP primary antibody) and the control group (-, without GFAP primary antibody) are shown in Figure 11b. The Venn diagram of the 689 endogenous wild-type biotinylated proteins and 795 D4-labeled proteins identified after enrichment is shown in Figure 11c. Further GO analysis was performed on the identified proteins, and the results are shown in 11d.

[0242] In the HTT disease model mouse, biotinylated labeling of HTT-adjacent proteins was shown in representative immunofluorescence images in Figure 12a. The total biotinylated protein content of the experimental group (+, with HTT primary antibody) and the control group (-, without HTT primary antibody) is shown in Figure 12b.

[0243] In summary, Nano-ID is compatible with FFPE sections. If the vascular endothelial marker PECAM1 antibody is used, PECAM1 and its interacting proteins can be identified in FFPE sections. In addition, Nano-ID is also compatible with OCT sections. If the astrocyte intermediate filament cytoskeleton protein marker GFAP antibody is used, GFAP and its interacting proteins can be identified. In addition to being part of the intermediate filament cytoskeleton, these interacting proteins are also involved in the maintenance of synapses, axons and dendrites.

[0244] In addition, Nano-ID is also suitable for localization markers of immune-related proteins and histone post-modification in FFPE and OCT sections. In vitro biotinylation labeling was performed on FFPE sections of lung adenocarcinoma tissue. Paraffin-embedded lung adenocarcinoma sections were dewaxed, rehydrated, and then subjected to in vitro biotinylation with PDL1 antibody (a) or H3K27Ac antibody (b) and Nano-ID. Alexa560 conjugate antibody labeling of PDL1 (a) or H3K27Ac (b) was performed using Alexa Fluor. TM 488 conjugated with streptavidin to label biotinylated protein. Images were acquired using a PANNORAMIC 1000 scanner; fluorescence localization results are shown in Figures 13a and 13b.

[0245] Biotin-labeled PAX6 neighboring protein was obtained from OCT slices of mouse brains from mice at 12.5 days of development. After fixation and permeabilization, PAX6 antibody and Nano-ID were added sequentially for in vitro biotinylation. The samples were then incubated with Alexa Fluor after the reaction. TM 560 conjugated antibody labeled PAX6, Alexa Fluor TM 488-conjugated streptavidin-labeled biotinylated protein, fluorescence localization results are shown in Figure 13c. In mouse 8-cell embryos, fluorescence results of Nano-ID labeling of the apical domain are shown in Figure 13d.

[0246] Example 11: Nano-ID can achieve dual labeling of a sample.

[0247] A schematic diagram of the dual-labeling experimental procedure is shown in Figure 14a. Intracellular ROI1 and ROI2 were labeled with antibodies from rabbit and mouse species, respectively. For example, in the first round of reaction, Nano-ID(Rb) recognized the rabbit primary antibody, labeling the rabbit-labeled ROI1 with wild-type biotin. After the first round of reaction, TEV enzyme was added for cleavage, cutting Nano-ID-(Rb), and the free TurboID was washed away. In the second round of reaction, Nano-ID(Ms) was added to recognize the mouse primary antibody, and then the biotin in the reaction solution was labeled with D4, performing the second round of reaction to label the mouse-labeled ROI2 with D4. After cell lysis, the resulting protein precipitate was digested into peptides, and biotinylation enrichment was performed at the peptide level, ultimately yielding wild-type and D4-labeled peptides, thus distinguishing the proteins labeled in the first and second rounds.

[0248] As mentioned earlier, the first round used rabbit-derived primary antibody anti-TFAM wild-type biotin to label mitochondria, and the second round used mouse-derived primary antibody anti-NPM1 isotope D4 biotin to label the nucleolus. The biotinylated proteins labeled in the first and second rounds were analyzed using Alexa Fluor. TM 488 and Alexa Fluor TM The streptavidin conjugated with 560 was observed, and the detection results are shown in Figure 14b. The immunoblotting results of TFAM and NPM1 labeling alone, as well as double TFAM and NPM1 labeling, are shown in Figure 14c.

[0249] In summary, the Nano-ID disclosed herein can achieve dual labeling of the same sample.

[0250] Example 12: Nano-ID can be used to label cell lines or model organisms expressing GFP fusion proteins.

[0251] To better adapt to cell lines with GFP overexpression or GFP gene knock-in, as well as model animals such as nematodes and fruit flies, we replaced the nanobody that recognizes rabbit or mouse IgG in Nano-ID with a nanobody that recognizes GFP, resulting in Nb2-ID. Nb2 is a nanobody that specifically recognizes GFP, and its amino acid sequence is shown in SEQ ID NO.46. The amino acid sequence of Nb2-ID is shown in SEQ ID NO.47, and the nucleotide sequence of Nb2-ID is shown in SEQ ID NO.48.

[0252] Figures 15a-c show the in vitro biotin labeling results of cell lines (b) overexpressing mEmerald-SRSF3 (a), mEmerald-ensconsin (c), or GFP knocked into the SC35 gene locus using CRISPR after incubation with Nb2-ID, using Alexa Fluor.TM 560-conjugated streptavidin was used to detect biotinylated proteins. Figure 12d shows nematodes stably expressing ajm-1-GFP at a developmental stage of 1.8 fold. After incubation with Nb2-ID, in vitro biotinylation was performed. The above samples were analyzed using Alexa Fluor. TM 560-conjugated streptavidin detection of biotinylated proteins.

[0253] In summary, Nano-ID can also be used to label cell lines or model organisms that express GFP.

[0254] Example 13: Validation of the biotinylation labeling capability of nanobody-photocontrolled proximity labeling enzyme fusion protein

[0255] In this embodiment, the biotinylation labeling capability of the nanobody-photocontrolled proximity labeling enzyme fusion protein was further verified.

[0256] After fixing, permeabilizing, and blocking U-2OS cells, they were incubated with rabbit-derived anti-PEX14 antibody (a) or mouse-derived anti-Tubulin antibody (b), and nanobodies of the corresponding species-based photosensitive adjacent labeling enzymes were added. The experimental and control groups were treated with light and without light, respectively, followed by biotinylation. After the reaction, immunofluorescence staining and imaging were performed.

[0257] The results are shown in Figure 15. In the control group without light treatment, the biotinylate signal was almost invisible, indicating that the fusion protein was in a state of inhibited enzyme activity. After light treatment, Alexa Fluor TM 647-coupled secondary antibody signal and Alexa Fluor TM Co-localization of the biotinylated protein signal by 488-conjugated streptavidin demonstrated the high localization accuracy of the fusion protein and the recovery of enzyme activity after light irradiation.

[0258] The technical solutions disclosed herein can reveal protein interaction networks and modification patterns in cells and tissues in greater detail and comprehensiveness. This will help to deepen our understanding of the regulatory mechanisms of biological processes and provide more precise targets and strategies for drug development and disease treatment. At the same time, this technology also has broad application prospects and can play an important role in the field of spatial proteomics.

[0259] The above description is only a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the scope of protection of this application.

Claims

1. A fusion protein for use with a proximity label, wherein, The fusion protein is operatively linked together by (a) a nanobody and (b) a neighboring labeling enzyme; a peptide linker is also included between the nanobody and the neighboring labeling enzyme.

2. The fusion protein according to claim 1, wherein, The adjacent labeling enzyme comprises peroxidase and / or biotin ligase; Preferably, the peroxidase is horseradish peroxidase (HRP) or ascorbate peroxidase; Preferably, the ascorbic acid peroxidase is APEX or APEX2; Preferably, the biotin ligase is selected from any one of Mini TurboID, TurboID, AirID, BioID, BASU, or BirA; Preferably, the biotin ligase is TurboID; Preferably, the bio-ligase is an engineered, light-controlled TurboID; Preferably, the amino acid sequence of the light-controlled TurboID is as shown in any one of SEQ ID NO.25-29.

3. The fusion protein according to claim 1 or 2, wherein, The nanobody is a primary antibody that targets a target protein or a secondary antibody that targets an immunoglobulin; Preferably, the immunoglobulin is rabbit immunoglobulin or mouse immunoglobulin; Preferably, the immunoglobulin is selected from IgG, IgM, IgD, IgE, IgA, or IgY; Preferably, the nanobody is a secondary antibody targeting IgG, and the secondary antibody specifically binds to a primary antibody targeting the target protein; Preferably, the amino acid sequence of the nanobody is as shown in SEQ ID NO.1, SEQ ID NO.11 or SEQ ID NO.

18.

4. The fusion protein according to claim 1 or 2, wherein, The nanobody is a nanobody that recognizes GFP; Preferably, the amino acid sequence of the nanobody that recognizes GFP is shown in SEQ ID NO.

46.

5. The fusion protein according to any one of claims 1-4, wherein, The peptide linker can be a flexible linker or a rigid linker. Preferably, the peptide linker is a flexible linker; Preferably, the amino acid sequence of the flexible connector is as shown in SEQ ID NO.

4.

6. The fusion protein according to any one of claims 1-5, wherein, The nanobody and the adjacent labeled enzyme are linked via a click chemistry reaction.

7. The fusion protein according to any one of claims 1-6, wherein, The nanobody and the adjacent labeled enzyme are fused together in any of the following ways: (a) The C-terminus of the nanobody is linked to the N-terminus of the adjacent labeled enzyme; or (b) The N-terminus of the nanobody is linked to the C-terminus of the adjacent labeled enzyme.

8. The fusion protein according to any one of claims 1-7, wherein, The amino acid sequence of the fusion protein is selected from: (a) A polypeptide having an amino acid sequence as shown in any one of SEQ ID NO. 5, 6, 12, 13, 19, 20, 30-39, 47; or (b) A polypeptide that is homologous to or has at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity with an amino acid sequence of any one of SEQ ID NO. 5, 6, 12, 13, 19, 20, 30-39, or 47, and is capable of labeling a neighboring protein molecule that interacts with the target protein; or (c) A protein or polypeptide derived from the insertion, substitution or deletion of one or more amino acids in the amino acid sequence of (a) or (b), which can be used to label neighboring protein molecules that interact with the target protein.

9. The fusion protein according to any one of claims 1-8, wherein, The fusion protein further includes a protein tag selected from any one of GST, 6x-His, MBP, Flag, HA, cMyc, GFP, eGFP, eYFP, mCherry, AviTag, or SUMO tags. Preferably, the protein tag is a 6xHis and a Flag tag.

10. The fusion protein according to any one of claims 1-9, wherein, The target protein is any intracellular or extracellular protein that can be recognized by nanobodies or immunoglobulins.

11. A nucleic acid molecule encoding the fusion protein of any one of claims 1-10.

12. A carrier comprising the nucleic acid molecule of claim 11.

13. A kit comprising the fusion protein according to any one of claims 1-10; Preferably, the kit further comprises a primary antibody targeting the target protein; Preferably, the kit further comprises a biotinylation reaction solution; Preferably, the kit further comprises streptavidin conjugated with a fluorescent agent; Preferably, the kit further comprises a fluorescently conjugated secondary antibody; Preferably, the biotinylation reaction solution comprises PBS, MgCl2, ATP, and biotin; Preferably, the biotinylation reaction solution contains biotin, phenol, and hydrogen peroxide.

14. A proximity marking method, characterized in that, Biotin labeling of a protein interacting with a target protein using the fusion protein according to any one of claims 1-10 or the kit according to claim 13 comprises the following steps: Add a primary antibody derived from rabbit or mouse species that targets the target protein to the cells and incubate them to allow the primary antibody to bind to the target protein; According to the species of the primary antibody, the corresponding fusion protein is added to the cell and incubated to form a neighboring labeling enzyme-nano secondary antibody-primary antibody-target protein complex in the cell; Add biotinylation reaction solution to cells, incubate, and biotinylate the proteins that interact with the target protein; After washing, streptavidin and secondary antibody conjugated with fluorescent dye were added separately, incubated, washed, and detected.

15. A method for analyzing intermolecular interactions, the method comprising the following steps: Biotin labeling of the protein interacting with the target protein is performed using the fusion protein according to any one of claims 1-10 or the kit according to claim 13; Biotin-labeled protein molecules can be enriched or fluorescently localized using magnetic beads or fluorescent agents coupled with streptavidin. The enriched biotin-labeled proteins were analyzed and identified by LC-MS / MS.

16. The use of the fusion protein according to any one of claims 1-10, the kit according to claim 13, or the method according to claim 14 or 15, wherein, The applications include: (a) Biotinylated proximity markers and component analysis of the cytoskeleton and organelles; (b) Biotinylated proximity markers and component analysis of post-modified histones; (c) Biotinylated proximity markers and component analysis of different nucleolar substructures; (d) Biotinylated proximity markers and interaction protein analysis of proteins in FFPE and OCT slices; (e) Double biotinylated neighbor markers in the same sample; or (f) Biotinylated proximity labeling and interaction protein analysis of proteins in model organisms.

Images