Phase-separated scaffold driven novel report system for biomolecular interaction, and use thereof

Abstract

A method for screening a biomolecule interaction regulator, a composition for screening a biomolecule interaction inhibitor, a genetically engineered cell for screening a biomolecule interaction inhibitor, and an apparatus for screening a biomolecule interaction regulator. The method for screening a biomolecule interaction regulator comprises: using an engineered cell, and detecting whether a ternary complex is present in the engineered cell as well as the increase and decrease changes in the number, so as to screen out an interaction regulator. The method has high detection speed and high sensitivity.

Description

Phase-separation backbone-driven novel biomolecular interaction reporter system and its applications Technical Field

[0001] The present invention belongs to the field of biological detection, and in particular, relates to a novel biomolecular interaction reporting system driven by a phase separation skeleton and applications thereof. Background Art

[0002] Monitoring biomolecular interactions in living cells plays a crucial role in biological and medical research. A variety of research tools have been developed for studying these interactions in vivo, including fluorescence resonance energy transfer (FRET) reporter assays and bimolecular fluorescence complementation assays. However, these tools have so far presented several complexities in imaging dispersed fluorescent proteins in living cells and animals. Key challenges include low fluorescence variability, complex signal patterns, tissue autofluorescence, light scattering, and rapid cell movement and shape changes, all of which complicate signal measurement and interpretation.

[0003] Currently, relevant detection methods in this field are prone to interfere with the expression, subcellular localization or biological function of interacting protein pairs and cannot be widely applied to various interacting molecules, especially proteins with larger molecular weight.

[0004] Therefore, there is still a need in this field to develop detection methods with low interference, especially suitable for large molecular weight interacting proteins, so as to more efficiently report interactions between biomolecules. Summary of the Invention

[0005] One object of the present invention is to provide a detection method and application thereof that has little interference and is particularly suitable for large molecular weight interacting proteins.

[0006] Another object of the present invention is to provide a product for screening biomolecular interaction modulators.

[0007] Another object of the present invention is to provide a device for screening biomolecular interaction modulators.

[0008] In a first aspect of the present invention, a method for screening a biomolecular interaction modulator is provided, comprising the following steps:

[0009] a. Providing an engineered cell, wherein the cell contains an aggregate, the aggregate contains a first fusion protein and a second fusion protein, and the cell further contains a third fusion protein; wherein the first fusion protein, the second fusion protein, and the third fusion protein form a ternary complex;

[0010] Wherein, the first fusion protein comprises a first connecting element L1, a first tag protein element C and a phase separation skeleton protein R;

[0011] The second fusion protein comprises a second connecting element L2, a second tag protein element A and a first interacting molecular element X;

[0012] The third fusion protein comprises a third tag protein element B and a second interaction molecule element X L (and / or E, F…);

[0013] Wherein, the first connecting element L1 and the second connecting element L2 have an interaction, and the first interacting molecular element X and the second interacting molecular element X L have interactions;

[0014] b. incubating the engineered cells in the presence of a candidate compound and detecting the presence and / or amount of the ternary complex;

[0015] Wherein, when the ternary complex exists and the amount of the ternary complex does not change significantly, the candidate compound is not the first interacting molecular element X and the second interacting molecular element X L Interaction moderators of

[0016] When the ternary complex does not exist or the amount of the ternary complex is significantly reduced, the candidate compound is the first interacting molecular element X and the second interacting molecular element X. L Interaction inhibitors of

[0017] When the ternary complex exists and the number of the ternary complex increases significantly, the candidate compound is the first interacting molecular element X and the second interacting molecular element X. L interaction enhancers.

[0018] In another preferred embodiment, the engineered cells are produced by transfecting (transiently transfecting) DNA sequence construct 1, DNA sequence construct 2 and DNA sequence construct 3 into cells, so that the cells co-express the aggregate and the third fusion protein;

[0019] Wherein, the DNA sequence construct 1 includes a first tag protein element C sequence, a phase separation backbone protein R sequence and a first connecting element L1 sequence;

[0020] The DNA sequence construct 2 includes a first interactive molecular element X sequence, a second tag protein element A sequence and a second connecting element L2 sequence; and

[0021] The DNA sequence construct 3 includes a second interactive molecular element X L sequence and the third tag protein element B sequence.

[0022] In another preferred embodiment, the sequence of the first fusion protein includes a sequence-phase separation scaffold protein R sequence-first connecting element L1 sequence;

[0023] The second fusion protein includes a second tag protein element A sequence-a first interaction molecule element X sequence-and a second linker element L2 sequence;

[0024] The third fusion protein includes a second interaction molecular element X L Sequence - the third tag protein element B sequence.

[0025] In another preferred embodiment, in step b, the ternary complex is detected under the same detection parameters.

[0026] In another preferred embodiment, the first connecting element L1 and the second connecting element L2 can form specific binding spontaneously or under induced stimulation.

[0027] In another preferred embodiment, the second tag protein element A and the second linking element L2 are the same.

[0028] In another preferred embodiment, the first tag protein element C and the first linking element L1 are the same.

[0029] In another preferred embodiment, the biomolecular interaction modulator refers to the first interaction molecular element X and the second interaction molecular element X L biomolecular interactions.

[0030] In another preferred embodiment, the first interacting molecular element X and the second interacting molecular element X L The interactions between them are constitutive or activatable.

[0031] In another preferred embodiment, the label protein is a fluorescent protein.

[0032] In another preferred example, the first tag protein element C, the second tag protein element A, and the third tag protein element B each independently emit different and distinguishable detectable signals.

[0033] In another preferred embodiment, before step b, it is detected whether the first fusion protein, the second fusion protein and the third fusion protein in the cell form the ternary complex.

[0034] In another preferred embodiment, during the detection process, when the signals of the first label protein element C, the second label protein element A and the third label protein element B overlap, it indicates that the ternary complex is formed, and the overlapping area is the location where the ternary complex exists.

[0035] In another preferred embodiment, during the detection process, when only the signals of the first label protein element C and the second label protein element A overlap, it indicates that the ternary complex is not formed, or the ternary complex does not exist, and the overlapping area is the location where the ternary complex exists.

[0036] In another preferred embodiment, the phase separation scaffold protein R has a sequence shown in SEQ ID NO: 1 to SEQ ID NO: 16.

[0037] In another preferred embodiment, the first interacting molecular element X and the second interacting molecular element X L (and / or E, F, ...) are each independently selected from the group consisting of a full-length protein, a mature protein, an intact protein, a protein fragment, a protein domain, or a nucleic acid.

[0038] In another preferred embodiment, the first interacting molecular element X has a homology of ≥70% (preferably ≥80%, more preferably ≥90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) with the complete sequence of an interacting molecular element X.

[0039] In another preferred embodiment, the second interactive molecular element X L For the second interaction molecular element X L The homology of the entire sequence is ≥70% 70% (preferably ≥80%, more preferably ≥90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%).

[0040] In another preferred embodiment, the first linking element L1 is a fluorescent tag protein or an artificially designed epitope tag.

[0041] In another preferred embodiment, the fluorescent tag protein is selected from GFP, EGFP, GFP variants, and mCherry, and the epitope tag is selected from LFA and BC2.

[0042] In another preferred embodiment, the second connecting element L2 is selected from an antigen-binding fragment (Fab), a single-chain variable fragment (scFv), a single-domain antibody fragment (VHH or nanobody), a polypeptide or an artificially designed binding fragment.

[0043] In a second aspect of the present invention, a composition for screening biomolecular interaction inhibitors is provided, the composition comprising:

[0044] A. a first fusion protein or its coding sequence or its expression vector, wherein the first fusion protein comprises a first connecting element L1, a first tag protein element C and a phase separation scaffold protein R;

[0045] B. a second fusion protein or its coding sequence or its expression vector, wherein the second fusion protein comprises a second connecting element L2, a second tag protein element A and a first interacting molecule element X; and

[0046] C. A third fusion protein or its coding sequence or its expression vector, wherein the third fusion protein comprises a third tag protein element B and a second interaction molecule element X L (and / or E, F…);

[0047] Wherein, the first connecting element L1 and the second connecting element L2 have an interaction, and the first interacting molecular element X and the second interacting molecular element X L Have interaction.

[0048] In another preferred embodiment, the first interacting molecular element X and the third interacting molecular element E interact with each other.

[0049] In another preferred embodiment, the first interacting molecular element X and the fourth interacting molecular element F interact with each other.

[0050] In a third aspect of the present invention, a genetically engineered cell for screening biomolecular interaction inhibitors is provided, the cell comprising:

[0051] The cell contains an aggregate, the aggregate contains a first fusion protein and a second fusion protein, and the cell also contains a third fusion protein; wherein the first fusion protein, the second fusion protein and the third fusion protein form a ternary complex;

[0052] Wherein, the first fusion protein comprises a first connecting element L1, a first tag protein element C and a phase separation skeleton protein R;

[0053] The second fusion protein comprises a second connecting element L2, a second tag protein element A and a first interacting molecular element X;

[0054] The third fusion protein comprises a third tag protein element B and a second interaction molecule element X L (and / or E, F…);

[0055] Wherein, the first connecting element L1 and the second connecting element L2 have an interaction, and the first interacting molecular element X and the second interacting molecular element X L (and / or E, F, ...) have interactions.

[0056] In another preferred embodiment, the cells are somatic cells.

[0057] In another preferred embodiment, the cells include prokaryotic cells and eukaryotic cells.

[0058] In another preferred embodiment, the cells are selected from the group consisting of bacteria, yeast cells, insect cells, mammalian cells, and plant cells.

[0059] In a fourth aspect of the present invention, there is provided an apparatus for screening a biomolecular interaction modulator, comprising:

[0060] a1. A test module, wherein the test module is used to place the test sample and apply the candidate compound, wherein the test sample contains the engineered cells as described above; the engineered cells contain a ternary complex;

[0061] a2. A data acquisition module configured to collect information on the presence and / or quantity of the ternary complex of the engineered cells;

[0062] a3. An interaction evaluation module, wherein the interaction evaluation module is configured to: evaluate whether the candidate compound is an interaction modulator based on the presence and / or amount of the ternary complex and provide an evaluation result;

[0063] Wherein, when the ternary complex exists and the amount of the ternary complex does not change significantly, the candidate compound is not the first interacting molecular element X and the second interacting molecular element X L Interaction moderators of

[0064] When the ternary complex does not exist or the amount of the ternary complex is significantly reduced, the candidate compound is the first interacting molecular element X and the second interacting molecular element X. L Interaction inhibitors of

[0065] When the ternary complex exists and the number of the ternary complex increases significantly, the candidate compound is the first interacting molecular element X and the second interacting molecular element X. L interaction enhancers;

[0066] a4. Output module, the output module is used for the evaluation results of the interaction evaluation module.

[0067] In another preferred embodiment, the sample to be tested is an organism or a cell.

[0068] In another preferred embodiment, the data acquisition module includes a fluorescence acquisition submodule, and the fluorescence acquisition submodule is configured to acquire detectable fluorescence signals respectively from the first fusion protein, the second fusion protein and the third fusion protein.

[0069] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1 is a schematic diagram of a new reporting system for biomolecular interactions driven by a phase separation framework.

[0071] FIG2 is an image showing the interaction between β-Catenin and BCL9 in HEK293T cells using GFP binding elements with different treatments (systems 1-4).

[0072] FIG3 is an image showing the interaction between β-Catenin and BCL9 in HEK293T cells using GFP binding elements with different treatments (systems 5-7).

[0073] FIG4 is an image showing the interaction between β-Catenin and BCL9 in HEK293T cells using GFP binding elements with different treatments (systems 8-11).

[0074] FIG5 is an image showing the interaction between P53 and MDM2 in HEK293T cells using GFP binding elements with different treatments (systems 12-15).

[0075] FIG6 is an image showing the interaction between P53 and MDM2 in HEK293T cells using GFP binding elements with different treatments (systems 16-19).

[0076] FIG7 is a quantitative analysis of the interaction between P53 and MDM2 in HEK293T cells using a GFP binding element and inhibitor treatment.

[0077] Figure 8 shows the interaction between IL-17 receptors and IL-17A in HEK293T cells using PDZ / KKETPV to connect different treatments.

[0078] FIG9 is a quantitative analysis of the interaction between IL-17 receptor and IL-17A in HEK293T cells using PDZ / KKETPV inhibitor-treated cells. DETAILED DESCRIPTION

[0079] After extensive and in-depth research, the inventors have developed a novel biomolecular interaction reporting system. This system utilizes specialized components to connect a phase-separated backbone with the biomolecules being tested, enabling independent and accurate detection of these interactions. This ensures that these interactions occur in a more natural manner within cells, offering the advantages of non-interference, high efficiency, and rapidity, providing a novel solution for biomolecular interaction research. This has led to the completion of the present invention.

[0080] the term

[0081] As used herein, the term "phase-separating scaffold protein R" is a natural or artificially synthesized protein (preferably an intrinsically disordered protein / region) that readily forms phase-separated aggregates or droplets spontaneously.

[0082] As used herein, the term "aggregates" are stable protein structures formed by the expression of phase-separating scaffold proteins and have dynamic fluidity.

[0083] As used herein, the term "interaction" or "interaction" refers to enzyme-substrate interactions, receptor-ligand interactions, and other intracellular protein-protein interactions with affinity. Inhibitors / promoters can participate in various post-translational modifications, such as phosphorylation, acetylation, dephosphorylation, and acetylation; they can also act directly at the structural interface of non-enzymatic macromolecules, such as blocking receptor-ligand binding.

[0084] As used herein, the terms "interacting biomolecules A" and "interacting biomolecules B" refer to a group of biomacromolecules that interact with each other, such as proteins, enzymes, and polypeptides. The biomacromolecules that can be used as "interacting biomolecules A" and "interacting biomolecules B" in this application can be signaling pathway proteins, for example, apoptosis pathway proteins: Bcl-2 family: including Bcl-2, Bcl-xl, etc., which regulate cell survival and apoptosis. Caspases: such as caspase-3, caspase-8, are key proteins that execute apoptosis. MDM2-p53 signaling pathway. Cell proliferation and survival signaling pathway proteins: PI3K-AKT-mTOR pathway: including PI3 kinase, Akt and mTOR, involved in cell growth, survival and metabolic regulation. MAPK pathway: including ERK, JNK and p38, involved in cell proliferation, differentiation and stress response. Cell surface receptors: receptor tyrosine kinase (RTK): such as EGFR, Insulin receptor, regulate growth factor signals. G protein-coupled receptors (GPCRs), such as the β-adrenergic receptor, regulate cell signaling. Wnt / β-catenin signaling pathway: β-catenin is degraded under normal conditions but accumulates upon Wnt signaling activation, interacting with proteins such as BCL9 and TCF4. JAK-STAT3 signaling pathway.

[0085] The interactions for which inhibitors can be screened using the methods of the present application can be protein-protein interactions known in the art, for example, common macromolecular interaction pairs and their corresponding diseases and inhibitors.

[0086] As used herein, the term "fluorescent protein" refers to a class of luminescent proteins with stable chemical properties, strong penetrability, and colors that are easy to observe under a microscope, including autofluorescent proteins and tagged proteins excited by fluorescent ligands. Among them, autofluorescent proteins include but are not limited to GFP, EGFP, TagBFP, mNeon, mOrange, mK02, tdTomato, mCherry, FusionRed, mApple, IFP2.0, mIFP and TagRFP657 and their respective mutants; tagged proteins excited by fluorescent ligands are preferably Halo and SNAP. As used herein, tagged protein F1, tagged protein F2, and tagged protein F3 respectively label phase separation scaffold protein R, interacting biomolecule A, and interacting biomolecule B, and the three emit different fluorescence colors.

[0087] A novel reporting system for biomolecular interactions driven by a phase-separating backbone

[0088] The present invention discloses a novel reporter system for measuring interactions between biomolecules to meet the demand for efficient and universal detection methods in this field. The system uses natural or synthetic proteins as the skeleton of phase-separated coacervates, which are independently expressed in cells and form stable and independent phase-separated coacervates. Through specific elements, the separately expressed phase-separated skeleton proteins are connected to the interacting biomolecules and visualized using fluorescent tags. When protein-protein interaction occurs or the interaction between biomolecules is activated, the combination of interaction factors drives the formation of a multi-component complex in the phase-separated coacervate, resulting in local phase-change coacervates with high concentrations of fluorescent tags. The results are characterized by the separation and aggregation of different molecular signals in highly concentrated local spots.

[0089] Specifically, as shown in FIG1 , it is known that there are biomolecules X and X that interact with each other. L , where X and X L It can be a protein, nucleic acid or polysaccharide; R is a natural or synthetic protein or fragment that easily forms phase-separated aggregates or droplets; A, B, and C are reporter groups, including fluorescent reporter groups; L1 and L2 are connecting elements, and L1 and L2 have an interaction, including a PDZ / KKETPV combination; L3 is a binding element that interacts with A / B / C, including a GFP binding element (GBP).

[0090] The present invention uses a phase separation skeleton protein based on intrinsically disordered proteins / regions as a reporter protein. To achieve this goal, two different types of connecting elements are designed, each type is respectively bound to a phase separation skeleton protein and a partner of protein-protein interaction, and at least one fluorescent protein is further introduced for visualization. In living cells, the expression of the reporter protein leads to the spontaneous formation of highly concentrated local spots, each of which contains multiple interacting factors and at least one pair of copies of a fluorescent protein reporter gene through the presence of a connecting element. When the interaction between biomolecules is activated, the multiple copies in the spot will be connected to the interacting protein through complementary interactions to form a multi-complex, enriching the fluorescent marker in the phase-separated intracellular droplets. The kinetics of multi-complex formation are fast and efficient. Therefore, these local point structures with strong signals can be easily observed and quantified, realizing expression and imaging in cells and living organisms.

[0091] This invention utilizes a phase-separating scaffold based on intrinsically disordered proteins / regions as a reporter element, cleverly separating it from biomolecular interactions by improving existing technologies, avoiding potential contaminants and achieving specific detection. This novel strategy offers numerous advantages, providing a highly efficient and versatile system for monitoring biomolecular interactions.

[0092] In one embodiment, the methods of the present invention include novel tools for determining interactions between transcription factors.

[0093] In one embodiment, the methods of the present invention can be used to observe and / or quantify target biomolecular interactions and provide methods for elucidating factors that modulate target biomolecular interactions or for testing modulators of selected interactions.

[0094] In one embodiment, the method of the present invention is used for high-throughput screening of modulators targeting biomolecular interactions.

[0095] In one embodiment, the method of the present invention can be widely applied to cells and whole organisms, providing a new solution for studying interactions between biomolecules.

[0096] In one embodiment, the methods, products, and associated devices of the present invention are suitable for detecting and imaging biomolecular interactions under various conditions, including in vivo imaging of living animals and in vitro imaging of cells.

[0097] Specifically, the phase separation scaffold protein of the present invention has an amino acid sequence selected from SEQ ID NO: 1 to SEQ ID NO: 15, wherein LCD represents low complexity domain, and SEQ ID NO: 16 is the nucleotide sequence of SEQ ID NO: 1.

[0098] Compared with the prior art, the main advantages of the present invention include:

[0099] 1. No interference: This method uses specific elements to connect the phase-separated backbone and interacting biomolecules, avoiding the potential interference with the interaction caused by direct fusion expression. Compared to existing technologies, this method ensures the accuracy and specificity of the interaction, resulting in more reliable experimental results.

[0100] 2. Native State: The independent expression of the phase-separated framework and interacting biomolecules allows interactions to occur in a more natural state within the cell. Phase-separated aggregates serve only as a detection site for interactions, more closely resembling the actual interactions in vivo and improving the reliability and comparability of experimental results.

[0101] 3. Efficient and Rapid: The independent expression of the phase-separated backbone enables more efficient and rapid detection of different biomolecular interactions. Simply expressing the test biomolecule combination within cells using traditional methods eliminates the need for designing complex fusion expression constructs, significantly improving experimental convenience and efficiency, saving research time and costs.

[0102] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples, for which specific conditions are not specified, were generally performed under conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0103] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those familiar to those skilled in the art. Furthermore, any methods and materials similar or equivalent to those described herein can be applied to the methods of the present invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0104] Material:

[0105] Fetal bovine serum: purchased from GIBCO, ThermoFisher Scientific

[0106] Activated carbon-treated fetal bovine serum: purchased from VivaCell

[0107] Pen-Strep: purchased from GIBCO, ThermoFisher Scientific

[0108] MI-773 small molecule: purchased from MCE

[0109] RG-7388 small molecule: purchased from MCE

[0110] DMEM: purchased from GIBCO, ThermoFisher Scientific

[0111] 96-well flat-bottom imaging plate (black bottom and transparent cover): purchased from Perkin Elmer

[0112] The human renal epithelial cell line HEK293T was purchased from the National Model and Specialty Experimental Cell Resource Bank (https: / / www.cellbank.org.cn / ) and maintained in DMEM containing 10% fetal bovine serum and 100 units / ml Pen-Strep at 37°C and 5% CO2.

[0113] Example 1: Application of Novel Linker Design in Detection of β-Catenin / BCL9 Interaction

[0114] 1. Preparation of recombinant vector

[0115] Artificially synthesized encoding BFP-NUP98N-GBP, encoding BFP-NUP98N, encoding BFP-GBP, encoding mCherry-β-Catenin, encoding GFP-BCL9, encoding GFP-BCL9 340-400aa DNA molecules (as shown in sequence numbers 1 to 6 in Table 1 below). Among them, the GFP binding protein is preferably a high-affinity single-domain antibody (sequence number 7) that specifically binds to GFP. The obtained DNA molecules were inserted into the pcDNA3.1 vector (Invitrogen), and the DNA fragment (containing enzyme recognition sequence) between the HindIII and EcoRI restriction enzyme site recognition sequences was replaced to obtain recombinant eukaryotic expression vectors pcDNA3.1-BFP-NUP98N-GBP, pcDNA3.1-BFP-NUP98N, pcDNA3.1-BFP-GBP, pcDNA3.1-mCherry-β-Catenin, pcDNA3.1-GFP-BCL9, pcDNA3.1-GFP-BCL9 340-400aa Plasmids were extracted using an endotoxin-free plasmid extraction kit (purchased from Jiangsu Kangwei Century Biotechnology Co., Ltd.) according to the manufacturer's instructions and stored at -20°C.

[0116] Table 1: Constructs used in Example 1

[0117] The NUP98N scaffold protein has an amino acid sequence as shown in SEQ ID NO: 1, and a nucleotide sequence as shown in SEQ ID NO: 16.

[0118] 2. Expression of recombinant DNA

[0119] Using the liposome transfection method generally known in the art, the purified DNA prepared in Example 1 was transiently expressed in cells as described below.

[0120] (1) Liposome transfection:

[0121] Prepare cell culture medium and seed HEK293T cells into a culture dish to achieve an appropriate cell density. According to the instructions of the liposome transfection reagent, the purified DNA prepared in Example 1 and the transfection reagent are mixed in a specified ratio to form a transfection complex.

[0122] Add the transfection complex to the cell culture medium, gently shake the culture dish to evenly distribute the complex on the cells, place the culture dish in a constant temperature incubator, and culture the cells for a certain period of time under appropriate conditions (such as 37° C., 5% CO 2 ).

[0123] (2) Expression detection:

[0124] Depending on the target protein being expressed, select an appropriate detection method. For example, you can use a fluorescence microscope to observe the expression of blue fluorescent protein (BFP), green fluorescent protein (GFP), or red fluorescent protein (mCherry) in cells.

[0125] 3. Verification of β-Catenin / BCL9 interaction

[0126] After culturing the transfected cell lines obtained in step 3 for 12-18 hours, images were collected using a laser confocal scanning microscope. The results (Figure 2 System 1-2) showed that the transfected cells had blue fluorescent signals (fluorescent signals emitted by BFP) that aggregated to form droplets. In addition, the transfected cells simultaneously expressed mCherry-β-Catenin and GFP-BCL9 or GFP-BCL9. 340-400aa Corresponding red fluorescent signals (fluorescent signals emitted by mCherry) and green fluorescent signals (fluorescent signals emitted by GFP) appeared and co-localized with the blue fluorescent signals.

[0127] In the control experiment, (1) the scaffold fusion protein linker deletion mutant BFP-NUP98N was expressed simultaneously with mCherry-β-Catenin and GFP-BCL9 or GFP-BCL9 340-400aa In the three-component control group, the three components did not co-localize due to the lack of the connecting element of the scaffold fusion protein (Figure 2 System 3-4);

[0128] (2) Simultaneous expression of the backbone fusion protein deletion mutant BFP-GBP with mCherry-β-Catenin and GFP-BCL9 or GFP-BCL9 340-400aa In the three-component control group, due to the simultaneous presence of specific interactions between GFP and GFP-binding protein (GBP), β-Catenin and BCL9, local co-localization of the three occurred (Figure 3, systems 5-6);

[0129] (3) In the two-component control group expressing both normal scaffold fusion protein and mCherry-β-Catenin, no co-localization of the two proteins occurred due to the absence of specific interaction (Figure 3, system 7);

[0130] (4) Simultaneous expression of normal backbone fusion protein and GFP-BCL9 or GFP-BCL9 340-400aa In the two-component control group, the specific interaction between GFP and GFP-binding protein resulted in the co-localization of the two in the droplets (Figure 4, systems 8-9);

[0131] (5) Simultaneous expression of mCherry-β-Catenin and GFP-BCL9 or GFP-BCL9 340-400aa In the two-component control group, the co-localization phenomenon of β-Catenin and BCL9 occurred due to the specific interaction between the two (Figure 4, systems 10-11).

[0132] Table 2: Detection system of β-Catenin / BCL9 interaction

[0133] Example 2: Application of novel linker design in P53 / MDM2 interaction detection

[0134] 1. Preparation of recombinant vector

[0135] Artificially synthesized DNA molecules encoding mCherry-P53 and GFP-MDM2 (shown in SEQ ID NOs. 8-9 in Table 3 below) were inserted into the pcDNA3.1 vector (Invitrogen), replacing the DNA fragment (containing the restriction enzyme recognition sequence) between the HindIII and EcoRI restriction enzyme recognition sites to generate the recombinant eukaryotic expression vectors pcDNA3.1-mCherry-P53 and pcDNA3.1-GFP-MDM2, respectively.

[0136] The plasmids were extracted using an endotoxin-free plasmid extraction kit (purchased from Jiangsu Kangwei Century Biotechnology Co., Ltd.) according to the manufacturer's instructions and stored at -20°C.

[0137] Table 3: Constructs used in Example 2

[0138] 2. Expression of recombinant DNA

[0139] Using the liposome transfection method generally known in the art, the purified DNA prepared in Example 2 was transiently expressed in cells as described below.

[0140] (1) Liposome transfection:

[0141] Prepare cell culture medium and seed HEK293T cells into a culture dish to achieve an appropriate cell density. According to the instructions of the liposome transfection reagent, the purified DNA prepared in Example 1 and the transfection reagent are mixed in a specified ratio to form a transfection complex.

[0142] Add the transfection complex to the cell culture medium, gently shake the culture dish to evenly distribute the complex on the cells, place the culture dish in a constant temperature incubator, and culture the cells for a certain period of time under appropriate conditions (such as 37° C., 5% CO 2 ).

[0143] (2) Expression detection:

[0144] Depending on the target protein being expressed, select an appropriate detection method. For example, you can use a fluorescence microscope to observe the expression of blue fluorescent protein (BFP), green fluorescent protein (GFP), or red fluorescent protein (mCherry) in cells.

[0145] 3. Verification of P53 / MDM2 interaction

[0146] After culturing the transfected cell line obtained in step 3 for 12-18 hours, images were collected using a laser confocal scanning microscope. The results (Figure 5, system 12) showed that the transfected cells had blue fluorescent signals (fluorescent signals emitted by BFP) that aggregated to form droplets. In addition, corresponding red fluorescent signals (fluorescent signals emitted by mCherry) and green fluorescent signals (fluorescent signals emitted by GFP) appeared when mCherry-P53 and GFP-MDM2 were simultaneously expressed, and these signals co-localized with the blue fluorescent signals.

[0147] In the control experiments, (1) the three-component control group that simultaneously expressed the scaffold fusion protein junction element deletion mutant BFP-NUP98N, mCherry-P53, and GFP-MDM2 did not show the co-localization phenomenon of the three components due to the lack of the junction element of the scaffold fusion protein (Figure 5 System 13);

[0148] (2) In the control group, which simultaneously expressed the backbone fusion protein deletion mutant BFP-GBP, mCherry-P53, and GFP-MDM2, local co-localization of the three occurred due to the specific interaction between GFP and GFP-binding protein, and between P53 and MDM2 (Figure 5, system 14);

[0149] (3) In the two-component control group expressing both normal scaffold fusion protein and mCherry-P53, no co-localization of the two proteins occurred due to the absence of specific interaction (Figure 5 System 15);

[0150] (4) In the dual-component control group, which simultaneously expressed the normal backbone fusion protein and GFP-MDM2, the co-localization phenomenon of the two occurred due to the specific interaction between GFP and GFP-binding protein (Figure 6, system 16);

[0151] (5) In the dual-component control group expressing mCherry-P53 and GFP-MDM2, co-localization of P53 and MDM2 occurred due to the specific interaction between P53 and MDM2 (Figure 6, system 17);

[0152] (7) After adding MI-773 or RG-7388, an antagonist of the interaction between P53 and MDM2, to system 12 (the final concentration of MI-773 or RG-7388 was 10 μM), similar image acquisition was performed using a laser confocal scanning microscope.

[0153] Figure 6, Systems 18-19, show that compared to before treatment (Figure 6, System 12), the number of red fluorescent signal clusters in droplets containing both blue and green fluorescent signals decreased, while the distribution and colocalization of the blue and green fluorescent signals remained unchanged. This indicates that MI-773 or RG-7388 significantly inhibits the interaction between P53 and MDM2.

[0154] Table 4: Detection system of P53 / MDM2 interaction

[0155] discuss

[0156] In natural systems, phase-separated droplets formed by multivalent proteins form unique structures within cells, creating specialized microenvironments. The key innovation of this invention lies in the use of specific components to connect the phase-separated framework to the interacting biomolecules to be tested. Compared with traditional methods of direct fusion expression, this method effectively avoids the potential interference of fusion expression with the interactions to be tested.

[0157] Compared to existing technologies, the independent expression of the phase-separated scaffold and interacting biomolecules allows for interactions to occur in a more natural state within the cell. The phase-separated aggregates serve solely as a site for detecting biomolecular interactions, thus ensuring the authenticity and specificity of these interactions. By providing a strong and specific signal as a unique indicator of biomolecular interactions, the method of this invention offers flexible customization, high observability, specificity without interference, and efficient quantification.

[0158] These innovations give the present invention important differences and significant advantages in the field of biomolecular interaction detection.

[0159] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. A method for screening biomolecular interaction modulators, characterized in that: The steps include: a. Providing an engineered cell, wherein the cell contains an aggregate, the aggregate contains a first fusion protein and a second fusion protein, and the cell further contains a third fusion protein; wherein the first fusion protein, the second fusion protein, and the third fusion protein form a ternary complex; Wherein, the first fusion protein comprises a first connecting element L1, a first tag protein element C and a phase separation skeleton protein R; The second fusion protein comprises a second connecting element L2, a second tag protein element A and a first interacting molecular element X; The third fusion protein comprises a third tag protein element B and a second interaction molecule element X L (and / or E, F…); Wherein, the first connecting element L1 and the second connecting element L2 have an interaction, and the first interacting molecular element X and the second interacting molecular element X L have interactions; b. incubating the engineered cells in the presence of a candidate compound and detecting the presence and / or amount of the ternary complex; Wherein, when the ternary complex exists and the amount of the ternary complex does not change significantly, the candidate compound is not the first interacting molecular element X and the second interacting molecular element X L Interaction modulators of When the ternary complex does not exist or the amount of the ternary complex is significantly reduced, the candidate compound is the first interacting molecular element X and the second interacting molecular element X. L Interaction inhibitors of When the ternary complex exists and the number of the ternary complex increases significantly, the candidate compound is the first interacting molecular element X and the second interacting molecular element X. L interaction enhancers.

2. The method according to claim 1, wherein The first tag protein element C, the second tag protein element A, and the third tag protein element B each independently emit different and distinguishable detectable signals.

3. The method according to claim 1, wherein Before step b, detecting whether the first fusion protein, the second fusion protein and the third fusion protein in the cell form the ternary complex.

4. The method according to claim 1, wherein The phase separation skeleton protein R has a sequence shown in SEQ ID NO: 1 to SEQ ID NO:

16.

5. The method according to claim 1, wherein The first interacting molecular element X and the second interacting molecular element X L (and / or E, F, ...) are each independently selected from the group consisting of a full-length protein, a mature protein, an intact protein, a protein fragment, a protein domain, or a nucleic acid.

6. The method according to claim 1, wherein The first connecting element L1 is a fluorescent tag protein or an artificially designed epitope tag.

7. The method according to claim 1, wherein The second connecting element L2 is selected from an antigen binding fragment (Fab), a single-chain variable fragment (scFv), a single-domain antibody fragment (VHH or nanobody), a polypeptide or an artificially designed binding fragment.

8. A composition for screening biomolecular interaction inhibitors, characterized in that: The composition comprises: A. a first fusion protein or its coding sequence or its expression vector, wherein the first fusion protein comprises a first connecting element L1, a first tag protein element C and a phase separation scaffold protein R; B. a second fusion protein or its coding sequence or its expression vector, wherein the second fusion protein comprises a second connecting element L2, a second tag protein element A and a first interacting molecule element X; and C. A third fusion protein or its coding sequence or its expression vector, wherein the third fusion protein comprises a third tag protein element B and a second interaction molecule element X L (and / or E, F…); Wherein, the first connecting element L1 and the second connecting element L2 have an interaction, and the first interacting molecular element X and the second interacting molecular element X L (and / or E, F, ...) have interactions.

9. A genetically engineered cell for screening biomolecular interaction inhibitors, characterized in that: The cells include: The cell contains an aggregate, the aggregate contains a first fusion protein and a second fusion protein, and the cell also contains a third fusion protein; wherein the first fusion protein, the second fusion protein and the third fusion protein form a ternary complex; Wherein, the first fusion protein comprises a first connecting element L1, a first tag protein element C and a phase separation skeleton protein R; The second fusion protein comprises a second connecting element L2, a second tag protein element A and a first interacting molecular element X; The third fusion protein comprises a third tag protein element B and a second interaction molecule element X L (and / or E, F…); Wherein, the first connecting element L1 and the second connecting element L2 have an interaction, and the first interacting molecular element X and the second interacting molecular element X L Have interaction.

10. A device for screening biomolecular interaction modulators, characterized in that: include: a1. A test module, wherein the test module is used to place the test sample and apply the candidate compound, wherein the test sample contains the engineered cells as described above; the engineered cells contain a ternary complex; a2. A data acquisition module configured to collect information on the presence and / or quantity of the ternary complex of the engineered cells; a3. An interaction evaluation module, wherein the interaction evaluation module is configured to: evaluate whether the candidate compound is an interaction modulator based on the presence and / or amount of the ternary complex and provide an evaluation result; Wherein, when the ternary complex exists and the amount of the ternary complex does not change significantly, the candidate compound is not the first interacting molecular element X and the second interacting molecular element X L Interaction modulators of When the ternary complex does not exist or the amount of the ternary complex is significantly reduced, the candidate compound is the first interacting molecular element X and the second interacting molecular element X. L Interaction inhibitors of When the ternary complex exists and the number of the ternary complex increases significantly, the candidate compound is the first interacting molecular element X and the second interacting molecular element X. L interaction enhancers; a4. Output module, the output module is used for the evaluation results of the interaction evaluation module.

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