Novel phase separation skeleton driven biomolecular interaction reporting system and application thereof

Abstract

The invention discloses a novel phase separation skeleton driven biomolecule interaction reporting system and application thereof. Specifically, the invention provides a method for screening the biomolecule interaction regulator, and the method comprises the following steps: using engineered cells, and detecting whether ternary complexes exist in the engineered cells and the increase and decrease change of the number of the ternary complexes so as to screen out the interaction regulator. The method disclosed by the invention is high in detection speed and high in sensitivity.

Description

Technical Field

[0001] The present invention belongs to the field of biological detection. Specifically, the present invention relates to a novel reporting system for biomolecular interactions driven by a phase separation scaffold and its applications. Background Art

[0002] Monitoring biomolecular interactions in living cells plays an important role in biological and medical research. Currently, a variety of research tools have been developed for studying in vivo interactions, including fluorescence resonance energy transfer (FRET) reporters and bimolecular fluorescence complementation assays. However, to date, these tools have some complexities in monitoring the imaging of fluorescent proteins dispersed in living cells and animals. The main challenges include low fluorescence changes, complex signal patterns, tissue autofluorescence, light scattering, as well as rapid cell movement and shape changes, which all make signal measurement and interpretation difficult.

[0003] Currently, related detection methods in this field are prone to interfering with the expression, subcellular localization or biological functions of interacting protein pairs, and cannot be widely applied to various interacting molecules, especially proteins with large molecular weights.

[0004] Therefore, there is still a need in this field to develop detection methods with little interference, especially suitable for detecting interacting proteins with large molecular weights, in order to more efficiently report biomolecular interactions. Summary of the Invention

[0005] One object of the present invention is to provide a detection method with little interference, especially suitable for detecting interacting proteins with large molecular weights, and its applications.

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

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

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

[0009] a. Provide an engineered cell containing condensates, wherein the condensates contain a first fusion protein and a second fusion protein, and a third fusion protein also exists in the cell; 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 linker element L1, a first tag protein element C and a phase separation scaffold protein R;

[0011] The second fusion protein described above comprises a second linking element L2, a second tag protein element A, and a first interacting molecule element X;

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

[0013] Wherein, the first linking element L1 and the second linking element L2 interact with each other, and the first interacting molecule element X and the second interacting molecule element X L Interact with each other;

[0014] b. Incubate the engineered cells in the presence of a candidate compound, and detect the presence and / or the quantity of the ternary complex;

[0015] Wherein, when the ternary complex is present and the quantity of the ternary complex does not change significantly, then the candidate compound is not an interaction regulator of the first interacting molecule element X and the second interacting molecule element X L ;

[0016] When the ternary complex is absent, or the quantity of the ternary complex decreases significantly, then the candidate compound is an interaction inhibitor of the first interacting molecule element X and the second interacting molecule element X L ;

[0017] When the ternary complex is present and the quantity of the ternary complex increases significantly, then the candidate compound is an interaction promoter of the first interacting molecule element X and the second interacting molecule element X L ;

[0018] In another preferred example, the engineered cells are obtained by transfecting (transient transfection) DNA sequence construct 1, DNA sequence construct 2, and DNA sequence construct 3 into cells, such that the cells co-express condensates and the third fusion protein;

[0019] Wherein, the DNA sequence construct 1 comprises a first tag protein element C sequence, a phase separation scaffold protein R sequence, and a first linking element L1 sequence;

[0020] The DNA sequence construct 2 comprises a first interacting molecule element X sequence, a second tag protein element A sequence, and a second linking element L2 sequence; and

[0021] The DNA sequence construct 3 comprises a second interacting molecule element X L sequence and a third tag protein element B sequence.

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

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

[0024] The third fusion protein includes the second interacting molecule element X L sequence - third tag protein element B sequence.

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

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

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

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

[0029] In another preferred example, the biomolecular interaction regulator refers to the biomolecular interaction between the first interacting molecule element X and the second interacting molecule element X L therebetween.

[0030] In another preferred example, the first interacting molecule element X and the second interacting molecule element X L have a constitutive or activatable interaction therebetween.

[0031] In another preferred example, the tag 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 example, 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 example, during the detection, when the signals of the first tag protein element C, the second tag protein element A, and the third tag protein element B overlap, it indicates that the ternary complex is formed, and the overlapping region is the location where the ternary complex exists.

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

[0036] In another preferred embodiment, the phase separation scaffold protein R has the sequences shown in SEQ ID NO: 1 - 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: full-length protein, mature protein, intact protein, protein fragment, protein domain, or 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 interacting molecular element X L 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 the second interacting molecular element X. L

[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, mCherry, and the epitope tag is selected from LFA, BC2. <d000113>

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

[0043] In the second aspect of the present invention, there is provided a composition for screening inhibitors of biomolecular interactions, 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 linking 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 linker 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 interacting molecule element X L (and / or E, F...);

[0047] wherein, the first linker element L1 and the second linker element L2 interact with each other, and the first interacting molecule element X and the second interacting molecule element X L interact with each other.

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

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

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

[0051] The cell contains condensates, the condensates contain a first fusion protein and a second fusion protein, and a third fusion protein is also present in the cell; 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 linker element L1, a first tag protein element C, and a phase separation scaffold protein R;

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

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

[0055] wherein, the first linker element L1 and the second linker element L2 interact with each other, and the first interacting molecule element X and the second interacting molecule element X L (and / or E, F...) interact with each other.

[0056] In another preferred embodiment, the cell is a somatic cell.

[0057] In another preferred embodiment, the cell includes prokaryotic cells, eukaryotic cells.

[0058] In another preferred example, 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 a device for screening regulators of biomolecular interactions, comprising:

[0060] a1. A test module for placing a sample to be tested and applying a candidate compound, wherein the sample to be tested contains the engineered cells as described above; and the engineered cells contain a ternary complex;

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

[0062] a3. An interaction evaluation module configured to: evaluate whether the candidate compound is an interaction regulator based on the presence and / or the quantity of the ternary complex and give an evaluation result;

[0063] Wherein, when the ternary complex is present and the quantity of the ternary complex does not change significantly, then the candidate compound is not an interaction regulator of the first interaction molecular element X and the second interaction molecular element X L ;

[0064] When the ternary complex is absent, or the quantity of the ternary complex decreases significantly, then the candidate compound is an interaction inhibitor of the first interaction molecular element X and the second interaction molecular element X L ;

[0065] When the ternary complex is present and the quantity of the ternary complex increases significantly, then the candidate compound is an interaction promoter of the first interaction molecular element X and the second interaction molecular element X L ;

[0066] a4. An output module for the evaluation result of the interaction evaluation module.

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

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

[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 specifically described 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 elaborated one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0071] Figure 2 It is the imaging observation of the interaction between β-Catenin and BCL9 treated differently using a GFP binding element in HEK293T cells (Systems 1-4).

[0072] Figure 3 It is the imaging observation of the interaction between β-Catenin and BCL9 treated differently using a GFP binding element in HEK293T cells (Systems 5-7).

[0073] Figure 4 It is the imaging observation of the interaction between β-Catenin and BCL9 treated differently using a GFP binding element in HEK293T cells (Systems 8-11).

[0074] Figure 5 It is the imaging observation of the interaction between P53 and MDM2 treated differently using a GFP binding element in HEK293T cells (Systems 12-15).

[0075] Figure 6 It is the imaging observation of the interaction between P53 and MDM2 treated differently using a GFP binding element in HEK293T cells (Systems 16-19).

[0076] Figure 7 It is the quantitative analysis of the interaction between P53 and MDM2 treated with an inhibitor using a GFP binding element in HEK293T cells.

[0077] Figure 8 It is the imaging observation of the interaction between the IL-17 receptor and IL-17A treated differently using a PDZ / KKETPV linker in HEK293T cells.

[0078] Figure 9 It is the quantitative analysis of the interaction between the IL-17 receptor and IL-17A treated with an inhibitor using a PDZ / KKETPV in HEK293T cells. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0079] After extensive and in-depth research, the inventors have provided, for the first time, a brand-new biomolecular interaction reporting system. The present invention uses specific elements to connect the phase separation scaffold with the biomolecule to be detected, achieving independent and accurate detection of the interaction, ensuring that the interaction occurs in a more natural state within the cell, and having the advantages of no interference effect and high efficiency and speed, providing a new solution for the study of biomolecular interactions. Based on this, the inventors have completed the present invention.

[0080] Term

[0081] As used herein, the term "phase separation scaffold protein R" is a natural or synthetic protein (preferably intrinsically disordered protein / region) that is prone to spontaneously form phase separation condensates or droplets.

[0082] As used herein, the term "condensate" refers to a stable protein structure formed by the expression of a phase separation scaffold protein and having dynamic fluidity.

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

[0084] As used herein, the terms "interacting biomolecule A" and "interacting biomolecule B" refer to a group of biomacromolecules with interactions, such as proteins, enzymes, polypeptides. The biomacromolecules that can be used as "interacting biomolecule A" and "interacting biomolecule B" in the present application can be signal 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, which are key proteins for executing apoptosis. MDM2-p53 signal pathway. Cell proliferation and survival signal pathway proteins: PI3K-AKT-mTOR pathway: including PI3 kinase, Akt, and mTOR, which participate in the regulation of cell growth, survival, and metabolism. MAPK pathway: including ERK, JNK, and p38, which participate in cell proliferation, differentiation, and stress response. Cell surface receptors: receptor tyrosine kinases (RTKs): such as EGFR, Insulin receptor, which regulate growth factor signals. G protein-coupled receptors (GPCRs): such as β-adrenergic receptor, which regulate cell signal transduction. Wnt / β-catenin signal pathway: β-catenin: is degraded under normal conditions, but accumulates when Wnt signal is activated and interacts with proteins such as BCL9, TCF4, etc. JAK-STAT3 signal pathway.

[0085] The interactions for screening inhibitors by the method of the present application can be protein-protein interactions known in the art

[0086] interactions, for example, common macromolecular interaction pairs and their corresponding diseases and inhibitors.

[0087]

[0088] 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 tag 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; tag proteins excited by fluorescent ligands are preferably Halo and SNAP. As used herein, tag protein F1, tag protein F2 and tag protein F3 label the phase separation scaffold protein R, interacting biomolecule A and interacting biomolecule B respectively, and the fluorescence colors emitted by the three are different.

[0089] Novel reporting system for biomolecular interactions driven by phase separation scaffolds

[0090] The present invention discloses a novel reporting system for measuring interactions between biomolecules to meet the needs of the field for efficient and universal detection methods. The described system uses natural or synthetic proteins as the scaffolds of phase separation condensates, enabling them to be independently expressed in cells and form stable and independent phase separation condensates. Through specific elements, the separately expressed phase separation scaffold proteins are connected to interacting biomolecules, and fluorescence tags are used for visualization. When protein-protein interactions or biomolecular interactions are activated, the combination of interacting factors drives the formation of multi-component complexes in the phase separation condensates, resulting in local phase transition condensates of high-concentration fluorescence tags, and the result is characterized by the separation and aggregation of different molecular signals in highly concentrated local spots.

[0091] Specifically, as Figure 1 , it is known that there are interacting biomolecules X and X L , where X and X LIt can be a protein, nucleic acid or polysaccharide; R is a natural or synthetic protein or fragment that is prone to spontaneously form phase-separated condensates or droplets; A, B, and C are reporter groups, including fluorescent reporter groups, etc.; L1 and L2 are linking elements, and there is an interaction between L1 and L2, including the PDZ / KKETPV combination, etc.; L3 is a binding element, and there is an interaction with A / B / C, including the GFP binding element (GBP), etc.

[0092] The present invention uses a phase-separating scaffold protein based on intrinsically disordered proteins / regions as a reporter protein. To achieve this goal, two different types of linking elements are designed, each type binds to a phase-separating scaffold protein and a partner of protein-protein interaction respectively, 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 spot containing multiple interacting factors and at least a pair of copies containing the fluorescent protein reporter gene through the presence of the linking element. When the interaction between biomolecules is activated, multiple copies in the spot will be linked to the interacting protein through complementary interactions to form a multi-component complex, enriching the fluorescent label in the phase-separated intracellular droplets. The kinetics of multi-component complex formation is fast and efficient. Therefore, these strongly-signaling local punctate structures can be easily observed and quantified, enabling expression and imaging in cells and living organisms.

[0093] The present invention uses a phase-separating scaffold protein based on intrinsically disordered proteins / regions as a reporter element. By improving the existing technology, it cleverly separates it from the interaction between biomolecules, avoiding potential interfering factors and achieving specific detection. Adopting this new strategy has many advantages, providing an efficient and universal system for monitoring the interaction between biomolecules.

[0094] In one embodiment, the method of the present invention includes a new tool for determining the interaction between transcription factors.

[0095] In one embodiment, the method of the present invention can be used to observe and / or quantify the interaction between target biomolecules, and provide a method for elucidating the factors regulating the interaction between target biomolecules or testing the modulators of the selected interaction.

[0096] In one embodiment, the method of the present invention is used for high-throughput screening of modulators targeting the interaction between biomolecules.

[0097] In one embodiment, the method of the present invention can be widely applied to cells and whole organisms, providing a brand-new solution for studying the interaction between biomolecules.

[0098] In one embodiment, the method, product and associated equipment of the present invention are applicable to detecting and imaging biomolecular interactions under various conditions, including in vivo imaging of living animals and in vitro cell imaging.

[0099] Specifically, the phase separation scaffold protein in the present invention has an amino acid sequence selected from SEQ ID NO: 1 - SEQ ID NO: 15, where LCD represents low complexity domain, a low complexity sequence. SEQ ID NO: 16 is the nucleotide sequence of SEQ ID NO: 1.

[0100]

[0101]

[0102] Compared with the prior art, the main advantages of the present invention include:

[0103] 1. No interference effect: The present invention uses specific elements to connect the phase separation scaffold and the interacting biomolecules, avoiding the interference with the interaction that may be caused by direct fusion expression. Compared with the existing technology, the method of the present invention ensures the accuracy and specificity of the interaction, making the experimental results more reliable.

[0104] 2. Natural state: The independent expression of the phase separation scaffold and the interacting biomolecules enables the interaction to occur in a more natural state within the cell. The phase separation condensate only serves as the detection site for the interaction, which is closer to the real situation of the interaction in the organism, improving the reliability and comparability of the experimental results.

[0105] 3. High efficiency and speed: The independent expression of the phase separation scaffold makes it more efficient and rapid to detect different biomolecular interactions. Just express the combination of biomolecules to be tested in the cell according to the traditional method, without the need to design a cumbersome fusion expression structure, greatly improving the operational convenience and efficiency of the experiment, and saving research time and costs.

[0106] The present invention will be further illustrated below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are generally carried out 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.

[0107] Unless otherwise defined, all professional and scientific terms used herein have the same meaning as those familiar to persons skilled in the art. In addition, any methods and materials similar or equivalent to those described may be applied to the method of the present invention. The preferred methods and materials described herein are for illustrative purposes only.

[0108] Materials:

[0109] Fetal bovine serum: Purchased from GIBCO, ThermoFisher Scientific

[0110] Charcoal-treated fetal bovine serum: Purchased from VivaCell

[0111] Pen-Strep: Purchased from GIBCO, ThermoFisher Scientific

[0112] MI-773 small molecule: Purchased from MCE

[0113] RG-7388 small molecule: Purchased from MCE

[0114] DMEM: Purchased from GIBCO, ThermoFisher Scientific

[0115] 96-well flat-bottom imaging plate (black bottom with clear lid): Purchased from Perkin Elmer

[0116] The human kidney epithelial cell line HEK293T was purchased from the National Resource Bank of Model and Characteristic Experimental Cells (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.

[0117] Example 1: Application of novel linker design in the detection of β-Catenin / BCL9 interaction

[0118] 1. Preparation of recombinant vectors

[0119] Synthetically encode BFP-NUP98N-GBP, encode BFP-NUP98N, encode BFP-GBP, encode mCherry-β-Catenin, encode GFP-BCL9, encode GFP-BCL9 340-400aaDNA molecules (shown as SEQ ID NOs: 1-6 in Table 1 below). Among them, the GFP-binding protein is preferably a high-affinity single-domain antibody (SEQ ID NO: 7) that specifically binds to GFP. The obtained DNA molecules were inserted into the pcDNA3.1 vector (Invitrogen), replacing the DNA fragment (including the restriction enzyme recognition sequence) between the HindIII and EcoRI restriction enzyme recognition sites, 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 According to the manufacturer's instructions, the plasmid was extracted using an endotoxin-free plasmid extraction kit (purchased from Nanjing Novoprotein Scientific Inc.), and stored at -20°C.

[0120] Table 1: Constructs used in Example 1

[0121]

[0122] Among them, the NUP98N backbone protein has the amino acid sequence shown in SEQ ID NO: 1, and its nucleotide sequence is as shown in SEQ ID NO: 16 below.

[0123] 2. Expression of recombinant DNA

[0124] Using the liposome transfection method commonly known in the art, the purified DNA prepared in Example 1 was used for transient cell expression as described below.

[0125] (1) Liposome transfection:

[0126] Prepare cell culture medium, and inoculate HEK293T cells into a culture dish to reach an appropriate cell density. According to the instructions of the liposome transfection reagent, the purified DNA prepared in Example 1 was mixed with the transfection reagent in a specified ratio to form a transfection complex.

[0127] The transfection complex was added to the cell culture medium, and the culture dish was gently shaken to evenly distribute the complex on the cells. The culture dish was placed in a constant temperature incubator and the cells were cultured under appropriate conditions (such as 37°C, 5% CO2) for a certain period of time.

[0128] (2) Expression detection:

[0129] According to the target protein to be expressed, select an appropriate method for detection. For example, fluorescence microscopy can be used to observe the expression of blue fluorescent protein (BFP) or / and green fluorescent protein (GFP) or / and red fluorescent protein (mCherry) in cells.

[0130] 3. Verification of β-Catenin / BCL9 interaction

[0131] After culturing the transfected cell line obtained in step 3 for 12 - 18 hours, image acquisition is performed using a laser confocal scanning microscope. The results ( Figure 2 Systems 1 - 2) show that there are droplets formed by the aggregation of blue fluorescent signals (fluorescent signals emitted by BFP) in the transfected cells. In addition, when mCherry-β-Catenin and GFP-BCL9 or GFP-BCL9 are co-expressed 340-400aa corresponding red fluorescent signals (fluorescent signals emitted by mCherry) and green fluorescent signals (fluorescent signals emitted by GFP) appear, and they co-localize with the blue fluorescent signals.

[0132] In the control experiment, (1) in the three-component control group co-expressing the deletion mutant BFP-NUP98N of the scaffold fusion protein linker element, mCherry-β-Catenin, and GFP-BCL9 or GFP-BCL9 340-400aa no co-localization of the three components occurs due to the deletion of the linker element of the scaffold fusion protein ( Figure 2 Systems 3 - 4);

[0133] (2) In the three-component control group co-expressing the deletion mutant BFP-GBP of the scaffold fusion protein, mCherry-β-Catenin, and GFP-BCL9 or GFP-BCL9 340-400aa local co-localization of the three components occurs due to the specific interactions between GFP and GFP-binding protein (GBP), and between β-Catenin and BCL9 ( Figure 3 Systems 5 - 6);

[0134] (3) In the two-component control group co-expressing the normal scaffold fusion protein and mCherry-β-Catenin, no co-localization of the two occurs due to the absence of specific interaction ( Figure 3 System 7);

[0135] (4) In the two-component control group co-expressing the normal scaffold fusion protein and GFP-BCL9 or GFP-BCL9 340-400aa droplet co-localization of the two occurs due to the specific interaction between GFP and GFP-binding protein ( Figure 4 Systems 8 - 9);

[0136] (5) Co - express mCherry - β - Catenin with GFP - BCL9 or GFP - BCL9 340-400aa of the two - component control group, due to the specific interaction between β - Catenin and BCL9, co - localization of the two appears ( Figure 4 System 10 - 11).

[0137] Table 2: Detection system for β - Catenin / BCL9 interaction

[0138]

[0139]

[0140] Example 2: Application of novel linker design in the detection of P53 / MDM2 interaction

[0141] 1. Preparation of recombinant vectors

[0142] Synthetic DNA molecules encoding mCherry - P53 and encoding GFP - MDM2 (shown as sequence numbers 8 - 9 in Table 3 below) are prepared. The obtained DNA molecules are inserted into the pcDNA3.1 vector (Invitrogen), replacing the DNA fragment (including the restriction enzyme recognition sequence) between the HindIII and EcoRI restriction enzyme recognition sites, respectively obtaining recombinant eukaryotic expression vectors pcDNA3.1 - mCherry - P53 and pcDNA3.1 - GFP - MDM2.

[0143] According to the manufacturer's instructions, use an endotoxin - free plasmid extraction kit (purchased from Kangwei Century Biotechnology Co., Ltd., Jiangsu) to extract the plasmids and store them at - 20°C.

[0144] Table 3: Constructs used in Example 2

[0145]

[0146] 2. Expression of recombinant DNA

[0147] Using the liposome transfection method commonly known in the art, transient cell expression is carried out for the purified DNA prepared in Example 2 as described below.

[0148] (1) Liposome transfection:

[0149] Prepare cell culture medium and seed HEK293T cells into a culture dish to reach an appropriate cell density. According to the instructions of the liposome transfection reagent, mix the purified DNA prepared in Example 1 with the transfection reagent in a specified ratio to form a transfection complex.

[0150] Add the transfection complex to the cell culture medium and gently shake the culture dish to evenly distribute the complex over the cells. Place the culture dish in an incubator and culture the cells under appropriate conditions (such as 37 °C, 5% CO2) for a certain period of time.

[0151] (2) Expression detection:

[0152] Select an appropriate method for detection according to the target protein to be expressed. For example, fluorescence microscopy can be used to observe the expression of blue fluorescent protein (BFP) or / and green fluorescent protein (GFP) or / and red fluorescent protein (mCherry) in cells.

[0153] 3. Verification of the P53 / MDM2 interaction

[0154] After culturing the transfected cell line obtained in step 3 for 12 - 18 hours, image acquisition is performed using a laser confocal scanning microscope. The results ( Figure 5 System 12) show that there are droplets formed by the aggregation of blue fluorescent signals (fluorescent signals emitted by BFP) in the transfected cells. In addition, the simultaneous expression of mCherry-P53 and GFP-MDM2 shows corresponding red fluorescent signals (fluorescent signals emitted by mCherry) and green fluorescent signals (fluorescent signals emitted by GFP), and they are co-localized with the blue fluorescent signals.

[0155] In the control experiment, (1) in the three-component control group that simultaneously expresses the deletion mutant BFP-NUP98N of the scaffold fusion protein linker element, mCherry-P53, and GFP-MDM2, no co-localization of the three occurs due to the deletion of the linker element of the scaffold fusion protein ( Figure 5 System 13);

[0156] (2) In the three-component control group that simultaneously expresses the deletion mutant BFP-GBP of the scaffold fusion protein, mCherry-P53, and GFP-MDM2, local co-localization of the three occurs due to the specific interactions between GFP and the GFP-binding protein, and between P53 and MDM2 ( Figure 5 System 14);

[0157] (3) In the two-component control group that simultaneously expresses the normal scaffold fusion protein and mCherry-P53, no co-localization of the two occurs due to the absence of specific interaction ( Figure 5 System 15);

[0158] (4) In the two-component control group that simultaneously expresses the normal scaffold fusion protein and GFP-MDM2, co-localization of the two occurs due to the specific interaction between GFP and the GFP-binding protein ( Figure 6 System 16);

[0159] (5) The two-component control group co-expressing mCherry-P53 and GFP-MDM2 showed co-localization of the two due to the specific interaction between P53 and MDM2 ( Figure 6 System 17);

[0160] (7) After adding the P53-MDM2 interaction antagonist MI-773 or RG-7388 (final concentration of MI-773 or RG-7388 was 10 μM) to System 12, similar image acquisition was performed using a laser confocal scanning microscope.

[0161] Figure 6 Systems 18-19, and the results showed that compared with before treatment ( Figure 6 System 12), the number of red fluorescence signals aggregated in the droplets containing both blue and green fluorescence signals decreased, while the distribution and co-localization of blue and green fluorescence signals showed no obvious changes. It was indicated that MI-773 or RG-7388 could significantly inhibit the interaction between P53 and MDM2.

[0162] Table 4: Detection systems for P53 / MDM2 interaction

[0163]

[0164] Discussion

[0165] In natural systems, phase-separated droplets formed by multivalent proteins form unique structures within cells, creating special microenvironments. The greatest innovation of the present invention lies in using specific components to connect the phase-separation scaffold with the interacting biomolecules to be detected. Compared with the traditional method of direct fusion expression, this method can effectively avoid the interference of fusion expression on the interaction to be detected.

[0166] Compared with the prior art, the independent expression of the phase-separation scaffold and the interacting biomolecules enables the interaction to occur in a more natural state within cells. The phase-separation condensate only serves as a site for detecting biomolecular interactions, thus ensuring the authenticity and specificity of the interaction. Through strong and specific signals, as unique indicators of biomolecular interactions, the method of the present invention has the characteristics of being flexible and customizable, highly observable, specifically non-interfering, and efficiently quantifiable.

[0167] These innovations make the present invention have important differences and significant advantages in the field of biomolecular interaction detection.

[0168] All documents mentioned in this invention are cited herein for reference as if each individual document was cited for reference. In addition, it should be understood that after reading the above teachings of this invention, those skilled in the art can make various changes or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims

1. A method for screening a modulator of biomolecular interaction, characterized in that, Comprising the following steps: a. Provide an engineered cell containing condensates, the condensates containing a first fusion protein and a second fusion protein, and a third fusion protein also present in the cell; 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 linker element L1, a first tag protein element C and a phase separation scaffold protein R; The second fusion protein comprises a second linker element L2, a second tag protein element A and a first interaction molecule element X; The third fusion protein described above comprises a third tag protein element B and a second interacting molecule element X L (and / or E, F...); Among them, the first connecting element L1 and the second connecting element L2 interact with each other, and the first interacting molecular element X and the second interacting molecular element X L interact with each other; b. Incubate the engineered cell in the presence of a candidate compound and detect the presence and / or the quantity 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 an interaction regulator of the first interacting molecular element X and the second interacting molecular element X L ; When the ternary complex does not exist or the amount of the ternary complex is significantly reduced, the candidate compound is an interaction inhibitor of the first interacting molecular element X and the second interacting molecular element X L ; When the ternary complex exists and the quantity of the ternary complex increases significantly, then the candidate compound is an interaction promoter for the first interacting molecular element X and the second interacting molecular element X L 。 2. The method according to claim 1, characterized in that, 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, characterized in that, Before step b, detect 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, characterized in that The phase separation scaffold protein R has the sequences shown in SEQ ID NO:1 - 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 described above L (and / or E, F, …) are each independently selected from the group consisting of: full-length protein, mature protein, intact protein, protein fragment, protein domain, or nucleic acid.

6. The method according to claim 1, characterized in that The first linker element L1 is a fluorescent tag protein or an artificially designed epitope tag.

7. The method according to claim 1, wherein The second linker element L2 is selected from antigen-binding fragments (Fab), single-chain variable fragments (scFv), single-domain antibody fragments (VHH or nanobodies), polypeptides or artificially designed binding fragments.

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

9. A genetically engineered cell for screening inhibitors of biomolecular interactions, characterized in that, The cell comprises: The cell contains condensates, the condensates containing a first fusion protein and a second fusion protein, and a third fusion protein also present in the cell; 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 linker element L1, a first tag protein element C and a phase separation scaffold protein R; The second fusion protein comprises a second linker element L2, a second tag protein element A and a first interaction molecule element X; The third fusion protein described above comprises a third tag protein element B and a second interacting molecule element X L (and / or E, F...); Among them, the first connecting element L1 and the second connecting element L2 interact with each other, and the first interacting molecular element X and the second interacting molecular element X L interact with each other.

10. An apparatus for screening a modulator of biomolecular interaction, characterized in that, Comprises: a1. A test module for placing a sample to be tested and applying a candidate compound, wherein the sample to be tested contains the engineered cell as described above; the engineered cell contains a ternary complex; a2. A data acquisition module configured to acquire the presence and / or the quantity of the ternary complex of the engineered cell; a3. An interaction evaluation module configured to: evaluate whether the candidate compound is an interaction regulator based on the presence and / or the quantity of the ternary complex and give 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 an interaction regulator of the first interacting molecular element X and the second interacting molecular element X L ; When the ternary complex does not exist or the amount of the ternary complex is significantly reduced, the candidate compound is an interaction inhibitor of the first interacting molecular element X and the second interacting molecular element X L ; When the ternary complex exists and the number of the ternary complex increases significantly, the candidate compound is an interaction promoter for the first interacting molecular element X and the second interacting molecular element X L ; a4. Output module, which is used for the evaluation results of the interaction evaluation module.

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