High-efficiency RNA interaction proteome identification method based on heterozygous proximity marker tool

Abstract

The invention relates to a high-efficiency RNA (Ribonucleic Acid) interaction proteome identification method based on a heterozygous proximity marker tool. A brand-new peroxidase APEX2 compound is designed, the peroxidase APEX2 is combined to mRNA, the peroxidase APEX2 can catalyze biotin-phenol under the action of hydrogen peroxide to form biotin-phenoxy free radicals to carry out covalent labeling on proximity proteins, and then technologies such as affinity purification and mass spectrum identification can be utilized to carry out covalent labeling on the proximity proteins. The mRNA interaction protein is efficiently and specifically identified; then mRNA candidate interacting protein is obtained through bioinformatics analysis, finally, the interaction relation between RNA and the candidate protein is verified by combining CLIP, WB and other technologies, and efficient identification of RNA interaction proteome is achieved.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a high-efficiency RNA interaction proteome identification method based on a hybrid proximity labeling tool. Background Art

[0002] The genome enables a complex manifestation of cellular function through the dynamic expression of distinct RNAs. RNAs do not function in isolation in the busy cellular environment, but rather interact with RNA-binding proteins (RBPs) that either influence or are influenced by the fate and function of RNA. Furthermore, RNA-RBP interactions can trigger liquid-liquid phase separation and the formation of biomolecular condensates, thereby compartmentalizing cellular processes. Given the large number of RNAs and RBPs within cells, combinatorial interaction networks often drive complex regulatory outcomes.

[0003] Many RNA-binding proteins (RBPs) that have been discovered and studied share two common features: they contain structural features that explain their RNA binding, called RNA-binding domains (RBDs), and they have functional annotations relevant to RNA biology, such as involvement in RNA processing or expression, or as components of ribonucleoprotein (RNP) complexes such as the ribosome, spliceosome, or telomerase. These RBPs are often referred to as “conventional” or “orthodox” RBPs. Conventional RBPs also include proteins with enzymatic functions that chemically modify or structurally remodel RNA, as well as those that simply bind to RNA. The latter often recruit other effector molecules to interact with RNA. The interaction of RBPs with RNA is mediated by structurally well-defined RBDs, such as RNA recognition motifs (RRMs) or DEAD-DEAH helicase domains. Depending on their function, conventional RBPs have a wide range of substrate affinities and specificities. Overall, these RBPs can control the entire life cycle of their substrates, including mRNA processing, trafficking, translation, and degradation.

[0004] However, hundreds of newly discovered RNA-binding proteins (RBPs) lack neither RNA-binding domains (RBDs) nor functional annotations related to RNA biology; instead, these RBPs are often well-studied proteins with very different functional annotations, including metabolic enzymes, ion channels or solute carriers, cytoskeletal proteins, or effector molecules of intracellular trafficking. Some of these RBPs are abundant cellular proteins, while others are not. These RBPs (or candidate RBPs before independent validation) are often referred to as unconventional, atypical, or non-classical RBPs. This suggests that the interaction between RNA and proteins is more complex than expected. Given the importance of messenger ribonucleoprotein (mRNP) complexes in almost all biological processes, their dysregulation can lead to a variety of diseases (including cancer and neurodegenerative diseases). For example, the human antigen R protein (HuR) binds to UDP-glucose, thereby affecting its binding to specific mRNAs; mutations in HuR can lead to increased mRNA stability in cancer cells and promote cancer cell invasiveness. FUS is involved in multiple aspects of RNA metabolism, and mutations can lead to protein mislocalization and aggregation, which in turn trigger neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). Therefore, in-depth research on the diversity and mechanism of RNA-protein interactions is of great significance for understanding gene expression regulation, cell fate determination and disease mechanisms.

[0005] There are currently two main strategies for detecting the interaction between RNA and protein. One is a protein-centric strategy, which is used to study RNA bound to the target protein; the other is an RNA-centric strategy, which is used to study proteins bound to the target RNA. Among them, the method based on UV cross-linking of RNA and protein can only cross-link directly interacting RNA and protein, and cannot effectively identify RBPs that interact indirectly. In addition, due to the low efficiency of UV cross-linking, a large amount of cell material is required, which is not suitable for rare cell samples. The method of using embedded nucleotide analogs to label newly transcribed RNA requires a long labeling time and is not suitable for studying transient interactions and dynamic changes of RNA-proteins. The method using phase-separated RNPs is not suitable for RNAs with low abundance, and unstable RNA-protein interactions may also be destroyed during the separation process.

[0006] In summary, how to achieve efficient identification of RNA-interacting proteomes is still one of the urgent problems to be solved in the field of RNA-protein interaction research. Summary of the invention

[0007] In order to solve the problem of how to achieve efficient identification of RNA interaction proteome, the present invention provides a high-efficiency RNA interaction proteome identification method based on a hybrid proximity labeling tool. The present invention designs an engineered peroxidase APEX2, and further develops proximity labeling complexes and RNA interaction protein labeling technology to achieve the establishment of a highly efficient and widely applicable RNA interaction protein research method.

[0008] To achieve this object, the present invention adopts the following technical solutions:

[0009] In a first aspect, the present invention provides an engineered peroxidase APEX2, wherein the structural formula of the engineered peroxidase APEX2 is as shown in Formula I;

[0010] AL-APEX2 formula I;

[0011] Wherein, A represents a non-natural amino acid containing an azide group, L represents a linker, and APEX2 represents peroxidase APEX2.

[0012] In the present invention, peroxidase APEX2 (Ascorbate Peroxidase 2) is redesigned, and a non-natural amino acid containing an azide group is connected to its N-terminus through a linker to obtain an engineered peroxidase APEX2, wherein the azide group can undergo a click chemistry (Click) reaction with dibenzocyclooctyne (DBCO) and the like. Therefore, a substance modified with dibenzocyclooctyne can be used to connect with the engineered peroxidase APEX2 to expand the application of peroxidase APEX2, such as connecting with a complementary sequence of mRNA, so that peroxidase APEX2 is connected to mRNA, and under the action of hydrogen peroxide, APEX2 catalyzes biotin phenol to form a biotin-phenoxy free radical to biotinylate proteins adjacent to mRNA.

[0013] It can be understood that the peroxidase APEX2 in the present invention refers to a tool enzyme for proximity labeling technology, and the APEX2 enzyme is used to catalyze the conversion of substrates (such as biotin phenol) into active free radicals, which can covalently bind to adjacent proteins, thereby achieving labeling of adjacent proteins. Peroxidase APEX2 with similar functions in the art is suitable for the present invention, for example, its amino acid sequence is shown in SEQ ID NO.1.

[0014] SEQ ID NO.1:

[0015] GKSYPTVSADYQDAVEKAKKKLRGFIAEKRCAPLMLRLAFHSAGTFDKGTKTGGPFGTIKHPAELAHSANNGLDIAVRLLEPLKAEFPILSYADFYQLAGVVAVEVTGGPKVPFHPGREDKPEPP PEGRLPDPTKGSDHLRDVFGKAMGLTDQDIVALSGGHTIGAAHKERSGFEGPWTSNPLIFDNSYFTELLSGEKEGLLQLPSDKALLSDPVFRPLVDKYAADEDAFFADYAEAHQKLSELGFADA.

[0016] Preferably, the non-natural amino acid containing an azide group includes a phenylalanine analog containing an azide group (p-azido-phenylalanine).

[0017] Preferably, the phenylalanine analog containing an azide group comprises at least one of p-azidophenylalanine, 3-azido-L-phenylalanine or 4-azido-L-phenylalanine.

[0018] Preferably, the linker comprises (Gly-Gly-Gly-Gly-Ser)n, Gly is glycine, Ser is serine, and n is a positive integer, for example, 1, 2, 3 or 4, etc.

[0019] In a second aspect, the present invention provides a method for preparing the engineered peroxidase APEX2 according to the first aspect, the preparation method comprising:

[0020] An amber stop codon is introduced into the peroxidase APEX2 coding gene, and an expression vector is inserted to obtain a recombinant vector, and the recombinant vector is introduced into a host cell, and the host cell also expresses an orthogonal aminoacyl tRNA synthetase and tRNA to obtain a recombinant cell, and the recombinant cell is cultured, and a non-natural amino acid containing an azide group is added to the culture medium. After the culture is completed, the culture fluid is collected, separated and purified to obtain the engineered peroxidase APEX2.

[0021] In the present invention, an amber stop codon (TAG) is first introduced into the APEX2 gene sequence at a specific site, and based on the site-specific insertion of non-natural amino acids, a system of engineered aminoacyl tRNA synthetases and corresponding tRNA orthogonal pairs (such as products developed by Ambrx) is used to precisely embed non-natural amino acids containing an azide group into the TAG position of the APEX2 protein.

[0022] Preferably, the culture temperature is 23-26°C, for example, it can be 23.5, 24, 24.5, 25 or 25.5°C, etc., preferably 24.5-25.5°C, and the culture time is 22-26h, for example, it can be 22.5, 23, 23.5, 24, 24.5, 25 or 25.5h, etc., preferably 23.5-24.5h.

[0023] In a third aspect, the present invention provides a proximity labeling complex, which includes the engineered peroxidase APEX2 described in the first aspect and a complementary nucleic acid sequence covalently linked thereto; the complementary nucleic acid sequence is complementary to the target RNA.

[0024] In the present invention, a nucleic acid sequence complementary to the target RNA is designed to be connected to the engineered peroxidase APEX2, so that the engineered peroxidase APEX2 can be bound to the RNA to label the protein interacting with the RNA.

[0025] It is understandable that the length and specific composition of the complementary nucleic acid sequence can be selected according to actual needs, as long as it can be complementary and paired with the target RNA.

[0026] Preferably, the length of the complementary nucleic acid sequence is 20 to 40 nt, for example, it can be 21, 22, 23, 24, 25, 30, 35, 36, 37, 38 or 39 nt.

[0027] Preferably, the complementary nucleic acid sequence comprises a polydT sequence.

[0028] In the present invention, the polydT sequence can complementarily pair with the polyA tail of mRNA.

[0029] In a fourth aspect, the present invention provides a method for preparing the proximity labeled complex according to the third aspect, the preparation method comprising:

[0030] The engineered peroxidase APEX2 described in the first aspect is mixed with the complementary nucleic acid sequence modified with dibenzocyclooctyne to obtain the proximity labeling complex.

[0031] Preferably, the molar ratio of the engineered peroxidase APEX2 to the complementary nucleic acid sequence modified with dibenzocyclooctyne is 1:(1-20), for example, it can be 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:10, 1:12, 1:14, 1:15, 1:18 or 1:19, etc., preferably 1:(9-11).

[0032] Preferably, the mixing temperature is 4 to 40°C, for example, it can be 5, 6, 7, 8, 10, 15, 20, 25, 30, 35, 36, 37, 38 or 39°C, etc., preferably 36 to 38°C, and the time is 1 to 5h, for example, it can be 1.5, 2, 2.5, 3, 3.5, 4 or 4.5h, etc., preferably 1.5 to 2.5h.

[0033] In the present invention, specific conditions are designed to further improve the binding efficiency of the engineered peroxidase APEX2 with the complementary nucleic acid sequence.

[0034] In a fifth aspect, the present invention provides a method for labeling RNA-interacting proteins, the method for labeling RNA-interacting proteins comprising:

[0035] The cells are pretreated, the pretreated cells are mixed and incubated with bovine serum albumin, the incubated cells are mixed with the proximity labeling complex described in the third aspect, biotin phenol and hydrogen peroxide are added, and the reaction is carried out.

[0036] Preferably, the pretreatment includes formaldehyde cross-linking treatment and cell perforation treatment.

[0037] The present invention further designs a method for labeling RNA-interacting proteins, wherein cells in a culture dish are cross-linked with formaldehyde to fix RNA-protein interactions under physiological conditions, the cells are perforated by disrupting the cell membrane with Triton or the like, and then bovine serum albumin (BSA) is added for incubation to block non-specific binding sites, and a neighboring labeled complex is added for incubation to allow it to specifically bind to mRNA. After completion of the incubation, the cells are washed to remove unbound complexes, and H2O2 is added to activate biotin phenol free radicals to allow it to covalently bind to neighboring proteins, thereby completing specific labeling of RNA-binding proteins.

[0038] Preferably, the reaction temperature is 2 to 30°C, for example, 3, 4, 5, 6, 7, 8, 10, 15, 20, 25, 26, 27, 28 or 29°C, etc., preferably 3 to 5°C, and the reaction time is 1 to 12 min, for example, 2, 3, 4, 5, 6, 7, 8 or 9 min, etc., preferably 11 to 12 min.

[0039] The specific labeling conditions designed in the present invention can further improve the labeling efficiency.

[0040] In a sixth aspect, the present invention provides a method for identifying an RNA interacting protein group, the RNA interacting protein group identification method comprising:

[0041] The RNA-interacting proteins are labeled using the method for labeling RNA-interacting proteins described in the fifth aspect, the labeled RNA-interacting proteins are separated and purified, and the separated and purified RNA-interacting proteins are identified.

[0042] Preferably, the separation and purification method includes a purification method based on biotin labeling, such as affinity purification using streptavidin agarose beads.

[0043] Preferably, the identification method includes Western Blot detection, liquid chromatography-mass spectrometry (LC-MS) and liquid chromatography-mass spectrometry (LC-MS / MS).

[0044] Preferably, based on the identified interacting protein spectrum data, the mass spectrometry data can be analyzed using MaxQuant and ProteomeDiscoverer software to obtain protein identification (ID) and quantitative information; the Limma package in R / Bioconductor is used to perform statistical analysis and screening of differential proteins, and the proteins that interact with mRNA are inferred through functional and interaction network analysis.

[0045] Compared with the prior art, the present invention has at least the following beneficial effects:

[0046] The present invention designs a new peroxidase APEX2 complex, and binds the peroxidase APEX2 to mRNA. The peroxidase APEX2 can catalyze biotin phenol to form biotin-phenoxy free radicals under the action of hydrogen peroxide to covalently label adjacent proteins, and can biotinylate the protein bound to the mRNA. Then, the mRNA interacting proteins can be efficiently and specifically identified by affinity purification and mass spectrometry identification and other technologies. Subsequently, the mRNA candidate interacting proteins are obtained through bioinformatics analysis, and finally, CLIP, WB and other technologies are combined to verify the interaction relationship between mRNA and candidate proteins, thereby achieving efficient identification of RNA interacting proteomes. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 This is a schematic diagram of the labeling principle of the proximity labeling complex of the present invention;

[0048] Figure 2 This is a schematic diagram of the process of RNA-interacting protein group identification;

[0049] Figure 3 This is a schematic diagram of the engineered peroxidase APEX2 expression vector;

[0050] Figure 4 The graphs are the results of (G4S)2-APEX2 and (G4S)3-APEX2 protein expression;

[0051] Figure 5 The graphs are the results of (G4S)1-APEX2 and (G4S)4-APEX2 protein expression;

[0052] Figure 6 This is the result of eluting (G4S)4-APEX2 protein on a nickel column with imidazole;

[0053] Fig. 7A The results of the labeling efficiency of Azf-APEX2 with different linker lengths are shown in the figure below. The left figure is a fluorescence image, and the right figure is a Coomassie Brilliant Blue staining image.

[0054] Figure 7B This is a diagram showing the results of the engineered peroxidase activity test;

[0055] Figure 8 The diagram shows the binding effect of engineered peroxidase and DBCO-modified polydT under different conditions;

[0056] Fig. 9 It is the gel electrophoresis diagram of the eluate of the anion exchange column of the proximity labeling complex;

[0057] Fig.10 The gel electrophoresis diagram of protein samples in each process of ultrafiltration concentration of proximity labeling complex;

[0058] Fig.11 This is the result of Western Blot detection of biotin protein under different labeling conditions;

[0059] Fig.12 This is the result of Western Blot testing the effect of BSA on labeling;

[0060] Fig.13 The result diagram of the labeling effect of different addition amounts of the proximity labeling complex;

[0061] Fig.14 This is the result of fluorescent staining to detect RNA and biotin in cells;

[0062] Fig.15 It is a bar graph showing the distribution of the number of RNA-interacting proteins;

[0063] Fig.16A This is the result diagram of hierarchical clustering analysis;

[0064] Fig. 16B It is a two-dimensional plane diagram of PCA analysis, with the abscissa being the first principal component and the ordinate being the second principal component. The distance between the points is approximately the sum of the differences in the abundance of each protein between the samples;

[0065] Fig.17A This is a graph of 1224 PrimAPEX identified proteins;

[0066] Fig. 17B Comparison of the Venn diagram for PrimAPEX identified proteins and annotated RBP database;

[0067] Fig.18 GO analysis of proteins identified by PrimAPEX. DETAILED DESCRIPTION

[0068] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and through specific implementation methods. However, the following examples are only simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0069] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0070] In order to achieve efficient identification of RNA-interacting proteomes, firstly, the present invention utilizes gene codon expansion technology to site-specifically introduce non-natural amino acids containing azide groups into peroxidase APEX2; APEX2 is combined with DBCO-modified complementary sequences (such as polydT) through click chemistry reaction, and then APEX2 is specifically bound to the vicinity of mRNA through complementary pairing of DBCO-modified complementary sequences and mRNA sequences to complete biotinylation labeling, and then affinity purification and mass spectrometry identification and other technologies are used to efficiently and specifically identify mRNA-interacting proteins; then, mRNA candidate interacting proteins are obtained through bioinformatics analysis, and finally, CLIP, WB and other technologies are combined to verify the interaction relationship between mRNA and candidate proteins.

[0071] (1) Development of “PrimAPEX” RNA-interacting protein labeling technology

[0072] In order to achieve APEX2 specific labeling of mRNA interacting proteins, the present invention uses Escherichia coli BL21 strain as a chassis cell, and on this basis constructs a system comprising an engineered aminoacyl tRNA synthetase and a corresponding tRNA orthogonal pair. The amber stop codon (TAG) is introduced into the APEX2 gene sequence by homologous recombination technology. The engineered aminoacyl tRNA synthetase can specifically connect the phenylalanine analog Azf (p-azido-phenylalanine, a non-natural amino acid) containing an azide group to its corresponding tRNA, and then accurately embed Azf in the TAG position of the APEX2 protein. Since the azide group contained in Azf can be used as a reactive group for bioorthogonal chemistry, the present invention can utilize click chemistry to couple Azf-APEX2 with a DBCO-modified complementary sequence through a cycloaddition reaction; and then, through the principle of base complementary pairing, the coupled complex is bound to mRNA, so that APEX2 can be efficiently and specifically guided to the vicinity of the polyA tail of mRNA. Under the action of hydrogen peroxide, APEX2 will catalyze biotin-phenol to form biotin-phenoxy free radicals to covalently label adjacent proteins (the technical principle diagram is shown in FIG. Figure 1 as shown), named “PrimAPEX”.

[0073] APEX2 protein containing Azf modification was obtained by protein purification, and the peroxidase activity of Azf-APEX2 and the embedding efficiency of Azf were tested. The binding efficiency of DBCO-modified polydT to Azf-APEX2 to form a polydT-APEX2 complex and the complementary pairing efficiency with mRNA were tested.

[0074] (2) Establishing RNA-interacting proteome identification method based on PrimAPEX technology

[0075] In order to efficiently identify RNA-interacting proteins, the present invention uses the commonly used laboratory cell line HELA as a model to establish a PrimAPEX method. Figure 2As shown in the figure, first, cells in the culture dish were cross-linked with formaldehyde to fix the RNA-protein interaction under physiological conditions, and the cells were perforated by disrupting the cell membrane with Triton, and then BSA was added for incubation to block non-specific binding sites, and polydT-APEX complex was added for incubation to make it specifically bind to mRNA. After the incubation, the cells were washed to remove the unbound polydT-APEX complex, and H2O2 was added to activate the biotin-phenol free radical, so that it covalently binds to the adjacent protein, thereby completing the specific labeling of RNA-binding protein. The cells successfully labeled with biotin were added to the lysis buffer, ultrasonically disrupted, and cross-linking was removed by high temperature, and then the protein concentration was determined by the BCA method. Streptavidin agarose beads were used to affinity purify the possible interacting proteins of mRNA, and a part of the agarose beads were used for Western Blot detection; the other part of the agarose beads were added to the trypsin buffer for digestion, cut into polypeptide fragments, and identified by LC-MS / MS.

[0076] Based on the above identified interacting protein spectrum data, the present invention uses MaxQuant and ProteomeDiscoverer software to analyze LC-MS / MS data to obtain protein identification (ID) and quantitative information. The Limma package in R / Bioconductor is used to perform statistical analysis and screening of differential proteins, and proteins that interact with mRNA are inferred through functional and interaction network analysis.

[0077] Example 1

[0078] In this example, the peroxidase APEX2 complex was prepared and the RNA-interacting protein labeling technology "PrimAPEX" was designed.

[0079] In order to precisely embed the phenylalanine analog Azf into the APEX2 protein, the Escherichia coli BL21 strain with an efficient expression system was selected as the chassis cell, and the pET28b(+) plasmid was used as the expression vector. A series of expression vectors containing TAG-linker of different lengths-APEX2 were constructed by homologous recombination technology ( Figure 3), with ATG connected as a control, and 6 histidine tags fused at the end to facilitate subsequent protein purification, and at the same time, the plasmid containing the aminoacyl-tRNA synthetase / tRNA orthogonal pair (aminoacyl-tRNA synthetase nucleic acid sequence is SEQ ID NO.2, amino acid sequence is SEQ ID NO.3; tRNA nucleic acid sequence is SEQ ID NO.4) was transferred into BL21 cells to form a strain expression system that can specifically recognize and insert pAzf (para-azidophenylalanine). A single colony of the protein expression vector was inoculated into 5 mL LB liquid culture medium (containing 50 μg / mL spe (spectinomycin), 100 μg / mL Amp (ampicillin), and placed at 37°C, 200 rpm shaker overnight culture, 4 mL of overnight culture solution was transferred to 500 mL LB liquid culture medium containing 1 mM pAzf for expansion culture, and the strain grew to OD 600 When the pH reached 0.6, IPTG was added to a final concentration of 1 mM, and the culture was continued at different expression temperatures and times to promote protein expression. The cells were collected by centrifugation, and the lysis solution (NaCl 0.5 mol / L, Tris-HCl 20 mmol / L, pH 8.0) was added to resuspend the cells, ultrasonically disrupted on ice, and filtered through a 0.45 μm filter membrane. APEX2 with a 6×His tag was purified using a nickel affinity column and eluted with an imidazole gradient. The purity of the purified protein was detected by SDS-PAGE. The protein concentration was determined by the BCA method.

[0080] SEQ ID NO.2:

[0081] 。

[0082] SEQ ID NO.3:

[0083] MDEFEMIKRNTSEIISEEELREVLKKDEKSAVIGFEPSGKIHLGHYLQIKKMIDLQNAGFDIIIYLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSEHGLDKDYTLNVYRLALKTTLKRARRSMELIAREDENPKVAEVIYPI MQVNGIHYEGVDVAVGGMEQRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSSKGNFIAVDDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTIKRPEKFGGDLTVNSYEELESLFKNKELHPMRLKNAVAEELIKILEPIRKRL.

[0084] SEQ ID NO.4:

[0085] ccggcggtagttcagcagggcagaacggcggactctaaatccgcatggcaggggttcaaatcccctccgccggacca.

[0086] The expression results of APEX2 protein at different expression temperatures and times are shown in Figure 2. Figure 4 and Figure 5 As shown, the expression level was higher when cultured at 25°C for 24 h.

[0087] The results of imidazole gradient elution are shown in 6, and relatively pure pAzF-APEX2 protein was obtained.

[0088] Detection of the embedding efficiency of pAzf in APEX2: Use the fluorescent dye DBCO-Cy5 for detection. Add 10μM DBCO-Cy5 to the purified pAzf-APEX2, incubate at room temperature for 30 minutes, and then perform SDS-PAGE. Use HEK293T cell protein lysate as a negative control for fluorescence development observation. HEK293T cell protein lysate does not fluoresce, while Azf-APEX2 with four different linker lengths all fluoresce ( Fig. 7A ), indicating that APEX2 is successfully embedded in Azf.

[0089] Detection of the catalytic activity of Azf-APEX2 with different linker lengths: Collect HEK293T cell protein lysate, add 1μM pAzf-APEX2, 500μM biotin-phenol and 1mM hydrogen peroxide to 160μg cell lysate for 1min, add 50mM sodium ascorbate to terminate the reaction, and develop the protein on Western Blot using streptavidin-horseradish peroxidase. The catalytic activity of the four Azf-APEX2 proteins was detected at the same time, and the results showed that the APEX2 labeling effects of the four different linkers were basically the same ( Figure 7B ). Based on the theory that the longer the linker length, the larger the labeling space formed, the Azf-APEX2 of (G4S)4 can be selected for subsequent experiments.

[0090] Optimizing the binding of Azf-APEX2 to DBCO-modified polydT: Azf-APEX2 was co-incubated with DBCO-modified polydT, and different molar ratios, incubation times, and temperatures were designed to select the conditions with the best binding effect. Figure 8 As shown, under the incubation conditions of 37°C, 1h incubation time and 1:10 molar ratio, the binding efficiency of pAzf-APEX2 protein and DBCO modified primer was the highest.

[0091] In order to remove proteins and primers that are not involved in the formation of polydT-APEX2 complex, anion exchange column was used to bind polydT-APEX2 complex, unbound proteins were eluted by low salt ion concentration, and then the polydT-APEX2 complex was eluted by increasing the salt ion concentration. The eluted protein (500-900mM NaCl) was selected by SDS-PAGE. Fig. 9 Finally, ultrafiltration was used to remove salt ions from the protein solution and concentrate the protein to obtain the final polydT-APEX2 complex ( Fig.10 ).

[0092] Example 2

[0093] This example uses the PrimAPEX method designed in Example 1 to identify RNA-interacting proteomes.

[0094] (1) Cell culture

[0095] About 8 × 10 6 HeLa cells (per sample) were incubated at 37°C and 5% CO2 until the cells reached 80% confluence (usually 1-2 days).

[0096] (2) Formaldehyde cross-linking and cell perforation

[0097] The cells were operated in a fume hood, and the DMEM medium was removed, 5 mL of 4% (v / v) formaldehyde was added, and the cells were incubated on a shaker at room temperature for 10 min. The formaldehyde was removed, 5 mL of 50 mM glycine was added, and the cells were incubated on a shaker at room temperature for 5 min. The cross-linking was stopped, the formaldehyde was removed, and the cells were washed three times with PBS with gentle shaking, 5 mL of 0.3% Triton-X100 was added, and the cells were incubated at room temperature for 10 min. The cell membranes were punctured, Triton-X100 was discarded, and the cells were washed three times with PBS with gentle shaking.

[0098] (3) APEX2 marker

[0099] Add 5mL 3% BSA to PBS, shake and incubate at room temperature for 1h, remove BSA, add 1μg polydT-APEX2 complex (diluted with 5mL 3% BSA in PBS) to each sample. Gently shake and incubate at room temperature for 1h, discard the supernatant, wash three times with PBST, wash twice with PBS, dissolve biotin phenol in PBS to a final concentration of 250μM, incubate the cells at room temperature for 30min, add H2O2 to a final concentration of 1mM to induce biotinylation, and test different temperatures (4℃, 25℃) and times (1min, 5min, 10min) for incubation.

[0100] Pour out the culture medium, add 50mM ascorbic acid, incubate at room temperature for 5min to terminate the reaction. Discard the supernatant, wash twice with PBS, add 150μL 4℃ RIPA lysis buffer (containing 50mM Tris-HCl (pH 7.5), 150mM NaCl, 1% (wt / vol) SDS, 0.5% (wt / vol) sodium deoxycholate, 1% (vol / vol) Triton X-100, 1mM EDTA and 1× protease inhibitor), incubate at 4℃ for 10min, and collect the cells with a cell scraper.

[0101] (4) Protein high temperature cross-linking

[0102] Ultrasonicate for 5 min (3 s on, 3 s off) at 30% power, shake and incubate at 95°C for 1 h to decrosslink. Centrifuge at 10,000×g for 10 min at 4°C, collect the supernatant (30 μL of supernatant from each sample was used as “input” for western blot analysis), and use the BCA kit to determine the concentration of each sample.

[0103] (5) Enrichment of biotinylated proteins with streptavidin agarose beads

[0104] Streptavidin agarose beads ("beads", 30 μL for each sample) were added to a centrifuge tube, centrifuged at 300 × g at 4°C for 2 min, the supernatant was discarded, and the beads were washed twice with RIPA buffer 2 (50 mM Tris-HCl (pH 7.5), 50 mM NaCl, 0.25% (w / v) SDS, 0.125% (w / v) sodium deoxycholate, 0.25% (v / v) Triton, 1 mM EDTA and 1× protease inhibitor). For each sample, 1 mg of protein was taken, and TBS was added to dilute the 1% SDS in the RIPA lysis buffer to 0.25%, and then placed on ice. Each sample was added to 30 μL of streptavidin agarose beads, incubated with rotation at 4°C for 2 h, centrifuged at 300 × g at 4°C for 2 min, washed twice with RIPA buffer 2, washed three times with 4 M Urea (dissolved in 50 mM Tris-HCl, pH 7.5), and washed three times with 30% After washing twice with ACN and three times with 50 mM TEAB, 1 mL was used for each wash. A portion of the agarose beads was used for Western Blot, and the other portion was digested with trypsin buffer, cut into peptide fragments, and identified by LC-MS / MS.

[0105] (6) Quality control analysis

[0106] For streptavidin Western Blot, 5× protein loading buffer was added to “beads” and boiled to elute biotinylated proteins. 4-20% SDS-PAGE was run using “Input” and “beads” proteins. The proteins were transferred to a 0.22 μm PVDF membrane (Millipore), blocked in 5% BSA in TBST for 1 h at room temperature, streptavidin-HRP (Beyotime, A0303, 1:5000 dilution) was added to 5% BSA in TBST, incubated at room temperature for 1 h, washed three times with TBST buffer, 5 min each time, developed using Clarity Western ECL substrate, and imaged using the ChemiDoc MP imaging system (Bio-Rad).

[0107] (7) Proteome analysis

[0108] For LC-MS / MS analysis, after enrichment and washing, the beads were resuspended in 200 μL of on-bead digestion buffer, 10 mM Tris(2-carboxyethyl)phosphine(TCEP) and 40 mM chloroacetamide(CAA) were added, and the beads were incubated at room temperature for 30 min. The beads were washed with 1 mL of on-bead digestion buffer, and then digestion buffer (50 mM Tris-HCl, 1 mM CaCl2 and 2% ACN, pH 8.0) containing 1 μg of mass spectrometry grade trypsin was added, and digestion was carried out at 37°C for 16 h. After digestion, centrifuge at 600 × g for 2 min, and the supernatant was collected into a new centrifuge tube. Buffer A (0.1% formic acid) was added to a final concentration of 0.01% to terminate the reaction. Before LC-MS / MS analysis, the peptide samples were desalted using StageTips, and the C18 material was loaded into a 200 μL pipette tip to make StageTips (6 layers).

[0109] (8) Processing of biological mass spectrometry data

[0110] Maxquant software was used to search the database and quantify proteins based on the mass spectrometry identification results. The reference database was the human protein database Swiss-Prot Human fasta. Variable modifications were set to methionine oxidation and N-terminal acetylation. Fixed modifications were set according to the experimental design, such as setting iodoacetamide alkylation of cysteine ​​as a fixed modification. The error ranges of the primary and secondary mass spectra were set according to the type of mass spectrometer used in the experiment (such as QE-HF mass spectrometry data, 20 ppm and 50 mmu, respectively). The minimum length of the peptide was 7, and the minimum number of independent peptides was 2. A positive and negative mixed library search strategy was used to evaluate the false positive rate of peptides, and peptides and proteins with FDRs less than 0.01 at the peptide and protein levels were retained. The iBAQ method was used for protein quantification.

[0111] (9) Use biostatistics and bioinformatics to analyze omics data

[0112] First, the data obtained from the search database were filtered for contaminant proteins, missing values ​​were filled, and intensity-based absolute quantification (iBAQ) was used as the quantitative basis. The Limma package in R / Bioconductor was used to perform statistical analysis and screening of differential proteins to infer protein complexes that specifically interact with specific RNAs. Secondly, the obtained differential proteins were annotated with functional, pathway, and network information: the DAVID tool (http: / / david.abcc.ncifcrf.gov / ) was used to extract the annotation information of the Biological process, Molecular function, and Cellular component categories of GO (Gene Ontology). The Ingenuity Pathway Analysis (IPA) analysis tool was used to annotate pathways and known regulatory networks. Cluster analysis was performed based on the K-mean algorithm, which was implemented using the Matlab tool; co-expression analysis was performed based on the WGCNA algorithm, which was implemented using the R toolkit.

[0113] By verifying the biotinylated protein by Western Blot, it was found that the biotinylated protein labeled at 25℃ for 10min increased significantly, followed by the group labeled at 4℃ for 10min. The labeling effects of the remaining conditions were basically the same ( Fig.11 ). In order to avoid nonspecific labeling caused by long labeling time and ensure labeling efficiency, the labeling condition of 4℃10min can be selected.

[0114] In order to explore the effect of adding BSA to incubate before labeling and blocking nonspecific sites on the labeling effect, a control group was further designed, including a group with pAzf-APEX2 alone or a group with DBCO-modified polydT alone. The experimental group was a group with polydT-APEX2 complexes, and the effect of adding or not adding BSA was tested respectively. The Western Blot results are shown in Figure 2. Fig.12 As shown, "-" indicates that the corresponding substance is not added, and "+" indicates that the corresponding substance is added, indicating that the nonspecific labeling is significantly reduced after the addition of BSA blocking, and the labeling effect of the polydT-APEX2 group is enhanced.

[0115] In order to verify the minimum amount of polydT-APEX2 complex, 0.5 μg, 1 μg, 2 μg, and 3 μg of polydT-APEX2 were added for labeling, and Streptavidin-HRP antibody was used to bind to biotinylated labeled protein. Western Blot results ( Fig.13 ) showed that adding 1 μg of the complex could achieve a good labeling effect.

[0116] In order to explore whether RNA and biotinylated protein co-localize, RNA and biotin were stained with fluorescent staining, respectively. The results are shown in Fig.14 As shown, it indicates that the positions of RNA and biotinylated protein overlap, and the biotinylated protein in the polydT-APEX2 group is significantly more than that in the control group.

[0117] The proteomic identification results were further analyzed.

[0118] 1) Identification of biotin-labeled proteins by mass spectrometry

[0119] RNA interacting proteins were labeled based on the PrimAPEX method. The experimental group was the polydT-APEX group (repeated twice), and the control group was the APEX2 group or the polydT group. Each group had 3 biological replicates, for a total of 12 samples. The mass spectrometry identification results were searched and protein quantified using Maxquant software. The minimum peptide length was 7, the minimum number of independent peptides was 2, and peptides and proteins with FDR less than 0.01 at the peptide and protein levels were retained. The data obtained from the search were filtered for contaminated proteins and missing values ​​were filled. A total of 631 proteins were identified in the APEX2 group, 814 proteins in the polydT group, 2436 proteins in polydT-APEX2Rep1, and 2477 proteins in polydT-APEX2 Rep2. The distribution of protein quantity in each sample is shown in Fig.15 .

[0120] 2) Proteomics data analysis

[0121] Unsupervised clustering was used to analyze the identified proteins. Samples in the same group could be clustered together, and the differences between groups were obvious ( Fig.16A To evaluate the differences between groups and the duplication of samples within a group, we performed principal component analysis (PCA) in an unsupervised mode on all identified proteins, and each group could be clustered together ( Fig. 16B ).

[0122] 3) PrimAPEX identified 1,224 RNA-interacting proteins

[0123] The differentially up-regulated proteins were screened under the conditions of P value < 0.05 and log2 (fold change) > 1 between the experimental group and the control group. A total of 307 proteins were identified. The proteins that were identified in each replicate of the experimental group but not in the control group were defined as unique proteins in the experimental group, a total of 917 proteins. A total of 1224 proteins were defined as RNA-interacting proteins identified by PrimAPEX ( Fig.17A). Based on the RBP2GO database (https: / / rbp2go.dkfz.de / ), we supplemented the literature on RBP research in the past five years and screened out proteins identified in more than two papers to form a new database called Annotated RBPs. This database contains 4659 proteins. The 1224 RNA-interacting proteins identified by PrimAPEX technology were compared with the Annotated RBPs database. 87% of the proteins identified by PrimAPEX overlapped with the database ( Fig. 17B ).

[0124] 4) Most of the proteins identified by PrimAPEX are related to RNA biological processes

[0125] Metascape was used to perform GO analysis on 1224 PrimAPEX-identified RNA-interacting proteins. The results showed that most of the 1224 PrimAPEX-identified proteins were related to RNA biological processes ( Fig.18 ). This indicates that the PrimAPEX technique has good specificity and can be used for RNA-interacting proteome identification.

[0126] In summary, the present invention designs a new peroxidase APEX2 complex, introduces a non-natural amino acid containing an azide group into the oxidase APEX2 at a fixed point, binds APEX2 to a DBCO-modified complementary sequence through a click chemistry reaction, and the complementary sequence can bind to the target RNA, thereby binding the peroxidase APEX2 to the mRNA. Under the action of hydrogen peroxide, the peroxidase APEX2 can catalyze biotin-phenol to form a biotin-phenoxy free radical to covalently label adjacent proteins, and can biotinylate the protein bound to the mRNA, and then use affinity purification and mass spectrometry identification and other technologies to efficiently and specifically identify mRNA interacting proteins; then, bioinformatics analysis is used to obtain mRNA candidate interacting proteins, and finally CLIP, WB and other technologies are combined to verify the interaction relationship between mRNA and candidate proteins, thereby achieving efficient identification of RNA interacting proteomes.

[0127] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. An engineered peroxidase APEX2, characterized in that: The structural formula of the engineered peroxidase APEX2 is shown in Formula I; AL-APEX2 formula I; Wherein, A represents an unnatural amino acid containing an azide group, L represents a linker, and APEX2 represents peroxidase APEX2.

2. The engineered peroxidase APEX2 according to claim 1, characterized in that The non-natural amino acid containing an azide group includes a phenylalanine analog containing an azide group; Preferably, the phenylalanine analog containing an azide group comprises at least one of p-azidophenylalanine, 3-azido-L-phenylalanine or 4-azido-L-phenylalanine; Preferably, the linker comprises (Gly-Gly-Gly-Gly-Ser)n, where n is a positive integer.

3. A method for preparing the engineered peroxidase APEX2 according to claim 1 or 2, characterized in that: The preparation method comprises: An amber stop codon is introduced into the peroxidase APEX2 coding gene, and an expression vector is inserted to obtain a recombinant vector, and the recombinant vector is introduced into a host cell, and the host cell also expresses an orthogonal aminoacyl tRNA synthetase and tRNA to obtain a recombinant cell, and the recombinant cell is cultured, and a non-natural amino acid containing an azide group is added to the culture medium. After the culture is completed, the culture fluid is collected, separated and purified to obtain the engineered peroxidase APEX2.

4. The method for preparing the engineered peroxidase APEX2 according to claim 3, characterized in that: The culture temperature is 23-26° C. and the culture time is 22-26 hours.

5. A proximity labeling complex, characterized in that The complex comprises the engineered peroxidase APEX2 according to claim 1 or 2 and a complementary nucleic acid sequence covalently linked thereto; The complementary nucleic acid sequence is complementary to the target RNA.

6. The proximity labeling complex according to claim 5, characterized in that The length of the complementary nucleic acid sequence is 20 to 40 nt; Preferably, the complementary nucleic acid sequence comprises a polydT sequence.

7. A method for preparing the proximity labeled complex according to claim 5 or 6, characterized in that: The preparation method comprises: The engineered peroxidase APEX2 according to claim 1 or 2 is mixed with the complementary nucleic acid sequence having dibenzocyclooctyne modification to obtain the proximity labeling complex.

8. The method for preparing a proximity labeled complex according to claim 7, characterized in that: The molar ratio of the engineered peroxidase APEX2 to the complementary nucleic acid sequence modified with dibenzocyclooctyne is 1:(1-20); Preferably, the mixing temperature is 4 to 40° C. and the mixing time is 1 to 5 hours.

9. A method for labeling RNA-interacting proteins, characterized in that: The method for labeling RNA-interacting proteins comprises: Pre-treating the cells, mixing the pre-treated cells with bovine serum albumin and incubating them, mixing the incubated cells with the proximity labeling complex of claim 5 or 6, adding biotin phenol and hydrogen peroxide, and reacting; Preferably, the pretreatment includes formaldehyde cross-linking treatment and cell perforation treatment; Preferably, the reaction temperature is 2 to 30° C. and the reaction time is 1 to 12 min.

10. A method for identifying RNA-interacting protein groups, characterized in that: The RNA interacting protein group identification method comprises: The RNA-interacting protein is labeled using the method for labeling RNA-interacting proteins described in claim 9, the labeled RNA-interacting protein is separated and purified, and the separated and purified RNA-interacting protein is identified.

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