Method for capturing in-situ interaction protein of ready-to-use RNA (Ribonucleic Acid)

Abstract

The invention belongs to the technical field of biology, and discloses a capturing method of ready-to-use RNA (Ribonucleic Acid) in-situ interaction protein. The method comprises the following steps: S1, adding a biotin-labeled FISH probe into a cell suspension; s2, then adding streptavidin to be labeled with horse radish peroxidase (Streptavidin-HRP), and carrying out enzyme deactivation on the horse radish peroxidase; s3, washing off non-specific binding; and S4, adding biotin phenol and a hydrogen peroxide substrate to trigger a biotinylation labeling reaction. According to the method, a ready-to-use RNA in-situ proximity biotinylation labeling technology without genetic manipulation is established through fusion RNA-FISH in-situ hybridization and proximity labeling technologies, proximity labeling enzyme can be directly guided to interested RNA through an RNA hybridization probe in a fixed cell through the technology, then a proximity biotinylation labeling reaction is carried out, and the RNA in-situ proximity biotinylation labeling technology is established. The method comprises the following steps of: adding a biotin label to interacting protein adjacent to RNA (Ribonucleic Acid), so that the protein can be enriched by streptavidin-beads for mass spectrum identification.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to a method for capturing interacting proteins of RNA in situ. Background Art

[0002] RNA-FISH in situ hybridization is widely used in scientific research. During experiments, some proteins exhibit properties such as insolubility, transient behavior, or weak interactions, which are often difficult to address using traditional biochemical methods. To address these challenges, proximity labeling technology has been introduced, which allows identification of nearby interacting proteins in situ by adding biotin tags. This technology has been successfully applied to address a variety of biological problems, initially expanding from the study of protein-protein interactions to the study of RNA-protein interactions.

[0003] However, existing methods for capturing RNA-interacting proteins in situ require genetic manipulation to introduce RNA-targeting systems and proximity-labeling enzymes into living cells for expression, but this approach is inoperable in fixed cells or clinical samples. Furthermore, existing RNA-in situ proximity biotinylation workflows often require genetic manipulation or purification steps.

[0004] Therefore, there is an urgent need to develop a method that can overcome the above technical limitations and apply proximity labeling technology in fixed cells to achieve convenient and rapid capture of interacting proteins of specific RNA in situ. Summary of the Invention

[0005] To overcome the above-mentioned technical problems, the present invention utilizes commercially available streptavidin-labeled horseradish peroxidase (Streptavidin-HRP) as a proximity biotinylation labeling enzyme. This enzyme is composed of covalently linked streptavidin protein and horseradish peroxidase. Streptavidin can bind biotin, and the coupling with horseradish peroxidase provides proximity biotinylation labeling enzyme activity. Using biotin-phenol and hydrogen peroxide (H2O2) as substrates, a ready-to-use biotin labeling system is constructed for detecting proximity-interacting proteins of RNA in situ, without the need for genetic manipulation.

[0006] In a first aspect, the present invention provides a method for capturing RNA-interacting proteins in situ, comprising the following steps:

[0007] S1, add biotin-labeled FISH probe to the cell suspension;

[0008] S2, add avidin-labeled peroxidase;

[0009] S3, wash away nonspecific binding;

[0010] S4. Add biotin phenol and hydrogen peroxide substrate to trigger the biotinylation reaction.

[0011] Using this technical solution, by integrating RNA-FISH in situ hybridization and proximity labeling, a ready-to-use RNA in situ proximity biotinylation labeling technique has been established that does not require genetic manipulation. This technique allows the direct introduction of a proximity labeling enzyme to the RNA of interest via an RNA hybridization probe in fixed cells. This proximity biotinylation reaction then proceeds, biotin-labeling the interacting proteins adjacent to the RNA. These proteins can then be enriched with streptavidin beads for mass spectrometry identification. This method, without the need for complex genetic manipulation experimental steps, allows for the rapid and convenient capture of interacting proteins in situ from RNA.

[0012] Furthermore, the FISH probe is capable of binding to a specific RNA in the cell through hybridization.

[0013] Furthermore, the avidin is selected from streptavidin, egg white avidin, egg yolk avidin or avidin-like substance, preferably streptavidin.

[0014] Furthermore, the peroxidase is selected from horseradish peroxidase, BioID, BioID2, AirID, BASU, APEX or APEX2, preferably horseradish peroxidase.

[0015] Furthermore, before step S1, a step of permeabilizing the cells to be treated is also included.

[0016] Furthermore, after step S4, the method further includes step S5 of detecting the biotinylation labeling signal, and performing imaging detection on the adjacent biotinylated protein and RNA bound to the hybridization probe.

[0017] Furthermore, after step S5, step S6 is further included to enrich the biotinylated RNA adjacent proteins and perform mass spectrometry identification to verify the interacting proteins identified by mass spectrometry.

[0018] In a second aspect, the present invention also provides a ready-to-use kit for capturing RNA-interacting proteins in situ, the kit comprising a biotin-labeled FISH probe capable of binding to specific RNA, avidin-labeled peroxidase, biotin phenol, hydrogen peroxide, and a buffer.

[0019] Furthermore, the avidin is selected from streptavidin, egg white avidin, egg yolk avidin or avidin-like substance, preferably streptavidin.

[0020] Furthermore, the peroxidase is selected from horseradish peroxidase, BioID, BioID2, AirID, BASU, APEX or APEX2, preferably horseradish peroxidase.

[0021] Using the above technical solution, the biotin-labeled FISH probe can bind to specific RNA in the cell. At the same time, horseradish peroxidase labeled with streptavidin can bind to biotin through streptavidin, and the coupled horseradish peroxidase can provide the activity of the adjacent biotinylated labeling enzyme. With the help of the extremely strong binding affinity between streptavidin and biotin and the biotin-labeled FISH hybridization probe, after cell permeabilization, Strep-HRP can be located on the target RNA through hybridization of the biotin-labeled FISH probe.

[0022] In a third aspect, the present invention also provides the use of the above kit in capturing RNA interacting proteins in situ.

[0023] Compared with the prior art, the present invention utilizes the extremely strong binding affinity between streptavidin and biotin (Kd is about 10^ -15 M) and biotin-labeled FISH hybridization probes, after cell permeabilization, Streptavidin-HRP is successfully located on the RNA of interest through hybridization of the biotin-labeled FISH probe. Subsequently, by washing away nonspecific binding, biotin phenol and hydrogen peroxide substrate are added to trigger the biotinylation reaction, and the adjacent interacting proteins of the RNA in situ are biotin-tagged. The interacting proteins adjacent to the biotin-tagged RNA can be enriched by streptavidin-beads for mass spectrometry identification. This method integrates RNA FISH in situ hybridization and proximity labeling technology, using commercially available streptavidin-labeled horseradish peroxidase (Streptavidin-HRP) as the proximity biotinylation labeling enzyme. During the capture process, no complicated genetic experimental operations are required, and the labeling of adjacent proteins in the RNA in situ can be achieved conveniently and quickly. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Schematic diagram of the capture method of the present invention.

[0025] Figure 2 Biotinylation activity was detected by streptavidin-labeled horseradish peroxidase.

[0026] Figure 3 Biotinylation of 45S RNA-associated proteins.

[0027] Figure 4Identification of proteins bound to 45S RNA in situ in HeLa cells. The upper left corner shows a biotinylated image. Red signals represent Cy3, and blue represents DAPI. Scale bar: 10 μm. The volcano plot displays the proteome data for 45S RNA binding. Significant differences were considered with a p-value < 0.01 and a fold change > 2.

[0028] Figure 5 Identification of proteins binding to Neat1 RNA in situ in Hela cells. The upper left corner shows a biotinylated image, with red representing Cy3 and blue representing DAPI. Scale bar: 10 μm. The volcano plot shows proteomic data for Neat1 RNA binding. Significant differences were identified with a p-value < 0.01 and a fold change > 2.

[0029] Figure 6 Identification of Xist-interacting proteins in 293T cells. Biotinylated images are shown in the upper left corner. Red signals represent Cy3, and blue represents DAPI. Scale bar: 10 μm. Volcano plots display Xist-binding proteome data. Significant differences were considered with a p-value < 0.01 and a fold change > 2.

[0030] Figure 7 Characterization of proteins that interact with Xist RNA. DETAILED DESCRIPTION

[0031] The present invention will be further described below with reference to specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. Experimental procedures in the following examples, where specific conditions are not specified, were generally performed under conventional conditions, conditions described in laboratory manuals, or conditions recommended by the manufacturer.

[0032] Streptavidin-HRP (strep-HRP): Biyuntian (A0305-1ml).

[0033] The present invention provides a biotin labeling system that does not require genetic manipulation, and the system can be used to label RNA in situ and capture RNA-adjacent interacting proteins.

[0034] like Figure 1As shown, streptavidin in streptavidin-labeled horseradish peroxidase (Strep-HRP) can bind to biotin, and after coupling with horseradish peroxidase, it can provide adjacent biotinylated labeling enzyme activity. Leveraging the extremely strong binding affinity between streptavidin and biotin and a biotin-labeled FISH hybridization probe, after cell fixation and permeabilization, Strep-HRP is localized to the target RNA through hybridization with the biotin-labeled FISH probe. Then, after eluting nonspecific binding, the biotinylation labeling reaction is terminated by adding a substrate to biotrigger the reaction. The biotinylation labeling signal is detected, and imaging of the biotinylated protein and RNA bound to the hybridization probe is performed.

[0035] like Figure 2 Figure 2. RNA-interacting protein capture in situ in fixed cells by binding streptavidin to a biotinylated probe. The biotinylated oligonucleotide probe recruits streptavidin-horseradish peroxidase to the RNA of interest, and the RNA is visualized after proximity labeling. Scale bar: 5 μm. The proteome associated with the RNA spot is identified by mass spectrometry (red dots).

[0036] Example 1 In vitro testing of biotinylation activity of streptavidin-HRP

[0037] In a 1.5 ml EP tube, add 0.1, 1, and 10 μl of 1 mg / ml streptavidin-HRP (strep-HRP) to 100 μl of 293T cell lysate, supplement with biotin-phenol (Iris Biotech GmbH, LS-3500) to 0.5 mM, and supplement with H2O2 (Sigma 88597) to 1 mM to trigger the proximity labeling reaction. After 1 minute, terminate the reaction with cold quenching buffer. Then add protein loading buffer and boil at 95°C for 3-5 minutes to fully denature the protein. Take 20 μl of the above-treated protein sample and run SDS-PAGE electrophoresis. The biotinylated signal of the protein in the cell lysate can be detected using anti-biotin antibody (such as Figure 3 This indicates that the streptavidin-HRP used has the activity of biotinylating proteins.

[0038] Example 2 Identification of 45S-related proteins in Hela cells

[0039] The well-known 45S RNA was selected to test the establishment of RNA proximity biotinylation labeling technology, and the specificity of the 45S hybridization probe was verified by RNA FISH targeting 45S.

[0040] 1. Synthesis of biotin-labeled FISH probes

[0041] The working concentration of 45S probe was 25 nM, which was synthesized by GeneWeiZ.

[0042] 2. Probe Hybridization

[0043] 2.1 Fix HeLa cells with 4% PFA (paraformaldehyde) for 10 minutes and wash three times with 1X PBS;

[0044] 2.2 Add 0.1% TritonX-100 to permeabilize for 15 minutes, and wash twice with PBS, 5 minutes each time;

[0045] 2.3 Add blocking buffer and incubate at 37°C for 30 minutes;

[0046] 2.4 Prehybridization step: Incubate cells in 10% formamide / 2X SSC at room temperature for 10 min;

[0047] 2.5 Add the biotinylated FISH probe to 10 μl of PBS, then add 400 μl of dissolved hybridization buffer (dissolve 1 g of 10% dextran sulfate in 1 ml of 20x SSC, 1 ml of 100% formamide, and add 10 ml of nuclease-free water) and denature at 73°C for 10 min.

[0048] 2.6 Hybridize at 37°C for 16 h;

[0049] 2.7 Wash with ~1 ml 10% formamide / 2xSSC at 37°C for 30 minutes, then wash with 1xSSC at room temperature for 15 minutes.

[0050] 3. Streptavidin-HRP (strep-HRP) was added to the above Hela cells. Using the binding force between streptavidin and biotin, streptavidin-labeled horseradish peroxidase (Strep-HRP) was recruited to 45S RNA through hybridization positioning of the biotin-labeled FISH probe.

[0051] 4. After washing away nonspecific binding, biotin-phenol and H2O2 were added to trigger the biotinylation labeling reaction and terminated after 1 minute (the specific operation was the same as in Example 1).

[0052] 5. Detect the biotinylation labeling signal and perform imaging detection on the biotinylated protein adjacent to 45S and the 45S bound by the hybridization probe.

[0053] 6. Enrich biotinylated 45S neighboring proteins and identify them by mass spectrometry.

[0054] 7. Verify the 45S interacting proteins identified by mass spectrometry.

[0055] like Figure 4Shown are visualization of 45S RNA (scale bar: 10 μm) and a volcano plot of 45S-associated proteins identified in HeLa cells. The FISH+ method was tested by targeting 45S pre-ribosomal RNA in HeLa cells (45S probe working concentration: 5 nM, conventionally synthesized by GENEWIZ). The biotinylated probe guided streptavidin-HRP to 45S RNA for proximity labeling. Biotinylated markers were visualized as bright spots within the nucleus (near 45S RNA spots) by detection with streptavidin-Cy3. Simultaneously, biotinylated proteins were enriched with streptavidin beads, and the protein group interacting with 45S RNA was analyzed by label-free quantitative mass spectrometry (LFQ). Significantly enriched proteins (p value < 0.01, fold change > 2) are shown as black dots (45S and non-targeted controls, n = 3). Red dots represent known 45S RBPs. We list proteins that interact with 45S RNA from reported literature and the StarBase v2.0 database, including known nucleolar markers such as NOP2, NOP58, NOP56, TCOF1, and PDCD11. These results demonstrate that the FISH+ method can identify 45S RNA-binding proteins in situ.

[0056] Example 3 Capturing the interacting proteome of Neat1 RNA in situ

[0057] The application scenarios of the FISH+ method were expanded by targeting Neat1 RNA in Hela cells.

[0058] 1. Synthesis of biotin-labeled FISH probes

[0059] Neat1 probe working concentration: 25 nM, conventionally synthesized by GeneWeiZ.

[0060] 2. The specific operations are the same as in Example 2.

[0061] 3. Streptavidin-HRP (strep-HRP) was added to the above Hela cells. Using the binding force between streptavidin and biotin, streptavidin-labeled horseradish peroxidase (Strep-HRP) was recruited to Neat1 RNA through hybridization positioning of the biotin-labeled FISH probe.

[0062] 4. After washing away nonspecific binding, biotin-phenol and H2O2 were added to trigger the biotinylation labeling reaction and terminated after 1 minute (the specific operation was the same as in Example 1).

[0063] 5. Detect the biotinylation labeling signal and perform imaging detection on the biotinylated protein adjacent to the Neat1 RNA and the Neat1 RNA bound by the hybridization probe.

[0064] 6. Enrich biotinylated Neat1 RNA adjacent proteins and identify them by mass spectrometry.

[0065] 7. Verify the Neat1 RNA-interacting proteins identified by mass spectrometry.

[0066] A biotinylated probe directs streptavidin-HRP to Neat1 RNA for proximity labeling. Biotinylated markers are visualized as bright spots within the nucleus (near Neat1 RNA spots) by detection with streptavidin-Cy3. Proteins interacting with Neat1 RNA were also characterized. Biotinylated proteins were enriched using streptavidin beads, and the proteome targeting Neat1 RNA interactions was analyzed by label-free quantitative mass spectrometry (LFQ).

[0067] like Figure 5 As shown, Neat1 was imaged adjacent to biotinylation markers. Scale bar: 10 μm. Strep-Cy3 was used to detect biotinylation markers, and clear, bright RNA spots were observed. Biotinylation markers of the proteome associated with Neat1 RNA spots are shown in red. A volcano plot of Neat1-associated proteins identified in Hela cells is also shown. Significantly enriched proteins (p-value < 0.01, fold change > 2) are shown as black dots (Neat1 and non-targeting control, n = 3). Red dots represent known Neat1 RBPs. A combination of known Neat1-interacting RBPs from the literature and the StarBase v2.0 database is listed, including NONO, KHSRP, RBM14, and SFPQ. Numerous paraspeckle body markers were also identified, including RNA-binding proteins such as AKAP8, ZC3H14, and PSPC1. This is consistent with reports that Neat1 functions as a structural component of paraspeckle bodies. Overall, these experimental results demonstrate that FISH+ is an effective RNA proximity biotinylation labeling method that can capture and identify RNA-binding proteins in situ.

[0068] Example 4

[0069] Xist RNA was used to test the establishment of RNA proximity biotinylation labeling technology, and the specificity of Xist RNA hybridization probes was verified by RNA FISH targeting Xist RNA.

[0070] 1. Synthesis of biotin-labeled FISH probes

[0071] Xist probe working concentration: 20 nM, conventionally synthesized by GeneWeiZ.

[0072] 2. The specific operations are the same as in Example 2.

[0073] 3. Streptavidin-HRP (strep-HRP) was added to the above HEK 293T cells. Using the binding force between streptavidin and biotin, streptavidin-labeled horseradish peroxidase (Strep-HRP) was recruited to Neat1 RNA through hybridization positioning of the biotin-labeled FISH probe.

[0074] 4. After washing away nonspecific binding, biotin-phenol and H2O2 were added to trigger the biotinylation labeling reaction and terminated after 1 minute (the specific operation was the same as in Example 1).

[0075] 5. Detect the biotinylation labeling signal and perform imaging detection of the biotinylated protein adjacent to the Xist RNA and the Xist RNA bound to the hybridization probe.

[0076] 6. Enrich biotinylated Xist RNA adjacent proteins and identify them by mass spectrometry.

[0077] 7. Verify the Xist RNA-interacting proteins identified by mass spectrometry.

[0078] Xist plays a key role in X chromosome inactivation during early embryonic development, and Xist dysregulation is associated with a variety of diseases. A biotinylated probe guides streptavidin-HRP to Xist RNA for proximity labeling. By detecting streptavidin-Cy3, the biotinylated label is visualized as bright spots within the cell nucleus (near the Xist RNA). Figure 6 As shown in the figure, in HEK293T cells, Xist expression appears as 1 to 2 bright spots per cell in the nucleus. Proteomic experiments using the FISH+ method identified 2,785 proteins with at least 2 unique peptides. For enrichment analysis, label-free quantitative mass spectrometry analysis was applied, and a cutoff value of enrichment greater than two times and an adjusted p-value <0.01 was set. In Xist FISH+, 219 candidate proteins were significantly enriched, shown as black dots. Among them, 59 proteins were reported as Xist-interacting proteins in the literature 5, 6, 16 and the StarBase v2.0 database, shown as red dots (such as Figure 6 volcano map).

[0079] At the same time, proteins that interact with Xist RNA were characterized. Biotinylated proteins were enriched by streptavidin beads, and the protein group targeting XistRNA interaction was analyzed by label-free quantitative mass spectrometry (LFQ). Many are well-known Xist-interacting proteins, such as the iconic Xist-binding protein SPEN, as well as CIZ1 and RBM15, indicating the strong reproducibility of the FISH+ method. Gene Ontology (GO) pathway enrichment analysis was performed for the significantly different Xist-binding proteins, and the top 23 Xist-interacting proteins with significant differences were listed. The analysis showed (such as Figure 7 Xist-interacting proteins are mainly involved in RNA splicing and chromatin remodeling.

[0080] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make non-creative modifications to the present embodiment as needed, but as long as they are within the scope of the claims of the present application, they are protected by the patent law.

Claims

1. A ready-to-use method for capturing RNA-interacting proteins in situ, characterized in that: The capture method comprises the following steps: S1, add biotin-labeled FISH probe to the cell suspension; S2, add avidin-labeled peroxidase; S3, wash away nonspecific binding; S4. Add biotin phenol and hydrogen peroxide substrate to trigger the biotinylation reaction.

2. The capture method according to claim 1, characterized in that The FISH probe binds to specific RNA in the cell through hybridization.

3. The capture method according to claim 1, characterized in that The avidin is selected from streptavidin, egg albumin avidin, egg yolk avidin or avidin-like substance, preferably streptavidin.

4. The capture method according to claim 1, characterized in that The peroxidase is selected from horseradish peroxidase, BioID, BioID2, AirID, BASU, APEX or APEX2, preferably horseradish peroxidase.

5. The capture method according to claim 1, characterized in that Before step S1, a step of permeabilizing the cells to be treated is also included.

6. The capture method according to claim 1, characterized in that After step S4, the method further includes step S5 of detecting the biotinylation labeling signal and performing imaging detection on the adjacent biotinylated protein and RNA bound to the hybridization probe.

7. The capture method according to claim 6, characterized in that After step S5, the method further includes step S6 of enriching the biotinylated RNA adjacent proteins and performing mass spectrometry identification to verify the interacting proteins identified by mass spectrometry.

8. A ready-to-use kit for capturing RNA-interacting proteins in situ, characterized in that: The kit comprises a biotin-labeled FISH probe capable of binding to a specific RNA, streptavidin-labeled horseradish peroxidase, biotin phenol, hydrogen peroxide and a buffer solution.

9. The kit according to claim 8, characterized in that The avidin is selected from streptavidin, egg white avidin, egg yolk avidin or avidin-like, preferably streptavidin; the peroxidase is selected from horseradish peroxidase, BioID, BioID2, AirID, BASU, APEX or APEX2, preferably horseradish peroxidase.

10. Use of the kit according to any one of claims 8 to 9 for capturing RNA interacting proteins in situ.

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