A proximity labeling-based RNA in situ imaging method and imaging kit

Abstract

The application belongs to the technical field of biology, and discloses a RNA in situ imaging method and imaging kit based on adjacent labeling. The imaging method comprises the following steps: 1) imaging the RNA and the adjacent protein captured by the RNA; and 2) combining the imaging information of the RNA and the adjacent protein and imaging again. The application realizes the visualization of dark and weak RNA based on “double-layer signal superposition”, and realizes extremely high sensitivity. The signal intensity of the traditional FISH technology is limited by the number of fluorescent groups directly carried by the probe. When facing medium and low abundance RNA, the signal is extremely weak and difficult to be captured by optical instruments. The application utilizes the “direct binding signal” of the target RNA in situ probe and a large number of deposited fluorescence amplification signals generated by the adjacent labeling reaction around the probe to perform double-layer superposition. This exponential signal multiplication can form extremely clear and bright RNA spots at low abundance RNA sites, and improves the signal-to-noise ratio and sensitivity of imaging.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically to a method and kit for in situ RNA imaging based on proximity markers. Background Technology

[0002] In the field of RNA in situ imaging, traditional fluorescence in situ hybridization (FISH) is the core technique for detecting the spatial distribution of RNA within cells. However, when faced with RNA targets of low abundance, traditional FISH often suffers from weak fluorescence signals, low signal-to-noise ratios, and difficulty in achieving clear imaging. To amplify RNA imaging signals, existing techniques attempt to combine specific targeting systems with labeling enzymes (such as adjacent labeling enzymes).

[0003] However, existing methods and their workflows have the following significant drawbacks and shortcomings: (1) High dependence on live-cell genetic manipulation, limiting applicability: Existing enzymatic amplification or proximity labeling methods usually require complex genetic modification operations (such as plasmid transfection, viral infection, etc.) in a live-cell system to introduce RNA targeting systems and labeling enzymes into cells for expression. This method, which is highly dependent on live-cell operation and genetic manipulation, cannot be implemented on fixed cells, tissue sections, or even precious clinical pathological samples, which greatly limits its application in clinical diagnosis and retrospective studies.

[0004] (2) The operation is cumbersome and requires additional purification steps: The existing biotinylation labeling and other workflows are not only limited to live cells, but also often require complex genetic engineering operations or are limited to cumbersome probe / enzyme purification steps in vitro. There is a lack of simple and efficient technical systems that can be directly applied to fixed samples.

[0005] Therefore, there is an urgent need to develop a method for in situ RNA imaging using proximity markers that requires no genetic manipulation or complex reagent synthesis. Summary of the Invention

[0006] To overcome the aforementioned technical problems, this invention utilizes a hybridization probe with specific modifications (such as biotinylation) to specifically bind to target RNA in a fixed sample; then, a commercially available coupling enzyme (such as Streptavidin-HRP) is directly introduced; finally, in situ signal deposition is generated through an enzymatic cascade reaction (such as adjacent biotinylation or TSA reaction), achieving double-layer signal superposition and enhanced imaging of the target RNA.

[0007] This invention provides a method for in situ RNA imaging based on proximity markers, the method comprising the following steps: 1) Imaging the RNA and imaging the neighboring proteins captured by the RNA; 2) Superimpose the signals from the two layers of the RNA and the adjacent protein for imaging.

[0008] In this application, a "double-layer signal superposition" limiting sensitivity imaging method is used to solve the problem of traditional FISH's difficulty in detecting low-abundance RNA. After the probe binds to the target RNA, it initiates a neighboring biotinylation or tyramine signal amplification (TSA) reaction. This double-layer signal superposition is achieved through the "direct signal from the RNA in situ probe" and the "abundant deposited fluorescent signal generated by the enzymatic cascade reaction around the probe." This signal superposition can form extremely clear and bright "RNA spots" visible to the naked eye at low-abundance RNA sites.

[0009] Furthermore, the RNA is bound to a biotin-labeled FISH probe and a streptavidin-peroxidase conjugate.

[0010] Furthermore, the adjacent protein is bound with biotin-phenol oxide free radicals.

[0011] Furthermore, the avidin is selected from streptavidin, egg albumin, egg yolk avidin, or avidin-like substances, preferably streptavidin.

[0012] Furthermore, the peroxidase is selected from horseradish peroxidase, BioID, BioID2, AirID, BASU, APEX, or APEX2, preferably horseradish peroxidase.

[0013] Secondly, the present invention also provides a proximity-labeled RNA in situ imaging kit, the kit comprising a biotin-labeled FISH probe capable of binding to target RNA, streptavidin-labeled horseradish peroxidase, biotinylate, hydrogen peroxide, and buffer.

[0014] Furthermore, the avidin is selected from streptavidin, egg albumin, egg yolk avidin or avidin-like substances, preferably streptavidin; the peroxidase is selected from horseradish peroxidase, BioID, BioID2, AirID, BASU, APEX or APEX2, preferably horseradish peroxidase.

[0015] Thirdly, the present invention also provides the application of the kit described in the second aspect in RNA in situ imaging based on proximity markers.

[0016] Compared with the prior art, the present invention has the following technical effects.

[0017] 1) This invention achieves visualization of faint RNA based on "double-layer signal superposition," resulting in extremely high sensitivity. The signal intensity of traditional FISH technology is limited by the number of fluorescent groups directly carried by the probe, leading to extremely weak signals at low-to-medium abundance RNA sites, making them difficult for optical instruments to capture. This invention utilizes a double-layer superposition of the "direct binding signal" of the target RNA in situ probe and the "amplified signal" of the abundant depositional fluorescence generated by neighboring labeling reactions around the probe. This exponential signal multiplication forms extremely clear and bright "RNA spots" (puncta RNA) at low-abundance RNA sites, improving the signal-to-noise ratio and sensitivity of the imaging.

[0018] 2) Conventional hapten-based recognition amplification strategies have weak affinity. When washing away non-specifically bound probes, harsh elution conditions can lead to the loss of the true signal, while mild conditions can leave extremely high background noise. The high-affinity system of this invention can withstand extremely harsh elution conditions, effectively washing away all non-specifically bound free probes. This not only completely eliminates non-specific background noise but also endows the method with extremely high stability when processing complex, immobilized samples.

[0019] 3) Traditional in vitro probe-enzyme coupling amplification techniques require complex, customized chemical synthesis and biochemical purification steps such as high-performance liquid chromatography (HPLC), resulting in high technical barriers, high costs, and low yields. This method eliminates the complex reagent synthesis and probe purification steps, achieving "ready-to-use." This significantly reduces the economic cost and time cycle of a single experiment, enabling any ordinary laboratory with standard facilities to conduct high-level, high-sensitivity in-situ RNA imaging. Attached Figure Description

[0020] Figure 1 This is a flowchart of the RNA in situ imaging method based on proximity markers of the present invention.

[0021] Figure 2 The signal intensity of RNA-adjacent biotinylation labeling using different doses of adjacency labeling enzymes is given.

[0022] Figure 3 This illustrates the co-localization of NEAT1 RNA with NONO and SFPQ proteins in Example 2.

[0023] Figure 4 In situ imaging of PNCTR and MALAT1 RNA in Example 3.

[0024] Figure 5 This is an in situ imaging of adjacent biotinylated markers of PNCTR in Example 4. Detailed Implementation

[0025] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions, conditions described in a laboratory manual, or conditions recommended by the manufacturer. Streptavidin-HRP: Beyotime (A0305-1mL).

[0026] The flowchart of the RNA in situ imaging method based on proximity markers of the present invention is as follows: Figure 1 As shown, in fixed cells or tissues, commercially available streptavidin-peroxidase conjugates (Streptavidin-HRP) and other mediated enzymes are precisely guided directly to the target RNA of interest using modularly designed oligonucleotide RNA hybridization probes, subsequently initiating a reaction based on tyramine signal amplification (TSA) and proximity biotinylation. This is because biotin has an extremely high binding affinity to streptavidin (Kd≈10). 15 This system (M) can withstand extremely harsh elution conditions, thus completely eliminating non-specific background. More importantly, it achieves "two-level signal superposition" through the direct signal of oligonucleotide probe binding to target RNA and the signal of a large number of nearby biotinylated proteins / fluorescent deposition generated by enzymatic reactions near the site. This not only makes the target (such as Neat1 or PNCTR) appear as extremely clear and bright RNA spots, but also, through this cascade amplification of dual signals, enables the in-situ generation of clear and bright RNA fluorescent spots, significantly improving the imaging signal-to-noise ratio, making it particularly suitable for in-situ imaging of RNA with low abundance. It also avoids complex protein purification steps and improves the accessibility of high-sensitivity in-situ imaging of RNA in routine laboratories.

[0027] Example 1: The signal intensity of RNA-near biotinylation labeling is affected by the dosage of the adjacent labeling enzyme. To determine the optimal Strep-HRP concentration for NEAT1 RNA FISH+ assay, different Strep-HRP concentration treatment groups were established. The Strep-HRP concentrations were set to 5 ng / mL, 10 ng / mL, and 20 ng / mL.

[0028] 1. Synthetic biotin-labeled FISH probes The working concentration of the NEAT1 probe was 25 nM, and it was synthesized by Genewiz using conventional methods.

[0029] 2. Probe hybridization 2.1 Fix HeLa cells with 4% PFA (paraformaldehyde) for 10 minutes, then wash 3 times with 1X PBS; 2.2 Add 0.1% Triton X-100 and permeate for 15 minutes, then wash twice with PBS. 5 min; 2.3 Add blocking buffer and incubate at 37°C for 30 min; 2.4 Prehybridization procedure: Cells were incubated in 10% formamide / 2X SSC at room temperature for 10 min; 2.5 Add the biotin-labeled FISH probe to 10 μL of nuclease-free water, then... Add 400 μL of hybridization buffer containing 1 mM RNase inhibitor: Dissolve 1 g of 10% dextran sulfate in 1 mL of 20xSSC and 1 mL of 100% formamide, add 10 mL of nuclease-free water, and denature at 73 °C for 10 min. 2.6 Hybridization at 37℃ for 16 hours; 2.7 Wash with 1 mL of 10% formamide / 2xSSC at 37°C for 30 minutes, then wash with 1xSSC. Wash for 15 minutes at room temperature.

[0030] 3. Different concentrations of streptavidin-HRP (strep-HRP) were added to the HeLa cells. Utilizing the binding force between streptavidin and biotin, streptavidin-labeled horseradish peroxidase (Strep-HRP) was recruited to NEAT1 RNA via hybridization localization using biotin-labeled FISH probes.

[0031] 4. After eluting off non-specific binding, add biotin-phenol and H2O2 to trigger the biotinylation labeling reaction and terminate the reaction after 1 minute.

[0032] HRP-mediated biotinylation deposition signals were detected using fluorescently labeled streptavidin, and images were acquired under the same microscopic imaging conditions. Quantitative analysis was performed on NEAT1 RNA-related spot signals from different Strep-HRP concentration treatment groups. The experimental results are shown below. Figure 2 The results showed that the NEAT1 RNA marker signal intensity increased with increasing Strep-HRP concentration; however, excessively high concentrations of Strep-HRP led to an expansion of the HRP-mediated free radical deposition area, resulting in larger fluorescent spot radii, blurred boundaries, and potential nonspecific neighbor-to-neighbor markers or false-positive neighbor-to-neighbor interactions. These results indicate that 10 ng / mL strep-HRP provides the optimal signal intensity and resolution. Scale bar: 50 µm.

[0033] Example 2: Colocalization of NEAT1 RNA with NONO and SFPQ proteins The well-known NEAT1 RNA was selected to test the establishment of RNA proximity biotinylation labeling technology, and the specificity of the NEAT1 hybridization probe was verified by RNA FISH targeting NEAT1.

[0034] 1. Synthetic biotin-labeled FISH probes The working concentration of the NEAT1 probe was 25 nM, and it was synthesized by Genewiz using conventional methods.

[0035] 2. Probe hybridization 2.1 Fix HeLa cells with 4% PFA (paraformaldehyde) for 10 minutes, then wash 3 times with 1X PBS; 2.2 Add 0.1% Triton X-100 and permeate for 15 minutes, then wash twice with PBS. 5 min; 2.3 Add blocking buffer and incubate at 37°C for 30 min; 2.4 Prehybridization procedure: Cells were incubated in 10% formamide / 2X SSC at room temperature for 10 min; 2.5 Add the biotin-labeled FISH probe to 10 μL of nuclease-free water, then... Add 400 μL of hybridization buffer containing 1 mM RNase inhibitor: Dissolve 1 g of 10% dextran sulfate in 1 mL of 20xSSC and 1 mL of 100% formamide, add 10 mL of nuclease-free water, and denature at 73 °C for 10 min. 2.6 Hybridization at 37℃ for 16 hours; 2.7 Wash with 1 mL of 10% formamide / 2xSSC at 37°C for 30 minutes, then wash with 1xSSC. Wash for 15 minutes at room temperature.

[0036] 3. When streptavidin-HRP (strep-HRP) at a concentration of 10 ng / mL was added to the HeLa cells, streptavidin-labeled horseradish peroxidase (Strep-HRP) was recruited to NEAT1 RNA via hybridization localization through biotin-labeled FISH probes, utilizing the binding force between streptavidin and biotin.

[0037] 4. After eluting off non-specific binding, add biotin-phenol and H2O2 to trigger the biotinylation labeling reaction and terminate the reaction after 1 minute.

[0038] 5. Immunofluorescence (IF) of NONO / SFPQ protein Blocking: Add PBS blocking solution containing 1% BSA (bovine serum albumin) and 1 mM RNase inhibitor, and incubate at room temperature in the dark for 30 minutes to block non-specific binding sites.

[0039] 6. Primary antibody incubation: Add pre-diluted anti-NONO protein primary antibody or anti-SFPQ protein primary antibody to the previously prepared experimental groups, incubate at room temperature for 1-2 hours, and then wash 3 times with 1X PBS.

[0040] 7. Secondary antibody incubation: Add FITC-fluorescently labeled secondary antibody, incubate at room temperature in the dark for 1 hour, then wash 3 times with 1X PBS.

[0041] 8. Mounting and Imaging Inspection The slides were mounted using anti-fluorescence quenching mounting medium and placed under a laser confocal microscope for multi-channel high-resolution imaging. The red channel (NEAT1 RNA), green channel (NONO or SFPQ protein), and co-localization of NEAT1 RNA and protein were acquired.

[0042] like Figure 3 As shown, the co-localization results of NEAT1 RNA and NONO protein demonstrate the co-localization of NEAT1 RNA and the parafoil marker protein NONO in HeLa cells. Using the imaging method of this invention, NEAT1 RNA (red fluorescent signal) exhibits a highly bright and discrete speckled localization distribution in HeLa cells, with extremely low nonspecific background signal in the nucleoplasm. Simultaneously, immunofluorescence imaging of NONO protein (green fluorescent signal) shows extensive nucleoplasmic signal and aggregated strong fluorescent spots. Observation of the combined image (overlapping areas are shown as yellow signals) reveals that a large number of NEAT1 RNA fluorescent spots detected by the method of this invention achieve precise co-localization with the NONO protein antibody-rich areas, accurately indicating the location of parafoil subcellular structures.

[0043] like Figure 3 As shown, the co-localization results of NEAT1 RNA and SFPQ protein demonstrate the co-localization of NEAT1 RNA and another parafoil marker protein, SFPQ, in HeLa cells. The discrete NEAT1 RNA clusters (red fluorescent signals) detected by the method of this invention also show a high degree of spatial overlap and precise co-localization with the SFPQ protein antibody-rich region (green fluorescent signal) indicating the parafoil structure of HeLa cells (yellow overlapping signal in the co-localization image).

[0044] Example 3: In situ imaging of PNCTR and MALAT1 RNA The present invention establishes a FISH probe-based technique for the visualization of RNA that is programmable, highly specific, and highly sensitive.

[0045] 1. Synthetic biotin-labeled FISH probes PNCTR probe working concentration: 50 nM, MALAT1: 10 nM, determined by Genewiz. Synthesis.

[0046] 2. Probe hybridization 2.1 Fix HeLa cells with 4% PFA (paraformaldehyde) for 10 minutes, then wash 3 times with 1X PBS; 2.2 Add 0.1% Triton X-100 and permeate for 15 minutes, then wash twice with PBS. 5 min; 2.3 Add blocking buffer and incubate at 37°C for 30 min; 2.4 Prehybridization procedure: Cells were incubated in 10% formamide / 2X SSC at room temperature for 10 min; 2.5 Add the biotin-labeled FISH probe to 10 μL of nuclease-free water, then... Then add 400 μL of hybridization buffer containing 1 mM RNase inhibitor (dissolve 1 g of 10% dextran sulfate in 1 ml of 20xSSC, 1 ml of 100% formamide, and add 10 mL of nuclease-free water), and denature at 73°C for 10 min. 2.6 Hybridization at 37℃ for 16 hours; 2.7 Wash with 1 mL of 10% formamide / 2xSSC at 37°C for 30 minutes, then use... Wash with 1xSSC for 15 minutes at room temperature.

[0047] 3. Add streptavidin-HRP at a concentration of 10 ng / mL. In HeLa cells, streptavidin-labeled horseradish peroxidase (Strep-HRP) is recruited to target RNA via hybridization localization using biotin-labeled FISH probes, utilizing the binding force between streptavidin and biotin.

[0048] 4. After eluting off non-specific binding, add biotin-phenol and H2O2 to trigger bio-activation. The reaction was labeled and terminated after 1 minute.

[0049] 5. Detect biotinylation labeling signals, targeting biotinylated proteins and impurities near PNCTR. Imaging and detection are performed using a cross-probe-coupled PNCTR. The MALAT procedure is the same as that for PNCTR.

[0050] like Figure 4 As shown, the proximity-labeled RNA in situ imaging method was used for in situ imaging of PNCTR and MALAT1 RNA. RNAs with low expression levels in cells were selected to test the proximity-labeled RNA in situ imaging method. PNCTR is a nucleolar RNA rich in the PTBP1 binding motif, with an expression level below 50 copies / cell, affecting alternative splicing. The intracellular copy number of MALAT1 is ~1000 copies / cell.

[0051] Example 4 To verify that the method of this invention captures a stable "protein footprint," rather than a signal solely dependent on free RNA, a Benzonase-treated control group was set up. After step 4 in Example 3, Benzonase enzyme solution was added to the cells before antibody incubation to effectively degrade most of the RNA scaffold. Subsequent fluorescence observation was then performed after treatment.

[0052] like Figure 5 As shown, FISH hybridization experiments were performed in fixed HeLa cells to test the specificity of the PNCTR probe, and Strep-Cy3 was used to detect the FISH signal. The middle row shows the imaging of biotinylated PNCTRs after the adjacent biotinylation labeling reaction, as indicated by RNA spots in the figure. The bottom row shows the degradation of all forms of nucleic acids by treating the adjacent biotinylated RNA with Benzonase nuclease. At this point, DAPI staining showed no signal, but the biotinylated proteins adjacent to the PNCTR remained, and Strep-Cy3 was used to detect and image the biotinylated proteins. Scale bar: 10 µm.

[0053] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A method for in situ RNA imaging based on proximity markers, characterized in that, The method includes the following steps: 1) Imaging the RNA and imaging the neighboring proteins captured by the RNA; 2) Superimpose the signals from the two layers of the RNA and the adjacent protein for imaging.

2. The RNA in situ imaging method according to claim 1, characterized in that, The RNA is bound to a biotin-labeled FISH probe and a streptavidin-peroxidase conjugate.

3. The RNA in situ imaging method according to claim 1, characterized in that, The adjacent protein is bound with biotin-phenolic radicals.

4. The RNA in situ imaging method according to claim 1, characterized in that, The avidin is selected from streptavidin, egg albumin, egg yolk avidin or avidin-like substances, preferably streptavidin.

5. 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.

6. A marker-based RNA in situ imaging kit, characterized in that, The kit includes a biotin-labeled FISH probe capable of binding to target RNA, streptavidin-labeled horseradish peroxidase, biotinylate, hydrogen peroxide, and buffer.

7. The reagent kit according to claim 6, characterized in that, The avidin is selected from streptavidin, egg albumin, egg yolk avidin or avidin-like substances, preferably streptavidin; the peroxidase is selected from horseradish peroxidase, BioID, BioID2, AirID, BASU, APEX or APEX2, preferably horseradish peroxidase.

8. The use of the kit according to any one of claims 6-7 in in situ RNA imaging based on proximity markers.

Images