Immune principle-based ribosome group or selective ribosome group detection and analysis method

Abstract

The invention discloses a ribosome group or selective ribosome group detection and analysis method based on an immune principle, and relates to the field of molecular biology and genomics. Comprising the following steps: sample preparation and cross-linking: treating cells by using a translation inhibitor to suspend ribosome translation, and then cross-linking RNA-protein in the cells by using a membrane permeable cross-linking agent; then cracking the cells, digesting RNA of a cracking product by using ribonuclease, and retaining ribosome and mRNA fragments protected by the ribosome; immune enrichment: carrying out specific immune enrichment on the digested sample by using an antibody combined with a carrier, and eluting to obtain a ribosome-mRNA compound; then decrosslinking and removing protein, extracting mRNA fragments and carrying out phosphorylation treatment so as to add joints, build a library and carry out sequencing; and finally, library construction, sequencing and bioinformatics analysis are carried out. The method has the advantages of high-specificity enrichment, accurate positioning of translation sites, wide application range and accurate sequence information.

Description

Technical Field

[0001] This invention relates to the fields of molecular biology and genomics, and in particular to methods for detecting and analyzing ribosomal or selective ribosomal sequences based on the principle of immunity. Background Technology

[0002] Traditional methods mainly involve enriching ribosomes and their translating mRNA sequences by sucrose density gradient centrifugation for sequencing. However, this method has low specificity and makes it difficult to distinguish ribosome populations with different functions.

[0003] Furthermore, ribosome-binding proteins have the function of regulating mRNA translation, but there is currently a lack of effective methods to directly analyze the mRNA translation regulated by specific ribosomal proteins. Existing technologies (such as application number CN202510039919) provide a method for extracting RNA fragments, but they do not solve the problem of highly specific enrichment for specific ribosome populations and subsequent accurate analysis.

[0004] Therefore, there is an urgent need in this field for a method that can specifically enrich ribosome-mRNA complexes and accurately analyze their sequence information.

[0005] Therefore, this invention proposes a ribosomal or selective ribosomal detection and analysis method based on the principle of immunity. Summary of the Invention

[0006] The purpose of this invention is to address the shortcomings of existing technologies by proposing a ribosome or selective ribosome detection and analysis method based on the principle of immunity.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: Ribosome or selective ribosome detection and analysis methods based on the principle of immunity include the following steps: S1: Sample preparation and crosslinking In step S1, the sample is treated with a translation inhibitor to suppress translation and immobilize the binding state of ribosomes to mRNA, thereby preserving the ribosome-mRNA complex formed during translation. Subsequently, a reversible cross-linking agent is used to cross-link and immobilize the intracellular ribosome complex to improve the stability of the ribosome-bound RNA fragments in subsequent processing. After cross-linking, the sample is lysed, and the unprotected RNA regions are selectively digested using ribonuclease to obtain ribosome-protected fragments.

[0008] Preferably, the treatment concentration of the translation inhibitor actinomycete ketone can be 0.05–0.5 mg / mL, and the treatment time is 1–10 minutes; Preferably, the concentration of the reversible crosslinking agent DSP can be 1–10 mM, and the crosslinking conditions are 37°C or room temperature for 30–90 minutes. Preferably, the reaction conditions for pyrolyzing NP-40 in the sample can be a reaction at 4°C for 5-30 minutes.

[0009] Preferably, the digestion concentration of the ribonuclease RNase I can be 0.01–1 U / μL, and the digestion conditions are a reaction at room temperature or 4°C for 0.5–3 hours.

[0010] S2: Immunoenrichment In step S2, the carrier is incubated with a specific antibody to immobilize the antibody; and the binding of the carrier and antibody is cross-linked using a cross-linking agent to improve stability during immunoprecipitation. The digested sample from step S1 is then used to perform an immunoprecipitation reaction with the cross-linked antibody bound to the carrier. The antibody used can be an antibody targeting ribosomal structural proteins or an antibody targeting ribosomal binding proteins, thereby achieving enrichment of the ribosomal group or selective ribosomal group, respectively. After immunoprecipitation enrichment, the immunoprecipitation complex is washed to remove non-specifically bound components, and eluted with an elution buffer to obtain the ribosome-mRNA complex.

[0011] Preferably, the carrier for immobilizing antibodies can be magnetic beads.

[0012] Preferably, the immunoprecipitation conditions are incubation at room temperature or 4°C for 0.5-24 hours; the washing step includes washing with NP40 buffer and ultrapure water; S3: RNA Extraction and Processing In step S3, the cross-linked structure of the ribosome-mRNA complex obtained by elution in step S2 is dissociated and the protein is removed; then the RNA is extracted, enriched and purified; and the extracted RNA fragments are subjected to terminal dephosphorylation and 5′ terminal phosphorylation to adapt to the requirements of subsequent adapter ligation and sequencing, and the RNA is purified again.

[0013] Preferably, the final concentration of the decrosslinking agent reducing agent DTT is 20–60 mM, and the reaction is carried out at 37°C or room temperature for 10–60 minutes. The concentration of proteinase K digestion for protein removal is 1–5 mg / mL, and the reaction is carried out at 37°C or room temperature for 5–30 minutes. Preferably, RNA fragment dephosphorylation is achieved by T4 polynucleotide kinase (PNK) catalysis in a phosphate-free system, with a working concentration of 0.1-1 U / µl. The reaction conditions are 37°C or room temperature for 0.5-3 hours. Phosphorylation is achieved by T4 PNK catalysis in a phosphate-supported system, with the reaction conditions being 37°C or room temperature for 0.5-3 hours, followed by incubation at 50-80°C for 5-60 minutes to inactivate the PNK enzyme. An RNase inhibitor is added to the reaction system to protect the RNA.

[0014] S4: Library Construction and Sequencing Analysis In step S4, the RNA fragments processed in step S3 are used to construct a sequencing library and perform sequencing. The raw sequencing data is then subjected to quality control filtering to remove adapter sequences, low-quality reads, and abnormal sequences, as well as sequencing reads shorter than a preset minimum length threshold or longer than a preset maximum length threshold, in order to obtain high-quality ribosome-binding RNA fragment sequence data.

[0015] Subsequently, the obtained RNA fragment sequence structure and nucleic acid or amino acid arrangement characteristics were analyzed, and compared with the reference transcriptome and / or reference genome to analyze their position on the complete transcript and / or gene, and to quantitatively assess the translation level of the gene or transcript.

[0016] Preferably, a small RNA library construction method can be used, including steps such as 3' adapter ligation, redundant adapter blocking, 5' adapter ligation, PAGE gel purification, reverse transcription, library amplification, and PCR product purification, to finally construct a DNA library suitable for high-throughput sequencing.

[0017] Preferably, in the library sorting step after library amplification, the PCR products are separated by electrophoresis using PAGE gel, and the target band of about 15-60 bp (excluding adapters and other additional sequences) is cut out and purified to obtain fragments of 50%-90% or more.

[0018] Preferably, the filtering criteria include: removing adapter sequences from reads; filtering reads containing more than 5 N bases; and filtering reads shorter than about 15 bp and longer than 60 bp.

[0019] When performing selective ribosome detection and analysis, the ribosome detection described in this patent or other ribosome detection methods are performed simultaneously. In step S5, the alignment results of selective ribosomics are intersected with those of ribosomics to ensure that the transcripts or genes identified in selective ribosomics are present in the ribosomics detection results.

[0020] Applications of ribosomal or selective ribosomal analysis methods based on immune principles in the identification and analysis of ribosome-related mRNA translation profiles, translation regulation studies, analysis of disease-related protein synthesis, or screening of biomarkers.

[0021] A kit for ribosomal or selective ribosomal analysis based on the principle of immunity, characterized in that it comprises at least: reagents related to translational state fixation and cross-linking; reagents related to ribonuclease digestion; reagents related to immunoprecipitation; antibodies against ribosomal structural proteins or ribosomal binding proteins; a carrier and reagents for cross-linking with antibodies; reagents related to de-cross-linking of ribosome-mRNA complexes and protein removal; reagents related to RNA extraction and purification; reagents related to RNA end modification; and reagents related to sequencing library construction.

[0022] An electronic device, characterized in that it comprises: A processor; a memory having a computer program stored thereon; wherein, when the computer program is run on the processor, the electronic device performs the ribosome or selective ribosome detection and analysis method based on the immune principle as described in any one of claims 1–5.

[0023] A computer-readable storage medium having a computer program stored thereon. When the computer program is executed by the processor, it implements the ribosome or selective ribosome detection and analysis method based on the immune principle as described in any one of claims 1–5.

[0024] The beneficial effects of this invention are as follows: High-specificity enrichment: Using antibody immunoprecipitation instead of sucrose density gradient centrifugation significantly improves the specificity of ribosome-mRNA complex enrichment.

[0025] Precisely locate translation sites: By pausing translation and RNA-protein crosslinking, the position of ribosomes on mRNA is effectively fixed.

[0026] Wide range of applications: By selecting different antibodies (ribosomal structural protein antibodies or ribosomal binding protein antibodies), non-discriminatory ribosomal analysis or targeted selective ribosomal analysis can be achieved.

[0027] Precise sequence information: Ribonuclease digestion removes non-protected regions, and combined with small fragment library construction and bioinformatics analysis, more accurate ribosome-protected mRNA sequence information can be obtained. Attached Figure Description

[0028] Figure 1This image shows the amplification curve and CT value of RNA enriched using the immunologically based ribosomal or selective ribosomal detection and analysis method proposed in this invention, validated by RT-qPCR. The amplification product is 18S RNA of the ribosome, with a CT value of approximately 27, indicating that the ribosomal complex has been enriched. Figure 2 The ribosomal complex enriched by the ribosomal or selective ribosomal analysis method based on the immune principle proposed in this invention is decrosslinked and purified by mRNA. After adding adapters to the RNA, the capillary electrophoresis image obtained is 120bp (adapter) + 15-60bp (RNA fragment), which is in line with expectations.

[0029] Figure 3 This is a partial alignment result between the sequencing-based ribosomal or selective ribosomal detection and analysis method proposed in this invention and a reference transcriptome database. Detailed Implementation

[0030] The technical solution of the present invention will be further described in detail below with reference to specific embodiments.

[0031] In the description of this invention, it should be noted that, unless otherwise explicitly specified and limited, the terms "installation," "connection," "linking," and "setting" should be interpreted broadly. For example, they can refer to a fixed connection or setting, a detachable connection or setting, or an integral connection or setting. Those skilled in the art can understand the specific meaning of the above terms in this invention according to the specific circumstances.

[0032] Example 1: I. Preparation of Immunoprecipitation Magnetic Beads Take 25 μL of magnetic beads and wash twice with 500 μL of 1xPBS containing 0.05% NP40.

[0033] Add the antibody to 20xPBS containing 1% NP40, then dilute with ultrapure water to a final antibody concentration of 5 μg / 100 μL, and dissolve in 1xPBS containing 0.05% NP40.

[0034] Incubate 100 μL of the diluted antibody solution with magnetic beads at room temperature for 15 minutes, then remove the liquid.

[0035] Wash once with 100 μL and 300 μL of 1xPBS containing 0.05% NP40.

[0036] Cross-linked antibody: Dilute DSS to 0.25 mM with DMSO.

[0037] Remove the liquid from the magnetic beads, and add 2.5 μL of 20xPBS, 4 μL of 0.25 mM DSS and 43.5 μL of ultrapure water (final volume 50 μL, DSS 20 μM) to the magnetic beads. React at room temperature for 30 minutes.

[0038] Wash twice with 100 μL of 0.1 M glycine hydrochloride (pH=2), then wash twice with 200 μL of pre-cooled 1X NP40 lysis buffer. The prepared magnetic beads can be stored at 4 °C.

[0039] II. Cell Collection and Immunoprecipitation Experimental procedure: Cell cross-linking: Add actinomycete ketone (final concentration 0.1 mg / mL) to cell culture dishes and incubate for 5 minutes. Discard the supernatant, wash twice with pre-chilled PBS containing 0.1 mg / mL actinomycete ketone, and remove the supernatant. Add 2-3 mL of 2.5 mM DSP solution and cross-link at 37°C for 30 minutes. Inactivate the cells with Tris-HCl (final concentration 20 mM) for 10 minutes, and then wash 1-2 times with pre-chilled PBS containing 0.1 mg / mL actinomycete ketone.

[0040] Cell lysis and RNase I digestion: every 5 × 10 6 Add 400 μL of NP40 lysis buffer containing 0.1 mg / mL actinomycin to the cells, incubate on ice for 5 minutes, scrape off the cells, and repeatedly freeze and thaw three times in a liquid nitrogen metal bath. Centrifuge at 17000g for 10 minutes at 4°C, and collect the supernatant. Add lysis buffer containing actinomycin to a final volume of 500 μL, add 0.5 μL of RNase I (final concentration 0.1 U / μL), and incubate at 4°C for 1 hour. After the reaction is complete, add 2.5 μL of RNase inhibitor.

[0041] Ribosome immunoenrichment: Incubate 500 μL of sample with prepared antibody magnetic beads overnight at 4°C. Collect the beads using a magnetic rack and discard the supernatant. Wash twice with 500 μL of NP40 lysis buffer, then once with 500 μL of ultrapure water. Elute with 100 μL of 0.1 M glycine hydrochloride (pH=2) at room temperature for 5 minutes, collect the supernatant, and add 10 μL of neutralization buffer (1M Tris-HCl, pH 8.5).

[0042] III. RNA Fragment Extraction and Processing Reagent preparation: Prepare a 20 mg / mL proteinase K working solution using PBS.

[0043] Experimental procedure: Ribosome digestion and dissociation: Add 10 μL of proteinase K (final concentration 2 mg / mL) to the eluent and incubate at room temperature for 10 minutes. Add 1 μL of 1M DTT (final concentration 5 mM) and incubate for 10 minutes. Add another 10 μL of 1M DTT (final concentration 50 mM) and incubate at 37°C for 10 minutes to decrosslink. Add 3 volumes of Trizol, extract with chloroform, and precipitate with isopropanol overnight. Centrifuge at 20,000 g for 30 minutes at 4°C, discard the supernatant, wash with 75% ethanol, air dry, and dissolve the RNA in 11 μL of DEPC water.

[0044] PNK dephosphorylation and phosphorylation: Adjust RNA concentration to be consistent. Denature at 80℃ for 90 seconds, recover at 37℃. Add 5 μL PNK buffer, 1 μL RNase inhibitor, and 2 μL PNK, and react at 37℃ for 1 hour. Add 5 μL ATP (10 mM), react at 37℃ for another hour, and inactivate at 70℃ for 10 minutes. Purify RNA and finally dissolve in 10 μL of enzyme-free water.

[0045] IV. RNA Library Construction and Sequencing Library construction was performed using the Novizan VAHTS Small RNA Library Prep Kit for Illumina V2.

[0046] 3' Adapter Ligation: Take 1-6 μL of RNA, add RL3 Adaptor to 7 μL, incubate at 70°C for 2 minutes, then on ice for 2 minutes. Add 10 μL of RL3 Buffer V2 and 3 μL of RL3 Enzyme Mix V2, incubate at 25°C for 1 hour.

[0047] Excess connector sealing: Add 1 μL RT Primer, incubate at 70°C for 5 minutes, then in an ice bath.

[0048] 5' Connector Connection: Add 1μL RL5 Adaptor, 1μL RL5 Buffer, and 2.5μL RL5 Enzyme Mix, incubate at 25°C for 1 hour.

[0049] Reverse transcription: Add 8 μL RT Buffer and 2 μL RT Enzyme mix V2, incubate at 50°C for 1 hour, then at 80°C for 5 minutes.

[0050] Library enrichment: Add 50 μL Amplification Mix 3, 2.5 μL Universal Primer, 2.5 μL Lindex Primer, and 5 μL water, for a total volume of 100 μL. PCR program: 94℃ for 3 minutes; 94℃ for 15 seconds, 65℃ for 15 seconds, 72℃ for 15 seconds, N cycles; 72℃ for 1 minute.

[0051] PCR product purification and sorting: Purification was performed using VAHTS DNA Clean Beads (1.8x volume). Target bands (approximately 130-190 bp, containing adapter sequences) were further sorted by non-denaturing PAGE gel electrophoresis, followed by gel extraction, precipitation, and purification.

[0052] Sequencing and Analysis: Sequencing was performed using the Illumina platform. FastP software was used for quality control filtering, and fragment lengths were selected to retain fragments between 15-60 bp (excluding adapters and other additional sequences) to obtain clean data. Clean reads were aligned to a reference genome, and gene expression quantification analysis was performed using FPKM values.

[0053] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for detecting and analyzing ribosomalomes or selective ribosomalomes based on the principle of immunity, characterized in that, Includes the following steps: S1: Sample preparation and cross-linking: Biological samples were treated with translation inhibitors to pause ribosome translation, followed by cross-linking of intracellular RNA-proteins to stabilize the ribosome-mRNA complex; then the cells were lysed and the lysate was digested with ribonuclease to degrade the unprotected mRNA regions. S2: Immunoprecipitation: Immunoprecipitation is performed on the sample digested in step S1 using an antibody bound to the carrier. The antibody is an antibody targeting ribosomal structural proteins or an antibody targeting ribosomal binding proteins, thereby achieving specific enrichment of the ribosomal group or selective ribosomal group, respectively. After enrichment, the vector was washed and eluted to obtain the ribosome-mRNA complex; S3: RNA extraction and processing: The complex obtained by elution in step S2 is deproteinized and decrosslinked, and then RNA fragments are extracted, purified and enriched; the extracted RNA fragments are dephosphated at the 3′ end and phosphated at the 5′ end, and RNA fragments with lengths outside the preset threshold are removed by separation and purification, and the RNA is purified again; S4: Library Construction and Sequencing Analysis: RNA fragments processed in step S3 were used to construct a library using RNA library construction reagents. The library was then sorted to obtain fragments within the target length range for sequencing. After sequencing, the raw data was quality controlled and filtered to remove rRNA sequences and select fragments with lengths within a preset threshold to obtain the sequence information of ribosome-binding mRNA fragments. Finally, the sequence structure characteristics of the mRNA fragments and their corresponding peptides were analyzed, and the sequences were aligned to a reference transcriptome and / or genome for localization and quantification.

2. The ribosome or selective ribosome detection and analysis method based on the principle of immunity according to claim 1, characterized in that, In step S1, the treatment concentration of the translation inhibitor actinomycete ketone can be 0.05–0.5 mg / mL, and the treatment time is 1–10 minutes; the concentration of the reversible crosslinking agent DSP can be 1–10 mM, and the crosslinking conditions are 37°C or room temperature for 30–90 minutes; the digestion concentration of ribonuclease RNase I can be 0.01–1 U / μL, and the digestion conditions are room temperature or 4°C for 0.5–3 hours.

3. The ribosome or selective ribosome detection and analysis method based on the principle of immunity according to claim 1, characterized in that, In step S2, antibodies against ribosomal structural proteins essential for translation are used for ribosomalomics, and antibodies against ribosomal binding proteins that selectively regulate translation are used for selective ribosomalomics.

4. The ribosome or selective ribosome detection and analysis method based on the principle of immunity according to claim 1, characterized in that, In step S3, the final concentration of the decrosslinking reducing agent DTT is 20–60 mM, and the reaction is carried out at 37°C or room temperature for 10–60 minutes. The concentration of proteinase K for protein removal is 1–5 mg / mL, and the reaction is carried out at 37°C or room temperature for 5–30 minutes. RNA fragment dephosphorylation is achieved by T4 polynucleotide kinase (PNK) catalysis in a phosphate-free system, with a working concentration of 0.1–1 U / µl. The reaction conditions are 37°C or room temperature for 0.5–3 hours. Phosphorylation is achieved by T4 PNK catalysis in a phosphate-supported system, with the reaction conditions being 37°C or room temperature for 0.5–3 hours, followed by incubation at 50–80°C for 5–60 minutes to inactivate the PNK enzyme. An RNase inhibitor is added to the reaction system to protect the RNA.

5. The ribosome or selective ribosome detection and analysis method based on the principle of immunity according to claim 1, characterized in that, In step S4, a small RNA library construction method is used, including operations such as adding adapters to the 3' and 5' ends; the library is sorted by fragment size and raw data is screened with a length of approximately 15-60 bp (excluding adapters and other additional sequences) to enrich sequences around 30 bp; the mRNA fragment sequence analysis includes at least one of the following: the compositional characteristics of the amino acid or nucleic acid sequence, the CG content characteristics of the sequence, and the location characteristics of the sequence on the corresponding transcript or gene.

6. The ribosome or selective ribosome detection and analysis method based on the principle of immunity according to claim 1, characterized in that, When performing selective ribosome detection and analysis, the ribosome detection described in this patent or other methods of ribosome detection are performed simultaneously. In step S4, when performing transcriptome and / or genome alignment, the alignment results of selective ribosomics are intersected with the alignment results of ribosomics to ensure that the transcripts or genes identified in selective ribosomics are present in the ribosomics detection results.

7. The application of the ribosomal or selective ribosomal analysis method based on the immune principle as described in any one of claims 1-6 in the identification and analysis of ribosome-related mRNA translation profiles, translation regulation studies, disease-related protein synthesis analysis, or biomarker screening.

8. A kit for implementing the ribosome or selective ribosome detection and analysis method based on the immune principle as described in any one of claims 1-6, characterized in that, It should include at least: reagents for translation state fixation and cross-linking; reagents for ribonuclease digestion; reagents for immunoprecipitation, and antibodies against ribosomal structural proteins or ribosomal binding proteins; vectors and antibody cross-linking reagents; reagents for decross-linking ribosome-mRNA complexes and protein removal; reagents for RNA extraction and purification; reagents for RNA end modification; and reagents for sequencing library construction.

9. An electronic device, characterized in that, include: processor; A memory having a computer program stored thereon; wherein, when the computer program is run on the processor, the electronic device performs the ribosome or selective ribosome detection and analysis method based on the immune principle as described in any one of claims 1–6.

10. A computer-readable storage medium having a computer program stored thereon, When the computer program is executed by the processor, it implements the ribosome or selective ribosome detection and analysis method based on the immune principle as described in any one of claims 1–6.

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