Live cell imaging system for RNA modification and its composition and application

Abstract

The present invention belongs to the field of biomolecular technology, and in particular to a kind of RNA-modified live cell imaging system and its composition and application. The RNA-modified live cell imaging system comprises an RNA reporter system and a FRET probe, wherein the RNA reporter system comprises a tetracycline-induced expression system, an mMaroon1 fluorescent protein, an NLS-nuclear localization sequence and a 22-repeat m6A-MS2 reporter sequence; the FRET imaging probe comprises a YTH domain, a YPet fluorescent protein, a long-chain flexible linker, an ECFP fluorescent protein, an MCP protein and an NLS nuclear localization signal. The m6A-modified live cell imaging system developed by this patent can perform high-resolution real-time dynamic imaging of m6A modification in living cells, and can realize triple dynamic monitoring of m6A modification levels and RNA localization and translation, thereby building a research platform and drug screening platform for m6A modification pathways.

Description

Technical Field

[0001] The present invention belongs to the field of biomolecule technology, and in particular relates to an RNA-modified living cell imaging system and a composition and application thereof. Background Art

[0002] RNA epigenetic modification is a key process in regulating posttranscriptional gene expression. N6-Adenylate methylation (m6A) is the most abundant known RNA modification and is widely involved in regulating RNA metabolism, three-dimensional genome structure, and DNA damage. Approximately 50% of RNAs have four or fewer m6A modifications, and the modified sequences share the common characteristic of RRACH (R = A / G; H = A / U / C). RNA m6A modification is dynamically regulated by the methyltransferase METTL3 / 14 complex and the demethylases FTO and ALKBH5, with recognition and functional implementation by the binding proteins YTHDF1 / 2 / 3, YTHDC1 / 2, and IGF2BP1 / 2 / 3. The regulation of RNA fate by m6A modification is currently a hot topic and a challenge in basic research. Existing studies suggest that m6A modification is involved in regulating RNA transcription, transport, degradation, and translation, but the specific mechanisms have yet to be agreed upon.

[0003] Multiple proteins involved in the m6A modification pathway have been shown to promote cancer. METTL3 has been shown to be abnormally elevated in acute myeloid leukemia (AML) and is closely associated with the development and progression of AML. The METTL3-specific inhibitor STM2457 effectively inhibits the proliferation of human AML tissue in mouse models. Furthermore, levels of the demethylase ALKBH5 have been shown to be negatively correlated with clinical efficacy in glioblastoma. Detailed studies have shown that ALKBH5 removes the m6A modification from the cancer marker FOXM1, thereby stabilizing FOXM1 and promoting its translation, thereby promoting cancerization. Furthermore, recent studies have found that m6A modifications and their recognition protein, YTHDF2, are enriched in patients with amyotrophic lateral sclerosis (ALS), and that knocking down YTHDF2 can alleviate TDP43- and C9ORF72-mediated neurotoxicity. Furthermore, over 90% of TDP43-bound RNAs have been shown to harbor m6A modifications. Therefore, it is crucial to develop small molecule regulators targeting proteins related to the m6A modification pathway, such as METTL3 / 14, FTO, and ALKBH5.

[0004] To address the current challenges and difficulties in m6A modification research, developing methods for live-cell imaging of m6A modification with temporal and spatial precision is a key breakthrough in uncovering the physiological and pathological functions of m6A modification, yet it also represents a gap in m6A modification research. Existing m6A modification detection methods primarily rely on colorimetric imaging of fixed cell samples using m6A antibodies, making it difficult to achieve dynamic observation and establish live-cell drug screening platforms.

[0005] The invention patent with publication number CN110567906A discloses a method for characterizing RNA methylation modification. This invention targets the RNA m6A methylation modification pathway, applies theoretical calculation methods to obtain the characteristic absorption spectra of RNA sequences and RNA m6A methylation sequences, and establishes a spectroscopy-based method for characterizing dynamic RNA methylation modification. This invention does not involve live cell imaging of RNA modification. Summary of the Invention

[0006] In view of this, the present invention innovatively constructed an m6A-modified RNA reporter system and a FRET imaging system, which can perform high-resolution real-time dynamic imaging of m6A modification in living cells.

[0007] One of the objectives of the present invention is to provide a composition for preparing a live cell imaging system for RNA modification.

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

[0009] A composition for preparing a live-cell imaging system for RNA modification, comprising an RNA reporter system and a FRET probe; the RNA reporter system comprises an inducible expression system, a protein tag, a subcellular organelle localization sequence, an RNA epigenetic modification sequence and / or an RNA aptamer sequence; and the FRET probe comprises an RNA epigenetic modification binding domain, a FRET pair and / or an RNA aptamer binding protein.

[0010] Furthermore, in the RNA reporter system, the inducible expression system is a tetracycline-inducible expression system; the protein tag is mCherry fluorescent protein, CFP fluorescent protein, GFP fluorescent protein, mMaroon1 fluorescent protein, Halo tag, SNAP tag and / or luciferase; the subcellular organelle localization sequence is SKL-peroxisome localization sequence, NLS-nuclear localization sequence and / or NES-nuclear export localization sequence; the RNA epigenetic modification sequence and the RNA aptamer sequence are 22 repeats of mA-MS2 reporter sequence and / or PP7-BoxB sequence.

[0011] Furthermore, in the FRET probe, the RNA epigenetic modification binding domain is a YTH domain; the FRET pair is YPet fluorescent protein and ECFP fluorescent protein, GFP fluorescent protein and mCherry fluorescent protein and / or BFP fluorescent protein and GFP fluorescent protein; and the RNA aptamer binding protein is MCP protein, PCP protein and / or N22 protein.

[0012] Furthermore, the RNA reporter system further includes a connecting sequence; and the FRET probe further includes a structural sequence and a connecting sequence.

[0013] A second object of the present invention is to provide a living cell imaging system for RNA modification.

[0014] To achieve the above object, the present invention adopts the following technical solutions:

[0015] A living cell imaging system for RNA modification, wherein the living cell imaging system contains the composition described in purpose one.

[0016] Furthermore, the RNA modification includes modifications of different nucleotide types, specifically adenine ribonucleotides, guanine ribonucleotides, cytosine ribonucleotides and uracil ribonucleotides;

[0017] Furthermore, the RNA modification includes methylation modification, acetylation modification, phosphorylation modification, hydroxymethylation modification and / or 2'-O-methylation modification.

[0018] Furthermore, the RNA modification includes m6A modification, m4C modification, m1A modification, m5C modification, hm5C modification, m7G, 2'-O-methylation modification, ac4C acetylation modification and / or m3U modification.

[0019] Currently, no literature has been found regarding live-cell imaging of RNA m6A modifications. Known detection methods for m6A modifications primarily include in vitro assays or the most common immunofluorescence assay. A search revealed that researchers in this field have employed antibody detection and rolling circle amplification techniques on fixed cell samples to image specific m6A modifications. However, this method struggles to achieve dynamic observation and establish a live-cell drug screening platform.

[0020] Furthermore, the m6A-modified RNA reporter system consists of a tetracycline-inducible expression system with a nucleotide sequence as shown in SEQ ID NO.1, an mMaroon1 fluorescent protein with a nucleotide sequence as shown in SEQ ID NO.2, an NLS-nuclear localization sequence with a nucleotide sequence as shown in SEQ ID NO.3, and an m6A-MS2 reporter sequence with 22 repeated nucleotide sequences as shown in SEQ ID NO.4.

[0021] Furthermore, the PCR primers for amplifying the mMaroon1 fluorescent protein include a forward primer mMaroon1 F1 and a reverse primer mMarron1 R1. The nucleotide sequence of the mMaroon1 F1 is shown in SEQ ID NO.5, and the nucleotide sequence of the mMarron1 R1 is shown in SEQ ID NO.6.

[0022] Furthermore, the nucleotide sequence of the RNA reporter system is shown in SEQ ID NO.7.

[0023] Furthermore, the m6A-modified FRET probe consists of a YTH domain as shown in SEQ ID NO.8, a YPet fluorescent protein as shown in SEQ ID NO.9, a long-chain flexible linker as shown in SEQ ID NO.10, an ECFP fluorescent protein as shown in SEQ ID NO.11, an MCP protein as shown in SEQ ID NO.12, and an NLS nuclear localization signal as shown in SEQ ID NO.13.

[0024] Furthermore, the nucleotide sequence of the FRET probe is shown in SEQ ID NO.14.

[0025] A third object of the present invention is to provide a stable cell line obtained by transfecting the living cell imaging system.

[0026] A fourth object of the present invention is to provide a method for detecting RNA modification levels using the living cell imaging system.

[0027] To achieve the above object, the present invention adopts the following technical solutions:

[0028] The method for detecting RNA modification levels using the living cell imaging system comprises: co-expressing a reporter RNA and a FRET probe in mammalian cells in vitro, and detecting RNA modification levels by measuring the FRET / ECFP ratio.

[0029] When the RNA reporter system lacks m6A modification, the long flexible linker effectively separates YPet and ECFP proteins, reducing the FRET background signal. When m6A modification is present, the MCP binds to the MS2 aptamer, and the YTH domain binds to the adjacent m6A modification, bringing YPet and ECFP closer together and enhancing the FRET signal.

[0030] A fifth object of the present invention is to provide an application of the combined use of the RNA reporter system and the FRET probe in dynamically monitoring m6A modification levels, RNA localization and translation in living cells.

[0031] Furthermore, the living cells are mammalian living cells cultured in vitro.

[0032] A sixth object of the present invention is to provide an application of the composition, the living cell imaging system and / or the stably transfected cell line in dynamically monitoring m6A modification levels, RNA localization and translation in living cells.

[0033] Furthermore, the living cells are mammalian living cells cultured in vitro.

[0034] Furthermore, the composition and / or the live cell imaging system are used in constructing a research platform for RNA modification pathways and a drug screening platform.

[0035] Furthermore, the RNA modification is m6A modification, m4C modification and / or m3U modification.

[0036] Furthermore, the composition and / or the RNA-modified living cell imaging system are used in the living cell drug screening of modification-related enzymes.

[0037] Furthermore, the related enzymes are methylase METTL3 / 14 complex, demethylase FTO and / or ALKBH5.

[0038] The inventive method encompasses reporter systems and FRET probes for all RNA epigenetic modifications (including but not limited to m6A modifications) using similar design principles. Specifically, in the reporter system, it includes using an inducible expression system different from tetracycline, fluorescent proteins or other label proteins different from mMaroon1, other subcellular organelle localization sequences different from NLS, m6A modification motifs or other RNA epigenetic modification sequences different from GGACC, other RNA aptamer sequences different from MS2, and connection sequences different from the patent display. In the FRET probe, it includes using m6A recognition domains and other RNA epigenetic modification binding domains different from YTH, FRET pairs different from YPet / ECFP, RNA aptamer binding proteins different from MCP, and different structural orders and connection sequences. The application of this patent is not limited to the research platform and drug screening platform for constructing m6A modification pathways mentioned in this patent. Those of ordinary skill in the art should understand that the technical solution of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solution of the present invention, and it should all be encompassed within the scope of the claims of the present invention.

[0039] The beneficial effects of the present invention are:

[0040] 1. The main innovation of this patent lies in the construction of an m6A modification reporter system with the function of inducible expression. It also has an original m6A modification indicator sequence connected to a 22X m6A modification motif and an MS2 aptamer. At the same time, this patent is the first to construct an m6A-modified FRET probe for high-resolution dynamic tracking in living cells.

[0041] 2. The m6A-modified live cell imaging system developed in this patent includes an m6A-modified RNA reporter system and a FRET imaging system. It can perform high-resolution, real-time dynamic imaging of m6A modification in living cells, allowing dynamic observation and a wider range of detection dimensions.

[0042] 3. This patent combines the use of an m6A-modified live-cell reporter RNA system and a FRET probe to achieve triple dynamic monitoring of RNA localization, m6A modification levels, and RNA translation, thereby establishing a research platform and drug screening platform for the m6A modification pathway;

[0043] 4. The design concept of this patent can be used to develop live cell imaging systems for other RNA modifications to study their physiological and pathological functions, as well as for drug development;

[0044] 5. The m6A-modified live cell imaging system developed in this patent does not rely on m6A antibodies and is less expensive;

[0045] 6. The m6A modification live cell imaging system developed in this patent can be used to construct stably transfected cell lines, making it easy to build a live cell drug screening platform for m6A modification-related enzymes. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 Schematic diagram of the detection principle of the live cell imaging system for m6A modification;

[0047] Figure 2 This is a graph showing the expression of m6A-modified FRET probes in mammalian cells;

[0048] Figure 3 This is the result of using the live cell imaging system for specific detection of m6A modification levels;

[0049] Figure 4 This is the live cell dynamic detection result of wild-type probe (WT BS) on m6A reporter system;

[0050] Figure 5 This is the result of dynamic detection of the m6A reporter system in living cells using the mutant probe (Mutant BS). DETAILED DESCRIPTION

[0051] The technical solutions of the present invention will be further described in detail below with reference to specific embodiments. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Therefore, all other embodiments derived by those skilled in the art based on the embodiments of the present invention without inventive effort are intended to fall within the scope of protection of the present invention.

[0052] In this application:

[0053] DOX: Doxycycline, a tetracycline derivative;

[0054] 22X m6A-MS2 sequence: 22 repeats of the m6A modification motif and the MS2 aptamer combination reporter sequence;

[0055] mMaroon1 fluorescent protein is a red fluorescent dye widely used in biotechnology as a tracer, including labeling of molecules and localization of cellular components;

[0056] NLS nuclear localization signal: a type of polypeptide sequence that helps the labeled protein be transported into the cell nucleus;

[0057] YTH domain: The YTH domain is a protein domain that specifically binds to m6A-modified RNA and is widely present in m6A-modified binding proteins;

[0058] YPet fluorescent protein: YPet is the brightest yellow fluorescent protein developed and has excellent photostability;

[0059] Long-chain flexible linker: A long-chain flexible linker is a commonly used linker sequence for constructing intramolecular FRET probes. This sequence, mainly composed of glycine and serine, has high flexibility and a relatively large length, which can reduce the FRET background.

[0060] ECFP fluorescent protein: enhanced cyan fluorescent protein;

[0061] MCP protein: a binding protein for the MS2 aptamer that can bind to the MS2 aptamer with high specificity and binding affinity;

[0062] Example 1. Live cell imaging method of m6A modification

[0063] The following steps are involved:

[0064] 1) Fuse the tetracycline-inducible expression system, mMaroon1 fluorescent protein, NLS localization sequence, and 22 repeats of the m6A modification reporter sequence to obtain the m6A modified reporter RNA plasmid;

[0065] 2) Fuse the YTH domain, YPet fluorescent protein, long-chain flexible linker, ECFP fluorescent protein, MCP protein, and NLS nuclear localization signal to obtain the m6A-modified FRET probe plasmid;

[0066] 3) Fuse the YTH domain mutant, YPet fluorescent protein, long-chain flexible linker, ECFP fluorescent protein, MCP protein, and NLS nuclear localization signal to obtain a loss-of-function plasmid of the m6A-modified FRET probe;

[0067] 4) In mammalian cells, the reporter RNA and FRET probe were co-expressed and the effect of tetracycline on the FRET / ECFP ratio was observed.

[0068] In step 1), the nucleotide sequence of the tetracycline-inducible expression system is shown in SEQ ID NO.1, the nucleotide sequence of the mMaroon1 fluorescent protein is shown in SEQ ID NO.2, the nucleotide sequence of the NLS positioning sequence is shown in SEQ ID NO.3, and the nucleotide sequence of the m6A-MS2 reporter sequence is shown in SEQ ID NO.4.

[0069] In step 2), the nucleotide sequence of the YTH domain is shown in SEQ ID NO.8, the nucleotide sequence of the YPet fluorescent protein is shown in SEQ ID NO.9, the nucleotide sequence of the long-chain flexible linker is shown in SEQ ID NO.10, the nucleotide sequence of the ECFP fluorescent protein is shown in SEQ ID NO.11, the nucleotide sequence of the MCP protein is shown in SEQ ID NO.12, and the nucleotide sequence of the NLS nuclear localization signal is shown in SEQ ID NO.13.

[0070] The specific principles and processes are as follows Figure 1As shown. The core of this patent lies in the design of the m6A-modified RNA reporter system and FRET imaging probe. The RNA reporter system is an artificially designed and assembled gene sequence that is spliced ​​together using molecular biology techniques to form a tetracycline-inducible expression system, mMaroon1 fluorescent protein, an NLS-nuclear localization sequence, and 22 repeats of the m6A-MS2 reporter sequence. Under the induction of tetracycline or DOX, this reporter system binds to rtTA-VP64 to initiate RNA transcription. After RNA transcription, the 22X m6A-MS2 sequence recruits the METTL3 / 14 methylase complex to perform m6A modification on specific sites. After the m6A-modified RNA is transported to the cytoplasm, it binds to the ribosome and is translated to form the mMaroon1 red fluorescent protein, which carries an NLS-nuclear localization sequence that guides the localization of mMaroon1 to the cell nucleus. The FRET imaging probe, on the other hand, comprises a YTH domain, a YPet fluorescent protein, a long flexible linker, an ECFP fluorescent protein, an MCP protein, and an NLS nuclear localization signal. The YTH domain can recognize the m6A modification of RNA, YPet and ECFP form a FRET pair with high FRET conversion efficiency, and the MCP protein can bind to the MS2 aptamer.

[0071] When the FRET probe is co-present with an m6A-modified reporter RNA, the YTH domain binds to the m6A modification site, and the MCP protein binds to the adjacent MS2 sequence, thereby shortening the distance between YPet and ECFP and enhancing the FRET signal. Conversely, if the YTH domain is mutated and unable to bind to the m6A modification site, YPet and ECFP are separated by the long flexible linker arm, resulting in a lower FRET background.

[0072] Example 2. Construction and evaluation of plasmids for reporter RNA system

[0073] The 22X m6A-MS2 sequence was designed and the gene fragment of 22X m6A-MS2 was obtained using custom gene synthesis services and inserted into the pUC Kan+ plasmid. The pUC Kan+ plasmid was digested with BamH1 and KpN1, and the target fragment containing 22X m6A-MS2 was recovered. The mMaroon1 sequence was amplified from pmMaroon1 (a non-commercial plasmid) using primers mMaroon1 F1 and mMaroon1 R1. The amplified sequence was digested with Nhe1 and KpN1, and the target fragment containing mMaroon1 was recovered. The pLD1112 plasmid was digested with Nhe1 and BamH1, and the ends were treated with CIP enzyme to recover the vector sequence. The pLD1112 vector, mMaroon1, and 22X m6A-MS2 were ligated using T4 ligase at 16°C overnight. The ligation product was transfected into Stbl3 competent cells, and single colonies were picked by plating. Sanger sequencing was used to identify and select the correct plasmid, resulting in the pLD1112NLS-mMaroon1-NLS-22X m6A-MS2 plasmid, hereinafter referred to as the reporter RNA plasmid. Its nucleotide sequence is shown in SEQ ID NO. 7. The reporter RNA plasmid was transiently transfected into HeLa cells using the lipo3000 transfection kit. 36 hours after transfection, 1 μg / mL DOX was added to induce mMaroon1 expression, and the fluorescence intensity of mMaroon1 was compared before and 12 hours after DOX induction. After 12 hours of DOX induction, the fluorescence intensity of mMaroon1 was significantly enhanced.

[0074] PCR primers for mMaroon1 amplification: The nucleotide sequence of mMaroon1 F1 is shown in SEQ ID NO.5, the nucleotide sequence of mMaroon1 R1 is shown in SEQ ID NO.2, and the nucleotide sequence after amplification is shown in SEQ ID NO.6.

[0075] Example 3. Construction of wild-type m6A-modified FRET probe plasmid

[0076] Search and compare the gene sequence of the YTH domain, obtain the gene fragment of the YTH domain using custom fund synthesis service, and insert it into the pUC Kan+ plasmid. Use YTH F and YTH R primers to amplify the YTH sequence from the above pUC Kan+. Use YPet F and ECFP R to amplify the YPet-linker-ECFP sequence from pSin H3K9Me3 FRET BS (non-commercial plasmid). Use MCP F and MCP R6 to amplify the MCP-NLS sequence from addgene 75383 (commercial plasmid). Use Nhe1 and EcoR1 to cut the pSin H3K9Me3 FRET BS, then use CIP enzyme to treat the end and recover the vector sequence. Use HiFi DNA Assembly was used to ligate the pSin vector, YTH, YPet-linker-ECFP, and MCP-NLS at 50°C for 1 hour. The ligation product was transfected into a DH5α competent cell, and single colonies were picked by plating. Sanger sequencing was used to identify and select the correct plasmid. The wild-type m6A FRET probe plasmid, referred to as the WT BS plasmid, was obtained. Its nucleotide sequence is shown in SEQ ID NO. 14.

[0077] PCR primers for YTH amplification: the nucleotide sequence of YTH F is shown in SEQ ID NO.15, and the nucleotide sequence of YTH R is shown in SEQ ID NO.16.

[0078] PCR primers for YPet-linker-ECFP amplification: the nucleotide sequence of YPet F is shown in SEQ ID NO.17, and the nucleotide sequence of ECFP R is shown in SEQ ID NO.18.

[0079] PCR primers for MCP-NLS amplification: the nucleotide sequence of MCP F is shown in SEQ ID NO.19, and the nucleotide sequence of MCP R6 is shown in SEQ ID NO.20.

[0080] Example 4. Construction of mutant m6A-modified FRET probe plasmid

[0081] The gene sequence of the mutant YTH domain was searched and compared, and the gene fragment of the YTH domain was obtained using a custom fund synthesis service and inserted into the pUC Kan + plasmid. The mutant YTH sequence was amplified from the above pUC Kan + using YTH F and YTH R primers. The other fragments were consistent with Example 3. HiFi DNA Assembly was used to connect the pSin vector, mutant YTH, YPet-linker-ECFP, and MCP-NLS at 50°C for 1 hour. The ligation product was transfected into DH5α competent medium, single clones were picked by plating, and Sanger sequencing was used to identify and select the correct plasmid. Finally, the mutant m6A FRET probe plasmid was obtained, hereinafter referred to as Mutant BS plasmid. The reporter plasmid was transiently transfected into Hela cells using the lipo3000 transfection kit. After 36 hours of transfection, the expression of the probe was observed. The specific experimental results are shown in the figure. Figure 2 Both WT BS and Mutant BS were mainly expressed in the cytoplasm, and WT BS formed a small amount of granular structures, while Mutant BS did not.

[0082] Example 5. Feasibility and specificity evaluation of the m6A-modified live cell imaging system

[0083] The reporter RNA plasmid and WT BS or Mutant BS plasmid were transiently transfected into Hela cells using the lipo3000 transfection kit. 36 hours after transfection, Hela cells were induced with DOX. 5 hours after induction, ECFP, YPet, FRET, and mMaroon1 fluorescence images were captured using a spinning disk confocal microscope with FRET function, and the differences in FRET / ECFP fluorescence intensity ratios in the WT BS or Mutant BS groups were analyzed. The experimental results are shown in Figure 2. Figure 3 As shown in the figure, when WT BS was co-expressed with the reporter RNA plasmid, the cells showed a higher FRET / ECFP ratio. In addition, the fluorescence intensity of ECFP, YPet, FRET and mMaroon1 was monitored in real time using a time series imaging mode. It was found that after DOX induction in the WT BS group, both mMaroon1 and the FRET / ECFP ratio increased gradually over time, and the increase in the FRET / ECFP ratio was earlier than that of mMaroon1. In the Mutant BS group, only the increase in the fluorescence intensity of mMaroon1 was observed, and there was no significant change in the FRET / ECFP fluorescence intensity ratio. The specific experimental results are shown in the figure. Figure 4 and Figure 5 shown.

[0084] Finally, it should be noted that the above implementation content is only used to illustrate the technical solution of the present invention and is not intended to be limiting. The inventive method encompasses reporter systems and FRET probes for all RNA epigenetic modifications (including but not limited to m6A modifications) using similar design principles. Specifically, in the reporter system, it includes using an inducible expression system different from tetracycline, fluorescent proteins or other tag proteins different from mMaroon1, other subcellular organelle localization sequences different from NLS, m6A modification motifs or other RNA epigenetic modification sequences different from GGACC, other RNA aptamer sequences different from MS2, and connection sequences different from the patent display. In the FRET probe, it includes using an m6A recognition domain different from YTH and other RNA epigenetic modification binding domains, a FRET pair not used for YPet / ECFP, an RNA aptamer binding protein different from MCP, and different structural orders and connection sequences. The application of this patent is not limited to the research platform and drug screening platform for constructing the m6A modification pathway mentioned in this patent. Those of ordinary skill in the art should understand that the technical solution of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solution of the present invention, and it should all be encompassed within the scope of the claims of the present invention.

Claims

1. A composition for preparing a living cell imaging system for m6A modification, characterized in that The composition includes an m6A-modified RNA reporter system and an m6A-modified FRET probe; the m6A-modified RNA reporter system includes a tetracycline-inducible expression system, an mMaroon1 fluorescent protein, an NLS-nuclear localization sequence, and a 22-repeated m6A-MS2 reporter sequence, and its nucleotide sequence is shown in SEQ ID NO.7; the m6A-modified FRET probe includes a YTH domain, a YPet fluorescent protein, a long-chain flexible connecting arm, an ECFP fluorescent protein, an MCP protein, and an NLS nuclear localization signal, and its nucleotide sequence is shown in SEQ ID NO.

14. 2.m6A modified live cell imaging system, characterized in that The living cell imaging system comprises the composition of claim 1.

3. Use of the composition of claim 1 and / or the live cell imaging system of claim 2 in dynamically monitoring m6A modification levels, RNA localization, and translation in living cells.

4. Use of the composition of claim 1 and / or the live cell imaging system of claim 2 in live cell drug screening for modification-related enzymes.

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