RNA-protein complex, expression vector, kit and application thereof

Abstract

The application discloses an RNA-protein complex, an expression vector, a kit and application thereof. The RNA-protein complex comprises: a chimeric RNA having a molecular structure represented as N1-N2 or N2-N1; wherein N1 is a target RNA sequence, and N2 is an RNA aptamer configured to activate a fluorophore molecule to emit spectral fluorescence; and a recombinant fusion protein having a molecular structure represented as P1-P2 or P2-P1; wherein P1 is a target protein, and P2 is a fluorescent protein, and the target protein recognizes and binds to the target RNA sequence.

Description

Technical Field

[0001] This invention relates to the technical field of bioengineering, specifically to an RNA-protein complex, its expression vector, kit, and applications. Background Technology

[0002] RNA is one of the most important biological macromolecules in life activities. It not only transmits genetic information from DNA to proteins but also plays a crucial role in the regulation of various functions in life processes. Besides RNAs with known functions, many other RNAs exist whose mechanisms of action and functions remain to be elucidated. Studying the spatiotemporal distribution and dynamics of specific RNAs can reveal the mechanisms of diseases caused by imbalances in RNA regulation. Therefore, developing tools for studying the spatiotemporal distribution of RNA is essential. Furthermore, the function of intracellular RNA largely involves RNA-binding proteins, and the loss of function or imbalance in the regulation of these proteins is associated with many diseases. Therefore, studying RNA-protein interactions at the molecular or living-cell level is crucial for understanding RNA functional mechanisms and for researching human physiology and diseases.

[0003] In the field of life science research, live-cell imaging is the most intuitive and realistic method for obtaining information about life activities at the cellular level. In RNA research, the most intuitive method is to directly observe its synthesis, transport, and localization within cells, which requires labeling and imaging the target RNA within the cell. However, the transient nature and low copy number of RNA (some RNAs may even have only one copy per cell) pose significant challenges to RNA research, making it difficult for researchers to accurately map the life processes of RNA from synthesis to degradation. Fluorescent proteins (FPs) can be used to locate and label target proteins in living cells and organisms, making them powerful tools for monitoring the dynamic processes of protein expression and interactions. However, due to the lack of high-quality natural fluorescent RNAs, scientists find it difficult to directly study RNA in living cells. Therefore, high-quality RNA imaging tools will play a crucial role in the detection and tracing of RNA in living cells.

[0004] Currently, techniques for imaging specific RNAs in cells include fluorescence in situ hybridization (FISH), covalent labeling, RNA binding protein-fluorescent protein (RBP-FP), and fluorescent RNA. Existing RNA imaging techniques have some limitations. For example, FISH can directly label endogenous RNA without requiring additional RNA targets, but due to the low nucleic acid permeability of cell membranes, it is generally only used for imaging RNA in fixed cells or tissues, and is difficult to use for imaging RNA in living cells. Covalent labeling only requires covalently labeling the target RNA with a very small fluorophore (<1 kDa), has minimal impact on the target RNA, and exhibits good labeling stability; however, existing covalent labeling techniques are also generally only applicable to fixed cells. RBP-FP technology can image RNA in live cells. However, free fused fluorescent proteins cause high background fluorescence. In order to improve the signal-to-noise ratio, multiple binding modules need to be inserted into the target RNA. However, the binding of multi-molecule fused fluorescent proteins will interfere with the normal physiological processes and functions of the target RNA, such as synthesis, transport and localization. Moreover, the protein takes a long time to mature, resulting in a lag in the detection information of RNA.

[0005] Fluorescent RNA (RNA) has many advantages as a fluorescent tag for RNA: (1) As a genetic coding tag, its synthesis is synchronized with the transcription of the target RNA, and the labeling of RNA is real-time; (2) Its molecular weight is small, the labeling system is simple, and it has little interference with the target RNA; (3) Its binding to the dye is reversible, and the dye can be removed directly by rinsing; (4) The dye molecule can only emit fluorescence after binding to a specific RNA aptamer, so the background fluorescence is very low; (5) The operation is simple, and it is only necessary to directly fuse the RNA aptamer to the target RNA and then add the dye molecule for labeling, which overcomes the defects of the above three RNA imaging techniques. However, many fluorescent dyes, such as cyanine dyes, Hoechst analogs, and fluorophore-quenchers, have strong background fluorescence in living cells or are difficult to penetrate the cell membrane, so they are not suitable for live cell RNA labeling and imaging. The dye molecules of fluorescent RNAs such as Spinach, Broccoli, and Corn are derived from the fluorophores of natural fluorescent proteins and have the advantages of low background fluorescence, good cell permeability, and low cytotoxicity. Unfortunately, these fluorescent RNAs exhibit low cellular fluorescence intensity and poor thermal and photostability, making it difficult to image low-abundance RNAs. Fluorescent RNAs containing G-tetramer structures, such as Spinach, Broccoli, and Mango, show poor dye specificity and can activate dyes of other fluorescent RNAs; for example, Spinach and Broccoli can activate Mango's dye TO1. This limitation prevents their simultaneous intracellular use, hindering simultaneous imaging of multiple RNAs. Furthermore, G-tetramers are unstable in mammalian cells, meaning that only a small fraction of correctly folded fluorescent RNA molecules with this structure can effectively label RNA in mammalian cells, resulting in poor thermal and photostability. Some fluorescent RNAs, such as Corn, require dimer formation to activate dye fluorescence, which may interfere with the molecular independence of the target RNA and affect its function.

[0006] Förster (or fluorescent) Resonance Energy Transfer (FRET) is a nonradiative energy transfer process that occurs between two fluorescent molecules (chromophores). For FRET to occur, the "Förster triad" must be met: the emission spectrum of the donor and the absorption spectrum of the acceptor must have a significant overlap; FRET occurs within a donor-acceptor distance of 1–10 nm; and the relative orientation of the donor's emission dipole and the acceptor's absorption dipole must be appropriate, with the highest efficiency when parallel and zero efficiency when perpendicular. The relationship between the energy transfer efficiency of FRET and the distance between the two molecules is as follows: E = R 06 / ( R 6 + R 0 6 Here, "E" represents the energy transfer efficiency, "R" represents the actual distance between the donor and acceptor, and "R0" represents the distance between the two molecules when the energy transfer efficiency is 50%. When R is less than R0, the transfer efficiency increases; when R is greater than R0, the transfer efficiency decreases.

[0007] FRET technology has been applied to research in multiple life sciences. At the single-molecule level, it is used to study the folding pathways, conformational heterogeneity, and dynamics of proteins and nucleic acids, revealing transient intermediate states that are difficult to capture using traditional structural biology methods. In cell signal transduction, it is used to visualize in real time the spatiotemporal fluctuations of second messengers (such as cAMP and IP3), GPCR activation, small G protein switching cycles, and the activity maps of various kinase / phosphatase networks. In gene regulation, it is used to monitor the dynamic processes of DNA damage and repair, RNA transcription and splicing, and the interaction between transcription factors and chromatin. In drug development and screening, it is widely used as a high-throughput screening platform for developing enzyme activity inhibitors and detecting protein-protein interaction regulators.

[0008] Although fluorescent RNA has been successfully used for imaging coding RNA and non-coding RNA in vivo or in vitro, most current methods for studying protein-RNA interactions are conducted in vitro, and there are few methods that can be used in living cells.

[0009] Therefore, it is necessary to provide an RNA-protein complex, its expression vector, kit, and application that can utilize FRET activation to generate fluorescence and provide the possibility of in vivo studies, in order to solve the problems existing in the prior art. Summary of the Invention

[0010] The main objective of this invention is to provide an RNA-protein complex, its expression vector, a kit, and its applications. The RNA-protein complex has advantages such as low background, high signal-to-noise ratio, and a wide dynamic response range, and can be used to detect RNA-protein interactions in living cells.

[0011] To achieve the above objectives, one embodiment of the present invention provides an RNA-protein complex comprising: (a) a chimeric RNA having a molecular structure represented by N1-N2 or N2-N1; wherein N1 is a target RNA sequence and N2 is an RNA aptamer configured to activate a fluorophore molecule to emit spectral fluorescence; and (b) a recombinant fusion protein having a molecular structure represented by P1-P2 or P2-P1; wherein P1 is a target protein and P2 is a fluorescent protein, and the target protein recognizes and binds to the target RNA sequence.

[0012] In one embodiment of the present invention, N1 and N2 of the chimeric RNA are connected by a nucleic acid sequence that does not interfere with the binding of the target RNA to the target protein, and / or P1 and P2 of the recombinant fusion protein are connected by a polypeptide that does not interfere with the binding of the target RNA to the target protein and maintains the fluorescence function of the fluorescent protein.

[0013] In one embodiment of the present invention, the fluorescent protein generates a first fluorescence under excitation light.

[0014] In one embodiment of the present invention, the RNA aptamer is selected from Pepper RNA aptamer (SEQ ID NO: 1), Clivia RNA aptamer (SEQ ID NO: 2), Okra RNA aptamer (SEQ ID NO: 3), RhoBAST RNA aptamer (SEQ ID NO: 4), Broccoli RNA aptamer (SEQ ID NO: 5), Corn RNA aptamer (SEQ ID NO: 6), Spinach RNA aptamer (SEQ ID NO: 7), Spinach2 RNA aptamer (SEQ ID NO: 8), DNB RNA aptamer (SEQ ID NO: 9), SRB-2 RNA aptamer (SEQ ID NO: 10), 13-2 min RNA aptamer (SEQ ID NO: 11), DIR-Apt1 RNA aptamer (SEQ ID NO: 15), DIR2s-Apt RNA aptamer (SEQ ID NO: 16), and Mango RNA aptamer (SEQ ID NO: 17). 17), or a group consisting of any of its biologically active variants or fragments.

[0015] In one embodiment of the present invention, the RNA aptamer recognizes and binds to the fluorophore molecule, and the fluorophore molecule is excited by the first fluorescence to emit the spectral fluorescence.

[0016] In one embodiment of the present invention, the fluorescent protein is: a blue fluorescent protein selected from the group consisting of Azurite, Sirius, TagBFP, mTagBFP, mTagBFP2, EBFP, EBFP2, Electra1, and Electra2; a cyan fluorescent protein selected from the group consisting of ECFP, SCFP3A, Cerulean, mTFP1, mTurquoise2, and mAmetrine; or a green fluorescent protein selected from the group consisting of GFP, EGFP, mNeonGreen, mWasabi, Clover, Venus, any one of their bioactive mutants or fragments, and chimeras of two or more of them.

[0017] In one embodiment of the present invention, under excitation light, the fluorescent protein generates first fluorescence, and the binding of the target protein to the target RNA sequence causes fluorescence resonance energy transfer between the fluorescent protein and the fluorophore molecule, thereby activating the fluorophore molecule to generate the spectral fluorescence.

[0018] Another embodiment of the present invention provides an expression vector that expresses the RNA-protein complex as described above.

[0019] In one embodiment of the present invention, the expression vector contains a nucleotide sequence encoding the chimeric RNA, and / or the expression vector contains a nucleotide sequence encoding the recombinant fusion protein.

[0020] Another embodiment of the present invention provides a method for detecting the interaction between target RNA and target protein in host cells, the method comprising the following steps: (a) Construct a prokaryotic or eukaryotic plasmid expression vector that expresses the RNA-protein complex as described above; (b) Introduce the prokaryotic or eukaryotic expression vector obtained in step (a) into a host cell; (c) Add fluorophore molecules separately and apply excitation light; and (d) Detect the fluorescence intensity of the fluorophore molecule and the fluorescence intensity of the fluorescent protein under the excitation light.

[0021] Another embodiment of the present invention provides a method for detecting the interaction between extracellular target RNA and target protein, the method comprising the following steps: (a) Prepare the chimeric RNA and recombinant fusion protein in the RNA-protein complex as described above; (b) Mix the chimeric RNA and the recombinant fusion protein; (c) Add fluorophore molecules separately and apply excitation light; and (d) Detect the fluorescence intensity of the fluorophore molecule and the fluorescence intensity of the fluorescent protein under the excitation light.

[0022] In another embodiment of the present invention, a kit is provided, the kit containing the expression vector as described above, and / or the kit contains prokaryotic or eukaryotic cells expressing the expression vector of the RNA-protein complex as described above. Attached Figure Description

[0023] Figure 1 A to 1C are schematic diagrams illustrating the basic generation principle of the FRET system.

[0024] Figures 2A to 2D It is mTagBFP2-λ N FRET detection of Clivia-boxB with mTagBFP2-MCP and Clivia-MS2 interaction pairs.

[0025] Figures 3A to 3B It is Clivia-boxB and mTagBFP2-λ in host cells N Live-cell imaging of complex interactions.

[0026] Figures 4A to 4D It is a detection method for intracellular RNA-protein interactions based on the Clivia-mTagBFP2 FRET system.

[0027] Figures 5A to 5E It is a live-cell imaging system based on the Clivia-mTagBFP2 FRET system.

[0028] Figure 6 It is based on the in vitro detection of Clivia-mTagBFP2 FRET system, mTagBFP2-YTHDF3 and Clivia-GGm 6 Fluorescence spectra of ACU or Clivia-GGUCU mixture. Detailed Implementation

[0029] To make the above and other objects, features, and advantages of the present invention more apparent and understandable, preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings. Furthermore, the singular forms "a," "an," and "described" used in this invention include multiple references unless the context clearly specifies otherwise. Numerical ranges (e.g., 10% to 11% of A) include upper and lower limits unless otherwise specified (i.e., 10% ≤ A ≤ 11%); if a numerical range does not define a lower limit (e.g., less than 0.2% of B, or B below 0.2%), then the lower limit may be 0 (i.e., 0% ≤ B ≤ 0.2%). The above terms are used to illustrate and understand the present invention, and not to limit it.

[0030] This invention provides an RNA-protein complex comprising at least two parts: the first part is a chimeric RNA, denoted by N1-N2 or N2-N1, where N1 is any target RNA sequence of interest, and N2 is an RNA aptamer. The RNA aptamer is any RNA sequence capable of activating a fluorophore molecule to emit suitable spectral fluorescence; the second part is a recombinant fusion protein, denoted by P1-P2 or P2-P1, where P1 is the target protein of interest, and P2 is a fluorescent protein. Furthermore, P1, representing the target protein, can specifically recognize and bind to the target RNA sequence represented by N1, and P2, representing the fluorescent protein, can produce fluorescence of a specific wavelength under excitation light of a specific wavelength.

[0031] Reference Figure 1 In step a, the fluorescent protein is fused with the target protein of interest, and the target RNA sequence is directly attached to the 5' end of the RNA aptamer to form a chimeric RNA; in step b, the fluorescent protein is fused with the target protein, and the target RNA sequence is directly attached to the 3' end of the RNA aptamer to form a chimeric RNA; in step c, the fluorescent protein is fused with the target protein, and the target RNA sequence is directly inserted into the non-functional region of the RNA aptamer to form a chimeric RNA.

[0032] In one embodiment, N1 and N2 in the chimeric RNA can be directly linked, i.e., the 3′ end of N1 is directly linked to the 5′ end of the RNA aptamer or the 5′ end of the N1 sequence is directly linked to the 3′ end of the RNA aptamer; N1 and the RNA aptamer sequence can also be operatively linked, i.e., one or more nucleotides are added or removed between the 3′ end of the N1 sequence and the 5′ end of the RNA aptamer, or the N1 sequence is inserted into the non-functional region sequence of the RNA aptamer, or the N1 sequence directly replaces the non-functional region in the RNA aptamer.

[0033] In one embodiment, in the recombinant fusion protein, P1 and P2 can be directly linked, that is, the C-terminus of P1 is directly linked to the N-terminus of P2 or the N-terminus of P1 is directly linked to the C-terminus of P2; P1 and P2 can also be operatively linked, that is, one or more amino acid linker peptides are added between P1 and P2 or one or more terminal amino acids that do not affect the protein function are truncated, or P2 is inserted into the flexible non-functional peptide chain of P1.

[0034] The RNA aptamer may be selected from Pepper RNA aptamer (SEQ ID NO: 1), Clivia RNA aptamer (SEQ ID NO: 2), Okra RNA aptamer (SEQ ID NO: 3), RhoBAST RNA aptamer (SEQ ID NO: 4), Broccoli RNA aptamer (SEQ ID NO: 5), Corn RNA aptamer (SEQ ID NO: 6), Spinach RNA aptamer (SEQ ID NO: 7), Spinach2 RNA aptamer (SEQ ID NO: 8), DNB RNA aptamer (SEQ ID NO: 9), SRB-2 RNA aptamer (SEQ ID NO: 10), 13-2 min RNA aptamer (SEQ ID NO: 11), DIR-Apt1 RNA aptamer (SEQ ID NO: 15), DIR2s-Apt RNA aptamer (SEQ ID NO: 16), Mango RNA aptamer (SEQ ID NO: 17), and others. 17), or any of its biologically active variants or fragments, or chimeras of the said RNA aptamers. The RNA aptamers can specifically recognize and bind to the fluorophore molecule and significantly enhance the fluorescence signal produced by the fluorophore molecule under excitation light of a suitable wavelength. The Pepper RNA aptamer can specifically recognize and bind to the fluorophore molecule HBC and a series of its derivatives, such as HBC485, HBC497, HBC508, HBC514, HBC525, HBC599, and HBC620. In one embodiment, the Pepper RNA aptamer-HBC complex Pepper530 has an optimal excitation wavelength of 485 nm and an optimal emission wavelength of 530 nm. Table 1 below lists the usable RNA aptamers and their corresponding fluorophore molecules, fluorescence excitation wavelengths, and emission wavelengths.

[0035] Table 1: Fluorescence properties of RNA aptamers and their corresponding fluorophore molecules

[0036] The structures of the various fluorophore molecules that can be activated by the Clivia aptamer are shown in Table 1:

[0037] In one embodiment, the fluorescent protein can produce fluorescence of a specific wavelength under excitation light of a specific wavelength.

[0038] The fluorescent protein is known in the art, and preferably selected from blue fluorescent proteins: Azurite, Sirius, TagBFP, mTagBFP, mTagBFP2, EBFP, EBFP2, Electra1, Electra2; cyan fluorescent proteins: ECFP, SCFP3A, Cerulean, mTFP1, mTurquoise2, mAmetrine; green fluorescent proteins: GFP, EGFP, mNeonGreen, mWasabi, Clover, Venus, or any bioactive variant or fragment thereof, or a chimera of two or more thereof. The properties and sequence sources of the fluorescent proteins listed above can be found in the FPbase database, and will not be elaborated further here.

[0039] In the RNA-protein complex, the binding of P1 and N1 brings P2 and N2 closer together in space to a suitable relative position. Furthermore, the specific wavelength light generated by P2 under specific excitation light can activate the fluorophore molecule bound to the RNA aptamer to produce spectral fluorescence via fluorescence resonance energy transfer (FRET).

[0040] The present invention also provides an expression vector comprising such an RNA-protein complex. The expression vector contains the coding nucleotide sequence of the chimeric RNA and / or the coding nucleotide sequence of the recombinant fusion protein, wherein the N1 and P1 coding nucleotide sequences are vacant to facilitate subsequent replacement with the target protein and target RNA for detection, thereby enabling the detection of the interaction between the target protein and target RNA sequences.

[0041] This invention provides a method for detecting RNA-protein interactions in host cells. The method includes the following steps: constructing a prokaryotic or eukaryotic expression plasmid expressing chimeric RNA (N1-N2 or N2-N1); constructing a prokaryotic or eukaryotic expression plasmid expressing a recombinant fusion protein (P1-P2 or P2-P1); introducing the plasmids into host cells for expression; adding a fluorophore molecule and applying excitation light to the fluorescent protein as required; and detecting the fluorescence intensity of the fluorophore molecule at a predetermined wavelength and the fluorescence intensity of the fluorescent protein under the excitation light. After N1 binds to P1, the RNA aptamer approaches the fluorescent protein. After adding the fluorophore molecule and applying excitation light of a specific wavelength to the fluorescent protein, a fluorescence resonance energy transfer (FRET) signal can be detected. The occurrence of the FRET signal reflects the interaction between the target RNA and the target protein.

[0042] The present invention also provides a method for detecting the interaction between extracellular target RNA and target protein using the above-mentioned RNA-protein complex. The method includes the steps of: a) preparing chimeric RNA and recombinant fusion protein of the RNA-protein complex; b) mixing the chimeric RNA and recombinant fusion protein; c) adding fluorophore molecules separately, applying excitation light, and detecting the fluorescence intensity of the fluorophore molecules at a predetermined wavelength and the fluorescence intensity of the fluorescent protein at the predetermined wavelength under the excitation light.

[0043] Definitions and explanations of terms used in this article: In this invention, "host cell" refers to prokaryotic bacterial cells or eukaryotic mammalian cells, which can be primitive, unmodified cells or genetically modified commercial cells. Preferred bacterial cells in this invention are Escherichia coli BL21, and preferred mammalian cells are human HEK293T cells, HeLa cells, and Cos-7 cells.

[0044] The term "target (or purpose) protein," also known as "protein of interest," refers to any protein used for research. In embodiments of this invention, this includes λ phage capsid proteins and a range of human RNA-binding proteins, such as LIN28A, Pumilio, YTHDF3, and SRSF1. It can also refer to proteins that are hypothesized to interact with a particular RNA, or proteins used to screen for RNAs that can interact with them. These proteins can be natural proteins or proteins that have undergone genetic recombination, artificial modification, or mutation.

[0045] "Target RNA," also known as "RNA of interest" or "target RNA," refers to any RNA used for research. In the embodiments of this invention, it includes λ phage RNA box B and a series of human-derived RNA motifs that can bind to proteins, such as LIN28A-binding AGGAGAU RNA, Pumilio-binding UGUUGUAUA RNA, YTHDF3-binding GGACU RNA, and SRSF1-binding UGAAGGAC RNA. It can also be RNA that is hypothesized to interact with a specific protein, or RNA used to screen for proteins that can interact with it. These RNAs can be natural RNA or RNA that has undergone genetic recombination, artificial modification, or mutation.

[0046] Fluorescent proteins (GFPs) are a class of proteins that emit fluorescence under specific excitation light. Their luminescence depends on an internal chromophore, which absorbs light of a specific wavelength, causing electrons to transition to a higher energy state, releasing energy and emitting longer wavelength light. The most famous example is green fluorescent protein (GFP), derived from the jellyfish *Aequorea Victoria*. Its internal chromophore is p-hydroxybenzoic acid imidazolide, a structure that forms spontaneously in the presence of oxygen without requiring an additional substrate. GFPs have ubiquitous applications. Through gene editing and fusion with target proteins, they can be used to track gene expression, protein localization, and cellular dynamics in living cells or tissues in real time, becoming a core tool in life science research. They are also ideal reporter genes, placed under a specific gene promoter, directly reflecting the on / off state and intensity of gene expression through fluorescence intensity. Even more ingeniously, scientists utilize their sensitivity to the microenvironment to design biosensors that can sense cellular "emotions," such as the calcium ion probe GCaMP, which monitors neuronal activity. Furthermore, through multicolor labeling techniques such as the "brain rainbow" mouse and molecular interaction detection based on fluorescence resonance energy transfer, fluorescent proteins provide unparalleled insights into depicting complex biological networks and revealing the dynamic mysteries of life.

[0047] "RNA aptamer," also known as "nucleic acid aptamer" or "aptamer molecule," refers to an RNA sequence capable of binding to and activating the fluorescence of a "fluorophore molecule." The preferred RNA aptamer Clivia in this invention can specifically recognize and bind to the fluorophore molecule NBSI and its derivatives, enabling them to emit strong fluorescent signals under appropriate excitation light. "Fluorescent RNA" refers to the complex formed by the RNA aptamer binding to the corresponding fluorophore molecule.

[0048] "Fluorophore molecules" are also called "fluorophores." A "fluorophore molecule" refers to a class of chemical molecules that can emit fluorescence at a specific wavelength when excited by light of a suitable wavelength. A "fluorophore molecule" can be a normally bright fluorescent molecule or dye, such as rhodamine, fluorescein, Cy3, Cy5, etc., or it can be a conditionally activated fluorophore molecule. Such conditionally activated fluorophore molecules exhibit a low quantum yield in the absence of a nucleic acid aptamer, but when bound by a nucleic acid aptamer, their fluorescence intensity under specific wavelength excitation can be significantly increased. In specific embodiments, when not bound to a specific aptamer, the quantum yield of the fluorophore is less than 0.1, more preferably less than 0.01, and optimally less than 0.001; when the fluorophore is bound to a specific aptamer, the quantum yield of the fluorophore increases by more than 2 times, more preferably more than 10 times, and optimally more than 100 times. The fluorophore molecule is preferably water-soluble, non-toxic to cells, and easily permeates the cell membrane. The fluorophore of the present invention is preferably capable of entering the cytoplasm or periplasm through the cell membrane or cell wall via active transport or passive diffusion. In embodiments of the present invention, the fluorophore can penetrate the outer and inner membranes of Gram-negative bacteria, the cell wall and cell membrane of plant cells, the cell wall and cell membrane of fungi, the cell membrane of animal cells, and the GI and endothelial cell membranes of living animals. A preferred fluorophore molecule in this invention is NBSI618, which exhibits strong fluorescence after binding to the RNA aptamer Clivia, with excitation and emission peaks at 510 nm and 618 nm, respectively. Examples of a series of fluorophore molecules in this invention are detailed in Table 1.

[0049] "Operational linking" refers to adding one or more amino acid linker peptides between P1 and P2, or removing one or more terminal amino acids that do not affect protein function, or inserting P2 into the flexible non-functional peptide chain of P1. It also involves adding or removing one or more nucleotides between the 3′ end of sequence N1 and the 5′ end of the RNA aptamer, or inserting sequence N1 into the non-functional region of the RNA aptamer, or directly replacing the non-functional region in the RNA aptamer with N1.

[0050] "Fusion protein," also known as "recombinant protein" or "recombinant fusion protein," refers to a recombinant protein obtained by genetic recombination of two or more proteins. Fusion proteins can be direct links between different proteins or operative links. The fusion protein in this invention comprises a fluorescent protein and an RNA-binding protein. A preferred embodiment is mTagBFP2-Lambda N(λ N ).

[0051] "Fusion RNA" refers to recombinant RNA obtained by genetic manipulation of two or more RNAs. Fusion RNA is a direct ligation of different RNAs (i.e., the target RNA is directly ligated to the 5' or 3' end of a fluorescent RNA aptamer). The fusion RNA of this invention contains one fluorescent RNA aptamer and one target RNA.

[0052] "Chimeric RNA" refers to recombinant RNA obtained through genetic manipulation of two or more RNAs. Chimeric RNA is formed when one RNA is inserted into the sequence of another RNA. The chimeric RNA of this invention comprises a fluorescent RNA aptamer and a target RNA. In a preferred embodiment of this invention, boxB RNA is inserted into the non-functional loop of the RNA aptamer Clivia.

[0053] The terms "expression vector," "vector," "gene expression vector," "recombinant plasmid," or "plasmid" have the same meaning in this article and can be used interchangeably. They refer to plasmid vectors that can express target proteins, or their recombinant proteins, target RNAs, RNA aptamers, or their fusion RNAs and chimeric RNAs in host cells.

[0054] "FRET" stands for Fluorescence Resonance Energy Transfer, which is the phenomenon where energy emitted by a fluorescent protein excites the fluorescence of a receptor fluorophore. This invention uses the energy emitted by the fluorescent protein mTagBFP2 to activate the fluorescence of the fluorescent RNA Clivia-NBSI618.

[0055] The conventional molecular biology methods used in the embodiments of this invention are briefly described below: (a) Polymerase chain reaction (PCR): 1. Short fragment amplification PCR:

[0056] 2. Long fragment (>2500bp) amplification PCR:

[0057] The DNA polymerase used in this vector amplification PCR was PrimeSTAR. ® Max DNA polymerase. The plasmid template usually needs to be diluted to reduce the proportion of the original plasmid vector in the PCR product, thereby improving the positive rate during plasmid identification. Refer to the table above for sample loading system and reaction procedure settings.

[0058] (ii) Homologous recombination: When constructing plasmids using homologous recombination, during PCR amplification of the target DNA fragment, a 15-25 bp sequence homologous to the ends of the linearized vector is introduced at the 5′ end of both the forward and reverse primers. Both the target DNA fragment and the linearized vector have repetitive sequences at both ends. Under the action of homologous recombinase, the ligation of the DNA fragment to the linearized vector can be completed. The homologous recombinase used in this article was purchased from Nanjing Novizan Biotechnology Co., Ltd. or Shanghai Yisheng Biotechnology Co., Ltd. After mixing thoroughly according to the instructions, it was placed in a PCR instrument at 50℃ for 20 min. The ligation product was placed on ice and can be directly transformed. If temporarily stored at -20℃, the transformation efficiency will decrease.

[0059]

[0060] (III) Plasmid Transformation Plasmid transformation: a) Take 100 μl of competent cells and thaw them on an ice bath.

[0061] b) Add an appropriate volume of ligation product, gently mix by pipetting, and incubate on ice for 30 minutes. The volume of ligation product added is usually less than 1 / 10 of the competent cell volume.

[0062] c) Place the bacterial culture in a 42°C water bath for 45 seconds to heat shock, then quickly transfer it to an ice bath and place it for 2 minutes.

[0063] d) Add 100 μl LB and incubate on a shaker at 37°C for 30 min.

[0064] e) Spread the bacterial suspension evenly by pipetting, and then spread it evenly on the surface of an agar plate containing an appropriate amount of antibiotics. Incubate the plate upside down in a 37°C incubator overnight.

[0065] (iv) Protein purification a) Collect the induced bacterial culture by centrifugation and discard the culture medium; b) The bacterial culture was resuspended using Buffer A and then ultrasonically disrupted using an ultrasonic disruptor, ensuring that the bacterial culture remained in an ice-water mixture throughout the ultrasonic process; c) Sonicate the bacterial solution until it is translucent, then centrifuge it at 10,000 rpm for 30 min at 4°C. Transfer the supernatant to a new 50 mL centrifuge tube and place it on ice. d) Clean Ni with ultrapure water 2+ The column volume needs to be cleaned approximately 10 column volumes, and then Buffer A is used to balance the 10 column volumes after cleaning. e) Ni after processing 2+ The column can be used for protein loading; an appropriate amount of sample can be transferred using a dropper. f) After protein loading, use a washing buffer to elute any remaining proteins. Use a Bradford assay until no protein is detected in the eluent; then stop washing. The washing buffer is a mixture of Buffer A and Buffer B in a specific ratio, typically 19:1, with an imidazole concentration of 34 mM. The proportion of Buffer A can be increased depending on the amount of remaining proteins. g) After washing, the target protein is eluted using an elution buffer. The buffer is prepared with a Buffer A:Buffer B ratio of 2:3 and an imidazole concentration of 300 mM. h) After elution, the protein is loaded onto the desalting column, which has been pre-washed with ultrapure water for 10 column volumes. i) Once Bradford detected the presence of the protein, he immediately collected the droplet using centrifuge tubes and stored the protein in a -80°C freezer. j) Ni after use 2+ The column needs to be cleaned with Buffer B for 2 column volumes, followed by 10 column volumes of ultrapure water. The desalting column also needs to be cleaned with ultrapure water for 10 column volumes. The cleaned Ni... 2+ The columns and desalination columns were sealed with 20% ethanol and stored in a cold storage at 4°C.

[0066] Conventional cell experimental methods used in the examples: (a) Mammalian cell culture All cells used in this study were adherent cells. Cell culture experiments were performed in a sterile laminar flow hood. All consumables used in cell culture were sterilized by high-temperature, high-pressure autoclaving (121℃, 20 min) (except for purchased sterile consumables). All cells were cultured in a constant-temperature CO2 incubator (37℃, 5% CO2). High-glucose medium (DMEM) was commonly used, and all media contained 10% fetal bovine serum (FBS).

[0067] 1. Cell passage: a) Use a vacuum pump to remove the cell culture medium from a culture dish that is confluent with cells (when the area of ​​adherent cells occupies 80%~90% of the bottom area of ​​the culture dish); b) Wash away residual culture medium with an appropriate amount of PBS (this can be repeated once); c) Add an appropriate amount of trypsin to digest the cells, causing them to detach from the bottom of the culture dish, and resuspend the cells in an appropriate amount of fresh culture medium. d) Dispense fresh culture medium into new petri dishes; e) According to the cell density required for passage, transfer an appropriate amount of cell suspension to fresh culture medium, mix well, and place in a 37°C constant temperature CO2 incubator for culture.

[0068] 2. Cell cryopreservation: a) Remove the culture medium from a culture dish that has grown cells; b) Wash away residual culture medium with an appropriate amount of PBS; c) Trypsin digestion removes the cells from the bottom of the culture dish, and the cells are resuspended in an appropriate amount of fresh culture medium; d) Transfer the cell suspension to a 15 mL centrifuge tube and centrifuge (800 rpm, 5 min); e) Discard the culture medium and resuspend the cells in 10% DMSO medium; f) Aliquot the cell suspension into cryovials, 1 mL / tube, and immediately place in a -80°C freezer; g) After storing overnight in a -80°C refrigerator, transfer to a liquid nitrogen container for preservation.

[0069] 3. Cell resuscitation: a) Remove the frozen cells from liquid nitrogen and thaw them rapidly in a 37°C water bath; b) Transfer 15 mL of fresh culture medium into a new petri dish; c) Remove the liquid from the upper half of the cryovial and resuspend the cells in an appropriate amount of fresh culture medium; d) Transfer the cell suspension to fresh culture medium, mix well, and place in a 37°C incubator.

[0070] (II) Cell transfection Cell transfection first requires preparing cells at a certain density, usually by plating the cells the night before transfection. To ensure better cell adhesion and spread, facilitating imaging of intracellular subcellular structures, the cell culture plate is typically coated with poly-L-lysine before plating.

[0071] 1. Poly-L-lysine coating Poly-L-Lysine commonly has molecular weights of 70,000–150,000, 150,000–300,000, and >300,000. Larger molecular weights result in stronger adhesion but are more difficult to dissolve. For preparation, a 1 mg / mL stock solution is typically prepared with PBS and stored at -20°C. When using, it is diluted 10-fold with PBS (0.1 mg / mL) and added directly to a glass-bottomed petri dish, then incubated at 37°C for 30 minutes. Poly-L-Lysine decomposes easily in aqueous solutions; therefore, the working solution is best prepared fresh or stored at 4°C and used as soon as possible.

[0072] 2. Cell plating: a) Remove the culture medium and wash away any remaining culture medium with an appropriate amount of PBS; b) Trypsin digestion removes the cells from the bottom of the culture dish, and the cells are resuspended in an appropriate amount of fresh culture medium; c) After resuspending the cells, perform cell counting. Mix 50 μL of cell suspension with 50 μL of bromophenol blue, add it to the wells of the cell counting plate, and count the cells using a cell counter. d) The required dilution factor is equal to the live cell density shown in the cell count results divided by the cell density required for plating (i.e., the number of cells required per well divided by the volume of cell suspension per well); for example, if a 24-well plate needs to be plated at a density of 100,000 cells / well and a volume of 0.5 mL of cell suspension / well, then the required cell suspension density is 200,000 / mL. If the cell count result is A0,000 / mL, then the required dilution factor is A / 20. e) Dispense fresh culture medium into new culture dishes, and according to the dilution factor, transfer an appropriate amount of cell suspension to fresh culture medium and mix well; f) Dispense the diluted cell suspension into well plates.

[0073] 3. Transient cell transfection: The transfection reagents used in this experiment were Lipofectamine 3000, purchased from Yisheng Biotechnology and Invitrogen. Typically, Hieff Trans™ is used for 293T / 17 cells, and Lipofectamine 3000 is used for COS-7 cells.

[0074] 2. Experimental procedures for transfection using Hieff Trans™ (taking 24-well plate transfection as an example) a) Dispense OM into a 96-well plate, 50 μL per well; b) Add 400 ng of plasmid to a well-packed OM and mix well; c) Add 1 μL of Hieff Trans™ transfection reagent (hereinafter referred to as "HT") to another well containing OM and mix well; d) After incubating the two mixtures separately for 5 min, mix the two mixtures together thoroughly and let stand for 20 min. e) Add the mixture to one well of a 24-well plate containing cells, and gently shake the plate to distribute the transfection reagent evenly in the well.

[0075] (III) Cell Imaging Cell imaging is used to visually obtain information about molecules within cells. The cell imaging technique used in this study is fluorescence microscopy. Fluorescence imaging is a technique that involves fluorescently labeling target molecules or cells and then observing the target molecules or cells by collecting the fluorescence signals.

[0076] Fluorescence microscopy: The fluorescence microscope used in this article is a Leica SP8 laser confocal microscope.

[0077] Sample preparation: a) Prepare cell samples according to the experimental procedures for cell transfection. For cell samples used in laser confocal imaging, this study typically uses glass-bottomed cell culture plates for cell plating and transfection; b) Dye labeling: Dyes are usually prepared directly using fresh culture medium. Remove the culture medium from the cell culture plate, add the prepared dye, and incubate for 5-10 minutes.

[0078] Microscope startup preparation: Follow the microscope startup procedure to turn on the microscope. If kinetic imaging is required, the live cell workstation needs to be turned on to provide the cells with suitable temperature and CO2 concentration.

[0079] Imaging settings: Since this study usually requires observation of subcellular localization within cells, HCXPLAPO 63.0×1.47 oil immersion lens and HyD detector are commonly used. An appropriate laser is selected for imaging according to the fluorescence properties of the sample. Data processing: The resulting images were processed using ImageJ software.

[0080] Example 1: Recombinant fusion protein mTagBFP2-λ N Design of chimeric RNA Clivia-boxB λ phage N protein (λ N Fusion of mTagBFP2 (SEQ ID NO: 18) with blue fluorescent protein mTagBFP2 (SEQ ID NO: 19) yields mTagBFP2-λ N First, primers P1 (SEQ ID NO: 20) and P2 (SEQ ID NO: 21) containing multiple cloning sites were designed. Using the pcDNA3.1-mTagBFP2 plasmid as a template, a linearized vector was obtained. A fragment was obtained using primers P3 (SEQ ID NO: 22), P4 (SEQ ID NO: 23), and P5 (SEQ ID NO: 24). The linearized vector and the fragment were ligated via infusion to obtain the fusion protein plasmid pcDNA3.1-mTagBFP2-λ. N (SEQ ID No: 25), such as Figure 2B As shown.

[0081] By replacing the non-functional region ring GAAA of Clivia (SEQ ID NO: 2) with the boxB sequence (SEQ ID NO: 26), it will be related to λ NThe interacting boxB sequence is infused into Clivia to obtain Clivia-boxB. Then, using pU6-Clivia as a vector template, the boxB sequence is amplified using primers P6 (SEQ ID NO: 27) and P7 (SEQ ID NO: 28). Finally, pU6-Clivia-boxB (SEQ ID No: 29) is obtained through self-infusion. Figure 2A As shown.

[0082] like Figure 2C As shown, Clivia-boxB and mTagBFP2-λ are co-expressed. N HEK293T cells were imaged with 2 μM NBSI618 and co-expressed Clivia and mTagBFP2-λ. N HEK293T cells were used as a control, and flow cytometry experiments were performed. The results showed that interacting Clivia-boxB and mTagBFP2-λ molecules were detectable in the cytoplasm. N The FRET signal, without the interaction of mTagBFP2-λ N In contrast, no obvious FRET signal was detected in Clivia. Therefore, it can be concluded that the recombinant fusion protein mTagBFP2-λ... N The high FRET ratio with the chimeric RNA Clivia-boxB demonstrates that the Clivia-mTagBFP2 FRET system can image RNA-protein interactions in living cells, such as... Figure 3A , 3B As shown.

[0083] Back Figure 2D The diagram shows orthogonal plots of flow cytometry experiments performed using the same method as in Example 1 to prepare other fluorescent proteins and different Clivia-NBSI complexes. In the experimental examples, all samples used fluorescent protein-λN and Clivia-boxB, while the control RNA sequences were all Clivia aptamers (SEQ ID No: 2). A series of fluorescent proteins selected for the experiments included: TagBFP, Electra2, ECFP, mTFP1, mTurquoise2, mAmetrine, EGFP, mNeonGreen, and mWasabi. The prepared fusion protein sequences were TagBFP-λN and Clivia-NBSI complexes. N (SEQ ID NO: 12), Electra2-λ N (SEQ ID NO: 13), ECFP-λ N (SEQ ID NO: 14), mTFP1-λ N(SEQ ID NO: 69), mTurquoise2-λ N (SEQ ID NO: 70), mAmetrine-λ N (SEQ ID NO: 71), EGFP-λ N (SEQ ID NO: 72), mNeonGreen-λ N (SEQ ID NO:73), mWasabi-λ N (SEQ ID NO: 74). Figure 2D The Y-axis value represents the ratio of the peak fluorescence value of the six different Clivia-NBSI complexes to the peak emission value of the fluorescent protein. The data represent the average value of three replicate experiments.

[0084] from Figure 2D It can be seen that fluorescent proteins with different spectra and Clivia-NBSI complexes with different spectra can form corresponding detection systems. The FRET ratio is increased in the system containing the boxB sequence, while the FRET ratio is weaker in the control group.

[0085] Example 2: Detection of the interaction between LIN28A protein and its target RNA sequence in live cells using the Clivia-mTagBFP2-based FRET system. LIN28A is an important RNA-binding protein. Its main mechanism involves binding to the precursors of let-7 family miRNAs via its zinc finger domain, inhibiting their maturation, and thus further influencing the expression of downstream target genes of let-7. Known genes regulated by LIN28A include... c-Myc , Ras , Cyclin D1 as well as Lin28A Themselves, etc. (Refer to...) Figure 4A As shown, LIN28A and AGGAGAU (denoted as: RNA) are displayed. LIN28A The crystal structure of the complex (PDB ID: 2LI8).

[0086] The mTagBFP2 protein was tandemly linked to the C-terminus of the target protein LIN28A to obtain a recombinant fusion protein. Using the pcDNA3.1-mTagBFP2 plasmid as a template, a linearized vector was obtained through amplification using primers P8 (SEQ ID NO: 30) and P9 (SEQ ID NO: 31). The LIN28A protein fragment was then amplified using primers P10 (SEQ ID NO: 32) and P11 (SEQ ID NO: 33). The amplified vector and fragment were infused and ligated to obtain the fusion protein plasmid pcDNA3.1-LIN28A-mTagBFP2 (SEQ ID No: 34). Figure 4BAs shown.

[0087] Inserting the AGGAGAU sequence into the 3' end of Clivia yields the RNA sequence Clivia-AGGAGAU (SEQ ID NO: 35). Using pU6-Clivia as a vector template, amplification was performed using primers P12 (SEQ ID NO: 36) and P13 (SEQ ID NO: 37), ultimately yielding pU6-Clivia-AGGAGAU (SEQ ID No: 38) through self-infusion. Figure 4C As shown.

[0088] like Figure 5A , 5E As shown, HEK293T cells co-expressing Clivia-AGGAGAU and LIN28A-mTagBFP2 were imaged with 2 μM NBSI618. HEK293T cells co-expressing Clivia and LIN28A-mTagBFP2 served as a control. The scale bar was 10 μm. The results showed that FRET signals from the interacting Clivia-AGGAGAU and LIN28A-mTagBFP2 complexes were detectable in the cytoplasm, while no significant FRET signal was detected from the non-interacting LIN28A-mTagBFP2 complexes. This indicates that the RNA-protein complex composed of Clivia-AGGAGAU and LIN28A-mTagBFP2 has a high FRET ratio.

[0089] Example 3: Detection of the interaction between Pumilio protein and its target RNA sequence in live cells using the Clivia-mTagBFP2 FRET system Pumilio (PUM) proteins belong to the highly conserved PUF (Pumilio-Fem3 binding factor) family of RNA-binding proteins. In most cases, PUM proteins bind to the 3'UTR of target mRNA and recruit chaperone proteins to reduce mRNA stability and / or inhibit its translation. Engineered nine-repeat PUF proteins can recognize RNA targets of varying lengths with high affinity; their structure can be found in [reference needed]. Figure 4A The diagram shows Pumilio and UUGUUGUAUA (denoted as: RNA). PUM The crystal structure of the complex (PDB ID: 5YKI).

[0090] The mTagBFP2 protein was tandemly linked to the C-terminus of the target protein PUM to obtain a fusion protein. Using the pcDNA3.1-mTagBFP2 plasmid as a template, a linearized vector was obtained by amplification using primers P14 (SEQ ID NO: 39) and P15 (SEQ ID NO: 40). The Pumilio protein fragment was amplified using primers P16 (SEQ ID NO: 41) and P17 (SEQ ID NO: 42). The amplified vector and fragment were infused and ligated to obtain the fusion protein plasmid pcDNA3.1-PUM-mTagBFP2 (SEQ ID No: 43). Figure 4B As shown.

[0091] The sequence UGUUGUAUA was inserted into the 3' end of Clivia to obtain the RNA fragment Clivia-UGUUGUAUA (SEQ ID NO: 44). Using pU6-Clivia as a vector template, it was amplified using primers P18 (SEQ ID NO: 45) and P19 (SEQ ID NO: 46), and finally, through self-infusion, pU6-Clivia-UGUUGUAUA (SEQ ID No: 47) was obtained. Figure 4C As shown.

[0092] like Figure 5B , 5C As shown, HEK293T cells co-expressing Clivia-UGUUGUAUA and PUM-mTagBFP2 were imaged with a 2 μM NBSI618 microscope. HEK293T cells co-expressing Clivia and PUM-mTagBFP2 served as a control. The scale bar was 10 μm. The results showed that FRET signals of interacting Clivia-UGUUGUAUA and PUM-mTagBFP2 could be detected in the cytoplasm, while no obvious FRET signal could be detected for non-interacting PUM-mTagBFP and Clivia. It can be concluded that PUM-mTagBFP2 and Clivia-UGUUGUAUA RNA have a high FRET ratio.

[0093] Example 4: Detection of the interaction between YTHDF3 protein and its target RNA sequence in live cells using the Clivia-mTagBFP2 FRET system Three YTH domain family proteins (YTHDF1, YTHDF2, and YTHDF3) in cells can recognize N6-methyladenosine (m3) in mRNA. 6 A) Modification: YTHDF3 works closely with YTHDF1 and YTHDF2 to precisely regulate m. 6 Gene expression and metabolic rate of A-modified mRNA.6 YTHDF3 modification and its reader are crucial in the nervous system. Dysfunction of the YTHDF3 protein can lead to neurodevelopmental abnormalities and neurodegenerative diseases. Aberrant expression of the YTHDF3 protein is also closely related to various cancers, such as breast cancer, prostate cancer, and liver cancer. YTHDF3 and GGACU (denoted as: RNA) YTHDF3 The crystal structure of the complex (PDB ID: 6ZOT) is as follows: Figure 4A As shown.

[0094] The mTagBFP2 protein was tandemly linked to the N-terminus of the target protein YTHDF3 to obtain a recombinant fusion protein. Using the pcDNA3.1-mTagBFP2 plasmid as a template, amplification was performed using primers P20 (SEQ ID NO: 48) and P21 (SEQ ID NO: 49) to obtain a linearized vector. The YTHDF3 protein fragment was then amplified using primers P22 (SEQ ID NO: 50) and P23 (SEQ ID NO: 51). The amplified vector and fragment were infused and ligated to obtain the fusion protein plasmid pcDNA3.1-mTagBFP2-YTHDF3 (SEQ ID No: 52). Figure 4B As shown.

[0095] Inserting the GGACU sequence into the 3' end of Clivia yielded RNA Clivia-GGACU (SEQ ID NO: 53). Using pU6-Clivia as a vector template, it was amplified using primers P24 (SEQ ID NO: 54) and P25 (SEQ ID NO: 55), and finally, through self-infusion, pU6-Clivia-GGACU (SEQ ID No: 56) was obtained. Replacing the base "A" in the target sequence GGACU with "T" eliminates false positives caused by methylation of this site, as thymine T is already a methylated product and becomes "U" in the RNA sequence after transcription. Clivia-GGUCU was designed as a control RNA and amplified using primers P26 (SEQ ID NO: 57) and P27 (SEQ ID NO: 58), and finally, through self-infusion, pU6-Clivia-GGUCU (SEQ ID No: 59) was obtained. Figure 4C As shown.

[0096] like Figure 5C , 5EAs shown, HEK293T cells co-expressing Clivia-GGACU and mTagBFP2-YTHDF3 were imaged with a 2μm NBS1618 microscope, while HEK293T cells co-expressing Clivia-GGUCU and mTagBFP2-YTHDF3 served as a control. The scale bar was 10μm. The results showed that FRET signals from interacting Clivia-GGACU and mTagBFP2-YTHDF3 could be detected in the cytoplasm, while no obvious FRET signal could be detected from mTagBFP2-YTHDF3 and Clivia-GGUCU that did not interact, demonstrating that mTagBFP2-YTHDF3 and Clivia-GGACU RNA have a high FRET ratio.

[0097] Figure 4D A series of chimeric RNAs (Clivia-RNAs) were shown. LIN28A Clivia-RNA PUM Clivia-RNA YTHDF3 Clivia-RNA SRSF1 LIN28A and its corresponding recombinant fusion proteins (LIN28A-mTagBFP2, PUM-mTagBFP2, mTagBFP2-YTHDF3 / mTagBFP2-SRSF1) were co-expressed in HEK293T cells and then subjected to flow cytometry experiments. The Y-axis value is the ratio of the peak value at 610 nm to the peak value at 450 nm in the emission spectrum, and the data represents the average value of three replicate experiments; the X-axis represents the name of the different chimeric RNAs detected (the co-expressed proteins are their respective corresponding fusion proteins).

[0098] from Figure 4D It can be seen that the FRET ratio is higher only in the system containing the target RNA sequence, while the FRET ratio is weaker in the control group lacking the target sequence.

[0099] Example 5: Detection of the interaction between SRSF1 protein and its target RNA sequence in living cells using the Clivia-mTagBFP2 FRET system.

[0100] Serine / arginine cleavage factor 1 (SRSF1) is a highly conserved serine / arginine-rich family protein. As an important cleavage factor, SRSF1 plays a crucial role in important biological processes such as pre-mRNA cleavage, mRNA stability, transcriptional elongation, and post-transcriptional translation. SRSF1 is related to UGAAGGAC (denoted as: RNA...) SRSF1 The crystal structure of the complex (PDB ID: 2M8D) is as follows: Figure 4A As shown.

[0101] The mTagBFP2 protein was tandemly linked to the N-terminus of the target protein SRSF1 to obtain a fusion protein. Using the pcDNA3.1-mTagBFP2 plasmid as a template, a linearized vector was obtained by amplification using primers P28 (SEQ ID NO: 60) and P29 (SEQ ID NO: 61). The SRSF1 protein fragment was then amplified using primers P30 (SEQ ID NO: 62) and P31 (SEQ ID NO: 63). The amplified vector and fragment were infused and ligated to obtain the fusion protein plasmid pcDNA3.1-mTagBFP2-SRSF1 (SEQ ID No: 64). Figure 4B As shown.

[0102] The sequence UGAAGGAC was inserted into the stem-loop of Clivia to obtain the chimeric RNA Clivia-UGAAGGAC (SEQ ID NO: 65). Using pU6-Clivia as a vector template, it was amplified by primers P32 (SEQ ID NO: 66) and P33 (SEQ ID NO: 67), and finally, pU6-Clivia-UGAAGGAC (SEQ ID No: 68) was obtained through self-infusion. Figure 4C As shown.

[0103] like Figure 5D , 5E As shown, HEK293T cells co-expressing Clivia-UGAAGGAC and mTagBFP2-SRSF1 were imaged with a 2 μm NBSI618 microscope. HEK293T cells co-expressing Clivia and mTagBFP2-SRSF1 served as a control. The scale bar was 10 μm. The results showed that FRET signals of interacting Clivia-UGAAGGAC and mTagBFP2-SRSF1 could be detected in the cytoplasm, while no obvious FRET signal could be detected for mTagBFP2-SRSF1 and Clivia, which do not interact. This demonstrates that mTagBFP2-SRSF1 and Clivia-UGAAGGAC RNA have a high FRET ratio.

[0104] Reference Figure 6 This demonstrates the in vitro detection of mTagBFP2-YTHDF3 and Clivia-GGm based on the Clivia-mTagBFP2 FRET system. 6 Fluorescence spectra of ACU or Clivia-GGUCU mixtures. From Figure 6It can be seen that the chimeric RNA with methylation modification showed the characteristic emission signal of fluorescent RNA Clivia at 620 nm, while the unmodified chimeric RNA showed almost no obvious signal at 620 nm.

[0105] The present invention has been described by the above-described embodiments; however, these embodiments are merely examples for implementing the present invention. It must be noted that the disclosed embodiments do not limit the scope of the present invention. Conversely, modifications and equivalents contained within the spirit and scope of the claims are all included within the scope of the present invention.

Claims

1. An RNA-protein complex, characterized in that: The RNA-protein complex includes: (a) A chimeric RNA having a molecular structure represented as N1-N2 or N2-N1; wherein N1 is the target RNA sequence and N2 is an RNA aptamer configured to activate a fluorophore molecule to emit spectral fluorescence; (b) A recombinant fusion protein having a molecular structure represented as P1-P2 or P2-P1; wherein P1 is a target protein, P2 is a fluorescent protein, and the target protein recognizes and binds to the target RNA sequence.

2. The RNA-protein complex according to claim 1, characterized in that: The N1 and N2 of the chimeric RNA are linked by a nucleic acid sequence that does not interfere with the binding of the target RNA to the target protein, and / or the P1 and P2 of the recombinant fusion protein are linked by a polypeptide that does not interfere with the binding of the target RNA to the target protein and maintains the fluorescence function of the fluorescent protein.

3. The RNA-protein complex according to claim 1, characterized in that: The fluorescent protein generates a first fluorescence under excitation light, the RNA aptamer recognizes and binds to the fluorophore molecule, and the fluorophore molecule is excited by the first fluorescence to emit the spectral fluorescence.

4. The RNA-protein complex according to claim 3, characterized in that: The RNA aptamers are selected from the group consisting of Pepper RNA aptamer (SEQ ID NO: 1), Clivia RNA aptamer (SEQ ID NO: 2), Okra RNA aptamer (SEQ ID NO: 3), RhoBAST RNA aptamer (SEQ ID NO: 4), Broccoli RNA aptamer (SEQ ID NO: 5), Corn RNA aptamer (SEQ ID NO: 6), Spinach RNA aptamer (SEQ ID NO: 7), Spinach2 RNA aptamer (SEQ ID NO: 8), DNB RNA aptamer (SEQ ID NO: 9), SRB-2 RNA aptamer (SEQ ID NO: 10), 13-2 min RNA aptamer (SEQ ID NO: 11), DIR-Apt1 RNA aptamer (SEQ ID NO: 15), DIR2s-Apt RNA aptamer (SEQ ID NO: 16), Mango RNA aptamer (SEQ ID NO: 17), or any of the biologically active variants or fragments thereof.

5. The RNA-protein complex according to claim 1, characterized in that: The fluorescent protein is: Blue fluorescent protein, selected from the group consisting of Azurite, Sirius, TagBFP, mTagBFP, mTagBFP2, EBFP, EBFP2, Electra1 and Electra2; Cyan fluorescent protein, wherein the cyan fluorescent protein is selected from the family consisting of ECFP, SCFP3A, Cerulean, mTFP1, mTurquoise2, and mAmetrine; or Green fluorescent protein, wherein the green fluorescent protein is selected from GFP, EGFP, mNeonGreen, mWasabi, Clover, Venus, any one of their bioactive mutants or fragments, and a group consisting of two or more of their chimeras.

6. An expression vector, characterized in that: The expression vector expresses the RNA-protein complex as described in any one of claims 1 to 5.

7. The expression vector according to claim 6, characterized in that: The expression vector contains a nucleotide sequence encoding the chimeric RNA, and / or the expression vector contains a nucleotide sequence encoding the recombinant fusion protein.

8. A method for detecting the interaction between target RNA and target protein in host cells, characterized in that: The method includes the following steps: (a) Constructing a prokaryotic or eukaryotic plasmid expression vector, wherein the prokaryotic or eukaryotic plasmid expression vector expresses the RNA-protein complex as described in any one of claims 1 to 5; (b) Introduce the prokaryotic or eukaryotic expression vector obtained in step (a) into a host cell; (c) Add fluorophore molecules separately and apply excitation light; and (d) Detect the fluorescence intensity of the fluorophore molecule and the fluorescence intensity of the fluorescent protein under the excitation light.

9. A method for detecting the interaction between extracellular target RNA and target protein, characterized in that: The method includes the following steps: (a) Preparing the chimeric RNA and recombinant fusion protein in the RNA-protein complex as described in any one of claims 1 to 5; (b) Mix the chimeric RNA and the recombinant fusion protein; (c) Add fluorophore molecules separately and apply excitation light; and (d) Detect the fluorescence intensity of the fluorophore molecule and the fluorescence intensity of the fluorescent protein under the excitation light.

10. A reagent kit, characterized in that: The kit contains the expression vector as described in any one of claims 6 to 7, and / or the kit contains prokaryotic or eukaryotic cells expressing the expression vector of the RNA-protein complex as described in any one of claims 1 to 5.

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