Cascade activated fluorescent RNA probe, expression vector and detection method thereof

Abstract

The application discloses a cascade-activated fluorescent RNA probe for detecting target nucleic acid molecules, and an expression vector and a detection method thereof. The application comprises a design method and preparation of the probe, and the probe is used for labeling and detecting target nucleic acid molecules in a solution sample, labeling and detecting target nucleic acid molecules in living cells, and labeling and detecting target nucleic acid molecules in fixed cells. The application can solve the problems of current commercial chemical synthesis FISH, molecular beacon and other probes, such as non-hereditary coding, difficult preparation, high price and the like. The cascade-activated fluorescent RNA probe for detecting target nucleic acid molecules can be obtained in a high-efficiency, simple and rapid way through hereditary coding or in vitro transcription, and excellent detection sensitivity is achieved through signal amplification effect, which provides a practical tool for life science basic problem research and instant diagnosis or real-time diagnosis of diseases, and has wide scientific significance and social significance.

Description

Technical Field

[0001] This application relates to a cascade-activated fluorescent RNA probe based on fluorescent RNA, its expression vector, and a method for labeling and detecting target nucleic acid molecules, which falls under the field of biomedical engineering technology. Background Technology

[0002] RNA, as a crucial biological macromolecule, participates extensively in various key life activities, including intracellular genetic information transmission, gene expression regulation, and signal transduction. The functional characteristics, expression regulation patterns, and mediated physiological / pathological processes of different RNA molecules are closely related to human health. Studies have shown a significant correlation between the biological function of RNA and its subcellular localization and dynamic distribution characteristics. Therefore, developing high spatiotemporal resolution real-time RNA imaging and dynamic tracking technologies not only helps to reveal the spatiotemporal expression regulation patterns of RNA but also provides a deeper understanding of RNA-mediated cellular spatial organization mechanisms, possessing significant scientific and applied value.

[0003] Fluorescence in situ hybridization (FISH) enables the visualization and detection of specific RNA transcripts at single-molecule resolution by designing fluorescently labeled DNA / RNA oligonucleotide probes. However, this technique relies on cell fixation and washing steps for unbound probes, thus only acquiring static spatiotemporal information. In contrast, molecular beacons (MBs), as dual-labeled oligonucleotide probes, can activate fluorescence signals through target-dependent conformational changes, theoretically supporting live-cell RNA imaging. However, both FISH and MB technologies rely on the covalent fluorophore molecular labeling of oligonucleotide probes, a crucial step with the following technical limitations: First, covalently modified probes cannot achieve dynamic tracking of genetically encoded RNA, severely restricting their application in real-time live-cell RNA monitoring; second, probe preparation must rely on commercially available custom synthesis, leading to significantly extended experimental cycles and high costs. These inherent limitations greatly restrict the widespread application of FISH and MB technologies in RNA research.

[0004] Fluorescent RNA (FR) is a class of RNA aptamers that specifically bind to and activate fluorophore molecules. It can be expressed in cells through genetic coding, providing a novel and effective analytical technique for live-cell RNA labeling. Since 2011, scientists have reported a series of fluorescent RNAs for live-cell RNA imaging, including Spinach (Page JS et al., Science 2011. 333(6042):642-6), Broccoli (Filonov GS et al., J Am Chem Soc 2014. 136(46):16299-308), Corn (Warner KD et al., Nat Chem Biol 2017.13(11):1195-1201), Mango (Dolgöshena EV et al., ACS Chem Biol 2014.9(10):2412-20), SRB2 (Shunfo M et al., Angew Chem Int Ed Engl 2013. 52(50):13401-4), and Okra (Nature Chemical Biology, 2024, 20, (Examples include 1272–1281.), Myosotis (Small, 2025, 21, e2405165.), etc. A significant characteristic of this type of FR (Fluorescence Imaging) is that it does not require a covalent link between the RNA aptamer and the fluorophore molecule; instead, the transcribed RNA aptamer and a specific fluorophore form a complex through molecular recognition to generate a fluorescence signal. This characteristic makes it the most direct RNA molecular imaging technique currently available.

[0005] However, when using fluorescent RNA to label target nucleic acid molecules, researchers need to perform genetic manipulation on the target nucleic acid molecules, that is, fuse the fluorescent RNA aptamer with the target nucleic acid molecule, and then add a fluorophore molecule for specific labeling. However, this process is cumbersome, especially for labeling and detecting intracellular RNA, which requires genetic manipulation of the genome, making it difficult to implement. Therefore, there is an urgent need to develop novel RNA labeling and detection technologies. Summary of the Invention

[0006] The purpose of this application is to provide a cascade-activated fluorescent RNA probe and its applications. This invention combines the advantages of high brightness and low background of fluorescent RNA to develop a cascade-activated fluorescent RNA probe based on Pepper and Clivia, referred to as the CaFR probe. When labeling and imaging target nucleic acid molecules (e.g., multicolor imaging analysis, high-resolution in situ imaging analysis), it is not necessary to fuse the CaFR probe with the target nucleic acid molecule for expression. The CaFR probe can be directly expressed in cells, and the addition of a fluorophore molecule can achieve specific labeling and detection of target nucleic acid molecules in living cells; alternatively, the CaFR probe can be prepared by in vitro transcription, mixed with the target nucleic acid molecule in solution, and the addition of a fluorophore molecule can achieve labeling and detection of the target nucleic acid molecule. Therefore, this technology overcomes the complex process of genetic manipulation of target nucleic acid molecules required when using fluorescent RNA, and has excellent practicality and advanced features.

[0007] To achieve the above objectives, the present invention provides a cascade activation fluorescent RNA probe, wherein the probe sequence comprises a sequence, b sequence, c sequence, c* sequence, d sequence, e sequence, e* sequence, f sequence, and f* sequence; The a sequence is a repressor sequence. In the absence of the b sequence binding to the target nucleic acid molecule, the a sequence can form a first stem structure with the c sequence through complementary base pairing. The b sequence is a target nucleic acid molecule recognition and binding sequence, and the b sequence can bind to the target nucleic acid molecule through complementary base pairing; The c sequence is a regulatory sequence. When the b sequence does not bind to the target nucleic acid molecule, the c sequence can form the first stem structure by base pairing with the a sequence. When the b sequence binds to the target nucleic acid molecule, the c sequence can form the second stem structure by base pairing with the c* sequence. The c* sequence is a stable sequence. When the b sequence binds to the target nucleic acid molecule, the c* sequence can form the second stem structure with the c sequence through base complementary pairing, thereby promoting the formation of a stable three-dimensional structure of the f sequence. The d sequence is a linker sequence that, when the b sequence binds to the target nucleic acid molecule, makes it less likely for the f sequence and the f* sequence, which restore fluorescence activation activity, to interfere with each other; and when the b sequence does not bind to the target nucleic acid molecule, makes it easier for the c* sequence and the e sequence to bind. The e sequence is a regulatory sequence. When the b sequence does not bind to the target nucleic acid molecule, the e sequence can form a third stem structure by base pairing with the c* sequence; when the b sequence binds to the target nucleic acid molecule, the e sequence can form a fourth stem structure by base pairing with the e* sequence. The e* sequence is a stable sequence. When the b sequence binds to the target nucleic acid molecule, the e* sequence can form the fourth stem structure through base complementary pairing with the e sequence, thereby promoting the formation of a stable three-dimensional structure of the f* sequence; and The f sequence and the f* sequence are fluorescent RNA aptamer sequences. When the c sequence and the c* sequence or the e sequence and the e* sequence are complementary to form the second stem structure or the fourth stem structure, the f sequence and the f* sequence will form a stable three-dimensional fluorescent RNA aptamer structure.

[0008] In some embodiments, the a sequence, the b sequence, the c sequence, the f sequence, the c* sequence, the d sequence, the e sequence, the f* sequence, and the e* sequence are connected in the order abcfc*-def*-e*.

[0009] In some embodiments, the a sequence and the c sequence are preferably sequences of 12 nucleotides in length (nt).

[0010] In some embodiments, the b sequence is preferably a sequence of 18 to 28 nucleotides in length.

[0011] In some embodiments, the b sequence may be a nucleotide sequence that is completely complementary to the target nucleic acid molecule, or it may be a nucleotide sequence that is not completely complementary to the target nucleic acid molecule and contains one, two or more nucleotide mutations.

[0012] In some embodiments, the c* sequence is preferably a sequence of 9 nucleotides in length.

[0013] In some embodiments, the d sequence is preferably a -CAAAA- sequence.

[0014] In some embodiments, the e sequence is preferably a sequence of 6 nucleotides in length.

[0015] In some embodiments, the e* sequence is preferably a sequence of 6 nucleotides in length.

[0016] In some embodiments, the f sequence and the f* sequence are fluorescent RNA aptamer sequences. When the c sequence and the c* sequence and the e sequence and the e* sequence are complementary to form the second stem structure and the fourth stem structure, the f sequence and the f* sequence will form a stable three-dimensional structure of fluorescent RNA aptamer, which will then bind to fluorophore molecules to form a first probe cascade activated fluorophore molecule and enhance the fluorescence intensity of the fluorophore molecule in the first probe cascade activated fluorophore molecule.

[0017] In some embodiments, the f sequence and the f* sequence are preferably derived from the Pepper fluorescent RNA aptamer or the Clivia fluorescent RNA aptamer.

[0018] In some embodiments, when the target nucleic acid molecule is absent, the cascade-activated fluorescent RNA probe cannot bind to the fluorophore molecule or has a weak binding ability to form a second probe cascade-activated fluorophore molecule; when the target nucleic acid molecule is present, the cascade-activated fluorescent RNA probe has a strong binding ability to the fluorophore molecule to form a first probe cascade-activated fluorophore molecule, wherein the fluorescence intensity produced by the second probe cascade-activated fluorophore molecule is weaker than the fluorescence intensity produced by the first probe cascade-activated fluorophore molecule.

[0019] In some embodiments, the target nucleic acid molecule may be a single-stranded DNA molecule or an RNA molecule.

[0020] The present invention also provides an expression vector comprising the cascade-activated fluorescent RNA probe, wherein the expression vector comprises the encoding gene of the cascade-activated fluorescent RNA probe.

[0021] This invention also provides a method for labeling and detecting target nucleic acid molecules in host cells using the cascade-activated fluorescent RNA probe, comprising the following steps: (a) The cascaded activation fluorescent RNA probe is constructed in a prokaryotic plasmid expression vector or a eukaryotic plasmid expression vector to obtain a recombinant plasmid; (b) Introducing the recombinant plasmid into the host cell; and (c) Adding fluorophore molecules for labeling and detection.

[0022] This invention also provides a method for labeling and detecting target nucleic acid molecules in host cells using the cascade-activated fluorescent RNA probe, comprising the following steps: (a) The cascade-activated fluorescent RNA probe was prepared by in vitro transcription or chemical synthesis; (b) Fixing the host cells; and (c) Add the cascade-activated fluorescent RNA probe and fluorophore molecule for labeling and detection.

[0023] This invention also provides a method for labeling and detecting target nucleic acid molecules in solution using the cascade-activated fluorescent RNA probe, comprising the following steps: (a) The cascade-activated fluorescent RNA probe is prepared by in vitro transcription or chemical synthesis; and (b) The cascade-activated fluorescent RNA probe and fluorophore molecule are added to the solution for labeling and detection.

[0024] Compared with existing technologies, the advantages of this invention are as follows: This invention constructs a cascade-activated fluorescent RNA probe technology based on fluorescent RNA, which can be expressed in cells in real time through genetic encoding, without the need for subsequent genome modification steps of the target nucleic acid molecule, nor for long-term dynamic tracking analysis of the target nucleic acid molecule; the cascade-activated fluorescent RNA probe of this invention can also be obtained through in vitro transcription, solving the problems of expensive preparation costs and complex synthesis processes of commercial hybridization probes, and obtaining high-throughput analysis of target nucleic acids in fixed cells efficiently, simply, quickly, and cost-effectively. Furthermore, due to its rapid and convenient synthesis, the transcribed probe can also be widely used in the real-time detection of target nucleic acid molecules in solution samples. Attached Figure Description

[0025] To make the objectives, technical solutions, and advantages of the present invention clearer, the preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings, wherein: Figure 1 This is a schematic diagram of the structure of the Pepper-based CaFR probe of the present invention; Figure 2 This is a schematic diagram illustrating the working principle of the Pepper-based CaFR probe of the present invention; Figure 3 This invention relates to the detection of changes in the a, b, c, c*, d, e, and e* sequences of a Pepper-based CaFR probe. Figure 4 This invention relates to the labeling and detection of target nucleic acid molecules containing single-point nucleotide mutations and multi-nucleotide site mutations using Pepper-based CaFR probes. Figure 5 The present invention relates to a Pepper-based CaFR probe for labeling and detection of target nucleic acid molecules in solution; Figure 6 The present invention relates to a Clivia-based CaFR probe for labeling and detection of target nucleic acid molecules in solution; Figure 7The CaFR probe transcribed in vitro in this invention is used for labeling and imaging of target nucleic acid molecules in immobilized cells; Figure 8 The present invention relates to the Pepper-based CaFR probe for labeling and imaging target nucleic acid molecules in living cells; Figure 9 The present invention relates to a Pepper and Clivia-based CaFR probe for labeling and detecting target nucleic acid molecules in solution and for labeling and multicolor imaging of target nucleic acid molecules in live cells. Detailed Implementation

[0026] The technical solutions in the embodiments of this application will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of this application, and not all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application. Unless otherwise stated herein, the singular forms "a" and "the" as used in this specification and claims include the plural forms. In this document, the term "or" includes the meaning of "and / or".

[0027] This invention provides a cascade-activated fluorescent RNA probe, wherein the probe sequence comprises sequences a, b, c, c*, d, e, e*, f, and f*, with the general structural formula as shown below. Figure 1As shown; wherein sequence a is a repressor sequence, and when sequence b does not bind to the target nucleic acid molecule, sequence a can form a first stem structure with sequence c through base pairing; sequence b is a target nucleic acid molecule recognition and binding sequence, and sequence b can bind to the target nucleic acid molecule through base pairing; sequence c is a regulatory sequence, and when sequence b does not bind to the target nucleic acid molecule, sequence c can form the first stem structure with sequence a through base pairing; when sequence b binds to the target nucleic acid molecule, sequence c can form a second stem structure with sequence c* through base pairing; sequence c* is a stabilizing sequence, and when sequence b binds to the target nucleic acid molecule, sequence c* can form the second stem structure with sequence c through base pairing, thereby promoting the formation of a stable three-dimensional structure of sequence f; sequence d is a linker sequence, and when sequence b binds to the target nucleic acid molecule, it makes the fluorescence-activated sequence f and sequence f* less likely to interfere; when sequence b does not bind to the target nucleic acid molecule, it makes it easier for sequence c* to bind to sequence e; The e sequence is a regulatory sequence. When the b sequence does not bind to the target nucleic acid molecule, the e sequence can form a third stem structure through base pairing with the c* sequence. When the b sequence binds to the target nucleic acid molecule, the e sequence can form a fourth stem structure through base pairing with the e* sequence. The e* sequence is a stable sequence. When the b sequence binds to the target nucleic acid molecule, the e* sequence can form the fourth stem structure through base pairing with the e sequence, thereby promoting the formation of a stable three-dimensional structure of the f* sequence. Furthermore, the f sequence and the f* sequence are fluorescent RNA aptamer sequences. When the c sequence and the c* sequence, or the e sequence and the e* sequence, complementarily pair to form the second or fourth stem structure, the f sequence and the f* sequence will form a stable three-dimensional fluorescent RNA aptamer structure. The complementary pairing can be complete or partial base pairing, as long as it forms a stem structure.

[0028] In the absence of a target nucleic acid molecule, the fluorescent RNA aptamer cannot form the active conformation of the activating fluorophore molecule due to interference from the blocking sequence. However, when a target nucleic acid molecule is present, the probe's target nucleic acid molecule recognition-binding sequence binds to the target nucleic acid molecule, inducing a cascade of activating fluorescent RNA probe conformational recombination. This restores the active conformation of the fluorescent RNA aptamer to the activating fluorophore molecule, thereby generating a fluorescent signal that responds to the target nucleic acid molecule, which can be used for the content analysis of the target nucleic acid molecule. Figure 2 ).

[0029] In the cascaded activated fluorescent RNA probe, the sequences a, b, c, f, c*, d, e, f*, and e* are linked in the order abcfc*-def*-e*. Preferably, the a and c sequences are 12 nucleotides in length. Preferably, the b sequence is 18 to 28 nucleotides in length. In the cascaded activated fluorescent RNA probe, the b sequence can be a nucleotide sequence that is completely complementary to the target nucleic acid molecule, or a nucleotide sequence that is not completely complementary to the target nucleic acid molecule and contains one, two, or more nucleotide mutations. Preferably, the c* sequence is 9 nucleotides in length. Preferably, the d sequence is a -CAAAA- sequence. Preferably, the e sequence is 6 nucleotides in length. Preferably, the e* sequence is 6 nucleotides in length.

[0030] In the cascaded activated fluorescent RNA probe, the f sequence and the f* sequence are fluorescent RNA aptamer sequences. When the c sequence and the c* sequence, and the e sequence and the e* sequence, complementarily pair to form the second stem structure and the fourth stem structure, the f sequence and the f* sequence form a stable three-dimensional fluorescent RNA aptamer structure. This structure then binds to a fluorophore molecule to form a first probe cascade activated fluorophore molecule, enhancing the fluorescence intensity of the fluorophore molecule within the first probe cascade activated fluorophore molecule. Preferably, the f sequence and the f* sequence are selected from Pepper fluorescent RNA aptamers or Clivia fluorescent RNA aptamers.

[0031] The nucleotides of the cascade-activated fluorescent RNA probe may be selected from, but are not limited to, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. The f sequence and the f* sequence may be selected from, but are not limited to, the SEQ ID NO: 2 fluorescent RNA aptamer sequence (Pepper fluorescent RNA-based CaFR probe) or the SEQ ID NO: 5 fluorescent RNA aptamer sequence (Clivia fluorescent RNA-based CaFR probe), and may be any fluorescent RNA aptamer.

[0032] In the cascade-activated fluorescent RNA probe, when the target nucleic acid molecule is absent, the cascade-activated fluorescent RNA probe cannot bind to the fluorophore molecule or has a weak binding ability to form a second probe cascade-activated fluorophore molecule; when the target nucleic acid molecule is present, the cascade-activated fluorescent RNA probe has a strong binding ability to the fluorophore molecule to form a first probe cascade-activated fluorophore molecule, wherein the fluorescence intensity produced by the second probe cascade-activated fluorophore molecule is weaker than the fluorescence intensity produced by the first probe cascade-activated fluorophore molecule.

[0033] In the cascade-activated fluorescent RNA probe, the b sequence is used for RNA-DNA hybridization or RNA-RNA hybridization and can recognize the nucleotide sequence of the target nucleic acid molecule, which can be a DNA molecule or an RNA molecule.

[0034] The cascade-activated fluorescent RNA probe of the present invention can be obtained by genetic encoding or by in vitro transcription.

[0035] The present invention also provides an expression vector comprising the cascade-activated fluorescent RNA probe, wherein the expression vector comprises the encoding gene of the cascade-activated fluorescent RNA probe.

[0036] This invention also provides a method for labeling and detecting target nucleic acid molecules in host cells using the cascade-activated fluorescent RNA probe, comprising the following steps: (a) The cascaded activation fluorescent RNA probe is constructed in a prokaryotic plasmid expression vector or a eukaryotic plasmid expression vector to obtain a recombinant plasmid; (b) Introducing the recombinant plasmid into the host cell; and (c) Adding fluorophore molecules for labeling and detection.

[0037] This invention also provides a method for labeling and detecting target nucleic acid molecules in host cells using the cascade-activated fluorescent RNA probe, comprising the following steps: (a) The cascade-activated fluorescent RNA probe was prepared by in vitro transcription or chemical synthesis; (b) Fixing the host cells; and (c) Add the cascade-activated fluorescent RNA probe and fluorophore molecule for labeling and detection.

[0038] This invention also provides a method for labeling and detecting target nucleic acid molecules in solution using the cascade-activated fluorescent RNA probe, comprising the following steps: (a) The cascade-activated fluorescent RNA probe is prepared by in vitro transcription or chemical synthesis; and (b) The cascade-activated fluorescent RNA probe and fluorophore molecule are added to the solution for labeling and detection.

[0039] In one specific embodiment of the present invention, the method for preparing CaFR probes by in vitro transcription includes: 1) constructing a series of probe libraries that respond to different target RNAs by substituting the b sequence encoding the target nucleic acid molecule recognition sequence according to the probe design principle. First, a cDNA library is synthesized, and a T7 promoter sequence is added to the upstream primer portion of the library for in vitro transcription into an RNA library. By designing forward primers (containing the T7 promoter), reverse primers, and template strands, an overlap extension PCR reaction is used to obtain the T7-abcfc*-def*-e* probe template sequence for solution transcription; 2) the PCR product is identified by agarose gel electrophoresis; 3) using the T7-abcfc*-def*-e* sequence as a transcription template, and then using a kit containing T7 RNA polymerase to transcribe and synthesize the CaFR probe; and 4) the transcribed RNA is purified by phenol-chloroform purification. A large number of CaFR probes can be prepared simply, rapidly, and cost-effectively using in vitro transcription. These probes can be used not only for high-throughput imaging analysis of target RNA in immobilized cells, but also for detecting RNA content in solution samples, providing rapid and efficient information on RNA concentration. Furthermore, a large number of prepared CaFR probes (such as SEQ ID NO: 6) can be used for imaging analysis of target nucleic acid molecules in immobilized cells, and can also be used to image and detect specific states of intracellular RNA under certain conditions, enabling the localization and distribution analysis of target nucleic acid molecules in cells.

[0040] In this invention, the CaFR probe prepared by in vitro transcription is used for imaging detection of target nucleic acid molecules in immobilized cells. The reagent kit used for transcription is considered to be any kit that can obtain RNA transcript products, and the methods used for sample fixation, denaturation, and hybridization are considered to be general methods for preparing samples for immobilized cell hybridization detection.

[0041] In one specific embodiment of the present invention, a method for preparing a genetically encoded CaFR probe that can be expressed in target nucleic acid molecules in living cells for a long time includes: 1) linearizing the pLKO.1-puro vector by digesting it with XbaI and EcoRI; 2) amplifying the abcfc*-def*-e* sequence by PCR using primer synthesis; 3) expressing the chimeric probe sequence mediated by the U6 promoter, and recombining the abcfc*-def*-e* sequence with the linearized pLKO.1-puro vector using In-Fusion seamless cloning technology to obtain a CaFR probe encoding system, as shown in SEQ ID NO: 7; and 4) transforming the recombinant plasmid, extracting the plasmid, sequencing and identifying it, and then expressing it by cell transcription to obtain the genetically encoded CaFR probe.

[0042] The CaFR probe prepared by the above-mentioned genetic coding method can be continuously expressed in living cells and used to analyze the expression distribution of target nucleic acid molecules in cells. It can also be used to image and track the dynamic changes of target nucleic acid molecules in single cells, and to study the imaging monitoring of target nucleic acid molecules in living cells over long time scales, as well as to indicate the state of cells.

[0043] In this invention, the CaFR probe obtained through genetic encoding is used for imaging studies of target nucleic acid molecules in living cells, and can perform imaging analysis on the expression degree, spatial distribution, dynamic behavior, etc. of the target molecules.

[0044] Definitions and explanations of terms used in this article: In this invention, a "fluorescent RNA aptamer" refers to an RNA aptamer sequence capable of binding to a specific "fluorophore molecule" and significantly increasing the fluorescence intensity of the fluorophore molecule under excitation light of a suitable wavelength after binding. Examples of a series of preferred fluorophore molecules and their corresponding fluorescent RNA aptamers in this invention are detailed in Table 1. In another preferred embodiment of this invention, the fluorescent RNA aptamer is Pepper, which can specifically recognize and bind to the fluorophore molecule HBC and its derivatives, such as HBC530 and HBC620. In a preferred embodiment of this invention, the fluorescent RNA aptamer is Clivia, which can specifically recognize and bind to the fluorophore molecule NBSI and its derivatives, such as NBSI565, NBSI574, NBSI580, NBSI595, NBSI618, and NBSI624.

[0045] Table 1: Fluorescent RNA aptamers and their corresponding fluorophore molecules of the present invention

[0046] The preferred structures of the fluorescent RNA aptamers and their corresponding fluorophore molecules of this invention are as follows:

[0047]

[0048]

[0049]

[0050] "Fluorescent RNA" refers to the RNA-fluorophore complex formed when the fluorescent RNA aptamer binds to the corresponding fluorophore molecule.

[0051] "Fluorescent molecules," also known as "fluorophores," "dyes," or "dye molecules," refer in this invention to a class of dye molecules that can be specifically bound and activated by fluorescent RNA aptamers. These fluorophore molecules exhibit low fluorescence intensity when not bound by a fluorescent RNA aptamer, but their fluorescence intensity at a specific wavelength increases significantly upon binding. In specific embodiments, the quantum yield of the fluorophore molecule is less than 0.1 when not bound to a specific fluorescent RNA aptamer, more preferably less than 0.01, and optimally less than 0.001; when bound to a specific fluorescent RNA aptamer, the quantum yield increases by more than 2 times, more preferably more than 10 times, and optimally more than 100 times. The fluorophore molecules are preferably water-soluble, non-toxic to cells, and easily permeate membranes. Preferably, the fluorophores of this invention can enter the cytoplasm or periplasm through the cell membrane or cell wall via active transport or passive diffusion. In embodiments of the present invention, fluorophore molecules can pass through the outer and inner membranes of Gram-negative bacteria, the cell walls and cell membranes of plant cells, the cell walls and cell membranes of fungi, the cell membranes of animal cells, and the GI and endothelial cell membranes of living animals.

[0052] The "expression vector" of the present invention comprises a DNA molecule integrated with an expression nucleic acid aptamer, which may be a plasmid or a viral particle.

[0053] The "host cell" of this invention includes, but is not limited to, bacteria, yeast, mammalian cells, insect cells, plant cells, zebrafish cells, fruit fly cells, and nematode cells. More preferably, the host cell is a cultured in vitro cell or a whole in vivo living tissue. The mammalian cells included in the host cell of this invention include, but are not limited to, 297T, COS-7, BHK, CHO, HEK293, HeLa, H1299, fertilized egg stem cells, induced pluripotent stem cells, and primary cells directly isolated from mammalian tissues.

[0054] Example 1: In vitro transcribed cascade-activated fluorescent RNA probes for labeling and detection of target nucleic acid molecules in solution. 1.1 Preparation of CaFR probes transcribed in vitro 1.1.1 amplification of cDNA Since the target nucleic acid sequence is relatively short, the template strand is usually a chemically synthesized nucleic acid. The upstream primer F (CaFR-F) and the downstream primer R (CaFR-R) are mixed and then subjected to PCR amplification to obtain cDNA.

[0055] It should be noted that since the transcription kit used in this embodiment is initiated by T7 RNA polymerase, a T7 promoter sequence needs to be added to the 5' end when designing the cDNA primers.

[0056] Table 2: cDNA amplification system

[0057] Add samples according to the system shown in Table 2, using the Taq system, with 2 tubes for each sample, 50 μL per tube.

[0058] PCR program: ① 95°C for 5 min; ② 98°C for 10 s; ③ 55°C for 30 s; ④ 72°C for 35 s (repeat ②-④ 30 times); ⑤ 72°C for 5 min; ⑥ 10°C forever.

[0059] Electrophoresis on a 2% agarose gel was performed, the gel was cut and recovered, and eluted with 20 μL of DEPC water.

[0060] 1.1.2 Transcription To avoid contamination of RNA during in vitro preparation and detection by RNase, all operations in this invention, unless otherwise specified, must be performed in an RNase-free laminar flow hood or biosafety cabinet, and all consumables used must be RNase-free and sterile. This embodiment primarily uses the T7 RNA polymerase kit manufactured by Thermo Fisher Scientific for RNA transcription.

[0061] Table 3: Transcription System

[0062] (1) Take PCR tubes that do not contain RNase (RNase-free), prepare the system as shown in Table 3, and incubate them in a PCR instrument at 37°C for 4 hours; (2) Add 0.5 μL DNase I and incubate at 37°C for 30 min.

[0063] It is important to note that the transcription reaction system needs to be prepared fresh for each use. Since T7 PCR polymerase is quite sensitive to temperature, all steps of the mixed reaction system must be completed on ice.

[0064] 1.1.3 Recycling (1) Preparation of organic solvent: Mix phenol, chloroform and isopropanol in a volume ratio of 25:24:1; Table 4: RNA recovery system

[0065] (2) Add the sample according to the system shown in Table 4, shake to mix, centrifuge at 10,000 rpm for 1 min, and transfer the supernatant to a new 1.5 mL nuclease-free centrifuge tube; (3) Add 88 μL of chloroform, shake to mix, centrifuge at 10,000 rpm for 1 min, transfer the supernatant to a new 1.5 mL nuclease-free centrifuge tube, and repeat this step twice; (4) Add 200 μL of anhydrous ethanol, gently invert, centrifuge at 12,000 rpm for 1 min, and discard the supernatant; (5) Add 800 μL of ice-cold 70% ethanol, centrifuge at 12,000 rpm for 1 min, discard the supernatant, and after the ethanol has evaporated, add 150 μL of DEPC water and mix well. (6) The RNA recovered from each tube is transferred to a new PCR tube for quantification.

[0066] (7) The recovered CaFR probe was quantified and aliquoted. The synthesized probe was stored at -80°C for later use, avoiding repeated freeze-thaw cycles.

[0067] 1.2 In vitro transcribed CaFR probes for labeling and detection of target nucleic acid molecules in solution. 1.2.1 Test Methods The probe used for detecting the target nucleic acid molecule in solution was synthesized using the same method as in Example 2. The detection solution contained 40 mM HEPES, 125 mM KCl, 5 mM MgCl2, and 5% DMSO, with a pH of 7.4.

[0068] Analysis of the relationship between fluorescence intensity and the molecular weight of the target nucleic acid: Different concentrations of the target ACTB gene were reacted with the CaFR probe to detect the dose-response relationship between fluorescence intensity and the target ACTB gene, and to evaluate its detection linear range.

[0069] CaFR probes are used for real-time detection of target nucleic acid molecules: by detecting the kinetics of the CaFR probe's response to the target nucleic acid molecule in response to the fluorescence intensity of the target miRNA 21 molecule, the response changes of the probe when it generates a signal to the target nucleic acid molecule can be determined.

[0070] 1.2.2 Experimental Results The CaFR-Pepper-miRNA 21 probe was incubated with miRNA 21, and then... Figure 3 A and Figure 3 B shows that when sequences a and c are 12 nucleotides long, c* is 9 nucleotides long, d is -CAAAA-, and e and e* are 6 nucleotides long, the CaFR-Pepper-miRNA 21 probe shows a clear specific labeling signal for miRNA 21, and the corresponding cascade-activated fluorescent RNA probe sequence is SEQ ID NO: 2. Furthermore, as... Figure 3 As shown in C, the CaFR probe can respond to both target nucleic acid molecules and target DNA molecules, and the corresponding cascade-activated fluorescent RNA probe sequence is SEQ ID NO: 2. Furthermore, as... Figure 3 D and Figure 3 As shown in E, when the b sequence is 18, 22, 25, and 28 nucleotides in length (SEQ ID NO: 9 to SEQ ID NO: 12), the probes all produce strong specific fluorescent signals against the target nucleic acid molecules. The corresponding cascade-activated fluorescent RNA probe sequences are SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. These experimental results demonstrate that different variations of the CaFR probe sequences a, b, c, c*, d, e, or e* can effectively label and detect target nucleic acid molecules.

[0071] When using the CaFR-Pepper-miRNA 21 probe to detect target nucleic acid molecules (SEQ ID NO: 13 to SEQ ID NO: 17) containing one or two nucleotide mutations, such as Figure 4 A and Figure 4 As shown in B, the CaFR probe can effectively label and detect target nucleic acid molecules containing one or two nucleotide mutations, and the corresponding cascade activation fluorescent RNA probe sequence is SEQ ID NO: 2.

[0072] The CaFR-Pepper-miRNA 21 probe was incubated with miRNA 21, and then... Figure 5 A shows that the fluorescence signal of the specifically labeled RNA reaches its maximum at an incubation time of approximately 40 minutes. The corresponding cascade-activated fluorescent RNA probe sequence is SEQ ID NO: 2. Using CaFR-Pepper-miRNA 21 incubated with different concentrations of miRNA 21 gene, dose-response analysis of the probe and target miRNA 21 revealed a linear relationship between the probe's labeling of the target nucleic acid molecule and the target RNA. Figure 5 B), and its signal can remain stable for more than 7 days (B). Figure 5C), the corresponding cascaded activation fluorescent RNA probe sequence is SEQ ID NO: 2.

[0073] The CaFR-Clivia probe was incubated with different target nucleic acid molecules, and then... Figure 6 A shows that the CaFR-Clivia probe can generate specific fluorescent signals for all target nucleic acid molecules. When using different fluorophore molecules for labeling, the CaFR-Clivia probe can activate different fluorophore molecules after binding to the target nucleic acid molecules (…). Figure 6 B). The above experimental results demonstrate that the prepared CaFR probe, obtained by replacing the f and f* sequences with different fluorescent RNA aptamer sequences, can effectively detect and analyze target nucleic acid molecules. The corresponding cascade-activated fluorescent RNA probe sequence is SEQ ID NO: 5.

[0074] Example 2: Cascade-activated fluorescent RNA probes prepared by in vitro transcription for labeling and imaging of target nucleic acid molecules immobilized in cells. 2.1 Preparation of CaFR probes transcribed in vitro The method for preparing the CaFR probe transcribed in vitro is the same as that in Example 1.

[0075] 2.2 Cascade-activated fluorescent RNA probes prepared by in vitro transcription for labeling and imaging of target nucleic acid molecules immobilized in cells. 2.2.1 Test Methods Fluorescence in situ hybridization: Cell samples were fixed with 4% paraformaldehyde at room temperature for 20 minutes and washed three times with PBS. Cells were then permeabilized with 0.3% (v / v) Triton X-100, followed by permeabilization with 70% ethanol overnight at 4°C. The next day, cells were washed twice with 2×SSC buffer (300 mM NaCl, 30 mM sodium citrate, pH 7.0) for 5 minutes each time, and then immersed in 2×SSC buffer containing 50% formamide for 30 minutes. Hybridization was performed for 12 hours at 37°C with 1 μM Cy3 / FAM-labeled DNA probes and CaFR probes (probes were prepared using hybridization buffer: 2×SSC, 50% formamide, 10% dextran sulfate, 2 mM vanadate-ribonucleoside complex, 0.01% RNase-free BSA, and 1 mg / ml E. coli tRNA). Cell samples were then washed three times with 2×SSC buffer preheated to 42°C for 5 minutes each time. Cell nuclei were stained with DAPI. Cells were then washed with 2×SSC and counterstained with 2×SSC containing 0.5 μM HBC620 dye and 5 mM MgCl2.

[0076] To image exogenous ACTB, plasmids expressing ACTB mRNA were transfected into HeLa cells. HeLa cells were fixed 36 hours post-transfection, and hybridization was performed using a CaFR probe according to the FISH protocol.

[0077] When imaging endogenous ACTB in cells, HeLa cells were cultured, fixed after 36 hours, and hybridization was detected using the CaFR probe according to the FISH procedure.

[0078] To label and dynamically image the stress state of cells after stimulation with endogenous ACTB, HeLa cells expressing G3BP1 plasmids were used. After 36 hours, the original medium was replaced with phenol red-free medium containing 0.5 mM sodium arsenite, and the cells were stimulated for 1 hour. Hybridization was then performed using a CaFR probe according to the FISH procedure. Immunofluorescence staining was then performed using G3BP1 antibodies: anti-G3BP1: 1 / 100 dilution; Alexa Fluor 488-labeled antibody: 1 / 400 dilution. The immunofluorescence staining procedure was as follows: cells were incubated with a blocking solution of PBS containing 0.5% bovine serum albumin at room temperature for 1 hour, followed by washing twice with PBS buffer for 5 minutes each time. Next, cells were incubated with G3BP1 antibody overnight, followed by incubation with Alexa Fluor 488 antibody at room temperature for 2 hours. Cells were then washed three times with 2×SSC for 5 minutes each time and incubated with 0.5 μM HBC620 containing 5 mM MgCl2.

[0079] Imaging was performed using a 60x oil immersion lens under the following conditions: DAPI: EX 405 nm, 2×2 Binning, 100 ms exposure time, 30% laser intensity; FAM and Alexa Fluor 488: EX 488 nm, 2×2 Binning, 100 ms exposure time, 20% laser intensity; HBC620: EX 561 nm, 2×2 Binning, 100 ms exposure time, 40% laser intensity. Image results were analyzed using ImageJ software, and statistical data were processed, fitted, and plotted using GraphPad software.

[0080] 2.2.2 Experimental Results The ACTB gene was overexpressed in HeLa cells, and the cells were fixed after 36 hours. The ACTB mRNA was then labeled and imaged using a CaFR probe. Figure 7 A) A distinct and specific fluorescent signal was observed, which was further confirmed by a FAM-labeled FISH probe, demonstrating that the CaFR probe can label and fix ACTB mRNA in cells with a significant correlation, while no response signal was observed in the control group. Figure 7 B). HeLa cells overexpressed the G3BP1 gene. After 36 hours, the HeLa cells were stimulated with 0.5 μM sodium arsenite and then fixed. Granular RNA molecules were observed in the cells, which were further confirmed by G3BP1 immunostaining. This demonstrates that the target gene produced a stress response when the cells were stimulated, indicating that the CaFR probe can reflect the state changes of the target nucleic acid molecules in the cells. Figure 7 C and Figure 7 D). The above experimental results demonstrate that the CaFR probe can perform imaging analysis on target nucleic acid molecules in fixed cells, and the corresponding cascade-activated fluorescent RNA probe sequence is SEQ ID NO: 6.

[0081] Example 3: Genetically encoded cascade-activated fluorescent RNA probes for labeling and imaging target nucleic acid molecules in living cells. 3.1 Preparation of the genetically encoded CaFR expression plasmid 3.1.1 cDNA amplification Because the required target cDNA sequence is relatively short, the template strand is obtained through chemical synthesis. The template, upstream primer F, and downstream primer R are then mixed and subjected to PCR amplification to obtain cDNA. In this embodiment, the DNA polymerase used for amplifying the target gene is PrimeSTAR® Max high-fidelity DNA polymerase.

[0082] upstream primer Table 5: PS Max Polymerase Reaction System

[0083] Table 6: PS Max Polymerase Reaction Procedure

[0084] 3.1.2 Ligation of the amplified fragment with the linearized vector Using a homologous recombination kit, during PCR amplification, primers are used to introduce homologous arms of 15 to 20 nucleotides in length at both ends of the linearized vector and the target fragment, respectively. Then, the homologous recombination enzyme catalyzes the ligation of the target fragment to the vector. The mixture needs to be reacted at 50°C for 20 minutes.

[0085] Table 7: Reaction systems for homologous recombination linkage

[0086] Transformation: The recombinant plasmid was added to 20 μL of prepared competent cells and placed on ice for 30 minutes. Then, it was placed in a water bath preheated to 42°C for 45 to 60 seconds for heat shock, and then placed back on ice for 3 to 5 minutes. 500 μL of antibiotic-free LB medium was added and the cells were shaken at 37°C for 45 to 60 minutes to activate the competent cells. The bacterial culture was collected by centrifugation at 4500 rpm for 5 minutes. Most of the medium was discarded, and 50 μL of the culture was resuspended and spread on solid LB medium containing antibiotics. The cells were incubated overnight at 37°C. After single colonies grew, single colonies were picked for colony PCR. After identification as positive colonies, single colony sequencing was performed.

[0087] 3.1.3 Plasmid Extraction Since endotoxins must be removed from plasmids expressed in eukaryotic cells, endotoxin-free plasmid extraction kits manufactured by Omega are used for plasmids used for transfection into eukaryotic systems.

[0088] 3.2 Genetically encoded CaFR probes for labeling and imaging target nucleic acid molecules in living cells. 3.2.1 Cell Culture and Transfection HeLa (TCHu187) cells were purchased from the Cell Bank of the Chinese Academy of Sciences and cultured in Dulbecco modified Eagle medium (DMEM, high glucose) containing 10% fetal bovine serum (FBS). Cells were cultured in a 37°C cell culture incubator (95% air, 5% CO2). When HeLa cells reached 80-90% confluence, transfection was performed using Hieff Trans liposome transfection reagent (YEASEN).

[0089] 3.2.2 Fluorescence Microscopy Imaging To image exogenous ACTB, the pU6-CaFR-ACTB plasmid expressing ACTB mRNA (transfection ratio 1:1, total 0.5 μg) was transfected into HeLa cells cultured in 35 mm 4-well glass dishes. After 36 hours, the original medium was replaced with phenol red-free medium containing 10% FBS, 0.5 μM HBC620 and 5 mM MgCl2. After incubation for 10 minutes, imaging analysis was performed.

[0090] To image endogenous ACTB, the pU6-CaFR-ACTB plasmid (0.5 μg) was transfected into HeLa cells cultured in 35 mm 4-well glass dishes. After 36 hours, the original medium was replaced with phenol red-free medium containing 10% FBS, 0.5 μM HBC620 and 5 mM MgCl2. After incubation for 10 minutes, imaging analysis was performed.

[0091] For multicolor imaging of endogenous ACTB and GAPDH, the plasmid vector expressing pU6-CaFR-ACTB-Pepper and the pU6-CaFR-GAPDH-Clivia probe plasmid (transfection ratio 1:1, total 0.5 μg) were transfected into HeLa cells cultured in 35 mm 4-well glass dishes. After 36 hours, the original medium was replaced with phenol red-free medium containing 0.5 μM HBC620, 0.5 mM sodium arsenite and 5 mM MgCl2. Imaging was performed after 20 minutes of stimulation.

[0092] Imaging was performed using a 60x oil immersion lens under the following conditions: Hoechst 33342 and BFP: EX 405 nm, 2×2 binning, 100 ms exposure time, 30% laser intensity; GFP: EX 488 nm, 2×2 binning, 100 ms exposure time, 20% laser intensity; HBC620: EX 561 nm, 2×2 binning, 100 ms exposure time, 40% laser intensity. Image results were analyzed using ImageJ software, and statistical data were processed, fitted, and plotted using GraphPad software.

[0093] 3.2.3 Experimental Results In HeLa cells, when the ACTB mRNA expression plasmid was co-transfected with the CaFR probe expression plasmid, a significant specific fluorescent signal was observed in the cells, while no significant specific fluorescent signal was observed when the control probe was transfected. Figure 8 A), and can effectively distinguish signals activated by endogenous target nucleic acid molecules (A), and can effectively distinguish signals of activation by endogenous target nucleic acid molecules ( Figure 8 B), and the specific fluorescent signal generated by activation can remain stable (B). Figure 8 C and Figure 8 D). The above experimental results demonstrate that CaFR probes can label and image target nucleic acid molecules in living cells. The corresponding cascade-activated fluorescent RNA probe sequence is SEQ ID NO: 7.

[0094] The CaFR-Pepper and CaFR-Clivia probes were incubated with their corresponding target nucleic acid molecules. Figure 9A shows that both the CaFR-Pepper and CaFR-Clivia probes can generate specific fluorescent signals for their corresponding target nucleic acid molecules. Furthermore, the spectra are orthogonal. The experimental results indicate that the combined detection of CaFR-Pepper and CaFR-Clivia probes can effectively detect and analyze these two target nucleic acid molecules. The corresponding cascade-activated fluorescent RNA probe sequences are SEQ ID NO: 7 and SEQ ID NO: 8. In HeLa cells, when the CaFR-Pepper probe expression plasmid and the CaFR-GAPDH-Clivia probe expression plasmid are co-transfected ( Figure 9 B), two different specific fluorescent signals can be observed in the cells. When transfecting the CaFR probe expression plasmid or the CaFR-GAPDH probe expression plasmid alone, only the fluorescent signal corresponding to each probe can be observed. However, when transfecting the control probe, no specific fluorescent signal is observed. Figure 9 (C) and can effectively distinguish activation signals of target nucleic acid molecules. The above experimental results demonstrate that CaFR probes can label and perform multicolor imaging analysis of target nucleic acid molecules in living cells.

[0095] 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 included within the scope of the present invention.

[0096] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference in their entirety, to the same extent as if each individual publication, patent, or patent application were specifically and individually identified and incorporated herein by reference. Furthermore, any references cited or indicated should not be construed as an admission that such references are prior art to the present invention. Heading portions in this application are used herein to facilitate understanding of the specification and should not be construed as necessary limitations.

Claims

1. A cascade-activated fluorescent RNA probe, characterized in that: The probe sequence includes sequence a, sequence b, sequence c, sequence c*, sequence d, sequence e, sequence e*, sequence f, and sequence f*. The a sequence is a repressor sequence. In the absence of the b sequence binding to the target nucleic acid molecule, the a sequence and the c sequence form a first stem structure through complementary base pairing. The b sequence is a target nucleic acid molecule recognition and binding sequence, and the b sequence binds to the target nucleic acid molecule through complementary base pairing; The c sequence is a regulatory sequence. When the b sequence does not bind to the target nucleic acid molecule, the c sequence and the a sequence form the first stem structure through complementary base pairing. When the b sequence binds to the target nucleic acid molecule, the c sequence and the c* sequence form the second stem structure through complementary base pairing. The c* sequence is a stable sequence. When the b sequence binds to the target nucleic acid molecule, the c* sequence and the c sequence form the second stem structure through complementary base pairing, thereby promoting the formation of a stable three-dimensional structure of the f sequence. The d sequence is a linker sequence that, when the b sequence binds to the target nucleic acid molecule, makes it less likely for the f sequence and the f* sequence, which restore fluorescence activation activity, to interfere with each other; and when the b sequence does not bind to the target nucleic acid molecule, makes it easier for the c* sequence and the e sequence to bind. The e sequence is a regulatory sequence. When the b sequence does not bind to the target nucleic acid molecule, the e sequence and the c* sequence form a third stem structure through complementary base pairing. When the b sequence binds to the target nucleic acid molecule, the e sequence and the e* sequence form a fourth stem structure through complementary base pairing. The e* sequence is a stable sequence. When the b sequence binds to the target nucleic acid molecule, the e* sequence and the e sequence form the fourth stem structure through complementary base pairing, thereby promoting the formation of a stable three-dimensional structure of the f* sequence; and The f sequence and the f* sequence are fluorescent RNA aptamer sequences. When the c sequence and the c* sequence or the e sequence and the e* sequence are complementary to form the second stem structure or the fourth stem structure, the f sequence and the f* sequence form a stable three-dimensional fluorescent RNA aptamer structure.

2. The cascade-activated fluorescent RNA probe according to claim 1, characterized in that: The sequences a, b, c, f, c*, d, e, f*, and e* are connected in the order abcfc*-def*-e*.

3. The cascade-activated fluorescent RNA probe according to claim 1, characterized in that: The a sequence and the c sequence are 12 nucleotides in length; the b sequence is 18 to 28 nucleotides in length; the c* sequence is 9 nucleotides in length; the e sequence is 6 nucleotides in length; the e* sequence is 6 nucleotides in length; the d sequence is a -CAAAA- sequence; the f sequence and the f* sequence include a Pepper fluorescent RNA aptamer or a Clivia fluorescent RNA aptamer; or the target nucleic acid molecule includes a single-stranded DNA molecule or an RNA molecule.

4. The cascade-activated fluorescent RNA probe according to claim 1, characterized in that: The b sequence is a nucleotide sequence that is completely complementary to the target nucleic acid molecule, or a nucleotide sequence that is not completely complementary to the target nucleic acid molecule and contains one, two or more nucleotide mutations.

5. The cascade-activated fluorescent RNA probe according to claim 1, characterized in that: The f sequence and the f* sequence are fluorescent RNA aptamer sequences. When the c sequence and the c* sequence and the e sequence and the e* sequence are complementary to form the second stem structure and the fourth stem structure, the f sequence and the f* sequence form a stable three-dimensional structure of the fluorescent RNA aptamer, which then binds to fluorophore molecules to form a first probe cascade activated fluorophore molecule and enhances the fluorescence intensity of the fluorophore molecule in the first probe cascade activated fluorophore molecule.

6. The cascade-activated fluorescent RNA probe according to claim 1, characterized in that: In the absence of the target nucleic acid molecule, the cascade-activated fluorescent RNA probe cannot bind to the fluorophore molecule or has a weak binding ability to form a second probe cascade-activated fluorophore molecule; in the presence of the target nucleic acid molecule, the cascade-activated fluorescent RNA probe has a strong binding ability to the fluorophore molecule to form a first probe cascade-activated fluorophore molecule, wherein the fluorescence intensity produced by the second probe cascade-activated fluorophore molecule is weaker than the fluorescence intensity produced by the first probe cascade-activated fluorophore molecule.

7. An expression vector comprising a cascade-activated fluorescent RNA probe according to any one of claims 1 to 6, characterized in that: The expression vector contains the encoding gene of the cascaded activated fluorescent RNA probe.

8. A method for detecting target nucleic acid molecules in host cells using a cascade-activated fluorescent RNA probe according to any one of claims 1 to 6, characterized in that: The method includes the following steps: (a) The cascaded activation fluorescent RNA probe is constructed in a prokaryotic plasmid expression vector or a eukaryotic plasmid expression vector to obtain a recombinant plasmid; (b) Introducing the recombinant plasmid into the host cell; and (c) Adding fluorophore molecules for labeling and detection.

9. A method for detecting target nucleic acid molecules in host cells using a cascade-activated fluorescent RNA probe according to any one of claims 1 to 6, characterized in that: The method includes the following steps: (a) The cascade-activated fluorescent RNA probe was prepared by in vitro transcription or chemical synthesis; (b) Fixing the host cells; and (c) Add the cascade-activated fluorescent RNA probe and fluorophore molecule for labeling and detection.

10. A detection method for labeling target nucleic acid molecules in solution using a cascade-activated fluorescent RNA probe according to any one of claims 1 to 6, characterized in that: The method includes the following steps: (a) The cascade-activated fluorescent RNA probe is prepared by in vitro transcription or chemical synthesis; and (b) The cascade-activated fluorescent RNA probe and fluorophore molecule are added to the solution for labeling and detection.

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