RNA biosensor, annular double-locked RNA biosensor and application of annular double-locked RNA biosensor

Abstract

The invention relates to the field of biological detection, in particular to an RNA biosensor, a circular double-locked RNA biosensor and application thereof. The invention provides an RNA biosensor. The RNA biosensor sequentially comprises a right lock module, an RNA sensing module, a first fluorescent aptamer module, a left lock module, a complementary module and a second fluorescent aptamer module from a 5'end to a 3 'end, the RNA sensing module and the complementary module are partially complementary; the right lock module and the second fluorescent aptamer module are partially complementary; and the left lock module and the first fluorescent aptamer module are partially complementary. The invention provides the double-locked RNA biosensor, the performance is greatly improved, and the nucleoplasm shuttle process of mitochondrial lncRNA is successfully monitored, which is not reported in the prior art at present. Other related substances and applications are developed based on the technology, and accurate visualization of RNA abundance can be used for disease diagnosis and treatment and drug effect and drug screening; the RNA concentration fluctuation and movement can be monitored in real time, and the method can be used for disease monitoring and RNA function research.

Description

Technical Field

[0001] The present invention relates to the field of biological detection, and in particular to an RNA biosensor, a circular double-locked RNA biosensor and applications thereof. Background Art

[0002] The abundance and distribution of RNA are tightly regulated, and aberrant expression and localization are associated with numerous diseases. Visualizing RNA localization and dynamics in living cells provides crucial insights into RNA processing, folding, function, degradation, and trafficking. This is crucial for exploring the mechanisms by which RNA function and abnormalities contribute to the pathogenesis of various diseases and for identifying potential therapeutic targets. High-resolution imaging techniques for detecting mRNA and its translational status, the spatial localization of lncRNAs, and RNA-protein interactions are currently in high demand in biomedicine and biotechnology.

[0003] RNA single-molecule fluorescence in situ hybridization (smFISH) is most commonly used to visualize RNA in fixed cells. However, traditional FISH techniques are not suitable for tracking RNA in living cells. Current methods for RNA imaging in living cells fall into two main categories: fluorescent protein-based methods and DNA-based hybridization probes. Fluorescent protein-based methods primarily utilize fluorescent protein-tagged RNA-binding proteins (such as the MS2-MCP system, PP7, λN22, or the CRISPR / Cas system in which RCas9 / dCas13 is fused to GFP). Among these, by inserting a 24× MS2 stem-loop cassette into the 3' UTR of the β-actin gene, β-actin mRNA was labeled, enabling spatiotemporal dynamic imaging of engineered β-actin mRNA nucleocytoplasmic transport. However, these methods have limitations, such as high background from overexpressed fluorescent proteins. Several approaches have also been developed to reduce protein background, such as Cas6-based switch platforms and RNA-stabilized fluorescent proteins. However, concerns remain regarding whether protein-based methods, including fluorescent proteins, will affect RNA structure, dynamics, expression, or function. DNA-based hybridization probes primarily utilize fluorescent oligonucleotide probes. However, these methods are expensive and often require duplication within the transcript to improve the detected fluorescence signal. To enhance fluorescence, molecular beacon-based enzymatic amplification has been used for spatially resolved in vivo imaging of inflammation-related IL-6 mRNA. However, this approach is limited to the specific target of IL-6. Therefore, universal methods are needed to systematically and precisely study RNA in living cells.

[0004] Jaffrey and his colleagues reported fluorescent RNA aptamers, providing a powerful tool for revealing the biological functions of RNA. Emerging genetically encoded molecular tools based on fluorescent RNA aptamers have created a useful platform for understanding the dynamic molecular biology of RNA in living cells. A common approach is to insert the corresponding fluorescent RNA aptamer sequence into the target RNA. However, this imaging method involves imaging engineered RNA, resulting in overexpression and failing to reflect the true state of RNA. Direct imaging of natural RNA is a challenge. Recently, RNA sensors designed based on cleavage or conformational transition principles, such as the AiFC method, the NP-miLS system, the iPepper technology, and the SSA technology, have been developed. These technologies essentially utilize one binding site on a target RNA to activate one fluorescent RNA aptamer. The closest existing technologies are the NP-miLS system (which activates one fluorescent RNA aptamer per binding site on a target, resulting in low fluorescence intensity) and the iPepper technology (which requires finding eight binding sites on a target to activate eight fluorescent RNA aptamers, resulting in design difficulties and high background). These existing technologies have largely achieved imaging of natural RNA within living cells. However, they suffer from low fluorescence intensity and high background, resulting in insufficient performance and still failing to achieve spatiotemporal dynamic monitoring of natural RNA within living cells. Furthermore, there are no reports of designs that simultaneously activate two fluorescent RNA aptamers using one binding site per target. Summary of the Invention

[0005] In view of this, the present invention provides an RNA biosensor, a circular double-locked RNA biosensor and its applications. The present invention provides a double-locked RNA biosensor that only requires one binding site on a target RNA to open two fluorescent RNA aptamers. Compared with the NP-miLS system, the fluorescence intensity is significantly improved, and compared with the iPepper technology, the background is significantly reduced, and it has the advantage of simple design. Functionally, the double-locked RNA biosensor realizes the visualization of the localization, abundance and movement of natural RNA in living cells, has good universality, and corresponding double-locked RNA biosensors can be designed according to different targets. The applicable RNA length range is greater than 20nt. Compared with the existing technology, the performance is greatly improved, and the nucleocytoplasmic shuttling process of mitochondrial lncRNA is successfully monitored, which is not reported in the current existing technology. Other related substances and applications are developed based on this technology, among which the precise visualization of RNA abundance can be used for disease diagnosis and treatment, drug efficacy and drug screening; real-time monitoring of RNA concentration fluctuations and movement can be used for disease monitoring and RNA function research.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides an RNA biosensor, which comprises, from the 5' end to the 3' end, a right lock module, an RNA sensing module, a first fluorescent aptamer module, a left lock module, a complementary module and a second fluorescent aptamer module;

[0008] The RNA sensing module is partially complementary to the complementary module;

[0009] The right lock module and the second fluorescent aptamer module are partially complementary;

[0010] The left-lock module and the first fluorescent aptamer module are partially complementary.

[0011] The base lengths of the left lock module and the right lock module are no less than 2 nt.

[0012] In some embodiments of the present invention, in the RNA biosensor, the base length of the left lock module is 3-7 nt; the base length of the right lock module is 3-5 nt.

[0013] The present invention also provides a circular double-locked RNA biosensor, comprising: the above RNA biosensor and a cyclization element.

[0014] The present invention also provides the use of the above RNA biosensor and / or the above circular double-locked RNA biosensor in any of the following items:

[0015] (I) RNA molecule detection; and / or (II) RNA drug screening; and / or (III) RNA drug efficacy evaluation; and / or (IV) preparation of products for RNA drug screening and / or RNA drug efficacy evaluation; and / or (V) RNA imaging; and / or (VI) preparation of products for diagnosing and / or treating RNA-related diseases; and / or (VII) preparation of products for studying RNA function.

[0016] The present invention also provides a method for detecting RNA molecules, which comprises mixing the above-mentioned RNA biosensor and / or the above-mentioned circular double-locked RNA biosensor with a detection target and a fluorescent dye for detection.

[0017] In some embodiments of the present invention, in the above detection method, when the detection target is complementary to the RNA sensing module, the first fluorescent aptamer module and the second fluorescent aptamer module are bound to the fluorescent dye to generate fluorescence.

[0018] In some embodiments of the present invention, the base length of the detection target in the above detection method is not less than 15 nt.

[0019] The present invention also provides a product, comprising: the RNA biosensor according to claim 1 or 2 and / or the circular double-locked RNA biosensor according to claim 3. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for describing the embodiments or the prior art.

[0021] Figure 1 Schematic diagram showing the principle of the double-locked RNA biosensor;

[0022] Figure 2 Figure 3. Evaluation of the sensitivity and specificity of the dual-lock RNA biosensor for in vitro RNA detection. Figure 3. Optimization of the dual-lock length. Figure 3. Spectral scanning results of the Ducker (250 nM, in vitro transcribed RNA) for survivin mRNA target detection. Figure 4. Linear correlation analysis between fluorescence intensity and the logarithmic value of survivin RNA concentration. The inset shows the linear correlation between fluorescence intensity and the logarithmic value of target RNA concentration in the low concentration range of 10 pM-5 nM. Figure 4. Target RNA visualization in vitro by Ducker. Figure 4. Specific fluorescence detection results of survivin target detection in total RNA by Ducker.

[0023] Figure 3 Figure 3. Evaluation of the ability of the dual-locked RNA biosensor to detect RNA in complex biological samples. Figure 3. A Ducker detection flow chart for target RNA in NIH-3T3 cell lysate in vitro. Figure 3. B Ducker fluorescence spectrum scanning results for detecting different concentrations of target Survivin mRNA in NIH-3T3 cell lysate. Figure 3. C Ducker fluorescence signal intensity versus the logarithm of Survivin mRNA concentration in NIH-3T3 cell lysate.

[0024] Figure 4 Figure 3. Evaluation of the imaging capability of circular double-locked RNA biosensors for native mRNA in living cells. (a) Schematic diagram of the design and in-vivo imaging of circDucker. (b) Confocal laser imaging of A549 cells transfected with plasmids expressing circular RNA biosensors and treated with Docetaxel (increases survivin mRNA) and YM155 (downregulates survivin mRNA). Scale bar represents 20 μm. (c) Comparison of target RNA imaging using circDucker and MB molecular beacon probes in living cells. Scale bar represents 10 μm.

[0025] Figure 5Figure 3 compares the performance advantages of circular double-locked RNA biosensors by comparing them with iPepper technology; a is a schematic diagram of the imaging principle of the iPepper system, in which one target binding site is designed to activate one fluorescent RNA aptamer, and fluorescence is enhanced in series, that is, eight binding sites are designed on one target to activate eight fluorescent RNA aptamers (8xiPepper); b is the optimization of the double-lock length; c is the comparison of the mRNA imaging capabilities of circDucker and 8xiPepper by laser confocal imaging analysis; d is the flow cytometry comparison of the mRNA imaging capabilities of circDucker and 8xiPepper;

[0026] Figure 6 Figure 3: Evaluation of the imaging capabilities of circular double-locked RNA biosensors for natural miRNAs in living cells and comparison with NP-miLS technology. Figure 3: Schematic diagram of the imaging principle of the NP-miLS system, where a target is designed to open a fluorescent RNA aptamer. Figure 4: Optimization of double-locked RNA length. Figure 5: Comparison of the imaging capabilities of circDucker and NP-miLS for miR-122 using confocal laser scanning imaging. Figure 6: Comparison of the imaging capabilities of circDucker and NP-miLS for miR-122 using flow cytometry.

[0027] Figure 7 Figure 3. Evaluation of the ability of fluorescence ratiometric RNA sensors to accurately quantify the abundance of natural mRNA in living cells and their application in drug evaluation and screening. a. Schematic diagram of the design principle of the fluorescence ratiometric RNA sensor circDucker-dBroccoli. b. Fluorescence ratiometric imaging results of circDucker-dBroccoli on Survivin mRNA in different cells. Scale bar represents 20 μm. c. Correlation analysis between qPCR quantitative results of Survivin mRNA expression levels in different cells and ratiometric imaging fluorescence signals. The horizontal axis represents the Survivin expression levels in different cells, and the vertical axis represents the relative red fluorescence signal intensity of the five cell types in fluorescence ratiometric imaging. d. Response of the logarithm of the YM155 inhibitor concentration to the Survivin mRNA expression level and the change in fluorescence ratiometric signal. e. Fluorescence ratiometric imaging results of circDucker-dBroccoli on endogenous Survivin mRNA in HeLa cells treated with different concentrations of YM155. Scale bar represents 20 μm.

[0028] Figure 8Figure 3. Evaluation of the ability of circular double-locked RNA biosensors to monitor concentration fluctuations of natural mRNA in living cells in real time and their application in disease diagnosis and treatment. a shows the optimization of double-locked RNA length. b shows circDucker imaging of IL-6 mRNA in RAW264.7 cells at different time points of LPS stimulation, with the scale bar representing 10 μm. c shows the fluorescence of flow cytometry quantification in b. d shows qPCR quantitative detection of IL-6 mRNA content in RAW264.7 cells at different time points of LPS stimulation. e shows real-time monitoring of IL-6 mRNA concentration fluctuations in RAW264.7 cells stimulated by LPS by circDucker, with the scale bar representing 10 μm. f shows the quantitative analysis results of the overall average fluorescence signal intensity of cells at each time point in e.

[0029] Figure 9 Figure 3 shows the evaluation of the spatiotemporal monitoring capability of circular double-locked RNA biosensors for the movement of natural lncRNAs in living cells and their application in RNA function research; a shows the optimization of double locks; b shows the three-dimensional confocal imaging results of mtlncCytb in A549 cells using circDucker, with the scale bar representing 10 μm; c shows the co-localization of mtlncCytb imaging using circDucker and smFISH, with the scale bar representing 10 μm; d shows the real-time monitoring imaging results of the nucleocytoplasmic bidirectional shuttling of the target RNA mtlncCytb in A549 cells using circDucker, where the white arrows indicate the movement process of mtlncCytb from the cytoplasm to the nucleus, and the pink arrows indicate the movement process of mtlncCytb from the nucleus to the cytoplasm, with the scale bar representing 5 μm; e. Schematic diagram of the movement trajectory of mtlncCytb bidirectional shuttling between the nucleus and the cytoplasm. DETAILED DESCRIPTION

[0030] The invention discloses an RNA biosensor, a circular double-locked RNA biosensor and applications thereof.

[0031] It should be understood that the expression "one or more of" includes individually each of the items recited after the expression and various combinations of two or more of the recited items, unless otherwise apparent from the context and usage. The expression "and / or" in conjunction with three or more recited items should be understood to have the same meaning, unless otherwise apparent from the context.

[0032] The terms "comprising", "having" or "containing", including their grammatical synonyms, should generally be understood as open and non-restrictive, e.g., not excluding other unrecited elements or steps, unless otherwise specifically stated or understood from the context.

[0033] It should be understood that the order of steps or the order in which certain actions are performed are not important as long as the present invention remains operable. Additionally, two or more steps or actions may be performed simultaneously.

[0034] The use of any and all examples or exemplary language, such as "such as" or "including," herein is intended merely to better illustrate the invention and does not limit the scope of the invention unless otherwise claimed. No language in this specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0035] In addition, the numerical ranges and parameters used to define the present invention are approximate values. The relevant numerical values ​​in the specific examples have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual testing methods. Therefore, unless otherwise expressly stated, all ranges, amounts, values, and percentages used in this disclosure should be understood to be modified by the word "about." As used herein, "about" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a specified value or range.

[0036] The present invention specifically adopts the following technical solutions:

[0037] In one aspect, the present invention provides a dual-locked RNA biosensor for RNA imaging, wherein the dual-locked RNA biosensor comprises or consists of the following four modules:

[0038] (1) An RNA sensing module: This part selects an RNA of about 20 nt in length as a binding sequence based on the sequence of the target RNA for specific recognition of the target RNA; (2) Two fluorescent RNA aptamer modules: As fluorescent reporter elements, a variety of fluorescent RNA aptamers have been reported, including Pepper, Broccoli, Corn, Mango, Spinach, Clivia, etc. The present invention uses Pepper as an example of a double-locked RNA biosensor. Different fluorescent reporter elements only need to select different fluorescent RNA aptamers; (3) A double-locked module, which is complementary to part of the fluorescent RNA aptamer and part of the RNA sensing module, presents two stem-like conformations, and is used to block the fluorescent RNA aptamer; (4) A sequence that is partially complementary to the RNA sensing module maintains the closed state of the double-locked RNA biosensor.

[0039] In the absence of target RNA, the fluorescent RNA aptamer is enclosed by the double-lock module and adopts an inactive conformation. When target RNA is present, it binds to the RNA sensing module, mediating the conformational switch of the RNA sensing module, releasing the two enclosed fluorescent RNA aptamers. These refold into an active conformation and bind to dyes to produce fluorescence (these dyes only "emit" light when bound to correctly folded fluorescent RNA aptamers). This enables highly sensitive and specific detection of RNA in vitro and imaging of RNA in living cells, including precise quantification of RNA localization and abundance and dynamic monitoring of movement.

[0040] Compared with the current design idea of ​​using one target to open one fluorescent RNA aptamer, the design innovation of the present invention lies in using one target RNA to simultaneously open two fluorescent RNA aptamers, significantly improving their fluorescence intensity in cells. Compared with RNA imaging technology based on fluorescent proteins (RCas9 / dCas13) and the tandem method using fluorescent RNA aptamers (such as iPepper), it has the advantage of low background, realizing the spatiotemporal dynamic monitoring of RNA.

[0041] For example, a person skilled in the art can delete or add some complementary nucleotides to the nucleotide sequence in (1)-(4) above, or replace part of the sequence according to the conventional technology in the art, so that the modified sequence or the modified sequence can play the same function as the original sequence.

[0042] Or a nucleotide sequence that performs the same function when transcribed in vivo or in vitro from any one of the nucleotide sequences (1)-(4).

[0043] Furthermore, based on the above-described dual-locked RNA biosensor, it is understood that those skilled in the art can modify the aforementioned dual-locked RNA biosensor using conventional techniques in the art to achieve the same functionality. For example, the aforementioned dual-locked RNA biosensor can be modified to have substantially the same or similar molecular structure, physicochemical properties, and functionality as the original dual-locked RNA biosensor, and to image RNA; the chemical modification can be any one or more of the following: phosphorylation, amination, methylation, sulfhydrylation, isotopization, substitution of oxygen for sulfur, or substitution of oxygen for selenium.

[0044] Secondly, the double-locked RNA biosensor of the present invention has good universality. Corresponding double-locked RNA biosensors can be designed according to different targets, and the applicable RNA length range is greater than 20 nt.

[0045] In a third aspect, the present invention further provides a combination of a double-locked RNA biosensor, comprising the above-mentioned double-locked RNA biosensor and a connecting substance connected thereto.

[0046] Furthermore, the connecting substance is a detection, labeling, diagnosis or treatment substance;

[0047] Alternatively, the conjugate is connected by any one or more of the following methods: T4 ligase connection, PCR connection, and biosynthetic connection;

[0048] Alternatively, the connecting substance may be any one or more of the following: chemical small molecules, biological macromolecules, and biological materials.

[0049] Furthermore, the double-locked RNA biosensor is coupled with a cyclization element or other functional nucleic acid to form a circular double-locked RNA biosensor or a fluorescence ratio biosensor for imaging RNA.

[0050] Furthermore, the cyclization element is an element that forms a circular double-locked RNA biosensor;

[0051] Furthermore, the fluorescence ratio biosensor is a double-locked RNA biosensor coupled with other fluorescent substances.

[0052] In a fourth aspect, the present invention further provides a derivative of a double-locked RNA biosensor, which is a tool capable of imaging RNA and is modified based on the above double-locked RNA biosensor or the above double-locked RNA biosensor combination;

[0053] The condition is that the derivatives all have substantially the same or similar molecular structure, physicochemical properties and functions as the double-locked RNA biosensor.

[0054] In a fifth aspect, the present invention further provides a product comprising the aforementioned double-locked RNA biosensor or a combination or derivative of the double-locked RNA biosensor; the product is a reagent, a kit or a drug.

[0055] In a fifth aspect, the present invention further provides an application of the double-locked RNA biosensor, characterized in that the application is any one or more of the following:

[0056] (1) Preparation of RNA-targeted drug efficacy evaluation or drug screening reagents;

[0057] (2) Preparation of reagents for RNA labeling, quantitative or qualitative detection, and RNA imaging;

[0058] (3) Preparation of reagents or drugs for disease diagnosis and treatment of RNA-related diseases.

[0059] (4) Preparation of reagents for RNA functional studies

[0060] In Examples 1 to 9 of the present invention, all the raw materials and reagents used can be purchased from the market.

[0061] The present invention will be further described below in conjunction with the embodiments:

[0062] Example 1 Dual-lock RNA biosensor design

[0063] Currently, a variety of fluorescent RNA aptamers have been reported, including Pepper, Broccoli, Corn, Mango, Spinach, Clivia, etc. In this invention, Pepper is used as an example of a double-locked RNA biosensor. Different fluorescent reporter elements only need to select different fluorescent RNA aptamers, such as Figure 1 As shown, it consists of four modules: (1) an RNA sensing module: this part is designed according to the sequence of the target RNA, and an RNA of about 20 nt in length is selected to be complementary to the target RNA for specific recognition of the target RNA; (2) two fluorescent RNA aptamer modules: as fluorescent reporter elements, this example uses the fluorescent RNA aptamer Pepper as an example; (3) a double lock module, which is complementary to part of the fluorescent RNA aptamer and part of the RNA sensing module, presenting two stem-like conformations, used to block the fluorescent RNA aptamer. Here, certain optimization is required depending on the target; (4) a sequence that is partially complementary to the RNA sensing module to maintain the closed state of the double lock RNA biosensor. The specific combination order of the above four modules is as follows: 5'-right lock + RNA sensing module + fluorescent RNA aptamer + left lock + partial complementary sequence of the RNA sensing module + fluorescent RNA aptamer-3'.

[0064] When there is no target RNA, the fluorescent RNA aptamer is enclosed by the double-lock module and has an inactive conformation; when the target RNA is present, it will bind to the RNA sensing module, mediate the conformational switch of the RNA sensing module, release the two enclosed fluorescent RNA aptamers, refold into an active conformation, and bind to dyes to produce fluorescence (these dyes only "emit light" when bound to correctly folded fluorescent RNA aptamers), realizing high-sensitivity and specific detection of RNA in vitro and imaging of RNA in living cells, including precise quantification of RNA localization and abundance and dynamic monitoring of movement.

[0065] Example 2 Evaluation of the sensitivity and specificity of the dual-locked RNA biosensor for in vitro RNA detection

[0066] Survivin mRNA was selected as the target and the corresponding double-lock RNA biosensor Ducker was designed. After screening and optimizing the double-lock length of Sur-Ducker (such as Figure 2The sensitivity and specificity of RNA detection were investigated in vitro using the best signal-to-noise ratio. First, Ducker (in vitro transcribed RNA) was used to detect Survivin mRNA targets at different concentration gradients. The results showed that as the target concentration increased, the fluorescence gradually increased (as shown in Figure 1). Figure 2 There is a good linear relationship between the fluorescence signal intensity and the logarithmic value of the target concentration (as shown in point b) and Figure 2 (c). Based on LOD = 3Std / σ (Std refers to the standard deviation of the control group without target RNA, and σ represents the slope of the linear regression equation), the detection limit of Ducker for Survivin mRNA was calculated to be 0.15 pM, indicating that Ducker has good sensitivity for in vitro RNA detection. At the same time, the visual detection ability of Ducker for targets was evaluated in vitro. The results showed that with the increase of Survivin mRNA target concentration, the fluorescence gradually increased, indicating that Ducker is suitable for visual detection of RNA (e.g. Figure 2 d).

[0067] To evaluate the specificity of Ducker for RNA detection in vitro, cell lysate was used for analysis, Ducker was incubated with total RNA from NIH-3T3 cells (overexpressing and not expressing Survivin mRNA) and fluorescence was detected, as shown in Figure 2 . Figure 2 The e showed that the cell group that did not express Survivin was basically the same as the negative control group, while in the cell group that overexpressed Survivin mRNA (Sur-OE), Ducker showed a gradually and significantly enhanced fluorescence signal as the quality of total RNA increased, indicating that Ducker was highly specific to the target RNA in vitro.

[0068] The sequence information:

[0069] Survivin mRNA (as shown in SEQ ID NO: 1):

[0070]

[0071]

[0072]

[0073] (The bold part represents the site where the RNA sensing module binds to the target, the same applies below)

[0074] Ducker of Survivin mRNA (as shown in SEQ ID No: 2):

[0075]

[0076] (The underlined bold part represents the right lock, the bold italic part represents the RNA sensing module, the underlined part represents the left lock, the bold part represents the partial complementary sequence of the sensing module, and the lowercase part represents the fluorescent RNA aptamer).

[0077] Example 3 Evaluation of the ability of the dual-locked RNA biosensor to detect RNA in complex biological samples

[0078] In order to evaluate the applicability of Ducker in the detection of complex biological samples, NIH-3T3 cell lysate (without Survivin mRNA) was selected as the test substrate, and different concentrations of Survivin mRNA were added to it and detected and analyzed using Ducker. Figure 3 As shown in the a detection flow chart, exogenous Survivin mRNA was added to the NIH3T3 cell lysate and detected by Ducker. The results showed that the fluorescence intensity was positively correlated with the Survivin mRNA concentration (as shown in Figure 3 b and Figure 3 c). Furthermore, the fluorescence signals of target RNA at different concentrations were substituted into the resulting linear regression equation to calculate the corresponding concentrations measured by Ducker. The calculated spike recovery rates ranged from 86% to 113%, demonstrating that Ducker can quantitatively detect target RNA concentrations in cell lysates without interference from other intracellular substances.

[0079] Example 4 Evaluation of the imaging capability of circular double-locked RNA biosensors for native mRNA in living cells

[0080] In order to realize Ducker imaging of natural RNA in living cells, the stability problem of the double-locked RNA biosensor needs to be solved first. Therefore, the present invention introduces the "Tornado" system, such as Figure 4 As shown in a, the double-locked RNA biosensor was connected to the F30 RNA scaffold and the Twister ribozyme to construct a circular double-locked RNA biosensor (circDucker) (as shown in Figure 4 As shown in a), a plasmid expressing circDucker was constructed and transfected into cells to evaluate the ability of circDucker to image natural mRNA in living cells. A circular single-lock RNA biosensor circSicker and a mutant circDucker (Mut-circDucker) were set as negative controls. The results showed that the dual-lock fluorescence was significantly better than the single-lock design (as shown in a). Figure 4(b), and after treatment with Docetaxel (increases Survivin mRNA) and YM155 (downregulates Survivin mRNA), the changes in fluorescence intensity were positively correlated with the effects of the drugs.

[0081] To further verify the accuracy of the results, molecular beacons (MBs) were designed as Yangshen verification, such as Figure 4 As shown in Figure c, in the absence of a complementary RNA target, the fluorescence signal of one end of the labeled fluorescent group will be quenched by the quenching group at the other end. When the target RNA is present, the MB hybridizes with the complementary RNA target, resulting in the separation of the fluorescent group and the quenching group, and the restoration of fluorescence. Figure 4 As shown in Figure c, the red fluorescence signal of Cy5-labeled MB is highly colocalized with the green fluorescence signal of Ducker, indicating that circDucker has good accuracy in imaging native RNA in living cells.

[0082] The sequence information:

[0083] circDucker of Survivin mRNA (as shown in SEQ ID No: 3):

[0084]

[0085] (The bold and underlined parts represent Twister's tornado system, the bold and underlined parts are the F30 bracket, and the lowercase part represents its Ducker).

[0086] circSicker of Survivin mRNA (as shown in SEQ ID No: 4):

[0087]

[0088] (The bold part represents the Tornado system and F30 bracket, the underlined part represents the single lock, the italic part represents the RNA sensing module, and the lowercase part represents the fluorescent RNA aptamer).

[0089] Mut-circDucker of Survivin mRNA (as shown in SEQ ID No: 5):

[0090]

[0091] (The bold part represents its Ducker, the lowercase part represents the Tornado system and F30 scaffold, and the underlined part represents the mutated RNA sensing module).

[0092] Survivin-MB (as shown in SEQ ID No: 6):

[0093] 5'-Cy5-CGCACCGAGAAAGGGCTGCCAGGTGCG-BHQ2-3'.

[0094] Example 5: Comparison with iPepper Technology to Evaluate the Performance Advantages of Circular Double-Locked RNA Biosensors

[0095] In order to evaluate the performance advantages of the circular double-locked RNA biosensor, the present invention was compared with the closest existing technology iPepper, and β-actin mRNA was selected as the target. The 8×iPepper reported in the literature was used (8 binding sites need to be found and designed separately. For this target, the binding site sequence is the complementary sequence of the target complementary sequence in SEQ ID No: 9) (such as Figure 5 As shown in a), after optimizing the double lock length (as shown in Figure 5 b), the corresponding circDucker was designed (finding a binding site design). The results showed that cells transfected with 8×iPepper or circDucker showed bright fluorescence. However, the background fluorescence intensity of 8×iPepper was significantly higher than that of circDucker (as shown in Figure 5 c), and the flow cytometry results were highly consistent with the laser confocal microscopy results (as shown in Figure 5 This shows that compared with the design difficulty and background control difficulty of 8×iPepper, circDucker has the advantages of high fluorescence intensity and low background.

[0096] The sequence information:

[0097] β-actin mRNA (as shown in SEQ ID No: 7):

[0098]

[0099]

[0100] (The bold part is Ducker's target)

[0101] circDucker of β-actin mRNA (as shown in SEQ ID No: 8):

[0102]

[0103] (The underlined portion represents the Tornado system and F30 scaffold, the bold portion represents the double lock, the italicized portion represents the RNA sensing module, the bold underlined portion represents the partial complementary sequence of the sensing module, and the lowercase portion represents the fluorescent RNA aptamer.) 8×iPepper for β-actin mRNA (as shown in SEQ ID No: 9):

[0104]

[0105] (The underlined part is iPepper, and the bold part is the target complementary sequence).

[0106] Example 6 Evaluation of the imaging capability of circular double-locked RNA biosensors for native miRNA in living cells and comparison with NP-miLS technology

[0107] To evaluate the substrate adaptability of the circular double-locked RNA biosensor, the present invention verified the imaging ability of circDucker for short RNA fragments, selected miR-122 as the target, and compared it with the closest prior art NP-miLS system reported in 2023 (e.g. Figure 6 After optimizing the double lock length, the corresponding circDucker (as shown in a) is designed. Figure 6 The results showed that cells transfected with circDucker showed bright fluorescence, and the fluorescence intensity was significantly higher than that of cells transfected with NP-miLS (as shown in b). Figure 6 Both of them have low fluorescence background, and the flow cytometry results are highly consistent with the laser confocal microscopy results (as shown in c). Figure 6 This indicates that circDucker is not only suitable for imaging long-sequence RNA, but also for imaging short-sequence RNA, and has the advantages of high fluorescence intensity and low background.

[0108] The sequence information:

[0109] miR-122 (as shown in SEQ ID No: 10): 5'-UGGAGUGUGACAAUGGUGUUUG-3';

[0110] circDucker of miR-122 (as shown in SEQ ID No: 11)

[0111]

[0112] (The underlined portion represents the Tornado system and F30 scaffold, the bold portion represents the double lock, the italic portion represents the RNA sensing module, the underlined and bold portion represents the partial complementary sequence of the sensing module, and the lowercase portion represents the fluorescent RNA aptamer.) NP-miLS (as shown in SEQ ID No: 12):

[0113]

[0114] (The bold part represents the target complementary sequence).

[0115] Example 7 Evaluation of the Ability of Fluorescence Ratiometric RNA Sensors to Accurately Quantify Native mRNA Abundance in Living Cells and Their Application in Drug Evaluation and Screening

[0116] Accurately measuring the intracellular RNA concentration is the key to exploring RNA. To avoid interference from factors unrelated to the analyte (such as fluorescence intensity, instrument parameters, etc.), the present invention connected an internal reference (the internal reference fluorescence is unrelated to the target) on the basis of circDucker and designed a fluorescence ratio RNA sensor (circDucker-dBroccoli) (such as Figure 7 (as shown in a), where dBroccoli performs a calibration function to achieve accurate quantification of RNA abundance in living cells. Subsequently, circDucker-dBroccol was used to image five cell types (HeLa, A549, MCF7, HepG2, and L-02) with different Survivin mRNA expression levels. The results showed that dBroccol (green) in the cells showed the same fluorescence intensity, while the fluorescence of circDucker (red) varied significantly in different cells (as shown in Figure 7 (as shown in b), the order of fluorescence intensity is HeLa>A549>MCF7>HepG2>L-02. To verify the accuracy of this result, qPCR was used to quantify Survivin mRNA in cells, showing the same trend. By comparing the qPCR quantitative results with the ratio imaging fluorescence quantitative results, the two were highly correlated (as shown in Figure 7 (as shown in c), indicating the accuracy of circDucker-dBroccoli's quantitative imaging results of Survivin mRNA abundance in different cells.

[0117] In order to expand the application of fluorescence ratio imaging technology based on circDucker-dBroccoli, the present invention uses it in drug efficacy evaluation and drug screening. Subsequently, cells were treated with different concentrations of YM155 and fluorescence ratio imaging was performed. The results showed that with the increase of the incubation concentration of the YM155 inhibitor, the fluorescence intensity of circDucker (red) showed a gradually weakening trend, while dBroccoli showed a stable green fluorescence signal, and the overlapping image of the fluorescence ratio (Red / Green) of circDucker and dBroccoli underwent obvious color shift. Based on the change of the fluorescence ratio signal of circDucker-dBroccoli, the dose-response curve of the YM155 inhibitor was drawn and the IC50 (8.526nM) was calculated. This is highly consistent with the inhibitory efficiency of YM155 calculated by RT-qPCR (IC50 is 8.832nM) (such as Figure 7 d and Figure 7 This further verified the accurate evaluation of drug efficacy by circDucker-dBroccoli and its application in drug screening.

[0118] The sequence information:

[0119] circDucker-dBroccoli (as shown in SEQ ID No: 13):

[0120]

[0121] (The underlined parts are the Tornado system and the F30 bracket, the italicized parts are the dBroccoli sequence, and the bold parts are Ducker).

[0122] Example 8 Evaluation of the Real-Time Monitoring Capability of Circular Double-Locked RNA Biosensors for Concentration Fluctuations of Natural mRNA in Living Cells and Their Application in Disease Diagnosis and Treatment

[0123] To evaluate whether circDucker can monitor the concentration fluctuation of natural mRNA in living cells in real time, the present invention selected the inflammation-related mouse IL-6 mRNA as the target, and optimized the double lock length (such as Figure 8 The corresponding circDucker was designed using RAW264.7 mouse mononuclear macrophage leukemia cells, one of the commonly used inflammatory cell models. RAW264.7 cells do not express inflammatory factors in the absence of stimulation. However, when stimulated by lipopolysaccharide (LPS), they produce IL-6 mRNA. The results of fluorescence imaging and flow cytometry were consistent with the results of RT-qPCR for IL-6 mRNA (see Figure 2). Figure 8 b and Figure 8 d), indicating that circDucker was successfully used for imaging of IL-6 mRNA.

[0124] In order to better visualize and understand the release of inflammatory factors when the body produces an inflammatory response and to grasp the dynamic expression process of IL-6 mRNA in RAW264.7 cells under LPS stimulation, the present invention designed a series of time points, covering the period before and after 3 hours of LPS stimulation of cells, and imaged IL-6 mRNA using circDucker under a fluorescence confocal microscope. The results showed that with the extension of LPS stimulation time, the fluorescence signal in the cells gradually increased, and the dynamic change process of fluorescence of RAW264.7 cells from dark to bright was recorded (such as Figure 8 e and Figure 8 This shows that circDucker can monitor the concentration fluctuation of natural mRNA in living cells in real time and has application prospects in disease diagnosis and treatment.

[0125] The sequence information:

[0126] IL-6 mRNA (as shown in SEQ ID No: 14):

[0127]

[0128] circDucker of IL-6 mRNA (as shown in SEQ ID No: 15):

[0129]

[0130] (The underlined part represents the Tornado system and F30 bracket, the bold part represents the double lock, the italic part represents the RNA sensing module, the bold and underlined parts represent the partial complementary sequence of the sensing module, and the lowercase part is the fluorescent RNA aptamer).

[0131] Example 9 Evaluation of the Spatiotemporal Monitoring Capability of Circular Double-Locked RNA Biosensors for the Movement of Natural lncRNAs in Living Cells and Their Application in RNA Function Studies

[0132] To evaluate the spatiotemporal monitoring of the movement of natural RNA in living cells by circular double-locked RNA biosensors, the present invention selected mitochondrial-encoded lncRNA Cyt b (mtlncCyt b) that is localized in the cell nucleus as a target in order to observe the nucleocytoplasmic shuttling process of mtlncCyt b. Figure 9First, circDucker was used to verify the nuclear localization ability of mitochondrial lncRNA in living cells, as shown in a. Figure 9 As shown in (b), cells were imaged layer by layer using a laser scanning confocal microscope. Three-dimensional reconstruction techniques were used to obtain more precise information about the distribution of intracellular fluorescence signals. The 3D reconstructions of cells from different angles clearly displayed the structure of the nucleus and intuitively highlighted the fluorescent signals embedded in the nucleus, demonstrating the ability of circDucker to effectively detect mitochondrial RNA nuclear localization in living cells.

[0133] Secondly, in order to verify the accuracy of circDucker, the present invention used single-molecule fluorescence in situ hybridization (smFISH) as a positive reference. The results showed that circDucker (green) and smFISH (red) were highly co-localized (e.g. Figure 9 c), indicating that circDucker has good accuracy in imaging native RNA in living cells.

[0134] In order to capture the nuclear-cytoplasmic transport process of mtlncCyt b, a series of multiple time periods were selected for real-time imaging of cells. The results showed that the dynamic process of mtlncCyt b transporting from the cytoplasm to the nucleus was tracked (e.g. Figure 9 d and Figure 9 e, the white arrow indicates the direction of transport of mtlncCyt b from the cytoplasm to the nucleus), providing direct evidence for the nuclear translocation of mtlncCyt b. Importantly, it was also observed that mitochondrial mtlncCyt b can not only enter the nucleus, but also be released from the nucleus and shuttle back to the cytoplasm (as shown in Figure 5). Figure 9 d and Figure 9 As shown in Figure e, the pink arrow clearly indicates the transport of mtlncCyt b from the nucleus to the cytoplasm), indicating that circDucker successfully monitored the dynamic process of mtlncCyt b's bidirectional shuttling between the nucleus and the cytoplasm, which has not been reported by traditional technologies and provides a new tool for discovering unknown functions of RNA.

[0135] mtlncCyt b (shown in SEQ ID No: 16):

[0136]

[0137]

[0138] circDucker of mtlncCyt b (shown in SEQ ID No: 17):

[0139]

[0140] (The underlined part represents the Tornado system and F30 bracket, the bold part represents the double lock, the italic part represents the RNA sensing module, the underlined and bold part represents the partial complementary sequence of the sensing module, and the lowercase part is the fluorescent RNA aptamer).

[0141] mtlncCyt b smFISH probe (Cy3 modified):

[0142] 5'-GCAGACCTCCTCATTCTAAC-3'; (as shown in SEQ ID No: 18)

[0143] 5'-CTCCTATTCTTGCACGAAAC-3'; (as shown in SEQ ID No: 19)

[0144] 5'-CATACATTGGGACAGACCTA-3'. (as shown in SEQ ID No: 20)

[0145] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. RNA biosensor, characterized in that From the 5' end to the 3' end, it includes: a right lock module, an RNA sensing module, a first fluorescent aptamer module, a left lock module, a complementary module and a second fluorescent aptamer module; The RNA sensing module is partially complementary to the complementary module; The right lock module and the second fluorescent aptamer module are partially complementary; The left lock module and the first fluorescent aptamer module are partially complementary.

2. The RNA biosensor according to claim 1, characterized in that The base lengths of the left lock module and the right lock module are no less than 2nt.

3. A circular double-locked RNA biosensor, characterized in that: include: The RNA biosensor and circularization element as claimed in claim 1 or 2.

4. Use of the RNA biosensor according to claim 1 or 2 and / or the circular double-locked RNA biosensor according to claim 3 in any of the following items; (I), RNA molecule detection; and / or (II), screening of RNA drugs; and / or (III) Evaluation of the efficacy of RNA drugs; and / or (IV) preparing products for RNA drug screening and / or RNA drug efficacy evaluation; and / or (V), RNA imaging; and / or (VI), preparing products for diagnosis and / or treatment of RNA-related diseases; and / or (VII) Preparation of products for studying RNA function.

5. A method for detecting RNA molecules, characterized in that: The RNA biosensor as claimed in claim 1 or 2 and / or the circular double-locked RNA biosensor as claimed in claim 3 is mixed with a detection target and a fluorescent dye for detection.

6. The detection method according to claim 5, characterized in that When the detection target is complementarily bound to the RNA sensing module, the first fluorescent aptamer module and the second fluorescent aptamer module are bound to the fluorescent dye to generate fluorescence.

7. The detection method according to claim 6, characterized in that The base length of the detection target is not less than 15 nt.

8. A product, characterized in that include: The RNA biosensor according to claim 1 or 2 and / or the circular double-locked RNA biosensor according to claim 3.

Images