RNA-protein heterodimerization probe and application thereof

Abstract

The invention relates to the technical field of molecular biology, in particular to an RNA-protein heterodimerization probe and application thereof. The RNA-protein heterodimerization probe disclosed by the invention is formed by heterodimerization of an RNA monomer and a protein monomer. Wherein the RNA monomer comprises a three-way stem-loop structure scaffold, and the three-way stem-loop structure scaffold comprises a loop structure, and a first stem structure, a second stem structure and a third stem structure which are respectively connected with the loop structure; the first stem structure is connected with Boxb; the second stem structure is connected with a fluorescent RNA module; the third stem structure is connected with a self-cyclization structure; the protein monomer is EGFP (Enhanced Green Fluorescent Protein)-lambda N, and the protein monomer EGFP-lambda N is in affinity connection with Boxb. The RNA-protein heterodimerization probe provided by the invention can be at least applied to detection of SAM and miRNA, and has the advantages of good specificity, high stability and good sensitivity.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology, and particularly relates to an RNA-protein heterodimerization probe and its application. Background Art

[0002] With the rapid development of precision medicine, biomarkers play an increasingly important role in the early diagnosis of diseases, drug development, and efficacy evaluation. Developing in-situ real-time analysis tools for the quantity and dynamics of biomarkers in living cells is conducive to clarifying disease mechanisms and revealing drug targets. However, existing analysis and detection methods mainly rely on traditional endpoint detection methods, which have problems such as complex operation, poor specificity, and insufficient sensitivity, limiting their application in real-time monitoring and high-throughput screening. Gene-encoded fluorescent probes, especially fluorescent "turn-on" RNA aptamer (FLAP) probes, have extremely high flexibility and target diversity, can be continuously expressed in living cells and monitor target molecules in real time, and are helpful for molecular dynamics analysis in a complex cellular environment. However, when RNA probes are used for intracellular analysis, there are problems such as large signal fluctuations, poor probe stability, and lack of specific organelle targeting parts. Therefore, it is necessary to further design the probes to address these problems. Summary of the Invention

[0003] In view of the deficiencies of the above-mentioned prior art, the purpose of the present invention is to provide an RNA-protein heterodimerization probe and its application, aiming to improve the stability and specificity of the probe.

[0004] To achieve the above purpose, the present invention adopts the following technical solutions:

[0005] The present invention provides an RNA-protein heterodimerization probe, comprising: an RNA monomer, the RNA monomer comprising a three-way stem-loop structure scaffold, the three-way stem-loop structure scaffold comprising a loop structure and a first stem structure, a second stem structure, and a third stem structure respectively connected to the loop structure; the first stem structure is connected with Boxb; the second stem structure is connected with a fluorescent RNA module; the third stem structure is connected with a self-cyclization structure; a protein monomer EGFP-λN, the protein monomer EGFP-λN being affinity-connected with Boxb.

[0006] The RNA-protein heterodimerization probe as described above, wherein the three-way stem-loop structure scaffold is an F30 three-way stem-loop structure; the self-cyclization structure is a Tornado structure.

[0007] The RNA-protein heterodimerization probe, wherein the fluorescent RNA module is a Pepper sequence with multiple base sequences replaced by a nuclear switch sequence or a miRNA response sequence; the nuclear switch sequence is used to form a nuclear switch structure; the nuclear switch structure or miRNA response sequence is used to restore the fluorescent molecule binding site in the Pepper sequence after binding to the detection substrate.

[0008] The present invention also provides the use of the RNA-protein heterodimerization probe described above in detecting SAM or miRNA.

[0009] The present invention also provides the use of the RNA-protein heterodimerization probe described above in detecting SAM in vitro, in cells, or in subcellular compartments, wherein the Pepper sequence is as shown in SEQ ID No. 1;

[0010] SEQ ID No. 1:

[0011] CCAAUCGUGGCGUGUCGGCCUGCUUCGGCAGGCACUGGCGCCG

[0012] The fluorescent RNA module is a Pepper sequence with the 18th to 33rd bases replaced by a nuclear switch sequence.

[0013] The use of the RNA-protein heterodimerization probe in detecting SAM in vitro, in cells or in subcellular space, wherein the nucleic acid sequence of the fluorescent RNA module is as shown in SEQ ID No. 2 or SEQ ID No. 3;

[0014] SEQ ID No. 2:

[0015] CCAAUCGUGGCGUGUCGACGAAAGGAUGGCGGAAACGCCAGAUGCCUUGUAACCGAAAGGGUACUGGCGCCG

[0016] SEQ ID No.3:

[0017] CCAAUCGUGGCGUGUCGACCGAAAGGAUGGCGGAAACGCCAGAUGCCUUGUAACCGAAAGGGGGUACUGGCGCCG

[0018] Use of the RNA-protein heterodimerization probe in detecting SAM in vitro, in cells, or in subcellular compartments, wherein the nucleic acid sequence of the RNA monomer of the RNA-protein heterodimerization probe is as shown in SEQ ID No. 4 or SEQ ID No. 5;

[0019] SEQ ID No.4:

[0020] UUGCCAUGUGUAUGUGGGGAAUUCCCAAUCGUGGCGUGUCGACGAAAGGAUGGCGGAAACGCCAGAUGCCUUGUAACCGAAAGGGUACUGGCGCCGGAAUUUCCCACAUACUCUGAUGAUCCGCUAGCGGCGCCCUGAAAAAGGGCGCCGCUAGUGGAUCAUUCAUGGCAA

[0021] SEQ ID No.5:

[0022] UUGCCAUGUGUAUGUGGGGAAUUCCCAAUCGUGGCGUGUCGACCGAAAGGAUGGCGGAAACGCCAGAUGCCUUGUAACCGAAAGGGGUACUGGCGCCGGAAUUUCCCACAUACUCUGAUGAUCCGCUAGCGGCGCCCUGAAAAAGGGCGCCGCUAGUGGAUCAUUCAUGGCAA

[0023] The present invention also provides the use of the RNA-protein heterodimerization probe as described above for detecting miRNA in vitro or in cells, wherein the Pepper sequence is as shown in SEQ ID No.1; the fluorescent RNA module is obtained by replacing the bases at positions 19-32 in the Pepper sequence with a miRNA response sequence.

[0024] The use of the RNA-protein heterodimerization probe for detecting miRNA in vitro or in cells, wherein when the miRNA is miRNA-21, the nucleic acid sequence of the fluorescent RNA module is as shown in SEQ ID No.6;

[0025] SEQ ID No.6:

[0026] CCAAUCGUGGCGUGUCGGAUAAGUCAACAUCAGUCUGAUAAGCUAUGUUCGCAUAGCUUAUCACUGGCGCCG

[0027] When the miRNA is miRNA-122, the nucleic acid sequence of the fluorescent RNA module is as shown in SEQ ID No.7

[0028] SEQ ID No.7:

[0029] CCAAUCGUGGCGUGUCGGUCACACAAACACCAUUGUCACACUCCAGGAAACUGGAGUGUGACACUGGCCCG

[0030] The use of the RNA-protein heterodimerization probe in detecting miRNA in vitro or in cells, wherein, when the miRNA is miRNA-21, the nucleic acid sequence of the RNA monomer of the RNA-protein heterodimerization probe is shown in SEQ ID No. 8;

[0031] SEQ ID No.8:

[0032] UUGCCAUGUGUAUGUGGGGAAUUCCCAAUCGUGGCGUGUCGGAUAAGUCAACAUCAGUCUGAUAAGCUAUGUUCGCAUAGCUUAUCACUGGCGCCGGAAUUUCCCACAUACUCUGAUGAUCCGCUAGCGGCGCCCUGAAAAAGGGCGCCGCUAGUGGAUCAUUCAUGGCAA

[0033] When the miRNA is miRNA-122, the nucleic acid sequence of the RNA monomer of the RNA-protein heterodimerization probe is shown in SEQ ID No. 9;

[0034] SEQ ID No.9:

[0035] UUGCCAUGUGUAUGUGGGGAAUUCCCAAUCGUGGCGUGUCGGUCACACAAACACCAUUGUCACACUCCAGGAAACUGGAGUGUGACACUGGCCCGGAAUUUCCCACAUACUCUGAUGAUCCGCUAGCGGCGCCCUGAAAAAGGGCGCCGCUAGUGGAUCAUUCAUGGCAA

[0036] Beneficial effects: The present invention provides an RNA-protein heterodimerization probe and its applications. By utilizing the specific affinity of λN-Boxb, the RNA-protein heterodimerization probe drives the self-assembly of EGFP-λN protein monomers and RNA monomers. The heterodimerized probe can effectively reduce the degradation of RNA monomers by RNase, improve the stability of the RNA structure, and thus enhance its applicability in complex biological environments. In addition, by binding different fluorescent RNA modules, the RNA-protein heterodimerization probe of the present invention can be used for the detection of SAM and the ratio fluorescence detection of multiple miRNAs respectively. The results show that the RNA-protein heterodimerization probe of the present invention has high stability, modular characteristics, and subcellular targeting ability, and can be extended to the detection of various biomarkers, providing new tools and perspectives for the efficient imaging of biomarkers, intracellular metabolism research, and drug efficacy evaluation. Description of the Drawings

[0037] Figure 1 It is a schematic structural diagram of the RNA-protein heterodimerization probe. Figure 1 In it, 1 is a loop structure, 2 is the first stem structure, 3 is the second stem structure, 4 is the third stem structure, 5 is Boxb, 6 is a fluorescent RNA module, 7 is a self-cyclized structure, and 8 is a protein monomer.

[0038] Figure 2 It shows the change of fluorescence of RNA monomers and the RNA-protein heterodimerization probe over time.

[0039] Figure 3 It is a schematic diagram of the detection principle of the RNA monomer for detecting SAM.

[0040] Figure 4 It is the comparison of fluorescence intensities of different Pepper-SAM detection modules.

[0041] Figure 5 It is the sequence and secondary structure of Pepper-SAM detection module 1.

[0042] Figure 6 It is the specific detection result of Pepper-SAM detection module 1.

[0043] Figure 7 It is the in vitro quantitative analysis result of the heterodimerization probe for SAM.

[0044] Figure 8 It is the magnesium ion dependence result of the SAM detection module based on Pepper.

[0045] Figure 9 It is a schematic diagram of plasmid construction and the fluorescence specificity result of the heterodimerization probe for SAM imaging.

[0046] Figure 10 For the results of monitoring the dynamic changes of SAM in living cells, the SAM synthesis pathway, and the mechanism of action of inhibitors using heterodimerization probes.

[0047] Figure 11 For the results and mechanism of monitoring the dynamic changes of SAM at the subcellular level using heterodimerization probes.

[0048] Figure 12 Schematic diagram of the detection principle and detection results of RNA monomers for detecting miRNA.

[0049] Figure 13 For the in vitro quantitative detection results of miRNA-21 using heterodimerization probes.

[0050] Figure 14 For the in vitro quantitative detection results of miRNA-122 using heterodimerization probes.

[0051] Figure 15 Schematic diagram of the imaging analysis of miRNA-21 in living cells and plasmid construction using heterodimerization probes. Specific implementation manners

[0052] The present invention provides an RNA-protein heterodimerization probe and its application. To make the purpose, technical solution and effects of the present invention clearer and more definite, the following further describes the present invention in detail with reference to the accompanying drawings and by way of examples. It should be understood that the specific examples described herein are only used to explain the present invention and are not used to limit the present invention.

[0053] Please refer to Figure 1 , the present invention provides a brand-new RNA-protein heterodimerization probe, including:

[0054] An RNA monomer, the RNA monomer includes a three-way stem-loop structure scaffold, the three-way stem-loop structure scaffold includes a loop structure and a first stem structure, a second stem structure, and a third stem structure respectively connected to the loop structure; the first stem structure is connected with Boxb; the second stem structure is connected with a fluorescent RNA module; the third stem structure is connected with a self-cyclization structure;

[0055] A protein monomer EGFP-λN, the protein monomer EGFP-λN is affinity-connected with Boxb.

[0056] Preferably, the F30 three-way stem-loop structure can be used as the backbone to design the RNA monomer to promote the correct folding of RNA and increase stability.

[0057] Preferably, the self-cyclization structure is a Tornado structure.

[0058] Preferably, the fluorescent RNA module is obtained by replacing multiple base sequences in the Pepper sequence with riboswitch sequences or miRNA response sequences. The riboswitch sequences are used to form riboswitch structures; after the riboswitch structures or miRNA response sequences bind to the detection substrates, the fluorescent molecule binding sites in the Pepper sequence are restored.

[0059] Specifically, the riboswitch sequence is an RNA sequence for binding to SAM (SAM riboswitch), and the corresponding fluorescent RNA module can be used for detecting SAM in vitro, in cells or in subcellular compartments at least.

[0060] The miRNA response sequence is a miRNA complementary sequence, and the corresponding fluorescent RNA module can be used for detecting miRNA in vitro or in cells at least.

[0061] The following examples are given to further illustrate the present invention.

[0062] Example 1. Stability analysis of RNA-protein heterodimerization probes

[0063] In this example, the template DNA (SEQ ID No. 10) of the RNA monomer of the RNA-protein heterodimerization probe is as follows:

[0064] 5’-TAATACGACTCACTATAGGTTGCCATGTGTATGTGGGGAATTCCCAATCGTGGCGTGTCG GCCTGCTTCGGCAGGCACTGGCGCCGGAATTTCCCACATACTCTGATGATCCGCTAGCGGCGCC CTGAAAAAGGGCGCCGCTAGTGGATCATTCATGGCAA-3’

[0065] The method for obtaining the RNA monomer is as follows: First, a double-stranded DNA template for in vitro transcription is obtained by polymerase chain reaction (PCR); subsequently, the obtained double-stranded DNA is transcribed using T7 RNA polymerase reaction; after the transcription ends, it is treated with DNase I for 15 minutes, and then the transcription product RNA is extracted by column purification.

[0066] In this example, the amino acid sequence (SEQ ID No.11) of the EGFP-λN protein monomer is as follows: MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKSGGSNARTRRRERRAEKQAQWKAAN

[0067] The method for obtaining the protein monomer EGFP-λN is as follows: Transform the EGFP-λN expression plasmid into E. coli BL21(DE3) cells, and culture them in LB medium containing 50 μg / mL kanamycin at 37 °C with shaking at 220 rpm. When the OD 600 reaches 0.6 - 0.8, add 0.2 mM IPTG to induce protein expression. After inducing for 16 hours at 16 °C, collect the cells, and use a nickel ion rapid purification column to extract and purify the product to obtain the protein monomer EGFP-λN.

[0068] To evaluate the stability of the heterodimerization probe, the effect of ribonuclease A (RNaseA) on the fluorescence of the RNA monomer was measured. The heterodimerization probe (0.5 μM EGFP-λN, 0.5 μM F30-Pepper-Boxb, 5 μM HBC620) was incubated at 37 °C for 1 h, then 10 ng / μL RNaseA was added, and the fluorescence change was recorded every 5 minutes for 20 minutes. To evaluate the degradation difference, the sample without EGFP-λN was used as a control.

[0069] Fluorescence measurements were performed in a buffer solution containing 125 mM KCl, 1 mM MgCl2, and 40 mM HEPES (pH 7.4), and measured using a Synergy H1 multimode microplate reader. The excitation and emission wavelengths of Pepper are 580 nm / 620 nm respectively, and the excitation and emission wavelengths of EGFP are 475 nm / 510 nm respectively.

[0070] Figure 2 Shows the fluorescence changes of the RNA monomer and the RNA-protein heterodimerization probe over time. From Figure 2As can be seen from the results, after the F30-Pepper-Boxb monomer was treated with 10 ng / μL RNase A nuclease, the fluorescence decreased rapidly, and the signal intensity dropped to 29% within 10 minutes, indicating that the naked RNA structure was extremely vulnerable to nuclease degradation. In contrast, when the RNA monomer formed a heterodimeric probe with the protein monomer EGFP-λN, the fluorescence decay rate decreased significantly, and 44% of the initial fluorescence was still retained after 10 minutes. This result shows that the full complexation of the protein monomer and the RNA monomer not only stabilizes the RNA monomer structure, but also inhibits the degradation of the RNA monomer by nucleases through steric hindrance effects. The RNA-protein heterodimeric probe of the present invention has good stability.

[0071] Example 2: Construction and performance evaluation of the SAM detection module in the heterodimeric probe

[0072] To develop a Pepper-based SAM detection module, the SAM riboswitch aptamer was fused with Pepper's Stem-loop1 through transducers of different lengths and thermodynamic stabilities, and named Pepper-SAM detection modules 1-5. Please refer to Figure 3 , the transducer domain is a thermodynamically unstable helical structure. The binding of the target SAM will induce the folding of the riboswitch aptamer and further stabilize the transducer stem structure; the stable transducer stem structure can promote the correct folding of Pepper and enhance its binding to the HBC fluorescent dye, thus activating the fluorescence signal.

[0073] 2.1 Optimization of the transducer in the Pepper-SAM detection module of the heterodimeric probe

[0074] The following table shows the DNA templates of the RNA monomers respectively containing Pepper-SAM detection modules 1-5

[0075]

[0076] Five corresponding RNA monomers were prepared according to the above DNA templates.

[0077] In the presence or absence of 0.1 mM SAM, 0.5 μM RNA monomer was incubated with 5 μM HBC620 at 37 °C for 1 h. The fluorescence emission spectral data were recorded from 600 nm to 700 nm at 5 nm increments using a Synergy H1 multimode microplate reader to evaluate the response of the Pepper-SAM detection module in the RNA monomer.

[0078] Figure 4A transducer comparison and fluorescence detection results for five SAM detection modules are shown. Comparison of the different transducers reveals that the Pepper-SAM detection modules designed with transducers 1 and 2 exhibit excellent sensitivity and signal-to-noise ratio after incubation with SAM. The Pepper-SAM detection module designed with transducer 1 exhibits a 34.1-fold fluorescence enhancement at 620 nm.

[0079] Figure 5 is the sequence and secondary structure of Pepper-SAM detection module 1.

[0080] 2.2 Selectivity Experiment of SAM Detection Module in Heterodimerization Probe

[0081] To evaluate the selectivity of Pepper-SAM detection module 1, three SAM analogs were used: S-(5′-Adenosyl)-L-homocysteine (SAH), methionine, and adenosine. Under identical conditions, 0.5 μM F30-PepperSAM detection module 1-Boxb, 5 μM HBC620, and 0.1 mM SAM analogs were incubated at 37°C for 1 hour. The fluorescence intensity of each sample was measured. Selectivity was assessed by comparing the response of the SAM detection module to each analyte.

[0082] like Figure 6 As shown in the figure, Pepper-SAM detection module 1 exhibits high selectivity for SAM. This result demonstrates that the Pepper-SAM detection module can accurately recognize and specifically bind to SAM without interference from structurally similar metabolites or precursors, and ensures the reliability of its subsequent highly selective detection of SAM in complex cellular environments.

[0083] Example 3: Heterodimerization probe for in vitro quantitative analysis of SAM

[0084] To determine the dynamic response of the heterodimerized probe to SAM, the probe (0.5 μM EGFP-λN, 0.5 μM F30-Pepper SAM detection module-Boxb, 5 μM HBC620) was incubated with various concentrations of SAM (1-100 μM) at 37°C for 1 hour, and the fluorescence emission spectrum of each sample was measured. The ratio of the fluorescence intensity at 620 nm to 505 nm was plotted against the SAM concentration, and the data were fitted to a nonlinear fitting model using GraphPad Prism 9 to calculate the apparent dissociation constant (Kd).

[0085] Compared with traditional single-fluorescence signal probes, the heterodimerization probe of the present invention uses EGFP-λN as an internal reference, enabling the detection signal to be presented in a ratio form, thereby effectively reducing the influence of background noise and experimental condition changes on the detection results and improving the reliability of the data. The results show that the green fluorescence signal of EGFP-λN remains constant at different SAM concentrations ( Figure 7 a), while the red fluorescence signal of the F30-Pepper SAM detection module 1-Boxb increases correspondingly with the increase of the SAM concentration ( Figure 7 b). Further linear regression analysis shows that in the concentration range of 10 - 100 μM, the logarithm of the SAM concentration has a good linear relationship with the ratio fluorescence signal, indicating that the probe has good quantitative detection ability (R 2 = 0.9293, Figure 7 c). In addition, the dissociation constant (Kd) between the probe and SAM is 31.35 μM, which can be used to detect the dynamic changes of SAM concentration (10 - 100 μM) in various mammalian cell lines ( Figure 7 d). These results together indicate that the RNA-protein heterodimerization probe can be used as a ratio fluorescence probe to quantitatively detect SAM concentration in vitro, and also lay a foundation for further research on intracellular SAM.

[0086] Example 4: Fluorescence specificity analysis of the heterodimerization probe for SAM imaging in living cells

[0087] To express the Pepper-SAM detection module in mammalian cells, template DNAs encoding Tornado-F30-Pepper SAM detection module1-Boxb and Tornado-F30-Pepper SAM detection module2-Boxb were synthesized and cloned into the pAV-U6+27 plasmid using SalI and XbaI restriction enzyme sites.

[0088] As Figure 8 shown in a, when the Pepper-SAM detection module 1 was incubated with HBC 620 and then imaged by confocal microscopy, the fluorescence signal was weak, while the Pepper-SAM detection module 2 had a stronger fluorescence signal.

[0089] Furthermore, the Mg 2+ dependency of the original Pepper, Pepper-SAM detection module 1, and Pepper-SAM detection module 2 was compared ( Figure 8 b). The results show that the original Pepper has a 2+The EC50 of Pepper-SAM Detection Module 2 is 0.5 mM, while that of Pepper-SAM Detection Module 1 is 1.4 mM. In contrast, the EC50 of Pepper-SAM Detection Module 2 remains at 0.5 mM, similar to the original Pepper, making it more suitable for free Mg in mammalian cells. 2+ Concentration range (0.2-1.5mM). In addition, Pepper-SAM detection module 2 can also highly activate SAM and show good fluorescence response characteristics.

[0090] The protein monomer EGFP-λN and the RNA monomer F30-Pepper SAM detection module2-Boxb were assembled into a heterodimerization probe for in vitro quantitative detection of SAM. The experimental results showed that the logarithm of SAM concentration and the ratio fluorescence intensity (F620 / F505) showed a good linear relationship in the concentration range of 10-100 μM (R 2 =0.9620, Figure 8 c), further verifying its quantitative detection capability. Therefore, in subsequent cell experiments, we selected Pepper-SAM detection module 2 as the fluorescent RNA sensing unit to image SAM.

[0091] Based on the above, a plasmid with dual promoters was further designed and constructed to regulate the protein and RNA expression levels in a consistent manner, ensuring that the two monomers of the probe self-assemble in the same cell, thereby achieving ratiometric fluorescence imaging of SAM in living cells. The pRNAT-U6.1_Neo plasmid was used for construction. The two promoters U6 and miniCMV of the plasmid were used to express F30-Pepper SAM detection module2-Boxb and EGFP-λN, respectively. Figure 9 a). Next, to evaluate the imaging capability of the heterodimeric probe in living cells, HEK293T cells were transfected with the dual-promoter plasmid and incubated with HBC620 fluorescent dye (0.5 μM) for imaging. The experimental results showed that green and red fluorescence could be successfully detected, and the ratio of red to green fluorescence was calculated to obtain the ratio signal ( Figure 9 b).

[0092] To further verify the specificity of the fluorescence reaction, cells were treated with SAHA, a histone acetylation inhibitor that can upregulate intracellular SAM levels; in addition, cells were treated with cycloleucine to inhibit the biosynthesis of SAM.

[0093] The results showed that cells treated with agonists showed stronger red fluorescence than untreated cells, while cells treated with inhibitors showed weak red fluorescence, while green fluorescence remained almost unchanged ( Figure 9 b andFigure 9 c). In addition, the ratio image also changes color according to the SAM concentration. The experimental results show that the heterodimerization probe we constructed can specifically image endogenous SAM in living cells.

[0094] Example 5: Dynamic Monitoring of SAM in Living Cells Using a Heterodimerization Probe

[0095] 5.1 Plasmid Construction

[0096] To achieve the co-expression of the Pepper-SAM detection module and the λN-fused fluorescent protein, a miniCMV promoter was constructed for EGFP-λN expression. The specific steps are as follows: Synthesize the template DNA encoding miniCMV and clone it into the pRNAT-U6.1_Neo vector through the HindIII and AgeI sites to form the pRNAT-miniCMV plasmid. Then, use PCR to amplify the template DNA encoding EGFP-λN and insert it downstream of miniCMV to form the pRNAT-miniCMV-EGFP-λN plasmid. Subsequently, clone the Tornado-F30-Pepper SAM detection module2-Boxb sequence under the U6 promoter to obtain the pRNAT-U6-Pepper SAM detection module2-miniCMV-EGFP-λN plasmid.

[0097] 5.2 Transformation and Extraction of Plasmids

[0098] Competent Escherichia coli DH5α was used for transformation. After culturing, the plasmid was extracted using a plasmid extraction kit.

[0099] 5.3 Cell Culture and Transfection

[0100] HEK293T cells were cultured and transfected according to the instructions of the Lipo3000 transfection reagent.

[0101] 5.4 Cell Imaging Experiment

[0102] HEK293T cells expressing the heterodimerization probe for SAM detection were incubated with 0.5 μM HBC620 and then imaged. The cells were treated with 40 mM cyclo-leucine to block SAM biosynthesis, and then the medium was replaced with a medium without cyclo-leucine after 30 minutes. The change in SAM level was evaluated in real time using ratio fluorescence. During image acquisition, the cells were maintained at a constant temperature of 37 °C.

[0103] As Figure 10As shown in Figure a, within 30 minutes after the addition of 40 mM cycloleucine, the ratiometric signal of the cells significantly decreased, indicating that the inhibitor can effectively reduce intracellular SAM levels. Subsequently, the culture medium was replaced to remove cycloleucine, and the ratiometric signal gradually recovered within 40 minutes of cycloleucine removal, eventually returning to the initial level, indicating that the cells' SAM synthesis capacity had been restored. This experimental result demonstrates that the designed RNA-protein heterodimerization probe can not only sensitively detect dynamic changes in SAM levels, but can also be used to monitor the effects of drugs on cellular metabolism in real time, with good reversibility. This not only provides an innovative tool for dynamic imaging of SAM in cells, but also provides a new detection strategy for drug efficacy evaluation and drug development.

[0104] Figure 10 b is a schematic diagram of the synthesis pathway of SAM. Cycloleucine inhibits the catalytic activity of MATase and thus inhibits SAM synthesis.

[0105] Figure 10 c is a graph showing the temporal changes in SAM levels in cells. The red-green fluorescence ratio of at least 10 ROIs was analyzed at each time point.

[0106] Example 6: Dynamic monitoring of SAM at the subcellular level using heterodimerization probes

[0107] Mitochondrial SAM (mitoSAM) was selected as the detection target.

[0108] A dual-promoter plasmid containing U6 and miniCMV promoters was redesigned and constructed for the expression of F30-PepperSAM detection module2-Boxb and Tomm20-EGFP-λN, respectively, enabling the heterodimerization probe to be precisely targeted to the mitochondrial outer membrane.

[0109] To explore the dynamic effects of a SAM synthesis inhibitor (cycloleucine) on the levels of cytoplasmic SAM and mitoSAM, HEK293T cells were transfected with the dual-promoter plasmid and imaged after incubation with HBC 620 fluorescent dye.

[0110] Figure 11 a is a schematic diagram of the construction of dual promoter expression: F30-Pepper SAM detection module2-Boxb is expressed by the U6 promoter, and Tomm20-EGFP-λN is expressed by the miniCMV promoter.

[0111] Figure 11 c is a schematic diagram of the construction of dual promoter expression: F30-Pepper SAM detection module2-Boxb is expressed by the U6 promoter, and EGFP-λN is expressed by the miniCMV promoter.

[0112] Figure 11 e is a schematic diagram of the cytoplasmic synthesis and mitochondrial transport and utilization of SAM. In human cells, SAM is synthesized in the cytoplasm and transported to the mitochondria by the mitoSAM carrier for utilization.

[0113] The results showed that after adding cycloleucine to inhibit SAM synthesis, the probe fluorescence ratio signal on the mitochondrial outer membrane gradually decreased ( Figure 11 b); In contrast, the fluorescence ratio signal of the probe in the cytoplasm decreased more rapidly ( Figure 11 d and Figure 11 f). This phenomenon suggests that SAM synthesis inhibitors primarily affect cytoplasmic SAM levels, with a delayed effect on mitochondrial SAM. This may be because SAM synthesis primarily occurs in the cytoplasm, so inhibition rapidly decreases cytoplasmic SAM levels. Mitochondrial SAM, on the other hand, is transported into mitochondria via cytoplasmic SAM, and its concentration changes are influenced by the balance between transport rate and consumption, resulting in a delayed effect. These results demonstrate the feasibility of heterodimerization probes for small molecule imaging analysis at the organelle level and provide new tools and insights for studying the role of SAM in cellular metabolism, mitochondrial function, and related diseases.

[0114] Example 7. Construction and performance testing of the Pepper-miRNA detection module in the heterodimerization probe

[0115] Construction method: The miRNA complementary sequence is fused to the Stem-loop1 of Pepper to disrupt the correct folding of Pepper; when the target miRNA binds to the complementary miRNA sequence, the complementary sequence will separate from the stem of Pepper and restore its folding, thereby achieving detection (such as Figure 12 a).

[0116] Specificity test

[0117] We selected miRNA-21 as the target, which is a common overexpressed biomarker in various cancers. In the absence of miRNA-21, the Pepper-miRNA21 detection module showed almost no fluorescence after incubation with HBC620 ( Figure 12 b), indicating that the folding of Pepper was successfully disrupted. In the presence of miRNA-21, the fluorescence intensity at 620 nm increased by about 93 times ( Figure 12b) demonstrated the successful construction of the Pepper-miRNA21 detection module. To investigate its selectivity, we selected three different miRNAs: miRNA-122, miRNA-222, and miRNA-378. The results showed that the detection module exhibited high selectivity for miRNA-21 ( Figure 12 c).

[0118] In addition, using the same strategy, the complementary sequence of miRNA-122 was fused with Pepper's Stem-loop1 to design a Pepper-based miRNA-122 detection module. In the presence of miRNA-122, the fluorescence intensity of the Pepper-miRNA122 detection module at 620 nm increased by approximately 286-fold ( Figure 12 d). This demonstrated the successful construction of the detection module and its efficient response ability. Meanwhile, its specific detection results further confirmed the high selectivity of the detection module ( Figure 12 e). This study indicated that the Pepper-based miRNA detection module can not only detect target miRNAs with high sensitivity and specificity but also has generality and can be customized for different miRNAs.

[0119] The following table shows the DNA templates of RNA monomers containing miRNA detection modules

[0120]

[0121] The following table shows the nucleic acid sequences of different miRNAs

[0122]

[0123] Example 8: Heterodimerization Probes for In Vitro Quantitative Analysis of miRNAs

[0124] Heterodimerization Probes for In Vitro Quantitative Analysis of miRNA21

[0125] The protein monomer EGFP-λN and the RNA monomer F30-Pepper miRNA21 detection module-Boxb were mixed in vitro to test their ratio fluorescence response to different concentrations of miRNA-21. In this ratio-type probe, EGFP-λN served as the reference signal, and its green fluorescence was not affected by the concentration of miRNA-21 ( Figure 13 a), while the Pepper-based detection module F30-Pepper miRNA21 detection module-Boxb showed an obvious concentration dependence, and the red fluorescence signal gradually increased with the increase in the concentration of miRNA-21 ( Figure 13b) The linear regression equation in the figure shows that within the concentration range of 1-30nM, the target concentration and the ratio fluorescence intensity (F620 / F505) show a significant linear relationship (R 2 =0.9927, Figure 13 c), the limit of detection (LOD) was as low as 0.2 nM, indicating that the heterodimerization probe has extremely high sensitivity for the detection of miRNA-21.

[0126] Heterodimerization probe for in vitro quantitative analysis of miRNA122

[0127] After the protein monomer EGFP-λN and the RNA monomer F30-Pepper miRNA122 detection module-Boxb were assembled in vitro, the same method was used to test their ratiometric fluorescence response to different concentrations of miRNA-122. As the concentration of miRNA-122 increased, the fluorescence signal of each monomer changed as shown in the following figure: Figure 14 a and Figure 14 b. In the range of 1-50 nM, the concentration of miRNA-122 and the ratio fluorescence intensity (F620 / F505) also showed a significant linear relationship (R 2 =0.9943, Figure 14 c), with a detection limit of 1.6 nM, demonstrating excellent sensitivity. These results demonstrate the broad applicability of the heterodimerization probe strategy, enabling the detection of diverse miRNA targets through modular design.

[0128] Example 9: Imaging analysis of miRNA in living cells using heterodimerization probes

[0129] miR-21 was selected as the target, and a plasmid with dual promoters was designed and constructed to achieve ratiometric fluorescence imaging of miRNA in living cells. Figure 15 (a) Schematic diagram of plasmid construction. Caco-2 cells were co-transfected with the plasmid and miRNA-21 mimic, followed by imaging analysis using confocal microscopy. Caco-2 cells are derived from a human colorectal adenocarcinoma cell line and have high endogenous expression levels of miRNA-21.

[0130] The results showed that under different experimental conditions, the EGFP green fluorescence signal remained constant, while cells treated with miRNA-21mimic showed stronger red fluorescence than the untreated control group ( Figure 15b). Furthermore, fluorescence ratiometric imaging revealed distinct color shifts in cells under different treatment conditions, demonstrating that the probe dynamically responds to changes in miRNA-21 levels. This probe not only provides a new imaging tool for studying miRNA function but also offers potential applications in the diagnosis of miRNA-related diseases and drug efficacy evaluation.

[0131] In the description of the present invention, it should be understood that the terms "first," "second," and "third" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of the technical features being referred to. Therefore, a feature specified as "first," "second," or "third" may explicitly or implicitly include one or more of such features. In the description of the present invention, unless otherwise specified, "plurality" means more than two.

[0132] It is understandable that those skilled in the art can make equivalent substitutions or changes based on the technical solution and inventive concept of the present invention, and all these changes or substitutions should fall within the scope of protection of the claims attached to the present invention.

Claims

1. An RNA-protein heterodimerization probe, characterized in that, include: An RNA monomer, wherein the RNA monomer comprises a three-way stem-loop structure scaffold, wherein the three-way stem-loop structure scaffold comprises a ring structure and a first stem structure, a second stem structure, and a third stem structure respectively connected to the ring structure; the first stem structure is connected to Boxb; the second stem structure is connected to a fluorescent RNA module; and the third stem structure is connected to a self-cyclization structure; The protein monomer EGFP-λN is affinity-linked to Boxb.

2. The RNA-protein heterodimerization probe according to claim 1, wherein The three-way stem-loop structure scaffold is an F30 three-way stem-loop structure; and the self-cyclization structure is a Tornado structure.

3. The RNA-protein heterodimerization probe according to claim 1, wherein The fluorescent RNA module is based on the Pepper sequence, with multiple base sequences replaced by a nuclear switch sequence or a miRNA response sequence; the nuclear switch sequence is used to form a nuclear switch structure; the nuclear switch structure or miRNA response sequence is used to restore the fluorescent molecule binding site in the Pepper sequence after binding to the detection substrate.

4. Use of the RNA-protein heterodimerization probe according to any one of claims 1 to 3 in detecting SAM or miRNA.

5. Use of the RNA-protein heterodimerization probe according to claim 3 for detecting SAM in vitro, in cells or in subcellular space, wherein the Pepper sequence is as shown in SEQ ID No. 1; The fluorescent RNA module is a Pepper sequence with the 18th to 33rd bases replaced by a nuclear switch sequence.

6. Use of the RNA-protein heterodimerization probe according to any one of claims 1 to 3 for detecting SAM in vitro, in cells or in subcellular compartments, wherein the nucleic acid sequence of the fluorescent RNA module is as shown in SEQ ID No. 2 or SEQ ID No.

3.

7. Use of the RNA-protein heterodimerization probe according to any one of claims 1 to 3 for detecting SAM in vitro, in cells or in subcellular compartments, wherein the nucleic acid sequence of the RNA monomer of the RNA-protein heterodimerization probe is shown as SEQ ID No. 4 or SEQ ID No.

5.

8. Use of the RNA-protein heterodimerization probe according to claim 3 for detecting miRNA in vitro or in cells, wherein the Pepper sequence is as shown in SEQ ID No. 1; The fluorescent RNA module is based on the Pepper sequence, with the 19th to 32nd bases replaced by miRNA response sequences.

9. Use of the RNA-protein heterodimerization probe according to any one of claims 1 to 3 in detecting miRNA in vitro or in cells, When the miRNA is miRNA-21, the nucleic acid sequence of the fluorescent RNA module is as shown in SEQ ID No. 6; When the miRNA is miRNA-122, the nucleic acid sequence of the fluorescent RNA module is shown as SEQ ID No.

7.

10. Use of the RNA-protein heterodimerization probe according to any one of claims 1 to 3 in detecting miRNA in vitro or in cells, When the miRNA is miRNA-21, the nucleic acid sequence of the RNA monomer of the RNA-protein heterodimerization probe is shown as SEQ ID No. 8; When the miRNA is miRNA-122, the nucleic acid sequence of the RNA monomer of the RNA-protein heterodimerization probe is shown as SEQ ID No.9.

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