RNA editing system based on multivalent RNA aptamer recruiting endogenous ADAR1 and application thereof

Abstract

The present application relates to the technical field of biological medicine, and particularly relates to an RNA editing system based on multivalent RNA aptamer recruiting endogenous ADAR1 and application thereof. The system realizes efficient recruitment of endogenous ADAR1 p150 through optimization of the multivalent aptamer of ADAR1-Zalpha domain, and then completes the gene editing of target RNA. The verification result proves that the multivalent RNA aptamer in the system can specifically combine with ADAR1-Zalpha with high affinity, and can realize high RNA editing efficiency in cells and animal models. The editing system module constructed by the present application has high modular degree and good targeting specificity, and can be used as an alternative technical platform for precise treatment type RNA editing; the system has good programmable expansion space and potential clinical transformation value, and is helpful to promote the research progress of targeted RNA therapeutic drugs, gene function research tools and disease models.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to an RNA editing system based on the recruitment of endogenous ADAR1 by multivalent RNA aptamers and its applications. Background Technology

[0002] RNA editing is a promising biotechnology that can expand transcriptome diversity through precise codon recoding and splicing regulation, without altering the genomic DNA sequence. This site-specific technology utilizes engineered mechanisms to mediate RNA-modifying enzymes targeting specific transcripts, achieving reversible and controllable gene expression regulation. Compared to permanent DNA editing, its significant advantages lie in its greater safety and ethical compliance, avoiding heritable gene alterations and effectively reducing the probability of off-target effects. These characteristics make RNA editing a potential therapeutic research tool for various diseases, with applications ranging from single-gene genetic disorders to complex multifactorial diseases. Therefore, as a promising technology platform in precision medicine, site-directed RNA editing is currently in a phase of rapid development, offering new ideas and directions for disease treatment due to its high specificity and controllable risks.

[0003] Currently, site-directed RNA base editing mainly revolves around two strategies. The first strategy involves the exogenous expression of engineered editing proteins that bind to guide RNA (gRNA). While this strategy achieves high targeted editing efficiency, it still carries certain safety risks, including potential tumorigenicity and off-target effects across the entire transcriptome caused by persistent protein overexpression. In contrast, the second strategy involves the artificial synthesis and design of gRNAs to activate endogenously expressed RNA adenosine deaminases (ADARs), enabling programmable adenine-hypoxanthine (AI) base conversion without the introduction of exogenous transgenic elements. This strategy utilizes chemically modified antisense oligonucleotides (such as RESTORE) or long nuclear-retained transcripts (such as LEAPER and CLUSTER) to complete the editing process, effectively improving biosafety and reducing immunogenicity. However, this method is often hampered by poor editing efficiency and significant bystander editing effects, thus limiting its therapeutic applications. To overcome these limitations, novel gRNA designs have been developed. Related studies have shown that introducing G•U swing base pairs into the circular CLUSTER structure can effectively alleviate bystander editing and improve the accuracy and effectiveness of in vivo RNA base editing, but its editing efficiency still needs further improvement. The MIRROR system, through engineered gRNA to mimic the endogenous inverted Alu repeat sequence—a natural high-affinity ADAR substrate—effectively improves RNA editing efficiency; however, the editing performance of this system is easily affected by the action background, requiring extensive debugging and personalized design for different targets, and its general programmability needs improvement. These advances highlight the urgent need for a simplified, efficient, and widely applicable RNA editing platform equipped with standardized gRNA design to achieve robust and specific recruitment of endogenous ADAR, thereby accelerating its translation into clinical therapy.

[0004] The ADAR enzyme family mainly comprises three isoforms: ADAR1, ADAR2, and ADAR3. ADAR3 lacks catalytic function, ADAR2 is specifically expressed only in neurons and vascular tissues, while ADAR1 is widely expressed in various cell types and is an endogenous editing enzyme with high potential for broad-spectrum therapeutic RNA base editing. ADAR1 mainly includes two isoforms: the stably expressed p110 isoform and the interferon-induced p150 isoform. The p150 isoform contains an N-terminal Zα domain absent in the p110 isoform. This structural difference results in different subcellular localization characteristics: the p110 isoform is mainly distributed in the nucleus, while the p150 isoform is mainly distributed in the cytoplasm. Under normal physiological conditions, the p110 isoform has a higher expression abundance within cells, but the p150 isoform has unique advantages in RNA editing technology research. Its cytoplasmic localization allows direct contact with exogenously delivered guide RNA, avoiding the technical difficulties of nuclear transport during RNA editing.

[0005] While existing methods for exogenous overexpression of ADAR can improve editing efficiency to some extent, ADAR overexpression is prone to causing unintended whole-transcriptome RNA over-editing, leading to a significant off-target risk. Furthermore, the delivery and expression of exogenous proteins also pose potential immunogenicity risks, significantly hindering clinical translation. Therefore, there is an urgent need to develop a more efficient and safer editing strategy. Summary of the Invention

[0006] This invention aims to at least solve one of the technical problems existing in related technologies. Therefore, the first objective of this invention is to provide an RNA editing system based on the recruitment of endogenous ADAR1 by multivalent RNA aptamers; the second objective of this invention is to provide applications of the RNA editing system based on the recruitment of endogenous ADAR1 by multivalent RNA aptamers.

[0007] To achieve the first objective, the technical solution adopted by this invention is as follows: An RNA editing system based on recruiting endogenous ADAR1 using multivalent RNA aptamers includes at least one RNA aptamer selected from any one or more of SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, and SEQ ID NO.8.

[0008] Furthermore, the binding domain of the RNA aptamer is ADAR1-Zα.

[0009] Furthermore, the secondary structure of the RNA aptamer includes an L2 loop structure, as shown in SEQ ID NO.6, SEQ ID NO.7, and SEQ ID NO.8.

[0010] Furthermore, the circular structure of the RNA aptamer is constructed via ribozyme-mediated self-circularization.

[0011] Furthermore, the circular structure of the RNA aptamer is constructed via ribozyme-mediated self-circularization.

[0012] Furthermore, the expression framework of the RNA aptamer comprises the following elements connected in sequence: (I) U6 promoter; (II) P3 type twister ribozyme; (III) A functional RNA sequence, wherein the functional RNA sequence contains the RNA aptamer; (Ⅳ) P1 type twister ribozyme.

[0013] To achieve the second objective, the technical solution adopted by this invention is as follows: Applications of RNA editing systems based on multivalent RNA aptamers recruiting endogenous ADAR1, including the preparation of one or more of the following: drugs, gene function research tools, and disease models using RNA editing systems based on multivalent RNA aptamers recruiting endogenous ADAR1 as described above.

[0014] Furthermore, the drug includes drugs used in gene mutation-related genetic diseases.

[0015] Furthermore, the gene mutation is a G>A point mutation.

[0016] Furthermore, the drug may also include pharmaceutically acceptable carriers or excipients.

[0017] Furthermore, the carrier includes an AAV carrier.

[0018] The above-described one or more technical solutions in the embodiments of the present invention have at least one of the following technical effects: This invention provides an RNA editing system based on multivalent RNA aptamers for recruiting endogenous ADAR and its applications. This RNA editing system achieves efficient recruitment of endogenous ADAR1 p150 through optimized multivalent aptamers targeting the ADAR1-Zα domain, thereby completing gene editing of the target RNA. Binding activity was verified by surface plasmon resonance (SPR) analysis, demonstrating that the multivalent RNA aptamers in this system can specifically bind to ADAR1-Zα with high affinity. Cellular and animal model experimental data show that this RNA editing system can achieve high RNA editing efficiency in both cell and animal models. The editing system constructed in this invention has a high degree of modularity and good targeting specificity, making it a potential technology platform for precision therapeutic RNA editing. This system possesses good programmability and potential clinical translational value, contributing to the advancement of research on targeted RNA therapeutics, gene function research tools, and disease models.

[0019] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0020] Figure 1 This is the secondary structure of the RNA aptamer provided in Embodiment 1 of the present invention.

[0021] Figure 2The results of AP performance evaluation provided in Example 2 of this invention are as follows: Figure a is the surface plasmon resonance (SPR) fitting curve; Figure b is a visualization of the ADAR1-Zα docking structure with AP molecules; Figure c is a schematic diagram of guide RNA (gRNA); Figure d is a schematic diagram of a dual-fluorescence reporter system (TagBFP–mNeonGreen) used to evaluate the efficiency of premature UAG stop codon editing; Figure e is a confocal image of HEK293T cells expressing the reporter gene after treatment with p8-AP gRNA; Figure f is a quantitative statistical chart of mNeonGreen / TagBFP positive cells; Figure g is a bar chart of target site RNA editing efficiency; and Figure h is a representative Sanger sequencing curve of the reporter gene region edited by p8-AP(i)gRNA.

[0022] Figure 3 The results of system performance characterization and molecular simulation analysis of the interaction between RNA aptamer (AP) and ADAR1 Zα domain provided in Example 2 of this invention are as follows: Figure a is a schematic diagram of the surface plasmon resonance (SPR) experiment principle; Figure b is the SPR spectrum of the binding of RNA aptamer and ADAR1-Zα at different concentrations; Figure c is a graph showing the root mean square deviation (RMSD) results of the AP–ADAR1-Zα complex in the molecular dynamics simulation process; Figure d is the residue correlation analysis result; Figure e is the change of hydrogen bonds in the AP–ADAR1-Zα complex over time; and Figure f is a visualization of the molecular dynamics simulation.

[0023] Figure 4 This invention provides an optimized structure of the RNA aptamer in Example 2 and the results of evaluating the editing efficiency of the optimized RNA aptamer. Figure a shows a schematic diagram of gRNA design with different A / C mismatch positions in the target-specific domain; Figure b shows the RNA editing efficiency results obtained by fluorescence recovery detection for gRNAs with different A / C mismatch positions in the target-specific domain; Figure c shows the RNA editing efficiency results obtained by Sanger sequencing for gRNAs with different A / C mismatch positions in the target-specific domain; and Figure d shows the secondary structure of the truncated AP variant designed by changing the distance to the editing site. Figure e shows the editing efficiency results of gRNA containing truncated AP structures obtained by fluorescence recovery detection; Figure f shows the editing efficiency results of gRNA containing truncated AP structures obtained by sequencing detection; Figure g is a schematic diagram of the design architecture of split gRNA; Figure h is a confocal image of HEK293T cells expressing reporter genes after treatment with split gRNA (scale bar is 100μm); Figure i is a fluorescence quantitative analysis of the confocal imaging results in Figure h; Figure j shows the detection results of RNA editing efficiency mediated by split gRNA; Figure k is a representative Sanger sequencing map of RNA editing mediated by split gRNA.

[0024] Figure 5 The results of the evaluation of the editing efficiency of tandem L2 aptamers provided in Example 2 of this invention are shown in Figure a. Figure a is a schematic diagram of the secondary structure of RNA aptamers with different numbers of tandem L2 circular structures obtained by MXfold2 modeling; Figure b is a confocal image (scale bar 100) of HEK293T cells expressing the TagBFP-mNeonGreen reporter gene after transfection with dividing gRNA containing different numbers of L2 circular structures. Figure c shows representative Sanger sequencing chromatograms of split gRNA editing results containing different numbers of L2 circular structures; Figure d shows the results of quantitative analysis of RNA editing efficiency of split gRNAs containing different numbers of L2 circular structures based on Sanger sequencing; Figure e shows a detailed structural visualization of the hydrogen bond interaction between ADAR1-Zα and AP-2×L2; Figure f shows a detailed structural visualization of the hydrogen bond interaction between ADAR1-Zα and AP-3×L2; Figure g shows a detailed structural visualization of the hydrogen bond interaction between ADAR1-Zα and AP-4×L2; Figure h shows a schematic diagram of the MARRS gRNA structure with systematically altered flanking nucleotides around the A–C mismatch (5′-N1CN2 motif) and the editing efficiency detection results of all 16 N1CN2 combinations; Figure i shows the results of editing adenosine and its corresponding MARRS. A schematic diagram of gRNA reverse motif (5′-N2'CN1') design, reporter gene carrying variable triplet context (5′-N1AN2), and adenosine editing efficiency for each 5′-N1AN2 context pair.

[0025] Figure 6 The following figures illustrate the performance characterization and efficiency verification results of the truncated aptamer and tandem L2 provided in Example 2 of this invention: Figure a shows the sensor spectrum obtained from surface plasmon resonance (SPR) analysis; Figure b shows the fluorescence analysis results of HEK293T cells expressing the TagBFP-mNeonGreen reporter gene after transfection with segmented gRNAs carrying different numbers of L2 circular structures; Figure c shows a schematic diagram of the sensor used in the surface plasmon resonance (SPR) analysis; Figure d shows a representative SPR sensor spectrum; Figure e shows a schematic diagram of gRNA design and a bar chart of editing efficiency; and Figure f shows the fluorescence recovery measured using 16 different 5′-N1CN2 combinations.

[0026] Figure 7The following are the evaluation results of the MARRS system (Ap-3xL2 recruiting ADAR1 system) provided in Example 2 of this invention on the efficiency of endogenous RNA editing; wherein, Figure a is a bar chart of the RNA editing efficiency of the MARRS system on the UAG stop codon of the TP53 gene in different cell lines, Figure b is a bar chart of the RNA editing efficiency of the MARRS system on the UAG stop codon of the KRAS gene in different cell lines, Figure c is a bar chart of the RNA editing efficiency of the MARRS system on the UAG stop codon of the EGFR gene in different cell lines, Figure d is a representative Sanger sequencing chromatogram, Figure e is a bar chart of by-stander editing analysis based on NGS read data, Figure f is a heatmap of the distribution of MARRS editing efficiency in the full-length TP53 mRNA sequence, Figure g is a heatmap of the distribution of MARRS editing efficiency in the full-length KRAS mRNA sequence, and Figure h is a scatter plot of gene expression levels in the MARRS-treated group and the untreated group.

[0027] Figure 8 The results of in vivo evaluation of RNA editing efficiency using the MARRS system (Ap-3xL2 recruiting ADAR1 system) provided in Example 2 of this invention are shown below: Figure a is a schematic diagram of AAV-mediated in vivo editing in mice; Figure b is a representative image of bioluminescence in mouse liver; Figure c is the quantitative analysis results of photon flux of firefly luciferase activity; Figure d is the expression ratio of firefly luciferase to Renilla luciferase in mouse liver; Figure e is the RNA editing efficiency of dual-luciferase reporter gene transcripts in mouse liver; and Figure f is the Sanger sequencing spectrum of liver RNA from different experimental groups. Detailed Implementation

[0028] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below in conjunction with specific embodiments. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention. The following embodiments are used to illustrate this invention, but cannot be used to limit the scope of this invention.

[0029] In the following embodiments, unless otherwise specified, the experimental methods used are conventional methods, and the materials and reagents used are commercially available, unless otherwise specified, and are carried out in accordance with the techniques or conditions described in the literature in this field or in accordance with the product instructions.

[0030] Example 1 Construct RNA aptamers.

[0031] The amino acid sequence of the ADAR1-Zα (134~198) fragment domain protein (SEQ ID NO.1) is shown below: SIYQDQEQRILKFLEELGEGKATTAHDLSGKLGTPKKEINRVLYSLAKKGKLQKEAGTPPLWKIA.

[0032] DNA aptamers with high affinity for the Zα domain of ADAR1 p150 were screened according to the method disclosed in patent CN121592654A and transcribed into RNA aptamers for structure-guided design.

[0033] The RNA aptamer sequence is as follows: The nucleotide sequence of RNA Aptamer (SEQ ID NO.2) is shown below: UUCAGCACUCCACGCAUAGCACGCCCGCCACCCCCUCUCCUGGCCAUCCGCGCACACCUAUGCGUGCUACCGUGAA; The nucleotide sequence of Aptamer-10 (SEQ ID NO.3) is shown below: CACGCAUAGCACGCCCGCCACCCCCUCUCCUGGCCAUCCGCGCACACCUAUGCGUG; The nucleotide sequence of Aptamer-15 (SEQ ID NO.4) is shown below: AUAGCACGCCCGCCACCCCCUCUCCUGGCCAUCCGCGCACACCUAU; The nucleotide sequence of Aptamer-17 (SEQ ID NO.5) is shown below: AGCACGCCCGCCACCCCCUCUCCUGGCCAUCCGCGCACACCU; The nucleotide sequence of Aptamer-2xL2 (SEQ ID NO.6) is shown below: AUAGCACGCGCAGCCACCCCCUCUCCUGGCCACACCGCGCACACCUAU; The nucleotide sequence of Aptamer-3xL2 (SEQ ID NO.7) is shown below: AUAGCACGCGCACGCGCAGCCACCCCCUCUCCUGGCCACACCGCGCACACCGCGCACACCUAU. The nucleotide sequence of Aptamer-AP-4xL2 (SEQ ID NO.8) is shown below: AUAGCACGCGCACGCGCACGCGCAGCCACCCCCUCUCCUGGCCACACCGCGCACACCGCGCACACGCGCACACCUAU; The RNA aptamers described in this application can be prepared using conventional nucleic acid synthesis methods, including but not limited to solid-phase chemical synthesis and in vitro transcription, or obtained through commercial nucleic acid synthesis services.

[0034] The secondary structure of the above RNA aptamers was predicted using Mfold2, and the results are as follows: Figure 1 As shown.

[0035] Example 2 I. Preparation of aptamer RNA.

[0036] Using DNA containing the T7 promoter as a template, linear RNA was synthesized in an in vitro transcription system using T7 RNA polymerase. The DNA template concentration in the transcription system was 1 μg, the NTP concentration was 10 mM, and the enzyme dosage was 20-50 U. The reaction was carried out at 37 °C for 4-8 hours. After the reaction, the template DNA was removed by DNase I treatment to obtain the aptamer RNA (denoted as AP).

[0037] Monomeric RNA aptamers (capable of binding ADAR1-Zα) have limited ability to recruit ADAR1. By tandemly linking multiple ADAR1-Zα-recognizing RNA aptamer units to create multivalent RNA aptamers, target protein capture is optimized through multiple binding sites. Multiple binding sites can synergistically enhance local ADAR1 enrichment and improve RNA editing efficiency. Experimental results show that editing efficiency is significantly improved when the number of repeat units (L2) in the RNA aptamer is 3 (Aptamer-3xL2); however, as the number of repeat units increases further, editing efficiency decreases. Combined with secondary structure prediction and molecular simulation analysis, this phenomenon is speculated to be related to the increased complexity of RNA folding and enhanced steric hindrance. Therefore, the aforementioned repeat range can achieve superior technical performance.

[0038] II. Optimize the RNA aptamer editing system.

[0039] To improve RNA stability and optimize the editing system, this invention further employs a construction strategy based on self-cleaving ribozymes to prepare circular RNA. Specifically, the construction method involves inserting an RNA aptamer and a guide sequence between two types of twister ribozymes, P3 and P1, and using the U6 promoter to drive transcription, thus constructing a U6 promoter–P3 twister ribozyme–functional RNA sequence (including the RNA aptamer)–P1 twister ribozyme expression framework. After transcription is completed in the cell, the twister ribozyme mediates RNA self-cleavage, generating an RNA intermediate with 5′-hydroxyl and 2′,3′-cyclic phosphate ends. This intermediate can be circularized using endogenous RNA ligases (such as RtcB) to form circular RNA. Compared to conventional linear RNA, circular RNA lacks free nucleic acid ends, exhibits stronger resistance to nucleases, and demonstrates superior molecular stability. Meanwhile, the circular structure of the multivalent RNA editing system can constrain the spatial range of the editing complex within a certain range, which can reduce the occurrence of non-specific editing and improve the sustained editing effect and the specificity of the entire editing system.

[0040] III. AP performance evaluation results, such as Figure 2 As shown; Figure a shows the surface plasmon resonance (SPR) fitting curve, which indicates that there is a high affinity interaction between AP and the Zα domain (Kd = 4.6 ± 0.4 nM). This result suggests that AP can achieve efficient and specific protein recruitment. Figure b is a visualization of the docking structure of ADAR1-Zα and AP molecules. From this figure, we can see that the L2 segment in the secondary structure of the RNA aptamer contributes the main binding interface and forms about 9 hydrogen bonds, indicating that this region plays a key role in stabilizing the complex formation. Figure c is a schematic diagram of guide RNA (gRNA). From the figure, we can see that it contains a 20-nucleotide specific domain (with an A–C mismatch at position 8), three 20-nucleotide RS repeat sequences, and a recruitment domain (RNA aptamer), which can effectively integrate targeted recognition and enzyme recruitment functions in space. Figure d shows a schematic diagram of a dual-fluorescence reporter system (TagBFP–mNeonGreen) used to evaluate the efficiency of premature UAG stop codon editing; Figure e shows a confocal image of HEK293T cells expressing the reporter gene after treatment with p8-AP gRNA. This image indicates that the system exhibits significant RNA editing activity in HEK293T cells. Figure f shows the quantitative statistical graph of mNeonGreen / TagBFP positive cells; Figure g is a bar chart of RNA editing efficiency at the target site, which shows that the editing efficiency has been significantly improved. Figure h shows a representative Sanger sequencing curve of the reporter gene region edited by p8-AP(i)gRNA. The figure shows that a clear A-to-G conversion occurred at the target site, indicating that the system can achieve efficient and precise RNA editing.

[0041] The above results demonstrate that the RNA aptamer and its engineering modification strategy provided by this invention can effectively recruit endogenous ADAR1 p150 and significantly improve RNA editing efficiency, exhibiting good specificity and application potential.

[0042] IV. This invention characterizes the system performance and performs molecular simulation analysis on the interaction between the RNA aptamer (AP) and the ADAR1 Zα domain. The results are as follows: Figure 3 As shown; Figure a shows a schematic diagram of the surface plasmon resonance (SPR) experiment, which illustrates the combination of AP and immobilized ADAR1-Zα. Figure b shows the SPR spectrum of the binding of RNA aptamers to ADAR1-Zα at different concentrations. This figure shows that AP can stably bind to immobilized ADAR1-Zα in a concentration-dependent manner, and the response signal is significantly enhanced with increasing aptamer concentration, further verifying its good binding characteristics. Figure c shows the root mean square deviation (RMSD) results of the AP–ADAR1-Zα complex in the molecular dynamics simulation. As can be seen from the figure, the root mean square deviation (RMSD) of the AP–ADAR1-Zα complex remained within a low fluctuation range throughout the simulation, indicating that the complex has high structural stability. Figure d shows the results of residue correlation analysis. From this figure, it can be seen that there is a significant dynamic interaction between the ADAR1-Zα domain (residues 1–70) and AP (residues 70–160), indicating that the two have a close coupling relationship in spatial conformation. Figure e shows the changes in hydrogen bonds in the AP–ADAR1-Zα complex over time. From this figure, we can see that the complex can maintain multiple stable hydrogen bond interactions during the simulation, which provides the molecular basis for its high affinity binding. Figure f is a visualization of molecular dynamics simulation. From this figure, we can see that the key bases of AP can interact strongly with specific sites in the ADAR1-Zα domain. Among them, the bases with outstanding interaction effects are marked as high contribution sites.

[0043] In summary, the RNA aptamer (AP) designed in this invention can form a stable and specific complex with the ADAR1 Zα domain; the relevant results, from the perspectives of structure and dynamics, verify that AP has a high-efficiency recruitment capability, and also lay an important foundation for achieving efficient RNA editing.

[0044] V. Optimized structure of RNA aptamers and evaluation of the editing efficiency of the optimized RNA aptamers, the results are as follows: Figure 4 As shown; Figure a shows a schematic diagram of gRNA design with different A / C mismatch positions in the target-specific domain; Figure b shows the RNA editing efficiency results obtained by fluorescence recovery detection for gRNAs with different A / C mismatch positions in the target-specific domain; Figure c shows the RNA editing efficiency results obtained by Sanger sequencing of gRNAs with different A / C mismatch positions in the target-specific domain; Figure d shows the secondary structure of a truncated AP variant designed to alter the distance to the editing site; Figure e shows the editing efficiency results of gRNA containing truncated AP structures obtained by fluorescence recovery detection. Figure f shows the editing efficiency results of gRNA containing truncated AP structures, obtained by sequencing detection. Figure g is a schematic diagram of the design architecture for splitting gRNA; Image h is a confocal image of HEK293T cells expressing reporter genes after treatment with splitting gRNA, with a scale bar of 100 μm. Figure i shows the fluorescence quantitative analysis of the confocal imaging results in Figure h; Figure j shows the results of RNA editing efficiency detection mediated by splitting gRNA; Figure k shows a representative Sanger sequencing map of split gRNA-mediated RNA editing.

[0045] Depend on Figure 4It is evident that the RNA editing efficiency is significantly improved after optimizing the gRNA structure in this invention. This invention first systematically designs the gRNA: introducing A / C mismatches at different positions in the target-specific domain and using a dual-fluorescence reporter system for functional evaluation. Experiments confirm that the mismatch positions significantly affect editing efficiency. Fluorescence detection results and Sanger sequencing results corroborate each other, showing that A / C mismatches at specific positions can significantly improve the editing efficiency of the target site, demonstrating that precise design of the editing window is crucial for improving system performance. Based on this, this invention truncates the RNA aptamer (AP) to adjust its spatial distance from the editing site, constructing multiple truncated variants. Experimental results show that after appropriate AP truncation, it still retains the ability to recruit ADAR1, while the spatial configuration is optimized, further improving editing efficiency. Fluorescence analysis and sequencing results both verify the effectiveness of this optimization scheme. This invention also proposes a split gRNA design strategy: splitting the ADAR recruitment domain (AP-15) into two fragments, each connected to a connecting stem structure. This design promotes RNA molecule circularization and achieves precise spatial alignment between the recruitment domain and the specific recognition domain. After treating HEK293T cells with splitting gRNA, the reporter gene fluorescence signal was significantly restored, and quantitative detection results showed that RNA editing efficiency was further improved. Sanger sequencing also clearly detected A-to-G base conversion at the target site, confirming that this structural optimization method can effectively enhance RNA editing activity. In summary, by optimizing mismatch sites, truncating aptamers, and designing splitting and circularization structures for synergistic regulation, the recruitment efficiency and editing accuracy of ADAR1 can be significantly improved at the spatial structure level, comprehensively improving the overall performance of the system of this invention.

[0046] VI. Results of the evaluation of the editing efficiency of tandem L2 aptamers, such as Figure 5 As shown; Figure a shows a schematic diagram of the secondary structure of RNA aptamers with different numbers of L2 circular structures obtained by MXfold2 modeling. Figure b shows a confocal image of HEK293T cells expressing the TagBFP-mNeonGreen reporter gene after being transfected with cleaving gRNA containing different numbers of L2 circular structures. The scale bar is 100 μm. Figure c shows a representative Sanger sequencing chromatogram of segmented gRNA editing results containing different numbers of L2 circular structures; Figure d shows the results of quantitative analysis of RNA editing efficiency of splitting gRNAs containing different numbers of L2 circular structures based on Sanger sequencing; Figure e is a detailed structural visualization of the hydrogen bond interaction between ADAR1-Zα and AP-2×L2. The red numbers in the figure represent the number of hydrogen bonds in each ADAR1-Zα, and the light orange shading represents the binding region of each ADAR1-Zα. Figure f shows a detailed structural visualization of the hydrogen bond interaction between ADAR1-Zα and AP-3×L2. The red numbers in the figure represent the number of hydrogen bonds in each ADAR1-Zα, and the light orange shading represents the binding region of each ADAR1-Zα. Figure g shows a detailed structural visualization of the hydrogen bond interaction between ADAR1-Zα and AP-4×L2. The red numbers in the figure represent the number of hydrogen bonds in each ADAR1-Zα, and the light orange shading represents the binding region of each ADAR1-Zα. Figure h shows a schematic diagram of the MARRS gRNA structure with systematically altered flanking nucleotides around the A–C mismatch (5′-N1CN2 motif) and the results of editing efficiency detection for all 16 N1CN2 combinations; Figure i shows a schematic diagram of a reporter gene designed for editable adenosine and its corresponding MARRS gRNA reverse motif (5′-N2'CN1'), carrying a variable triplet context (5′-N1AN2), and the adenosine editing efficiency of each 5′-N1AN2 context.

[0047] from Figure 5It is evident that introducing tandem L2 aptamers can significantly improve the RNA editing efficiency of this invention. This invention first utilizes MXfold2 to model the secondary structure of tandem L2 aptamers. The results show that multiple L2 modules, while maintaining overall structural stability, can form a spatial conformation conducive to ADAR1-Zα binding. After transfecting HEK293T cells with splitting gRNAs carrying different numbers of L2 circular structures, confocal imaging results showed that as the number of L2 repeat structures increased, the fluorescence recovery effect of the reporter gene significantly improved, indicating that RNA editing activity increased accordingly. Sanger sequencing results further verified this conclusion: the number of L2 repeat structures significantly affects the A-to-G editing efficiency at the target site. Quantitative analysis shows that, within a certain range, the editing efficiency increases significantly with the increase in the number of L2 repeats; however, when the number of repeats is too high, the efficiency improvement gradually plateaus and even slightly declines, proving that this multivalent design has an optimal copy number range. Molecular structure visualization analysis revealed that increasing the number of L2 repeats significantly increased the number of hydrogen bonds formed between ADAR1-Zα and the aptamer, and broadened the binding region, effectively improving the stability of the complex and enzyme recruitment ability. This structurally explains the improved editing efficiency. Furthermore, this invention systematically altered the flanking nucleotide sequences on both sides of the A–C mismatch site to examine the impact of sequence background on editing efficiency. Results showed that different N1CN2 combinations significantly regulated editing efficiency, confirming the crucial role of the local sequence environment in RNA editing. Systematic testing of adenosine editing efficiency using different 5′-N1AN2 triplet sequences revealed that the editing system provided by this invention maintains stable and efficient editing activity under various sequence backgrounds. In summary, constructing a multivalent RNA structure by tandemly connecting L2 aptamers and combining it with sequence environment optimization strategies effectively enhances the recruitment and editing capabilities of ADAR1, comprehensively improving the overall performance of the RNA editing system of this invention.

[0048] VII. Performance characterization and efficiency verification results of truncated aptamers and tandem L2, such as... Figure 6 As shown; Figure a shows the sensor spectrum obtained from surface plasmon resonance (SPR) analysis. Figure b shows the fluorescence analysis results of HEK293T cells expressing the TagBFP-mNeonGreen reporter gene after transfection with segmented gRNA carrying different numbers of L2 circular structures; Figure c shows a schematic diagram of the sensor used in surface plasmon resonance (SPR) analysis; Figure d shows a representative SPR sensor spectrum. Figure e shows a schematic diagram of gRNA design and a bar chart of editing efficiency; Figure f shows the fluorescence recovery measured using 16 different combinations of 5′-N1CN2.

[0049] Depend on Figure 6 It was found that the truncated AP aptamer could still bind stably to the ADAR1-Zα domain, maintaining high affinity while optimizing the molecular conformation, thus providing favorable conditions for improving RNA editing efficiency. Transfection of splitting gRNAs containing different numbers of L2 circular structures into HEK293T cells showed that fluorescence recovery of the reporter gene gradually increased with the increase in the number of L2 repeat units, indicating a significant improvement in RNA editing efficiency. SPR detection further verified this trend: by immobilizing aptamers with different numbers of L2 circular structures and detecting their binding to ADAR1-Zα, it was shown that the multivalent L2 structure significantly enhanced the protein binding signal, and the binding response increased with the increase of repeat number, but tended to saturate after reaching a certain number, proving that this multivalent design has an optimal range. The representative SPR curve also corroborated the synergistic effect of the multivalent aptamer in enhancing binding strength. Furthermore, gRNAs containing different triplet sequences (5′-CNA, where N is A, U, C, or G) were constructed, and the effect of changes in the contralateral base pairing of the target adenine on editing efficiency was systematically analyzed. The results showed that the local sequence environment has a significant regulatory effect on editing efficiency. Fluorescence recovery analysis of 16 5′-N1CN2 combinations indicated that different sequence combinations resulted in significant differences in editing efficiency. The system of this invention can achieve fine-tuning of editing efficiency through sequence optimization.

[0050] The above experimental results demonstrate that by truncating the aptamer to optimize its spatial configuration and combining it with L2 tandem multivalent design, ADAR1 binding ability can be maintained and enhanced, and RNA editing efficiency can be significantly improved. At the same time, combined with sequence environment optimization strategies, precise regulation of editing activity can be achieved, thereby further improving the overall performance of the system of this invention.

[0051] 8. Evaluation results of the MARRS system (Ap-3xL2 recruiting ADAR1 system) on the efficiency of endogenous RNA editing, such as... Figure 7 As shown; Figure a shows the bar chart of the RNA editing efficiency of the MARRS system on the UAG stop codon of the TP53 gene in different cell lines; Figure b shows the bar chart of RNA editing efficiency of the MARRS system on the UAG stop codon of the KRAS gene in different cell lines; Figure c shows the bar chart of the RNA editing efficiency of the MARRS system on the UAG stop codon of the EGFR gene in different cell lines; Figure d shows a representative Sanger sequencing chromatogram; Figure e is a bar chart of by-stander editing analysis based on NGS data; Figure f is a heatmap showing the distribution of MARRS editing efficiency in the full-length TP53 mRNA sequence; Figure g is a heatmap showing the distribution of MARRS editing efficiency in the full-length KRAS mRNA sequence; Figure h is a scatter plot of gene expression levels in the MARRS-treated and untreated groups; from Figure 7 It is evident that the MARRS system constructed in this invention exhibits excellent editing efficiency and specificity in endogenous RNA editing. The editing effect of MARRS gRNA in various cell lines was examined, and the results showed that the system can achieve high-efficiency RNA editing under different cellular backgrounds, demonstrating good applicability and stability. Representative Sanger sequencing data further confirmed that a clear A-to-G base conversion occurred at the target site in MCF7 cells, proving that the system can achieve precise editing at the endogenous transcriptional level. Bystander editing off-target analysis based on NGS sequencing data showed that the MARRS system had a low level of non-specific off-target editing, confirming its high editing specificity. Analysis of the editing efficiency of MARRS gRNA targeting adenine sites in TP53 and KRAS mRNA showed a significantly high editing ratio, suggesting that the system can achieve efficient editing of function-related genes. Furthermore, comparison of gene expression levels between the MARRS-treated and untreated groups showed that the expression levels of most genes remained stable, with only a few genes showing slight upregulation, proving that the system does not induce significant global transcriptome perturbations. The above results demonstrate that the MARRS system constructed in this invention can achieve efficient, precise, and low-off-target endogenous RNA editing in various cell types, while possessing both good safety profile and broad application potential.

[0052] IX. Results of in vivo assessment of RNA editing efficiency using the MARRS system (Ap-3xL2 recruiting ADAR1 system), such as... Figure 8 As shown; Figure a shows a schematic diagram of AAV-mediated in vivo editing in mice. Figure b shows a representative image of bioluminescence in mouse liver; Figure c shows the results of quantitative analysis of photon flux of firefly luciferase activity; Figure d shows the expression ratio of firefly luciferase to kidney luciferase in mouse liver; Figure e shows the RNA editing efficiency of dual-luciferase reporter gene transcripts in mouse liver; Figure f shows the Sanger sequencing patterns of liver RNA from different experimental groups.

[0053] from Figure 8It is evident that the MARRS system (Ap-3xL2 recruiting ADAR1 system) constructed in this invention can achieve highly efficient RNA editing in vivo. Using an AAV-mediated in vivo delivery strategy, dual-luciferase reporter genes (Rluc and Fluc) containing TAG stop codons and MARRS gRNA driven by the CMV promoter were introduced into mice. Subsequent analysis of mouse liver tissue revealed that the firefly luciferase signal in the liver of mice treated with the MARRS system was significantly enhanced, indicating that the stop codon at the target site was successfully edited and the expression of the functional protein was effectively restored. Photon flux quantitative analysis showed that the luciferase activity in the MARRS-treated group was significantly increased compared to the control group. Furthermore, statistical analysis of the expression ratio of firefly luciferase to Renilla luciferase revealed a significant increase in the MARRS-treated group, further confirming the effective improvement in RNA editing efficiency. At the molecular level, sequencing analysis of reporter gene transcripts in mouse liver showed that significant RNA editing occurred at the target site with high efficiency. The corresponding Sanger sequencing map clearly showed A-to-G base conversion signals, and the editing effect was consistent with the positive control group, further confirming that the system of the present invention can achieve precise and effective RNA editing in vivo.

[0054] The above experimental results show that the MARRS system provided by this invention not only has excellent editing performance in vitro, but also has efficient RNA editing ability and good functional recovery effect in vivo. This result indicates that it has broad application prospects and potential value in the field of RNA targeted therapy.

[0055] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. An RNA editing system based on the recruitment of endogenous ADAR1 using multivalent RNA aptamers, characterized in that, It includes at least one RNA aptamer, said RNA aptamer being selected from any one or more of SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, and SEQ ID NO.

8.

2. The RNA editing system based on the recruitment of endogenous ADAR1 using multivalent RNA aptamers as described in claim 1, characterized in that, The binding domain of the RNA aptamer is ADAR1-Zα.

3. The RNA editing system based on the recruitment of endogenous ADAR1 by multivalent RNA aptamers as described in any one of claims 1 or 2, characterized in that, The secondary structure of the RNA aptamer includes an L2 circular structure, as shown in SEQ ID NO.6, SEQ ID NO.7, and SEQ ID NO.

8.

4. The RNA editing system based on the recruitment of endogenous ADAR1 using multivalent RNA aptamers as described in claim 3, characterized in that, The circular structure of the RNA aptamer is constructed via ribozyme-mediated self-circularization.

5. The RNA editing system based on the recruitment of endogenous ADAR1 using multivalent RNA aptamers as described in claim 4, characterized in that, The expression framework of the RNA aptamer comprises the following elements connected in sequence: (I) U6 promoter; (II) P3 type twister ribozyme; (III) A functional RNA sequence, wherein the functional RNA sequence contains the RNA aptamer; (Ⅳ) P1 type twister ribozyme.

6. The application of an RNA editing system based on the recruitment of endogenous ADAR1 by multivalent RNA aptamers, characterized in that, The RNA editing system based on the recruitment of endogenous ADAR1 by multivalent RNA aptamers as described in any one of claims 1 to 5 is used to prepare one or more of the following: drugs, gene function research tools, and disease models.

7. The application of the RNA editing system based on the recruitment of endogenous ADAR1 by multivalent RNA aptamers as described in claim 6, characterized in that, The drugs include those used in gene mutation-related genetic diseases.

8. The application of the RNA editing system based on the recruitment of endogenous ADAR1 by multivalent RNA aptamers as described in claim 7, characterized in that, The gene mutations include G>A point mutations.

9. The application of the RNA editing system based on the recruitment of endogenous ADAR1 by multivalent RNA aptamers as described in claim 6, characterized in that, The drug may also include pharmaceutically acceptable carriers or excipients.

10. The application of the RNA editing system based on the recruitment of endogenous ADAR1 by multivalent RNA aptamers as described in claim 8, characterized in that, The carrier includes an AAV carrier.

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