Method for site-specific modification of cyclization efficiency of anabaena ribozyme PIE carrier based on cryoelectron microscope structure

Abstract

The invention discloses a method for site-specific modification of the cyclization efficiency of an anabaena ribozyme PIE carrier based on a cryoelectron microscope structure. The RNA (Ribonucleic Acid) of the Anabaena mutant comprises at least one of an RNA (Ribonucleic Acid) of an Anabaena intron G37 mutant and an RNA of an Anabaena PIE POLR2A mutant. The invention also provides an application of the mutant in research of increasing the yield of circRNA. The method has the beneficial effects that cryo-EM is utilized to analyze a tertiary structure of Anabaena ribozyme in a cyclization reaction process, a complete molecular structure mechanism for forming circRNA is researched through a cryoelectron microscope technology, the Anabaena mutant RNA with improved cyclization efficiency as well as the preparation method and application thereof are provided accordingly, the provided mutant RNA can increase the yield of circRNA by 1-2 times, and the yield of circRNA is increased by 1-2 times. The mutant is obviously superior to a wild type and other site mutants.

Description

Technical Field

[0001] This invention relates to the field of RNA structural biology and cryo-electron microscopy, specifically to a method for site-specific structural modification of the circularization efficiency of the Anabaena ribozyme PIE vector based on cryo-electron microscopy. Background Technology

[0002] Circular RNAs (circRNAs) are a novel class of non-coding RNA molecules characterized by a single-stranded, covalently closed topological structure. Due to their unique circular structure, they are less susceptible to degradation by exonucleases and possess greater stability. Over the past decade, thanks to advancements in high-throughput RNA sequencing technology and bioinformatics tools, research in the field of circular RNAs has increased significantly, and more functions of circular RNAs have been discovered, such as serving as RNA aptamers, molecular sponges for miRNAs and proteins, antisense RNAs, regulating intracellular innate immune responses, protein translation vectors, and disease-related molecular biomarkers. These functions also make circular RNAs promising for applications in disease diagnosis and treatment, making them a promising target for drug development and gene therapy.

[0003] Currently, the in vitro synthesis of circular RNA mainly employs three technical approaches: chemical synthesis, ligase catalysis, and ribozyme catalysis. Among these, ribozyme catalysis based on self-splicing introns has become the most widely studied cyclization strategy due to its advantages such as mild reaction conditions and high cyclization efficiency. This technique utilizes the self-catalytic splicing properties of Group I and Group II introns to achieve efficient RNA cyclization by constructing a PIE (Permuted Intron-Exon) system. The main idea is to engineer introns using the PIE system, whose core structure, in the 5'→3' direction, contains the following functional elements: a 3' intron (as shown in SEQ ID NO.1), a truncated exon E1 (as shown in SEQ ID NO.3), the target gene, a truncated exon E2 (as shown in SEQ ID NO.4), and a 5' intron (as shown in SEQ ID NO.2). The cyclization process of this system involves two key steps: First, the guanosine cofactor attacks the E1 splice site, breaking the connection between E1 and the intron; then, the 3'-OH end of E1 attacks the E2 splice site, completing the cyclization of RNA.

[0004] However, some existing circular vector modifications are based on the molecular mechanisms of splicing reactions and the base pairing characteristics in primary and secondary sequences. These optimization methods mainly rely on empirical strategies such as multi-site combination mutations at splice sites, sequence insertions, or deletions. Furthermore, sequence-based modifications are often unpredictable and it is difficult to foresee the structural changes and functional impacts after modification. Additionally, they suffer from low circularization efficiency and low circRNA yield. Summary of the Invention

[0005] The technical problem to be solved by this invention is how to address the blindness and unpredictability of traditional circular vector modification, which leads to low circularization efficiency of mutants and low yield of circRNA.

[0006] The present invention solves the above-mentioned technical problems through the following technical means:

[0007] The first aspect of this invention proposes a method for improving cyclization efficiency. Anabaena mutant RNA, including Anabaena Intron G37 mutant RNA, Anabaena At least one of the PIE POLR2A mutant RNAs; Anabaena The sequence of the intron G37 mutant RNA is shown in SEQ ID NO: 11 or SEQ ID NO: 12. Anabaena The sequence of the PIE POLR2A mutant RNA is shown in SEQ ID NO: 17 or SEQ ID NO: 18.

[0008] The second aspect of the present invention is as described above. Anabaena Application of mutant RNA in studies on increasing circRNA production.

[0009] The third aspect of the present invention is as described above. Anabaena The method for preparing intron G37 mutant RNA includes the following steps: Anabaena A mutation is made at the 37th base G of the intron's wild-type DNA, replacing G with U or C, followed by transcription to obtain the desired result. Anabaena Intron G37U mutant RNA or Anabaena Intron G37C mutant RNA; Anabaena The sequence of the intron wild-type DNA is shown in SEQ ID NO.5.

[0010] The 37th base (G, U, C) is abbreviated as G37, U37, C37. The rest are similarly abbreviated as A57 and A84, respectively.

[0011] Single-point mutation: G37U (meaning the 37th base G is mutated to the base U; the rest are similar.) Double-point mutation: A57U / A84U (meaning that the base A at position 57 mutates to the base U, and the base A at position 84 mutates to the base U; the rest are similar.) The fourth aspect of the present invention is as described above. Anabaena The method for preparing the PIE POLR2A mutant includes the following steps: Anabaena A mutation is made at base G at position 564 of the wild-type DNA of PIE POLR2A, replacing G with U or C, followed by transcription to obtain the desired result. Anabaena The sequence of PIE POLR2A wild-type DNA is shown in SEQ ID NO.9.

[0012] The 564th bit is G, U, or C, abbreviated as G564, U564, or C564.

[0013] G564U (This means that the base G at position 564 is mutated to the base U; the rest are similar.) The fifth aspect of the present invention is as described above. Anabaena In the efficient preparation of G37 mutant RNA from introns Anabaena Applications in self-circular RNA.

[0014] The sixth aspect of the present invention is as described above. Anabaena Application of PIE POLR2A mutant RNA in the efficient preparation of POLR2A circular RNA.

[0015] The seventh aspect of the present invention provides a Anabaena An efficient method for preparing self-circular RNA includes the following steps: right Anabaena The G37 mutant RNA of the intron is heated (to denature it), a buffer solution is added, and it is incubated to obtain the product; the buffer solution includes HEPES-Na, MgCl2 and GTP.

[0016] Preferably, the heating conditions are: 88~92℃, 2~4min; more preferably 90℃, 3min.

[0017] Preferably, the final concentration of HEPES-Na is 48-52 mM, the final concentration of MgCl2 is 18-22 mM, and the final concentration of GTP is 1-3 mM. More preferably, they are 50 mM, 20 mM, and 2 mM, respectively.

[0018] Preferably, the incubation conditions are 36~38℃ for 0.6~1.2h, and more preferably 37℃ for 0.75h.

[0019] The eighth aspect of this invention provides an efficient method for preparing POLR2A circular RNA, comprising the following steps:Anabaena PIE POLR2A mutant RNA was heated, cooled, and then incubated with Tris-HCl solution, MgCl2 solution, and GTP solution to obtain the final product.

[0020] Preferably, the heating conditions are 68~72℃ for 2~4 minutes, and more preferably 70℃ for 3 minutes.

[0021] Preferably, the temperature is lowered to 52~57℃, and more preferably to 55℃.

[0022] Preferably, the final concentration of the Tris-HCl solution is 48~52mM, and more preferably 50mM.

[0023] Preferably, the final concentration of the MgCl2 solution is 8-12 mM, and more preferably 10 mM.

[0024] Preferably, the final concentration of the GTP solution is 1~3 mM, more preferably 2 mM.

[0025] Preferably, the incubation conditions are 52~57℃ for 1~7 min; more preferably 55℃ for 5 min.

[0026] The beneficial effects of this invention are as follows: 1. This invention utilizes cryo-EM for parsing. Anabaena The tertiary structure of ribozymes during cyclization was investigated. Key nucleotide residues were screened and mapped onto the PIE vector for site-specific structural optimization to improve the in vitro cyclization efficiency of target RNA and the yield of circular RNA. The complete molecular structure mechanism of circRNA formation was studied using cryo-electron microscopy, and based on this, methods to improve cyclization efficiency were proposed. Anabaena Mutant RNA, its preparation method and application: The proposed mutant RNA can increase circRNA production by 1-2 times, which is significantly better than wild type and other site mutants.

[0027] 2. This invention reveals for the first time using cryo-electron microscopy technology... Anabaena The mechanism by which class I introns self-splice to form the complete molecular structure of circRNA was discovered. The study found that the mechanism is similar to the first step of splicing but with independent site selection. By comparing the active sites before and after class I intron splicing, it was found that... AnabaenaIntrons achieve self-cyclization through unique structural rearrangements. First, the GG nucleotide added at the 5' end mimics the natural cofactor exoG, inducing a rearrangement of the G37 base in the J2 / 3 region, stabilizing the A1-GG cyclization site. Second, the P9.0 domain undergoes a significant conformational change, precisely locating the 3' ωG through A206 / G242 base stacking. Simultaneously, the conserved A1-U8 base pair in the P1 extension region and the wobbling acceptor motif composed of A57 / A84 jointly stabilize the cyclization site. Finally, the removal of GG completes the end-to-end ligation. This discovery provides a structural basis for the rational design of efficient cyclization vectors.

[0028] 3. This invention innovatively utilizes cryo-electron microscopy technology to... Anabaena The high-resolution atomic structure of class I introns was presented visually, and a novel strategy for site-specific optimization of molecular structure based on cryo-electron microscopy was proposed. This strategy involves analyzing the conformational changes of key residues in class I introns during cyclization using cryo-electron microscopy, and then precisely mutagenesing key sites in the cyclization process. This approach aims to further enhance molecular structure while preserving low immunogenicity. Anabaena The efficiency of PIE vector in in vitro synthesis of circRNA.

[0029] 3. Comparison of structural optimizations Anabaena The performance of the PIE POLR2A mutant and the TRIC-V2 cyclization system was compared, revealing that the TRIC-V2 system exhibited a 1.8-fold faster cyclization advantage in the initial stage of the reaction (within 1 minute). Anabaena PIE POLR2A achieves a higher final product yield after a 5-minute reaction. Anabaena PIE POLR2A ~65% vs TRIC-V2 ~57%). TRIC-V2 achieves rapid dynamics by minimizing the stent sequence, while... Anabaena PIE POLR2A, based on full-length structural optimization, retains the low immunogenicity advantage of the 27nt exogenous sequence and can be directly applied without complex vector reconstruction. More importantly, Anabaena PIE POLR2A, through rational mutations targeting key nucleotides (such as G564), not only significantly improves circulation efficiency but also maintains the simplicity of system operation, providing a highly efficient and practical solution for the large-scale preparation of circRNA.

[0030] 4. The innovation of this invention lies in the first-time analysis of... Anabaena A high-resolution atomic structure model of intron cyclization was developed, key functional residues were identified, and efficient [technology / method] was developed based on this model. Anabaena The PIE POLR2A mutant. This technology improves circRNA synthesis efficiency by approximately 1-2 times while preserving the 27nt exogenous sequence (ensuring low immunogenicity), providing a breakthrough solution for the large-scale preparation of circRNA.

[0031] Of course, implementing any product or method of the present invention does not necessarily require achieving all of the advantages described above at the same time. Attached Figure Description

[0032] Figure 1 In Embodiment 1 of the present invention Anabaena High-resolution atomic structure diagrams of multiple reaction conformations of ribozymes; Figure 2 In Embodiment 1 of the present invention Anabaena Diagram showing changes in key residues during ribozyme splicing; Figure 3 In Embodiment 2 of the present invention Anabaena Analysis of intron key nucleotide mutation results and circularization effects, specifically including denaturing gel electrophoresis images (A) and statistical results (B) of intron circularization effects of mutants such as WT and G37; denaturing gel electrophoresis images (C) and statistical results (D) of pre-tRNA self-splicing circularization effects of mutants such as WT and G37. Figure 4 In Embodiment 3 of the present invention Anabaena PIE POLR2A site-directed mutagenesis results analysis diagram; Figure 5 In Embodiment 3 of the present invention Anabaena Figure showing the comparison results of PIE POLR2A mutant RNA and TRIC circularization system. Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise defined, the technical terms used below have the same meaning as understood by those skilled in the art.

[0034] Unless otherwise specified, the test materials and reagents used in the following examples are commercially available or prepared by known methods.

[0035] Unless otherwise specified, all techniques or conditions described in the embodiments can be performed in accordance with the techniques or conditions described in the literature in this field or in the product manual. Unless otherwise specified, the quantitative experiments in the following embodiments are all repeated three times or more, and the results are averaged.

[0036] The sequence involved in this invention is as follows: 3' Intron Nucleotide Sequence (SEQ ID NO.1) AACAACAGAUAACUUACAGCUAGUCGGAAGGUGCAGAGACUCGACGGGAGCUACCCUAACGUCAAGACGAGGGUAAAGAGAGAGUCCAAUUCUCAAAGCCAAUAGGCAGUAGCGAAAGCUGCGGGAGAAUG 5' intron nucleotide sequence (SEQ ID NO.2) AAAUAAUUGAGCCUUAGAGAAGAAAUUCUUUAAGUGGAUGCUCUCAAACUCAGGGAAACCUAAAUCUAGCUAUAGACAAGGCAAUCCUGAGCCAAGCCGAAGUAGUAAUUAGUAAGUU E1 nucleotide sequence (SEQ ID NO.3) AGACGCUACGGACUU E2 nucleotide sequence (SEQ ID NO.4) AAAAUCCGUUGA Anabaena Intron wild-type DNA sequence (SEQ ID NO.5) AAATAATTGAGCCTTAGAGAAGAAATTCTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGCTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTTAACAACAGATAACTTACAGCTAGTCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCGGGAGAAATG Anabaena Intron wild-type RNA sequence (SEQ ID NO.6) AAAUAAUUGAGCCUUAGAGAAGAAAUUCUUUAAGUGGAUGCUCUCAAACUCAGGGAAACCUAAAUCUAGCUAUAGACAAGGCAAUCCUGAGCCAAGCCGAAGUAGUAAUUAGUAAGUUAACAACAGAUAACUUACAGCUAGUCGGAAGGUGCAGAGACUCGACGGGAGCUACCCUAACGUCAAGACGAGGGUAAAGAGAGAGUCCAAUUCUCAAAGCCAAUAGGCAGUAGCGAAAGCUGCGGGAGAAUG Anabaena Full-length nucleotide sequence of wild-type pre-tRNA (SEQ ID NO.7) GGGGGUGUGGCGGAAUGGUAGACGCUACGGACUUAAAUAAUUGAGCCUUAGAGAAGAAAUUCUUUAAGUGGAUGCUCUCAAACUCAGGGAAACCUAAAUCUAGCUAUAGACAAGGCAAUCCUGAGCCAAGCCGAAGUAGUAAUUAGUAAGUUAACAACAGAUAACUUACAGCUAGUCGGAAGGUGCAGAGACUCGACGGGAGCUACCCUAACGUCAAGACGAGGGUAAAGAGAGAGUCCAAUUCUCAAAGCCAAUAGGCAGUAGCGAAAGCUGCGGGAGAAUGAAAAUCCGUUGACCUUAAACGGUCGUGUGGGUUCAAGUCCCUCCACCCCCA Anabaena PIE nucleotide sequence (SEQ ID NO.8) AACAACAGAUAACUUACAGCUAGUCGGAAGGUGCAGAGACUCGACGGGAGCUACCCUAACGUCAAGACGGGUAAAGAGAGAGUCCAAUUCUCAAAGCCAAUAGGCAGUAGCGAAAGCUGCGGGAGAAUGAAAUCCGUUGACCUAACGUACGUGGUGGGUUCAAG UCCCUCCACCCCCAGGGGGUGUGGCGAAUGGUAGACGCUACGGACUUAAAUAUUGAGCCUUAGAGAAAAUUCUUUAAGUGGAUGCUCUCAAACUCAGGGAAACCUAAAUCUAGCUAUAGACAAGGCAAUCCUGAGCCAAGCCGAAGUAGUAAUUAGUAAGUUGGA GACCCUCGACCGUCGAUUGUCCACUGGUCAACAAUAGAUGACUUACAACUAAUCGGAAGGUGCAGAGACUCGACGGGAGCUACCCUACUCAAGACGAGGUAAAGAGAGAGUCCAAUUCUCAAAGCCAAUAGGCAGUAGCGAAAGCUGCAAGAGAAUGAAAAUCCGU UGAAGACGCUACGGACUUAAAUAAUUGAGCCUUAAAGAAGAAAUUCUUUAAGUGGAUGCUCUCAAACUCAGGGAAACCUAAAUCUAGUUAGACAAGGCAAUCCUGAGCCAAGCCGAAGUAGUAAUUAGUAAGACCAGUGGACAAUCGACGGAUAACAGCAUAUCUAG Anabaena PIE POLR2A cross-linked DNA (SEQ ID NO.9) GGGAGACCCTCGACCGTCGATTGTCCACTGGTCAACAATAGATGACTTACAACTAATCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCAAGAGAATGAAAAT CCGTTGACCGGCAGCCAACTCTGCACAAAATGTCCATGATGGGGCATCGGGTCCGCATTCTCCCATGGTCTACCTTTCGCTTGAATCTTAGTGACAACTCCGTACAATGCAGACTTTGACGGGGATGAGATGAACTTGCACCTGCCAGTCTCTGGAGACGCGAGCA GAGATCCAGGAGCTGGCCATGGTTCCTCGCATGATTGTCACCCCCCAGAGCAATCGGCCTGTCATGGGTATTGTGCAGGACACTCACAGCAGTGCGCAAATTCACCAAGAGAGACGTCTTCCTGGAGCGGGTGGAACGGCACATGTGATGGGGACATTGTTATCT TCAAAGACGCTACGGACTTAAATTGAGCCTTAAAGAAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGCAGTGGACAATCCGACGGATAACAGCATCATTCTAG Anabaena PIE POLR2A RNA(SEQ ID NO.10) GGGAGACCCUCGACCGUCGAUUGUCCACUGGUCAACAAUAGAUGACUUACAACUAAUCGGAAGGUGCAGAGACUCGACGGGAGCUACCCUAACGUCAAGACGAGGGUAAAGAGAGAGUCCAAUUCUCAAAGCCAAUAGGCAGUAGCGAAAGCUGCAAGAGAAUGAAAAUCCGUUGACCGGCAGCCAACUCUGCACAAAAUGUCCAUGAUGGGGCAUCGGGUCCGCAUUCUCCCAUGGUCUACCUUUCGCUUGAAUCUUAGUGUGACAACUCCGUACAAUGCAGACUUUGACGGGGAUGAGAUGAACUUGCACCUGCCACAGUCUCUGGAGACGCGAGCAGAGAUCCAGGAGCUGGCCAUGGUUCCUCGCAUGAUUGUCACCCCCCAGAGCAAUCGGCCUGUCAUGGGUAUUGUGCAGGACACACUCACAGCAGUGCGCAAAUUCACCAAGAGAGACGUCUUCCUGGAGCGGGUGGAACGGCACAUGUGUGAUGGGGACAUUGUUAUCUUCAAAGACGCUACGGACUUAAAUAAUUGAGCCUUAAAGAAGAAAUUCUUUAAGUGGAUGCUCUCAAACUCAGGGAAACCUAAAUCUAGUUAUAGACAAGGCAAUCCUGAGCCAAGCCGAAGUAGUAAUUAGUAAGACCAGUGGACAAUCGACGGAUAACAGCAUAUCUAG Anabaena Intron G37U mutant RNA sequence (SEQ ID NO.11) AAAUAAUUGAGCCUUAGAGAAGAAAUUCUUUAAGUG UAUGCUCUCAAACUCAGGGAAACCUAAAUCUAGCUAUAGACAAGGCAAUCCUGAGCCAAGCCGAAGUAGUAAUUAGUAAGUAAACAACAGAUAACUUACAGCUAGUCGGAAGGUGCAGAGACUCGACGGGAGCUACCCUACUAGACGAGGGUAAGAGAGAGAGAGUCCAAUUCUCAAAGCCAAAGGAGAGGAGAGAGGAGAGAGAGAGACAAGGAGCUACCUACAGCUAGUCGGAAGGUGCAGAGACUCGACGGGAGCUACCUAACGAGAGAGGAGAGAGAGAGAGAGUCCAAUUCUCAAAGCCAAAGGAGAGAGGAGGAGAGAGAGAGAGACAACAGAUAACUACAGCUAGUCGGAAGGUGCAGAGACUCGACGGGA Anabaena G37C RNA (SEQ ID NO.12) AAAUAAUUGAGCCUUAGAGAAGAAAUUCUUUAAGUG C AUGCUCUCAAACUCAGGGAAACCUAAAUCUAGCUAUAGACAAGGCAAUCCUGAGCCAAGCCGAAGUAGUAAUUAGUAAGUAAACAACAGAUAACUUACAGCUAGUCGGAAGGUGCAGAGACUCGACGGGAGCUACCCUACUAGACGAGGGUAAGAGAGAGAGAGUCCAAUUCUCAAAGCCAAAGGAGAGGAGAGAGGAGAGAGAGAGACAAGGAGCUACCUACAGCUAGUCGGAAGGUGCAGAGACUCGACGGGAGCUACCUAACGAGAGAGGAGAGAGAGAGAGAGUCCAAUUCUCAAAGCCAAAGGAGAGAGGAGGAGAGAGAGAGAGACAACAGAUAACUACAGCUAGUCGGAAGGUGCAGAGACUCGACGGGA Anabaena A57U / A84U RNA sequence (SEQ ID NO.13) AAAUAAUUGAGCCUUAGAGAAGAAAUUCUUUAAGUGGAUGCUCUCAAACUCAGGGA U ACCUAAAUCUAGCUAGACAAGGCA U UCCUGAGCCAAGCCGAAGUAGUAAUUAGUAAGUAAACAACAGAUAACUACAGCUAGUCGGAAGGUGCAGAGACUCGACGGGAGCUACCCUACUCAAGACGAGGUAAAGAGAGAGUCCAAUUCUCAAAGCCAAUAGGCAGUAGCGAAAGCUGCGGGAGAAUG Anabaena A57C / A84C RNA sequence (SEQ ID NO.14) AAAUAAUUGAGCCUUAGAGAAGAAAUUCUUUAAGUGGAUGCUCUCAAACUCAGGGA C ACCUAAAUCUAGCUAUAGACAAGGCA C UCCUGAGCCAAGCCGAAGUAGUAAUUAGUAAGUUAACAACAGAUAACUUACAGCUAGUCGGAAGGUGCAGAGACUCGACGGGAGCUACCCUAACGUCAAGACGAGGGUAAAGAGAGAGUCCAAUUCUCAAAGCCAAUAGGCAGUAGCGAAAGCUGCGGGAGAAUG Anabaena Intron A206U mutant RNA sequence (SEQ ID NO.15) AAAUAAUUGAGCCUUAGAGAAGAAAUUCUUUAAGUGGAUGCUCUCAAACUCAGGGAAACCUAAAUCUAGCUAUAGACAAGGCAAUCCUGAGCCAAGCCGAAGUAGUAAUUAGUAAGUUAACAACAGAUAACUUACAGCUAGUCGGAAGGUGCAGAGACUCGACGGGAGCUACCCUAACGUCAAGACGAGGGUAAAGAGAGAGUCC U AUUCUCAAAGCCAAUAGGCAGUAGCGAAAGCUGCGGGAGAAUG Anabaena Intron A206C mutant RNA sequence (SEQ ID NO.16) AAAUAAUUGAGCCUUAGAGAAGAAAUUCUUUAAGUGGAUGCUCUCAAACUCAGGGAAACCUAAAUCUAGCUAUAGACAAGGCAAUCCUGAGCCAAGCCGAAGUAGUAAUUAGUAAGUUAACAACAGAUAACUUACAGCUAGUCGGAAGGUGCAGAGACUCGACGGGAGCUACCCUAACGUCAAGACGAGGGUAAAGAGAGAGUCC C AUUCUCAAAGCCAAUAGGCAGUAGCGAAAGCUGCGGGAGAAUG Anabaena PIE POLR2A G564U mutant RNA sequence (SEQ ID NO.17) GGGAGACCCUCGACCGUCGAUUGUCCACUGGUCAACAAUAGAUGACUUACAACUAAUCGGAAGGUGCAGAGACUCGACGGGAGCUACCCUAACGUCAAGACGAGGGUAAAGAGAGAGUCCAAUUCUCAAAGCCAAUAGGCAGUAGCGAAAGCUGCAAGAGAAUGAAAAUCCGUUGACCGGCAGCCAACUCUGCACAAAAUGUCCAUGAUGGGGCAUCGGGUCCGCAUUCUCCCAUGGUCUACCUUUCGCUUGAAUCUUAGUGUGACAACUCCGUACAAUGCAGACUUUGACGGGGAUGAGAUGAACUUGCACCUGCCACAGUCUCUGGAGACGCGAGCAGAGAUCCAGGAGCUGGCCAUGGUUCCUCGCAUGAUUGUCACCCCCCAGAGCAAUCGGCCUGUCAUGGGUAUUGUGCAGGACACACUCACAGCAGUGCGCAAAUUCACCAAGAGAGACGUCUUCCUGGAGCGGGUGGAACGGCACAUGUGUGAUGGGGACAUUGUUAUCUUCAAAGACGCUACGGACUUAAAUAAUUGAGCCUUAAAGAAGAAAUUCUUUAAGUG U AUGCUCUCAAACUCAGGGAAACCUAAAUCUAGUUAUAGACAAGGCAAUCCUGAGCCAAGCCGAAGUAGUAAUUAGUAAGACCAGUGGACAAUCGACGGAUAACAGCAUAUCUAG Anabaena PIE POLR2A G564C mutant RNA sequence (SEQ ID NO.18) GGGAGACCCUCGACCGUCGAUUGUCCACUGGUCAACAAUAGAUGACUUACAACUAAUCGGAAGGUGCAGAGACUCGACGGGAGCUACCCUAACGUCAAGACGAGGGUAAAGAGAGAGUCCAAUUCUCAAAGCCAAUAGGCAGUAGCGAAAGCUGCAAGAGAAUGAAAAUCCGUUGACCGGCAGCCAACUCUGCACAAAAUGUCCAUGAUGGGGCAUCGGGUCCGCAUUCUCCCAUGGUCUACCUUUCGCUUGAAUCUUAGUGUGACAACUCCGUACAAUGCAGACUUUGACGGGGAUGAGAUGAACUUGCACCUGCCACAGUCUCUGGAGACGCGAGCAGAGAUCCAGGAGCUGGCCAUGGUUCCUCGCAUGAUUGUCACCCCCCAGAGCAAUCGGCCUGUCAUGGGUAUUGUGCAGGACACACUCACAGCAGUGCGCAAAUUCACCAAGAGAGACGUCUUCCUGGAGCGGGUGGAACGGCACAUGUGUGAUGGGGACAUUGUUAUCUUCAAAGACGCUACGGACUUAAAUAAUUGAGCCUUAAAGAAGAAAUUCUUUAAGUG C AUGCUCUCAAACUCAGGGAAACCUAAAUCUAGUUAUAGACAAGGCAAUCCUGAGCCAAGCCGAAGUAGUAAUUAGUAAGACCAGUGGACAAUCGACGGAUAACAGCAUAUCUAG TRIC-V2 POLR2A nucleotide sequence (SEQ ID NO.19) GAUAAUACGGUCAAUCAGUUGGCUUCCGAAAUAAUUGAGCCUUAGAGAAGAAAUUCUUUAAGUGGAUGCUCUCAAACUCAGGGAAACCUAAAUCUAGCUAUAGACAAGGCAAUCCUGAGCCAAGCCGAAGUAGUAAUUAGUAAGUUAACAACAGAUAACUUACAGCUAAUCGGAAGGUGCAGAGACUCGACGGGAGCUACCCUAACGUCAAGACGAGGGUAAAGAGAGAGUCCAAUUCUCAAAGCCAAUAGGCAGUAGCGAAAGCUGCGGGAGAAUGAAAAUCCGUAGCGUCUCGCCGGUAACGCAGAAAAACAAAAAACAAAAAAAACAAAAAAAAAACCAAAAAAACAAAACACACCGGCAGCCAACUCUGCACAAAAUGUCCAUGAUGGGGCAUCGGGUCCGCAUUCUCCCAUGGUCUACCUUUCGCUUGAAUCUUAGUGUGACAACUCCGUACAAUGCAGACUUUGACGGGGAUGAGAUGAACUUGCACCUGCCACAGUCUCUGGAGACGCGAGCAGAGAUCCAGGAGCUGGCCAUGGUUCCUCGCAUGAUUGUCACCCCCCAGAGCAAUCGGCCUGUCAUGGGUAUUGUGCAGGACACACUCACAGCAGUGCGCAAAUUCACCAAGAGAGACGUCUUCCUGGAGCGGGUGGAACGGCACAUGUGUGAUGGGGACAUUGUUAUCUUCAAAAAAAACAAAAAACAAAACGGCUAUUAUGCGUUACCGGCGAGACGCUACGGACUUAAACACCCGGAAGCCAACUGAUUGACCAAAAAAAAAAAA Example 1: 1.1 Anabaena Ribozyme (i.e., Anabaena intron) cryo-EM structure analysis In a reaction environment of 4℃ and 100% humidity, 8-22μM RNA samples (SEQ ID NO.7) were used to control the reaction rate and intermediate content by adjusting the magnesium ion concentration (0-20 mM) and reaction time (5-60 minutes). Then, by changing different specifications of 2 / 1 or 1.2 / 13, different metal properties of Au or Cu grids, and different filter paper adsorption times, frozen samples with uniform thickness, uniform particle distribution, and complete RNA folding were further prepared for 300kV cryo-electron microscopy data acquisition.

[0037] The system was reconstructed using Relion and CryoSPARC software. The process was as follows: Motion Correction to eliminate the influence of sample drift, Contrast Transfer Function Correction (CTF) to compensate for phase reversal, and Deep Learning-assisted automatic particle selection. High-quality particles were screened through multiple rounds of 2D classification. Different conformational states were separated by 3D classification, an initial 3D density map was established, and iterative optimization was performed.

[0038] Finally, an initial atomic model was obtained based on the Alphafold3 prediction model or by de novo modeling using EMRNA / Auto-DRAFFTER. The model was then dynamically matched to the electron microscopy density map using molecular dynamics flexible fitting (MDFF). Finally, manual adjustments were made using Coot, and geometric constraint optimization and B-factor refinement were performed using Phenix, ultimately yielding an atomic model with accurate stereochemical parameters. This workflow ensures end-to-end quality control from raw data to a high-precision atomic model.

[0039] 1.2 Anabaena Ribozyme (i.e.) Anabaena Dynamic conformational changes during intron cyclization The cryo-electron microscopy technique was successfully used to resolve the [the following]. Anabaen Atomic resolution structures of four key functional states during RNA cyclization: the Apo state (3.38 Å) fully reveals the native folded conformation of the ribozyme; the Pre-1S state (3.46 Å) reveals the specific binding site of the exoG nucleotide and the induced conformational changes; the Linear intron state (3.43 Å) captures the dynamic process of 5' splice site activation and G37 base rearrangement; and the Circular intron state (3.45 Å) demonstrates the structural features of covalent cyclization. This study is the first to resolve the multi-state dynamic conformational changes of the entire RNA cyclization process at the atomic level, elucidating the... Anabaena The molecular mechanism of ribozyme self-splicing provides a precise structural basis for the rational design of efficient cyclization systems.

[0040] like Figure 1The image shows cryo-electron microscopy three-dimensional reconstructions of pre-tRNA (Leu) and its introns at different reaction stages. The first image from the left shows the ligand-free Apo state, where the magenta region indicates the internal guide sequence and its extension region IGS / IGSext, and circles indicate the positions of the 3' and 5' ends. The second image from the left shows the Pre-1S state, where the magenta region indicates the P1 helix formed by substrate pairing and the P1ext extension region formed by intron self-pairing, and the 3' and 5' ends are marked. The second image from the right shows the Linear intron after pre-tRNA (Leu) releases tRNA, where the magenta region indicates the P1 / P1ext region, and circles indicate the positions of the 5' and 3' ends. The first image from the right shows the Circular intron after further circularization of the Linear intron, where the magenta region indicates the P1ext region, and circles indicate the positions of the covalently linked 5' and 3' ends. Anabaena Changes in key residues during ribozyme splicing, such as Figure 2 As shown in the figure, the three-dimensional structural comparison between the Pre-1S state (gray) and the linear intron (blue) demonstrates the conformational differences during splicing. The smaller figures show magnified structural details of the P1 extension region (P1ext) in the linear intron. The schematic diagram of the reorientation of G37 residue in the J2 / 3 region shows its conformational adjustment that stabilizes the P1ext structure and promotes intron cyclization through base stacking. The detailed structural changes of the guanosine binding site (G-bindingsite) show the conformational shifts of key residues (such as G249, A207, etc.).

[0041] Example 2: Anabaena Ribozymes (i.e.) Anabaena Key nucleotide mutations at intron cyclization sites 2.1 Anabaena Preparation of intron mutant RNA: Anabaena The method for preparing intron G37U mutant RNA includes the following steps: Using cryo-electron microscopy structure-guided systematic mutation analysis, in vitro point mutation primers were designed to target... AnabaenaA mutation was made at base G at position 37 of the wild-type intron DNA (SEQ ID NO. 5). The mutant nucleotide G37U (representing the mutation of base G at position 37 to base U) was introduced via plasmid PCR, resulting in a plasmid containing the mutated U. This plasmid was then re-transformed into DH5α competent cells, and sequencing was performed using universal primers to confirm the correct introduction of the mutated residue. Subsequently, the plasmid was extracted, and the DNA template was amplified by in vitro PCR. Alcohol precipitation yielded a single DNA band. Further in vitro transcription and purification of the DNA yielded a single RNA band; thus, the RNA was obtained. Anabaena Intron G37U mutant RNA (SEQ ID NO.11).

[0042] Similarly, the preparation of the remaining mutants is as follows: Replace "G37U" with "G37C", leaving everything else unchanged, and you will get the result. Anabaena Intron G37U mutant RNA (SEQ ID NO. 12).

[0043] Replace “G37U” with “A206U”, leaving the rest unchanged, and you will get the result. Anabaena Intron A206U mutant RNA (SEQ ID NO.15).

[0044] Replace “G37U” with “A206C”, leaving the rest unchanged, and you will get the result. Anabaena Intron A206C mutant RNA (SEQ ID NO.16).

[0045] Replace “G37U” with “A57U / A84U”, leaving the rest unchanged, and you will get the result. Anabaena Intron A57U / A84U mutant RNA (SEQ ID NO.13).

[0046] Replace “G37U” with “A57C / A84C”, leaving the rest unchanged, and you will get the result. Anabaena Intron A57C / A84C mutant RNA (SEQ ID NO.14).

[0047] 2.2 The above-prepared Anabaena Intron mutant RNA in efficient preparation Anabaena Applications in self-circular RNA (applications in studies to increase circRNA yield): by Anabaena Taking intron G37U mutant RNA as an example, an in vitro circularization reaction was performed to prepare... Anabaena Self-circular RNA (G37U), including the following steps: right AnabaenaIntron G37U mutant RNA was heated to 90°C for 3 minutes (to denature it), then a buffer solution consisting of 50 mM HEPES-Na (pH 7.8), 20 mM MgCl2, and 2 mM GTP was added, and the mixture was incubated at 37°C for 45 minutes. (Incubation times can also be 1 minute, 5 minutes, 15 minutes, 36 minutes, 60 minutes, or 72 minutes, etc.) Similarly, Will" Anabaena Replace "intron G37U mutant RNA" with " Anabaena The intron wild-type RNA sequence (denoted as WT) remains unchanged, thus obtaining the RNA prepared using WT. Anabaena Self-circular RNA (as control group) Will" Anabaena Replace the intron G37U mutant RNA with other mutant RNAs, leaving everything else unchanged, to obtain RNAs obtained using other mutant methods. Anabaena Self-circular RNA.

[0048] Compare Anabaena Intron wild-type RNA and Anabaena The levels of self-circularization products of intron mutant RNA were as follows: Figure 3 As shown, it reveals Anabaena The functional mechanisms of key nucleotides such as G37, A57 / A84, and A206 during ribozyme cyclization were investigated. The study found that base rearrangement of G37 promotes cyclization by stabilizing the P1 extension region. A single-point mutation (G37U / C) in G37, compared to the wild type, can increase circRNA yield by 1-2 times, while other single-point mutations (A206U / C) or double-point mutations (A57U / A84U, A57C / A84C) do not significantly increase circRNA yield. The wobbling receptor motif A57 / A84 precisely locates the cyclization site by recognizing the A1-U8 base pair, while the A206 mutation in the P9.0 region significantly affects the localization efficiency of ωG.

[0049] Example 3: Anabaena Preparation, application, and comparison with the TRIC cyclization system of PIE POLR2A mutant RNA. 3.1 Anabaena Preparation method of PIE POLR2A mutant RNA by Anabaena Taking the PIE POLR2A G564U mutant RNA as an example, the following steps are included: right AnabaenaA mutation was made at base G at position 564 of the wild-type DNA of PIE POLR2A (SEQ ID NO.9). The mutant nucleotide G564U (representing the mutation of base G at position 564 to base U) was introduced via plasmid PCR, resulting in a plasmid containing the mutated U. This plasmid was then re-transformed into DH5α competent cells, and sequencing was performed using universal primers to confirm the correct introduction of the mutant residue. Subsequently, the plasmid was extracted, and the DNA template was amplified by in vitro PCR. Alcohol precipitation yielded a single DNA band. Further in vitro transcription and purification of the DNA yielded a single RNA band; thus, the RNA was obtained. Anabaena PIE POLR2A G564U mutant RNA (SEQ ID NO.17).

[0050] Similarly, replacing "G564U" with "G564C" while keeping everything else unchanged, yields the result. Anabaena PIE POLR2A G564C mutant RNA (SEQ ID NO.18).

[0051] 3.2 Anabaena Application of PIE POLR2A mutant RNA in the efficient preparation of POLR2A circular RNA (Application in research on increasing circRNA yield): by Anabaena Taking the PIE POLR2A G564U mutant RNA as an example, an in vitro circularization reaction was performed to prepare POLR2A circular RNA (G564U), including the following steps: Will Anabaena PIE POLR2A G564U mutant RNA was heated at 70 °C for 3 min, cooled to 55 °C and incubated for 1 min. A buffer solution consisting of 50 mM Tris-HCl (pH 7.4), 10 mM MgCl2, 1 mM DTT, and 2 mM GTP was added, and the mixture was incubated for 5 min. (Incubation times can also be 1 min, 3 min, 5 min, or 7 min).

[0052] Similarly, Will" Anabaena Replace "PIE POLR2A G564U mutant RNA" with " Anabaena "PIE POLR2A G564C mutant RNA", with everything else unchanged, to obtain POLR2A circular RNA (G564C).

[0053] Will" Anabaena Replace "PIE POLR2A G564U mutant RNA" with " Anabaena PIE POLR2A wild-type RNA”, with everything else unchanged, yielded POLR2A circular RNA (wild-type WT), which served as a control.

[0054] Anabaena PIE POLR2A wild type and Anabaena Denaturing gel electrophoresis images and statistical results of the effects of PIE POLR2A mutants on the generation of cyclic POLR2A are shown below. Figure 4 As shown in the figure, it can be seen that the following is adopted: Anabaena The circRNA production of the PIE POLR2A mutant RNA was significantly better than that of the PIE POLR2A mutant RNA. Anabaena PIE POLR2A wild-type RNA.

[0055] 3.3 Performance comparison with TRIC circulatory system: The in vitro cyclization reaction was performed under the following conditions: denaturation at 95 °C for 2 min, followed by immediate cooling on ice for 3 min. Then, a reaction solution of 50 mM Tris-HCl (pH 7.4), 10 mM MgCl2, 1 mM DTT, and 2 mM GTP was added, and the mixture was incubated at 55 °C for 0, 1, and 5 min to compare the optimized structures. Anabaena Performance of the PIE POLR2A mutant with the TRIC-V2 cyclization system.

[0056] TRIC-V2 and Anabaena Denaturing gel electrophoresis images and statistical results of the effect of PIE POLR2A mutant in generating cyclic POLR2A are shown below. Figure 5 As shown, Anabaena Compared to the TRIC-V2 system, the PIE POLR2A mutant RNA showed a significant increase in circRNA yield and better product uniformity. Furthermore, the TRIC-V2 system exhibited a 1.8-fold faster circularization advantage in the initial reaction phase (within 1 minute). Anabaena PIE POLR2A achieves a higher final product yield after a 5-minute reaction. Anabaena PIE POLR2A ~65% vs TRIC-V2 ~57%). TRIC-V2 achieves rapid dynamics by minimizing the stent sequence, while... Anabaena PIE POLR2A, based on full-length structural optimization, retains the low immunogenicity advantage of the 27nt exogenous sequence and can be directly applied without complex vector reconstruction. More importantly, Anabaena PIE POLR2A, through rational mutations targeting key nucleotides (such as G564), not only significantly improves circulation efficiency but also maintains the simplicity of system operation, providing a highly efficient and practical solution for the large-scale preparation of circRNA.

[0057] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. 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. Such 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. A method for improving cyclization efficiency Anabaena Mutant RNA, characterized by, include Anabaena Intron G37 mutant RNA, Anabaena At least one of the PIE POLR2A mutant RNAs; Anabaena The sequence of the intron G37 mutant RNA is shown in SEQ ID NO: 11 or SEQ ID NO:

12. Anabaena The sequence of the PIE POLR2A mutant RNA is shown in SEQ ID NO: 17 or SEQ ID NO:

18.

2. The claim 1 Anabaena Application of mutant RNA in studies on increasing circRNA production.

3. A kind Anabaena A method for preparing intron G37 mutant RNA, characterized in that, Includes the following steps: right Anabaena The 37th base G of the intron's wild-type DNA is mutated to U or C, and then transcribed to obtain the desired result; Anabaena The sequence of the intron wild-type DNA is shown in SEQ ID NO. 5; Anabaena The sequence of the intron G37 mutant RNA is shown in SEQ ID NO: 11 or SEQ ID NO:

12.

4. A kind Anabaena The method for preparing the PIE POLR2A mutant is characterized by, Includes the following steps: right Anabaena A mutation is made at base G at position 564 of the wild-type DNA of PIE POLR2A, replacing G with U or C, followed by transcription to obtain the desired result. Anabaena The sequence of PIE POLR2A wild-type DNA is shown in SEQ ID NO. 9; Anabaena The sequence of the PIEPOLR2A mutant RNA is shown in SEQ ID NO: 17 or SEQ ID NO:

18.

5. The preparation method according to claim 3 yields... Anabaena In the efficient preparation of G37 mutant RNA from introns Anabaena Applications in self-circular RNA.

6. The preparation method according to claim 4 yields... Anabaena Application of PIE POLR2A mutant RNA in the efficient preparation of POLR2A circular RNA.

7. A kind Anabaena A method for preparing self-circular RNA, characterized in that, Includes the following steps: right Anabaena The intron G37 mutant RNA was heated, buffer solution was added, and incubated to obtain the desired result. The buffer solution comprises HEPES-Na, MgCl2, and GTP; Anabaena The sequence of the intron G37 mutant RNA is shown in SEQ ID NO: 11 or SEQ ID NO:

12.

8. The preparation method according to claim 7, characterized in that, The heating conditions are: 88~92℃, 2~4min; the final concentration of HEPES-Na is 48~52mM, the final concentration of MgCl2 is 18~22mM, and the final concentration of GTP is 1~3mM; the incubation conditions are: 36~38℃, 0.6~1.2h.

9. A method for preparing POLR2A circular RNA, characterized in that, Includes the following steps: right Anabaena The PIEPOLR2A mutant RNA was heated, cooled, and then incubated with Tris-HCl solution, MgCl2 solution, and GTP solution to obtain the final product.

10. The preparation method according to claim 9, characterized in that, The heating conditions are 68~72℃ for 2~4 min; cooling to 52~57℃; the final concentration of the Tris-HCl solution is 48~52 mM; the final concentration of the MgCl2 solution is 8~12 mM; the final concentration of the GTP solution is 1~3 mM; the incubation conditions are 52~57℃ for 1~7 min.

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