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CN-122012500-A - Method for improving cyclization efficiency of anabaena ribozyme PIE vector based on fixed-point refrigeration electron microscope structure

CN122012500ACN 122012500 ACN122012500 ACN 122012500ACN-122012500-A

Abstract

The invention discloses a method for improving cyclization efficiency of anabaena ribozyme PIE vector based on fixed-point of a freeze electron microscope structure. The Anabaena mutant RNA comprises at least one of Anabaena intron G37 mutant RNA and AnabaenaPIE POLR A mutant RNA. The application of the mutant in the research of improving the circRNA yield is also proposed. The invention has the beneficial effects that the three-level structure of cryo-EM analysis Anabaena ribozyme in the cyclization reaction process is utilized, the complete molecular structure mechanism of the formed circRNA is researched by a freeze electron microscope technology, anabaena mutant RNA with improved cyclization efficiency is provided according to the complete molecular structure mechanism, and a preparation method and application thereof, and the provided mutant RNA can improve the output of the circRNA by 1-2 times and is obviously superior to wild type mutants and other site mutants.

Inventors

  • ZHANG KAIMING
  • LI SHANSHAN
  • ZHANG XIAOJING

Assignees

  • 合肥综合性国家科学中心大健康研究院
  • 中国科学技术大学

Dates

Publication Date
20260512
Application Date
20260213

Claims (10)

  1. 1. The Anabaena mutant RNA with improved cyclization efficiency is characterized by comprising at least one of Anabaena intron G37 mutant RNA and AnabaenaPIE POLR A mutant RNA, wherein the sequence of the Anabaena intron G37 mutant RNA is shown as SEQ ID NO. 11 or SEQ ID NO. 12, and the sequence of the AnabaenaPIE POLR2A mutant RNA is shown as SEQ ID NO. 17 or SEQ ID NO. 18.
  2. 2. Use of the Anabaena mutant RNA of claim 1 in a study to enhance the yield of circRNA.
  3. 3. A preparation method of Anabaena intron G37 mutant RNA is characterized by comprising the following steps of mutating 37 th base G of Anabaena intron wild type DNA, mutating G into U or C, and then carrying out transcription, wherein the sequence of Anabaena intron wild type DNA is shown as SEQ ID NO.5, and the sequence of Anabaena intron G37 mutant RNA is shown as SEQ ID NO. 11 or SEQ ID NO. 12.
  4. 4. A preparation method of AnabaenaPIE POLR A mutant is characterized by comprising the steps of mutating 564 th base G of AnabaenaPIE POLR A wild type DNA, mutating G into U or C, and then transcribing, wherein the sequence of AnabaenaPIE POLR A wild type DNA is shown as SEQ ID NO.9, and the sequence of AnabaenaPIE POLR A mutant RNA is shown as SEQ ID NO. 17 or SEQ ID NO. 18.
  5. 5. Use of Anabaena intron G37 mutant RNA produced by the production method of claim 3 for efficient production of Anabaena self circular RNA.
  6. 6. The use of AnabaenaPIE POLR a mutant RNA prepared by the method of claim 4 for the efficient preparation of POLR2A circular RNA.
  7. 7. A method for preparing Anabaena self-loop RNA, comprising the steps of: heating Anabaena intron G37 mutant RNA, adding a buffer solution, and incubating to obtain the recombinant DNA; The buffer solution comprises HEPES-Na, mgCl 2 and GTP, and the sequence of the Anabaena intron G37 mutant RNA is shown as SEQ ID NO. 11 or SEQ ID NO. 12.
  8. 8. The method according to claim 7, wherein the heating is performed at 88-92 ℃ for 2-4 min, the final concentration of HEPES-Na is 48-52 mM, the final concentration of MgCl 2 is 18-22 mM, the final concentration of GTP is 1-3 mM, and the incubation is performed at 36-38 ℃ for 0.6-1.2 h.
  9. 9. A preparation method of POLR2A annular RNA is characterized by comprising the following steps of heating AnabaenaPIE POLR A mutant RNA, cooling, adding Tris-HCl solution, mgCl 2 solution and GTP solution, and incubating.
  10. 10. The preparation method according to claim 9, wherein the heating condition is 68-72 ℃ for 2-4 min, the temperature is reduced to 52-57 ℃, the final concentration of Tris-HCl solution is 48-52 mM, the final concentration of MgCl 2 solution is 8-12 mM, the final concentration of GTP solution is 1-3 mM, and the incubation condition is 52-57 ℃ for 1-7 min.

Description

Method for improving cyclization efficiency of anabaena ribozyme PIE vector based on fixed-point refrigeration electron microscope structure Technical Field The invention relates to the technical field of RNA structure biology and frozen electron microscopy, in particular to a method for improving cyclization efficiency of anabaena ribozyme PIE vector based on frozen electron microscope structure fixed point. Background Circular RNAs (circRNAs) are a novel class of non-coding RNA molecules characterized by a single-stranded, covalently closed topology, which are less susceptible to exonuclease degradation due to their unique circular structure, with higher stability. Over the last decade, with the benefit of advances in high-throughput RNA sequencing technology and bioinformatics tools, the field of circular RNAs has been increasingly studied, and more circular RNA functions have been explored, for example as RNA aptamers, molecular sponges for mirnas and proteins, antisense RNAs, regulatory intracellular innate immune responses, protein translation vectors, and disease-related molecular markers. The functions also lead the annular RNA to have wide application prospect in the diagnosis and treatment directions of diseases, and lead the annular RNA to be a promising target for drug development and gene therapy application. Currently, three types of technical schemes, namely chemical synthesis method, ligase catalysis method and ribozyme catalysis method, are mainly adopted for in-vitro synthesis of the circular RNA. Among them, the ribozyme catalytic method based on the self-splicing intron has become the most widely studied cyclization strategy at present because of the advantages of mild reaction conditions, high cyclization efficiency and the like. The technology utilizes the autocatalytic splicing characteristics of Group I introns and Group II introns to realize efficient cyclization of RNA by constructing PIE (Permuted Intron-Exon) system. The main idea is to use PIE system to make intronic engineering transformation, its core structure includes the following functional elements in sequence according to 5 '. Fwdarw.3' direction, 3 'intron (shown as SEQ ID NO. 1), truncated exon E1 (shown as SEQ ID NO. 3), target gene, truncated exon E2 (shown as SEQ ID NO. 4) and 5' intron (shown as SEQ ID NO. 2). The cyclization process of the system comprises two key steps, namely, firstly, a guanosine cofactor attacks the E1 splice site to disconnect the E1 from an intron, and then, the 3' -OH end of the E1 attacks the E2 splice site to complete cyclization of RNA. However, some existing cyclization vector transformation is based on modification of base pairing characteristics in a splicing reaction molecular mechanism, a primary sequence and a secondary structure, and the optimization methods mainly depend on empirical strategies such as multi-site combination mutation, sequence insertion or deletion of splice sites, and the transformation based on the sequence is blind and difficult to predict structural change and functional influence after the transformation. And the cyclization efficiency is low, and the circRNA yield is low. Disclosure of Invention The technical problem to be solved by the invention is how to solve the problems of blindness and unpredictability existing in the transformation of the traditional cyclization vector, which results in low cyclization efficiency of mutants and low yield of prepared circRNA. The invention solves the technical problems by the following technical means: The first aspect of the invention provides Anabaena mutant RNA with improved cyclization efficiency, which comprises at least one of Anabaena intron G37 mutant RNA and AnabaenaPIE POLR A mutant RNA, wherein the sequence of Anabaena intron G37 mutant RNA is shown as SEQ ID NO. 11 or SEQ ID NO. 12, and the sequence of AnabaenaPIE POLR A mutant RNA is shown as SEQ ID NO. 17 or SEQ ID NO. 18. The second aspect of the invention provides the application of Anabaena mutant RNA in the study of improving the circRNA yield. The third aspect of the invention provides a preparation method of the Anabaena intron G37 mutant RNA, which comprises the following steps of mutating the 37 th base G of Anabaena intron wild type DNA, mutating G into U or C, and then transcribing to obtain Anabaena intron G37U mutant RNA or Anabaena intron G37C mutant RNA, wherein the sequence of the Anabaena intron wild type DNA is shown as SEQ ID NO. 5. Base G, U, C at position 37, abbreviated as G37, U37, C37. The rest are the same, and the 57 th base A and the 84 th base A are called A57 and A84 for short, and the rest are the same. Single point mutation G37U (meaning that the base G at position 37 is mutated to base U; the rest of the same are the same.) Double point mutation A57U/A84U (meaning that the base A at position 57 is mutated to base U and the base A at position 84 is mutated to base U; the rest are the same.) The fourth aspect of the invention