CN-121986173-A - Method for producing protein or peptide, method for producing 5' -capped polynucleotide, and reagent for use in these methods

Abstract

The present invention aims to further promote the translation efficiency of a technique for translating a protein by using RNA encoding the protein and a capping polynucleotide capable of binding thereto by complementary base pairing, and to improve the capping efficiency of the production of a 5' capped polynucleotide. This problem is solved by providing a 5' -capped polynucleotide and/or the single-stranded RNA with a translation efficiency promoting structure, and performing base insertion into the promoter so that the base sequence of the cap-analogue polynucleotide is complementary to the base sequence upstream from the transcription initiation point of the promoter in a specific positional relationship.

Inventors

• ABE HIROSHI
• INAGAKI MASAHITO
• Nakaka Yuko
• ABE NAOKO
• Toya Aya
• Kimura Kangming

Assignees

• 国立大学法人东海国立大学机构

Dates

Publication Date

20260505

Application Date

20240906

Priority Date

20230906

Claims (20)

1. 1. A method for producing or expressing a protein or peptide, characterized by: Comprising the step of carrying out a translation reaction in a reaction system, The reaction system comprises: a 5' -capped polynucleotide comprising an arbitrary base sequence A, and A single-stranded RNA comprising a base sequence B capable of binding to the base sequence A by complementary base pairing and a protein or peptide coding sequence, The 5' capped polynucleotide and/or the single stranded RNA has a translation efficiency promoting structure.
2. 2. The method of manufacturing or expressing according to claim 1, wherein: The 5 'capped polynucleotide is a 5' capped RNA.
3. 3. The method of manufacturing or expressing according to claim 1, wherein: The 5' capped polynucleotide or single stranded RNA is a polynucleotide endogenous to an organism or cell.
4. 4. A method of manufacturing or expressing as claimed in claim 3 wherein: The organism or cell endogenous polynucleotide is lncRNA or mRNA.
5. 5. The method of manufacturing or expressing according to claim 1, wherein: The translation efficiency promoting structure is a stabilizing structure that stabilizes the binding of the 5' capped polynucleotide to the single stranded RNA.
6. 6. The method of manufacturing or expressing according to claim 1, wherein: And (b) that the 5' capped polynucleotide and/or the single stranded RNA has a nuclease-binding structure, and that the reaction system comprises the nuclease, The nuclease is RISC or Cas, and The 5' capping polynucleotide is siRNA or miRNA, or The 5' capped polynucleotide is lncRNA or mRNA and the single stranded RNA comprises a tracrRNA sequence.
7. 7. The method of manufacturing or expressing according to claim 1, wherein: Satisfying at least 1 condition selected from (a) and (c), (A) The single-stranded RNA is circular RNA, (C) The 5' capped polynucleotide and/or the single stranded RNA has a structure that allows the two to be covalently bound.
8. 8. The method of manufacturing or expressing according to claim 7, wherein: satisfying the condition (a), and the circular RNA does not contain a stop codon, and/or The condition (c) is satisfied, and the structure is a photocrosslinkable structure.
9. 9. A composition of matter comprising a blend of two or more of the above, characterized by comprising: 5' -capped polynucleotide comprising any of the base sequences A, and/or A single-stranded RNA comprising a base sequence B capable of binding to the base sequence A by complementary base pairing and a protein or peptide coding sequence, The 5' capped polynucleotide and/or the single stranded RNA has a translation efficiency promoting structure.
10. 10. The composition of claim 9, wherein: The production or expression method according to any one of claims 1 to 8.
11. 11. A method for producing or expressing a protein or peptide, characterized by: Comprising the step of carrying out a translation reaction in a reaction system, The reaction system comprises a single-stranded circular RNA having a base length of 500 or less, which is formed by ligating 5' -capped polynucleotides via a linker.
12. 12. A composition characterized by: A single-stranded circular RNA having a base length of 500 or less, which is obtained by ligating 5' -capped polynucleotides via a linker.
13. 13. The composition of claim 12, wherein: A method of manufacture or expression as claimed in claim 11.
14. 14. A method of making a 5' capped polynucleotide, comprising: Comprising the step of carrying out a translation reaction in a reaction system, The reaction system comprises: a double-stranded polynucleotide comprising a phage promoter adjacent to the upstream side of the transcription initiation site and having a base insertion mutation sequence, and an antisense strand comprising a base sequence Y (upstream side) -Y2-Y3 or (upstream side) -Y1-Y2-Y3, wherein Y1 and Y2 represent bases within the base insertion mutation sequence and Y3 represents a base of the transcription initiation site, and 5' Cap analogues having a polynucleotide comprising the base sequence X (cap side) -X2-X3 or (cap side) -X1-X2-X3, X3 is a complementary base to Y3, and X1 is a complementary base of Y1 and/or X2 is a complementary base of Y2.
15. 15. The method of manufacturing as set forth in claim 14, wherein: The antisense strand of the phage promoter comprises the base sequence Y (upstream side) -Y1-Y2-Y3, X1 is the complementary base of Y1, and X2 is the complementary base of Y2.
16. 16. The method of manufacturing as set forth in claim 14, wherein: The 5 'capped polynucleotide is a 5' capped RNA.
17. 17. The method of manufacturing as set forth in claim 14, wherein: The phage promoter is a T7 promoter.
18. 18. The method of manufacturing as set forth in claim 14, wherein: The base sequence X is (cap side) -CUG, CAG, GUG or GAG, and Y3 is C.
19. 19. A composition of matter comprising a blend of two or more of the above, characterized by comprising: a double-stranded polynucleotide comprising a phage promoter adjacent to the upstream side of the transcription initiation site and having a base insertion mutation sequence, and an antisense strand comprising a base sequence Y (upstream side) -Y2-Y3 or (upstream side) -Y1-Y2-Y3, wherein Y1 and Y2 represent bases within the base insertion mutation sequence, Y3 represents a base of the transcription initiation site, and/or 5' Cap analogues having a polynucleotide comprising the base sequence X (cap side) -X2-X3 or (cap side) -X1-X2-X3, X3 is a complementary base to Y3, and X1 is a complementary base of Y1 and/or X2 is a complementary base of Y2.
20. 20. The composition of claim 19, wherein: The production method according to any one of claims 14 to 18.

Description

Method for producing protein or peptide, method for producing 5' -capped polynucleotide, and reagent for use in these methods Technical Field The present invention relates to a method for producing a protein or peptide, a method for producing a 5' -capped polynucleotide, a reagent used in the production methods, and the like. Background In recent years, the use of mRNA vaccines has become widespread. The structure of the mRNA vaccine is divided into a cap structure, a 5' untranslated region, a protein coding region, a 3' untranslated region, and a PolyA tail region from the 5' end. In order for protein translation to occur from an mRNA vaccine, a cap structure is required. However, in the conventional mRNA synthesis technology, the proportion of mRNA having a cap structure relative to the total amount of mRNA (capping efficiency) is low, and immune reaction by non-cap mRNA becomes a problem. In such a situation, non-patent document 1 reports that using a hydrophobic purification tag capable of being removed by light as a cap analogue, only capped mRNA is separated and purified by reverse phase chromatography after transcription reaction, and then the hydrophobic tag is removed by photoreaction, thereby realizing production of capped mRNA with high purity. Prior art literature Non-patent literature Non-patent document 1, nat Commun. 2023 May 11, 14 (1): 2657 doi: 10.1038/s41467-023-38244-8. Disclosure of Invention Problems to be solved by the invention In addition, translation efficiency is also important in order to improve the performance of mRNA vaccines. In the course of research, the present inventors developed a technique for protein translation using an RNA encoding a protein and a capped RNA capable of binding thereto by complementary base pairing, focusing on further promotion of translation efficiency of the technique. The present invention aims to further promote the translation efficiency of a technique for translating a protein or peptide by using RNA encoding the protein or peptide and a capping polynucleotide capable of binding thereto by complementary base pairing and to improve the capping efficiency of the production of a 5' capped polynucleotide. Means for solving the problems The present inventors have made intensive studies in view of the above problems, and as a result, have found that the structure of a 5' -capped polynucleotide and/or a single-stranded RNA (RNA encoding a protein or peptide) contributes to promotion of translation efficiency, and have found that the capping efficiency is improved by base insertion into a promoter such that the base sequence of the polynucleotide of the cap analogue is complementary to the base sequence upstream from the transcription initiation point of the promoter in a specific positional relationship. The inventors of the present invention have further studied based on these findings, and as a result, completed the present invention. That is, the present invention includes the following. A method for producing or expressing a protein or peptide, comprising the step of carrying out a translation reaction in a reaction system, The reaction system comprises: a 5' -capped polynucleotide comprising an arbitrary base sequence A, and A single-stranded RNA comprising a base sequence B capable of binding to the base sequence A by complementary base pairing and a protein or peptide coding sequence, The 5' capped polynucleotide and/or the single stranded RNA has a translation efficiency promoting structure. The method of claim 1, wherein the 5 'capped polynucleotide is a 5' capped RNA. The method of claim 1 or 2, wherein the 5' capped polynucleotide is a polynucleotide endogenous to an organism or cell. The method of claim 4, wherein the polynucleotide endogenous to the organism or cell is lncRNA or mRNA. The method according to any one of items 1 to 4, wherein the translation efficiency promoting structure is a stabilizing structure for stabilizing binding between the 5' -capped polynucleotide and the single-stranded RNA. The method according to any one of items 1 to 5, wherein (b) the 5' -capped polynucleotide and/or the single-stranded RNA has a nuclease-binding structure, and the reaction system contains the nuclease, The nuclease is RISC or Cas, and The 5' capping polynucleotide is siRNA or miRNA, or The 5' capped polynucleotide is lncRNA or mRNA, and the single stranded RNA comprises a tracrRNA sequence. The production or expression method according to any one of items 1 to 6, which satisfies at least 1 condition selected from the group consisting of (a) and (c), (A) The single-stranded RNA is a circular RNA, (C) The 5' capped polynucleotide and/or the single stranded RNA has a structure in which the two are covalently bonded. The method of producing or expressing according to item 7, which satisfies the above condition (a) and the above circular RNA does not contain a stop codon, and/or The above condition (c) is satisfied, and the above structure is a