Circular RNA and method for producing the same

Abstract

The present invention relates to the biomedical field, and more specifically to engineered circular RNA molecules, pharmaceutical compositions containing the same, and their uses. The present invention further relates to vectors for producing the above-mentioned circular RNA molecules and methods for producing them.

Description

[Technical Field]

[0001] The present invention relates to the biomedical field, and more specifically to engineered circular RNA molecules, pharmaceutical compositions containing them, and their uses. The present invention further relates to vectors and methods for producing said engineered circular RNA molecules. [Background technology]

[0002] Circular RNA (RNA) is a common type of RNA in eukaryotes. Naturally occurring circular RNA is primarily produced through an intracellular molecular mechanism called "back splicing." Currently, it has been discovered that eukaryotic circular RNA possesses various molecular and cellular regulatory functions. For example, circular RNA can regulate the expression of target genes by binding to microRNAs. Circular RNA can also further regulate gene expression by directly binding to target proteins.

[0003] Because circular RNA has a longer half-life than linear mRNA due to its circular nature, it is hypothesized that in vitro synthesized circular RNA may have good stability. Methods for forming circular RNA in vitro include chemical methods, enzyme-catalyzed methods, and ribozyme-catalyzed methods. Chemical methods are costly and the size of the resulting circular RNA molecule is limited. Enzyme-catalyzed methods mainly utilize T4 RNA ligases to catalyze the circularization of linear RNA, and the size of the RNA payload that can be circularized is also limited. Ribozyme-catalyzed methods (e.g., based on group I introns) are a promising method for producing circular RNA.

[0004] The natural group I intron system can undergo cleavage and ligation reactions to form circular intron RNA. A specific cleavage site-conserved sequence located at the 5' end exon E1 is cleaved by nucleophilic attack from the 3' hydroxyl group of a free guanosine triphosphate, generating an exposed 3' hydroxyl group, which then binds to the cleaved 5' exon E1. Subsequently, the exposed 3' hydroxyl group at the 5' end of the intron attacks the conserved sequence between the 3' end of the intron and exon E2, cleaving exon E2. The intron then undergoes a cyclization reaction, yielding circular intron RNA. Currently, an improved ribozyme-catalyzed method derived from the tRNA intron of Anabaena, applied to the in vitro formation of circular RNA, has been reported and is called the "Group I permuted intron-exon self-splicing system" (PIE system). This method has the potential to form expressible circular RNA because it can excise introns and form circular RNA containing exons. The basic design principle of the PIE system is to ligate exon E1 and E2 sequences by molecular cloning to form a continuous circular plasmid. The introns are cleaved with restriction endonucleases to obtain a linear plasmid. Further in vitro transcription is performed with a T7 promoter upstream of the inverted 3' intron to obtain linear RNA containing a 3' intron-E2-E1-5' intron structure. Similar to the natural group I intron system, the conserved sequence at a specific cleavage site of exon E1 is cleaved by nucleophilic attack of the 3' hydroxyl group of a free guanylic acid, and exon E1 produces an exposed 3' hydroxyl group, while the guanylic acid binds to the cleaved 5' intron. Subsequently, the exposed 3' hydroxyl group of exon E1 attacks the conserved sequence between the 3' intron and exon E2, cleaving the 3' intron. However, exons E2 and E1 undergo a cyclization reaction, yielding circular E1-E2 RNA.

[0005] However, conventional techniques have limited access to group I introns usable for in vitro cyclization, and only a very small number of introns have been demonstrated to be usable for circular RNA formation in the PIE system. Therefore, this field needs to develop new group I introns that can be used for efficient RNA cyclization in vitro. [Overview of the project]

[0006] In one embodiment, the object of the present invention is to provide a circular RNA precursor which is oriented in the direction from 5' to 3'. a) 3' self-splicing intron fragment, b) Includes a 5' self-splicing intron fragment, Here, the combination of the 3' self-splicing intron fragment and the 5' self-splicing intron fragment preserves the self-splicing activity of the self-splicing intron and allows for the production of circular RNA by the self-splicing of the circular RNA precursor. Here, the self-splicing intron is selected from group I introns and group II introns, for example, the self-splicing intron is selected from group I introns, Here, the 3' self-splicing intron fragment originates from the 3' terminal portion of the self-splicing intron, starting from the intron's internal split region I and extending to the 3' end of the self-splicing intron, and the 5' self-splicing intron fragment originates from the 5' terminal portion of the self-splicing intron, starting from the 5' end of the self-splicing intron and extending to the intron's internal split region II.

[0007] In aspects and embodiments of the present invention, the self-splicing intron is selected from the group consisting of cyanobacterium Anabaena Group I Intron, Azoarcus Group I Intron (Azo), Scytalidium dimidiatum Group I Intron (Sd), Staphylococcus phage Twort Group I Intron (Twort), Scytonema - hoffmanni tRNA fMet Group I Intron (Sh), or Agrobacterium - tumefaciens Group I Intron (At).

[0008] In some aspects of the present invention, the circular RNA precursor further comprises a homology arm sequence I located upstream of the 3' self-splicing intron fragment and a homology arm sequence II located downstream of the 5' self-splicing intron fragment.

[0009] In one aspect, one object of the present invention is to provide a circular RNA precursor, which in the 5' to 3' direction, a) a 3' self-splicing intron fragment, b) a first residue cyclization element, c) a nucleotide sequence of interest, d) a second residue cyclization element, e) a 5' self-splicing intron fragment, and where the combination of the 3' self-splicing intron fragment and the 5' self-splicing intron fragment maintains the self-splicing activity of the self-splicing intron and allows the production of a circular RNA containing the first residue cyclization element, the nucleotide sequence of interest, and the second residue cyclization element by self-splicing of the circular RNA precursor.

[0010] In one aspect, one object of the present invention is to provide a circular RNA precursor, which, in the 5' to 3' direction, a) a 3' self-splicing intron fragment, b) a nucleotide sequence of interest, c) a 5' self-splicing intron fragment, and where the combination of the 3' self-splicing intron fragment and the 5' self-splicing intron fragment maintains the self-splicing activity of the self-splicing intron and allows the production of a circular RNA containing the nucleotide sequence of interest by self-splicing of the circular RNA precursor, where the nucleotide sequence of interest, in the 5' to 3' direction, comprises a circularization sequence fragment II containing a ribozyme recognition site II at its 5' end and a circularization sequence fragment I containing a ribozyme recognition site I at its 3' end, where the circularization sequence fragment I is derived from the 5' end portion of the circularization sequence, and the circularization sequence fragment II is derived from the 3' end portion of the circularization sequence, and where in the generated circular RNA, the ribozyme recognition site I and the ribozyme recognition site II are ligated, and thereby the circularization sequence fragment I and the circularization sequence fragment II are ligated to form the circularization sequence.

[0011] In some embodiments, the circularization sequence is a protein coding sequence, a non-coding sequence, a translation initiation element (TIE) or a non-TIE functional element.

[0012] In some embodiments, the nucleotide sequence of interest further comprises one or more sequences selected from the group consisting of at least one protein coding sequence, a translation initiation element (TIE), a non-TIE functional element, at least one non-coding sequence, or any combination thereof, located between the circularization sequence fragment II and the circularization sequence fragment I.

[0013] In the embodiment, the ribozyme recognition site I includes or consists of the following nucleotide motifs in the direction from 5' to 3', wherein the nucleotide motif consists of one sequence from SEQ ID NO: 95 to 116, and specifically, the nucleotide motif consists of one sequence from CUU, CUC, or CAU.

[0014] In one embodiment, one object of the present invention is to provide a circular RNA precursor which is oriented in the direction from 5' to 3'. a) 3' self-splicing intron fragment, b) The target nucleotide sequence and c) Includes a 5' self-splicing intron fragment, Here, the combination of the 3' self-splicing intron fragment and the 5' self-splicing intron fragment preserves the self-splicing activity of the self-splicing intron and allows the self-splicing of the circular RNA precursor to produce a circular RNA containing the target nucleotide sequence. Here, the target nucleotide sequence includes, in the direction from 5' to 3', a protein-coding sequence fragment II, a translation initiation element such as an internal ribosome entry site (IRES), and a protein-coding sequence fragment I. Here, the protein-coding sequence fragment I is derived from the 5' end of at least one protein-coding sequence, and the protein-coding sequence fragment II is derived from the 3' end of the at least one protein-coding sequence. The protein-coding sequence fragment I includes a ribozyme recognition site I located at its 3' end, and the protein-coding sequence fragment II includes a ribozyme recognition site II located at its 5' end. In the resulting circular RNA, the ribozyme recognition site I and the ribozyme recognition site II are linked together, thereby linking the protein-coding sequence fragment I and the protein-coding sequence fragment II to form the at least one protein-coding sequence.

[0015] In one embodiment, one object of the present invention is to provide a circular RNA precursor which is oriented in the direction from 5' to 3'. a) 3' self-splicing intron fragment, b) The target nucleotide sequence and c) Includes a 5' self-splicing intron fragment, Here, the combination of the 3' self-splicing intron fragment and the 5' self-splicing intron fragment preserves the self-splicing activity of the self-splicing intron and allows the self-splicing of the circular RNA precursor to produce a circular RNA containing the target nucleotide sequence. Here, the target nucleotide sequence includes, in the direction from 5' to 3', a protein-coding sequence fragment II, a translation initiation element such as an internal ribosome entry site (IRES), and a protein-coding sequence fragment I. Here, the protein-coding sequence fragment I is the 5' end portion of the protein-coding sequence, and the protein-coding sequence fragment II is the 3' end portion of the protein-coding sequence, and The protein-coding sequence fragment I includes a ribozyme recognition site I located at its 3' end, and the protein-coding sequence fragment II includes a ribozyme recognition site II located at its 5' end. In the resulting circular RNA, the ribozyme recognition site I and the ribozyme recognition site II are linked together, thereby linking the protein-coding sequence fragment I and the protein-coding sequence fragment II to form the protein-coding sequence.

[0016] In embodiments, the at least one protein coding sequence is a sequence that codes for a diagnostic or therapeutic protein. In embodiments, the protein coded by the protein coding sequence is a mammalian protein, such as a human protein. In embodiments, the at least one protein coding sequence comprises at least two protein coding sequences. In embodiments, the at least one protein coding sequence is a sequence that codes for two or more proteins. In embodiments, the at least one protein coding sequence is a sequence that codes for two or more diagnostic or therapeutic proteins. Such at least two protein coding sequences may be operably linked to each other via a spacer, a 2A sequence, or another translation initiation element. In embodiments, another translation initiation element is operably linked between any two adjacent protein coding sequences. In embodiments, the translation initiation element is an IRES sequence, a 5'UTR sequence, a Kozak sequence, m 6 The sequence is selected from an A-modified sequence, a sequence complementary to ribosomal 18S rRNA, or any combination thereof. In one embodiment, the translation initiation element is an IRES sequence.

[0017] In the embodiment, the ribozyme recognition site I includes or consists of the following nucleotide motifs in the direction from 5' to 3', wherein the nucleotide motif consists of one sequence from SEQ ID NO: 95 to 116, and specifically, the nucleotide motif consists of one sequence from CUU, CUC, or CAU.

[0018] In one embodiment, one object of the present invention is to provide a circular RNA precursor which is oriented in the direction from 5' to 3'. a) 3' self-splicing intron fragment, b) The target nucleotide sequence and c) Includes a 5' self-splicing intron fragment, Here, the combination of the 3' self-splicing intron fragment and the 5' self-splicing intron fragment preserves the self-splicing activity of the self-splicing intron and allows the self-splicing of the circular RNA precursor to produce a circular RNA containing the target nucleotide sequence. Here, the target nucleotide sequence includes, in the direction from 5' to 3', a translation initiation element fragment II, at least one protein coding sequence, and a translation initiation element fragment I. Here, the translation initiation element fragment I originates from the 5' end portion of the translation initiation element, and the translation initiation element fragment II originates from the 3' end portion of the translation initiation element, and The translation initiation element fragment I includes a ribozyme recognition site I located at its 3' end, and the translation initiation element fragment II includes a ribozyme recognition site II located at its 5' end. In the generated circular RNA, the ribozyme recognition site I and the ribozyme recognition site II are linked together, thereby linking the translation initiation element fragment I and the translation initiation element fragment II to form a translation initiation element that can recruit a ribosome and initiate translation.

[0019] In one embodiment, one object of the present invention is to provide a circular RNA precursor which is oriented in the direction from 5' to 3'. a) 3' self-splicing intron fragment, b) The target nucleotide sequence and c) Includes a 5' self-splicing intron fragment, Here, the combination of the 3' self-splicing intron fragment and the 5' self-splicing intron fragment preserves the self-splicing activity of the self-splicing intron and allows the self-splicing of the circular RNA precursor to produce a circular RNA containing the target nucleotide sequence. Here, the target nucleotide sequence includes, in the direction from 5' to 3', a translation initiation element fragment II, at least one protein coding sequence, and a translation initiation element fragment I. Here, the translation initiation element fragment I is the 5' end portion of the translation initiation element, and the translation initiation element fragment II is the 3' end portion of the translation initiation element, and The translation initiation element fragment I includes a ribozyme recognition site I located at its 3' end, and the translation initiation element fragment II includes a ribozyme recognition site II located at its 5' end. In the resulting circular RNA, the ribozyme recognition site I and the ribozyme recognition site II are linked together, thereby linking the translation initiation element fragment I and the translation initiation element fragment II to form a translation initiation element that can recruit a ribosome and initiate translation.

[0020] In embodiments, the at least one protein coding sequence is a sequence that codes for a diagnostic or therapeutic protein. In embodiments, the protein coded by the protein coding sequence is a mammalian protein, such as a human protein. In embodiments, the at least one protein coding sequence comprises at least two protein coding sequences. In embodiments, the at least one protein coding sequence is a sequence that codes for two or more proteins. In embodiments, the at least one protein coding sequence is a sequence that codes for two or more diagnostic or therapeutic proteins. Such at least two protein coding sequences can be operably linked to each other via a spacer, a 2A sequence, or another translation initiation element. In embodiments, the translation initiation element is an IRES sequence, a 5'UTR sequence, a Kozak sequence, m 6 The sequence is selected from an A-containing sequence, a sequence complementary to ribosomal 18S rRNA, or any combination thereof. In one embodiment, the translation initiation element is an IRES sequence.

[0021] In the embodiment, the ribozyme recognition site I includes or comprises the following nucleotide motifs in the direction from 5' to 3', wherein the nucleotide motif consists of one sequence from SEQ ID NO: 95 to 116, and specifically, the nucleotide motif consists of one sequence from CUU, CUC, or CAU.

[0022] In one embodiment, one object of the present invention is to provide a circular RNA precursor which is oriented in the direction from 5' to 3'. a) 3' self-splicing intron fragment, b) The target nucleotide sequence and c) Includes a 5' self-splicing intron fragment, Here, the combination of the 3' self-splicing intron fragment and the 5' self-splicing intron fragment preserves the self-splicing activity of the self-splicing intron and allows the self-splicing of the circular RNA precursor to produce a circular RNA containing the target nucleotide sequence. Here, the target nucleotide sequence includes, in the direction from 5' to 3', a non-TIE functional element fragment II, a translation initiation element, at least one protein coding sequence, and a non-TIE functional element fragment I. Here, the non-TIE functional element fragment I originates from the 5' end of the non-TIE functional element, and the non-TIE translation initiation element fragment II originates from the 3' end of the non-TIE functional element. The non-TIE functional element fragment I includes a ribozyme recognition site I located at its 3' end, and the non-TIE functional element fragment II includes a ribozyme recognition site II located at its 5' end. In the resulting circular RNA, the ribozyme recognition site I and the ribozyme recognition site II are linked together, thereby linking the non-TIE functional element fragment I and the non-TIE functional element fragment II to form a non-TIE functional element.

[0023] In embodiments, the non-TIE functional element is a 3'UTR or a replicon. In embodiments, the at least one protein-coding sequence comprises at least two protein-coding sequences. Such at least two protein-coding sequences may be linked to each other via a spacer, a 2A sequence, or another translation initiation element. In embodiments, the translation initiation element is an IRES sequence, a 5'UTR sequence, a Kozak sequence, m 6 The sequence is selected from an A-modified sequence, a sequence complementary to ribosomal 18S rRNA, or any combination thereof. In one embodiment, the translation initiation element is an IRES sequence.

[0024] In one embodiment, the ribozyme recognition site I includes or comprises the following nucleotide motifs in the 5' to 3' direction, wherein the nucleotide motif consists of any one sequence from SEQ ID NO: 95 to 116, specifically, the nucleotide motif consists of any one sequence from CUU, CUC, or CAU. In one embodiment, one object of the present invention is to provide a circular RNA precursor which, in the 5' to 3' direction, a) 3' self-splicing intron fragment, b) The target nucleotide sequence and c) Includes a 5' self-splicing intron fragment, Here, the combination of the 3' self-splicing intron fragment and the 5' self-splicing intron fragment preserves the self-splicing activity of the self-splicing intron and allows the self-splicing of the circular RNA precursor to produce a circular RNA containing the target nucleotide sequence. Here, the target nucleotide sequence includes a non-coding sequence fragment II and a non-coding sequence fragment I in the direction from 5' to 3', Here, the non-coding sequence fragment I originates from the 5' end of the non-coding sequence, and the non-coding sequence fragment II originates from the 3' end of the non-coding sequence, and the non-coding sequence fragment I includes a ribozyme recognition site I located at its 3' end, and the non-coding sequence fragment II includes a ribozyme recognition site II located at its 5' end, and in the resulting circular RNA, the ribozyme recognition site I and the ribozyme recognition site II are linked together, thereby linking the non-coding sequence fragment I and the non-coding sequence fragment II to form a complete non-coding sequence.

[0025] In one embodiment, the non-coding sequence is an endogenous circular RNA. In another embodiment, the nucleotide sequence of interest further comprises one or more non-coding sequences located between the non-coding sequence fragment I and the non-coding sequence fragment II.

[0026] In the embodiment, the ribozyme recognition site I includes or comprises the following nucleotide motifs in the direction from 5' to 3', wherein the nucleotide motif consists of one sequence from SEQ ID NO: 95 to 116, and specifically, the nucleotide motif consists of one sequence from CUU, CUC, or CAU.

[0027] In aspects of the present invention, the protein-coding sequence is an aquaporin, for example, AQP-1 protein, or a sequence that has the same activity as AQP-1 protein and encodes a protein derived from AQP-1 protein by substituting, deleting, or adding 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids in the amino acid sequence of AQP-1 protein. In embodiments, the AQP-1 protein is a mammalian AQP-1 protein, specifically a human AQP-1 protein.

[0028] In another embodiment, an object of the present invention is to provide a nucleic acid vector for producing a circular RNA molecule, the vector comprising a coding sequence for a circular RNA precursor as described above.

[0029] In another embodiment, an object of the present invention is to provide a circular RNA polynucleotide, which is produced by a circular RNA precursor or nucleic acid vector as described above. In embodiments, the circular RNA polynucleotide of the present invention has an in vivo expression duration of at least 24 hours (1 day), for example, 36 hours, 48 ​​hours, 72 hours, 96 hours, 5 days, 10 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days or more. In another embodiment, an object of the present invention is to provide the circular RNA precursor and / or circular RNA as described above for use as an expression vector. In embodiments, the expression vector is used to express one or more diagnostic or therapeutic proteins.

[0030] In another embodiment, an object of the present invention is to provide a pharmaceutical composition comprising a circular RNA precursor and / or a nucleic acid vector and / or a circular RNA as described above, and a pharmaceutically acceptable carrier.

[0031] In another embodiment, an object of the present invention is to provide a method for producing circular RNA, the method being 1) Providing a circular RNA precursor as described above, or obtaining a circular RNA precursor by transcription from a nucleic acid vector as described above, 2) A step of incubating a cyclic RNA precursor at a temperature at which RNA cyclization occurs in the presence of a divalent metal cation, 3) The step of recovering the circular RNA obtained in step 2).

[0032] In another embodiment, one object of the present invention is to provide a method for producing circular RNA, the method being a) A step of providing a nucleic acid vector containing an RNA circular element based on intronic self-splicing as a transcription template, b) The nucleic acid vector is incubated for a first time period in an in vitro transcription system containing a divalent metal cation and RNA polymerase, during which the linear RNA produced by in vitro transcription undergoes self-circulation under the action of an RNA circulating element to produce circular RNA.

[0033] In another aspect, an object of the present invention is to provide a method for purifying circular RNA, the method being a) A step of contacting a mixture containing circular RNA and a circular RNA precursor with a circular RNA-specific probe under conditions that allow the circular RNA-specific probe to specifically bind to the circular RNA and form a complex, b) A step of separating the complex from one or more components in the mixture that are not bound to the cyclic RNA-specific probe, c) The step of releasing circular RNA from the complex.

[0034] In another aspect, an object of the present invention is to provide a method for purifying circular RNA, the method being i) A step of contacting a mixture containing circular RNA and a circular RNA precursor with a linear circular RNA precursor-specific probe under conditions that allow the linear circular RNA precursor-specific probe to specifically bind to the linear circular RNA precursor and form a complex, ii) A step of removing the complex formed by the linear circular RNA precursor-specific probe and the linear circular RNA precursor from the mixture, iii) the step of collecting the cyclic RNA-containing mixture obtained in step ii), Optionally, steps i) to iii) may be performed multiple times, for example, two, three, four, or more times.

[0035] In another aspect, one object of the present invention is to provide a method for purifying circular RNA, the method being i) A step of adding a linear RNA-specific tag to linear RNA in a mixture containing circular RNA and linear RNA, ii) A step of contacting a mixture containing circular RNA and linear RNA with a linear RNA probe that specifically binds to a tag, under conditions that allow the probe to specifically bind to the linear RNA and form a complex; iii) The step of removing the complex formed by the linear RNA probe and linear RNA from the mixture, iv) The step of collecting the circular RNA-containing mixture obtained in step iii), Optionally, perform steps ii) to iv) multiple times, for example, two, three, four, or more times.

[0036] In another aspect, one object of the present invention is to provide a product expressed by the circular RNA described above, the circular RNA produced by the method described above, or the circular RNA purified by the method described above.

[0037] In another embodiment, one object of the present invention is to provide a pharmaceutical composition comprising a circular RNA precursor and / or a nucleic acid vector and / or a circular RNA and / or a circular RNA produced by the method described above and / or a circular RNA purified by the method described above, and a pharmaceutically acceptable carrier.

[0038] In another embodiment, an object of the present invention is to provide a method for preventing or treating a disease or disorder in a subject, the method comprising administering to a subject a circular RNA as described above, a circular RNA produced by the method described above, or a circular RNA purified by the method described above, or a pharmaceutical composition as described above.

[0039] In another aspect, an object of the present invention is to provide applications for circular RNA as described above, circular RNA produced by the method described above, or circular RNA purified by the method described above, or pharmaceutical compositions as described above, in the manufacture of pharmaceuticals for preventing or treating diseases or disorders in subjects.

[0040] In some embodiments, an object of the present invention is to provide a method for screening nucleotide motifs of a target open reading frame (ORF) sequence encoding a target polypeptide, the method being 1) A step of extracting amino acid units in the direction from the N-terminus to the C-terminus of a target polypeptide containing m amino acids, wherein each group of amino acid units contains n amino acids, one amino acid is used as the step size, and the steps are slid along the N-terminus to the C-terminus, where n is an integer and n ≥ 2, and m is an integer and ≥ 1. 2) The step of listing all nucleotide sequences for each group of amino acid units according to the degenerate codon, 3) A step of recognizing a nucleotide sequence that matches the nucleotide motif obtained in step 2), The nucleotide motif consists of one sequence from SEQ ID NO:95~116, preferably consisting of CUU, CUC, or CAU, 4) The step of optionally selecting a target ORF as the circularization sequence of a circular RNA precursor.

[0041] In some embodiments, one object of the present invention is to provide a system for screening nucleotide motifs of a target open reading frame (ORF), wherein the target open reading frame (ORF) is a nucleotide sequence encoding a target polypeptide, and the system is a) An amino acid extraction module for extracting amino acid units in the direction from the N-terminus to the C-terminus of a target polypeptide containing m amino acids, wherein each group of amino acid units contains n amino acids, one amino acid is used as the step size, sliding along the N-terminus to the C-terminus, and n is an integer and n ≥ 2, and m is an integer and ≥ 1; b) A convertible module for determining the codon sequence of a group of amino acid units, wherein the codon sequence of each group includes all degenerate codon sequences corresponding to the amino acid units of each group, c) A target codon sequence filtering module for determining whether the target codon sequence contains a nucleotide sequence that precisely matches the nucleotide motif, Here, the nucleotide motif consists of one nucleotide sequence from SEQ ID NO:95~116, preferably consisting of CUU, CUC, or CAU.

[0042] In some embodiments, an object of the present invention is to provide a method for screening nucleotide motifs of target internal ribosome entry sites (IRESs), the method being 1) A step of identifying non-conserved sequence regions of a target IRES by comparing the target IRES with other IRES sequences from the same viral subgenus, wherein the comparison is performed using a multiplex sequence alignment tool, such as Clustal Omega or MUSCLE. 2) Based on the results of multiple sequence alignment, the low-structure region of the target IRES is determined using secondary structure prediction software, such as RNAfold, Mfold, RNAstruct, or RNAalifold. 3) The step of editing 0, 1, or 2 bases in a low-structure region of a target IRES to match a nucleotide motif without affecting the translation initiation activity of the IRES, Here, the nucleotide motif consists of one nucleotide sequence from SEQ ID NO:95~116, and preferably consists of CUU, CUC, or CAU.

[0043] In some embodiments, an object of the present invention is to provide a system for screening nucleotide motifs of target internal ribosome entry sites (IRESs), wherein the system is a) A multi-sequence alignment module for recognizing non-conserved sequence regions by comparing the target IRES with other IRES sequences from the same viral subgenus, b) A low-structure prediction module for determining low-structure regions within the target IRES based on the multiple sequence alignment results of the target IRES, c) A base editing module for editing one or more bases at any position within the region to obtain an edited region that matches a nucleotide motif, Here, the nucleotide motif consists of one nucleotide sequence from SEQ ID NO:95~116, and preferably consists of CUU, CUC, or CAU.

[0044] In some embodiments, the object of the present invention is to provide a screening method for screening for targeted non-TIE functional elements against a nucleotide motif in a ribozyme recognition site I, the method being a) The step of editing 0, 1, or 2 bases in a non-TIE functional element to include an edited region matching the nucleotide motif in the non-TIE functional element sequence (e.g., 3'UTR or replicon) while maintaining the non-TIE function, Here, the nucleotide motif consists of one nucleotide sequence from SEQ ID NO:95~116, and preferably consists of CUU, CUC, or CAU.

[0045] In some embodiments, an object of the present invention is to provide a system for screening nucleotide motifs of target region sequences in non-TIE functional element sequences (e.g., 3'UTR or replicon), wherein the screening system is a) Includes a base editing module for editing one or more bases at any position within a region to obtain an edited region that matches a nucleotide motif, Here, the nucleotide motif consists of one nucleotide sequence from SEQ ID NO:95~116, and preferably consists of CUU, CUC, or CAU.

[0046] It should be understood that each embodiment of the preventive or therapeutic method described herein may also be represented as an embodiment of the applicable use type. [Brief explanation of the drawing]

[0047] [Figure 1] These are different PIE designs for the Azoarcus group I intron (Azop). [Figure 2] This shows the cyclization efficiency of Azop in different PIEs. [Figure 3] This is a PIE design for the Scytalidium dimidiatum group I intron (Sd). [Figure 4] This is the cyclization efficiency of Sd in different PIEs. [Figure 5] These are different PIE designs of the Staphylococcus phage Twort group I intron (Twort). [Figure 6] This shows the cyclic efficiency of Twort in different PIEs. [Figure 7] This is a different PIE design for the Scytonema hofmanii group I intron (Sh). [Figure 8] This is the cyclization efficiency of Scytonema hofmanii (Sh). [Figure 9] This is the cyclic cyclization efficiency of IL2 in different group I introns using the present invention. [Figure 10] This is the RNA circularization efficiency of Agrobacterium tumefaciens group I introns (At). [Figure 11] This involves predicting the structure of precursors with different homology arms (HAs) in homology arm (HA) design. [Figure 12] This is the cyclic circulation efficiency of AnaX-IL2 with different homology arms. [Figure 13] This is a correlation analysis between homology arm cyclization efficiency and average base MFE. [Figure 14] This is a clean-PIE design of ORF or IRES based on PIE. [Figure 15] This is a clean-PIE design based on PIE functional elements. [Figure 16] This is the arrangement and structure of the CLEAN-PIE design for IL2. [Figure 17] This is the cyclization efficiency of AnaX Clean PIE for IL2. [Figure 18] This is a CLEAN-PIE design (CLEAN-PIE circAQP1) that uses a different circular RNA polynucleotide to express the AQP1 protein. [Figure 19] This describes the cyclization efficiency, translation, and immunogenicity of circAQP1 Clean PIE. [Figure 20] This is the effect of miRNA target sites in circRNA on expression levels. [Figure 21] This is the effect of UTR on circRNA translation. [Figure 22] This is CLEAN-PIE circAQP1 expression in rat salivary glands. [Figure 23] This is a representative image of IHC staining of AQP1 expression in salivary glands. [Modes for carrying out the invention]

[0048] In this invention, unless otherwise specified, the scientific and technical terms used have meanings that are generally understood by those skilled in the art. Furthermore, all terms and experimental methods related to protein and nucleotide chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein belong to the terms and methods commonly used in the art. For example, the standard DNA recombination and molecular cloning techniques used herein are well known to those skilled in the art and are described in detail in the reference Sambrook, J., Fritsch, Efland Maniatis, T., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 1989. In addition, to better understand this invention, definitions and interpretations of relevant terms are provided below.

[0049] As used herein, the singular forms “one,” “one,” and “the” include multiple referents unless specifically indicated otherwise in the context. It should also be noted that claims may be written excluding any optional elements. For example, “circular RNA precursor” refers to one or more circular RNA precursors. Therefore, the terms “one,” “one,” “one or more,” and “the last one” are interchangeable. For example, the term “at least one” refers to one, two, or more. This description is intended to form the basis for any use of exclusive terms relating to the description of elements of a claim, such as “only,” “only,” etc., or for limiting them using “negative.” Similarly, the terms “include,” “inclusive,” and “have” are interchangeable.

[0050] As used herein, the term "and / or" covers all combinations of items connected by the term, and each combination should be considered to be listed separately herein. For example, "A and / or B" covers "A", "A and B", and "B". For example, "A, B and / or C" covers "A", "B", "C", "A and B", "A and C", "B and C", and "A, B and C".

[0051] As used herein, “about,” “approximately,” “substantially,” and “significantly” are understood by those skilled in the art and vary to some extent depending on the context in which they are used. Unless the use of these terms is clarified in the context in which those skilled in the art use them, “about” and “approximately” mean adding or subtracting <10% from the particular term, and “substantially” and “significantly” mean adding or subtracting >10% from the particular term.

[0052] In this application, “optional” or “optionally” means that the events or circumstances described below may or may not occur, and such description includes both the occurrence and non-occurrence of such events or circumstances.

[0053] "Polynucleotide," "nucleic acid sequence," "nucleotide sequence," or "nucleic acid fragment" are interchangeable terms and refer to single-stranded or double-stranded RNA or DNA polymers that may optionally contain synthetic, unnatural, or modified nucleotide bases. Nucleotides (usually existing in the form of 5'-monophosphate) are represented by their single-letter names as follows: "A" represents adenosine or deoxyadenosine (corresponding to RNA or DNA, respectively), "C" represents cytidine or deoxycytidine, "G" represents guanosine or deoxyguanosine, "U" represents uridine, "T" represents deoxythymidine, "R" represents purine (A or G), "Y" represents pyrimidine (C or T), "K" represents G or T, "H" represents A, C, or T, "I" represents inosine, and "N" represents any nucleotide. In this specification, nucleotide sequences may be represented as DNA sequences (including T), but when RNA is referred to, those skilled in the art can easily determine the corresponding RNA sequence (i.e., by replacing T with U).

[0054] For example, the target nucleotide sequence in the present invention may be a non-coding sequence, such as antisense RNA, aptamer, guide RNA, or any non-coding RNA present in a living organism.

[0055] Sequence "identity" has a meaning known in the art, and the percentage of sequence identity between two nucleic acid or polypeptide molecules or regions can be calculated using the techniques disclosed. Sequence identity may be measured along the entire length of a polynucleotide or polypeptide, or by a region of the molecule. (See, for example, Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., Stockton Press, New York, 1991). Many methods are available for determining sequence identity. One example of an algorithm suitable for determining the percentage of sequence identity is the algorithm used in the Basic Local Alignment Search Tool (hereinafter referred to as "BLAST"). See, for example, Altschul et al., J.Mol.Biol.215:403-410, 1990 and Altschul et al., Nucleic Acids Res., 15:3389-3402, 1997. Software for performing BLAST analysis is publicly available from the National Center for Biotechnology Information (hereinafter referred to as "NCBI"). Default parameters used to determine sequence identity using software available from NCBI (e.g., BLASTN for nucleic acid sequences) are described in McGinnis et al., Nucleic Acids Res., 32:W20-W25, 2004.

[0056] A polynucleotide sequence "derived" from a specified polynucleotide sequence (e.g., an RNA polynucleotide sequence or fragment thereof) means that the latter polynucleotide sequence is derived from the former. In some embodiments, a polynucleotide sequence derived from a particular polynucleotide sequence has the same, substantially the same, or homologous polynucleotide sequence as the particular sequence or fragment thereof. A polynucleotide sequence derived from a particular polynucleotide sequence may be a variant of the particular sequence or fragment thereof. For example, those skilled in the art should understand that circular RNA molecules applied herein may be modified to be sequence-different from the sequence from which they are derived while retaining the desired activity. In this specification, "circular RNA precursor" refers to a linear RNA molecule that can form a covalently linked closed circular RNA molecule, for example, by self-splicing. A circular RNA precursor may be produced by transcription from a nucleic acid vector containing the coding sequence of the circular RNA precursor. Alternatively, a circular RNA precursor may be obtained by chemical synthesis.

[0057] In some embodiments, a circular RNA precursor can form a covalently linked closed circular RNA molecule by self-splicing under the action of self-splicing intron fragments and residue cyclization elements.

[0058] As used herein, the term “self-splicing intron” refers to an intron that has self-splicing ribozyme activity and can excise itself and link two exons of flanking. In some embodiments, the splicing is autocatalytic splicing.

[0059] "Self-splicing introns" include, but are not limited to, group I introns and group II introns. Group I introns include 14 subgroups, but most group I introns belong to the IC3 subgroup. For example, the group I intron may be a group I intron from the cyanobacterium genus Anabaena belonging to the IC3 subgroup, or a group I intron derived from a T4 phage belonging to the IA2 subgroup, or a group I intron derived from Azoarcus sp. BH72 belonging to the IC3 subgroup.Examples of additional self-splicing introns available for use in this invention include Enterobacteriophage T4, phage Twort, phage SPO1, phage S3b, Bacillus anthracis, Clostridium botulinum, Tetrahymena thermophila, Dunaliella parva, Pneumocystis carinii, Physarum polycephalum, Anabaena PCC7120, Scytonema hofmanni, Agrobacterium tumefaciens, Synechocystis PCC 6803, Synechococcus elongatus PCC 6301, and Neurospora crassae. This includes, but is not limited to, self-splicing introns derived from living organisms such as *Candida albicans*, *Scytalidium dimidiatum*, *Pediadiaces Chlamydomonas nivalis*, *Chlorella vulgaris*, *Amoebidium parasiticum*, *Neurospora crassa*, *Emericella nidulans*, *Saccharomyces cerevisiae*, *Schizosaccharomyces pombe*, *Neochloris aquatica*, *Dunaliella parva*, *Symkania negevensis*, and *Emericella nidulans*.For example, see Vicens, Q., et al., (2008). Toward predicting self-splicing and protein-facilitated splicing of group I introns. RNA 14:2013-2029, and Tanner, AM, et al., (1996). Activity and thermostability of the small self-splicing group I intron in the pre-tRNAIIe of the purple bacterium Azoarcus. RNA 2:74-83.

[0060] In some embodiments, the self-splicing intron is selected from the group of cyanobacteria anabaena group I introns such as AnaX, azoarcus group I introns (Azo), Scytalidium dimidiatum group I introns (Sd), staphylococcal phage Twort group I introns (Twort), Scytonema-hofmani tRNA fMet group I introns (Sh), or Agrobacterium tumefaciens group I introns (At). For example, a self-splicing intron is selected from one nucleotide sequence among SEQ ID NO:1 to 6, or from a nucleotide sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with one of SEQ ID NO:1 to 6.

[0061] In some embodiments, the 3' self-splicing intron fragment and the 5' self-splicing intron fragment originate from the same self-splicing intron. In some embodiments, the 3' self-splicing intron fragment originates from or includes the 3' terminal portion of the self-splicing intron (a naturally occurring self-splicing intron), and accordingly, the 5' self-splicing intron fragment originates from or includes the 5' terminal portion of the self-splicing intron (a naturally occurring self-splicing intron). In some embodiments, the combination of the 3' self-splicing intron fragment and the 5' self-splicing intron fragment retains the self-splicing activity of the self-splicing intron (a naturally occurring self-splicing intron).

[0062] In some embodiments, the self-splicing intron is a group I intron. In some embodiments, the self-splicing intron is a group I intron of the IA2 or IC3 subgroup, preferably the IC3 subgroup.

[0063] In some embodiments, the 3' self-splicing intron fragment is a 3' group I intron fragment. In some embodiments, the 5' self-splicing intron fragment is a 5' group I intron fragment.

[0064] As used herein, a 3' self-splicing intron fragment (e.g., a 3' group I intron fragment) is sequence-identical to at least 75%, e.g., at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the 3' terminal portion of a natural self-splicing intron (e.g., a group I intron). A 5' self-splicing intron fragment (e.g., a 5' group I intron fragment) is sequence-identical to at least 75%, e.g., at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the 5' terminal portion of a natural self-splicing intron (e.g., a group I intron).

[0065] Generally, to achieve cyclic formation, it is considered necessary to split the natural group I intron at an internal site to form a so-called "inverted group I intron-exon self-splicing system" (PIE system). The internal split site used herein refers to the position where the natural group I intron splits into two parts, and the internal split site is selected so that even after rearrangement (permutation), the two independent parts (3' terminal and 5' terminal) of the resulting natural group I intron can both maintain the ribozyme activity necessary for self-splicing. It is considered that the two independent parts of the natural group I intron that maintain the overall conformation of the natural group I intron can maintain the ribozyme activity necessary for self-splicing. As used herein, an internal split site is located between intron internal split site I and intron internal split site II, where intron internal split site I refers to the first nucleotide downstream of the internal split site, and intron internal split site II refers to the first nucleotide upstream of the internal split site. As used herein, “PIE” or “PIE method” or “PIE system” refers to the “reorganized group I intron-exon self-splicing system, PIE system” described above.

[0066] In some embodiments, the 3' self-splicing intron fragment originates from the 3' terminal portion of the self-splicing intron, starting from the intron's internal split region I and extending to the 3' end of the self-splicing intron, and the 5' self-splicing intron fragment originates from the 5' terminal portion of the self-splicing intron, starting from the 5' end of the self-splicing intron and extending to the intron's internal split region II.

[0067] Only a portion of the internal splitting sites of group I introns can be determined by those skilled in the art, and these can be determined by referring to literature such as Puttaraju M., et al., (1992). Group I permuted intron-exon (PIE) sequences self-splice to produce circular exons; and Puttaraju M., et al., (1996). Circular ribozymes generated in Escherichia coli using group I self-splicing permuted intron-exon sequences. For example, group I introns, and especially anabaena group I introns, can generally split at specific sites within their P6 region to form a PIE system.

[0068] The length of the 3' end of a natural group I intron can be approximately 5% to 95% of the total length of the natural group I intron, for example, approximately 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%. Accordingly, the length of the 5' end of a natural group I intron can be approximately 5% to 90% of the total length of the natural group I intron, for example, approximately 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%.

[0069] In some embodiments, the combination of the 3' group I intron fragment and the 5' group I intron fragment substantially exhibits the self-splicing activity of the corresponding native group I intron.

[0070] As used herein, “cyclization efficiency” may refer to the ratio of the circular RNA output to the input precursor within a given time period. Alternatively, as used herein, “cyclization efficiency” may refer to the ratio of the desired circular RNA in the final product to the linear circRNA precursor, or the ratio of the circRNA to the sum of the circRNA and the linear circRNA precursor, within a given time period. Cyclization efficiency may be determined by methods well known in the art, for example, the methods described in Examples 2, 4, 6, and 8 of the present invention.

[0071] In some embodiments, the cyclization efficiency of the circular RNA increases by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, or more.

[0072] As used herein, “reduced immunogenicity” may mean that circular RNA causes a reduced immune response after contact with cells, i.e., the level of immune response is lower than that of control circular RNA or control linear RNA. For example, a reduced immune response refers to reduced cytokine expression. The cytokines include, but are not limited to, IFNβ, TNFα, IL6, and / or RIG-I. In some embodiments, the immunogenicity of the circular RNA is reduced by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more.

[0073] As used herein, “spacer” refers to any consecutive nucleotide sequence that does not adversely affect the function of the elements linked by it. Generally, a spacer may be inserted between two elements when it is desirable to avoid interaction between two adjacent or neighboring elements. The spacer sequences described herein can perform two functions: (1) to facilitate cyclization and (2) to facilitate functionality by allowing the correct folding of the residue cyclization element and the nucleotide sequence of interest (e.g., IRES). In some embodiments, the length of the spacer is 150 or less, 100 or less, 50 or less, 30 or less, 10 or less, 5 or less, or 3 or less nucleotides. In some embodiments, the length of the spacer is 5 nucleotides. In some embodiments, the length of the spacer is 4 nucleotides. In some embodiments, the length of the spacer is 3 nucleotides. In some embodiments, the first spacer may be omitted. In some embodiments, the second spacer may be omitted. In some embodiments, the first and second spacers may be omitted.

[0074] In one embodiment, the object of the present invention is to provide a circular RNA precursor which is oriented in the direction from 5' to 3'. a) 3' self-splicing intron fragment, b) Includes a 5' self-splicing intron fragment, Here, the combination of a 3' self-splicing intron fragment and a 5' self-splicing intron fragment preserves the self-splicing activity of the self-splicing intron and allows for the production of circular RNA by self-splicing of the circular RNA precursor. Here, the self-splicing intron is selected from group I introns and group II introns; for example, the self-splicing intron is selected from group I introns.

[0075] In embodiments of the present invention, the self-splicing intron is selected from the group of cyanobacteria anabaena group I introns such as AnaX, Azoarcus group I introns (Azo), Scytalidium dimidiatum group I introns (Sd), Staphylococcus phage Twort group I introns (Twort), Scytonema-hofmani tRNA fMet group I introns (Sh), or Agrobacterium tumefaciens group I introns (At).

[0076] In some embodiments of the present invention, the 3' self-splicing intron fragment originates from the 3' terminal portion of the self-splicing intron, starting from the intron internal split site I and extending to the 3' end of the self-splicing intron, and the 5' self-splicing intron fragment originates from the 5' terminal portion of the self-splicing intron, starting from the 5' end of the self-splicing intron and extending to the intron internal split site II.

[0077] In some embodiments of the present invention, when the self-splicing intron is a cyanobacterial anabaena group I intron such as AnaX, the intron internal split site II and the intron internal split site I are nucleotide A at position 115 and nucleotide A at position 116, respectively, derived from the 5' end of the AnaX intron (SEQ ID NO:1), and form the nucleotide sequence AA upon ligation.

[0078] In some embodiments of the present invention, when the self-splicing intron is an Azoarcus group I intron (Azo) (SEQ ID NO:2), 1) Intron internal split site II and intron internal split site I are nucleotides A at the 24th and 25th positions of the 5' end of the Azo intron, respectively, and when ligated, they form the nucleotide sequence AA(P2). 2) Intron internal split site II and intron internal split site I are nucleotides A at position 85 and 86 of the 5' end of the Azo intron, respectively, and when ligated, they form the nucleotide sequence AA(P4-5). 3) Intron internal split site II and intron internal split site I are nucleotides C at the 71st and 72nd positions of the 5' end of the Azo intron, respectively, and when ligated, they form the nucleotide sequence CC(P5). 4) Intron internal split site II and intron internal split site I are nucleotides C and U at the 108th and 109th positions of the 5' end of the Azo intron, respectively, and when ligated, they form the nucleotide sequence CU(P6). 5) Intron internal split site II and intron internal split site I are nucleotide A at position 154 and nucleotide A at position 155 of the 5' end of the Azo intron, respectively, and when ligated, they form the nucleotide sequence AA(P8), or 6) Intron internal split site II and intron internal split site I are nucleotides C at position 198 and A at position 199 of the 5' end of the Azo intron, respectively, and form the nucleotide sequence CA(P9) when ligated.

[0079] In some embodiments of the present invention, when the self-splicing intron is a Scytalidium dimidiatum group I intron (Sd) (SEQ ID NO: 4), 1) Intron internal split site II and intron internal split site I are nucleotides A at position 88 and G at position 89 of the 5' end of the Sd intron, respectively, and when ligated, they form the nucleotide sequence AG(P2). 2) Intron internal split site II and intron internal split site I are nucleotides U (173rd) and G (174th) at the 5' end of the Sd intron, respectively, and when ligated, they form the nucleotide sequence UG(P5). 3) Intron internal split site II and intron internal split site I are nucleotides A and G at the 204th and 205th positions of the 5' end of the Sd intron, respectively, and when ligated, they form the nucleotide sequence AG(P6). 4) Intron internal split site II and intron internal split site I are nucleotides C and G at the 252nd and 253rd positions of the 5' end of the Sd intron, respectively, and when ligated, form the nucleotide sequence CG(P8), or 5) Intron internal split site II and intron internal split site I are nucleotides C (370th) and G (371st) at the 5' end of the Sd intron, respectively, and form the nucleotide sequence CG(P9) when ligated.

[0080] In some embodiments of the present invention, when the self-splicing intron is a Staphylococcus phage Twort group I intron (Twort) (SEQ ID NO:3), 1) Intron internal split site II and intron internal split site I are nucleotides U (70th) and A (71st) at the 5' end of the Twort intron, respectively, and when ligated, they form the nucleotide sequence UA(P5). 2) Intron internal split site II and intron internal split site I are nucleotides U (103rd) and A (104th) at the 5' end of the Twort intron, respectively, and when ligated, they form the nucleotide sequence UA(P6). 3) Intron internal split site II and intron internal split site I are nucleotides A and U at the 132nd and 133rd positions of the 5' end of the Twort intron, respectively, and when ligated, they form the nucleotide sequence AU(P7). 4) Intron internal split site II and intron internal split site I are nucleotides U (173rd) and C (174th) at the 5' end of the Twort intron, respectively, and when ligated, they form the nucleotide sequence UC(P8). 5) Intron internal split site II and intron internal split site I are nucleotide A at position 206 and nucleotide A at position 207 of the 5' end of the Twort intron, respectively, and when ligated, they form the nucleotide sequence AA(P9). 6) Intron internal split site II and intron internal split site I are nucleotides G at position 226 and U at position 227 of the 5' end of the Twort intron, respectively, and when ligated, they form the nucleotide sequence GU(P9.1). 7) Intron internal split site II and intron internal split site I are the 23rd nucleotide U and the 24th nucleotide A at the 5' end of the Twort intron, respectively, and when ligated, they form the nucleotide sequence UA(P2), or 8) Intron internal split site II and intron internal split site I are nucleotides 82 and 83 of the 5' end of the Twort intron, respectively, and form the nucleotide sequence AA(P4-5) when ligated.

[0081] In some embodiments of the present invention, when the self-splicing intron is a Scytonema-hofmani tRNA fMet group I intron (Sh) (SEQ ID NO: 5), 1) Intron internal split site II and intron internal split site I are nucleotide A at position 129 and nucleotide A at position 130 of the 5' end of the Sh intron, respectively, and form the nucleotide sequence AA(P6) when ligated. 2) Intron internal split site II and intron internal split site I are nucleotide A at position 60 and nucleotide A at position 61 of the 5' end of the Sh intron, respectively, and form the nucleotide sequence AA(P2) when ligated. 3) Intron internal split site II and intron internal split site I are nucleotide A at position 109 and nucleotide A at position 110 of the 5' end of the Sh intron, respectively, and form the nucleotide sequence AA(P4-5) when ligated. 4) Intron internal split site II and intron internal split site I are nucleotide A at position 213 and nucleotide A at position 214 of the 5' end of the Sh intron, respectively, and form the nucleotide sequence UA(P9) when ligated.

[0082] In some embodiments of the present invention, when the self-splicing intron is an Agrobacterium tumefaciens group I intron (At) (SEQ ID NO: 6), the intron internal split site II and the intron internal split site I are nucleotide A at position 107 and nucleotide A at position 116, respectively, derived from the 5' end of the At intron, and form the nucleotide sequence AA(P6) upon ligation.

[0083] In some embodiments of the present invention, the 3' self-splicing intron fragment has at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the 3' terminal portion of the self-splicing intron. The sequence is such that the 5' self-splicing intron fragment has at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the 5' terminal portion of the self-splicing intron.

[0084] In some embodiments of the present invention, the self-splicing intron is a cyanobacterial anabaena group I intron, e.g., AnaX, and the 3' self-splicing intron fragment contains or comprises a nucleotide sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO:7, and the 5' self-splicing intron fragment contains a nucleotide sequence having at least 99% identity with SEQ ID NO:8 or SEQ ID It contains or consists of a nucleotide sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with NO:8.

[0085] In some embodiments of the present invention, the self-splicing intron is AnaX (SEQ ID NO:1), where the 3' self-splicing intron fragment contains or consists of a nucleotide sequence of SEQ ID NO:7, and the 5' self-splicing intron fragment contains or consists of a nucleotide sequence of SEQ ID NO:8.

[0086] In some embodiments of the present invention, the self-splicing intron is an Azoarcus group I intron (Azo), and the 3' self-splicing intron fragment contains or comprises one nucleotide sequence from SEQ ID NO: 9, 11, 13, 15, 17, or 19, or a nucleotide sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 9, 11, 13, 15, 17, or 19, and the 5' self-splicing intron fragment is SEQ ID It contains or consists of a nucleotide sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the nucleotide sequence of NO:10, 12, 14, 16, 18, or 20.

[0087] In some embodiments of the present invention, the self-splicing intron is an Azoarcus group I intron (Azo) (SEQ ID NO:2), where the 3' self-splicing intron fragment and the 5' self-splicing intron fragment are SEQ ID NO:9 and SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18, respectively.

[0088] In some embodiments of the present invention, the self-splicing intron is a Scytalidium dimidiatum group I intron (Sd) (SEQ ID NO: 4), and the 3' self-splicing intron fragment contains or comprises a nucleotide sequence of SEQ ID NO: 37, 39, 41, 43 or 45 or a nucleotide sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 37, 39, 41, 43 or 45, and the 5' self-splicing intron fragment is SEQ ID It contains or consists of a nucleotide sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the nucleotide sequence of NO:38, 40, 42, 44, or 46.

[0089] In some embodiments of the present invention, the self-splicing intron is a Scytalidium dimidiatum group I intron (Sd) (SEQ ID NO: 4), where the 3' self-splicing intron fragment and the 5' self-splicing intron fragment are SEQ ID NO: 37 and SEQ ID NO: 38, SEQ ID NO: 39 and SEQ ID NO: 40, SEQ ID NO: 41 and SEQ ID NO: 42, SEQ ID NO: 43 and SEQ ID NO: 44, SEQ ID NO: 45 and SEQ ID NO: 46, respectively.

[0090] In some embodiments of the present invention, the self-splicing intron is a Staphylococcus phage Twort group I intron (Twort) (SEQ ID NO:3), and the 3' self-splicing intron fragment is a nucleotide sequence or SEQ ID NO:21, 23, 25, 27, 29, 31, 33 or 35. NO:21, 23, 25, 27, 29, 31, 33 or 35 contains or comprises a nucleotide sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity, and the 5' self-splicing intron fragment contains or comprises SEQ ID NO:22, 24, 26, 28, 30, 32, 34 or 36 or SEQ ID NO: Contains or consists of nucleotide sequences having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with NO:22, 24, 26, 28, 30, 32, 34, or 36.

[0091] In some embodiments of the present invention, the self-splicing intron is a Staphylococcus phage Twort group I intron (Twort) (SEQ ID NO:3), where the 3' self-splicing intron fragment and the 5' self-splicing intron fragment are SEQ ID NO:21 and SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:24, SEQ ID NO:25 and SEQ ID NO:26, SEQ ID NO:27 and SEQ ID NO:28, SEQ ID NO:29 and SEQ ID NO:30, SEQ ID NO:31 and SEQ ID NO:32, SEQ ID NO:33 and SEQ ID NO:34, SEQ ID NO:35 and SEQ ID NO:36, respectively.

[0092] In some embodiments of the present invention, the self-splicing intron is a Scytonema-hofmani Trna fMet group I intron (Sh) (SEQ ID NO: 5), and the 3' self-splicing intron fragment contains or consists of a nucleotide sequence of SEQ ID NO: 47, 49, 51, 53 or a nucleotide sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 47, 49, 51, 53, and the 5' self-splicing intron fragment is SEQ ID It contains or consists of nucleotide sequences having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the nucleotide sequences of NO:48, 50, 52, 54 or SEQ ID NO:48, 50, 52, 54.

[0093] In some embodiments of the present invention, the self-splicing intron is a Scytonema-hofmani Trna fMet group I intron (Sh) (SEQ ID NO: 5), where the 3' self-splicing intron fragment and the 5' self-splicing intron fragment are SEQ ID NO: 47 and SEQ ID NO: 48, SEQ ID NO: 49 and SEQ ID NO: 50, SEQ ID NO: 51 and SEQ ID NO: 52, SEQ ID NO: 53 and SEQ ID NO: 54, respectively.

[0094] In some embodiments of the present invention, the self-splicing intron is an Agrobacterium tumefaciens group I intron (At) (SEQ ID NO: 6), and the 3' self-splicing intron fragment contains or comprises a nucleotide sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 55, and the 5' self-splicing intron fragment contains a nucleotide sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 8. It contains or consists of a nucleotide sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with NO:56.

[0095] In some embodiments of the present invention, the self-splicing intron is an Agrobacterium tumefaciens group I intron (At) (SEQ ID NO: 6), where the 3' self-splicing intron fragment and the 5' self-splicing intron fragment are SEQ ID NO: 55 and SEQ ID NO: 56, respectively.

[0096] In one embodiment, the present invention provides a circular RNA precursor which is oriented in the direction from 5' to 3'. a) 3' self-splicing intron fragment, b) The first residue cyclization element, c) Selectively select the target nucleotide sequence, d) The second residue cyclization element, e) Includes a 5' self-splicing intron fragment, Here, the combination of the 3' self-splicing intron fragment and the 5' self-splicing intron fragment preserves the self-splicing activity of the self-splicing intron, allowing the self-splicing of the circular RNA precursor to generate a circular RNA containing a first residue cyclization element, the desired nucleotide sequence, and a second residue cyclization element. Here, the total length of the first residue cyclic element and the second residue cyclic element is approximately 2 to 200 nucleotides.

[0097] As used herein, “residue cyclization element” refers to an exon-inducing sequence that is introduced separately to facilitate the cyclization process. Residue cyclization elements are sequences derived from the native exons of self-splicing introns (exons of self-splicing intron flanking) that are involved in or required for the cyclization of self-splicing introns, and that participate in cyclization together with the self-splicing intron but are retained in the final circular RNA. Specifically, residue cyclization elements are required for cyclization because they can be recognized and / or spliced ​​by the self-splicing intron (or a combination of a first self-splicing intron fragment and a second self-splicing intron fragment).

[0098] As used herein, the “exon region” in the residue cyclization element is a sequence derived from the native exon of a self-splicing intron (an exon of a self-splicing intron flanking) and is necessary for cyclization because it can be recognized and / or spliced ​​by a self-splicing intron (or a combination of a first self-splicing intron fragment and a second self-splicing intron fragment). The “exon region” as used herein may also be called a “splicing site sequence.”

[0099] In some embodiments, the first residue cyclization element comprises or consists of a 3' exon region oriented from 5' to 3' and an optional first spacer. In the cyclic RNA precursor, the 5' end of the 3' exon region is directly ligated to the 3' end of a 3' self-splicing intron fragment.

[0100] In some embodiments, the 3' exon region is derived from the natural 3' exon of the self-splicing intron (the 3'-terminal flanking (downstream) exon of the self-splicing intron) or a continuous fragment beginning with the 5' terminal nucleotide. In some embodiments, the total length of the first residue cyclization element and the second residue cyclization element is about 500 nucleotides or less, for example, about 200, about 100, about 90, about 80, about 70, about 60, about 50, about 40, about 30, about 20, about 15, about 10, about 5 nucleotides, about 4 nucleotides, about 3 nucleotides, about 2 nucleotides, about 1 nucleotide, or 0 or fewer nucleotides.

[0101] In some embodiments, the total length of the first residue cyclization element and the second residue cyclization element is about 2 to about 200 nucleotides, or any integer number of nucleotides in that range. In some embodiments, the total length of the first residue cyclization element and the second residue cyclization element is about 2 to about 30 nucleotides, or any integer number of nucleotides in that range. In some embodiments, the total length of the first residue cyclization element and the second residue cyclization element is about 2 to about 20 nucleotides, or any integer number of nucleotides in that range. In some embodiments, the total length of the first residue cyclization element and the second residue cyclization element is about 2 to about 15 nucleotides, or any integer number of nucleotides in that range. In some embodiments, the total length of the first residue cyclization element and the second residue cyclization element is about 2 to about 10 nucleotides, or any integer number of nucleotides in that range.

[0102] In some embodiments, the total length of the first residue cyclization element and the second residue cyclization element is at least 5 nucleotides. In some embodiments, the total length of the first residue cyclization element and the second residue cyclization element is at least 10 nucleotides. In some embodiments, the total length of the first residue cyclization element and the second residue cyclization element is at least 15 nucleotides.

[0103] In some embodiments, the total length of the first residue cyclization element and the second residue cyclization element is about 5 to about 100 nucleotides, or any integer number of nucleotides in that range. In some embodiments, the total length of the first residue cyclization element and the second residue cyclization element is about 10 to about 100 nucleotides, or any integer number of nucleotides in that range. In some embodiments, the total length of the first residue cyclization element and the second residue cyclization element is about 15 to about 100 nucleotides, or any integer number of nucleotides in that range.

[0104] In some embodiments, the total lengths of the first residue cyclization element and the second residue cyclization element are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46 These are 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, and 200 nucleotides.

[0105] In some embodiments, the first residue cyclization element and the second residue cyclization element are configured to give the circular RNA containing them reduced immunogenicity.

[0106] In some embodiments, the first residue cyclization element and the second residue cyclization element are configured to have a cyclization efficiency equivalent to or increased by the cyclization efficiency of the cyclization RNA or cyclization RNA precursor containing them.

[0107] In some embodiments, the first residue cyclization element comprises or consists of a 3' exon region oriented from 5' to 3' and an optional first spacer. In the cyclic RNA precursor, the 5' end of the 3' exon region is directly ligated to the 3' end of a 3' self-splicing intron fragment.

[0108] As used herein, the “exon region” in the residue cyclization element is a sequence derived from the native exon of a self-splicing intron (an exon of a self-splicing intron flanking) and is necessary for cyclization because it can be recognized and / or spliced ​​by a self-splicing intron (or a combination of a first self-splicing intron fragment and a second self-splicing intron fragment). The “exon region” as used herein may also be called a “splicing site sequence.”

[0109] In some embodiments, the 3' exon region is derived from the natural 3' exon of the self-splicing intron (the 3' terminal flanking (downstream) exon of the self-splicing intron) or a contiguous fragment beginning with the 5' terminal nucleotide.

[0110] In some embodiments, the 3' exon region is all the natural 3' exons of the self-splicing intron, or has at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with all the natural 3' exons of the self-splicing intron, or has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nucleotide substitutions, deletions, or additions compared to all the natural 3' exons of the self-splicing intron.

[0111] In some embodiments, the 3' exon region is a contiguous fragment beginning with the 5' terminal nucleotide of the natural 3' exon. In some embodiments, the 3' exon region has at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the contiguous fragment beginning with the 5' terminal nucleotide of the natural 3' exon. In some embodiments, the 3' exon region has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nucleotide substitutions, deletions, or additions compared to the contiguous fragment beginning with the 5' terminal nucleotide of the natural 3' exon.

[0112] In some embodiments, the contiguous fragment described above, starting from the 5' terminal nucleotide of the natural 3' exon, contains or comprises at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the nucleotides of the natural 3' exon. In some embodiments, the length of the contiguous fragment described above, starting from the 5' terminal nucleotide of the natural 3' exon, is at least 1 nucleotide, for example, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 50, or more nucleotides. In some embodiments, the length of the continuous fragment starting from the 5' terminal nucleotide of the natural 3' exon is one nucleotide or up to two, three, four, five, six, seven, eight, nine, ten, fifteen, twenty, twenty-five, fifty nucleotides or the full length of the natural 3' exon.

[0113] In some embodiments, the self-splicing intron is a group I intron, and the 3' exon region includes at least one sequence (e.g., a sequence of about 1 to about 20 nucleotides) at its 5' end, the sequence which can pair with the P1 region of the group I intron to form a P10 double-stranded region.

[0114] Regarding the self-splicing of group I introns, one or more consecutive nucleotides (e.g., at least about 1 to about 7 nucleotides) starting from the 5' end of the natural 3' exon can pair with the P1 region to form a P10 double-stranded region, and are therefore considered to play an important role in self-splicing. The definitions of the P1 and P10 regions of group I introns are known in this field and may be determined by referring to literature such as Burke, JM, et al., (1987). Structural conventions for group I introns, Stahley, RM, et al (2006). RNA splicing: group I intron crystal structures reveal the basis of splice site selection and metal ion catalysis, and Woodson, AS, (2005). Structure and assembly of group I introns.

[0115] In some embodiments, the second residue cyclization element includes or consists of a 5' exon region oriented from 3' to 5' and an optional second spacer. In the cyclic RNA precursor, the 3' end of the 5' exon region is directly ligated to the 5' end of the second self-splicing intron fragment.

[0116] In some embodiments, the 5' exon region is derived from the natural 5' exon of the self-splicing intron (the 5' terminal flanking (downstream) exon of the self-splicing intron) or a contiguous fragment beginning with the 3' terminal nucleotide.

[0117] In some embodiments, the 5' exon region is all the natural 5' exons of the self-splicing intron, or has at least 50%, at least 60%, at least 70%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 99%, or at least 99% sequence identity with all the natural 5' exons of the group I intron, or has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nucleotide substitutions, deletions, or additions compared to all the natural 5' exons of the self-splicing intron.

[0118] In some embodiments, the 5' exon region is a contiguous fragment beginning with the 3' terminal nucleotide of the natural 5' exon. In some embodiments, the 5' exon region has at least 50%, at least 60%, at least 70%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the contiguous fragment beginning with the 3' terminal nucleotide of the natural 5' exon. In some embodiments, the 5' exon region has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nucleotide substitutions, deletions, or additions compared to the contiguous fragment beginning with the 3' terminal nucleotide of the natural 5' exon.

[0119] In some embodiments, the contiguous fragment described above, starting from the 3' terminal nucleotide of the natural 5' exon, contains or comprises at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the nucleotides of the natural 5' exon. In some embodiments, the length of the contiguous fragment described above, starting from the 3' terminal nucleotide of the natural 5' exon, is at least 1 nucleotide, for example, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 50, or more nucleotides. In some embodiments, the length of the continuous fragment starting from the 3' terminal nucleotide of the natural 5' exon is one nucleotide or up to two, three, four, five, six, seven, eight, nine, ten, fifteen, twenty, twenty-five, fifty nucleotides or the full length of the natural 5' exon.

[0120] In some embodiments, the self-splicing intron is a group I intron, and its 5' exon region includes a sequence (e.g., a sequence of about 3 to about 8 consecutive nucleotides) at its 3' end, which can pair with an internal guide sequence (IGS) of the group I intron to form a P1 double-stranded region.

[0121] Regarding the self-splicing of group I introns, approximately 3 to 8 consecutive nucleotides starting from the 3' end of the aforementioned natural 5' exon can pair with an internal guide sequence (IGS) to form a P1 double-stranded region, and are therefore considered to play an important role in self-splicing. The definition of the IGS and / or P1 region of group I introns is known in this field and may be determined by referring to literature such as Burke, JM, et al., (1987). Structural conventions for group I introns, Stahley, RM, et al (2006). RNA splicing: group I intron crystal structures reveal the basis of splice site selection and metal ion catalysis, and / or Woodson, AS, (2005). Structure and assembly of group I introns.

[0122] In some embodiments, the first residue cyclization element and the second residue cyclization element include spacers with different sequences, or one of them includes a spacer while the other does not.

[0123] In some embodiments, the first residue cyclization element and the second residue cyclization element are configured to form a stem-loop structure. In some embodiments, the splicing junction is included in the loop of the stem-loop structure.

[0124] In some embodiments, the presence of the stem-loop structure is predicted and / or confirmed by the nucleotide sequences of the 3' self-splicing intron fragment involved in cyclization (e.g., the 3' group I intron fragment), the first residue cyclization element, the second residue cyclization element, and the 5' self-splicing intron fragment (e.g., the 5' group I intron fragment). In some embodiments, the presence of the stem-loop structure is predicted and / or confirmed based on the nucleotide sequences by an RNA structure prediction tool, such as RNAfold (http: / / rna.tbi.univie.ac.at / cgi-bin / RNAWebSuite / RNAfold.cgi) or RNAstructure (https: / / rna.urmc.rochester.edu / RNAstructureWeb / index.html).

[0125] In some embodiments, the first residue cyclization element includes a sequence structure described by the formula 5'-first loop sequence-first paired sequence-first unpaired sequence-3', and the second residue cyclization element includes a sequence structure described by the formula 5'-second unpaired sequence-second paired sequence-second loop sequence-3', Here, the first unpaired sequence or the second unpaired sequence may or may not exist independently. Here, the first paired array and the second paired array can pair complementaryly to form the stem of the stem-loop structure, and the first loop array and the second loop array can form the loop of the stem-loop structure, for example, by self-splicing for annularization.

[0126] Generally, the sequence forming the loop in the stem-loop structure originates from the 3' exon region and / or the 5' exon region.

[0127] In some embodiments, the self-splicing intron is a group I intron, and the first loop sequence contains or consists of one or more nucleotides (e.g., about 1 to about 20 nucleotides), which can pair with the P1 region of the group I intron in question (or a structure consisting of a 3' self-splicing intron fragment and a 5' self-splicing intron fragment) to form a P10 double-stranded region during the cyclization process.

[0128] In some embodiments, the first loop sequence may include or consist of (N)n nucleotide sequences, where N represents any nucleotide (A, G, U, or C), and n is an integer between 1 and 20, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some specific embodiments, n is 2, 4, or 5.

[0129] In some embodiments, the first loop sequence comprises or consists of about 1 to about 7 consecutive nucleotides starting from the 5' terminal nucleotide of the native 3' exon of the group I intron.

[0130] In some embodiments, the first loop array includes, or consists of, for example, AAAA, AA, UUUU, CAAA, or GAAA.

[0131] In some embodiments, the self-splicing intron is a group I intron, and the second loop sequence contains or consists of one or more nucleotides (e.g., about 3 to about 8 nucleotides), which can pair with the internal guide sequence (IGS) (or a structure consisting of a 3' self-splicing intron fragment and a 5' self-splicing intron fragment) of the group I intron to form a P1 double-stranded region during the cyclization process.

[0132] In some embodiments, the second loop sequence comprises or consists of about 3 to about 8 consecutive nucleotides starting from the 3' terminal nucleotide of the native 5' exon of the group I intron.

[0133] In some embodiments, the second loop array includes, for example, CUU or CUC, or consists of them.

[0134] In some specific embodiments, after circularization, a loop having the sequences CUUAAAA, CUUUUUU, CUUAA, CUUGAAA, CUUUAAA, CUUCAAA, or CUCAAAA can be formed.

[0135] In some embodiments, the first loop sequence includes or consists of AAAA, and the second loop sequence includes or consists of CUU. In some specific embodiments, a loop having a CUUAAAA sequence can be formed after circularization.

[0136] The paired arrangement forming the stem of the stem-loop structure may originate from the exon region, but may also originate from the spacer arrangement. Alternatively, the paired arrangement may originate from both the exon region and the spacer arrangement, i.e., the paired arrangement includes at least a portion of the exon region and at least a portion of the spacer.

[0137] While not limited to any particular theory, the efficiency of RNA cyclization based on intronic self-splicing (e.g., group I intronic self-splicing) is related to the number, type, or composition of base pairs in the stem portion of the stem-loop structure formed by the residue cyclization element. The stability of the stem-loop structure (which can be predicted, for example, by calculated free energy) may also affect the cyclization efficiency.

[0138] In some embodiments, the stem portion of the stem-loop structure includes at least two base pairs, for example, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, or more base pairs, preferably consecutive base pairs. In some embodiments, the stem portion of the stem-loop structure includes 2 to 15 or more consecutive base pairs. In some embodiments, the stem portion of the stem-loop structure includes 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 base pairs, preferably consecutive base pairs. In some embodiments, the stem portion of the stem-loop structure includes five base pairs, preferably consecutive base pairs. In some embodiments, the stem portion of the stem-loop structure includes six base pairs, preferably consecutive base pairs. In some embodiments, the stem portion of the stem-loop structure includes seven base pairs, preferably consecutive base pairs.

[0139] In some embodiments, the stem portion of the stem-loop structure contains at most two base mismatches, or at most one base mismatch, and preferably, the stem portion does not contain any base mismatches.

[0140] In some embodiments, the first pairing sequence includes only G, and the second pairing sequence includes only C. In some embodiments, the first pairing sequence includes only C, and the second pairing sequence includes only G. In some embodiments, the first pairing sequence includes only A, and the second pairing sequence includes only U.

[0141] In some embodiments, the circular RNA precursor further comprises a 5' homology arm sequence and a 3' homology arm sequence that can complementarily pair to form a homology arm double-stranded region. In some embodiments, the 5' homology arm sequence is located upstream of the 5' end of the 3' self-splicing intron fragment, and the 3' homology arm sequence is located downstream of the 3' end of the 5' self-splicing intron fragment.

[0142] In some embodiments, the desired nucleotide sequence comprises at least one protein-coding sequence and a translation initiation element, such as an internal ribosome entry site (IRES), which is operably ligated thereto. Here, "operably ligated" means that the translation initiation element, such as the IRES, is capable of guiding the translation of the encoded protein. In some embodiments, in the circular RNA precursor, the translation initiation element, such as the IRES, is located upstream of the 5' end of at least one protein-coding sequence, or the translation initiation element, such as the IRES, is located downstream of the 3' end of at least one protein-coding sequence.

[0143] The protein-coding sequence may encode a protein of eukaryotic, prokaryotic, or viral origin. In some embodiments, the protein may be any protein for therapeutic or diagnostic purposes. For example, the protein-coding region may encode a human protein, antigen, antibody, gene-editing enzyme, such as a CRISPR nuclease. For example, the encoded protein may be a chimeric antigen receptor, an immunomodulatory protein, and / or a transcription factor. Some specific examples include, but are not limited to, EGF, FGF1, RBD, G6PC, PAH, and HGF.

[0144] In some embodiments, the at least one protein-coding sequence may encode IL-2 or an aquaporin, such as the AQP-1 protein, or a protein derived from the naturally occurring AQP-1 protein by substituting, deleting, or adding one, two, three, four, five, six, seven, eight, nine, ten or more amino acids in the amino acid sequence of the naturally occurring AQP-1 protein, while having the same activity as the naturally occurring AQP-1 protein. In embodiments, the protein-coding sequence of the present invention encodes the AQP-1 protein. In embodiments, the AQP-1 protein is a mammalian AQP-1 protein, specifically, a human AQP-1 protein.

[0145] In some embodiments, at least one protein coding sequence of the present invention comprises one nucleotide sequence from SEQ ID NO:117~118, or comprises a nucleotide sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with one of SEQ ID NO:117~118.

[0146] In some embodiments, the translation initiation element of the present invention is an IRES sequence, a 5'UTR sequence, a Kozak sequence, m 6 It contains A-containing sequences, sequences complementary to ribosomal 18S rRNA, or any combination thereof.

[0147] IRES sequences include Taura syndrome virus, assassin bug virus, Tyler's encephalomyelitis virus, Simian virus 40, fire ant virus 1, wheat aphid virus, reticuloendotheliosis virus, Forman's poliovirus 1, soybean looper virus, Casimir wasp virus, human rhinovirus 2, leafhopper virus-1, human immunodeficiency virus type 1, leafhopper virus-1, pygmy kite P virus, hepatitis C virus, hepatitis A virus, hepatitis GB virus, foot-and-mouth disease virus, human enterovirus 71, horse rhinovirus, white-winged geometrid virus, encephalomyocarditis virus (EMCV), Drosophila C virus, Brassicaceae tobamovirus, cricket paralysis virus, bovine viral diarrhea virus 1, black queen bee larva virus, aphid lethal paralysis virus, avian encephalomyelitis virus, acute wasp paralysis virus, hibiscus chlorotic ring spot virus, swine fever virus, human FGF2, Human SFTPA1, Human AMLI / RUNXLI, Drosophila Antennapedia, Human AQP4, Human AT1R, Human BAG-1, Human BCL2, Human BiP, Human c-IAPl, Human c-myc, Human eIF4G, Mouse NDST4L, Human LEF1, Mouse HIF1α, Human n-myc, Mouse Gtx, Human p27kipl, Human PDGF2 / c-sis, Human p53, Human Pim-1, Mouse Rbm3, Drosophila reaper, Dog Sc IRES sequences may be selected from, but are not limited to, amper, Drosophila Ubx, human UNR, mouse UtrA, human VEGF-A, human XIAP, Drosophila hairless, budding yeast TFIID, budding yeast YAP1, human c-src, human FGF-1, salpicomavirus, cab crinkle virus, aptamers for eIF4G, coxsackievirus B3 (CVB3), or coxsackievirus A (CVA1 / 2). Wild-type IRES sequences may also be modified and used in the present invention. Preferably, the IRES is CVB3, BRAV-1_L, PV1_L, CAV2_L, BRAV-1, PV1, or CAV2.

[0148] For example, IRES contains a nucleotide sequence shown in one of SEQ ID NO: 120-526, or contains a nucleotide sequence having at least 75%, for example, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with one of SEQ ID NO: 120-526. In some embodiments, the circular RNA precursor further comprises homology arm sequence I and homology arm sequence II, which can complementaryally pair with each other to form homology arm double-stranded regions.

[0149] In some embodiments, homology arm array I is located upstream of the 3' self-splicing intron fragment, and homology arm array II is located downstream of the 5' self-splicing intron fragment.

[0150] In some embodiments, the length of homology arm sequence I or homology arm sequence II is approximately 5 to 500, 5 to 450, 5 to 400, 5 to 350, 5 to 300, 5 to 250, 5 to 200, 5 to 150, 5 to 100, 5 to 80, 5 to 60, 5 to 50, 5 to 40, 5 to 30, 5 to 25, 5 to 20, 5 to 15, or 5 to 10 nucleotides, preferably with a length of approximately 20, 25, 40, 50, 100, or 300 nucleotides.

[0151] In some embodiments, the two homologous arm arrays may be polyA and polyT, or polyG and polyC, respectively.

[0152] In some embodiments, one of homology arm sequences I and homology arm sequences II of the present invention includes one nucleotide sequence from SEQ ID NO: 71 to 94, or includes a nucleotide sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with one of SEQ ID NO: 71 to 94, while the other homology arm sequence includes the corresponding complementary sequence.

[0153] In some embodiments, the cyclic RNA precursor of the present invention comprises a nucleotide sequence selected from one of SEQ ID NO: 527, 532, and 537-542, or comprises a nucleotide sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with one of SEQ ID NO: 527, 532, and 537-542.

[0154] In some embodiments, the target nucleotide sequence is a non-protein-coding sequence. For example, the non-protein-coding sequence may be POLR2A, antisense RNA, aptamer, guide RNA, or any non-protein-coding RNA present in a living organism. The non-protein-coding sequence may or may not include a specific secondary structure.

[0155] In one embodiment, the present invention provides a nucleic acid vector for generating a circular RNA molecule, the vector comprising a circular RNA precursor coding sequence described in the present invention.

[0156] As used herein, “vector” refers to a single segment of DNA extracted from the cells of a virus, plasmid, or higher organism into which an exogenous DNA fragment may or may have been inserted for the purpose of cloning and / or expression. In some embodiments, the vector may be stably maintained in vivo. The vector may include, for example, an origin of replication, a selection marker or reporter gene, such as antibiotic resistance or GFP, and / or a multicloning site (MCS). The term includes linear DNA fragments (e.g., PCR products, linear plasmid fragments), plasmid vectors, viral vectors, cosmids, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), and the like.

[0157] In some embodiments, the nucleic acid vector further comprises an RNA polymerase promoter sequence operably ligated to the circular RNA precursor coding sequence. The operably ligated promoter allows in vivo and / or in vitro transcription of the circular RNA precursor. The promoter is, for example, a T7 RNA polymerase promoter, a T6 viral RNA polymerase promoter, an SP6 viral RNA polymerase promoter, a T3 viral RNA polymerase promoter, or a T4 viral RNA polymerase promoter.

[0158] This invention provides a novel Clean PIE system for producing circular RNA, which can reduce the immunogenicity of circular RNA, increase the stability of intracellular circular RNA, and lower safety risks for clinical applications by improving the accuracy of the circular RNA sequence and reducing alterations to the circular RNA secondary structure.

[0159] In one embodiment, the present invention provides a circular RNA precursor which is oriented in the direction from 5' to 3'. a) 3' self-splicing intron fragment, b) The target nucleotide sequence and c) Includes a 5' self-splicing intron fragment, Here, the combination of a 3' self-splicing intron fragment and a 5' self-splicing intron fragment preserves the self-splicing activity of the self-splicing intron, allowing the production of a circular RNA containing the target nucleotide sequence by self-splicing of the circular RNA precursor. Here, the target nucleotide sequence includes a circular sequence fragment II containing a ribozyme recognition site II at its 5' end and a circular sequence fragment I containing a ribozyme recognition site I at its 3' end, in the direction from 5' to 3'. Here, the circularized sequence fragment I originates from the 5' end portion of the circularized sequence, and the circularized sequence fragment II originates from the 3' end portion of the circularized sequence, and Here, in the generated circular RNA, the ribozyme recognition site I and the ribozyme recognition site II are linked, thereby linking the circularized sequence fragment I and the circularized sequence fragment II to form the circularized sequence.

[0160] In one embodiment, the present invention provides a circular RNA precursor which is oriented in the direction from 5' to 3'. a) 3' self-splicing intron fragment, b) The target nucleotide sequence and c) Includes a 5' self-splicing intron fragment, Here, the combination of the 3' self-splicing intron fragment and the 5' self-splicing intron fragment preserves the self-splicing activity of the self-splicing intron and allows for the production of a circular RNA containing the target nucleotide sequence by self-splicing of the circular RNA precursor. Here, the target nucleotide sequence includes a circular sequence fragment II containing a ribozyme recognition site II at its 5' end and a circular sequence fragment I containing a ribozyme recognition site I at its 3' end, in the direction from 5' to 3'. Here, the circularized sequence fragment I is the 5' end portion of the circularized sequence, and the circularized sequence fragment II is the 3' end portion of the circularized sequence, and Here, in the generated circular RNA, ribozyme recognition site I and ribozyme recognition site II are linked, thereby linking circularized sequence fragment I and circularized sequence fragment II to form the circularized sequence.

[0161] In the embodiment, the target nucleotide sequence includes, between the circularized sequence fragment II and the circularized sequence fragment I, one or more sequences selected from the group consisting of at least one protein-coding sequence, a translation initiation element (TIE), a non-TIE functional element, or any combination thereof.

[0162] As used herein, in a Clean-PIE system, “circularized sequence” refers to a nucleotide sequence that is to be part of a target nucleotide sequence. The circularized sequence is involved in or required for the circularization process by a self-splicing intron and achieves circularization together with the self-splicing intron. In embodiments, the circularized sequence is a protein-coding sequence, a non-coding sequence, a translation initiation element (TIE), or a non-TIE functional element. As used herein, in a Clean-PIE system, “circularized sequence fragment I” and “circularized sequence fragment II” are each part of a circularized sequence. In the Clean-PIE system of the present invention, circularized sequence fragments I and II are linked to form the circularized sequence, and such a circularized sequence retains its biological function or activity.

[0163] As used herein, in the Clean-PIE system, “ribozyme recognition site” refers to a nucleotide sequence fragment belonging to a part of the circularized sequence. The ribozyme recognition site is involved in or required for the circularization process by self-splicing introns, and together with the self-splicing introns, it achieves circularization, but is retained as part of the circularized sequence in the final circular RNA. In embodiments, ribozyme recognition site I and ribozyme recognition site II are linked to form a ribozyme recognition site. In embodiments, the ribozyme recognition site comprises 2 to 50 nucleotides, for example, 2 to 40, 2 to 30, 2 to 25, 2 to 20, 2 to 15, 2 to 10 nucleotides, specifically comprising 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more nucleotides. In the embodiment, ribozyme recognition site I consists of 1 to 10 nucleotides, specifically 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In the embodiment, ribozyme recognition site II consists of 1 to 10 nucleotides, specifically 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides.

[0164] As used herein, “nucleotide motif” refers to a nucleotide sequence mode related to the biological function of a ribozyme recognition site. In the present invention, the nucleotide motif is located at the 3' end of ribozyme recognition site I.

[0165] In the embodiment, the ribozyme recognition site I includes or consists of a nucleotide motif consisting of one of SEQ ID NO:95~116 in the direction from 5' to 3', specifically, the nucleotide motif consists of one of CUU, CUC, or CAU.

[0166] For the sole purpose of illustrating the present invention, the following examples of the “Clean-PIE System” of the present invention are provided herein. These specific examples include the “Clean-PIE-Cargo System,” the “Clean-PIE-TIE System,” the “Clean-PIE-NonTIE System,” and the “Clean-PIE-Non-coding Cargo System.” Those skilled in the art should understand that any changes, deviations, modifications, etc., made to the “Clean-PIE System” described below are all within the scope of the present invention and do not depart from the present invention.

[0167] Regarding the Clean-PIE-Cargo system, the present invention provides a circular RNA precursor which is oriented from 5' to 3'. a) 3' self-splicing intron fragment, b) The target nucleotide sequence and c) Includes a 5' self-splicing intron fragment, Here, the combination of a 3' self-splicing intron fragment and a 5' self-splicing intron fragment preserves the self-splicing activity of the self-splicing intron, allowing the production of a circular RNA containing the target nucleotide sequence by self-splicing of the circular RNA precursor. Here, the target nucleotide sequence includes protein-coding sequence fragment II, a translation initiation element such as an internal ribosome entry site (IRES), and protein-coding sequence fragment I, in the direction from 5' to 3'. Here, protein-coding sequence fragment I originates from the 5' end of at least one protein-coding sequence, and protein-coding sequence fragment II originates from the 3' end of at least one protein-coding sequence. Protein coding sequence fragment I includes a ribozyme recognition site I located at its 3' end, and protein coding sequence fragment II includes a ribozyme recognition site II located at its 5' end. In the resulting circular RNA, ribozyme recognition site I and ribozyme recognition site II are linked together, thereby linking protein coding sequence fragment I and protein coding sequence fragment II to form the aforementioned at least one protein coding sequence.

[0168] The present invention provides a circular RNA precursor which is oriented in the direction from 5' to 3'. a) 3' self-splicing intron fragment, b) The target nucleotide sequence and c) Includes a 5' self-splicing intron fragment, Here, the combination of a 3' self-splicing intron fragment and a 5' self-splicing intron fragment preserves the self-splicing activity of the self-splicing intron and allows the self-splicing of the circular RNA precursor to produce a circular RNA containing the desired nucleotide sequence, wherein the desired nucleotide sequence comprises, in the 5' to 3' direction, a protein-coding sequence fragment II, a translation initiation element such as an internal ribosome entry site (IRES), and a protein-coding sequence fragment I. Here, protein-coding sequence fragment I is the 5' end portion of at least one protein-coding sequence, and protein-coding sequence fragment II is the 3' end portion of at least one protein-coding sequence, and The protein-coding sequence fragment I includes a ribozyme recognition site I located at its 3' end, and the protein-coding sequence fragment II includes a ribozyme recognition site II located at its 5' end. In the resulting circular RNA, the ribozyme recognition site I and the ribozyme recognition site II are linked together, thereby linking the protein-coding sequence fragment I and the protein-coding sequence fragment II to form the at least one protein-coding sequence.

[0169] In some embodiments, at least one protein coding sequence is a sequence that codes for a diagnostic or therapeutic protein. In some embodiments, the protein coded by the protein coding sequence is a mammalian protein, such as a human protein. In some embodiments, at least one protein coding sequence comprises at least two protein coding sequences. In some embodiments, the at least one protein coding sequence is a sequence that codes for two or more proteins. In some embodiments, the at least one protein coding sequence comprises at least two protein coding sequences, and another translation initiation element may be ligated between any two adjacent protein coding sequences, and such another translation initiation element may be an IRES sequence, a 5'UTR sequence, a Kozak sequence, m 6 It contains A-containing sequences, sequences complementary to ribosomal 18S rRNA, or any combination thereof.

[0170] In some embodiments, the at least one protein-coding sequence includes one nucleotide sequence from SEQ ID NO: 117 to 118, or includes a nucleotide sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with one of SEQ ID NO: 117 to 118. In the Clean-PIE-Cargo system of the present invention, it should be understood that ribozyme recognition site I and ribozyme recognition site II are each nucleotide sequences of a single segment belonging to a part of the cargo sequence (Cargo), and are configured to reduce the immunogenicity of the circular RNA containing them. In some embodiments, ribozyme recognition sites I and II are configured to have a cyclization efficiency equivalent to or increased by the cyclization efficiency of the cyclization RNA precursor containing them.

[0171] In some embodiments, the total length of the ribozyme recognition site I and ribozyme recognition site II is approximately 0 to approximately 100 nucleotides, approximately 0 to approximately 90 nucleotides, approximately 0 to approximately 80 nucleotides, approximately 0 to approximately 70 nucleotides, approximately 0 to approximately 60 nucleotides, approximately 0 to approximately 50 nucleotides, approximately 0 to approximately 40 nucleotides, approximately 0 to approximately 35 nucleotides, approximately 2 to approximately 100 nucleotides, approximately 5 to approximately 100 nucleotides, approximately 10 to approximately 100 nucleotides, approximately 20 to approximately 100 nucleotides, approximately 30 to approximately 100 nucleotides, or approximately 30 to approximately 100 nucleotides, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 ,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,5 These are 7, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

[0172] In some embodiments, the total length of the ribozyme recognition site I is approximately 0 to approximately 100 nucleotides, approximately 0 to approximately 90 nucleotides, approximately 0 to approximately 80 nucleotides, approximately 0 to approximately 70 nucleotides, approximately 0 to approximately 60 nucleotides, approximately 0 to approximately 50 nucleotides, approximately 0 to approximately 40 nucleotides, approximately 0 to approximately 35 nucleotides, approximately 2 to approximately 100 nucleotides, approximately 5 to approximately 100 nucleotides, approximately 10 to approximately 100 nucleotides, approximately 20 to approximately 100 nucleotides, approximately 30 to approximately 100 nucleotides, or approximately 30 to approximately 100 nucleotides, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 ,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,5 These are 8, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

[0173] In some embodiments, the total length of the ribozyme recognition site II is approximately 0 to approximately 100 nucleotides, approximately 0 to approximately 90 nucleotides, approximately 0 to approximately 80 nucleotides, approximately 0 to approximately 70 nucleotides, approximately 0 to approximately 60 nucleotides, approximately 0 to approximately 50 nucleotides, approximately 0 to approximately 40 nucleotides, approximately 0 to approximately 35 nucleotides, approximately 2 to approximately 100 nucleotides, approximately 5 to approximately 100 nucleotides, approximately 10 to approximately 100 nucleotides, approximately 20 to approximately 100 nucleotides, approximately 30 to approximately 100 nucleotides, or approximately 30 to approximately 100 nucleotides, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 5 These are 8, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

[0174] In some embodiments, the ribozyme recognition site I includes a sequence structure of the formula 5'-third unpaired sequence-third paired sequence-third loop sequence-3', and the ribozyme recognition site II includes a sequence structure of the formula 5'-fourth loop sequence-fourth paired sequence-fourth unpaired sequence-3'.

[0175] In some examples, the third or fourth unpaired sequence may or may not exist independently.

[0176] In some embodiments, the ribozyme recognition site I includes or consists of the “nucleotide motif” defined above. In embodiments, the ribozyme recognition site I includes or consists of a nucleotide motif oriented from 5' to 3', where the nucleotide motif consists of one of SEQ ID NO: 95 to 116, specifically, the nucleotide motif consists of one of CUU, CUC, or CAU.

[0177] In some embodiments, the ribozyme recognition site I includes or consists of a nucleotide sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with one nucleotide sequence from SEQ ID NO:95-116.

[0178] In some embodiments, the ribozyme recognition site I includes or comprises a nucleotide sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with one of the nucleotide sequences selected from SEQ ID NO: 58, 60, 62, 64, 66, 68, or 70, preferably the ribozyme recognition site I includes the nucleotide sequences GAGGUCUU, AGAGACUU, UCCAGCUU, or UCUGGCAU.

[0179] In some embodiments, the ribozyme recognition site II includes or comprises a nucleotide sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with one of the nucleotide sequences selected from SEQ ID NO: 57, 59, 61, 63, 65, 67, or 69, and preferably the ribozyme recognition site II includes the nucleotide sequence AAACCUC, AAUCUCU, AACUGGA, or AACCAGC.

[0180] In some embodiments, the ribozyme recognition site I includes or consists of one nucleotide sequence from SEQ ID NO: 58, 60, 62, 64, 66, 68, or 70, and the ribozyme recognition site II includes or consists of one nucleotide sequence from SEQ ID NO: 57, 59, 61, 63, 65, 67, or 69.

[0181] In one embodiment, the ribozyme recognition site I is selected from the nucleotide sequences UGUAACUU, GAGGUCUU, AGAGACUU, UCCAGCUU, or UCUGGCAU.

[0182] In the embodiment, the ribozyme recognition site II is selected from the nucleotide sequences AGAAGUAAC, AAAAUUACA, AAACCUC, AAUCUCU, AACUGGA, or AACCAGC.

[0183] In this embodiment, the ribozyme recognition site I is UGUAACUU, and the ribozyme recognition site II is AGAAGUAAC.

[0184] In this embodiment, the ribozyme recognition site I is UGUAACUU, and the ribozyme recognition site II is AAAAUUACA.

[0185] In this embodiment, the ribozyme recognition site I is GAGGUCUU, and the ribozyme recognition site II is AAACCUC.

[0186] In this embodiment, the ribozyme recognition site I is AGAGACUU, and the ribozyme recognition site II is AAUCUCU.

[0187] In this embodiment, the ribozyme recognition site I is UCCAGCUU, and the ribozyme recognition site II is AACUGGA.

[0188] In this embodiment, the ribozyme recognition site I is UCUGGCAU, and the ribozyme recognition site II is AACCAGC.

[0189] Regarding the Clean-PIE-TIE system, the present invention provides a circular RNA precursor which is oriented from 5' to 3'. a) 3' self-splicing intron fragment, b) The target nucleotide sequence and c) Includes a 5' self-splicing intron fragment, Here, the combination of a 3' self-splicing intron fragment and a 5' self-splicing intron fragment preserves the self-splicing activity of the self-splicing intron, allowing the production of a circular RNA containing the target nucleotide sequence by self-splicing of the circular RNA precursor. Here, the target nucleotide sequence includes, in the direction from 5' to 3', a translation initiation element fragment II, at least one protein coding sequence, and a translation initiation element fragment I. Here, translation initiation element fragment I originates from the 5' end portion of the translation initiation element, and translation initiation element fragment II originates from the 3' end portion of the translation initiation element, and The translation initiation element fragment I includes a ribozyme recognition site I located at its 3' end, and the translation initiation element fragment II includes a ribozyme recognition site II located at its 5' end. In the resulting circular RNA, the ribozyme recognition site I and the ribozyme recognition site II are linked together, thereby linking the translation initiation element fragment I and the translation initiation element fragment II to form a translation initiation element that can recruit a ribosome and initiate translation.

[0190] One object of the present invention is to provide a circular RNA precursor which is oriented in the direction from 5' to 3'. a) 3' self-splicing intron fragment, b) The target nucleotide sequence and c) Includes a 5' self-splicing intron fragment, Here, the combination of the 3' self-splicing intron fragment and the 5' self-splicing intron fragment preserves the self-splicing activity of the self-splicing intron and allows for the production of a circular RNA containing the target nucleotide sequence by self-splicing of the circular RNA precursor. Here, the target nucleotide sequence includes, in the direction from 5' to 3', a translation initiation element fragment II, at least one protein coding sequence, and a translation initiation element fragment I. Here, the translation initiation element fragment I is the 5' end portion of the translation initiation element, and the translation initiation element fragment II is the 3' end portion of the translation initiation element, and The translation initiation element fragment I includes a ribozyme recognition site I located at its 3' end, and the translation initiation element fragment II includes a ribozyme recognition site II located at its 5' end. Here, the ribozyme recognition site I and the ribozyme recognition site II are linked in the generated circular RNA, thereby linking the translation initiation element fragment I and the translation initiation element fragment II to form a translation initiation element that can recruit a ribosome and initiate translation.

[0191] In an embodiment, the at least one protein coding sequence is a sequence that codes for a diagnostic or therapeutic protein. In an embodiment, the protein coded by the protein coding sequence is a mammalian protein, such as a human protein. In an embodiment, the at least one protein coding sequence comprises at least two protein coding sequences. In an embodiment, the at least one protein coding sequence is a sequence that codes for two or more proteins. In an embodiment, the at least one protein coding sequence is a sequence that codes for two or more diagnostic or therapeutic proteins. In an embodiment, the at least one protein coding sequence is a sequence that codes for two or more proteins, and another translation initiation element is operably linked between any two adjacent protein coding sequences, and the other translation initiation element is an IRES sequence, a 5'UTR sequence, a Kozak sequence, m 6 The sequence contains A-modified sequences, sequences complementary to ribosomal 18S rRNA, or any combination thereof.

[0192] In some embodiments, the at least one protein coding sequence described in the present invention comprises one nucleotide sequence from SEQ ID NO: 117 to 118, or comprises a nucleotide sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with one of SEQ ID NO: 117 to 118.

[0193] In the Clean-PIE-TIE system of the present invention, ribozyme recognition site I and ribozyme recognition site II are each nucleotide sequences of a single segment belonging to a part of the IRES sequence, and are configured to reduce the immunogenicity of the circular RNA containing them. In some embodiments, ribozyme recognition site I and ribozyme recognition site II are configured to have a cyclization efficiency equivalent to or increased by the circular RNA precursor containing them.

[0194] In some embodiments, the total length of the ribozyme recognition site I and ribozyme recognition site II is approximately 0 to approximately 100 nucleotides, approximately 0 to approximately 90 nucleotides, approximately 0 to approximately 80 nucleotides, approximately 0 to approximately 70 nucleotides, approximately 0 to approximately 60 nucleotides, approximately 0 to approximately 50 nucleotides, approximately 0 to approximately 40 nucleotides, approximately 0 to approximately 35 nucleotides, approximately 2 to approximately 100 nucleotides, approximately 5 to approximately 100 nucleotides, approximately 10 to approximately 100 nucleotides, approximately 20 to approximately 100 nucleotides, approximately 30 to approximately 100 nucleotides, or approximately 30 to approximately 100 nucleotides, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 ,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,5 These are 7, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

[0195] In some embodiments, the total length of the ribozyme recognition site I is approximately 0 to approximately 100 nucleotides, approximately 0 to approximately 90 nucleotides, approximately 0 to approximately 80 nucleotides, approximately 0 to approximately 70 nucleotides, approximately 0 to approximately 60 nucleotides, approximately 0 to approximately 50 nucleotides, approximately 0 to approximately 40 nucleotides, approximately 0 to approximately 35 nucleotides, approximately 2 to approximately 100 nucleotides, approximately 5 to approximately 100 nucleotides, approximately 10 to approximately 100 nucleotides, approximately 20 to approximately 100 nucleotides, approximately 30 to approximately 100 nucleotides, or approximately 30 to approximately 100 nucleotides, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 ,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,5 These are 8, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

[0196] In some embodiments, the total length of the ribozyme recognition site II is approximately 0 to approximately 100 nucleotides, approximately 0 to approximately 90 nucleotides, approximately 0 to approximately 80 nucleotides, approximately 0 to approximately 70 nucleotides, approximately 0 to approximately 60 nucleotides, approximately 0 to approximately 50 nucleotides, approximately 0 to approximately 40 nucleotides, approximately 0 to approximately 35 nucleotides, approximately 2 to approximately 100 nucleotides, approximately 5 to approximately 100 nucleotides, approximately 10 to approximately 100 nucleotides, approximately 20 to approximately 100 nucleotides, approximately 30 to approximately 100 nucleotides, or approximately 30 to approximately 100 nucleotides, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 5 These are 8, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

[0197] In some embodiments, ribozyme recognition site I includes a sequence structure of the formula 5'-third unpaired sequence-third paired sequence-third loop sequence-3', and ribozyme recognition site II includes a sequence structure of the formula 5'-fourth loop sequence-fourth paired sequence-fourth unpaired sequence-3'.

[0198] In some embodiments, the third unpaired sequence or the fourth unpaired sequence may or may not be present independently.

[0199] In some embodiments, ribozyme recognition site I includes a nucleotide sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with one nucleotide sequence from SEQ ID NO:95-116.

[0200] In some embodiments, the ribozyme recognition site I includes or comprises one nucleotide motif from SEQ ID NO:95~116 in the 5' to 3' direction, specifically, the nucleotide motif consists of one of CUU, CUC, or CAU.

[0201] In some embodiments, the ribozyme recognition site II may be selected from a variety of sequences.

[0202] In some embodiments, the ribozyme recognition site II includes or consists of the 3' exon region defined above, and the ribozyme recognition site I includes or consists of the 5' exon region defined above.

[0203] In some embodiments, ribozyme recognition site I includes or comprises a nucleotide sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with one of SEQ ID NO: 58, 60, 62, 64, 66, 68, or 70, preferably ribozyme recognition site I includes the nucleotide sequence GAGGUCUU, AGAGACUU, UCCAGCUU, or UCUGGCAU.

[0204] In some embodiments, ribozyme recognition site II includes or comprises a nucleotide sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with one of SEQ ID NO: 57, 59, 61, 63, 65, 67, or 69, preferably ribozyme recognition site II includes the nucleotide sequence AAACCUC, AAUCUCU, AACUGGA, or AACCAGC.

[0205] In some embodiments, the ribozyme recognition site I includes or consists of one nucleotide sequence from SEQ ID NO: 58, 60, 62, 64, 66, 68, or 70, and the ribozyme recognition site II includes or consists of one nucleotide sequence from SEQ ID NO: 57, 59, 61, 63, 65, 67, or 69.

[0206] In an embodiment, the ribozyme recognition site I is selected from the nucleotide sequences UGUAACUU, UGUAACUU, GAGGUCUU, AGAGACUU, UCCAGCUU or UCUGGCAU.

[0207] In an embodiment, the ribozyme recognition site II is selected from the nucleotide sequences AGAAGUAAC, AAAAUUACA, AAACCUC, AAUCUCU, AACUGGA or AACCAGC.

[0208] In an embodiment, the ribozyme recognition site I is UGUAACUU, and the ribozyme recognition site II is AGAAGUAAC.

[0209] In an embodiment, the ribozyme recognition site I is UGUAACUU, and the ribozyme recognition site II is AAAAUUACA.

[0210] In an embodiment, the ribozyme recognition site I is GAGGUCUU, and the ribozyme recognition site II is AAACCUC.

[0211] In an embodiment, the ribozyme recognition site I is AGAGACUU, and the ribozyme recognition site II is AAUCUCU.

[0212] In an embodiment, the ribozyme recognition site I is UCCAGCUU, and the ribozyme recognition site II is AACUGGA.

[0213] In an embodiment, the ribozyme recognition site I is UCUGGCAU, and the ribozyme recognition site II is AACCAGC.

[0214] In one aspect, one object of the present invention is to provide a circular RNA precursor, which, in the 5' to 3' direction, a) a 3' self-splicing intron fragment, and b) The target nucleotide sequence and c) Includes a 5' self-splicing intron fragment, Here, the combination of a 3' self-splicing intron fragment and a 5' self-splicing intron fragment preserves the self-splicing activity of the self-splicing intron, allowing the production of a circular RNA containing the target nucleotide sequence by self-splicing of the circular RNA precursor. Here, the target nucleotide sequence comprises, in the direction from 5' to 3', a non-TIE functional element fragment II, a translation initiation element, at least one protein coding sequence, and a non-TIE functional element fragment I, wherein the non-TIE functional element fragment I originates from the 5' terminal portion of the non-TIE functional element, and the non-TIE translation initiation element fragment II originates from the 3' terminal portion of the non-TIE functional element, and The non-TIE functional element fragment I includes a ribozyme recognition site I located at its 3' end, and the non-TIE functional element fragment II includes a ribozyme recognition site II located at its 5' end. In the generated circular RNA, the ribozyme recognition site I and the ribozyme recognition site II are linked together, thereby linking the non-TIE functional element fragment I and the non-TIE functional element fragment II to form a non-TIE functional element.

[0215] As used herein, the term “non-TIE functional element” refers to a functional element that is not involved in translation initiation, such as a nucleotide sequence for regulating RNA translation, RNA cyclization, or RNA transcription.

[0216] In embodiments, the non-TIE functional element is a 3'UTR or a replicon. In embodiments, the at least one protein-coding sequence comprises at least two protein-coding sequences. Such at least two protein-coding sequences may be linked to each other via a spacer or another translation initiation element. In embodiments, the translation initiation element is an IRES sequence, a 5'UTR sequence, a Kozak sequence, m 6The sequence is selected from an A-modified sequence, a sequence complementary to ribosomal 18S rRNA, or any combination thereof. In one embodiment, the translation initiation element is an IRES sequence.

[0217] In some embodiments, the total length of the ribozyme recognition site I and ribozyme recognition site II is approximately 0 to approximately 100 nucleotides, approximately 0 to approximately 90 nucleotides, approximately 0 to approximately 80 nucleotides, approximately 0 to approximately 70 nucleotides, approximately 0 to approximately 60 nucleotides, approximately 0 to approximately 50 nucleotides, approximately 0 to approximately 40 nucleotides, approximately 0 to approximately 35 nucleotides, approximately 2 to approximately 100 nucleotides, approximately 5 to approximately 100 nucleotides, approximately 10 to approximately 100 nucleotides, approximately 20 to approximately 100 nucleotides, approximately 30 to approximately 100 nucleotides, or approximately 30 to approximately 100 nucleotides, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 ,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,5 These are 7, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

[0218] In some embodiments, the total length of the ribozyme recognition site I is approximately 0 to approximately 100 nucleotides, approximately 0 to approximately 90 nucleotides, approximately 0 to approximately 80 nucleotides, approximately 0 to approximately 70 nucleotides, approximately 0 to approximately 60 nucleotides, approximately 0 to approximately 50 nucleotides, approximately 0 to approximately 40 nucleotides, approximately 0 to approximately 35 nucleotides, approximately 2 to approximately 100 nucleotides, approximately 5 to approximately 100 nucleotides, approximately 10 to approximately 100 nucleotides, approximately 20 to approximately 100 nucleotides, approximately 30 to approximately 100 nucleotides, or approximately 30 to approximately 100 nucleotides, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 ,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,5 These are 8, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

[0219] In some embodiments, the total length of the ribozyme recognition site II is approximately 0 to approximately 100 nucleotides, approximately 0 to approximately 90 nucleotides, approximately 0 to approximately 80 nucleotides, approximately 0 to approximately 70 nucleotides, approximately 0 to approximately 60 nucleotides, approximately 0 to approximately 50 nucleotides, approximately 0 to approximately 40 nucleotides, approximately 0 to approximately 35 nucleotides, approximately 2 to approximately 100 nucleotides, approximately 5 to approximately 100 nucleotides, approximately 10 to approximately 100 nucleotides, approximately 20 to approximately 100 nucleotides, approximately 30 to approximately 100 nucleotides, or approximately 30 to approximately 100 nucleotides, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 5 These are 8, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

[0220] In some embodiments, the ribozyme recognition site I includes a nucleotide sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with one nucleotide sequence from SEQ ID NO:95-116.

[0221] In some embodiments, the ribozyme recognition site II may be selected from a plurality of sequences.

[0222] In some embodiments, the ribozyme recognition site I includes or consists of the nucleotide motif defined above. In embodiments, the ribozyme recognition site I includes or consists of a nucleotide motif in the 5' to 3' direction, where the nucleotide motif consists of one of SEQ ID NO: 95 to 116, specifically, the nucleotide motif consists of one of CUU, CUC, or CAU.

[0223] In some embodiments, the ribozyme recognition site I includes or comprises a nucleotide sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with one of SEQ ID NO: 58, 60, 62, 64, 66, 68, or 70, preferably the ribozyme recognition site I includes the nucleotide sequence GAGGUCUU, AGAGACUU, UCCAGCUU, or UCUGGCAU.

[0224] In some embodiments, the ribozyme recognition site II includes or comprises a nucleotide sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with one of SEQ ID NO: 57, 59, 61, 63, 65, 67, or 69, preferably the ribozyme recognition site II includes the nucleotide sequence AAACCUC, AAUCUCU, AACUGGA, or AACCAGC.

[0225] In some embodiments, the ribozyme recognition site I includes or consists of one nucleotide sequence from SEQ ID NO: 58, 60, 62, 64, 66, 68, or 70, and the ribozyme recognition site II includes or consists of one nucleotide sequence from SEQ ID NO: 57, 59, 61, 63, 65, 67, or 69.

[0226] In one embodiment, the ribozyme recognition site I is selected from the nucleotide sequences UGUAACUU, UGUAACUU, GAGGUCUU, AGAGACUU, UCCAGCUU, or UCUGGCAU.

[0227] In the embodiment, the ribozyme recognition site II is selected from the nucleotide sequences AGAAGUAAC, AAAAUUACA, AAACCUC, AAUCUCU, AACUGGA, or AACCAGC.

[0228] In this embodiment, the ribozyme recognition site I is UGUAACUU, and the ribozyme recognition site II is AGAAGUAAC.

[0229] In an embodiment, the ribozyme recognition site I is UGUAACUU, and the ribozyme recognition site II is AAAAUUACA.

[0230] In an embodiment, the ribozyme recognition site I is GAGGUCUU, and the ribozyme recognition site II is AAACCUC.

[0231] In an embodiment, the ribozyme recognition site I is AGAGACUU, and the ribozyme recognition site II is AAUCUCU.

[0232] In an embodiment, the ribozyme recognition site I is UCCAGCUU, and the ribozyme recognition site II is AACUGGA.

[0233] In an embodiment, the ribozyme recognition site I is UCUGGCAU, and the ribozyme recognition site II is AACCAGC.

[0234] In one aspect, one object of the present invention is to provide a circular RNA precursor, which, in the 5' to 3' direction, a) a 3' self-splicing intron fragment, b) a nucleotide sequence of interest, c) a 5' self-splicing intron fragment, and where the combination of the 3' self-splicing intron fragment and the 5' self-splicing intron fragment maintains the self-splicing activity of the self-splicing intron and allows the production of a circular RNA containing the nucleotide sequence of interest by self-splicing of the circular RNA precursor, where the nucleotide sequence of interest includes a non-coding sequence fragment II and a non-coding sequence fragment I in the 5' to 3' direction, Here, non-coding sequence fragment I originates from the 5' end of a non-coding sequence, and non-coding sequence fragment II originates from the 3' end of a non-coding sequence. Non-coding sequence fragment I includes a ribozyme recognition site I located at its 3' end, and non-coding sequence fragment II includes a ribozyme recognition site II located at its 5' end. In the resulting circular RNA, ribozyme recognition site I and ribozyme recognition site II are linked together, thereby linking non-coding sequence fragment I and non-coding sequence fragment II to form a complete non-coding sequence.

[0235] In this embodiment, the non-coding sequence is an endogenous circular RNA.

[0236] In some embodiments, the total length of ribozyme recognition site I and ribozyme recognition site II is approximately 0 to approximately 100 nucleotides, approximately 0 to approximately 90 nucleotides, approximately 0 to approximately 80 nucleotides, approximately 0 to approximately 70 nucleotides, approximately 0 to approximately 60 nucleotides, approximately 0 to approximately 50 nucleotides, approximately 0 to approximately 40 nucleotides, approximately 0 to approximately 35 nucleotides, approximately 2 to approximately 100 nucleotides, approximately 5 to approximately 100 nucleotides, approximately 10 to approximately 100 nucleotides, approximately 20 to approximately 100 nucleotides, approximately 30 to approximately 100 nucleotides, or approximately 30 to approximately 100 nucleotides, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57 These are 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

[0237] In some embodiments, the total length of ribozyme recognition site I is approximately 0 to approximately 100 nucleotides, approximately 0 to approximately 90 nucleotides, approximately 0 to approximately 80 nucleotides, approximately 0 to approximately 70 nucleotides, approximately 0 to approximately 60 nucleotides, approximately 0 to approximately 50 nucleotides, approximately 0 to approximately 40 nucleotides, approximately 0 to approximately 35 nucleotides, approximately 2 to approximately 100 nucleotides, approximately 5 to approximately 100 nucleotides, approximately 10 to approximately 100 nucleotides, approximately 20 to approximately 100 nucleotides, approximately 30 to approximately 100 nucleotides, or approximately 30 to approximately 100 nucleotides, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58 These are 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

[0238] In some embodiments, the total length of ribozyme recognition site II is approximately 0 to 100 nucleotides, approximately 0 to 90 nucleotides, approximately 0 to 80 nucleotides, approximately 0 to 70 nucleotides, approximately 0 to 60 nucleotides, approximately 0 to 50 nucleotides, approximately 0 to 40 nucleotides, approximately 0 to 35 nucleotides, approximately 2 to 100 nucleotides, approximately 5 to 100 nucleotides, approximately 10 to 100 nucleotides, approximately 20 to 100 nucleotides, approximately 30 to 100 nucleotides, or approximately 30 to 100 nucleotides, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 ,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,5 These are 8, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

[0239] In some embodiments, ribozyme recognition site I includes a nucleotide sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with one nucleotide sequence from SEQ ID NO:95-116.

[0240] In some embodiments, the ribozyme recognition site II may be selected from a plurality of sequences.

[0241] In some embodiments, ribozyme recognition site I includes or consists of the nucleotide motif defined above. In embodiments, ribozyme recognition site I includes or consists of a nucleotide motif oriented from 5' to 3', where the nucleotide motif is one of SEQ ID NO: 95 to 116, specifically, the nucleotide motif is one of CUU, CUC, or CAU.

[0242] In some embodiments, ribozyme recognition site I includes or comprises a nucleotide sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with one of SEQ ID NO: 58, 60, 62, 64, 66, 68, or 70, preferably ribozyme recognition site I includes the nucleotide sequence GAGGUCUU, AGAGACUU, UCCAGCUU, or UCUGGCAU.

[0243] In some embodiments, ribozyme recognition site II includes or comprises a nucleotide sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with one of SEQ ID NO: 57, 59, 61, 63, 65, 67, or 69, preferably ribozyme recognition site II includes the nucleotide sequence AAACCUC, AAUCUCU, AACUGGA, or AACCAGC.

[0244] In some embodiments, ribozyme recognition site I includes or consists of one nucleotide sequence from SEQ ID NO: 58, 60, 62, 64, 66, 68, or 70, and ribozyme recognition site II includes or consists of one nucleotide sequence from SEQ ID NO: 57, 59, 61, 63, 65, 67, or 69.

[0245] In one embodiment, the ribozyme recognition site I is selected from the nucleotide sequences UGUAACUU, UGUAACUU, GAGGUCUU, AGAGACUU, UCCAGCUU, or UCUGGCAU.

[0246] In this embodiment, the ribozyme recognition site II is selected from the nucleotide sequences AGAAGUAAC, AAAAUUACA, AAACCUC, AAUCUCU, AACUGGA, or AACCAGC.

[0247] In the embodiment, the ribozyme recognition site I includes or comprises a nucleotide motif oriented from 5' to 3', the nucleotide motif being one of SEQ ID NO: 95 to 116, and specifically, the nucleotide motif being one of CUU, CUC, or CAU.

[0248] In this embodiment, ribozyme recognition site I is UGUAACUU, and ribozyme recognition site II is AGAAGUAAC.

[0249] In one embodiment, ribozyme recognition site I is UGUAACUU, and ribozyme recognition site II is AAAAUUACA.

[0250] In one embodiment, ribozyme recognition site I is GAGGUCUU, and ribozyme recognition site II is AAACCUC.

[0251] In one embodiment, ribozyme recognition site I is AGAGACUU, and ribozyme recognition site II is AAUCUCU.

[0252] In one embodiment, ribozyme recognition site I is UCCAGCUU, and ribozyme recognition site II is AACUGGA.

[0253] In this embodiment, ribozyme recognition site I is UCUGGCAU, and ribozyme recognition site II is AACCAGC.

[0254] In some embodiments, the length of the desired nucleotide sequence is at least 10, 20, 40, 60, 80, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 10000, or 20000 nucleotides.

[0255] In some embodiments, the length of the circular RNA is at least 10, 20, 40, 60, 80, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 10000, or 20000 nucleotides. In some embodiments, the length of the circular RNA is at least about 10 nucleotides. In some embodiments, the circular RNA is about 50 nt or less. In some embodiments, the circular RNA is about 80 nt or less. In some embodiments, the circular RNA is about 100 nt or less. In some embodiments, the circular RNA is about 150 nt or less. In some embodiments, the circular RNA is about 200 nt or less. In some embodiments, the circular RNA is about 250 nt or less. In some embodiments, the circular RNA is about 300 nt or less. In some embodiments, the circular RNA is approximately 350 nt or less. In some embodiments, the circular RNA is approximately 400 nt or less. In some embodiments, the circular RNA is approximately 450 nt or less. In some embodiments, the circular RNA is approximately 500 nt or less. In some embodiments, the circular RNA is approximately 600 nt or less. In some embodiments, the circular RNA is approximately 700 nt or less. In some embodiments, the circular RNA is approximately 800 nt or less. In some embodiments, the circular RNA is approximately 900 nt or less. In some embodiments, the circular RNA is at least approximately 1000 nt.

[0256] In another embodiment, the present invention provides a pharmaceutical composition comprising a cyclic RNA precursor and / or a nucleic acid vector and / or a cyclic RNA as described in the present invention, and a pharmaceutically acceptable carrier.

[0257] In some embodiments, the pharmaceutically acceptable carrier may include, but is not limited to, buffers, excipients, stabilizers or preservatives. Examples of pharmaceutically acceptable carriers include physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, such as salts, buffers, sugars, antioxidants, aqueous or non-aqueous carriers, preservatives, wetting agents, surfactants or emulsifiers or combinations thereof. Based on the activity of the carrier and the properties required for the formulation, such as stability and / or minimal oxidation, the amount of the pharmaceutically acceptable carrier in the pharmaceutical composition can be determined experimentally.

[0258] In another aspect, the present invention provides a method for producing circular RNA, the method comprising: 1) providing the circular RNA precursor described in the present invention, or obtaining a circular RNA precursor by transcription from the nucleic acid vector described in the present invention; 2) incubating the circular RNA precursor at a temperature at which RNA circularization occurs in the presence of a divalent metal cation; 3) recovering the circular RNA obtained in step 2).

[0259] In some embodiments, the divalent metal cation is Mg 2+ and / or Mn 2+ .

[0260] In some embodiments, the concentration of the divalent metal cation is at least about 5 mM, such as about 5 mM to about 550 mM, such as at least about 5 mM, about 10 mM, about 15 mM, at least about 20 mM, at least about 30 mM, at least about 40 mM, at least about 50 mM, at least about 60 mM, at least about 70 mM, at least about 80 mM, at least about 90 mM, at least about 100 mM, at least about 125 mM, at least about 150 mM, at least about 175 mM, at least about 200 mM, at least about 250 mM, at least about 300 mM, at least about 350 mM, at least about 400 mM, at least about 450 mM, at least about 500 mM, at least about 550 mM or more.

[0261] In another aspect, the present invention provides a method for producing circular RNA, the method being a) A step of providing a nucleic acid vector containing RNA circularization elements based on self-splicing introns to be used as a transcription template, b) The nucleic acid vector is incubated for a first time period in an in vitro transcription system containing a divalent metal cation and RNA polymerase, during which linear RNA produced by in vitro transcription self-circulates under the action of the RNA circulating element to produce circular RNA.

[0262] In some embodiments, the nucleic acid vector is the nucleic acid vector described in the present invention.

[0263] In some embodiments, the divalent metal cation in the in vitro transfer system is Mg 2+ That is the case.

[0264] In some embodiments, the in vitro transfer system further comprises a monovalent metal cation and / or a monovalent anion.

[0265] In some embodiments, the monovalent metal cation is Na + or K + That is the case.

[0266] In some embodiments, the monovalent metal anion is Cl - Or CH3COO - (OAc)

[0267] In some embodiments, the concentration of the divalent metal cation in the system during the first time period is about 5 mM to about 50 mM, for example, about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, and about 50 mM. In some embodiments, the concentration of the divalent metal cation in the system during the first time period is 30 mM.

[0268] In some embodiments, the concentration of the monovalent metal cation in the system within the first time period is about 5 mM to about 50 mM, for example, about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, and about 100 mM. In some embodiments, the monovalent metal cation is Na + The concentration in the system within the first time period is 15 mM. In some embodiments, the monovalent metal cation is K + Therefore, the concentration in the system during the first time period is 90 mM.

[0269] In some embodiments, the concentration of the monovalent anion in the system within the first time period is about 5 mM to about 50 mM, for example, about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 100 mM, about 150 mM. In some embodiments, the monovalent anion is Cl - The concentration in the system within the first time period is 90 mM. In some embodiments, the monovalent anion is CH3COO - Therefore, the concentration in the system during the first time period is 125 mM.

[0270] In some embodiments, the in vitro transcription and autocyclization are carried out in the same reaction system.

[0271] In some embodiments, the method does not include the step of separating and / or purifying the linear RNA produced by in vitro transcription.

[0272] Those skilled in the art will recognize that the in vitro transcription system further includes various components necessary for transcription, such as buffers, rATP, rCTP, rUTP, rGTP, and the like.

[0273] In some embodiments, the buffer for the in vitro transfer system is Tris hydrochloride buffer, HEPES buffer, MES buffer, citrate buffer, or phosphate buffer. In some embodiments, the buffer for the in vitro transfer system is HEPES buffer.

[0274] In some embodiments, the pH range of the in vitro transfer system is about 5 to about 8, for example, pH is about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, or about 8. In some embodiments, the pH of the in vitro transfer system is 7.5.

[0275] The RNA polymerase is dependent on a transcription-driving promoter used on the nucleic acid vector. The RNA polymerase may include, but is not limited to, T7 RNA polymerase, T6 viral RNA polymerase, SP6 viral RNA polymerase, T3 viral RNA polymerase, or T4 viral RNA polymerase. In some embodiments, the RNA polymerase is T7 RNA polymerase.

[0276] In some embodiments, the first time period is at least 0.5 hours, for example, about 0.5 hours to about 24 hours, for example, about 0.5 hours, about 1 hour, about 1.5 hours, about 2 hours, about 2.5 hours, about 3 hours, about 3.5 hours, about 4 hours, about 5 hours, about 10 hours, or about 24 hours. In some embodiments, the first time period is 3 hours.

[0277] In some embodiments, the reaction in the first time period is carried out at approximately 16°C to approximately 60°C, for example, approximately 16°C, approximately 17°C, approximately 18°C, approximately 19°C, approximately 20°C, approximately 21°C, approximately 22°C, approximately 23°C, approximately 24°C, approximately 25°C, approximately 26°C, approximately 27°C, approximately 28°C, approximately 29°C, approximately 30°C, approximately 31°C, approximately 32°C, approximately 33°C, approximately 34°C The reaction is carried out at approximately 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, or 60°C. In some embodiments, the reaction in the first time period is carried out at approximately 37°C.

[0278] In some embodiments, after the reaction in the first time period of step b), the method By adding an additional amount of metal cation to the in vitro transfer system and allowing the reaction to proceed only during the second time period, or The method further includes step c) changing the buffer solution of the system, adding a metal cation, and allowing the reaction to proceed only during the second time period.

[0279] In some embodiments, the metal cation added to the reaction in the second time zone is a divalent metal cation, such as Mg 2+ or Mn 2+ That is the case.

[0280] In some embodiments, during the reaction in the second time period, the metal cation is added such that the final concentration is at least about 5 mM, for example, about 5 mM to about 550 mM, for example, at least about 5 mM, at least about 10 mM, at least about 15 mM, at least about 20 mM, at least about 30 mM, at least about 40 mM, at least about 50 mM, at least about 60 mM, at least about 70 mM, at least about 80 mM, at least about 90 mM, at least about 100 mM, at least about 125 mM, at least about 150 mM, at least about 175 mM, at least about 200 mM, at least about 250 mM, at least about 300 mM, at least about 350 mM, at least about 400 mM, at least about 450 mM, at least about 500 mM, at least about 550 mM or more.

[0281] In some embodiments, during the reaction in the second time period, the buffer in the system is Tris hydrochloride buffer, HEPES buffer, MES buffer, citrate buffer, or phosphate buffer.

[0282] In some embodiments, the pH range of the reaction system during the second time period is 5 to 8, for example, pH 5, pH 5.5, pH 6, pH 6.5, pH 7, pH 7.5, or pH 8.

[0283] In some embodiments, the second time period is at least 5 minutes, for example about 5 minutes to about 2 hours, for example about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes, about 120 minutes or longer.

[0284] In some embodiments, the reaction in the second time period is carried out at a temperature of about 25°C to about 75°C, for example, about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, about 30°C, about 31°C, about 32°C, about 33°C, about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, about 39°C, about 40°C, about 41°C, about 42°C, about 43°C, about 44°C, about 45°C, about 50°C, about 55°C, about 60°C, about 65°C, about 70°C, or about 75°C.

[0285] In some embodiments, the method further includes the step of recovering or purifying the circular RNA produced.

[0286] In one embodiment, the present invention provides circular RNA produced by the method of the present invention.

[0287] In one embodiment, the present invention provides an in vitro transfer method, the method being a) A step of providing a nucleic acid vector to be used as a template for in vitro transcription, b) A step of incubating the nucleic acid vector in an in vitro transcription system containing a divalent metal cation and RNA polymerase, and reacting it only for a first time period, c)i) adding an additional amount of metal cation to the system, or ii) changing the buffer solution of the system, adding metal cation to the system, incubating the system, and allowing the reaction to proceed for a second time period only.

[0288] In some embodiments, the in vitro transfer system in the reaction of the first time period further comprises a monovalent metal cation and / or a monovalent anion.

[0289] In some embodiments, the divalent metal cation in the in vitro transfer system during the reaction in the first time period is Mg 2+ That is the case.

[0290] In some embodiments, the monovalent metal cation in the reaction during the first time period is Na + , or K + That is the case.

[0291] In some embodiments, the monovalent anion in the reaction during the first time period is Cl - , or CH3COO - (OAc)

[0292] The RNA polymerase is dependent on a transcription-driving promoter used on the nucleic acid vector. The RNA polymerase may include, but is not limited to, T7 RNA polymerase, T6 viral RNA polymerase, SP6 viral RNA polymerase, T3 viral RNA polymerase, or T4 viral RNA polymerase. In some embodiments, the RNA polymerase is T7 RNA polymerase.

[0293] In some embodiments, the concentration of the divalent metal cation in the reaction during the first time period is about 5 mM to about 50 mM, for example, about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, and about 50 mM.

[0294] In some embodiments, the concentration of the monovalent metal cation in the reaction during the first time period is about 5 mM to about 100 mM, for example, about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, and about 100 mM.

[0295] In some embodiments, the concentration of the monovalent anion in the reaction during the first time period is about 5 mM to about 150 mM, for example, about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 100 mM, and about 150 mM.

[0296] In some embodiments, the buffer for the in vitro transfer system in the reaction during the first time period is Tris hydrochloride buffer, or HEPES buffer, or MES buffer, or citrate buffer, or phosphate buffer.

[0297] In some embodiments, the pH range of the in vitro transfer system in the reaction during the first time period is 5 to 8, for example, pH 5, pH 5.5, pH 6, pH 6.5, pH 7, pH 7.5, or pH 8.

[0298] In some embodiments, the first time period is at least 0.5 hours, for example, about 0.5 hours to about 24 hours, for example, about 0.5 hours, about 1 hour, about 1.5 hours, about 2 hours, about 2.5 hours, about 3 hours, about 3.5 hours, about 4 hours, about 5 hours, about 10 hours, or about 24 hours.

[0299] In some embodiments, the reaction in the first time period is carried out at approximately 16°C to approximately 60°C, for example, approximately 16°C, approximately 17°C, approximately 18°C, approximately 19°C, approximately 20°C, approximately 21°C, approximately 22°C, approximately 23°C, approximately 24°C, approximately 25°C, approximately 26°C, approximately 27°C, approximately 28°C, approximately 29°C, approximately 30°C, approximately 31°C, approximately 32°C, approximately 33°C, approximately 34°C The process is carried out at approximately 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, or 60°C.

[0300] In some embodiments, the metal cation added to the system for the second time zone reaction is a divalent metal cation, such as Mg 2+ or Mn 2+ That is the case.

[0301] In some embodiments, the metal cation in the reaction during the second time period is added such that the final concentration is at least about 5 mM, for example, about 5 mM to about 550 mM, for example, at least about 5 mM, about 10 mM, about 15 mM, at least about 20 mM, at least about 30 mM, at least about 40 mM, at least about 50 mM, at least about 60 mM, at least about 70 mM, at least about 80 mM, at least about 90 mM, at least about 100 mM, at least about 125 mM, at least about 150 mM, at least about 175 mM, at least about 200 mM, at least about 250 mM, at least about 300 mM, at least about 350 mM, at least about 400 mM, at least about 450 mM, at least about 500 mM, at least about 550 mM or more.

[0302] In some embodiments, the second time period is at least 5 minutes, for example about 5 minutes to about 2 hours, for example about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes, about 120 minutes or longer.

[0303] In some embodiments, the reaction in the second time period is carried out at a temperature of about 25°C to about 75°C, for example, about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, about 30°C, about 31°C, about 32°C, about 33°C, about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, about 39°C, about 40°C, about 41°C, about 42°C, about 43°C, about 44°C, about 45°C, about 50°C, about 55°C, about 60°C, about 65°C, about 70°C, or about 75°C.

[0304] In some embodiments, the method further includes the step of recovering and / or purifying the RNA obtained in step c).

[0305] In one embodiment, the present invention provides RNA produced by the method of the present invention.

[0306] In another aspect, the present invention provides a method for purifying circular RNA, the method being a) A step of contacting a mixture containing circular RNA and a linear circular RNA precursor with a circular RNA-specific probe under conditions that allow the circular RNA-specific probe to specifically bind to the circular RNA and form a complex, b) A step of separating the complex from one or more components in the mixture that are not bound to the cyclic RNA-specific probe, c) the step of releasing the circular RNA from the complex.

[0307] In some embodiments, the circular RNA is produced by cyclization of a linear circular RNA precursor. In some embodiments, the circular RNA is produced by ligating both ends of a linear circular RNA precursor with an RNA ligase, such as T4 RNA ligase. In some embodiments, the circular RNA is produced by the self-splicing ribozyme activity of a group I intron-based cyclization element contained in the linear circular RNA precursor, for example, the circular RNA is the circular RNA described in Section I of this Specification or any one of the following items, and / or produced by the method described in Section II of this Specification or any one of the following items.

[0308] In some embodiments, the circular RNA-specific probe is a single-stranded DNA probe.

[0309] In some embodiments, the circular RNA-specific probe is a single-stranded RNA probe.

[0310] In some embodiments, the circular RNA-specific probe specifically hybridizes to the regions on both sides of the circularization junction of the circular RNA.

[0311] In some embodiments, the length of the circular RNA-specific probe is at least 10 nucleotides, at least 12 nucleotides, at least 14 nucleotides, at least 16 nucleotides, at least 18 nucleotides, at least 20 nucleotides, at least 25 nucleotides, at least 30 nucleotides, at least 35 nucleotides or more, for example, the length of the circular RNA-specific probe is 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 25, 27, 28, 29, or 30 nucleotides.

[0312] In some embodiments, the circular RNA-specific probe is immobilized on a support, such as a solid support; for example, the circular RNA-specific probe is immobilized on the support after binding to the circular RNA, or the circular RNA-specific probe is immobilized on the support beforehand.

[0313] In some embodiments, the conditions in step a) include denaturing the RNA at a temperature of about 60°C to about 95°C (e.g., about 60°C, about 62°C, about 64°C, about 66°C, about 68°C, about 70°C, about 75°C, about 80°C, about 85°C, about 90°C, about 95°C) for about 2 minutes to about 10 minutes (e.g., about 2, 3, 4, 5, 6, 7, 8, 9 or 10 minutes), and then gradually reducing the temperature to below about 40°C (e.g., below about 35°C, below about 30°C, below about 25°C, below about 20°C or less) to anneal the circular RNA and the circular RNA-specific probe.

[0314] In some embodiments, the conditions in step a) include a high salt concentration range of 0.25 M to 2 M, for example, 0.25 M, 0.5 M, 0.75 M, 1 M, 1.25 M, 1.5 M, 1.75 M, or 2 M. In some embodiments, the salt is NaCl or a guanidine salt (for example, guanidine hydrochloride).

[0315] In some embodiments, step b) hereby removes one or more of the components by washing the complex with a washing buffer.

[0316] In some embodiments, here in step c), the circular RNA is released by raising the temperature to approximately 60°C to approximately 95°C (e.g., approximately 60°C, approximately 62°C, approximately 64°C, approximately 66°C, approximately 68°C, approximately 70°C, approximately 75°C, approximately 80°C, approximately 85°C, approximately 90°C, approximately 95°C).

[0317] In some embodiments, step c) hereby releases the circular RNA by eluting with an elution buffer. In some embodiments, the elution buffer is a low-salt buffer, for example, a buffer with a salt concentration lower than 0.5 M. In some embodiments, the elution buffer is Tris-EDTA buffer (TE buffer) or water.

[0318] In some embodiments, the method is i) A step of contacting a mixture containing circular RNA and a linear circular RNA precursor with a linear circular RNA precursor-specific probe under conditions that allow the linear circular RNA precursor-specific probe to specifically bind to the linear circular RNA precursor and form a complex; ii) The step of removing the complex formed by the linear circular RNA precursor-specific probe and the linear circular RNA precursor from the mixture, iii) further comprises the step of collecting the cyclic RNA-containing mixture obtained in step ii).

[0319] In some embodiments, steps i) to iii) are performed before step a), for example, steps i) to iii) are performed multiple times, for example, two, three, four or more times, before step a). In some embodiments, steps i) to iii) and steps a) to c) are performed simultaneously.

[0320] In one aspect, the present invention provides a method for purifying circular RNA, the method being i) A step of contacting a mixture containing circular RNA and a linear circular RNA precursor with a linear circular RNA precursor-specific probe under conditions that allow the linear circular RNA precursor-specific probe to specifically bind to the linear circular RNA precursor and form a complex; ii) The step of removing the complex formed by the linear circular RNA precursor-specific probe and the linear circular RNA precursor from the mixture, iii) the step of collecting the cyclic RNA-containing mixture obtained in step ii), Optionally, steps i) to iii) may be performed multiple times, for example, two, three, four or more times.

[0321] In some embodiments, the linear circular RNA precursor-specific probe specifically binds to the linear circular RNA precursor but does not substantially bind to the circular RNA.

[0322] In some embodiments, the linear circular RNA precursor-specific probe is immobilized on a support, such as a solid support; for example, the linear circular RNA precursor-specific probe is immobilized on the support after binding to the linear circular RNA precursor, or the linear circular RNA precursor-specific probe is immobilized on the support beforehand.

[0323] In some embodiments, the circular RNA-specific probe specifically hybridizes to at least a portion of the first residue cyclization element and a portion of the second residue cyclization element.

[0324] In some embodiments, the linear circular RNA precursor-specific probe hybridizes to the portion of the linear circular RNA precursor other than the first residue cyclization element, the target nucleotide sequence, and the second residue cyclization element.

[0325] In some embodiments, the linear circular RNA precursor-specific probe hybridizes to the 3' self-splicing intron fragment or a portion thereof or its 5' flanking sequence, or to the 5' self-splicing intron fragment or a portion thereof or its 3' flanking sequence.

[0326] In some embodiments, the linear circular RNA precursor comprises a sequence selected from SEQ ID NO: 600-610 or a complementary sequence thereof, preferably SEQ ID NO: 606 or a complementary sequence thereof, which lies outside the first residue cyclization element, the target nucleotide sequence, and the second residue cyclization element, and the linear circular RNA precursor-specific probe specifically hybridizes thereto.

[0327] In some embodiments of the method for purifying circular RNA described in the present invention, the molar ratio of the probe to the RNA molecules in the mixture is about 1:1 to about 100,000:1.

[0328] In some embodiments, the linear circular RNA precursor-specific probe hybridizes to a 3' homology arm sequence or a portion thereof on the linear circular RNA precursor, or to a 5' homology arm sequence or a portion thereof on the linear circular RNA precursor. In some embodiments, the homology arm sequence comprises polyA, polyU, polyC, or polyG. In some embodiments, the length of the homology arm sequence is about 10 to about 200 nt. Accordingly, in some embodiments, the linear circular RNA precursor-specific probe comprises polyU, polyA, polyG, or polyC. In some embodiments, the length of the linear circular RNA precursor-specific probe is about 10 to about 200 nt.

[0329] In one aspect, the present invention provides a method for purifying circular RNA, the method being i) A step of adding a linear RNA-specific tag to linear RNA in a mixture containing circular RNA and linear RNA, ii) A step of contacting a mixture containing circular RNA and linear RNA with a linear RNA probe that specifically binds to the tag, under conditions that allow the probe to specifically bind to the linear RNA and form a complex; iii) The step of removing the complex formed by the linear RNA probe and linear RNA from the mixture, iv) The step of collecting the circular RNA-containing mixture obtained in step ii), Optionally, steps ii) to iv) above may be performed multiple times, for example, two, three, four or more times.

[0330] In some embodiments, the tag includes a polyA, polyG, polyU, or polyC sequence. Accordingly, the probe includes a polyT / polyU, polyC, polyA, or polyG sequence. Alternatively, the tag may be a random sequence, and the probe includes a sequence complementary to the random sequence.

[0331] In some embodiments, the length of the tag may be about 10 to 200 nt. In some embodiments, the length of the probe may be about 10 to 200 nt.

[0332] In some embodiments, the tag is added to the ends of the linear RNA by adding PolyA / T / C / G polymerase or ligase to the mixture.

[0333] In some embodiments, the mixture further includes adding rATP, rGTP, rUTP, rCTP, or rNTP, or a 10-200 nt random tag sequence.

[0334] In some embodiments, the linear RNA probe specifically binds to the added tag but does not substantially bind to the circular RNA. In some embodiments, the linear RNA probe is a single-stranded DNA probe or a single-stranded RNA probe.

[0335] In some embodiments, the linear RNA probe is immobilized on a support, such as a solid support, for example, the linear RNA probe is immobilized on the support after binding to the linear RNA, or the linear RNA probe is immobilized on the support beforehand.

[0336] In some embodiments, the present invention provides a method for screening nucleotide motifs of a target open reading frame (ORF) sequence encoding a target polypeptide, the method being: 1) A step of extracting amino acid units in the direction from the N-terminus to the C-terminus of a target polypeptide containing m amino acids, wherein each group of amino acid units contains n amino acids, one amino acid is used as the step size, and the steps are slid along the N-terminus to the C-terminus, where n is an integer and n ≥ 2, and m is an integer and ≥ 1. 2) A step of listing all nucleotide sequences corresponding to the amino acid units of each group according to the degenerate codon, 3) A step of identifying a nucleotide sequence that matches the nucleotide motif obtained in step 2), The step involves setting a nucleotide motif consisting of one of SEQ ID NO:95~116, preferably consisting of CUU, CUC, or CAU. 4) The step of optionally selecting a target ORF as the circularization sequence of a circular RNA precursor.

[0337] In some embodiments, the present invention provides a system for screening targeted open reading frames (ORFs) of nucleotide motifs, wherein the targeted open reading frame (ORF) is a nucleotide sequence encoding a target polypeptide, and the system is a) An amino acid extraction module for extracting amino acid units in the direction from the N-terminus to the C-terminus of a target polypeptide containing m amino acids, wherein each group of amino acid units contains n amino acids, one amino acid is used as the step size, sliding along the N-terminus to the C-terminus, and n is an integer and n ≥ 2, and m is an integer and ≥ 1; b) A conversion module for determining the codon sequence of a group of amino acid units, wherein the codon sequence of each group includes all degenerate codon sequences corresponding to the amino acid units of each group, c) A target codon sequence filtering module for determining whether the target codon sequence contains a nucleotide sequence that precisely matches the nucleotide motif, Here, the nucleotide motif consists of one of SEQ ID NO:95~116, and preferably consists of CUU, CUC, or CAU.

[0338] In some embodiments, the present invention provides a method for screening nucleotide motifs in a target non-coding sequence, the method being 1) A step of editing 0, 1, or 2 bases in a non-coding sequence to include an edited region that matches a nucleotide motif in the non-coding sequence, while maintaining the function of the non-coding sequence, The nucleotide motif consists of one nucleotide sequence from SEQ ID NO: 95-116, preferably consisting of CUU, CUC, or CAU. 2) The step of optionally selecting a target non-coding sequence, which is an endogenous circular RNA, as the circularization sequence of the circular RNA precursor.

[0339] In some embodiments, the present invention provides a system for screening target non-coding sequences of nucleotide motifs, wherein the target non-coding sequence is an endogenous circular RNA, and the system is a) Includes a base editing module for editing one or more bases at any position in a target non-coding sequence to obtain an edited region that matches a nucleotide motif, Here, the nucleotide motif consists of one of SEQ ID NO:95~116, and preferably consists of CUU, CUC, or CAU.

[0340] In some embodiments, the present invention provides a method for screening target internal ribosome entry sites (IRESs) of nucleotide motifs, the method being 1) A step of identifying non-conserved sequence regions of the target IRES by comparing the target IRES with other IRES sequences from the same viral subgenus using a multiplex sequence alignment tool, such as Clustal Omega or MUSCLE. 2) Based on the results of multiple sequence alignment, the low-structure region of the target IRES is determined using secondary structure prediction software, such as RNAfold, Mfold, RNAstruct, or RNAalifold. 3) The step of editing 0, 1, or 2 bases in a low-structure region of a target IRES to match a nucleotide motif without affecting the translation initiation activity of the IRES, Here, the nucleotide motif consists of one nucleotide sequence from SEQ ID NO:95~116, and preferably consists of CUU, CUC, or CAU.

[0341] As used herein, the term “low-structured region” refers to a region that does not have a stem-loop structure, a region with high free energy, or a single-chain region.

[0342] In some embodiments, the present invention provides a system for screening target internal ribosome entry sites (IRESs) of nucleotide motifs, wherein the system is: a) A multi-sequence alignment module for recognizing non-conserved sequence regions by comparing the target IRES with other IRES sequences from the same viral subgenus, b) A low-structure prediction module for determining low-structure regions within the target IRES based on the multiple sequence alignment results of the target IRES, c) A base editing module for editing one or more bases at any position within the region to obtain an edited region that matches a nucleotide motif, Here, the nucleotide motif consists of one nucleotide sequence from SEQ ID NO:95~116, and preferably consists of CUU, CUC, or CAU.

[0343] In some embodiments, the present invention provides a screening method for screening for targeted non-TIE functional elements against a nucleotide motif in a ribozyme recognition site I, the method being a) The step of editing 0, 1, or 2 bases in a non-TIE functional element to include an edited region matching a nucleotide motif in the non-TIE functional element sequence (e.g., 3'UTR or replicon) while maintaining the non-TIE functional element sequence, Here, the nucleotide motif consists of one nucleotide sequence from SEQ ID NO:95~116, and preferably consists of CUU, CUC, or CAU.

[0344] In some embodiments, the present invention provides a system for screening nucleotide motifs in target region sequences in non-TIE functional element sequences (e.g., 3'UTR or replicon), wherein the screening system is: a) Includes a base editing module for editing one or more bases at any position within a region to obtain an edited region that matches a nucleotide motif, Here, the nucleotide motif consists of one nucleotide sequence from SEQ ID NO:95~116, and preferably consists of CUU, CUC, or CAU.

[0345] In another embodiment, a method is provided for preventing or treating a disease or disorder in a subject, the method comprising administering the subject to the cyclic RNA of the present invention or the pharmaceutical composition of the present invention.

[0346] In some embodiments, the disease or disorder includes, but is not limited to, xerostomia, dry eye, and Sjögren's syndrome.

[0347] Sjögren's syndrome is a systemic autoimmune disease in which immune cells attack and destroy the exocrine glands that produce saliva and tears. Sjögren's syndrome also affects multiple organs, including the kidneys and lungs. Sjögren's syndrome can occur in women and men of any age. Sjögren's syndrome can develop independently and is called primary Sjögren's syndrome, or it can develop several years after the onset of an associated rheumatic disease and is called secondary Sjögren's syndrome.

[0348] Xerostomia (dry mouth) and dry eye (keratoconjunctivitis sicca, dry eye) are characteristic features of Sjögren's syndrome. In susceptible individuals, immune-activated or apoptotic glandular epithelial cells exposed to autoantigens can lead to autoimmune-mediated tissue damage. Immune activation generally manifests as the infiltration of focal monocytes (T, B, and macrophages) near ductal epithelial cells (epitheliitis), forming sialadenitis. While the pathogenesis of such autoimmune exocrine disorders is not yet fully understood, it has been revealed that CD4+ T lymphocytes account for 60-70% of the monocytes infiltrating the glands. Reports indicate that abnormal activation of pro-inflammatory Th1 and Th17 cells is crucial for the induction of SS in human or animal models.

[0349] This invention provides a novel therapy for preventing or treating xerostomia associated with Sjögren's syndrome in subjects. The inventors discovered that administering a circular RNA containing a nucleotide sequence encoding aquaporin-1 (AQP-1) protein or a variant thereof to subjects can prevent or treat xerostomia associated with Sjögren's syndrome in subjects.

[0350] In embodiments, the protein available for use in the present invention is the AQP-1 protein or a variant thereof, where the AQP-1 protein or a variant thereof refers to the naturally occurring AQP-1 protein or a protein having the same activity as the naturally occurring AQP-1, which is derived from the AQP-1 protein by substituting, deleting, or adding 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids in the amino acid sequence of the naturally occurring AQP-1 protein.

[0351] In some embodiments, the AQP-1 protein or its variants contain a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the entire amino acid sequence of the naturally occurring AQP-1 protein. In embodiments, the AQP-1 protein is a portion of the amino acid sequence of the AQP-1 protein, where the portion of the AQP-1 protein retains the ability to form water-permeable channels in the cell membrane. It is also known in the art that several isoforms of the AQP-1 protein exist. Therefore, in embodiments, the AQP-1 protein is an isoform of the AQP-1 protein, where such isoform retains the ability to form water-permeable channels. In embodiments, the AQP-1 protein is a portion of an isoform of the AQP-1 protein or another naturally occurring variant, where the portion retains the ability to form water-permeable channels in a membrane. Methods for producing functional portions and variants of the AQP-1 protein (e.g., conserved variants of the AQP-1 protein) are known to those skilled in the art.

[0352] In some embodiments, the present invention provides a circular RNA precursor which is oriented in the direction from 5' to 3'. a) 3' self-splicing intron fragment, b) A target nucleotide sequence comprising protein coding sequence fragment II, a translation initiation element such as an internal ribosome entry site (IRES), and protein coding sequence fragment I, in the direction from 5' to 3', c) Includes a 5' self-splicing intron fragment, Here, the combination of the 3' self-splicing intron fragment and the 5' self-splicing intron fragment preserves the self-splicing activity of the self-splicing intron, allowing the self-splicing of the circular RNA precursor to produce a circular RNA containing a first residue cyclization element, the desired nucleotide sequence, and a second residue cyclization element. Here, protein-coding sequence fragment I originates from the 5' end of at least one protein-coding sequence, and protein-coding sequence fragment II originates from the 3' end of at least one protein-coding sequence. The protein-coding sequence fragment I includes a ribozyme recognition site I located at its 3' end, and the protein-coding sequence fragment II includes a ribozyme recognition site II located at its 5' end, wherein in the generated circular RNA, the ribozyme recognition site I and the ribozyme recognition site II are linked, thereby linking the protein-coding sequence fragment I and the protein-coding sequence fragment II to form at least one protein-coding sequence. Here, the at least one protein-coding sequence encodes an aquaporin, for example, the AQP-1 protein or a variant thereof.

[0353] In some embodiments, the present invention provides a circular RNA precursor which is oriented in the direction from 5' to 3'. a) 3' self-splicing intron fragment, b) A target nucleotide sequence comprising protein coding sequence fragment II, a translation initiation element such as an internal ribosome entry site (IRES), and protein coding sequence fragment I, in the direction from 5' to 3', c) Includes a 5' self-splicing intron fragment, Here, the combination of the 3' self-splicing intron fragment and the 5' self-splicing intron fragment preserves the self-splicing activity of the self-splicing intron, allowing the self-splicing of the circular RNA precursor to produce a circular RNA containing protein-coding sequence fragment II, a translation initiation element, and protein-coding sequence fragment I. Here, protein-coding sequence fragment I originates from the 5' end of at least one protein-coding sequence, and protein-coding sequence fragment II originates from the 3' end of at least one protein-coding sequence. The protein-coding sequence fragment I includes a ribozyme recognition site I located at its 3' end, and the protein-coding sequence fragment II includes a ribozyme recognition site II located at its 5' end, wherein in the generated circular RNA, the ribozyme recognition site I and the ribozyme recognition site II are linked, thereby linking the protein-coding sequence fragment I and the protein-coding sequence fragment II to form at least one protein-coding sequence. Here, the at least one protein-coding sequence encodes an aquaporin, for example, the AQP-1 protein or a variant thereof.

[0354] In embodiments, the present invention provides a circular RNA precursor, in the direction from 5' to 3', a) 3' self-splicing intron fragment, b) Translation start element (TIE) fragment II, c) A target nucleotide sequence encoding the aquaporin-1 (AQP-1) protein or a variant thereof, d) Translation start element fragment I and, e) Includes a 5' self-splicing intron fragment, Here, the combination of a 3' self-splicing intron fragment and a 5' self-splicing intron fragment preserves the self-splicing activity of the self-splicing intron, allowing the self-splicing of the circular RNA precursor to produce a circular RNA containing the desired nucleotide sequence. Here, TIE fragment I originates from the 5' end of the TIE sequence, and TIE fragment II originates from the 3' end of the TIE sequence, and The TIE fragment I includes a ribozyme recognition site I located at its 3' end, and the TIE fragment II includes a ribozyme recognition site II located at its 5' end. In the resulting circular RNA, the ribozyme recognition site I and the ribozyme recognition site II are linked, thereby linking TIE I and TIE II to form a TIE sequence that can recruit ribosomes and initiate translation.

[0355] In an embodiment, the present invention provides a circular RNA precursor which is oriented in the direction from 5' to 3'. a) 3' self-splicing intron fragment, b) Translation start element (TIE) fragment II, c) A target nucleotide sequence encoding the aquaporin-1 (AQP-1) protein or a variant thereof, d) Translation start element fragment I and, e) Includes a 5' self-splicing intron fragment, Here, the combination of a 3' self-splicing intron fragment and a 5' self-splicing intron fragment preserves the self-splicing activity of the self-splicing intron, allowing the self-splicing of the circular RNA precursor to produce a circular RNA containing the desired nucleotide sequence. Here, TIE fragment I originates from the 5' end of the TIE sequence, and TIE fragment II originates from the 3' end of the TIE sequence, and The TIE fragment I includes a ribozyme recognition site I located at its 3' end, and the TIE fragment II includes a ribozyme recognition site II located at its 5' end. In the resulting circular RNA, the ribozyme recognition site I and the ribozyme recognition site II are linked, thereby linking TIE I and TIE II to form a TIE sequence that can recruit ribosomes and initiate translation.

[0356] In some embodiments, the self-splicing intron is selected from the group of cyanobacteria anabaena group I introns such as AnaX, azoarcus group I introns (Azo), Scytalidium dimidiatum group I introns (Sd), staphylococcal phage Twort group I introns (Twort), Scytonema-hofmani tRNA fMet group I introns (Sh), or Agrobacterium tumefaciens group I introns (At).

[0357] In some embodiments, the self-splicing intron is selected from a nucleotide sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with one of SEQ ID NO:1 to 6.

[0358] In some embodiments, the self-splicing intron is selected from a nucleotide sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with one of SEQ ID NO:1 and 3.

[0359] In some embodiments of the present invention, the self-splicing intron is a cyanobacterial anabaena group I intron, such as AnaX, and the 3' self-splicing intron fragment contains or comprises a nucleotide sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO:7, and the 5' self-splicing intron fragment contains a nucleotide sequence having at least 99% identity with SEQ ID NO:8 or SEQ ID It contains or consists of a nucleotide sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with NO:8.

[0360] In some embodiments of the present invention, the self-splicing intron is a Staphylococcus phage Twort group I intron (Twort), and the 3' self-splicing intron fragment contains or consists of a nucleotide sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO:27, and the 5' self-splicing intron fragment contains a nucleotide sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 82%, at least 83%, at least 84%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO:27, and the 5' self-splicing intron fragment contains a nucleotide sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO:27. It contains or consists of a nucleotide sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with NO:28.

[0361] In some embodiments, the total length of ribozyme recognition site I and ribozyme recognition site II is approximately 0 to approximately 100 nucleotides, approximately 0 to approximately 90 nucleotides, approximately 0 to approximately 80 nucleotides, approximately 0 to approximately 70 nucleotides, approximately 0 to approximately 60 nucleotides, approximately 0 to approximately 50 nucleotides, approximately 0 to approximately 40 nucleotides, approximately 0 to approximately 35 nucleotides, approximately 2 to approximately 100 nucleotides, approximately 5 to approximately 100 nucleotides, approximately 10 to approximately 100 nucleotides, approximately 20 to approximately 100 nucleotides, approximately 30 to approximately 100 nucleotides, or approximately 30 to approximately 100 nucleotides, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57 These are 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

[0362] In some embodiments, the total length of ribozyme recognition site I is approximately 0 to approximately 100 nucleotides, approximately 0 to approximately 90 nucleotides, approximately 0 to approximately 80 nucleotides, approximately 0 to approximately 70 nucleotides, approximately 0 to approximately 60 nucleotides, approximately 0 to approximately 50 nucleotides, approximately 0 to approximately 40 nucleotides, approximately 0 to approximately 35 nucleotides, approximately 2 to approximately 100 nucleotides, approximately 5 to approximately 100 nucleotides, approximately 10 to approximately 100 nucleotides, approximately 20 to approximately 100 nucleotides, approximately 30 to approximately 100 nucleotides, or approximately 30 to approximately 100 nucleotides, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58 These are 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

[0363] In some embodiments, the total length of ribozyme recognition site II is approximately 0 to 100 nucleotides, approximately 0 to 90 nucleotides, approximately 0 to 80 nucleotides, approximately 0 to 70 nucleotides, approximately 0 to 60 nucleotides, approximately 0 to 50 nucleotides, approximately 0 to 40 nucleotides, approximately 0 to 35 nucleotides, approximately 2 to 100 nucleotides, approximately 5 to 100 nucleotides, approximately 10 to 100 nucleotides, approximately 20 to 100 nucleotides, approximately 30 to 100 nucleotides, or approximately 30 to 100 nucleotides, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 ,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,5 These are 8, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

[0364] In some embodiments, ribozyme recognition site I includes a nucleotide sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with one of the nucleotide sequences selected from SEQ ID NO: 58, 60, or 64.

[0365] In some embodiments, the ribozyme recognition site I includes the nucleotide sequence UCCAGCUU, UCUGGCAU, or UGUAACUU.

[0366] In some embodiments, the ribozyme recognition site II includes the nucleotide sequence AACUGGA, AACCAGC, or AAAAUUACA.

[0367] In one embodiment, ribozyme recognition site I is UCCAGCUU, and ribozyme recognition site II is AACUGGA.

[0368] In this embodiment, ribozyme recognition site I is UCUGGCAU, and ribozyme recognition site II is AACCAGC.

[0369] In some embodiments, ribozyme recognition site I is UGUAACUU, and ribozyme recognition site II is AAAAUUACA. In some embodiments, the circular RNA precursor further comprises homology arm sequence I and homology arm sequence II, which can complementaryally pair with each other to form homology arm double-stranded regions.

[0370] In some embodiments, homology arm array I is located upstream of the 3' self-splicing intron fragment, and homology arm array II is located downstream of the 5' self-splicing intron fragment.

[0371] In some embodiments, the length of homology arm sequence I or homology arm sequence II is about 5 to 200, about 5 to 150, about 5 to 100, about 5 to 80, about 5 to 50, about 5 to 40, about 5 to 30, about 5 to 20, or about 5 to 10 nucleotides, preferably with a length of about 5, 10, 15, 20, 25, 30, 35, or 40 nucleotides.

[0372] In some embodiments, one of homology arm sequences I and homology arm sequences II of the present invention includes one nucleotide sequence from SEQ ID NO: 83 to 84, or includes a nucleotide sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with one of SEQ ID NO: 83 to 84, while the other homology arm sequence includes the corresponding complementary sequence.

[0373] In some embodiments, the translation initiation element is an internal ribosome entry site (IRES), which is selected from CVB3, HRV-B3, BRAV-1_L, PV1_L, CAV2_L, BRAV-1, PV1, or CAV2, and is preferably CVB3.

[0374] In an embodiment, IRES includes a nucleotide sequence shown in one of SEQ ID NO:120~526, or includes a nucleotide sequence having at least 75%, for example, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% sequence identity with one of SEQ ID NO:120~526.

[0375] In some embodiments, IRES comprises the nucleotide sequence of SEQ ID NO:121, or comprises a nucleotide sequence having at least 75%, for example, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:121.

[0376] In some embodiments, the at least one protein-coding sequence includes the AQP-1 protein-coding sequence, or a protein-coding sequence that has the same activity as the AQP-1 protein and is obtained by substituting, deleting, or adding 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids to the amino acid sequence of the AQP-1 protein.

[0377] In some embodiments, the AQP-1 protein coding sequence comprises the nucleotide sequence of SEQ ID NO:118, or comprises a nucleotide sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:118. Specifically, the AQP-1 protein coding sequence is SEQ ID NO:118, or a nucleotide sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:118.

[0378] In some embodiments, the circular RNA precursor of the present invention comprises a nucleotide sequence selected from one of SEQ ID NO: 533 to 536, or a nucleotide sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with one of SEQ ID NO: 533 to 536.

[0379] In the examples, the present invention provides a circular RNA precursor which is oriented in the direction from 5' to 3'. a) 3' self-splicing intron fragment, b) Translation start element (TIE) fragment II, c) A target nucleotide sequence encoding the aquaporin-1 (AQP-1) protein or a variant thereof, d) Translation start element fragment I and, e) Includes a 5' self-splicing intron fragment, Here, the combination of a 3' self-splicing intron fragment and a 5' self-splicing intron fragment preserves the self-splicing activity of the self-splicing intron, allowing the self-splicing of the circular RNA precursor to produce a circular RNA containing the desired nucleotide sequence. Here, non-TIE fragment I originates from the 5' end of the non-TIE sequence, and non-TIE II originates from the 3' end of the non-TIE sequence, and The non-TIE fragment I includes a ribozyme recognition site I located at its 3' end, and the non-TIE fragment II includes a ribozyme recognition site II located at its 5' end. In the resulting circular RNA, the ribozyme recognition site I and the ribozyme recognition site II are linked together, and the non-TIE fragment I and the non-TIE fragment II are linked to form a non-TIE sequence. In this embodiment, the non-TIE sequence is a 3'UTR sequence.

[0380] Furthermore, this invention relates to the following themes.

[0381] [Item 1] A circular RNA precursor, in the direction from 5' to 3', a) 3' self-splicing intron fragment, b) Includes a 5' self-splicing intron fragment, Here, the combination of the 3' self-splicing intron fragment and the 5' self-splicing intron fragment preserves the self-splicing activity of the self-splicing intron and allows for the production of circular RNA by self-splicing of the circular RNA precursor. Here, the self-splicing intron is selected from group I introns and group II introns, for example, the self-splicing intron is selected from group I introns, and is a circular RNA precursor.

[0382] [Item 2] The self-splicing intron is selected from the following groups, for example: cyanobacterium anabaena group I intron (Azo), azoarcus group I intron (Azo), Scytalidium dimidiatum group I intron (Sd), staphylococcal phage twort group I intron (Twort), Scytonema-hofmani tRNA fMet group I intron (Sh), or Agrobacterium-tumefaciens group I intron (At). For example, the self-splicing intron is a nucleotide sequence selected from one of SEQ ID NO: 1 to 6, or SEQ ID A circular RNA precursor as described in item 1, which is a nucleotide sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with one of NO:1-6.

[0383] [Item 3] The circular RNA precursor according to Item 1 or 2, wherein the 3' self-splicing intron fragment originates from the 3' terminal portion of the self-splicing intron, starting from the internal split site I of the intron and extending to the 3' end of the intron, and the 5' self-splicing intron fragment originates from the 5' terminal portion of the self-splicing intron, starting from the 5' end of the self-splicing intron and extending to the internal split site II of the intron.

[0384] [Item 4] The circular RNA precursor described in any one of items 1 to 3, wherein the self-splicing intron is a cyanobacterial anabaena group I intron, for example, AnaX, and the intron internal split site II and the intron internal split site I are nucleotides 115A and 116A located at the 5' end of the AnaX intron (SEQ ID NO:1), respectively, and when they are ligated together they form the nucleotide sequence AA.

[0385] [Item 5] If the self-splicing intron is an Azoarcus group I intron (Azo) (SEQ ID NO:2), 1) Intron internal split site II and intron internal split site I are nucleotides A at position 24 and 25 at position 25 of the 5' end of the Azo intron, respectively, and when they are joined together they form the nucleotide sequence AA(P2). 2) Intron internal split site II and intron internal split site I are nucleotides A at position 85 and 86 of the 5' end of the Azo intron, respectively, and when they are joined together they form the nucleotide sequence AA(P4-5). 3) Intron internal split site II and intron internal split site I are nucleotides C at the 71st and 72nd positions of the 5' end of the Azo intron, respectively, and when they are joined together they form the nucleotide sequence CC(P5). 4) Intron internal split site II and intron internal split site I are nucleotides C and U at the 108th and 109th positions of the 5' end of the Azo intron, respectively, and when they are joined together they form the nucleotide sequence CU(P6). 5) Intron internal split site II and intron internal split site I are nucleotides A at position 154 and 155 of the 5' end of the Azo intron, respectively, and when they are ligated together they form the nucleotide sequence AA(P8), or 6) A circular RNA precursor as described in any one of items 1 to 4, wherein the intron internal split site II and the intron internal split site I are nucleotides C at the 198th and A at the 5' end of the Azo intron, respectively, and when they are ligated together they form the nucleotide sequence CA(P9).

[0386] [Item 6] If the self-splicing intron is a Scytalidium dimidiatum group I intron (Sd) (SEQ ID NO: 4), 1) Intron internal split site II and intron internal split site I are nucleotides A and G at the 88th and 89th positions of the 5' end of the Sd intron, respectively, and when they are joined together they form the nucleotide sequence AG(P2). 2) Intron internal split site II and intron internal split site I are nucleotides U (173rd) and G (174th) of the 5' end of the Sd intron, respectively, and when they are joined together they form the nucleotide sequence UG(P5). 3) Intron internal split site II and intron internal split site I are nucleotides A and G at the 204th and 205th positions of the 5' end of the Sd intron, respectively, and when they are joined together they form the nucleotide sequence AG(P6). 4) Intron internal split site II and intron internal split site I are nucleotides C and G at the 252nd and 253rd positions of the 5' end of the Sd intron, respectively, and when they are linked together they form the nucleotide sequence CG(P8), or 5) A circular RNA precursor as described in any one of items 1 to 4, wherein the intron internal split site II and the intron internal split site I are nucleotides C at position 370 and G at position 371 of the 5' end of the Sd intron, respectively, and when they are ligated together they form the nucleotide sequence CG(P9).

[0387] [Item 7] If the self-splicing intron is a Staphylococcus phage Twort group I intron (Twort) (SEQ ID NO:3), 1) Intron internal split site II and intron internal split site I are nucleotides U (70th) and A (71st) at the 5' end of the Twort intron, respectively, and when they are joined together they form the nucleotide sequence UA(P5). 2) Intron internal split site II and intron internal split site I are nucleotides U (103rd) and A (104th) at the 5' end of the Twort intron, respectively, and when they are joined together they form the nucleotide sequence UA(P6). 3) Intron internal split site II and intron internal split site I are nucleotides A and U at the 132nd and 133rd positions of the 5' end of the Twort intron, respectively, and when they are joined together they form the nucleotide sequence AU(P7). 4) Intron internal split site II and intron internal split site I are nucleotides U (173rd) and C (174th) at the 5' end of the Twort intron, respectively, and when they are joined together they form the nucleotide sequence UC(P8). 5) Intron internal split site II and intron internal split site I are nucleotide A at position 206 and nucleotide A at position 207 of the 5' end of the Twort intron, respectively, and when they are joined together they form the nucleotide sequence AA(P9). 6) Intron internal split site II and intron internal split site I are nucleotides G at position 226 and U at position 227 of the 5' end of the Twort intron, respectively, and when they are joined together they form the nucleotide sequence GU(P9.1). 7) Intron internal split site II and intron internal split site I are the 23rd nucleotide U and the 24th nucleotide A at the 5' end of the Twort intron, respectively, and when they are linked together they form the nucleotide sequence UA(P2), or 8) A circular RNA precursor as described in any one of items 1 to 4, wherein the intron internal split site II and the intron internal split site I are nucleotides 82 and 83 of the 5' end of the Twort intron, respectively, and when they are ligated together they form the nucleotide sequence AA(P4-5).

[0388] [Item 8] If the self-splicing intron is a Scytonema-hofmani tRNA fMet group I intron (Sh) (SEQ ID NO: 5), 1) Intron internal split site II and intron internal split site I are nucleotide A at position 129 and nucleotide A at position 130 of the 5' end of the Sh intron, respectively, and when they are joined together they form the nucleotide sequence AA(P6). 2) Intron internal split site II and intron internal split site I are nucleotide A at position 60 and nucleotide A at position 61 of the 5' end of the Sh intron, respectively, and when they are joined together they form the nucleotide sequence AA(P2). 3) Intron internal split site II and intron internal split site I are nucleotide A at position 109 and nucleotide A at position 110 of the 5' end of the Sh intron, respectively, and when they are linked together they form the nucleotide sequence AA(P4-5), or 4) A circular RNA precursor as described in any one of items 1 to 4, wherein the intron internal split site II and the intron internal split site I are nucleotides A at position 213 and 214 of the 5' end of the Sh intron, respectively, and when they are ligated together they form the nucleotide sequence UA(P9).

[0389] [Item 9] A circular RNA precursor as described in any one of Items 1 to 4, wherein the self-splicing intron is an Agrobacterium tumefaciens group I intron (At) (SEQ ID NO: 6), and the internal intron split site II and internal intron split site I are nucleotides A at position 107 and 108 at position 108 of the 5' end of the At intron, respectively, and when they are ligated together they form the nucleotide sequence AA(P6).

[0390] [Item 10] The 3' self-splicing intron fragment is a sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the 3' terminal portion of the self-splicing intron, and the 5' self-splicing intron fragment is a sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 885%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the 5' self-splicing intron. A circular RNA precursor according to any one of items 1 to 9, wherein the slicing intron fragment is a sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the 5' terminal portion of the self-splicing intron.

[0391] [Item 11] The self-splicing intron is, for example, a cyanobacterial anabaena group I intron such as AnaX, and the 3' self-splicing intron fragment contains or consists of a nucleotide sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO:7, and the 5' self-splicing intron fragment contains a nucleotide sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 8. A circular RNA precursor according to any one of items 1 to 10, comprising or consisting of a nucleotide sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with NO:8.

[0392] [Item 12] The self-splicing intron is an Azoarcus group I intron (Azo), and the 3' self-splicing intron fragment contains or consists of a nucleotide sequence of SEQ ID NO: 9, 11, 13, 15, 17 or 19 or a nucleotide sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 9, 11, 13, 15, 17 or 19, and the 5' self-splicing intron fragment is SEQ ID A circular RNA precursor according to any one of items 1 to 10, comprising or consisting of a nucleotide sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the nucleotide sequence of NO:10, 12, 14, 16, 18, or 20.

[0393] [Item 13] The self-splicing intron is a Scytalidium dimidiatum group I intron (Sd), and the 3' self-splicing intron fragment contains or consists of a nucleotide sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 37, 39, 41, 43, or 45, and the 5' self-splicing intron fragment contains a nucleotide sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 38, 40, 42, 44, or 46 A circular RNA precursor according to any one of items 1 to 10, comprising or consisting of a nucleotide sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with ID NO: 38, 40, 42, 44, or 46.

[0394] [Item 14] The self-splicing intron is a Staphylococcus phage Twort group I intron (Twort), and the 3' self-splicing intron fragment contains or consists of a nucleotide sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 21, 23, 25, 27, 29, 31, 33, or 35, and the 5' self-splicing intron fragment is SEQ ID A circular RNA precursor according to any one of items 1 to 10, comprising or consisting of a nucleotide sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with NO:22, 24, 26, 28, 30, 32, 34, or 36.

[0395] [Item 15] The self-splicing intron is a Scytonema-hofmani tRNA fMet group I intron (Sh), and the 3' self-splicing intron fragment contains a nucleotide sequence of SEQ ID NO: 47, 49, 51 or 53 or a nucleotide sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 47, 49, 51 or 53, and the 5' self-splicing intron fragment contains a nucleotide sequence of SEQ ID NO: 48, 50, 52 or 54 or SEQ ID A circular RNA precursor according to any one of items 1 to 10, comprising a nucleotide sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with NO:48, 50, 52, or 54.

[0396] [Item 16] The self-splicing intron is an Agrobacterium tumefaciens group I intron (At), and the 3' self-splicing intron fragment contains or consists of the nucleotide sequence of SEQ ID NO: 55 or a nucleotide sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 55, and the 5' self-splicing intron fragment contains the nucleotide sequence of SEQ ID NO: 56 or SEQ ID A circular RNA precursor according to any one of items 1 to 10, comprising or consisting of a nucleotide sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with NO:56.

[0397] [Item 17] Further comprising an optional first residue cyclization element in the direction from 5' to 3', the nucleotide sequence of interest, and an optional second residue cyclization element located between a) and b), wherein the total length of the first and second residue cyclization elements is approximately 0 to approximately 500 nucleotides, or The circular RNA precursor is a circular RNA precursor described in any one of items 1 to 16, further comprising homology arm sequence I upstream of a 3' self-splicing intron fragment and homology arm sequence II downstream of a 5' self-splicing intron fragment.

[0398] [Item 18] The cyclic RNA precursor according to any one of items 1 to 17, wherein the first residue cyclization element comprises or consists of a 3' exon region oriented from 5' to 3' and an optional first spacer, and the second residue cyclization element comprises or consists of a 5' exon region oriented from 3' to 5' and an optional second spacer.

[0399] [Item 19] The circular RNA precursor according to Item 18, wherein the 3' exon region is derived from the native 3' exon of a self-splicing intron, the 5' exon region is derived from the native 5' exon of a self-splicing intron, and the 3' and 5' exon regions can be recognized and / or spliced ​​by a self-splicing intron or a combination of a 3' self-splicing intron fragment and a 5' self-splicing intron fragment.

[0400] [Item 20] The 3' exon region is a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the natural 3' exon, or a continuous fragment of about 1 to about 50 nucleotides starting from the 5' terminal nucleotide of the natural 3' exon. The 5' exon region is a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the natural 5' exon, or a continuous fragment of about 1 to about 50 nucleotides starting from the 3' terminal nucleotide of the natural 5' exon, as described in item 19.

[0401] [Item 21] A circular RNA precursor, in the direction from 5' to 3', a) 3' self-splicing intron fragment, b) The first residue cyclization element, c) The target nucleotide sequence, d) The second residue cyclization element, e) Includes a 5' self-splicing intron fragment, Here, the combination of the 3' self-splicing intron fragment and the 5' self-splicing intron fragment preserves the self-splicing activity of the self-splicing intron, allowing the self-splicing of the circular RNA precursor to produce a circular RNA containing a first residue cyclization element, the desired nucleotide sequence, and a second residue cyclization element. Here, the total length of the first residue cyclic element and the second residue cyclic element is approximately 0 to 500 nucleotides, forming a cyclic RNA precursor.

[0402] [Item 22] The self-splicing intron is selected from group I introns and group II introns, for example, the self-splicing intron is selected from group I introns, as described in Item 21, the circular RNA precursor.

[0403] [Item 23] The self-splicing introns are selected from the following groups: cyanobacteria anabaena group I introns (such as AnaX), azoarcus group I introns (Azo), Scytalidium dimidiatum group I introns (Sd), staphylococcal phage Twort group I introns (Twort), Scytonema-hofmani tRNA fMet group I introns (Sh), or Agrobacterium tumefaciens group I introns (At). For example, a self-splicing intron is one nucleotide sequence from SEQ ID NO: 1 to 6, or SEQ ID A circular RNA precursor as described in item 21 or 22, selected from nucleotide sequences having at least 50%, at least 60%, at least 70%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with one of NO:1-6.

[0404] [Item 24] The circular RNA precursor described in any one of items 21 to 23, wherein the 3' self-splicing intron fragment is derived from the 3' terminal portion of a self-splicing intron that starts from the internal split site I of the intron and extends to the 3' end of the intron, and the 5' self-splicing intron fragment is derived from the 5' terminal portion of a self-splicing intron that starts from the 5' end of the self-splicing intron and extends to the internal split site II of the intron.

[0405] [Item 25] The circular RNA precursor according to any one of items 21 to 23, wherein the self-splicing intron is a cyanobacterial anabaena group I intron, for example AnaX, and the intron internal split site II and the intron internal split site I are nucleotides A at the 115th and 116th positions located at the 5' end of the AnaX intron (SEQ ID NO:1), respectively, and are ligated to form the nucleotide sequence AA.

[0406] [Item 26] If the self-splicing intron is an Azoarcus group I intron (Azo) (SEQ ID NO:2), 1) Intron internal split site II and intron internal split site I are nucleotides A at position 24 and 25 at position 25 of the 5' end of the Azo intron, respectively, and when they are joined together they form the nucleotide sequence AA(P2). 2) Intron internal split site II and intron internal split site I are nucleotides A at position 85 and 86 of the 5' end of the Azo intron, respectively, and when they are joined together they form the nucleotide sequence AA(P4-5). 3) Intron internal split site II and intron internal split site I are nucleotides C at the 71st and 72nd positions of the 5' end of the Azo intron, respectively, and when they are joined together they form the nucleotide sequence CC(P5). 4) Intron internal split site II and intron internal split site I are nucleotides C and U at the 108th and 109th positions of the 5' end of the Azo intron, respectively, and when they are joined together they form the nucleotide sequence CU(P6). 5) Intron internal split site II and intron internal split site I are nucleotides A at position 154 and 155 of the 5' end of the Azo intron, respectively, and when they are ligated together they form the nucleotide sequence AA(P8), or 6) A circular RNA precursor as described in any one of items 21-24, wherein the intron internal split site II and the intron internal split site I are nucleotides C at the 5' end and A at the 199th end of the Azo intron, respectively, and when they are ligated together they form the nucleotide sequence CA(P9).

[0407] [Item 27] ​​If the self-splicing intron is a Scytalidium dimidiatum group I intron (Sd) (SEQ ID NO: 4), 1) Intron internal split site II and intron internal split site I are nucleotides A and G at the 88th and 89th positions of the 5' end of the Sd intron, respectively, and when they are joined together they form the nucleotide sequence AG(P2). 2) Intron internal split site II and intron internal split site I are nucleotides U (173rd) and G (174th) of the 5' end of the Sd intron, respectively, and when they are joined together they form the nucleotide sequence UG(P5). 3) Intron internal split site II and intron internal split site I are nucleotides A and G at the 204th and 205th positions of the 5' end of the Sd intron, respectively, and when they are joined together they form the nucleotide sequence AG(P6). 4) Intron internal split site II and intron internal split site I are nucleotides C and G at the 252nd and 253rd positions of the 5' end of the Sd intron, respectively, and when they are linked together they form the nucleotide sequence CG(P8), or 5) A circular RNA precursor as described in any one of items 21 to 24, wherein the intron internal split site II and the intron internal split site I are nucleotides C at position 370 and G at position 371 of the 5' end of the Sd intron, respectively, and when they are ligated together they form the nucleotide sequence CG(P9).

[0408] [Item 28] If the self-splicing intron is a Staphylococcus phage Twort group I intron (Twort) (SEQ ID NO:3), 1) Intron internal split site II and intron internal split site I are nucleotides U (70th) and A (71st) at the 5' end of the Twort intron, respectively, and when they are joined together they form the nucleotide sequence UA(P5). 2) Intron internal split site II and intron internal split site I are nucleotides U (103rd) and A (104th) at the 5' end of the Twort intron, respectively, and when they are joined together they form the nucleotide sequence UA(P6). 3) Intron internal split site II and intron internal split site I are nucleotides A and U at the 132nd and 133rd positions of the 5' end of the Twort intron, respectively, and when they are joined together they form the nucleotide sequence AU(P7). 4) Intron internal split site II and intron internal split site I are nucleotides U (173rd) and C (174th) at the 5' end of the Twort intron, respectively, and when they are joined together they form the nucleotide sequence UC(P8). 5) Intron internal split site II and intron internal split site I are nucleotide A at position 206 and nucleotide A at position 207 of the 5' end of the Twort intron, respectively, and when they are joined together they form the nucleotide sequence AA(P9). 6) Intron internal split site II and intron internal split site I are nucleotides G at position 226 and U at position 227 of the 5' end of the Twort intron, respectively, and when they are joined together they form the nucleotide sequence GU(P9.1). 7) Intron internal split site II and intron internal split site I are the 23rd nucleotide U and the 24th nucleotide A at the 5' end of the Twort intron, respectively, and when they are linked together they form the nucleotide sequence UA(P2), or 8) A circular RNA precursor as described in any one of items 21 to 24, wherein the intron internal split site II and the intron internal split site I are nucleotides 82 and 83 of the 5' end of the Twort intron, respectively, and when they are ligated together they form the nucleotide sequence AA(P4-5).

[0409] [Item 29] If the self-splicing intron is a Scytonema-hofmani tRNA fMet group I intron (Sh) (SEQ ID NO: 5), 1) Intron internal split site II and intron internal split site I are nucleotide A at position 129 and nucleotide A at position 130 of the 5' end of the Sh intron, respectively, and when they are joined together they form the nucleotide sequence AA(P6). 2) Intron internal split site II and intron internal split site I are nucleotide A at position 60 and nucleotide A at position 61 of the 5' end of the Sh intron, respectively, and when they are joined together they form the nucleotide sequence AA(P2). 3) Intron internal split site II and intron internal split site I are nucleotide A at position 109 and nucleotide A at position 110 of the 5' end of the Sh intron, respectively, and when they are linked together they form the nucleotide sequence AA(P4-5), or 4) A circular RNA precursor as described in any one of items 21-24, wherein the intron internal split site II and the intron internal split site I are nucleotides A at position 213 and 214 of the 5' end of the Sh intron, respectively, and when they are ligated together they form the nucleotide sequence UA(P9).

[0410] [Item 30] A circular RNA precursor as described in any one of items 21-24, wherein the self-splicing intron is an Agrobacterium tumefaciens group I intron (At) (SEQ ID NO: 6), and the internal intron split site II and internal intron split site I are nucleotides A at position 107 and 108 at position 108 of the 5' end of the At intron, respectively, and when they are ligated together they form the nucleotide sequence AA(P6).

[0411] [Item 31] The 3' self-splicing intron fragment is a sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the 3' terminal portion of the self-splicing intron, and is a 5' self-splicing intron. A circular RNA precursor as described in any one of items 21 to 30, wherein the sing-intron fragment is a sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the 5' terminal portion of a self-splicing intron.

[0412] [Item 32] The self-splicing intron is, for example, a cyanobacterial anabaena group I intron such as AnaX, and the 3' self-splicing intron fragment contains or consists of the nucleotide sequence of SEQ ID NO:7 or a nucleotide sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO:7, and the 5' self-splicing intron fragment contains the nucleotide sequence of SEQ ID NO:8 or SEQ ID A circular RNA precursor according to any one of items 21 to 31, comprising or consisting of a nucleotide sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with NO:8.

[0413] [Item 33] The self-splicing intron is an Azoarcus group I intron (Azo), and the 3' self-splicing intron fragment contains or consists of a nucleotide sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 9, 11, 13, 15, 17, or 19, and the 5' self-splicing intron fragment is SEQ ID A circular RNA precursor according to any one of items 21 to 31, comprising or consisting of a nucleotide sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the nucleotide sequence of NO:10, 12, 14, 16, 18, or 20.

[0414] [Item 34] The self-splicing intron is a Scytalidium dimidiatum group I intron (Sd), and the 3' self-splicing intron fragment contains or consists of a nucleotide sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 37, 39, 41, 43, or 45, and the 5' self-splicing intron fragment contains a nucleotide sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 38, 40, 42, 44, or 46 or SEQ A circular RNA precursor as described in any one of items 21 to 31, comprising or consisting of a nucleotide sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with ID NO: 38, 40, 42, 44, or 46.

[0415] [Item 35] The self-splicing intron is a Staphylococcus phage Twort group I intron (Twort), and the 3' self-splicing intron fragment contains or consists of a nucleotide sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 21, 23, 25, 27, 29, 31, 33, or 35, and the 5' self-splicing intron fragment is SEQ ID A circular RNA precursor as described in any one of items 21 to 31, comprising or consisting of a nucleotide sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with NO:22, 24, 26, 28, 30, 32, 34, or 36.

[0416] [Item 36] The self-splicing intron is a Scytonema-hofmani tRNA fMet group I intron (Sh), and the 3' self-splicing intron fragment contains a nucleotide sequence of SEQ ID NO: 47, 49, 51 or 53 or a nucleotide sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 47, 49, 51 or 53, and the 5' self-splicing intron fragment contains a nucleotide sequence of SEQ ID NO: 48, 50, 52 or 54 or SEQ ID A circular RNA precursor according to any one of items 21 to 31, comprising a nucleotide sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with NO:48, 50, 52, or 54.

[0417] [Item 37] The self-splicing intron is an Agrobacterium tumefaciens group I intron (At), and the 3' self-splicing intron fragment contains or consists of the nucleotide sequence of SEQ ID NO: 55 or a nucleotide sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 55, and the 5' self-splicing intron fragment contains the nucleotide sequence of SEQ ID NO: 56 or SEQ ID A circular RNA precursor according to any one of items 21 to 31, comprising or consisting of a nucleotide sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with NO:56.

[0418] [Item 38] The circular RNA precursor according to any one of items 21 to 37, wherein the first residue cyclization element and the second residue cyclization element are configured to give the circular RNA containing them reduced immunogenicity.

[0419] [Item 39] The cyclic RNA precursor according to any one of items 21 to 38, wherein the first residue cyclization element and the second residue cyclization element are configured to have a cyclization efficiency equivalent to or increased by the cyclic RNA precursor containing them.

[0420] [Item 40] A cyclic RNA precursor as described in any one of items 21 to 39, wherein the total length of the first residue cyclic element and the second residue cyclic element is approximately 0 to approximately 100 nucleotides, approximately 0 to approximately 90 nucleotides, approximately 0 to approximately 80 nucleotides, approximately 0 to approximately 70 nucleotides, approximately 0 to approximately 60 nucleotides, approximately 0 to approximately 50 nucleotides, approximately 0 to approximately 40 nucleotides, approximately 0 to approximately 35 nucleotides, approximately 2 to approximately 100 nucleotides, approximately 5 to approximately 100 nucleotides, approximately 10 to approximately 100 nucleotides, approximately 20 to approximately 100 nucleotides, approximately 30 to approximately 100 nucleotides, or approximately 30 to approximately 100 nucleotides.

[0421] [Item 41] The cyclic RNA precursor according to any one of items 21 to 40, wherein the first residue cyclization element and the second residue cyclization element are configured to form a stem-loop structure, for example, by self-splicing cyclization.

[0422] [Item 42] The loop of the stem-loop structure contains a splicing binding site, as described in Item 41, which is a circular RNA precursor.

[0423] [Item 43] The first residue cyclization element includes a sequence structure of the formula 5'-first loop sequence-first paired sequence-first unpaired sequence-3', and the second residue cyclization element includes a sequence structure of the formula 5'-second unpaired sequence-second paired sequence-second loop sequence-3', Here, the first unpaired sequence or the second unpaired sequence may or may not exist independently. The first pairing sequence and the second pairing sequence can pair complementaryly to each other to form the stem of a stem-loop structure, where the first loop sequence and the second loop sequence can form the loop of the stem-loop structure, for example, by self-splicing cyclization, as described in any one of items 41-42.

[0424] [Item 44] The first residue cyclization element may be selected from a variety of sequences, provided that the first loop sequence of the first residue cyclization element does not pair with any sequence of the second residue cyclization element, as described in any one of items 41-43.

[0425] [Item 45] A circular RNA precursor as described in Item 43 or 44, wherein the first loop sequence comprises or consists of one or more nucleotides that can pair with the P1 region of the self-splicing intron to form a P10 double-stranded region during circularization.

[0426] [Item 46] A circular RNA precursor as described in any one of items 43-45, wherein the first loop sequence consists of a (N)n nucleotide sequence, where N represents any nucleotide (A, G, U, or C) and n represents an integer from 1 to 20, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0427] [Item 47] The circular RNA precursor according to any one of items 43 to 46, wherein the first loop sequence comprises or consists of the sequences AAAA, AGAA, AA, UUUU, UAAA, CAAA, or GAAA, preferably AAAA.

[0428] [Item 48] The circular RNA precursor according to any one of items 43 to 47, wherein the second loop sequence comprises or consists of one or more nucleotides that can pair with the internal guide sequence (IGS) of the self-splicing intron to form a double-stranded region during P1 circularization.

[0429] [Item 49] The circular RNA precursor according to any one of items 43 to 48, wherein the second loop sequence comprises or consists of one nucleotide sequence from SEQ ID NO: 95 to 116, for example CUU, CAU, or CUC, and preferably CUU. [Item 50] The loop of the stem-loop structure has the sequence CUUAAAA, CUUUUUU, CUUAA, CUUGAAA, CUUUAAA, CUUAGAA, CUUCAAA, or CUCAAAA, preferably CUUAAAA, as described in any one of items 43 to 49.

[0430] [Item 51] The cyclic RNA precursor according to any one of items 43 to 50, wherein the stem portion of the stem-loop structure comprises 2 to 15 or more consecutive matching base pairs, preferably the stem portion of the stem-loop structure comprises 5, 6 or 7 consecutive matching base pairs.

[0431] [Item 52] The circular RNA precursor according to any one of items 43 to 51, wherein the stem portion in the stem-loop structure contains at most two base pair mismatches, or contains only one base pair mismatch, and preferably the stem portion does not have any base pair mismatches.

[0432] [Item 53] A circular RNA precursor according to any one of items 43 to 52, wherein the first pairing sequence contains only G and the second pairing sequence contains only C, or the first pairing sequence contains only C and the second pairing sequence contains only G.

[0433] [Item 54] A circular RNA precursor according to any one of items 43 to 53, wherein the first pairing sequence contains only A, and the second pairing sequence contains only U.

[0434] [Item 55] A circular RNA precursor according to any one of items 43 to 54, wherein the first pairing sequence contains only U, and the second pairing sequence contains only A.

[0435] [Item 56] The cyclic RNA precursor according to any one of items 21 to 55, wherein the first residue cyclization element comprises or consists of a 3' exon region oriented from 5' to 3' and an optional first spacer, and the second residue cyclization element comprises or consists of a 5' exon region oriented from 3' to 5' and an optional second spacer.

[0436] [Item 57] The circular RNA precursor according to Item 56, wherein the 3' exon region is derived from the native 3' exon of a self-splicing intron, the 5' exon region is derived from the native 5' exon of a self-splicing intron, and the 3' and 5' exon regions can be recognized and / or spliced ​​by a self-splicing intron or a combination of a 3' self-splicing intron fragment and a 5' self-splicing intron fragment.

[0437] [Item 58] The 3' exon region is a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with a natural 3' exon or a fragment having a length of about 1 to about 50 nucleotides starting from the 5' terminal nucleotide of a natural 3' exon. The circular RNA precursor described in item 56 or 57 is a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with a fragment that is approximately 1 to about 50 nucleotides long and starts from the 3' terminal nucleotide of a natural 5' exon.

[0438] [Item 59] The cyclic RNA precursor according to any one of items 21 to 58, wherein the first residue cyclization element comprises or consists of a nucleotide sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 57, 59, 61, 63, 65, 67, or 69, and is preferably a first residue cyclization element selected from the nucleotide sequence AAAAUCCGUUGA or AAAACCCCCCCA.

[0439] [Item 60] The cyclic RNA precursor according to any one of items 21 to 59, wherein the second residue cyclization element comprises or consists of a nucleotide sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 58, 60, 62, 64, 66, 68, or 70, and is preferably a second residue cyclization element selected from the nucleotide sequence AGACGCUACGGACUU or AGACGGGGGGGGCUU.

[0440] [Item 61] The circular RNA precursor according to any one of items 21 to 60, wherein the first residue cyclization element contains or consists of one nucleotide sequence from SEQ ID NO: 57, 59, 61, 63, 65, 67, or 69, and the second residue cyclization element contains or consists of one nucleotide sequence from SEQ ID NO: 58, 60, 62, 64, 66, 68, or 70.

[0441] [Item 62] 1) The first residue cyclization element contains or consists of the nucleotide sequence of SEQ ID NO: 57, and the second residue cyclization element contains or consists of the nucleotide sequence of SEQ ID NO: 58. 2) The first residue cyclization element contains or consists of the nucleotide sequence of SEQ ID NO: 59, and the second residue cyclization element contains or consists of the nucleotide sequence of SEQ ID NO: 60. 3) The first residue cyclization element contains or consists of the nucleotide sequence of SEQ ID NO:61, and the second residue cyclization element contains or consists of the nucleotide sequence of SEQ ID NO:62. 4) The first residue cyclization element contains or consists of the nucleotide sequence of SEQ ID NO:63, and the second residue cyclization element contains or consists of the nucleotide sequence of SEQ ID NO:64. 5) The first residue cyclization element contains or consists of the nucleotide sequence of SEQ ID NO:65, and the second residue cyclization element contains or consists of the nucleotide sequence of SEQ ID NO:66. 6) The first residue cyclization element contains or consists of the nucleotide sequence of SEQ ID NO:67, and the second residue cyclization element contains or consists of the nucleotide sequence of SEQ ID NO:68, or 7) The first residue cyclization element contains or consists of the nucleotide sequence of SEQ ID NO:69, and the second residue cyclization element contains or consists of the nucleotide sequence of SEQ ID NO:70. Preferably, the first residue cyclization element is selected from the nucleotide sequence AAAAUCCGUUGA and the second residue cyclization element is selected from the nucleotide sequence AGACGCUACGGACUU, or the first residue cyclization element is selected from the nucleotide sequence AAAACCCCCCCA and the second residue cyclization element is selected from the nucleotide sequence AGACGGGGGGGGCUU, as described in any one of items 21 to 61.

[0442] [Item 63] The circular RNA precursor according to any one of items 21 to 62, further comprising homology arm sequence I and homology arm sequence II, which can complementarily pair with each other to form homology arm double-stranded regions.

[0443] [Item 64] A circular RNA precursor as described in Item 63, wherein homology arm sequence I is located upstream of the 3' self-splicing intron fragment, and homology arm sequence II is located downstream of the 5' self-splicing intron fragment.

[0444] [Item 65] A circular RNA precursor according to any one of items 61 to 62, wherein the length of homology arm sequence I or homology arm sequence II is about 5 to 500, about 5 to 450, about 5 to 400, about 5 to 350, about 5 to 300, about 5 to 250, about 5 to 200, about 5 to 150, about 5 to 100, about 5 to 80, about 5 to 60, about 5 to 50, about 5 to 40, about 5 to 30, about 5 to 25, about 5 to 20, about 5 to 15 or about 5 to 10 nucleotides, preferably about 20, 25, 40, 50, 100 or 300 nucleotides.

[0445] [Item 66] The circular RNA precursor according to any one of items 61 to 65, wherein the two homologous arm sequences may each be polyA and polyU, or polyG and polyC.

[0446] [Item 67] A circular RNA precursor as described in any one of Items 61 to 66, wherein one of homology arm sequence I and homology arm sequence II contains a nucleotide sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with one of SEQ ID NO: 71 to 94, and the other homology arm sequence contains the corresponding complementary sequence.

[0447] [Item 68] The cyclic RNA precursor according to any one of items 21 to 67, wherein the nucleotide sequence of the object of interest comprises at least one protein coding sequence and a translation initiation element operably ligated thereto, such as an internal ribosome entry site (IRES).

[0448] [Item 69] A translation initiation element, e.g., IRES, is a circular RNA precursor as described in Item 68, located upstream of the 5' end of at least one protein-coding sequence.

[0449] [Item 70] The translation initiation element, e.g., IRES, is a circular RNA precursor as described in Item 68, located downstream of the 3' end of at least one protein-coding sequence.

[0450] [Item 71] The translation initiation element is the internal ribosome entry site (IRES), which is used in Taura syndrome virus, assassin bug virus, Tyler's encephalomyelitis virus, Simian virus 40, fire ant virus 1, wheat aphid virus, reticuloendotheliosis virus, Forman's poliovirus 1, soybean looper virus, Casimir wasp virus, human rhinovirus 2, leafhopper virus-1, human immunodeficiency virus type 1, leafhopper virus-1, pygmy flea P virus, hepatitis C virus, hepatitis A virus Viruses, Hepatitis GB virus, Foot-and-mouth disease virus, Human enterovirus 71, Horse rhinovirus, White-winged geometrid virus, Encephalomyocarditis virus (EMCV), Drosophila C virus, Brassicaceae tobamovirus, Cricket paralysis virus, Bovine viral diarrhea virus 1, Black queen bee larva virus, Aphid lethal paralysis virus, Avian encephalomyelitis virus, Acute bee paralysis virus, Hibiscus chlorotic ring spot virus, Swine fever virus, Human FGF2, Human SFTPA1, Human AM1 / RUNX1 Drosophila Antennapedia, Human AQP4, Human AT1R, Human BAG-1, Human BCL2, Human BiP, Human c-IAPl, Human c-myc, Human eIF4G, Mouse NDST4L, Human LEF1, Mouse HIF1α, Human n.myc, Mouse Gtx, Human p27kipl, Human PDGF2 / c-sis, Human p53, Human Pim-1, Mouse Rbm3, Drosophila reaper, Canine Scamper, Drosophila Ubx, Human UNR, Mouse UtrA, Human VEGF-A, Human XIAP, A circular RNA precursor as described in any one of items 68 to 70, selected from IRES sequences such as Drosophila hairless, budding yeast TFIID, budding yeast YAP1, human c-src, human FGF-1, salpicomavirus, cab crinkle virus, aptamer for eIF4G, coxsackievirus B3 (CVB3), or coxsackievirus A (CVA1 / 2), preferably the IRES being CVB3, BRAV-1_L, PV1_L, CAV2_L, BRAV-1, PV1, or CAV2.

[0451] [Item 72] IRES is a circular RNA precursor according to any one of items 68 to 71, comprising a nucleotide sequence shown in one of SEQ ID NO: 120 to 526, or comprising a nucleotide sequence having at least 75%, for example, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% sequence identity with one of SEQ ID NO: 120 to 526.

[0452] [Item 73] The at least one protein coding sequence codes for a protein of eukaryotic, prokaryotic, or viral origin, for example, a protein for therapeutic or diagnostic purposes. Preferably, the cyclic RNA precursor according to any one of items 68 to 72, wherein the at least one protein-coding sequence encodes a protein having the same activity as the AQP-1 protein and derived from the AQP-1 protein by substituting, deleting, or adding 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids in the amino acid sequence of the AQP-1 protein.

[0453] [Item 74] The circular RNA precursor according to any one of items 68 to 73, wherein the at least one protein-coding sequence comprises one nucleotide sequence from SEQ ID NO: 117 to 118, or comprises a nucleotide sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with one of SEQ ID NO: 117 to 118.

[0454] [Item 75] The circular RNA precursor according to any one of items 68 to 74, wherein the target nucleotide sequence is a non-protein-coding sequence, for example, the non-protein-coding sequence may be an antisense RNA, an aptamer, a guide RNA, or any non-protein-coding RNA present in a living organism.

[0455] [Item 76] A circular RNA precursor according to any one of items 21 to 75, comprising a nucleotide sequence selected from one of SEQ ID NO: 527, 532, and 537-542, or comprising a nucleotide sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with one of SEQ ID NO: 527, 532, and 537-542.

[0456] [Item 77] A circular RNA precursor, in the direction from 5' to 3', a) 3' self-splicing intron fragment, b) The target nucleotide sequence and c) Includes a 5' self-splicing intron fragment, Here, the combination of the 3' self-splicing intron fragment and the 5' self-splicing intron fragment preserves the self-splicing activity of the self-splicing intron, allowing the production of a circular RNA containing the target nucleotide sequence by self-splicing of the circular RNA precursor, and Here, the target nucleotide sequence includes a circular sequence fragment II containing a ribozyme recognition site II at its 5' end and a circular sequence fragment I containing a ribozyme recognition site I at its 3' end, in the direction from 5' to 3'. Here, circularized sequence fragment I is the 5' end portion of the circularized sequence, and circularized sequence fragment II is the 3' end portion of the circularized sequence, and Here, in the generated circular RNA, ribozyme recognition site I and ribozyme recognition site II are linked, thereby linking circularized sequence fragment I and circularized sequence fragment II to form the circularized sequence, thus forming a circular RNA precursor.

[0457] [Item 78] The cyclic RNA precursor according to Item 77, wherein the target nucleotide sequence comprises the sequences of one or more circularized sequence fragments II and cyclicized sequence fragments I selected from the group consisting of at least one protein-coding sequence, a non-coding sequence, a translation initiation element (TIE), a non-TIE functional element, or any combination thereof.

[0458] [Item 79] The circularized sequence is selected from at least one protein-coding sequence, non-coding sequence, translation initiation element (TIE), or non-TIE functional element, as described in Item 77 or 78, and is a circular RNA precursor.

[0459] [Item 80] A circular RNA precursor, in the direction from 5' to 3', a) 3' self-splicing intron fragment, b) The target nucleotide sequence and c) Includes a 5' self-splicing intron fragment, Here, the combination of a 3' self-splicing intron fragment and a 5' self-splicing intron fragment preserves the self-splicing activity of the self-splicing intron, allowing the production of a circular RNA containing the target nucleotide sequence by self-splicing of the circular RNA precursor. Here, the target nucleotide sequence includes protein-coding sequence fragment II, a translation initiation element such as an internal ribosome entry site (IRES), and protein-coding sequence fragment I, in the direction from 5' to 3'. Here, protein-coding sequence fragment I is the 5' end portion of at least one protein-coding sequence, and protein-coding sequence fragment II is the 3' end portion of at least one protein-coding sequence, and Protein coding sequence fragment I includes a ribozyme recognition site I located at its 3' end, and protein coding sequence fragment II includes a ribozyme recognition site II located at its 5' end, where, in the generated circular RNA, ribozyme recognition site I and ribozyme recognition site II are linked, thereby linking protein coding sequence fragment I and protein coding sequence fragment II to form at least one protein coding sequence. Optionally, the at least one protein coding sequence may include at least two protein coding sequences, and any two adjacent protein coding sequences may be linked together by another translation initiation element, 2A sequence, or spacer, and the translation initiation element may be an IRES sequence, a 5'UTR sequence, a Kozak sequence, or m 6 A circular RNA precursor containing an A-containing sequence, a sequence complementary to ribosomal 18S rRNA, or any combination thereof.

[0460] [Item 81] The self-splicing intron is selected from group I introns and group II introns, for example, the self-splicing intron is selected from group I introns, as described in any one of items 77 to 80.

[0461] [Item 82] Self-splicing introns are selected from the following groups: cyanobacteria anabaena group I introns (such as AnaX), azoarcus group I introns (Azo), Scytalidium dimidiatum group I introns (Sd), staphylococcal phage Twort group I introns (Twort), Scytonema-hofmani tRNA fMet group I introns (Sh), or Agrobacterium tumefaciens group I introns (At). For example, a self-splicing intron is one nucleotide sequence from SEQ ID NO: 1-6, or SEQ ID A circular RNA precursor as described in any one of items 77-81, selected from nucleotide sequences having at least 50%, at least 60%, at least 70%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with one of NO:1-6.

[0462] [Item 83] The circular RNA precursor according to any one of items 77 to 82, wherein the 3' self-splicing intron fragment is derived from the 3' terminal portion of a self-splicing intron that starts from the internal split site I of the intron and extends to the 3' end of the intron, and the 5' self-splicing intron fragment is derived from the 5' terminal portion of a self-splicing intron that starts from the 5' end of the self-splicing intron and extends to the internal split site II of the intron.

[0463] [Item 84] The circular RNA precursor according to any one of items 77-83, wherein the self-splicing intron is a cyanobacterial anabaena group I intron, e.g., AnaX, and the intron internal split site II and the intron internal split site I are nucleotides A at the 115th and 116th positions located at the 5' end of the AnaX intron (SEQ ID NO:1), respectively, and they form the nucleotide sequence AA when ligated together.

[0464] [Item 85] If the self-splicing intron is an Azoarcus group I intron (Azo) (SEQ ID NO:2), 1) Intron internal split site II and intron internal split site I are nucleotides A at position 24 and 25 at position 25 of the 5' end of the Azo intron, respectively, and when they are joined together they form the nucleotide sequence AA(P2). 2) Intron internal split site II and intron internal split site I are nucleotides A at position 85 and 86 of the 5' end of the Azo intron, respectively, and when they are joined together they form the nucleotide sequence AA(P4-5). 3) Intron internal split site II and intron internal split site I are nucleotides C at the 71st and 72nd positions of the 5' end of the Azo intron, respectively, and when they are joined together they form the nucleotide sequence CC(P5). 4) Intron internal split site II and intron internal split site I are nucleotides C and U at the 108th and 109th positions of the 5' end of the Azo intron, respectively, and when they are joined together they form the nucleotide sequence CU(P6). 5) Intron internal split site II and intron internal split site I are nucleotides A at position 154 and 155 of the 5' end of the Azo intron, respectively, and when they are ligated together they form the nucleotide sequence AA(P8), or 6) A circular RNA precursor as described in any one of items 77-83, wherein the intron internal split site II and the intron internal split site I are nucleotides C at the 5' end and A at the 199th end of the Azo intron, respectively, and when they are ligated together they form the nucleotide sequence CA(P9).

[0465] [Item 86] If the self-splicing intron is a Scytalidium dimidiatum group I intron (Sd) (SEQ ID NO: 4), 1) Intron internal split site II and intron internal split site I are nucleotides A and G at the 88th and 89th positions of the 5' end of the Sd intron, respectively, and when they are joined together they form the nucleotide sequence AG(P2). 2) Intron internal split site II and intron internal split site I are nucleotides U (173rd) and G (174th) of the 5' end of the Sd intron, respectively, and when they are joined together they form the nucleotide sequence UG(P5). 3) Intron internal split site II and intron internal split site I are nucleotides A and G at the 204th and 205th positions of the 5' end of the Sd intron, respectively, and when they are joined together they form the nucleotide sequence AG(P6). 4) Intron internal split site II and intron internal split site I are nucleotides C and G at the 252nd and 253rd positions of the 5' end of the Sd intron, respectively, and when they are linked together they form the nucleotide sequence CG(P8), or 5) A circular RNA precursor as described in any one of items 77 to 83, wherein the intron internal split site II and the intron internal split site I are nucleotides C at position 370 and G at position 371 of the 5' end of the Sd intron, respectively, and when they are ligated together they form the nucleotide sequence CG(P9).

[0466] [Item 87] If the self-splicing intron is a Staphylococcus phage Twort group I intron (Twort) (SEQ ID NO:3), 1) Intron internal split site II and intron internal split site I are nucleotides U (70th) and A (71st) at the 5' end of the Twort intron, respectively, and when they are joined together they form the nucleotide sequence UA(P5). 2) Intron internal split site II and intron internal split site I are nucleotides U (103rd) and A (104th) at the 5' end of the Twort intron, respectively, and when they are joined together they form the nucleotide sequence UA(P6). 3) Intron internal split site II and intron internal split site I are nucleotides A and U at the 132nd and 133rd positions of the 5' end of the Twort intron, respectively, and when they are joined together they form the nucleotide sequence AU(P7). 4) Intron internal split site II and intron internal split site I are nucleotides U (173rd) and C (174th) at the 5' end of the Twort intron, respectively, and when they are joined together they form the nucleotide sequence UC(P8). 5) Intron internal split site II and intron internal split site I are nucleotide A at position 206 and nucleotide A at position 207 of the 5' end of the Twort intron, respectively, and when they are joined together they form the nucleotide sequence AA(P9). 6) Intron internal split site II and intron internal split site I are nucleotides G at position 226 and U at position 227 of the 5' end of the Twort intron, respectively, and when they are joined together they form the nucleotide sequence GU(P9.1). 7) Intron internal split site II and intron internal split site I are the 23rd nucleotide U and the 24th nucleotide A at the 5' end of the Twort intron, respectively, and when they are linked together they form the nucleotide sequence UA(P2), or 8) A circular RNA precursor as described in any one of items 77 to 83, wherein the intron internal split site II and the intron internal split site I are nucleotides A at position 82 and 83 of the 5' end of the Twort intron, respectively, and when they are ligated together they form the nucleotide sequence AA(P4-5).

[0467] [Item 88] If the self-splicing intron is a Scytonema-hofmani tRNA fMet group I intron (Sh) (SEQ ID NO: 5), 1) Intron internal split site II and intron internal split site I are nucleotide A at position 129 and nucleotide A at position 130 of the 5' end of the Sh intron, respectively, and when they are joined together they form the nucleotide sequence AA(P6). 2) Intron internal split site II and intron internal split site I are nucleotide A at position 60 and nucleotide A at position 61 of the 5' end of the Sh intron, respectively, and when they are joined together they form the nucleotide sequence AA(P2). 3) Intron internal split site II and intron internal split site I are nucleotide A at position 109 and nucleotide A at position 110 of the 5' end of the Sh intron, respectively, and when they are linked together they form the nucleotide sequence AA(P4-5), or 4) A circular RNA precursor as described in any one of items 77-83, wherein the intron internal split site II and the intron internal split site I are nucleotides A at position 213 and 214 at position 214 of the 5' end of the Sh intron, respectively, and when they are ligated together they form the nucleotide sequence UA(P9).

[0468] [Item 89] A circular RNA precursor as described in any one of items 77-83, wherein the self-splicing intron is an Agrobacterium tumefaciens group I intron (At) (SEQ ID NO: 6), and the internal intron split site II and internal intron split site I are nucleotides A at position 107 and A at position 108 of the 5' end of the At intron, respectively, and when they are ligated together they form the nucleotide sequence AA(P6).

[0469] [Item 90] The 3' self-splicing intron fragment is a sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the 3' terminal portion of the self-splicing intron, and the 5' self-splicing intron fragment is a self-splicing intron A circular RNA precursor according to any one of items 77 to 89, which is a sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the 5' terminal portion of tron.

[0470] [Item 91] The self-splicing intron is, for example, a cyanobacterial anabaena group I intron such as AnaX, and the 3' self-splicing intron fragment contains or consists of the nucleotide sequence of SEQ ID NO:7 or a nucleotide sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO:7, and the 5' self-splicing intron fragment contains the nucleotide sequence of SEQ ID NO:8 or SEQ ID A circular RNA precursor according to any one of items 77 to 90, comprising or consisting of a nucleotide sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with NO:8.

[0471] [Item 92] The self-splicing intron is an Azoarcus group I intron (Azo), and the 3' self-splicing intron fragment contains or consists of a nucleotide sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 9, 11, 13, 15, 17, or 19, and the 5' self-splicing intron fragment is SEQ ID A circular RNA precursor according to any one of items 77 to 90, comprising or consisting of a nucleotide sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the nucleotide sequence of NO:10, 12, 14, 16, 18, or 20.

[0472] [Item 93] The self-splicing intron is a Scytalidium dimidiatum group I intron (Sd), and the 3' self-splicing intron fragment contains or consists of a nucleotide sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 37, 39, 41, 43, or 45, and the 5' self-splicing intron fragment contains a nucleotide sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 38, 40, 42, 44, or 46 or SEQ A circular RNA precursor according to any one of items 77 to 90, comprising or consisting of a nucleotide sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with ID NO: 38, 40, 42, 44, or 46.

[0473] [Item 94] The self-splicing intron is a Staphylococcus phage Twort group I intron (Twort), and the 3' self-splicing intron fragment contains or consists of a nucleotide sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 21, 23, 25, 27, 29, 31, 33, or 35, and the 5' self-splicing intron fragment is SEQ ID A circular RNA precursor as described in any one of items 77 to 90, comprising or consisting of a nucleotide sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with NO:22, 24, 26, 28, 30, 32, 34, or 36.

[0474] [Item 95] The self-splicing intron is a Scytonema-hofmani tRNA fMet group I intron (Sh), and the 3' self-splicing intron fragment contains a nucleotide sequence of SEQ ID NO: 47, 49, 51 or 53 or a nucleotide sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 47, 49, 51 or 53, and the 5' self-splicing intron fragment contains a nucleotide sequence of SEQ ID NO: 48, 50, 52 or 54 or SEQ ID A circular RNA precursor according to any one of items 77 to 90, comprising a nucleotide sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with NO:48, 50, 52, or 54.

[0475] [Item 96] The self-splicing intron is an Agrobacterium tumefaciens group I intron (At), and the 3' self-splicing intron fragment contains or consists of the nucleotide sequence of SEQ ID NO: 55 or a nucleotide sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 55, and the 5' self-splicing intron fragment contains the nucleotide sequence of SEQ ID NO: 56 or SEQ ID A circular RNA precursor according to any one of items 77 to 90, comprising or consisting of a nucleotide sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with NO:56.

[0476] [Item 97] The circular RNA precursor according to any one of items 77 to 96, wherein the first residue cyclization element and the second residue cyclization element are configured to give the circular RNA containing them reduced immunogenicity.

[0477] [Item 98] The cyclic RNA precursor according to any one of items 77 to 97, wherein the first residue cyclization element and the second residue cyclization element are configured to have a cyclization efficiency equivalent to or increased by the cyclic RNA precursor containing them.

[0478] [Item 99] The total length of the ribozyme recognition site I and ribozyme recognition site II is approximately 0 to 100 nucleotides, approximately 0 to 90 nucleotides, approximately 0 to 80 nucleotides, approximately 0 to 70 nucleotides, approximately 0 to 60 nucleotides, approximately 0 to 50 nucleotides, approximately 0 to 40 nucleotides, approximately 0 to 35 nucleotides, approximately 2 to 100 nucleotides, approximately 5 to 100 nucleotides, approximately 10 to 100 nucleotides, approximately 20 to 100 nucleotides, approximately 30 to 100 nucleotides, or approximately 30 to 100 nucleotides, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 ,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64, A circular RNA precursor as described in any one of items 77-98, comprising 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

[0479] [Item 100] The total length of the ribozyme recognition site I is approximately 0 to 100 nucleotides, approximately 0 to 90 nucleotides, approximately 0 to 80 nucleotides, approximately 0 to 70 nucleotides, approximately 0 to 60 nucleotides, approximately 0 to 50 nucleotides, approximately 0 to 40 nucleotides, approximately 0 to 35 nucleotides, approximately 2 to 100 nucleotides, approximately 5 to 100 nucleotides, approximately 10 to 100 nucleotides, approximately 20 to 100 nucleotides, approximately 30 to 100 nucleotides, or approximately 30 to 100 nucleotides, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 1 7, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65 A circular RNA precursor as described in any one of items 77-99, consisting of 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

[0480] [Item 101] The cyclical RNA precursor described in any one of items 77 to 100, wherein the ribozyme recognition site I and ribozyme recognition site II are configured to form a stem-loop structure, for example, during self-splicing cyclization.

[0481] [Item 102] The loop of the stem-loop structure contains a splicing binding site, as described in Item 101, which is a circular RNA precursor.

[0482] [Item 103] Ribozyme recognition site I includes a sequence structure of the form 5'-third unpaired sequence-third paired sequence-third loop sequence-3', and ribozyme recognition site II includes a sequence structure of the form 5'-fourth loop sequence-fourth paired sequence-fourth unpaired sequence-3', Here, the third unpaired sequence or the fourth unpaired sequence may or may not exist independently. The third pairing sequence and the fourth pairing sequence can pair complementaryly to each other to form the stem of a stem-loop structure, where the third loop sequence and the fourth loop sequence can form the loop of the stem-loop structure by self-splicing to achieve circularity, as described in any one of items 77-102.

[0483] [Item 104] Ribozyme recognition site II is a circular RNA precursor as described in any one of items 77-102, which may be selected from multiple sequences.

[0484] [Item 105] The circular RNA precursor according to Item 103 or 104, wherein the fourth loop sequence comprises or consists of one or more nucleotides, the one or more nucleotides being able to pair with the P1 region of the corresponding self-splicing intron to form a P10 double-stranded region during circularization.

[0485] [Item 106] A circular RNA precursor as described in any one of items 103-105, wherein the fourth loop sequence consists of a (N)n nucleotide sequence, where N represents any nucleotide (A, G, U, or C) and n represents an integer from 1 to 20, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0486] [Item 107] The circular RNA precursor according to any one of items 103 to 106, wherein the fourth loop sequence comprises or consists of the sequence AAAA, AGAA, AA, UUUU, UAAA, CAAA, or GAAA, preferably AAAA.

[0487] [Item 108] A circular RNA precursor according to any one of items 103 to 107, wherein ribozyme recognition site I comprises a nucleotide sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with one nucleotide sequence from SEQ ID NO: 95 to 116.

[0488] [Item 109] A circular RNA precursor according to any one of items 103 to 108, wherein the third loop sequence comprises or consists of one or more nucleotides, the nucleotides being able to pair with the internal guide sequence (IGS) of the corresponding self-splicing intron to form a P1 double-stranded region during circularization.

[0489] [Item 110] The circular RNA precursor according to any one of items 103 to 109, wherein the third loop sequence comprises or consists of one nucleotide sequence from SEQ ID NO: 95 to 116, for example CUU, CAU, or CUC, and preferably CUU.

[0490] [Item 111] The loop of the stem-loop structure has the sequence CUUAAAA, CUUUUUU, CUUAA, CUUGAAA, CUUUAAA, CUUAGAA, CUUCAAA, or CUCAAAA, preferably CUUAAAA, as described in any one of Items 103 to 110.

[0491] [Item 112] The cyclic RNA precursor according to any one of items 103 to 111, wherein the stem portion of the stem-loop structure comprises 2 to 15 or more consecutive matching base pairs, preferably the stem portion of the stem-loop structure comprises 5, 6 or 7 consecutive matching base pairs.

[0492] [Item 113] The circular RNA precursor according to any one of items 103 to 112, wherein the stem portion of the stem-loop structure contains at most two base pair mismatches, or the stem portion contains only one base pair mismatch, and preferably the stem portion does not contain any base pair mismatches.

[0493] [Item 114] A circular RNA precursor as described in any one of items 103 to 113, wherein the third pairing sequence contains only G and the fourth pairing sequence contains only C, or the third pairing sequence contains only C and the fourth pairing sequence contains only G.

[0494] [Item 115] A circular RNA precursor as described in any one of items 103 to 114, wherein the third pairing sequence contains only A and the fourth pairing sequence contains only U, or the third pairing sequence contains only U and the fourth pairing sequence contains only A.

[0495] [Item 116] A circular RNA precursor as described in any one of items 77 to 115, wherein ribozyme recognition site II includes or consists of a 3' exon region, and ribozyme recognition site I includes or consists of a 5' exon region.

[0496] [Item 117] The circular RNA precursor according to Item 116, wherein the 3' exon region is derived from the native 3' exon of a self-splicing intron, the 5' exon region is derived from the native 5' exon of a self-splicing intron, and the 3' and 5' exon regions can be recognized and / or spliced ​​by a self-splicing intron or a combination of a 3' self-splicing intron fragment and a 5' self-splicing intron fragment.

[0497] [Item 118] The 3' exon region is a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with a fragment that is about 1 to about 50 nucleotides in length, starting from the natural 3' exon or the 5' terminal nucleotide of a natural 3' exon. The circular RNA precursor described in item 116 or 117 is a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with a fragment that is approximately 1 to about 50 nucleotides long and starts from the 3' terminal nucleotide of a natural 5' exon.

[0498] [Item 119] Ribozyme recognition site I comprises or consists of a nucleotide sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with one of SEQ ID NO: 58, 60, 62, 64, 66, 68, or 70, preferably ribozyme recognition site I comprises the nucleotide sequence GAGGUCUU, AGAGACUU, UCCAGCUU, or UCUGGCAU, as described in any one of items 77 to 118.

[0499] [Item 120] A circular RNA precursor according to any one of items 77 to 119, wherein the ribozyme recognition site I includes or comprises a nucleotide motif oriented from 5' to 3', the nucleotide motif being one of SEQ ID NO: 95 to 116, and in particular the nucleotide motif being one of CUU, CUC, or CAU.

[0500] [Item 121] The ribozyme recognition site II comprises or consists of a nucleotide sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with one of SEQ ID NO: 57, 59, 61, 63, 65, 67, or 69, preferably the ribozyme recognition site II comprises the nucleotide sequence AAACCUC, AAUCUCU, AACUGGA, or AACCAGC, as describe...

Claims

1. A circular RNA precursor, in the direction from 5' to 3', a) 3' self-splicing intron fragment, b) Including a 5' self-splicing intron fragment, Here, the combination of the 3' self-splicing intron fragment and the 5' self-splicing intron fragment maintains the self-splicing activity of the self-splicing intron, allows for the production of circular RNA by the self-splicing of the circular RNA precursor, and Here, the self-splicing intron is a circular RNA precursor selected from group I introns and group II introns.

2. The self-splicing intron is selected from the group of cyanobacteria anabaena group I introns such as AnaX, azoarcus group I introns (Azo), Scytalidium dimidiatum group I introns (Sd), staphylococcal phage Twort group I introns (Twort), Scytonema-hofmani tRNA fMet group I introns (Sh), or Agrobacterium tumefaciens group I introns (At). For example, the self-splicing intron has a nucleotide sequence selected from one of SEQ ID NO: 1 to 6, or SEQ ID The circular RNA precursor according to claim 1, having a nucleotide sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with one of NO: 1 to 6.

3. The cyclic RNA precursor according to claim 1 or 2, wherein the 3' self-splicing intron fragment is the 3' terminal portion of a self-splicing intron, starting from the internal split site I of the intron and extending to the 3' end of the self-splicing intron, and the 5' self-splicing intron fragment is the 5' terminal portion of a self-splicing intron, starting from the 5' end of the self-splicing intron and extending to the internal split site II of the intron.

4. The self-splicing intron is a cyanobacterial anabaena group I intron, for example, AnaX having the nucleotide sequence shown in SEQ ID NO: 1, and the intron internal split site II and the intron internal split site I correspond to the 115th nucleotide A and the 116th nucleotide A at the 5' end of SEQ ID NO: 1, respectively, according to any one of claims 1 to 3.

5. The self-splicing intron is an Azoarcus group I intron (Azo), which has the nucleotide sequence shown in SEQ ID NO: 2, and here, 1) Intron internal split region II and intron internal split region I correspond to the 24th nucleotide A and the 25th nucleotide A at the 5' end of SEQ ID NO: 2, respectively, or 2) Intron internal split region II and intron internal split region I correspond to nucleotide A at position 85 and nucleotide A at position 86 of the 5' end of SEQ ID NO: 2, respectively, or 3) Intron internal split region II and intron internal split region I correspond to nucleotide C at position 71 and position 72 of the 5' end of SEQ ID NO: 2, respectively, or 4) Intron internal split region II and intron internal split region I correspond to nucleotide C at position 108 and nucleotide U at position 109 of the 5' end of SEQ ID NO: 2, respectively, or 5) Intron internal split region II and intron internal split region I correspond to nucleotide A at position 154 and nucleotide A at position 155 of the 5' end of SEQ ID NO: 2, respectively, or 6) The circular RNA precursor according to any one of claims 1 to 3, wherein the intron internal split region II and the intron internal split region I correspond to the 198th nucleotide C and the 199th nucleotide A at the 5' end of SEQ ID NO: 2, respectively.

6. The self-splicing intron is a Staphylococcus phage Twort group I intron (Twort), which has the nucleotide sequence shown in SEQ ID NO: 3, and 1) Intron internal split region II and intron internal split region I correspond to nucleotide U at the 5' end of SEQ ID NO: 3 and nucleotide A at the 71st position, respectively, or 2) Intron internal split region II and intron internal split region I correspond to nucleotide U at the 5' end and nucleotide A at the 104th position of SEQ ID NO: 3, respectively, or 3) Intron internal split region II and intron internal split region I correspond to nucleotide A at the 5' end of SEQ ID NO: 3 and nucleotide U at the 132nd and 133rd positions, respectively, or 4) Intron internal split region II and intron internal split region I correspond to nucleotide 173 U and nucleotide 174 C at the 5' end of SEQ ID NO: 3, respectively, or 5) Intron internal split region II and intron internal split region I correspond to nucleotide A at position 206 and nucleotide A at position 207 of the 5' end of SEQ ID NO: 3, respectively, or 6) Intron internal split region II and intron internal split region I correspond to nucleotide 226 G and nucleotide 227 U at the 5' end of SEQ ID NO: 3, respectively, or 7) Intron internal split region II and intron internal split region I correspond to the 23rd nucleotide U and the 24th nucleotide A at the 5' end of SEQ ID NO: 3, respectively, or 8) The circular RNA precursor according to any one of claims 1 to 3, wherein the intron internal split region II and the intron internal split region I correspond to the 82nd nucleotide A and the 83rd nucleotide A at the 5' end of SEQ ID NO: 3, respectively.

7. The self-splicing intron is a Scytalidium diffidiatum group I intron (Sd), which has the nucleotide sequence shown in SEQ ID NO: 4, and here, 1) Intron internal split region II and intron internal split region I correspond to nucleotide A at the 88th position and nucleotide G at the 5' end of SEQ ID NO: 4, respectively, or 2) Intron internal split region II and intron internal split region I correspond to nucleotide U at the 5' end of SEQ ID NO: 4 and nucleotide G at the 174th nucleotide, respectively, or 3) Intron internal split region II and intron internal split region I correspond to nucleotide A at position 204 and nucleotide G at position 205 of the 5' end of SEQ ID NO: 4, respectively, or 4) Intron internal split region II and intron internal split region I correspond to nucleotide 252 C and nucleotide 253 G at the 5' end of SEQ ID NO: 4, respectively, or 5) The circular RNA precursor according to any one of claims 1 to 3, wherein the intron internal split region II and the intron internal split region I correspond to the 370th nucleotide C and the 371st nucleotide G at the 5' end of SEQ ID NO: 4, respectively.

8. The self-splicing intron is a Scytonema-hofmani tRNA fMet group I intron (Sh), which has the nucleotide sequence shown in SEQ ID NO: 5, and 1) Intron internal split region II and intron internal split region I correspond to nucleotide A at position 129 and nucleotide A at position 130 of the 5' end of SEQ ID NO: 5, respectively, or 2) Intron internal split region II and intron internal split region I correspond to nucleotide A at position 60 and nucleotide A at position 61 of the 5' end of SEQ ID NO: 5, respectively, or 3) Intron internal split region II and intron internal split region I correspond to nucleotide A at position 109 and nucleotide A at position 110 of the 5' end of SEQ ID NO: 5, respectively, or 4) The circular RNA precursor according to any one of claims 1 to 3, wherein the intron internal split region II and the intron internal split region I correspond to the 213th nucleotide A and the 214th nucleotide A at the 5' end of SEQ ID NO: 5, respectively.

9. The self-splicing intron is an Agrobacterium tumefaciens group I intron (At), which has the nucleotide sequence shown in SEQ ID NO: 6, and the internal split site II and the internal split site I of the intron correspond to the 107th nucleotide A and the 108th nucleotide A at the 5' end of SEQ ID NO: 6, respectively, according to any one of claims 1 to 4.

10. The self-splicing intron is a cyanobacterial anabaena group I intron such as AnaX, and the 3' self-splicing intron fragment contains or consists of a nucleotide sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 7, and the 5' self-splicing intron fragment contains a nucleotide sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 8. The circular RNA precursor according to claim 1, comprising or consisting of a nucleotide sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with NO:

8.

11. The self-splicing intron is an Azoarcus group I intron (Azo), and the 3' self-splicing intron fragment contains or consists of a nucleotide sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 9, 11, 13, 15, 17, or 19, and the 5' self-splicing intron fragment is SEQ ID A circular RNA precursor according to claim 1, comprising or consisting of a nucleotide sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with nucleotide sequence NO: 10, 12, 14, 16, 18, or 20.

12. The self-splicing intron is a Scytalidium dimidiatum group I intron (Sd), and the 3' self-splicing intron fragment contains or consists of a nucleotide sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 37, 39, 41, 43, or 45, and the 5' self-splicing intron fragment is SEQ ID A circular RNA precursor according to claim 1, comprising or consisting of a nucleotide sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the nucleotide sequence of NO: 38, 40, 42, 44, or 46.

13. The self-splicing intron is a Staphylococcus phage Twort group I intron (Twort), and the 3' self-splicing intron fragment is a nucleotide sequence or SEQ ID NO: 21, 23, 25, 27, 29, 31, 33 or 35. NO: 21, 23, 25, 27, 29, 31, 33 or 35 contains or comprises a nucleotide sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity, and the 5' self-splicing intron fragment contains or comprises SEQ ID NO: 22, 24, 26, 28, 30, 32, 34 or 36 or SEQ ID A circular RNA precursor according to claim 1, comprising or consisting of a nucleotide sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with NO: 22, 24, 26, 28, 30, 32, 34, or 36.

14. The self-splicing intron is a Scytonema-hofmani tRNA fMet group I intron (Sh), and the 3' self-splicing intron fragment contains or consists of a nucleotide sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 47, 49, 51, or 53, and the 5' self-splicing intron fragment is SEQ ID A circular RNA precursor according to claim 1, comprising or consisting of a nucleotide sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the nucleotide sequence of NO: 48, 50, 52, or 54.

15. The self-splicing intron is an Agrobacterium tumefaciens group I intron (At), and the 3' self-splicing intron fragment contains or consists of a nucleotide sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 55, and the 5' self-splicing intron fragment contains a nucleotide sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 8. The circular RNA precursor according to claim 1, comprising or consisting of a nucleotide sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with NO:

56.

16. Further comprising an optional first residue cyclization element in the direction from 5' to 3', the target nucleotide sequence, and an optional second residue cyclization element located between a) and b), wherein the total length of the first and second residue cyclization elements is approximately 0 to approximately 500 nucleotides, and / or The circular RNA precursor according to any one of claims 1 to 15, further comprising homology arm sequence I upstream of a 3' self-splicing intron fragment and homology arm sequence II downstream of a 5' self-splicing intron fragment.

17. From 5' to 3', i) 3' self-splicing intron fragment, ii) The first residue cyclization element, iii) The target nucleotide sequence, iv) The second residue cyclization element, v) The circular RNA precursor according to claim 16, comprising a 5' self-splicing intron fragment.

18. The cyclic RNA precursor according to claim 16 or 17, wherein the first residue cyclization element comprises or consists of a 3' exon region oriented from 5' to 3' and an optional first spacer, and the second residue cyclization element comprises or consists of a 5' exon region oriented from 3' to 5' and an optional second spacer.

19. The cyclic RNA precursor according to claim 18, wherein the 3' exon region is derived from the native 3' exon of a self-splicing intron, the 5' exon region is derived from the native 5' exon of a self-splicing intron, and the 3' and 5' exon regions can be recognized and / or spliced ​​by a self-splicing intron or a combination of a 3' self-splicing intron fragment and a 5' self-splicing intron fragment.

20. The 3' exon region is a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with a fragment that is approximately 1 to 50 nucleotides in length, starting from the natural 3' exon or the 5' terminal nucleotide of a natural 3' exon. The circular RNA precursor according to claim 19, wherein the 5' exon region is a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with a natural 5' exon or a fragment having a length of about 1 to about 50 nucleotides starting from the 3' terminal nucleotide of a natural 5' exon.

21. The circular RNA precursor according to any one of claims 16 to 20, wherein the target nucleotide sequence comprises at least one protein coding sequence and a translation initiation element, such as an internal ribosome entry site (IRES), operably ligated thereto.

22. The aforementioned translation initiation element is an internal ribosome entry site (IRES), where the IRES is Taura syndrome virus, assassin bug virus, Tyler's encephalomyelitis virus, Simian virus 40, fire ant virus 1, wheat aphid virus, reticuloendotheliosis virus, Forman's poliovirus 1, soybean looper virus, Casimir wasp virus, human rhinovirus 2, leafhopper virus-1, human immunodeficiency virus type 1, leafhopper virus-1, pygmy flea P virus, hepatitis C virus, hepatitis A virus, hepatitis GB virus, foot-and-mouth disease virus, human enterovirus 71, horse rhinovirus, and white-winged dace virus. Jak-like virus, encephalomyocarditis virus (EMCV), Drosophila C virus, Brassicaceae tobamovirus, cricket paralysis virus, bovine viral diarrhea virus 1, black queen bee brood virus, aphid lethal paralysis virus, avian encephalomyelitis virus, acute bee paralysis virus, hibiscus chlorotic ring spot virus, swine fever virus, human FGF2, human SFTPA1, human AML1 / RUNXL, Drosophila Antennapedia, human AQP4, human AT1R, human BAG-1, human BCL2, human BiP, human c-IAP1, human c-myc, human eIF4G, mouse NDST4L, human LEF1, mouse HIF1α, human n. myc, mouse Gtx, human p27kipl, human PDGF2 / c-sis, human p53, human Pim-1, mouse Rbm3, fruit fly reaper, canine Scamper, fruit fly Ubx, human UNR, mouse UtrA, human VEGF-A, human XIAP, fruit fly hairless, budding yeast TFIID, budding yeast YAP1, human c-src, human FGF -1, selected from an IRES sequence of salpicomavirus, cab crinkle virus, eIF4G, coxsackievirus B3 (CVB3), or coxsackievirus A (CVA1 / 2), preferably the IRES is CVB3, BRAV-1_L, PV1_L, CAV2_L, BRAV-1, PV1, or CAV2, according to claim 21.

23. The circular RNA precursor according to claim 22, wherein IRES comprises a nucleotide sequence shown in one of SEQ ID NO: 120 to 526, or comprises a nucleotide sequence having at least 75%, for example, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% sequence identity with one of SEQ ID NO: 120 to 526.

24. The aforementioned at least one protein-coding sequence codes for a protein of eukaryotic, prokaryotic, or viral origin, for example, a protein for therapeutic or diagnostic purposes. For example, the cyclic RNA precursor according to any one of claims 21 to 23, wherein the at least one protein coding sequence codes for IL-2 or an aquaporin, such as AQP-1 protein, or a protein having the same activity as AQP-1 protein and derived from AQP-1 protein by substituting, deleting, or adding 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids in the amino acid sequence of AQP-1 protein.

25. The circular RNA precursor according to any one of claims 21 to 24, wherein the at least one protein coding sequence comprises one nucleotide sequence from SEQ ID NO: 117 to 118, or comprises a nucleotide sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with one of SEQ ID NO: 117 to 118.

26. The target nucleotide sequence is a non-protein-coding sequence, for example, the non-protein-coding sequence may be an antisense RNA, an aptamer, a guide RNA, or any non-protein-coding RNA present in a living organism, according to any one of claims 16 to 23.

27. In the direction from 5' to 3', the nucleotide sequence of the target and the 5' self-splicing intron fragment are included. Here, the combination of the 3' self-splicing intron fragment and the 5' self-splicing intron fragment preserves the self-splicing activity of the self-splicing intron and allows for the production of a circular RNA formed from the target nucleotide sequence by self-splicing of the circular RNA precursor. Here, the target nucleotide sequence includes a cyclic sequence fragment II containing a ribozyme recognition site II at its 5' end and a cyclic sequence fragment I containing a ribozyme recognition site I at its 3' end, in the direction from 5' to 3'. Here, the circularized sequence fragment I is the 5' end portion of the circularized sequence, and the circularized sequence fragment II is the 3' end portion of the circularized sequence, and Herein, the ribozyme recognition site I and the ribozyme recognition site II in the generated circular RNA are linked, thereby linking the circularized sequence fragment I and the circularized sequence fragment II to form the circularized sequence, as described in any one of claims 1 to 15.

28. The circular RNA precursor according to claim 27, wherein the target nucleotide sequence comprises one or more sequences selected from the group consisting of at least one protein-coding sequence, a non-coding sequence, a translation initiation element (TIE), a non-TIE functional element, or any combination thereof.

29. The circular RNA precursor according to claim 27 or 28, wherein the circularized sequence is selected from at least one protein-coding sequence, a non-coding sequence, a translation initiation element (TIE), or a non-TIE functional element.

30. In the direction from 5' to 3', the nucleotide sequence of the target and the 5' self-splicing intron fragment are included. Here, the combination of the 3' self-splicing intron fragment and the 5' self-splicing intron fragment maintains the self-splicing activity of the self-splicing intron and allows for the production of the following circular RNA, which is formed by self-splicing of a circular RNA precursor from a target nucleotide sequence and contains at least one protein-coding sequence. Here, the target nucleotide sequence includes, in the direction from 5' to 3', a protein-coding sequence fragment II, a translation initiation element such as an internal ribosome entry site (IRES), and a protein-coding sequence fragment I. Here, protein-coding sequence fragment I is the 5' end portion of at least one protein-coding sequence, and protein-coding sequence fragment II is the 3' end portion of at least one protein-coding sequence, and The circular RNA precursor according to any one of claims 1 to 15, wherein the protein coding sequence fragment I includes a ribozyme recognition site I located at its 3' end, and the protein coding sequence fragment II includes a ribozyme recognition site II located at its 5' end, and in the resulting circular RNA, the ribozyme recognition site I and the ribozyme recognition site II are linked, thereby linking the protein coding sequence fragment I and the protein coding sequence fragment II to form the at least one protein coding sequence.

31. The generated circular RNA comprises at least two protein-coding sequences, and may have another translation initiation element, 2A sequence, or spacer ligated between any two adjacent protein-coding sequences, and the translation initiation element may be an IRES sequence, a 5'UTR sequence, a Kozak sequence, or m ６ The circular RNA precursor according to claim 30, comprising an A-containing sequence, a sequence complementary to ribosomal 18S rRNA, or any combination thereof.

32. The cyclical RNA precursor according to any one of claims 29 to 31, wherein the ribozyme recognition site I and ribozyme recognition site II are configured such that the cyclical RNA precursor containing them has high cyclization efficiency.

33. The total length of the ribozyme recognition site I and ribozyme recognition site II is approximately 0 to 100 nucleotides, approximately 0 to 90 nucleotides, approximately 0 to 80 nucleotides, approximately 0 to 70 nucleotides, approximately 0 to 60 nucleotides, approximately 0 to 50 nucleotides, approximately 0 to 40 nucleotides, approximately 0 to 35 nucleotides, approximately 2 to 100 nucleotides, approximately 5 to 100 nucleotides, approximately 10 to 100 nucleotides, approximately 20 to 100 nucleotides, approximately 30 to 100 nucleotides, or approximately 30 to 100 nucleotides, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 ,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65 A circular RNA precursor according to any one of claims 29 to 32, comprising 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

34. The total length of the ribozyme recognition site I is approximately 0 to 100 nucleotides, approximately 0 to 90 nucleotides, approximately 0 to 80 nucleotides, approximately 0 to 70 nucleotides, approximately 0 to 60 nucleotides, approximately 0 to 50 nucleotides, approximately 0 to 40 nucleotides, approximately 0 to 35 nucleotides, approximately 2 to 100 nucleotides, approximately 5 to 100 nucleotides, approximately 10 to 100 nucleotides, approximately 20 to 100 nucleotides, approximately 30 to 100 nucleotides, or approximately 30 to 100 nucleotides, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 ,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66 A circular RNA precursor according to any one of claims 29 to 33, comprising 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

35. The cyclic RNA precursor according to any one of claims 29 to 34, wherein the ribozyme recognition site I and ribozyme recognition site II are configured to form a stem-loop structure when, for example, self-splicing is performed to create a circular structure.

36. The circular RNA precursor according to claim 35, wherein the loop of the stem-loop structure includes a splicing binding site.

37. The ribozyme recognition site I includes a sequence structure of the form 5'-third unpaired sequence-third paired sequence-third loop sequence-3', and the ribozyme recognition site II includes a sequence structure of the form 5'-fourth loop sequence-fourth paired sequence-fourth unpaired sequence-3'. Here, the third unpaired sequence or the fourth unpaired sequence may or may not exist independently. The third pairing sequence and the fourth pairing sequence can pair complementaryly with each other to form a stem of a stem-loop structure, wherein the third loop sequence and the fourth loop sequence can form a loop of the stem-loop structure, for example, by self-splicing to achieve circularization, according to any one of claims 29 to 36.

38. The cyclic RNA precursor according to any one of claims 29 to 37, wherein the ribozyme recognition site II may be selected from a variety of sequences.

39. The cyclic RNA precursor according to claim 37 or 38, wherein the fourth loop sequence comprises or consists of one or more nucleotides, and the one or more nucleotides can pair with the P1 region of the corresponding self-splicing intron to form a P10 double-stranded region during cyclization.

40. The circular RNA precursor according to any one of claims 37 to 39, wherein the fourth loop sequence consists of a (N)n nucleotide sequence, where N represents any nucleotide (A, G, U, or C) and n represents an integer from 1 to 20, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

41. The circular RNA precursor according to any one of claims 37 to 40, wherein the fourth loop sequence includes or consists of the sequence AAAA, AGAA, AA, UUUU, UAAA, CAAA, or GAAA, and is preferably AAAA.

42. The circular RNA precursor according to any one of claims 37 to 41, wherein the ribozyme recognition site I comprises a nucleotide sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with one nucleotide sequence from among SEQ ID NO: 95 to 116.

43. The circular RNA precursor according to any one of claims 37 to 42, wherein the third loop sequence comprises or consists of one or more nucleotides, the nucleotides being able to pair with the internal guide sequence (IGS) of the corresponding self-splicing intron to form a P1 double-stranded region during circularization.

44. The circular RNA precursor according to any one of claims 37 to 43, wherein the third loop sequence includes or consists of one nucleotide sequence from SEQ ID NO: 95 to 116, for example CUU, CAU, or CUC, and preferably CUU.

45. The loop of the stem-loop structure has the sequence CUUAAAAAA, CUUUUUU, CUUAA, CUUGAAA, CUUUAAAAAA, CUUAGAA, CUUCAAA, or CUCAAAA, preferably CUUAAAAAA, according to any one of claims 37 to 44.

46. The aforementioned translation initiation element is an internal ribosome entry site (IRES), where the IRES is Taura syndrome virus, assassin bug virus, Tyler's encephalomyelitis virus, Simian virus 40, fire ant virus 1, wheat aphid virus, reticuloendotheliosis virus, Forman's poliovirus 1, soybean looper virus, Casimir wasp virus, human rhinovirus 2, leafhopper virus-1, human immunodeficiency virus type 1, leafhopper virus-1, pygmy flea P virus, hepatitis C virus, hepatitis A virus, hepatitis GB virus, foot-and-mouth disease virus, human enterovirus 71, horse rhinovirus, and white-winged dace virus. Jak-like virus, encephalomyocarditis virus (EMCV), Drosophila C virus, Brassicaceae tobamovirus, cricket paralysis virus, bovine viral diarrhea virus 1, black queen bee brood virus, aphid lethal paralysis virus, avian encephalomyelitis virus, acute bee paralysis virus, hibiscus chlorotic ring spot virus, swine fever virus, human FGF2, human SFTPA1, human AML1 / RUNXL, Drosophila Antennapedia, human AQP4, human AT1R, human BAG-1, human BCL2, human BiP, human c-IAP1, human c-myc, human eIF4G, mouse NDST4L, human LEF1, mouse HIF1α, human n. myc, mouse Gtx, human p27kipl, human PDGF2 / c-sis, human p53, human Pim-1, mouse Rbm3, fruit fly reaper, dog Scamper, fruit fly Ubx, human UNR, mouse UtrA, human VEGF-A, human XIAP, fruit fly hairless, budding yeast TFIID, budding yeast YAP1, human c-src, human FGF-1, monkey A cyclic RNA precursor according to any one of claims 29 to 45, selected from an IRES sequence of picomavirus, kabukurinkle virus, an aptamer for eIF4G, coxsackievirus B3 (CVB3), or coxsackievirus A (CVA1 / 2), preferably the IRES being CVB3, BRAV-1_L, PV1_L, CAV2_L, BRAV-1, PV1, or CAV2.

47. The circular RNA precursor according to any one of claims 29 to 46, wherein IRES comprises a nucleotide sequence shown in one of SEQ ID NO: 120 to 526, or comprises a nucleotide sequence having at least 75%, for example, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% sequence identity with one of SEQ ID NO: 120 to 526.

48. The circular RNA precursor according to any one of claims 29 to 47, wherein the at least one protein-coding sequence encodes a protein of eukaryote, prokaryote or viral origin, for example, a protein for therapeutic or diagnostic purposes, specifically a mammalian or human protein.

49. A cyclic RNA precursor according to any one of claims 29 to 48, wherein at least one protein-coding sequence encodes IL-2 or an aquaporin.

50. The cyclic RNA precursor according to claim 49, wherein the aquaporin is the AQP-1 protein, or a protein having the same activity as the AQP-1 protein and obtained by substituting, deleting, or adding 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids to the amino acid sequence of the AQP-1 protein.

51. The circular RNA precursor according to any one of claims 29 to 50, wherein the at least one protein coding sequence comprises one nucleotide sequence from SEQ ID NO: 117 to 118, or comprises a nucleotide sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with one of SEQ ID NO: 117 to 118.

52. A circular RNA precursor according to any one of claims 29 to 51, comprising a nucleotide sequence selected from one of SEQ ID NO: 530, 531, and 533 to 534, or comprising a nucleotide sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with one of SEQ ID NO: 530, 531, and 533 to 534.

53. A nucleic acid vector for producing a circular RNA molecule, wherein the vector comprises a coding sequence for a circular RNA precursor as described in any one of claims 1 to 52.

54. The nucleic acid vector according to claim 53, further comprising an RNA polymerase promoter sequence operably linked to the coding sequence of the cyclic RNA precursor.

55. The nucleic acid vector according to claim 53 or 54, wherein the promoter is a T7 RNA polymerase promoter, a T6 viral RNA polymerase promoter, an SP6 viral RNA polymerase promoter, a T3 viral RNA polymerase promoter, or a T4 viral RNA polymerase promoter, and preferably a T7 RNA polymerase promoter.

56. A circular RNA, which is produced by a circular RNA precursor according to any one of claims 1 to 52, and preferably the circular RNA has an in vivo expression duration of at least 24 hours, for example, 36 hours, 48 ​​hours, 72 hours, 96 hours, 5 days, 10 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days or longer.

57. The nucleic acid vector according to claim 56, comprising or having one nucleotide sequence from among SEQ ID NO: 543 to 558, or a nucleotide sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with one of SEQ ID NO: 543 to 558.

58. Use of the circular RNA precursor described in any one of claims 1 to 52 and / or the circular RNA described in claim 56 or 57 as an expression vector.

59. A pharmaceutical composition comprising a cyclic RNA precursor according to any one of claims 1 to 52 and / or a nucleic acid vector according to any one of claims 53 to 55 and / or a cyclic RNA according to claim 56 or 57, and a pharmaceutically acceptable carrier.

60. A product expressed by the circular RNA described in claim 56 or 57.

61. The product according to claim 60, comprising IL-2, POLR2A, or aquaporin, for example, an AQP-1 protein having the sequence shown in SEQ ID NO: 118, or a protein having the same activity as the AQP-1 protein and derived from the AQP-1 protein by substituting, deleting, or adding 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids in the amino acid sequence of the AQP-1 protein.

62. A method for preventing or treating a disease or disorder in a subject, comprising administering to the subject the circular RNA described in claim 56 or 57 or the pharmaceutical composition described in claim 59.

63. The method according to claim 62, wherein the disease or disorder includes, but is not limited to, xerostomia, dry eye, and Sjögren's syndrome.

64. Uses of the circular RNA described in claim 56 or 57 or the pharmaceutical composition described in claim 59 in the manufacture of a pharmaceutical for preventing or treating a disease or disorder of a subject.

65. The use according to claim 64, wherein the disease or disorder is xerostomia, dry eye, or Sjögren's syndrome.

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