Construct based on group i intron of cob gene of fusarium oxysporum and construction method therefor, RNA circularization method, circular RNA, and use

Abstract

A construct based on a group I intron of a CoB gene of Fusarium oxysporum and a construction method therefor, an RNA circularization method, a circular RNA, and a use. In the construct used for in vitro RNA circularization, a group I intron derived from a CoB gene of Fusarium oxysporum is used as a self-splicing ribozyme for mediating in vitro RNA circularization and at least a P1 domain of the group I intron is retained, so that in vitro RNA circularization can be achieved by introducing a small amount of exon fragments or without introducing exon fragments, and the residual exogenous sequence in the resulting circular RNA is only 5 nt, thereby effectively solving the problem of immune responses caused by large residual exogenous sequences in existing circular RNAs synthesized in vitro.

Description

Constructs of type I introns based on the *Fusarium oxysporum* CoB gene, their construction methods, RNA circularization methods, circular RNA, and their applications. Technical Field

[0001] This invention belongs to the field of biotechnology, and particularly relates to a construct of a type I intron based on the CoB gene of Fusarium oxysporum, its construction method, RNA circularization method, circular RNA, and its applications. Background Technology

[0002] Circular RNA (circRNA) is a class of non-coding RNA molecules found in eukaryotic cells that lack a 5'-terminal cap and a 3'-terminal poly(A) tail, forming a circular structure through covalent bonds. It can influence gene expression by affecting transcription initiation, elongation, and splicing, and participate in controlling the function of other molecules. It exists in eukaryotic organisms.

[0003] Currently, the most commonly used method for in vitro RNA circularization is the ribozyme method. This method primarily involves constructing a PIE (Permutated Intron-Exon) construct with the target sequence inserted into different types of introns. This PIE construct utilizes an intron ribozyme autocatalytic cleavage reaction—the intron fragment falls off during self-cleavage, and the exons on both sides align, thus achieving in vitro circularization of the target sequence. However, the ribozyme method leaves a relatively long exogenous sequence residue after circularization. This residue may not only induce significant immunogenicity in vivo but also potentially affect RNA translation, leading to reduced expression of the target protein, thus presenting significant limitations. Summary of the Invention

[0004] The first objective of this invention is to provide a construct for in vitro RNA circularization. When this construct is applied to in vitro RNA circularization, it can maintain RNA circularization efficiency with only a small amount of exogenous sequence (5nt), enrich existing RNA circularization methods, and overcome the defects of long exogenous sequence residues and strong immunogenicity in existing RNA circularization methods.

[0005] A second objective of this invention is to provide a method for constructing a construct for in vitro circularization of RNA.

[0006] A third objective of this invention is to provide a construct obtained by the construction method for in vitro circularization of RNA described above.

[0007] A fourth objective of this invention is to provide a method for circularizing RNA.

[0008] The fifth objective of this invention is to provide a circular RNA.

[0009] A sixth objective of this invention is to provide the use of the above-described constructs and / or circular RNAs in the preparation of drugs, vaccines, and / or cells that highly express target genes.

[0010] Specifically, the construct for in vitro RNA circularization provided by this invention comprises, along the 5'-3' direction, a 3'-intron, exon 2, the gene to be circularized, exon 1, and a 5'-intron; wherein the 3'-intron and 5'-intron are derived from type I introns of the *Fusarium oxysporum* *cob* gene with nucleotide sequences as shown in SEQ ID NO:1, and the 5'-intron includes a P1 domain, the P1 domain including a nucleotide fragment with a sequence as shown in SEQ ID NO:38; exon 1 is the exon fragment immediately adjacent to the 5' end of the type I intron, and the length of exon 1 is 0–393 nt; exon 2 is the exon fragment immediately adjacent to the 3' end of the type I intron, and the length of exon 2 is 0–780 nt.

[0011] Furthermore, the nucleotide sequence of the type I intron is shown in SEQ ID NO:2.

[0012] Furthermore, the 5'-intron and 3'-intron are obtained by breaking type I introns, with the break site on the type I intron located at positions 18-1020 of the sequence shown in SEQ ID NO:2.

[0013] Furthermore, the nucleotide sequence of the 3'-intron is shown in SEQ ID NO:29, the nucleotide sequence of the 5'-intron is shown in SEQ ID NO:6, and the lengths of exon 1 and exon 2 are 0 nt.

[0014] Furthermore, the construct includes a T7 promoter located at the 5' end of the 3'-intron.

[0015] Furthermore, the nucleotide sequence of the T7 promoter is shown in SEQ ID NO:11.

[0016] Furthermore, the construct includes a CVB3 IRES sequence located between the gene to be circularized and exon 1.

[0017] Furthermore, the nucleotide sequence of the CVB3 IRES sequence is shown in SEQ ID NO:13.

[0018] The method for constructing a construct for in vitro RNA circularization provided by the present invention includes: performing structural analysis on the type I introns of the Fusarium oxysporum cob gene to obtain break sites related to the enzyme activity of the type I introns, and constructing a construct framework; introducing the gene to be circularized based on the construct framework to construct the construct.

[0019] Furthermore, the nucleotide sequence of the type I intron is shown in SEQ ID NO:2.

[0020] Furthermore, the break site is positions 18-1020 of the sequence shown in SEQ ID NO:2.

[0021] This invention provides a construct obtained by constructing a construct for in vitro circularization of RNA as described above.

[0022] Furthermore, the construct comprises, along the 5'-3' direction, the following components in sequence: 3'-intron, exon 2, gene to be circularized, exon 1, and 5'-intron.

[0023] Furthermore, the nucleotide sequence of the 3'-intron is shown in SEQ ID NO:29, the nucleotide sequence of the 5'-intron is shown in SEQ ID NO:6, and the lengths of exon 1 and exon 2 are 0 nt.

[0024] The RNA circularization method provided by the present invention includes: transcribing the construct according to any one of claims 1 to 5 and 11 to obtain a linear RNA molecule; and performing a circularization reaction on the linear RNA molecule to obtain the circular RNA.

[0025] The circular RNA provided by this invention is prepared using the RNA circularization method described above. This invention also provides the application of the above-described constructs and / or circular RNA in the preparation of drugs, vaccines, and / or cells that highly express target genes. Beneficial effects:

[0026] The construct for in vitro RNA circularization provided by this invention uses a type I intron from the cob gene of Fusarium oxysporum as a cleavage ribozyme to mediate in vitro RNA circularization. It can effectively catalyze in vitro RNA circularization with or without the introduction of a small number of exon fragments, and the resulting circular RNA contains only 5 nt of foreign sequence residue. This invention can effectively solve the problem of immune response caused by existing in vitro synthesized circular RNA and has good application prospects. Attached Figure Description

[0027] Figure 1 is a schematic diagram of the cob gene structure provided in Example 1 of the present invention;

[0028] Figure 2 is a schematic diagram of the secondary structure of the type I intron provided in Embodiment 1 of the present invention;

[0029] Figure 3 is a schematic diagram of the structure of the construct and its circular RNA provided in Example 1 of the present invention;

[0030] Figure 4 shows the experimental results of urea-PAGE gel electrophoresis provided in Example 1 of this invention;

[0031] Figure 5 shows a partial Sanger sequencing result of circular RNA2 provided in Example 1 of this invention;

[0032] Figure 6 is a schematic diagram of nucleotide deletion of start exon 2 provided in Example 2 of the present invention;

[0033] Figure 7 is a schematic diagram of nucleotide deletion of start exon 1 provided in Example 2 of the present invention;

[0034] Figure 8 shows the experimental results of urea-PAGE gel electrophoresis (in vitro transcription) provided in Example 2 of this invention;

[0035] Figure 9 shows the experimental results of urea-PAGE gel electrophoresis (in vitro circularization) provided in Example 2 of this invention;

[0036] Figure 10 shows the base sequence and secondary structure of the type I intron P1 domain provided in this invention;

[0037] Figure 11 shows the base sequences and secondary structures of the type I intron P9 and P10 domains provided in this invention;

[0038] Figure 12 is a schematic diagram of the modification of the F2-3'-intron provided in Embodiment 3 of the present invention;

[0039] Figure 13 is a schematic diagram of the modification of the F2-5'-intron provided in Embodiment 3 of the present invention;

[0040] Figure 14 shows the experimental results of urea-PAGE gel electrophoresis provided in Example 3 of this invention (constructions 11-13);

[0041] Sequencing results showing the circumduction induced by altered P1 and P10 structures in ribozymes;

[0042] Figure 15 is a partial Sanger sequencing result of circular RNA11 provided in Example 3 of the present invention;

[0043] Figure 16 is a schematic diagram of the modification of the F2-5'-intron provided in Embodiment 3 of the present invention (mutation of the P1 domain);

[0044] Figure 17 shows the experimental results of urea-PAGE gel electrophoresis provided in Example 3 of this invention (constructions 14-16);

[0045] Figure 18 shows the experimental results of urea-PAGE gel electrophoresis provided in Example 4 of this invention. Detailed Implementation

[0046] The nucleotide sequences involved in this invention are shown in Table 1:

[0047] Table 1.

[0048] Based on in-depth and extensive research on in vitro RNA circularization reactions, the inventors of this invention discovered that type I introns of the *Fusarium oxysporum* *cob* gene possess excellent potential for catalyzing in vitro RNA circularization. Further research was conducted, and the type I introns were modified, and the gene to be circularized was introduced to construct the construct claimed in this invention. When this construct is applied to the in vitro circularization of RNA, in vitro RNA circularization can be achieved, and the resulting circular RNA retains only 5 nt of the exogenous sequence, exhibiting low immunogenicity.

[0049] In this invention, the Gene ID number of the Fusarium oxysporum cob gene is LT906358, and the specific nucleotide sequence is shown in SEQ ID NO:1; wherein, the nucleotide sequence of the type I intron contained in the cob gene is shown in SEQ ID NO:2.

[0050] In this invention, the construct comprises, sequentially along the 5'-3' direction, a 3'-intron, exon 2, the gene to be circularized, exon 1, and a 5'-intron. More specifically, the 3'-intron and 5'-intron are formed by breaking type I introns, and the break sites on the type I introns that can break to form 3'-introns and 5'-introns with self-cleaving catalytic activity are located at positions 18-1020 of the sequence shown in SEQ ID NO:2, such as positions 18, 25, 108, 114, 737, 1000, 1020, or any sites therein, and the 5'-intron includes a P1 domain, which includes a nucleotide fragment with the sequence shown in SEQ ID NO:38.

[0051] In some specific embodiments, the break site is preferably located at position 144 of the sequence shown in SEQ ID NO:2, that is, the nucleotide sequence of the 3'-intron in the construct is shown in SEQ ID NO:5, and the nucleotide sequence of the 5'-intron is shown in SEQ ID NO:6. In this case, the obtained 3'-intron and 5'-intron are used in the construct for in vitro RNA circularization, exhibiting relatively good in vitro circularization efficiency and enabling in vitro RNA circularization.

[0052] In this invention, exon 1 is the exon segment immediately adjacent to the 5' end of a type I intron, with a length specifically ranging from 0 to 393 nt, such as 0 nt, 5 nt, 50 nt, 218 nt, 253 nt, 350 nt, 393 nt, or any integer value between them; exon 2 is the exon segment immediately adjacent to the 3' end of a type I intron, with a length specifically ranging from 0 to 780 nt, such as 0 nt, 58 nt, 93 nt, 218 nt, 324 nt, 630 nt, 736 nt, 748 nt, 780 nt, or any value between them. The introduction of exon 1 and exon 2 into the construct is to maintain or enhance the self-cleavage catalytic activity of the type I intron.

[0053] In some specific embodiments, the lengths of exon 1 and exon 2 in the construct are preferably 0 nt, meaning that no additional exon fragments adjacent to the type I introns are introduced into the construct. In this case, the type I introns on the *Fusarium oxysporum* *cob* gene also possess self-cleaving catalytic activity, maintaining a certain level of self-cleaving catalytic activity even without introducing exon fragments, and effectively solving the problem of long exogenous sequences remaining in existing RNA in vitro circularization processes.

[0054] In this invention, the 3'-intron preferably does not include the P10 domain, as shown in SEQ ID NO:29. While the deletion of the P10 domain in the 3'-intron reduces the self-cleavage catalytic activity of the ribozyme, more importantly, it achieves the excellent effect of reducing residual foreign sequences in the circular RNA.

[0055] In some specific embodiments, the preferred nucleotide sequences of the 3'-introns in the construct are shown in SEQ ID NO:29, and the nucleotide sequences of the 5'-introns are shown in SEQ ID NO:6. In this case, the construct enables in vitro circularization of RNA, and the resulting circular RNA contains only 5 nt of the exogenous sequence, exhibiting low immunogenicity.

[0056] In this invention, the construct preferably further includes a promoter located at the 5' end of the 3'-intron. This promoter serves as an important initiator for in vitro transcription, enabling the in vitro transcription of the construct. In some specific embodiments, examples of the promoter include, but are not limited to, one or more of the T7 promoter, T3 promoter, and SP6 promoter. In some preferred embodiments, the construct includes a T7 promoter located at the 5' end of the 3'-intron. This T7 promoter is derived from T7 bacteriophage and is used to initiate transcription of the construct. The specific nucleotide sequence is shown in SEQ ID NO:11.

[0057] In this invention, the construct preferably further includes an IRES sequence and / or an IRES-like sequence located between the gene to be circularized and exon 1. The IRES sequence and / or IRES-like sequence serves as an internal ribosome entry site to initiate the translation of the circular RNA obtained from the in vitro circularization reaction of the construct, which is beneficial to the realization of the biological function of the gene to be circularized. More specifically, specific examples of the IRES sequence and / or IRES-like sequence include, but are not limited to, one or more of the following: CVB3IRES sequence, EMCV IRES sequence, and EV29IRES sequence.

[0058] In some specific embodiments, the IRES sequence is preferably the CVB3 IRES sequence, and the specific nucleotide sequence is shown in SEQ ID NO:13. In this case, the circular RNA obtained by in vitro circularization of the construct has high translation efficiency, enabling efficient and specific gene expression.

[0059] In this invention, the construct can specifically be DNA and / or RNA. When the construct is DNA, it can be loaded onto a plasmid vector, and the linear RNA obtained after transcription can undergo an in vitro circularization reaction to obtain circular RNA. When the construct is RNA, it can be directly used as a raw material for an in vitro circularization reaction to obtain circular RNA. In some specific embodiments, the construct is preferably DNA. In this case, the construct exhibits higher stability.

[0060] The present invention also provides a method for constructing a construct for in vitro RNA circularization. This method is based on the potential of the type I intron of the aforementioned Fusarium oxysporum cob gene to catalyze in vitro RNA circularization. The method involves analyzing and designing a construct framework, and then introducing the gene to be circularized and optional functional sequences into the construct framework to finally obtain a construct for in vitro RNA circularization.

[0061] In this invention, the construction method specifically includes: performing structural analysis on the type I introns of the Fusarium oxysporum cob gene to obtain break sites related to the enzyme activity of the type I introns, and constructing a construct framework; introducing the gene to be circularized based on the construct framework to construct the construct.

[0062] In this invention, the construct preferably further includes functionalized sequences; specific examples of the functionalized sequences include, but are not limited to, promoters and / or IRES sequences. The promoters and IRES sequences described above are, but are not limited to, the promoters and IRES sequences mentioned above, and are a commonly used type of functionalized sequences. Those skilled in the art can select appropriate sequences according to their needs, and this invention does not impose any particular limitations on them.

[0063] The present invention also provides a construct obtained by the above-described construction method, wherein the construct comprises, along the 5'-3' direction, a 3'-intron, exon 2, a gene to be circularized, exon 1, and a 5'-intron, and the construct may be partially the same as or completely identical to the construct described above.

[0064] This invention provides a method for circularizing RNA, wherein the construct used in the circularization method is DNA, specifically comprising: taking the construct and transcribing it to obtain a linear RNA molecule; taking the linear RNA molecule and performing a circularization reaction to obtain the circular RNA.

[0065] In this invention, the transcription is preferably in vitro transcription, specifically referring to the process of generating RNA in vitro using DNA as a template and four ribonucleotides (A / G / C / U) as raw materials, in a suitable buffer system, mimicking the in vivo reaction process, through the catalytic reaction of RNA polymerase. The reagents and conditions used are conventional techniques in the field, limited to achieving extracellular transcription of DNA, and this invention does not impose any particular limitations on them.

[0066] In this invention, the cyclization reaction is achieved based on the self-cleavage catalytic activity of the 3'-intron and 5'-intron in the construct. The reagents and conditions used are conventional techniques in the art, limited to achieving in vitro cyclization of linear RNA. This invention does not impose any particular limitations on these techniques.

[0067] This invention also provides a circular RNA, which is prepared by the above-described RNA circularization method. The circular RNA contains only a small amount of exogenous sequence (5 nt), exhibits low immunogenicity, and when introduced into the body, it can achieve efficient expression of the target protein without triggering a severe immune response, demonstrating high biosafety.

[0068] This invention also provides the application of the above-described constructs and / or circular RNA in the preparation of drugs, vaccines, and / or cells that highly express target genes.

[0069] In this invention, the circular RNA obtained by in vitro circularization of the above-mentioned construct and / or the circular RNA containing only a small amount of exogenous sequence (5nt) has low immunogenicity. When the circular RNA is introduced into the body, it can achieve efficient expression of the target protein without triggering a severe immune response in the body, and has high biosafety. Therefore, it can be well applied in the fields of preparing drugs, vaccines and / or cells that highly express the target gene.

[0070] The embodiments of the present invention are described in detail below. These embodiments are intended to explain the present invention and should not be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0071] Example 1

[0072] This embodiment illustrates a construct and its construction method. The construct is obtained through analysis and design based on the type I intron of the *Fusarium oxysporum* cob gene (Gene ID: LT906358, SEQ ID NO: 1), as shown in Figure 1. Its construction specifically includes:

[0073] 1. The secondary structure of type I introns (nucleotide sequence as shown in SEQ ID NO:2) was calculated and analyzed, and multiple break sites and adjacent exon fragments that are highly related to the self-cleavage catalytic activity of type I introns were screened. The specific secondary structure of type I introns and the break sites thereon are shown in Figure 2.

[0074] Sequence inversion was performed based on the break site and the adjacent exon fragment to obtain 3'-intron, 5'-intron, start exon 1 and start exon 2, and construct framework 1 to 3 (nucleotide sequences as shown in SEQ ID NO: 11 to 13), as shown in Table 2.

[0075] Table 2.

[0076] 2. Referring to Figure 3, the T7 promoter (nucleotide sequence as shown in SEQ ID NO:11), the gene to be circularized (GFP gene, nucleotide sequence as shown in SEQ ID NO:12), and the CVB3 IRES sequence (nucleotide sequence as shown in SEQ ID NO:13) are introduced into the corresponding positions in the construct frameworks 1 to 3 to construct constructs 1 to 3 (nucleotide sequences as shown in SEQ ID NO:14 to 16 in sequence); and the partial sequences of the obtained constructs 1 to 3 are entrusted to General Biol for synthesis.

[0077] 3. (1) Construction of recombinant plasmids: Using the Gibson cloning kit (vazyme, catalog number C112-02) and following the instructions, constructs 1 to 3 were ligated into the pUC57-KanR plasmid to construct recombinant plasmids.

[0078] (2) In vitro transcription: using The T7 High-Efficiency RNA Synthesis Kit (NEB, catalog number E2040S) was used to perform in vitro transcription on constructs 1-3 according to the instructions to obtain in vitro transcription products.

[0079] The in vitro transcription products were processed with Dnase I enzyme and purified by precipitation with 3M LiCl solution to obtain linear RNA1-3.

[0080] (3) In vitro circularization: Linear RNA 1-3 was added to a circularization buffer (including 20 mM Tris-HCl, 5 mM MgCl2 and 25 mM NaCl) according to the final concentration of linear RNA of 50 nM. GTP was added to make the concentration of GTP in the solution 2 mM. The mixture was circularized in vitro at 55 °C for 15 min to obtain 1-3 cyclized products.

[0081] The cyclized products were purified using an RNA purification kit (NEB, catalog number T2040L) and the instructions were followed to obtain purified cyclized products 1-3.

[0082] The purified cyclized product was digested at 37°C for 1 h using Rnase-R (Hanhai New Enzyme, catalog number HBP004600-1) to obtain circular RNA1-3, the structures of which are shown in Figure 3.

[0083] 4. The linear RNAs 1-3, purified circularized products 1-3, and circular RNAs 1-3 obtained in step “3” were subjected to 4% urea-PAGE gel electrophoresis, with the same sample addition amount and conditions during electrophoresis. The circularization efficiency (%) of constructs 1-3 was calculated according to the following formula. The results are shown in Figure 4 and Table 3. Circulation efficiency (%) = X1 / X2 × 100%

[0084] Where X1 is the band intensity of circular RNA in the gel electrophoresis diagram; X2 is the band intensity of linear RNA in the gel electrophoresis diagram.

[0085] Table 3.

[0086] As shown in Figure 4 and Table 3, the cyclization efficiency of constructs 1 to 3 is all above 45%, indicating high cyclization efficiency. Furthermore, the strength of the ring band in construct 2 is slightly higher than that in constructs 1 and 3.

[0087] The circular band in construct 2 was gel-cleaved and recovered, then reverse transcribed and sequenced using Sanger sequencing. The results are shown in Figure 5. As shown in Figure 5, the circular RNA2 molecule is fused end-to-end, further confirming that it is a circular RNA product.

[0088] Example 2

[0089] This embodiment illustrates a construct and its construction method. The construct is based on construct 2 obtained in Example 1. Nucleotides are deleted from the start exons 1 and 2. Constructs 4 to 10 are constructed according to the construction method provided in Example 1. The obtained constructs 4 to 10 are obtained by homologous PCR amplification, as shown in Figures 6 and 7 and Table 4.

[0090] Table 4.

[0091] The constructs 4-10 were transcribed and circularized in vitro according to the method provided in Example 1 to obtain linear RNA 4-10 and circular RNA 4-10; and the linear RNA 4-10 and circular RNA 4-10 were subjected to 4% urea-PAGE gel electrophoresis. The results are shown in Figures 8 and 9.

[0092] As shown in Figures 8 and 9, construct 4 does not contain circular RNA bands in its in vitro transcripts without the introduction of additional exon fragments. However, after induction under circularization conditions, it can still achieve good in vitro RNA circularization even without the introduction of additional exons (i.e., exon length of 0 nt).

[0093] Example 3

[0094] This embodiment illustrates a construct and its construction method. The constructs 11-16 provided in this embodiment are obtained by modifying the 3'-intron and 5'-intron according to the construction method provided in Embodiment 1. Specifically, they include:

[0095] 1. Construction of constructs 11-13: (1) Based on construct 4 obtained in Example 2, the 3'-intron and 5'-intron were modified according to Table 5 and Figures 10-13 and obtained by homologous PCR amplification.

[0096] Table 5.

[0097] Following the method provided in Example 1, constructs 11-13 were subjected to in vitro transcription and in vitro circularization to obtain linear RNA 11-13, purified circularized products 11-13, and circular RNA 11-13. The purified circularized products 11-13 and circular RNA 11-13 were then subjected to 4% urea-PAGE gel electrophoresis, with the same sample addition amount and conditions during electrophoresis. The circularization efficiency (%) was calculated. The results are shown in Figure 14 and Table 6.

[0098] Table 6.

[0099] As shown in Figure 14 and Table 6, constructs 12 and 13 could not undergo in vitro circularization to obtain circular RNA, while construct 11, even with the p10 domain removed, could still achieve in vitro circularization of RNA after induction under circularization conditions, thus obtaining circular RNA 11.

[0100] (2) The circular RNA11 (obtained by in vitro circularization of construct 11) was gel-cleaved and recovered, and then reverse transcribed and Sanger sequenced. The results are shown in Figure 15.

[0101] As shown in Figure 15, the circular RNA obtained by using construct 11 for in vitro circularization reaction contains only 5 nucleotides (TGGGT) remaining in the F2-3'-intron P1 domain. This effectively solves the problem of large immune responses caused by the introduction of large exogenous sequences in existing in vitro synthesized circular RNA, and has good application prospects.

[0102] 2. Construction of constructs 14-16: Based on construct 11 obtained above, the P1 domain of the 5'-intron was modified according to Table 7 and Figure 16, and obtained by homologous PCR amplification.

[0103] Table 7.

[0104] The constructs 14-16 were transcribed and circularized in vitro according to the method provided in Example 1 to obtain linear RNA 14-16, purified circularized products 14-16, and circular RNA 14-16. The purified circularized products 14-16 and circular RNA 14-16 were subjected to 4% urea-PAGE gel electrophoresis, and the sample addition amount and conditions were the same during the electrophoresis. The circularization efficiency (%) was calculated, and the results are shown in Figure 17 and Table 8.

[0105] Table 8.

[0106] As shown in Figure 17 and Table 8, constructs 14–16 could not undergo in vitro circularization to obtain circular RNA, which proves that the P1 domain is the key sequence for the catalytic effect of type I introns in achieving in vitro RNA circularization.

[0107] Example 4

[0108] This embodiment illustrates a construct and its construction method. The construct is based on construct 12 obtained in Example 3. The 3'-intron and 5'-intron in constructs 1 and 3 are modified (Figures 11 and 12). Constructs 17 and 18 are constructed according to the construction method provided in Example 3. The obtained constructs 17 and 18 are obtained by homologous PCR amplification, as shown in Table 9.

[0109] Table 9.

[0110] Constructions 17 and 18 were transcribed and circularized in vitro according to the method provided in Example 1 to obtain linear RNAs 17 and 18, purified circularized products 17 and 18, and circular RNAs 17 and 18. The purified circularized products 17 and 18 and the circular RNAs 17 and 18 were subjected to 4% urea-PAGE gel electrophoresis under the same sample addition amounts and conditions, and the circularization efficiency (%) was calculated. The results are shown in Figure 18 and Table 10.

[0111] Table 10.

[0112] As shown in Figure 18 and Table 10, although the in vitro circularization efficiency of constructs 17 and 18 is not as high as that of construct 11, circular RNA can still be obtained. This proves that the P1 domain is the key sequence for the catalytic role of type I introns in the in vitro circularization of RNA.

[0113] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention without departing from the principles and spirit of the present invention.

Claims

1. A construct for in vitro circularization of RNA, characterized in that, The construct comprises, in the order given from 5' to 3': a 3'-intron, an exon 2, a gene to be circularized, an exon 1 and a 5'-intron. The 3'-intron and the 5'-intron are derived from a type I intron of a cob gene of Fusarium oxysporum, and the 5'-intron comprises a P1 domain comprising a nucleotide fragment as set forth in SEQ ID NO: 38; the exon 1 is an immediately adjacent exon fragment at the 5' end of the type I intron, and the length of the exon 1 is 0-393 nt; the exon 2 is an immediately adjacent exon fragment at the 3' end of the type I intron, and the length of the exon 2 is 0-780 nt.

2. Construct for in vitro circularization of RNA according to claim 1, characterized in that, The nucleotide sequence of the type I intron is as set forth in SEQ ID NO:

2.

3. Construct for in vitro circularization of RNA according to claim 1, characterized in that, The 5'-intron and the 3'-intron are obtained by cleaving a type I intron, and the cleavage site on the type I intron is located at positions 18-1020 of the sequence as set forth in SEQ ID NO:

2.

4. Construct for in vitro circularization of RNA according to claim 1, characterized in that, The nucleotide sequence of the 3'-intron is as set forth in SEQ ID NO: 29, the nucleotide sequence of the 5'-intron is as set forth in SEQ ID NO: 6, and the lengths of the exon 1 and the exon 2 are 0 nt.

5. Construct for in vitro circularization of RNA according to claim 1, characterized in that, The construct comprises a T7 promoter, and the T7 promoter is located at the 5' end of the 3'-intron.

6. Construct for in vitro circularization of RNA according to claim 5, characterized in that, The nucleotide sequence of the T7 promoter is as set forth in SEQ ID NO:

11.

7. Construct for in vitro circularization of RNA according to claim 1, characterized in that, The construct comprises a CVB3 IRES sequence.

8. Construct for in vitro circularization of RNA according to claim 7, characterized in that, The nucleotide sequence of the CVB3 IRES sequence is as set forth in SEQ ID NO:

13.

9. A method for the construction of a construct for RNA in vitro circularization, characterized in that, The construction method comprises: performing structural analysis on a type I intron of a cob gene of Fusarium oxysporum to obtain a cleavage site related to the enzymatic activity of the type I intron, and constructing a construct framework; and introducing a gene to be circularized based on the construct framework to construct the construct.

10. The method for constructing a construct for in vitro circularization of RNA according to claim 9, characterized in that, The nucleotide sequence of the type I intron is as set forth in SEQ ID NO:

2.

11. The method for constructing a construct for in vitro circularization of RNA according to claim 9, characterized in that, The cleavage site is at positions 18-1020 of the sequence as set forth in SEQ ID NO:

2.

12. A construct constructed by the construction method for the construct for RNA in vitro circularization according to claim 9.

13. Construct for in vitro circularization of RNA according to claim 12, characterized in that, The construct comprises, in the order given from 5' to 3': a 3'-intron, an exon 2, a gene to be circularized, an exon 1 and a 5'-intron.

14. Construct for in vitro circularization of RNA according to claim 13, characterized in that, The nucleotide sequence of the 3'-intron is as set forth in SEQ ID NO: 29, the nucleotide sequence of the 5'-intron is as set forth in SEQ ID NO: 6, and the lengths of the exon 1 and the exon 2 are 0 nt.

15. A method of circularizing an RNA, characterized by, The circularization method specifically comprises: transcribing the construct according to claim 1 to obtain a linear RNA molecule; and performing a circularization reaction on the linear RNA molecule to obtain the circular RNA.

16. A circular RNA, characterized in that, The circular RNA is prepared by the circularization method for RNA according to claim 15.

17. Use of the circular RNA of claim 15 in the preparation of a medicament, a vaccine and / or a cell with high expression of a target gene.

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