Constructs Based on the Group I Intron of the CoB Gene of Fusarium oxysporum, Their Construction Methods, RNA Cyclization Methods, Circular RNAs, and Applications

Abstract

The present invention belongs to the field of biotechnology, and discloses a construct based on the group I intron of the CoB gene of Fusarium oxysporum, a construction method thereof, an RNA cyclization method, a circular RNA, and applications. The construct for in vitro RNA cyclization provided by the present invention uses the group I intron on the cob gene of Fusarium oxysporum as a ribozyme for mediating in vitro RNA cyclization, and at least retains the P1 domain of the group I intron. In the case of introducing a small amount of exon fragments or without introducing exon fragments, it can achieve in vitro cyclization of RNA, and the residual foreign sequences in the obtained circular RNA are as low as 5 nt, effectively solving the problem of immune response caused by the large residual foreign sequences in the existing in vitro synthesized circular RNA, and having good application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a construct based on the group I intron of Fusarium oxysporum CoB gene, a construction method thereof, an RNA cyclization method, a circular RNA, and applications thereof. Background Art

[0002] Circular RNA (circRNA) is a class of non-coding RNA molecules existing in eukaryotic cells that do not have a 5'-terminal cap and a 3'-terminal poly(A) tail, and form a circular structure by covalent bonds. It can affect gene expression by influencing transcription initiation, elongation, and splicing, etc., and participate in controlling the functions of other molecules in eukaryotes.

[0003] Currently, the most commonly used method for in vitro RNA cyclization is the ribozyme method, which mainly constructs a PIE (Permutated Intron-Exon) construct inserted with a target sequence based on different types of introns. This PIE construct utilizes the self-catalytic cleavage reaction of the intron ribozyme - the intron fragment falls off during self-cleavage, and the two flanking exons are connected to achieve the in vitro cyclization of the target sequence. However, when cyclizing the target sequence by the ribozyme method, there will be a residue of a relatively long foreign sequence. The remaining foreign sequence may not only cause a relatively large immunogenicity in vivo, but also may affect the translation of RNA, resulting in a decrease in the expression of the target protein, and has great limitations. Summary of the Invention

[0004] The first object of the present invention is to provide a construct for in vitro RNA cyclization. When this construct is applied to in vitro RNA cyclization, it can maintain the RNA cyclization efficiency on the premise of only introducing a small amount of foreign sequence (5 nt), enrich the existing RNA cyclization methods, and overcome the defects of long foreign sequence residue and strong immunogenicity existing in the existing RNA cyclization.

[0005] The second object of the present invention is to provide a construction method of a construct for in vitro RNA cyclization.

[0006] The third object of the present invention is to provide a construct obtained by the construction method of the construct for in vitro RNA cyclization described above.

[0007] The fourth object of the present invention is to provide a method for cyclizing RNA.

[0008] The fifth object of the present invention is to provide a circular RNA.

[0009] The sixth object of the present invention is to provide the applications of the construct and / or circular RNA described above in the preparation of drugs, vaccines, and / or cells with high expression of target genes.

[0010] Specifically, the construct for in vitro RNA circularization provided by the present invention sequentially includes, 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 the type I intron of the cob gene of Fusarium oxysporum with the nucleotide sequence as shown in SEQ ID NO:1, and the 5'-intron includes a P1 domain, and the P1 domain includes a nucleotide fragment with the sequence as shown in SEQ ID NO:38; exon 1 is the exon fragment 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 adjacent to the 3' end of the type I intron, and the length of exon 2 is 0 - 780 nt.

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

[0012] Further, the 5'-intron and 3'-intron are obtained by cleaving the type I intron, and the cleavage site on the type I intron is located at positions 18 - 1020 of the sequence shown in SEQ ID NO:2.

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

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

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

[0016] Further, the construct includes a CVB3 IRES sequence, and the CVB3 IRES sequence is located in the middle of the gene to be circularized and exon 1.

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

[0018] The construction method of the construct for in vitro RNA circularization provided by the present invention includes: performing a structural analysis on the type I intron of the cob gene of Fusarium oxysporum to obtain the cleavage site related to the enzyme activity of the type I intron, 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 Group I intron is as shown in SEQ ID NO:2.

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

[0021] The present invention provides a construct obtained by constructing the construct for in vitro RNA cyclization described above.

[0022] Furthermore, the construct sequentially includes, along the 5'-3' direction: 3'-intron, exon 2, gene to be cyclized, exon 1, and 5'-intron.

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

[0024] The method for cyclizing RNA provided by the present invention includes: transcribing any one of the constructs described in claims 1 - 5 and 11 to obtain a linear RNA molecule; subjecting the linear RNA molecule to a cyclization reaction to obtain the circular RNA.

[0025] The circular RNA provided by the present invention is prepared by the method for cyclizing RNA described above. The present invention also provides the application of the construct and / or circular RNA described above in the preparation of drugs, vaccines, and / or cells with high expression of target genes.

[0026] Beneficial effects:

[0027] The construct for in vitro RNA cyclization provided by the present invention uses a Group I intron from the cob gene of Fusarium oxysporum as a self-splicing ribozyme for mediating in vitro RNA cyclization. Without introducing exon fragments or introducing only a small amount of exon fragments, it can well catalyze in vitro RNA cyclization, and the residual foreign sequence in the obtained circular RNA is only 5 nt, which can effectively solve the problem of immune response caused by existing in vitro synthesized circular RNA and has good application prospects. Description of the drawings

[0028] Figure 1 It is a schematic diagram of the cob gene structure provided in Example 1 of the present invention;

[0029] Figure 2 It is a schematic diagram of the secondary structure of the Group I intron provided in Example 1 of the present invention;

[0030] Figure 3Schematic diagram of the construct and its circular RNA provided in Example 1 of the present invention;

[0031] Figure 4 Experimental result diagram of urea-PAGE gel electrophoresis provided in Example 1 of the present invention;

[0032] Figure 5 Partial sanger sequencing result diagram of circular RNA 2 provided in Example 1 of the present invention;

[0033] Figure 6 Schematic diagram of nucleotide deletion of start exon 2 provided in Example 2 of the present invention;

[0034] Figure 7 Schematic diagram of nucleotide deletion of start exon 1 provided in Example 2 of the present invention;

[0035] Figure 8 Experimental result diagram of urea-PAGE gel electrophoresis provided in Example 2 of the present invention (in vitro transcription);

[0036] Figure 9 Experimental result diagram of urea-PAGE gel electrophoresis provided in Example 2 of the present invention (in vitro cyclization);

[0037] Figure 10 Base sequence and secondary structure of type I intron P1 domain provided in the present invention;

[0038] Figure 11 Base sequence and secondary structure of type I intron P9 and P10 domains provided in the present invention;

[0039] Figure 12 Schematic diagram of the modification of F2-3'-intron provided in Example 3 of the present invention;

[0040] Figure 13 Schematic diagram of the modification of F2-5'-intron provided in Example 3 of the present invention;

[0041] Figure 14 Experimental result diagram of urea-PAGE gel electrophoresis provided in Example 3 of the present invention (constructs 11-13);

[0042] Sequencing result diagram of the cyclization induced by changing the P1 and P10 structures in the ribozyme;

[0043] Figure 15 Partial sanger sequencing result diagram of circular RNA 11 provided in Example 3 of the present invention;

[0044] Figure 16Schematic diagram of the modification of F2-5'-intron provided in Example 3 of the present invention (mutating the P1 domain);

[0045] Figure 17 Experimental result diagram of urea-PAGE gel electrophoresis provided in Example 3 of the present invention (constructs 14-16);

[0046] Figure 18 Experimental result diagram of urea-PAGE gel electrophoresis provided in Example 4 of the present invention. Detailed implementation manners

[0047] The nucleotide sequences involved in the present invention are shown in Table 1:

[0048] Table 1.

[0049]

[0050]

[0051]

[0052]

[0053]

[0054]

[0055]

[0056]

[0057] Based on in-depth and extensive research on the in vitro RNA cyclization reaction, the inventors of the present invention found that the group I intron of the cob gene of Fusarium oxysporum has excellent potential for catalyzing in vitro RNA cyclization. Further research was carried out to modify the group I intron and introduce the gene to be cyclized, and the construct claimed in the present invention was constructed. At this time, when the construct is applied to the in vitro cyclization of RNA, the in vitro cyclization of RNA can be achieved, and only 5 nt of the foreign sequence remains in the obtained circular RNA, with low immunogenicity.

[0058] In the present invention, the Gene ID number of the cob gene of Fusarium oxysporum is LT906358, and the specific nucleotide sequence is as shown in SEQ ID NO: 1; among them, the nucleotide sequence of the group I intron contained in the cob gene is as shown in SEQ ID NO: 2.

[0059] In the present invention, the construct sequentially includes, along the 5'-3' direction: a 3'-intron, exon 2, a gene to be circularized, exon 1, and a 5'-intron. More specifically, the 3'-intron and 5'-intron are formed by cleaving a group I intron, and the cleavage site on the group I intron that can be cleaved to form a 3'-intron and a 5'-intron retaining self-cleavage catalytic activity is 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 position therebetween, and the 5'-intron includes a P1 domain, and the P1 domain includes a nucleotide fragment having a sequence as shown in SEQ ID NO:38.

[0060] In some specific embodiments, the cleavage 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 as shown in SEQ ID NO:5, and the nucleotide sequence of the 5'-intron is as shown in SEQ ID NO:6. At this time, the obtained 3'-intron and 5'-intron are applied to the construct for in vitro RNA circularization, and have relatively good in vitro circularization efficiency, and can achieve in vitro circularization of RNA.

[0061] In the present invention, exon 1 is an exon fragment adjacent to the 5'-end of the group I intron, and its length is specifically 0-393 nt, such as 0 nt, 5 nt, 50 nt, 218 nt, 253 nt, 350 nt, 393 nt or any integer value therebetween; exon 2 is an exon fragment adjacent to the 3'-end of the group I intron, and its length is specifically 0-780 nt, such as 0 nt, 58 nt, 93 nt, 218 nt, 324 nt, 630 nt, 736 nt, 748 nt, 780 nt or any value therebetween. At this time, the introduction of exon 1 and exon 2 in the construct is to maintain or improve the self-cleavage catalytic activity of the group I intron.

[0062] In some specific embodiments, the lengths of exon 1 and exon 2 in the construct are preferably 0 nt, that is, no additional exon fragments adjacent to the group I intron are introduced into the construct. At this time, the group I intron on the cob gene of Fusarium oxysporum also has self-cleavage catalytic activity. Even without the introduction of exon fragments, it can still maintain a certain self-cleavage catalytic activity, and well solves the problem of residual long foreign sequences in existing in vitro RNA circularization.

[0063] In the present invention, the 3'-intron preferably does not include the P10 domain, and the specific nucleotide sequence is as shown in SEQ ID NO: 29. At this time, although the deletion of the P10 domain in the 3'-intron will reduce the self-cleavage catalytic activity of the ribozyme, more importantly, it can achieve the excellent effect of reducing the residual foreign sequences in the circular RNA.

[0064] In some specific embodiments, the nucleotide sequence of the preferred 3'-intron in the construct is as shown in SEQ ID NO: 29, and the nucleotide sequence of the 5'-intron is as shown in SEQ ID NO: 6. At this time, the construct can achieve the in vitro circularization of RNA, and only 5 nt of foreign sequences remain in the obtained circular RNA, with low immunogenicity.

[0065] In the present invention, the construct preferably further includes a promoter located at the 5'-end of the 3'-intron. The promoter is an important starting element for in vitro transcription to achieve the in vitro transcription of the construct. In some specific embodiments, specific 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 the T7 promoter located at the 5'-end of the 3'-intron. The T7 promoter is derived from bacteriophage T7 and is used to initiate the transcription of the construct, and the specific nucleotide sequence is as shown in SEQ ID NO: 11.

[0066] In the present 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 by 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 CVB3 IRES sequence, EMCV IRES sequence, and EV29 IRES sequence.

[0067] In some specific embodiments, the IRES sequence is preferably the CVB3 IRES sequence, and the specific nucleotide sequence is as shown in SEQ ID NO: 13. At this time, the circular RNA obtained by the in vitro circularization of the construct has high translation efficiency and can achieve efficient and specific gene expression.

[0068] In the present invention, the construct may specifically be DNA and / or RNA. Among them, when the construct is DNA, it can be loaded on a plasmid vector, and the linear RNA obtained after its transcription can undergo an in vitro cyclization reaction to obtain circular RNA. When the construct is RNA, it can directly be used as a raw material for the in vitro cyclization reaction to obtain circular RNA. In some specific embodiments, the construct is preferably DNA. At this time, the construct has higher stability.

[0069] The present invention also provides a method for constructing a construct for in vitro RNA cyclization. This construction method is based on the potential of the group I intron of the Fusarium oxysporum cob gene to catalyze in vitro RNA cyclization, analyzes and designs to obtain a construct framework, and introduces a gene to be cyclized and optionally a functionalized sequence on the basis of the construct framework, so as to finally obtain a construct for in vitro RNA cyclization.

[0070] In the present invention, the construction method specifically includes: performing a structural analysis on the group I intron of the Fusarium oxysporum cob gene to obtain cleavage sites related to the enzyme activity of the group I intron, and constructing a construct framework; introducing a gene to be cyclized based on the construct framework to construct the construct.

[0071] In the present invention, the construct preferably further includes a functionalized sequence; specific examples of the functionalized sequence include but are not limited to: a promoter and / or an IRES sequence. The promoter and IRES sequence may be, but are not limited to, the promoter and IRES sequence described above, which are a type of commonly used functionalized sequence. Those skilled in the art can select a suitable sequence according to needs, and the present invention does not particularly limit it.

[0072] The present invention also provides a construct obtained by the above-described construction method. This construct sequentially includes, along the 5'-3' direction: a 3'-intron, exon 2, a gene to be cyclized, exon 1, and a 5'-intron, and the construct may be partially or completely the same as the construct described above.

[0073] The present invention provides a method for cyclizing RNA. The construct used in this cyclization method is DNA, and specifically includes: transcribing the construct to obtain a linear RNA molecule; performing a cyclization reaction on the linear RNA molecule to obtain the circular RNA.

[0074] In the present invention, the transcription is preferably in vitro transcription, specifically referring to a process that is separated from the cell system, uses DNA as a template in vitro, uses four ribonucleotides A / G / C / U as raw materials, and in an appropriate buffer system, mimics the in vivo reaction process and generates RNA through the catalytic reaction of RNA polymerase; the reagents and conditions used are conventional technical means in the art, limited to achieving extracellular transcription of DNA, and the present invention does not specifically limit them.

[0075] In the present invention, the cyclization reaction is achieved based on the self-cleaving catalytic activities of the 3'-intron and 5'-intron in the construct, and the reagents and conditions used are conventional technical means in the art, limited to achieving in vitro cyclization of linear RNA, and the present invention does not specifically limit them.

[0076] The present invention also provides a circular RNA, which is prepared by the above-described RNA cyclization method. The circular RNA contains only a small amount of foreign sequences (5 nt), has low immunogenicity, and when this circular RNA is introduced into an organism, it can achieve high-efficiency expression of the target protein and will not trigger a serious immune response in the organism, and has high biosafety.

[0077] The present invention also provides the application of the above-described construct and / or circular RNA in the preparation of drugs, vaccines, and / or cells with high expression of the target gene.

[0078] In the present invention, the circular RNA obtained by in vitro cyclization of the above-described construct and / or the circular RNA contains only a small amount of foreign sequences (5 nt), has low immunogenicity, and when this circular RNA is introduced into an organism, it can achieve high-efficiency expression of the target protein and will not trigger a serious immune response in the organism, and has high biosafety, and thus can be well applied in fields such as the preparation of drugs, vaccines, and / or cells with high expression of the target gene.

[0079] The following details the embodiments of the present invention. The examples are intended to explain the present invention and should not be construed as limiting the present invention. For those not specifying specific techniques or conditions in the examples, they shall be carried out according to the techniques or conditions described in the literature in the art or according to the product specifications. For reagents or instruments not indicating the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0080] Example 1

[0081] This example is used to illustrate a construct and its construction method. The construct has a structure as Figure 1Based on the group I intron on the cob gene (Gene ID: LT906358, SEQ ID NO: 1) of Fusarium oxysporum, it is obtained through analysis and design. Its construction specifically includes:

[0082] 1. Calculate and analyze the secondary structure of the group I intron (nucleotide sequence shown in SEQ ID NO: 2), screen to obtain multiple cleavage sites and exon fragments adjacent to the self-splicing catalytic activity of the group I intron. The secondary structure of the group I intron and the cleavage sites thereon are specifically as Figure 2 shown;

[0083] Based on the cleavage sites and adjacent exon fragments, perform sequence inversion to obtain 3'-intron, 5'-intron, starting exon 1 and starting exon 2, and construct constructs 1-3 (nucleotide sequences shown in SEQ ID NO: 11-13), as shown in Table 2 specifically.

[0084] Table 2.

[0085]

[0086] 2. Refer to Figure 3 , introduce the T7 promoter (nucleotide sequence shown in SEQ ID NO: 11), the gene to be circularized (GFP gene, nucleotide sequence shown in SEQ ID NO: 12), and the CVB3 IRES sequence (nucleotide sequence shown in SEQ ID NO: 13) into the corresponding positions in constructs 1-3 respectively, and construct constructs 1-3 (nucleotide sequences are shown in SEQ ID NO: 14-16 in sequence); and part of the sequences of the obtained constructs 1-3 are entrusted to General Biol for synthesis.

[0087] 3. (1) Construction of recombinant plasmids: Use the Gibson cloning kit (vazyme, product number C112-02) and connect constructs 1-3 to the pUC57-KanR plasmid respectively according to the instructions of the manual to construct recombinant plasmids.

[0088] (2) In vitro transcription: Use the T7 High Yield RNA Synthesis Kit (NEB, product number E2040S) and perform in vitro transcription on constructs 1-3 according to the instructions of the manual to obtain in vitro transcription products;

[0089] Take Dnase I enzyme to treat the in vitro transcription products, and precipitate with a 3M LiCl solution to purify and obtain linear RNAs 1-3.

[0090] (3) In vitro cyclization: Linear RNAs 1 - 3 were added to the cyclization buffer (including 20 mM Tris-HCl, 5 mM MgCl2, and 25 mM NaCl) at an addition amount with a final concentration of 50 nM for linear RNA, and GTP was added to a concentration of 2 mM in the solution. In vitro cyclization was carried out at 55 °C for 15 min to obtain cyclized products 1 - 3;

[0091] After purifying the cyclized products using an RNA purification kit (NEB, catalog number T2040L) and operating according to the instructions, purified cyclized products 1 - 3 were obtained;

[0092] Purified cyclized products were digested with Rnase-R (HanHaiXinMei, catalog number HBP004600 - 1) at 37 °C for 1 h to obtain circular RNAs 1 - 3. The structures of circular RNAs 1 - 3 are as Figure 3 shown.

[0093] 4. 4% urea-PAGE gel electrophoresis was performed on the linear RNAs 1 - 3, purified cyclized products 1 - 3, and circular RNAs 1 - 3 obtained in "3", and the sample addition amounts and conditions were the same during electrophoresis. The cyclization efficiency (%) of constructs 1 - 3 was calculated according to the following formula. The results are as Figure 4 and Table 3 show.

[0094] Cyclization efficiency (%) = X1 / X2 × 100%

[0095] where X1 is the band intensity of circular RNA in the gel electrophoresis pattern; X2 is the band intensity of linear RNA in the gel electrophoresis pattern.

[0096] Table 3.

[0097]

[0098] From Figure 4 and the results shown in Table 3, it can be seen that the cyclization efficiencies of constructs 1 - 3 are all above 45%, with relatively high cyclization efficiencies, and the intensity of the circular band in construct 2 is slightly higher than that in constructs 1 and 3.

[0099] The circular band in construct 2 was excised and recovered, followed by reverse transcription and Sanger sequencing. The results are as Figure 5 shown. From Figure 5 it can be seen that the circular RNA 2 molecules are connected end to end, further proving that it is a circular RNA product.

[0100] Example 2

[0101] This example is used to illustrate a construct and its construction method. The construct is based on the construct 2 obtained in Example 1, and nucleotides are deleted from the starting exons 1 and 2. Referring to the construction method provided in Example 1, constructs 4-10 are constructed, and the obtained constructs 4-10 are obtained by homologous PCR amplification, specifically as Figure 6 and 7 as well as shown in Table 4.

[0102] Table 4.

[0103]

[0104]

[0105] Referring to the method provided in Example 1, constructs 4-10 are subjected to in vitro transcription and in vitro circularization to obtain linear RNAs 4-10 and circular RNAs 4-10; and the above linear RNAs 4-10 and circular RNAs 4-10 are subjected to 4% urea-PAGE gel electrophoresis, and the results are as Figure 8 and 9 shown.

[0106] From Figure 8 and 9 the results shown, it can be seen that for construct 4, without introducing additional exon fragments, the in vitro transcription product basically does not contain circular RNA bands, but after induction by circularization conditions, even without additional introduction of exons (i.e., exon length is 0 nt), RNA can still be well circularized in vitro.

[0107] Example 3

[0108] This example is used to illustrate a construct and its construction method. The constructs 11-16 provided in this example are obtained by referring to the construction method provided in Example 1 and modifying the 3'-intron and 5'-intron, specifically including:

[0109] 1. Construction of constructs 11-13: (1) Based on the construct 4 obtained in Example 2, and referring to Table 5 as well as Figures 10 - 13 modify the 3'-intron and 5'-intron, and obtain them by homologous PCR amplification.

[0110] Table 5.

[0111]

[0112] Perform in vitro transcription and in vitro cyclization on constructs 11-13 according to the method provided in Example 1 to obtain linear RNAs 11-13, purified cyclized products 11-13, and circular RNAs 11-13; perform 4% urea-PAGE gel electrophoresis on the above purified cyclized products 11-13 and circular RNAs 11-13, and the sample addition amount and conditions are the same during electrophoresis, and calculate the cyclization efficiency (%). The results are as Figure 14 shown in Table 6.

[0113] Table 6.

[0114] Construct Intron Circularization efficiency (%) Construct 11 F2 - 3’ - Intron - 2 and F2 - 5’ - Intron 6.0 Construct 12 F2 - 3’ - Intron - 2 and F2 - 5’ - Intron - 2 - Construct 13 F2 - 3’ - Intron - 3 and F2 - 5’ - Intron - 3 -

[0115] As can be seen from Figure 14 the results shown in Table 6, constructs 12 and 13 cannot undergo in vitro cyclization reaction to obtain circular RNAs, while construct 11 can still achieve in vitro cyclization of RNA after being induced by cyclization conditions even after deleting the P1 domain, so as to obtain circular RNA 11.

[0116] (2) Perform gel cutting and recovery on circular RNA 11 (obtained by performing in vitro cyclization reaction on construct 11), and perform reverse transcription and Sanger sequencing. The results are as Figure 15 shown.

[0117] As can be seen from Figure 15 the results shown, only 5 nucleotides (TGGGT) in the P1 domain of the F2-3'-intron remain in the circular RNA obtained by performing in vitro cyclization reaction with construct 11, which can effectively solve the problem of large immune response caused by introducing large foreign sequences in the existing in vitro synthesis of circular RNA, and has good application prospects.

[0118] 2. Construction of constructs 14-16: Based on construct 11 constructed above, and referring to Table 7 and Figure 16 modify the P1 domain of the 5'-intron, and obtain it by homologous PCR amplification.

[0119] Table 7.

[0120]

[0121]

[0122] Perform in vitro transcription and in vitro cyclization on constructs 14 - 16 according to the method provided in Example 1 to obtain linear RNAs 14 - 16, purified cyclized products 14 - 16, and circular RNAs 14 - 16; perform 4% urea - PAGE gel electrophoresis on the above - mentioned purified cyclized products 14 - 16 and circular RNAs 14 - 16, with the same sample addition amount and conditions during electrophoresis, and calculate the cyclization efficiency (%). The results are as Figure 17 shown in Table 8.

[0123] Table 8.

[0124] Construct Intron Circularization efficiency (%) Construct 14 F2 - 3’ - Intron - 2 and F2 - 5’ - Intron - 4 - Construct 15 F2 - 3’ - Intron - 2 and F2 - 5’ - Intron - 5 - Construct 16 F2 - 3’ - Intron - 3 and F2 - 5’ - Intron - 6 -

[0125] As can be seen from Figure 17 the results shown in Table 8, constructs 14 - 16 cannot undergo in vitro cyclization reaction to obtain circular RNAs, which proves that the P1 domain is the key sequence for the catalytic function of type I intron to achieve in vitro RNA cyclization.

[0126] Example 4

[0127] This example is used to illustrate a construct and its construction method. This construct is based on construct 12 obtained in Example 3, and the 3'-intron and 5'-intron in constructs 1 and 3 are modified ( Figure 11 and Figure 12 ), and referring to the construction method provided in Example 3, constructs 17 and 18 are constructed, and the obtained constructs 17 and 18 are obtained by homologous PCR amplification, as shown in Table 9 specifically.

[0128] Table 9.

[0129]

[0130] Perform in vitro transcription and in vitro cyclization on constructs 17 and 18 according to the method provided in Example 1 to obtain linear RNAs 17 and 18, purified cyclized products 17 and 18, and circular RNAs 17 and 18; perform 4% urea - PAGE gel electrophoresis on the above - mentioned purified cyclized products 17 and 18 and circular RNAs 17 and 18, with the same sample addition amount and conditions during electrophoresis, and calculate the cyclization efficiency (%). The results are as Figure 18 shown in Table 10.

[0131] Table 10.

[0132] Construct Intron Circularization efficiency (%) Construct 17 F1 - 3’ - Intron - 2 and F1 - 5’ - Intron 2.6 Construct 18 F3 - 3’ - Intron - 2 and F3 - 5’ - Intron 2.3

[0133] As can be seen from Figure 18As can be seen from the results shown in Table 10, for constructs 17 and 18, although the cyclization efficiency is lower than that of construct 11 during the in vitro cyclization reaction, circular RNAs can still be obtained, which proves that the P1 domain is the key sequence for the catalytic action of group I introns to achieve in vitro RNA cyclization.

[0134] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Without departing from the principles and spirit of the present invention, those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention.

Claims

1. A construct for in vitro RNA circularization, characterized in that, Compared with a construct that sequentially includes a 3'-intron, exon 2, the gene to be circularized, exon 1, and a 5'-intron in the 5'-3' direction; the lengths of exon 1 and exon 2 of the construct for in vitro RNA circularization are 0 nt; the nucleotide sequence of the 3'-intron of the construct for in vitro RNA circularization is as shown in SEQ ID NO:29, and the nucleotide sequence of the 5'-intron of the construct for in vitro RNA circularization is as shown in SEQ ID NO:

6.

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

3. The construct for in vitro RNA cyclization according to claim 2, wherein, The nucleotide sequence of the T7 promoter is as shown in SEQ ID NO:

11.

4. The construct for in vitro RNA cyclization according to claim 1, characterized in that, The construct includes a CVB3 IRES sequence, and the CVB3 IRES sequence is located in the middle of the gene to be circularized and exon 1.

5. The construct for in vitro RNA cyclization according to claim 4, characterized in that, The nucleotide sequence of the CVB3 IRES sequence is as shown in SEQ ID NO:

13.

6. The method for constructing the construct for in vitro RNA cyclization according to claim 1, characterized in that, The construction method includes: performing a structural analysis on the group I intron of the cob gene of Fusarium oxysporum ( Fusarium oxysporum ), obtaining cleavage sites related to the enzymatic activity of the group I intron, and constructing a construct framework; introducing a gene to be cyclized based on the construct framework to construct the construct; the nucleotide sequence of the group I intron is as shown in SEQ ID NO: 2, and the cleavage sites are between the 143rd and 144th positions of the sequence shown in SEQ ID NO:

2.

7. A method for circularizing RNA, characterized in that, The circularization method specifically includes: transcribing any one of the constructs described in claims 1 to 5 to obtain a linear RNA molecule; performing a circularization reaction on the linear RNA molecule to obtain a circular RNA.

8. A circular RNA, characterized in that, Obtained by the RNA circularization method described in claim 7.

9. Use of any one of the constructs described in claims 1 to 5 and / or the circular RNA described in claim 8 in the preparation of drugs, vaccines, and / or cells with high expression of target genes.

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