A method for RNA circularization in vitro

Abstract

The present invention belongs to the field of biomedicine, and specifically relates to a method for RNA cyclization in vitro. Specifically, the present invention provides a nucleic acid molecule, wherein the nucleic acid molecule contains a first matching sequence, a ribozyme upstream end sequence, a sequence to be cyclized (GOI) or a restriction site, a ribozyme downstream end sequence, and a second matching sequence; RNA is prepared by the above nucleic acid molecule and cyclized to obtain circular RNA.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and specifically relates to a method for RNA cyclization in vitro. Background Art

[0002] Circular RNA was first discovered in the 1970s. With the development of related technologies, people have gradually deepened their research on this type of molecule. From the perspective of species, circular RNA has been found in viruses, yeast, archaea, humans, primates, and mice. In terms of quantity, circular RNA was only discovered sporadically in the early days. Due to the advancement of new generation high-throughput sequencing technology and molecular biology technology, it is currently predicted based on the results of bioinformatics computational analysis that the number of circular RNA is equivalent to about 1% of mRNA, or even more. They have been found in different cell lines and are species-specific and tissue-specific.

[0003] Linear RNA has the special characteristics of high difficulty in precise synthesis, easy degradation, and difficult preservation, which makes linear RNA drugs face challenges in process control, engineering assurance, large-scale preparation process, quality control and quality system. Although most endogenous circular RNAs cannot express proteins, they can play various regulatory roles in cells. Recent studies have shown that in vitro engineered circular RNA can express target proteins at high levels. At the same time, compared with linear RNA, circular RNA has a more stable structure, simpler process, more flexible delivery, and lower production and transportation storage costs. Therefore, the advantages of industrialization are more obvious, and it is an ideal platform for the development of the next generation of new drugs.

[0004] Circular RNA has obvious advantages, but its industrialization also faces many problems. At present, the circularization method and purification strategy of circular RNA are still immature, and the impact of its potential immunogenicity on vaccine development is unclear. Many unknown factors restrict the research and development and application of circular RNA. This field urgently needs an efficient, accurate and simple method for synthesizing circular RNA. Summary of the invention

[0005] The main purpose of the present invention is to provide a method for RNA cyclization in vitro. Specifically, the present invention provides the following technical solutions:

[0006] In one aspect, the present invention provides a nucleic acid molecule, wherein the nucleic acid molecule comprises a first matching sequence, a ribozyme upstream sequence, a sequence to be cyclized (GOI) or a restriction site, a ribozyme downstream sequence, and a second matching sequence;

[0007] The first matching sequence and at least 75%, 80%, 85%, 90%, 95%, 99%, 100% of the second matching sequence are reverse complementary sequences to each other;

[0008] The upstream sequence of the ribozyme has the sequence shown in SEQ ID NO.6, 8, 10, 12, 14, 16;

[0009] The downstream end sequence of the ribozyme has the sequence shown in SEQ ID NO.7, 9, 11, 13, 15, 17.

[0010] Preferably, the first matching sequence is completely complementary to the second matching sequence.

[0011] Preferably, the length of the first matching sequence or the second matching sequence can be at least 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 or more nt.

[0012] Preferably, the length of the first matching sequence or the second matching sequence can be 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 nt.

[0013] Preferably, the length of the first matching sequence or the second matching sequence may be at least 5 nt.

[0014] Preferably, the length of the first matching sequence or the second matching sequence may be 5-50 nt.

[0015] In the present invention, the "sequence to be cyclized" may contain IRES, target RNA or other elements, including but not limited to expression-promoting elements (such as kozak), spacers (such as polyA, polyAC), 5'UTR, 3'UTR and the like.

[0016] The "target RNA (target gene)" includes any naturally occurring sequence or artificially synthesized sequence, such as any viral antigen, chimeric antigen receptor or T cell receptor. The technical solution to be solved by the present invention is to obtain a circular "sequence to be cyclized" (circular RNA) more efficiently, so that the target RNA (target gene) can be stably and continuously highly expressed. In some embodiments, the target RNA may have an actual function, or may not have an actual function.

[0017] Preferably, the IRES sequence of the present invention includes an IRES sequence from any source, and the IRES sequence may include but is not limited to Taura syndrome virus, blood-sucking assassin bug virus, Theile's encephalomyelitis virus, simian virus type 40, red fire ant virus type 1, cereal aphid virus, reticuloendotheliosis virus, Forman polio virus type 1, soybean looper virus, Kashmir bee virus, human rhinovirus type 2, human immunodeficiency virus type 1, glass leafhopper virus type 1, lice P virus, hepatitis C virus, hepatitis A virus, GB hepatitis virus, foot-and-mouth disease virus, human enterovirus, equine rhinovirus, tea looper-like virus, encephalomyocarditis virus, fruit fly C virus, crucifer tobacco virus, cricket paralysis virus, bovine viral diarrhea virus type 1, black queen cell disease Virus, aphid lethal paralysis virus, avian encephalomyelitis virus, acute bee paralysis virus, hibiscus yellow ringspot virus, swine fever virus, human FGF2, human SFTPA1, human AML1, fruit fly antennae, 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, human p53, human Pim-1, mouse Rbm3, fruit fly reaper, dog Scamper, fruit fly Ubx, human UNR, mouse UtrA, human VEGF-A, human XIAP. Or the IRES type disclosed in CN112399860A and US11203767B2 patent applications. The wild-type IRES sequence can also be modified or mutated and applied to the present invention.

[0018] The term "restriction enzyme cutting site" refers to a specific sequence of bases on DNA, which can be recognized by a restriction enzyme and cut into two segments. The restriction enzyme cutting site in the present invention includes a specific sequence recognized by a restriction enzyme, and also includes a sequence that can be cut after being recognized by two restriction enzymes. For example, the restriction enzyme cutting site in a specific embodiment is GGATCCTAAGTCGAC, which can be replaced by other target sequences after being cut by BamHI and SalI.

[0019] The term "nucleic acid molecule" used interchangeably herein includes polymeric forms of nucleotides of any length, including ribonucleotides, deoxyribonucleotides or their analogs or modified forms, including single-stranded or double-stranded DNA or RNA. At the same time, the nucleic acid of the present invention (including the circular RNA prepared by the present invention) may contain modified nucleotide bases. Preferably, the modification includes phosphate backbone modification, base modification, and ribose modification.

[0020] On the other hand, the present invention also provides a vector carrying the aforementioned nucleic acid molecule.

[0021] Preferably, the vector is an expression vector.

[0022] Preferably, the vector comprises a bacterial plasmid vector, a bacteriophage vector, a yeast plasmid vector, an adenoviral vector, a retroviral vector or a lentiviral vector.

[0023] Preferably, the expression vector also includes an operably linked promoter and transcription termination sequence.

[0024] Preferably, the promoter includes T7, Sp6, T3, T7lac, araBAD, trp, lac, Ptac, pL, CMV, EF1a, SV40, PGK1, Ubc, human beta actin, CAG, TRE, UAS, Ac5, Polyhedrin, CaMKIIa, GAL1, GAL10, TEF1, GDS, ADH1, CaMV35S, Ubi, H1, and U6 promoters.

[0025] Preferably, the promoter may be an in vitro promoter, such as T7, Sp6, or T3 promoter.

[0026] In another aspect, the present invention also provides a cell comprising the above nucleic acid molecule or vector.

[0027] In the present application, the term "cell" generally refers to an individual cell, cell line or cell system that may or already contain a nucleic acid molecule or vector according to the present invention. The cell also includes its progeny cells, which may not necessarily be completely identical to the original parent cell in morphology or genome due to natural, accidental or intentional mutations, but only need to contain the nucleic acid molecule or vector according to the present invention. The cell can be obtained by in vitro transfection of cells using the vectors described in the present application.

[0028] Specifically, the cells include CHO cells, 293 cells, CHO-K1 cells, Caco2 cells, U2-OS cells, NIH3T3 cells, NSO cells, SP2 cells, CHO-S cells, DG44 cells, K-562 cells, U-937 cells, MRC5 cells, IMR90 cells, Jurkat cells, HepG2 cells, HeLa cells, HT-1080 cells, HCT-116 cells, Hu-h7 cells, Huvec cells, Molt 4 cells, etc.

[0029] In a specific embodiment, the cell is an isolated cell or a commercial cell line.

[0030] In another aspect, the present invention provides a composition comprising the above-mentioned nucleic acid molecule, vector, cell or circular RNA.

[0031] On the other hand, the present invention provides the use of any one of the above nucleic acid molecules, vectors, and cells in preparing sequences to be cyclized, and increasing the content or expression of target RNA in cells.

[0032] More specifically, a circular sequence to be cyclized is prepared.

[0033] Specifically, the amount or expression is increased by at least 1.5 times, 5 times, 10 times, 50 times, 100 times, 500 times, 1000 times, 1500 times, 2000 times, 2500 times, 3000 times, 3500 times, 4000 times, 5000 times or more of the original amount or expression.

[0034] On the other hand, the present invention provides the use of any one of the above nucleic acid molecules, vectors, and cells in the preparation of a product for increasing the content or expression of a target RNA in a cell.

[0035] On the other hand, the present invention provides a method for preparing circular RNA, wherein the method circularizes RNA containing a first matching sequence, a ribozyme upstream end sequence, a sequence to be circularized (GOI) or a restriction site, a ribozyme downstream end sequence, and a second matching sequence;

[0036] Alternatively, the aforementioned vector is transcribed; or the aforementioned cell is cultured.

[0037] More specifically, the RNA is prepared by constructing the aforementioned vector and performing an in vitro transcription reaction; obtaining a linear DNA template containing a promoter and performing in vitro transcription with a corresponding RNA polymerase is conventionally known in the art.

[0038] Preferably, the cyclization treatment is carried out in the presence of GTP and / or magnesium ions (Mg 2+ ) in a buffer solution; or, incubating in a buffer solution containing excess GMP and / or magnesium ions;

[0039] More specifically, the cyclization treatment was performed by incubation in the following buffer containing GTP: 50 mM Tris-HCl, 10 mM MgCl2, 1 mM DTT, pH 7.5.

[0040] More preferably, the final concentration of GTP is 2 mM.

[0041] Most preferably, the incubation step is at 70°C for 5 min, followed by incubation at 55°C for 15 min.

[0042] Preferably, the method further comprises the step of purifying the product to improve its purity.

[0043] Preferably, the cells are ex vivo cells.

[0044] Preferably, the method is performed in vitro.

[0045] Preferably, the method is for non-therapeutic purposes.

[0046] Preferably, the method further comprises the step of purifying the product, which can be done by any purification method, such as HPLC.

[0047] The "circular" or "cyclization" mentioned in the present invention can express the same meaning in the present invention. Any RNA presenting a circular structure can be called "circular RNA" or "circularized RNA". Whether the sequence to be cyclized is circular can be confirmed by detecting whether it is connected end to end. For example, in a specific embodiment of the present invention, the connection point is confirmed after primer amplification and sequencing, which means that it is determined that the target gene is connected end to end to form a circle.

[0048] On the other hand, the present invention provides circular RNA (circular sequence to be cyclized) prepared by the above method.

[0049] In another aspect, the present invention provides a pharmaceutical composition comprising the above circular RNA.

[0050] Preferably, the pharmaceutical composition also includes pharmaceutically acceptable adjuvants, carriers, excipients, glidants, sweeteners, diluents, preservatives, dyes, colorants, flavor enhancers, wetting agents, dispersants, suspending agents, stabilizers, isotonic agents, surfactants or emulsifiers that can be used for humans or livestock.

[0051] Preferably, the pharmaceutical composition can be formulated for parenteral or non-parenteral administration in a variety of ways. In one embodiment, the composition can be formulated for infusion or intravenous administration. The compositions disclosed herein can be provided, for example, as a sterile liquid preparation, such as an isotonic aqueous solution, emulsion, suspension, dispersion or viscous composition, which can be buffered to the required pH. Preparations suitable for oral administration can include liquid solutions, capsules, sachets, tablets, lozenges and lozenges, liquid suspension powders and emulsions in suitable liquids.

[0052] Preferably, the diseases targeted by the pharmaceutical composition include diseases caused by low expression of one or more genes, including proliferative diseases, autoimmune diseases, allergic diseases, inflammatory diseases, transplant rejection, viral infectious diseases or cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 This is a schematic diagram of the structure of the RNA cyclization system.

[0054] Figure 2 This is a diagram showing the results of RNA cyclization system cyclization efficiency detection. A is a cyclization system designed using the T4 phage intron sequence, and B is a cyclization system designed using the Anabaena intron sequence.

[0055] Figure 3 This is the separation pattern of the cyclization products of the intron RNA cyclization system of Anabaena by HPLC.

[0056] Figure 4 This is a graph showing the purity analysis results of the collected main peak samples.

[0057] Figure 5 This is the result of DNA sequencing to verify the circular RNA splicing site.

[0058] Figure 6 This is a graph showing the expression results of circular RNA prepared by the RNA cyclization system. DETAILED DESCRIPTION

[0059] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes based on the technical solution and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

[0060] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources. Method for preparing RNA:

[0061] 1. For plasmid extraction, refer to the instructions of Tiangen Plasmid Extraction Kit.

[0062] 2. Plasmid linearization can be performed using a single restriction site reserved in the plasmid DNA. Please refer to the instructions for Takara restriction endonuclease.

[0063] 3. Linearized plasmid purification:

[0064] 1) Add water to 500 μL of the digested product, add an equal volume of organic solvent (phenol / chloroform / isoamyl alcohol 25:24:1), mix well, and centrifuge at 4°C, 12,000 rpm for 15 min.

[0065] 2) After centrifugation, transfer the DNA in the upper aqueous phase to a new EP tube, aspirate about 400 μL. Add an equal volume of chloroform, a total of 800 μL, vortex to mix. Centrifuge at 4°C, 12,000 rpm for 15 minutes.

[0066] 3) Take 300 μL of the supernatant and put it into a new centrifuge tube, add 30 μL of 3M sodium acetate, and then add twice the volume (660 μL) of pre-cooled anhydrous ethanol. Mix well and precipitate at -20℃ for at least 30 minutes.

[0067] 4) Centrifuge at 14000 rpm for 15 min at 4°C to collect the DNA precipitate.

[0068] 5) After removing the supernatant, slowly add 750 μL of ice-cold 70% ethanol to the EP tube, shake gently, and centrifuge at 4°C, 14,000 rpm for 5 minutes to collect the DNA precipitate.

[0069] 6) Repeat step 5);

[0070] 7) Discard the supernatant, invert the centrifuge tube, and air dry. Add 20 μL of RNase-free water to dissolve.

[0071] 4. In vitro transcription:

[0072] 1) Prepare the following system in an EP tube using NEB's in vitro transcription kit:

[0073] Element Quantity (μL) 10X Reaction buffer 2 ATP 2 GTP 2 UTP 2 CTP 2 T7 RNA polymerase Mix 2 Template DNA (1 μg / μL) 1 RNase-free water 7 Total 20

[0074] 2) Gently mix and centrifuge to the bottom of the tube. Place the reactants in a PCR instrument and incubate at 37°C for 2 hours.

[0075] 3) Add DNase I (Takara) to degrade the DNA template, prepare the reaction system according to the table below, mix well, and place in a 37°C metal bath for 20-30 minutes.

[0076] Element Quantity (μL) Total RNA 20 10X DNase I Buffer 10 Nuclease free water 60 Recombinant DNAse I 8 RNase inhibitor 2 Total Rection Volume 100

[0077] 4) After the incubation, add 2.5 μL of 0.5 M EDTA to the reaction mixture, mix well, centrifuge, place in an 80°C water bath for 2 min to inactivate DNase I, and then perform RNA column purification to obtain RNA.

[0078] Example 1: Preparation of circular RNA

[0079] The P9.1 position of the I-type intron of the tRNA gene of Anabaena (ANA) or the td gene of T4 bacteriophage was opened according to Figure 1 The schematic diagram shows the design of the Anabaena RNA cyclization system and the T4 phage RNA cyclization system, wherein a BamHI-SalI restriction site (GGATCCTAAGTCGAC) is retained, and the DNA sequences are shown in SEQ ID NOs. 1 and 2. The restriction sites in SEQ ID NOs. 1 and 2 are replaced with CVB3-kozak-EGFP for subsequent experiments, and the sequences of CVB3, kozak, and EGFP are shown in SEQ ID NOs. 3-5, respectively.

[0080] More specifically, the SEQ ID NO.1 is composed of: a first matching sequence-Ana-P9.1_upstream shown in SEQ ID NO.6-polyAC-BamHI-SalI restriction site-polyAC-Ana-P9.1_downstream shown in SEQ ID NO.7-a second matching sequence complementary to the first matching sequence;

[0081] The SEQ ID NO.2 is composed of the first matching sequence-T4-P9.1 shown in SEQ ID NO.8_upstream-polyAC-BamHI-SalI restriction site-polyAC-T4-P9.1 shown in SEQ ID NO.9_downstream-the second matching sequence complementary to the first matching sequence.

[0082] Opening the loop at the P9 position of the type I intron of Anabaena (ANA) or T4 can obtain a cyclization system with SEQ ID NO.10-11, SEQ ID NO.12-13 as the core structure. Opening the loop at the P9.2 position of the type I intron of Anabaena (ANA) or T4 can obtain a cyclization system with SEQ ID NO.14-15, SEQ ID NO.16-17 as the core structure.

[0083] After RNA is prepared according to the aforementioned method for preparing RNA, a cyclization reaction is performed. The specific steps are to first prepare 100 μL of the cyclization system, add cyclization buffer (final concentration of 50 mM Tris-HCl, 10 mM MgCl2, 1 mM DTT, pH 7.5) and GTP (final concentration of 2 mM) to 50-100 μg of precursor RNA. The insufficient part is made up with RNase-free water. Incubate at 70°C for 5 minutes, and then incubate at 55°C for 15 minutes. Finally, RNA column purification is performed to obtain a reaction product containing circular RNA.

[0084] The electrophoresis analysis results showed that circular RNA was successfully obtained, and grayscale analysis showed that the circularization efficiency of the T4 phage RNA circularization system was about 30% ( Figure 2 A). The cyclization efficiency of the Anabaena RNA cyclization system is about 90% ( Figure 2 B).

[0085] Example 2: Purity analysis of circular RNA

[0086] The cyclization products obtained by the RNA cyclization system of Anabaena were separated and purified by high performance liquid chromatography (HPLC). The separation and purification adopted a 150mM phosphate isogradient elution method, and the separation spectrum showed that the cyclization products could be effectively separated ( Figure 3 ).

[0087] collect Figure 3 The corresponding main peak sample was analyzed by HPLC, and the results showed that the impurities in the cyclization reaction had been basically removed ( Figure 4 ).

[0088] Example 3: Splice site verification

[0089] The circular RNA collected in Example 2 was subjected to a reverse transcription experiment, and the reverse transcription was performed with reference to the full-form gold reverse transcription kit. The product obtained by PCR amplification was inserted between EcoR1 and HidIII of the PUC57-kan vector by homologous recombination, and DNA sequencing was performed. The results showed that the circular RNA splicing reaction occurred at the expected position ( Figure 5 ).

[0090] Example 4: Circular RNA Expression Analysis

[0091] After desalting and precipitation, the circular RNA obtained in Example 2 was transformed into 293F cells and cultured for 24 hours to observe the expression of fluorescent protein. The results showed that circular RNA can efficiently express green fluorescent protein ( Figure 6 ).

[0092] The above contents are further detailed descriptions of the present invention in combination with specific preferred embodiments, and it cannot be determined that the specific implementation of the present invention is limited to these descriptions. For ordinary technicians in the technical field to which the present invention belongs, several simple deductions or substitutions can be made without departing from the concept of the present invention, which should be regarded as falling within the protection scope of the present invention.

Claims

1. A nucleic acid molecule, which is composed of a first matching sequence, a ribozyme upstream sequence, a sequence to be cyclized or a restriction site, a ribozyme downstream sequence, and a second matching sequence complementary to the first matching sequence; The nucleic acid molecule comprising the restriction site is shown in SEQ ID NO.1 or SEQ ID NO.2; The nucleic acid molecule comprising the sequence to be cyclized is a nucleic acid molecule obtained by replacing the restriction site in the nucleic acid molecule shown in SEQ ID NO.1 or SEQ ID NO.2 with the sequence to be cyclized; The sequence to be cyclized contains target RNA.

2. The nucleic acid molecule according to claim 1, wherein the sequence to be cyclized further comprises one or more of an IRES, an expression-promoting element, a spacer, a 5'UTR, and a 3'UTR.

3. The nucleic acid molecule of claim 2, wherein the IRES comprises a nucleic acid from Taura syndrome virus, blood-sucking assassin bug virus, Theile's encephalomyelitis virus, simian virus type 40, fire ant virus type 1, aphid virus, reticuloendotheliosis virus, Forman polio virus type 1, soybean looper virus, Kashmir bee virus, human rhinovirus type 2, human immunodeficiency virus type 1, glass leafhopper virus type 1, lice P virus, hepatitis C virus, hepatitis A virus, GB hepatitis virus, foot-and-mouth disease virus, human enterovirus, equine rhinovirus, tea looper-like virus, encephalomyocarditis virus, fruit fly C virus, crucifer tobacco virus, cricket paralysis virus, bovine viral diarrhea virus type 1, black queen cell virus, aphid lethal paralysis virus, avian encephalitis virus, IRES of myelitis virus, acute bee paralysis virus, hibiscus yellow ringspot virus, classical swine fever virus, human FGF2, human SFTPA1, human AML1, Drosophila antennae, 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, human p53, human Pim-1, mouse Rbm3, Drosophila reaper, canine Scamper, Drosophila Ubx, human UNR, mouse UtrA, human VEGF-A, or human XIAP.

4. A vector carrying the nucleic acid molecule according to any one of claims 1 to 3. The vector according to claim 4 , which is an expression vector.

6. The vector according to claim 4, wherein the vector is a bacterial plasmid vector, a phage vector, a yeast plasmid vector, an adenovirus vector, a retrovirus vector or a lentivirus vector.

7. The vector according to claim 5, further comprising an operably linked promoter and transcription termination sequence.

8. The vector according to claim 7, wherein the promoter is T7, Sp6 or T3 promoter.

9. A cell comprising the nucleic acid molecule according to any one of claims 1 to 3 or the vector according to any one of claims 4 to 8.

10. The cell according to claim 9, wherein the cell is a CHO cell, a 293 cell, a CHO-K1 cell, a Caco2 cell, a U2-OS cell, a NIH 3T3 cell, a NSO cell, a SP2 cell, a CHO-S cell, a DG44 cell, a K-562 cell, a U-937 cell, a MRC5 cell, an IMR90 cell, a Jurkat cell, a HepG2 cell, a HeLa cell, a HT-1080 cell, a HCT-116 cell, a Hu-h7 cell, a Huvec cell or a Molt 4 cell. The cell according to claim 9 , which is an isolated cell or a commercial cell line.

12. A composition comprising the nucleic acid molecule of any one of claims 1 to 3, the vector of any one of claims 4 to 8, or the cell of any one of claims 9 to 11.

13. Use of any one of the nucleic acid molecules of claims 1 to 3, the vectors of claims 4 to 8, and the cells of claims 9 to 11 in preparing sequences to be cyclized, or in increasing the target RNA content or expression in cells for non-therapeutic purposes.

14. A method for preparing circular RNA, the method comprising circularizing an RNA comprising the first matching sequence, a ribozyme upstream sequence, a sequence to be cyclized or a restriction site, a ribozyme downstream sequence, and a second matching sequence as claimed in claim 1; Alternatively, the vector according to any one of claims 4 to 8 is transcribed; or the cell according to any one of claims 9 to 11 is cultured.

15. The method of claim 14, wherein the cyclization treatment is performed by incubating in a buffer containing GTP and / or magnesium ions; Alternatively, the incubation is carried out in a buffer containing excess GMP and / or magnesium ions.

16. The method of claim 15, wherein the buffer is 50 mM Tris-HCl, 10 mM MgCl2, 1 mM DTT, pH 7.

5.

17. The method of claim 15, wherein the final concentration of GTP is 2 mM.

18. The method of claim 15, wherein the incubation step is performed at 70°C for 5 min and then at 55°C for 15 min.

19. The method of claim 15, further comprising the step of purifying the product to increase its purity.

20. The circular RNA prepared by the method according to any one of claims 14 to 19.

21. A pharmaceutical composition comprising the circular RNA according to claim 20.

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