Novel circular RNA synthesis method using RNA ligase

Abstract

The present invention relates to a novel circular RNA synthesis method using RNA ligase. More specifically, using a circular RNA precursor having a specific structure according to the present invention, the 5' end triphosphate is converted into a hydroxyl group (-OH) by phosphatase and cyclic phosphate is introduced into the 3' end (2',3'-cyclic phosphate) by ribozyme. Subsequently, the ends are linked using RtcB RNA ligase, thereby enabling the production and synthesis of longer circular RNAs with significantly higher efficiency compared to conventional methods.

Description

A novel circular RNA synthesis method using RNA ligase

[0001] The present invention relates to a novel circular RNA synthesis method using RNA ligase.

[0002] Circular RNA (Circular RNA) is a single-stranded RNA with a covalent bond between the 5' and 3' ends. It is known to be generated through "back splicing" in various eukaryotes. Due to its structural characteristic of having no ends, circular RNA is not easily degraded by exonucleases, so it exists in the body much more stably than messenger RNA (mRNA) of similar size. Through RNA-seq data and newly developed bioinformatics approaches, tens of thousands of types of circRNAs have been identified in various organisms, and eukaryotic circular RNAs have been found to have various molecular and cellular regulatory functions (Chen LL. The biogenesis and emerging roles of circular RNAs. Nat Rev Mol Cell Biol. 2016 Apr;17(4):205-11.). For example, circular RNAs are known to regulate gene expression by binding to microRNAs or directly binding to target proteins.

[0003] Meanwhile, mRNA holds great potential in various fields, including vaccines and therapeutics, and thus, pharmaceutical development utilizing it has been active recently. However, mRNA is easily degraded in vivo by exonucleases and has a relatively short half-life, limiting its effectiveness. To overcome these limitations, research is being conducted to alter the terminal structure of mRNA or to enhance stability by adding a poly(A) tail. In a similar vein, US 10,953,033 discloses circular RNA for gene expression purposes, based on its excellent in vivo stability.

[0004] Circular RNA can be synthesized artificially, both in vitro and in vitro. Known methods for in vitro synthesis include "chemical methods," "methods using Group I intron ribozymes," and "methods using RNA ligases" (Obi and Chen. The design and synthesis of circular RNAs. Methods. 196:85-103. 2021). Chemical methods have the disadvantage of being expensive and limiting the size of circular RNA that can be produced. Methods using ribozymes, such as Group I introns, have the advantage of achieving relatively high circularization efficiency, but can be problematic when used as therapeutics due to the disadvantage of inducing stronger immunogenicity than methods using RNA ligases (Liu et al., RNA circles with minimized immunogenicity as potent PKR inhibitors, Mol Cell. 82(2):420-434. 2021). In addition, the ribozyme method has the disadvantage of generating a high proportion of open (nicked) circular RNA during the synthesis process and making it difficult to remove open circular RNA during the purification process (Wesselhoeft et al., RNA circularization diminishes immunogenicity and can extend translation duration in vivo, Mol Cell. 74(3):508-520. 2019). The RNA ligase method mainly uses T4 RNA ligase 1, T4 RNA ligase 2, and tRNA ligase to induce circularization of linear RNA, but has the disadvantage of showing a superior circularization efficiency than chemical methods but a lower circularization efficiency than the ribozyme method. On the other hand, the RNA ligase method has the advantage of not generating open circular RNA and showing relatively low immunogenicity.

[0005] Accordingly, the present inventors studied a method for significantly improving circularization efficiency while not generating open circular RNA and inducing low immunogenicity in the production of circular RNA for the purpose of protein expression, and completed the present invention.

[0006] An object of the present invention is to provide a circular RNA precursor.

[0007] In addition, the present invention provides a recombinant nucleic acid vector for producing a circular RNA precursor.

[0008] In addition, the present invention provides a method for producing circular RNA.

[0009] In addition, the present invention provides a use of a circular RNA precursor.

[0010] To achieve the above purpose, the present invention provides a circular RNA precursor having a structure of 5'-5' homology region - gene of interest (GOI) - 3' homology region - ribozyme - 3'.

[0011] Additionally, the present invention provides a recombinant nucleic acid vector for producing the circular RNA precursor.

[0012] In addition, the present invention provides a method for producing the circular RNA.

[0013] Additionally, the present invention provides the use of a circular RNA precursor for use in the production of circular RNA.

[0014] By using a circular RNA precursor having a specific structure according to the present invention, a 5'-terminal triphosphate is converted into a hydroxyl group (-OH) by a phosphatase, a 3'-terminal cyclic phosphate (2', 3'-cyclic phosphate) is induced by a ribozyme, and this is linked by RtcB RNA ligase, thereby enabling the production / synthesis of a longer circular RNA with significantly higher efficiency than before.

[0015] Figure 1 is a schematic diagram showing a method for synthesizing circular RNA of the present invention.

[0016] Figure 2 is a diagram showing the structure of the precursor RNA of the present invention.

[0017] Figure 3 is a diagram showing the structure of a precursor RNA (racRNA) for making a conventional circular RNA.

[0018] Figure 4 is a diagram analyzing the transcription efficiency according to the starting sequence at the 5' end of the 5' homology region of the precursor rpcRNA template structure at the transcription stage.

[0019] Figure 5 is a diagram showing the circular RNA synthesis process of rpcRNA and racRNA.

[0020] Figure 6 is a diagram showing the results of circular RNA synthesis and the synthesis efficiency synthesized through the process of Figure 4.

[0021] Figure 7 is a diagram showing the circular RNA synthesis efficiency according to the concentration of GTP when synthesizing circular RNA using E. coli-derived RtcB RNA ligase.

[0022] Figure 8 is a diagram showing the circular RNA synthesis efficiency according to the concentration of Mn (GTP concentration: 20 nM) when synthesizing circular RNA using E. coli-derived RtcB RNA ligase.

[0023] Figure 9 is a diagram showing the circular RNA synthesis efficiency according to the concentration of GTP and Mn when synthesizing circular RNA using E. coli-derived RtcB RNA ligase.

[0024] Figure 10 is a diagram showing the circular RNA synthesis efficiency according to the ligation reaction time when synthesizing circular RNA using E. coli-derived RtcB RNA ligase.

[0025] Figure 11 is a diagram showing the efficiency of circular RNA synthesis according to the ligation reaction temperature when synthesizing circular RNA using E. coli-derived RtcB RNA ligase.

[0026] Figure 12 is a diagram verifying the synthesis of circular RNA by treating circular RNA synthesized using E. coli-derived RtcB RNA ligase with RNase R.

[0027] Figure 13 is a diagram showing the efficiency of circular RNA synthesis according to the concentration of GTP during circular RNA synthesis using human-derived RtcB RNA ligase.

[0028] Figure 14 is a diagram showing the efficiency of circular RNA synthesis according to the concentration of NaCl during circular RNA synthesis using human-derived RtcB RNA ligase.

[0029] Figure 15 is a diagram showing the circular RNA synthesis efficiency according to the concentration of Mn when synthesizing circular RNA using human-derived RtcB RNA ligase.

[0030] Figure 16 is a diagram showing the efficiency of circular RNA synthesis according to the concentration of DTT during circular RNA synthesis using human-derived RtcB RNA ligase.

[0031] Figure 17 is a diagram confirming the circular RNA synthesis efficiency according to the concentration of a 1:1 mixture of human-derived RtcB RNA ligase and Archease during circular RNA synthesis.

[0032] Figure 18 is a diagram confirming the circular RNA synthesis efficiency according to the concentration combination of human-derived RtcB RNA ligase and Archease during circular RNA synthesis.

[0033] Figure 19 is a diagram showing the efficiency of circular RNA synthesis according to the ligation reaction temperature when synthesizing circular RNA using RtcB RNA ligase derived from Thermus thermophilus.

[0034] Figure 20 is a diagram showing the efficiency of circular RNA synthesis according to ligation reaction time when synthesizing circular RNA using RtcB RNA ligase derived from Thermus thermophilus.

[0035] Figure 21 is a diagram showing the efficiency of circular RNA synthesis using E. coli-derived RtcB RNA ligase according to the size (nt) of precursor RNA.

[0036] Figure 22 is a diagram showing the efficiency of circular RNA synthesis using human-derived RtcB RNA ligase according to the size (nt) of precursor RNA.

[0037] Figure 23 is a diagram showing the efficiency of circular RNA synthesis using RtcB RNA ligase derived from Thermus thermophilus according to the size (nt) of precursor RNA.

[0038] Figure 24 is a diagram showing the efficiency of circular RNA synthesis using E. coli-derived RtcB RNA ligase according to the type of ribozyme contained in the precursor RNA.

[0039] Figure 25 is a diagram confirming the efficiency of circular RNA synthesis using human-derived RtcB RNA ligase according to the type of ribozyme included in the precursor RNA.

[0040] Figure 26 is a diagram showing the efficiency of circular RNA synthesis using RtcB RNA ligase derived from Thermus thermophilus according to the type of ribozyme included in the precursor RNA.

[0041] Figure 27 is a diagram confirming the circular RNA synthesis efficiency according to the sequence of the homologous region in the structure of rpcRNA.

[0042] Figure 28 is a diagram showing the structure of a precursor RNA template (rpc-sp-IH-sp template) that additionally includes an internal homology region and a spacer in rpcRNA.

[0043] Figure 29 shows the efficiency of circular RNA synthesis using E. coli, human or Thermus thermophilus-derived RtcB RNA ligase from precursor RNAs that additionally contain internal homology regions and spacers to rpcRNA.

[0044] Figure 30 is a diagram showing in vitro confirmation of the expression of the target protein of the CDS contained in rpcRNA and circular RNA.

[0045] Figure 31 is a diagram confirming in vivo the expression of the target protein of the CDS contained in rpcRNA and circular RNA.

[0046] Hereinafter, the present invention will be described in detail with reference to the attached drawings, using exemplary embodiments of the present invention. However, the following exemplary embodiments are provided as examples of the present invention. If a detailed description of a technology or configuration well known to those skilled in the art is judged to unnecessarily obscure the gist of the present invention, such detailed description may be omitted, and the present invention is not limited thereby. The present invention is capable of various modifications and applications within the scope of the following claims and equivalents interpreted therefrom.

[0047] Additionally, the terminology used in this specification is intended to appropriately express preferred embodiments of the present invention, and may vary depending on the intent of the user or operator, or the customs of the field to which the present invention pertains. Therefore, the definitions of these terms should be determined based on the contents throughout this specification. Throughout this specification, when a part is said to "include" a certain component, unless specifically stated otherwise, this does not mean that other components are excluded, but rather that other components may be included.

[0048] Unless otherwise indicated, nucleic acids are written in a 5'→3' orientation from left to right. Numerical ranges recited within the specification are inclusive of the numbers defining the range and include each integer or any non-integer fraction within the defined range.

[0049] Unless otherwise defined, all technical terms used in this invention have the same meaning as commonly understood by those skilled in the art. While preferred methods and samples are described herein, similar or equivalent methods are also included within the scope of the present invention. The contents of all publications cited as references herein are incorporated herein by reference.

[0050]

[0051] In one aspect, the present invention relates to a circular RNA precursor having a structure of 5'-5' homology region-gene of interest (GOI)-3' homology region-ribozyme-3', wherein the 5' homology region includes a sequence that is reverse complementary to the 3' homology region.

[0052] In one embodiment, the 5' homology region may be reverse complementary to the 3' homology region.

[0053] In one embodiment, the reverse complementary portion of the sequence of the 5' homology region and the 3' homology region may be at least 50%, at least 60%, at least 70%, or at least 80% of the sequence of the 5' homology region.

[0054] In one embodiment, the 5' homology region may comprise a sequence that is reverse complementary to the sequence of the 3' homology region, and optionally may comprise 1 to 5 mismatched nucleotide pairs in the reverse complementary sequence.

[0055] In one embodiment, the 5' homology region may comprise a sequence that is reverse complementary to the sequence of the 3' homology region, and the length of the reverse complementary sequence may be 5 to 50 nt.

[0056] In one embodiment, the 5' homology region may additionally comprise GGX (wherein X is A, U, C or G) at the 5' end, and GGX may be GGC or GGG (Guanine).

[0057] In one embodiment, the addition of GGX to the 5' end can increase the transcription efficiency from the template encoding it to the precursor RNA.

[0058] In one embodiment, the 5' homology region may comprise the base sequence of SEQ ID NO: 1.

[0059] In one embodiment, the 5' homology region may be reverse complementary to the 3' homology region, excluding the GGX at the 5' end.

[0060] In one embodiment, the 5' homology region may include any one sequence selected from the group consisting of SEQ ID NOs: 1 to 4, and may include any one sequence selected from the group consisting of SEQ ID NOs: 2 to 4 in the 3' direction of GGX present at the 5' end of the 5' homology region.

[0061] In one embodiment, the 3' homology region may comprise any one sequence selected from the group consisting of SEQ ID NOs: 5 to 10.

[0062] In one embodiment, the circular RNA precursor may comprise a 5' homology region comprising the base sequence of SEQ ID NO: 2 and a 3' homology region comprising the base sequence of SEQ ID NO: 5; a 5' homology region comprising the base sequence of SEQ ID NO: 2 and a 3' homology region comprising the base sequence of SEQ ID NO: 6; a 5' homology region comprising the base sequence of SEQ ID NO: 2 and a 3' homology region comprising the base sequence of SEQ ID NO: 7; a 5' homology region comprising the base sequence of SEQ ID NO: 3 and a 3' homology region comprising the base sequence of SEQ ID NO: 8; or a 5' homology region comprising the base sequence of SEQ ID NO: 4 and a 3' homology region comprising the base sequence of SEQ ID NO: 9.

[0063] In one embodiment, the circular RNA precursor may have a triphosphate (PPP) at the 5' end.

[0064] In one embodiment, the gene of interest (GOI) may include a gene encoding a gene or protein of interest, for example, a coding sequence (CDS), and the length of the gene of interest may be 100 to 9000 nt.

[0065] In one embodiment, the target gene may include an IRES (internal ribosome entry site) region at the 5' end, and the IRES may be Taura syndrome virus, Triatoma virus, Theiler's encephalomyelitis virus, Simian Virus 40, Solenopsis invicta virus 1, Rhopalo- siphum padi virus, Reticuloendotheliosis virus, Human poliovirus 1, Plautia stali intestine virus, Kashmir bee virus, Human rhinovirus 2, Homalodisca coagulata virus-1, Human Immunodeficiency Virus type 1, Homalodisca coagulata virus-1, Himetobi P virus, Hepatitis C virus, Hepatitis A virus, Hepatitis GB virus, Foot and mouth disease virus, Human enterovirus 71, Equine rhinitis virus, Ectropis obliqua picorna-like virus, Encephalomyocarditis virus, Drosophila C Virus, Human coxsackievirus B3, Crucifer tobamovirus, Cricket paralysis virus, Bovine viral diarrhea virus 1, Black Queen Cell Virus, Aphid lethal paralysis virus, Avian encephalomyelitis virus, Acute bee paralysis virus, Hibiscus chlorotic ringspot virus, Classical swine fever virus, Human FGF2, Human SFTPA1,Human AML1 / RUNX1, Drosophila antennapedia, Human AQP4, Human ATIR, Human BAG-1, Human BCL2, Human BiP, Human c-IAP1, Human c-myc, Human eIF4G, Mouse NDST4L, Human LEF1, Mouse HIF1 alpha, Human n.myc, Mouse Gtx, Human p27kip1, Human PDGF2 / c-sis, Human p53, Human Pim-1, Mouse Rbm3, Drosophila reaper, Canine Scamper, Drosophila Ubx, Human UNR, Mouse UtrA, Human VEGF-A, Human XIAP, Drosophila hairless, S. cerevisiae TFIID, S. cerevisiae YAP1, tobacco etch virus, turnip crinkle virus, EMCV-A, EMCV-B, EMCV-Bf, EMCV-Cf, EMCV pEC9, Picobirnavirus, HCV QC64, Human Cosavirus E / D, Human Cosavirus F, Human Cosa- virus JMY, Rhinovirus NAT001, HRV14, HRV89, HRVC- 02, HRV-A21, Salivirus A SH1, Salivirus FHB, Salivirus NG-J1, Human Parechovirus 1, Crohivirus B, Ye-3, Rosa- virus M-7, Shanbavirus A, Pasivirus A, Pasivirus A 2, Echovirus E14, Human Parechovirus 5, Aichi Virus, Hepa- titis A Virus HA16, Phopivirus, CVA10, Enterovirus C, Enterovirus D, Enterovirus J, Human Pegivirus 2, GBV-C GT110,GBV-C K1737, GBV-C Iowa, Pegivirus A 1220, Pasivirus A 3, Sapelovirus, Rosavirus B, Bakunsa Virus, Tremovirus A, Swine Pasivirus 1, PLV-CHN, Pasivirus A, Sicinivirus, Hepacivirus K, Hepacivirus A, BVDV1, Border Disease Virus, BVDV2, CSFV-PK15C, SF573 Dicistrovi-rus, Hubei Picoma-like Virus, CRPV, Salivirus A BN5, Salivirus A BN2, Salivirus A 02394-01, Salivirus A GUT, Salivirus A CH, Salivirus A SZ1, Salivirus FHB, Salivirus NG-J1, Salivirus A YZ01, Salivirus A YZ02, Salivirus A YZ03, Salivirus It may be the whole, part or variant of one or more virus-derived sequences selected from the group consisting of A FUAN-01, Salivirus sp isolate ETH, Salivirus A Hu / Baja-5332, Salivirus sp isolate CHN / XJ / env, CVB3, CVB1, Echovirus 7, CVBS, EVA71, CVA3, CVA12 and EV24.

[0066] In one embodiment, the IRES may be an aptamer for elF4G.

[0067] In one embodiment, the circular RNA precursor may further include a 5' internal homology region in the 3' direction of the 5' homology region, and a 3' internal homology region in the 5' direction of the 3' homology region, wherein the 5' internal homology region may include a sequence that is reverse complementary to the 3' internal homology region, thereby increasing the efficiency of synthesizing a long circular RNA.

[0068] In one embodiment, the 5' internal homology region may comprise the base sequence of SEQ ID NO: 10.

[0069] In one embodiment, a spacer region may be additionally included in the 5' and 3' directions of the internal homology region, and the spacer may include a base sequence of SEQ ID NO: 11 or 12.

[0070] In one embodiment, the ribozyme may be a small ribozyme of 30 to 150 nt, and may be Twister, Twister Sister, Hatchet, Pistol, Hammerhead, Hairpin, Hepatitis Delta Virus (HDV), Neurospora Varkud Satellite (VS), Vg1, glucosamine-6-phosphate synthase (glmS) or an engineered synthetic ribozyme, or a variant thereof, and may include the base sequence of SEQ ID NO: 13.

[0071] In one embodiment, the circular RNA precursor may additionally comprise a label at the 3' end, which may be a PolyA, PolyG, PolyU or PolyC sequence.

[0072] In one embodiment, the circular RNA precursor may be about 250 to 10,000 nt in size.

[0073] In one embodiment, the circular RNA precursor may comprise a nucleotide analogue in which the sugar or backbone of one or more nucleotides is modified.

[0074] In one embodiment, the modification of the sugar may be such that the hydroxyl group (-OH) at the 2' carbon position of ribose is replaced with a methyl group (-CH3) (OMe), a methoxy group (-OCH3), an amine group (-NH2), a fluorine (-F) (fluoro), an O-2-methoxyethyl group (methoxyethyl, MOE), an O-propyl group, an O-2-methylthioethyl group, an O-3-aminopropyl group, an O-3-dimethylaminopropyl group, an ON-methylacetamido group, or an O-dimethylamidooxyethyl group, and it is more preferable that the 2'-hydroxyl group of ribose is replaced with 2'-MOE (2'-O-methoxyethyl), 2'-OMe (2'-O-Methyl), or 2'-F (fluoro).

[0075] In one embodiment, the modification of the backbone may be such that the phosphate backbone of the nucleotide is modified with phosphorothioate, phosphorodithioate, methyl phosphonate, alkylphosphonate, phosphoroamidate or boranophosphate, with phosphorothioate being more preferred.

[0076] The term "reverse complementary sequence" used in the present invention refers to a sequence in which a base sequence indicated in the 5' to 3' direction and a base sequence indicated in the 3' to 5' direction are complementary sequences indicated in the 5' to 3' direction. Generally, the base sequence of a gene is indicated in the 5' to 3' direction from the position of the promoter, and during transcription, the complementary sequence of the base sequence indicated in the 5' to 3' direction, that is, the 3' to 5' sequence, is used as a template, and as a result, the 5' to 3' sequence from the direction of the promoter position becomes a sequence that matches the sequence of the mRNA transcript. At this time, the sequence identical to the mRNA is called the sense sequence, and the sequence complementary to the sense sequence is called the antisense sequence. At this time, the sequence of the strand used as a template is called the reverse complementary sequence or the reverse complementation sequence in the 5' to 3' direction. That is, when the base sequence is indicated as 5'-AAC ACG GAC-3', its reverse complement sequence is 5'-GUC CGU GUU-3'.

[0077] The term "coding sequence (CDS)" used in the present invention means a sequence that is translated into a protein.

[0078] The term "circular RNA precursor" as used in the present invention is a linear single-stranded ribonucleotide before circularization.

[0079] In one aspect, the present invention relates to a recombinant nucleic acid vector for producing a circular RNA precursor of the present invention.

[0080] In one embodiment, the recombinant nucleic acid vector may further comprise an RNA polymerase promoter sequence operably linked to the coding sequence of the circular RNA precursor.

[0081] In one embodiment, the recombinant nucleic acid vector may be a recombinant vector that transcribes a circular RNA precursor.

[0082] In one embodiment, the recombinant vector may comprise a promoter operably linked to a gene encoding a circular RNA precursor.

[0083] In one embodiment, the recombinant vector may comprise a template DNA of a circular RNA precursor.

[0084] The recombinant nucleic acid vector of the present invention can be produced by a recombinant DNA method known in the art, and may additionally include regulatory sequences including a leader sequence, a polyadenylation sequence, a promoter, an enhancer, an upstream activating sequence, a signal peptide sequence, and a transcription terminator, as needed for efficient transcription of a circular RNA precursor.

[0085] In one aspect, the present invention relates to a composition for preparing circular RNA, comprising a circular RNA precursor of the present invention, a phosphatase, and an RtcB RNA ligase.

[0086] In one aspect, the present invention relates to a kit for preparing circular RNA, comprising a circular RNA precursor of the present invention, a phosphatase, and an RtcB RNA ligase.

[0087] In one aspect, the present invention relates to a method for producing circular RNA, comprising the steps of synthesizing precursor RNA by in vitro transcription (IVT) of the vector of the present invention; treating the synthesized precursor RNA with phosphatase; and treating it with RtcB RNA ligase.

[0088] In one embodiment, the phosphatase and RtcB RNA ligase can be processed simultaneously or sequentially.

[0089] In one embodiment, the triphosphate at the 5' end of the circular RNA precursor can be converted into a hydroxyl group (-OH) by the phosphatase treatment.

[0090] In one embodiment, a 2', 3' cyclic phosphate can be induced at the 3' end of the transcribed precursor RNA by a ribozyme.

[0091] In one embodiment, the RtcB RNA ligase can circularize a circular RNA precursor by linking a hydroxyl group at the 5' end of the circular RNA precursor to a cyclic phosphate at the 3' end.

[0092] In one embodiment, the method of the present invention can produce circular RNA having a size of about 200 to 9950 nt.

[0093] In one embodiment, the RtcB RNA ligase may be of bacterial or mammalian origin, and may be an RtcB RNA ligase from E. coli, Thermus thermophilus, or human.

[0094] In one embodiment, the method of the present invention can additionally treat an archease in the step of treating RtcB RNA ligase, in which case the circularization efficiency of a larger / longer circular RNA precursor can be improved.

[0095] In one embodiment, the method of the present invention may further comprise a step of treating with an exoribonuclease after the RtcB RNA ligase treatment step.

[0096] In one aspect, the present invention relates to the use of a circular RNA precursor for use in the production of circular RNA.

[0097] The present invention is described in more detail through the following examples. However, the following examples are intended only to concretize the content of the present invention and are not intended to limit the present invention.

[0098]

[0099] Example 1. Precursor RNA synthesis

[0100] 1-1. Precursor RNA structure design

[0101] To utilize the RtcB RNA ligase, which recognizes the -OH at the 5' end and the cyclic phosphate (2', 3') at the 3' end, a precursor rpcRNA template structure was designed that included a 5' homology region-IRES-CDS (target sequence)-3' homology region-3' ribozyme from the 5' end (Fig. 2). In addition, for comparison with rpcRNA, a conventional precursor racRNA template structure that included a ribozyme at the 5' end was designed (Fig. 3).

[0102]

[0103] 1-2. Precursor RNA template synthesis

[0104] In vitro transcription (IVT) template DNA for synthesizing precursor RNA was synthesized using the structure designed in Example 1-1, and then transformed into DH5α chemically competent E. coli cells (Engenomics, CP010), plated on 50 μg / ml kanamycin LB plates, and cultured overnight in an incubator at 37°C. One colony formed on the plate was inoculated into 50 μg / ml kanamycin LB broth, and cultured with agitation at 200 rpm at 37°C. Afterwards, the cells were harvested and disrupted, and the IVT template DNA was purified using a QIAfilter plasmid Midi kit (Qiagen, 12243), and the purified IVT template was linearized using restriction enzyme XbaI under the conditions of Table 1. Linearized IVT template DNA was purified using the Qiaquick PCR purification kit (Qiagen, 28106).

[0105] ComponentConditionIVT template DNA15 ug10 x buffer10 ulRestrction enzyme (20 U / ul)4 ulD.WUp to 100 ul

[0106] 1-3. Precursor RNA synthesis

[0107] Linearized IVT template DNA synthesized in the above Example 1-2 and EZ TMPrecursor RNA was synthesized using the MEGA T7 transcription kit (Engenomics, EZ039S). Specifically, 1 μg of IVT template DNA, 2 μl of MEGA T7 enzyme mix, 2 μl of 10x MEGA T7 reaction buffer, 2 μl of 100 mM rATP, 2 μl of 100 mM rCTP, 2 μl of 100 mM rGTP, and 2 μl of 100 mM rUTP were added to a final volume of 20 μl and mixed in a SimpliAmp Thermal Cycler (Thermo). After incubation at 37°C for 2 h using a SimpliAmp Thermal Cycler (Thermo), 2 μl of DNase I was added, mixed, and incubated at 37°C for 15 min. 80 ul of NFW was added to the reaction mixture, mixed, and the synthesized precursor RNA was purified using RNeasy Mini Kit (Qiagen, 74106), thereby obtaining two racRNA precursors (racRNA and racRNA2) and two rpcRNA precursors (rpc-1 and rpc-3`rib) containing the sequences in Table 2 below.

[0108]

[0109]

[0110] 1-4. Analysis of transcription efficiency of precursor RNA according to sequence of homologous region

[0111] In order to confirm the transcription efficiency according to the starting sequence of the 5' end of the 5' homology region of the precursor rpcRNA template structure in the precursor RNA synthesis of the above Examples 1-3, i.e., the transcription step, the synthesis amount per IVT 1 reaction of templates encoding two types of racRNA, which are conventional precursor RNAs that cannot help but have a GXX sequence due to the ribozyme sequence located at 5' (racRNA and racRNA2 having a GCC sequence as the starting sequence of the 5' end of the 5' homology region and having different sequences of the subsequent homology region) and two types of rpcRNA of the present invention having a GGX sequence (rpc-3'rib having a GGC sequence as the starting sequence of the 5' end of the 5' homology region and rpc-1 having a GGG sequence) was analyzed.

[0112] As a result, the synthesis amounts per IVT 1 reaction of racRNA, racRNA2, rpc-3`rib, and rpc-1 were 38.8 ug, 29.3 ug, 137.6 ug, and 171.5 ug, respectively (Fig. 4), indicating that the transcription efficiency of rpcRNA containing the GGX sequence was approximately 3.55 to 4.42 times higher than that of racRNA containing the GXX sequence. Therefore, it was confirmed that the efficiency of the in vitro transcription (IVT) step was increased by the sequence configuration, thereby improving the overall circular RNA production efficiency.

[0113]

[0114] Example 2. Dephosphorylation of the 5'-terminus of precursor RNA

[0115] Since RtcB RNA ligase recognizes and acts on the hydroxyl group at the 5' end and the cyclic phosphate at the 3' end, the triphosphate (PPP) present at the 5' end of the precursor RNA was dephosphorylated using a phosphatase to create a hydroxyl group (-OH) at the 5' end. Specifically, 10 x antarctic phosphatase phosphatase buffer and 50 units of antarctic phosphatase phosphatase (NEB, M026L) were added to 10 ul of rpc-1 and rpc-3'rib, which had no ribozyme at the 5' end among the precursor RNAs synthesized in Example 1, to make a final volume of 100 ul, mixed, and dephosphorylated at 37°C for 30 minutes. Dephosphorylated precursor RNA was purified using the RNeasy Mini Kit (Qiagen, 74106).

[0116]

[0117] Example 3. Circular RNA synthesis using RtcB RNA ligase

[0118] 3-1. Circular RNA synthesis

[0119] The racRNA synthesized in Example 1 and the precursor RNA dephosphorylated using phosphatase in Example 2 were each circularized using RtcB RNA ligase (Engenomics Cat#. M064L) (Fig. 5). Specifically, 5 pmol of precursor RNA racRNA or dephosphorylated precursor RNA (rpc-1 or rpc-3'rib), 2 μl of 10x RtcB reaction buffer, 20 nM of GTP, 0.2 mM of MnCl2, and 15 pmol of RtcB ligase were added to a PCR strip-tube to make a final volume of 20 μl and mixed. The final volume was made up to 20 μl and mixed. After reacting for 15 minutes at 37°C using a SimpliAmp Thermal Cycler (Thermo), the synthesized circular RNA was purified using RNA clean & concentrator-5 (Zymo research, R1015).

[0120]

[0121] 3-2. Analysis of circular RNA

[0122] The circular RNA (rpc-1, rpc-3`rib and racRNA) (R) synthesized in the above Example 3-1, the precursor RNA (rpc-1, rpc-3`rib and racRNA) (P) synthesized in the above Example 1 and the precursor RNA (rpc-1 and rpc-3`rib) (AP) dephosphorylated in the above Example 2 were mixed with E-Gel. TM Electrophoresis was performed using the Power Snap Electrophoresis System (thermo). Specifically, 200 ng of each RNA, 10 μl of DW, and 10 μl of formamide (Sigma, F9037) were added to a PCR strip tube and mixed. The mixture was incubated at 70°C for 5 minutes using a SimpliAmp Thermal Cycler (Thermo), cooled on ice for 3 minutes, and E-GelTM The sample and ladder (1 μl of RiboRular High Range RNA ladder (Thermo, SM1821), 9 μl of DW, and 10 μl of formamide mixture) were loaded onto a 2% E-gel EX (Thermo, G401002) mounted on a Power Snap Electrophoresis Device (thermo, G8100) and run using the E-Gel EX 1-2% method. After running, the gel was cooled for 15 minutes and CehmiDoc TM Bands were imaged using the XRS+ System (Bio-rad, 1708265). As a result, the circular RNA ratios of rpc-1 (47%) and rpc-3'rib (45%) were significantly higher than that of the precursor RNA racRNA (15%) (Fig. 6), confirming that hydroxylation of the 5' end of the precursor RNA using a phosphatase is important for circularization of the precursor RNA.

[0123]

[0124] Example 4. Analysis of circular RNA synthesis efficiency according to ligation reaction conditions.

[0125] 4-1. Analysis of circular RNA synthesis efficiency using E. coli-derived RtcB RNA ligase

[0126] 4-1-1. Analysis of circularization efficiency according to synthetic reaction conditions

[0127] In Example 2, the precursor RNA rpcRNA (rpc-1) (IRES: Salivirus FHB and CDS: Gaussia luciferase) dephosphorylated using phosphatase was circularized with E. coli-derived RtcB RNA ligase. The circularization efficiency (circular RNA synthesis efficiency) according to the conditions of GTP concentration (10 to 100 nM), Mn concentration (0.05 to 0.5 nM), Mn and GTP concentration (GTP: 20 to 100 nM, MnCl2: 0.1 to 1 mM), and incubation temperature (25 to 37°C) and time (0 to 60 min) during the reaction was confirmed. Specifically, 5 pmol of rpcRNA, 2 ul of 10x RtcB reaction buffer, 15 pmol of E. E. coliRtcB ligase (Cat#. M064L, Engenomics), 10–100 nM GTP, and 0.05–0.5 nM MnCl2 were mixed in a total volume of 20 μl. The reaction was incubated at 25–37°C for 0–60 min using a SimpliAmp Thermal Cycler (Thermo). Afterwards, the synthesized circular RNA was purified using RNA clean & concentrator-5 (Zymo research, R1015), and 200 ng of the purified circular RNA was confirmed by gel electrophoresis.

[0128] As a result, the circular RNA synthesis efficiency was found to be the best under the conditions of reaction at 37°C for 15 minutes with a composition of 20 nM GTP and 0.2 mM MnCl2 (Figs. 7 to 11).

[0129]

[0130] 4-1-2. Confirmation of circular RNA synthesis

[0131] To confirm whether the synthesized circular RNA is actually a circular RNA that is not degraded by exoribonuclease, RNase R was treated with the circular RNA synthesized in Example 4-1-1 for 0 to 60 minutes, and this was confirmed by electrophoresis.

[0132] As a result, it was found that the newly synthesized RNA was not degraded even after RNaseR treatment (Fig. 12), confirming that the synthesized RNA was circular RNA.

[0133]

[0134] 4-2. Analysis of circular RNA synthesis efficiency using human-derived RtcB RNA ligase

[0135] 4-2-1. Analysis of circularization efficiency according to synthetic reaction conditions

[0136] In Example 2, the precursor RNA rpcRNA (rpc-1) (IRES: Salivirus FHB and CDS: IgG HC) dephosphorylated using phosphatase was circularized with human-derived RtcB RNA ligase, and the circularization efficiency (circular RNA synthesis efficiency) according to the conditions of GTP concentration (1.0 to 1 mM), NaCl concentration (70 to 300 mM), MnCl2 concentration (0.2 to 1 mM), and DTT concentration (0 to 10 mM) was confirmed. Specifically, 12.5 pmol rpcRNA, 5 μl of 10x RtcB reaction buffer, 500 nM human RtcB ligase (Sinobio), 1.0–1 mM GTP, 70–300 mM NaCl, 0.2–1 mM MnCl2, and 0–10 mM DTT were mixed in a total volume of 50 μl in a PCR strip-tube (Axygen, PCR-0208-CP-C). The reaction was carried out by incubation at 37°C for 30 min using a SimpliAmp Thermal Cycler (Thermo). Afterwards, the synthesized circular RNA was purified using RNA clean & concentrator-5 (Zymo research, R1015), and 200 ng of the purified circular RNA was confirmed by electrophoresis.

[0137] As a result, it was found that the circular RNA synthesis efficiency was the best under the condition of a composition of 0.5 mM GTP and 0.5 mM MnCl2 (Figs. 13 to 16).

[0138]

[0139] 4-2-2. Combination of human-derived RtcB RNA ligase and Archease

[0140] We analyzed the circular RNA synthesis efficiency by combining Archease as an enhancer of RNA ligase. Specifically, human RtcB RNA ligase and Archease were mixed in a 1:1 (molar ratio) and treated at a concentration of 250 nM to 4 μM, or 250 or 500 nM human RtcB RNA ligase was combined with 1 to 5 μM Archease.

[0141] As a result, the circular RNA synthesis efficiency was found to be the best under the combined conditions of 500 nM human RtcB ligase and 500 nM human archease (Figs. 17 and 18).

[0142]

[0143] 4-3. Analysis of circular RNA synthesis efficiency using RtcB RNA ligase from Thermus thermophilus

[0144] In Example 2, the precursor RNA rpcRNA (rpc-1) (IRES: Salivirus FHB and CDS: Gaussia luciferase) dephosphorylated using phosphatase was circularized with RtcB RNA ligase from Thermus thermophilus, and the circularization efficiency (circular RNA synthesis efficiency) according to the incubation temperature and time conditions during the reaction was confirmed. Specifically, 10 pmol rpcRNA, 2 μl of 5x RtcB reaction buffer, 0.15 mM GTP, 0.25 mM MnCl2, 2.5 uM T. thermophilus RtcB ligase, and T. thermophilus archease were mixed in a total volume of 20 μl in a PCR strip tube (Axygen, PCR-0208-CP-C). The reaction was carried out by incubation at a reaction temperature of 50 to 60°C for 10 to 60 minutes using a SimpliAmp Thermal Cycler (Thermo). Afterwards, the synthesized circular RNA was purified using RNA clean & concentrator-5 (Zymo research, R1015), and 200 ng of the purified circular RNA was confirmed by electrophoresis.

[0145] As a result, it was found that the circular RNA synthesis efficiency was the best under the condition of reaction at 55°C for 20 minutes (Figs. 19 to 20).

[0146]

[0147] Example 5. Analysis of circular RNA synthesis efficiency according to circular RNA size.

[0148] 5-1. Analysis of circular RNA synthesis efficiency using E. coliRtcB RNA ligase

[0149] In order to analyze the circular RNA synthesis efficiency according to the size of precursor RNA, 1390, 1489, 2176 or 3016 nt rpcRNA (rpc-1) dephosphorylated using phosphatase by the method of the present invention was synthesized into circular RNA using E. coli-derived RtcB RNA ligase under the conditions optimized in Example 4-1-1, and compared with circular RNA synthesized using 1390 nt racRNA.

[0150] As a result, when rpcRNA was synthesized using the method of the present invention, the circular RNA synthesis efficiency was found to be significantly increased compared to conventional racRNA, and the circular RNA synthesis efficiency was found to be significantly increased even when the size of the precursor RNA increased (Fig. 21).

[0151]

[0152] 5-2. Analysis of circular RNA synthesis efficiency using human RtcB RNA ligase

[0153] In order to analyze the circular RNA synthesis efficiency according to the size of precursor RNA, circular RNA was synthesized using 1390, 1489, 2176 or 3016 nt rpcRNA (rpc-1) dephosphorylated using phosphatase by the method of the present invention and human-derived RtcB RNA ligase under the conditions optimized in Example 4-2, and compared with circular RNA synthesized using 1390 nt racRNA.

[0154] As a result, when rpcRNA was synthesized using the method of the present invention, the circular RNA synthesis efficiency was found to be significantly increased compared to conventional racRNA, and the circular RNA synthesis efficiency was found to be significantly increased even when the size of the precursor RNA increased (Fig. 22).

[0155]

[0156] 5-3. Analysis of circular RNA synthesis efficiency using Thermus thermophilus RtcB RNA ligase

[0157] In order to analyze the circular RNA synthesis efficiency according to the size of precursor RNA, 1390, 1489, 2176 or 3016 nt rpcRNA (rpc-1) dephosphorylated using phosphatase by the method of the present invention was synthesized into circular RNA using RtcB RNA ligase derived from Thermus thermophilus under the conditions optimized in Example 4-3, and compared with circular RNA synthesized using 1390 nt racRNA.

[0158] As a result, when rpcRNA was synthesized using the method of the present invention, the circular RNA synthesis efficiency was found to be significantly increased compared to conventional racRNA, and the circular RNA synthesis efficiency was found to be significantly increased even when the size of the precursor RNA increased (Fig. 23).

[0159]

[0160] Example 6. Analysis of circular RNA synthesis efficiency according to the type of 3'-terminal ribozyme.

[0161] 6-1. Analysis of circular RNA synthesis efficiency using E. coliRtcB RNA ligase

[0162] In order to analyze the circular RNA synthesis efficiency according to the type of ribozyme located at the 3' end of the precursor RNA, rpcRNA (IRES: FHB and CDS: Gaussia luciferase) containing Twister P1, sHDV (short HDV (hepatitis delta virus-derived ribozyme) ribozyme), Termite HHR (hamerhead ribozyme), or T3H48 HHR at the 3' end was dephosphorylated using phosphatase, and circular RNA was synthesized using E. coli-derived RtcB RNA ligase under the conditions optimized in Example 4-1-1. At this time, for comparison, circular RNA was synthesized using racRNA (IRES: Salivirus FHB and CDS: Gaussia luciferase) containing Twister P3 U2A at the 5' end and Twister P1 ribozyme at the 3' end.

[0163] As a result, when rpcRNA (rpc-1) was synthesized using the method of the present invention, the circular RNA synthesis efficiency was found to be significantly increased in all ribozymes compared to conventional racRNA (Fig. 24).

[0164]

[0165] 6-2. Analysis of circular RNA synthesis efficiency using human RtcB RNA ligase

[0166] To analyze the circular RNA synthesis efficiency according to the type of ribozyme located at the 3' end of precursor RNA, rpcRNA (rpc-1) (IRES: Salivirus FHB and CDS: Gaussia luciferase) containing Twister P1, sHDV, Termite HHR or T3H48 HHR at the 3' end was dephosphorylated using phosphatase, and circular RNA was synthesized using human-derived RtcB RNA ligase under the conditions optimized in Example 4-2. At this time, for comparison, circular RNA was synthesized using racRNA (IRES: FHB and CDS: Gaussia luciferase) containing Twister P3 U2A at the 5' end and Twister P1 ribozyme at the 3' end.

[0167] As a result, when rpcRNA was synthesized using the method of the present invention, the circular RNA synthesis efficiency was found to be significantly increased in all ribozymes compared to conventional racRNA (Fig. 25).

[0168]

[0169] 6-3. Analysis of circular RNA synthesis efficiency using Thermus thermophilus RtcB RNA ligase

[0170] To analyze the circular RNA synthesis efficiency according to the type of ribozyme located at the 3' end of precursor RNA, rpcRNA (rpc-1) (IRES: Salivirus FHB and CDS: Gaussia luciferase) containing Twister P1, sHDV, Termite HHR or T3H48 HHR at the 3' end was dephosphorylated using phosphatase, and circular RNA was synthesized using human-derived RtcB RNA ligase under the conditions optimized in Example 4-2. At this time, for comparison, circular RNA was synthesized using racRNA (IRES: FHB and CDS: Gaussia luciferase) containing Twister P3 U2A at the 5' end and Twister P1 ribozyme at the 3' end.

[0171] As a result, when rpcRNA was synthesized using the method of the present invention, the circular RNA synthesis efficiency was found to be significantly increased in all ribozymes compared to conventional racRNA (Fig. 26).

[0172]

[0173] Example 7. Analysis of circular RNA synthesis efficiency according to homology region sequence

[0174] In order to analyze the circular RNA synthesis efficiency according to the sequence of the homology region in the structure of the rpcRNA of the present invention, the 5' homology region of rpcRNA (rpc-1) excluding GGX (GGC or GGG) at the 5' end of the 5' homology region was constructed with the sequence of Table 3 below, and the sequence of the 3' homology region was changed so that 1 to 2 nucleotides were mismatched with the 5' homology region (M1 and M2), or the lengths of the 5' homology region and the 3' homology region were each constructed to be shortened by 3 to 5 nucleotides (D6 and D10), and the circular RNA synthesis efficiency was compared.

[0175] Homology sequence5` Homology region3` Homology regionrpcAAC ACG GAC UCA AGC UCC UACGUA GGA GCU UGA GUC CGU GUUM1AAC ACG GAC UCA AGC UCC UACGUA GGA GAU UGA GUC CGU GUUM2AAC ACG GAC UCA AGC UCC UACGUA GGA GAU UGA GUACGU GUUD6AAC ACG GAC UCA AGC UACGUA GCU UGA GUC CGU GUUD10AAC ACG GAC UCA AUA CGUA UUG AGU CCG UGU U

[0176] As a result, it was confirmed that even if there was a mismatch in the sequence of the 5' homology region and the 3' homology region, the circular RNA synthesis efficiency was not affected, and even if the length of the 5' homology region and the 3' homology region were short, the circular RNA synthesis efficiency was not affected (Fig. 27).

[0177] Example 8. Analysis of circular RNA synthesis efficiency of precursor RNA containing additional complementary sequences

[0178] A precursor RNA template (rpc-sp-IH-sp template) was designed by adding complementary 5' internal homology region and 3' internal homology region to the 3' end of the 5' homology region and the 5' end of the 3' homology region, respectively, to the structure of the rpcRNA (rpc-1) of the present invention, and adding spacers on both sides of the internal homology region (Fig. 28). Using the above template, precursor RNA (IRES: Salivirus FHB and CDS: IgG HC) was synthesized, followed by dephosphorylation using phosphatase, and circular RNA was prepared using E. coli-derived RtcB RNA ligase, human-derived RtcB RNA ligase, and T. Thermophilus-derived RtcB RNA ligase, respectively.

[0179] sp-IH-sp sequenceSpacer 1AAAAAACAAAAAACAAAA5' Internal Homology regionGCCGGAAACGC AATAGCCGSpacer 2AAAC AAAC AAAC3' Internal Homology region5' Complementary sequence of Internal Homology region

[0180] As a result, in the case of Human and T. Thermophilus RtcB, the circular RNA synthesis efficiency by precursor RNA with spacers added on both sides of the internal homology region did not increase, but in the case of E. coli RtcB, it was confirmed that there was an effect of increasing the circular RNA synthesis efficiency (Fig. 29).

[0181] Example 9. Expression analysis of circular RNA

[0182] 9-1. In vitro expression analysis

[0183] Circular RNA containing the light or heavy chain of Salivirus FHB IRES and IgG as CDS was synthesized by the method of the present invention or by the method using RNA ligase2. In this case, in the case of the method using RNA ligase2, EZ TMPrecursor RNA was synthesized using MEGAT7 transcription kit (Engenomics, EZ039S), circular RNA was synthesized using NEB T4 RNA Ligase 2 (Cat#. M043L), and then dephosphorylated using Antarctic phosphatase (NEB, M026L). Specifically, 1 μg of linearized IVT template, 2 μl of MEGA T7 enzyme Mix, 2 μl of 10x MEGA T7 reaction buffer, 2 μl of 100 mM rATP, 2 μl of 100 mM rCTP, 2 μl of 100 mM rGTP, 2 μl of 100 mM rUTP, and 1.5 μl of 400 mM GMP were added to a PCR strip-tube (Axygen, PCR-0208-CP-C), and mixed to a total volume of 20 μl (GMP: GTP = 3:1). Then, the reaction was performed at 37°C for 2 h using a SimpliAmp Thermal Cycler (Thermo). 2 μl of DNaseⅠ was added to the IVT mixture, mixed, and then further reacted at 37°C for 15 min. After the additional reaction, 80 μl of NFW was added, and the synthesized precursor RNA was purified using the Rneasy Mini Kit (Qiagen, 74106) according to the manufacturer's manual. 75 pmol of the purified precursor RNA, 10 μl of 10x T4 RNA Ligase 2 reaction buffer, and 37.5 U of RNA ligase 2 were added to a PCR strip-tube (Axygen, PCR-0208-CP-C), and mixed to a total volume of 100 μl. The reaction was performed at 25°C for 18 hours (O / N) using a SimpliAmp Thermal Cycler (Thermo). After the reaction, 80 μl of NFW was added, and the synthesized circular RNA was purified using the Rneasy Mini Kit (Qiagen, 74106) according to the manufacturer's manual.Ligated circular RNA (50 μg), 10 μl of 10x antarctic phosphatase buffer, and 50 units of antarctic phosphatase were added and mixed to a total volume of 100 μl. The mixture was reacted at 37°C for 1 hour and 30 minutes, and the dephosphorylated circular RNA was purified using 7.5 M LiCl. Specifically, LiCl corresponding to 0.5 volume of the reaction solution volume was added to the reaction solution, mixed, and incubated at -20°C for 30 minutes. The mixture was centrifuged at 14,000 rpm for 20 minutes. The supernatant was removed, 700 μl of 70% EtOH was added, inverted, and centrifuged at 14,000 rpm for 20 minutes. After centrifugation, the supernatant was removed and air-dried to remove the remaining 70% EtOH, and an RNA pellet was obtained. The obtained RNA pellet was dissolved in nuclease-free water. The purified dephosphorylated circular RNA was loaded onto E-Gel EX 2% to confirm the band. The circular RNAs containing the light and heavy chains, each synthesized by the method of the present invention and the method using RNA ligase 2, were mixed at a 1:1 ratio (molar ratio) and then introduced into 293T cells using lipofectamine 3000. Thereafter, the expression of the target protein, the antibody, was confirmed by Western blot analysis using an 8% SDS-PAGE gel.

[0184] As a result, it was confirmed that circular RNAs synthesized by two methods effectively produce antibodies. In particular, it was confirmed that the method of the present invention produced approximately 1.4 times more antibodies than the method using RNA ligase 2 (Fig. 30).

[0185]

[0186] 9-2. In vivo expression analysis

[0187] Circular RNAs containing the light or heavy chain of Salivirus FHB IRES and IgG as CDS were synthesized using the method of the present invention and a method using RNA ligase2, respectively. The circular RNAs containing the light and heavy chains were mixed at a ratio (molar ratio) of 1:1 and then formulated into LNPs (lipid nanoparticles). The manufactured LNPs were intravenously injected into mice at a concentration of 0.5 mg / kg, and the blood antibody concentration was measured 24 hours later.

[0188] As a result, for both methods, sufficient amounts of antibodies were detected in the mouse blood (Fig. 31), confirming that antibodies were sufficiently expressed from the circular RNA administered to the mice. In particular, the circular RNA synthesized by the method of the present invention exhibited a blood antibody concentration approximately 1.6 times higher than that of the circular RNA synthesized by the RNA ligase 2 method (Fig. 31).

Claims

1. As a circular RNA precursor, The above precursor has a structure of 5'-5' homology region-gene of interest (GOI)-3' homology region-ribozyme-3', and A circular RNA precursor, wherein the 5' homology region contains a sequence that is reverse complementary to the 3' homology region.

2. A circular RNA precursor in claim 1, wherein the 5' homology region additionally includes a GGX sequence at the 5' end, wherein X is A (adenine), U (uracil), C (cytosine), or (guanine).

3. A circular RNA precursor according to claim 1, wherein the 5' homology region comprises any one sequence selected from the group consisting of SEQ ID NOs: 1 to 4.

4. A circular RNA precursor having a triphosphate (PPP) at the 5' end in the first paragraph.

5. In the first paragraph, the target gene is a circular RNA precursor comprising an IRES (internal ribosome entry site) region at the 5' end. 6.제 5항에 있어서, IRES는 Taura syndrome virus, Triatoma virus, Theiler's encephalomyelitis virus, Simian Virus 40, Solenopsis invicta virus 1, Rhopalo- siphum padi virus, Reticuloendotheliosis virus, Human poliovirus 1, Plautia stali intestine virus, Kashmir bee virus, Human rhinovirus 2, Homalodisca coagulata virus-1, Human Immunodeficiency Virus type 1, Homalodisca coagulata virus-1, Himetobi P virus, Hepatitis C virus, Hepatitis A virus, Hepatitis GB virus, Foot and mouth disease virus, Human enterovirus 71, Equine rhinitis virus, Ectropis obliqua picorna-like virus, Encephalomyocarditis virus, Drosophila C Virus, Human coxsackievirus B3, Cru- cifer tobamovirus, Cricket paralysis virus, Bovine viral diarrhea virus 1, Black Queen Cell Virus, Aphid lethal paralysis virus, Avian encephalomyelitis virus, Acute bee paralysis virus, Hibiscus chlorotic ringspot virus, Classical swine fever virus, Human FGF2, Human SFTPA1, Human AML1 / RUNX1, Drosophila antennapedia, Human AQP4, Human ATIR,Human BAG-1, Human BCL2, Human BiP, Human c-IAP1, Human c-myc, Human eIF4G, Mouse NDST4L, Human LEF1, Mouse HIF1 alpha, Human n.myc, Mouse Gtx, Human p27kip1, Human PDGF2 / c-sis, Human p53, Human Pim-1, Mouse Rbm3, Drosophila reaper, Canine Scamper, Drosophila Ubx, Human UNR, Mouse UtrA, Human VEGF-A, Human XIAP, Drosophila hairless, S. cerevisiae TFIID, S. cerevisiae YAP1, tobacco etch virus, turnip crinkle virus, EMCV-A, EMCV-B, EMCV-Bf, EMCV-Cf, EMCV pEC9, Picobirnavirus, HCV QC64, Human Cosavirus E / D, Human Cosavirus F, Human Cosa- virus JMY, Rhinovirus NAT001, HRV14, HRV89, HRVC- 02, HRV-A21, Salivirus A SH1, Salivirus FHB, Salivirus NG-J1, Human Parechovirus 1, Crohivirus B, Ye-3, Rosa- virus M-7, Shanbavirus A, Pasivirus A, Pasivirus A 2, Echovirus E14, Human Parechovirus 5, Aichi Virus, Hepa- titis A Virus HA16, Phopivirus, CVA10, Enterovirus C, Enterovirus D, Enterovirus J, Human Pegivirus 2, GBV-C GT110, GBV-C K1737, GBV-C Iowa, Pegivirus A 1220, Pasivirus A 3, Sapelovirus,Rosavirus B, Bakunsa Virus, Tremovirus A, Swine Pasivirus 1, PLV-CHN, Pasivirus A, Sicinivirus, Hepacivirus K, Hepacivirus A, BVDV1, Border Disease Virus, BVDV2, CSFV-PK15C, SF573 Dicistrovirus, Hubei Picoma-like Virus, CRPV, Salivirus A BN5, Salivirus A BN2, Salivirus A 02394-01, Salivirus A GUT, Salivirus A CH, Salivirus A SZ1, Salivirus FHB, Salivirus NG-J1, Salivirus A YZ01, Salivirus A YZ02, Salivirus A YZ03, Salivirus A FUAN-01, Salivirus sp isolate ETH, Salivirus A Hu / Baja-5332, Salivirus sp isolate A circular RNA precursor which is all, part or a variant of one or more viral sequences selected from the group consisting of CHN / XJ / env, CVB3, CVB1, Echovirus 7, CVBS, EVA71, CVA3, CVA12 and EV24.

7. A circular RNA precursor in claim 5, wherein the IRES is an aptamer for elF4G.

8. A circular RNA precursor according to claim 1, further comprising a 5' internal homology region in the 3' direction of the 5' homology region, and a 3' internal homology region in the 5' direction of the 3' homology region, wherein the 5' internal homology region comprises a sequence that is reverse complementary to the 3' internal homology region.

9. A circular RNA precursor in claim 8, wherein the 5' internal homology region comprises the base sequence of SEQ ID NO:

10.

10. A circular RNA precursor according to claim 8, further comprising a spacer region in the 5' and 3' directions of the internal homology region.

11. A circular RNA precursor according to claim 10, wherein the spacer comprises a base sequence of SEQ ID NO: 11 or 12.

12. In claim 1, the ribozyme is a circular RNA precursor which is Twister, Twister Sister, Hatchet, Pistol, Hammerhead, Hairpin, Hepatitis Delta Virus (HDV), Neurospora Varkud Satellite (VS), Vg1, glucosamine-6-phosphate synthase (glmS) or an engineered synthetic ribozyme, or a variant thereof.

13. A circular RNA precursor according to claim 1, further comprising a label.

14. In claim 13, a circular RNA precursor comprising a PolyA, PolyG, PolyU or PolyC sequence.

15. A circular RNA precursor having a size of 250 to 10,000 nt in the first paragraph.

16. A recombinant nucleic acid vector for producing the circular RNA precursor of paragraph 1.

17. A recombinant nucleic acid vector comprising a promoter operably linked to a gene encoding a circular RNA precursor according to claim 16. 18.1) A step of synthesizing precursor RNA by in vitro transcription (IVT) of the recombinant vector of Article 16; 2) A step of treating the synthesized precursor RNA with phosphatase; and 3) A method for producing circular RNA, comprising a step of treating RtcB RNA ligase.

19. A method for producing circular RNA, wherein phosphatase and RtcB RNA ligase are treated simultaneously or sequentially in claim 18.

20. A method for producing circular RNA, comprising producing circular RNA having a size of 200 to 9950 nt in accordance with claim 18.

21. A method for producing circular RNA in claim 18, wherein the RtcB RNA ligase is derived from bacteria or mammals.

22. A method for producing circular RNA, wherein archease is additionally treated in the step of treating RtcB RNA ligase in claim 18.

23. A method for producing circular RNA, comprising, in claim 18, additionally comprising a step of treating exoribonuclease after step 3).

24. Use of a circular RNA precursor for the production of circular RNA.

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