Preparation method of self-cyclized RNA (Ribonucleic Acid)

Abstract

The present application provides a method of preparing a circular RNA, the method comprising transcribing a vector to form a precursor RNA, wherein the vector comprises the following elements operably linked to each other and arranged in the following order: a) a 5 'element, b) a group 3' I self-splicing intron fragment comprising a 3 'splicing site dinucleotide, c) an element free of or containing an internal ribosome entry site (IRES) and a protein coding region or an element containing a non-coding region, d) a 5 'Group I self-splicing intron fragment comprising a 5' splicing site dinucleotide, and e) a 3 'element wherein the 5' element and the 3 'element form a stable structure having a Gibbs free energy ([Delta] G) of-190 kcal / mol to-9.0 kcal / mol, with the proviso that the stable structure is not a double strand having at least 95% base pairing between the 5' element and the 3 'element, wherein the 3 'I group self-splicing intron fragment and the 5' I group self-splicing intron fragment form a self-cleaving and self-ligation RNA molecule, thereby producing a circular RNA.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is an International (PCT) application claiming priority to U.S. Provisional Patent Application No. 63 / 476,864, filed on December 22, 2022, the entire contents of which are hereby incorporated by reference for all purposes. Reference to a sequence listing submitted as a compliant ASCII text file (.xml)

[0002] In accordance with the EFS-Web legal framework and 37 C.FR §1.821-825 (see MPEP §2442.03(a)), a sequence listing submitted in the form of an ASCII-compliant text file (titled "3000076-009977_sequence_listing_ST26.xml", created on December 14, 2023, and 110,393 bytes in size) is submitted concurrently with this application, and the entire contents of the sequence listing are incorporated herein by reference. Background of the Invention

[0003] Messenger RNA (mRNA) has broad potential in a range of therapeutic and engineering applications. Perhaps a significant limitation to its use is its relatively short half-life in biological systems. Therefore, there is a need to extend the duration of protein expression from full-length RNA messages. Summary of the Invention

[0004] In one aspect, the present disclosure relates to a method for preparing a circular RNA, the method comprising: transcribing a vector to form a precursor RNA, wherein the vector comprises the following elements operably linked to each other and arranged in the following order: a) a 5' element that does not comprise or comprises at least one stem-loop structure, b) a 3' Group I self-splicing intron fragment comprising a 3' splice site dinucleotide, c) an element that does not comprise an internal ribosome entry site (IRES) and a protein coding region or comprises a non-coding region, d) a 5' Group I self-splicing intron fragment comprising a 5' splice site dinucleotide, e) a 3' element that does not comprise or comprises at least one stem-loop structure; provided that, when the 5' element does not comprise a stem-loop structure, the 3' element comprises at least one stem-loop structure; and, when the 3' element does not comprise a stem-loop structure, the 5' element comprises at least one stem-loop structure, wherein the 5' element and the 3' element form a thermodynamically stable multi-directional junction RNA structure, wherein the precursor RNA is capable of forming a circular RNA that is translatable and / or biologically active in a cell.

[0005] On the other hand, the thermodynamically stable multi-way junction RNA structure can be a 3-way junction (3WJ), a 4-way junction (4WJ), a 5-way junction (5WJ), a hand-in-hand interaction, a kissing loop or a pseudo knot. In the hand-in-hand interaction structure, one loop sequence may have a high affinity with another loop sequence to form a closed interaction dimer structure (Shu et al., "Stable RNA nanoparticles as potential new generation drugs for cancer therapy". Advanced drug delivery reviews. 66 (2014) 74-89; the contents of which are hereby incorporated by reference in their entirety). The pseudo knot structure may comprise at least two stem-loop structures, wherein one half of one stem may interact between the two halves of the other stem (Staple et al., "Pseudoknots: RNA structures with diverse functions". PLOSBio 3 (6): e213; the contents of which are hereby incorporated by reference in their entirety).

[0006] On the other hand, 3WJ may include a first branch of the 3WJ domain, which may be formed by the 5' portion of the 3WJa sequence and the 3' portion of the 3WJc sequence and may include a first helical region; a second branch of the 3WJ domain, which may be formed by the 3' portion of the 3WJa sequence and the 5' portion of the 3WJb sequence and may include a second helical region; and a third branch of the 3WJ domain, which may be formed by the 3' portion of the 3WJb sequence and the 5' portion of the 3WJc sequence and may include a third helical region, wherein each of the helical regions may include multiple RNA nucleotide pairs that form a canonical Watson-Crick bond.

[0007] On the other hand, the 3WJa, 3WJb and 3WJc sequences may be as follows: 3WJa comprises or consists of SEQ ID NO: 1, 3WJb comprises or consists of SEQ ID NO: 2, and 3WJc comprises or consists of SEQ ID NO: 3; or 3WJa comprises or consists of SEQ ID NO: 4, 3WJb comprises or consists of SEQ ID NO: 5, and 3WJc comprises or consists of SEQ ID NO: 6; or 3WJa comprises or consists of SEQ ID NO: 10, 3WJb comprises or consists of SEQ ID NO: 11, and 3WJc comprises or consists of SEQ ID NO: 12; or 3WJa comprises or consists of SEQ ID NO: 13, 3WJb comprises or consists of SEQ ID NO: 2, and 3WJc comprises or consists of SEQ ID NO: 13; or 3WJa comprises or consists of SEQ ID NO: 13, 3WJb comprises or consists of SEQ ID NO: 14, and 3WJc comprises or consists of SEQ ID NO: 3; or 3WJa comprises or consists of SEQ ID NO: 13, 3WJb comprises or consists of SEQ ID NO: 15, and 3WJc comprises or consists of SEQ ID NO: 3; or 3WJa comprises or consists of SEQ ID NO: 13, 3WJb comprises or consists of SEQ ID NO: 16, and 3WJc comprises or consists of SEQ ID NO: 3; or 3WJa comprises or consists of SEQ ID NO: 1, 3WJb comprises or consists of SEQ ID NO: 14, and 3WJc comprises or consists of SEQ ID NO: 3; or 3WJa comprises or consists of SEQ ID NO: 1, 3WJb comprises or consists of SEQ ID NO: 15, and 3WJc comprises or consists of SEQ ID NO: 3; or 3WJa comprises or consists of SEQ ID NO: 1, 3WJb comprises or consists of SEQ ID NO: 16, and 3WJc comprises or consists of SEQ ID NO: 3; or 3WJa comprises or consists of SEQ ID NO: 17, 3WJb comprises or consists of SEQ ID NO: 15, and 3WJc comprises or consists of SEQ ID NO: 18; or 3WJa comprises or consists of SEQ ID NO: 19, 3WJb comprises or consists of SEQ ID NO: 20, and 3WJc comprises or consists of SEQ ID NO: 18; or 3WJa comprises or consists of SEQ ID NO: 19, 3WJb comprises or consists of SEQ ID NO: 21, and 3WJc comprises or consists of SEQ ID NO: 22; or 3WJa comprises or consists of SEQ ID NO: 23, 3WJb comprises or consists of SEQ ID NO: 21, and 3WJc comprises or consists of SEQ ID NO: 24; or 3WJa comprises or consists of SEQ ID NO: 25, 3WJb comprises or consists of SEQ ID NO: 26, and 3WJc comprises or consists of SEQ ID NO: 24; or 3WJa comprises or consists of SEQ ID NO: 25, 3WJb comprises or consists of SEQ ID NO: 27, and 3WJc comprises or consists of SEQ ID NO: 28; or 3WJa comprises or consists of SEQ ID NO: 29, 3WJb comprises or consists of SEQ ID NO: 27, and 3WJc comprises or consists of SEQ ID NO: 30; or 3WJa comprises or consists of SEQ ID NO:31, 3WJb comprises or consists of SEQ ID NO:32, and 3WJc comprises or consists of SEQ ID NO:30; or 3WJa comprises or consists of SEQ ID NO:31, 3WJb comprises or consists of SEQ ID NO:33, and 3WJc comprises or consists of SEQ ID NO:34; or 3WJa comprises or consists of SEQ ID NO:41, 3WJb comprises or consists of SEQ ID NO:11, and 3WJc comprises or consists of SEQ ID NO:42; or 3WJa comprises or consists of SEQ ID NO:43, 3WJb comprises or consists of SEQ ID NO:44, and 3WJc comprises or consists of SEQ ID NO:42; or 3WJa comprises or consists of SEQ ID NO:43, 3WJb comprises or consists of SEQ ID NO:45, and 3WJc comprises or consists of SEQ ID NO:46; or 3WJa comprises or consists of SEQ ID NO:47, 3WJb comprises or consists of SEQ ID NO:45, and 3WJc comprises or consists of SEQ ID NO:48; or 3WJa comprises or consists of SEQ ID NO:49, 3WJb comprises or consists of SEQ ID NO:50, and 3WJc comprises or consists of SEQ ID NO:48; or 3WJa comprises or consists of SEQ ID NO:49, 3WJb comprises or consists of SEQ ID NO:51, and 3WJc comprises or consists of SEQ ID NO:52; or 3WJa comprises or consists of SEQ ID NO:53, 3WJb comprises or consists of SEQ ID NO:51, and 3WJc comprises or consists of SEQ ID NO:54; or 3WJa comprises or consists of SEQ ID NO:55, 3WJb comprises or consists of SEQ ID NO:56, and 3WJc comprises or consists of SEQ ID NO:54; or 3WJa comprises or consists of SEQ ID NO:55, 3WJb comprises or consists of SEQ ID NO:57, and 3WJc comprises or consists of SEQ ID NO:58; or 3WJa comprises or consists of SEQ ID NO:59, 3WJb comprises or consists of SEQ ID NO:57, and 3WJc comprises or consists of SEQ ID NO:60; or 3WJa comprises or consists of SEQ ID NO: 61, 3WJb comprises or consists of SEQ ID NO: 63, and 3WJc comprises or consists of SEQ ID NO: 64; or 3WJa comprises or consists of SEQ ID NO: 65, 3WJb comprises or consists of SEQ ID NO: 66, and 3WJc comprises or consists of SEQ ID NO: 64; or 3WJa comprises or consists of SEQ ID NO: 65, 3WJb comprises or consists of UGOCACGGG, and 3WJc comprises or consists of SEQ ID NO: 68; or 3WJa comprises or consists of SEQ ID NO:43, 3WJb comprises or consists of SEQ ID NO:69, and 3WJc comprises or consists of SEQ ID NO:46; or 3WJa comprises or consists of SEQ ID NO:47, 3WJb comprises or consists of SEQ ID NO:70, and 3WJc comprises or consists of SEQ ID NO:52; or 3WJa comprises or consists of SEQ ID NO:55, 3WJb comprises or consists of SEQ ID NO:71, and 3WJc comprises or consists of SEQ ID NO:72; or 3WJa comprises or consists of SEQ ID NO: 76, 3WJb comprises or consists of SEQ ID NO: 8, and 3WJc comprises or consists of SEQ ID NO: 9; or 3WJa comprises or consists of SEQ ID NO: 77, 3WJb comprises or consists of SEQ ID NO: 78, and 3WJc comprises or consists of SEQ ID NO: 79; or 3WJa comprises or consists of SEQ ID NO:80, 3WJb comprises or consists of SEQ ID NO:81, and 3WJc comprises or consists of SEQ ID NO:82; or 3WJa comprises or consists of SEQ ID NO:83, 3WJb comprises or consists of SEQ ID NO:84, and 3WJc comprises or consists of SEQ ID NO:85; or 3WJa comprises or consists of SEQ ID NO: 7, 3WJb comprises or consists of SEQ ID NO: 89, and 3WJc comprises or consists of SEQ ID NO: 9; or 3WJa comprises or consists of SEQ ID NO:90, 3WJb comprises or consists of SEQ ID NO:91, and 3WJc comprises or consists of SEQ ID NO:79; or 3WJa comprises or consists of SEQ ID NO:76, 3WJb comprises or consists of SEQ ID NO:89, and 3WJc comprises or consists of SEQ ID NO:9; or 3WJa comprises or consists of SEQ ID NO: 77, 3WJb comprises or consists of SEQ ID NO: 91, and 3WJc comprises or consists of SEQ ID NO: 79; or 3WJa comprises or consists of SEQ ID NO:80, 3WJb comprises or consists of SEQ ID NO:93, and 3WJc comprises or consists of SEQ ID NO:82; or 3WJa comprises or consists of SEQ ID NO:83, 3WJb comprises or consists of SEQ ID NO:95, and 3WJc comprises or consists of SEQ ID NO:85; or 3WJa comprises or consists of SEQ ID NO: 112, 3WJb comprises or consists of SEQ ID NO: 113, and 3WJc comprises or consists of SEQ ID NO: 114; or 3WJa comprises or consists of SEQ ID NO: 119, 3WJb comprises or consists of SEQ ID NO: 120, and 3WJc comprises or consists of SEQ ID NO: 121; or 3WJa contains AUGUGUA, 3WJb contains UACUUUG, and 3WJc contains AUCAUG; or 3WJa contains GCGUU, 3WJb contains UUCGC, and 3WJc contains GCCAUAGCG; or 3WJa contains GUAUGGCAC, 3WJb contains GUCACGG, and 3WJc contains CUCUUAC; or 3WJa contains AUGGUA, 3WJb contains ACUUUGU, and 3WJc contains AUCA; or 3WJa contains UGGU, 3WJb contains ACUUGU, and 3WJc contains AUCA; or 3WJa contains UGGU, 3WJb contains ACUGU, and 3WJc contains AUCA; or 3WJa contains UGGU, 3WJb contains ACGUU, and 3WJc contains AAUCA; or 3WJa contains UGUGU, 3WJb contains ACUUGU, and 3WJc contains AUCA; or 3WJa contains UGUGU, 3WJb contains ACUGU, and 3WJc contains AUCA; or 3WJa contains UGUGU, 3WJb contains ACGUU, and 3WJc contains AAUCA; or 3WJa contains UGGU, 3WJb contains ACUGU, and 3WJc contains AUCA; or 3WJa contains UAUGGCAC, 3WJb contains GUCACGG, and 3WJc contains CUCUUA; or 3WJa contains UAUGG, 3WJb contains UCAGG, and 3WJc contains CCUCUUA; or 3WJa contains UAUGGCAC, 3WJb contains GUCACGG, and 3WJc contains CUCUUA; or 3WJa contains UAUG, 3WJb contains CAGGGG, and 3WJc contains CUUG; or 3WJa contains UAUGU, 3WJb contains GCAGG, and 3WJc contains UCUUG; or 3WJa contains UAUGU, 3WJb contains GCAGGG, and 3WJc contains CUUG; or 3WJa contains UAUGU, 3WJb contains GCAGG, and 3WJc contains UCUUG; or 3WJa contains UGUGU, 3WJb contains ACUUUGU, and 3WJc contains AUCA; or 3WJa contains UGUGU, 3WJb contains ACUUU, and 3WJc contains AAAUCA.

[0008] On the other hand, 4WJ may include a first branch of the 4WJ domain, which may be formed by the 5' portion of the 4WJa sequence and the 3' portion of the 4WJd sequence and may include a first helical region; a second branch of the 4WJ domain, which may be formed by the 3' portion of the 4WJa sequence and the 5' portion of the 4WJb sequence and may include a second helical region; a third branch of the 4WJ domain, which may be formed by the 3' portion of the 4WJb sequence and the 5' portion of the 4WJc sequence and may include a third helical region; and a fourth branch of the 4WJ domain, which may be formed by the 3' portion of the 4WJc sequence and the 5' portion of the 4WJd sequence and may include a fourth helical region, wherein each of the helical regions may include multiple RNA nucleotide pairs that form canonical Watson-Crick bonds.

[0009] On the other hand, the 4WJa, 4WJb, 4WJc, and 4WJd sequences may be as follows: 4WJa comprises or consists of SEQ ID NO: 7, 4WJb comprises or consists of SEQ ID NO: 8, 4WJc comprises or consists of SEQ ID NO: 9, and 4WJd comprises or consists of SEQ ID NO: 102; or 4WJa comprises or consists of SEQ ID NO: 103, 4WJb comprises or consists of SEQ ID NO: 104, 4WJc comprises or consists of SEQ ID NO: 105, and 4WJd comprises or consists of SEQ ID NO: 106; or 4WJa comprises or consists of SEQ ID NO: 115, 4WJb comprises or consists of SEQ ID NO: 116, 4WJc comprises or consists of SEQ ID NO: 117, and 4WJd comprises or consists of SEQ ID NO: 118; or 4WJa comprises UGCAGGUG, 4WJb comprises ACGGGC, 4WJc comprises CCAGCA, and 4WJd comprises SEQ ID NO: 67; or 4WJa comprises SEQ ID NO:74, 4WJb comprises AACUG, 4WJc comprises SEQ ID NO:75, and 4WJd comprises AUCAUG; or 4WJa comprises SEQ ID NO: 122, 4WJb comprises GAACU, 4WJc comprises SEQ ID NO: 123, and 4WJd comprises AAUCA.

[0010] On the other hand, 5WJ may include a first branch of the 5WJ domain, which may be formed by the 5' portion of the 5WJa sequence and the 3' portion of the 5WJe sequence and may include a first helical region; a second branch of the 5WJ domain, which may be formed by the 3' portion of the 5WJa sequence and the 5' portion of the 5WJb sequence and may include a second helical region; a third branch of the 5WJ domain, which may be formed by the 3' portion of the 5WJb sequence and the 5' portion of the 5WJc sequence and may include a third helical region; a fourth branch of the 5WJ domain, which may be formed by the 3' portion of the 5WJc sequence and the 5' portion of the 5WJd sequence and may include a fourth helical region; and a fifth branch of the 5WJ domain, which may be formed by the 3' portion of the 5WJd sequence and the 5' portion of the 5WJe sequence and may include a fifth helical region, wherein each of the helical regions may include multiple RNA nucleotide pairs that form a canonical Watson-Crick bond.

[0011] On the other hand, 5WJa comprises or consists of SEQ ID NO:107, 5WJb comprises or consists of SEQ ID NO:108, 5WJc comprises or consists of SEQ ID NO:109, 5WJd comprises or consists of SEQ ID NO:110, and 5WJe comprises or consists of SEQ ID NO:111; or, 5WJa comprises GUGA, 5WJb comprises UUGC, 5WJc comprises GUGU, 5WJd comprises AUGC, and 5WJe comprises GUGC.

[0012] In another aspect, the vector may further comprise an internal ribosome entry site (IRES) at the 5′ end of c), wherein the IRES may be selected from an IRES sequence from a virus or gene selected from the group consisting of Taura syndrome virus, Triatoma virus, Theiler's encephalomyelitis virus, Simian virus 40, Solenopsis invicta virus 1, Rhopalosiphum padi virus, Reticuloendothelioma virus, Human poliovirus 1, Plautiastaliintestine 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 G virusvirus), foot-and-mouth disease virus, human enterovirus 71, equine rhinitis virus, tea geometrid picorna-like virus, encephalomyocarditis virus (EMCV), Drosophila C virus, crucifer tobacco mosaic virus, cricket paralysis virus, bovine viral diarrhea virus 1, black queen cell virus, aphid lethal paralysis virus, avian encephalomyelitis virus, honeybee acute paralysis enterovirus, hibiscus chlorotic ringspot virus, classical swine fever virus, human fibroblast growth factor 2 (FGF2), human surfactant protein A1 (SFTPA1), human acute myeloid leukemia virus (HALV), Leukemia protein 1 / runt-related transcription factor 1 (AML1 / RUNX1), Drosophila Antennapedia, human aquaporin-4 (AQP4), human angiotensin II receptor type 1 (AT1R), human BCL2-associated immortality gene 1 (BAG-1), human B-cell lymphoma 2 (BCL2), human immunoglobulin binding protein (BiP), human inhibitor of apoptosis family protein 1 (c-IAP1), human c-myc, human eukaryotic translation initiation factor 4G (eIF4G), mouse N-deacetylase and N-sulfonylase NDST4L, human lymphoid enhancer binding factor-1 (LEF1), mouse hypoxia-inducible factor 1 subunit alpha (HIF1α), human N-myc, mouse glial cell and testis-specific homeobox protein (Gtx), human cyclin-dependent kinase inhibitor 1B (p27kip1), human platelet-derived growth factor B / simian sarcoma virus human homolog (PDGF2 / c-sis), human p53, human proviral integration site of Moloney murine leukemia virus-1 (Pim-1), small Mouse RNA-binding protein 3 (Rbm3), Drosophila reaper, canine Scamper, Drosophila Ultrabithorax (Ubx), Salivirus, coronavirus, parechovirus, human N-ras upstream (UNR), mouse utrophin A (UtrA), human vascular endothelial growth factor A (VEGF-A), human X-linked inhibitor of apoptosis (XIAP), Drosophila hairless, Saccharomyces cerevisiae transcription factor IID (TFIID), Saccharomyces cerevisiae Yes1-associated transcription regulator (YAP1), human proto-oncogene tyrosine-protein kinase Src (c-src), human fibroblast growth factor 1 (FGF-1), simian picornavirus, turnip crinkle virus, coxsackievirus B3 (CVB3), and coxsackievirus A (CVB1 / 2).

[0013] In another aspect, the vector may further comprise an RNA polymerase promoter.

[0014] In another aspect, the RNA polymerase promoter can be a T7 viral RNA polymerase promoter, a T6 viral RNA polymerase promoter, an SP6 viral RNA polymerase promoter, a T3 viral RNA polymerase promoter, or a T4 viral RNA polymerase promoter.

[0015] On the other hand, the 3' I-group self-splicing intron fragment and the 5' I-group self-splicing intron fragment may be derived from the Pre-tRNA-Leu gene of the cyanobacterium Cyanobacterium anabaena sp.

[0016] On the other hand, the 3' I group self-splicing intron fragment and the 5' I group self-splicing intron fragment may be derived from the T4 phage Td gene.

[0017] In another aspect, the methods of the present disclosure may further comprise forming circular RNA by splint-mediated ligation of precursor RNAs.

[0018] In another aspect, the vector can be transfected into cells prior to transcription using lipofection or electroporation.

[0019] In another aspect, nanovectors can be used to transfect vectors into cells prior to transcription.

[0020] In another aspect, the nanocarrier can be a lipid, a polymer, or a lipid-polymer hybrid.

[0021] In another aspect, the methods of the disclosure can further comprise forming the circular RNA and purifying the circular RNA using a size exclusion chromatography column in tris-EDTA or ion-pair reverse phase HPLC.

[0022] In another aspect, the method of the present disclosure may further comprise forming the circular RNA and purifying the circular RNA in a high performance liquid chromatography (HPLC) system in a triethylammonium acetate (TEAA)-acetonitrile buffer having a pH range of about 4-10 at a flow rate of about 0.01-5 mL / min.

[0023] In another aspect, the methods of the disclosure may further comprise forming the circular RNA and purifying the circular RNA using phosphatase treatment.

[0024] In another aspect, the methods of the present disclosure may further comprise incubating the precursor RNA in the presence of (i) magnesium ions and / or (ii) guanosine nucleotides or guanosine nucleosides.

[0025] In another aspect, the incubation of the precursor RNA can be performed at a temperature between about 20°C and about 60°C.

[0026] In another aspect, transcription of the vector can occur in the presence of a nucleoside or nucleotide monophosphate or diphosphate for incorporation as the first nucleotide of a precursor RNA transcribed from the vector.

[0027] In another aspect, the precursor RNA may comprise a monophosphate 5' end capable of being ligated using a ligase.

[0028] In another aspect, transcription of the vector can occur in the presence of: a) guanosine nucleoside or mono- or diphosphate nucleotides; b) cytidine nucleoside or mono- or diphosphate nucleotides; c) uridine nucleoside or mono- or diphosphate nucleotides; d) adenosine nucleoside or mono- or diphosphate nucleotides; or e) a combination thereof, so as to incorporate the nucleoside or mono- or diphosphate nucleotide as the first nucleotide of an RNA chain transcribed from the vector or a transcript produced from the vector.

[0029] In another aspect, the protein coding region may encode a non-natural protein comprising one or more synthetic protein elements.

[0030] In another aspect, the vector may comprise a 5' spacer element located at the 3' end of b).

[0031] In another aspect, the vector may comprise a 3' spacer element located at the 5' end of d).

[0032] In another aspect, the 5' spacer element or the 3' spacer element can comprise a polyA sequence or a polyA-C sequence.

[0033] In another aspect, the noncoding region may comprise an element encoding one or more RNAs selected from the group consisting of antisense RNA, transfer RNA (tRNA), transfer-messenger RNA (tmRNA), ribosomal RNA (rRNA), signal recognition particle RNA (7SLRNA or SRP RNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), SmY RNA (SmY), small Cajal body-specific RNA (scaRNA), guide RNA (gRNA), Y RNA, splicing leader RNA (SL RNA), microRNA (miRNA), small interfering RNA (siRNA), cis-natural antisense transcript (cis-NAT), CRISPR RNA (crRNA), long noncoding RNA (lncRNA), Piwi-interacting RNA (piRNA), short hairpin RNA (shRNA), trans-acting siRNA (tasiRNA), repeat-associated siRNA (rasiRNA), 7SK RNA (7SK), telomerase RNA component (TERC), vault RNA (vRNA, vtRNA), and enhancer RNA (eRNA).

[0034] In one aspect, the present disclosure relates to a precursor RNA comprising the following elements operably linked to each other and arranged in the following order: a) a 5' element that does not comprise or comprises at least one stem-loop structure, b) a 3' Group I self-splicing intron fragment comprising a 3' splice site dinucleotide, c) a protein coding region or a non-coding region, d) a 5' Group I self-splicing intron fragment comprising a 5' splice site dinucleotide, e) a 3' element that does not comprise or comprises at least one stem-loop structure; provided that, when the 5' element does not comprise a stem-loop structure, the 3' element comprises at least one stem-loop structure; and, when the 3' element does not comprise a stem-loop structure, the 5' element comprises at least one stem-loop structure, wherein the 5' element and the 3' element form a thermodynamically stable multi-directional junction, wherein the precursor RNA is capable of forming a circular RNA that is translatable and / or biologically active in a cell.

[0035] In one aspect, the vector may encode a precursor RNA of the disclosure.

[0036] In another aspect, the vector can be a plasmid, a viral vector, a polymerase chain reaction (PCR) product, a cosmid, a bacterial artificial chromosome (BAC), or a yeast artificial chromosome (YAC).

[0037] In one aspect, the present disclosure relates to a method for preparing a circular ribonucleic acid (RNA), the method comprising transcribing a vector to form a precursor RNA comprising the following elements operably linked to each other and arranged in tandem in a 5' to 3' orientation: a) a 5' element, b) a 3' Group I self-splicing intron fragment comprising a 3' splice site dinucleotide, c) an element without or comprising an internal ribosome entry site (IRES) and a protein coding region, or an element comprising a non-coding region, d) a 5' Group I self-splicing intron fragment comprising a 5' splice site dinucleotide, and e) a 3' element; wherein the 5' element and the 3' element form a stable structure having a Gibbs free energy (ΔG) of -190 kcal / mol to -9.0 kcal / mol, provided that the stable structure is not a duplex with at least 95% base pairing between the 5' element and the 3' element, wherein the 3' Group I self-splicing intron fragment and the 5' Group I self-splicing intron fragment form a self-cleaved and self-ligated RNA molecule, thereby generating a circular RNA.

[0038] In one aspect, the disclosure relates to a precursor RNA comprising the following elements operably linked to each other and arranged in tandem in a 5' to 3' orientation: a) a 5' element, b) a 3' Group I self-splicing intron fragment comprising a 3' splice site dinucleotide, c) an element without or comprising an IRES and a protein coding region or an element comprising a non-coding region, d) a 5' Group I self-splicing intron fragment comprising a 5' splice site dinucleotide, and e) a 3' element, wherein the 5' element and the 3' element form a stable structure having a Gibbs free energy (ΔG) of -190 kcal / mol to -9.0 kcal / mol, provided that the stable structure is not a duplex with at least 95% base pairing between the 5' element and the 3' element, wherein the 3' Group I self-splicing intron fragment and the 5' Group I self-splicing intron fragment form a self-cleaving and self-ligating RNA molecule, thereby generating a circular RNA.

[0039] In one aspect, the present disclosure relates to a method for producing a protein in a cell, the method comprising introducing a precursor RNA containing a protein coding region or a vector containing a protein coding region of the present disclosure into the cell, and producing the protein.

[0040] In one aspect, the present disclosure relates to a method for editing a gene in a cell, the method comprising introducing a precursor RNA containing a non-coding region capable of editing a gene or a vector containing a non-coding region capable of editing a gene of the present disclosure into the cell, and editing the gene. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 Shown is a "three-way junction" ("3WJ") domain according to one embodiment of the present disclosure.

[0042] Figure 2 Shown is a 3WJ domain according to another embodiment of the present disclosure.

[0043] Figure 3 Shown is a 3WJ domain according to another embodiment of the present disclosure.

[0044] Figure 4 A method for preparing circularized RNA (circRNA) according to one embodiment of the present disclosure is shown.

[0045] Figure 5 A method for preparing circularized RNA (circRNA) according to another embodiment of the present disclosure is shown.

[0046] Figure 6 Shown is a 4WJ domain according to one embodiment of the present disclosure.

[0047] Figure 7 Shown is a 4WJ domain according to another embodiment of the present disclosure.

[0048] Figure 8 A method for preparing circularized RNA (circRNA) according to one embodiment of the present disclosure is shown.

[0049] Figure 9 Shown is a 5WJ domain according to one embodiment of the present disclosure.

[0050] Figure 10 A method for preparing circularized RNA (circRNA) according to one embodiment of the present disclosure is shown.

[0051] Figure 11 A method for preparing circularized RNA (circRNA) according to another embodiment of the present disclosure is shown.

[0052] Figure 12 The purity of circRNA prepared by the method according to some embodiments of the present disclosure is shown.

[0053] Figure 13 Shown are the circRNA / precursor RNA ratios of circRNAs prepared by the methods according to some embodiments of the present disclosure.

[0054] Figure 14A Shown is an HPLC-purified circRNA prepared by a method according to one embodiment of the present disclosure.

[0055] Figure 14B The purity of circRNA prepared by the method according to one embodiment of the present disclosure is shown.

[0056] Figure 15A Shown is an HPLC-purified circRNA prepared by a method according to another embodiment of the present disclosure.

[0057] Figure 15B The purity of circRNA prepared by the method according to another embodiment of the present disclosure is shown.

[0058] Figure 16 Shown are gene expressions of circRNAs prepared by the methods according to some embodiments of the present disclosure.

[0059] Figure 17 Shown are gene expressions of circRNAs prepared by the methods according to some embodiments of the present disclosure.

[0060] Figure 18 Shown is the gene expression of circRNAs prepared by the methods according to some embodiments of the present disclosure over time.

[0061] Figure 19Shown is the gene expression of circRNAs prepared by the methods according to some embodiments of the present disclosure over time.

[0062] Figure 20 The purity of circRNA prepared by the method according to some embodiments of the present disclosure is shown.

[0063] Figure 21A Shown are RNA circularization efficiencies determined by agarose gel electrophoresis and densitometry analysis according to some embodiments of the present disclosure.

[0064] Figure 21B Shown are RNA circularization efficiencies determined by high performance liquid chromatography (HPLC) according to some embodiments of the present disclosure.

[0065] Figure 21C Shown are RNA circularization efficiencies determined by HPLC according to some embodiments of the present disclosure.

[0066] Figure 22A Shown is the gene expression of circRNAs prepared by the methods according to some embodiments of the present disclosure over time.

[0067] Figure 22B Shows Figure 22A Fold changes in gene expression.

[0068] Figure 23A Shown is the gene expression of circRNAs prepared by the methods according to some embodiments of the present disclosure over time.

[0069] Figure 23B Shows Figure 23A Fold changes in gene expression.

[0070] Figure 24A Shown are the copy numbers of circRNAs produced by the methods according to some embodiments of the present disclosure over time.

[0071] Figure 24B Shows Figure 24A Fold change in copy number. DETAILED DESCRIPTION

[0072] mRNA has been shown to be a promising platform for the development of RNA-based therapeutics and vaccines. Development of mRNA vaccines for human use may require both a 5' cap and a 3' polyA component for efficient mRNA expression in vivo, which may require a cap-dependent mechanism to recruit ribosomes for translation. These requirements may pose a challenge to the production of high-quality mRNA, as the polyA tail may be lost during the plasmid preparation step. Furthermore, the stability of the linear mRNA sequence may be another limitation to its in vitro preservation and in vivo half-life due to degradation by exonucleases.

[0073] Exogenous circular RNAs have been developed to extend the duration of protein expression from full-length RNA sequences. Three general strategies are possible for circularizing exogenous RNAs: chemical methods using cyanogen bromide or similar condensing agents, enzymatic methods using RNA or DNA ligases, and ribozyme methods using self-splicing introns. A ribozyme approach utilizing a displaced group I catalytic intron may be more suitable for circularizing long RNAs and may require only the addition of GTP and Mg2+ as cofactors. Functional proteins can be produced from these circRNAs in eukaryotic cells, and translation can be maximized by incorporating distinct internal ribosome entry sites (IRES) and internal polyadenylation tracts. This displaced intron-exon (PIE) splicing strategy may involve fusions of partial exons flanked by hemi-intronic sequences. In vitro, these constructs undergo a transesterification reaction specific to the group I catalytic intron, but because the exons are already fused, they are excised as covalent 5' to 3' linked loops. Using this strategy as a starting point for creating protein-coding circular RNAs, Wesselhoeft (“Engineering circular RNA for potent and stable translation in eukaryotic cells.” Nat Commun. 2018 Jul 6;9(1):2629) described a method for making circular RNAs in vitro using a design that replaces introns and exons and utilizes homology arms within the sequence. However, this circularization reaction may not be very efficient. Therefore, extensive HPLC purification and RNaseR digestion purification may be required to remove unreacted precursor linear RNA to obtain relatively pure circular RNA for drug development.

[0074] PIE (Placing Intron-Exon) can be used to design circular RNAs with sequences on both the 5' and 3' ends to form highly thermodynamically stable scaffolds with low Gibbs free energy (<-40 kcal / mol). The lower free energy uses intron sequences on the 5' and 3' ends of the circular RNA to drive the formation of ribozymes. The thermodynamically stable motifs on both sides of the 5' and 3' ends of the precursor circular RNA may have high affinity to associate with each other to form complex structures, thereby promoting the formation of multidirectional junction motifs and ribozyme structures. For example, a 3-way junction (3WJ) may have a fast "on" state to form a 3WJ structure with a 1.37×10 5The three-way junctions have an association rate constant of M-1s-1 (Binzel et al., "Mechanism of three-component collision to produce ultrastable pRNA three-way junction of Phi29 DNA-packaging motor by kinetic assessment. RNA. 2016 Nov; 22(11): 1710-1718, the contents of which are hereby incorporated by reference in their entirety) and a very low dissociation constant. They do not dissociate in the pmol range (Shu et al., "Thermodynamically stable RNA three-way junction for constructing multifunctional nanoparticles for delivery of therapeutics." Nature Nanotechnology 2011; 6(10): 658-67; the contents of which are hereby incorporated by reference in their entirety). This property may enable the formation of ribozymes from both ends of long precursor RNAs. In addition, the thermodynamic motifs formed by this design can further form 3D structures with high affinity (Zhang et al., "Crystal Structure of 3WJ Core Revealing Divalent Ion-promoted Thermostability and Assembly of the Phi29 Hexameric Motor pRNA.” RNA 2013 Sep;19(9):1226-37; the contents of which are hereby incorporated by reference in their entirety), thereby further ensuring the correct folding of its adjacent ribozyme structure through intron formation.

[0075] As used herein, a "homology arm" or "homology region" or "duplex-forming region" can be any contiguous sequence that: 1) is predicted to form base pairs with at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, about 100%, 100% of another sequence (e.g., another homology arm) in an RNA, 2) is at least about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 47,48,49, or 50 nucleotides and is no longer than 250 nt, 3) is located before, adjacent to, or contained within a 3' intronic fragment, and / or is located after, adjacent to, or contained within a 5' intronic fragment, and optionally, 4) is predicted to have less than 50% (e.g., less than 45%, less than 40%, less than 35%, less than 30%, less than 25%) base pairing with an unexpected sequence in the RNA (e.g., a non-homologous arm sequence). In some embodiments, the homology arm or homology region or duplex-forming region can be about 9 to about 50 nucleotides in length. In one embodiment, the length of the homology arms or homology regions or duplex-forming regions may be about 9 to about 19 nucleotides. In some embodiments, the length of the homology arms or homology regions or duplex-forming regions may be about 20 to about 40 nucleotides. In certain embodiments, the length of the homology arms or homology regions or duplex-forming regions may be about 30 nucleotides.

[0076] 5' and 3' homology arms or homology regions or duplex forming district can be synthetic sequences, and are different from internal homology regions but function similarly.The length of homology arms or homology regions or duplex forming district can be, for example, about 5-50 nucleotides, about 9-19 nucleotides, for example, about 5, about 10, about 20, about 30, about 40 or about 50 nucleotides. In another embodiment, the length of homology arms or homology regions or duplex forming district can be 9 nucleotides. In a further embodiment, the length of homology arms or homology regions or duplex forming district can be 19 nucleotides. In certain embodiments, the length of homology arms or homology regions or duplex forming district is at least 5,6,7,8,9,10,11,12,13,14,15,16,17,18 or 19 nucleotides. In certain embodiments, the length of homology arms or homology regions or duplex forming district is no more than 50,45,40,35,30,25 or 20 nucleotides. In some embodiments, the homology arms or homology regions or duplex-forming regions are 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, or 50 nucleotides in length.

[0077] Unlike conventional 5' and 3' homology arms or homology regions or duplex-forming regions as described, for example, in WO 2021236855 and US11447796 (the contents of which are incorporated herein by reference in their entirety), the 5' and 3' regions of the present disclosure can hybridize to form multidirectional junctions (WJs), such as 3WJs, 4WJs and 5WJs, as described below.

[0078] As used herein, a 3' group I intron segment is a contiguous sequence that is at least 75% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, 100%) homologous to the 3' proximal segment of a native group I intron, including the 3' splice site dinucleotide, and optionally, the length of the adjacent exon sequence is at least 1 nucleotide (e.g., at least 5 nucleotides, at least 10 nucleotides, at least 15 nucleotides, at least 20 nucleotides, at least 25 nucleotides, at least 50 nucleotides). In one embodiment, the included adjacent exon sequence is about the length of a native exon. In some embodiments, the 5' group I intron segment is a contiguous sequence at least 75% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, 100%) homologous to the 5' proximal segment of a native group I intron, including the 5' splice site dinucleotide, and optionally, the adjacent exon sequence is at least 1 nucleotide in length (e.g., at least 5 nucleotides, at least 10 nucleotides, at least 15 nucleotides, at least 20 nucleotides, at least 25 nucleotides, at least 50 nucleotides). In one embodiment, the included adjacent exon sequence is about the length of a native exon.

[0079] Examples of Group I intron self-splicing sequences include, but are not limited to, self-splicing replacement intron-exon sequences derived from the T4 bacteriophage gene td or the cyanobacterium Anabaena sp. pre-tRNA-Leu gene.

[0080] As used herein, "spacer" refers to any contiguous nucleotide sequence that: 1) is predicted to avoid interfering adjacent structure, such as from an IRES, coding or noncoding region, or intron, 2) is at least 7 nucleotides long (and optionally no longer than 100 nucleotides), 3) is located downstream and adjacent to a 3' intronic segment and / or upstream and adjacent to a 5' intronic segment, and / or 4) contains one or more of: a) an unstructured region of at least 5 nt in length, b) a region of at least 5 nt in length that is predicted to base pair with a distal (i.e., non-adjacent) sequence (including another spacer), and / or c) a structured region of at least 1 nt in length that is limited in scope to the spacer sequence.

[0081] As used herein, "interfering" with respect to a sequence refers to a sequence that is predicted or empirically determined to alter the folding of other structures in the RNA, such as an IRES or group I intron-derived sequence.

[0082] As used herein, "unstructured" with respect to RNA refers to an RNA sequence that is not predicted by RNAFold software or similar prediction tools to form a structure (e.g., a hairpin loop) with itself or other sequences in the same RNA molecule.

[0083] As used herein, "structured" with respect to RNA refers to an RNA sequence that is predicted by RNAFold software or similar prediction tools to form a structure (e.g., a hairpin loop) with itself or other sequences in the same RNA molecule.

[0084] In some embodiments, the length of the spacer sequence can be, for example, at least 10 nucleotides, at least 15 nucleotides, or at least 30 nucleotides. In some embodiments, the length of the spacer sequence is at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, or 30 nucleotides. In some embodiments, the length of the spacer sequence is no more than 100, 90, 80, 70, 60, 50, 45, 40, 35, or 30 nucleotides. In some embodiments, the length of the spacer sequence is between 20 and 50 nucleotides. In certain embodiments, the spacer sequence is 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, or 50 nucleotides in length.

[0085] The spacer sequence can be a polyA sequence, a polyA-C sequence, a polyC sequence, or a poly-U sequence, or the spacer sequence can be specifically engineered based on the IRES. The spacer sequence as described herein can have two functions: (1) promote circularization and (2) promote functionality by allowing the intron and IRES to fold correctly. More specifically, the engineering of the spacer sequence as described herein has three priorities: 1) be inert with respect to the folding of the proximal intron and IRES structure; 2) adequately separate the intron and IRES secondary structure; and 3) contain a region of spacer-spacer complementarity to promote the formation of a "splicing bubble". In one embodiment, the vector is compatible with many possible IRES and coding or non-coding regions and two spacer sequences.

[0086] In one embodiment, the vector may comprise a 5' spacer sequence but not a 3' spacer sequence. In another embodiment, the vector may comprise a 3' spacer sequence but not a 5' spacer sequence. In another embodiment, the vector may not comprise a 5' spacer sequence and a 3' spacer sequence. In another embodiment, the vector does not comprise an IRES sequence. In another embodiment, the vector does not comprise an IRES sequence, a 5' spacer sequence, or a 3' spacer sequence.

[0087] As used herein, " vector " means a section of DNA, which is synthesized (e.g., using PCR), or taken from a virus, plasmid or a cell of a higher organism, into which an exogenous DNA fragment can be inserted or has been inserted for cloning and / or expression purposes. In certain embodiments, the vector can be stably maintained in an organism. The vector can include, for example, an origin of replication, a selective marker or a reporter gene, such as antibiotic resistance or GFP, and / or a multiple cloning site (MCS). The term can include linear DNA fragments (e.g., PCR products, linearized plasmid fragments), plasmid vectors, viral vectors, clays, bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC) and the like. In one embodiment, the vector provided herein includes a multiple cloning site (MCS). In another embodiment, the vector provided herein does not include an MCS.

[0088] Unless otherwise indicated, the cells described herein may include any cells in which an exogenous nucleic acid as described herein can be introduced or expressed. It should be understood that the basic concepts of the present disclosure described herein are not limited by cell type. The cells described herein may include somatic cells, stem cells, eukaryotic cells, prokaryotic cells, animal cells, plant cells, fungal cells, archaeal cells, eubacterial cells, etc. Cells may include eukaryotic cells, such as yeast cells, plant cells, and animal cells. Specific cells may include mammalian cells, such as human cells. In addition, cells may include any cells in which expression of circRNA would be beneficial or desirable.

[0089] The protein coding region of the gene of interest (GOI) can encode a protein of eukaryotic or prokaryotic origin. In some embodiments, the protein can be any protein for therapeutic use or diagnostic use. For example, the protein coding region can encode a human protein or antibody. In some embodiments, the protein can be selected from, but is not limited to, chimeric antigen receptor (CAR), T cell receptor (TCR), antibody, human factor IX (hFIX), lung-associated surfactant protein B (SP-B), vascular endothelial growth factor A (VEGF-A), human methylmalonyl-CoA mutant enzyme (hMUT), CF- transmembrane transduction regulator (CFTR), cancer autoantigens, and other gene editing enzymes, such as regularly spaced clustered short palindromic repeats (CRISPR) related (Cas) proteins (e.g., Cas9 and Cpf1), zinc finger nucleases (ZFNs), and transcription activator-like effector nucleases (TALENs). In some embodiments, the vector or circRNA lacks a protein coding sequence. In some embodiments, precursor RNA is a necessary intermediate between plasmid and circRNA.

[0090] In some embodiments, the vector may comprise an IRES sequence located at the 5' end of the target gene (GOI) encoding the protein. The IRES sequence may be selected from, but is not limited to, the following IRES sequences: Taura syndrome virus, Triatomine bug virus, Theiler encephalomyelitis virus, Simian virus 40, Fire ant virus 1, Grain aphid virus, Reticuloendothelioma virus, Human poliovirus 1, Small stink bug enterovirus, Kashmir bee virus, Human rhinovirus 2, Green leafhopper virus-1, Human immunodeficiency virus type 1, Green leafhopper virus-1, Himetobi P virus, hepatitis C virus, hepatitis A virus, hepatitis G virus and foot-and-mouth disease virus, human enterovirus 71, equine rhinitis virus, tea geometrid picorna-like virus, encephalomyocarditis virus (EMCV), Drosophila C virus, crucifer tobacco mosaic virus, cricket paralysis virus, bovine viral diarrhea virus 1, black queen cell virus, aphid lethal paralysis virus, avian encephalomyelitis virus, honeybee acute paralysis enterovirus, hibiscus chlorotic ringspot virus, classical swine fever virus, human FGF2, human SFTPA1, human AML1 / RUNX1, Drosophila antennapedia, human AQP4, human AT1R, human BAG-1, human BCL2, human BiP, human c-IAP1, human c- Myc, human eIF4G, mouse NDST4L, human LEF1, mouse HIF1α, human N-myc, mouse Gtx, human 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, Saccharomyces cerevisiae TFIID, Saccharomyces cerevisiae YAP1, human c-src, human FGF-1, simian picornavirus, turnip crinkle virus, eIF4G aptamer, Coxsackievirus B3 (CVB3) or Coxsackievirus A (CVB1 / 2). The wild-type IRES sequence can also be modified and is effective in the present invention. In some embodiments, the IRES sequence is about 50 nucleotides in length.

[0091] In some embodiments, the gene of interest (GOI) can be replaced by a nucleic acid encoding one or more RNA molecules, including but not limited to antisense RNA, transfer RNA (tRNA), transfer-messenger RNA (tmRNA), ribosomal RNA (rRNA), signal recognition particle RNA (7SL RNA or SRP RNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), SmY RNA (SmY), small Cajal body-specific RNA (scaRNA), guide RNA (gRNA), Y RNA, splicing leader RNA (SLRNA), microRNA (miRNA), small interfering RNA (siRNA), cis-natural antisense transcript (cis-NAT), CRISPR RNA (crRNA), long noncoding RNA (lncRNA), Piwi-interacting RNA (piRNA), short hairpin RNA (shRNA), trans-acting siRNA (tasiRNA), repeat-associated siRNA (rasiRNA), 7SK RNA (7SK), telomerase RNA component (TERC), vault RNA (vRNA, vtRNA), and enhancer RNA (eRNA). In some embodiments, a vector comprising a nucleic acid encoding one or more RNA molecules may or may not contain an IRES sequence.

[0092] Circular RNA can be purified by passing the RNA through a reverse phase chromatography column in a high performance liquid chromatography (HPLC) system in a triethylammonium acetate (TEAA) buffer. In one embodiment, the RNA can be passed through a reverse phase HPLC column in a TEAA-acetonitrile buffer having a pH range of about 4-10 at a flow rate of about 0.01-5 mL / min.

[0093] In certain embodiments, provided herein are methods for generating precursor RNA by in vitro transcription using a vector provided herein as a template (eg, a vector provided herein having an RNA polymerase promoter positioned upstream of the 5' homology arm).

[0094] In some embodiments, the nucleotides, nucleosides, or chemically modified nucleotides or nucleosides used in the in vitro transcription reactions described herein can be in excess concentration relative to the similar nucleotide triphosphates. "Excess concentration" is defined as a concentration greater than the similar nucleotide triphosphate, the purpose of which is to modify the 5' terminal nucleotide, particularly to reduce the immunogenicity of the circRNA preparation by preventing the inclusion of a 5' triphosphate motif or to allow enzymatic cyclization of the precursor molecule by including the necessary 5' monophosphate motif.

[0095] In some embodiments, the nucleotide used in excess can be guanosine monophosphate (GMP). In other embodiments, the nucleotide used in excess can be GDP, ADP, CDP, UDP, AMP, CMP, UMP, guanosine, adenosine, cytidine, uridine, or any chemically modified nucleotide or nucleoside. In some embodiments, the excess can be about 10 times excess. In some embodiments, the excess can be about 12.5 times excess.

[0096] In one embodiment, nucleotides, nucleosides, or chemically modified nucleotides or nucleosides can be used in an in vitro transcription reaction at a concentration that is at least about 10-fold greater than the concentration of the analogous nucleotide triphosphate.

[0097] In some embodiments, circRNAs generated from precursor RNAs synthesized in an in vitro transcription reaction where nucleotides, nucleosides, or chemically modified nucleotides or nucleosides are present in at least about a 10-fold excess over the similar nucleotide triphosphates are then purified by HPLC to minimize immunogenicity.

[0098] Since residual precursor linear RNA or nicked RNA can compete with circular RNA for ribosome recruitment, resulting in reduced expression efficiency, the purity of the circular RNA product appears to be crucial for expression. Methods for preparing high-purity circular RNA in vitro may help translate circular RNA technology into applications in RNA therapeutics and vaccine development. The embodiments disclosed herein provide solutions for the in vitro design and preparation of circular RNA with high self-circularization efficiency.

[0099] Embodiments of the present disclosure may include methods for preparing precursor RNAs with thermodynamically stable polydirectional junctions next to the splicing bubble to more efficiently prepare self-circularizing RNAs in vitro.

[0100] The efficiency of pre-RNA self-circularization reactions designed using intron-exon replacement can be highly dependent on the efficiency of self-splicing bubble formation. Traditional methods rely on the formation of a duplex RNA structure by homology arms at the 5' and 3' ends of the pre-RNA, allowing the splicing bubble to fold into its secondary structure to achieve splicing function. Due to the respiratory dynamics of RNA duplexes, self-circularization efficiencies can often be less than 60%.

[0101] To address the problem of efficiently forming the secondary structure of the self-splicing bubble, embodiments of the present disclosure may include a stable RNA motif consisting of at least 2 stems and 1 loop to lock the splicing bubble in its secondary structure, thereby promoting its correct folding and splicing efficiency. For example, the stable RNA motif may comprise an asymmetric three-way junction (3WJ) structure to lock the splicing bubble and thus promote correct folding and splicing efficiency, such as the thermodynamically stable multidirectional junction structure of RNA described in the packaging RNA of the phi29 DNA packaging motor (Shu et al., "Thermodynamically stable RNA three-way junction forconstructing multifunctional nanoparticles for delivery of therapeutics." Nat Nanotechnol. 2011 Sep 11;6(10):658-67, the contents of which are hereby incorporated by reference in their entirety), and the three segments 3WJa, 3WJb and 3WJc rapidly and automatically assemble into a three-way junction (3WJ) structure to lock folding (Binzel et al., "Mechanism of three-component collision to produce ultrastable pRNA three-way junction of Phi29 DNA-packaging motor by kinetic assessment.RNA. 2016 Nov;22(11):1710-1718”, the contents of which are hereby incorporated by reference in their entirety).

[0102] The polyhedral junction structure can be formed by cleaving from one stem-loop position and assigning sequences to the 5' and 3' ends of the precursor RNA sequence, respectively. Three-way junction (3WJ) domain

[0103] The packaging (or proto) RNA (pRNA) three-way junction (3WJ) motif can be applied in biotechnology, for example, for targeting human immunodeficiency virus (HIV) and cancer. For example, the phi29 phage pRNA 3WJ nanomotif has been successfully used as a building block in the rational design of nanostructures with functions such as cancer targeting (U.S. Patent No. 9,297,013, the contents of which are hereby incorporated by reference in their entirety).

[0104] As used herein, the term "three-way junction" ("3WJ") or "trifurcated" scaffold (or domain) refers to a structure assembled from three RNA sequences. Figure 1It was shown (10) that the 3WJ domain can be constructed from three (5'→3') RNA strands (designated 3WJa, 3WJb, and 3WJc) that base-pair with each other. A first (5'→3') RNA oligonucleotide sequence designated as 3WJa, a second (5'→3') RNA oligonucleotide sequence designated as 3WJb, and a third (5'→3') RNA oligonucleotide sequence designated as 3WJc can be combined and base-paired to form a trifurcated 3WJ domain (10), wherein a first branch (11) of the 3WJ domain is formed by the 5' portion of the 3WJa sequence and the 3' portion of the 3WJc sequence, a second branch (12) of the 3WJ domain is formed by the 3' portion of the 3WJa sequence and the 5' portion of the 3WJb sequence, and a third branch (13) of the 3WJ domain is formed by the 3' portion of the 3WJb sequence and the 5' portion of the 3WJc sequence, wherein each of the first (11), second (12), and third (13) branches can comprise a helical region having a plurality of RNA nucleotide pairs that form canonical Watson-Crick bonds. One, two and / or three branches of 3WJ of the present disclosure may also include non-Watson-Crick nucleotide pairs or protrusions, such as, but not limited to, GU wobble base pairs, or protrusions with a small amount of additional non-paired bases. In certain embodiments, the 3' end of 3WJb may be connected to the 5' end of 3WJc via a joint sequence (14). In certain embodiments, the joint sequence (14) connecting 3WJb and 3WJc may not be paired at all, for example, forming a stem-loop structure. In certain embodiments, the joint sequence (14) may be paired with its sequence portion, for example, forming a loop and a stem. In certain embodiments, the joint sequence (14) may be paired completely with its sequence, for example, forming a stem. In certain embodiments, the joint sequence (14) may not exist, for example, and the 3' end of 3WJb may be directly connected to the 5' end of 3WJc.

[0105] As used herein, stem-loop structure can appear in single-stranded RNA.This structure can also be referred to as hairpin or hairpin loop, and can comprise stem and (end) loop in continuous sequence, wherein stem can be formed by two adjacent complete or partial reverse complementary sequences, and complementary sequence is separated by the short sequence as the spacer of forming loop stem-loop structure.Two adjacent complete or partial reverse complementary sequences can be defined as the element of for example stem-loop structure sequence 1 and sequence 2.When these two adjacent complete or partial reverse complementary sequences (for example, stem-loop structure element sequence 1 and sequence 2) form base pair each other, stem-loop structure can be formed, this causes formation double-stranded nucleic acid sequence, and this double-stranded nucleic acid sequence comprises the unpaired loop formed by the short sequence between the element of stem-loop structure sequence 1 and sequence 2 in continuous sequence at its end.Therefore, unpaired loop is typically the nucleic acid region that can not be paired with any of these elements of stem-loop structure.The multidirectional connection domain thus produced folds into the compact structure with low Gibbs free energy (Δ G), thereby increasing the thermodynamic stability of this structural formula, and promoting splicing bubble to form its real structure. The stability of the paired elements of the stem-loop structure can be determined by the length and GC ratio, the number of inconsistencies or loops contained therein (a small amount of inconsistencies is generally tolerated, especially in large double-stranded regions), and the base composition of the paired regions. In some embodiments of the present disclosure, a loop length of 3 to 15 bases is tolerated, but a more preferred loop length may be 3 to 20 bases, preferably 3 to 19, 3 to 18, 3 to 17, 3 to 16, 3 to 15, 3 to 14, 3 to 13, 3 to 12, 3 to 11, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, or more preferably 4 to 5 bases, most preferably 4 bases. The length of the stem sequence forming the double-stranded structure may be 5 to 20 bases, preferably 5 to 19, 5 to 18, 5 to 17, 5 to 16, 5 to 15, 5 to 14, 5 to 13, 5 to 12, 5 to 11, 5 to 10, 5 to 9, 5 to 8, 5 to 7 or 5 to 6 bases.

[0106] The Gibbs free energy (ΔG) for each RNA scaffold formation was calculated using Mfold (MFold), a folding library for RNA, available from the UNAFold web server (Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res. 31(13), 3406-15, (2003)). ΔG was calculated based on RNA folding at 37°C in 1 M NaCl.

[0107] The multidirectional linkages of the present disclosure may have a ΔG of about -200 kcal / mol to about -5 kcal / mol, about -195 kcal / mol to about -6 kcal / mol, about -190 kcal / mol to about -7 kcal / mol, about -190 kcal / mol to about -8 kcal / mol, about -190 kcal / mol to about -9 kcal / mol, about -189 kcal / mol to about -9.5 kcal / mol, about -188.7 kcal / mol to about -9.8 kcal / mol, about -188.7 kcal / mol to about -2 7.1 kcal / mol, about -188.7 kcal / mol to about -35.8 kcal / mol, about -188.7 kcal / mol to about -143.7 kcal / mol, about -188.7 kcal / mol to about -62.7 kcal / mol, about -188.7 kcal / mol to about -35.4 kcal / mol, about -188.7 kcal / mol to about -31.9 kcal / mol, about -188.7 kcal / mol to about -31.7 kcal / mol, about -188.7 kcal / mol to about -31.4 kcal / mol, about -188.7 kcal / mol to about -32 .5 kcal / mol, about -188.7 kcal / mol to about -32.3 kcal / mol, about -188.7 kcal / mol to about -32.0 kcal / mol, about -188.7 kcal / mol to about -29.2 kcal / mol, about -188.7 kcal / mol to about -25.9 kcal / mol, about -188.7 kcal / mol to about -22.6 kcal / mol, about -188.7 kcal / mol to about -21.7 kcal / mol, about -188.7 kcal / mol to about -17.8 kcal / mol, about -188.7 kcal / mol to about -15.0 kcal / mol, about -188.7 kcal / mol to about -13.5 kcal / mol, about -188.7 kcal / mol to about -13.3 kcal / mol, about -188.7 kcal / mol to about -10.10 kcal / mol, about -188.7 kcal / mol to about -32.0 kcal / mol, about -188.7 kcal / mol to about -28.10 kcal / mol, about -188.7 kcal / mol to about -29.30 kcal / mol, about -188.7 kcal / mol to about -26.70 kcal / mol, about -188.7 kcal / mol to about -23.kcal / mol, about -188.7 kcal / mol to about -20.10 kcal / mol, about -188.7 kcal / mol to about -19.20 kcal / mol, about -188.7 kcal / mol to about -18.20 kcal / mol, about -188.7 kcal / mol to about -13.10 kcal / mol, about -188.7 kcal / mol to about -10.15 kcal / mol, about -188.7 kcal / mol to about -13.0 kcal / mol, about -188.7 kcal / mol to about -16.0 kcal / mol, about -188.7 kcal / mol to about -12.7 kcal / mol, about -188.7 kcal / mol to about -23.65 kcal / mol, about -188.7 kcal / mol to about -19.40 kcal / mol mol, about -188.7 kcal / mol to about -15.40 kcal / mol, about -188.7 kcal / mol to about -34.40 kcal / mol, about -188.7 kcal / mol to about -25.30 kcal / mol, about -188.7 kcal / mol to about -16.40 kcal / mol, about -188.7 kcal / mol to about -12.60 kcal / mol, about -188.7 kcal / mol to about -30.50 kcal / mol, about -188.7 kcal / mol to about -21.40 kcal / mol, about -188.7 kcal / mol to about -34.30 kcal / mol, about -188.7 kcal / mol to about -25.20 kcal / mol, or about -188.7 kcal / mol to about -16.30 kcal / mol.

[0108] In certain non-limiting embodiments, each of the 3WJa, 3WJb, and 3WJc oligonucleotide sequences of the 3WJ scaffolds or domains of the present disclosure may independently comprise 8 to 36 nucleotides (e.g., 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, or 36 nucleotides), which do not include an RNA linker or an RNA portion conjugated to a biologically active portion of the 3WJ scaffold.

[0109] A four-way junction (4WJ) domain can be constructed from four (5'→3') RNA strands (designated 4WJa, 4WJb, 4WJc, and 4WJd) that are base-paired with each other. A first (5'→3') RNA oligonucleotide sequence designated 4Wja, a second (5'→3') RNA oligonucleotide sequence designated 4WJb, a third (5'→3') RNA oligonucleotide sequence designated 4WJc, and a fourth (5'→3') RNA oligonucleotide sequence designated 4WJd can be combined and base-paired to form a 4WJ domain. For example, Figure 6 The first branch (1BR) of the 4WJ domain is formed by the 5' portion of the 4Wja sequence and the 3' portion of the 4WJd sequence, the second branch (2BR) of the 4WJ domain is formed by the 3' portion of the 4Wja sequence and the 5' portion of the 4WJb sequence, the third branch (3BR) of the 4WJ domain is formed by the 3' portion of the 4WJb sequence and the 5' portion of the 4WJc sequence, and the fourth branch (4BR) of the 4WJ domain is formed by the 3' portion of the 4WJc sequence and the 5' portion of the 4WJd sequence, wherein each of the first, second, third and fourth branches may include a helical region having a plurality of RNA nucleotide pairs that form a canonical Watson-Crick bond. One, two, three and / or four branches of each 4WJ of the present disclosure may also include non-Watson-Crick nucleotide pairs, such as, but not limited to, GU. In certain embodiments, the 3' end of 4Wja can be connected to the 5' end of 4WJb via a joint sequence, and the 3' end of 4WJc can be connected to the 5' end of 4WJd via a joint sequence. In certain embodiments, the one or more joint sequences connecting 4Wja and 4WJb and the joint sequence connecting 4WJc and 4WJd can not be paired completely, for example, forming a stem-loop structure. In certain embodiments, one or more joint sequences can be paired with their sequence moieties, for example, forming a ring and a stem. In certain embodiments, one or more joint sequences can be paired completely with their sequence, for example, forming a stem. In certain embodiments, one or more joint sequences may not exist, for example, the 3' end of 4Wja can be directly connected to the 5' end of 4WJb and the 3' end of 4WJc can be directly connected to the 5' end of 4WJd.

[0110] A five-way junction (5WJ) domain can be constructed from five (5'→3') RNA strands (designated 5WJa, 5WJb, 5WJc, 5WJd, and 5WJe) that are base-paired with one another. The first (5'→3') RNA oligonucleotide sequence designated 5WJa, the second (5'→3') RNA oligonucleotide sequence designated 5WJb, the third (5'→3') RNA oligonucleotide sequence designated 5WJc, the fourth (5'→3') RNA oligonucleotide sequence designated 5WJd, and the fifth (5'→3') RNA oligonucleotide sequence designated 5WJe can be combined and base-paired to form a 5WJ domain. For example, Figure 9 The first branch (1BR) of the 5WJ domain is formed by the 5' portion of the 5WJa sequence and the 3' portion of the 5WJe sequence, the second branch (2BR) of the 5WJ domain is formed by the 3' portion of the 5WJa sequence and the 5' portion of the 5WJb sequence, the third branch (3BR) of the 5WJ domain is formed by the 3' portion of the 5WJb sequence and the 5' portion of the 5WJc sequence, the fourth branch (4BR) of the 5WJ domain is formed by the 3' portion of the 5WJc sequence and the 5' portion of the 5WJd sequence, and the fifth branch (5BR) of the 5WJ domain is formed by the 3' portion of the 5WJd sequence and the 5' portion of the 5WJe sequence, wherein each of the first, second, third, fourth and fifth branches may include a helical region having a plurality of RNA nucleotide pairs that form a canonical Watson-Crick bond. One, two, three, four and / or five branches of each 5WJ of the present disclosure may also include non-Watson-Crick nucleotide pairs, such as, but not limited to, GU. In certain embodiments, the 3' end of 5WJa can be connected to the 5' end of 5WJb via a joint sequence, the 3' end of 5WJc can be connected to the 5' end of 5WJd via a joint sequence, and the 3' end of 5WJd can be connected to the 5' end of 5WJe via a joint sequence. In certain embodiments, the one or more joint sequences connecting 5WJa and 5WJb, the joint sequence connecting 5WJc and 5WJd, and the joint sequence connecting 5WJd and 5WJe can be completely unpaired, for example, forming a stem-loop structure. In certain embodiments, one or more joint sequences can be paired with their sequence moieties, for example, forming a loop and a stem. In certain embodiments, one or more joint sequences can be paired completely with their sequences, for example, forming a stem. In certain embodiments, one or more joint sequences may not exist, for example, the 3' end of 5WJa can be directly connected to the 5' end of 5WJb, the 3' end of 5WJc can be directly connected to the 5' end of 5WJd, and / or the 3' end of 5WJd can be directly connected to the 5' end of 5WJe.

[0111] In certain embodiments, each multidirectional connection (for example, 3WJ, 4WJ and 5WJ) of the present disclosure may include a core structure. The duplex formed by each multidirectional connection may not affect the formation of the core structure (Khisamutdinov et al., " Enhancing immunomodulation on innate immunity by shape transition among RNA triangle, square and pentagon nanovehicles. " Nucleic Acids Res. 2014 November 1; 42 (15): 9996-10004; its content is hereby incorporated by reference in its entirety). Changing nucleotides (N) while forming a base pairing duplex with the corresponding arm may not affect the connection formation. For example, 3WJ can form a 60 degree angle between the two arms, and the duplex sequences on both sides can be designed to different lengths to form a triangle, quadrilateral or pentagonal connection structure with good flexibility, thereby achieving a thermodynamically stable 3WJ structure.

[0112] Table A shows the core structure sequences of 3WJ, 4WJ and 5WJ according to some embodiments of the present disclosure. Table A

[0113] Table 1 shows examples of 3WJ, 4WJ, and 5WJ sequences according to some embodiments of the present disclosure. 3WJ, 4WJ, and 5WJ motifs can be designed using bold sequences to form a core structure and to extend the arms with duplexes to enhance their thermodynamic stability. The three fragments co-assemble into a 3WJ structure with remarkable speed and affinity via a two-step reaction mechanism [ref1]. For example, Figure 2 and Figure 3 The 3WJ motif formed by SEQ ID NOs: 1-3 (Construct #1, ΔG = -27.3 Kcal / mol) and the 3WJ motif formed by SEQ ID NOs: 4-6 (Construct #2, ΔG = -35.8 Kcal / mol) are shown, respectively. Figure 6 and Figure 7 Shown are the 4WJ motif formed by SEQ ID NOs: 7-9 and 102 (Construct #3, AG = -143.7 Kcal / mol) and the 4WJ motif formed by SEQ ID NOs: 103-106 (Construct #57, AG = -62.7 Kcal / mol). Figure 9The 5WJ motif formed by SEQ ID NOs: 107-111 is shown (construct #58, AG = -188.7 Kcal / mol). Table 1 (Nucleotides involved in the core structure are shown in bold.) Example 1 Production of 3WJ-circRNA

[0114] Figure 4 It was shown that by using 3WJa at the 5' end of the precursor circular RNA sequence and 3WJb and 3WJc at the 3' end, a thermodynamically stable 3WJ (ΔG = -27.7 Kcal / mol) can be formed, followed by a self-splicing reaction to efficiently generate a circular RNA with an exon scar inside the circRNA. An internal ribosome entry site (IRES) (e.g., from encephalomyocarditis virus (EMCV)), a gene of interest (GOI) (e.g., enhanced green fluorescent protein (eGFP)) can be inserted, and the two sides can be two short regions that correspond to the exon fragments (E1 and E2) of the replacement intron-exon (PIE) construct between the 3' and 5' introns (I1 and I2) of the group I catalytic intron, such as the thymidylate synthase (Td) gene from T4 phage. Similarly, as Figure 5 As shown, thermodynamically stable 3WJ can be formed by using 3WJa and 3WJb at the 5' end and 3WJc at the 3' end.

[0115] The precursor RNA is synthesized by in vitro transcription under conditions known in the art using a plasmid DNA linearized after the 3' end of the precursor RNA design, and then heated in the presence of magnesium ions and GTP to promote self-cyclization before or after purification of the crude IVT reaction product. During self-cyclization, the first branch (1st BR) of the 3WJ domain can be formed by the 5' portion of the 3WJa sequence and the 3' portion of the 3WJc sequence, the second branch (2nd BR) of the 3WJ domain can be formed by the 3' portion of the 3WJa sequence and the 5' portion of the 3WJb sequence, and the third branch (3rd BR) of the 3WJ domain can be formed by the 3' portion of the 3WJb sequence and the 5' portion of the 3WJc sequence. Therefore, the 3WJ domain can lock the splicing bubble to promote correct folding and splicing efficiency. In certain embodiments, the 3rd BR can be formed before self-cyclization. For example, the 3' portion of the 3WJb sequence and the 5' portion of the 3WJc sequence can base pair to form the 3rd BR before cyclization occurs. In certain embodiments, a third branch may be formed during self-cyclization. Each branch may contain multiple RNA nucleotide pairs that form canonical Watson-Crick bonds. During splicing, the 3' hydroxyl group of the guanosine nucleotide participates in an ester exchange reaction at the 5' splice site. The 5' intron half (I1) is excised, and the free hydroxyl group at the end of the intermediate participates in a second ester exchange at the 3' splice site, resulting in cyclization of the middle region and excision of the 3' intron (I2) and the 3WJ domain together. 4WJ-circRNA production

[0116] Figure 8 We show that by using 4WJa and 4WJb at the 5' end and 4WJc and 4WJd at the 3' end of the precursor circular RNA sequence, a thermodynamically stable 4WJ (construct #57, ΔG = -62.7 Kcal / mol) can be formed, followed by a self-splicing reaction, to efficiently generate circular RNAs with exon scars within the circRNA. An internal ribosome entry site (IRES) (e.g., from encephalomyocarditis virus (EMCV)), a gene of interest (GOI) (e.g., enhanced green fluorescent protein (eGFP)) can be inserted, and flanked by two short regions corresponding to the exon segments (E1 and E2) of a replacement intron-exon (PIE) construct between the 3' and 5' introns (I1 and I2) of a group I catalytic intron, such as the thymidylate synthase (Td) gene from T4 phage, can be inserted. Production of 5WJ-circRNA

[0117] Figure 10It was shown that by using 5WJa, 5WJb and 5WJc at the 5' end of the precursor circular RNA sequence and 5WJd and 5WJe at the 3' end, a thermodynamically stable 5WJ (construct #58, ΔG = -188.7 Kcal / mol) can be formed, followed by a self-splicing reaction to efficiently generate a circular RNA with an exon scar inside the circRNA. An internal ribosome entry site (IRES) (e.g., from encephalomyocarditis virus (EMCV)), a gene of interest (GOI) (e.g., enhanced green fluorescent protein (eGFP)) can be inserted, and can be flanked by two short regions corresponding to the exon fragments (E1 and E2) of the replacement intron-exon (PIE) construct between the 3' and 5' introns (I1 and I2) of the group I catalytic intron, such as the thymidylate synthase (Td) gene from T4 phage. Similarly, as Figure 11 As shown, thermodynamically stable 5WJ can be formed by using 5WJa and 5WJb at the 5' end and 5WJc, 5WJd, and 5WJe at the 3' end. Example 2 3WJ-circRNA design improves RNA circularization efficiency

[0118] To compare the RNA circularization efficiency of the 3WJ-circRNA design and the conventional homology RNA design, the precursor RNA ( Figure 4 ) and precursor RNA synthesized by in vitro transcription from a vector encoding eGFP with a 5' homology arm at the 5' end and a 3' homology arm at the 3' end were analyzed.

[0119] Figure 12 The results show that the crude circular RNA formed by conventional homologous RNA design without column purification (lanes 1 and 3) (3WJ-) produced approximately 30% unreacted precursor RNA, as shown by the distinct middle band, and approximately 50% circular RNA, as shown by the top band. In contrast, the circular RNA formed by 3WJ-circular RNA design without column purification (lane 5) (3WJ+) produced approximately 80% circularized RNA, as shown by the top band, and produced approximately 5% unreacted precursor RNA and approximately 15% nicked RNA, as shown by the middle and lower bands, respectively. In addition, 3WJ-circular RNA (lane 6), which produced less unreacted precursor RNA, was more efficiently purified using ion-pairing reversed-phase HPLC compared to the homologous circular RNA (lanes 2 and 4), which produced more unreacted precursor RNA. Figure 13It was shown that, as determined by a bioanalyzer, the ratio of circRNA / precursor RNA produced by the 3WJ-circRNA design (3WJ+) was higher than that of the conventional homology circular RNA design (3WJ-). The circRNA generated by the 3WJ-circRNA design can be easily purified by reversed-phase HPLC. In order to compare the purity of circRNA produced with or without 3WJ, the homology arms (e.g., without 3WJ (3WJ (-))) were used to generate circular RNA encoding eGFP, and the crude product was fractionated using reversed-phase HPLC ( Figure 14A ), and then analyzed by agarose gel electrophoresis ( Figure 14B ). In contrast, 3WJ (3WJ(+)) was used to generate circular RNA encoding eGFP, and the crude product was fractionated using reverse phase HPLC ( Figure 15A ) and then analyzed by agarose gel electrophoresis ( Figure 15B ). These results show that when produced using 3WJ, the abundance of the precursor RNA (the third peak, at approximately 17.3 min) is reduced and the purity of the circular RNA fraction is improved (see Sections 3 and 4). These results indicate that the 3WJ-circRNA design improves RNA circularization and purification efficiency compared to the conventional homology arm design.

[0120] To compare the purity of circRNAs produced with or without 3WJ and purified with or without HPLC, circular RNA encoding Gaussia luciferase (G-Luc) was generated using homology arms (e.g., without 3WJ), and the crude product was fractionated using reverse-phase HPLC and then analyzed by agarose gel electrophoresis; and circular RNA encoding G-Luc was generated using 3WJ, and the crude product was fractionated using reverse-phase HPLC and then analyzed by agarose gel electrophoresis. Figure 20 The purity of the final crude circular RNA encoding the G-Luc sequence using 3WJ was shown to be 69%-79% crude (3WJ+ and purified-) (lane 3), and 81%-91% (3WJ+ and purified+) after HPLC purification (lane 4). In contrast, the purity of the final crude circular RNA encoding the G-Luc sequence without 3WJ was 55%-67% crude (3WJ- and purified-) (lane 1), and 72%-83% (3WJ- and purified+) after HPLC purification (lane 2). These results indicate that the use of multiple ligations (e.g., 3WJ) can produce circular RNAs of higher purity than without the use of multiple ligations (e.g., conventional homology arm methods). Highly pure circular RNAs can achieve therapeutic uses of such circular RNAs. Example 3 3WJ-circRNA design improves circular RNA expression

[0121] To test whether the improved RNA circularization efficiency by 3WJ-circRNA design is associated with enhanced gene expression, Figure 12 The eGFP-encoding vector used in the study was introduced into A549 cells, and then eGFP expression was analyzed using a fluorescence microplate reader. Figure 16 It was shown that the higher purity circRNAs produced by 3WJ-circRNA design in A549 cells exhibited superior circRNA expression levels (3WJ+, Purified+), as indicated by higher relative fluorescence units (RFU) compared to circRNAs designed with conventional homology arms (3WJ-, Purified+). Figure 12 The eGFP-encoding vector used in the study was introduced into 293 cells, and then eGFP expression was analyzed using a fluorescence microplate reader. Figure 17 It was shown that the higher purity circular RNA produced by 3WJ-circular RNA design in 293 cells exhibited excellent circular RNA expression levels (3WJ+, purified+), as shown by higher relative fluorescence units (RFU) compared to the circular RNA designed with conventional homology arms (3WJ-, purified+). Mock transfection (mock) served as a control. To compare the changes in protein expression levels over time, A549 cells were transfected with eGFP-encoding circRNA produced by - / +3WJ or with eGFP-encoding mRNA. Fluorescence was analyzed by flow cytometry for up to 6 days after transfection (10,000 cells were analyzed under each condition at each time point). Figure 18 and Figure 19 showed that the samples were consistent with those purified in HPLC ( Figure 14A 、 14B , 15A and 15B), circRNA generated by 3WJ (3WJ+) had better expression than that generated by homology arms (3WJ-) and mRNA. Example 4 RNA circularization using 3WJ and 4WJ Table 2 (Nucleotides involved in the core structure are shown in bold.)

[0122] Circularization of RNAs containing the coxsackievirus B3 (CVB3) IRES and eGFP sequence and RNAs containing the CVB3 IRES and eSpCas9 nuclease (which is a mutant form of the Cas9 nuclease) sequence using (1) 3WJ (Table 3, SEQ ID NOs: 119-121), (2) extended 3WJ (Ex3WJ, ΔG = -53.5 Kcal / mol) (Table 2), or (3) 4WJ (Construct 57, ΔG = -62.7 Kcal / mol) was compared. Circularization was performed using agarose gel electrophoresis and densitometry analysis ( Figure 21A ) or HPLC( Figure 21B and Figure 21C These results show that RNA with (+) 3WJ, Ex3WJ, or 4WJ produces higher RNA circularization than RNA without (-) 3WJ, Ex3WJ, or 4WJ. Time-course expression of circular RNA-encoded Gaussia luciferase (Gluc)

[0123] After transfection of A549 cells with Lipofectamine MessengerMAX reagent, the expression of Gluc encoding circRNA (unmodified) and linear mRNA (generated by in vitro transcription using Cap1 (ribose methylation of adjacent nucleotides of m7G) generated by constructs with 3WJ (Table 3, SEQ ID NO: 119-121, ΔG = -27.7 Kcal / mol) was compared. Luminescence was analyzed by microplate reader (20 μL of culture medium ( Figure 22A ) and normalized to pore volume)( Figure 22B These results showed that on day 3 after transfection, cells transfected with circRNA produced approximately 4.5-fold higher Gluc expression than cells transfected with linear mRNA. Time-course expression of circular RNA-encoded eGFP

[0124] After transfection of A549 cells with Lipofectamine MessengerMAX reagent, the expression of eGFP-encoding circRNA (unmodified) and linear mRNA (Cap1, 100% N1-methyl-pseudo-UTP modification) produced by constructs with 3WJ (Table 3, SEQ ID NO: 119-121) was compared. Fluorescence was analyzed by flow cytometry after transfection (10,000 cells were analyzed per condition at each time point ( Figure 23A ) and normalized to the pore volume ( Figure 23BThese results showed that on day 3 post-transfection, cells transfected with circRNA produced approximately 3.5-fold higher eGFP expression than cells transfected with linear mRNA.

[0125] The abundance of eGFP-encoding circRNA (unmodified) and linear mRNA (Cap1, 100% N1-methyl-pseudo-UTP modification) in A549 cells after transfection with Lipofectamine MessengerMAX reagent was compared. RNA copy number was determined by quantitative PCR (cDNA synthesized from extracted cellular RNA was analyzed using the standard curve method ( Figure 24A ) and normalized to the pore volume ( Figure 24B These results showed that on day 3 post-transfection, cells transfected with circRNA produced approximately four times the number of RNA copies compared to cells transfected with linear mRNA. Table 3 (Nucleotides involved in the core structure are shown in bold.)

[0126] The present invention can be defined by the following aspects: 1. A method for preparing circular RNA, comprising: The vector is transcribed to form a precursor RNA, wherein the vector comprises the following elements operably linked to each other and arranged in the following order: a) a 5' element comprising no or a sequence forming at least one stem-loop structure, b) a 3' group I self-splicing intron fragment containing a 3' splice site dinucleotide, c) protein coding or non-coding regions, d) a 5' group I self-splicing intron fragment containing a 5' splice site dinucleotide, e) a 3' element that contains no or a sequence that forms at least one stem-loop structure; provided that, when the 5' element does not comprise a stem-loop structure, the 3' element comprises at least one stem-loop structure; and, when the 3' element does not comprise a stem-loop structure, the 5' element comprises at least one stem-loop structure, wherein the 5' element and the 3' element form a thermodynamically stable multidirectional junction, The precursor RNA can form a circular RNA that is translatable and / or biologically active in the cell. 2. The method of aspect 1, wherein the thermodynamically stable multi-way junction is a 3-way junction (3WJ), a 4-way junction (4WJ) or a 5-way junction (5WJ). 3. The method according to aspect 2, wherein the 3WJ comprises The first branch of the 3WJ domain is formed by the 5' portion of the 3WJa sequence and the 3' portion of the 3WJc sequence and comprises the first helical region, a second branch of the 3WJ domain, the second branch being formed by the 3' portion of the 3WJa sequence and the 5' portion of the 3WJb sequence and comprising a second helical region, and The third branch of the 3WJ domain is formed by the 3' portion of the 3WJb sequence and the 5' portion of the 3WJc sequence and contains the third helical region. Each of the helical regions comprises a plurality of RNA nucleotide pairs that form canonical Watson-Crick bonds. 4. The method according to claim 3, wherein 3WJa comprises or consists of SEQ ID NO: 1, 3WJb comprises or consists of SEQ ID NO: 2, and 3WJc comprises or consists of SEQ ID NO: 3; or 3WJa comprises or consists of SEQ ID NO: 4, 3WJb comprises or consists of SEQ ID NO: 5, and 3WJc comprises or consists of SEQ ID NO: 6; or 3WJa comprises or consists of SEQ ID NO: 10, 3WJb comprises or consists of SEQ ID NO: 11, and 3WJc comprises or consists of SEQ ID NO: 12; or 3WJa comprises or consists of SEQ ID NO: 13, 3WJb comprises or consists of SEQ ID NO: 2, and 3WJc comprises or consists of SEQ ID NO: 13; or 3WJa comprises or consists of SEQ ID NO: 13, 3WJb comprises or consists of SEQ ID NO: 14, and 3WJc comprises or consists of SEQ ID NO: 3; or 3WJa comprises or consists of SEQ ID NO: 13, 3WJb comprises or consists of SEQ ID NO: 15, and 3WJc comprises or consists of SEQ ID NO: 3; or 3WJa comprises or consists of SEQ ID NO: 13, 3WJb comprises or consists of SEQ ID NO: 16, and 3WJc comprises or consists of SEQ ID NO: 3; or 3WJa comprises or consists of SEQ ID NO: 1, 3WJb comprises or consists of SEQ ID NO: 14, and 3WJc comprises or consists of SEQ ID NO: 3; or 3WJa comprises or consists of SEQ ID NO: 1, 3WJb comprises or consists of SEQ ID NO: 15, and 3WJc comprises or consists of SEQ ID NO: 3; or 3WJa comprises or consists of SEQ ID NO: 1, 3WJb comprises or consists of SEQ ID NO: 16, and 3WJc comprises or consists of SEQ ID NO: 3; or 3WJa comprises or consists of SEQ ID NO: 17, 3WJb comprises or consists of SEQ ID NO: 15, and 3WJc comprises or consists of SEQ ID NO: 18; or 3WJa comprises or consists of SEQ ID NO: 19, 3WJb comprises or consists of SEQ ID NO: 20, and 3WJc comprises or consists of SEQ ID NO: 18; or 3WJa comprises or consists of SEQ ID NO: 19, 3WJb comprises or consists of SEQ ID NO: 21, and 3WJc comprises or consists of SEQ ID NO: 22; or 3WJa comprises or consists of SEQ ID NO: 23, 3WJb comprises or consists of SEQ ID NO: 21, and 3WJc comprises or consists of SEQ ID NO: 24; or 3WJa comprises or consists of SEQ ID NO: 25, 3WJb comprises or consists of SEQ ID NO: 26, and 3WJc comprises or consists of SEQ ID NO: 24; or 3WJa comprises or consists of SEQ ID NO: 25, 3WJb comprises or consists of SEQ ID NO: 27, and 3WJc comprises or consists of SEQ ID NO: 28; or 3WJa comprises or consists of SEQ ID NO: 29, 3WJb comprises or consists of SEQ ID NO: 27, and 3WJc comprises or consists of SEQ ID NO: 30; or 3WJa comprises or consists of SEQ ID NO:31, 3WJb comprises or consists of SEQ ID NO:32, and 3WJc comprises or consists of SEQ ID NO:30; or 3WJa comprises or consists of SEQ ID NO:31, 3WJb comprises or consists of SEQ ID NO:33, and 3WJc comprises or consists of SEQ ID NO:34; or 3WJa comprises or consists of SEQ ID NO:41, 3WJb comprises or consists of SEQ ID NO:11, and 3WJc comprises or consists of SEQ ID NO:42; or 3WJa comprises or consists of SEQ ID NO:43, 3WJb comprises or consists of SEQ ID NO:44, and 3WJc comprises or consists of SEQ ID NO:42; or 3WJa comprises or consists of SEQ ID NO:43, 3WJb comprises or consists of SEQ ID NO:45, and 3WJc comprises or consists of SEQ ID NO:46; or 3WJa comprises or consists of SEQ ID NO:47, 3WJb comprises or consists of SEQ ID NO:45, and 3WJc comprises or consists of SEQ ID NO:48; or 3WJa comprises or consists of SEQ ID NO:49, 3WJb comprises or consists of SEQ ID NO:50, and 3WJc comprises or consists of SEQ ID NO:48; or 3WJa comprises or consists of SEQ ID NO:49, 3WJb comprises or consists of SEQ ID NO:51, and 3WJc comprises or consists of SEQ ID NO:52; or 3WJa comprises or consists of SEQ ID NO:53, 3WJb comprises or consists of SEQ ID NO:51, and 3WJc comprises or consists of SEQ ID NO:54; or 3WJa comprises or consists of SEQ ID NO:55, 3WJb comprises or consists of SEQ ID NO:56, and 3WJc comprises or consists of SEQ ID NO:54; or 3WJa comprises or consists of SEQ ID NO:55, 3WJb comprises or consists of SEQ ID NO:57, and 3WJc comprises or consists of SEQ ID NO:58; or 3WJa comprises or consists of SEQ ID NO:59, 3WJb comprises or consists of SEQ ID NO:57, and 3WJc comprises or consists of SEQ ID NO:60; or 3WJa comprises or consists of SEQ ID NO: 61, 3WJb comprises or consists of SEQ ID NO: 63, and 3WJc comprises or consists of SEQ ID NO: 64; or 3WJa comprises or consists of SEQ ID NO: 65, 3WJb comprises or consists of SEQ ID NO: 66, and 3WJc comprises or consists of SEQ ID NO: 64; or 3WJa comprises or consists of SEQ ID NO: 65, 3WJb comprises or consists of UGOCACGGG, and 3WJc comprises or consists of SEQ ID NO: 68; or 3WJa comprises or consists of SEQ ID NO:43, 3WJb comprises or consists of SEQ ID NO:69, and 3WJc comprises or consists of SEQ ID NO:46; or 3WJa comprises or consists of SEQ ID NO:47, 3WJb comprises or consists of SEQ ID NO:70, and 3WJc comprises or consists of SEQ ID NO:52; or 3WJa comprises or consists of SEQ ID NO:55, 3WJb comprises or consists of SEQ ID NO:71, and 3WJc comprises or consists of SEQ ID NO:72; or 3WJa comprises or consists of SEQ ID NO: 76, 3WJb comprises or consists of SEQ ID NO: 8, and 3WJc comprises or consists of SEQ ID NO: 9; or 3WJa comprises or consists of SEQ ID NO: 77, 3WJb comprises or consists of SEQ ID NO: 78, and 3WJc comprises or consists of SEQ ID NO: 79; or 3WJa comprises or consists of SEQ ID NO:80, 3WJb comprises or consists of SEQ ID NO:81, and 3WJc comprises or consists of SEQ ID NO:82; or 3WJa comprises or consists of SEQ ID NO:83, 3WJb comprises or consists of SEQ ID NO:84, and 3WJc comprises or consists of SEQ ID NO:85; or 3WJa comprises or consists of SEQ ID NO: 7, 3WJb comprises or consists of SEQ ID NO: 89, and 3WJc comprises or consists of SEQ ID NO: 9; or 3WJa comprises or consists of SEQ ID NO:90, 3WJb comprises or consists of SEQ ID NO:91, and 3WJc comprises or consists of SEQ ID NO:79; or 3WJa comprises or consists of SEQ ID NO:76, 3WJb comprises or consists of SEQ ID NO:89, and 3WJc comprises or consists of SEQ ID NO:9; or 3WJa comprises or consists of SEQ ID NO: 77, 3WJb comprises or consists of SEQ ID NO: 91, and 3WJc comprises or consists of SEQ ID NO: 79; or 3WJa comprises or consists of SEQ ID NO:80, 3WJb comprises or consists of SEQ ID NO:93, and 3WJc comprises or consists of SEQ ID NO:82; or 3WJa comprises or consists of SEQ ID NO:83, 3WJb comprises or consists of SEQ ID NO:95, and 3WJc comprises or consists of SEQ ID NO:85; or 3WJa comprises or consists of SEQ ID NO: 112, 3WJb comprises or consists of SEQ ID NO: 113, and 3WJc comprises or consists of SEQ ID NO: 114; or 3WJa comprises or consists of SEQ ID NO: 119, 3WJb comprises or consists of SEQ ID NO: 120, and 3WJc comprises or consists of SEQ ID NO: 121; or 3WJa contains AUGUGUA, 3WJb contains UACUUUG, and 3WJc contains AUCAUG; or 3WJa contains GCGUU, 3WJb contains UUCGC, and 3WJc contains GCCAUAGCG; or 3WJa contains GUAUGGCAC, 3WJb contains GUCACGG, and 3WJc contains CUCUUAC; or 3WJa contains AUGGUA, 3WJb contains ACUUUGU, and 3WJc contains AUCA; or 3WJa contains UGGU, 3WJb contains ACUUGU, and 3WJc contains AUCA; or 3WJa contains UGGU, 3WJb contains ACUGU, and 3WJc contains AUCA; or 3WJa contains UGGU, 3WJb contains ACGUU, and 3WJc contains AAUCA; or 3WJa contains UGUGU, 3WJb contains ACUUGU, and 3WJc contains AUCA; or 3WJa contains UGUGU, 3WJb contains ACUGU, and 3WJc contains AUCA; or 3WJa contains UGUGU, 3WJb contains ACGUU, and 3WJc contains AAUCA; or 3WJa contains UGGU, 3WJb contains ACUGU, and 3WJc contains AUCA; or 3WJa contains UAUGGCAC, 3WJb contains GUCACGG, and 3WJc contains CUCUUA; or 3WJa contains UAUGG, 3WJb contains UCAGG, and 3WJc contains CCUCUUA; or 3WJa contains UAUGGCAC, 3WJb contains GUCACGG, and 3WJc contains CUCUUA; or 3WJa contains UAUG, 3WJb contains CAGGGG, and 3WJc contains CUUG; or 3WJa contains UAUGU, 3WJb contains GCAGG, and 3WJc contains UCUUG; or 3WJa contains UAUGU, 3WJb contains GCAGGG, and 3WJc contains CUUG; or 3WJa contains UAUGU, 3WJb contains GCAGG, and 3WJc contains UCUUG; or 3WJa contains UGUGU, 3WJb contains ACUUUGU, and 3WJc contains AUCA; or 3WJa contains UGUGU, 3WJb contains ACUUU, and 3WJc contains AAAUCA. 5. The method according to aspect 2, wherein the 4WJ comprises The first branch of the 4WJ domain is formed by the 5' portion of the 4WJa sequence and the 3' portion of the 4WJd sequence and comprises the first helical region, a second branch of the 4WJ domain, the second branch being formed by the 3' portion of the 4WJa sequence and the 5' portion of the 4WJb sequence and comprising a second helical region, and The third branch of the 4WJ domain, the third branch is formed by the 3' portion of the 4WJb sequence and the 5' portion of the 4WJc sequence and contains the third helical region, and The fourth branch of the 4WJ domain is formed by the 3' portion of the 4WJc sequence and the 5' portion of the 4WJd sequence and includes the fourth helical region, Each of the helical regions comprises a plurality of RNA nucleotide pairs that form canonical Watson-Crick bonds. 6. The method according to claim 5, wherein 4WJa comprises or consists of SEQ ID NO: 7, 4WJb comprises or consists of SEQ ID NO: 8, 4WJc comprises or consists of SEQ ID NO: 9, and 4WJd comprises or consists of SEQ ID NO: 102; or 4WJa comprises or consists of SEQ ID NO: 103, 4WJb comprises or consists of SEQ ID NO: 104, 4WJc comprises or consists of SEQ ID NO: 105, and 4WJd comprises or consists of SEQ ID NO: 106; or 4WJa comprises or consists of SEQ ID NO: 115, 4WJb comprises or consists of SEQ ID NO: 116, 4WJc comprises or consists of SEQ ID NO: 117, and 4WJd comprises or consists of SEQ ID NO: 118; or 4WJa comprises UGCAGGUG, 4WJb comprises ACGGGC, 4WJc comprises CCAGCA, and 4WJd comprises SEQ ID NO: 67; or 4WJa comprises SEQ ID NO:74, 4WJb comprises AACUG, 4WJc comprises SEQ ID NO:75, and 4WJd comprises AUCAUG; or 4WJa comprises SEQ ID NO: 122, 4WJb comprises GAACU, 4WJc comprises SEQ ID NO: 123, and 4WJd comprises AAUCA. 7. The method according to aspect 2, wherein the 5WJ comprises The first branch of the 5WJ domain is formed by the 5' portion of the 5WJa sequence and the 3' portion of the 5WJe sequence and comprises the first helical region, The second branch of the 5WJ domain is formed by the 3' portion of the 5WJa sequence and the 5' portion of the 5WJb sequence and contains the second helical region. The third branch of the 5WJ domain is formed by the 3' portion of the 5WJb sequence and the 5' portion of the 5WJc sequence and contains the third helical region. a fourth branch of the 5WJ domain, the fourth branch being formed by the 3' portion of the 5WJc sequence and the 5' portion of the 5WJd sequence and comprising a fourth helical region, and The fifth branch of the 5WJ domain is formed by the 3' portion of the 5WJd sequence and the 5' portion of the 5WJe sequence and comprises the fifth helical region. Each of the helical regions comprises a plurality of RNA nucleotide pairs that form canonical Watson-Crick bonds. 8. The method according to aspect 5, wherein 5WJa comprises or consists of SEQ ID NO: 107, 5WJb comprises or consists of SEQ ID NO: 108, 5WJc comprises or consists of SEQ ID NO: 109, 5WJd comprises or consists of SEQ ID NO: 110, and 5WJe comprises or consists of SEQ ID NO: 111; or 5WJa contains GUGA, 5WJb contains UUGC, 5WJc contains GUGU, 5WJd contains AUGC, and 5WJe contains GUGC. 9. The method of any one of aspects 1 to 8, wherein the vector further comprises an internal ribosome entry site (IRES) at the 5' end of c),wherein the IRES is selected from an IRES sequence from a virus or gene selected from the group consisting of: Taura syndrome virus, Triatomine bug virus, Theiler encephalomyelitis virus, Simian virus 40, Fire ant virus 1, Grain aphid virus, Reticuloendothelioma virus, Human poliovirus 1, Small stink bug enterovirus, Kashmir bee virus, Human rhinovirus 2, Cibotium virus-1, Human immunodeficiency virus type 1, Cibotium virus-1, Himetobi virus P virus, hepatitis C virus, hepatitis A virus, hepatitis G virus, foot-and-mouth disease virus, human enterovirus 71, equine rhinitis virus, tea geometrid picorna-like virus, encephalomyocarditis virus (EMCV), Drosophila C virus, crucifer tobacco mosaic virus, cricket paralysis virus, bovine viral diarrhea virus 1, black queen cell virus, aphid lethal paralysis virus, avian encephalomyelitis virus, honeybee acute paralysis enterovirus, hibiscus chlorotic ringspot virus, classical swine fever virus, human fibroblast growth factor 2 (FGF2), human surfactant protein A1 (SFTPA1), human acute myeloid leukemia protein 1 / r unt-related transcription factor 1 (AML1 / RUNX1), Drosophila Antennapedia, human aquaporin-4 (AQP4), human angiotensin II receptor type 1 (AT1R), human BCL2-associated immortality gene 1 (BAG-1), human B-cell lymphoma 2 (BCL2), human immunoglobulin binding protein (BiP), human inhibitor of apoptosis family protein 1 (c-IAP1), human c-myc, human eukaryotic translation initiation factor 4G (eIF4G), mouse N-deacetylase and N-sulfotransferase 4 (NDST4L), human lymphoid enhancer binding factor-1 (LEF1), Mouse hypoxia-inducible factor 1 subunit alpha (HIF1α), human N-myc, mouse glial cell and testis-specific homeobox protein (Gtx), human cyclin-dependent kinase inhibitor 1B (p27kip1), human platelet-derived growth factor B / simian sarcoma virus human homolog (PDGF2 / c-sis), human p53, Moloney murine leukemia virus human proviral integration site-1 (Pim-1), mouse RNA-binding protein 3 (Rbm3), Drosophila reaper, canine Scamper, Drosophila ultra-bithorax gene (Ubx), Saari virus, coronavirus, parasite Enterovirus, human N-ras upstream (UNR), mouse dystrophin-related protein A (UtrA), human vascular endothelial growth factor A (VEGF-A), human X-linked inhibitor of apoptosis (XIAP), Drosophila hairless, Saccharomyces cerevisiae transcription factor IID (TFIID), Saccharomyces cerevisiae Yes1-associated transcription regulator (YAP1), human proto-oncogene tyrosine-protein kinase Src (c-src), human fibroblast growth factor 1 (FGF-1), simian picornavirus, turnip crinkle virus, coxsackievirus B3 (CVB3) and coxsackievirus A (CVB1 / 2). 10. The method of any one of aspects 1-9, wherein the vector further comprises an RNA polymerase promoter. 11. The method of aspect 10, wherein the RNA polymerase promoter is a T7 viral RNA polymerase promoter, a T6 viral RNA polymerase promoter, an SP6 viral RNA polymerase promoter, a T3 viral RNA polymerase promoter, or a T4 viral RNA polymerase promoter. 12. The method of any one of aspects 1-11, wherein the 3' Group I self-splicing intron fragment and the 5' Group I self-splicing intron fragment are from the cyanobacterium Anabaena sp. Pre-tRNA-Leu gene. 13. The method of any one of aspects 1-12, wherein the 3' Group I self-splicing intron fragment and the 5' Group I self-splicing intron fragment are from the T4 bacteriophage Td gene. 14. The method of any one of aspects 1-13, further comprising forming circular RNA by splint-mediated ligation of precursor RNAs. 15. The method of any one of aspects 1-14, wherein the vector is transfected into the cell using lipofection or electroporation prior to transcription. 16. The method of any one of aspects 1-15, wherein the vector is transfected into the cell using a nanovector prior to transcription. 17. The method of aspect 16, wherein the nanocarrier is a lipid, a polymer, or a lipid-polymer hybrid. 18. The method of any one of aspects 1-17, further comprising forming the circular RNA and purifying the circular RNA using a size exclusion chromatography column in tris-EDTA or ion-pair reverse phase HPLC. 19. The method of any one of aspects 1-17, further comprising forming the circular RNA and purifying the circular RNA in a high performance liquid chromatography (HPLC) system in a triethylammonium acetate (TEAA)-acetonitrile buffer having a pH range of about 4-10 at a flow rate of about 0.01-5 mL / min. 20. The method of any one of aspects 1-17, further comprising forming a circular RNA and purifying the circular RNA using phosphatase treatment. 21. The method of aspect 20, further comprising incubating the precursor RNA in the presence of (i) magnesium ions and / or (ii) guanosine nucleotides or guanosine nucleosides. 22. The method of aspect 21, wherein the incubation of the precursor RNA occurs at a temperature between about 20°C and about 60°C. 23. The method of any one of aspects 1 to 22, wherein transcription of the vector occurs in the presence of a nucleoside or a nucleotide monophosphate or diphosphate for incorporation of the nucleoside or nucleotide as the first nucleotide of a precursor RNA transcribed from the vector. 24. The method of aspect 23, wherein the precursor RNA comprises a monophosphate 5' end capable of being ligated using a ligase. 25. The method of aspect 24, wherein transcription of the vector occurs in the presence of a) guanosine nucleoside or mono- or diphosphate nucleotides; b) cytidine nucleoside or mono- or diphosphate nucleotide; c) uridine or mono- or diphosphate nucleotides; d) an adenosine nucleoside or a mono- or diphosphate nucleotide; or e) combinations thereof, The nucleoside or mono- or diphosphate nucleotide is incorporated as the first nucleotide of an RNA chain transcribed from the vector or a transcript produced from the vector. 26. The method of any one of aspects 1-25, wherein the protein coding region encodes a non-natural protein comprising one or more synthetic protein elements. 27. The method of any one of aspects 1-26, wherein the precursor RNA comprises nucleoside modifications. 28. The method of aspect 27, wherein the nucleoside modification is selected from the group consisting of: N 6 -methyladenosine (m6A), pseudouridine (Ψ), N 1 -methylpseudouridine (m1Ψ) and 5-methoxyuridine (5moU). 29. The method of any one of aspects 1-28, wherein the vector comprises a 5' spacer element located at the 3' end of b). 30. The method of any one of aspects 1-29, wherein the vector comprises a 3' spacer element located at the 5' end of d). 31. The method of aspect 29 or 30, wherein the 5' spacer element or the 3' spacer element comprises a polyA sequence or a polyA-C sequence. 32. The method of any one of aspects 1 to 31, wherein the noncoding region comprises an element encoding one or more RNAs selected from the group consisting of antisense RNA, transfer RNA (tRNA), transfer-messenger RNA (tmRNA), ribosomal RNA (rRNA), signal recognition particle RNA (7SL RNA or SRP RNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), SmY RNA (SmY), small Cajal body-specific RNA (scaRNA), guide RNA (gRNA), Y RNA, splicing leader RNA (SLRNA), microRNA (miRNA), small interfering RNA (siRNA), cis-natural antisense transcript (cis-NAT), CRISPR RNA (crRNA), long noncoding RNA (lncRNA), Piwi-interacting RNA (piRNA), short hairpin RNA (shRNA), trans-acting siRNA (tasiRNA), repeat-associated siRNA (rasiRNA), 7SK RNA (7SK), telomerase RNA component (TERC), vault RNA (vRNA, vtRNA), and enhancer RNA (eRNA). 33. A precursor RNA comprising the following elements operably linked to each other and arranged in the following order: a) a 5' element comprising no or at least one stem-loop structure, b) a 3' group I self-splicing intron fragment containing a 3' splice site dinucleotide, c) protein coding or non-coding regions, d) a 5' group I self-splicing intron fragment containing a 5' splice site dinucleotide, e) a 3' element that contains no or at least one stem-loop structure; provided that, when the 5' element does not comprise a stem-loop structure, the 3' element comprises at least one stem-loop structure; and, when the 3' element does not comprise a stem-loop structure, the 5' element comprises at least one stem-loop structure, wherein the 5' element and the 3' element form a thermodynamically stable multidirectional junction, The precursor RNA can form a circular RNA that is translatable and / or biologically active in the cell. 34. The precursor RNA of aspect 33, wherein the thermodynamically stable multi-way junction is a 3-way junction (3WJ), a 4-way junction (4WJ) or a 5-way junction (5WJ). 35. The precursor RNA of aspect 34, wherein the 3WJ comprises The first branch of the 3WJ domain is formed by the 5' portion of the 3WJa sequence and the 3' portion of the 3WJc sequence and comprises the first helical region, a second branch of the 3WJ domain, the second branch being formed by the 3' portion of the 3WJa sequence and the 5' portion of the 3WJb sequence and comprising a second helical region, and The third branch of the 3WJ domain is formed by the 3' portion of the 3WJb sequence and the 5' portion of the 3WJc sequence and contains the third helical region. Each of the helical regions comprises a plurality of RNA nucleotide pairs that form canonical Watson-Crick bonds. 36. The precursor RNA of aspect 35, wherein 3WJa comprises or consists of SEQ ID NO: 1, 3WJb comprises or consists of SEQ ID NO: 2, and 3WJc comprises or consists of SEQ ID NO: 3; or 3WJa comprises or consists of SEQ ID NO: 4, 3WJb comprises or consists of SEQ ID NO: 5, and 3WJc comprises or consists of SEQ ID NO: 6; or 3WJa comprises or consists of SEQ ID NO: 10, 3WJb comprises or consists of SEQ ID NO: 11, and 3WJc comprises or consists of SEQ ID NO: 12; or 3WJa comprises or consists of SEQ ID NO: 13, 3WJb comprises or consists of SEQ ID NO: 2, and 3WJc comprises or consists of SEQ ID NO: 13; or 3WJa comprises or consists of SEQ ID NO: 13, 3WJb comprises or consists of SEQ ID NO: 14, and 3WJc comprises or consists of SEQ ID NO: 3; or 3WJa comprises or consists of SEQ ID NO: 13, 3WJb comprises or consists of SEQ ID NO: 15, and 3WJc comprises or consists of SEQ ID NO: 3; or 3WJa comprises or consists of SEQ ID NO: 13, 3WJb comprises or consists of SEQ ID NO: 16, and 3WJc comprises or consists of SEQ ID NO: 3; or 3WJa comprises or consists of SEQ ID NO: 1, 3WJb comprises or consists of SEQ ID NO: 14, and 3WJc comprises or consists of SEQ ID NO: 3; or 3WJa comprises or consists of SEQ ID NO: 1, 3WJb comprises or consists of SEQ ID NO: 15, and 3WJc comprises or consists of SEQ ID NO: 3; or 3WJa comprises or consists of SEQ ID NO: 1, 3WJb comprises or consists of SEQ ID NO: 16, and 3WJc comprises or consists of SEQ ID NO: 3; or 3WJa comprises or consists of SEQ ID NO: 17, 3WJb comprises or consists of SEQ ID NO: 15, and 3WJc comprises or consists of SEQ ID NO: 18; or 3WJa comprises or consists of SEQ ID NO: 19, 3WJb comprises or consists of SEQ ID NO: 20, and 3WJc comprises or consists of SEQ ID NO: 18; or 3WJa comprises or consists of SEQ ID NO: 19, 3WJb comprises or consists of SEQ ID NO: 21, and 3WJc comprises or consists of SEQ ID NO: 22; or 3WJa comprises or consists of SEQ ID NO: 23, 3WJb comprises or consists of SEQ ID NO: 21, and 3WJc comprises or consists of SEQ ID NO: 24; or 3WJa comprises or consists of SEQ ID NO: 25, 3WJb comprises or consists of SEQ ID NO: 26, and 3WJc comprises or consists of SEQ ID NO: 24; or 3WJa comprises or consists of SEQ ID NO: 25, 3WJb comprises or consists of SEQ ID NO: 27, and 3WJc comprises or consists of SEQ ID NO: 28; or 3WJa comprises or consists of SEQ ID NO: 29, 3WJb comprises or consists of SEQ ID NO: 27, and 3WJc comprises or consists of SEQ ID NO: 30; or 3WJa comprises or consists of SEQ ID NO:31, 3WJb comprises or consists of SEQ ID NO:32, and 3WJc comprises or consists of SEQ ID NO:30; or 3WJa comprises or consists of SEQ ID NO:31, 3WJb comprises or consists of SEQ ID NO:33, and 3WJc comprises or consists of SEQ ID NO:34; or 3WJa comprises or consists of SEQ ID NO:41, 3WJb comprises or consists of SEQ ID NO:11, and 3WJc comprises or consists of SEQ ID NO:42; or 3WJa comprises or consists of SEQ ID NO:43, 3WJb comprises or consists of SEQ ID NO:44, and 3WJc comprises or consists of SEQ ID NO:42; or 3WJa comprises or consists of SEQ ID NO:43, 3WJb comprises or consists of SEQ ID NO:45, and 3WJc comprises or consists of SEQ ID NO:46; or 3WJa comprises or consists of SEQ ID NO:47, 3WJb comprises or consists of SEQ ID NO:45, and 3WJc comprises or consists of SEQ ID NO:48; or 3WJa comprises or consists of SEQ ID NO:49, 3WJb comprises or consists of SEQ ID NO:50, and 3WJc comprises or consists of SEQ ID NO:48; or 3WJa comprises or consists of SEQ ID NO:49, 3WJb comprises or consists of SEQ ID NO:51, and 3WJc comprises or consists of SEQ ID NO:52; or 3WJa comprises or consists of SEQ ID NO:53, 3WJb comprises or consists of SEQ ID NO:51, and 3WJc comprises or consists of SEQ ID NO:54; or 3WJa comprises or consists of SEQ ID NO:55, 3WJb comprises or consists of SEQ ID NO:56, and 3WJc comprises or consists of SEQ ID NO:54; or 3WJa comprises or consists of SEQ ID NO:55, 3WJb comprises or consists of SEQ ID NO:57, and 3WJc comprises or consists of SEQ ID NO:58; or 3WJa comprises or consists of SEQ ID NO:59, 3WJb comprises or consists of SEQ ID NO:57, and 3WJc comprises or consists of SEQ ID NO:60; or 3WJa comprises or consists of SEQ ID NO: 61, 3WJb comprises or consists of SEQ ID NO: 63, and 3WJc comprises or consists of SEQ ID NO: 64; or 3WJa comprises or consists of SEQ ID NO: 65, 3WJb comprises or consists of SEQ ID NO: 66, and 3WJc comprises or consists of SEQ ID NO: 64; or 3WJa comprises or consists of SEQ ID NO: 65, 3WJb comprises or consists of UGOCACGGG, and 3WJc comprises or consists of SEQ ID NO: 68; or 3WJa comprises or consists of SEQ ID NO:43, 3WJb comprises or consists of SEQ ID NO:69, and 3WJc comprises or consists of SEQ ID NO:46; or 3WJa comprises or consists of SEQ ID NO:47, 3WJb comprises or consists of SEQ ID NO:70, and 3WJc comprises or consists of SEQ ID NO:52; or 3WJa comprises or consists of SEQ ID NO:55, 3WJb comprises or consists of SEQ ID NO:71, and 3WJc comprises or consists of SEQ ID NO:72; or 3WJa comprises or consists of SEQ ID NO: 76, 3WJb comprises or consists of SEQ ID NO: 8, and 3WJc comprises or consists of SEQ ID NO: 9; or 3WJa comprises or consists of SEQ ID NO: 77, 3WJb comprises or consists of SEQ ID NO: 78, and 3WJc comprises or consists of SEQ ID NO: 79; or 3WJa comprises or consists of SEQ ID NO:80, 3WJb comprises or consists of SEQ ID NO:81, and 3WJc comprises or consists of SEQ ID NO:82; or 3WJa comprises or consists of SEQ ID NO:83, 3WJb comprises or consists of SEQ ID NO:84, and 3WJc comprises or consists of SEQ ID NO:85; or 3WJa comprises or consists of SEQ ID NO: 7, 3WJb comprises or consists of SEQ ID NO: 89, and 3WJc comprises or consists of SEQ ID NO: 9; or 3WJa comprises or consists of SEQ ID NO:90, 3WJb comprises or consists of SEQ ID NO:91, and 3WJc comprises or consists of SEQ ID NO:79; or 3WJa comprises or consists of SEQ ID NO:76, 3WJb comprises or consists of SEQ ID NO:89, and 3WJc comprises or consists of SEQ ID NO:9; or 3WJa comprises or consists of SEQ ID NO: 77, 3WJb comprises or consists of SEQ ID NO: 91, and 3WJc comprises or consists of SEQ ID NO: 79; or 3WJa comprises or consists of SEQ ID NO:80, 3WJb comprises or consists of SEQ ID NO:93, and 3WJc comprises or consists of SEQ ID NO:82; or 3WJa comprises or consists of SEQ ID NO:83, 3WJb comprises or consists of SEQ ID NO:95, and 3WJc comprises or consists of SEQ ID NO:85; or 3WJa comprises or consists of SEQ ID NO: 112, 3WJb comprises or consists of SEQ ID NO: 113, and 3WJc comprises or consists of SEQ ID NO: 114; or 3WJa comprises or consists of SEQ ID NO: 119, 3WJb comprises or consists of SEQ ID NO: 120, and 3WJc comprises or consists of SEQ ID NO: 121; or 3WJa contains AUGUGUA, 3WJb contains UACUUUG, and 3WJc contains AUCAUG; or 3WJa contains GCGUU, 3WJb contains UUCGC, and 3WJc contains GCCAUAGCG; or 3WJa contains GUAUGGCAC, 3WJb contains GUCACGG, and 3WJc contains CUCUUAC; or 3WJa contains AUGGUA, 3WJb contains ACUUUGU, and 3WJc contains AUCA; or 3WJa contains UGGU, 3WJb contains ACUUGU, and 3WJc contains AUCA; or 3WJa contains UGGU, 3WJb contains ACUGU, and 3WJc contains AUCA; or 3WJa contains UGGU, 3WJb contains ACGUU, and 3WJc contains AAUCA; or 3WJa contains UGUGU, 3WJb contains ACUUGU, and 3WJc contains AUCA; or 3WJa contains UGUGU, 3WJb contains ACUGU, and 3WJc contains AUCA; or 3WJa contains UGUGU, 3WJb contains ACGUU, and 3WJc contains AAUCA; or 3WJa contains UGGU, 3WJb contains ACUGU, and 3WJc contains AUCA; or 3WJa contains UAUGGCAC, 3WJb contains GUCACGG, and 3WJc contains CUCUUA; or 3WJa contains UAUGG, 3WJb contains UCAGG, and 3WJc contains CCUCUUA; or 3WJa contains UAUGGCAC, 3WJb contains GUCACGG, and 3WJc contains CUCUUA; or 3WJa contains UAUG, 3WJb contains CAGGGG, and 3WJc contains CUUG; or 3WJa contains UAUGU, 3WJb contains GCAGG, and 3WJc contains UCUUG; or 3WJa contains UAUGU, 3WJb contains GCAGGG, and 3WJc contains CUUG; or 3WJa contains UAUGU, 3WJb contains GCAGG, and 3WJc contains UCUUG; or 3WJa contains UGUGU, 3WJb contains ACUUUGU, and 3WJc contains AUCA; or 3WJa contains UGUGU, 3WJb contains ACUUU, and 3WJc contains AAAUCA. 37. The precursor RNA of aspect 34, wherein the 4WJ comprises The first branch of the 4WJ domain is formed by the 5' portion of the 4WJa sequence and the 3' portion of the 4WJd sequence and comprises the first helical region, a second branch of the 4WJ domain, the second branch being formed by the 3' portion of the 4WJa sequence and the 5' portion of the 4WJb sequence and comprising a second helical region, and The third branch of the 4WJ domain, the third branch is formed by the 3' portion of the 4WJb sequence and the 5' portion of the 4WJc sequence and contains the third helical region, and The fourth branch of the 4WJ domain is formed by the 3' portion of the 4WJc sequence and the 5' portion of the 4WJd sequence and includes the fourth helical region, Each of the helical regions comprises a plurality of RNA nucleotide pairs that form canonical Watson-Crick bonds. 38. The precursor RNA of aspect 37, wherein 4WJa comprises or consists of SEQ ID NO: 7, 4WJb comprises or consists of SEQ ID NO: 8, 4WJc comprises or consists of SEQ ID NO: 9, and 4WJd comprises or consists of SEQ ID NO: 102; or 4WJa comprises or consists of SEQ ID NO: 103, 4WJb comprises or consists of SEQ ID NO: 104, 4WJc comprises or consists of SEQ ID NO: 105, and 4WJd comprises or consists of SEQ ID NO: 106; or 4WJa comprises or consists of SEQ ID NO: 115, 4WJb comprises or consists of SEQ ID NO: 116, 4WJc comprises or consists of SEQ ID NO: 117, and 4WJd comprises or consists of SEQ ID NO: 118; or 4WJa comprises UGCAGGUG, 4WJb comprises ACGGGC, 4WJc comprises CCAGCA, and 4WJd comprises SEQ ID NO: 67; or 4WJa comprises SEQ ID NO:74, 4WJb comprises AACUG, 4WJc comprises SEQ ID NO:75, and 4WJd comprises AUCAUG; or 4WJa comprises SEQ ID NO: 122, 4WJb comprises GAACU, 4WJc comprises SEQ ID NO: 123, and 4WJd comprises AAUCA. 39. The precursor RNA of aspect 34, wherein the 5WJ comprises The first branch of the 5WJ domain is formed by the 5' portion of the 5WJa sequence and the 3' portion of the 5WJe sequence and comprises the first helical region, The second branch of the 5WJ domain is formed by the 3' portion of the 5WJa sequence and the 5' portion of the 5WJb sequence and contains the second helical region. The third branch of the 5WJ domain is formed by the 3' portion of the 5WJb sequence and the 5' portion of the 5WJc sequence and contains the third helical region. a fourth branch of the 5WJ domain, the fourth branch being formed by the 3' portion of the 5WJc sequence and the 5' portion of the 5WJd sequence and comprising a fourth helical region, and The fifth branch of the 5WJ domain is formed by the 3' portion of the 5WJd sequence and the 5' portion of the 5WJe sequence and comprises the fifth helical region. Each of the helical regions comprises a plurality of RNA nucleotide pairs that form canonical Watson-Crick bonds. 40. The precursor RNA of aspect 39, wherein 5WJa comprises or consists of SEQ ID NO: 107, 5WJb comprises or consists of SEQ ID NO: 108, 5WJc comprises or consists of SEQ ID NO: 109, 5WJd comprises or consists of SEQ ID NO: 110, and 5WJe comprises or consists of SEQ ID NO: 111; or 5WJa contains GUGA, 5WJb contains UUGC, 5WJc contains GUGU, 5WJd contains AUGC, and 5WJe contains GUGC. 41. The precursor RNA of any of aspects 33-40, wherein the noncoding region comprises one or more RNAs selected from the group consisting of antisense RNA, transfer RNA (tRNA), transfer-messenger RNA (tmRNA), ribosomal RNA (rRNA), signal recognition particle RNA (7SL RNA or SRP RNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), SmY RNA (SmY), small Cajal body-specific RNA (scaRNA), guide RNA (gRNA), Y RNA, splicing leader RNA (SLRNA), microRNA (miRNA), small interfering RNA (siRNA), cis-natural antisense transcript (cis-NAT), CRISPR RNA (crRNA), long noncoding RNA (lncRNA), Piwi-interacting RNA (piRNA), short hairpin RNA (shRNA), trans-acting siRNA (tasiRNA), repeat-associated siRNA (rasiRNA), 7SK RNA (7SK), telomerase RNA component (TERC), vault RNA (vRNA, vtRNA), and enhancer RNA (eRNA). 42. A vector encoding the precursor RNA of any one of aspects 33-41. 43. The vector of aspect 42 is a plasmid, a viral vector, a polymerase chain reaction (PCR) product, a cosmid, a bacterial artificial chromosome (BAC) or a yeast artificial chromosome (YAC).

[0127] Advantages of the present disclosure may include integrating RNA nanotechnology designs (e.g., thermodynamically stable 3WJ, 4WJ, and 5WJ structures) into the artificial circular RNA self-splicing process to promote correct folding and improve self-splicing efficiency, thereby paving the way for mass production of high-quality circular RNA for therapeutic development.

[0128] All references cited in this specification are incorporated herein by reference as if each reference were specifically and individually indicated to be incorporated herein by reference. Citation of any reference is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such reference by virtue of prior invention.

[0129] It will be understood that each of the above elements, or two or more of them, may also find useful application in other types of methods than those described above. Without further analysis, the foregoing will sufficiently reveal the gist of the present disclosure to enable others to readily adapt it to various applications by applying current knowledge, without omitting features that, from the perspective of the prior art, fairly constitute essential features of the general or specific aspects of the present disclosure as set forth in the appended claims. The foregoing embodiments are presented by way of example only; the scope of the present disclosure is limited only by the following claims.

Claims

1. A method for preparing circular ribonucleic acid (RNA), the method comprising: The vector is transcribed to form a precursor RNA comprising the following elements operably linked to each other and arranged in tandem in a 5' to 3' orientation: a) 5' element, b) 3′ group I self-splicing intron fragment, c) lack or contain an internal ribosome entry site (IRES) and a protein coding region or contain a non-coding region, d) a 5' group I self-splicing intron fragment, and e) 3′ element; wherein the 5' element and the 3' element form a stable structure having a Gibbs free energy (ΔG) of -190 kcal / mol to -9.0 kcal / mol, provided that the stable structure is not a duplex having at least 95% base pairing between the 5' element and the 3' element, The 3'I group self-splicing intron fragment and the 5'I group self-splicing intron fragment form self-cleaving and self-ligating RNA molecules, thereby generating circular RNA.

2. The method of claim 1 , wherein the 5' element does not contain or contains a sequence that forms at least one stem-loop structure, and the 3' element does not contain or contains a sequence that forms at least one stem-loop structure, provided that when the 5' element does not contain a stem-loop structure, the 3' element contains at least one stem-loop structure; and when the 3' element does not contain a stem-loop structure, the 5' element contains at least one stem-loop structure, and wherein the 5' element and the 3' element form a thermodynamically stable multi-directional junction RNA structure.

3. The method according to claim 1 or 2, wherein the precursor RNA is capable of forming a circular RNA that is translatable and / or biologically active in a cell.

4. The method of any one of claims 1 to 3, wherein the 5' element and the 3' element form a thermodynamically stable multidirectional junction.

5. The method of claim 1, wherein the thermodynamically stable multi-way junction RNA structure is a 3-way junction (3WJ), a 4-way junction (4WJ), a 5-way junction (5WJ), a hand-in-hand interaction, a kissing loop, or a pseudoknot.

6. The method of claim 5, wherein the 3WJ comprises The first branch of the 3WJ domain is formed by the 5' portion of the 3WJa sequence and the 3' portion of the 3WJc sequence and comprises a first helical region, a second branch of the 3WJ domain, the second branch being formed by the 3' portion of the 3WJa sequence and the 5' portion of the 3WJb sequence and comprising a second helical region, and The third branch of the 3WJ domain is formed by the 3' portion of the 3WJb sequence and the 5' portion of the 3WJc sequence and comprises a third helical region, Each of the helical regions comprises a plurality of RNA nucleotide pairs that form canonical Watson-Crick bonds.

7. The method of claim 6, wherein said 3WJa comprises or consists of SEQ ID NO: 1, 3WJb comprises or consists of SEQ ID NO: 2, and 3WJc comprises or consists of SEQ ID NO: 3; or said 3WJa comprises or consists of SEQ ID NO: 4, 3WJb comprises or consists of SEQ ID NO: 5, and 3WJc comprises or consists of SEQ ID NO: 6; or said 3WJa comprises or consists of SEQ ID NO: 10, 3WJb comprises or consists of SEQ ID NO: 11, and 3WJc comprises or consists of SEQ ID NO: 12; or said 3WJa comprises or consists of SEQ ID NO: 13, 3WJb comprises or consists of SEQ ID NO: 2, and 3WJc comprises or consists of SEQ ID NO: 13; or said 3WJa comprises or consists of SEQ ID NO: 13, 3WJb comprises or consists of SEQ ID NO: 14, and 3WJc comprises or consists of SEQ ID NO: 3; or said 3WJa comprises or consists of SEQ ID NO: 13, 3WJb comprises or consists of SEQ ID NO: 15, and 3WJc comprises or consists of SEQ ID NO: 3; or said 3WJa comprises or consists of SEQ ID NO: 13, 3WJb comprises or consists of SEQ ID NO: 16, and 3WJc comprises or consists of SEQ ID NO: 3; or said 3WJa comprises or consists of SEQ ID NO: 1, 3WJb comprises or consists of SEQ ID NO: 14, and 3WJc comprises or consists of SEQ ID NO: 3; or said 3WJa comprises or consists of SEQ ID NO: 1, 3WJb comprises or consists of SEQ ID NO: 15, and 3WJc comprises or consists of SEQ ID NO: 3; or said 3WJa comprises or consists of SEQ ID NO: 1, 3WJb comprises or consists of SEQ ID NO: 16, and 3WJc comprises or consists of SEQ ID NO: 3; or said 3WJa comprises or consists of SEQ ID NO: 17, 3WJb comprises or consists of SEQ ID NO: 15, and 3WJc comprises or consists of SEQ ID NO: 18; or said 3WJa comprises or consists of SEQ ID NO: 19, 3WJb comprises or consists of SEQ ID NO: 20, and 3WJc comprises or consists of SEQ ID NO: 18; or said 3WJa comprises or consists of SEQ ID NO: 19, 3WJb comprises or consists of SEQ ID NO: 21, and 3WJc comprises or consists of SEQ ID NO: 22; or said 3WJa comprises or consists of SEQ ID NO: 23, 3WJb comprises or consists of SEQ ID NO: 21, and 3WJc comprises or consists of SEQ ID NO: 24; or said 3WJa comprises or consists of SEQ ID NO: 25, 3WJb comprises or consists of SEQ ID NO: 26, and 3WJc comprises or consists of SEQ ID NO: 24; or said 3WJa comprises or consists of SEQ ID NO: 25, 3WJb comprises or consists of SEQ ID NO: 27, and 3WJc comprises or consists of SEQ ID NO: 28; or the 3WJa comprises or consists of SEQ ID NO: 29, 3WJb comprises or consists of SEQ ID NO: 27, and 3WJc comprises or consists of SEQ ID NO: 30; or said 3WJa comprises or consists of SEQ ID NO: 31, 3WJb comprises or consists of SEQ ID NO: 32, and 3WJc comprises or consists of SEQ ID NO: 30; or said 3WJa comprises or consists of SEQ ID NO: 31, 3WJb comprises or consists of SEQ ID NO: 33, and 3WJc comprises or consists of SEQ ID NO: 34; or said 3WJa comprises or consists of SEQ ID NO:41, 3WJb comprises or consists of SEQ ID NO:11, and 3WJc comprises or consists of SEQ ID NO:42; or said 3WJa comprises or consists of SEQ ID NO:43, 3WJb comprises or consists of SEQ ID NO:44, and 3WJc comprises or consists of SEQ ID NO:42; or said 3WJa comprises or consists of SEQ ID NO:43, 3WJb comprises or consists of SEQ ID NO:45, and 3WJc comprises or consists of SEQ ID NO:46; or said 3WJa comprises or consists of SEQ ID NO:47, 3WJb comprises or consists of SEQ ID NO:45, and 3WJc comprises or consists of SEQ ID NO:48; or said 3WJa comprises or consists of SEQ ID NO:49, 3WJb comprises or consists of SEQ ID NO:50, and 3WJc comprises or consists of SEQ ID NO:48; or said 3WJa comprises or consists of SEQ ID NO:49, 3WJb comprises or consists of SEQ ID NO:51, and 3WJc comprises or consists of SEQ ID NO:52; or said 3WJa comprises or consists of SEQ ID NO: 53, 3WJb comprises or consists of SEQ ID NO: 51, and 3WJc comprises or consists of SEQ ID NO: 54; or said 3WJa comprises or consists of SEQ ID NO: 55, 3WJb comprises or consists of SEQ ID NO: 56, and 3WJc comprises or consists of SEQ ID NO: 54; or the 3WJa comprises or consists of SEQ ID NO: 55, 3WJb comprises or consists of SEQ ID NO: 57, and 3WJc comprises or consists of SEQ ID NO: 58; or the 3WJa comprises or consists of SEQ ID NO: 59, 3WJb comprises or consists of SEQ ID NO: 57, and 3WJc comprises or consists of SEQ ID NO: 60; or said 3WJa comprises or consists of SEQ ID NO: 61, 3WJb comprises or consists of SEQ ID NO: 63, and 3WJc comprises or consists of SEQ ID NO: 64; or said 3WJa comprises or consists of SEQ ID NO: 65, 3WJb comprises or consists of SEQ ID NO: 66, and 3WJc comprises or consists of SEQ ID NO: 64; or said 3WJa comprises or consists of SEQ ID NO: 65, 3WJb comprises or consists of UGOCACGGG, and 3WJc comprises or consists of SEQ ID NO: 68; or the 3WJa comprises or consists of SEQ ID NO:43, 3WJb comprises or consists of SEQ ID NO:69, and 3WJc comprises or consists of SEQ ID NO:46; or the 3WJa comprises or consists of SEQ ID NO:47, 3WJb comprises or consists of SEQ ID NO:70, and 3WJc comprises or consists of SEQ ID NO:52; or the 3WJa comprises or consists of SEQ ID NO: 55, 3WJb comprises or consists of SEQ ID NO: 71, and 3WJc comprises or consists of SEQ ID NO: 72; or the 3WJa comprises or consists of SEQ ID NO: 76, 3WJb comprises or consists of SEQ ID NO: 8, and 3WJc comprises or consists of SEQ ID NO: 9; or said 3WJa comprises or consists of SEQ ID NO: 77, 3WJb comprises or consists of SEQ ID NO: 78, and 3WJc comprises or consists of SEQ ID NO: 79; or said 3WJa comprises or consists of SEQ ID NO: 80, 3WJb comprises or consists of SEQ ID NO: 81, and 3WJc comprises or consists of SEQ ID NO: 82; or said 3WJa comprises or consists of SEQ ID NO:83, 3WJb comprises or consists of SEQ ID NO:84, and 3WJc comprises or consists of SEQ ID NO:85; or said 3WJa comprises or consists of SEQ ID NO: 7, 3WJb comprises or consists of SEQ ID NO: 89, and 3WJc comprises or consists of SEQ ID NO: 9; or said 3WJa comprises or consists of SEQ ID NO:90, 3WJb comprises or consists of SEQ ID NO:91, and 3WJc comprises or consists of SEQ ID NO:79; or the 3WJa comprises or consists of SEQ ID NO: 76, 3WJb comprises or consists of SEQ ID NO: 89, and 3WJc comprises or consists of SEQ ID NO: 9; or the 3WJa comprises or consists of SEQ ID NO: 77, 3WJb comprises or consists of SEQ ID NO: 91, and 3WJc comprises or consists of SEQ ID NO: 79; or said 3WJa comprises or consists of SEQ ID NO: 80, 3WJb comprises or consists of SEQ ID NO: 93, and 3WJc comprises or consists of SEQ ID NO: 82; or the 3WJa comprises or consists of SEQ ID NO: 83, 3WJb comprises or consists of SEQ ID NO: 95, and 3WJc comprises or consists of SEQ ID NO: 85; or said 3WJa comprises or consists of SEQ ID NO: 112, 3WJb comprises or consists of SEQ ID NO: 113, and 3WJc comprises or consists of SEQ ID NO: 114; or the 3WJa comprises or consists of SEQ ID NO: 119, 3WJb comprises or consists of SEQ ID NO: 120, and 3WJc comprises or consists of SEQ ID NO: 121; or The 3WJa contains AUGUGUA, the 3WJb contains UACUUUG, and the 3WJc contains AUCAUG; or The 3WJa comprises GCGUU, the 3WJb comprises UUCGC, and the 3WJc comprises GCCAUAGCG; or wherein 3WJa comprises GUAUGGCAC, 3WJb comprises GUCACGG, and 3WJc comprises CUCUUAC; or The 3WJa comprises AUGGUA, the 3WJb comprises ACUUUGU, and the 3WJc comprises AUCA; or The 3WJa comprises UGGU, the 3WJb comprises ACUUGU, and the 3WJc comprises AUCA; or The 3WJa comprises UGGU, the 3WJb comprises ACUGU, and the 3WJc comprises AUCA; or The 3WJa comprises UGGU, the 3WJb comprises ACGUU, and the 3WJc comprises AAUCA; or The 3WJa comprises UGUGU, the 3WJb comprises ACUUGU, and the 3WJc comprises AUCA; or The 3WJa comprises UGUGU, the 3WJb comprises ACUGU, and the 3WJc comprises AUCA; or The 3WJa comprises UGUGU, the 3WJb comprises ACGUU, and the 3WJc comprises AAUCA; or The 3WJa comprises UGGU, the 3WJb comprises ACUGU, and the 3WJc comprises AUCA; or wherein 3WJa comprises UAUGGCAC, 3WJb comprises GUCACGG, and 3WJc comprises CUCUUA; or The 3WJa comprises UAUGG, the 3WJb comprises UCAGG, and the 3WJc comprises CCUCUUA; or wherein 3WJa comprises UAUGGCAC, 3WJb comprises GUCACGG, and 3WJc comprises CUCUUA; or The 3WJa comprises UAUG, the 3WJb comprises CAGGGG, and the 3WJc comprises CUUG; or The 3WJa comprises UAUGU, the 3WJb comprises GCAGG, and the 3WJc comprises UCUUG; or The 3WJa comprises UAUGU, the 3WJb comprises GCAGGG, and the 3WJc comprises CUUG; or The 3WJa comprises UAUGU, the 3WJb comprises GCAGG, and the 3WJc comprises UCUUG; or The 3WJa comprises UGUGU, the 3WJb comprises ACUUUGU, and the 3WJc comprises AUCA; or The 3WJa contains UGUGU, 3WJb contains ACUUU, and 3WJc contains AAAUCA.

8. The method of claim 5, wherein the 4WJ comprises The first branch of the 4WJ domain is formed by the 5' portion of the 4WJa sequence and the 3' portion of the 4WJd sequence and comprises a first helical region, a second branch of the 4WJ domain, the second branch being formed by the 3' portion of the 4WJa sequence and the 5' portion of the 4WJb sequence and comprising a second helical region, and The third branch of the 4WJ domain, the third branch is formed by the 3' portion of the 4WJb sequence and the 5' portion of the 4WJc sequence and comprises a third helical region, and The fourth branch of the 4WJ domain is formed by the 3' portion of the 4WJc sequence and the 5' portion of the 4WJd sequence and comprises a fourth helical region, Each of the helical regions comprises a plurality of RNA nucleotide pairs that form canonical Watson-Crick bonds.

9. The method of claim 8, wherein said 4WJa comprises or consists of SEQ ID NO: 7, 4WJb comprises or consists of SEQ ID NO: 8, 4WJc comprises or consists of SEQ ID NO: 9, and 4WJd comprises or consists of SEQ ID NO: 102; or said 4WJa comprises or consists of SEQ ID NO: 103, 4WJb comprises or consists of SEQ ID NO: 104, 4WJc comprises or consists of SEQ ID NO: 105, and 4WJd comprises or consists of SEQ ID NO: 106; or said 4WJa comprises or consists of SEQ ID NO: 115, 4WJb comprises or consists of SEQ ID NO: 116, 4WJc comprises or consists of SEQ ID NO: 117, and 4WJd comprises or consists of SEQ ID NO: 118; or wherein 4WJa comprises UGCAGGUG, 4WJb comprises ACGGGC, 4WJc comprises CCAGCA, and 4WJd comprises SEQ ID NO: 67; or wherein 4WJa comprises SEQ ID NO: 74, 4WJb comprises AACUG, 4WJc comprises SEQ ID NO: 75, and 4WJd comprises AUCAUG; or The 4WJa comprises SEQ ID NO: 122, 4WJb comprises GAACU, 4WJc comprises SEQ ID NO: 123, and 4WJd comprises AAUCA.

10. The method of claim 5, wherein the 5WJ comprises The first branch of the 5WJ domain is formed by the 5' portion of the 5WJa sequence and the 3' portion of the 5WJe sequence and comprises a first helical region, The second branch of the 5WJ domain, the second branch is formed by the 3' portion of the 5WJa sequence and the 5' portion of the 5WJb sequence and comprises a second helical region, The third branch of the 5WJ domain is formed by the 3' portion of the 5WJb sequence and the 5' portion of the 5WJc sequence and comprises a third helical region, The fourth branch of the 5WJ domain, the fourth branch is formed by the 3' portion of the 5WJc sequence and the 5' portion of the 5WJd sequence and comprises a fourth helical region, and The fifth branch of the 5WJ domain is formed by the 3' portion of the 5WJd sequence and the 5' portion of the 5WJe sequence and comprises a fifth helical region, Each of the helical regions comprises a plurality of RNA nucleotide pairs that form canonical Watson-Crick bonds.

11. The method of claim 10, wherein the 5WJa comprises or consists of SEQ ID NO: 107, the 5WJb comprises or consists of SEQ ID NO: 108, the 5WJc comprises or consists of SEQ ID NO: 109, the 5WJd comprises or consists of SEQ ID NO: 110, and the 5WJe comprises or consists of SEQ ID NO: 111; or The 5WJa contains GUGA, the 5WJb contains UUGC, the 5WJc contains GUGU, the 5WJd contains AUGC, and the 5WJe contains GUGC.

12. The method according to any one of claims 1 to 11,wherein the IRES is selected from an IRES sequence from a virus or gene selected from the group consisting of: Taura syndrome virus, Triatomine bug virus, Theiler encephalomyelitis virus, Simian virus 40, Fire ant virus 1, Grain aphid virus, Reticuloendothelioma virus, Human poliovirus 1, Small stink bug enterovirus, Kashmir bee virus, Human rhinovirus 2, Cibotium virus-1, Human immunodeficiency virus type 1, Cibotium virus-1, Himetobi virus P virus, hepatitis C virus, hepatitis A virus, hepatitis G virus, foot-and-mouth disease virus, human enterovirus 71, equine rhinitis virus, tea geometrid picorna-like virus, encephalomyocarditis virus (EMCV), Drosophila C virus, crucifer tobacco mosaic virus, cricket paralysis virus, bovine viral diarrhea virus 1, black queen cell virus, aphid lethal paralysis virus, avian encephalomyelitis virus, honeybee acute paralysis enterovirus, hibiscus chlorotic ringspot virus, classical swine fever virus, human fibroblast growth factor 2 (FGF2), human surfactant protein A1 (SFTPA1), human acute myeloid leukemia protein 1 / r unt-related transcription factor 1 (AML1 / RUNX1), Drosophila Antennapedia, human aquaporin-4 (AQP4), human angiotensin II receptor type 1 (AT1R), human BCL2-associated immortality gene 1 (BAG-1), human B-cell lymphoma 2 (BCL2), human immunoglobulin binding protein (BiP), human inhibitor of apoptosis family protein 1 (c-IAP1), human c-myc, human eukaryotic translation initiation factor 4G (eIF4G), mouse N-deacetylase and N-sulfotransferase 4 (NDST4L), human lymphoid enhancer binding factor-1 (LEF1), Mouse hypoxia-inducible factor 1 subunit alpha (HIF1α), human N-myc, mouse glial cell and testis-specific homeobox protein (Gtx), human cyclin-dependent kinase inhibitor 1B (p27kip1), human platelet-derived growth factor B / simian sarcoma virus human homolog (PDGF2 / c-sis), human p53, Moloney murine leukemia virus human proviral integration site-1 (Pim-1), mouse RNA-binding protein 3 (Rbm3), Drosophila reaper, canine Scamper, Drosophila ultra-bithorax gene (Ubx), Saari virus, coronavirus, parasite Enterovirus, human N-ras upstream (UNR), mouse dystrophin-related protein A (UtrA), human vascular endothelial growth factor A (VEGF-A), human X-linked inhibitor of apoptosis (XIAP), Drosophila hairless, Saccharomyces cerevisiae transcription factor IID (TFIID), Saccharomyces cerevisiae Yes1-associated transcription regulator (YAP1), human proto-oncogene tyrosine-protein kinase Src (c-src), human fibroblast growth factor 1 (FGF-1), simian picornavirus, turnip crinkle virus, coxsackievirus B3 (CVB3) and coxsackievirus A (CVB1 / 2).

13. The method of any one of claims 1 to 12, wherein the vector further comprises an RNA polymerase promoter.

14. The method of claim 13, wherein the RNA polymerase promoter is a T7 viral RNA polymerase promoter, a T6 viral RNA polymerase promoter, an SP6 viral RNA polymerase promoter, a T3 viral RNA polymerase promoter, or a T4 viral RNA polymerase promoter.

15. The method of any one of claims 1 to 14, wherein the 3' Group I self-splicing intron fragment and the 5' Group I self-splicing intron fragment are derived from the cyanobacterium Anabaena sp. Pre-tRNA-Leu gene. 16 . The method according to claim 1 , wherein the 3′ group I self-splicing intron fragment and the 5′ group I self-splicing intron fragment are derived from the T4 bacteriophage Td gene.

17. The method of any one of claims 1-16, further comprising forming the circular RNA by splint-mediated ligation of precursor RNAs.

18. The method of any one of claims 1 to 17, wherein the vector is transfected into the cell using lipofection or electroporation prior to transcription.

19. The method of any one of claims 1-18, wherein the vector is transfected into the cell using a nanovector prior to transcription.

20. The method of claim 19, wherein the nanocarrier is a lipid, a polymer, or a lipid-polymer hybrid.

21. The method of any one of claims 1-20, further comprising forming the circular RNA and purifying the circular RNA using a size exclusion chromatography column in tris-EDTA or ion-pair reverse phase HPLC.

22. The method of any one of claims 1-20, further comprising forming the circular RNA and purifying the circular RNA in a high performance liquid chromatography (HPLC) system in a triethylammonium acetate (TEAA)-acetonitrile buffer having a pH range of about 4-10 at a flow rate of about 0.01-5 mL / min.

23. The method of any one of claims 1-20, further comprising forming the circular RNA and purifying the circular RNA using phosphatase treatment.

24. The method of claim 23, further comprising incubating the precursor RNA in the presence of (i) magnesium ions and / or (ii) guanosine nucleotides or guanosine nucleosides.

25. The method of claim 24, wherein the incubation of the precursor RNA occurs at a temperature between about 20°C and about 60°C.

26. The method of any one of claims 1 to 25, wherein transcription of the vector occurs in the presence of a nucleoside or a nucleotide monophosphate or diphosphate for incorporation of the nucleoside or nucleotide as the first nucleotide of a precursor RNA transcribed from the vector.

27. The method of claim 26, wherein the precursor RNA comprises a monophosphate 5' end capable of being ligated using a ligase.

28. The method of claim 27, wherein transcription of the vector occurs in the presence of: a) guanosine nucleoside or mono- or diphosphate nucleotides; b) cytidine nucleoside or mono- or diphosphate nucleotide; c) uridine or mono- or diphosphate nucleotides; d) an adenosine nucleoside or a mono- or diphosphate nucleotide; or e) combinations thereof, The nucleoside or mono- or diphosphate nucleotide is incorporated as the first nucleotide of an RNA chain transcribed from the vector or a transcript produced from the vector.

29. The method of any one of claims 1-28, wherein the protein coding region encodes a non-natural protein comprising one or more synthetic protein elements.

30. The method of any one of claims 1-31, wherein the vector comprises a 5' spacer element located at the 3' end of b).

31. The method of any one of claims 1-32, wherein the vector comprises a 3' spacer element located at the 5' end of d).

32. The method of claim 32 or 33, wherein the 5' spacer element or the 3' spacer element comprises a polyA sequence or a polyA-C sequence.

33. The method of any one of claims 1 to 34, wherein the noncoding region comprises an element encoding one or more RNAs selected from the group consisting of antisense RNA, transfer RNA (tRNA), transfer-messenger RNA (tmRNA), ribosomal RNA (rRNA), signal recognition particle RNA (7SL RNA or SRP RNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), SmY RNA (SmY), small Cajal body-specific RNA (scaRNA), guide RNA (gRNA), Y RNA, splicing leader RNA (SL RNA), microRNA (miRNA), small interfering RNA (siRNA), cis-natural antisense transcript (cis-NAT), CRISPR RNA (crRNA), long noncoding RNA (lncRNA), Piwi-interacting RNA (piRNA), short hairpin RNA (shRNA), trans-acting siRNA (tasiRNA), repeat-associated siRNA (rasiRNA), 7SK RNA (7SK), telomerase RNA component (TERC), vault RNA (vRNA, vtRNA), and enhancer RNA (eRNA).

34. A precursor RNA comprising the following elements operably linked to each other and arranged in tandem in a 5' to 3' orientation: a) 5' element, b) 3′ group I self-splicing intron fragment, c) without or containing an IRES and a protein coding region or containing a non-coding region, d) a 5' group I self-splicing intron fragment, and e) 3′ element wherein the 5' element and the 3' element form a stable structure having a Gibbs free energy (ΔG) of -190 kcal / mol to -9.0 kcal / mol, provided that the stable structure is not a duplex having at least 95% base pairing between the 5' element and the 3' element, The 3'I group self-splicing intron fragment and the 5'I group self-splicing intron fragment form self-cleaving and self-ligating RNA molecules, thereby generating circular RNA.

35. The precursor RNA of claim 36, wherein the 5' element does not contain or contains a sequence that forms at least one stem-loop structure, and the 3' element does not contain or contains a sequence that forms at least one stem-loop structure, provided that when the 5' element does not contain a stem-loop structure, the 3' element contains at least one stem-loop structure; and when the 3' element does not contain a stem-loop structure, the 5' element contains at least one stem-loop structure, and wherein the 5' element and the 3' element form a thermodynamically stable multi-directional junction RNA structure.

36. The precursor RNA according to claim 36 or 37, wherein the precursor RNA is capable of forming a circular RNA that is translatable and / or biologically active in a cell.

37. The precursor RNA of any one of claims 36-38, wherein the 5' element and the 3' element form a thermodynamically stable multidirectional junction.

38. The precursor RNA of claim 39, wherein the thermodynamically stable multi-way junction RNA structure is a 3-way junction (3WJ), a 4-way junction (4WJ), a 5-way junction (5WJ), a hand-in-hand interaction, a kissing loop, or a pseudoknot.

39. The precursor RNA of claim 40, wherein the 3WJ comprises The first branch of the 3WJ domain is formed by the 5' portion of the 3WJa sequence and the 3' portion of the 3WJc sequence and comprises a first helical region, a second branch of the 3WJ domain, the second branch being formed by the 3' portion of the 3WJa sequence and the 5' portion of the 3WJb sequence and comprising a second helical region, and The third branch of the 3WJ domain is formed by the 3' portion of the 3WJb sequence and the 5' portion of the 3WJc sequence and comprises a third helical region, Each of the helical regions comprises a plurality of RNA nucleotide pairs that form canonical Watson-Crick bonds.

40. The precursor RNA of claim 41, wherein said 3WJa comprises or consists of SEQ ID NO: 1, 3WJb comprises or consists of SEQ ID NO: 2, and 3WJc comprises or consists of SEQ ID NO: 3; or said 3WJa comprises or consists of SEQ ID NO: 4, 3WJb comprises or consists of SEQ ID NO: 5, and 3WJc comprises or consists of SEQ ID NO: 6; or said 3WJa comprises or consists of SEQ ID NO: 10, 3WJb comprises or consists of SEQ ID NO: 11, and 3WJc comprises or consists of SEQ ID NO: 12; or said 3WJa comprises or consists of SEQ ID NO: 13, 3WJb comprises or consists of SEQ ID NO: 2, and 3WJc comprises or consists of SEQ ID NO: 13; or said 3WJa comprises or consists of SEQ ID NO: 13, 3WJb comprises or consists of SEQ ID NO: 14, and 3WJc comprises or consists of SEQ ID NO: 3; or said 3WJa comprises or consists of SEQ ID NO: 13, 3WJb comprises or consists of SEQ ID NO: 15, and 3WJc comprises or consists of SEQ ID NO: 3; or said 3WJa comprises or consists of SEQ ID NO: 13, 3WJb comprises or consists of SEQ ID NO: 16, and 3WJc comprises or consists of SEQ ID NO: 3; or said 3WJa comprises or consists of SEQ ID NO: 1, 3WJb comprises or consists of SEQ ID NO: 14, and 3WJc comprises or consists of SEQ ID NO: 3; or said 3WJa comprises or consists of SEQ ID NO: 1, 3WJb comprises or consists of SEQ ID NO: 15, and 3WJc comprises or consists of SEQ ID NO: 3; or said 3WJa comprises or consists of SEQ ID NO: 1, 3WJb comprises or consists of SEQ ID NO: 16, and 3WJc comprises or consists of SEQ ID NO: 3; or said 3WJa comprises or consists of SEQ ID NO: 17, 3WJb comprises or consists of SEQ ID NO: 15, and 3WJc comprises or consists of SEQ ID NO: 18; or said 3WJa comprises or consists of SEQ ID NO: 19, 3WJb comprises or consists of SEQ ID NO: 20, and 3WJc comprises or consists of SEQ ID NO: 18; or said 3WJa comprises or consists of SEQ ID NO: 19, 3WJb comprises or consists of SEQ ID NO: 21, and 3WJc comprises or consists of SEQ ID NO: 22; or said 3WJa comprises or consists of SEQ ID NO: 23, 3WJb comprises or consists of SEQ ID NO: 21, and 3WJc comprises or consists of SEQ ID NO: 24; or said 3WJa comprises or consists of SEQ ID NO: 25, 3WJb comprises or consists of SEQ ID NO: 26, and 3WJc comprises or consists of SEQ ID NO: 24; or said 3WJa comprises or consists of SEQ ID NO: 25, 3WJb comprises or consists of SEQ ID NO: 27, and 3WJc comprises or consists of SEQ ID NO: 28; or the 3WJa comprises or consists of SEQ ID NO: 29, 3WJb comprises or consists of SEQ ID NO: 27, and 3WJc comprises or consists of SEQ ID NO: 30; or said 3WJa comprises or consists of SEQ ID NO: 31, 3WJb comprises or consists of SEQ ID NO: 32, and 3WJc comprises or consists of SEQ ID NO: 30; or said 3WJa comprises or consists of SEQ ID NO: 31, 3WJb comprises or consists of SEQ ID NO: 33, and 3WJc comprises or consists of SEQ ID NO: 34; or said 3WJa comprises or consists of SEQ ID NO:41, 3WJb comprises or consists of SEQ ID NO:11, and 3WJc comprises or consists of SEQ ID NO:42; or said 3WJa comprises or consists of SEQ ID NO:43, 3WJb comprises or consists of SEQ ID NO:44, and 3WJc comprises or consists of SEQ ID NO:42; or said 3WJa comprises or consists of SEQ ID NO:43, 3WJb comprises or consists of SEQ ID NO:45, and 3WJc comprises or consists of SEQ ID NO:46; or said 3WJa comprises or consists of SEQ ID NO:47, 3WJb comprises or consists of SEQ ID NO:45, and 3WJc comprises or consists of SEQ ID NO:48; or said 3WJa comprises or consists of SEQ ID NO:49, 3WJb comprises or consists of SEQ ID NO:50, and 3WJc comprises or consists of SEQ ID NO:48; or said 3WJa comprises or consists of SEQ ID NO:49, 3WJb comprises or consists of SEQ ID NO:51, and 3WJc comprises or consists of SEQ ID NO:52; or said 3WJa comprises or consists of SEQ ID NO: 53, 3WJb comprises or consists of SEQ ID NO: 51, and 3WJc comprises or consists of SEQ ID NO: 54; or said 3WJa comprises or consists of SEQ ID NO: 55, 3WJb comprises or consists of SEQ ID NO: 56, and 3WJc comprises or consists of SEQ ID NO: 54; or the 3WJa comprises or consists of SEQ ID NO: 55, 3WJb comprises or consists of SEQ ID NO: 57, and 3WJc comprises or consists of SEQ ID NO: 58; or the 3WJa comprises or consists of SEQ ID NO: 59, 3WJb comprises or consists of SEQ ID NO: 57, and 3WJc comprises or consists of SEQ ID NO: 60; or said 3WJa comprises or consists of SEQ ID NO: 61, 3WJb comprises or consists of SEQ ID NO: 63, and 3WJc comprises or consists of SEQ ID NO: 64; or said 3WJa comprises or consists of SEQ ID NO: 65, 3WJb comprises or consists of SEQ ID NO: 66, and 3WJc comprises or consists of SEQ ID NO: 64; or said 3WJa comprises or consists of SEQ ID NO: 65, 3WJb comprises or consists of UGOCACGGG, and 3WJc comprises or consists of SEQ ID NO: 68; or the 3WJa comprises or consists of SEQ ID NO:43, 3WJb comprises or consists of SEQ ID NO:69, and 3WJc comprises or consists of SEQ ID NO:46; or the 3WJa comprises or consists of SEQ ID NO:47, 3WJb comprises or consists of SEQ ID NO:70, and 3WJc comprises or consists of SEQ ID NO:52; or the 3WJa comprises or consists of SEQ ID NO: 55, 3WJb comprises or consists of SEQ ID NO: 71, and 3WJc comprises or consists of SEQ ID NO: 72; or the 3WJa comprises or consists of SEQ ID NO: 76, 3WJb comprises or consists of SEQ ID NO: 8, and 3WJc comprises or consists of SEQ ID NO: 9; or said 3WJa comprises or consists of SEQ ID NO: 77, 3WJb comprises or consists of SEQ ID NO: 78, and 3WJc comprises or consists of SEQ ID NO: 79; or said 3WJa comprises or consists of SEQ ID NO: 80, 3WJb comprises or consists of SEQ ID NO: 81, and 3WJc comprises or consists of SEQ ID NO: 82; or said 3WJa comprises or consists of SEQ ID NO:83, 3WJb comprises or consists of SEQ ID NO:84, and 3WJc comprises or consists of SEQ ID NO:85; or said 3WJa comprises or consists of SEQ ID NO: 7, 3WJb comprises or consists of SEQ ID NO: 89, and 3WJc comprises or consists of SEQ ID NO: 9; or said 3WJa comprises or consists of SEQ ID NO:90, 3WJb comprises or consists of SEQ ID NO:91, and 3WJc comprises or consists of SEQ ID NO:79; or the 3WJa comprises or consists of SEQ ID NO: 76, 3WJb comprises or consists of SEQ ID NO: 89, and 3WJc comprises or consists of SEQ ID NO: 9; or the 3WJa comprises or consists of SEQ ID NO: 77, 3WJb comprises or consists of SEQ ID NO: 91, and 3WJc comprises or consists of SEQ ID NO: 79; or said 3WJa comprises or consists of SEQ ID NO: 80, 3WJb comprises or consists of SEQ ID NO: 93, and 3WJc comprises or consists of SEQ ID NO: 82; or the 3WJa comprises or consists of SEQ ID NO: 83, 3WJb comprises or consists of SEQ ID NO: 95, and 3WJc comprises or consists of SEQ ID NO: 85; or said 3WJa comprises or consists of SEQ ID NO: 112, 3WJb comprises or consists of SEQ ID NO: 113, and 3WJc comprises or consists of SEQ ID NO: 114; or the 3WJa comprises or consists of SEQ ID NO: 119, 3WJb comprises or consists of SEQ ID NO: 120, and 3WJc comprises or consists of SEQ ID NO: 121; or The 3WJa contains AUGUGUA, the 3WJb contains UACUUUG, and the 3WJc contains AUCAUG; or The 3WJa comprises GCGUU, the 3WJb comprises UUCGC, and the 3WJc comprises GCCAUAGCG; or wherein 3WJa comprises GUAUGGCAC, 3WJb comprises GUCACGG, and 3WJc comprises CUCUUAC; or The 3WJa comprises AUGGUA, the 3WJb comprises ACUUUGU, and the 3WJc comprises AUCA; or The 3WJa comprises UGGU, the 3WJb comprises ACUUGU, and the 3WJc comprises AUCA; or The 3WJa comprises UGGU, the 3WJb comprises ACUGU, and the 3WJc comprises AUCA; or The 3WJa comprises UGGU, the 3WJb comprises ACGUU, and the 3WJc comprises AAUCA; or The 3WJa comprises UGUGU, the 3WJb comprises ACUUGU, and the 3WJc comprises AUCA; or The 3WJa comprises UGUGU, the 3WJb comprises ACUGU, and the 3WJc comprises AUCA; or The 3WJa comprises, the 3WJb comprises ACGUU, and the 3WJc comprises AAUCA; or The 3WJa comprises UGGU, the 3WJb comprises ACUGU, and the 3WJc comprises AUCA; or wherein 3WJa comprises UAUGGCAC, 3WJb comprises GUCACGG, and 3WJc comprises CUCUUA; or The 3WJa comprises UAUGG, the 3WJb comprises UCAGG, and the 3WJc comprises CCUCUUA; or wherein 3WJa comprises UAUGGCAC, 3WJb comprises GUCACGG, and 3WJc comprises CUCUUA; or The 3WJa comprises UAUG, the 3WJb comprises CAGGGG, and the 3WJc comprises CUUG; or The 3WJa comprises UAUGU, the 3WJb comprises GCAGG, and the 3WJc comprises UCUUG; or The 3WJa comprises UAUGU, the 3WJb comprises GCAGGG, and the 3WJc comprises CUUG; or The 3WJa comprises UAUGU, the 3WJb comprises GCAGG, and the 3WJc comprises UCUUG; or The 3WJa comprises UGUGU, the 3WJb comprises ACUUUGU, and the 3WJc comprises AUCA; or The 3WJa contains UGUGU, 3WJb contains ACUUU, and 3WJc contains AAAUCA.

41. The precursor RNA of claim 40, wherein the 4WJ comprises The first branch of the 4WJ domain is formed by the 5' portion of the 4WJa sequence and the 3' portion of the 4WJd sequence and comprises a first helical region, a second branch of the 4WJ domain, the second branch being formed by the 3' portion of the 4WJa sequence and the 5' portion of the 4WJb sequence and comprising a second helical region, and The third branch of the 4WJ domain, the third branch is formed by the 3' portion of the 4WJb sequence and the 5' portion of the 4WJc sequence and comprises a third helical region, and The fourth branch of the 4WJ domain is formed by the 3' portion of the 4WJc sequence and the 5' portion of the 4WJd sequence and comprises a fourth helical region, Each of the helical regions comprises a plurality of RNA nucleotide pairs that form canonical Watson-Crick bonds.

42. The precursor RNA of claim 43, wherein said 4WJa comprises or consists of SEQ ID NO: 7, 4WJb comprises or consists of SEQ ID NO: 8, 4WJc comprises or consists of SEQ ID NO: 9, and 4WJd comprises or consists of SEQ ID NO: 102; or said 4WJa comprises or consists of SEQ ID NO: 103, 4WJb comprises or consists of SEQ ID NO: 104, 4WJc comprises or consists of SEQ ID NO: 105, and 4WJd comprises or consists of SEQ ID NO: 106; or said 4WJa comprises or consists of SEQ ID NO: 115, 4WJb comprises or consists of SEQ ID NO: 116, 4WJc comprises or consists of SEQ ID NO: 117, and 4WJd comprises or consists of SEQ ID NO: 118; or wherein 4WJa comprises UGCAGGUG, 4WJb comprises ACGGGC, 4WJc comprises CCAGCA, and 4WJd comprises SEQ ID NO: 67; or wherein 4WJa comprises SEQ ID NO: 74, 4WJb comprises AACUG, 4WJc comprises SEQ ID NO: 75, and 4WJd comprises AUCAUG; or The 4WJa comprises SEQ ID NO: 122, 4WJb comprises GAACU, 4WJc comprises SEQ ID NO: 123, and 4WJd comprises AAUCA.

43. The precursor RNA of claim 40, wherein the 5WJ comprises The first branch of the 5WJ domain is formed by the 5' portion of the 5WJa sequence and the 3' portion of the 5WJe sequence and comprises a first helical region, The second branch of the 5WJ domain, the second branch is formed by the 3' portion of the 5WJa sequence and the 5' portion of the 5WJb sequence and comprises a second helical region, The third branch of the 5WJ domain is formed by the 3' portion of the 5WJb sequence and the 5' portion of the 5WJc sequence and comprises a third helical region, The fourth branch of the 5WJ domain, the fourth branch is formed by the 3' portion of the 5WJc sequence and the 5' portion of the 5WJd sequence and comprises a fourth helical region, and The fifth branch of the 5WJ domain is formed by the 3' portion of the 5WJd sequence and the 5' portion of the 5WJe sequence and comprises a fifth helical region, Each of the helical regions comprises a plurality of RNA nucleotide pairs that form canonical Watson-Crick bonds.

44. The precursor RNA of claim 45, wherein the 5WJa comprises or consists of SEQ ID NO: 107, the 5WJb comprises or consists of SEQ ID NO: 108, the 5WJc comprises or consists of SEQ ID NO: 109, the 5WJd comprises or consists of SEQ ID NO: 110, and the 5WJe comprises or consists of SEQ ID NO: 111; or The 5WJa contains GUGA, the 5WJb contains UUGC, the 5WJc contains GUGU, the 5WJd contains AUGC, and the 5WJe contains GUGC.

45. The precursor RNA of any one of claims 36-46, wherein the noncoding region comprises one or more RNAs selected from the group consisting of antisense RNA, transfer RNA (tRNA), transfer-messenger RNA (tmRNA), ribosomal RNA (rRNA), signal recognition particle RNA (7SL RNA or SRP RNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), SmY RNA (SmY), small Cajal body-specific RNA (scaRNA), guide RNA (gRNA), Y RNA, splicing leader RNA (SLRNA), microRNA (miRNA), small interfering RNA (siRNA), cis-natural antisense transcript (cis-NAT), CRISPR RNA (crRNA), long noncoding RNA (lncRNA), Piwi-interacting RNA (piRNA), short hairpin RNA (shRNA), trans-acting siRNA (tasiRNA), repeat-associated siRNA (rasiRNA), 7SK RNA (7SK), telomerase RNA component (TERC), vault RNA (vRNA, vtRNA), and enhancer RNA (eRNA).

46. A vector encoding the precursor RNA according to any one of claims 36 to 47.

47. The vector of claim 48, which is a plasmid, a viral vector, a polymerase chain reaction (PCR) product, a cosmid, a bacterial artificial chromosome (BAC), or a yeast artificial chromosome (YAC).

48. A method for producing a protein in a cell, comprising introducing into the cell a precursor RNA as described in any one of claims 36 to 47, 50 and 51 comprising a protein coding region or a vector as described in claim 48 or 49 comprising a protein coding region, and producing the protein.

49. A method for editing a gene in a cell, the method comprising introducing into the cell a precursor RNA as described in any one of claims 36-47, 50 and 51 comprising a non-coding region capable of editing the gene, or a vector as described in claim 48 or 49 comprising a non-coding region capable of editing the gene, and editing the gene.

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