Vector used for preparing circular RNA containing few non-target sequences, and method

Abstract

A vector used for preparing a circular RNA, comprising: a 3' self-splicing intron fragment containing a 3' cleavage site, a sequence encoding an internal ribosome entry site (IRES), a target sequence, and a 5' self-splicing intron fragment containing a 5' cleavage site, wherein all the elements are operably linked in sequence, and the 3' self-splicing intron fragment containing the 3' cleavage site and the 5' self-splicing intron fragment containing the 5' cleavage site are derived from introns of Bacillus anthracis recA gene, Coxiella burnetii 23S ribosome gene, Scytonema hofmanni tRNA-fMet gene, or Thermotoga naphthophila 23S rRNA gene. The vector does not contain a spacer sequence.

Description

Vectors and methods for preparing circular RNAs containing fewer non-target sequences

[0001] Cross-reference to related applications

[0002] This application claims priority to Chinese patent application No. 202410666661.1, filed on May 27, 2024, the entire contents of which are incorporated herein by reference. Invention Field

[0003] This application relates to a vector for preparing circular RNA, comprising: an optional promoter, an optional sequence encoding a 5' homologous arm, a 3' self-splicing intron fragment containing a 3' splice site, an optional sequence encoding an internal ribosome entry site (IRES), a target sequence, a 5' self-splicing intron fragment containing a 5' splice site, and an optional sequence encoding a 3' homologous arm, wherein the elements are operatively linked in sequence, wherein the 3' self-splicing intron fragment and the 5' self-splicing intron fragment may originate from the same self-splicing intron, for example from an intron of the *Coxiella belladonna* 23S ribosome gene, the *Pseudococcus hoffmannii* tRNA-fMet gene, the *Bacillus anthracis* recA gene, or the *Thermophyton naphthaleneophilus* 23S rRNA gene. The vector may omit spacer sequences, such as a 5' spacer sequence between the 3' self-splicing intron fragment and the IRES, and / or a 3' spacer sequence between the target sequence and the 5' self-splicing intron fragment. The 5' self-splicing intron fragment in this vector can contain 5-40, especially 5-20 nucleotide exon sequences, and the 3' self-splicing intron fragment can contain 3-40, especially 3-20 nucleotide exon sequences. Background Technology

[0004] With technological breakthroughs, RNA therapy has emerged as a promising new field within the biopharmaceutical industry, encompassing the use of messenger RNA (mRNA), small interfering RNA (siRNA), and microRNA (miRNA). For example, it was recently used in the treatment of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), which experienced a widespread global pandemic. [1,2] This has drawn attention to mRNA vaccines and given people hope. The rapid pace of the entire process from design and production to testing of mRNA vaccines undoubtedly represents a new and promising medical approach for dealing with rapidly mutating viral types.

[0005] With the success of mRNA drugs, improving mRNA stability and thus prolonging protein translation duration has attracted considerable attention and interest. Previously, efforts mainly focused on mimicking the structure of natural mRNA, using methods such as untranslated regions, methylguanosine cap analogs, nucleoside modifications, and codon optimization to improve mRNA stability to some extent. [3,4–7] Another possible approach is to convert mRNA into circular RNA (circRNA), leveraging the natural structural advantages of circRNA to stabilize RNA and prolong protein translation. circRNA is a class of covalently linked, closed circular single-stranded RNA molecules. 8 Compared to mRNA, circRNA is ligatured end-to-end and lacks 5' and 3' ends, thus exhibiting stronger resistance to exonuclease degradation and a longer half-life. [12,17] circRNAs are widely distributed in eukaryotes, primarily in the form of non-coding RNAs. [9,10,11] Most are produced by reverse splicing. [12–14] With the discovery of more and more circRNAs, certain specific circRNA molecules have also been shown to have protein-coding functions. [15,16] .

[0006] Gene fragments typically contain introns and exons. After RNA splicing, the exon sequences are spliced ​​together to form mature mRNA. Most intron removal requires multiple protein interactions, but some special introns are removed through self-splicing. These self-splicing introns can be classified into two categories based on their structure and splicing mechanism: Class I and Class II. The processing of Class I introns is initiated by a transesterification reaction mediated by exogenous guanosine cofactor (GTP). [18,19] The splicing of class II introns is similar to that of eukaryotic mRNA precursors. The 5' splice site is attacked by the -OH group of a nucleotide protruding at the branching site, initially creating an intron lasso structure. Subsequently, the -OH group released from the 5' exon attacks the 3' splice site, ultimately splicing the two exons together. [19,20] Utilizing the self-splicing function of class I and class II introns, researchers designed sequences to split the self-splicing introns into two halves, which were then loaded onto both ends of the RNA sequence to be circularized. RNA circularization was then achieved through in vitro catalysis. [21-24] Because circular RNAs are covalently closed at both ends and lack a naked 5' end, they lack a cap structure to initiate RNA translation. To address the translation challenges of circular RNAs, researchers added ribosome entry site sequences (IRES) to them, enabling them to perform stable in vitro and in vivo translation. [8,10,14] .

[0007] Daniel G. Anderson et al. [21-24] In vitro circularization of RNA was achieved using introns of class I self-splicing introns, such as the pre-tRNA-Leu gene from Anabaena or the Td gene (T4td) from T4 phage. Since the splicing sites of the replaced intron-exon elements (PIEs) are located near the IRES, and both sequences are highly structured, the IRES sequence may interfere with the folding of splicing ribozymes. To enable these structures to fold independently, a series of spacer sequences were designed between the PIE splicing sites and the IRES. By increasing the number of spacer sequences, the splicing efficiency was nearly doubled. While this artificial addition of exogenous spacer sequences increased splicing efficiency to some extent, it also increased the subsequent risks related to immunogenicity. Furthermore, PIE-based circRNA synthesis inevitably incorporates the exon fragments (E1 and E2) flanking the self-splicing introns, which are necessary for self-splicing, into the nascent circRNA, potentially limiting the application scope of circRNA synthesis.

[0008] Reference to any document in this application is not an admission that such document is prior art. Summary of the Invention

[0009] In order to synthesize precise, effective and low-immunogenic circular RNA as potential RNA vaccines and therapies, the inventors of this application attempted to reduce the foreign sequences introduced in the in vitro synthesized circular RNA.

[0010] First, the inventors of this application tested introns from different sources and found that in RNA with the 5' and 3' spacer sequences removed, circularization could still be achieved when containing introns from certain self-splicing sources, such as those derived from the Bacillus anthracis recA gene, the Coxiella burnetii 23S ribosome gene, and the Thermotoga subterranea 23S rRNA. Specifically, when containing introns from certain self-splicing sources, such as those derived from the Bacillus anthracis recA gene and the Coxiella burnetii 23S ribosome gene, the RNA without the spacer sequences still exhibited higher circularization efficiency than RNA containing introns with the 5' and 3' spacer sequences, and those derived from the Anabaena pre-tRNA-Leu gene; that is, the resulting circular RNA had higher purity.

[0011] Secondly, the inventors of this application truncated the exons flanking the introns required for self-splicing. They found that RNA circularization efficiency remained high even when fewer exon sequences were retained flanking the introns derived from, for example, the *Coxiella belladonna* 23S ribosome gene. Specifically, when 10 and 10 nucleotides, 10 and 6 nucleotides, or 9 and 3 nucleotides of exon sequences were retained flanking the introns derived from, for example, the *Coxiella belladonna* 23S ribosome gene, the RNA circularization efficiency was comparable to that of RNA containing 5' spacer sequences, 3' spacer sequences, and introns derived from the *Anabaena pre-tRNA-Leu* gene. However, when introns derived from, for example, the Coxiella beckonii 23S ribosomal gene retain 20 nucleotides and 10 nucleotides of exon sequences on either side, RNA circularization efficiency may still be higher than that of RNA containing 5' spacer sequences, 3' spacer sequences, and introns derived from the untruncated Anabaena pre-tRNA-Leu gene.

[0012] Furthermore, RNA from the *Scytonema hofmannii* tRNA-fMet gene introns, containing truncated exons after the removal of the 5' and 3' spacer sequences, was found to be capable of circularization, with a circularization efficiency similar to that of RNA containing the 5' and 3' spacer sequences and RNA derived from the untruncated *Anabaena* pre-tRNA-Leu gene introns. RNA from the *Bacillus anthracis* recA gene introns, containing truncated exons after the removal of the 5' and 3' spacer sequences, was also capable of circularization, albeit with slightly lower efficiency.

[0013] Therefore, in a first aspect, this application provides a single-stranded DNA molecule for preparing circular RNA, which may sequentially comprise from the 5' end to the 3' end: a 3' self-splicing intron fragment containing a 3' splice site, a sequence encoding an internal ribosome entry site (IRES), a target sequence, and a 5' self-splicing intron fragment containing a 5' splice site, wherein the elements are operatively linked, and the 3' self-splicing intron fragment containing the 3' splice site and the 5' self-splicing intron fragment containing the 5' splice site may originate from the same self-splicing intron, wherein the single-stranded DNA molecule does not contain a spacer sequence.

[0014] The spacer sequences include the spacer sequence between a 3' self-splicing intron containing a 3' splice site and a sequence encoding the internal ribosome entry site (IRES), and the spacer sequence between the target sequence and a 5' self-splicing intron containing a 5' splice site.

[0015] The 3' self-splicing intron fragment containing a 3' splice site and the 5' self-splicing intron fragment containing a 5' splice site can be derived from the *Bacillus anthracis* recA gene, the *Coxiella behneckii* 23S ribosome gene, the *Pseudococcus hummusae* tRNA-fMet gene, or the *Thermophyton naphthaleneophilus* 23S rRNA gene. In some embodiments, the 3' self-splicing intron fragment containing a 3' splice site and the 5' self-splicing intron fragment containing a 5' splice site can be derived from the *Coxiella behneckii* 23S ribosome gene, the *Pseudococcus hummusae* tRNA-fMet gene, or the *Bacillus anthracis* recA gene.

[0016] Introns derived from the *Bacillus anthracis* recA gene, *Coxiella belladonna* 23S ribosome gene, *Pseudococcus hoffmannii* tRNA-fMet gene, and *Thermophyton naphthaleneophilus* 23S rRNA gene may contain nucleotide sequences having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 13, 12, 35, and 14, respectively. In some embodiments, the nucleotide sequences of introns derived from the *Bacillus anthracis* recA gene, *Coxiella belladonna* 23S ribosome gene, *Pseudococcus hoffmannii* tRNA-fMet gene, and *Thermophyton naphthaleneophilus* 23S rRNA gene may be as shown in SEQ ID NO: 13, 12, 35, and 14, respectively.

[0017] The 3' self-splicing intron fragment may contain a naturally adjacent exon or a sequence corresponding to the naturally adjacent exon at its 3' end. In some embodiments, the 3' self-splicing intron fragment may contain a naturally adjacent exon at its 3' end. The length of the naturally adjacent exon or the sequence corresponding to the naturally adjacent exon in the 3' self-splicing intron fragment may be about 3-40 nucleotides, about 3-20 nucleotides, about 3-10 nucleotides, about 3-8 nucleotides, or about 3-6 nucleotides. In some embodiments, the length of the naturally adjacent exon or the sequence corresponding to the naturally adjacent exon in the 3' self-splicing intron fragment may be about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides.

[0018] The 5' self-splicing intron fragment may contain a naturally adjacent exon or a sequence corresponding to the naturally adjacent exon at its 5' end. In some embodiments, the 5' self-splicing intron fragment may contain a naturally adjacent exon at its 5' end. The length of the naturally adjacent exon or the sequence corresponding to the naturally adjacent exon in the 5' self-splicing intron fragment may be about 5-40 nucleotides, about 5-20 nucleotides, about 5-10 nucleotides, about 5-9 nucleotides, about 5-8 nucleotides, or about 5-6 nucleotides. In some embodiments, the length of the naturally adjacent exon or the sequence corresponding to the naturally adjacent exon in the 5' self-splicing intron fragment may be about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides.

[0019] In some embodiments, the 3' self-splicing intron fragment containing the 3' splice site includes a naturally adjacent exon or a sequence corresponding to the naturally adjacent exon at its 3' end, wherein the length of the naturally adjacent exon or the sequence corresponding to the naturally adjacent exon can be 3-40 nucleotides, 3-20 nucleotides, 3-10 nucleotides, 3-8 nucleotides, or 3-6 nucleotides, and / or

[0020] A 5' self-splicing intron fragment containing a 5' splice site contains a naturally adjacent exon or a sequence corresponding to the naturally adjacent exon at its 5' end, wherein the length of the naturally adjacent exon or the sequence corresponding to the naturally adjacent exon can be 5-40 nucleotides, 5-20 nucleotides, 5-10 nucleotides, 5-9 nucleotides, 5-8 nucleotides, or 5-6 nucleotides.

[0021] The 3' self-splicing intron fragment containing a 3' splice site may contain a nucleotide sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:15, 26, 27, or 28, and the 5' self-splicing intron fragment containing a 5' splice site may contain a nucleotide sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:16, 24, or 25.

[0022] The 3' self-splicing intron fragment containing a 3' cleavage site may contain a nucleotide sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:44 or 49, and the 5' self-splicing intron fragment containing a 5' cleavage site may contain a nucleotide sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:45 or 50.

[0023] The 3' self-splicing intron fragment containing a 3' cleavage site may contain a nucleotide sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:17 or 40, and the 5' self-splicing intron fragment containing a 5' cleavage site may contain a nucleotide sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:18 or 41.

[0024] The 3' self-splicing intron fragments containing 3' cleavage sites and the 5' self-splicing intron fragments containing 5' cleavage sites may respectively contain nucleotide sequences having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with i) SEQ ID NO:26 and 16, ii) SEQ ID NO:28 and 25, iii) SEQ ID NO:27 and 24, iv) SEQ ID NO:26 and 24, v) SEQ ID NO:44 and 45, vi) SEQ ID NO:40 and 41, vii) SEQ ID NO:15 and 16, viiii) SEQ ID NO:17 and 18, ix) SEQ ID NO:49 and 50, x) SEQ ID NO:19 and 20, or xi) SEQ ID NO:42 and 43. In some embodiments, the 3' self-splicing intron fragments containing 3' cleavage sites and the 5' self-splicing intron fragments containing 5' cleavage sites may respectively contain nucleotide sequences having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with i) SEQ ID NO:26 and 16, ii) SEQ ID NO:28 and 25, iii) SEQ ID NO:27 and 24, iv) SEQ ID NO:26 and 24, v) SEQ ID NO:44 and 45, vi) SEQ ID NO:40 and 41, vii) SEQ ID NO:15 and 16, viiii) SEQ ID NO:17 and 18, or ix) SEQ ID NO:49 and 50.

[0025] The 3' and 5' cleavage sites can be dinucleotides, encoding, for example, AG and GU, or AC and AU, respectively.

[0026] Internal ribosome entry sites (IRES) can be found on Coxsackievirus B3 (CVB3), Coxsackievirus A (CVB1 / 2), Taura syndrome virus, blood-sucking assassin bug virus, Tyrell's encephalomyelitis virus, simian virus 40, red imported fire ant virus 1, cereal constrictor aphid virus, reticuloendotheliosis virus, Forman poliovirus 1, soybean inchworm virus, Kashmir wasp virus, human rhinovirus 2, glass leafhopper virus-1, and human immunodeficiency virus type 1. Leafhopper virus-1, louse P virus, hepatitis C virus, hepatitis A virus, GB hepatitis virus, foot-and-mouth disease virus, human enterovirus 71, equine rhinovirus, tea geometrid moth-like virus, encephalocarditis virus (EMCV), fruit fly C virus, cruciferous tobacco virus, cricket paralysis virus, bovine viral diarrhea virus 1, black queen cell virus, aphid lethal paralysis virus, avian encephalomyelitis virus, acute bee paralysis virus, hibiscus yellow spot virus, classical swine fever virus, human FGF2 Human SFTPA1, human AML1 / RUNX1, Drosophila antennae, human AQP4, human AT1R, human BAG-1, human BCL2, human BiP, human c-IAPl, human c-myc, human eIF4G, mouse NDST4L, human LEF1, mouse HIF1α, human n.myc, mouse Gtx, human p27kipl, human PDGF2 / c-sis, human p53, human Pim-1, mouse Rbm3, Drosophila reaper, canine scamper, Drosophila Ubx, salivary virus, Coxsackievirus, bi-echovirus, human UNR, mouse UtrA, human VEGF-A, human XIAP, Drosophila hairless, Saccharomyces cerevisiae TFIID, Saccharomyces cerevisiae YAP1, human c-src, human FGF-1, simian microRNA virus, turnip shrunkenness virus, or IRES of eIF4G aptamer. In some implementations, the IRES can be the IRES of Coxsackievirus B3 (CVB3). The sequence encoding the IRES may contain a nucleotide sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:5.

[0027] The target sequence may contain an open reading frame (ORF), a single cloning site, or a multiple cloning site encoding a target peptide or protein. The length of the target sequence may be 50-8000 nucleotides. The target peptide or protein may be of eukaryotic or prokaryotic origin. The target peptide or protein may be human or non-human. In some embodiments, the target peptide or protein may be an antigen protein, an antibody, or a Cas9 endonuclease, etc. In some embodiments, the target peptide or protein may be firefly luciferase, long-horned leptospirase, Gaussian luciferase, green fluorescent protein, etc. In some embodiments, the target sequence may contain one or more ORFs encoding a target peptide or protein. In some embodiments, the target sequence may be one or more ORFs encoding a target peptide or protein. In some embodiments, the target sequence may contain a single cloning site or a multiple cloning site. In some embodiments, the target sequence may be a single cloning site or a multiple cloning site.

[0028] The single-stranded DNA molecule for preparing circular RNA according to this application may further include a sequence encoding a 5' homologous arm upstream of or at the 5' end of the 3' self-splicing intron fragment, and a sequence encoding a 3' homologous arm downstream of or at the 3' end of the 5' self-splicing intron fragment. In some embodiments, the single-stranded DNA molecule may sequentially include, from the 5' end to the 3' end, a sequence encoding a 5' homologous arm, a 3' self-splicing intron fragment containing a 3' splice site, a sequence encoding an internal ribosome entry site (IRES), a target sequence, a 5' self-splicing intron fragment containing a 5' splice site, and a sequence encoding a 3' homologous arm.

[0029] The 5' and 3' homologous arms can be complementary, for example, forming at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% base pairing. In some embodiments, the 5' and 3' homologous arms can complementarily form a structure containing a palindromic stem and a stem-loop, wherein the palindromic stem is connected to the stem portion of the stem-loop, and the 5' end of the 5' homologous arm and the 3' end of the 3' homologous arm are adjacent at the palindromic stem portion. In some embodiments, the 5' and 3' homologous arms can complementarily form a structure containing a palindromic stem and two stem-loops, wherein the palindromic stem is connected to the stem portions of two stem-loops, and the stem portions of two stem-loops are connected, wherein the 5' end of the 5' homologous arm and the 3' end of the 3' homologous arm are adjacent at the loop portion of the palindromic stem or one of the stem-loops. In some embodiments, the sequences encoding the 5' homologous arm and the sequences encoding the 3' homologous arm may respectively comprise nucleotide sequences having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:2 and 10.

[0030] The single-stranded DNA molecule for preparing circular RNA according to this application may contain an RNA polymerase promoter at its 5' end. The RNA polymerase promoter may be an RNA polymerase promoter derived from T7 virus, T6 virus, SP6 virus, T3 virus, or T4 virus. In some embodiments, the RNA polymerase promoter may be a T7 virus promoter, which may contain a nucleotide sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:1.

[0031] The single-stranded DNA molecule used to prepare circular RNA according to this application may also contain a restriction endonuclease site at its 3' end. The restriction endonuclease site may be, for example, EcoRI, EcoRV, etc.

[0032] In some embodiments, the single-stranded DNA molecule for preparing circular RNA according to this application may sequentially comprise, from the 5' end to the 3' end: an RNA polymerase promoter, a sequence encoding a 5' homologous arm, a 3' self-splicing intron fragment containing a 3' cleavage site, a sequence encoding an internal ribosome entry site (IRES), a target sequence, a 5' self-splicing intron fragment containing a 5' cleavage site, and a sequence encoding a 3' homologous arm, wherein the elements are operatively linked. The single-stranded DNA molecule for preparing circular RNA according to this application may omit spacer sequences between the aforementioned elements, particularly the spacer sequence between the 3' self-splicing intron fragment containing a 3' cleavage site and the sequence encoding the internal ribosome entry site (IRES), and the spacer sequence between the target sequence and the 5' self-splicing intron fragment containing a 5' cleavage site. In some embodiments, the single-stranded DNA molecule for preparing circular RNA of this application may be composed, from the 5' end to the 3' end, of an RNA polymerase promoter, a sequence encoding a 5' homologous arm, a 3' self-splicing intron fragment containing a 3' splice site, a sequence encoding an internal ribosome entry site (IRES), a target sequence, a 5' self-splicing intron fragment containing a 5' splice site, and a sequence encoding a 3' homologous arm, wherein the elements are operatively linked.

[0033] In some embodiments, the single-stranded DNA molecule for preparing circular RNA according to this application may sequentially comprise, from the 5' end to the 3' end: an RNA polymerase promoter, a sequence encoding a 5' homologous arm, a 3' self-splicing intron fragment containing a 3' cleavage site, a sequence encoding an internal ribosome entry site (IRES), a target sequence, a 5' self-splicing intron fragment containing a 5' cleavage site, a sequence encoding a 3' homologous arm, and a restriction endonuclease site, wherein the elements are operatively linked. The single-stranded DNA molecule for preparing circular RNA according to this application may omit spacer sequences between the aforementioned elements, particularly the spacer sequence between the 3' self-splicing intron fragment containing a 3' cleavage site and the sequence encoding the internal ribosome entry site (IRES), and the spacer sequence between the target sequence and the 5' self-splicing intron fragment containing a 5' cleavage site. In some embodiments, the single-stranded DNA molecule for preparing circular RNA of this application may be composed, from the 5' end to the 3' end, sequentially consisting of an RNA polymerase promoter, a sequence encoding a 5' homologous arm, a 3' self-splicing intron fragment containing a 3' cleavage site, a sequence encoding an internal ribosome entry site (IRES), a target sequence, a 5' self-splicing intron fragment containing a 5' cleavage site, a sequence encoding a 3' homologous arm, and a restriction endonuclease site, wherein the elements are operatively linked.

[0034] This application also provides a double-stranded DNA molecule, which may comprise i) the single-stranded DNA molecule of this application, and ii) a second strand complementary to the single-stranded DNA molecule. In some embodiments, the double-stranded DNA molecule of this application may comprise i) the single-stranded DNA molecule of this application, and ii) a second strand completely complementary to the single-stranded DNA molecule. In some embodiments, the double-stranded DNA molecule is a double-stranded DNA molecule used for preparing circular RNA.

[0035] This application also provides a vector comprising the single-stranded DNA molecule or double-stranded DNA molecule of this application. In some embodiments, the vector may be a vector for preparing circular RNA, comprising the single-stranded DNA molecule of this application for preparing circular RNA, or the double-stranded DNA molecule for preparing circular RNA. The vector may be circular or linear. In some embodiments, the vector may be linear. In some embodiments, the vector may be circular and processed to become linear. The vector of this application can transcribe circular RNA of about 500 to about 10,000 nucleotides. In some embodiments, the vector of this application can transcribe circular RNA of at least 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2500, 3000, 3500, 4000, 4500, or 5000 nucleotides.

[0036] In a second aspect, this application provides a circularizable RNA molecule that, from its 5' end to its 3' end, sequentially comprises: a 3' self-splicing intron fragment containing a 3' splice site, an internal ribosome entry site, a target sequence, and a 5' self-splicing intron fragment containing a 5' splice site, wherein the elements are operatively linked, and the 3' self-splicing intron fragment containing the 3' splice site and the 5' self-splicing intron fragment containing the 5' splice site may originate from the same self-splicing intron, wherein the RNA molecule does not contain a spacer sequence.

[0037] The spacer sequences include the spacer sequence between a 3' self-splicing intron containing a 3' splice site and a sequence encoding the internal ribosome entry site (IRES), and the spacer sequence between the target sequence and a 5' self-splicing intron containing a 5' splice site.

[0038] The 3' self-splicing intron fragment containing a 3' splice site and the 5' self-splicing intron fragment containing a 5' splice site can be derived from the *Coxiella benzi* 23S ribosome gene, the *Pseudococcus hummusae* tRNA-fMet gene, the *Bacillus anthracis* recA gene, or the *Thermophyton naphthaleneophilus* 23S rRNA gene. In some embodiments, the 3' self-splicing intron fragment containing a 3' splice site and the 5' self-splicing intron fragment containing a 5' splice site can be derived from the *Coxiella benzi* 23S ribosome gene, the *Pseudococcus hummusae* tRNA-fMet gene, or the *Bacillus anthracis* recA gene.

[0039] Introns derived from the 23S ribosomal gene of Coxiella behnea, the tRNA-fMet gene of Pseudobranchia huffmannii, the recA gene of Bacillus anthracis, and the 23S rRNA gene of Thermophyton naphthaleneophilus may contain nucleotide sequences having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:12, 35, 13, and 14, respectively.

[0040] The 3' self-splicing intron fragment may contain a naturally adjacent exon or a sequence corresponding to the naturally adjacent exon at its 3' end. In some embodiments, the 3' self-splicing intron fragment may contain a naturally adjacent exon at its 3' end. The length of the naturally adjacent exon or the sequence corresponding to the naturally adjacent exon in the 3' self-splicing intron fragment may be about 3-40 nucleotides, about 3-20 nucleotides, about 3-10 nucleotides, about 3-8 nucleotides, or about 3-6 nucleotides. In some embodiments, the length of the naturally adjacent exon or the sequence corresponding to the naturally adjacent exon in the 3' self-splicing intron fragment may be about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides.

[0041] The 5' self-splicing intron fragment may contain a naturally adjacent exon or a sequence corresponding to the naturally adjacent exon at its 5' end. In some embodiments, the 5' self-splicing intron fragment may contain a naturally adjacent exon at its 5' end. The length of the naturally adjacent exon or the sequence corresponding to the naturally adjacent exon in the 5' self-splicing intron fragment may be about 5-40 nucleotides, about 5-20 nucleotides, about 5-10 nucleotides, about 5-9 nucleotides, about 5-8 nucleotides, or about 5-6 nucleotides. In some embodiments, the length of the naturally adjacent exon or the sequence corresponding to the naturally adjacent exon in the 5' self-splicing intron fragment may be about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides.

[0042] The 3' self-splicing intron fragment containing a 3' splice site may contain a nucleotide sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:15, 26, 27, or 28, and the 5' self-splicing intron fragment containing a 5' splice site may contain a nucleotide sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:16, 24, or 25.

[0043] The 3' self-splicing intron fragment containing a 3' cleavage site may contain a nucleotide sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:44 or 49, and the 5' self-splicing intron fragment containing a 5' cleavage site may contain a nucleotide sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:45 or 50.

[0044] The 3' self-splicing intron fragment containing a 3' cleavage site may contain a nucleotide sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:17 or 40, and the 5' self-splicing intron fragment containing a 5' cleavage site may contain a nucleotide sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:18 or 41.

[0045] The 3' self-splicing intron fragments containing 3' cleavage sites and the 5' self-splicing intron fragments containing 5' cleavage sites may respectively contain nucleotide sequences having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with i) SEQ ID NO:26 and 16, ii) SEQ ID NO:28 and 25, iii) SEQ ID NO:27 and 24, iv) SEQ ID NO:26 and 24, v) SEQ ID NO:44 and 45, vi) SEQ ID NO:40 and 41, vii) SEQ ID NO:15 and 16, viiii) SEQ ID NO:17 and 18, ix) SEQ ID NO:49 and 50, x) SEQ ID NO:19 and 20, or xi) SEQ ID NO:42 and 43. In some embodiments, the 3' self-splicing intron fragments containing 3' cleavage sites and the 5' self-splicing intron fragments containing 5' cleavage sites may respectively contain nucleotide sequences having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with i) SEQ ID NO:26 and 16, ii) SEQ ID NO:28 and 25, iii) SEQ ID NO:27 and 24, iv) SEQ ID NO:26 and 24, v) SEQ ID NO:44 and 45, vi) SEQ ID NO:40 and 41, vii) SEQ ID NO:15 and 16, viiii) SEQ ID NO:17 and 18, or ix) SEQ ID NO:49 and 50.

[0046] The 3' and 5' cleavage sites can be dinucleotides, encoding, for example, AG and GU, or AC and AU, respectively.

[0047] Internal ribosome entry sites (IRES) can be found on Coxsackievirus B3 (CVB3), Coxsackievirus A (CVB1 / 2), Taura syndrome virus, blood-sucking assassin bug virus, Tyrell's encephalomyelitis virus, simian virus 40, red imported fire ant virus 1, cereal constrictor aphid virus, reticuloendotheliosis virus, Forman poliovirus 1, soybean inchworm virus, Kashmir wasp virus, human rhinovirus 2, glass leafhopper virus-1, and human immunodeficiency virus type 1. Leafhopper virus-1, louse P virus, hepatitis C virus, hepatitis A virus, GB hepatitis virus, foot-and-mouth disease virus, human enterovirus 71, equine rhinovirus, tea geometrid moth-like virus, encephalocarditis virus (EMCV), fruit fly C virus, cruciferous tobacco virus, cricket paralysis virus, bovine viral diarrhea virus 1, black queen cell virus, aphid lethal paralysis virus, avian encephalomyelitis virus, acute bee paralysis virus, hibiscus yellow spot virus, classical swine fever virus, human FGF2 Human SFTPA1, human AML1 / RUNX1, Drosophila antennae, human AQP4, human AT1R, human BAG-1, human BCL2, human BiP, human c-IAPl, human c-myc, human eIF4G, mouse NDST4L, human LEF1, mouse HIF1α, human n.myc, mouse Gtx, human p27kipl, human PDGF2 / c-sis, human p53, human Pim-1, mouse Rbm3, Drosophila reaper, canine scamper, Drosophila Ubx, salivary virus, Coxsackievirus, bi-echovirus, human UNR, mouse UtrA, human VEGF-A, human XIAP, Drosophila hairless, Saccharomyces cerevisiae TFIID, Saccharomyces cerevisiae YAP1, human c-src, human FGF-1, simian microRNA virus, turnip shrunkenness virus, or IRES of eIF4G aptamer. In some embodiments, the IRES can be an IRES of Coxsackievirus B3 (CVB3). The IRES may contain a nucleotide sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:5.

[0048] The target sequence may contain an open reading frame encoding a target peptide or protein. The length of the target sequence may be 50-8000 nucleotides. The target peptide or protein may be of eukaryotic or prokaryotic origin. The target peptide or protein may be human or non-human. In some embodiments, the target peptide or protein may be an antigen protein, antibody, or Cas9 endonuclease, etc. In some embodiments, the target peptide or protein may be firefly luciferase, long-horned beetle luciferase, Gaussian luciferase, green fluorescent protein, etc. In some embodiments, the target sequence may contain one or more open reading frames encoding a target peptide or protein. In some embodiments, the target sequence may be one or more open reading frames encoding a target peptide or protein.

[0049] The RNA molecule of this application may also include a 5' homologous arm upstream of or at the 5' end of the 3' self-splicing intron fragment, and a 3' homologous arm downstream of or at the 3' end of the 5' self-splicing intron fragment. In some embodiments, the RNA molecule may sequentially include a 5' homologous arm, a 3' self-splicing intron fragment containing a 3' splice site, an internal ribosome entry site (IRES), a target sequence, a 5' self-splicing intron fragment containing a 5' splice site, and a 3' homologous arm from the 5' end to the 3' end.

[0050] The 5' and 3' homologous arms can be complementary, for example, forming at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% base pairing. In some embodiments, the 5' and 3' homologous arms can complementarily form a structure containing a palindromic stem and a stem-loop, wherein the palindromic stem is connected to the stem portion of the stem-loop, and the 5' end of the 5' homologous arm and the 3' end of the 3' homologous arm are adjacent at the palindromic stem portion. In some embodiments, the 5' and 3' homologous arms can complementarily form a structure containing a palindromic stem and two stem-loops, wherein the palindromic stem is connected to the stem portions of two stem-loops, and the stem portions of two stem-loops are connected, wherein the 5' end of the 5' homologous arm and the 3' end of the 3' homologous arm are adjacent at the loop portion of the palindromic stem or one of the stem-loops. In some embodiments, the 5' homologous arm and the 3' homologous arm may respectively comprise nucleotide sequences having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:2 and 10.

[0051] In some embodiments, the RNA molecule of this application may sequentially comprise, from the 5' end to the 3' end: a 5' homologous arm, a 3' self-splicing intron fragment containing a 3' splice site, an IRES, a target sequence, a 5' self-splicing intron fragment containing a 5' splice site, and a 3' homologous arm, wherein the elements are operatively linked. The RNA molecule of this application may omit spacer sequences between the aforementioned elements, particularly the spacer sequence between the 3' self-splicing intron fragment containing a 3' splice site and the sequence encoding the internal ribosome entry site (IRES), and the spacer sequence between the target sequence and the 5' self-splicing intron fragment containing a 5' splice site. In some embodiments, the RNA molecule of this application may sequentially comprise, from the 5' end to the 3' end: a 5' homologous arm, a 3' self-splicing intron fragment containing a 3' splice site, an IRES, a target sequence, a 5' self-splicing intron fragment containing a 5' splice site, and a 3' homologous arm, wherein the elements are operatively linked.

[0052] In a third aspect, this application provides a single-stranded DNA molecule for preparing circular RNA, which may sequentially comprise from the 5' end to the 3' end: a 3' self-splicing intron fragment containing a 3' splice site, a target sequence, and a 5' self-splicing intron fragment containing a 5' splice site, wherein the elements are operatively linked, and the 3' and 5' self-splicing intron fragments may be derived from the same self-splicing intron. The 3' self-splicing intron fragment may contain a naturally adjacent exon or a sequence corresponding to the naturally adjacent exon at its 3' end, wherein the length of the naturally adjacent exon or the sequence corresponding to the naturally adjacent exon in the 3' self-splicing intron fragment may be about 3-20 nucleotides, about 3-10 nucleotides, about 3-8 nucleotides, or about 3-6 nucleotides. In some embodiments, the length of the naturally adjacent exon or the sequence corresponding to the naturally adjacent exon in the 3' self-splicing intron fragment can be about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides. The 5' self-splicing intron fragment may contain a naturally adjacent exon or the sequence corresponding to the naturally adjacent exon at its 5' end, wherein the length of the naturally adjacent exon or the sequence corresponding to the naturally adjacent exon in the 5' self-splicing intron fragment can be about 5-20 nucleotides, about 5-10 nucleotides, about 5-8 nucleotides, or about 5-6 nucleotides. In some embodiments, the length of the naturally adjacent exon or the sequence corresponding to the naturally adjacent exon in the 5' self-splicing intron fragment can be about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides.

[0053] The 3' and 5' self-splicing intron fragments can be derived from the same self-splicing intron, for example, from introns of the *Coxiella behneckii* 23S ribosome gene, the *Pseudococcus hummusae* tRNA-fMet gene, the *Bacillus anthracis* recA gene, and the *Thermophyton naphthaleneophilus* 23S rRNA gene. In some embodiments, the 3' and 5' self-splicing intron fragments can be derived from the *Coxiella behneckii* 23S ribosome gene, the *Pseudococcus hummusae* tRNA-fMet gene, or the *Bacillus anthracis* recA gene. Introns derived from the 23S ribosomal gene of Coxiella behnea, the tRNA-fMet gene of Pseudobranchia huffmannii, the recA gene of Bacillus anthracis, and the 23S rRNA gene of Thermophyton naphthaleneophilus may contain nucleotide sequences having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:12, 35, 13, and 14, respectively.

[0054] In some embodiments, the 3' self-splicing intron fragment may include a naturally adjacent exon at its 3' end. In some embodiments, the length of the naturally adjacent exon in the 3' self-splicing intron fragment may be about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides.

[0055] In some embodiments, the 5' self-splicing intron fragment may include a naturally adjacent exon at its 5' end. In some embodiments, the length of the naturally adjacent exon in the 5' self-splicing intron fragment may be about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides.

[0056] In some embodiments, the lengths of the naturally adjacent exons or sequences corresponding to the naturally adjacent exons in the 3' self-splicing intron fragment and the lengths of the naturally adjacent exons or sequences corresponding to the naturally adjacent exons in the 5' self-splicing intron fragment can be i) 20 nucleotides and 20 nucleotides, ii) 10 nucleotides and 20 nucleotides, iii) 10 nucleotides and 10 nucleotides, iv) 6 nucleotides and 10 nucleotides, v) 6 nucleotides and 8 nucleotides, vi) 3 nucleotides and 9 nucleotides, respectively.

[0057] In some embodiments, the self-splicing introns are derived from the Coxiella Behringer 23S ribosomal gene, and the lengths of the naturally adjacent exons or sequences corresponding to the naturally adjacent exons in the 3' self-splicing intron fragments and the lengths of the naturally adjacent exons or sequences corresponding to the naturally adjacent exons in the 5' self-splicing intron fragments are i) 3 nucleotides and 9 nucleotides, ii) 6 nucleotides and 10 nucleotides, iii) 10 nucleotides and 10 nucleotides, iv) 10 nucleotides and 20 nucleotides, or v) 20 nucleotides and 20 nucleotides, respectively.

[0058] The 3' self-splicing intron fragment containing a 3' splice site may contain a nucleotide sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:15, 26, 27, or 28, and the 5' self-splicing intron fragment containing a 5' splice site may contain a nucleotide sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:16, 24, or 25.

[0059] The 3' self-splicing intron fragment containing a 3' cleavage site may contain a nucleotide sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:44 or 49, and the 5' self-splicing intron fragment containing a 5' cleavage site may contain a nucleotide sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:45 or 50.

[0060] The 3' self-splicing intron fragment containing a 3' cleavage site may contain a nucleotide sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:17 or 40, and the 5' self-splicing intron fragment containing a 5' cleavage site may contain a nucleotide sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:18 or 41.

[0061] The 3' self-splicing intron fragments containing 3' cleavage sites and the 5' self-splicing intron fragments containing 5' cleavage sites may respectively contain nucleotide sequences having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with i) SEQ ID NO:26 and 16, ii) SEQ ID NO:28 and 25, iii) SEQ ID NO:27 and 24, iv) SEQ ID NO:26 and 24, v) SEQ ID NO:44 and 45, vi) SEQ ID NO:40 and 41, vii) SEQ ID NO:15 and 16, viiii) SEQ ID NO:17 and 18, ix) SEQ ID NO:49 and 50, or x) SEQ ID NO:42 and 43. In some embodiments, the 3' self-splicing intron fragments containing 3' cleavage sites and the 5' self-splicing intron fragments containing 5' cleavage sites may respectively contain nucleotide sequences having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with i) SEQ ID NO:26 and 16, ii) SEQ ID NO:28 and 25, iii) SEQ ID NO:27 and 24, iv) SEQ ID NO:26 and 24, v) SEQ ID NO:44 and 45, vi) SEQ ID NO:40 and 41, vii) SEQ ID NO:15 and 16, or viiii) SEQ ID NO:17 and 18.

[0062] The 3' and 5' cleavage sites can be dinucleotides, encoding, for example, AG and GU, or AC and AU, respectively.

[0063] The target sequence can be any sequence. The target sequence can contain an open reading frame (ORF), a single cloning site, or a multiple cloning site encoding the target peptide or protein. The length of the target sequence can be 50-8000 nucleotides. The target peptide or protein can be of eukaryotic or prokaryotic origin. The target peptide or protein can be human or non-human. In some embodiments, the target peptide or protein can be an antigen protein, an antibody, or a Cas9 endonuclease, etc. In some embodiments, the target peptide or protein can be firefly luciferase, long-horned leptospirase, Gaussian luciferase, green fluorescent protein, etc. In some embodiments, the target sequence can contain one or more ORFs encoding the target peptide or protein. In some embodiments, the target sequence can be one or more ORFs encoding the target peptide or protein. In some embodiments, the target sequence can contain a single cloning site or a multiple cloning site. In some embodiments, the target sequence can be a single cloning site or a multiple cloning site.

[0064] The single-stranded DNA molecule for preparing circular RNA of this application may include a sequence encoding an internal ribosome entry site (IRES) between a 3' self-splicing intron fragment containing a 3' cleavage site and a target sequence, wherein the elements are operatively linked. In some embodiments, the single-stranded DNA molecule for preparing circular RNA of this application may sequentially include from the 5' end to the 3' end: a 3' self-splicing intron fragment containing a 3' cleavage site, a sequence encoding an internal ribosome entry site (IRES), a target sequence, and a 5' self-splicing intron fragment containing a 5' cleavage site, wherein the elements are operatively linked.

[0065] Internal ribosome entry sites (IRES) can be found on Coxsackievirus B3 (CVB3), Coxsackievirus A (CVB1 / 2), Taura syndrome virus, blood-sucking assassin bug virus, Tyrell's encephalomyelitis virus, simian virus 40, red imported fire ant virus 1, cereal constrictor aphid virus, reticuloendotheliosis virus, Forman poliovirus 1, soybean inchworm virus, Kashmir wasp virus, human rhinovirus 2, glass leafhopper virus-1, and human immunodeficiency virus type 1. Leafhopper virus-1, louse P virus, hepatitis C virus, hepatitis A virus, GB hepatitis virus, foot-and-mouth disease virus, human enterovirus 71, equine rhinovirus, tea geometrid moth-like virus, encephalocarditis virus (EMCV), fruit fly C virus, cruciferous tobacco virus, cricket paralysis virus, bovine viral diarrhea virus 1, black queen cell virus, aphid lethal paralysis virus, avian encephalomyelitis virus, acute bee paralysis virus, hibiscus yellow spot virus, classical swine fever virus, human FGF2 Human SFTPA1, human AML1 / RUNX1, Drosophila antennae, human AQP4, human AT1R, human BAG-1, human BCL2, human BiP, human c-IAPl, human c-myc, human eIF4G, mouse NDST4L, human LEF1, mouse HIF1α, human n.myc, mouse Gtx, human p27kipl, human PDGF2 / c-sis, human p53, human Pim-1, mouse Rbm3, Drosophila reaper, canine scamper, Drosophila Ubx, salivary virus, Coxsackievirus, bi-echovirus, human UNR, mouse UtrA, human VEGF-A, human XIAP, Drosophila hairless, Saccharomyces cerevisiae TFIID, Saccharomyces cerevisiae YAP1, human c-src, human FGF-1, simian microRNA virus, turnip shrunkenness virus, or IRES of eIF4G aptamer. In some implementations, the IRES can be the IRES of Coxsackievirus B3 (CVB3). The sequence encoding the IRES may contain a nucleotide sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:5.

[0066] The single-stranded DNA molecule for preparing circular RNA according to this application may further include a sequence encoding a 5' homologous arm upstream of or at the 5' end of the 3' self-splicing intron fragment, and a sequence encoding a 3' homologous arm downstream of or at the 3' end of the 5' self-splicing intron fragment. In some embodiments, the single-stranded DNA molecule may sequentially include, from the 5' end to the 3' end, a sequence encoding a 5' homologous arm, a 3' self-splicing intron fragment containing a 3' splice site, a sequence encoding an internal ribosome entry site (IRES), a target sequence, a 5' self-splicing intron fragment containing a 5' splice site, and a sequence encoding a 3' homologous arm.

[0067] The 5' and 3' homologous arms can be complementary, for example, forming at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% base pairing. In some embodiments, the 5' and 3' homologous arms can complementarily form a structure containing a palindromic stem and a stem-loop, wherein the palindromic stem is connected to the stem portion of the stem-loop, and the 5' end of the 5' homologous arm and the 3' end of the 3' homologous arm are adjacent at the palindromic stem portion. In some embodiments, the 5' and 3' homologous arms can complementarily form a structure containing a palindromic stem and two stem-loops, wherein the palindromic stem is connected to the stem portions of two stem-loops, and the stem portions of two stem-loops are connected, wherein the 5' end of the 5' homologous arm and the 3' end of the 3' homologous arm are adjacent at the loop portion of the palindromic stem or one of the stem-loops. In some embodiments, the sequences encoding the 5' homologous arm and the sequences encoding the 3' homologous arm may respectively comprise nucleotide sequences having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:2 and 10.

[0068] The single-stranded DNA molecule for preparing circular RNA according to this application may further include a 5' spacer sequence between the 3' self-splicing intron fragment and the sequence encoding the internal ribosome entry site (IRES) or the target sequence, and / or include a 3' spacer sequence between the target sequence and the 5' self-splicing intron fragment. In one embodiment, the single-stranded DNA molecule may sequentially include, from the 5' end to the 3' end, a sequence encoding a 5' homologous arm, a 3' self-splicing intron fragment containing a 3' splice site, a 5' spacer sequence, a sequence encoding the internal ribosome entry site (IRES), the target sequence, a 3' spacer sequence, a 5' self-splicing intron fragment containing a 5' splice site, and a sequence encoding a 3' homologous arm. The length of the 5' spacer sequence and / or the 3' spacer sequence may be at least 10, 20, or 30 nucleotides, for example, about 40 nucleotides or about 70 nucleotides. In some embodiments, the single-stranded DNA molecule of this application for preparing circular RNA does not contain spacer sequences, particularly the 5' spacer sequence between the 3' self-splicing intron fragment and the sequence encoding the internal ribosome entry site (IRES) or the target sequence, and the 3' spacer sequence between the target sequence and the 5' self-splicing intron fragment.

[0069] The single-stranded DNA molecule for preparing circular RNA according to this application may contain an RNA polymerase promoter at its 5' end. The RNA polymerase promoter may be an RNA polymerase promoter derived from T7 virus, T6 virus, SP6 virus, T3 virus, or T4 virus. In some embodiments, the RNA polymerase promoter may be a T7 virus promoter, which may contain a nucleotide sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:1.

[0070] The single-stranded DNA molecule used to prepare circular RNA according to this application may also contain a restriction endonuclease site at its 3' end. The restriction endonuclease site may be, for example, EcoRI or EcoRV.

[0071] In some embodiments, the single-stranded DNA molecule for preparing circular RNA according to this application may sequentially comprise, from the 5' end to the 3' end: an RNA polymerase promoter, a sequence encoding a 5' homologous arm, a 3' self-splicing intron fragment containing a 3' cleavage site, a sequence encoding an internal ribosome entry site (IRES), a target sequence, a 5' self-splicing intron fragment containing a 5' cleavage site, and a sequence encoding a 3' homologous arm, wherein each element is operatively linked. In some embodiments, the single-stranded DNA molecule for preparing circular RNA according to this application may sequentially comprise, from the 5' end to the 3' end: an RNA polymerase promoter, a sequence encoding a 5' homologous arm, a 3' self-splicing intron fragment containing a 3' cleavage site, a 5' spacer sequence, a sequence encoding an internal ribosome entry site (IRES), a target sequence, a 3' spacer sequence, a 5' self-splicing intron fragment containing a 5' cleavage site, and a sequence encoding a 3' homologous arm, wherein each element is operatively linked.

[0072] In some embodiments, the single-stranded DNA molecule for preparing circular RNA of this application may sequentially comprise from the 5' end to the 3' end: an RNA polymerase promoter, a sequence encoding a 5' homologous arm, a 3' self-splicing intron fragment containing a 3' cleavage site, a sequence encoding an internal ribosome entry site (IRES), a target sequence, a 5' self-splicing intron fragment containing a 5' cleavage site, a sequence encoding a 3' homologous arm, and a restriction endonuclease site, wherein the elements are operatively linked. In some embodiments, the single-stranded DNA molecule for preparing circular RNA of this application may sequentially comprise from the 5' end to the 3' end: an RNA polymerase promoter, a sequence encoding a 5' homologous arm, a 3' self-splicing intron fragment containing a 3' cleavage site, a 5' spacer sequence, a sequence encoding an internal ribosome entry site (IRES), a target sequence, a 3' spacer sequence, a 5' self-splicing intron fragment containing a 5' cleavage site, a sequence encoding a 3' homologous arm, and a restriction endonuclease site, wherein the elements are operatively linked.

[0073] This application also provides a double-stranded DNA molecule, which may comprise i) the single-stranded DNA molecule of this application, and ii) a second strand complementary to the single-stranded DNA molecule. In some embodiments, the double-stranded DNA molecule of this application may comprise i) the single-stranded DNA molecule of this application, and ii) a second strand completely complementary to the single-stranded DNA molecule. In some embodiments, the double-stranded DNA molecule is a double-stranded DNA molecule used for preparing circular RNA.

[0074] This application also provides a vector comprising the single-stranded DNA molecule or double-stranded DNA molecule of this application. In some embodiments, the vector may be a vector for preparing circular RNA, comprising the single-stranded DNA molecule of this application for preparing circular RNA, or the double-stranded DNA molecule for preparing circular RNA. The vector may be circular or linear. In some embodiments, the vector may be linear. In some embodiments, the vector may be circular and processed to become linear. The vector of this application can transcribe circular RNA of about 500 to about 10,000 nucleotides. In some embodiments, the vector of this application can transcribe circular RNA of at least 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2500, 3000, 3500, 4000, 4500, or 5000 nucleotides.

[0075] In a fourth aspect, this application provides a circularizable RNA molecule that, from its 5' end to its 3' end, sequentially comprises: a 3' self-splicing intron fragment containing a 3' splice site, a target sequence, and a 5' self-splicing intron fragment containing a 5' splice site, wherein the elements are operatively linked, and the 3' and 5' self-splicing intron fragments can be derived from the same self-splicing intron. The 3' self-splicing intron fragment may contain a naturally adjacent exon or a sequence corresponding to a naturally adjacent exon at its 3' end, wherein the length of the naturally adjacent exon or the sequence corresponding to a naturally adjacent exon in the 3' self-splicing intron fragment can be about 3-20 nucleotides, about 3-10 nucleotides, about 3-8 nucleotides, or about 3-6 nucleotides. In some embodiments, the length of the naturally adjacent exon or the sequence corresponding to the naturally adjacent exon in the 3' self-splicing intron fragment can be about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides. The 5' self-splicing intron fragment may contain a naturally adjacent exon or the sequence corresponding to the naturally adjacent exon at its 5' end, wherein the length of the naturally adjacent exon or the sequence corresponding to the naturally adjacent exon in the 5' self-splicing intron fragment can be about 5-20 nucleotides, about 5-10 nucleotides, about 5-9 nucleotides, about 5-8 nucleotides, or about 5-6 nucleotides. In some implementations, the length of the naturally adjacent exon in the 5' self-splicing intron fragment or the sequence corresponding to the naturally adjacent exon can be about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides.

[0076] The 3' and 5' self-splicing intron fragments can be derived from the same self-splicing intron, for example, from introns of the *Coxiella behneckii* 23S ribosomal gene, the *Pseudococcus hoffmannii* tRNA-fMet gene, the *Bacillus anthracis* recA gene, and the *Thermophyton naphthaleneophila* 23S rRNA gene. In some embodiments, the 3' and 5' self-splicing intron fragments can be derived from the *Coxiella behneckii* 23S ribosomal gene, the *Pseudococcus hoffmannii* tRNA-fMet gene, the *Bacillus anthracis* recA gene, or *Thermophyton naphthaleneophila*. Introns derived from the 23S ribosomal gene of Coxiella behnea, the tRNA-fMet gene of Pseudobranchia huffmannii, the recA gene of Bacillus anthracis, and the 23S rRNA gene of Thermophyton naphthaleneophilus may contain nucleotide sequences having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:12, 35, 13, and 14, respectively.

[0077] In some embodiments, the 3' self-splicing intron fragment may include a naturally adjacent exon at its 3' end. In some embodiments, the length of the naturally adjacent exon in the 3' self-splicing intron fragment may be about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides.

[0078] In some embodiments, the 5' self-splicing intron fragment may include a naturally adjacent exon at its 5' end. In some embodiments, the length of the naturally adjacent exon in the 5' self-splicing intron fragment may be about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides.

[0079] In some embodiments, the lengths of the naturally adjacent exons or sequences corresponding to the naturally adjacent exons in the 3' self-splicing intron fragment and the lengths of the naturally adjacent exons or sequences corresponding to the naturally adjacent exons in the 5' self-splicing intron fragment can be i) 20 nucleotides and 20 nucleotides, ii) 10 nucleotides and 20 nucleotides, iii) 10 nucleotides and 10 nucleotides, iv) 6 nucleotides and 10 nucleotides, v) 6 nucleotides and 8 nucleotides, vi) 3 nucleotides and 9 nucleotides, respectively.

[0080] The 3' self-splicing intron fragment containing a 3' splice site may contain a nucleotide sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:15, 26, 27, or 28, and the 5' self-splicing intron fragment containing a 5' splice site may contain a nucleotide sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:16, 24, or 25.

[0081] The 3' self-splicing intron fragment containing a 3' cleavage site may contain a nucleotide sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:44 or 49, and the 5' self-splicing intron fragment containing a 5' cleavage site may contain a nucleotide sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:45 or 50.

[0082] The 3' self-splicing intron fragment containing a 3' cleavage site may contain a nucleotide sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:17 or 40, and the 5' self-splicing intron fragment containing a 5' cleavage site may contain a nucleotide sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:18 or 41.

[0083] The 3' self-splicing intron fragments containing 3' cleavage sites and the 5' self-splicing intron fragments containing 5' cleavage sites may respectively contain nucleotide sequences having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with i) SEQ ID NO:26 and 16, ii) SEQ ID NO:28 and 25, iii) SEQ ID NO:27 and 24, iv) SEQ ID NO:26 and 24, v) SEQ ID NO:44 and 45, vi) SEQ ID NO:40 and 41, vii) SEQ ID NO:15 and 16, viiii) SEQ ID NO:17 and 18, ix) SEQ ID NO:49 and 50, or x) SEQ ID NO:42 and 43. In some embodiments, the 3' self-splicing intron fragments containing 3' cleavage sites and the 5' self-splicing intron fragments containing 5' cleavage sites may respectively contain nucleotide sequences having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with i) SEQ ID NO:26 and 16, ii) SEQ ID NO:28 and 25, iii) SEQ ID NO:27 and 24, iv) SEQ ID NO:26 and 24, v) SEQ ID NO:44 and 45, vi) SEQ ID NO:40 and 41, vii) SEQ ID NO:15 and 16, viiii) SEQ ID NO:17 and 18, or ix) SEQ ID NO:49 and 50.

[0084] The 3' and 5' cleavage sites can be dinucleotides, encoding, for example, AG and GU, or AC and AU, respectively.

[0085] The target sequence can be any sequence. The target sequence can contain an open reading frame encoding a target peptide or protein. The length of the target sequence can be 50-8000 nucleotides. The target peptide or protein can be of eukaryotic or prokaryotic origin. The target peptide or protein can be human or non-human. In some embodiments, the target peptide or protein can be an antigen protein, antibody, or Cas9 endonuclease, etc. In some embodiments, the target peptide or protein can be firefly luciferase, long-horned beetle luciferase, Gaussian luciferase, green fluorescent protein, etc. In some embodiments, the target sequence can contain one or more open reading frames encoding a target peptide or protein. In some embodiments, the target sequence can be one or more open reading frames encoding a target peptide or protein.

[0086] The RNA molecule of this application may include an internal ribosome entry site (IRES) between a 3' self-splicing intron fragment containing a 3' splice site and a target sequence, wherein the elements are operatively linked. In some embodiments, the RNA molecule of this application may sequentially comprise from the 5' end to the 3' end: a 3' self-splicing intron fragment containing a 3' splice site, an internal ribosome entry site (IRES), a target sequence, and a 5' self-splicing intron fragment containing a 5' splice site, wherein the elements are operatively linked.

[0087] Internal ribosome entry sites (IRES) can be found on Coxsackievirus B3 (CVB3), Coxsackievirus A (CVB1 / 2), Taura syndrome virus, blood-sucking assassin bug virus, Tyrell's encephalomyelitis virus, simian virus 40, red imported fire ant virus 1, cereal constrictor aphid virus, reticuloendotheliosis virus, Forman poliovirus 1, soybean inchworm virus, Kashmir wasp virus, human rhinovirus 2, glass leafhopper virus-1, and human immunodeficiency virus type 1. Leafhopper virus-1, louse P virus, hepatitis C virus, hepatitis A virus, GB hepatitis virus, foot-and-mouth disease virus, human enterovirus 71, equine rhinovirus, tea geometrid moth-like virus, encephalocarditis virus (EMCV), fruit fly C virus, cruciferous tobacco virus, cricket paralysis virus, bovine viral diarrhea virus 1, black queen cell virus, aphid lethal paralysis virus, avian encephalomyelitis virus, acute bee paralysis virus, hibiscus yellow spot virus, classical swine fever virus, human FGF2 Human SFTPA1, human AML1 / RUNX1, Drosophila antennae, human AQP4, human AT1R, human BAG-1, human BCL2, human BiP, human c-IAPl, human c-myc, human eIF4G, mouse NDST4L, human LEF1, mouse HIF1α, human n.myc, mouse Gtx, human p27kipl, human PDGF2 / c-sis, human p53, human Pim-1, mouse Rbm3, Drosophila reaper, canine scamper, Drosophila Ubx, salivary virus, Coxsackievirus, bi-echovirus, human UNR, mouse UtrA, human VEGF-A, human XIAP, Drosophila hairless, Saccharomyces cerevisiae TFIID, Saccharomyces cerevisiae YAP1, human c-src, human FGF-1, simian microRNA virus, turnip shrunkenness virus, or IRES of eIF4G aptamer. In some embodiments, the IRES can be an IRES of Coxsackievirus B3 (CVB3). The IRES may contain a nucleotide sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:5.

[0088] The RNA molecule of this application may also include a 5' homologous arm upstream of or at the 5' end of the 3' self-splicing intron fragment, and a 3' homologous arm downstream of or at the 3' end of the 5' self-splicing intron fragment. In some embodiments, the RNA molecule may sequentially include a 5' homologous arm, a 3' self-splicing intron fragment containing a 3' splice site, an IRES, a target sequence, a 5' self-splicing intron fragment containing a 5' splice site, and a 3' homologous arm from the 5' end to the 3' end.

[0089] The 5' and 3' homologous arms can be complementary, for example, forming at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% base pairing. In some embodiments, the 5' and 3' homologous arms can complementarily form a structure containing a palindromic stem and a stem-loop, wherein the palindromic stem is connected to the stem portion of the stem-loop, and the 5' end of the 5' homologous arm and the 3' end of the 3' homologous arm are adjacent at the palindromic stem portion. In some embodiments, the 5' and 3' homologous arms can complementarily form a structure containing a palindromic stem and two stem-loops, wherein the palindromic stem is connected to the stem portions of two stem-loops, and the stem portions of two stem-loops are connected, wherein the 5' end of the 5' homologous arm and the 3' end of the 3' homologous arm are adjacent at the loop portion of the palindromic stem or one of the stem-loops. In some embodiments, the 5' homologous arm and the 3' homologous arm may respectively comprise nucleotide sequences having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:2 and 10.

[0090] The RNA molecule of this application may also include a 5' spacer sequence between the 3' self-splicing intron fragment and the IRES or target sequence, and / or a 3' spacer sequence between the target sequence and the 5' self-splicing intron fragment. In one embodiment, the RNA molecule may sequentially include, from the 5' end to the 3' end, a 5' homologous arm, a 3' self-splicing intron fragment containing a 3' splice site, a 5' spacer sequence, an IRES, the target sequence, a 3' spacer sequence, a 5' self-splicing intron fragment containing a 5' splice site, and a 3' homologous arm. The length of the 5' spacer sequence and / or the 3' spacer sequence may be at least 10, 20, or 30 nucleotides, for example, about 40 nucleotides or about 70 nucleotides. In some embodiments, the RNA molecule of this application does not include a spacer sequence, particularly a 5' spacer sequence between the 3' self-splicing intron fragment and the sequence encoding the internal ribosome entry site (IRES) or the target sequence, and a 3' spacer sequence between the target sequence and the 5' self-splicing intron fragment.

[0091] In some embodiments, the RNA molecule of this application may sequentially comprise, from the 5' end to the 3' end: a 5' homologous arm, a 3' self-splicing intron fragment containing a 3' splice site, an IRES, a target sequence, a 5' self-splicing intron fragment containing a 5' splice site, and a 3' homologous arm, wherein the elements are operatively linked. In some embodiments, the RNA molecule of this application may sequentially comprise, from the 5' end to the 3' end: a 5' homologous arm, a 3' self-splicing intron fragment containing a 3' splice site, a 5' spacer sequence, an IRES, a target sequence, a 3' spacer sequence, a 5' self-splicing intron fragment containing a 5' splice site, and a 3' homologous arm, wherein the elements are operatively linked.

[0092] In a fifth aspect, this application provides a method for preparing circular RNA, comprising i) in vitro transcription of precursor RNA from a single-stranded DNA molecule, double-stranded DNA molecule, or vector according to the first or third aspect of this application under suitable conditions, and ii) incubating the precursor RNA under suitable conditions, wherein the suitable conditions for step ii) include the presence of magnesium ions and guanosine triphosphate (GTP). In some embodiments, suitable conditions include the presence of guanosine triphosphate and magnesium... 2+ In the presence of [specific ingredient], incubate at approximately 37°C for approximately 2 hours. In some embodiments, suitable conditions include [specific conditions] in the presence of guanosine triphosphate and Mg [specific ingredient]. 2+ In the presence of [specific ingredient], incubate at approximately 37°C for about 2 hours, and then add DNase I and incubate at approximately 37°C for about 30 minutes. The concentration of GTP can be 2 mM, and the concentration of magnesium ions can be 10 mM-20 mM.

[0093] Alternatively, the method for preparing circular RNA according to this application may include incubating the circularizable RNA molecule of the second or fourth aspect of this application under suitable conditions, wherein suitable conditions include the presence of magnesium ions and guanosine triphosphate (GTP). In some embodiments, suitable conditions include the presence of guanosine triphosphate and magnesium... 2+ In the presence of [specific ingredient], incubate at approximately 37°C for approximately 2 hours. In some embodiments, suitable conditions include [specific conditions] in the presence of guanosine triphosphate and Mg [specific ingredient]. 2+ In the presence of [specific ingredient], incubate at approximately 37°C for about 2 hours, and then add DNase I and incubate at approximately 37°C for about 30 minutes. The concentration of GTP can be 2 mM, and the concentration of magnesium ions can be 10 mM-20 mM.

[0094] This application also protects circular RNA prepared from the vector or circularizable RNA molecule of this application, as well as circular RNA prepared by the method of this application.

[0095] Circular RNA molecules can be translated into proteins within cells or exist as biologically active non-coding RNAs. The length of a circular RNA can be at least 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2500, 3000, 3500, 4000, 4500, or 5000 nucleotides.

[0096] In a sixth aspect, this application provides a host cell that may contain the single-stranded DNA molecule, double-stranded DNA molecule, vector, circularizable RNA molecule, or circular RNA molecule of this application.

[0097] This application also provides a composition that may comprise the single-stranded DNA molecule, double-stranded DNA molecule, vector, circularizable RNA molecule, or host cell of this application. This composition can be used to prepare circularizable RNA and / or circular RNA, particularly to generate transmissible proteins or biologically active circular RNA in vitro or in vivo. Biologically active circular RNA may be, for example, miRNA sponges or non-coding RNA. The composition of this application (comprising single-stranded DNA molecules and double-stranded DNA molecules) can be used to prepare a vector for preparing circular RNA.

[0098] This application also provides a composition that may contain the circular RNA molecule of this application. The composition may be a pharmaceutical composition and may also contain a pharmaceutically acceptable carrier. The composition may be transfected into cells via, for example, liposome transfection, electroporation, or encapsulation with a nanocarrier.

[0099] This application also provides the use of the composition in the preparation of circular RNA or for in vivo therapy. For example, in one embodiment, this application provides a method for expressing a vaccine, therapeutic protein, or other type of protein in a subject in need, comprising administering the composition of this application containing circular RNA to the subject. The therapeutic protein may be, for example, an antibody, a fusion protein, etc.

[0100] In a seventh aspect, this application also provides the use of introns derived from the *Coxiella belladonna* 23S ribosome gene, the *Pseudoclade chrysophanum* tRNA-fMet gene, the *Bacillus anthracis* recA gene, or the *Thermophyton naphthaleneophilus* 23S rRNA gene in the preparation of circularizable RNA molecules. These introns can be split into 3' self-splicing intron fragments and 5' self-splicing intron fragments, and are located in the DNA vector or linear RNA molecule used to prepare the circularizable RNA molecule.

[0101] This application also provides the use of introns derived from the *Coxiella behneckii* 23S ribosomal gene, the *Pseudococcus hoffmannii* tRNA-fMet gene, the *Bacillus anthracis* recA gene, or the *Thermophyton naphthaleneophilus* 23S rRNA gene in the preparation of a vector for preparing circular RNA, wherein the vector for preparing circular RNA comprises a 3' self-splicing intron fragment containing a 3' splicing site and a 5' self-splicing intron fragment containing a 5' splicing site from the introns derived from the *Coxiella behneckii* 23S ribosomal gene, the *Pseudococcus hoffmannii* tRNA-fMet gene, the *Bacillus anthracis* recA gene, or the *Thermophyton naphthaleneophilus* 23S rRNA gene.

[0102] The 3' self-splicing intron fragment containing a 3' splice site may contain a nucleotide sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:15, 26, 27, or 28, and the 5' self-splicing intron fragment containing a 5' splice site may contain a nucleotide sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:16, 24, or 25.

[0103] The 3' self-splicing intron fragment containing a 3' cleavage site may contain a nucleotide sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:44 or 49, and the 5' self-splicing intron fragment containing a 5' cleavage site may contain a nucleotide sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:45 or 50.

[0104] The 3' self-splicing intron fragment containing a 3' cleavage site may contain a nucleotide sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:17 or 40, and the 5' self-splicing intron fragment containing a 5' cleavage site may contain a nucleotide sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:18 or 41.

[0105] The 3' self-splicing intron fragments containing 3' cleavage sites and the 5' self-splicing intron fragments containing 5' cleavage sites may respectively contain nucleotide sequences having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with i) SEQ ID NO:26 and 16, ii) SEQ ID NO:28 and 25, iii) SEQ ID NO:27 and 24, iv) SEQ ID NO:26 and 24, v) SEQ ID NO:44 and 45, vi) SEQ ID NO:40 and 41, vii) SEQ ID NO:15 and 16, viiii) SEQ ID NO:17 and 18, ix) SEQ ID NO:49 and 50, x) SEQ ID NO:19 and 20, or xi) SEQ ID NO:42 and 43. In some embodiments, the 3' self-splicing intron fragments containing 3' cleavage sites and the 5' self-splicing intron fragments containing 5' cleavage sites may respectively contain nucleotide sequences having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with i) SEQ ID NO:26 and 16, ii) SEQ ID NO:28 and 25, iii) SEQ ID NO:27 and 24, iv) SEQ ID NO:26 and 24, v) SEQ ID NO:44 and 45, vi) SEQ ID NO:40 and 41, vii) SEQ ID NO:15 and 16, viiii) SEQ ID NO:17 and 18, or x) SEQ ID NO:49 and 50.

[0106] In an eighth aspect, this application provides a method for treating or preventing a disease in a subject in need, comprising administering to the subject a pharmaceutical composition comprising a circular RNA molecule of this application. The circular RNA molecule comprises an open reading frame encoding a target peptide or protein. The target peptide or protein may be a disease-associated antigen or a therapeutic agent. Disease-associated antigens may be peptides or proteins located on the surface of microorganisms, such as viruses, bacteria, mycoplasma, etc., or tumor-associated antigens. The therapeutic agent may be, for example, an antibody.

[0107] When the target peptide or protein is a peptide or protein on the surface of a microorganism, such as a virus, bacteria, mycoplasma, etc., the method of this application can be used to treat or prevent diseases related to infection by that microorganism.

[0108] When the target peptide or protein is a tumor-associated antigen, or a protein such as an antibody that targets a tumor-associated antigen, the method of this application can be used to treat tumors associated with that tumor-associated antigen.

[0109] The target peptide or protein can also be a normal protein expressed in mammals, such as humans, which can be used to supplement subjects who lack this normal protein.

[0110] Subjects can be mammals, such as humans.

[0111] In this application, the same nucleotide sequence, such as the nucleotide sequence represented by the same SEQ ID NO, can represent both a DNA sequence and an RNA sequence, the only difference being the substitution of T and U.

[0112] Other features and advantages disclosed herein will become readily apparent from the following detailed description and embodiments, which should not be construed as limiting. All references, Genbank registration numbers, patents, and published patent applications cited in this specification are incorporated herein by reference. Attached Figure Description

[0113] The following detailed description, given by way of example but not intended to limit the invention to the specific embodiments described, can be better understood in conjunction with the accompanying drawings.

[0114] Figure 1 shows the structural schematic diagrams of the published strategy vector (control vector) (A) and the resulting circular RNA molecule (D), the spacerless sequence vector (B) of this application and the resulting circular RNA molecule (E), and the spacerless sequence and exon truncated vector (C) of this application and the resulting circular RNA molecule (F).

[0115] Figure 2 shows the E-Gel of in vitro transcripts and cyclization products generated by the vector of this application and the control vector. TM EX gel image (A), Gaussian luciferase expression of cyclized products after cell transfection (B), and capillary gel electrophoresis detection of cyclized products produced by control vector (C), vector containing introns of the 23S ribosomal gene from Coxiella behnea (D), vector containing introns of the recA gene from Bacillus anthracis (E), and vector containing introns of the 23S rRNA gene from Thermophyton floccosum (F).

[0116] Figure 3 shows the E-Gel of the in vitro transcription products and cyclization products generated by the vector of this application and the control vector. TMEX gel image (A), expression of green fluorescent protein after transfection of cyclized products into cells (B), and capillary gel electrophoresis detection of cyclized products produced by control vector (C), vector containing introns of the 23S ribosomal gene from Coxiella behnea (D), vector containing introns of the recA gene from Bacillus anthracis (E), and vector containing introns of the 23S rRNA gene from Thermophyton floccosum (F).

[0117] Figure 4 shows the E-Gel of the in vitro transcript and circularized product generated by the vector using the intron- and exon-truncated Coxiella behnken 23S ribosomal gene in this application. TM EX gel image (A), and capillary gel electrophoresis detection images of cyclized products generated by carriers with E1 length 20bp + E2 length 10bp (B), E1 length 10bp + E2 length 10bp (C), E1 length 10bp + E2 length 6bp (D), and E1 length 9bp + E2 length 3bp (E).

[0118] Figure 5 shows the E-Gel of in vitro transcripts and circularized products generated using vectors containing introns of the Bacillus anthracis recA gene, the Thermophilus naphthalene 23S rRNA gene, or the Pseudoclade Hoffmann tRNA-fMet gene with truncated exons. TM EX gel image (A), and capillary gel electrophoresis detection of circularized products generated by vectors using introns and truncated exons of the Bacillus anthracis recA gene (B), introns and truncated exons of the Thermophila naphthaleneophila 23S rRNA gene (C), or introns and truncated exons of the Pseudoclade Hoffmann tRNA-fMet gene (D). Detailed Implementation

[0119] Unless otherwise specified, the terms used herein have their common meanings as found in dictionaries, textbooks, and technical reference books, or as commonly understood by those skilled in the art. The following descriptions of some terms are for the purpose of understanding this application only and are not intended to impose any particular limitations on these terms, unless otherwise specified.

[0120] As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include the plural form of the object referred to, unless the context clearly specifies otherwise.

[0121] The term "or" refers to a single element among the listed selectable elements, unless the context explicitly indicates otherwise.

[0122] The terms "comprising" or "including" mean that the stated elements, integers, or steps are included, but do not exclude the inclusion of any other elements, integers, or steps. In this document, when the terms "comprising" or "including" are used, unless otherwise specified, they also cover combinations of the stated elements, integers, or steps. The terms "consisting of" or "comprises of" generally mean that only the stated elements, integers, or steps are included, without the addition of other elements, integers, or steps.

[0123] The term "operably linked" means that the arrangement of the elements enables the completion of the required function. For example, the DNA strands in which the elements are "operably linked" in this application can perform RNA transcription in vitro or in vivo, and the RNA molecules in which the elements are "operably linked" in this application can perform circularization, etc.

[0124] The 5' end of a nucleic acid molecule can be a terminal with a free phosphate group, and the 3' end can be a terminal with a free hydroxyl group. "Upstream" generally refers to a position relatively closer to the 5' end in the nucleic acid sequence, while "downstream" generally refers to a position relatively closer to the 3' end in the nucleic acid sequence.

[0125] "In vitro transcription" or "IVT" refers to the process of forming RNA using DNA as a template in a cell-free system under conditions containing RNA transcriptase, NTPs, etc., mimicking the in vivo transcription process. When using a plasmid vector as a DNA template, the plasmid line is linearized by enzyme digestion sites before in vitro transcription.

[0126] "Precursor RNA", "IVT transcript", "messenger RNA", "mRNA" or "circularizable RNA" refers to the RNA product transcribed from the vector of this application, which can be circularized under suitable conditions to form circular RNA.

[0127] Exons are sequences in DNA that appear on mature RNA molecules. Exons are separated by introns and are joined together after transcription by intron removal. Conversely, introns are sequences that separate exons; they are transcribed into precursor RNA, removed by splicing, and ultimately not displayed in mature RNA molecules. Intron removal typically requires the participation of splice bodies, ribonucleoprotein complexes dynamically composed of nuclear small RNAs (snRNAs, U1, U2, U4, U5, U6, etc.) and protein factors (approximately 100 types), which recognize splice sites on the RNA precursor and catalyze the splicing reaction. However, a very small number of introns that form ribozymes undergo self-splicing, functioning as spliceases in RNA alone.

[0128] In this application, "self-splicing introns" refers to introns capable of splicing themselves without the involvement of spliceosomes. Conditions for splicing of self-splicing introns include the presence of GTP and magnesium ions. In some embodiments, the conditions for splicing of self-splicing introns include incubation at approximately 70°C for 5 minutes, placement on ice for 3 minutes, and the addition of GTP and Mg. 2+ Incubate at approximately 55°C for 8 minutes. Based on their structure and splicing mechanism, self-splicing introns are generally classified into two types: Type I and Type II.

[0129] In this article, "complementary," "pairing," "complementary pairing," or "base pairing" refers to the ability of two nucleotides or two bases to pair and bind according to the AT, AU, CG base complementarity principle. When one nucleotide sequence is "complementary" to another nucleotide sequence, it can mean that the two nucleotide sequences are 100% complementary to each other, or it can mean that the two nucleotide sequences are highly complementary, such as more than 90% complementary.

[0130] In this article, a "promoter" refers to a sequence that controls transcriptomic synthesis by providing recognition and binding sites for RNA polymerase. The promoter region may also include recognition or binding sites for other factors involved in transcriptional regulation. Promoters can be inducible, initiating transcription in response to an induction signal, or constitutive. Inducible promoters, in the absence of an induction signal, elicit very little or no transcription.

[0131] An "open reading frame" or "ORF" is a continuous sequence of nucleotides that begins with a start codon and ends with a stop codon, encoding a complete polypeptide chain. In an mRNA sequence, every three consecutive nucleotides (i.e., a triplet "codon") encode a corresponding amino acid. There is one start codon (AUG) and three stop codons (UAA, UAG, and UGA). The ribosomes begin translation from the start codon, synthesizing the polypeptide chain along the mRNA sequence and continuously elongating it. The elongation process terminates when a stop codon is encountered.

[0132] "Internal ribosome entry site" or "IRES" refers to an RNA sequence that forms a secondary structure to attract the transcription initiation complex precursor to the translation start codon, such as AUG.

[0133] A "homologous arm" refers to an RNA sequence that can form at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% base pairs with another RNA (e.g., another homologous arm).

[0134] "Spacer sequences" are sequences added to prevent interference between adjacent, nearby, or even distant elements during transcription, RNA folding, and other processes. Whether vector elements will interfere with each other during transcription, RNA folding, etc., can be predicted using computer software such as RNA folding software (e.g., RNAFold). Spacer sequences can also be designed using computer software such as RNA folding software (e.g., RNAFold).

[0135] The term "identity" or "sequence identity" as used herein refers to the percentage of nucleotides / amino acids in a sequence that are identical to those in a reference sequence after sequence alignment. If necessary, spaces are introduced in the sequence alignment to achieve the maximum percentage of sequence similarity between the two sequences. Those skilled in the art can use various methods, such as computer software, to perform pairwise or multiple sequence alignments to determine the percentage of sequence similarity between two or more nucleic acid or amino acid sequences. Such computer software includes, for example, ClustalOmega, T-coffee, Kalign, and MAFFT.

[0136] The term “subject” includes any human or non-human animal. The term “non-human animal” includes all vertebrates, such as mammals and non-mammalians, such as non-human primates, sheep, dogs, cats, cattle, horses, chickens, amphibians, and reptiles, although mammals, such as non-human primates, sheep, dogs, cats, cattle, and horses, are preferred.

[0137] The term "vector" refers to a naturally occurring or synthetic fragment of DNA, including single-stranded and double-stranded DNA fragments, such as chemically synthesized DNA fragments, natural plasmids, or modified viral genomes. A foreign DNA fragment may be inserted into a vector for the cloning and / or expression of that foreign DNA fragment. Vectors may contain, for example, origins of replication, selectivity markers or reporter genes, multiple cloning sites (MCS), etc. The term includes linear DNA fragments (such as PCR products, linearized plasmid fragments, etc.), plasmid vectors, viral vectors, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), etc. When the vector is double-stranded DNA, the description of the element sequencing and the orientation of the element sequence is relative to one of the DNA strands. In a double-stranded vector, the two DNA strands are substantially complementary or completely complementary, meaning that at least approximately 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the bases are paired between the two DNA strands.

[0138] "Nucleoside" is a component of DNA and RNA, consisting of ribose (for RNA) or deoxyribose (for DNA) and a base. "Nucleotide" refers to a molecule composed of a nucleoside and a phosphate group, which may contain a hydroxyl group at the 5' position and a phosphate group at the 3' position, or vice versa. In this application, "nucleotide" and "base" may be used interchangeably in certain contexts.

[0139] When preparing circular RNA using self-cleaving introns, it is inevitable that some non-target sequences will be introduced.

[0140] For example, to avoid unnecessary interference between the highly structured IRES and the splicing site, which could affect normal function, 5' and 3' spacer sequences are typically placed between the IRES and the self-splicing intron fragment. The length of the 5' and 3' spacer sequences is at least 10, 20, or 30 nucleotides, and is usually about 40 or about 70 nucleotides.

[0141] In addition, a certain number of exon segments must be present on both sides of the self-splicing intron to ensure the normal self-splicing of the intron. The length of the naturally adjacent exon or the sequence corresponding to the naturally adjacent exon in the 3' self-splicing intron segment, and the length of the naturally adjacent exon or the sequence corresponding to the naturally adjacent exon in the 5' self-splicing intron segment, are typically about 10-40 nucleotides.

[0142] The aforementioned spacer sequences are artificially designed, non-natural sequences. The naturally adjacent exons flanking the self-splicing introns are sequences derived from microorganisms, and the sequences corresponding to the naturally adjacent exons are artificially designed, non-natural sequences. Therefore, when these sequences remain in intermediate products, such as in vitro transcribed linear RNA molecules, and in the final product, i.e., circular RNA molecules, they are likely to induce immunogenicity when applied, for example, to humans.

[0143] The inventors of this application have studied the preparation technology of circular RNA by removing spacer sequences and truncating exon sequences.

[0144] First, the inventors constructed vectors for preparing circular RNA using self-splicing introns derived from various organisms, without the spacer sequences typically used to enhance expression and circularization efficiency. The results showed that when containing self-splicing introns from certain sources, such as those derived from the *Bacillus anthracis* recA gene, *Coxiella belladonna* 23S ribosome gene, *Pseudococcus hoffmannii* tRNA-fMet gene, or *Thermophyton naphthaleneophilus* 23S rRNA, RNA with the 5' and 3' spacer sequences removed could still achieve circularization. Specifically, when containing self-splicing introns from certain sources, such as those derived from the *Bacillus anthracis* recA gene and *Coxiella belladonna* 23S ribosome gene, RNA without spacer sequences still exhibited higher circularization efficiency than RNA containing 5' and 3' spacer sequences, and RNA with introns derived from the *Anabaena* pre-tRNA-Leu gene.

[0145] Furthermore, the inventors conducted truncation tests on the exon fragments flanking introns that maintained good circularization efficiency after removing the spacer sequences. The results showed that when fewer exon sequences were retained flanking introns derived from self-splicing introns, such as the Coxiella benziii 23S ribosome gene, RNA circularization efficiency remained at a high level. Specifically, when 10 and 10 nucleotides, 10 and 6 nucleotides, or 9 and 3 nucleotides of exon sequences were retained flanking introns derived from, for example, the Coxiella benziii 23S ribosome gene, the RNA circularization efficiency was comparable to that of RNA containing 5' spacer sequences, 3' spacer sequences, and introns derived from the Anabaena pre-tRNA-Leu gene. However, when introns derived from, for example, the Coxiella beckonii 23S ribosomal gene retain 20 nucleotides and 10 nucleotides of exon sequences on either side, RNA circularization efficiency may still be higher than that of RNA containing 5' spacer sequences, 3' spacer sequences, and introns derived from the untruncated Anabaena pre-tRNA-Leu gene.

[0146] The inventors also found that RNA containing introns of the *Pseudococcus hoffmannii* tRNA-fMet gene with the 5' and 3' spacer sequences removed and flanking exons truncated could be circularized, with a circularization efficiency comparable to RNA containing the 5' and 3' spacer sequences and introns derived from the untruncated *Anabaena* pre-tRNA-Leu gene. This result indirectly suggests that when using introns of the *Pseudococcus hoffmannii* tRNA-fMet gene without exon truncation, the circularization efficiency of the vector-generated RNA is likely comparable to, or even higher than, RNA containing the 5' and 3' spacer sequences and introns derived from the untruncated *Anabaena* pre-tRNA-Leu gene. Furthermore, RNA containing introns of *Bacillus anthracis* with the 5' and 3' spacer sequences removed and flanking exons truncated could also be circularized, albeit with slightly lower efficiency.

[0147] When circular RNA is applied, for example, to humans, and where it is necessary to minimize immunogenicity, the methods described in this application, such as removing spacer sequences and / or truncating self-splicing introns adjacent to exons, can be employed. Furthermore, introns derived from the *Coxiella belladonna* 23S ribosome gene or the *Pseudococcus hoffmannii* tRNA-fMet gene can be selected, particularly intron fragments of the *Coxiella belladonna* 23S ribosome gene or the *Pseudococcus hoffmannii* tRNA-fMet gene truncated to adjacent exon segments, thereby maintaining high RNA circularization efficiency while reducing immunogenicity.

[0148] Therefore, this application provides a DNA molecule and vector for producing circular RNA without spacer sequences and / or truncated exon fragments flanking introns, as well as the circular RNA generated therefrom. The beneficial technical effects of this application include reducing the residue of undesirable nucleotides while maintaining high circularization efficiency, which has a positive impact on improving the yield of circular RNA, controlling costs, and reducing process residues.

[0149] In a first aspect, this application provides a single-stranded DNA molecule for preparing circular RNA, which may sequentially comprise from the 5' end to the 3' end: a 3' self-splicing intron fragment containing a 3' splice site, a sequence encoding an internal ribosome entry site (IRES), a target sequence, and a 5' self-splicing intron fragment containing a 5' splice site, wherein the elements are operatively linked, and the 3' self-splicing intron fragment containing the 3' splice site and the 5' self-splicing intron fragment containing the 5' splice site may be derived from introns of the *Coxiella belladonna* 23S ribosome gene, the *Pseudoclade Hoffmann* tRNA-fMet gene, the *Bacillus anthracis* recA gene, or the *Thermophyton naphthaleneophilus* 23S rRNA gene, wherein the single-stranded DNA molecule does not contain a spacer sequence.

[0150] Even after removing the 5' and 3' spacer sequences, RNA containing introns from certain sources, such as those derived from the *Bacillus anthracis* recA gene, *Coxiella bengal* 23S ribosome gene, *Pseudococcus hoffmannii* tRNA-fMet gene, and *Thermophyton naphthaleneophilus* 23S rRNA, can still circularize. Specifically, when containing introns from certain sources, such as those derived from the *Bacillus anthracis* recA gene and *Coxiella bengal* 23S ribosome gene, RNA without spacer sequences exhibits higher circularization efficiency than RNA containing 5' and 3' spacer sequences, and introns derived from the *Anabaena pre-tRNA-Leu* gene. Furthermore, the expression levels of exogenous proteins in the circular RNA are comparable to or higher than those in RNA without spacer sequences compared to RNA containing 5' and 3' spacer sequences, and introns derived from the *Anabaena pre-tRNA-Leu* gene. This is unexpected. Although there is no direct comparison, the inventors believe that the circularization efficiency of RNA containing introns of the Hoffmann pseudobranchia tRNA-fMet gene with the 5' and 3' spacer sequences removed but with untrunculated exons on both sides may be comparable to, or even slightly higher than, that of RNA containing the 5' and 3' spacer sequences and introns derived from the untrunculated Anabaena pre-tRNA-Leu gene.

[0151] When preparing circular RNA using self-cleaving introns, the introns are typically truncated at different sites. The 3' intron fragment and exon fragment are placed on the 5' side of the vector (relative to one strand of the vector's main DNA), and the 5' exon fragment and intron fragment are placed on the 3' side of the vector, with the target sequence placed between them. Thus, when intron self-cleavage occurs, the fragments other than the introns remain in the circular RNA. The exon sequence is preferably naturally present on the flanking side of the intron. The 3' intron fragment needs to contain a 3' cleavage site, and the 5' intron fragment needs to contain a 5' cleavage site. The 3' and 5' cleavage sites can be dinucleotides, encoding, for example, AG and GU, or AC and AU, respectively.

[0152] The target sequence in a single-stranded DNA molecule may contain an open reading frame (OPF), a single cloning site, or a multiple cloning site encoding a target peptide or protein. In preparing a universal vector, the target sequence can be a single cloning site or a multiple cloning site, particularly a multiple cloning site. Thus, the vector can, as needed, add an OPF encoding, for example, a target peptide or protein, at the cloning site. The target peptide or protein can be of eukaryotic or prokaryotic origin. The target peptide or protein can be human or non-human. In some embodiments, the target peptide or protein can be an antigen protein, an antibody, or a Cas9 endonuclease, etc. In preparing circular RNA for prevention or treatment, the circular RNA molecule contains an OPF encoding a target peptide or protein. The target peptide or protein can be a disease-associated antigen or a therapeutic agent. Disease-associated antigens can be peptides or proteins located on the surface of microorganisms, such as viruses, bacteria, mycoplasma, etc., or tumor-associated antigens. Therapeutic agents can be, for example, antibodies. When the target peptide or protein is located on the surface of microorganisms such as viruses, bacteria, mycoplasma, etc., the method of this application can be used to treat or prevent diseases associated with infection by such microorganisms. When the target peptide or protein is a tumor-associated antigen or a protein targeting a tumor-associated antigen, such as an antibody, the method of this application can be used to treat tumors associated with the tumor-associated antigen. The target peptide or protein can also be a normal protein expressed in mammals, such as humans, which can be used to supplement subjects lacking the normal protein.

[0153] The single-stranded DNA molecule used to prepare circular RNA may also contain sequences encoding 5' homologous arms upstream of or at the 5' end of the 3' self-splicing intron fragment, and sequences encoding 3' homologous arms downstream of or at the 3' end of the 5' self-splicing intron fragment. The presence of 5' and 3' homologous arms allows the two ends of the linear RNA to bind together, bringing the 3' and 5' self-splicing intron fragments closer together, facilitating intron splicing. In some embodiments, the 5' and 3' homologous arms may be part of an inverted terminal repeat (ITR) or an ITR-like structure, respectively. When the 5' and 3' homologous arms in the linear RNA are close together, an ITR or similar structure can be formed. Compared to forming linearly paired 5' and 3' homologous arms, forming an ITR or an ITR-like structure results in higher circularization efficiency.

[0154] Single-stranded DNA molecules used to prepare circular RNA may contain an RNA polymerase promoter at their 5' end for transcription of the DNA molecule. The RNA polymerase promoter can be any suitable RNA polymerase promoter, as long as it ensures transcription of the DNA molecule, especially an RNA polymerase promoter with high transcription efficiency.

[0155] Single-stranded DNA molecules used to prepare circular RNA may also include a restriction endonuclease site at the 3' end for linearization of the DNA vector. Vector linearization is necessary during transcription, especially in vitro transcription. This linearization can be achieved by digestion at the aforementioned restriction endonuclease site. In some embodiments, this restriction endonuclease site is unique within the vector.

[0156] This application also provides a corresponding double-stranded DNA molecule, which may comprise i) the single-stranded DNA molecule of this application, and ii) a second strand that is complementary to, for example, completely complementary to, the single-stranded DNA molecule.

[0157] This application also provides a vector containing the single-stranded DNA molecule or the double-stranded DNA molecule of this application.

[0158] Accordingly, this application also provides a circularizable RNA molecule, which may sequentially comprise from the 5' end to the 3' end: a 3' self-splicing intron fragment containing a 3' splice site, an internal ribosome entry site, a target sequence, and a 5' self-splicing intron fragment containing a 5' splice site, wherein the elements are operatively linked, and the 3' self-splicing intron fragment containing the 3' splice site and the 5' self-splicing intron fragment containing the 5' splice site may be derived from the introns of the Coxiella behnea 23S ribosome gene, the Pseudoclade Hoffmann tRNA-fMet gene, the Bacillus anthracis recA gene, or the Thermophyton naphthaleneophilus 23S rRNA gene, wherein the RNA molecule does not contain a spacer sequence.

[0159] The RNA molecule of this application may also contain a 5' homologous arm upstream of the 3' self-splicing intron fragment or at the 5' end, and a 3' homologous arm downstream of the 5' self-splicing intron fragment or at the 3' end.

[0160] In a second aspect, this application provides a single-stranded DNA molecule for preparing circular RNA, which may sequentially comprise from the 5' end to the 3' end: a 3' self-splicing intron fragment containing a 3' splice site, a target sequence, and a 5' self-splicing intron fragment containing a 5' splice site, wherein the elements are operatively linked, and the 3' and 5' self-splicing intron fragments may be derived from the same self-splicing intron. The 3' self-splicing intron fragment may contain a naturally adjacent exon or a sequence corresponding to the naturally adjacent exon at its 3' end, wherein the length of the naturally adjacent exon or the sequence corresponding to the naturally adjacent exon in the 3' self-splicing intron fragment may be about 3-20 nucleotides, about 3-10 nucleotides, about 3-8 nucleotides, or about 3-6 nucleotides. 5' self-splicing intron fragments may contain a naturally adjacent exon or a sequence corresponding to a naturally adjacent exon at the 5' end. The length of the naturally adjacent exon or the sequence corresponding to a naturally adjacent exon in the 5' self-splicing intron fragment may be about 5-20 nucleotides, about 5-10 nucleotides, about 5-8 nucleotides, or about 5-6 nucleotides.

[0161] RNA circularization efficiency remains high even when fewer exon sequences are retained flanked by introns derived from, for example, the Coxiella beckerle 23S ribosomal gene. Specifically, when 3-nucleotide and 9-nucleotide exon sequences are retained flanked by introns derived from, for example, the Coxiella beckerle 23S ribosomal gene, the RNA circularization efficiency is comparable to that of RNA containing 5' spacer sequences, 3' spacer sequences, and introns derived from the Anabaena pre-tRNA-Leu gene.

[0162] In some embodiments, when using introns derived from the Coxiella 23S ribosomal gene, the lengths of the naturally adjacent exons or sequences corresponding to the naturally adjacent exons in the 3' self-splicing intron fragment and the lengths of the naturally adjacent exons or sequences corresponding to the naturally adjacent exons in the 5' self-splicing intron fragment can be i) 20 nucleotides and 20 nucleotides, ii) 10 nucleotides and 20 nucleotides, iii) 10 nucleotides and 10 nucleotides, iv) 6 nucleotides and 10 nucleotides, v) 6 nucleotides and 9 nucleotides, vi) 3 nucleotides and 9 nucleotides.

[0163] The target sequence can be any sequence. The target sequence can contain an open reading frame, a single cloning site, or a multiple cloning site encoding the target peptide or protein.

[0164] Single-stranded DNA molecules used to prepare circular RNA may include a sequence encoding the internal ribosome entry site (IRES) between the 3' self-splicing intron fragment at the 3' splicing site and the target sequence; may include a sequence encoding a 5' homologous arm upstream of the 3' self-splicing intron fragment or at the 5' end, and a sequence encoding a 3' homologous arm downstream of the 5' self-splicing intron fragment or at the 3' end; may include a 5' spacer sequence between the 3' self-splicing intron fragment and the sequence encoding the internal ribosome entry site (IRES) or the target sequence, and / or include a 3' spacer sequence between the target sequence and the 5' self-splicing intron fragment; may include an RNA polymerase promoter at the 5' end; and / or may include a restriction endonuclease site at the 3' end.

[0165] In particular, single-stranded DNA molecules may not contain spacer sequences between the aforementioned elements, especially spacer sequences between 3' self-splicing intron fragments containing 3' splicing sites and sequences encoding internal ribosome entry sites (IRES), and spacer sequences between the target sequence and 5' self-splicing intron fragments containing 5' splicing sites.

[0166] This application also provides a double-stranded DNA molecule that may comprise i) the single-stranded DNA molecule of this application, and ii) a second strand that is complementary to, for example, completely complementary to, the single-stranded DNA molecule.

[0167] This application also provides a vector containing the single-stranded DNA molecule or the double-stranded DNA molecule of this application.

[0168] Accordingly, a circularizable RNA molecule is also provided, which may sequentially comprise from the 5' end to the 3' end: a 3' self-splicing intron fragment containing a 3' splice site, a target sequence, and a 5' self-splicing intron fragment containing a 5' splice site, wherein the elements are operatively linked, and the 3' and 5' self-splicing intron fragments may be derived from the same self-splicing intron. The 3' self-splicing intron fragment may contain a naturally adjacent exon or a sequence corresponding to the naturally adjacent exon at the 3' end, wherein the length of the naturally adjacent exon or the sequence corresponding to the naturally adjacent exon in the 3' self-splicing intron fragment may be approximately 3-20 nucleotides, approximately 3-10 nucleotides, approximately 3-8 nucleotides, or approximately 3-6 nucleotides. 5' self-splicing intron fragments may contain a naturally adjacent exon or a sequence corresponding to a naturally adjacent exon at the 5' end. The length of the naturally adjacent exon or the sequence corresponding to a naturally adjacent exon in the 5' self-splicing intron fragment may be about 5-20 nucleotides, about 5-10 nucleotides, about 5-8 nucleotides, or about 5-6 nucleotides.

[0169] The circularizable RNA molecules of this application can be obtained by transcription using the vector described in this application.

[0170] This application provides a composition that may comprise the single-stranded DNA molecule, double-stranded DNA molecule, vector, or circularizable RNA molecule of this application, or a cell comprising the aforementioned single-stranded DNA molecule, double-stranded DNA molecule, vector, or circularizable RNA molecule. This composition can be used to prepare circularizable RNA and / or circular RNA, particularly to generate translatable proteins or biologically active circular RNA in vitro or in vivo. Biologically active circular RNA may be, for example, miRNA sponges or non-coding RNA.

[0171] This application also provides a composition that may comprise the circular RNA molecule of this application or a cell comprising the circular RNA molecule. The composition may be a pharmaceutical composition and may also comprise a pharmaceutically acceptable carrier, excipient, or diluent.

[0172] Pharmaceutically acceptable carriers can be components in a composition other than the active ingredient (i.e., the carrier, cell, precursor RNA, or circular RNA of this application). Pharmaceutically acceptable carriers can include, but are not limited to, buffers, excipients, stabilizers, or preservatives. Examples of pharmaceutically acceptable carriers are physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delay agents, such as salts, buffers, sugars, antioxidants, aqueous or non-aqueous carriers, preservatives, wetting agents, surfactants, or emulsifiers, or combinations thereof. The amount of a pharmaceutically acceptable carrier in a drug composition can be determined experimentally based on the activity of the carrier and the desired properties of the formulation, such as stability and / or minimal oxidation.

[0173] In some embodiments, the compositions of this application may comprise buffer solutions such as acetic acid, citric acid, histidine, boric acid, formic acid, succinic acid, phosphoric acid, carbonic acid, malic acid, aspartic acid, Tris buffer, HEPPSO, HEPES, neutral buffered saline, phosphate buffered saline, etc.; carbohydrates such as glucose, sucrose, mannose or dextran, mannitol; proteins; peptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); antibacterial and antifungal agents; and preservatives.

[0174] This application also provides the use of the composition in the preparation of circular RNA or for in vivo therapy. For example, in one embodiment, this application provides a method for expressing a vaccine, therapeutic protein, or other type of protein in a subject in need, including administering the composition of this application containing circular RNA to the subject. The therapeutic protein may be, for example, an antibody, a fusion protein, etc. In one embodiment, the subject is a subject with a loss of a functional gene, and the circular RNA in the composition of this application may perform the function of the RNA transcribed from the missing gene, or continuously express the protein translated from the missing gene in vivo.

[0175] When the circular RNA is translated to express a therapeutic protein or exerts a therapeutic effect, a therapeutically effective amount of the composition containing the circular RNA is administered to the subject. The "therapeutically effective amount" of the composition preferably causes a reduction in the severity of disease symptoms, an increase in the frequency and duration of symptom-free periods, or prevention of damage or disability caused by the disease. For example, in the treatment of a tumor-bearing subject, a "therapeuticly effective amount" means, relative to an untreated subject, preferably, tumor growth is inhibited by at least about 40%, more preferably by at least about 60%, more preferably by at least about 80%, and more preferably by at least about 99%. The therapeutically effective amount of the fusion protein of this application can reduce tumor volume or alleviate symptoms in a subject (typically a human, or possibly another mammal).

[0176] When necessary, the precursor RNA or circular RNA in this application can be purified. For example, when administering the composition of this application to a subject, the circular RNA is preferably purified. Purification of the circular RNA or precursor RNA can be performed by applying a size exclusion column in a high-performance liquid chromatography (HPLC) system.

[0177] The specific administration of the drug composition can be determined by medical professionals, such as doctors, based on the subject's specific circumstances, such as gender, age, and medical history.

[0178] The pharmaceutical compositions of this application can be formulated for oral, intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or bolus). "Parenteral administration" refers to methods other than intestinal and topical application, typically administered by injection, including but not limited to intravenous, intramuscular, intra-arterial, intramembranous, intracystic, intraorbital, intracardiac, intradermal, intraperitoneal, tracheal, subcutaneous, subepidermal, intra-articular, sub-bursular, subarachnoid, intraspinal, supradural, and intrasternal injections and boluses. In one embodiment, the composition can be formulated for infusion or intravenous administration. The compositions disclosed herein can be provided, for example, as sterile liquid formulations, such as isotonic aqueous solutions, emulsions, suspensions, dispersions, or viscous compositions that can be buffered to the desired pH. In some embodiments, the composition can be administered in any manner, such as via parenteral or non-parenteral administration, including via aerosol inhalation, injection, infusion, ingestion, transfusion, implantation, or transplantation. For example, the compositions described herein can be administered to patients via intravenous injection, intranasal administration, intrathecal administration, intrasheath administration, intraperitoneal administration via artery, intradermal administration, subcutaneous administration, intratumoral administration, intramedullary administration, intranodal administration, or intramuscular administration.

[0179] The compositions of this application can be transfected into cells via, for example, liposome transfection, electroporation, or encapsulation with nanocarriers. Nanocarriers can be, for example, lipids, polymers, or lipid-polymer hybrids.

[0180] The compositions of this application can be delivered to a subject via a release delivery system. Release delivery systems include polymer-based systems such as polylactide-glycolic acid, copolyoxalate, polyesteramide, polyorthoester, polycaprolactone, polyhydroxybutyrate, and polyanhydride. Delivery systems also include non-polymer systems, which are lipids, including sterols such as cholesterol and cholesterol esters, and fatty acids or neutral fats such as monoglycerides and triglycerides; peptide-based systems; hydrogel release systems; wax coatings; tablets using conventional adhesives and excipients; partially fused implants; and so on. In some embodiments, lipid nanoparticles or polymers are used as delivery carriers for the therapeutic circular RNA described herein, including the delivery of RNA to tissues.

[0181] The pharmaceutical composition can be a sustained-release agent, including implants and microcapsule delivery systems. Biodegradable and biocompatible polymers such as ethylene-vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoester, and polylactic acid can be used. The pharmaceutical composition can be administered via medical devices, such as (1) needle-free subcutaneous injection devices (e.g., U.S. Patents 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; and 4,596,556); (2) microinfusion pumps (U.S. Patent 4,487,603); (3) transdermal drug delivery devices (U.S. Patent 4,486,194); (4) bolus injection devices (U.S. Patents 4,447,233 and 4,447,224); and (5) permeation devices (U.S. Patents 4,439,196 and 4,475,196).

[0182] The compositions of this application can be used in combination with other therapeutic agents, vaccines, etc. The combinations of therapeutic agents discussed herein can be administered simultaneously as a single composition in a pharmaceutically acceptable carrier, or as separate compositions, wherein each agent is contained in a pharmaceutically acceptable carrier. In another embodiment, the combinations of therapeutic agents can be administered sequentially.

[0183] Furthermore, if multiple combination therapies are administered and the drugs are administered sequentially, the order of administration at each time point can be reversed or kept the same, and sequential administration can be combined with simultaneous administration or any combination thereof.

[0184] This application will be further described with reference to the following non-limiting embodiments.

[0185] Example

[0186] Example 1. Construction of a vector for producing circularizable RNA without spacer sequences

[0187] Based on the public information of Daniel G. et al. [21 Based on this, intron sequences from three different sources—the 23S ribosomal RNA gene from Coxiella burnetii, the recA gene from Bacillus anthracis, and the 23S rRNA gene from Thermotoga subterranea—were used to replace introns from Anabaena to construct vectors for preparing circularizable RNA, while simultaneously removing spacer sequences.

[0188] The strategy of constructing vectors based on Daniel G.'s published content is called the "published strategy," while the vector construction strategy of this paper is called the "no-interval sequence strategy."

[0189] 1.1 Carrier Construction

[0190] Refer to Daniel G.'s published articles

[0021] We constructed a control vector and the vector used in this application for preparing circularizable RNA.

[0191] Specifically, for the construction of the control vector, the synthesized DNA fragment contains, from 5' to 3', the following sequence: T7 promoter (SEQ ID NO:1), sequence encoding 5' ITR homologous arm (SEQ ID NO:2), 3' intron & exon E2 (SEQ ID NO:3), 5' spacer sequence (SEQ ID NO:4), sequence encoding CVB3 IRES (SEQ ID NO:5), sequence encoding Gaussian luciferase (GLUC) (SEQ ID NO:6), 3' spacer sequence (SEQ ID NO:8), 5' intron & exon E1 (SEQ ID NO:9), sequence encoding 3' ITR homologous arm (SEQ ID NO:10), and EcoRV restriction enzyme site (GATATC) for plasmid linearization. The 3' intron & exon E2 and 5' intron & exon E1 are derived from Anabaena, and E1 and E2 are the exon sequences located at both ends of the intron that are required to achieve self-splicing function. The synthesized DNA fragment was cloned into the pUC57 vector (SEQ ID NO:11), and the linearized vector product was obtained by EcoRV digestion. The structure of the transcribed RNA can be seen in Figure 1(A).

[0192] For the vector used in this application to prepare circularizable RNA, the construction method is the same as above, but the 5' spacer sequence and the 3' spacer sequence are removed, and introns from the *Coxiella belladonna* 23S ribosomal RNA gene, *Bacillus anthracis* recA gene, or *Thermophyton floccosum* 23S rRNA gene are used. Specifically, each synthesized DNA fragment contains, from 5' to 3', the following sequence: T7 promoter (SEQ ID NO:1), sequence encoding 5' ITR homologous arm (SEQ ID NO:2), 3' intron & exon E2 (SEQ ID NO:15, 17, or 19), sequence encoding CVB3 IRES (SEQ ID NO:5), sequence encoding Gaussian luciferase (GLUC) (SEQ ID NO:6), 5' intron & exon E1 (SEQ ID NO:16, 18, or 20), sequence encoding 3' ITR homologous arm (SEQ ID NO:10), and EcoRV restriction enzyme site (GATATC) for plasmid linearization. The structure of the RNA transcribed by each vector can be seen in Figure 1(B).

[0193] The gene synthesis, vector construction, and plasmid linearization described above were all performed using GenScript.

[0194] 1.2 In vitro transcription

[0195] The linearized plasmid vector obtained above was subjected to in vitro transcription (IVT) to obtain RNA product. Specifically, the reaction system shown in Table 1 was incubated at 37°C for 2 h, then 2 μl of DNase I (RNase-free, ON-109, Hongene) was added, mixed well, and incubated at 37°C for 30 min.

[0196] Table 1. Reaction system for in vitro transcription

[0197] 1.3 LiCl precipitation purification of RNA

[0198] The RNA product obtained above was purified by LiCl precipitation. Specifically, 22 μl of enzyme-free water (AM9937, Thermo Fisher) and 20 μl of 8M LiCl solution (DNase and RNase-free, ST498-100 ml, Beyotime) were added to the IVT stock solution to make the LiCl concentration 2.5M. The mixture was then incubated at -20℃ for at least 30 min. The mixture was centrifuged at 12000g for 15 min at 4℃, and the supernatant was discarded. 500 μl of 70% ethanol was added, the mixture was inverted and mixed, and the mixture was centrifuged at 12000g for 3 min at 4℃, and the supernatant was discarded. This step was repeated. The mixture was centrifuged at 12000g for 1 min at 4℃, the supernatant was aspirated, and 100 μl of enzyme-free water was added to dissolve the RNA.

[0199] The concentration of purified RNA can be detected using a Nanodrop ONEC micro-volume UV-Vis spectrophotometer (ND-ONEC-W, Thermo Scientific).

[0200] 1.4 RNA cyclization (one-step cyclization method) and LiCl precipitation purification

[0201] The purified mRNA obtained above was then circularized. Specifically, 20 μg of purified mRNA was taken and adjusted to approximately 0.5-2 μg / μl with enzyme-free water, incubated at 70°C for 5 min, and then immediately placed on ice for 3 min. GTP (100 mM) was added to a final concentration of 2 mM, and then Mg2+ was added. 2+ T4 RNA ligase buffer (10x, BO216S, NEB) allows Mg to... 2+ The final concentration was 10 mM. Incubate at 55°C for 8 min.

[0202] The product was purified by LiCl precipitation. Specifically, 9.1 μl of 8M LiCl solution was added to the RNA circularization solution to make the LiCl concentration 2.5M. The concentration of the purified circularized RNA product could be detected by Nanodrop.

[0203] 1.5 RNase R digestion treatment

[0204] Take the purified RNA product obtained from LiCl in step 1.4, add 0.6 μl Tris-HCl, 1 μl KCl-3M, 0.003 μl MgCl2-1M, and 0.3 μl RNase R (48 U / μl), mix well by pipetting, and incubate at 37 °C for 15 min.

[0205] 1.6 Gel electrophoresis analysis

[0206] The purified RNA obtained in step 1.3, the LiCl-purified RNA obtained in step 1.4, and the RNA treated with RNase R obtained in step 1.5 were then processed using E-Gel. TM E-Gel electrophoresis was performed for analysis. Specifically, 150-200 ng of purified RNA was taken, adjusted to 10 μl with enzyme-free water, and then 10 μl of gel loading buffer II (AM8546G, Thermo Fisher) was added and mixed well. The E-Gel was then removed. TM EX agarose gel (2%, G401002, Thermo Fisher), remove the comb, place the gel into the gel cartridge of the electrophoresis system, and add 20 μl of well-mixed sample to each well. Set the electrophoresis time to 10-20 min and begin electrophoresis. After electrophoresis, turn on the filter and cool for 5-10 min. Use E-Gel. TM The EX device (G8300, ThermoFisher) has a built-in image acquisition system for observing electrophoresis results and acquiring images.

[0207] E-Gel of RNA prepared from control vector and spacerless sequence vector of this application TM The E-gel electrophoresis gel is shown in Figure 2(A), where lanes 1 to 3 for each vector are 1.3, 1.4, and 1.5, respectively, for the RNA obtained. Typically, in E-Gel... TM In the EX gel electrophoresis image, circular RNA migrated the slowest, followed by precursor linear RNA, and then nicked circular RNA migrated the fastest. It can be seen that the circular RNA prepared using the spacerless sequence strategy of this application produces a purer band (rightmost lane) after digestion with RNase R; that is, there is no obvious residual precursor linear RNA band, nor interference from other bands (including nicked circular RNA), which is beneficial for subsequent production.

[0208] 1.7 Capillary gel electrophoresis

[0209] To detect the efficiency of RNA circularization, capillary gel electrophoresis was performed on RNA before and after circularization using an Agilent 5200 fragment analyzer (M53100AA, Agilent) and an Agilent DNF-471 RNA kit (15nt, DNF-471-0500, Agilent).

[0210] Specifically, take the purified RNA obtained in steps 1.3 and 1.4 and adjust its concentration to 100-150 ng / μl with enzyme-free water. Add 22 μL of dilution standard (RNA dilution standard), 2 μL of the test sample (concentration between 100 ng / μl and 150 ng / μl), and 2 μL of RNA molecular weight standard to the sample plate. After mixing, incubate at 80°C for 2 min, then immediately place on an ice box at 4°C for instantaneous cooling. Capillary electrophoresis is then performed; refer to the Agilent 5200 fragment analyzer manual for specific operating procedures.

[0211] The results are shown in Figure 2 (CF). As can be seen from the band sizes, the band of the in vitro transcription product before circularization is slightly larger than the band of the circular RNA after circularization. This is because the linear precursor RNA contains self-splicing intron sequences, which are cleaved out during circularization, becoming free intron fragments. The purity of the circular RNA prepared from the control vector was 79.1% (C), while the purity of the circular RNA obtained from the vector using the Coxiella behnea 23S rRNA gene introns in this application was 80.6% (D), the purity of the circular RNA obtained using the Bacillus anthracis recA gene introns was 81.2% (E), and the purity of the circular RNA obtained using the Thermophyton floccosum 23S rRNA gene introns was 48.7% (F). It can be seen that when using the Coxiella 23S ribosomal RNA gene intron and the Bacillus anthracis recA gene intron, the purity of the transcribed circular RNA is still higher than that of the control vector transcribed using the Anabaena intron and containing the spacer sequence, even without the spacer sequence in the vector.

[0212] 1.8 Sequencing of circularized RNA

[0213] The circular RNA product obtained in step 1.4 was reverse transcribed, PCR was performed, and gel was recovered. The positions of the circular adapters were then sequenced.

[0214] use II. First-strand cDNA Synthesis Kit (+gDNA Removal Agent) (R212-01, Novizan) was used to reverse transcribe the purified circular RNA product obtained in step 1.4. Specifically, the reaction system was prepared as shown in Table 2, and the mixture was pipetted and heated at 42°C for 2 min.

[0215] Table 2. Reverse transcription reaction system

[0216] Then, add 4 μl of 5×HiScript II qRT SuperMix II to the above mixture, pipette and mix well, incubate at 50°C for 15 min, then at 85°C for 5 sec.

[0217] After reverse transcription, the obtained cDNA was added to the PCR reaction system shown in Table 3, mixed by pipetting, and the PCR reaction was carried out according to the procedure shown in Table 4.

[0218] Table 3. PCR reaction system

[0219] Table 4. PCR Program Settings

[0220] All reaction products were recovered by electrophoresis, and the target band was cut using a blue light gel cutter (OSE-470L, Tiangen Biotech Co., Ltd.) as a reference. Gel recovery was performed using the gel recovery kit (D2500-01 / D2500-02, omega) according to the instructions. The recovered product was sent for Sanger sequencing, and the sequences of the circular adapter sequencing primers are shown in SEQ ID NO:21 and 22.

[0221] The sequencing results of the circular linker positions of RNA transcribed from vectors containing introns of the Coxiella behnea 23S ribosomal RNA gene, the Bacillus anthracis recA gene, or the Thermophyton floccosum 23S rRNA gene are shown in SEQ ID NO:29, 30, and 31, respectively. The sequences encoding Gaussian luciferase (GLUC), E1, E2, and IRES are indicated by bold, underline, bold italic, and underline, respectively. It can be seen that all introns have been removed, and E1 and E2 are linked together.

[0222] The structures of the circular RNAs prepared from the control vector and the vector of this application are shown in Figure 1 (D and F), respectively.

[0223] 1.9 Cell Expression Assay

[0224] RNA transcribed from vectors with high RNA circularization efficiency, namely, vectors using introns of the Coxiella brevis 23S ribosomal RNA gene and the recA gene of Bacillus anthracis without spacers, was purified by 1.4 circularization + LiCl and digested by 1.5 RNase R, and then transfected into 293T cells (purchased from the American Center for Type Culture Collection) for eukaryotic expression detection.

[0225] Specifically, HEK293T cells were seeded into 24-well or 6-well plates using Dulbecco modified Eagle medium (DMEM, BI) supplemented with 10% fetal bovine serum (BI) and penicillin / streptomycin antibiotics (100 U / ml penicillin, 100 μg / ml streptomycin; Gibco). The density was 1 × 10⁶ cells per well in the 24-well plate. 5 10 cells, 4 × 10⁴ cells per well in a 6-well plate 5 Cells were cultured in 2000 μl culture medium at 37°C, 5% CO2, and 90% relative humidity. On the second day, the above-mentioned circular RNA treated with RNase R was added to 24-well or 6-well plates, 500 ng per well in 24-well plates and 2 μg per well in 6-well plates. The cells were then transfected with lipofectamine MessengerMax (Invitrogen, LRNA003). At this point, the cell culture must have >90% viability and reach 70% confluence.

[0226] Collect 10 μl of cell culture supernatant at 24, 48 and 72 hours after transfection, add it to a 96-well white or black plate, add 50 μl of Gaussian luciferase detection working solution (Beyotime_RG021S) to each well, mix well, and incubate at room temperature (about 25°C) for 5-10 minutes to allow the luminescence signal to stabilize. Perform chemiluminescence detection using a multi-functional microplate reader.

[0227] The results are shown in Figure 2(B). It can be seen that the circular RNA prepared by the spacerless sequence vector using the anthrax bacillus recA gene intron and the Coxiella behnea 23S ribosomal RNA gene intron in this application has a better expression effect in cells, namely the expression level of Gaussian luciferase (GLUC), than the circular RNA produced by the control vector.

[0228] Example 2. RNA circularization efficiency containing different target expression genes under a spacerless sequence strategy

[0229] To verify whether the removal of the spacer sequence has different effects on the circularization efficiency of RNA containing different target expression sequences, the target expression sequence in the vector was replaced with a sequence encoding Gaussian luciferase (SEQ ID NO:7).

[0230] Referring to sections 1.2 to 1.9, the obtained linearized plasmids were subjected to in vitro transcription and circularization treatment, and the resulting RNA was subjected to E-Gel processing. TM EX gel electrophoresis, capillary electrophoresis, sequencing, and intracellular expression were performed. In the sequencing step in 1.8, SEQ ID NO:21 and 23 were used as primer pairs for PCR and sequencing. In the intracellular expression assay in 1.9, the circular RNA treated with RNase R obtained in 1.5 was transfected into cells. Cells were photographed under an inverted fluorescence microscope at 24, 48, and 72 hours post-transfection, and fluorescence values ​​were then read using ImageJ.

[0231] E-Gel TM The results of EX gel electrophoresis are shown in Figure 3(A), where lanes 1 to 3 of each vector contain RNA obtained in lanes 1.3, 1.4, and 1.5, respectively. The circular RNA prepared from the spacerless vector of this application showed a purer band (rightmost lane) after RNase R digestion, with no obvious precursor linear RNA band residue and no interference from other bands, which is beneficial for subsequent production and transfection.

[0232] The results of capillary electrophoresis are shown in Figure 3 (CF). The purity of the circular RNA prepared from the control vector was 78.4% (C), while the purity of the circular RNA prepared from the vectors using the *Coxiella behnea* 23S ribosomal RNA gene introns, *Bacillus anthracis* recA gene introns, and *Thermophyton naphthaleneophilus* 23S rRNA gene introns in this application was 86.3% (D), 81.2% (E), and 48.1% (F), respectively. It can be seen that regardless of the target gene being expressed, when using the *Coxiella behnea* 23S ribosomal RNA gene introns and *Bacillus anthracis* recA gene introns, the purity of the transcribed circular RNA was higher than that of the circular RNA transcribed from the control vector using the *Anabaena* intron when the vector did not contain a spacer sequence.

[0233] RNA transcribed and circularized from vectors containing introns of the *Coxiella belladonna* 23S ribosomal RNA gene, *Bacillus anthracis* recA gene, or *Thermophyton naphthaleneophilus* 23S rRNA gene was sequenced, and the sequencing results of the circular linker positions are shown in SEQ ID NO: 32, 33, and 34, respectively. The sequences encoding green fluorescent protein (EGFP), E1, E2, and IRES are indicated by bold, underline, bold italic, and underline, respectively. It can be seen that all introns have been removed, and E1 and E2 are linked together.

[0234] The results of cell transfection expression are shown in Figure 3(B). The circular RNA prepared by the spacerless sequence vector containing the intron of the Coxiella 23S ribosomal RNA gene in this application showed the best EGFP expression in cells. The circular RNA produced by the vector containing the intron of the Bacillus anthracis recA gene and the control vector showed basically similar EGFP expression in cells.

[0235] In summary, the circular RNA prepared by the spacerless vector of this application has a significantly reduced length of the unwanted sequence (i.e., spacer sequence) compared to the circular RNA produced by the control vector, and its purity after circularization and purification is higher, resulting in slightly better protein expression in cells.

[0236] Example 3. Truncation of exon sequences flanking introns of the Coxiellar 23S ribosomal RNA gene

[0237] PIE-based circular RNA synthesis inevitably results in the presence of partially exon sequences (E1 and E2) flanking self-splicing introns in the newly synthesized circular RNA. This leads to increased immunogenicity of the circular RNA, limiting its application.

[0238] In this embodiment, the E1 and E2 vertices on both sides of the self-splicing intron are truncated to see if they affect the RNA circularization process or circularization efficiency.

[0239] Specifically, the exons E1 and E2 flanking the intron of the Coxsell 23S ribosomal RNA gene were progressively truncated, and a vector was constructed, the specific structure of which is shown in Figure 1(C), and is basically the same as the vector structure in Example 2. The sequences of the 3' introns & exons E2 with lengths of 20bp, 10bp, 6bp, and 3bp are shown in SEQ ID NO:15, 26, 27, and 28, respectively, and the sequences of the 5' introns & exons E1 with lengths of 20bp, 10bp, and 9bp are shown in SEQ ID NO:16, 24, and 25, where the sequences of E1 and E2 are shown in bold underline. Gene synthesis, vector construction, and plasmid linearization were all performed using GenScript.

[0240] The constructed E1 and E2 vectors were combined into lengths of 20+10, 10+10, 10+6, and 9+3. After linearization, in vitro transcription, circularization, E-Gel electrophoresis, capillary gel electrophoresis, and Sanger sequencing were performed according to a process of 1.2-1.8.

[0241] The E-Gel results of the circularized RNA generated by the E1 / E2 truncated vectors are shown in Figure 4(A), where lanes 1 to 3 of each vector yielded RNAs at positions 1.3, 1.4, and 1.5, respectively. It can be seen that despite the different degrees of truncation in E1 and / or E2, the RNAs prepared by these vectors all exhibited circularization, and their circularization abilities were similar.

[0242] The capillary electrophoresis results of circular RNA generated by the E1 / E2 truncated vectors are shown in Figure 4 (BE). The purities of circular RNA generated by vectors with E1 and E2 length combinations of 20+10 (B), 10+10 (C), 10+6 (D), and 9+3 (E) were 80.3%, 71.4%, 74.5%, and 70.2%, respectively, all above 70%. In particular, when E1 was truncated to 9 bp and E2 to 3 bp, the purity of the circular RNA was 70.2%, which was not much different from that before truncation (Figure 3 (D)), and was not much different from the purity of circular RNA prepared by vectors containing 5' spacer sequences, 3' spacer sequences, and introns derived from the Anabaena pre-tRNA-Leu gene (Figure 3 (C)).

[0243] Sequencing results of the adapter sites of circular RNAs generated by vectors with E1 and E2 length combinations of 20+10, 10+10, 10+6, and 9+3 are shown in SEQ ID NO:36-39, where the sequences encoding green fluorescent protein (EGFP), E1, E2, and IRES are indicated by bold, underline, italicized bold, and underlined text, respectively. It can be seen that the RNAs generated by all truncated vector combinations are normally circularized, with the shortest exon residual base reduced to 12.

[0244] The structure of the circular RNA prepared from the E1 / E2 truncated combination vectors of this application is shown in Figure 1(F).

[0245] Example 4. Truncation of exon sequences flanking other introns

[0246] To verify the universality of the E1 & E2 truncation method on both sides of the self-splicing intron, different self-splicing introns were selected and their E1 & E2 truncation was performed to see if it would affect the RNA circularization process or circularization efficiency.

[0247] Specifically, introns of the *Bacillus anthracis* recA gene, *Thermophilus naphthaleneophilus*, and the *Scytonema hofmannii* tRNA-fMet gene were selected, and the E1 and E2 regions on both sides of these introns were truncated, resulting in the same structure as in Example 3. When applying the *Scytonema hofmannii* tRNA-fMet gene introns, the 3' intron & exon E2 (SEQ ID NO:50) with a length of 13 bp and the 5' intron & exon E1 (SEQ ID NO:49) with a length of 13 bp are typically selected. The amino acid sequence of the recA gene of Bacillus anthracis, with E1 shortened to 9 bp and 5' intron & exon E1, is shown in SEQ ID NO:40; the amino acid sequence of E2 shortened to 3 bp and 3' intron & exon E2, is shown in SEQ ID NO:41. The amino acid sequence of the 23S rRNA gene of Thermophyton naphthaleneophilus, with E1 shortened to 10 bp and 5' intron & exon E1, is shown in SEQ ID NO:42; the amino acid sequence of E2 shortened to 5 bp and 3' intron & exon E2, is shown in SEQ ID NO:43. The amino acid sequence of the tRNA-fMet gene of Pseudobranchia hoffmannis, with E1 shortened to 7 bp and 5' intron & exon E1, is shown in SEQ ID NO:44; the amino acid sequence of E2 shortened to 6 bp and 3' intron & exon E2, is shown in SEQ ID NO:45.

[0248] The sequences described above are shown below, with E1 and E2 sequences highlighted in bold underline. Gene synthesis, vector construction, and plasmid linearization were all performed using GenScript.

[0249] Vectors containing E1 & E2 truncated introns flanking the *Bacillus anthracis* recA gene, *Thermophyton naphthaleneophilus* 23S rRNA gene, and *Pseudoclade chrysophanum* tRNA-fMet gene were linearized and then subjected to in vitro transcription, circularization, E-Gel electrophoresis, capillary gel electrophoresis, and Sanger sequencing according to steps 1.2–1.8. Step 1.5 was omitted, and gel electrophoresis was performed only on the purified RNA obtained in steps 1.3 and 1.4. The E-Gel results of the circular RNA generated by the E1 / E2 truncated vectors are shown in Figure 5(A), where lanes 1 and 2 of each vector contain RNA obtained in steps 1.3 and 1.4, respectively. It can be seen that despite varying degrees of truncation of E1 and / or E2, the RNA prepared by the vectors can all undergo circularization. The capillary electrophoresis results of the circular RNA generated by the E1 / E2 truncated vectors are shown in Figure 5(BD), with purities of 62.2%, 40.9%, and 69.8%, respectively.

[0250] Sequencing results of the adapter sites of circular RNAs generated from vectors flanking the E1 & E2 truncated introns of the *Bacillus anthracis* recA gene, *Thermophyton naphthaleneophilus* 23S rRNA gene, and *Pseudoclade Hoffmann* tRNA-fMet gene are shown in SEQ ID NO:46-48, where the sequences encoding green fluorescent protein (EGFP), E1, E2, and IRES are indicated by bold, underline, italicized bold, and underlined text, respectively. It can be seen that the RNAs generated from vectors with all E1 & E2 truncated introns are normally circularized.

[0251] Table 5. Some sequences involved in this application

[0252] Although this application has been described in conjunction with one or more embodiments, it should be understood that this application is not limited to these embodiments. The description in this application is intended to cover all variations and equivalents, all of which are included within the spirit and scope of the appended claims. All references cited herein are incorporated herein by reference in their entirety.

[0253] References

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[0258] 5.Kuhn,A.N.et al.Phosphorothioate cap analogs increase stability and translational efficiency of RNA vaccines in immature dendritic cells and induce superior immune responses in vivo.Gene Ther.17,961–971(2010).

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[0261] 8.Jeck,W.R.&Sharpless,N.E.Detecting and characterizing circular RNAs.Nat.Biotechnol.32,453–461(2014).

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Claims

1. A single-stranded DNA molecule comprising, from its 5' end to its 3' end, the following elements in sequence: a 3' self-splicing intron fragment containing a 3' splice site, a sequence encoding an internal ribosome entry site (IRES), a target sequence, and a 5' self-splicing intron fragment containing a 5' splice site, wherein the elements are operatively linked, wherein the 3' self-splicing intron fragment containing the 3' splice site and the 5' self-splicing intron fragment containing the 5' splice site are derived from introns of the *Bacillus anthracis* recA gene, *Coxiella belladonna* 23S ribosome gene, *Pseudobranchia hoffmanni* tRNA-fMet gene, or *Thermophyton naphthaleneophila* 23S rRNA gene, wherein the single-stranded DNA molecule does not contain a spacer sequence.

2. The single-stranded DNA molecule of claim 1, wherein the introns of the Coxiella 23S ribosomal gene comprise a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:

12. The introns of the *Pseudocladocerae* tRNA-fMet gene contain nucleotide sequences with at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:

35. The introns of the *Bacillus anthracis* recA gene contain a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:13, or The introns of the 23S rRNA gene of *Thermophyton flavus* contain nucleotide sequences that have at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:

14.

3. The single-stranded DNA molecule of claim 1, wherein the 3' self-splicing intron fragment containing the 3' splice site includes a naturally adjacent exon or a sequence corresponding to the naturally adjacent exon at its 3' end, wherein the length of the naturally adjacent exon or the sequence corresponding to the naturally adjacent exon is 3-40 nucleotides, 3-20 nucleotides, 3-10 nucleotides, 3-8 nucleotides, or 3-6 nucleotides, and / or A 5' self-splicing intron fragment containing a 5' splice site contains a naturally adjacent exon or a sequence corresponding to the naturally adjacent exon at its 5' end, wherein the length of the naturally adjacent exon or the sequence corresponding to the naturally adjacent exon is 5-40 nucleotides, 5-20 nucleotides, 5-10 nucleotides, 5-9 nucleotides, 5-8 nucleotides, or 5-6 nucleotides.

4. The single-stranded DNA molecule of claim 3, wherein the 3' self-splicing intron fragment containing a 3' cleavage site and the 5' self-splicing intron fragment containing a 5' cleavage site respectively comprise nucleotide sequences having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with i) SEQ ID NO:26 and 16, ii) SEQ ID NO:28 and 25, iii) SEQ ID NO:27 and 24, iv) SEQ ID NO:26 and 24, v) SEQ ID NO:44 and 45, vi) SEQ ID NO:40 and 41, vii) SEQ ID NO:15 and 16, viiii) SEQ ID NO:17 and 18, or ix) SEQ ID NO:49 and 50.

5. The single-stranded DNA molecule of claim 1, wherein the target sequence comprises an open reading frame, a single cloning site, or a multiple cloning site encoding a target peptide or protein.

6. The single-stranded DNA molecule as described in claim 1, wherein IRES is Coxsackievirus B3 (CVB3), Coxsackievirus A (CVB1 / 2), Taura syndrome virus, blood-sucking assassin bug virus, Tyrell's encephalomyelitis virus, simian virus 40, red imported fire ant virus 1, cereal constrictor aphid virus, reticuloendotheliosis virus, Forman poliovirus 1, soybean inchworm virus, Kashmir wasp virus, human rhinovirus 2, glass leafhopper virus-1, human immunodeficiency virus. Foot-and-mouth disease virus (FMDV) type 1, leafhopper virus-1, louse virus (P), hepatitis C virus, hepatitis A virus, GB virus, foot-and-mouth disease virus, human enterovirus 71, equine rhinovirus, tea geometrid moth-like virus, encephalomyelitis virus (EMCV), fruit fly virus (C), cruciferous tobacco virus, cricket paralysis virus, bovine viral diarrhea virus 1, black queen cell virus, aphid lethal paralysis virus, avian encephalomyelitis virus, acute bee paralysis virus, hibiscus yellow spot virus, classical swine fever virus, human... FGF2, human SFTPA1, human AML1 / RUNX1, Drosophila antennae, human AQP4, human AT1R, human BAG-1, human BCL2, human BiP, human c-IAPl, human c-myc, human eIF4G, mouse NDST4L, human LEF1, mouse HIF1α, human n.myc, mouse Gtx, human p27kipl, human PDGF2 / c-sis, human p53, human Pim-1, mouse Rbm3, Drosophila reaper, canine scamper, Drosophila Ubx, salivary viruses, Coxsackieviruses, bi-echoviruses, human UNR, mouse UtrA, human VEGF-A, human XIAP, Drosophila hairless, Saccharomyces cerevisiae TFIID, Saccharomyces cerevisiae YAP1, human c-src, human FGF-1, simian microRNA virus, turnip shrunkenness virus, or IRES of eIF4G aptamers.

7. The single-stranded DNA molecule of claim 1, further comprising a sequence encoding a 5' homologous arm at the 5' end of the 3' self-splicing intron fragment, and a sequence encoding a 3' homologous arm at the 3' end of the 5' self-splicing intron fragment, wherein the 5' homologous arm and the 3' homologous arm are complementary.

8. The single-stranded DNA molecule of claim 1 further comprises an RNA polymerase promoter at the 5' end; or further comprises a restriction endonuclease site at the 3' end.

9. The single-stranded DNA molecule of claim 1, comprising, from the 5' end to the 3' end, the following sequence: an RNA polymerase promoter, a sequence encoding a 5' homologous arm, a 3' self-splicing intron fragment containing a 3' cleavage site, a sequence encoding an internal ribosome entry site (IRES), a target sequence, a 5' self-splicing intron fragment containing a 5' cleavage site, and a sequence encoding a 3' homologous arm.

10. A single-stranded DNA molecule comprising, from its 5' end to its 3' end: a 3' self-splicing intron fragment containing a 3' cleavage site, a target sequence, and a 5' self-splicing intron fragment containing a 5' cleavage site, wherein the elements are operatively linked, wherein the 3' self-splicing intron fragment and the 5' self-splicing intron fragment are derived from the same self-splicing intron. The 3' self-splicing intron fragment contains a naturally adjacent exon or a sequence corresponding to the naturally adjacent exon at its 3' end. The length of the naturally adjacent exon or the sequence corresponding to the naturally adjacent exon is 3-20 nucleotides, 3-10 nucleotides, 3-8 nucleotides, or 3-6 nucleotides. The 5' self-splicing intron fragment contains a naturally adjacent exon or a sequence corresponding to the naturally adjacent exon at its 5' end, wherein the length of the naturally adjacent exon or the sequence corresponding to the naturally adjacent exon is 5-20 nucleotides, 5-10 nucleotides, 5-8 nucleotides, or 5-6 nucleotides.

11. The single-stranded DNA molecule of claim 10, wherein the self-cleaving intron is an intron derived from the 23S ribosomal gene of Coxiella behnea, the tRNA-fMet gene of Pseudobranchia hoffmannii, the recA gene of Bacillus anthracis, or the 23S rRNA gene of Thermophyton naphthaleneophilus.

12. The single-stranded DNA molecule of claim 11, wherein the introns of the Coxiella behnea 23S ribosomal gene comprise a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:

12. The introns of the *Pseudocladocerae* tRNA-fMet gene contain nucleotide sequences that have at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:

35. The introns of the *Bacillus anthracis* recA gene contain nucleotide sequences that have at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:

13. The introns of the 23S rRNA gene of *Thermophyton flavus* contain nucleotide sequences that have at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:

14.

13. The single-stranded DNA molecule of claim 12, wherein the 3' self-splicing intron fragment comprising a 3' cleavage site comprises a nucleotide sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 26, 27, 28, or 15, and the 5' self-splicing intron fragment comprising a 5' cleavage site comprises a nucleotide sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 24, 25, or 16. The 3' self-splicing intron fragment containing a 3' cleavage site comprises a nucleotide sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:45 or 50, and the 5' self-splicing intron fragment containing a 5' cleavage site comprises a nucleotide sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:44 or 49. The 3' self-splicing intron fragment containing a 3' cleavage site comprises a nucleotide sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:41 or 17, and the 5' self-splicing intron fragment containing a 5' cleavage site comprises a nucleotide sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:40 or 18. The 3' self-splicing intron fragment containing a 3' cleavage site comprises a nucleotide sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:43, and the 5' self-splicing intron fragment containing a 5' cleavage site comprises a nucleotide sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:

42.

14. The single-stranded DNA molecule of claim 10, wherein the self-splicing introns are derived from the Coxiellar 23S ribosomal gene, and the lengths of the naturally adjacent exons or sequences corresponding to the naturally adjacent exons in the 3' self-splicing intron fragments and the lengths of the naturally adjacent exons or sequences corresponding to the naturally adjacent exons in the 5' self-splicing intron fragments are i) 3 nucleotides and 9 nucleotides, ii) 6 nucleotides and 10 nucleotides, iii) 10 nucleotides and 10 nucleotides, iv) 10 nucleotides and 20 nucleotides, or v) 20 nucleotides and 20 nucleotides, respectively.

15. The single-stranded DNA molecule of claim 14, wherein the 3' self-splicing intron fragment containing a 3' cleavage site and the 5' self-splicing intron fragment containing a 5' cleavage site respectively comprise nucleotide sequences having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with i) SEQ ID NO:28 and 25, ii) SEQ ID NO:27 and 25, iii) SEQ ID NO:27 and 24, iv) SEQ ID NO:26 and 24, v) SEQ ID NO:26 and 16, or vi) SEQ ID NO:15 and 16.

16. The single-stranded DNA molecule of claim 10, wherein the target sequence comprises an open reading frame, a single cloning site, or a multiple cloning site encoding a target peptide or protein.

17. The single-stranded DNA molecule of claim 10, further comprising a sequence encoding an internal ribosome entry site (IRES) between the 3' self-splicing intron fragment at the 3' splicing site and the target sequence.

18. The single-stranded DNA molecule of claim 10, further comprising a sequence encoding a 5' homologous arm at the 5' end of the 3' self-splicing intron fragment, and a sequence encoding a 3' homologous arm at the 3' end of the 5' self-splicing intron fragment, wherein the 5' homologous arm and the 3' homologous arm are complementary.

19. The single-stranded DNA molecule of claim 10 or 17, further comprising an RNA polymerase promoter at the 5' end; It also contains a restriction endonuclease site at the 3' end; or It also includes a 5' spacer sequence between the 3' self-splicing intron and the sequence encoding the internal ribosome entry site or the target sequence, and / or includes a 3' spacer sequence between the target sequence and the 5' self-splicing intron.

20. The single-stranded DNA molecule of claim 10, wherein it does not contain a 5' spacer sequence between the 3' self-splicing intron fragment and the sequence encoding the internal ribosome entry site or the target sequence, or does not contain a 3' spacer sequence between the target sequence and the 5' self-splicing intron fragment.

21. The single-stranded DNA molecule of claim 10, comprising, from the 5' end to the 3' end, the following sequence: an RNA polymerase promoter, a sequence encoding a 5' homologous arm, a 3' self-splicing intron fragment containing a 3' cleavage site, a sequence encoding an internal ribosome entry site, a target sequence, a 5' self-splicing intron fragment containing a 5' cleavage site, and a sequence encoding a 3' homologous arm.

22. A double-stranded DNA molecule comprising i) a single-stranded DNA molecule according to any one of claims 1-21, and ii) a second strand complementary to the single-stranded DNA molecule.

23. A vector for preparing circular RNA, comprising a single-stranded DNA molecule according to any one of claims 1-21 or a double-stranded DNA molecule according to claim 22.

24. A method for preparing circular RNA, comprising i) transcribing precursor RNA in vitro from a single-stranded DNA molecule of any one of claims 1-21, a double-stranded DNA molecule of claim 22, or a vector of claim 23 under suitable conditions, and ii) incubating the precursor RNA under suitable conditions, wherein the suitable conditions for step ii) include the presence of magnesium ions and guanosine triphosphate (GTP).

25. A circularizable RNA molecule comprising, from its 5' end to its 3' end: a 3' self-splicing intron fragment containing a 3' cleavage site, an internal ribosome entry site, a target sequence, and a 5' self-splicing intron fragment containing a 5' cleavage site, wherein the elements are operatively linked, wherein the 3' self-splicing intron fragment containing the 3' cleavage site and the 5' self-splicing intron fragment containing the 5' cleavage site are derived from introns of the *Coxiella belladonna* 23S ribosome gene, the *Pseudoclade Hoffmann* tRNA-fMet gene, the *Bacillus anthracis* recA gene, or the *Thermophyton naphthalene* 23S rRNA gene, wherein the circularizable RNA molecule does not contain a spacer sequence.

26. The circularizable RNA molecule of claim 25, wherein the 3' self-splicing intron fragment containing a 3' cleavage site and the 5' self-splicing intron fragment containing a 5' cleavage site respectively comprise nucleotide sequences having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with i) SEQ ID NO:26 and 16, ii) SEQ ID NO:28 and 25, iii) SEQ ID NO:27 and 24, iv) SEQ ID NO:26 and 24, v) SEQ ID NO:44 and 45, vi) SEQ ID NO:40 and 41, vii) SEQ ID NO:15 and 16, viiii) SEQ ID NO:17 and 18, or ix) SEQ ID NO:49 and 50.

27. A circularizable RNA molecule comprising, from its 5' end to its 3' end: a 3' self-splicing intron fragment containing a 3' splice site, an internal ribosome entry site, a target sequence, and a 5' self-splicing intron fragment containing a 5' splice site, wherein the elements are operatively linked, wherein the 3' self-splicing intron fragment and the 5' self-splicing intron fragment are derived from the *Coxiella belladonna* 23S ribosome gene, the *Pseudococcus hoffmannii* tRNA-fMet gene, or the *Bacillus anthracis* recA gene, or the *Thermophyton naphthaleneophilus* 23S rRNA gene. The 3' self-splicing intron fragment contains a naturally adjacent exon or a sequence corresponding to the naturally adjacent exon at its 3' end. The length of the naturally adjacent exon or the sequence corresponding to the naturally adjacent exon is 3-20 nucleotides, 3-10 nucleotides, 3-8 nucleotides, or 3-6 nucleotides. The 5' self-splicing intron fragment contains a naturally adjacent exon or a sequence corresponding to the naturally adjacent exon at its 5' end. The length of the naturally adjacent exon or the sequence corresponding to the naturally adjacent exon is 5-20 nucleotides, 5-10 nucleotides, 5-8 nucleotides, or 5-6 nucleotides.

28. The circularizable RNA molecule of claim 27, wherein the 3' self-splicing intron fragment containing a 3' cleavage site and the 5' self-splicing intron fragment containing a 5' cleavage site respectively comprise nucleotide sequences having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with i) SEQ ID NO:28 and 25, ii) SEQ ID NO:27 and 25, iii) SEQ ID NO:27 and 24, iv) SEQ ID NO:26 and 24, v) SEQ ID NO:26 and 16, vi) SEQ ID NO:15 and 16, vii) SEQ ID NO:44 and 45, viiii) SEQ ID NO:49 and 50, ix) SEQ ID NO:40 and 41, or x) SEQ ID NO:17 and 18.

29. The circularizable RNA molecule of claim 25 or 27, further comprising a 5' homologous arm at the 5' end of the 3' self-splicing intron fragment and a 3' homologous arm at the 3' end of the 5' self-splicing intron fragment.

30. Use of introns derived from the *Coxiella belladonna* 23S ribosomal gene, the *Pseudococcus hoffmannii* tRNA-fMet gene, the *Bacillus anthracis* recA gene, or the *Thermophyton naphthaleneophila* 23S rRNA gene in the preparation of a vector for preparing circular RNA, wherein the vector for preparing circular RNA comprises a 3' self-splicing intron fragment containing a 3' splicing site and a 5' self-splicing intron fragment containing a 5' splicing site from the introns of the *Coxiella belladonna* 23S ribosomal gene, the *Pseudococcus hoffmannii* tRNA-fMet gene, the *Bacillus anthracis* recA gene, or the *Thermophyton naphthaleneophila* 23S rRNA gene. The 3' self-splicing intron fragment containing a 3' cleavage site and the 5' self-splicing intron fragment containing a 5' cleavage site contain nucleotide sequences having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with i) SEQ ID NO:26 and 16, ii) SEQ ID NO:28 and 25, iii) SEQ ID NO:27 and 24, iv) SEQ ID NO:26 and 24, v) SEQ ID NO:44 and 45, vi) SEQ ID NO:40 and 41, vii) SEQ ID NO:15 and 16, viiii) SEQ ID NO:17 and 18, ix) SEQ ID NO:19 and 20, x) SEQ ID NO:49 and 50, or xi) SEQ ID NO:42 and 43.

31. The use as described in claim 30, wherein the vector for preparing circular RNA does not contain a spacer sequence.

32. A host cell comprising a single-stranded DNA molecule according to any one of claims 1-21, a double-stranded DNA molecule according to claim 22, a vector according to claim 23, or a circularizable RNA molecule according to any one of claims 25-29.

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