Circular RNA vector and use thereof

Abstract

Provided are a circular RNA vector and the use thereof. Further provided is an RNA molecule, which comprises, in sequence, a self-splicing intron and a sequence to be circularized, wherein the elements are operably linked in sequence; the self-splicing intron comprises an internal guide sequence and is capable of causing circularization of the RNA molecule and being removed from the RNA molecule during circularization; and the sequence to be circularized comprises, at the 3' end, a nucleotide capable of being reverse complementary to the 3' end sequence of the internal guide sequence, and comprises, at the 5' end, a nucleotide capable of being reverse complementary to the 5' end sequence of the internal guide sequence. Further provided is a method for preparing a circular RNA.

Description

Circular RNA Vectors and Their Applications

[0001] Cross-citation of related applications

[0002] This application claims priority to Chinese Patent Application No. 202510108144.7, filed on January 22, 2025, entitled "Circular RNA Vector and Its Application", the entire contents of which are incorporated herein by reference. Technical Field

[0003] This application relates to an RNA molecule for preparing circular RNA, which may sequentially comprise an optional 5' homologous arm, a self-splicing intron, a sequence to be circularized, an optional 3' homologous arm spacer sequence, and an optional 3' homologous arm, wherein the elements are operatively linked in sequence, wherein the self-splicing intron contains an internal guide sequence and enables the RNA molecule to be circularized and removed from the RNA molecule during the circularization process, wherein the sequence to be circularized contains a nucleotide at its 3' end that is anticomplementary to the 3' end sequence of the internal guide sequence, and at its 5' end that is anticomplementary to the 5' end sequence of the internal guide sequence. This application also relates to a DNA molecule or vector for preparing the above-described RNA molecule, a method for preparing circular RNA using the above-described RNA molecule or DNA molecule or vector, and the use of the circular RNA. Background Technology

[0004] mRNA has been widely used as a protein replacement therapy. From the 5' end to the 3' end, mRNA consists of a cap structure, a 5' UTR, a protein-coding region, a 3' UTR, and a polyadenylated nucleotide sequence. mRNA can be prepared through in vitro transcription and delivered into cells via vector media, where it efficiently produces target proteins, thus enabling gene therapy for some diseases, or it can be translated in vivo to produce antigenic proteins, triggering the body to produce corresponding antibodies. Several mRNA vaccines targeting infectious diseases have already been developed.

[0005] However, linear mRNAs, with their exposed 5' and 3' ends, are vulnerable to nuclease attack and rapid degradation. In contrast, circular RNAs (circRNAs), lacking free 5' and 3' ends, are resistant to some RNase-mediated degradation, have a longer half-life, and, when engineered, can efficiently translate target peptides or proteins within cells. Furthermore, circRNAs induce lower host immune side effects compared to linear mRNA molecules.

[0006] Currently, circRNA is typically transcribed in vitro to produce linear RNA precursors (preRNA), which are then circulated via enzyme ligation or intron self-splicing systems (ribozyme method, also known as PIE). In enzyme ligation, complementary DNA sequences to the RNA ends are added as splice sequences, or the internal sequence of the RNA precursor is used as a splice sequence to form a local double-stranded structure. This allows the ligase to catalyze the formation of phosphodiester bonds between the 5' and 3' ends of the RNA strand, completing RNA circulation. This method requires a high level of skill from the user, the circulation rate depends on the rational design of the molecular structure, and the circulation efficiency is significantly affected by molecular length. The ribozyme method utilizes self-splicing introns and their adjacent exons to perform intron self-splicing, achieving circulation through a two-step transesterification reaction. It does not require an additional enzyme reaction system or auxiliary splice strands and offers a relatively higher circulation rate. During splicing, the internal guide sequence (IGS) of the self-splicing intron needs to recognize and complement each other with ribozyme recognition site 1 in the 5' side exon fragment and ribozyme recognition site 2 in the 3' side exon fragment, so as to promote the separation of the intron from the exon fragment at the 5' splice site and the 3' splice site.

[0007] Patent application CN112399860B discloses a method for preparing circRNA using an intron self-splicing system derived from the pre-tRNA-Leu gene of Anabaena or the Td gene of T4 bacteriophage. In this method, the self-splicing intron is divided into two fragments, with its 3' end fragment and adjacent 3' exon fragments, and its 5' end fragment and adjacent 5' exon fragments, respectively positioned upstream and downstream of the target sequence GOI. After the intron fragments assemble to form a complete ribozyme and complete reverse splicing, the GOI and the flanking exon sequences circularize, and the intron fragments are removed. This method can also place spacer sequences on both sides of the GOI to some extent shield the GOI from interference with the conformation of the self-splicing intron, thereby improving splicing and circularization efficiency, with a circularization rate reaching up to 84-95%. However, the circularization system disclosed in this patent application shows a significant increase in the amount of nicked RNA and linear RNA in the product when circularizing longer GOIs, leading to a significant decrease in the circularization rate. When linear RNA is transcribed in vitro, co-transcriptional folding occurs according to the "transcription-first, fold-first" principle. During RNA elongation, its folding conformation is constantly affected by internal and external factors. When the GOI length increases, especially above 2500 bases, 3' and 5' intron fragments, particularly the 5' intron downstream of the GOI, are likely to be interfered with by long GOI sequences, failing to fold into a catalytically active conformation. This reduces intron splicing activity and leads to a decrease in circularization rate. Correct intron folding presents even greater challenges in the large-scale preparation of circRNA. Therefore, there is an urgent need in this field for a more robust circularization system to address the common shortcomings of existing technologies.

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

[0009] The inventors of this application, by placing self-splicing introns upstream or at the 5' end of the sequence to be circularized, and based on the "first-transcription-first-folding" principle, avoid interference from the target sequence, especially longer ones, on the conformation of self-splicing introns, thus improving circularization efficiency. When a spacer sequence is placed in the 5' region of the target sequence, interference from the target sequence on the intron conformation is better shielded, further improving circularization efficiency. In particular, when spacer sequences are placed in both the 5' and 3' regions of the target sequence, and the two spacer sequences can pair in reverse complementary directions, a stem-loop structure is formed, further shielding the target sequence from interference on the intron conformation. Compared to existing vectors where two intron fragments are distributed on either side of the target sequence, the vector in this application, with introns located upstream of the target sequence, exhibits higher circularization efficiency, and this advantage becomes more pronounced as the length of the target sequence fragment increases.

[0010] Furthermore, to reduce the immunogenicity of circular RNA, the 5' and 3' ends of the sequence to be circularized can be designed to contain ribozyme recognition sites 2 (SS2) and 1 (SS1) complementary to the internal guide sequence (IGS) of the self-splicing intron, respectively. These sites replace the ribozyme recognition sites in the exons flanking the self-splicing intron, thus ensuring that the final circular RNA does not contain undesirable sequences such as exons. This can be achieved by finding sequences identical or similar to SS1 and SS2 in the sequence to be circularized and rearranging the fragments of that sequence. For example, the sequence to be circularized can be split into two parts in the middle of SS1 and SS2 or their approximate sequences, with the 3' end fragment starting with SS2 located at the 5' end and the 5' end fragment ending with SS1 located at the 3' end, thus assembling a new sequence fragment. In this case, after RNA circularization, the original sequence to be circularized will be present.

[0011] Therefore, in a first aspect, this application provides a single-stranded DNA molecule for preparing circular RNA, which may comprise, from the 5' end to the 3' end, a self-splicing intron and a sequence to be circularized, wherein the elements are operatively linked.

[0012] This self-splicing intron enables the circularization of RNA molecules transcribed from the single-stranded DNA molecule or its complementary strand.

[0013] The single-stranded DNA molecule can be linear, and the self-splicing intron enables the RNA molecule transcribed from the complementary strand of the single-stranded DNA molecule to become circular.

[0014] The single-stranded DNA molecule can be circular or closed. Circular or closed single-stranded DNA molecules allow RNA molecules transcribed from the closed single-stranded DNA molecule to become circular.

[0015] During the circularization of RNA molecules transcribed from the complementary strand of the single-stranded DNA molecule or the single-stranded closed DNA molecule, the self-splicing intron is removed from the RNA molecule.

[0016] The sequence to be circularized may contain the target sequence. The target sequence may contain i) an open reading frame encoding the target peptide or protein and translational elements, or ii) a sequence for transcribing non-coding RNA.

[0017] The self-splicing intron may contain an internal guide sequence (IGS). The internal guide sequence (IGS) may be derived from a natural self-splicing intron or be modified.

[0018] The self-splicing intron can be a class I self-splicing intron. In some embodiments, the self-splicing intron can be derived from the *E. coli* phage T4.A2.nrdB gene. In some embodiments, the self-splicing intron is an IGS-modified class I self-splicing intron, such as the IGS-modified *E. coli* phage T4.A2.nrdB gene self-splicing intron.

[0019] The self-splicing intron may include a 5' intron fragment, an optional linker element, and a 3' intron fragment. The 5' and 3' intron fragments may be derived from class I self-splicing introns, such as the self-splicing intron of the *E. coli* phage T4.A2.nrdB gene, and the 5' intron fragment may contain IGS. In some embodiments, the 5' intron fragment is the 5' intron fragment of the IGS-modified *E. coli* phage T4.A2.nrdB gene self-splicing intron.

[0020] In some embodiments, the linker element can form a stem-loop structure. The stem length in the stem-loop structure can be 6-16 bp. The loop length in the stem-loop structure can be 4-10 nt. In some embodiments, the 5' intron fragment can contain a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:13. In some embodiments, the 3' intron fragment can contain a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:12. In some embodiments, the linker element may comprise a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:38. In some embodiments, the self-splicing intron may comprise a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:11.

[0021] The sequence to be circularized may contain a 3' cleavage site at its 5' end. The sequence to be circularized may also contain a nucleotide at its 5' end that is anticomplementary to the 5' end sequence of the inner guide sequence. The number of nucleotides anticomplementary to the 5' end sequence of the inner guide sequence can be any number, for example, 1-4 nucleotides (e.g., 1, 2, 3, 4). This anticomplementation can be substantially complementary, forming, for example, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more complementarity, or complete complementarity.

[0022] The sequence to be circumscribed may contain a 5' cleavage site at its 3' end. The sequence to be circumscribed may contain a nucleotide at its 3' end that is anticomplementary to the 3' end sequence of the inner guide sequence. The number of nucleotides anticomplementary to the 3' end sequence of the inner guide sequence can be any number, for example, 2-4 nucleotides (e.g., 2, 3, 4). The sequence to be circumscribed may contain a T-base nucleotide at its 3' end. This anticomplementation can be substantially complementary, forming, for example, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more complementarity, or complete complementarity.

[0023] In some embodiments, the two nucleotides at the 5' end of the sequence to be circularized are reverse complementary to the 5' end of the internal guide sequence, and the four nucleotides at the 3' end of the sequence to be circularized are reverse complementary to the 3' end of the internal guide sequence.

[0024] In some embodiments, the IGS comprises 6 nucleotides. In some embodiments, the IGS may contain the nucleotide sequence shown in ATGCGC. In some embodiments, the nucleotide in the sequence to be circularized that is reverse complementary to the 3' end sequence of the IGS may contain the nucleotide sequence shown in GCGT. In some embodiments, the nucleotide in the sequence to be circularized that is reverse complementary to the 3' end sequence of the IGS is GCGT. In some embodiments, the nucleotide in the sequence to be circularized that is reverse complementary to the 5' end sequence of the IGS may contain the nucleotide sequence shown in GT. In some embodiments, the nucleotide in the sequence to be circularized that is reverse complementary to the 5' end sequence of the IGS is GT.

[0025] The single-stranded DNA molecule of this application may also include a 5' homologous arm sequence upstream or 5' to the self-splicing intron, and a 3' homologous arm sequence downstream or 3' to the sequence to be circularized. The 5' and 3' homologous arm sequences may be reverse complementary, for example, forming at least about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% base pairing. The length of each of the 5' and 3' homologous arm sequences may be 10-100 nt. In some embodiments, the 5' homologous arm sequence and the 3' homologous arm sequence 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:3 and 4, SEQ ID NO:5 and 6, or SEQ ID NO:7 and 8.

[0026] In some embodiments, the DNA molecule of this application may also include a 3' homologous arm spacer sequence between the sequence to be circularized and the 3' homologous arm. In some embodiments, the 5' end mononucleotide of the 3' homologous arm spacer sequence is complementary to the mononucleotide immediately adjacent to the 5' end of the 3' end sequence of the internal guide sequence in the self-splicing intron. The length of the 3' homologous arm spacer sequence may be at least 5 nt, for example 5-20 nt, or 5-10 nt. In some embodiments, the 3' homologous arm spacer sequence may be a flexible spacer sequence, for example, a poly(AC) or polyA sequence. In some embodiments, the folding energy of the 3' homologous arm spacer sequence is 0 kcal / mol. In some embodiments, the 3' homologous arm spacer sequence is, in principle, not complementary to other sequences in the preRNA after transcription, or the complementary nucleotides are controlled to be below 50%, for example 40%, 30%, 20%, 10%, or 0%, to avoid hindering intron self-splicing. In some embodiments, the 3' homologous arm spacer sequence may comprise 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 or SEQ ID NO:43. In some embodiments, the 3' homologous arm spacer sequence is adjacent to the 3' end of the 5' splice site (SS1).

[0027] The sequence to be circularized may contain the target sequence or be composed of the target sequence.

[0028] In some implementations, the target sequence may contain a 3' cleavage site at the 5' end, i.e., a nucleotide that is reverse complementary to the 5' end sequence of the internal guide sequence, and a 5' cleavage site at the 3' end, i.e., a nucleotide that is reverse complementary to the 3' end sequence of the internal guide sequence.

[0029] The target sequence may include an open reading frame encoding a target peptide or protein and a translational functional element. In some embodiments, the target sequence may include, or consist of, the translational functional element and the open reading frame encoding the target peptide or protein from the 5' end to the 3' end. In some embodiments, the target sequence is rearranged, for example, the target sequence may include, or be composed of, the open reading frame encoding the target peptide or protein and the translational functional element from the 5' end to the 3' end; or the target sequence may include, or be composed of, the 3' end sequence of the translational functional element, the open reading frame encoding the target peptide or protein, and the 5' end sequence of the translational functional element, wherein the 5' end sequence and the 3' end sequence of the translational functional element form the translational functional element when arranged in this order; or the target sequence may include, or be composed of, the 3' end sequence of the open reading frame encoding the target peptide or protein, the translational functional element, and the 5' end sequence of the open reading frame encoding the target peptide or protein, wherein the 5' end sequence and the 3' end sequence of the open reading frame encoding the target peptide or protein form the open reading frame encoding the target peptide or protein when arranged in this order.

[0030] The target sequence may contain a sequence for transcribing non-coding RNA. In some embodiments, the target sequence is rearranged; for example, the target sequence may contain a sequence for transcribing the non-coding RNA. The target sequence may contain, or be composed of, the 3' end sequence of the sequence for transcribing the non-coding RNA and the 5' end sequence of the sequence for transcribing the non-coding RNA, from the 5' end to the 3' end, wherein the 5' end sequence of the sequence for transcribing the non-coding RNA and the 3' end sequence of the sequence for transcribing the non-coding RNA, when arranged in this order, form the sequence for transcribing the non-coding RNA.

[0031] The target sequence may contain a single cloning site or a multiple cloning site, or be composed of both. Any desired sequence, such as an open reading frame encoding a target peptide or protein, and a translational functional element, can be inserted into the single-stranded DNA molecule of this application through a single cloning site or a multiple cloning site.

[0032] The target sequence may contain a single-cloning site or a multiple-cloning site, and a translational element, or be composed of thereof. An open reading frame encoding, for example, a target peptide or protein, can be inserted into a single-stranded DNA molecule via a single-cloning site or a multiple-cloning site. The target sequence may contain, from the 5' end to the 3' end, the 3' end sequence of the translational element, the single-cloning site or the multiple-cloning site, and the 5' end sequence of the translational element, or be composed of thereof, wherein the 5' end sequence and the 3' end sequence of the translational element, when arranged in this order, form the translational element.

[0033] The sequence to be circularized, in addition to the target sequence, may also include i) an exon naturally adjacent to the 3' side of the self-splicing intron or a sequence corresponding to the naturally adjacent exon, ii) an exon naturally adjacent to the 5' side of the self-splicing intron or a sequence corresponding to the naturally adjacent exon, iii) an optional 5' spacer sequence, and iv) an optional 3' spacer sequence. In some embodiments, the sequence to be circularized may include, from the 5' end to the 3' end, an exon naturally adjacent to the 3' side of the self-splicing intron or a sequence corresponding to the naturally adjacent exon, an optional 5' spacer sequence, a target sequence, an optional 3' spacer sequence, and an exon naturally adjacent to the 5' side of the self-splicing intron or a sequence corresponding to the naturally adjacent exon.

[0034] In some embodiments, the exon naturally adjacent to the 3' side of the self-splicing intron, or the sequence corresponding to the naturally adjacent exon, may contain a 3' cleavage site at its 5' end, i.e., a nucleotide that is anticomplementary to the 5' end sequence of the internal guide sequence. The exon naturally adjacent to the 5' side of the self-splicing intron, or the sequence corresponding to the naturally adjacent exon, may contain a 5' cleavage site at its 3' end, i.e., a nucleotide that is anticomplementary to the 3' end sequence of the internal guide sequence. The exon naturally adjacent to the 3' side of the self-splicing intron, or the sequence corresponding to the naturally adjacent exon, and the exon naturally adjacent to the 5' side of the self-splicing intron, or the sequence corresponding to the naturally adjacent exon, may respectively contain nucleotide sequences containing GT and GCGT, the nucleotide sequences shown in SEQ ID NO: 20 and 21, or the nucleotide sequences shown in SEQ ID NO: 18 and 19.

[0035] In some embodiments, a non-specific sequence within the sequence to be circularized is used as a cleavage site, and the IGS of the self-splicing intron is modified to be anticomplementary to the cleavage site. In some embodiments, the sequence to be circularized contains or consists solely of the rearranged target sequence. In some embodiments, the sequence to be circularized contains a 5' cleavage site (SS1) at its 3' end, i.e., a nucleotide that is anticomplementary to the 3' end sequence of the inner guide sequence: 5'-NNNT-3', and a 3' cleavage site (SS2) at its 5' end, i.e., a nucleotide that is anticomplementary to the 5' end sequence of the inner guide sequence: 5'-NN-3', where N is any nucleotide from A, T, C, and G. This can be achieved, for example, by rearranging the target sequence. This rearrangement can refer to dividing the target sequence (e.g., containing the translational element and the open reading frame encoding the target peptide or protein from the 5' end to the 3' end) into fragment 1 and fragment 2 at appropriate positions. Fragment 1 contains a 5' cleavage site (SS1) at its 3' end, and fragment 2 contains a 3' cleavage site (SS2) at its 5' end. The two fragments are then rearranged to form the target sequence consisting of fragment 2 and fragment 1 from the 5' end to the 3' end. This rearrangement can, for example, include dividing the open reading frame encoding the target peptide or protein into 5' and 3' end sequences at appropriate positions and inverting them at both ends of the translational element (e.g., IRES), or dividing the translational element (e.g., IRES) into 5' and 3' end sequences at appropriate positions and inverting them at both ends of the open reading frame encoding the target peptide or protein.

[0036] In some embodiments, the IGS sequence of the self-splicing intron is modified to 5'-NNGNNN-3', where N is any nucleotide from A, T, C, and G, such that the four bases at the 3' end (5'-GNNN-3') are anticomplementary to SS1, and the two bases at the 5' end (5'-NN-3') are anticomplementary or partially complementary to SS2.

[0037] In some embodiments, the 5' intron fragment of the self-splicing intron may comprise a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:44. In some embodiments, the 3' intron fragment of the self-splicing intron may comprise a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:12. In some embodiments, the linker element of the self-splicing intron may comprise a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:38. In some embodiments, the self-splicing intron may comprise a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:39.

[0038] In some embodiments, the IGS may comprise the nucleotide sequence shown in TTGCGT. In some embodiments, the nucleotide in the sequence to be circularized that is anticomplementary to the 3' end sequence of the IGS is ACGT. In some embodiments, the nucleotide in the sequence to be circularized that is anticomplementary to the 5' end sequence of the IGS is AA. In some embodiments, the 5' intron fragment of the self-splicing intron may comprise a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:45. In some embodiments, the self-splicing intron may comprise a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:40.

[0039] The 5' and 3' interval sequences can be any sequences. The length of each 5' and 3' interval sequence can be at least 10 nt, for example, 20-40 nt, or 20-30 nt. The folding energy of the 5' and 3' interval sequences can be 0 kcal / mol. In some embodiments, the 5' and 3' interval sequences can be flexible interval sequences. Flexible interval sequences can be poly(AC) or polyA sequences. Flexible interval sequences can be polyA, polyU, poly(CU), or other random sequences of length 10-50 nt.

[0040] In some embodiments, the 5' spacer sequence and the 3' spacer sequence may be reverse complementary, forming at least about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% base pairing. In some embodiments, the 5' spacer sequence and the 3' spacer sequence may be perfectly complementary sequences of 6-15 nt in length. In some embodiments, the 5' spacer sequence and the 3' spacer sequence may be incompletely complementary sequences of 18-25 nt in length. In some embodiments, the 5' spacer sequence may sequentially include a flexible spacer sequence and a 5' internal homologous arm sequence from the 5' end to the 3' end, and the 3' spacer sequence may sequentially include a 3' internal homologous arm sequence and a flexible spacer sequence from the 5' end to the 3' end. The 5' internal homologous arm sequence and the 3' spacer sequence can be reverse complementary, forming at least about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% base pairing. The lengths of the 5' internal homologous arm sequence and the 3' internal homologous arm sequence can each be 6-25 nt, such as 6-15 nt, 18-25 nt, etc. The 5' spacer sequence or the 3' spacer sequence may be complementary to fewer than five (e.g., four, three, two, one, or zero) adjacent nucleotides. In some embodiments, the sequence to be circularized may contain only the 5' spacer sequence and not the 3' spacer sequence. In some embodiments, the sequence to be circularized may contain only the 3' spacer sequence and not the 5' spacer sequence. In some embodiments, the sequence to be circularized may contain both the 5' spacer sequence and the 3' spacer sequence. In some embodiments, the 5' spacer sequence may contain a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:22. In some embodiments, the 3' spacer sequence may comprise a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:23.

[0041] Translational functional elements can be selected from translation initiation elements and translation enhancement elements. Translation initiation elements can be sequences used to initiate RNA translation, such as the internal ribosome entry site (IRES) or the 5' untranslated region (5'UTR). Translation enhancement elements can be sequences used to enhance RNA translation, such as poly(A), poly(C), poly(AC), poly(T / U), kozak sequences, 3'UTR, etc.

[0042] The target sequence may contain one or more (e.g., 1, 2, or 3) translation enhancement elements located on the 5' side, 3' side, or both sides of the open reading frame encoding the target peptide or protein.

[0043] In some implementations, the target sequence may include the following from the 5' end to the 3' end:

[0044] i) Translation initiation element and open reading frame encoding the target peptide or protein; 3' sequence of translation initiation element, open reading frame encoding the target peptide or protein, and 5' sequence of translation initiation element; 3' sequence of open reading frame encoding the target peptide or protein, translation initiation element, and 5' sequence of open reading frame encoding the target peptide or protein;

[0045] ii) Translation initiation element, open reading frame encoding the target peptide or protein, and (one or more, such as 1 or 2) translation enhancement elements; the 3' end sequence of the translation initiation element, open reading frame encoding the target peptide or protein, (one or more, such as 1 or 2) translation enhancement elements, and the 5' end sequence of the translation initiation element; the 3' end sequence of the open reading frame encoding the target peptide or protein, (one or more, such as 1 or 2) translation enhancement elements, translation initiation element, and the 5' end sequence of the open reading frame encoding the target peptide or protein; the 3' end sequence of the translation enhancement elements, translation initiation element, open reading frame encoding the target peptide or protein, and (one or more, such as 1 or 2) translation enhancement elements;

[0046] iii) Translation initiation element, translation enhancement element, and open reading frame encoding the target peptide or protein; the 3' end sequence of the translation initiation element, the translation enhancement element, the open reading frame encoding the target peptide or protein, and the 5' end sequence of the translation initiation element; the 3' end sequence of the translation enhancement element, the open reading frame encoding the target peptide or protein, the translation initiation element, and the 5' end sequence of the translation enhancement element; the 3' end sequence of the open reading frame encoding the target peptide or protein, the translation initiation element, the translation enhancement element, and the 5' end sequence of the open reading frame encoding the target peptide or protein; or

[0047] iv) Translation initiation element, translation enhancement element, open reading frame encoding the target peptide or protein, and (one or more, such as 1 or 2) translation enhancement elements; the 3' end sequence of the translation initiation element, the translation enhancement element, the open reading frame encoding the target peptide or protein, (one or more, such as 1 or 2) translation enhancement elements, and the 5' end sequence of the translation initiation element; the 3' end sequence of the translation enhancement element, the open reading frame encoding the target peptide or protein, (one or more, such as 1 or 2) translation enhancement elements, the translation initiation element, and the 5' end sequence of the translation enhancement element; the 3' end sequence of the open reading frame encoding the target peptide or protein, (one or more, such as 1 or 2) translation enhancement elements, the translation initiation element, the translation enhancement element, and the 5' end sequence of the open reading frame encoding the target peptide or protein; the 3' end sequence of the translation enhancement element, the translation initiation element, the translation enhancement element, the open reading frame encoding the target peptide or protein, and (one or more, such as 1 or 2) translation enhancement elements.

[0048] In some embodiments, the translation functional element may include a translation initiation element. In some embodiments, the translation functional element may be a translation initiation element. In some embodiments, the translation initiation element may be an IRES.

[0049] The IRES can be selected from Coxsackievirus B3 (CVB3), Coxsackievirus A (CVB1 / 2), Foot-and-mouth disease virus (FMDV), Human enterovirus 71 (IEV71), Human rhinovirus B3 (HRVB3), Encephalomyocarditis virus (EMCV), Simian enterovirus A (SimianEV-A), Echovirus E11 (EchoV-E11), etc. In some embodiments, the IRES can be Coxsackievirus B3 (CVB3). In some embodiments, the IRES can contain a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:27.

[0050] UTRs can be selected from human α-globin 3'UTR, human β-globin 3'UTR, murine α-globin 3'UTR, murine β-globin 3'UTR, IgG-Fc transport receptor protein α fragment, chemokine ligand 22 fragment, N-terminal cleavage enhancer fragment, Coxsackievirus B3 3'UTR, ribosomal protein S27a 3'UTR, Sindbis virus 3'UTR, human serum albumin 3'UTR, etc.

[0051] The length of the sequence to be circulated can be 50-8000 nucleotides. The length of the sequence to be circulated can be more than 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, or 6500 nucleotides.

[0052] The target peptide or protein can be of eukaryotic or prokaryotic origin. It can be human or non-human. In some embodiments, the target peptide or protein can be an antigen protein, antibody, or protease, etc. In some embodiments, the target peptide or protein can be firefly luciferase, long-bellied luciferase, Gaussian luciferase, green fluorescent protein, etc.

[0053] In some implementations, the sequence to be circularized may contain, or consist of, translational functional elements (e.g., IRES) and open reading frames encoding the target peptide or protein from the 5' end to the 3' end.

[0054] In some implementations, the sequence to be circularized may include, or be composed of, the 3' end sequence of the open reading frame encoding the target peptide or protein, a translational functional element (e.g., IRES), and the 5' end sequence of the open reading frame encoding the target peptide or protein from the 5' end to the 3' end.

[0055] In some implementations, the sequence to be circularized may include, or be composed of, the 3' end sequence of a translational functional element (e.g., IRES), an open reading frame encoding the target peptide or protein, and the 5' end sequence of a translational functional element (e.g., IRES) from the 5' end to the 3' end.

[0056] In some embodiments, the sequence to be circularized may include, from the 5' end to the 3' end, an exon naturally adjacent to the 3' side of the self-splicing intron or a sequence corresponding to the naturally adjacent exon, a 5' spacer sequence, a translational element (e.g., IRES), an open reading frame encoding the target peptide or protein, a 3' spacer sequence, and an exon naturally adjacent to the 5' side of the self-splicing intron or a sequence corresponding to the naturally adjacent exon; or may consist of, an exon naturally adjacent to the 3' side of the self-splicing intron or a sequence corresponding to the naturally adjacent exon, a 5' spacer sequence, a translational element (e.g., IRES), an open reading frame encoding the target peptide or protein, and an exon naturally adjacent to the 5' side of the self-splicing intron or a sequence corresponding to the naturally adjacent exon, or the sequence thereof.

[0057] The single-stranded DNA molecule of this application may also include an RNA polymerase promoter at its 5' end, for example, on the 5' side of a 5' homologous arm sequence. 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%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:1. The single-stranded DNA molecule of this application may also include a leader sequence on the 3' side of the RNA polymerase promoter. The leader sequence may contain a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:2.

[0058] The single-stranded DNA molecule of this application may also contain a restriction endonuclease site at its 3' end, for example, on the 3' side of the 3' homologous arm sequence. The restriction endonuclease site may be, for example, NdeI, HindIII, EcoRI, EcoRV, etc.

[0059] In some embodiments, the single-stranded DNA molecule of this application may sequentially comprise, from the 5' end to the 3' end: an RNA polymerase promoter, an optional leader sequence, a 5' homologous arm sequence, a self-splicing intron, a sequence to be circularized, a 3' homologous arm spacer sequence, a 3' homologous arm sequence, and an optional restriction endonuclease site, or be composed thereof, wherein the elements are operatively linked.

[0060] This application also protects the complementary strand of the aforementioned single-stranded DNA molecule.

[0061] This application also provides a double-stranded DNA molecule, which may comprise a first DNA strand and a second DNA strand complementary to the first DNA strand, wherein the first DNA strand may comprise or be composed of the single-stranded DNA molecule of this application. The first DNA strand and the second strand may be completely complementary or substantially complementary, for example, forming about 70%, 75%, 80%, 85%, 85%, 90%, or more than 95% complementarity.

[0062] 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. 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. In some embodiments, the vector may be a plasmid, a fragment obtained by PCR, etc.

[0063] In some embodiments, the single-stranded DNA molecule used to prepare circular RNA may have its self-splicing introns and the sequence to be circularized linked in order from the 3' end to the 5' end, wherein the elements are operatively linked.

[0064] In a second aspect, this application provides an RNA molecule that may sequentially comprise, from the 5' end to the 3' end: a self-splicing intron and a sequence to be circularized, wherein the elements are operatively linked.

[0065] The sequence to be circularized may contain the target sequence. The target sequence may contain i) an open reading frame encoding the target peptide or protein and translational elements, or ii) a non-coding RNA sequence.

[0066] This self-splicing intron enables the RNA molecule to circularize and be removed from the RNA molecule during the circularization process.

[0067] The self-splicing intron may contain an internal guide sequence (IGS). The internal guide sequence (IGS) may be derived from a natural self-splicing intron or be modified.

[0068] The self-splicing intron can be a class I self-splicing intron. In some embodiments, the self-splicing intron can be derived from the *E. coli* phage T4.A2.nrdB gene. In some embodiments, the self-splicing intron is an IGS-modified class I self-splicing intron, such as the IGS-modified *E. coli* phage T4.A2.nrdB gene self-splicing intron.

[0069] The self-splicing intron may include a 5' intron fragment, an optional linker element, and a 3' intron fragment. The 5' and 3' intron fragments may be derived from class I self-splicing introns, such as the self-splicing intron of the *E. coli* phage T4.A2.nrdB gene, and the 5' intron fragment may contain IGS. In some embodiments, the 5' intron fragment is the 5' intron fragment of the IGS-modified *E. coli* phage T4.A2.nrdB gene self-splicing intron.

[0070] In some embodiments, the linker element can form a stem-loop structure. The stem length in the stem-loop structure can be 6-16 bp. The loop length in the stem-loop structure can be 4-10 nt. In some embodiments, the 5' intron fragment can contain a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:13. In some embodiments, the 3' intron fragment can contain a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:12. In some embodiments, the linker element may comprise a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:38. In some embodiments, the self-splicing intron may comprise a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:11.

[0071] The sequence to be circularized may contain a 3' cleavage site at its 5' end. The sequence to be circularized may also contain a nucleotide at its 5' end that is anticomplementary to the 5' end sequence of the inner guide sequence. The number of nucleotides anticomplementary to the 5' end sequence of the inner guide sequence can be any number, for example, 1-4 nucleotides (e.g., 1, 2, 3, 4). This anticomplementation can be substantially complementary, forming, for example, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more complementarity, or complete complementarity.

[0072] The sequence to be circumscribed may contain a 5' cleavage site at its 3' end. The sequence to be circumscribed may contain a nucleotide at its 3' end that is anticomplementary to the 3' end sequence of the inner guide sequence. The number of nucleotides anticomplementary to the 3' end sequence of the inner guide sequence can be any number, for example, 2-4 nucleotides (e.g., 2, 3, 4). The sequence to be circumscribed may contain a U-terminal nucleotide. This anticomplementation can be substantially complementary, forming, for example, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more complementarity, or complete complementarity.

[0073] In some embodiments, the two nucleotides at the 5' end of the sequence to be circularized are anticomplementary to the 5' end sequence of the internal guide sequence, and the four nucleotides at the 3' end of the sequence to be circularized are anticomplementary to the 3' end sequence of the internal guide sequence. In some embodiments, the IGS comprises six nucleotides. In some embodiments, the IGS may comprise the nucleotide sequence shown as AUGCGC. In some embodiments, the nucleotides in the sequence to be circularized that are anticomplementary to the 3' end sequence of the IGS may comprise the nucleotide sequence shown as GCGU. In some embodiments, the nucleotides in the sequence to be circularized that are anticomplementary to the 3' end sequence of the IGS are GCGU. In some embodiments, the nucleotides in the sequence to be circularized that are anticomplementary to the 5' end sequence of the IGS may comprise the nucleotide sequence shown as GU. In some embodiments, the nucleotides in the sequence to be circularized that are anticomplementary to the 5' end sequence of the IGS are GU.

[0074] The RNA molecule of this application may also include a 5' homologous arm upstream or 5' to the self-splicing intron, and a 3' homologous arm downstream or 3' to the sequence to be circularized. The 5' and 3' homologous arms may be anticomplementary, for example, forming at least about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% base pairing. The length of each 5' and 3' homologous arm may be 10-100 nt. 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:3 and 4, SEQ ID NO:5 and 6, or SEQ ID NO:7 and 8.

[0075] In some embodiments, the RNA molecule of this application may also include a 3' homologous arm spacer sequence between the sequence to be circularized and the 3' homologous arm. In some embodiments, the 5' end mononucleotide of the 3' homologous arm spacer sequence is complementary to the mononucleotide immediately adjacent to the 5' end of the 3' end sequence of the internal guide sequence in the self-splicing intron. The length of the 3' homologous arm spacer sequence may be at least 5 nt, for example 5-20 nt, or 5-10 nt. In some embodiments, the 3' homologous arm spacer sequence may be a flexible spacer sequence, for example, a poly(AC) or polyA sequence. In some embodiments, the folding energy of the 3' homologous arm spacer sequence is 0 kcal / mol. In some embodiments, the 3' homologous arm spacer sequence is generally not complementary to other sequences in the preRNA, or the complementary nucleotides are controlled to less than 50%, for example 40%, 30%, 20%, 10%, or 0%, to avoid hindering intron self-splicing. In some embodiments, the 3' homologous arm spacer sequence may comprise 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 or SEQ ID NO:43. In some embodiments, the 3' homologous arm spacer sequence is adjacent to the 3' end of the 5' splice site (SS1).

[0076] The sequence to be circularized may contain the target sequence or be composed of the target sequence.

[0077] In some implementations, the target sequence may contain a 3' cleavage site at the 5' end, i.e., a nucleotide that is reverse complementary to the 5' end sequence of the internal guide sequence, and a 5' cleavage site at the 3' end, i.e., a nucleotide that is reverse complementary to the 3' end sequence of the internal guide sequence.

[0078] The target sequence may include an open reading frame encoding a target peptide or protein and a translational functional element. In some embodiments, the target sequence may include, or consist of, the translational functional element and the open reading frame encoding the target peptide or protein from the 5' end to the 3' end. In some embodiments, the target sequence is rearranged, for example, the target sequence may include, or be composed of, the open reading frame encoding the target peptide or protein and the translational functional element from the 5' end to the 3' end; or the target sequence may include, or be composed of, the 3' end sequence of the translational functional element, the open reading frame encoding the target peptide or protein, and the 5' end sequence of the translational functional element, wherein the 5' end sequence and the 3' end sequence of the translational functional element form the translational functional element when arranged in this order; or the target sequence may include, or be composed of, the 3' end sequence of the open reading frame encoding the target peptide or protein, the translational functional element, and the 5' end sequence of the open reading frame encoding the target peptide or protein, wherein the 5' end sequence and the 3' end sequence of the open reading frame encoding the target peptide or protein form the open reading frame encoding the target peptide or protein when arranged in this order.

[0079] The target sequence may contain a non-coding RNA sequence. In some embodiments, the target sequence is rearranged, for example, the target sequence may include, or be composed of, the 3' end sequence of the non-coding RNA sequence and the 5' end sequence of the non-coding RNA sequence from the 5' end to the 3' end, wherein the 5' end sequence of the non-coding RNA sequence and the 3' end sequence of the non-coding RNA sequence, when arranged in this order, form the non-coding RNA sequence.

[0080] The sequence to be circularized, in addition to the target sequence, may also include i) an exon naturally adjacent to the 3' side of the self-splicing intron or a sequence corresponding to the naturally adjacent exon, ii) an exon naturally adjacent to the 5' side of the self-splicing intron or a sequence corresponding to the naturally adjacent exon, iii) an optional 5' spacer sequence, and iv) an optional 3' spacer sequence. In some embodiments, the sequence to be circularized may include, from the 5' end to the 3' end, an exon naturally adjacent to the 3' side of the self-splicing intron or a sequence corresponding to the naturally adjacent exon, an optional 5' spacer sequence, a target sequence, an optional 3' spacer sequence, and an exon naturally adjacent to the 5' side of the self-splicing intron or a sequence corresponding to the naturally adjacent exon.

[0081] In some embodiments, the exon naturally adjacent to the 3' side of the self-splicing intron, or the sequence corresponding to the naturally adjacent exon, may contain a 3' cleavage site at its 5' end, i.e., a nucleotide that is anticomplementary to the 5' end sequence of the internal guide sequence. The exon naturally adjacent to the 5' side of the self-splicing intron, or the sequence corresponding to the naturally adjacent exon, may contain a 5' cleavage site at its 3' end, i.e., a nucleotide that is anticomplementary to the 3' end sequence of the internal guide sequence. The exon naturally adjacent to the 3' side of the self-splicing intron, or the sequence corresponding to the naturally adjacent exon, and the exon naturally adjacent to the 5' side of the self-splicing intron, or the sequence corresponding to the naturally adjacent exon, may respectively contain nucleotide sequences containing GU and GCGU, the nucleotide sequences shown in SEQ ID NO:20 and 21, or the nucleotide sequences shown in SEQ ID NO:18 and 19.

[0082] In some embodiments, a non-specific sequence within the sequence to be circularized is used as a cleavage site, and the IGS of the self-splicing intron is modified to be anticomplementary to the cleavage site. In some embodiments, the sequence to be circularized contains or consists solely of the rearranged target sequence. In some embodiments, the sequence to be circularized contains a 5' cleavage site (SS1) at its 3' end, i.e., a nucleotide that is anticomplementary to the 3' end sequence of the inner guide sequence: 5'-NNNU-3', and a 3' cleavage site (SS2) at its 5' end, i.e., a nucleotide that is anticomplementary to the 5' end sequence of the inner guide sequence: 5'-NN-3', where N is any nucleotide from A, U, C, and G. This can be achieved, for example, by rearranging the target sequence. This rearrangement can refer to dividing the target sequence (e.g., containing the translational element and the open reading frame encoding the target peptide or protein from the 5' end to the 3' end) into fragment 1 and fragment 2 at appropriate positions. Fragment 1 contains a 5' cleavage site (SS1) at its 3' end, and fragment 2 contains a 3' cleavage site (SS2) at its 5' end. The two fragments are then rearranged to form the target sequence consisting of fragment 2 and fragment 1 from the 5' end to the 3' end. This rearrangement can, for example, include dividing the open reading frame encoding the target peptide or protein into 5' and 3' end sequences at appropriate positions and inverting them at both ends of the translational element (e.g., IRES), or dividing the translational element (e.g., IRES) into 5' and 3' end sequences at appropriate positions and inverting them at both ends of the open reading frame encoding the target peptide or protein.

[0083] In some embodiments, the IGS sequence of the self-splicing intron is modified to 5'-NNGNNN-3', where N is any nucleotide from A, U, C, and G, such that the four bases at the 3' end (5'-GNNN-3') are anticomplementary to SS1, and the two bases at the 5' end (5'-NN-3') are anticomplementary or partially complementary to SS2.

[0084] In some embodiments, the 5' intron fragment of the self-splicing intron may comprise a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:44. In some embodiments, the 3' intron fragment of the self-splicing intron may comprise a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:12. In some embodiments, the linker element of the self-splicing intron may comprise a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:38. In some embodiments, the self-splicing intron may comprise a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:39.

[0085] In some embodiments, the IGS may comprise the nucleotide sequence shown as UUGCGU. In some embodiments, the nucleotide in the sequence to be circularized that is anticomplementary to the 3' end sequence of the IGS is ACGU. In some embodiments, the nucleotide in the sequence to be circularized that is anticomplementary to the 5' end sequence of the IGS is AA. In some embodiments, the 5' intron fragment of the self-splicing intron may comprise a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:45. In some embodiments, the self-splicing intron may comprise a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:40.

[0086] The 5' spacer sequence and the 3' spacer sequence can be any sequence. The length of each of the 5' spacer sequence and the 3' spacer sequence can be at least 10 nt, for example, 20-40 nt, or 20-30 nt. The folding energy of the 5' spacer sequence and the 3' spacer sequence can be 0 kcal / mol. In some embodiments, the 5' spacer sequence and the 3' spacer sequence can be flexible spacer sequences. The flexible spacer sequence can be a poly(AC) or polyA sequence. The flexible spacer sequence can be polyA, polyU, poly(CU), or other random sequences of 10-50 nt in length. In some embodiments, the 5' spacer sequence and the 3' spacer sequence can be reverse complementary, forming at least about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% base pairing. In some embodiments, the 5' spacer sequence and the 3' spacer sequence can be perfectly complementary pairs of 6-15 nt in length. In some embodiments, the 5' spacer sequence and the 3' spacer sequence can be partially complementary pairs of 18-25 nt in length. In some embodiments, the 5' spacer sequence may sequentially include a flexible spacer sequence and a 5' internal homologous arm sequence from the 5' end to the 3' end, and the 3' spacer sequence may sequentially include a 3' internal homologous arm sequence and a flexible spacer sequence from the 5' end to the 3' end, wherein the 5' internal homologous arm sequence and the 3' spacer sequence can be reverse complementary pairs, forming at least about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% base pairing. The lengths of the 5' internal homologous arm sequences and the 3' internal homologous arm sequences can each be 6-25 nt, such as 6-15 nt, 18-25 nt, etc. The 5' spacer sequence or the 3' spacer sequence can be complementary to fewer than 5 (e.g., 4, 3, 2, 1, or 0) adjacent nucleotides. In some embodiments, the sequence to be circularized may contain only the 5' spacer sequence and not the 3' spacer sequence. In some embodiments, the sequence to be circularized may contain only the 3' spacer sequence and not the 5' spacer sequence. In some embodiments, the sequence to be circularized may contain both the 5' spacer sequence and the 3' spacer sequence. In some embodiments, the 5' spacer sequence may contain a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:22.In some embodiments, the 3' spacer sequence may comprise a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:23.

[0087] Translational functional elements can be selected from translation initiation elements and translation enhancement elements. Translation initiation elements can be sequences used to initiate RNA translation, such as the internal ribosome entry site (IRES) or the 5' untranslated region (5'UTR). Translation enhancement elements can be sequences used to enhance RNA translation, such as poly(A), poly(C), poly(AC), poly(T / U), kozak sequences, 3'UTR, etc.

[0088] The target sequence may contain one or more (e.g., 1, 2, or 3) translation enhancement elements located on the 5' side, 3' side, or both sides of the open reading frame encoding the target peptide or protein.

[0089] In some implementations, the target sequence may include the following from the 5' end to the 3' end:

[0090] i) Translation initiation element and open reading frame encoding the target peptide or protein; 3' sequence of translation initiation element, open reading frame encoding the target peptide or protein, and 5' sequence of translation initiation element; 3' sequence of open reading frame encoding the target peptide or protein, translation initiation element, and 5' sequence of open reading frame encoding the target peptide or protein;

[0091] ii) Translation initiation element, open reading frame encoding the target peptide or protein, and (one or more, such as 1 or 2) translation enhancement elements; the 3' end sequence of the translation initiation element, open reading frame encoding the target peptide or protein, (one or more, such as 1 or 2) translation enhancement elements, and the 5' end sequence of the translation initiation element; the 3' end sequence of the open reading frame encoding the target peptide or protein, (one or more, such as 1 or 2) translation enhancement elements, translation initiation element, and the 5' end sequence of the open reading frame encoding the target peptide or protein; the 3' end sequence of the translation enhancement elements, translation initiation element, open reading frame encoding the target peptide or protein, and (one or more, such as 1 or 2) translation enhancement elements;

[0092] iii) Translation initiation element, translation enhancement element, and open reading frame encoding the target peptide or protein; the 3' end sequence of the translation initiation element, the translation enhancement element, the open reading frame encoding the target peptide or protein, and the 5' end sequence of the translation initiation element; the 3' end sequence of the translation enhancement element, the open reading frame encoding the target peptide or protein, the translation initiation element, and the 5' end sequence of the translation enhancement element; the 3' end sequence of the open reading frame encoding the target peptide or protein, the translation initiation element, the translation enhancement element, and the 5' end sequence of the open reading frame encoding the target peptide or protein; or

[0093] iv) Translation initiation element, translation enhancement element, open reading frame encoding the target peptide or protein, and (one or more, such as 1 or 2) translation enhancement elements; the 3' end sequence of the translation initiation element, the translation enhancement element, the open reading frame encoding the target peptide or protein, (one or more, such as 1 or 2) translation enhancement elements, and the 5' end sequence of the translation initiation element; the 3' end sequence of the translation enhancement element, the open reading frame encoding the target peptide or protein, (one or more, such as 1 or 2) translation enhancement elements, the translation initiation element, and the 5' end sequence of the translation enhancement element; the 3' end sequence of the open reading frame encoding the target peptide or protein, (one or more, such as 1 or 2) translation enhancement elements, the translation initiation element, the translation enhancement element, and the 5' end sequence of the open reading frame encoding the target peptide or protein; the 3' end sequence of the translation enhancement element, the translation initiation element, the translation enhancement element, the open reading frame encoding the target peptide or protein, and (one or more, such as 1 or 2) translation enhancement elements.

[0094] In some embodiments, the translation functional element may include a translation initiation element. In some embodiments, the translation functional element may be a translation initiation element. In some embodiments, the translation initiation element may be an IRES.

[0095] The IRES can be selected from Coxsackievirus B3 (CVB3), Coxsackievirus A (CVB1 / 2), Foot-and-mouth disease virus (FMDV), Human enterovirus 71 (IEV71), Human rhinovirus B3 (HRVB3), Encephalomyocarditis virus (EMCV), Simian enterovirus A (SimianEV-A), Echovirus E11 (EchoV-E11), etc. In some embodiments, the IRES can be Coxsackievirus B3 (CVB3). In some embodiments, the IRES can contain a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:27.

[0096] UTRs can be selected from human α-globin 3'UTR, human β-globin 3'UTR, murine α-globin 3'UTR, murine β-globin 3'UTR, IgG-Fc transport receptor protein α fragment, chemokine ligand 22 fragment, N-terminal cleavage enhancer fragment, Coxsackievirus B3 3'UTR, ribosomal protein S27a 3'UTR, Sindbis virus 3'UTR, human serum albumin 3'UTR, etc.

[0097] The length of the sequence to be circulated can be 50-8000 nucleotides. The length of the sequence to be circulated can be more than 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, or 6500 nucleotides.

[0098] The target peptide or protein can be of eukaryotic or prokaryotic origin. It can be human or non-human. In some embodiments, the target peptide or protein can be an antigen protein, antibody, or protease, etc. In some embodiments, the target peptide or protein can be firefly luciferase, long-bellied luciferase, Gaussian luciferase, green fluorescent protein, etc.

[0099] In some implementations, the sequence to be circularized may contain, or consist of, translational functional elements (e.g., IRES) and open reading frames encoding the target peptide or protein from the 5' end to the 3' end.

[0100] In some implementations, the sequence to be circularized may include, or be composed of, the 3' end sequence of the open reading frame encoding the target peptide or protein, a translational functional element (e.g., IRES), and the 5' end sequence of the open reading frame encoding the target peptide or protein from the 5' end to the 3' end.

[0101] In some implementations, the sequence to be circularized may include, or be composed of, the 3' end sequence of a translational functional element (e.g., IRES), an open reading frame encoding the target peptide or protein, and the 5' end sequence of a translational functional element (e.g., IRES) from the 5' end to the 3' end.

[0102] In some embodiments, the sequence to be circularized may include, from the 5' end to the 3' end, an exon naturally adjacent to the 3' side of the self-splicing intron or a sequence corresponding to the naturally adjacent exon, a 5' spacer sequence, a translational element (e.g., IRES), an open reading frame encoding the target peptide or protein, a 3' spacer sequence, and an exon naturally adjacent to the 5' side of the self-splicing intron or a sequence corresponding to the naturally adjacent exon; or may consist of, an exon naturally adjacent to the 3' side of the self-splicing intron or a sequence corresponding to the naturally adjacent exon, a 5' spacer sequence, a translational element (e.g., IRES), an open reading frame encoding the target peptide or protein, and an exon naturally adjacent to the 5' side of the self-splicing intron or a sequence corresponding to the naturally adjacent exon, or the sequence thereof.

[0103] The RNA molecule of this application may also include an RNA polymerase promoter at its 5' end, for example, on the 5' side of a 5' homologous arm. 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%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:1. The RNA molecule of this application may also include a leader sequence on the 3' side of the RNA polymerase promoter. The leader sequence may contain a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:2.

[0104] The RNA molecule of this application may also contain a restriction endonuclease site at its 3' end, such as on the 3' side of a 3' homologous arm. The restriction endonuclease site may be, for example, NdeI, HindIII, EcoRI, EcoRV, etc.

[0105] In some embodiments, the RNA molecule of this application may sequentially comprise, from the 5' end to the 3' end: an RNA polymerase promoter, an optional leader sequence, a 5' homologous arm, a self-splicing intron, a sequence to be circularized, a 3' homologous arm spacer sequence, a 3' homologous arm, and an optional restriction endonuclease site, or be composed thereof, wherein the elements are operatively linked.

[0106] In some embodiments, the RNA molecule of this application may be transcribed from the complementary strand of a single-stranded DNA molecule of the first aspect of this application, a double-stranded DNA molecule, or a vector.

[0107] In some embodiments, the RNA molecule provided in this application may have its self-splicing introns and the sequence to be circularized linked in a sequence from the 3' end to the 5' end, wherein the elements are operatively linked.

[0108] In a third aspect, this application provides a method for preparing circular RNA, comprising i) in vitro transcription of RNA molecules from a single-stranded DNA molecule or its complementary strand, a double-stranded DNA molecule, or a vector according to the first aspect of this application under suitable conditions, and ii) incubating the RNA molecule under suitable conditions, wherein the suitable conditions for step ii) include the presence of magnesium ions and guanosine. In some embodiments, suitable conditions include the presence of guanosine and magnesium ions. 2+ In the presence of [specific ingredient], incubate at approximately 45-60°C, for example, 50-55°C, for about 15 minutes. The concentration of guanosine can be 0.2 mM-4.0 mM, for example, 2.0 mM, and the concentration of magnesium ions can be 2.0 mM-40.0 mM, for example, 10.0 mM-37.0 mM. Guanosine can be guanosine triphosphate (GTP).

[0109] Alternatively, the method for preparing circular RNA according to this application may include incubating the circularizable RNA molecule of the second aspect of this application under suitable conditions, wherein suitable conditions include the presence of magnesium ions and guanosine. In some embodiments, suitable conditions include the presence of guanosine and magnesium ions. 2+ In the presence of [specific ingredient], incubate at approximately 45-60°C, for example, 50-55°C, for about 15 minutes. The concentration of guanosine can be 0.2 mM-4.0 mM, for example, 2.0 mM, and the concentration of magnesium ions can be 2.0 mM-40.0 mM, for example, 10.0 mM-37.0 mM. Guanosine can be guanosine triphosphate (GTP).

[0110] This application also protects circular RNA prepared from single-stranded DNA molecules or their complementary strands, double-stranded DNA molecules or vectors of the first aspect of this application or circularizable RNA molecules of the second aspect, as well as circular RNA prepared by the method for preparing circular RNA of this application.

[0111] 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.

[0112] In a fourth aspect, this application provides a host cell that may contain the single-stranded DNA molecule of this application or its complementary strand, double-stranded DNA molecule, vector, circularizable RNA molecule or circular RNA molecule.

[0113] This application also provides a composition that may comprise the single-stranded DNA molecule or its complementary strand, a double-stranded DNA molecule, a vector, a circularizable RNA molecule, or a host cell containing the single-stranded DNA molecule or its complementary strand, a double-stranded DNA molecule, a vector, or a 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. The composition of this application (comprising a single-stranded DNA molecule or its complementary strand, or a double-stranded DNA molecule) can be used to prepare a vector for preparing circular RNA.

[0114] This application also provides a composition that may comprise the circular RNA molecule of this application or a host cell containing the circular RNA molecule of this application. The composition may be a pharmaceutical composition and may also comprise a pharmaceutically acceptable carrier. The composition may be delivered to cells or animals via a delivery system. Delivery systems include, but are not limited to, liposomes, polyethyleneimine (PEI), nanoparticles (e.g., noble metal nanoparticles, quantum dots, silica microspheres), etc. The circular RNA can express the target protein within 1, 2, 3, 4, 5, 6, or 7 days after introduction into cells or animals.

[0115] 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.

[0116] In a fifth 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.

[0117] When the target peptide or protein is located 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 associated with infection by that microorganism.

[0118] 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.

[0119] 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.

[0120] Subjects can be mammals, such as humans.

[0121] 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 difference being only the substitution of T and U. Those skilled in the art can determine the type of sequence represented by the SEQ ID NO, i.e., a DNA sequence or an RNA sequence, based on the context.

[0122] In this application, unless the context clearly indicates otherwise, the nucleotide sequences are written in the order from left to right, from the 5' end to the 3' end.

[0123] 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

[0124] 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.

[0125] Figure 1 shows a schematic diagram of five vector backbones for preparing circular RNA designed based on T4.A2.nrdB self-splicing introns in this application.

[0126] Figure 2 shows the E-Gel characterization of IVT products and self-splicing products obtained from forward and reverse splicing vectors of T4.A2.nrdB self-splicing introns.

[0127] Figure 3 shows the Sanger sequencing maps of the splicing sites of the self-splicing products obtained from the forward and reverse splicing vectors of the T4.A2.nrdB self-splicing intron.

[0128] Figure 4 shows the E-Gel characterization of IVT products containing long GOI fragments, cyclized products, and products treated with RNase R.

[0129] Figure 5 shows the E-Gel characterization (A) and RNA circularization rate (B) of the IVT products and their circularized products prepared from the vector backbones T4.A2.nrdB1.0, T4.A2.nrdB2.0, T4.A2.nrdB3.0, and T4.A2.nrdB3.1.

[0130] Figure 6 shows the E-Gel characterization (A) and circularization rate (B) of RNA products containing target sequences of different lengths prepared from the T4.A2.nrdB3.1 vector backbone.

[0131] Figure 7 shows the E-Gel characterization of each step of the RNA products containing CVB3-EGFP prepared using the T4.A2.nrdB2.0 and T4.A2.nrdB3.1 vector backbones (A); the E-Gel characterization of each step of the RNA products containing CVB3-Fluc prepared using the T4.A2.nrdB2.0 and T4.A2.nrdB3.1 vector backbones (B); and the E-Gel characterization of each step of the RNA products containing CVB3-Cas9 prepared using the T4.A2.nrdB3.1 backbone vector (C).

[0132] Figure 8 shows the E-Gel characterization (A) and circularization rate (B) of the RNA products prepared from the T4.A2.nrdB3.1 and Ana2.0 vector backbones at each step.

[0133] Figure 9 shows the design of the GOI region in the T4.A2.nrdB3.2 vector backbone (A), a schematic diagram of the composition of the circular RNA prepared from the T4.A2.nrdB3.0 and T4.A2.nrdB3.2 vector backbones (B), and the E-Gel characterization of its IVT product and circularization product (C).

[0134] The top of Figure 10 shows a schematic diagram of the circularization of CVB3-EGFP using the T4.A2.nrdB3.2+ vector backbone. The bottom left of Figure 10 shows the secondary structure formed by the SS1, SS2, IGS, and 3' homologous arm spacer sequences. The bottom right of Figure 10 shows the E-Gel characterization of the RNA products at each step.

[0135] Figure 11 shows the Sanger sequencing map of the splice site after circularization of CVB3-EGFP using the T4.A2.nrdB3.2+ vector backbone.

[0136] Figure 12 shows the chromatographic map (A), E-Gel characterization of the chromatographic fraction (B), comparison of circRNA purity before dephosphorylation and after reverse chromatography purification (C), and expression activity in in vitro cells (D) of circRNA prepared by T4.A2.nrdB3.1. Detailed Implementation

[0137] 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.

[0138] 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.

[0139] The term "or" refers to a single element among the listed selectable elements, unless the context explicitly indicates otherwise.

[0140] 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.

[0141] The terms “optional,” “optional,” “optionally,” or “discretionarily” mean that certain ingredients or steps are not mandatory or required, meaning that certain ingredients or steps may be included in some cases and not in others.

[0142] "Operationally linked" means that the arrangement of the elements enables the completion of the desired function. For example, the DNA strand with "operationally linked" elements in this application can perform RNA transcription in vitro or in vivo, and the RNA molecule with "operationally linked" elements in this application can perform circularization, etc. 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. In the case of double-stranded DNA, "upstream" generally refers to a position closer to the 5' end of the coding strand, and relatively speaking, "downstream" refers to a position closer to the 3' end of the coding strand.

[0143] "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.

[0144] "Precursor RNA," "preRNA," "IVT transcript," "messenger RNA," "mRNA," or "circular RNA" refers to the RNA product transcribed from the vector or DNA of this application, which can be circularized under suitable conditions to form circular RNA. Alternatively, it may refer to the circularizable RNA of this application.

[0145] "Host cell" refers to a cell that has been introduced (e.g., transformed, infected, or transfected) with a segregated polynucleotide sequence or that can be introduced (e.g., transformed, infected, or transfected) with a segregated polynucleotide sequence. Host cells can be prokaryotic or eukaryotic cells, including but not limited to bacterial cells, fungal cells, yeast cells, microbial cells, insect cells, and mammalian cells, such as yeast cells, CHO cells, and HEK293 cells.

[0146] 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.

[0147] In this application, "self-splicing introns" refers to introns that can splice themselves without the participation of splice bodies. In this application, "self-splicing introns" may also be referred to as "ribozymes." Conditions for splicing of self-splicing introns include the presence of guanosine (e.g., GTP) and magnesium ions. Based on their structure and splicing mechanism, self-splicing introns are generally divided into two categories: Group I and Group II. In this application, the introns are selected from Group I introns. Group I introns are a class of very large ribozymes capable of self-splicing reactions, commonly found in many species, including but not limited to Tetrahymena sp., T4 phage, Anabaena sp., TpaCOX2, Ptu, etc. Group I introns are primarily involved in catalyzing the excision of mRNA, tRNA, and rRNA precursors. In some embodiments, the self-splicing introns are selected from the T4 phage nrdB or nrdD gene. Specifically, in this application, a “self-splicing intron” does not include its 5' or 3' side naturally adjacent exon.

[0148] In this document, the terms "exon," "exon fragment," and "naturally adjacent exon" related to self-splicing introns refer to the exons or fragments adjacent to the upstream and downstream sides of the self-splicing intron in its natural state, or sequences with the same function as the exons. "Sequence corresponding to the naturally adjacent exon" refers to a sequence with the same function as the exon. This exon can complementarily pair with the IGS sequence of the self-splicing intron, thereby enabling the intron to self-splice after transcription, removing itself from the mRNA. In some embodiments, the nucleic acid molecule of this application includes exon 1 and exon 2, where exon 2 is selected from the exon naturally adjacent to the 3' side of the self-splicing intron or an exon sequence with the same function as the naturally adjacent exon, and exon 1 is selected from the exon naturally adjacent to the 5' side of the self-splicing intron or an exon sequence with the same function as the naturally adjacent exon. In some embodiments, the sequences of exon 2 and exon 1 respectively contain nucleotide sequences of GTAC and GCGT, the nucleotide sequences shown in SEQ ID NO:20 and 21, or the nucleotide sequences shown in SEQ ID NO:18 and 19.

[0149] The "internal guide sequence" or "IGS" refers to a sequence near the 5' end of a class I self-splicing intron required for self-splicing. This sequence can form complementary pairs with the ribozyme recognition sequence (or "ribozyme recognition site" or "splicing site") immediately adjacent to the 5' side of the intron, enabling the intron to form the correct secondary and tertiary structures and thus advancing the intron splicing process. Specifically, a G in the IGS can form a G:U complementarity with the U at the end of the 3' side of the 5' side ribozyme recognition sequence. The ribozyme recognition sites of naturally self-splicing introns are located in the 5' and 3' side exons of the intron, pairing with the IGS. The splicing site of the self-splicing intron is at the junction of the intron and the ribozyme recognition site.

[0150] 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 base complementarity principles of A:T, A:U, C:G, and G:U. Specifically, it can refer to Watson-Crick base pairing, which forms hydrogen bonds between adenine (A) and thymine (T) or uracil (U), or guanine (G) and cytosine (C). It can also refer to swing base pairings between hypoxanthine and uracil (I:U), guanine and uracil (G:U), adenine and cytosine (A:C), hypoxanthine and adenine (I:A), or hypoxanthine and cytosine (I:C). Furthermore, as mentioned above, a G in the self-splicing intron IGS can form a G:U complementarity with the U at the 3' end of the 5' exon in the native state of the intron, which is one of the necessary conditions for the initiation of self-splicing by the self-splicing intron. When one nucleotide sequence is "complementary" to another, it can mean that the two nucleotide sequences are 100% complementary, highly complementary (e.g., more than 90%), or substantially complementary (e.g., more than 30%, 40%, 50%, 60%, 65%, 70%, 75%, or 80%). The two "complementary" strands are each other's "complementary strands." "Anti-complementarity" refers to two segments or two nucleotide chains on the same nucleotide chain that can pair complementarily when read from the 5' to 3' direction and from the 3' to 5' direction, respectively. For example, the GCGC and GCGU segments on the same nucleotide chain are anti-complementary.

[0151] 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.

[0152] The "lead sequence" refers to the nucleotide sequence downstream of the promoter from position +1 to +8, which is closely related to IVT production. In the RNA molecule of this application, the RNA fragment transcribed from the leader sequence is linked to the RNA fragment transcribed from the self-splicing intron and can be removed during circularization.

[0153] 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.

[0154] In this document, "translational functional element" refers to a sequence in an RNA molecule that plays a role in its own translation. This can include sequences that initiate RNA translation (also referred to herein as "translation initiation elements"), such as IRES and 5'UTR, and sequences that enhance translation (also referred to herein as "translation enhancement elements"), such as poly(A). In this application, "translational functional element" also refers to the DNA sequence used to transcribe these elements.

[0155] "Internal ribosome entry site" or "IRES" refers to an RNA sequence that forms a secondary structure to attract the transcription initiation complex precursor to a translation start codon such as AUG. In this application, "IRES" also refers to a DNA sequence used for transcription of the IRES.

[0156] A "homologous arm" or "homologous arm sequence" refers to an RNA or DNA sequence that can form at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% base pairings with another RNA or DNA (e.g., another homologous arm).

[0157] "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).

[0158] 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.

[0159] 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.

[0160] The term "vector" refers to a naturally occurring or synthetic nucleotide fragment, such as a DNA or RNA fragment, 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 refers 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% base pairing exists between the two DNA strands.

[0161] "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.

[0162] "Immunogenicity" refers to the property of a substance to elicit an immune response in cells or the body. Generally speaking, foreign substances all have a certain degree of immunogenicity, which can activate, proliferate, and differentiate immune cells, ultimately producing immune effector substances such as antibodies and sensitized lymphocytes.

[0163] In this article, "the sequence to be circularized" refers to the RNA sequence that remains in the circular RNA after RNA circularization, and its corresponding DNA sequence. "Target sequence" or "GOI" refers only to the sequence in the sequence to be circularized that is used to encode proteins or transcribe non-coding RNA, including translational elements.

[0164] The "conformation" of nucleic acids (such as introns) refers to their spatial structure. The conformation of nucleic acid molecules may change due to sequence variations or other environmental conditions, but such changes do not lead to the breaking or formation of covalent bonds.

[0165] Linear RNA is highly susceptible to degradation, making it difficult to preserve and utilize. Circular RNA, as the name suggests, is a type of nucleic acid molecule that is linked end-to-end. Because it lacks free 5' and 3' ends, it can avoid attack by some RNases, thus having a longer half-life.

[0166] In this field, linear RNA precursors (preRNAs) are typically generated through in vitro transcription, followed by the preparation of circular RNA using either enzyme ligation or the use of self-splicing introns. The method using self-splicing introns is more popular than enzyme ligation because it requires less complex procedures and achieves a higher circularization rate.

[0167] In methods utilizing self-splicing introns, the intron is typically divided into two fragments. The 3' end fragment and its adjacent 3' exons, and the 5' end fragment and its adjacent 5' exons, are positioned upstream and downstream of the target GOI sequence, respectively. After the intron fragments assemble into a complete ribozyme and complete backsplicing, the GOI and the flanking exons circularize, and the intron fragments are removed. During in vitro transcription of preRNA, RNA undergoes co-transcriptional folding according to the "first-to-fold" principle. As the GOI length increases, the folding or conformation of the 3' and 5' intron fragments, especially the 5' intron downstream of the GOI, may be interfered with by the long GOI sequence, preventing it from folding into a catalytically active conformation. This reduces the intron's splicing activity and leads to a decrease in circularization rate. Even with the use of spacer sequences between the GOI and the 5' or 3' intron fragments to partially shield the GOI from interference with the self-splicing intron conformation, the problem of reduced cyclization rate cannot be effectively solved.

[0168] The inventors of this application, by placing self-splicing introns upstream or on the 5' side of the sequence to be circularized, and based on the "first-transcription-first-folding" principle, avoid interference from the sequence to be circularized, especially long fragments, on the conformation of the self-splicing introns, thus improving circularization efficiency. When a spacer sequence is placed between the self-splicing intron and the target sequence, the interference of the target sequence on the intron conformation can be better shielded, further improving circularization efficiency. As shown in Examples 2 and 3, compared to existing vectors where two intron fragments are distributed on both sides of the sequence to be circularized, the vector in this application, with introns located upstream of the sequence to be circularized, has higher circularization efficiency, and this advantage becomes more pronounced as the length of the target sequence fragment increases.

[0169] In addition, when using self-cleaving introns to prepare circular RNA, some non-target sequences are inevitably introduced.

[0170] For example, self-splicing introns require adjacent exon sequences on both sides to form the necessary secondary and tertiary structures, thus advancing the self-splicing process. Specifically, the internal guide sequence IGS near the 5' end of the intron needs to form complementary pairings with the 5' and 3' exons of the intron, resulting in splicing at the splice site, intron removal, and ligation of the ribozyme recognition sites on both sides. When constructing vectors for preparing circular RNA using self-splicing introns, the intron is typically split in two, with the 3' intron fragment (and 3' exon) and the 5' intron fragment (and 5' exon) loaded onto the 5' and 3' sides of the GOI, respectively. When the RNA is circularized, the self-splicing intron and the adjacent exons on both sides undergo the aforementioned complementary pairing, the intron detaches from the overall structure, and the exons containing the ribozyme recognition sites on both sides ligate and remain in the circular RNA product. The adjacent exon sequences may increase immunogenicity, thus limiting the application range of circular RNA.

[0171] Furthermore, to shield the GOI from interference with the self-splicing intron conformation, spacer sequences may be placed between the two intron fragments and the GOI. These spacer sequences are also artificially designed sequences and possess a certain degree of immunogenicity.

[0172] To reduce the immunogenicity of circular RNA, the inventors of this application designed the 5' and 3' sequences of the sequence to be circularized to be complementary to the internal guide sequence (IGS) of a self-splicing intron, replacing the sequences complementary to the IGS in the exons flanking the self-splicing intron, thereby ensuring that the final circular RNA product does not contain the exon sequences flanking the intron. This can be achieved by finding sequences in the sequence to be circularized that are identical or similar to the IGS-paired sequences in the flanking exons and rearranging the fragment of the target sequence. It is important to note that one G in the 3' end sequence of the IGS needs to form a G:U complementarity with the U at the 3' end of the target sequence; therefore, the 3' end of the sequence to be circularized should, or preferably should, be a nucleotide containing the bases U or T. In Example 4 of this application, exon-free circular RNA with a high circularization rate was prepared.

[0173] To further shield the GOI from interference with the self-splicing intron conformation, a spacer sequence can be placed between the intron and the GOI. Compared to existing techniques that include two spacer sequences, this reduces the need for one spacer sequence, minimizing unwanted sequences in the final circular RNA while maintaining circularization efficiency.

[0174] Therefore, this application provides a single-stranded DNA molecule for preparing circular RNA, which sequentially comprises, from the 5' end to the 3' end: a self-splicing intron and a sequence to be circularized, wherein the elements are operatively linked. The self-splicing intron enables the circularization of RNA molecules transcribed from the single-stranded DNA molecule or its complementary strand. During the circularization of the RNA molecule transcribed from the single-stranded DNA molecule or its complementary strand, the self-splicing intron can be removed from the RNA molecule.

[0175] The self-splicing intron may contain an internal guide sequence (IGS). The 5' end sequence of the sequence to be circumscribed is anticomplementary to the 5' end sequence of the internal guide sequence. Any number of nucleotides at the 5' end of the sequence to be circumscribed can be anticomplementary to the 5' end sequence of the internal guide sequence. In some embodiments, 2-4 nucleotides, for example, 2 nucleotides, at the 5' end of the sequence to be circumscribed are anticomplementary to the 5' end sequence of the internal guide sequence. The sequence to be circumscribed may contain a T-terminated nucleotide at the 3' end, and be anticomplementary to the 3' end sequence of the internal guide sequence, wherein the 5' end of the 3' end sequence of the internal guide sequence is initiated by a G-terminated nucleotide. The 3' end sequence of the sequence to be circumscribed is anticomplementary to the 3' end sequence of the internal guide sequence. Any number of nucleotides at the 3' end of the sequence to be circumscribed can be anticomplementary to the 3' end sequence of the internal guide sequence. In some embodiments, 2-4 nucleotides, for example, 4 nucleotides, at the 3' end of the sequence to be circumscribed are anticomplementary to the 3' end sequence of the internal guide sequence.

[0176] This application may employ any self-splicing intron that can achieve self-splicing or circularization in the structure of this application, especially type I self-splicing introns, such as self-splicing introns derived from the Escherichia coli phage T4.A2.nrdB gene.

[0177] It is worth noting that the self-splicing introns used in this application are not complete introns, but rather intron segments that are important or critical to the intron's function (such as P1 at the 5' end and P10 at the 3' end). The important intron segments at the 5' and 3' ends can be connected by connecting elements or directly, as long as the intron can perform its self-splicing function normally. In some embodiments, the connecting elements can be sequences capable of forming stem-loop structures, thereby bringing the important intron segments at the 5' and 3' ends closer together, allowing the IGS to form a complementary pair with the 5' and 3' end sequences in the sequence to be looped.

[0178] To facilitate circularization, a 5' homologous arm sequence can be included upstream or 5' to the self-splicing intron, and a 3' homologous arm sequence can be included downstream or 3' to the sequence to be circularized. Through complementary pairing of the two homologous arms, the 5' and 3' ends of the linear preRNA can be physically close, facilitating intron self-splicing. Complementary pairing can be achieved between the two homologous arms. The length of the homologous arms can be 10-100 nucleotides, for example, 15-50 nucleotides. However, in principle, each homologous arm should not be complementary to other sequences in the preRNA, or the complementary nucleotides should be controlled below 50%, for example, 40%, 30%, 20%, 10%, or 0%, to avoid hindering intron self-splicing.

[0179] The sequence to be circularized can contain any desired sequence, as long as its 5' end sequence is anticomplementary to the 5' end sequence of the intron IGS (it can be basically complementary, preferably highly complementary or completely complementary) and its 3' end sequence is anticomplementary to the 3' end sequence of the intron IGS (it can be basically complementary, preferably highly complementary or completely complementary).

[0180] Therefore, the sequence to be circularized may include the target sequence, exon 2 (which is the 3' side natural neighbor exon of the self-splicing intron or a sequence with the same function as the natural neighbor exon), exon 1 (which is the 5' side natural neighbor exon of the self-splicing intron or a sequence with the same function as the natural neighbor exon), 5' spacer sequence, 3' spacer sequence, etc.

[0181] The target sequence may contain an open reading frame encoding a target peptide or protein and a translational element. The target sequence may contain, or consist of, the translational element and the open reading frame encoding the target peptide or protein from the 5' end to the 3' end. The target sequence may contain, or consist of, the open reading frame encoding the target peptide or protein and the translational element from the 5' end to the 3' end. The target sequence may contain, or consist of, the 3' end sequence of the translational element, the open reading frame encoding the target peptide or protein, and the 5' end sequence of the translational element, wherein the 5' end sequence and the 3' end sequence of the translational element, when arranged in this order, form the translational element. The target sequence may include, or be composed of, the 3' end sequence of the open reading frame encoding the target peptide or protein, the translational functional element, and the 5' end sequence of the open reading frame encoding the target peptide or protein, from the 5' end to the 3' end, wherein the 5' end sequence of the open reading frame encoding the target peptide or protein and the 3' end sequence of the open reading frame encoding the target peptide or protein, when arranged in this order, form the open reading frame encoding the target peptide or protein.

[0182] The target sequence may contain a sequence for transcribing non-coding RNA. The target sequence may contain, or be composed of, the 3' end sequence of the sequence for transcribing non-coding RNA and the 5' end sequence of the sequence for transcribing non-coding RNA, from the 5' end to the 3' end, wherein the 5' end sequence of the sequence for transcribing non-coding RNA and the 3' end sequence of the sequence for transcribing non-coding RNA, when arranged in this order, form the sequence for transcribing non-coding RNA.

[0183] The target sequence may contain a single cloning site or a multiple cloning site, or be composed of both. Any desired sequence, such as an open reading frame encoding a target peptide or protein, and a translational functional element, can be inserted into the single-stranded DNA molecule of this application through a single cloning site or a multiple cloning site.

[0184] The target sequence may contain a single-cloning site or a multiple-cloning site, and a translational element, or be composed of thereof. An open reading frame encoding, for example, a target peptide or protein, can be inserted into a single-stranded DNA molecule via a single-cloning site or a multiple-cloning site. The target sequence may contain, from the 5' end to the 3' end, the 3' end sequence of the translational element, the single-cloning site or the multiple-cloning site, and the 5' end sequence of the translational element, or be composed of thereof, wherein the 5' end sequence and the 3' end sequence of the translational element, when arranged in this order, form the translational element.

[0185] The sequence to be circularized may, in some embodiments, include exon 2, an optional 5' spacer sequence, the target sequence, an optional 3' spacer sequence, and exon 1 from the 5' end to the 3' end. While ensuring that exon 2 can pair complementaryly with the 5' end sequence of the IGS and that exon 1 can pair complementaryly with the 3' end sequence of the IGS, any desired sequence may be included between the two exons or corresponding sequences, such as an open reading frame encoding the target peptide or protein and optionally a sequence encoding a translational functional element, a sequence encoding non-coding RNA, a single cloning site, or a multiple cloning site. The order can be freely changed, as long as the circular RNA can perform non-coding RNA function or achieve protein expression.

[0186] The 5' spacer sequence and the 3' spacer sequence can be composed of flexible spacer sequences. The flexible spacer sequence can be a poly(AC) or polyA sequence. The length of the spacer sequence can be at least 10 nucleotides (e.g., 20-30) and the folding energy can be 0 kcal / mol. In some embodiments, the target sequence may contain only the 5' spacer sequence and not the 3' spacer sequence. In some embodiments, the 5' spacer sequence and the 3' spacer sequence can be reverse complementary, forming at least about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% base pairing. In some embodiments, the 5' spacer sequence may sequentially include a flexible spacer sequence and a 5' internal homologous arm sequence from the 5' end to the 3' end, and the 3' spacer sequence may sequentially include a 3' internal homologous arm sequence and a flexible spacer sequence from the 5' end to the 3' end. The 5' internal homologous arm sequence and the 3' spacer sequence can be reverse complementary, forming at least about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% base pairing. Thus, the two spacer sequences, together with the sequence between them, such as the target sequence, can form a stem-loop structure, thereby further reducing the impact on the intron's folding conformation.

[0187] In principle, the 5' spacer sequence or the 3' spacer sequence should not be complementary to other sequences in the preRNA, or the complementary nucleotide pairs should be controlled to be 4, 3, 2 or 1 or less, in order to avoid hindering intron self-splicing, etc.

[0188] 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, ribosome entry site sequences (IRES) or 5'UTRs need to be added to the circular RNA to enable it to perform translation both in vitro and in vivo.

[0189] When the sequence to be circularized does not contain naturally adjacent exons of introns or their corresponding sequences or spacer sequences, i.e., it only contains the target sequence, to ensure intron self-splicing with IGS, the coding frames and translational elements, or sequences used for transcription of non-coding RNA, in the target sequence can be appropriately truncated and rearranged so that the 5' and 3' ends of the sequence to be circularized can be complementary to IGS (basically complementary or completely complementary), forming the correct intron ribozyme conformation, successfully carrying out intron self-splicing, and resulting in the circularized RNA molecule forming a functionally normal and correctly arranged coding frames and translational elements, or non-coding RNA. These operations can be conveniently performed after studying the invention content and embodiments of this application. If a truncated and rearranged fragment complementary to IGS cannot be found, point mutation can be attempted, but the mutation should not affect subsequent protein translation or function, or the function of non-coding RNA. Alternatively, any other method known in the art can be used to design a sequence complementary to IGS on the target sequence, as long as it does not affect subsequent protein translation or function, or the function of non-coding RNA.

[0190] Accordingly, this application also provides circularizable RNA molecules. In some embodiments, they can be transcribed from the single-stranded DNA molecule of this application or its complementary strand.

[0191] A circularizable RNA molecule may contain, from the 5' end to the 3' end, a self-splicing intron and a sequence to be circularized, wherein the elements are operatively linked. The self-splicing intron enables the RNA molecule to be circularized and removed during the circularization process. The self-splicing intron may contain an internal guide sequence (IGS). The 5' end sequence of the sequence to be circularized is anticomplementary to the 5' end sequence of the internal guide sequence. The sequence to be circularized may contain a U-terminal nucleotide and be anticomplementary to the 3' end sequence of the internal guide sequence. The 5' end of the 3' end sequence of the internal guide sequence may, or preferably may, begin with a G-terminal nucleotide.

[0192] The DNA or RNA molecules of this application are particularly suitable for preparing circular RNA molecules of relatively long length, such as 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, especially at least 2000 or 2500 nucleotides.

[0193] Specifically, RNA molecules can be i) transcribed in vitro from the single-stranded DNA molecule or its complementary strand, double-stranded DNA molecule or vector of this application under suitable conditions, and ii) the RNA molecule can be incubated under suitable conditions, wherein suitable conditions in step ii) include the presence of magnesium ions and guanosine. In some embodiments, suitable conditions include the presence of guanosine and magnesium ions. 2+ In the presence of [the substance], incubate at approximately 45-60°C, for example, 50-55°C, for approximately 5 minutes to approximately 1 hour, for example, 8 minutes to 30 minutes, particularly for approximately 15 minutes. The concentration of guanosine can be 0.2 mM to 4.0 mM, for example, 2.0 mM, and the concentration of magnesium ions can be 2.0 mM to 40.0 mM, particularly 10.0 to 37.0 mM.

[0194] Alternatively, the cyclizable RNA molecule of this application can be incubated under suitable conditions, including the presence of magnesium ions and guanosine. In some embodiments, suitable conditions include the presence of guanosine and magnesium ions. 2+ In the presence of [the substance], incubate at approximately 45-60°C, for example, 50-55°C, for approximately 5 minutes to approximately 1 hour, for example, 8 minutes to 30 minutes, particularly for approximately 15 minutes. The concentration of guanosine can be 0.2 mM to 4.0 mM, for example, 2.0 mM, and the concentration of magnesium ions can be 10.0 mM to 40.0 mM, particularly 10.0 to 37.0 mM.

[0195] Guanosine can be guanosine triphosphate (GTP).

[0196] When performing RNA cyclization in a cyclization buffer, the cyclization buffer may contain 0 mM to 1 M ammonium ions, especially 0 mM to 500 mM ammonium ions.

[0197] In this application, E-Gel is used to separate and analyze IVT products and self-splicing products of linear DNA. The inventors of this application tested different lengths of sequences to be circularized and found that E-Gel can separate reaction components in various reaction systems, making it an effective method for analyzing and evaluating the efficiency of cyclization reactions.

[0198] This application also provides a composition that may comprise the single-stranded DNA molecule or its complementary strand, double-stranded DNA molecule, vector, or circularizable RNA molecule of this application, or a cell comprising the aforementioned single-stranded DNA molecule or its complementary strand, 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.

[0199] 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.

[0200] 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.

[0201] 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.

[0202] 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.

[0203] 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).

[0204] When necessary, the precursor RNA or circular RNA in this application can be purified. For example, the precursor RNA or circular RNA can be purified by RNase R treatment, chromatography, or electrophoresis to remove non-circular linear RNA precursors, dsRNA, nicked RNA, and other impurities. Chromatographic methods include, but are not limited to, nucleophilic chromatography, reversed-phase liquid chromatography, and size exclusion chromatography. Electrophoretic methods include, but are not limited to, agarose gel electrophoresis and capillary electrophoresis.

[0205] Circular RNA can also be treated with alkaline phosphatase to remove phosphate groups from the terminal ends of impurity RNA, thus preventing activation of the RIG-1 signaling pathway and the resulting strong immune response. The alkaline phosphatases used include, but are not limited to, thermosensitive phosphatase (HSP), bovine intestinal alkaline phosphatase (CIP), and shrimp alkaline phosphatase (SAP).

[0206] 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.

[0207] 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.

[0208] 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.

[0209] 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.

[0210] 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).

[0211] 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.

[0212] 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.

[0213] The technical solution of the present invention will be further described in detail below through embodiments and in conjunction with the accompanying drawings. Unless otherwise stated, the methods and materials of the embodiments described below are all conventional products that can be purchased from the market. Those skilled in the art will understand that the methods and materials described below are merely exemplary and should not be considered as limiting the scope of the present invention.

[0214] Example

[0215] Example 1. Forward and reverse splicing of self-splicing introns and E-Gel electrophoresis characterization of circRNA

[0216] Forward and backward splicing tests were performed on the self-splicing introns obtained from the Escherichia coli phage T4.A2.nrdB gene (Genbank accession number: X04140.1).

[0217] In forward splicing, the precursor RNA or preRNA sequentially contains a 5' natural adjacent exon, a self-splicing intron, and a 3' natural adjacent exon. In reverse splicing, the self-splicing intron is split into two segments: the 3' intron and its 3' natural adjacent exon are positioned on the 5' side, while the 5' intron and its 5' natural adjacent exon are positioned on the 3' side. The 5' natural adjacent exon contains ribozyme recognition site 1 (SS1, GCGT), and the 3' natural adjacent exon contains ribozyme recognition site 2 (SS2, GT).

[0218] Step 1: Vector design, construction, and amplification

[0219] A vector for forward self-splicing was constructed, and DNA containing the following interconnected elements in sequence was synthesized: a T7 promoter (SEQ ID NO:1), a leader sequence (SEQ ID NO:2), a 5' natural exon fragment containing SS1 (SEQ ID NO:9), an nrdB intron fragment (SEQ ID NO:11), a 3' natural exon fragment containing SS2 (SEQ ID NO:10), and restriction enzyme sites NdeI and HindIII (CATATG and AAGCTT). The above DNA sequence was inserted into an empty pUC57 vector (SEQ ID NO:37) via the NdeI and HindIII restriction sites.

[0220] A vector for reverse self-splicing was constructed, and DNA containing the following interconnected elements in sequence was synthesized: a T7 promoter (SEQ ID NO:1), a leader sequence (SEQ ID NO:2), a 5' homologous arm-1 (SEQ ID NO:3), a 3' intron fragment (SEQ ID NO:12), a 3' natural exon fragment containing SS2 (SEQ ID NO:10), a 5' natural exon fragment containing SS1 (SEQ ID NO:9), a 5' intron fragment (SEQ ID NO:13), a 3' homologous arm-1 (SEQ ID NO:4), and restriction enzyme sites NdeI and HindIII (CATATG and AAGCTT). The above DNA was then constructed into the pUC57-Kan empty vector (SEQ ID NO:37) via NdeI and HindIII restriction sites.

[0221] The two vectors mentioned above were prepared, validated, and amplified by Genscript Biotech Ltd.

[0222] Step 2: Vector linearization

[0223] The plasmid obtained in step 1 was digested with the restriction endonuclease HindIII to obtain a linearized vector.

[0224] Specifically, 150 μg of circular plasmid (1 mg / mL) and 18.75 μL of NE Buffer were added sequentially to the enzyme digestion reaction system. TM r2.1 (NEB#B7202), 5.625 μL HindIII (NEB#R3104S), and 13.13 μL of water for injection were incubated at 37±0.5℃ for 12 h, then inactivated at 80℃ for 20 min. The resulting product was purified using VAHTS RNA magnetic beads (Cellagen Technology, N412-02) according to the instructions and was ready for use.

[0225] Step 3: In vitro transcription (IVT)

[0226] The linearized vector obtained in step 2 was transcribed in vitro. Specifically, a 50 μL in vitro transcription system was prepared according to Table 1. The following were added sequentially to the in vitro transcription system: 20.05 μL of water for injection (WFI), 1.25 μL of murine nuclease inhibitor, 5 μL of each NTP, 0.2 μL of inorganic pyrophosphatase, 5 μL of 10× in vitro transcription buffer, 2.5 μL of linearized plasmid template (template concentration 1 mg / mL), and 1 μL of T7 ribonuclease. The in vitro transcription system was then incubated at 37 °C for 120 min ± 15 min in a metal thermostat mixer.

[0227] Table 1. In vitro transcription reaction system

[0228] Step 4: Remove template

[0229] In the above in vitro transcription reaction solution, deoxyribonuclease I was added to a working concentration of 0.2 U / μL, and the mixture was incubated for 20.0 min in a metal thermostat at 37.0 °C. 0.5 M EDTA was added to a final concentration of 30 mM to terminate the reaction. The mixture was purified by magnetic beads (N412-02, Cellagen Technology), and resuspended in WFI to a final concentration of 3 mg / mL for later use.

[0230] Step 5: Intron self-splicing (or RNA circularization)

[0231] For example, prepare a 50 μL reaction system according to Table 2. Specifically, add 27.3 μL WFI, 16.7 μL of the IVT sample obtained in step 4, 5 μL of 10× cyclization reaction buffer, and 1 μL of GTP to the reaction system sequentially. Transfer the reaction mixture to a thermostatic metal mixer and react at 55℃±2℃ for 15 min. After the reaction is complete, add 0.5M EDTA to a final concentration of 10 mM to terminate the reaction. The resulting products, namely linear RNA prepared from the forward splicing vector and circular RNA (circRNA) obtained from the reverse splicing vector, are purified by magnetic beads. The crude RNA product is resuspended in WFI to a final concentration of 1 mg / mL for later use.

[0232] Table 2. (Cycling) Reaction System

[0233] Step 6: RNase R digestion

[0234] The crude RNA product obtained in step 5 was digested with RNase R (Yisheng, 14606ES72), for example, a 50 μL digestion reaction system was prepared according to Table 3. 18.75 μL of WFI, 25 μL of crude RNA product, and 1.25 μL of RNase R were added sequentially to the digestion system. The mixture was transferred to a thermostatic metal reactor and digested at 37 °C for 60 min. EDTA was then added to a final concentration of 30 mM to terminate the reaction. The obtained RNA was purified by magnetic beads, resuspended in WFI, and treated at 70 °C for 10 min to inactivate any remaining RNase R.

[0235] Table 3. RNase R digestion reaction system

[0236] Step 7: E-Gel agarose gel electrophoresis

[0237] Using E-Gel iBase (Invitrogen) TM The intron splicing efficiency of RNA prepared from two vectors was determined using a 2% E-gel EX agarose gel electrophoresis method. Specifically, 200 ng (10 μL) of RNA products obtained from steps 4, 5, and 6 above were mixed with an equal volume of loading buffer (Invitrogen). TM After thorough mixing, denature at 70°C for 5 minutes, immediately cool on ice for 3 minutes, then spot the sample and perform electrophoresis for 20 minutes. The RNA obtained from the reverse splicing vector after step 5 may contain not only circular RNA but also nicked RNA, which is produced after the hydrolysis and breakage of the circular RNA.

[0238] Step 8: Sanger sequencing

[0239] For the RNA product obtained from step 5 or 6, cDNA double strands were prepared by reverse transcription PCR using the PCR primers shown in SEQ ID NO:14 and 15 according to the amplification system in Table 4. Then, the obtained cDNA was sequenced by Sanger sequencing to detect whether it contained the theoretical sequence.

[0240] nrdB-pF: 5'-GAAGCTATTATGAAACGTGCTGAGTCC-3' (SEQ ID NO: 14)

[0241] nrdB-pR: 5'-GATATAGGGTCGAAGCCG-3' (SEQ ID NO: 15)

[0242] Table 4. Reverse Transcription PCR Reaction System

[0243] Figure 2 shows the E-Gel detection map. Lanes 1 and 2 are the products of steps 4 and 5 prepared by the forward self-splicing vector, respectively, and lanes 3-5 are the products of steps 4, 5, and 6 prepared by the reverse self-splicing vector, respectively. It can be seen that the preRNA prepared by the two vectors, i.e., the products of step 4 (lanes 1 and 3), have almost the same nucleotide length, approximately 816 nucleotides. After self-splicing, the preRNA band in the preRNA prepared by the forward self-splicing vector disappears, producing the splicing main product band (i.e., linear RNA without introns) and the intron band, with theoretical lengths of 544 and 272 nucleotides, respectively (lane 2). After self-splicing of the preRNA prepared from the reverse self-splicing vector (lane 4), five bands were generated: a 5' intron fragment band, a 3' intron fragment band (theoretical lengths of 180 and 108 nucleotides, respectively), a nicked RNA band (theoretical length of 528 nucleotides, accounting for 8.5%), a splicing main product band (accounting for 90%), and a polymer band. After treatment with RNase R in step 6, the linear RNA, including the preRNA, nicked RNA, and intron fragment bands, disappeared, and only the splicing main product band was observed, indicating that it was composed of RNase R-resistant circRNA (lane 5).

[0244] Theoretically, the RNA product after step 5 prepared by the forward self-splicing vector is longer than the circular RNA prepared by the reverse self-splicing vector. This is because the RNA product produced by the forward splicing vector has restriction enzyme sites and leader sequences, leaving an additional 16 nucleotides, while the circular RNA product produced by the reverse indirect vector does not have these two types of nucleotide sequences.

[0245] Figure 3 shows the Sanger sequencing map. The sequence at the splice site is as follows: (SEQ ID NO:16, bold text represents the exon containing SS1, bold underline represents SS1, italics represent the exon containing SS2, italic underline represents SS2), which conforms to the theoretical splicing site sequence. This shows that self-splicing introns can be achieved through either forward or backward splicing, with the 5' and 3' exons connected after self-splicing.

[0246] Furthermore, the reverse splicing vector achieves highly efficient RNA circularization, with a splicing efficiency of approximately 98.5% and a circularization rate of 90%. Specifically, splicing efficiency = (circular RNA + nicked RNA) / (circular RNA + nicked RNA + preRNA), and circularization rate = circular RNA / (circular RNA + nicked RNA + preRNA).

[0247] In addition, using the circularization backbone from CN112399860B, longer fragments to be circularized (corresponding to the two exon fragments mentioned above) were circularized to obtain a 1475 nt preRNA. The E-Gel results are shown in Figure 4. Compared to Figure 2, the relative distances between the circRNA and preRNA, and between the nicked RNA bands, increased. This demonstrates that E-Gel can effectively separate the reaction components in the circularization reaction of GOIs of different lengths, and is an effective method for analyzing and evaluating the efficiency of the circularization reaction.

[0248] Example 2. Design and optimization of vectors for preparing circular RNA

[0249] Self-splicing introns obtained from the T4.A2.nrdB gene were used to circularize CVB3-EGFP RNA (SEQ ID NO:17) via forward or reverse splicing, and the circularization rate was tested.

[0250] Specifically, four vectors for preparing circular RNA were designed and synthesized, corresponding to the first four circularization systems based on the T4.A2.nrdB intron in Figure 1.

[0251] For the T4.A2.nrdB1.0 circularization system in Figure 1, which is the circularization system used for reverse splicing in Example 1, DNA containing the following interconnected elements in sequence was synthesized: T7 transcription promoter (SEQ ID NO:1), leader sequence (SEQ ID NO:2), 5' homologous arm-1 (SEQ ID NO:3), 3' intron fragment (SEQ ID NO:12), exon fragment containing SS2 (SEQ ID NO:18), target sequence GOI (SEQ ID NO:17), exon fragment containing SS1 (SEQ ID NO:19), 5' intron fragment (SEQ ID NO:13), 3' homologous arm-1 (SEQ ID NO:4), and restriction enzyme sites NdeI and HindIII (CATATG and AAGCTT). The above DNA was inserted into the NdeI and HindIII restriction sites of the pUC57-Kan vector (SEQ ID NO:37).

[0252] For the T4.A2.nrdB2.0 circularization system in Figure 1, DNA containing the following interconnected elements in sequence was synthesized: T7 transcription promoter (SEQ ID NO:1), leader sequence (SEQ ID NO:2), 5' homologous arm-2 (SEQ ID NO:5), 3' intron fragment (SEQ ID NO:12), exon fragment containing SS2 (SEQ ID NO:20), 5' spacer sequence (SEQ ID NO:22), target sequence GOI (SEQ ID NO:17), 3' spacer sequence (SEQ ID NO:23), exon fragment containing SS1 (SEQ ID NO:21), 5' intron fragment (SEQ ID NO:13), 3' homologous arm-2 (SEQ ID NO:6), and restriction enzyme sites (CATATG and AAGCTT). The synthesized DNA was inserted into the NdeI and HindIII restriction sites of the pUC57-Kan vector (SEQ ID NO:37). Among them, the 5' end sequence in the 5' spacer sequence can be complementary to the 3' end sequence in the 3' spacer sequence, so that the 5' spacer sequence, the target sequence GOI, and the 3' spacer sequence can form a splicing bubble, thereby reducing the influence of GOI on introns.

[0253] For the T4.A2.nrdB3.0 circularization system in Figure 1, DNA containing the following interconnected elements in sequence was synthesized: T7 transcription promoter (SEQ ID NO:1), leader sequence (SEQ ID NO:2), 5' homologous arm (SEQ ID NO:7), intron sequence (SEQ ID NO:11), exon fragment containing SS2 (SEQ ID NO:20), target sequence GOI (SEQ ID NO:17), exon fragment containing SS1 (SEQ ID NO:21), 3' homologous arm spacer sequence-2 (SEQ ID NO:42), 3' homologous arm-3 (SEQ ID NO:8), and restriction enzyme sites (CATATG and AAGCT). The synthesized DNA was inserted into the NdeI and HindIII restriction sites of the pUC57-Kan vector (SEQ ID NO:37).

[0254] For the T4.A2.nrdB3.1 circularization system in Figure 1, DNA containing the following interconnected elements in sequence was synthesized: T7 transcription promoter (SEQ ID NO:1), leader sequence (SEQ ID NO:2), 5' homologous arm-3 (SEQ ID NO:7), intron sequence (SEQ ID NO:11), exon fragment containing SS2 (SEQ ID NO:20), 5' spacer sequence (SEQ ID NO:22), target sequence GOI (SEQ ID NO:17), 3' spacer sequence (SEQ ID NO:23), exon fragment containing SS1 (SEQ ID NO:21), 3' homologous arm spacer sequence (SEQ ID NO:43), 3' homologous arm-3 (SEQ ID NO:8), and restriction enzyme sites (CATATG and AAGCT). The self-splicing DNA template sequence was inserted into the NdeI and HindIII restriction sites of the pUC57-Kan vector (SEQ ID NO:37).

[0255] Following the steps and parameters in Example 1, the obtained plasmid vector was linearized, transcribed in vitro, detemplated, spliced ​​with introns (circularized), digested with RNase R, and subjected to E-Gel electrophoresis.

[0256] Theoretically, the main RNA product of steps 3-4 is preRNA, containing a small amount of circular RNA and introns. The RNA product of step 5 mainly consists of introns, nicked RNA, intermediate products, preRNA, circular RNA, and aggregates. Among them, the intermediate products refer to the linear products that are spliced ​​at SS1 but not at SS2.

[0257] Figure 5(A) shows E-Gel images of the products obtained from each vector through steps 3-4 and 5, without showing intron fragment bands. The intermediate product differs from the preRNA by only 58 bases; its band may overlap with or be close to the preRNA. Furthermore, a small number of aggregates in the product have bands above the circular RNA, possibly due to the formation of multimeric circular RNA during splicing or insufficient E-Gel denaturation leading to hybridization between circular RNAs. The cleaved RNA has the same base composition as the circular RNA, arising from the hydrolysis of the phosphodiester bond between two bases in the circular RNA; its band is below both the preRNA and circRNA. When the circular RNA band is longer than 1400 bases, its position is above the preRNA, and its migration rate decreases significantly with increasing base number.

[0258] Figure 5(B) shows the circularization rate of RNA prepared by each vector. It can be seen that the RNA obtained by the T4.A2.nrdB3.1 vector has the highest circularization rate, followed by T4.A2.nrdB2.0.

[0259] As the length of the GOI sequence increased, the circularization rate of T4.A2.nrdB1.0 decreased from 90% in Example 1 to approximately 18%. T4.A2.nrd2.0 improved the circularization rate to approximately 65% ​​by optimizing the exon fragment and adding spacer sequences to spatially shield the GOI sequence from interference with ribozyme recognition sites and ribozyme / intron folding. T4.A2.nrd3.0 further improved the circularization rate to approximately 45% by optimizing the exon fragment and employing forward splicing, i.e., a "cis-ribozyme / self-splicing intron" design. Based on the "first-to-first-to-fold" co-transcriptional folding rule, introns are transcribed and folded first to form splicing-active ribozymes, thereby reducing the interference of GOI on their folding. T4.A2.nrd3.1 further improved the circularization rate to approximately 70% by adding spacer sequences.

[0260] The above results indicate that adding spacer sequences and using the cis-ribozyme strategy can optimize T4.A2.nrdB1.0 in terms of both spatial structure and folding sequence, thereby improving the efficiency of intron self-splicing to a certain extent.

[0261] Example 3. Cycling test of long GOI fragments

[0262] Using the T4.A2.nrdB2.0 and T4.A2.nrdB3.1 circulization systems in Example 2, GOI sequences of different lengths were circulized, and the circulization rate was compared with that of the published Ana2.0 (see patent CN112399860B for related description).

[0263] For the T4.A2.nrdB2.0 circularization system, DNA containing the following interconnected elements in sequence is synthesized: T7 transcription promoter (SEQ ID NO:1), leader sequence (SEQ ID NO:2), 5' homologous arm-2 (SEQ ID NO:5), 3' intron fragment (SEQ ID NO:12), exon fragment containing SS2 (SEQ ID NO:20), 5' spacer sequence (SEQ ID NO:22), target sequence GOI (CVB3-EGFP (SEQ ID NO:17), CVB3-Fluc (SEQ ID NO:24) or CVB3-Cas9 (SEQ ID NO:25)), 3' spacer sequence (SEQ ID NO:23), exon fragment containing SS1 (SEQ ID NO:21), 5' intron fragment (SEQ ID NO:13), 3' homologous arm-2 (SEQ ID NO:6), and restriction enzyme sites (CATATG and AAGCTT). The synthesized DNA was inserted into the NdeI and HindIII restriction sites of the pUC57 vector (SEQ ID NO:37).

[0264] For the T4.A2.nrdB3.1 circularization system, DNA containing the following interconnected elements in sequence is synthesized: T7 transcription promoter (SEQ ID NO:1), leader sequence (SEQ ID NO:2), 5' homologous arm (SEQ ID NO:7), intron sequence (SEQ ID NO:11), exon fragment containing SS2 (SEQ ID NO:20), 5' spacer sequence (SEQ ID NO:22), target sequence GOI (CVB3-EGFP (SEQ ID NO:17), CVB3-Fluc (SEQ ID NO:24), or CVB3-Cas9 (SEQ ID NO:25)), 3' spacer sequence (SEQ ID NO:23), exon fragment containing SS1 (SEQ ID NO:21), 3' homologous arm spacer sequence (SEQ ID NO:43), 3' homologous arm-3 (SEQ ID NO:8), and restriction enzyme sites (CATATG and AAGCTT). The synthesized DNA was inserted into the NdeI and HindIII restriction sites of the pUC57 vector (SEQ ID NO:37).

[0265] For the control Ana2.0 (circular backbone identical to SEQ ID NO:13 in CN112399860B), DNA containing the following interconnected elements in sequence was synthesized: T7 transcription promoter (SEQ ID NO:1), leader sequence (SEQ ID NO:28), 5' homologous arm (SEQ ID NO:29), 3' intron fragment (SEQ ID NO:35), exon fragment 2 (E2, SEQ ID NO:34), 5' spacer sequence (SEQ ID NO:31), target sequence GOI (CVB3-EGFP (SEQ ID NO:17), CVB3-Fluc (SEQ ID NO:24) or CVB3-Cas9 (SEQ ID NO:25)), 3' spacer sequence (SEQ ID NO:32), exon fragment 1 (E1, SEQ ID NO:33), 5' intron fragment (SEQ ID NO:36), 3' homologous arm (SEQ ID NO:30), and restriction enzyme sites (CATATG and GAATTC). The synthesized DNA was inserted into the NdeI and EcoRI restriction sites of the pUC57-Kan vector (SEQ ID NO:37).

[0266] The nucleic acid lengths of CVB3-EGFP, CVB3-Fluc, and CVB3-Cas9, which are GOIs, are 1464, 2397, and 4959 nucleotides, respectively.

[0267] Following the steps and parameters in Example 1, the obtained plasmid vector was linearized, transcribed in vitro, detemplated, spliced ​​with introns (circularized), digested with RNase R, and subjected to E-Gel electrophoresis.

[0268] Figure 6 shows E-Gel images (A) and RNA circularization rates (B) of circRNA products of different lengths obtained from T4.A2.nrdB3.1 after step 5, where 1603, 2536, and 5098 correspond to the lengths of CVB3-EGFP, CVB3-Fluc, and CVB3-Cas9 after adding the spacer sequence and exon, respectively.

[0269] Figure 7 shows E-Gel images of the products obtained from steps 3-4, 5, and 6 for GOIs CVB3-EGFP (A), CVB3-Fluc (B), and CVB3-Cas9 (C) with T4.A2.nrdB2.0 and T4.A2.nrdB3.1.

[0270] Figure 8 shows the E-Gel gel map (A) and cyclization rate (B) of the product from step 5 of the T4.A2.nrdB3.1 and Ana2.0 cyclization systems.

[0271] The E-Gel results above show that the circularization rates of CVB3-EGFP by the T4.A2.nrdB2.0, T4.A2.nrdB3.1, and Ana2.0 circularization systems were 66%, 68%, and 58.6%, respectively, and the circularization rates of CVB3-Fluc were 54%, 69%, and 54%, respectively. Notably, when circularizing CVB3-Fluc, the splicing efficiency of the control group Ana2.0 was only 67.8%, significantly lower than the 88.2% of T4.A2.nrdB3.1. This indicates that the self-splicing efficiency of Ana2.0 decreases with increasing GOI length, while the T4.A2.nrdB3.1 circularization system can address this issue. Furthermore, the circularization efficiency of CVB3-Cas9 by T4.A2.nrdB3.1 still reached 57.4%.

[0272] In other words, the length of the GOI has a relatively small impact on the cyclization efficiency of the T4.A2.nrdB3.1 cyclization system. When the GOI length is in the range of 1500-2500 nt, the cyclization rate remains stable at over 60%, while when the GOI length exceeds 5000 nt, a high cyclization rate of approximately 57% can still be achieved. Compared to traditional flanking intron designs such as the T4.A2.nrdB2.0 design, the novel cis-ribozyme design in the T4.A2.nrdB3.1 system can effectively reduce the influence of other sequences in the vector on intron folding and improve the cyclization efficiency of long GOI fragments.

[0273] Example 4. Vector for preparing base-free circular RNA

[0274] The circular RNAs prepared by the vectors in the above embodiments all retain natural exon fragments flanking the self-splicing introns.

[0275] To avoid immunogenicity issues caused by these exon fragments, the T4.A2.nrdB3.2 circularization system shown in Figure 1 was further designed, which can produce circular RNA without exon residues.

[0276] Specifically, DNA containing the following interconnected elements in sequence was synthesized: the T7 transcription promoter (SEQ ID NO:1), the leader sequence (SEQ ID NO:2), the 5' homologous arm-3 (SEQ ID NO:7), the intron sequence (SEQ ID NO:11), the target sequence GOI (SEQ ID NO:26), the 3' homologous arm spacer sequence (SEQ ID NO:43), the 3' homologous arm-3 (SEQ ID NO:8), and the restriction enzyme sites (CATATG and AAGCTT). The synthesized DNA was inserted into the NdeI and HindIII restriction sites of the pUC57-Kan vector (SEQ ID NO:37).

[0277] The GOI in the vector contains the EGFP coding region and the ribosome entry site (IRES). As shown in Figure 9(A), the nucleotide sequence at positions 86-91 of the EGFP coding region (SEQ ID NO:17 underlined) is an exon-like sequence. The nucleotide sequences at positions 86-89 and 90-91 are identical to the SS1 (GCGU) sequence contained in exon fragment 1 of T4.A2.nrdB and the SS2 (GU) sequence contained in exon fragment 2 of T4.A2.nrdB, respectively (only T and U are substituted). During vector construction, the EGFP coding region is truncated after nucleotide 89, dividing it into coding region fragment 1 (i.e., the 5' fragment) and coding region fragment 2 (i.e., the 3' fragment), which are placed on the 3' side and 5' side of the IRES, respectively, to obtain FP-CVB3-EG-1 (SEQ ID NO:26). Meanwhile, as a control, DNA containing the following interconnected elements was synthesized based on the T4.A2.nrdB3.0 circularization system: the T7 transcription promoter (SEQ ID NO:1), leader sequence (SEQ ID NO:2), 5' homologous arm-3 (SEQ ID NO:7), intron sequence (SEQ ID NO:11), exon fragment containing SS2 (SEQ ID NO:20), target sequence GOI (SEQ ID NO:17), exon fragment 1 containing SS1 (E1, SEQ ID NO:21), 3' homologous arm-3 (SEQ ID NO:8), and restriction enzyme sites (CATATG and AAGCT). The complete DNA template was inserted into the NdeI and HindIII restriction sites of the pUC57-Kan vector (SEQ ID NO:37). The GOI in this vector is the sequentially arranged IRES and EGFP coding regions, differing from the GOI in T4.A2.nrdB3.2 only in that the EGFP coding region was not truncated and rearranged.

[0278] To improve the versatility of the T4.A2.nrdB2.0 and 3.2 circularization systems, a 3.2+ circularization system was further designed, which can generate circular RNA without exon residues without using exon-like sequences.

[0279] In the T4.A2.nrdB3.2+ cyclization system, the intron ribozyme (SEQ ID NO:39) at positions 4-9 has a variable IGS sequence NNGNNN (N is any base of A, U, C, or G). Positions 4-5 (NN) represent the 5' end of the IGS, pairing with the 5' cyclization site SS2 of the GOI. Positions 6-9 (GNNN) represent the 3' end of the IGS, pairing with the 3' cyclization site SS1 of the GOI. Position 6 (G) is fixed and forms a swing pair with the 3' U of SS1. The 3' homologous arm spacer sequence is adjacent to the 3' end of SS1, and its first nucleotide sequence pairs with the 5th position sequence of the intron ribozyme. The SS1 and 3' homologous arm spacer sequences can form a 5-base complementary double helix structure P1 with the 3' end of the IGS. SS2 forms P10 with the 5' end of the IGS, either complementary or incompletely complementary.

[0280] The T4.A2.nrdB 3.2+ circularization system uses the sequence “ACGU” at positions 65-68 of the EGFP coding region (underlined in SEQ ID NO:17) as SS1 and the sequence “AA” at positions 69-70 as SS2 to circularize GOI CVB3-EGFP (SEQ ID NO:17). During vector construction, the EGFP coding region is truncated after nucleotide position 68, dividing it into coding region fragment 1 (i.e., the 5' fragment) and coding region fragment 2 (i.e., the 3' fragment), which are placed on the 3' and 5' sides of the IRES, respectively, to obtain FP-CVB3-EG-2 (SEQ ID NO:41).

[0281] Simultaneously, the original IGS sequence at positions 4-9 of the ribozyme (SEQ ID NO:40) in the T4.A2.nrdB3.2+ cyclization system was modified to UUGCGU. The "UU" at the 5' end of the IGS paired with SS2 (AA), and the "GCGU" at the 3' end of the IGS paired with SS1 (ACGU). The 3' homologous arm spacer sequence of the T4.A2.nrdB3.2+ cyclization system is ACAAAAAA (SEQ ID NO:42), and its adenosine at position 1 paired with uridine at position 5 of the intronic ribozyme. The final secondary structure formed by the SS1, SS2, IGS, and 3' homologous arm spacer sequences is shown in the lower left of Figure 10.

[0282] Specifically, the T4.A2.nrdB3.2+ circularization system synthesizes DNA containing the following interconnected elements in sequence during the circularization of CVB3-EGFP: the T7 transcription promoter (SEQ ID NO:1), the leader sequence (SEQ ID NO:2), the 5' homologous arm-3 (SEQ ID NO:7), the intron ribozyme sequence (SEQ ID NO:40), the target sequence GOI (SEQ ID NO:41), the 3' homologous arm spacer sequence-2 (SEQ ID NO:42), the 3' homologous arm-3 (SEQ ID NO:8), and restriction enzyme sites (CATATG and AAGCT). The synthesized DNA is then inserted into the NdeI and HindIII restriction sites of the pUC57-Kan vector (SEQ ID NO:37).

[0283] Following the steps and parameters in Example 1, the obtained plasmid vector was linearized, transcribed in vitro, detemplated, spliced ​​with introns (circularized), digested with RNase R, and subjected to E-Gel electrophoresis.

[0284] As shown in Figure 9(B), RNA circularization was successfully completed using both the T4.A2.nrdB3.0 and T4.A2.nrdB3.2 circularization systems, and the circular RNA obtained via T4.A2.nrdB3.2 did not contain any residual exon fragments. Figure 8(C) shows the E-Gel images of the products from steps 3 and 5 obtained from the two vectors. The circularization rate of the T4.A2.nrdB3.2 backbone was approximately 46.6%, comparable to that of the T4.A2.nrdB3.0 backbone (47.6%).

[0285] Figure 10, lower right, shows the E-Gel images of the products from steps 3-4, 5, and 6 obtained from the T4.A2.nrdB3.2+ vector. The T4.A2.nrdB3.2+ backbone showed a circularization rate of approximately 53.4% ​​for CVB3-EGFP, with 76.1% of the RNA remaining after digestion. In Figure 10, C represents the product of step 5, and C+R represents the product of step 6. Figure 11 shows that the circular RNA adapters obtained from T4.A2.nrdB3.2+ conform to the theoretical sequence and do not contain residual exon fragments. Some circular RNA is also generated during IVT, but the circularization rate is lower than that of step 5. Unless otherwise specified, the circularization rate in this application refers to the circularization rate of step 5.

[0286] Example 5. Preparation of high-purity circRNA and in vitro cell expression assay

[0287] Circular RNA was prepared based on the T4.A2.nrdB 3.1 circularization system, and its expression in cells was tested.

[0288] Specifically, DNA containing the following interconnected elements in sequence was synthesized: the T7 transcription promoter (SEQ ID NO:1), the leader sequence (SEQ ID NO:2), the 5' homologous arm-3 (SEQ ID NO:7), the intron sequence (SEQ ID NO:11), the exon containing SS2 (SEQ ID NO:20), the 5' spacer sequence (SEQ ID NO:22), the target sequence GOI (SEQ ID NO:24, total length 2397nt), the 3' spacer sequence (SEQ ID NO:23), the exon containing SS1 (SEQ ID NO:21), the 3' homologous arm spacer sequence (SEQ ID NO:43), the 3' homologous arm-3 (SEQ ID NO:8), and restriction enzyme sites (CATATG and AAGCT). The complete DNA template was inserted into the NdeI and HindIII restriction sites of the pUC57-Kan vector (SEQ ID NO:37).

[0289] Following the steps and parameters in Example 1, the obtained plasmid vector was linearized, transcribed in vitro, detemplated, and intron spliced ​​(circularized).

[0290] Subsequently, in order to minimize double-stranded RNA, preRNA, nicked RNA and other impurities, the crude circRNA obtained in step 5 was dephosphorylated and purified by HPLC.

[0291] Specifically, the crude circRNA obtained in step 5 was reacted with a thermosensitive phosphatase (NEB, MO289L) at 37°C for 1 hour, with a circRNA concentration of 0.5 μg / μL. The resulting sample was purified by magnetic beading and the solution was changed before use.

[0292] The dephosphorylated sample was then purified by reverse chromatography using a Sepax Technologies reverse chromatographic column and an Agilent 1260 series HPLC system. Gradient loading and elution were employed at a flow rate of 0.5 mL / min. Peaks were collected based on the retention time of each component by detecting the UV absorbance at 260 nm. The collected circRNA fraction was resuspended in WFI after buffer replacement and subjected to E-Gel electrophoresis according to step 7 of Example 1.

[0293] Figure 12 shows the reverse chromatography pattern of the RNA sample (A), E-Gel characterization of the chromatographic fraction (B), and a comparison of circRNA purity before dephosphorylation and after reverse chromatography purification (C). It can be seen that the RNA recovery rate of single-step chromatography is approximately 56%, and E-Gel analysis shows that the RNA purity after chromatographic purification is approximately 93%.

[0294] In addition, a linear mRNA control molecule encoding luciferase (Fluc) was prepared according to CN108291230B. Specifically, the 5'UTR of the mRNA backbone used the human α-globin 5'UTR, including the Koazk sequence, the Fluc coding sequence used was SEQ ID NO:24 of this paper, and the 3'UTR used was the chimeric 3'UTR formed by combining SEQ ID NO:86 and 115 of CN108291230B. The mRNA was prepared by in vitro co-transcription and purified by affinity chromatography.

[0295] Subsequently, the RNA obtained after the reverse chromatography and the control mRNA were expressed in vitro in cells.

[0296] HEK293T cells were used at a rate of 4 × 10⁻⁶ 4 Cells / well were seeded in DMEM high glucose medium (Gibco, 10566016) containing 10% fetal bovine serum and cultured in a 37°C, 5% CO2 incubator, and passaged every 2 to 3 days.

[0297] HEK293T cells were used at a rate of 1×10⁻⁶. 5 Cells were seeded per well in 24-well plates and cultured at 37°C in a 5% CO2 incubator. Once the cells reached 70-90% confluence, 500 ng of RNA was added to each well using Lipofectamine Messenger Max transfection reagent (Thermo Fisher LMRNA008), and the cells were incubated at 37°C in a 5% CO2 incubator. Samples were taken at 24h, 48h, 72h, and 96h of incubation, and cells were lysed using 1% Triton X-100 in PBS solution (AMRESCO, 0694-1L). Protein expression was then detected using a luciferase assay kit (Vazyme, DD1201-02-AB) according to the manufacturer's instructions.

[0298] As shown in Figure 12(D), the expression activity / level of Fluc-circRNA prepared based on the T4.A2.nrdB3.1 circularization system gradually increased from 24h to 72h, and from 48h to 96h, the expression activity / level was significantly higher than that of Fluc-linear mRNA, indicating that the circRNA prepared by the T4.A2.nrdB3.1 circularization system has a longer half-life in cells than mRNA.

[0299] In summary, the T4.A2.nrdB3.1 cyclization system exhibits high cyclization and purification recovery rates, meeting the requirements of industrial production. Furthermore, the purified circRNA demonstrates high purity and sustained high protein expression activity, highlighting its potential advantages in gene expression regulation and meeting the needs of clinical and other applications.

[0300] The embodiments of the present invention are not limited to those described above. Without departing from the spirit and scope of the present invention, those skilled in the art can make various changes and improvements to the present invention in form and detail, and these are all considered to fall within the protection scope of the present invention.

Claims

1. An RNA molecule comprising, from its 5' end to its 3' end: a self-splicing intron and a sequence to be circularized, wherein the elements are operatively linked in a sequential manner. The sequence to be circularized contains the target sequence. The target sequence includes i) an open reading frame and translational elements encoding the target peptide or protein, or ii) a non-coding RNA sequence. This self-splicing intron contains an internal guide sequence (IGS) that enables the RNA molecule to circularize and is removed from the RNA molecule during the circularization process. The sequence to be circumscribed contains a nucleotide at its 5' end that is reverse complementary to the 5' end sequence of the internal guide sequence, and at its 3' end that is reverse complementary to the 3' end sequence of the internal guide sequence.

2. The RNA molecule of claim 1, wherein the self-splicing intron is a class I self-splicing intron, preferably obtained from the Escherichia coli phage T4.A2.nrdB gene.

3. The RNA molecule of claim 1 or 2, wherein the self-splicing intron comprises a 5' intron sequence, an optional linker element, and a 3' intron sequence, wherein the 5' intron sequence comprises IGS; Preferably, the linker element can form a stem-loop structure, preferably the 5' intron sequence contains the nucleotide sequence shown in SEQ ID NO:13, preferably the 3' intron sequence contains the nucleotide sequence shown in SEQ ID NO:12, and preferably the linker element contains the nucleotide sequence shown in SEQ ID NO:

38.

4. The RNA molecule according to any one of claims 1-3, wherein 1-4 nucleotides at the 5' end of the sequence to be circularized are anticomplementary to the 5' end of the internal guide sequence, wherein 2-4 nucleotides at the 3' end of the sequence to be circularized are anticomplementary to the 3' end of the internal guide sequence, preferably 1-2 nucleotides at the 5' end of the sequence to be circularized are anticomplementary to the 5' end of the internal guide sequence, and preferably 4 nucleotides at the 3' end of the sequence to be circularized are anticomplementary to the 3' end of the internal guide sequence.

5. The RNA molecule according to any one of claims 1-4, wherein, The target sequence contains a nucleotide at its 5' end that is reverse complementary to the 5' end sequence of the internal guide sequence, and at its 3' end that is reverse complementary to the 3' end sequence of the internal guide sequence. i) The target sequence contains from the 5' end to the 3' end. (a) The translational functional element and the open reading frame encoding the target peptide or protein, (b) the open reading frame encoding the target peptide or protein, and the translational functional element. (c) The 3' end sequence of the translational element, the open reading frame encoding the target peptide or protein, and the 5' end sequence of the translational element, wherein the 5' end sequence and the 3' end sequence of the translational element are arranged in this order to form the translational element, or (d) The 3' end sequence of the open reading frame encoding the target peptide or protein, the translational functional element, and the 5' end sequence of the open reading frame encoding the target peptide or protein, wherein the 5' end sequence of the open reading frame encoding the target peptide or protein and the 3' end sequence of the open reading frame encoding the target peptide or protein are arranged in this order to form the open reading frame encoding the target peptide or protein. Preferably, the translation functional element is selected from translation initiation elements and translation enhancement elements, or ii) The target sequence contains a non-coding RNA sequence, which (a) Contains the non-coding RNA sequence, or (b) The non-coding RNA sequence comprises the 3' end sequence of the non-coding RNA sequence and the 5' end sequence of the non-coding RNA sequence from the 5' end to the 3' end, wherein the 5' end sequence of the non-coding RNA sequence and the 3' end sequence of the non-coding RNA sequence are arranged in this order to form the non-coding RNA sequence.

6. The RNA molecule according to any one of claims 1-4, wherein the sequence to be circularized further comprises: i) The exon naturally adjacent to the 3' side of the self-splicing intron, or the sequence corresponding to the naturally adjacent exon. ii) The exon naturally adjacent to the 5' side of the self-splicing intron, or the sequence corresponding to the naturally adjacent exon. iii) Optional 5' interval sequence, and iv) Optional 3' interval sequence, in, The exon naturally adjacent to the 3' side of the self-splicing intron, or the sequence corresponding to the naturally adjacent exon, contains a nucleotide at its 5' end that is reverse complementary to the 5' end sequence of the internal guide sequence.

7. The RNA molecule of claim 6, wherein the sequence to be circularized comprises, from the 5' end to the 3' end: The exon naturally adjacent to the 3' side of the self-splicing intron or the sequence corresponding to the naturally adjacent exon, an optional 5' spacer sequence, the target sequence, an optional 3' spacer sequence, and the exon naturally adjacent to the 5' side of the self-splicing intron or the sequence corresponding to the naturally adjacent exon. Preferably, the sequence to be circularized includes a 5' spacer sequence and a 3' spacer sequence; more preferably, the 5' spacer sequence and the 3' spacer sequence are capable of reverse complementary pairing.

8. The RNA molecule of claim 6 or 7, wherein the exon naturally adjacent to the 3' side of the self-splicing intron or the sequence corresponding to the naturally adjacent exon, and the exon naturally adjacent to the 5' side of the self-splicing intron or the sequence corresponding to the naturally adjacent exon, respectively contain nucleotide sequences of GU and GCGU, the nucleotide sequences shown in SEQ ID NO: 20 and 21, or the nucleotide sequences shown in SEQ ID NO: 18 and 19.

9. The RNA molecule of claim 1 or 2, wherein the target sequence contains SS2: 5'-NN-3' at the 5' end and SS1: 5'-NNNU-3' at the 3' end; the self-splicing intron contains a modified internal guide sequence (IGS) containing 5'-NNGNNN-3', such that the four bases 5'-GNNN-3' at the 3' end are anticomplementary to SS1, and the two bases 5'-NN-3' at the 5' end are anticomplementary to SS2; wherein N is any nucleotide from A, U, C, and G; Preferably, the self-splicing intron comprises a 5' end intron sequence, an optional connecting element, and a 3' end intron sequence, wherein the 5' end intron sequence comprises IGS; preferably, the connecting element is capable of forming a stem-loop structure; Preferably, the 5' intron fragment comprises a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:44; preferably, the 3' intron fragment comprises a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:12; preferably, the linker element comprises a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:44; preferably, the linker element comprises a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:

44. NO:38 is a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.

10. The RNA molecule of claim 9, wherein SS1 is 5'-AA-3', SS2 is 5'-ACGU-3', and the modified internal guide sequence is 5'-UUGCGU-3'; Preferably, the 5' intron fragment comprises a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:

45.

11. The RNA molecule according to any one of claims 1-10, further comprising a 5' homologous arm on the 5' side of the self-splicing intron, a 3' homologous arm on the 3' side of the sequence to be circularized, and a 3' homologous arm spacer sequence between the sequence to be circularized and the 3' homologous arm, wherein the elements are operatively connected, wherein the 5' homologous arm and the 3' homologous arm are capable of reverse complementary pairing; preferably, the 3' homologous arm spacer sequence is a poly(A) or poly(AC) sequence.

12. The RNA molecule of claim 11, wherein the 5' homologous arm and the 3' homologous arm comprise the nucleotide sequences shown in SEQ ID NO:3 and 4, SEQ ID NO:5 and 6, or SEQ ID NO:7 and 8, respectively.

13. The RNA molecule according to any one of claims 1-12, wherein the translational functional element is a translation initiation element, wherein the translation initiation element is preferably an internal ribosome entry site (IRES), wherein the IRES is preferably Coxsackievirus B3 (CVB3).

14. A DNA molecule capable of transcribing into an RNA molecule according to any one of claims 1-13.

15. The DNA molecule of claim 14, wherein it is a single-stranded linear DNA, a single-stranded closed DNA, or a double-stranded DNA.

16. The DNA molecule of claim 14 or 15, comprising an optional 5' homologous arm, a self-splicing intron, a sequence to be circularized, an optional 3' homologous arm spacer sequence, and an optional 3' homologous arm, wherein the elements are operatively linked in sequence.

17. The DNA molecule of claim 16, further comprising an RNA polymerase promoter and an optional leader sequence on the 5' side of the 5' homologous arm; or further comprising a restriction endonuclease site on the 3' side of the 3' homologous arm.

18. A vector for preparing circular RNA, comprising an RNA molecule according to any one of claims 1-12, or a DNA molecule according to any one of claims 14-17.

19. A method for preparing circular RNA, comprising: i) Incubate the RNA molecule of any one of claims 1-13 under suitable conditions, wherein suitable conditions include the presence of magnesium ions and guanosine, or (ii) (a) Transcribe RNA from the DNA molecule of any one of claims 14-17 under suitable conditions, and (b) Incubate the RNA under suitable conditions, wherein the suitable conditions in step (b) include the presence of magnesium ions and guanosine. Guanosine is preferably guanosine triphosphate.

20. A host cell comprising an RNA molecule according to any one of claims 1-13, a DNA molecule according to any one of claims 14-17, or a vector according to claim 18.

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