Recombinant nucleic acid molecule for preparing scar-free circular RNA based on stem-loop structure and use thereof

Abstract

Provided are a method for preparing scar-free circular RNA, and a recombinant nucleic acid molecule for preparing scar-free circular RNA and use thereof. The provided recombinant nucleic acid molecule based on the IRES stem-loop structure can be used for preparing a circular nucleic acid molecule completely free of exogenous sequences without altering any sequence of the IRES, thereby avoiding unpredictable effects on ribosome recruitment function caused by IRES mutations.

Description

A recombinant nucleic acid molecule for preparing scarless circular RNA based on stem-loop structure and its application.

[0001] This application claims priority to the earlier application filed on November 7, 2024, with patent application number 2024115848515, entitled "A Recombinant Nucleic Acid Molecule for Preparing Scarless Circular RNA Based on Stem-Loop Structure and Its Application". The entire contents of the earlier application are incorporated herein by reference. Technical Field

[0002] This disclosure relates to the fields of molecular biology, bioengineering technology, and gene recombination technology, and in particular to a method for preparing and applying a circular nucleic acid molecule with completely exogenous sequences eliminated. Background Technology

[0003] mRNA drugs, as a highly promising drug delivery method, can efficiently express proteins intracellularly, making them ideal for vaccines and protein supplementation therapies. However, several challenges remain, including mRNA stability and organ-specific expression. Unlike linear RNA, circular RNA (circRNA) lacks a 5′ cap and a 3′ polyA tail, exhibiting a covalently closed circular structure. This allows circRNA to be protected from exonuclease degradation, resulting in a relatively longer half-life and greater stability within cells. circRNA can also serve as transcripts for protein translation. Inserting an internal ribosome entry site (IRES) upstream of the protein-coding sequence (ORF) within circRNA can recruit ribosomes for protein translation. Comparative evaluations of known IRES activities and recombinant optimizations have been reported. The greater stability and protein translation capabilities of circRNA make it a potential alternative to mRNA molecules.

[0004] Currently, there are various chemical and biological methods for circularizing RNA. Biological methods include the use of T4 RNA ligase, group I intron ribozymes, group II intron ribozymes, and the Tornado expression system. Natural group I intron ribozymes can catalyze their own excision from the RNA precursor through two consecutive transesterification reactions. A permuted intron-exon (PIE) strategy has been developed using group I intron ribozymes. This PIE splicing strategy inverts the half-intron sequences flanking the group I ribozyme, resulting in linear RNA containing a 3' intron-E2-target RNA-E1-5' intron structure. Only the addition of GTP and Mg2+ is required. 2+As a cofactor, it enables intron splicing to obtain E2-target RNA-E1 circular RNA. Further engineering improvements have been made to the PIE method to enhance its circularization efficiency. Researchers such as Daniel G. Anderson used Anabaena tRNA ribozyme introns as the main component, adding complementary homologous arms to both ends of the "3' intron-E2-target RNA-E1-5' intron" to bring the linear RNA ends closer together. Spacer sequences (including polyA or polyAC and complementary homologous arms) were added to both sides of the target RNA and between E2 and E1 to ensure the ribozyme structure is not interfered with by other RNA sequences, forming an independent splice bubble. This allows the PIE method to achieve highly efficient circularization in vitro, making it more suitable for circularizing long RNAs. However, the E2 and E1 exons and the spacer sequences that assist circularization in the PIE method are redundant sequence structures and may have unnecessary effects, such as immunogenicity, interference with the circular RNA structure, and impact on translation.

[0005] Building upon the PIE method, to eliminate redundant exon sequences, researchers such as Zuo Chijian developed a systematic method for screening target protein coding region sequences using T4Td intron ribozymes. This method involves screening for exon sequences (5'-TTGGGTCT-3') or similar sequences at the T4Td ribozyme recognition site within the target coding region sequence, ensuring that the upstream and downstream sequences have few hairpin structures and low free energy. The exon sequence is then truncated at the T4Td ribozyme recognition site and reassembled to form a linear structure: "3' intron - coding region truncated fragment 1 - IRES - coding region truncated fragment 2 - 5' intron." This allows the intron to splice within the coding region, yielding circular RNA without redundant sequences. However, this method has limitations: the coding region sequence must contain the exon sequence (5'-TTGGGTCT-3') or similar sequences at the T4Td ribozyme recognition site, limiting its application. Furthermore, researchers such as Zuo Chijian employed a similar approach, screening for positions with fewer hairpin structures and lower free energy in the IRES sequences of Enterovirus A90, Caprine kobuvirus, and Echovirus E29. They then mutated a sequence at this position similar to the T4Td ribozyme recognition site into a 5'-TTGGGTCT-3' ribozyme recognition site, and truncated and reassembled it at this site to form a linear structure: "3' intron - IRES truncated fragment II - coding region - IRES truncated fragment I - 5' intron," allowing intron splicing within the IRES region. However, this method mutated the IRES, unpredictably affecting its function in recruiting ribosomes to initiate translation. Therefore, eliminating redundant exon sequences in the construction of circular RNA still requires the development of more efficient circularization strategies. Summary of the Invention

[0006] To address the problems of existing technologies, this disclosure provides a method for preparing scarless circular RNA based on a stem-loop structure, and a recombinant nucleic acid molecule for preparing scarless circular RNA based on the stem-loop structure. Taking the IRES sequence as an example, this disclosure uses software such as RNAFold to simulate its secondary structure, screens for stem-loop structures, designs breakpoints at the loop position, connects the broken IRES to group I introns, and mutates the P1 and P10 guide sequences of the group I introns to match the sequences at the IRES breakpoint. These sequences are then combined with other functional sequences to form a recombinant nucleic acid molecule for preparing scarless circular RNA. This disclosure, based on the IRES stem-loop structure, does not perform any base mutations on the IRES itself, but instead selects to mutate the group I introns to match the loop position sequence in the stem-loop position of the IRES, ensuring that the ribozyme splices at the predetermined IRES loop position. This disclosure ensures that the circular RNA product not only lacks redundant sequences from group I intron circularization residues, but also avoids mutations in the IRES sequence due to the need for splice sites during circularization. Furthermore, the presence of stems in the selected IRES ensures that the ribozyme forms independent splice vesicles to effectively circularize the target RNA.

[0007] On one hand, this disclosure provides a recombinant nucleic acid molecule for preparing scarless circular RNA, comprising elements arranged in the following order from the 5' to 3' direction:

[0008] a.3'I group introns or their mutant fragments (intron fragment II),

[0009] b. Unit I fragment II, whose 5' end includes the II ribozyme recognition fragment.

[0010] c. Functional units,

[0011] d. The first unit fragment I, whose 3' end includes the I ribozyme recognition fragment,

[0012] e.5'I group introns or their mutant fragments (intron fragment I);

[0013] The functional units include IRES, nucleic acid aptamers, protein binding sequences, protein coding regions, non-coding regions, etc., or combinations thereof;

[0014] The intron fragment II is located at the 3' end of the intron fragment I, that is, the complete group I intron or its mutant sequence includes: intron fragment I - intron fragment II, where "-" represents a phosphodiester bond;

[0015] The first unit fragment II is located at the 3' end of the first unit fragment I, that is, the complete first unit sequence includes: first unit fragment I - first unit fragment II, where "-" represents a phosphodiester bond;

[0016] Wherein, the 3' end of the first unit fragment I contains a first ribozyme recognition fragment, which is composed of a first predetermined number of nucleotides located at the 3' end of the first unit fragment I;

[0017] The 5' end of the first unit fragment II contains a second ribozyme recognition fragment, which is composed of a second predetermined number of nucleotides located at the 5' end of the first unit fragment II;

[0018] The intron mutants in group I recognize and covalently link the first ribozyme recognition fragment and the second ribozyme recognition fragment to obtain the circular nucleic acid molecule, that is, the complete first unit sequence in the scarless circular RNA contains the first ribozyme recognition fragment - the second ribozyme recognition fragment ("circular fragment"), where "-" represents a phosphodiester bond;

[0019] The first unit has a local stem structure, a local double-chain structure, or a local hairpin structure, wherein the local stem structure, local double-chain structure, or local hairpin structure is adjacent to or includes the cyclic segment.

[0020] Preferably, the first unit is a nucleic acid aptamer or a translation initiation element;

[0021] Preferably, the cyclic fragment is located in the ring of the stem-loop structure of the translation initiation element, and a double-stranded structure formed by complementary pairing sequences exists within 100 bases upstream and downstream of the cyclic fragment; preferably, the double-stranded structure contains at least 5 consecutive complementary pairing bases; even more preferably, the number of complementary and non-complementary pairing bases in the stem of the stem-loop structure exceeds 20 bp.

[0022] In one embodiment, the group I intron mutant contains a mutation in the guide region of P1 / P10, and the mutated guide region is complementary to the first / second ribozyme recognition fragment in the first unit fragment I / II, the first / second ribozyme recognition fragment being defined as a circular fragment.

[0023] In one embodiment, the 3' end base of the first ribozyme recognition fragment is T, the first predetermined number of nucleotides is selected from 3-6 nucleotides, and the second predetermined number of nucleotides is selected from 0-3 nucleotides;

[0024] Preferably, the group I introns are mutants of the Ana ribozyme, wherein the first ribozyme recognition fragment is 5'-N1N2N3T-3' or 5'-N2N3T-3', and the second ribozyme recognition fragment is 5'-N4N5N6-3' or 5'-N4N5-3'. The Ana ribozyme mutant contains the following mutant region N in the 5' intron fragment. 6’ N 7’ ATAAN5’ N 4’ GN 3’ N 2’ Where N is A, U, C, G, or T, or does not exist. 3’ N 2’ Pairing with N2N3 in reverse complementary direction, N 5’ N 4’ Pairing with N4N5 in reverse complementary direction, N 5’ N 4’ With N 6’ N 7’ Reverse complementary pairing, the base complementary pairing includes AU, GC, GU, AT, and GT base pairs;

[0025] Preferably, the group I introns are mutants of the T4td ribozyme, wherein the first ribozyme recognition fragment is 5'-N1N2N3N4N5T-3' or 5'-N2N3N4N5T-3' or 5'-N3N4N5T-3', and the second ribozyme recognition fragment is 5'-N6N7-3' or N6 or absent, wherein N is A, U, C, G or T. The T4td ribozyme mutant contains the following mutant region N in the 5' intron fragment. 8’ AATTGN 7’ N 6’ GN 5’ N 4’ N 3’ N 2’ N 1’ Where N is A, U, C, G, or T, or does not exist. 5’ N 4’ N 3’ N 2’ N 1’ Pairing with N1N2N3N4N5 in reverse complementary order, or N 5’ N 4’ N 3’ N 2’ Pairing with N2N3N4N5 in reverse complementary order, or N 5’ N 4’ N 3’ Pairing with N3N4N5 in reverse complementary direction; N 7’ N 6’ Pairing with N6N7 in reverse complementary direction, or N 6’ Complementary pairing with N6, or preferably, when N6N7 is absent, the 3' end T base of the I ribozyme recognition fragment is adjacent to the stem structure of the first unit, then the P10 guide sequence N 7’ N 6’ It can be without mutation; N 8’ With N 6’Complementary pairing, the complementary base pairing includes AU, GC, GU, AT, GT base pairs; preferably, N6 is U or C, N 6’ No mutations occur.

[0026] In one embodiment, the first unit fragment is an active sequence having the ability to initiate translation of the aforementioned functional unit, and the first unit fragment I and the first unit fragment II are translation initiation element fragment I and translation initiation element fragment II, respectively;

[0027] Optionally, the translation initiation element sequence comprises one or more of the following sequences: IRES sequence, 5'UTR sequence, Kozak sequence, sequence containing m6A modification, complementary sequence of ribosomal 18S rRNA, and aptamer sequence.

[0028] In one embodiment, the IRES sequence includes ribosome entry site sequences such as HRV-B3, HRV-B92, iHRV-B37, iHRV-B97, iHRV-B4, iHRV-C11, iPV2, Human XIAP, CVB3, and EMCV, as well as mutants thereof, and artificially recombined shuffledIRES#01, shuffledIRES#38, shuffledIRES#03, shuffledIRES#42, and mutants thereof;

[0029] Preferably, the nucleotide sequence of the translation initiation element is shown in any one of SEQ ID NO: 59, 60, 61, 62, 63, 64, 66, 68, 70 and 86;

[0030] Preferably, the nucleotide sequence of the translation initiation element fragment I is as shown in SEQ ID NO:72, and the nucleotide sequence of the translation initiation element fragment II is as shown in SEQ ID NO:73;

[0031] Preferably, the nucleotide sequence of the translation initiation element fragment I is shown in SEQ ID NO:98, and the nucleotide sequence of the translation initiation element fragment II is shown in SEQ ID NO:99;

[0032] Preferably, the nucleotide sequence of the translation initiation element fragment I is shown in SEQ ID NO:90, and the nucleotide sequence of the translation initiation element fragment II is shown in SEQ ID NO:91;

[0033] Preferably, the nucleotide sequence of the translation initiation element fragment I is shown in SEQ ID NO:102, and the nucleotide sequence of the translation initiation element fragment II is shown in SEQ ID NO:103;

[0034] Preferably, the nucleotide sequence of the translation initiation element fragment I is shown in SEQ ID NO:94, and the nucleotide sequence of the translation initiation element fragment II is shown in SEQ ID NO:95;

[0035] Preferably, the nucleotide sequence of the translation initiation element fragment I is shown in SEQ ID NO:72, and the nucleotide sequence of the translation initiation element fragment II is shown in SEQ ID NO:87.

[0036] Preferably, the nucleotide sequence of the translation initiation element fragment I is shown in SEQ ID NO:106, and the nucleotide sequence of the translation initiation element fragment II is shown in SEQ ID NO:107.

[0037] Preferably, the nucleotide sequence of the translation initiation element fragment I is shown in SEQ ID NO:108, and the nucleotide sequence of the translation initiation element fragment II is shown in SEQ ID NO:109.

[0038] Preferably, the nucleotide sequence of the translation initiation element fragment I is shown in SEQ ID NO:110, and the nucleotide sequence of the translation initiation element fragment II is shown in SEQ ID NO:111.

[0039] Preferably, the nucleotide sequence of the translation initiation element fragment I is shown in SEQ ID NO:112, and the nucleotide sequence of the translation initiation element fragment II is shown in SEQ ID NO:113.

[0040] In one embodiment, the nucleotide sequence of intron fragment I is shown in any one of SEQ ID NO:76, 77, 78, 92, 96, 100 and 104, and the nucleotide sequence of intron fragment II is shown in SEQ ID NO:74 or 75.

[0041] Preferably, the nucleotide sequence of intron fragment I is as shown in SEQ ID NO: 76 or 77, and the nucleotide sequence of intron fragment II is as shown in SEQ ID NO: 74;

[0042] Preferably, the nucleotide sequence of intron fragment I is shown in any one of 78, 92, 96, 100 and 104, and the nucleotide sequence of intron fragment II is shown in SEQ ID NO:75.

[0043] In one embodiment, the functional unit comprises at least one coding region; alternatively, the functional unit comprises at least two coding regions, each coding region independently encoding any type of target polypeptide.

[0044] In one implementation, the functional unit comprises at least two coding regions, wherein any two adjacent coding regions are connected by a connector;

[0045] Preferably, the linker is a polynucleotide encoding a 2A peptide.

[0046] In one implementation, the functional unit comprises at least two coding regions, wherein a translation initiation element is connected between any two adjacent coding regions;

[0047] Optionally, the translation initiation element located between any two adjacent coding regions contains one or more of the following sequences: IRES sequence, 5'UTR sequence, Kozak sequence, sequence containing m6A modification, complementary sequence of ribosomal 18S rRNA, and aptamer.

[0048] In one implementation, the functional unit is a coding sequence for a human or non-human protein;

[0049] Preferably, the human or non-human protein is selected from one or more of the following: antigen, antibody, antigen-binding fragment, therapeutic peptide, fluorescent protein, CAR-T molecule, 2A peptide, protein with disease therapeutic activity, and protein with gene editing activity.

[0050] Preferably, the human or non-human protein is a tandem tumor antigen peptide and a Fluc protein; more preferably, the amino acid sequence of the tandem tumor antigen peptide is shown in SEQ ID NO:13, and the nucleotide sequence of the Fluc protein is shown in SEQ ID NO:22.

[0051] In one embodiment, the recombinant nucleic acid molecule further includes an insertion element located between the coding element and the translation initiation element;

[0052] The insertion element is selected from at least one of the following groups (i)-(iii):

[0053] (i) transcriptional regulatory elements, (ii) translational regulatory elements, and (iii) purification elements;

[0054] Optionally, the insert element comprises a sequence of one or more combinations of the following:

[0055] Untranslated region sequences, polyA sequences, polyAC sequences, aptamer sequences, riboswitch sequences, sequences that bind transcription regulatory factors, antisense oligonucleotides (ASO), small interfering RNA (siRNA), miRNA, miRNA sponges, or lncRNA.

[0056] In one embodiment, the nucleotide sequence of the recombinant nucleic acid molecule is shown in any one of SEQ ID NO: 82, 83, 84, 88, 89, 93, 97, 101, 105, 114, 115, 116 and 117.

[0057] On the other hand, this disclosure provides a recombinant expression vector, wherein the recombinant expression vector comprises the aforementioned recombinant nucleic acid molecule;

[0058] Preferably, the vector contains a promoter upstream of the recombinant nucleic acid molecule;

[0059] More preferably, the promoter includes one or more promoters, including but not limited to T7 promoter, Sp6 promoter, T3 promoter, Ptac promoter, trp promoter, CMV promoter, PGK promoter, Ubc promoter, SV40 promoter, CAG promoter, U6 promoter and H1 promoter;

[0060] Preferably, the promoter is the T7 promoter, whose nucleotide sequence is shown in SEQ ID NO:85.

[0061] On the other hand, this disclosure provides the use of the aforementioned recombinant nucleic acid molecules or the aforementioned recombinant expression vectors in the in vitro preparation of circular RNA.

[0062] On the other hand, this disclosure provides a method for preparing circular RNA in vitro, which includes the following steps:

[0063] (1) The aforementioned recombinant nucleic acid molecules or the aforementioned recombinant expression vector are transcribed to form circularized precursor nucleic acid molecules;

[0064] (2) The circularized precursor nucleic acid undergoes a circularization reaction to obtain circular RNA;

[0065] Optionally, the method further includes the step of purifying the circular RNA.

[0066] On the other hand, this disclosure provides circular RNA obtained according to the aforementioned recombinant nucleic acid molecules, the aforementioned recombinant expression vectors, or the aforementioned methods.

[0067] On the other hand, this disclosure provides a host cell that expresses the aforementioned recombinant nucleic acid molecule or the aforementioned recombinant expression vector.

[0068] On the other hand, this disclosure provides a composition comprising the aforementioned recombinant nucleic acid molecule, the aforementioned recombinant expression vector or the aforementioned circular RNA and one or more pharmaceutically acceptable vectors.

[0069] In one embodiment, the pharmaceutically acceptable carrier is selected from lipids, polymers, or lipid-polymer complexes.

[0070] On the other hand, this disclosure provides a method for expressing functional units in cells for non-disease treatment purposes, wherein the method includes the step of transferring the aforementioned circular RNA or the aforementioned composition into cells.

[0071] On the other hand, this disclosure provides the use of the aforementioned circular RNA in the preparation of a medicament for preventing or treating a disease by means of administering the aforementioned circular RNA to a subject;

[0072] Preferably, the drug is an anti-tumor drug or an mRNA vaccine.

[0073] On the other hand, this disclosure provides the application of the aforementioned recombinant nucleic acid molecules, the aforementioned recombinant expression vectors, the aforementioned host cells, and the aforementioned compositions in improving the targeting of mRNA drugs and improving the expression efficiency of mRNA drugs.

[0074] On the other hand, this disclosure provides the application of the aforementioned recombinant nucleic acid molecules and recombinant expression vectors in expressing proteins in cells.

[0075] On the other hand, this disclosure provides a method for constructing the aforementioned recombinant nucleic acid molecule based on the stem-loop structure in the first unit sequence, and the method for T4td intron ribozymes includes:

[0076] (1) Based on the first unit sequence, the secondary structure was predicted using RNA structure prediction software;

[0077] (2) Based on the secondary structure, the stem-loop structure in the first unit sequence is screened. The loop position contains at least 4 free bases, including T or U bases. The upstream of the T or U base contains at least 3 free bases, and the downstream of the T or U base contains at least 0 free bases. The first unit sequence is split into two fragments at the 3' position of T or U in the screened loop position sequence. The 5' end fragment is the first unit fragment I, and the 3' end fragment is the first unit fragment II.

[0078] (3) Based on the splitting position of the first unit sequence, the P1 and P10 guide sequences of the group I intron ribozymes are mutated to make them complementary to the recognition fragments of the first and second ribozymes, forming the P1 and P10 structures of the group I ribozymes. Then, the first unit fragment I is connected to the 5' end of the mutated 5' group I intron, and the first unit fragment II is connected to the 3' end of the mutated 3' group I intron.

[0079] (4) The 5' end of the functional sequence is linked downstream of the first unit fragment II, and the 3' end is linked to the 5' end of the first unit fragment I to obtain a recombinant nucleic acid molecule.

[0080] In one embodiment, the ring position in step (2) contains at least 6 free bases, including T or U bases, with at least 4 free bases upstream of the T or U base and at least 1 free base downstream of the T base, and the ring position sequence is N2N3N4N5TN6 or N2N3N4N5UN6, where N2-N6 are A, G, C, U or T.

[0081] In a preferred embodiment, the loop position in step (2) contains at least 8 free bases, including a T or U base, with at least 5 free bases upstream of the T or U base and at least 2 free bases downstream of the T or U base. The loop position sequence is N1N2N3N4N5TN6N7 or N1N2N3N4N5UN6N7, where N1-N7 are A, G, C, U, or T, or are absent. The intron ribozyme in group I is a mutant of the T4td ribozyme, which contains the following mutant region N in the P1 / P10 guide sequence. 8’ AATTGN 7’ N 6’ GN 5’ N 4’ N 3’ N 2’ N 1’ , where N 1’ -N 8’ It is a mutant base, N 5’ N 4’ N 3’ N 2’ N 1’ Pairing with N1N2N3N4N5 in reverse complementary order, or N 5’ N 4’ N 3’ N 2’ Pairing with N2N3N4N5 in reverse complementary order, or N 5’ N 4’ N 3’ Pairing with N3N4N5 in reverse complementary direction; N 7’ N 6’ Pairing with N6N7 in reverse complementary direction, or N 6’ If the N6 base is complementary to N6, or if N6N7 is absent, and the 3' end T base of the I ribozyme recognition fragment is adjacent to the stem structure of the first unit, then the P10 guide sequence N... 7’ N 6’ It can be without mutation; N 8’ With N 6’ Complementary pairing, the complementary base pairing includes AU, GC, GU, AT, and GT base pairs.

[0082] On the other hand, this disclosure provides a method for constructing the aforementioned recombinant nucleic acid molecule based on the stem-loop structure of the sequence in the first unit, and the method for Ana intron ribozymes includes:

[0083] (1) Based on the first unit sequence, the secondary structure was predicted using RNA structure prediction software;

[0084] (2) Based on the secondary structure, the stem-loop structure in the first unit is screened. The loop position contains at least 5 free bases, including T or U bases. The upstream of the T or U base contains at least 2 free bases, and the downstream of the T or U base contains at least 2 free bases. The first unit sequence is split into two fragments at the 3' position of T or U in the screened loop position sequence. The 5' end fragment is the first unit fragment I, and the 3' end fragment is the first unit fragment II.

[0085] (3) Based on the splitting position of the first unit, the P1 and P10 guide sequences of the intron ribozyme of group I are mutated to make them complementary to the recognition fragment of ribozyme I and the recognition fragment of ribozyme II, forming the P1 and P10 structures of group I ribozyme. Then, the first unit fragment I is connected to the 5' end of the mutated 5' group I intron, and the first unit fragment II is connected to the 3' end of the mutated 3' group I intron.

[0086] (4) The 5' end of the functional sequence is linked downstream of the first unit fragment II, and the 3' end is linked to the 5' end of the first unit fragment I to obtain a recombinant nucleic acid molecule.

[0087] In one embodiment, the loop position in step (2) contains at least 7 free bases, including a T or U base, with at least 3 free bases upstream of the T or U base and at least 3 free bases downstream of the T or U base. The loop position sequence is N1N2N3TN4N5N6 or N1N2N3UN4N5N6, where N1-N6 are A, G, C, U, or T, or are absent. The intron ribozyme in group I is a mutant of the Ana ribozyme, which contains the following mutant region N in the P1 / P10 guide sequence. 6’ N 7’ ATAAN 5’ N 4’ GN 3’ N 2’ , where N 2’ -N 7’ It is a mutant base, in which N 3’ N 2’ Pairing with N2N3 in reverse complementary direction, N 5’ N 4’ Pairing with N4N5 in reverse complementary direction, N 6’ N 7’ With N 5’ N 4’Reverse complementary pairing, the base complementary pairing includes AU, GC, GU, AT, and GT base pairs.

[0088] In one implementation, the aforementioned first unit is a translation initiation element or a nucleic acid aptamer.

[0089] On the other hand, the present invention provides a computer-readable storage medium storing a program that implements the aforementioned construction method.

[0090] On the other hand, the present invention provides a computer terminal, the computer terminal including a processor and a readable storage medium as described in claim 30, the processor being configured to invoke a program stored in the readable storage medium to execute the construction method; preferably, the computer terminal further includes a display device for outputting visualization results. Beneficial effects

[0091] The recombinant nucleic acid molecule based on the stem-loop structure of the first unit disclosed herein can be used to prepare circular nucleic acid molecules with completely exogenous sequences eliminated without altering any sequence of the first unit, thus avoiding the unpredictable impact of mutations on the function of the first unit. IRES generally have a complex three-dimensional structure, including stem-loop structures. Using RNAFold to predict the secondary structure of IRES, the sequence at the loop position in the stem-loop structure of the IRES is split at the 3' end of the T base and spliced ​​with group I ribozyme. At the same time, the corresponding bases in the P1 and P10 guide sequences of group I introns are mutated, causing the linear RNA to circularize at the stem-loop position in the IRES. This ensures that only the functional sequence is retained in the circularized RNA product, without the redundant sequence of group I intron circularization residue. Furthermore, due to the presence of the stem at the selected stem-loop position in the IRES, it can be ensured that the ribozyme forms an independent splice vesicle, effectively promoting RNA circularization. Attached Figure Description

[0092] Figure 1 is a schematic diagram of the method for constructing recombinant nucleic acid molecules based on stem-loop structure to prepare scarless circular RNA.

[0093] Figure 2 is a schematic diagram of the recombinant nucleic acid molecular structure for preparing scarless circular RNA.

[0094] Figure 3 is a schematic diagram of the structures of wild-type T4td ribozyme and Ana ribozyme, wherein the nucleotides in italics belong to the intron ribozyme mutation region in the group I intron mutation method described in this disclosure.

[0095] Figure 4 shows the recombinant nucleic acid molecular structure of PIE-circularized RNA without a spacer (left), the recombinant nucleic acid molecular structure of PIE-circularized RNA with a polyAC spacer (middle), and the recombinant nucleic acid molecular structure of PIE-circularized RNA with an internal homologous arm (Arm) (right).

[0096] Figure 5 shows the circularization electrophoresis diagrams and circularization efficiency statistics of the RNA sequences (SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20) of the three RNA nucleic acid molecular structures in Figure 4. The electrophoresis was performed using Ana ribozyme and IRES. MP,75 And the antigen peptide coding sequence. IVT is the in vitro transcription product of recombinant nucleic acid molecules, C is the product after cyclization of the in vitro transcription product, and C+R is the product after RNase R cleavage of the cyclized product.

[0097] Figure 6 shows the circularization electrophoresis diagrams and circularization efficiency statistics of the RNA sequences (SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19) of the three RNA nucleic acid molecular structures in Figure 4. The electrophoresis was performed using T4td ribozyme and IRES. MP,75 And the antigen peptide coding sequence. IVT is the in vitro transcription product of recombinant nucleic acid molecules, C is the product after cyclization of the in vitro transcription product, and C+R is the product after RNase R cleavage of the cyclized product.

[0098] Figure 7 shows the circularization electrophoresis diagrams and circularization efficiency statistics of the RNA sequences (SEQ ID NO: 23-28) of the three RNA nucleic acid molecular structures in Figure 4, using T4td ribozyme or Ana ribozyme, CVB3 IRES, and Fluc coding sequences. IVT is the in vitro transcription product of the recombinant nucleic acid molecule, C is the product after the in vitro transcription product is circularized, and C+R is the product after the circularized product is cleaved by RNase R.

[0099] Figure 8 shows a schematic diagram of the shortened Ana ribozyme exon structure and an electrophoresis image of the circularized RNA (SEQ ID NO:29-36) obtained by the PIE method with internal homologous arms.

[0100] Figure 9 shows a schematic diagram of the shortened T4td ribozyme exon structure and an electrophoresis image of the circularized RNA (SEQ ID NO:37-40) obtained by the PIE method with internal homologous arms.

[0101] Figure 10 shows the sequencing validation results and circularization efficiency bar charts for shortening Ana ribozyme exons in the PIE method with internal homologous arms (SEQ ID NO:29-36).

[0102] Figure 11 shows the sequencing validation results and circularization efficiency bar chart of shortened T4td ribozyme exons in the PIE method with internal homologous arms (SEQ ID NO:37-40).

[0103] Figure 12 shows a schematic diagram of the shortened Ana ribozyme exon structure, an electrophoresis diagram of the circularized RNA, and a bar chart of the circularization efficiency (SEQ ID NO:41-45) in the PIE method with internal homologous arms.

[0104] Figure 13 shows a schematic diagram of the shortened T4td ribozyme exon structure, an electrophoresis diagram of the circularized RNA, and a bar graph of the circularization efficiency (SEQ ID NO:46-50) in the PIE method with internal homologous arms.

[0105] Figure 14 shows electrophoresis diagrams and circularization efficiency bar charts of Ana ribozymes and T4td ribozymes with internal homologous arms of different lengths (SEQ ID NO:51-58).

[0106] Figure 15 is a schematic diagram of the remaining stem-loop structure when the exon is shortest after splicing of Ana ribozyme and T4td ribozyme.

[0107] Figure 16 shows the analysis diagram of different IRES secondary structures and the optional stem-ring structure positions.

[0108] Figure 17 shows a schematic diagram of the secondary structure of HRV-B3 and HRV-B3-eIF4G IRES and the selected stem-ring position.

[0109] Figure 18 is a schematic diagram of the structure of scarless circular RNA prepared using nucleic acid molecules containing T4td intron mutants (P1 rigorous complementation).

[0110] Figure 19 is a schematic diagram of the preparation of scarless circular RNA structures using nucleic acid molecules containing T4td ribozyme mutants (P1 is not strictly complementary).

[0111] Figure 20 is a schematic diagram of the structure for preparing scarless circular RNA using nucleic acid molecules containing Ana intron mutants.

[0112] Figure 21 shows the electrophoresis diagram and sequencing results of the circular RNA splicing site for identification of scarless circular RNA (SEQ ID NO:82) expressing the antigenic peptide prepared using the mutated T4td intron (P1 rigorously complementary).

[0113] Figure 22 shows the electrophoresis diagram and sequencing results of the circular RNA splicing site for identification of scarless circular RNA (SEQ ID NO:83) expressing the antigenic peptide prepared using the mutated T4td intron (P1 non-strict complementation).

[0114] Figure 23 shows the electrophoresis diagram and sequencing results of the circular RNA splicing site for identification of scarless circular RNA (SEQ ID NO:84) expressing the antigenic peptide prepared using Ana ribozyme mutant.

[0115] Figure 24 shows the electrophoresis diagram and sequencing results of the circular RNA splicing site for identifying scarless circular RNA (SEQ ID NO:88) expressing Fluc prepared using the mutated T4td intron (P1 non-strict complementation).

[0116] Figure 25 shows the electrophoresis diagram of the scarless circular RNA (SEQ ID NO:89) expressing Fluc prepared using the Ana ribozyme mutant and the sequencing results of the circular RNA splicing site.

[0117] Figure 26 shows the results of the ELISpot experiment in which scarless circular RNA (SEQ ID NO:82 and SEQ ID NO:83) expressing antigenic peptides effectively presented antigenic peptides in PBMCs. The wells circled in the box are the control group with added PHA.

[0118] Figure 27 is a bar graph showing the activity of the scarless circular RNA (SEQ ID NO:88) expressing Fluc protein in cells.

[0119] Figure 28 shows the schematic diagram of the design structure of scarless circular RNA expressing the antigenic peptide prepared using HRV-B4 IRES and Ana ribozyme mutants, the identification electrophoresis image, and the sequencing results of the circular RNA splicing site. The top left shows the loop sequence in the stem-loop position selected by HRV-B4 IRES, with the arrow indicating the IRES truncation location. The bottom left shows the mutant sequences of the Ana ribozyme P1 and P10 regions corresponding to the HRV-B4 IRES truncation sites. In the center, the Ana ribozyme mutant maintains its intact structure in the predicted full-length structure of the recombinant nucleic acid molecule, unaffected by other sequences.

[0120] Figure 29 shows the schematic diagram of the design structure of scarless circular RNA expressing the antigenic peptide prepared using Human XIAP IRES and Ana ribozyme mutants, the identification electrophoresis image, and the sequencing results of the circular RNA splicing site. The top left shows the loop sequence in the stem-loop position selected by Human XIAP IRES, with the arrow indicating the IRES truncation location. The bottom left shows the mutant sequences of the Ana ribozyme P1 and P10 regions corresponding to the Human XIAP IRES truncation locations. In the center, the Ana ribozyme mutant maintains its intact structure in the predicted full-length structure of the recombinant nucleic acid molecule, unaffected by other sequences.

[0121] Figure 30 shows the schematic diagram of the design structure of scarless circular RNA expressing the antigenic peptide prepared using HRV-B97 IRES and Ana ribozyme mutants, the identification electrophoresis image, and the sequencing results of the circular RNA splicing site. The top left shows the loop sequence in the stem-loop position selected by HRV-B97 IRES, with the arrow indicating the IRES truncation location. The bottom left shows the mutant sequences of the Ana ribozyme P1 and P10 regions corresponding to the HRV-B97 IRES truncation locations. In the center, the Ana ribozyme mutant maintains its intact structure in the predicted full-length structure of the recombinant nucleic acid molecule, unaffected by other sequences.

[0122] Figure 31 shows the schematic diagram, identification electrophoresis image, and sequencing results of the circular RNA splicing site for preparing scarless circular RNA expressing the antigenic peptide using ShuffledIRES#42IRES and Ana ribozyme mutants. The top left shows the loop sequence in the stem-loop position selected by ShuffledIRES#42IRES, with the arrow indicating the IRES truncation location. The bottom left shows the mutant sequences of the Ana ribozyme P1 and P10 regions corresponding to the ShuffledIRES#42IRES truncation locations. In the center, the Ana ribozyme mutant maintains its intact structure in the predicted full-length structure of the recombinant nucleic acid molecule, unaffected by other sequences.

[0123] Figure 32 is a bar graph showing the circularization efficiency of scarless circular RNA expressing antigenic peptides prepared using HRV-B4, HRV-B97, Human XIAP, ShuffledIRES#42IRES, and Ana ribozyme mutants.

[0124] Figure 33 shows the electrophoresis diagrams for the transcription, circularization, and RNase R tolerance identification of scarless circular RNA expressing antigenic peptides prepared using HRV-B37, HRV-B92, ShuffledIRES#1, ShuffledIRES#38IRES, and Ana ribozyme mutants.

[0125] Figure 34 is a schematic diagram of the secondary structure of CVB3 IRES and its structural domains. The black ellipse circles the variable region and the irregular region between the V and VI structural domains.

[0126] Figure 35 shows the CVB3 IRES secondary structure predicted by RNAfold and the selected truncation sites S1, S2 and S3.

[0127] Figure 36 shows the electrophoresis diagrams for the identification of transcription, circularization, and RNase R tolerance in SEQ ID NO:119, SEQ ID NO:120, and SEQ ID NO:121. Detailed Implementation

[0128] The technical solutions of this disclosure will be further described in detail below with reference to specific embodiments. The following embodiments are merely illustrative and explanatory of this disclosure and should not be construed as limiting the scope of protection of this disclosure. All technologies implemented based on the above content of this disclosure are covered within the scope of protection intended by this disclosure.

[0129] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods. The experimental methods in the following examples are conventional experimental methods, and unless otherwise specified, they should be performed according to the technical conditions described in the literature within this disclosure or according to the product instructions.

[0130] In this disclosure, the human or non-human proteins include any polypeptides that can be used for therapeutic purposes, including but not limited to antibodies, intracellular antibodies, single-chain variable fragments (scFv), affinities, bispecific or multispecific antibodies or binders, receptors, ligands, enzymes for, for example, enzyme replacement therapy or gene editing, tumor inhibitors, viral or bacterial inhibitors, cellular component proteins, DNA and / or RNA-binding proteins, DNA repair inhibitors, nucleases, proteases, integrases, transcription factors, growth factors, apoptosis inhibitors and inducers, toxins (e.g., Pseudomonas exotoxin), structural proteins, neurotrophic factors such as NT3 / 4, brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) and their subunits such as the 2.5Sβ subunit, ion channels, membrane transport proteins, protein stabilizing factors, proteins involved in cell signal transduction, translation and transcription-related proteins, nucleotide-binding proteins, protein-binding proteins, lipid-binding proteins, glycosaminoglycans (GAG) and GAG-binding proteins, metabolic proteins, cellular stress-regulating proteins, inflammatory and immune system-regulating proteins, mitochondrial proteins and heat shock proteins, etc.

[0131] In this disclosure, the tumor-specific antigens include, but are not limited to, alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), carbohydrate antigen 125 (CA125), carbohydrate antigen 153 (CA153), carbohydrate antigen 19-9 (CA19-9), carbohydrate antigen 724 (CA724), carbohydrate antigen 242 (CA242), carbohydrate antigen 50 (CA50), CYFRA21-1 (Cy211), neuron-specific enolase (NSE), prostate-specific antigen (PSA), human chorionic gonadotropin (HCG), thyroglobulin (TG), ferritin (SF), β2-microglobulin (β2-MG), and squamous cell antigen (SCC).

[0132] In this disclosure, the pathogen antigens include, but are not limited to, tuberculosis antigen, anthrax antigen, hepatitis A virus (HAV) antigen, hepatitis B virus (HBV) antigen, hepatitis C virus (HCV) antigen, human immunodeficiency virus (HIV) antigen, influenza virus antigen, herpes simplex virus (HSV) antigen, Haemophilus influenzae type b (Hib) antigen, Neisseria meningitidis antigen, Corynebacterium diphtheria antigen, Bordetella pertussis antigen, Clostridium tetani antigen, and Varicella virus antigen. In one embodiment of this disclosure, the pathogen antigen is SARS-CoV-2 antigen, influenza virus antigen, herpes virus antigen, etc.

[0133] In this disclosure, "IRES" (Internal Ribosome Entry Site) is also known as the internal ribosome entry site. The internal ribosome entry site (IRES) is a translation control sequence, typically located at the 5' end of the gene of interest, enabling cap-independent RNA translation. Transcribed IRES directly binds to ribosomal subunits, allowing the mRNA start codon to be properly oriented within the ribosome for translation. The IRES sequence is typically located in the 5' UTR of the mRNA (directly upstream of the start codon). Functionally, the IRES replaces the need for various protein factors that interact with eukaryotic translation mechanisms.

[0134] In this disclosure, the IRES include, but are not limited to, the IRES sequences of the following viruses: Taura syndrome virus, tussock virus, Tiller's encephalomyelitis virus, simian virus 40, red imported fire ant virus 1, rice constrictor aphid virus, reticuloendotheliosis virus, human poliovirus 1, P. stearnivirus, Kashmir bee virus, human rhinovirus 2, glass leafhopper virus-1, human immunodeficiency virus type 1, and Himetobi virus. P virus, Hepatitis C virus, Hepatitis A virus, GB hepatitis virus, Foot-and-mouth disease virus, Human enterovirus 71, Equine rhinitis virus, Tea geometrid moth microRNA virus-like virus, Encephalomyocarditis virus, Drosophila C virus, Human Coxsackievirus B3, Tobacco mosaic virus, Cricket paralysis virus, Bovine viral diarrhea virus 1, Black bee queen cell virus, Aphid lethal paralysis virus, Avian encephalomyelitis virus, Acute bee paralysis virus, Hibiscus chlorosis and ringspot virus, Classical swine fever virus, Human FGF2, Human SFTPA1, Human AML1 / RUNX1, Drosophila antennae and legs, Human AQP4, Human AT1R, Human BAG-1, Human BCL2, Human BiP, Human c-IAPl, Human c-myc, Human eIF4G, Mouse NDST4L, Human LEF1, Mouse HIF1α, Human n.myc, mouse Gtx, human p27kipl, human PDGF2 / c-sis, human p53, human Pim-1, mouse Rbm3, Drosophila reaper, canine scamper, Drosophila ubx, human UNR, mouse UtrA, human VEGF-A, human XIAP, hairless Drosophila, Saccharomyces cerevisiae TFIID, Saccharomyces cerevisiae YAP1, tobacco etch virus, turnip shrunken virus, EMCV-A, EMCV-B, EMCV-Bf, EMCV-Cf, EMCV pEC9, small disegmented RNA virus, HCV QC64, human cosavirus E / D, human cosavirus F, human cosavirus JMY, rhinovirus NAT001, HRV14, HRV89, HRVC-02, HRV-A21, HRV-B3, Sasavirus A SH1, Sasavirus FHB, Sasavirus NG-J1, human paraenteric orphan virus 1, Crohivirus B, Yc-3, Rosavirus M-7, Shanbavirus A, Pasivirus A, Pasivirus A 2, Echovirus E14, Human Paraenterovirus 5, Aichi Virus, Hepatitis A Virus HA16, Phopivirus, CVA10, Enterovirus C, Enterovirus D, Enterovirus J, Human Hepatic Virus 2, GBV-C GT110, GBV-C K1737, GBV-C Iowa, Hepatic Virus A 1220, Pasivirus A 3, Sapellovirus, Rosavirus B, Bakunsa Virus, Tremor Virus A, Porcine Pasivirus 1, PLV-CHN, Pasivirus A, Sicinivirus, Hepatitis Virus K, Hepatitis Virus A, BVDV1, Border Disease Virus, BVDV2, CSFV-PK15C, SF573 Bicistronic Virus, Hubei MicroRNA Virus-like Virus, CRPV, Salivirus A BN5, Salivirus A BN2, Salivirus A The aptamers are 02394, Salivirus A GUT, Salivirus A CH, Salivirus A SZ1, Salivirus FHB, CVB3, CVB1, Echovirus 7, CVB5, EVA71, CVA3, CVA12, EV24, or eIF4G. In one embodiment of this disclosure, the IRES sequence is IRES. MP,75 HRV-B3-eIF4G.

[0135] In this disclosure, "vector" refers to a segment of DNA that is synthetic (e.g., using PCR) or extracted from a virus, plasmid, or cell of a higher organism, into which a foreign DNA fragment may be inserted or has been inserted for cloning and / or expression purposes. In some embodiments, the vector can be stably maintained in an organism. The vector may contain, for example, an origin of replication, a selection marker or reporter gene, such as antibiotic resistance or GFP, and / or a multiple cloning site (MCS). The term includes linear DNA fragments (e.g., PCR products, linear plasmid fragments), plasmid vectors, viral vectors, granules, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), etc. In one embodiment, the vector provided herein contains a multiple cloning site.

[0136] In this disclosure, "circular nucleic acid molecule" refers to a nucleic acid molecule that is in a closed circular shape. In some specific embodiments, the circular nucleic acid molecule is a circular RNA molecule. More specifically, the circular nucleic acid molecule is a circular mRNA molecule.

[0137] In this disclosure, "precursor nucleic acid molecule" refers to a linear nucleic acid molecule, preferably an RNA molecule, that can form a circular nucleic acid molecule through a cyclization reaction. It is generally formed by transcription of a linear DNA molecule (e.g., a vector containing a recombinant nucleic acid molecule).

[0138] In this disclosure, a “coding region” refers to a gene sequence that can be transcribed into messenger RNA and ultimately translated into a target polypeptide or protein.

[0139] In this disclosure, "expression" includes any step involved in polypeptide production, including but not limited to: transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0140] In this disclosure, "host cell" refers to a cell into which exogenous polynucleotides have been introduced, including progeny cells of this type. Host cells include "transformers" and "transformed cells," which include primary transformed cells and their derived progeny. Host cells can be any type of cell system that can be used to produce the nucleic acid molecules or proteins encoded by this disclosure, including eukaryotic cells, such as mammalian cells, insect cells, and yeast cells; and prokaryotic cells, such as *E. coli* cells. Host cells include cultured cells, as well as cells within transgenic animals, transgenic plants, or cultured plant or animal tissues. The term "recombinant host cell" encompasses a host cell that differs from its parent cell after the introduction of recombinant nucleic acid molecules, recombinant expression vectors, or circular RNA, specifically achieved through transformation. The host cells of this disclosure can be prokaryotic or eukaryotic cells, as long as they are capable of introducing the recombinant nucleic acid molecules, recombinant expression vectors, circular RNA, etc., of this disclosure.

[0141] In one specific embodiment of this disclosure, the group I introns are mutants of the Ana ribozyme, wherein the first ribozyme recognition fragment is 5'-N1N2N3T-3', the second ribozyme recognition fragment is 5'-N4N5N6-3', and the Ana ribozyme mutant contains the following mutant region N in the 5' intron fragment. 6’ N 7’ ATAAN 5’ N 4’ GN 3’ N 2’ (The original sequence is: 5'-AAAUAAUUGAG-3', with the underlined mutation site), where N is A, U, C, G, or T, and N2N3 is related to N. 3’ N 2’ Reverse complementary pairing, N4N5 and N 5’ N 4’ Reverse complementary pairing, N 6’ N 7’ With N 5’ N 4’ Reverse complementary pairing, the base complementary pairing includes AU, GC, GU, AT, and GT base pairs. The corresponding cyclic fragment in the first unit is N1N2N3TN4N5N6, and the cyclic site is N1N2N3T / N4N5N6.

[0142] In one specific embodiment of this disclosure, the group I introns are mutants of the T4td ribozyme, wherein the first ribozyme recognition fragment is 5'-N1N2N3N4N5T-3', and the second ribozyme recognition fragment is 5'-N6N7-3'. The T4td ribozyme mutant contains the following mutation region N in the 5' intron fragment. 8’ AATTGN 7’ N 6’ GN 5’ N 4’ N 3’ N 2’ N 1’ (The original sequence is: 5'-UAAUUGAGGCCUGA-3', with the underlined mutation site), where N is A, U, C, G, or T, or absent, and N1N2N3N4N5 is related to N. 5’ N 4’ N 3’ N 2’ N 1’ Reverse complementary pairing or N2N3N4N5 with N 5’ N 4’ N 3’ N 2’ Reverse complementary pairing, N 7’ N 6’ Pairing with N6N7 in reverse complementary direction, N 8’ With N6’ Complementary pairing, specifically the base pairings AU, GC, GU, AT, and GT base pairs. The corresponding cyclic fragment in the first unit is N1N2N3N4N5TN6N7, and the cyclic site is N1N2N3N4N5T / N6N7. In a further specific embodiment, N6 is U or C, and N... 6’ No mutations occur.

[0143] In this disclosure, free bases refer to bases in a cyclic sequence whose positions do not need to be predetermined. These positions can be formed by mutating the corresponding bases of the ribozyme recognition fragment to form complementary pairs. For example, N1-N6 in the Ana ribozyme cyclic fragment N1N2N3TN4N5N6 are free bases.

[0144] Example 1: Selection of Group I ribozyme spacer sequences in the PIE method

[0145] Circular RNA was prepared using the PIE method. Adding spacer sequences at the junction of the group I intron ribozyme and the functional sequence facilitated RNA circularization. The spacer sequences included stem structures formed by internal homologous arms and loose, disordered structures. T4td introns and Ana introns are two commonly used ribozymes in the PIE method, and their structures are shown in Figure 3. We evaluated the effects of these two spacer sequences on circularization efficiency using T4td ribozyme 3' introns (SEQ ID NO:1) and 5' introns (SEQ ID NO:2), and Ana ribozyme 3' introns (SEQ ID NO:3) and 5' introns (SEQ ID NO:4) as examples.

[0146] 1. Plasmid construction

[0147] For the functional sequence in RNA to be circularized, IRES derived from crTMV are selected. MP,75 (SEQ ID NO:5) Recruitment of ribosomes to initiate the translation of tumor antigen peptides. The antigen peptide encoding sequences were selected from seven previously reported tumor antigen peptides, including MAGE-1 (SEQ ID NO:6), TRP-2 (SEQ ID NO:7), gp100 (SEQ ID NO:8), IL13Rα2 (SEQ ID NO:9), Melan A (SEQ ID NO:10), BST2 (SEQ ID NO:11), and IMP2 (SEQ ID NO:12). These seven antigen peptides were combined in a specific order, with each pair separated by a 4-amino acid interval. The resulting polypeptide amino acid sequence is shown in SEQ ID NO:13. Codon optimization yielded the nucleic acid sequence encoding this polypeptide, as shown in SEQ ID NO:14.

[0148] First, a control plasmid without spacers was constructed, and then the T4td ribozyme 3' intron-IRES was ligated after the T7 promoter. MP,75 -Antigen peptide coding sequence- T4td ribozyme 5' intron obtained SEQ ID NO:15, Ana ribozyme 3' intron linked after T7 promoter-IRES MP,75 The antigen peptide encoding sequence-Ana ribozyme 5' intron was used to obtain SEQ ID NO:16 (Figure 4). Then, a plasmid containing a loosely structured polyAC spacer sequence was constructed, and the T4td ribozyme 3' intron-polyAC-IRES was ligated after the T7 promoter. MP,75 -Antigen peptide encoding sequence-polyAC-T4td ribozyme 5' intron obtained SEQ ID NO:17, Ana ribozyme 3' intron-polyAC-IRES ligated after T7 promoter. MP,75 The antigen peptide encoding sequence-polyAC-Ana ribozyme 5' intron was used to obtain SEQ ID NO:18 (Figure 4). Then, a plasmid containing the internal homologous arm spacer sequence was constructed, and the T4td ribozyme 3' intron-5' homologous arm-IRES was ligated after the T7 promoter. MP,75 -Antigen peptide coding sequence-3' homologous arm-T4td ribozyme 5' intron to obtain SEQ ID NO:19, ligated Ana ribozyme 3' intron-5' homologous arm-IRES after T7 promoter. MP,75 -Antigen peptide coding sequence-3' homologous arm-Ana ribozyme 5' intron to obtain SEQ ID NO:20 (Figure 4).

[0149] The IRES and antigen peptide coding sequences in the above sequences are relatively short. Therefore, the longer CVB3 IRES (SEQ ID NO:21) and FLUC protein coding sequences (SEQ ID NO:22) were used to further evaluate the effect of these two spacer sequences on cyclization efficiency.

[0150] First, a control plasmid without spacer sequences was constructed. Then, a T4td ribozyme 3' intron-CVB3 IRES-FLUC coding sequence-T4td ribozyme 5' intron was ligated after the T7 promoter to obtain SEQ ID NO:23. Next, an Ana ribozyme 3' intron-CVB3 IRES-FLUC coding sequence-Ana ribozyme 5' intron was ligated after the T7 promoter to obtain SEQ ID NO:24. Then, a plasmid containing a loosely structured polyAC spacer sequence was constructed. A T4td ribozyme 3' intron-polyAC-CVB3 IRES-FLUC coding sequence-polyAC-T4td ribozyme 5' intron was ligated after the T7 promoter to obtain SEQ ID NO:25. Finally, an Ana ribozyme 3' intron-polyAC-CVB3 IRES-FLUC coding sequence-polyAC-Ana ribozyme 5' intron was ligated after the T7 promoter to obtain SEQ ID NO:26. Then, a plasmid containing the internal homologous arm spacer sequence was constructed, and SEQ ID NO:27 was obtained by ligating the T4td ribozyme 3' intron-5' homologous arm-CVB3IRES-FLUC coding sequence-3' homologous arm-T4td ribozyme 5' intron after the T7 promoter. SEQ ID NO:28 was obtained by ligating the Ana ribozyme 3' intron-5' homologous arm-CVB3IRES-FLUC coding sequence-3' homologous arm-Ana ribozyme 5' intron after the T7 promoter.

[0151] 2. Plasmid linearization

[0152] The plasmid was digested with XbaI (NEB, #R0145L), incubated at 37°C for 1.5 h, and the reaction was terminated by heating at 65°C for 20 min. The digested product was purified using the phenol-chloroform method.

[0153] 3. In vitro transcription (IVT)

[0154] use The T7 Quick High Yield RNA Synthesis Kit (NEB, #E2050S) was used to synthesize circular RNA precursors from linearized plasmid templates via in vitro transpiration (IVT). Following the manufacturer's instructions, nuclease-free water, NTP Buffer Mix, linearized DNA template, and T7 RNA Polymerase Mix were added, and the mixture was gently mixed and incubated at 37°C for 3 hours. After IVT, the IVT product was treated with DNase I (RNase-free) (NEB, #M0303S) for 15-20 minutes to digest the DNA template. The RNA was then purified by column purification using the Monarch RNA Cleanup Kit (NEB, #T2050L).

[0155] 4. In vitro circulization

[0156] Add 10×T4 RNA Ligase Reaction Buffer (NEB, #B0216L) to the purified RNA solution, and make up the reaction volume with nuclease-free water. Gently mix all components and heat at 65°C for 5 min. Then place on ice for 5 min. Add 1.5 μL of GTP (NEB, #N0450S) to the sample and incubate at 55°C for 30–40 min. The reaction system is shown in Table 1. Column purification of RNA was performed using the Monarch RNA Cleanup Kit (NEB, #T2050L).

[0157] Table 1 In vitro cyclization reaction system

[0158] 5. Digestion of linear RNA

[0159] RNase R (Beyotime, #R7092M) is a Mg 2+ The 3' to 5' exonucleases were used to digest and remove linear RNA. The reaction system is shown in Table 2. The reaction system was treated at 37°C for 40-50 min, and then heated at 70°C for 10 min to terminate the reaction. The RNA was purified by column purification using the Monarch RNA Cleanup Kit (NEB, #T2040L or #T2050L).

[0160] Table 2. Circular RNA Enrichment Reaction System

[0161] 6. RNA circularization identification

[0162] Circular RNA was detected by formaldehyde denaturing electrophoresis. A 1.5%–2.0% (w / v) agarose-formaldehyde (Macklin, #F809702-500mL) gel was prepared and electrophoresed in 1×MOPS (Sangon Biotech, #C516042-0001) for 40–60 min. The electrophoretic bands were observed using a fully automated gel imaging system.

[0163] Figures 5, 6, and 7 show the electrophoresis diagrams of the transcription products, circularization products, and RNase R degradation products during the preparation of circular RNA from the above nucleotide sequences (SEQ ID NO: 15-20, SEQ ID NO: 23-28), and the circularization efficiency calculated using ImageJ software based on the electrophoresis results. The electrophoresis results show that for shorter RNAs, circularization efficiency (IRES) is significantly improved. MP,75For antigen peptide coding sequences (approximately 400 bases), a polyAC spacer sequence favors T4td ribozyme cyclization of RNA, while a stem spacer sequence promotes both T4td and Ana ribozymes. For longer RNA cyclization (CVB3 IRES-FLUC coding sequence, approximately 2400 bases), a stem spacer sequence favors both T4td and Ana ribozymes, while a polyAC spacer sequence discourages Ana ribozyme cyclization. Therefore, it can be concluded that regardless of the length of the RNA being cyclized, a stem spacer sequence favors both T4td and Ana ribozymes.

[0164] Example 2: Determination of Group I ribozyme exon length using the PIE method

[0165] Circular RNA was prepared using the PIE method. The circular RNA product typically contains exon sequences linked to group I intron ribozymes. Commonly used exons are 66 nt long (Ana) and 37 nt long (T4td). The length of these exon sequences affects circularization efficiency. The Chen Lingling research group believes that exons shorter than 27 nt for Ana ribozymes lead to a significant reduction in circularization efficiency. The Ana intron ribozyme used in Example 1 has a 66 nt exon, and the T4td intron ribozyme has a 37 nt exon. We have already confirmed in Example 1 that a stem-like spacer sequence is beneficial for T4td and Ana ribozymes to circulate RNA. Therefore, we further evaluated the effect of different exon lengths on circularization efficiency using the PIE method with internal homologous arms.

[0166] 1. Plasmid construction

[0167] Plasmids containing Ana ribozyme exons of different lengths were constructed, and Ana ribozyme 3' intron-5nt exon (AAAAT)-IRES was ligated after the T7 promoter. MP,75 The antigen peptide coding sequence-5nt exon (GACTT)-Ana ribozyme 5' intron yields SEQ ID NO:29. After splicing and circularization, a 10nt exon (GACTTAAAAT) remains, as shown in structural diagram 8. Following the T7 promoter, an Ana ribozyme 3' intron-4nt exon (AAAA)-IRES is ligated. MP,75 The antigen peptide coding sequence-4nt exon (ACTT)-Ana ribozyme 5' intron yields SEQ ID NO:30. After splicing and circularization, an 8nt exon (ACTTAAAA) remains, as shown in structural diagram 8. Following the T7 promoter, an Ana ribozyme 3' intron-3nt exon (AAA)-IRES is ligated. MP,75The antigen peptide coding sequence-4nt exon (ACTT)-Ana ribozyme 5' intron yields SEQ ID NO:31. After splicing and circularization, a 7nt exon (ACTTAAA) remains, as shown in structural diagram 8. Following the T7 promoter, an Ana ribozyme 3' intron-2nt exon (AA)-IRES is linked. MP,75 The antigen peptide coding sequence-4nt exon (ACTT)-Ana ribozyme 5' intron yields SEQ ID NO:32. After splicing and circularization, a 6nt exon (ACTTAA) residue is formed, as shown in structural diagram 8 (6nt-2). Following the T7 promoter, an Ana ribozyme 3' intron-3nt exon (AAA)-IRES is linked. MP,75 The antigen peptide coding sequence-3nt exon (CTT)-Ana ribozyme 5' intron yields SEQ ID NO:33. After splicing and circularization, a 6nt exon (CTTAAA) remains, as shown in structural diagram 8. Following the T7 promoter, an Ana ribozyme 3' intron-2nt exon (AA)-IRES is ligated. MP,75 The antigen peptide coding sequence-3nt exon (CTT)-Ana ribozyme 5' intron yields SEQ ID NO:34. After splicing and circularization, a 5nt exon (CTTAA) residue is formed, as shown in structural diagram 8. Following the T7 promoter, an Ana ribozyme 3' intron-1nt exon (A)-IRES is linked. MP,75 The antigen peptide coding sequence-3nt exon (CTT)-Ana ribozyme 5' intron yields SEQ ID NO:35. After splicing and circularization, a 4nt exon (CTTAA) residue is formed, as shown in structural diagram 8 (4nt-2). Following the T7 promoter, an Ana ribozyme 3' intron-2nt exon (AA)-IRES is ligated. MP,75 -Antigen peptide coding sequence-2nt exon (TT)-Ana ribozyme 5' intron to obtain SEQ ID NO:36, after splicing into a loop to form a 4nt exon (TTAA) residue, as shown in structural diagram 8 4nt.

[0168] Plasmids containing T4td ribozyme exons of different lengths were constructed, and T4td ribozyme 3' intron-3nt exon (CTA)-IRES was ligated after the T7 promoter. MP,75 The antigen peptide coding sequence-7nt exon (CTTGGGT)-T4td ribozyme 5' intron yielded SEQ ID NO:37. After splicing and circularization, a 10nt exon (CTTGGGTCTA) residue was formed, as shown in structural diagram 9. Following the T7 promoter, the T4td ribozyme 3' intron-2nt exon (CT)-IRES was ligated. MP,75The antigen peptide coding sequence-6nt exon (TTGGGT)-T4td ribozyme 5' intron yields SEQ ID NO:38. After splicing and circularization, an 8nt exon (TTGGGTCT) remains, as shown in structural diagram 9. Following the T7 promoter, a T4td ribozyme 3' intron-1nt exon (C)-IRES is ligated. MP,75 -Antigen peptide coding sequence-5nt exon (TGGGT)-T4td ribozyme 5' intron to obtain SEQ ID NO:39, splicing into a circular form to form a 6nt exon (TGGGTC) residue, as shown in structural diagram 9; after the T7 promoter, the T4td ribozyme 3' intron-IRES is linked. MP,75 The antigen peptide coding sequence -4nt exon (GGGT) -T4td ribozyme 5' intron obtained SEQ ID NO:40, after splicing into a loop, formed a 4nt exon (GGGT) residue, as shown in structural diagram 9.

[0169] The IRES and antigen peptide coding sequences in the above sequences are relatively short. Therefore, the longer CVB3 IRES (SEQ ID NO:21) and FLUC protein coding sequences (SEQ ID NO:22) were used to further evaluate the effect of exons of different lengths on the cyclization efficiency of Ana ribozymes. Plasmids with exons of different lengths of Ana ribozyme were constructed. Following the T7 promoter, an Ana ribozyme 3' intron-4nt exon (AAAA)-CVB3 IRES-FLUC coding sequence-4nt exon (ACTT)-Ana ribozyme 5' intron was ligated to obtain SEQ ID NO:41. After splicing and circularization, an 8nt exon (ACTTAAAA) residue was formed, as shown in structural diagram 8. Following the T7 promoter, an Ana ribozyme 3' intron-3nt exon (AAA)-CVB3 IRES-FLUC coding sequence-4nt exon (ACTT)-Ana ribozyme 5' intron was ligated to obtain SEQ ID NO:42. After splicing and circularization, a 7nt exon (ACTTAAA) residue was formed, as shown in structural diagram 8. Following the T7 promoter, an Ana ribozyme 3' intron-2nt exon (AA)-CVB3 was ligated to obtain SEQ ID NO:42. The IRES-FLUC coding sequence - 4nt exon (ACTT) - Ana ribozyme 5' intron yields SEQ ID NO:43. After splicing and circularization, a 6nt exon (ACTTAA) residue is formed, as shown in structural diagram 8 (6nt-2). Following the T7 promoter, the Ana ribozyme 3' intron - 4nt exon (AAAA) - CVB3 and the IRES-FLUC coding sequence - 3nt exon (CTT) - Ana ribozyme 5' intron are linked to obtain SEQ ID NO:44. After splicing and circularization, a 7nt exon (CTTAAAA) residue is formed, as shown in structural diagram 12 (7nt-2). Following the T7 promoter, the Ana ribozyme 3' intron - 3nt exon (AAA) - CVB3 and the IRES-FLUC coding sequence - 3nt exon (CTT) - Ana ribozyme 5' intron are linked to obtain SEQ ID NO:44. NO:45, after splicing into a ring, forms a 6nt exon (CTTAAA) residue, as shown in the structural diagram 8.

[0170] The effects of different exon lengths on the cyclization efficiency of T4td ribozymes were evaluated using the longer CVB3 IRES (SEQ ID NO:21) and FLUC protein coding sequences (SEQ ID NO:22). Plasmids with T4td ribozyme exons of different lengths were constructed. Following the T7 promoter, a sequence was ligated from the T4td ribozyme 3' intron to a 2nt exon (CT)-CVB3 IRES-FLUC coding sequence to a 6nt exon (TTGGGT)-T4td ribozyme 5' intron, yielding SEQ ID NO:46. After splicing and circularization, an 8nt exon (TTGGGTCT) remnant was formed, as shown in structural diagram 9. Following the T7 promoter, a sequence was ligated from the T4td ribozyme 3' intron to a 2nt exon (CT)-CVB3 IRES-FLUC coding sequence to a 5nt exon (TGGGT)-T4td ribozyme 5' intron, yielding SEQ ID NO:47. After splicing and circularization, a 7nt exon (TGGGTCT) remnant was formed, as shown in structural diagram 13. Following the T7 promoter, a sequence was ligated from the T4td ribozyme 3' intron to a 1nt exon (C)-CVB3. The IRES-FLUC coding sequence - 6nt exon (TTGGGT) - T4td ribozyme 5' intron yields SEQ ID NO:48. After splicing and circularization, a 7nt exon (TTGGGTC) residue is formed, as shown in structural diagram 13 (7nt-2). Following the T7 promoter, the T4td ribozyme 3' intron - 1nt exon (C) - CVB3 is linked to the IRES-FLUC coding sequence - 5nt exon (TGGGT) - T4td ribozyme 5' intron, yielding SEQ ID NO:49. After splicing and circularization, a 6nt exon (TGGGTC) residue is formed, as shown in structural diagram 9 (6nt). Following the T7 promoter, the T4td ribozyme 3' intron - CVB3 and the IRES-FLUC coding sequence - 4nt exon (GGGT) - T4td ribozyme 5' intron are linked to obtain SEQ ID NO:49. NO:50, after splicing into a ring, forms a 4nt exon (GGGT) residue, as shown in structural diagram 9.

[0171] 2. Preparation of circular RNA

[0172] The steps for plasmid linearization, in vitro transcription, in vitro circularization, and linear RNA digestion are as described in Example 1.

[0173] 3. RNA circularization identification

[0174] The method for detecting circular RNA by formaldehyde denaturation electrophoresis is as described in Example 1.

[0175] 4. Validation by sequencing of circular RNA splicing sites

[0176] Following the instructions of the TRUEscript RT MasterMix (OneStep gDNA Removal) kit (Catalog No.: PC7002, Beijing Adley Biotechnology Co., Ltd.), the prepared circular RNA was subjected to reverse transcription PCR to obtain cDNA products, and then... The Ultra-Rapid II HotStart PCR Master Mix (Catalog No.: 10167ES03, Yisheng Bio) kit instructions state that PCR is performed on the splice site sequence, and the obtained DNA product is sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing to verify whether the circular RNA has been spliced ​​and circularized as expected.

[0177] Figures 8 and 10 show the electrophoresis diagrams, circularization products, and RNase R degradation products of the nucleotide sequences constructed using Ana ribozyme exons (the nucleotide sequences are shown in SEQ ID NO: 29-36) during the preparation of circular RNA. Figures 9 and 11 show the electrophoresis diagrams, circularization products, and RNase R degradation products of the nucleotide sequences constructed using T4td ribozyme exons (the nucleotide sequences are shown in SEQ ID NO: 37-40) during the preparation of circular RNA. The sequencing results show that the above nucleotide sequences (SEQ ID NO: 29-35, SEQ ID NO: 37-40) were all circularized at the expected sequence positions. The electrophoresis results show that the short RNA circularization (IRES) is relatively short. MP,75 -The antigen peptide coding sequence (approximately 400 bases) requires only 5 nt exons for the Ana ribozyme to efficiently complete the cyclization reaction, while the T4td ribozyme requires only 4 nt exons to efficiently complete the cyclization reaction. Figure 12 shows the electrophoresis diagrams of the transcription products, cyclization products, and RNase R degradation products during the preparation of cyclic RNA from the nucleotide sequence constructed using the Ana ribozyme exons (its nucleotide sequence is shown in SEQ ID NO:41-45). Figure 13 shows the electrophoresis diagrams of the transcription products, cyclization products, and RNase R degradation products during the preparation of cyclic RNA from the nucleotide sequence constructed using the T4td ribozyme exons (its nucleotide sequence is shown in SEQ ID NO:46-50). The electrophoresis results show that for longer RNA cyclization (CVB3 IRES-FLUC coding sequence, approximately 2400 bases), the T4td ribozyme can efficiently complete RNA cyclization by retaining 4 nt exons, while the Ana ribozyme requires 6 nt exons (6nt-2) to efficiently complete RNA cyclization.

[0178] Example 3: IRES sequence structure prediction and stem-loop structure screening

[0179] Based on the above experimental results, we determined that in the design of PIE method for circularizing RNA, the stem structure of the spacer sequence is beneficial for T4td ribozyme and Ana ribozyme to perform RNA circularization. Furthermore, for the circularization of shorter RNA, T4td ribozyme and Ana ribozyme only need to retain 4nt and 5nt exons, respectively, to effectively complete RNA circularization. For the circularization of longer RNA, retaining 4nt and 6nt exons is sufficient to efficiently complete RNA circularization. We then examined the stem length. We tried different stem structures for Ana ribozymes, retaining 8 nt exons, and tested the cyclization efficiency of recombinant nucleic acid molecules with no stem structure (SEQ ID NO:51), with a 6 nt stem structure (SEQ ID NO:52), with an 8 nt stem structure (SEQ ID NO:53), and with a 20 nt stem structure (SEQ ID NO:54). The electrophoresis results and Image J statistical results are shown in Figure 14. Without the stem structure, the Ana intron ribozyme could not cyclize RNA. With the 6 nt stem structure, the Ana intron ribozyme could cyclize RNA. The 8 nt and 20 nt stem structures cyclized RNA better. In addition, the 5 nt stem structure in the 66 nt exon of the natural Ana ribozyme can also complete cyclization. When all T4td ribozyme exons retained 8nt, the cyclization efficiency of recombinant nucleic acid molecules was detected with no stem structure (SEQ ID NO:55), with a 6nt stem structure (SEQ ID NO:56), with a 10nt stem structure (SEQ ID NO:57), and with a 20nt stem structure (SEQ ID NO:58). The electrophoresis results and Image J statistical results are shown in Figure 14. The results show that T4td intron ribozymes can cyclize RNA with both no stem and stem structures, and T4td intron ribozymes with 10nt and 20nt stem structures can cyclize RNA better.

[0180] Based on the above experimental results, T4td ribozymes and Ana intron ribozymes, under the presence of stem structures and exon shortening (T4td ribozyme exons shortened to 4 nt, Ana ribozyme exons shortened to 5 nt), successfully completed splicing circularization, resulting in circular RNA with splicing scars at stem-loop structures. The exons form the loop position sequence, as shown in Figure 15. Therefore, we can assume that by truncating suitable stem-loop structures in the circular RNA at the loop position and splicing them with group I intron ribozymes, and correspondingly mutating group I ribozymes to maintain the required splicing structure, scarless circularization of RNA can be achieved. IRES generally have complex secondary structures, including stem-loop structures. Therefore, we plan to screen suitable stem-loop structures in known IRES, replace the exon sequences of T4td and Ana ribozymes with their loop position sequences, and use the stem structure to assist in the formation of splice vesicles, thus achieving the construction of scarless circular RNA.

[0181] Based on the above experimental results, the principles for screening IRES stem-loop structures include: under T4td ribozyme conditions, the loop position should contain at least 4 free bases, including either a T or U base, with at least 3 free bases upstream of the T or U base, and the downstream sequence of T forming the stem structure. More preferably, the loop position should contain at least 6 free bases, including either a T or U base, with at least 4 free bases upstream of the T or U base and at least 1 free base downstream of the T or U base. Even more preferably, the loop position should contain at least 8 free bases, including either a T or U base, with at least 5 free bases upstream of the T or U base and at least 2 free bases downstream of the T base. For convenience, this is denoted as N in the following description. 1N2N3N4N5TN6N7 or N1N2N3N4N5UN6N7, where N is A, G, C, U or T; under Ana ribozyme conditions, the ring position contains at least 5 free bases, including a T or U base, with at least 2 free bases upstream of the T or U base and at least 2 free bases downstream of the T or U base. More preferably, the ring position contains at least 7 free bases, including a T or U base, with at least 3 free bases upstream of the T or U base and at least 3 free bases downstream of the T or U base. For convenience, this is referred to below as N1N2N3TN4N5N6 or N1N2N3UN4N5N6, where N is A, G, C, U or T.

[0182] Based on the above experimental results, IRES stem-loop structures were screened. The stem is a double-stranded structure formed by complementary pairing sequences within 100 bases upstream and downstream of the T or U split position in the loop. The double-stranded structure contains at least 5 consecutive complementary pairing bases. Preferably, the number of complementary and non-complementary pairing bases in the stem exceeds 20 bp. More preferably, the complementary pairing sequence is adjacent to the upstream N1 and downstream N6 or N7 bases of the T or U split position.

[0183] Existing literature has evaluated the ability of various IRES to recruit ribosomal translation proteins, and a series of IRES with stronger translation capabilities were obtained by recombination using DNA shuffling technology. We used RNAfold to predict the secondary structure of these IRES and screened for stem-loop structures that met the above requirements. The IRES obtained after screening and their loop position base sequences (in bold) are shown in Table 3. Figure 16 shows the secondary structure and stem-loop structure positions of the IRES in Table 3.

[0184] Table 3 shows the IRES obtained through screening and their loop position base sequences.

[0185] Among these IRES, the stem-loop structures in HRV-B3, HRV-B92, HRV-B37, shuffledIRES#01, shuffledIRES#38, HRV-B97, shuffledIRES#03, HRV-B4, HRV-C11, and shuffledIRES#42 are all located in Domain I. The stem contains complementary and non-complementary base pairs, and the number of bases in the stem exceeds 20 bp. The stem-loop structures of HRV-B3 (SEQ ID NO:59), HRV-B92 (SEQ ID NO:60), HRV-B37 (SEQ ID NO:61), shuffledIRES#01 (SEQ ID NO:62), and shuffledIRES#38 (SEQ ID NO:63) are more regular and stable (Base-pair probabilities in RNAFold structure prediction, bases are marked in red). The stem-loop structures of HRV-B97 (SEQ ID NO:64), shuffledIRES#03 (SEQ ID NO:65), HRV-B4 (SEQ ID NO:66), HRV-C11 (SEQ ID NO:67), and shuffledIRES#42 (SEQ ID NO:68) are less regular and stable, especially the loop position sequence (Base-pair probabilities in RNAFold structure prediction, bases are marked in yellow, green, and blue). The secondary structures of iPV2 (SEQ ID NO:69) and Human XIAP (SEQ ID NO:70) differ significantly from the IRES structures described above.

[0186] Example 4: Preparation of scarless circular RNA based on stem-loop structure in HRV-B3 IRES and its mutants

[0187] 1. Plasmid construction

[0188] Since the loop position sequences in the IRES stem-loop structures of HRV-B3, HRV-B92, HRV-B37, shuffledIRES#01, and shuffledIRES#38 are the same, we take HRV-B3 IRES as an example for recombinant nucleic acid molecule design. As shown in Figure 17, based on the literature's partitioning of the HRV-B3 IRES domain and RNAFold's prediction of the secondary structure of the HRV-B3 IRES sequence (SEQ ID NO:59), stem-loop structures in HRV-B3 IRES were screened. A relatively long stem-loop structure exists in Domain I of HRV-B3 IRES. Its loop position sequence CCAAGTAAC (SEQ ID NO:71) contains a T base. Upstream of the T base are 5 free bases of CCAAG, and downstream are 3 free bases of AAC. This meets both the T4td ribozyme splicing requirements (N1N2N3N4N5TN6N7) and the Ana ribozyme splicing requirements (N1N2N3TN4N5N6). The loop position bases were split at the slash position (5'-CCAAGT / AAC-3'), yielding the fragment HRV-B3IRES I (nucleotide sequence as shown in SEQ ID NO:79) including CCAAGT and its upstream sequence. (as shown in NO:72) and the fragment HRV-B3IRES II (nucleotide sequence as shown in SEQ ID NO:73), which includes AAC bases and their downstream sequences.

[0189] HRV-B3 IRES II is linked to the 3' ends of the 3' intron of the T4td ribozyme (SEQ ID NO:74) and the 3' intron of the Ana ribozyme (SEQ ID NO:75), respectively. HRV-B3 IRES I is linked to the 5' ends of the T4td ribozyme 5' intron mutant (SEQ ID NO:76, P1 strictly complementary pair, as shown in Figure 18; SEQ ID NO:77, P1 not strictly complementary pair, as shown in Figure 19) and the Ana ribozyme 5' intron mutant (SEQ ID NO:78) (as shown in Figure 20).

[0190] The T4td intron ribozyme mutation region is mutated to AAATTGTTGCTTGG (SEQ ID NO:79, making P1 strictly complementary), in which the 5' intron P1 guide sequence is mutated to CTTGG (reverse complementary to CCAAG at the loop position), as shown in Figure 18; or the T4td intron ribozyme mutation region is mutated to AAATTGTTGCTTGT (SEQ ID NO:80, making P1 not strictly complementary), in which the 5' intron P1 guide sequence is mutated to CTTGT (CTTG reverse complementary to CAAG at the loop position, making P1 not strictly complementary), as shown in Figure 19; the T4td ribozyme 5' intron P10 guide sequence is mutated to TT (reverse complementary to AA at the loop position), and the corresponding bases in the T4td ribozyme 5' intron that are complementary to the P10 guide sequence are mutated to A. The 5' intron mutation region of Ana ribozyme is mutated to AAATAATTGCT (SEQ ID NO:81), in which the P1 guide sequence of the 5' intron is mutated to CT (reverse complementary to AG at the loop position), and the P10 guide sequence TT is reverse complementary to the AA base at the loop position, as shown in Figure 20.

[0191] The nucleotide sequence encoding the antigenic peptide protein (SEQ ID NO:14) was linked between the downstream of HRV-B3 IRES II and the upstream of HRV-B3 IRES I to obtain a nucleic acid molecule composed of T4td ribozyme 3' intron-HRV-B3 IRES II-antigen peptide ORF-HRV-B3IRES I-T4td ribozyme 5' intron (its nucleotide sequence is shown in SEQ ID NO:82, P1 is strictly complementary; SEQ ID NO:83, P1 is not strictly complementary), and a nucleic acid molecule composed of Ana ribozyme 3' intron-HRV-B3 IRES II-antigen peptide ORF-HRV-B3 IRES I-Ana ribozyme 5' intron (its nucleotide sequence is shown in SEQ ID NO:84). A T7 promoter (SEQ ID NO:85) was added to the 5' end of these three nucleic acid strands, and then the molecules were cloned into the pUC19 plasmid from 5' to 3' through the EcoRI and XbaI restriction sites.

[0192] Based on the literature's partitioning of the HRV-B3-eIF4G IRES domain and RNAFold's prediction of the secondary structure of the HRV-B3-eIF4G IRES sequence (SEQ ID NO:86), as shown in Figure 17, stem-loop structures in HRV-B3-eIF4G were screened. The loop position sequence CCAAGTAAC (SEQ ID NO:71) in the longer stem-loop structure in Domain I simultaneously met the requirements of T4td and Ana ribozyme splicing. The bases at the loop position were split at the slash position (5'-CCAAGT / AAC-3'), resulting in fragment HRV-B3-eIF4G IRES I (SEQ ID NO:72, identical to the HRV-B3 IRES I sequence) including CCAAGT and its upstream sequence, and fragment HRV-B3-eIF4G IRES II (SEQ ID NO:87) including AAC bases and its downstream sequence. HRV-B3-eIF4G IRES II is linked to the 3' end of the 3' intron of the T4td ribozyme (SEQ ID NO:74). HRV-B3-eIF4G IRES I is linked to the 5' end of the loosely paired 5' intron of the T4td ribozyme (SEQ ID NO:77).

[0193] Linking the Fluc protein coding sequence from 5' to 3' between the downstream of HRV-B3-eIF4G IRES II and the upstream of HRV-B3-eIF4G IRES I yielded a nucleic acid molecule (SEQ ID NO:88) composed of T4td ribozyme 3' intron - HRV-B3-eIF4G IRES II - Fluc ORF - HRV-B3-eIF4G IRES I - T4td ribozyme 5' intron; linking the Fluc protein coding sequence from 5' to 3' between the downstream of HRV-B3 IRES II and the upstream of HRV-B3 IRES I yielded a nucleic acid molecule (SEQ ID NO:89) composed of Ana ribozyme 3' intron - HRV-B3 IRES II - Fluc ORF - HRV-B3 IRES I - Ana ribozyme 5' intron. Adding a T7 promoter (SEQ ID NO:85) to the 5' end of these two nucleic acid strands, and then using EcoRI and XbaI in the 5' to 3' direction... The I restriction site was cloned onto the pUC19 plasmid.

[0194] To increase expression levels, a kozak sequence was added upstream of the ORF in the plasmid described above, and a poly AC sequence was added downstream.

[0195] 2. Preparation of circular RNA

[0196] The steps for plasmid linearization, in vitro transcription, in vitro circularization, and linear RNA digestion are as described in Example 1.

[0197] 3. RNA circularization identification

[0198] The method for detecting circular RNA by formaldehyde denaturation electrophoresis is as described in Example 1. The method for sequencing circular RNA splice sites is as described in Example 2.

[0199] Figure 21 shows the successful circularization of nucleic acid molecules with nucleotide sequences as shown in SEQ ID NO:82. Sequencing results show that the circularization occurred at the correct site, and the circularization efficiency was 98.3% as analyzed by Image J. Figure 22 shows the successful circularization of nucleic acid molecules with nucleotide sequences as shown in SEQ ID NO:83. Sequencing results show that the circularization occurred at the correct site, and the circularization efficiency was 96.4% as analyzed by Image J. Figure 23 shows the successful circularization of nucleic acid molecules with nucleotide sequences as shown in SEQ ID NO:84. Sequencing results show that the circularization occurred at the correct site, and the circularization efficiency was 97.5% as analyzed by Image J. Figure 24 shows the successful circularization of nucleic acid molecules with nucleotide sequences as shown in SEQ ID NO:88. Sequencing results show that the circularization occurred at the correct site, and the circularization efficiency was 97.2% as analyzed by Image J. Figure 25 shows the successful circularization of nucleic acid molecules with nucleotide sequences as shown in SEQ ID NO:89. Sequencing results show that the circularization occurred at the correct site, and the circularization efficiency was 97.4% as analyzed by Image J.

[0200] Example 5: Evaluation of the potency of circular mRNA encoding tumor antigen peptides

[0201] 1. Isolation of peripheral blood mononuclear cells (PBMCs):

[0202] PBMCs were isolated using Lymphocyte Separation Medium 1.077 (Yeasen Biotechnology, 40503ES). 2 ml of anticoagulated human blood was mixed and diluted with 2 ml of PBS. 3 ml of Lymphocyte Separation Medium (LSM) was added to a 15 ml centrifuge tube. The diluted blood was carefully added to the top layer of the LSM. The tube was centrifuged at 400×g for 30 min at room temperature. A white membrane of PBMCs formed on the LSM. The plasma above the PBMCs layer was aspirated, and the PBMCs layer was transferred to a new centrifuge tube. An equal volume of PBS was added, and the tube was centrifuged at 200×g for 10 min at room temperature. The PBMCs were washed once more with an appropriate amount of PBS. Finally, an appropriate amount of culture medium was used to resuspend the cells.

[0203] 2. Circular mRNA encoding tumor antigen peptides stimulates PBMCs

[0204] Take 100 μl (10 μL) of the separated PBMCs from each well. 5 100 μl of coated LNP-LNP cells (containing 0.1 μg of circRNA, whose nucleotide sequence is shown in SEQ ID NO:82 or SEQ ID NO:83) was added to each well of an untreated 96-well plate. Simultaneously, IL-2 and IL-7 were added to a final concentration of 20 U / ml and 20 ng / ml, respectively. After 72 h, half of the culture medium was aspirated from each well, and half of fresh culture medium was added. 0.1 μg of coated LNP-CircRNA was added to each well, and IL-2 and IL-7 were added to a final concentration of 20 U / ml and 20 ng / ml, respectively. After 72 h, the above steps were repeated. After 72 h, PBMCs were washed twice with PBS, and 100 μl of cells (10⁻⁶ cells / well) were added to each well. 5 (Number of cells) were transferred to a pre-coated IFN-γ ELISpot plate, stimulated with 10 μg / ml GP100 peptide, and supplemented with IL-2 and IL-7 to a final concentration of 20 U / ml and 20 ng / ml, respectively. After 16-18 hours, the plates were collected for ELISpot colorimetric assays. Two negative control groups were set up: group NC1 without circRNA, IL-2, and IL-7, and group NC2 without circRNA. Wells containing phytohemagglutinin (PHA) were also included to verify PBMC cell viability.

[0205] 3. ELISpot color development

[0206] ELISpot colorimetric assay was performed according to the experimental procedures of the ELISpot Plus: Human IFN-γ (HRP) kit (Dakewe, 3420-4HST). The culture medium in the plate was poured out, and pre-cooled deionized water was added (200 μl / well). The cells were incubated at 4°C for 10 min to lyse using hypotonic methods. The liquid in the wells was discarded, and the cells were washed 5 times with PBS, adding 200 μl of PBS to each well each time. Dilute biotin-labeled 7-B6-1 antibody to 1 μg / ml with PBS containing 0.5% fetal bovine serum (PBS-0.5% FCS), add 100 μl to each well, and incubate at 37°C for 2 h. Wash 5 times with PBS, dilute streptavidin-HRP (1:1000) in PBS-0.5% FCS, add 100 μl to each well, and incubate at 37°C for 1 h. Wash 5 times with PBS, add 100 μl of freshly prepared TMB substrate chromogenic solution to each well, and develop at room temperature in the dark for 5-30 min. Pour out the liquid in the wells, rinse with tap water to stop the chromogenic process, air dry, and analyze using an ELISpot spot analyzer. As shown in Figure 26, the circular RNA formed by SEQ ID NO:82 (SEQ 76 in the figure) and SEQ ID NO:83 (SEQ 77 in the figure) stimulated PBMCs and produced significantly more IFN-γ points than the negative control group. This indicates that the circular RNA formed by SEQ ID NO:82 and SEQ ID NO:83 can effectively express the encoded tumor antigen peptide in DC cells and activate T cells.

[0207] Example 6: Evaluation of the potency of circ mRNA encoding Fluc protein

[0208] According to Lipofectamine TM HEK 293T cells were transfected with the circular RNA formed by SEQ ID NO:88 according to the Thermo Fisher 3000 transfection kit (L3000008). Forty-eight hours after transfection, Fluc luciferase activity was detected according to the Thermo Fisher 16177 firefly luciferase glow detection kit (L3000008). A negative control group (NC) without transfection and a positive control group (PC) transfected with Fluc luciferase-expressing mRNA were set up. The results, shown in Figure 27, indicate that the amount of Fluc protein expressed by the circular RNA formed by SEQ ID NO:88 in HEK 293T cells was significantly higher than that in the negative and positive controls.

[0209] Example 7: Preparation of scarless circular RNA based on stem-loop structure in HRV-B4 IRES

[0210] 1. Plasmid construction

[0211] The secondary structure of the HRV-B4 IRES sequence (SEQ ID NO:66) was predicted using RNAFold. The stem-loop structure position in HRV-B4IRES was screened as shown in Figure 16. The CCGTAAC in its loop position sequence (Table 3) contains a T base. There are 3 free bases of CCG upstream of the T base and 3 free bases of AAC downstream, which meets the requirements of Ana ribozyme splicing (N1N2N3TN4N5N6). The bases in its loop position were split at the slash position (5'-CCGT / AAC-3') to obtain the fragment HRV-B4 IRES I (nucleotide sequence as shown in SEQ ID NO:90) including CCGT and its upstream sequence and the fragment HRV-B4 IRES II (nucleotide sequence as shown in SEQ ID NO:91) including the AAC base and its downstream sequence.

[0212] HRV-B4 IRES II is linked to the 3' end of the 3' intron of the Ana ribozyme (SEQ ID NO:75). HRV-B4 IRES I is linked to the 5' end of the 5' intron mutant of the Ana ribozyme (SEQ ID NO:92). The 5' intron mutant region of the Ana ribozyme is mutated to AAATAATTGCG, in which the P1 guide sequence of the 5' intron is mutated to CG (reverse complementary to CG at the loop position), and the P10 guide sequence TT is reverse complementary to the AA base at the loop position (as shown in Figure 28).

[0213] The antigen peptide protein-coding sequence (SEQ ID NO:14) was ligated between the downstream of HRV-B4 IRES II and the upstream of HRV-B4IRES I to obtain a nucleic acid molecule composed of Ana ribozyme 3' intron-HRV-B4 IRES II-antigen peptide ORF-HRV-B4 IRES I-Ana ribozyme 5' intron (its nucleotide sequence is shown in SEQ ID NO:93). RNAFold performed secondary structure prediction on this nucleic acid sequence, and the results showed that the Ana ribozyme structure was not interfered with by other sequences (Figure 28). A T7 promoter (SEQ ID NO:85) was added to the 5' end of this nucleic acid chain, and then it was cloned into the pUC19 plasmid from the 5' to 3' direction through the EcoRI and XbaI restriction sites.

[0214] To increase expression levels, a kozak sequence was added upstream of the ORF in the plasmid described above, and a poly AC sequence was added downstream.

[0215] 2. Preparation of circular RNA

[0216] The steps for plasmid linearization, in vitro transcription, in vitro circularization, and linear RNA digestion are as described in Example 1.

[0217] 3. RNA circularization identification

[0218] The method for detecting circular RNA by formaldehyde denaturation electrophoresis is as described in Example 1. The method for sequencing circular RNA splice sites is as described in Example 2.

[0219] Figure 28 shows that the nucleic acid molecule with the nucleotide sequence as shown in SEQ ID NO:93 was successfully circularized. The circularization efficiency was 37.8% as shown in column B4 of Figure 32. The circularized RNA was subjected to reverse transcription PCR. Primers were designed on both sides of the adapter for PCR. Sanger sequencing results showed that the nucleic acid molecule with the nucleotide sequence as shown in SEQ ID NO:93 was successfully circularized at the predetermined base positions.

[0220] Example 8: Preparation of scar-free circular RNA based on stem-loop structure in Human XIAP IRES

[0221] 1. Plasmid construction

[0222] Secondary structure prediction of the Human XIAP IRES sequence (SEQ ID NO:70) was performed using RNAFold. The stem-loop structure position in Human XIAP IRES was screened as shown in Figure 16. In the loop position sequence (Table 3), AATTAAT has 3 free AAT bases upstream and downstream of the second T base, which meets the requirements of Ana ribozyme splicing (N1N2N3TN4N5N6). The bases in the loop position were split at the slash position (5'-AATT / AAT-3') to obtain fragment XIAP IRES I (nucleotide sequence as shown in SEQ ID NO:94) including AAT and its upstream sequence, and fragment XIAP IRES II (nucleotide sequence as shown in SEQ ID NO:95) including AAT bases and its downstream sequence.

[0223] XIAP IRES II is linked to the 3' end of the 3' intron of the Ana ribozyme (SEQ ID NO:75). XIAP IRES I is linked to the 5' end of the 5' intron mutant of the Ana ribozyme (SEQ ID NO:96). The 5' intron mutant region of the Ana ribozyme is mutated to AAATAATTGAT, where the P1 guide sequence of the 5' intron is mutated to AT (reverse complementary to AT at the loop position), and the P10 guide sequence TT is reverse complementary to the AA base at the loop position (as shown in Figure 29).

[0224] The antigen peptide protein-coding sequence (SEQ ID NO:14) was ligated between the downstream of XIAP IRES II and the upstream of XIAP IRES I to obtain a nucleic acid molecule composed of Ana ribozyme 3' intron-HRV-B4 IRES II-antigen peptide ORF-HRV-B4 IRES I-Ana ribozyme 5' intron (its nucleotide sequence is shown in SEQ ID NO:97). RNAFold performed secondary structure prediction on this nucleic acid sequence, and the results showed that the Ana ribozyme structure was not interfered with by other sequences (Figure 29). A T7 promoter (SEQ ID NO:85) was added to the 5' end of this nucleic acid chain, and then it was cloned into the pUC19 plasmid from the 5' to 3' direction through the EcoRI and XbaI restriction sites.

[0225] To increase expression levels, a kozak sequence was added upstream of the ORF in the plasmid described above, and a poly AC sequence was added downstream.

[0226] 2. Preparation of circular RNA

[0227] The steps for plasmid linearization, in vitro transcription, in vitro circularization, and linear RNA digestion are as described in Example 1.

[0228] 3. RNA circularization identification

[0229] The method for detecting circular RNA by formaldehyde denaturation electrophoresis is as described in Example 1. The method for sequencing circular RNA splice sites is as described in Example 2.

[0230] Figure 29 shows that the nucleic acid molecule with the nucleotide sequence as shown in SEQ ID NO:97 was successfully circularized. The circularization efficiency was 62.6% as shown in the XIAP column of Figure 32. The circularized RNA was subjected to reverse transcription PCR. Primers were designed on both sides of the adapter for PCR. Sanger sequencing results showed that the nucleic acid molecule with the nucleotide sequence as shown in SEQ ID NO:97 was successfully circularized at the predetermined base positions.

[0231] Example 9: Preparation of scarless circular RNA based on the stem-loop structure in HRV-B97 IRES

[0232] 1. Plasmid construction

[0233] The secondary structure of the HRV-B97 IRES sequence (SEQ ID NO:64) was predicted using RNAFold. The stem-loop structure position in HRV-B97 IRES was screened as shown in Figure 16. In the CGTTAGA sequence of its loop position (Table 3), there are 3 free CGT bases upstream of the second T base and 3 free AGA bases downstream, which meets the requirements of Ana ribozyme splicing (N1N2N3TN4N5N6). The bases at the loop position were split at the slash position (5'-CGTT / AGA-3') to obtain the fragment HRV-B97 IRES I (nucleotide sequence as shown in SEQ ID NO:98) including CGTT and its upstream sequence and the fragment HRV-B97 IRES II (nucleotide sequence as shown in SEQ ID NO:99) including the AGA base and its downstream sequence.

[0234] HRV-B97 IRES II is linked to the 3' end of the 3' intron (SEQ ID NO:75) of the Ana ribozyme. HRV-B97IRES I is linked to the 5' end of the 5' intron mutant of the Ana ribozyme (SEQ ID NO:100). The 5' intron mutant region of the Ana ribozyme is mutated to AGATAACTGAC, in which the P1 guide sequence of the 5' intron is mutated to AC (reverse complementary to GT at the loop position), and the P10 guide sequence CT is reverse complementary to the AG base at the loop position (as shown in Figure 30).

[0235] The antigen peptide protein-coding sequence (SEQ ID NO:14) was ligated between the downstream of HRV-B97 IRES II and the upstream of HRV-B97 IRES I to obtain a nucleic acid molecule composed of Ana ribozyme 3' intron-HRV-B97 IRES II-antigen peptide ORF-HRV-B97 IRES I-Ana ribozyme 5' intron (its nucleotide sequence is shown in SEQ ID NO:101). RNAFold performed secondary structure prediction on this nucleic acid sequence, and the results showed that the Ana ribozyme structure was not interfered with by other sequences (Figure 30). A T7 promoter (SEQ ID NO:85) was added to the 5' end of this nucleic acid chain, and then it was cloned into the pUC19 plasmid from the 5' to 3' direction through the EcoRI and XbaI restriction sites.

[0236] To increase expression levels, a kozak sequence was added upstream of the ORF in the plasmid described above, and a poly AC sequence was added downstream.

[0237] 2. Preparation of circular RNA

[0238] The steps for plasmid linearization, in vitro transcription, in vitro circularization, and linear RNA digestion are as described in Example 1.

[0239] 3. RNA circularization identification

[0240] The method for detecting circular RNA by formaldehyde denaturation electrophoresis is as described in Example 1. The method for sequencing circular RNA splice sites is as described in Example 2.

[0241] Figure 30 shows that the nucleic acid molecule with the nucleotide sequence as shown in SEQ ID NO:101 was successfully circularized. The circularization efficiency was 64.3% as shown in column B97 of Figure 32. The circularized RNA was subjected to reverse transcription PCR. Primers were designed on both sides of the adapter for PCR. Sanger sequencing results showed that the nucleic acid molecule with the nucleotide sequence as shown in SEQ ID NO:101 was successfully circularized at the predetermined base positions.

[0242] Example 10: Preparation of scarless circular RNA based on stem-loop structure in ShuffledIRES#42IRES

[0243] 1. Plasmid construction

[0244] The secondary structure of the ShuffledIRES#42IRES sequence (SEQ ID NO:68) was predicted using RNAFold. The stem-loop structure position in ShuffledIRES#42IRES was screened as shown in Figure 16. In the loop position sequence (Table 3), there are 3 free bases of ATG upstream of the second T base and 3 free bases of AAC downstream, which meets the requirements of Ana ribozyme splicing (N1N2N3TN4N5N6). The bases in the loop position were split at the slash position (5'-ATGT / AAC-3') to obtain the fragment ShuffledIRES#42IRES I (nucleotide sequence as shown in SEQ ID NO:102) including ATGT and its upstream sequence and the fragment ShuffledIRES#42IRES II (nucleotide sequence as shown in SEQ ID NO:103) including the AGA base and its downstream sequence.

[0245] ShuffledIRES#42IRES II is linked to the 3' end of the 3' intron (SEQ ID NO:75) of the Ana ribozyme. ShuffledIRES#42IRES I is linked to the 5' end of the 5' intron mutant (SEQ ID NO:104) of the Ana ribozyme. The 5' intron mutant region of the Ana ribozyme is mutated to AAATAATTGCA, in which the P1 guide sequence of the 5' intron is mutated to CA (reverse complementary to TG at the loop position), and the P10 guide sequence TT is reverse complementary to the AA base at the loop position (as shown in Figure 31).

[0246] The antigen peptide protein-coding sequence (SEQ ID NO:14) was ligated between the downstream of ShuffledIRES#42IRES II and the upstream of ShuffledIRES#42IRES I to obtain a nucleic acid molecule composed of the Ana ribozyme 3' intron-ShuffledIRES#42IRES II-antigen peptide ORF-ShuffledIRES#42IRES I-Ana ribozyme 5' intron (its nucleotide sequence is shown in SEQ ID NO:105). RNAFold performed secondary structure prediction on this nucleic acid sequence, and the results showed that the Ana ribozyme structure was not interfered with by other sequences (Figure 31). A T7 promoter (SEQ ID NO:85) was added to the 5' end of this nucleic acid chain, and then it was cloned into the pUC19 plasmid from the 5' to 3' direction through the EcoRI and XbaI restriction sites.

[0247] To increase expression levels, a kozak sequence was added upstream of the ORF in the plasmid described above, and a poly AC sequence was added downstream.

[0248] 2. Preparation of circular RNA

[0249] The steps for plasmid linearization, in vitro transcription, in vitro circularization, and linear RNA digestion are as described in Example 1.

[0250] 3. RNA circularization identification

[0251] The method for detecting circular RNA by formaldehyde denaturation electrophoresis is as described in Example 1. The method for sequencing circular RNA splice sites is as described in Example 2.

[0252] Figure 31 shows that the nucleic acid molecule with the nucleotide sequence as shown in SEQ ID NO:105 was successfully circularized. The circularization efficiency was 94.5% as shown in column S42 of Figure 32. The circularized RNA was subjected to reverse transcription PCR. Primers were designed on both sides of the adapter for PCR. Sanger sequencing results showed that the nucleic acid molecule with the nucleotide sequence as shown in SEQ ID NO:105 was successfully circularized at the predetermined base positions.

[0253] Example 11: Regular stem-loop structures are more conducive to scarless RNA circularization.

[0254] Based on the experimental results above, we can conclude that the circumduction efficiency is relatively higher for more regular stem-loop structures such as HRV-B3 IRES and ShuffledIRES#42, while the circumduction efficiency is relatively lower for structures like HRV-B4 IRES and HRV-B97 IRES, where the loop position contains four consecutive base pairs, resulting in an unstable double-stranded structure. To further verify that regular stem-loop structures are more conducive to scarless RNA circumduction, we further selected four IRES with more regular stem-loop structures—HRV-B92 (SEQ ID NO:60), HRV-B37 (SEQ ID NO:61), shuffledIRES#01 (SEQ ID NO:62), and shuffledIRES#38 (SEQ ID NO:63)—to design and prepare scarless circular RNA.

[0255] 1. Plasmid construction

[0256] The four IRES ring position sequences HRV-B37, HRV-B92, shuffledIRES#01, and shuffledIRES#38 all contain CCAAGTAAC (SEQ ID NO:71). By splitting them at the slash positions of the ring bases (5'-CCAAGT / AAC-3'), the following fragments were obtained: HRV-B37 IRES I (SEQ ID NO:106), HRV-B37 IRES II (SEQ ID NO:107), HRV-B92 IRES I (SEQ ID NO:108), HRV-B92 IRES II (SEQ ID NO:109), shuffledIRES#01IRES I (SEQ ID NO:110), shuffledIRES#01IRES II (SEQ ID NO:111), shuffledIRES#38IRES I (SEQ ID NO:112), and shuffledIRES#38IRES II (SEQ ID NO:113). The split IRES II sequences were ligated to the 3' end of the 3' intron of the Ana ribozyme (SEQ ID NO:75), and IRES I sequences were ligated to the 5' end of the 5' intron mutant of the Ana ribozyme (SEQ ID NO:78). The nucleotide sequence encoding the antigenic peptide protein (SEQ ID NO:14) was ligated between the downstream of each IRES II and the upstream of each IRES I, respectively, to obtain the precursor sequences SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, and SEQ ID NO:117.

[0257] The T7 promoter (SEQ ID NO:85) was added to the 5' end of the above four precursor nucleic acid sequences, and then the sequences were cloned into the pUC19 plasmid from the 5' to 3' direction through the EcoRI and XbaI restriction sites.

[0258] To increase expression levels, a kozak sequence was added upstream of the ORF in the plasmid described above, and a poly AC sequence was added downstream.

[0259] 2. Preparation of circular RNA

[0260] The steps for plasmid linearization, in vitro transcription, in vitro circularization, and linear RNA digestion are as described in Example 1.

[0261] 3. RNA circularization identification

[0262] The formaldehyde denaturing electrophoresis method for detecting circular RNA was as described in Example 1. Figure 33 shows that circular RNAs prepared using regular stem-loop structures from HRV-B37, HRV-B92, shuffledIRES#01, and shuffledIRES#38 all achieved good circularization. Specifically, the circularization efficiency based on the HRV-B37 stem-loop structure was 91%, the HRV-B92 stem-loop structure was 87%, the shuffledIRES#01 stem-loop structure was 90%, and the shuffledIRES#38 stem-loop structure was 79%. This indicates that using a more regular stem-loop structure to prepare scar-free circular RNA is more conducive to improving RNA circularization efficiency.

[0263] Example 12: Scarless circular RNA is difficult to prepare without relying on the stem-loop structure rules in this disclosure.

[0264] In addition to the stable stem-loop structure, the secondary structure of IRES also contains other unstable random coils. To further demonstrate the necessity of the stem-loop structure rules in this disclosure during the design and preparation of scarless circular RNA, we took CVB3IRES (SEQ ID NO:118) as an example, selected sites in the random coil region of CVB3 IRES for truncation, spliced ​​it with the Ana intron ribozyme, and mutated the intron guide sequence according to the mutation rules in this disclosure, and performed in vitro transcription circularization verification.

[0265] 1. Plasmid construction

[0266] The CVB3 IRES contains eight main domains: I, II, III, IV, V, VI, VII, and a variable region (Figure 34). Secondary structure prediction of the CVB3 IRES sequence using RNAFold is shown in Figure 35. Besides the variable region, unstable random coils also exist between domains V and VI. First, two sites (S1 and S2) in the variable region were selected for truncation. The S1 site was truncated at the slash of the sequence CACTTAGCTT / AAAGAGGTTA, spliced ​​with the Ana ribozyme, and the Ana intron ribozyme guide sequence was mutated according to the rules in the invention. After assembly with the ORF of the Fluc protein, the precursor sequence SEQ ID NO:119 was obtained. The S2 site was truncated at the slash of the sequence GAGGTTACTT / AAAACATTAC, spliced ​​with the Ana ribozyme, and the Ana intron ribozyme guide sequence was mutated according to the rules in the invention. After assembly with the ORF of the Fluc protein, the precursor sequence SEQ ID NO:120 was obtained. A site S3 (CCGACTACTT / TGGGTGTCCG) is selected between the V and VI domains, truncated at the slash and spliced ​​with the Ana ribozyme, and the Ana intron ribozyme guide sequence is mutated according to the rules in the invention. After assembly with the ORF of the Fluc protein, the precursor sequence SEQ ID NO:121 is obtained.

[0267] The T7 promoter (SEQ ID NO:85) was added to the 5' end of the above three precursor nucleic acid sequences, and then the sequences were cloned into the pUC19 plasmid from the 5' to 3' direction via the EcoRI and XbaI restriction sites.

[0268] To increase expression levels, a kozak sequence was added upstream of the ORF in the plasmid described above, and a poly AC sequence was added downstream.

[0269] 2. Preparation of circular RNA

[0270] The steps for plasmid linearization, in vitro transcription, in vitro circularization, and linear RNA digestion are as described in Example 1.

[0271] 3. RNA circularization identification

[0272] The method for detecting circular RNA by formaldehyde denaturation electrophoresis is as described in Example 1. Figure 36 shows that nucleic acid molecules with nucleotide sequences as shown in SEQ ID NO:119, SEQ ID NO:120, and SEQ ID NO:121 failed to complete circularization well and were degraded into diffuse bands after RNase R treatment. This indicates that using the stem-loop structure rules based on this disclosure to design and prepare scarless circular RNA can effectively improve the success rate of RNA circularization.

[0273] The embodiments of this disclosure have been described above. However, this disclosure is not limited to the above embodiments. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this disclosure should be included within the protection scope of this disclosure.

[0274] Sequence information:

[0275] SEQ ID NO:1 (T4td ribozyme 3' intron, containing exon 21nt)

[0276] SEQ ID NO:2 (T4td ribozyme 5' intron, containing 16nt exon)

[0277] SEQ ID NO:3 (Ana ribozyme 3' intron, containing 51 nt exon)

[0278] SEQ ID NO:4 (Ana ribozyme 5' intron, containing 15nt exon)

[0279] SEQ ID NO:5(IRES MP,75 )

[0280] SEQ ID NO:6 Tumor Antigen Peptide MAGE-1

[0281] SEQ ID NO:7 Tumor Antigen Peptide TRP-2

[0282] SEQ ID NO:8 Tumor Antigen Peptide gp100

[0283] SEQ ID NO:9 Tumor antigen peptide IL13Rα2

[0284] SEQ ID NO:10 Tumor antigen peptide Melan A

[0285] SEQ ID NO:11 Tumor Antigen Peptide BST2

[0286] SEQ ID NO:12 Tumor Antigen Peptide IMP2

[0287] SEQ ID NO:13

[0288] SEQ ID NO:14

[0289] SEQ ID NO:15

[0290] SEQ ID NO:16

[0291] SEQ ID NO:17

[0292] SEQ ID NO:18

[0293] SEQ ID NO:19

[0294] SEQ ID NO:20

[0295] SEQ ID NO:21(CVB3 IRES)

[0296] SEQ ID NO:22(FLUC DNA sequence)

[0297] SEQ ID NO:23

[0298] SEQ ID NO:24

[0299] SEQ ID NO:25

[0300] SEQ ID NO:26

[0301] SEQ ID NO:27

[0302] SEQ ID NO:28

[0303] SEQ ID NO:29(10nt)

[0304] SEQ ID NO:30(8nt)

[0305] SEQ ID NO:31(7nt)

[0306] SEQ ID NO:32(6nt-2)

[0307] SEQ ID NO:33(6nt)

[0308] SEQ ID NO:34(5nt)

[0309] SEQ ID NO:35(4nt-2)

[0310] SEQ ID NO:36(4nt)

[0311] SEQ ID NO:37(10nt)

[0312] SEQ ID NO:38(8nt)

[0313] SEQ ID NO:39(6nt)

[0314] SEQ ID NO:40(4nt)

[0315] SEQ ID NO:41(8nt)

[0316] SEQ ID NO:42(7nt)

[0317] SEQ ID NO:43(6nt-2)

[0318] SEQ ID NO:44

[0319] SEQ ID NO:45(6nt)

[0320] SEQ ID NO:46(8nt)

[0321] SEQ ID NO:47(7nt)

[0322] SEQ ID NO:48(7nt-2)

[0323] SEQ ID NO:49(6nt)

[0324] SEQ ID NO:50(4nt)

[0325] SEQ ID NO:51(NoAa)

[0326] SEQ ID NO:52(6ntAa)

[0327] SEQ ID NO:53(8ntAa)

[0328] SEQ ID NO:54(20ntAa)

[0329] SEQ ID NO:55(NOAT)

[0330] SEQ ID NO:56(6ntAT)

[0331] SEQ ID NO:57(10ntAT)

[0332] SEQ ID NO:58(20ntAT)

[0333] SEQ ID NO:59 (HRV-B3 IRES)

[0334] SEQ ID NO:60 (HRV-B92 IRES)

[0335] SEQ ID NO:61 (HRV-B37 IRES)

[0336] SEQ ID NO:62 (shuffledIRES#01)

[0337] SEQ ID NO:63 (shuffledIRES#38)

[0338] SEQ ID NO:64 (HRV-B97)

[0339] SEQ ID NO:65 (shuffledIRES#03)

[0340] SEQ ID NO:66 (HRV-B4)

[0341] SEQ ID NO:67 (HRV-C11)

[0342] SEQ ID NO:68 (shuffledIRES#42)

[0343] SEQ ID NO:69 (iPV2)

[0344] SEQ ID NO:70 (Human XIAP)

[0345] SEQ ID NO:71 (HRV-B3 IRES loop position sequence)

[0346] SEQ ID NO:72 (HRV-B3 IRES I)

[0347] SEQ ID NO:73 (HRV-B3 IRES II)

[0348] SEQ ID NO:74 (T4td ribozyme 3' intron)

[0349] SEQ ID NO:75 (Ana ribozyme 3' intron)

[0350] SEQ ID NO:76 (T4td ribozyme 5' intron strict)

[0351] SEQ ID NO:77 (T4td ribozyme 5' intron is not rigorous)

[0352] SEQ ID NO:78 (Ana ribozyme 5' intron)

[0353] SEQ ID NO:79 (T4td intron ribozyme P1 and P10 region mutants are strictly defined)

[0354] SEQ ID NO:80 (T4td intron ribozyme P1 and P10 region mutants are not rigorous)

[0355] SEQ ID NO:81 (Ana intron ribozyme P1 and P10 region mutant)

[0356] SEQ ID NO:82 (P1 rigorous complementarity)

[0357] SEQ ID NO:83

[0358] SEQ ID NO:84

[0359] SEQ ID NO:85(T7 Promoter)

[0360] SEQ ID NO:86(HRV-B3-eIF4G IRES)

[0361] SEQ ID NO:87(HRV-B3-eIF4G IRES II)

[0362] SEQ ID NO:88

[0363] SEQ ID NO:89

[0364] SEQ ID NO:90(HRV-B4 IRES I)

[0365] SEQ ID NO:91(HRV-B4 IRES II)

[0366] SEQ ID NO:92 (5' intron of Ana ribozyme)

[0367] SEQ ID NO:93 (Ana ribozyme HRVB4 polypeptide)

[0368] SEQ ID NO:94(Human XIAP IRES I)

[0369] SEQ ID NO:95(Human XIAP IRES II)

[0370] SEQ ID NO:96 (5' intron mutant of Ana ribozyme)

[0371] SEQ ID NO:97 (Ana ribozyme XIAP polypeptide)

[0372] SEQ ID NO:98(HRV-B97 IRES I)

[0373] SEQ ID NO:99(HRV-B97 IRES II)

[0374] SEQ ID NO:100 (5' intron mutant of Ana ribozyme)

[0375] SEQ ID NO:101(HRV-B97 Ana peptide)

[0376] SEQ ID NO:102 (ShuffledIRES#42 IRES I)

[0377] SEQ ID NO:103 (ShuffledIRES#42 IRES II)

[0378] SEQ ID NO:104 (5’ intron mutant of Ana ribozyme)

[0379] SEQ ID NO:105 (Ana ribozyme ShuffledIRES#42 IRES polypeptide)

[0380] SEQ ID NO:106 (HRV - B37 IRES I)

[0381] SEQ ID NO:107 (HRV - B37 IRES II)

[0382] SEQ ID NO:108 (HRV - B92 IRES I)

[0383] SEQ ID NO:109 (HRV - B92 IRES II)

[0384] SEQ ID NO:110 (shuffledIRES#01 IRES I)

[0385] SEQ ID NO:111 (shuffledIRES#01 IRES II)

[0386] SEQ ID NO:112(shuffledIRES#38 IRES I)

[0387] SEQ ID NO:113 (shuffledIRES#38 IRES II)

[0388] SEQ ID NO:114 (B37 Ana polypeptide)

[0389] SEQ ID NO:115(B92 Ana polypeptide)

[0390] SEQ ID NO:116(S01 Ana polypeptide)

[0391] SEQ ID NO:117(S38 Ana polypeptide)

[0392] SEQ ID NO:118(CVB3 IRES)

[0393] SEQ ID NO:119 (S1 circular RNA precursor)

[0394] SEQ ID NO:120 (S2 circular RNA precursor)

[0395] SEQ ID NO:121 (S3 circular RNA precursor)

Claims

1. A recombinant nucleic acid molecule for preparing scarless circular RNA, comprising elements arranged in the following order from the 5' to 3' direction: a.3'I group introns or their mutant fragments (intron fragment II), b. Unit I fragment II, whose 5' end includes the II ribozyme recognition fragment. c. Functional units, d. The first unit fragment I, whose 3' end includes the I ribozyme recognition fragment, e.5'I group introns or their mutant fragments (intron fragment I); The functional units include IRES, nucleic acid aptamers, protein binding sequences, protein coding regions, non-coding regions, or combinations thereof; The intron fragment II is located at the 3' end of the intron fragment I, meaning that the complete group I introns or their mutant sequences include: Intron fragment I - Intron fragment II, where "-" represents a phosphodiester bond; The first unit fragment II is located at the 3' end of the first unit fragment I, that is, the complete first unit sequence includes: first unit fragment I - first unit fragment II, where "-" represents a phosphodiester bond; Wherein, the 3' end of the first unit fragment I contains a first ribozyme recognition fragment, which is composed of a first predetermined number of nucleotides located at the 3' end of the first unit fragment I; The 5' end of the first unit fragment II contains a second ribozyme recognition fragment, which is composed of a second predetermined number of nucleotides located at the 5' end of the first unit fragment II; The intron mutants in group I recognize and covalently link the first ribozyme recognition fragment and the second ribozyme recognition fragment to obtain the circular nucleic acid molecule. That is, the complete first unit sequence in the scarless circular RNA contains the first ribozyme recognition fragment - the second ribozyme recognition fragment ("circular fragment"), where "-" represents a phosphodiester bond. The first unit has a local stem structure, a local double-chain structure, or a local hairpin structure, wherein the local stem structure, local double-chain structure, or local hairpin structure is adjacent to or includes the ring-forming segment; Preferably, the first unit is a nucleic acid aptamer or a translation initiation element; Preferably, the cyclic fragment is located in the ring of the stem-loop structure of the translation initiation element, and a double-stranded structure formed by complementary pairing sequences exists within 100 bases upstream and downstream of the cyclic fragment; preferably, the double-stranded structure contains at least 5 consecutive complementary pairing bases; even more preferably, the number of complementary and non-complementary pairing bases in the stem of the stem-loop structure exceeds 20 bp.

2. The recombinant nucleic acid molecule according to claim 1, wherein the group I intron mutant contains a mutation in the guide region of P1 / P10, and the mutated guide region is complementary to the first / second ribozyme recognition fragment in the first unit fragment I / II, and the first / second ribozyme recognition fragment is defined as a circular fragment.

3. The recombinant nucleic acid molecule according to claim 1 or 2, wherein, The 3' end base of the first ribozyme recognition fragment is T, the first preset number of nucleotides is selected from 3-6 nucleotides, and the second preset number of nucleotides is selected from 0-3 nucleotides; Preferably, the group I introns are mutants of the Ana ribozyme, wherein the first ribozyme recognition fragment is 5'-N1N2N3T-3' or 5'-N2N3T-3', and the second ribozyme recognition fragment is 5'-N4N5N6-3' or 5'-N4N5-3'. The Ana ribozyme mutant contains the following mutant region N in the 5' intron fragment. 6’ N 7’ ATAAN 5’ N 4’ GN 3’ N 2’ Where N is A, U, C, G or T, where N 3’ N 2’ Pairing with N2N3 in reverse complementary direction, N 5’ N 4’ Pairing with N4N5 in reverse complementary direction, N 5’ N 4’ With N 6’ N 7’ Reverse complementary pairing, the base complementary pairing includes AU, GC, GU, AT, and GT base pairs; Preferably, the group I introns are mutants of the T4td ribozyme, wherein the first ribozyme recognition fragment is 5'-N1N2N3N4N5T-3' or 5'-N2N3N4N5T-3' or 5'-N3N4N5T-3', and the second ribozyme recognition fragment is 5'-N6N7-3' or N6 or absent. The T4td ribozyme mutant contains the following mutation region N in the 5' intron fragment. 8’ AATTGN 7’ N 6’ GN 5’ N 4’ N 3’ N 2’ N 1’ Where N is A, U, C, G, or T; N 5’ N 4’ N 3’ N 2’ N 1’ Pairing with N1N2N3N4N5 in reverse complementary order, or N 5’ N 4’ N 3’ N 2’ Pairing with N2N3N4N5 in reverse complementary order, or N 5’ N 4’ N 3’ Pairing with N3N4N5 in reverse complementary direction; N 7’ N 6’ Pairing with N6N7 in reverse complementary direction, or N 6’ Complementary pairing with N6, or preferably, when N6N7 is absent, the 3' end T base of the I ribozyme recognition fragment is adjacent to the stem structure of the first unit, then the P10 guide sequence N 7’ N 6’ It can be without mutation; N 8’ With N 6’ Complementary pairing, the complementary base pairing includes AU, GC, GU, AT, GT base pairs; preferably, N6 is U or C, N 6’ No mutations occur.

4. The recombinant nucleic acid molecule according to any one of claims 1-3, wherein, The first unit fragment is an active sequence having the function of initiating translation of the aforementioned functional unit, and the first unit fragment I and the first unit fragment II are translation initiation element fragment I and translation initiation element fragment II, respectively; Optionally, the translation initiation element sequence comprises one or more of the following sequences: IRES sequence, 5'UTR sequence, Kozak sequence, sequence containing m6A modification, complementary sequence of ribosomal 18S rRNA, and aptamer sequence.

5. The recombinant nucleic acid molecule according to claim 4, wherein the IRES sequence comprises HRV-B3, HRV-B92, iHRV-B37, iHRV-B97, iHRV-B4, iHRV-C11, iPV2, Human XIAP, CVB3, EMCV ribosome entry site sequences and their mutants, as well as artificially recombinant shuffledIRES#01, shuffledIRES#38, shuffledIRES#03, shuffledIRES#42 and their mutants; Preferably, the nucleotide sequence of the translation initiation element is shown in any one of SEQ ID NO: 59, 60, 61, 62, 63, 64, 66, 68, 70 and 86; Preferably, the nucleotide sequence of the translation initiation element fragment I is as shown in SEQ ID NO:72, and the nucleotide sequence of the translation initiation element fragment II is as shown in SEQ ID NO:73; Preferably, the nucleotide sequence of the translation initiation element fragment I is shown in SEQ ID NO:98, and the nucleotide sequence of the translation initiation element fragment II is shown in SEQ ID NO:99; Preferably, the nucleotide sequence of the translation initiation element fragment I is as shown in SEQ ID NO:90, and the nucleotide sequence of the translation initiation element fragment II is as shown in SEQ ID NO:91; Preferably, the nucleotide sequence of the translation initiation element fragment I is shown in SEQ ID NO:102, and the nucleotide sequence of the translation initiation element fragment II is shown in SEQ ID NO:103; Preferably, the nucleotide sequence of the translation initiation element fragment I is shown in SEQ ID NO:94, and the nucleotide sequence of the translation initiation element fragment II is shown in SEQ ID NO:95; Preferably, the nucleotide sequence of the translation initiation element fragment I is shown in SEQ ID NO:72, and the nucleotide sequence of the translation initiation element fragment II is shown in SEQ ID NO:87; Preferably, the nucleotide sequence of the translation initiation element fragment I is shown in SEQ ID NO:106, and the nucleotide sequence of the translation initiation element fragment II is shown in SEQ ID NO:107; Preferably, the nucleotide sequence of the translation initiation element fragment I is shown in SEQ ID NO:108, and the nucleotide sequence of the translation initiation element fragment II is shown in SEQ ID NO:109; Preferably, the nucleotide sequence of the translation initiation element fragment I is shown in SEQ ID NO:110, and the nucleotide sequence of the translation initiation element fragment II is shown in SEQ ID NO:111; Preferably, the nucleotide sequence of the translation initiation element fragment I is shown in SEQ ID NO:112, and the nucleotide sequence of the translation initiation element fragment II is shown in SEQ ID NO:

113.

6. The recombinant nucleic acid molecule according to any one of claims 1-5, wherein the nucleotide sequence of intron fragment I is shown in any one of SEQ ID NO: 76, 77, 78, 92, 96, 100 and 104, and the nucleotide sequence of intron fragment II is shown in SEQ ID NO: 74 or 75; Preferably, the nucleotide sequence of intron fragment I is as shown in SEQ ID NO: 76 or 77, and the nucleotide sequence of intron fragment II is as shown in SEQ ID NO: 74; Preferably, the nucleotide sequence of intron fragment I is shown in any one of 78, 92, 96, 100 and 104, and the nucleotide sequence of intron fragment II is shown in SEQ ID NO:

75.

7. The recombinant nucleic acid molecule according to any one of claims 1-6, wherein, The functional unit contains at least one coding region; alternatively, the functional unit contains at least two coding regions, each coding region independently encoding any type of target polypeptide.

8. The recombinant nucleic acid molecule according to claim 7, wherein the functional unit comprises at least two coding regions, wherein, Connectors are used to link any two adjacent coding regions; Preferably, the linker is a polynucleotide encoding a 2A peptide.

9. The recombinant nucleic acid molecule according to claim 7, wherein the functional unit comprises at least two coding regions, wherein, A translation start element is connected between any two adjacent coding regions; Optionally, the translation initiation element located between any two adjacent coding regions contains one or more of the following sequences: IRES sequence, 5'UTR sequence, Kozak sequence, sequence containing m6A modification, complementary sequence of ribosomal 18S rRNA, and aptamer.

10. The recombinant nucleic acid molecule according to any one of claims 1-9, wherein, The functional unit contains the coding sequence of a human protein or a non-human protein; Preferably, the human or non-human protein is selected from one or more of the following: antigen, antibody, antigen-binding fragment, therapeutic peptide, fluorescent protein, CAR-T molecule, protein with disease therapeutic activity, and protein with gene editing activity; Preferably, the human or non-human protein is a tandem tumor antigen peptide and a Fluc protein; more preferably, the amino acid sequence of the tandem tumor antigen peptide is shown in SEQ ID NO:13, and the nucleotide sequence of the Fluc protein is shown in SEQ ID NO:

22.

11. The recombinant nucleic acid molecule according to any one of claims 1-10, wherein, The recombinant nucleic acid molecule further includes an insertion element located between the coding element and the translation initiation element; The insertion element is selected from at least one of the following groups (i)-(iii): (i) transcriptional regulatory elements, (ii) translational regulatory elements, and (iii) purification elements; Optionally, the insert element comprises a sequence of one or more combinations of the following: polyA sequence, polyAC sequence, aptamer sequence, riboswitch sequence, sequence that binds transcription regulatory factors, antisense oligonucleotide (ASO), small interfering RNA (siRNA), miRNA, miRNA sponge, or lncRNA.

12. The recombinant nucleic acid molecule according to any one of claims 1-11, wherein, The interior of any one of the intron fragment I, the coding element, and the intron fragment II does not contain a nucleotide sequence derived from an exon; or, the interior of any two of the intron fragment I, the translation initiation element fragment II, the coding element, the translation initiation element fragment I, and the intron fragment II does not contain a nucleotide sequence derived from an exon.

13. The recombinant nucleic acid molecule according to any one of claims 1-12, wherein the nucleotide sequence of the recombinant nucleic acid molecule is shown in any one of SEQ ID NO: 82, 83, 84, 88, 89, 93, 97, 101, 105, 114, 115, 116 and 117.

14. A recombinant expression vector, wherein the recombinant expression vector comprises the recombinant nucleic acid molecule as described in any one of claims 1-13; Preferably, the vector contains a promoter upstream of the recombinant nucleic acid molecule; More preferably, the promoter includes one or more promoters, including but not limited to T7 promoter, Sp6 promoter, T3 promoter, Ptac promoter, trp promoter, CMV promoter, PGK promoter, Ubc promoter, SV40 promoter, CAG promoter, U6 promoter and H1 promoter; Preferably, the promoter is the T7 promoter, whose nucleotide sequence is shown in SEQ ID NO:

85.

15. Use of the recombinant nucleic acid molecule according to any one of claims 1-13, or the recombinant expression vector according to claim 14, for the in vitro preparation of circular RNA.

16. A method for preparing circular RNA in vitro, comprising the following steps: (1) The recombinant nucleic acid molecule as described in any one of claims 1-13 or the recombinant expression vector as described in claim 14 is transcribed to form a circularized precursor nucleic acid molecule; (2) The circularized precursor nucleic acid undergoes a circularization reaction to obtain circular RNA; Optionally, the method further includes the step of purifying the circular RNA.

17. The recombinant nucleic acid molecule according to any one of claims 1-13, the recombinant expression vector according to claim 14, or the circular RNA obtained by the method according to claim 16.

18. A host cell expressing the recombinant nucleic acid molecule of any one of claims 1-13, the recombinant expression vector of claim 14, or the circular RNA of claim 17.

19. A composition comprising a recombinant nucleic acid molecule as described in any one of claims 1-13, a recombinant expression vector as described in claim 14, or a circular RNA as described in claim 17, and one or more pharmaceutically acceptable vectors.

20. The composition according to claim 19, wherein, The pharmaceutically acceptable carrier is selected from lipids, polymers, or lipid-polymer complexes.

21. A method for expressing functional units within cells for non-disease therapeutic purposes, wherein, The method includes the step of transferring the circular RNA according to claim 17 or the composition according to any one of claims 19 or 20 into cells.

22. Use of the circular RNA of claim 17 in the preparation of a medicament for preventing or treating a disease by means of, wherein, The method includes administering the circular RNA according to claim 17 to a subject; Preferably, the drug is an anti-tumor drug or an mRNA vaccine.

23. The use of the recombinant nucleic acid molecule of any one of claims 1-13, the recombinant expression vector of claim 14, the circular RNA of claim 17, the host cell of claim 18, and the composition of claim 19 in improving mRNA drug targeting and improving mRNA drug expression efficiency.

24. The use of the recombinant nucleic acid molecule of any one of claims 1-13, the recombinant expression vector of claim 14, and the circular RNA of claim 17 in expressing proteins in cells.

25. A method for constructing the recombinant nucleic acid molecule of claim 1 based on the stem-loop structure in the first unit sequence, wherein the method is for T4td intron ribozymes, the method comprising: (1) Based on the first unit sequence, the secondary structure was predicted using RNA structure prediction software; (2) Based on the secondary structure, the stem-loop structure in the first unit sequence is screened. The loop position contains at least 4 free bases, including T or U bases. The upstream of the T or U base contains at least 3 free bases, and the downstream of the T or U base contains at least 0 free bases. The first unit sequence is split into two fragments at the 3' position of T or U in the screened loop position sequence. The 5' end fragment is the first unit fragment I, and the 3' end fragment is the first unit fragment II. (3) Based on the splitting position of the first unit sequence, the P1 and P10 guide sequences of the group I intron ribozymes are mutated to make them complementary to the recognition fragments of the first and second ribozymes, forming the P1 and P10 structures of the group I ribozymes. Then, the first unit fragment I is connected to the 5' end of the mutated 5' group I intron, and the first unit fragment II is connected to the 3' end of the mutated 3' group I intron. (4) The 5' end of the functional sequence is linked downstream of the first unit fragment II, and the 3' end is linked to the 5' end of the first unit fragment I to obtain a recombinant nucleic acid molecule.

26. The method according to claim 25, wherein in step (2), the loop position preferably contains at least 6 free bases, including a T or U base, with at least 4 free bases upstream of the T or U base and at least 1 free base downstream of the T or U base, and the loop position sequence is N2N3N4N5TN6 or N2N3N4N5UN6, where N2-N6 are A, G, C, U, or T; preferably, in step (2), the loop position preferably contains at least 8 free bases, including a T or U base, with at least 5 free bases upstream of the T or U base and at least 2 free bases downstream of the T or U base, and the loop position sequence is N1N2N3N4N5TN6N7 or N1N2N3N4N5UN6N7, where N1-N7 are A, G, C, U, T, or absent, and the intron ribozyme in group I is a mutant of the T4td ribozyme, which contains the following mutation region N in the P1 / P10 guide sequence. 8’ AATTGN 7’ N 6’ GN 5’ N 4’ N 3’ N 2’ N 1’ , where N 1’ -N 8’ It is a mutant base, N 5’ N 4’ N 3’ N 2’ N 1’ Pairing with N1N2N3N4N5 in reverse complementary order, or N 5’ N 4’ N 3’ N 2’ Pairing with N2N3N4N5 in reverse complementary order, or N 5’ N 4’ N 3’ Pairing with N3N4N5 in reverse complementary direction, N 7’ N 6’ Pairing with N6N7 in reverse complementary direction, or N 6’ If the N6 base is complementary to N6, or if N6N7 is absent, and the 3' end T base of the I ribozyme recognition fragment is adjacent to the stem structure of the first unit, then the P10 guide sequence N... 7’ N 6’ It is possible to avoid mutation, N 8’ With N 6’ Complementary pairing, the complementary base pairing includes AU, GC, GU, AT, and GT base pairs.

27. A method for constructing the recombinant nucleic acid molecule of claim 1 based on the stem-loop structure in the first unit sequence, the method for Ana intron ribozymes comprising: (1) Based on the first unit sequence, the secondary structure was predicted using RNA structure prediction software; (2) Based on the secondary structure, the stem-loop structure in the first unit sequence is screened. The loop position contains at least 5 free bases, including T or U bases. The T or U bases upstream contain at least 2 free bases, and the T or U bases downstream contain at least 2 free bases. The first unit sequence is split into two fragments at the 3' position of T or U in the screened loop position sequence. The 5' end fragment is the first unit fragment I, and the 3' end fragment is the first unit fragment II. (3) Based on the splitting position of the first unit, the P1 and P10 guide sequences of the intron ribozyme of group I are mutated to make them complementary to the recognition fragment of ribozyme I and the recognition fragment of ribozyme II, forming the P1 and P10 structures of group I ribozyme. Then, the first unit fragment I is connected to the 5' end of the mutated 5' group I intron, and the first unit fragment II is connected to the 3' end of the mutated 3' group I intron. (4) The 5' end of the functional sequence is linked downstream of the first unit fragment II, and the 3' end is linked to the 5' end of the first unit fragment I to obtain a recombinant nucleic acid molecule.

28. The method according to claim 27, wherein the loop position in step (2) contains at least 7 free bases, including a T or U base, with at least 3 free bases upstream of the T or U base and at least 3 free bases downstream of the T or U base, the loop position sequence being N1N2N3TN4N5N6 or N1N2N3UN4N5N6, N1-N6 being A, G, C, U, or T or absent, and the intron ribozyme in group I being a mutant of the Ana ribozyme, which contains the following mutant region N in the P1 / P10 guide sequence. 6’ N 7’ ATAAN 5’ N 4’ GN 3’ N 2’ , where N 2’ -N 7’ It is a mutant base, in which N 3’ N 2’ Pairing with N2N3 in reverse complementary direction, N 5’ N 4’ Pairing with N4N5 in reverse complementary direction, N 6’ N 7’ With N 5’ N 4’ Reverse complementary pairing, the base complementary pairing includes AU, GC, GU, AT, and GT base pairs.

29. The method of any one of claims 25-28, wherein the first unit is a translation initiation element or a nucleic acid aptamer.

30. A computer-readable storage medium storing a program implementing the construction method of any one of claims 25-29.

31. A computer terminal, the computer terminal comprising a processor and a readable storage medium as claimed in claim 30, the processor being configured to invoke a program stored in the readable storage medium to execute the construction method; preferably, the computer terminal further comprising a display device for outputting visualization results.

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