Recombinant nucleic acid molecule for preparing scar-free circular RNA based on stem-loop structure and application of recombinant nucleic acid molecule

Abstract

The invention provides a preparation method for preparing a scar-free circular RNA (Ribonucleic Acid), a recombinant nucleic acid molecule for preparing the scar-free circular RNA and application of the recombinant nucleic acid molecule. The recombinant nucleic acid molecule based on the IRES stem-loop structure provided by the invention can be used for preparing a circular nucleic acid molecule in which an exogenous sequence is completely eliminated, and any sequence of IRES is not changed, so that the unpredictable influence of IRES mutation on recruitment of ribosome functions is avoided.

Description

Technical Field

[0001] This invention relates to the fields of molecular biology, bioengineering technology, and gene recombination technology, and particularly to a method for preparing and applying a circular nucleic acid molecule with completely exogenous sequences eliminated. Background Technology

[0002] 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.

[0003] 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.

[0004] 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 ribozyme introns. 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. In addition, researchers such as Zuo Chijian used 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 similar to the T4Td ribozyme recognition site at this position by base substitution, transforming it into a ribozyme recognition site 5'-TTGGGTCT-3'. This site was then truncated and reassembled to form a linear structure of "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, potentially causing unpredictable effects on the IRES's function of recruiting ribosomes to initiate translation. [7] Therefore, to eliminate redundant exon sequences in the construction of circular RNA, it is still necessary to develop better circularization strategies. Summary of the Invention

[0005] To address the problems of existing technologies, this invention 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 invention 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 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 invention, 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 invention ensures that the circular RNA product not only lacks redundant sequences from the circularization of group I introns, 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.

[0006] On one hand, the present invention provides a recombinant nucleic acid molecule for preparing scarless circular RNA, comprising elements arranged in the following order from the 5' to 3' direction:

[0007] a.3'I group intron mutant fragment (intron fragment II),

[0008] b. Unit I fragment II, whose 5' end includes the II ribozyme recognition fragment.

[0009] c. Functional units,

[0010] e. The first unit fragment I, whose 3' end includes the I-th ribozyme recognition fragment.

[0011] f.5'I group intron mutant fragment (intron fragment I);

[0012] The functional units include IRES, RNA aptamers, protein-binding sequences, protein-coding regions, non-coding regions, etc., or combinations thereof;

[0013] The intron fragment II is located at the 3' end of the intron fragment I, that is, the complete group I intron mutant sequence includes: intron fragment I - intron fragment II, where "-" represents a phosphodiester bond;

[0014] 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;

[0015] 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;

[0016] 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;

[0017] 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;

[0018] The first unit has a local stem structure, a local double bond structure, or a local hairpin structure, wherein the local stem structure, local double bond structure, or local hairpin structure is adjacent to or includes the cyclic segment.

[0019] Preferably, the first unit is a nucleic acid aptamer or a translation initiation element;

[0020] 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.

[0021] In one implementation, the group I intron mutant contains a mutation in the guide region of P1 / P10, and the mutated guide region identifies the region of the original fragment in the first unit as a circular fragment.

[0022] 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;

[0023] 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', 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’ GN3’ 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;

[0024] 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.

[0025] 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;

[0026] 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.

[0027] 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;

[0028] Preferably, the nucleotide sequence of the translation initiation element is shown in any one of SEQ ID NO: 53, 54, 55, 56, 57, 58, 60, 62, 64 and 80;

[0029] Preferably, the nucleotide sequence of the translation initiation element fragment I is as shown in SEQ ID NO:66, and the nucleotide sequence of the translation initiation element fragment II is as shown in SEQ ID NO:67;

[0030] Preferably, the nucleotide sequence of the translation initiation element fragment I is shown in SEQ ID NO:92, and the nucleotide sequence of the translation initiation element fragment II is shown in SEQ ID NO:93;

[0031] Preferably, the nucleotide sequence of the translation initiation element fragment I is shown in SEQ ID NO:84, and the nucleotide sequence of the translation initiation element fragment II is shown in SEQ ID NO:85;

[0032] Preferably, the nucleotide sequence of the translation initiation element fragment I is shown in SEQ ID NO:96, and the nucleotide sequence of the translation initiation element fragment II is shown in SEQ ID NO:97;

[0033] Preferably, the nucleotide sequence of the translation initiation element fragment I is shown in SEQ ID NO:88, and the nucleotide sequence of the translation initiation element fragment II is shown in SEQ ID NO:89;

[0034] Preferably, the nucleotide sequence of the translation initiation element fragment I is shown in SEQ ID NO:66, and the nucleotide sequence of the translation initiation element fragment II is shown in SEQ ID NO:81.

[0035] Preferably, the nucleotide sequence of the translation initiation element fragment I is shown in SEQ ID NO:100, and the nucleotide sequence of the translation initiation element fragment II is shown in SEQ ID NO:101.

[0036] 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.

[0037] Preferably, the nucleotide sequence of the translation initiation element fragment I is shown in SEQ ID NO:104, and the nucleotide sequence of the translation initiation element fragment II is shown in SEQ ID NO:105.

[0038] 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.

[0039] In one embodiment, the nucleotide sequence of intron fragment I is shown in any one of SEQ ID NO: 70, 71, 72, 86, 90, 94 and 98, and the nucleotide sequence of intron fragment II is shown in SEQ ID NO: 68 or 69.

[0040] Preferably, the nucleotide sequence of intron fragment I is as shown in 70 or 71, and the nucleotide sequence of intron fragment II is as shown in SEQ ID NO:68;

[0041] Preferably, the nucleotide sequence of intron fragment I is shown in any one of 72, 86, 90, 94 and 98, and the nucleotide sequence of intron fragment II is shown in SEQ ID NO:69.

[0042] 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.

[0043] In one implementation, the functional unit comprises at least two coding regions, wherein any two adjacent coding regions are connected by a connector;

[0044] Preferably, the linker is a polynucleotide encoding a 2A peptide.

[0045] 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;

[0046] 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.

[0047] In one implementation, the functional unit is a coding sequence for a human or non-human protein;

[0048] 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.

[0049] 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.

[0050] In one embodiment, the recombinant nucleic acid molecule further includes an insertion element located between the coding element and the translation initiation element;

[0051] The insertion element is selected from at least one of the following groups (i)-(iii):

[0052] (i) transcriptional regulatory elements, (ii) translational regulatory elements, and (iii) purification elements;

[0053] Optionally, the insert element comprises a sequence of one or more combinations of the following:

[0054] 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.

[0055] In one embodiment, the nucleotide sequence of the recombinant nucleic acid molecule is shown in any one of SEQ ID NO: 76, 77, 78, 82, 83, 87, 91, 95, 99, 108, 109, 110 and 111.

[0056] On the other hand, the present invention provides a recombinant expression vector, wherein the recombinant expression vector comprises the recombinant nucleic acid molecule as described above;

[0057] Preferably, the vector contains promoters at both ends of the recombinant nucleic acid molecule;

[0058] 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;

[0059] Preferably, the promoter is the T7 promoter, whose nucleotide sequence is shown in SEQ ID NO:79.

[0060] On the other hand, the present invention provides the use of the aforementioned recombinant nucleic acid molecules or the aforementioned recombinant expression vectors in the in vitro preparation of circular RNA.

[0061] On the other hand, the present invention provides a method for preparing circular RNA in vitro, comprising the following steps:

[0062] (1) The aforementioned recombinant nucleic acid molecules or the aforementioned recombinant expression vector are transcribed to form circularized precursor nucleic acid molecules;

[0063] (2) The circularized precursor nucleic acid undergoes a circularization reaction to obtain circular RNA;

[0064] Optionally, the method further includes the step of purifying the circular RNA.

[0065] On the other hand, the present invention provides circular RNA prepared according to the aforementioned recombinant nucleic acid molecule, the aforementioned recombinant expression vector or the aforementioned method.

[0066] On the other hand, the present invention provides a host cell that expresses the aforementioned recombinant nucleic acid molecule or the aforementioned recombinant expression vector.

[0067] On the other hand, the present invention 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.

[0068] In one embodiment, the pharmaceutically acceptable carrier is selected from lipids, polymers, or lipid-polymer complexes.

[0069] On the other hand, the present invention 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.

[0070] On the other hand, the present invention provides the use of the aforementioned circular RNA in the preparation of a medicament for preventing or treating a disease by means of a method comprising administering the aforementioned circular RNA to a subject;

[0071] Preferably, the drug is an anti-tumor drug or an mRNA vaccine.

[0072] On the other hand, the present invention provides the application of the aforementioned recombinant nucleic acid molecule, the aforementioned recombinant expression vector, the aforementioned host cell, and the aforementioned composition in improving the targeting of mRNA drugs and improving the expression efficiency of mRNA drugs.

[0073] On the other hand, the present invention provides the application of the aforementioned recombinant nucleic acid molecules and the aforementioned recombinant expression vectors in expressing proteins in cells.

[0074] On the other hand, the present invention provides a method for constructing the aforementioned recombinant nucleic acid molecule based on the stem-loop structure in the first unit sequence, wherein the method for the T4td intron ribozyme includes:

[0075] (1) Based on the first unit sequence, the secondary structure was predicted using RNA structure prediction software;

[0076] (2) Based on the secondary structure, the stem-loop structure in the translation initiation element 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 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.

[0077] (3) Based on the splitting position of the first unit sequence, the P1 and P10 guide sequences of the group I ribozyme introns 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.

[0078] (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.

[0079] 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.

[0080] 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 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 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 can be without mutation; N 8’ With N 6’ Complementary pairing, the complementary base pairing includes AU, GC, GU, AT, and GT base pairs.

[0081] On the other hand, the present invention provides a method for constructing the aforementioned recombinant nucleic acid molecule based on a stem-loop structure of a first unit sequence, wherein the method for Ana intron ribozymes includes:

[0082] (1) Based on the first unit sequence, the secondary structure was predicted using RNA structure prediction software;

[0083] (2) Based on the secondary structure, the stem-loop structure in the first unit is screened. The loop position contains at least 6 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 3 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.

[0084] (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.

[0085] (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.

[0086] 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 in the P1 / P10 guide sequence.

[0087] N 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] Beneficial effects

[0090] The recombinant nucleic acid molecule based on the stem-loop structure of the first unit provided by this invention 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 a stem-loop structure. Using RNAFold to predict the secondary structure of the 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 a group I ribozyme. Simultaneously, the corresponding bases in the P1 and P10 guide sequences of the group I introns are mutated, causing the linear RNA to circularize at the stem-loop position in the IRES. This ensures that the circularized RNA product retains only the functional sequence, without the redundant sequence remaining from the circularization of the group I introns. Furthermore, due to the presence of the stem at the selected stem-loop position in the IRES, it ensures that the ribozyme forms an independent splicing vesicle, effectively promoting RNA circularization. Attached Figure Description

[0091] Figure 1 A schematic diagram of a method for constructing recombinant nucleic acid molecules with scarless circular RNA based on stem-loop structure (the first unit is represented by IRES).

[0092] Figure 2 A schematic diagram of the recombinant nucleic acid molecule structure for preparing scarless circular RNA (the first unit is represented by IRES).

[0093] Figure 3 The diagram shows the structures of wild-type T4td ribozyme and Ana ribozyme. The red and blue nucleotides represent the intron mutation regions of the ribozyme in the group I intron mutation method described in this invention. The red indicates the position of the major mutated base, and the black indicates the exon sequence.

[0094] Figure 4 The diagrams show the recombinant nucleic acid molecular structures of PIE-circulated RNA without a spacer (left), PIE-circulated RNA with a polyAC spacer (middle), and PIE-circulated RNA with an internal homologous arm (Arm) (right).

[0095] Figure 5 for Figure 4 Circulation electrophoresis diagrams and circularization efficiency statistical bar charts of RNA sequences (SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20) of three RNA nucleic acid molecular structures were obtained 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.

[0096] Figure 6 for Figure 4 Circulation electrophoresis diagrams and circularization efficiency statistical bar charts of RNA sequences (SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19) of three RNA nucleic acid molecular structures were obtained 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.

[0097] Figure 7 for Figure 4 Electrophoresis diagrams and bar graphs of circularization efficiency of RNA sequences (SEQ ID NO: 23-28) of three RNA nucleic acid molecular structures were generated 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.

[0098] Figure 8 This is a schematic diagram of the structure of Ana ribozyme exons shortened in the PIE method with internal homologous arms.

[0099] Figure 9 This is a schematic diagram of the structure of the T4td ribozyme exon shortened in the PIE method with internal homologous arms.

[0100] Figure 10 Electrophoresis diagrams, circularization efficiency bar graphs, and sequencing verification results for shortened Ana ribozyme exon circularized RNA using the PIE method with internal homologous arms (SEQ ID NO:29-32).

[0101] Figure 11 Electrophoresis diagrams, circularization efficiency bar graphs, and sequencing verification results for shortening T4td ribozyme exon circularized RNA using the PIE method with internal homologous arms (SEQ ID NO:33-36).

[0102] Figure 12 Electrophoretic images of shortened Ana ribozyme exon circularized RNA in the PIE method with internal homologous arms (SEQ ID NO:37-40).

[0103] Figure 13Electrophoretic images of T4td ribozyme exon circularized RNA shortened using the PIE method with internal homologous arms (SEQ ID NO:41-44).

[0104] Figure 14 Electrophoresis diagrams and circularization efficiency bar charts of RNA circularized by Ana ribozymes and T4td ribozymes with internal homologous arms of different lengths (SEQ ID NO:45-52)

[0105] Figure 15 This is a schematic diagram of the stem-loop structure remaining after splicing of Ana ribozyme and T4td ribozyme.

[0106] Figure 16 This diagram shows the secondary structure analysis of different IRES and the possible positions of stem-ring structures.

[0107] Figure 17 Schematic diagram of the secondary structure of HRV-B3 and HRV-B3-eIF4G IRES and the selected stem-ring position.

[0108] Figure 18 This is a schematic diagram of the structure for preparing scarless circular RNA using nucleic acid molecules containing the T4td intron mutant (P1 rigorous complementation). The red and blue nucleotides belong to the T4td ribozyme intron mutant, the red ones are the intron mutant bases, and the green ones are the IRES sequences.

[0109] Figure 19 This diagram illustrates the preparation of scarless circular RNA structures using nucleic acid molecules containing the T4td ribozyme mutant (P1 is not strictly complementary). The red and blue nucleotides represent the T4td ribozyme intron mutant, with red representing the intron mutant bases and green representing the IRES sequence.

[0110] Figure 20 This is a schematic diagram of the structure for preparing scarless circular RNA using nucleic acid molecules containing Ana intron mutants. The red and blue nucleotides belong to the Ana ribozyme intron mutants, with red representing intron mutant bases and green representing IRES sequences.

[0111] Figure 21 Electrophoresis images and sequencing results of circular RNA splicing sites were obtained to identify the preparation of scarless circular RNA (SEQ ID NO:76) expressing antigenic peptides using the mutated T4td intron (P1 rigorously complementary).

[0112] Figure 22 Electrophoresis images and sequencing results of circular RNA splicing sites were used to identify the preparation of scarless circular RNA (SEQ ID NO:77) expressing antigenic peptides using the mutated T4td intron (P1 non-strict complementation).

[0113] Figure 23Electrophoresis diagram and sequencing results of circular RNA splicing sites were obtained to identify the preparation of scarless circular RNA (SEQ ID NO:78) expressing antigenic peptides using Ana ribozyme mutants.

[0114] Figure 24 Electrophoresis images and sequencing results of circular RNA splicing sites were obtained to identify the preparation of scarless circular RNA (SEQ ID NO:82) expressing Fluc using the mutated T4td intron (P1 non-strict complementation).

[0115] Figure 25 Electrophoresis diagram and sequencing results of circular RNA splicing sites were obtained to identify the preparation of scarless circular RNA (SEQ ID NO:83) expressing Fluc using Ana ribozyme mutant.

[0116] Figure 26 The results of the ELISpot experiment show the effective expression of the antigenic peptide by scarless circular RNA (SEQ ID NO:76 and SEQ ID NO:77) in PBMC. The wells circled in blue are the control group with added PHA.

[0117] Figure 27 A bar graph showing the activity of the scarless circular RNA (SEQ ID NO:82) expressing the Fluc protein in cells.

[0118] Figure 28 This diagram illustrates the design, electrophoresis results, and sequencing results of the circular RNA splicing site for preparing scarless circular RNA expressing the antigenic peptide using HRV-B4 IRES and Ana ribozyme mutants. The top left shows the loop sequence within the stem-loop position selected by HRV-B4 IRES, with arrows 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. The center shows that the Ana ribozyme mutant maintains its intact structure in the predicted full-length structure of the recombinant nucleic acid molecule, unaffected by other sequences.

[0119] Figure 29 This diagram illustrates the design of a scarless circular RNA expressing an antigenic peptide using Human XIAP IRES and Ana ribozyme mutants, along with identification electrophoresis and sequencing results of the circular RNA splicing site. The top left shows the loop sequence within the stem-loop position selected by Human XIAP IRES, with arrows 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 sites. The center shows that 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 30This diagram illustrates the design, electrophoresis results, and sequencing results of the circular RNA splicing site for preparing scarless circular RNA expressing the antigenic peptide using HRV-B97 IRES and Ana ribozyme mutants. The top left shows the loop sequence within the stem-loop position selected by HRV-B97 IRES, with arrows 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 sites. The center shows that 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 31 This diagram illustrates the design of a scarless circular RNA expressing an antigenic peptide using ShuffledIRES#42IRES and Ana ribozyme mutants, along with electrophoresis images and sequencing results of the circular RNA splicing site. The top left shows the loop sequence within the stem-loop position selected by ShuffledIRES#42IRES, with arrows 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 sites. The center shows that 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 32 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.

[0123] Figure 33 Electrophoretic images for identifying transcription, circularization, and RNase R tolerance of scarless circular RNA expressing antigenic peptides prepared using HRV-B37, HRV-B92, ShuffledIRES#1, ShuffledIRES#38IRES, and Ana ribozyme mutants.

[0124] Figure 34 This is a schematic diagram of the secondary structure and domains of CVB3 IRES. The variable region and the irregular region between the V and VI domains are circled by black ellipses.

[0125] Figure 35 The predicted secondary structure of CVB3 IRES for RNAfold and the selected truncation sites S1, S2 and S3.

[0126] Figure 36 Electrophoresis images for the identification of transcription, circularization, and RNase R tolerance in SEQ ID NO:113, SEQ ID NO:114, and SEQ ID NO:115. Detailed Implementation

[0127] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. The following embodiments are merely illustrative and explanatory of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0128] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available 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 the field of this invention or according to the product instructions.

[0129] In this invention, 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.

[0130] In this invention, 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).

[0131] In this invention, 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 invention, the pathogen antigens are SARS-CoV-2 antigen, influenza virus antigen, herpes virus antigen, etc.

[0132] In this invention, "IRES" (Internal Ribosome Entry Site) 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, ensuring the proper orientation of the mRNA start codon within the ribosome for translation. The IRES sequence is usually located in the 5' UTR of the mRNA (directly upstream of the start codon). Functionally, IRES replaces the need for various protein factors that interact with eukaryotic translation mechanisms.

[0133] In this invention, the IRES includes, but is not limited to, the IRES sequences of the following viruses: Taura syndrome virus, stag beetle virus, Tiller's encephalomyelitis virus, simian virus 40, red imported fire ant virus 1, rice constrictor aphid virus, reticuloendotheliosis virus, human poliovirus 1, P. stollenella enterovirus, Kashmir bee virus, human rhinovirus 2, glass leafhopper virus-1, human immunodeficiency virus type 1, Himetobi virus, etc. 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, HCVQC64, 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-CGT110, GBV-C K1737, GBV-C Iowa, Hepatic Virus A1220, 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 ABN2, 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 the present invention, the IRES sequence is IRES. MP,75 HRV-B3-eIF4G.

[0134] In this invention, a "vector" refers to a segment of DNA that is synthetic (e.g., using PCR) or extracted from viruses, plasmids, or cells of higher organisms, into which foreign DNA fragments can be inserted or have already 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 vectors provided herein contain a multiple cloning site.

[0135] In this invention, "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.

[0136] In this invention, "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 recombinant nucleic acid molecules).

[0137] In this invention, the “coding region” refers to a gene sequence that can be transcribed into messenger RNA and ultimately translated into a target polypeptide or protein.

[0138] In this invention, "expression" includes any step involved in polypeptide production, including but not limited to: transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0139] In this invention, "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 encoded proteins of this invention, 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.

[0140] In one specific embodiment of the present invention, 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 mutation region N in the 5' intron fragment. 6’ N 7’ ATAAN 5’ N 4’ GN 3’ N 2’ (The original sequence is: 5'- AA AUAA UU G AG -3' (underlined to indicate 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.

[0141] In one specific embodiment of the present invention, group I introns are mutants of the T4td ribozyme, wherein the first ribozyme recognition fragment is 5'-N1N2N3N4N5T-3', the second ribozyme recognition fragment is 5'-N6N7-3', and 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’ (The original sequence is: 5'- U AAUUG AG G CCUGA -3' (underlined to indicate mutation site), where N is A, U, C, G, or T or absent, N1N2N3N4N5 and 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, N7’ N 6’ Pairing with N6N7 in reverse complementary direction, N 8’ With N 6’ 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.

[0142] Example 1: Selection of Group I ribozyme spacer sequences in the PIE method

[0143] Circular RNA is prepared using the PIE method. Adding a spacer sequence at the junction of the group I intron ribozyme and the functional sequence facilitates RNA circularization. The spacer sequence includes a stem structure formed by internal homologous arms and a loose, disordered structure. T4td and Ana introns are two commonly used ribozymes in the PIE method, and their structures are as follows: Figure 3 As shown, we evaluated the effects of these two spacer sequences on cyclization efficiency using the 3' intron (SEQ ID NO:1) and 5' intron (SEQ ID NO:2) of T4td ribozyme and the 3' intron (SEQ ID NO:3) and 5' intron (SEQ ID NO:4) of Ana ribozyme as examples.

[0144] 1. Plasmid construction

[0145] For the functional sequence in RNA to be circularized, IRES derived from crTMV were 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.

[0146] 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 -Antigen peptide encoding sequence-Ana ribozyme 5' intron obtained 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 linked after T7 promoter. MP,75 -Antigen peptide encoding sequence-polyAC-Ana ribozyme 5' intron obtained 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, after which Ana ribozyme 3' intron-5' homologous arm-IRES is linked after the T7 promoter. MP,75 -Antigen peptide encoding sequence-3' homologous arm-Anaribozyme 5' intron to obtain SEQ ID NO:20( Figure 4 ).

[0147] 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.

[0148] First, a control plasmid without spacer sequences was constructed. Then, a T4td ribozyme 3' intron-CVB3IRES-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-CVB3IRES-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-CVB3IRES-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-CVB3IRES-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.

[0149] 2. Plasmid linearization

[0150] 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.

[0151] 3. In vitro transcription (IVT)

[0152] 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 chromatography using the Monarch RNACleanup Kit (NEB, #T2050L).

[0153] 4. In vitro circulization

[0154] 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).

[0155] Table 1 In vitro cyclization reaction system

[0156]

[0157]

[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]

[0162] 6. RNA circularization identification

[0163] 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.

[0164] Figure 5 , Figure 6 , Figure 7Electrophoresis 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) are shown, along with the circularization efficiency calculated using ImageJ software based on the electrophoresis results. The electrophoresis results indicate that for shorter RNAs, circularization efficiency (IRES) is significantly improved. MP,75 For 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.

[0165] Example 2: Determination of Group I ribozyme exon length using the PIE method

[0166] Circular RNA was prepared using the PIE method. The circular RNA product typically contains exon sequences linked to group I ribozyme introns. Commonly used Ana exons are 66 nt long, and T4td exons are 37 nt long. The length of these exon sequences affects circularization efficiency. The Chen Lingling research group believes that Ana ribozyme exons shorter than 27 nt lead to a significant reduction in circularization efficiency. The Ana ribozyme introns used in Example 1 contained 66 nt exons, and the T4td ribozyme introns contained 37 nt exons. We have already confirmed in Example 1 that a stem-like spacer sequence is beneficial for T4td and Ana ribozymes to circularize RNA. Therefore, we further evaluated the effect of different exon lengths on circularization efficiency using the PIE method with internal homologous arms.

[0167] 1. Plasmid construction

[0168] 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 -Antigen peptide coding sequence-5nt exon (GACTT)-Ana ribozyme 5' intron to obtain SEQ ID NO:29, splicing into a circular form to form a 10nt exon (GACTTAAAAT) residue, as shown in the structural diagram. Figure 8 10nt in the middle; linked to the Ana ribozyme 3' intron-4nt exon (AAAA)-IRES after the T7 promoter. MP,75-Antigen peptide coding sequence-4nt exon (ACTT)-Ana ribozyme 5' intron to obtain SEQ ID NO:30, spliced ​​into a circular form to form an 8nt exon (ACTTAAAA) residue, as shown in the structural diagram. Figure 8 8nt; followed by the T7 promoter, an Ana ribozyme 3' intron-3nt exon (AAA)-IRES is linked. MP,75 -Antigen peptide coding sequence-3nt exon (CTT)-Ana ribozyme 5' intron to obtain SEQ ID NO:31, spliced ​​into a circular form to form a 6nt exon (CTTAAA) residue, as shown in the structural diagram. Figure 8 The middle 6nt; after the T7 promoter, the Ana ribozyme 3' intron-2nt exon (AA)-IRES is linked. MP,75 -Antigen peptide coding sequence-2nt exon (TT)-Ana ribozyme 5' intron to obtain SEQ ID NO:32, splicing into a circular form to form a 4nt exon (TTAA) residue, as shown in the structural diagram. Figure 8 Medium 4nt.

[0169] 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:33. After splicing and circularization, a 10nt exon (CTTGGGTCTA) residue was formed, as shown in the structural diagram. Figure 9 10nt in the middle; followed by the T4td ribozyme 3' intron-2nt exon (CT)-IRES after the T7 promoter. MP,75 -Antigen peptide coding sequence-6nt exon (TTGGGT)-T4td ribozyme 5' intron to obtain SEQ ID NO:34, splicing into a circular form to form an 8nt exon (TTGGGTCT) residue, as shown in the structural diagram. Figure 9 8nt; followed by the T4td ribozyme 3' intron-1nt exon (C)-IRES after the T7 promoter. MP,75 The antigen peptide coding sequence-5nt exon (TGGGT)-T4td ribozyme 5' intron yielded SEQ ID NO:35. After splicing and circularization, a 6nt exon (TGGGTC) residue was formed, as shown in the structural diagram. Figure 9 6nt; followed by the T4td ribozyme 3' intron-IRES after the T7 promoter. MP,75 -Antigen peptide coding sequence-4nt exon (GGGT)-T4td ribozyme 5' intron to obtain SEQ ID NO:36, after splicing into a circular form to form a 4nt exon (GGGT) residue, as shown in the structural diagram. Figure 9 Medium 4nt.

[0170] 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 different exon lengths on the cyclization efficiency of Ana ribozymes. Plasmids with Ana ribozyme exons of different lengths were constructed. After the T7 promoter, Ana ribozyme 3' intron-4nt exon (AAAA)-CVB3 IRES-FLUC coding sequence-4nt exon (ACTT)-Ana ribozyme 5' intron were ligated to obtain SEQ ID NO:37. After splicing and cyclization, an 8nt exon (ACTTAAAA) residue was formed, as shown in the structural diagram. Figure 8 The 8nt exon is linked after the T7 promoter to form the 3' intron of the Ana ribozyme, the 4nt exon (AAAA), the CVB3 IRES-FLUC coding sequence, the 3nt exon (CTT), and the 5' intron of the Ana ribozyme, resulting in SEQ ID NO:38. After splicing and circularization, a 7nt exon (CTTAAAATC) remains, as shown in the structural diagram. Figure 8 The 7nt-5' intron of the Ana ribozyme is linked after the T7 promoter to form the 3' intron-3nt exon (AAA)-CVB3 IRES-FLUC coding sequence-4nt exon (ACTT)-Ana ribozyme 5' intron, resulting in SEQ ID NO:39. After splicing and circularization, a 7nt exon (ACTTAAA) residue is formed, as shown in the structural diagram. Figure 8 The 7nt-3; after the T7 promoter, the Ana ribozyme 3' intron-3nt exon (AAA)-CVB3 IRES-FLUC coding sequence-3nt exon (CTT)-Ana ribozyme 5' intron is linked to obtain SEQ ID NO:40. After splicing into a circular shape, a 6nt exon (CTTAAA) residue is formed, as shown in the structural diagram. Figure 8 6nt.

[0171] The effects of exon lengths on the circularization efficiency of T4td ribozyme were evaluated using the relatively long CVB3 IRES (SEQ ID NO:21) and FLUC protein-coding sequences (SEQ ID NO:22). Plasmids containing T4td ribozyme exons of different lengths were constructed. Following the T7 promoter, a 2nt exon (CT) of the T4td ribozyme 3' intron was ligated into a 6nt exon (TTGGGT) of the CVB3 IRES-FLUC coding sequence, followed by a 5' exon (T4td ribozyme 5' intron), yielding SEQ ID NO:41. After splicing and circularization, an 8nt exon (CTTGGGTCTA) residue was formed, as shown in the structural diagram. Figure 9The 8nt exon is linked after the T7 promoter to the T4td ribozyme 3' intron - 2nt exon (CT) - CVB3 IRES-FLUC coding sequence - 5nt exon (TGGGT) - T4td ribozyme 5' intron to obtain SEQ ID NO:42. After splicing into a circular shape, a 7nt exon (TGGGTCT) residue is formed, as shown in the structural diagram. Figure 9 The 7nt-5' intron of the T4td ribozyme is linked to the T4td ribozyme 3' intron-1nt exon (C)-CVB3 IRES-FLUC coding sequence-6nt exon (TTGGGT)-T4td ribozyme 5' intron to obtain SEQ ID NO:43. After splicing into a circular shape, a 7nt exon (TTGGGTC) residue is formed, as shown in the structural diagram. Figure 9 The 7nt-3; after the T7 promoter, the T4td ribozyme 3' intron-1nt exon (C)-CVB3 IRES-FLUC coding sequence-5nt exon (TGGGT)-T4td ribozyme 5' intron is linked to obtain SEQ ID NO:44. After splicing into a circular shape, a 6nt exon (TGGGTC) residue is formed, as shown in the structural diagram. Figure 9 6nt.

[0172] 2. Preparation of circular RNA

[0173] The steps for plasmid linearization, in vitro transcription, in vitro circularization, and linear RNA digestion are as described in Example 1.

[0174] 3. RNA circularization identification

[0175] The method for detecting circular RNA by formaldehyde denaturation electrophoresis is as described in Example 1.

[0176] 4. Circular RNA splicing site sequencing verification

[0177] 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.

[0178] Figure 10This image shows electrophoresis diagrams of transcription products, cyclization products, and RNase R degradation products during the preparation of the circular RNA of the above nucleotide sequence (SEQ ID NO:29-32), a statistical bar graph of cyclization efficiency, and sequencing verification. Figure 11 This image shows electrophoresis diagrams of transcription products, circularization products, and RNase R degradation products during the preparation of circular RNA from the above nucleotide sequences (SEQ ID NO: 33-36), a bar graph of circularization efficiency, and sequencing verification. Sequencing results show that the above nucleotide sequences (SEQ ID NO: 29-32, SEQ ID NO: 33-36) were all circularized at the expected sequence positions. Electrophoresis results show relatively short RNA circularization (IRES). MP,75 -The antigen peptide encoding sequence (approximately 400 bases) can be effectively cyclized by retaining 6 exons of the Ana ribozyme, while the T4td ribozyme can also be effectively cyclized by retaining 4 exons. Figure 12 Electrophoretic images showing the transcription products, cyclization products, and RNase R degradation products during the preparation of the circular RNA of the above nucleotide sequence (SEQ ID NO:37-40) are displayed. Figure 13 Electrophoresis images of the transcription products, circularization products, and RNase R degradation products during the preparation of the above nucleotide sequence (SEQ ID NO: 41-44) circular RNA are shown. The electrophoresis results show that for longer RNA circularization (CVB3 IRES-FLUC coding sequence, approximately 2400 bases), T4td ribozyme can still efficiently complete RNA circularization by retaining a 6nt exon, while Ana ribozyme requires a 7nt exon (7nt-3) to efficiently complete RNA circularization.

[0179] Example 3: IRES sequence structure prediction and stem-loop structure screening

[0180] Based on the above experimental results, we determined that in the design of PIE-based RNA circularization, a stem-like spacer sequence is beneficial for T4td and Ana ribozymes to perform RNA circularization. Furthermore, for short RNAs, T4td and Ana ribozymes only need to retain 4nt and 6nt exons to effectively complete RNA circularization, while for longer RNAs, retaining 6nt and 7nt exons is sufficient. We then investigated the stem length, testing the circularization efficiency of recombinant nucleic acid molecules with 8nt exons retained by Ana ribozymes (SEQ ID NO:45), without a stem structure (SEQ ID NO:46), with an 8nt stem structure (SEQ ID NO:47), and with a 20nt stem structure (SEQ ID NO:48). Electrophoresis results and ImageJ statistical results are shown below. Figure 14As shown, without the stem structure, Ana ribozyme introns cannot circularize RNA. Adding a 6nt stem structure allows Ana ribozyme introns to circularize RNA, while adding an 8nt or 20nt stem structure improves RNA circularization. Furthermore, the presence of a 5nt stem structure in the 66nt exon of the native Ana ribozyme also enables circularization. When all T4td ribozyme exons retained 8nt, the circularization efficiency of recombinant nucleic acid molecules without a stem structure (SEQ ID NO:49), with a 6nt stem structure (SEQ ID NO:50), with an 8nt stem structure (SEQ ID NO:51), and with a 9nt stem structure (SEQ ID NO:52) was measured. Electrophoresis results and Image J statistical results are shown below. Figure 14 As shown, the results indicate that the T4td ribozyme intron can circulate RNA both with and without a stem, and the T4td ribozyme intron with a 9nt stem structure can circulate RNA better.

[0181] Based on the above experimental results, T4td ribozyme and Ana ribozyme introns, under the conditions of stem structure and exon shortening (T4td ribozyme exons shortened to 4 nt, Ana ribozyme exons shortened to 6 nt), successfully completed splicing circularization, resulting in circular RNA splicing scars with stem-loop structures, where the exons form the loop position sequence, such as... Figure 15 As shown, we can assume that by truncating suitable stem-loop structures in circular RNA at the loop position and splicing them with group I intron ribozymes, and then mutating the group I ribozymes accordingly to maintain the structure required for splicing, 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 bubbles, thereby constructing scarless circular RNA.

[0182] 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 loop position contains at least 6 free bases, including a T or U base, with at least 2 free bases upstream of the T or U base and at least 3 free bases downstream of the T or U base. More preferably, the loop 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.

[0183] 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.

[0184] 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 through DNA shuffling recombination. 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 The locations of the IRES secondary structure and stem-ring structure are shown in Table 3.

[0185] Table 3 shows the IRES obtained through screening and their loop position base sequences.

[0186]

[0187]

[0188]

[0189]

[0190] Among these IRES, HRV-B3, HRV-B92, HRV-B37, shuffledIRES#01, shuffledIRES#38, HRV-B97, shuffledIRES#03, HRV-B4, HRV-C11, and shuffledIRES#42 have similar stem-loop structures, with the stem containing complementary and non-complementary base pairs, and the number of bases in the stem exceeding 20 bp. The stem-loop structures of HRV-B3 (SEQ ID NO:53), HRV-B92 (SEQ ID NO:54), HRV-B37 (SEQ ID NO:55), shuffledIRES#01 (SEQ ID NO:56), and shuffledIRES#38 (SEQ ID NO:57) have the same loop position sequence. The stem-loop structures of HRV-B97 (SEQ ID NO:58) and shuffledIRES#03 (SEQ ID NO:59) have the same loop position sequence. The loop position sequences of HRV-B4 (SEQ ID NO:60), HRV-C11 (SEQ ID NO:61), and shuffledIRES#42 (SEQ ID NO:62) are different. The secondary structures of iPV2 (SEQ ID NO:63) and Human XIAP (SEQ ID NO:64) differ significantly from the aforementioned IRES structures.

[0191] Example 4: Preparation of scarless circular RNA based on stem-loop structure in HRV-B3 IRES and its mutants

[0192] 1. Plasmid construction

[0193] Because the loop position sequences in the IRES stem-loop structures of HRV-B3, HRV-B92, HRV-B37, shuffledIRES#01, and shuffledIRES#38 are identical, we used HRV-B3 IRES as an example for recombinant nucleic acid molecule design. Figure 17As shown, 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:53), 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:65) 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 bases at the loop position were split at the slash position (5'-CCAAGT / AAC-3'), yielding the fragment HRV-B3IRES I (nucleotide sequence as shown in SEQ ID NO:53) including CCAAGT and its upstream sequence. (as shown in NO:66) and the fragment HRV-B3IRES II (nucleotide sequence as shown in SEQ ID NO:67), which includes AAC bases and their downstream sequences.

[0194] HRV-B3 IRES II is linked to the 3' ends of the 3' intron of the T4td ribozyme (SEQ ID NO:68) and the 3' intron of the Ana ribozyme (SEQ ID NO:69), respectively. HRV-B3 IRES I is linked to the 5' intron mutant of the T4td ribozyme (SEQ ID NO:70, P1) with strict complementary pairing, such as... Figure 18 ; SEQ ID NO:71, P1 is not strictly complementary, such as Figure 19 The 5' end of the 5' intron mutant of Ana ribozyme (SEQ ID NO:72) is linked to the 5' end (e.g.) Figure 20 ).

[0195] The T4td ribozyme intron mutation region is mutated to... A AATTG TT G CTTGG (SEQ ID NO:73, ensuring strict complementary pairing of P1), where the 5' intron P1 guide sequence is mutated to CTTGG (reverse complementary pairing with CCAAG at the loop position), such as Figure 18 ; or the T4td ribozyme intron mutation region is mutated to A AATTG TT G CTTGT (SEQ ID NO:74, making P1 non-strictly complementary), where the 5' intron P1 guide sequence is mutated to CTTGT (CTTG is reverse complementary to CAAG at the loop position, making P1 non-strictly complementary), such as Figure 19As shown; the P10 guide sequence of the 5' intron of the T4td ribozyme is mutated to TT (reverse complementary to the AA base at the loop position), and the corresponding bases in the 5' intron of the T4td ribozyme that are complementary to the P10 guide sequence are mutated to A. The mutation region of the 5' intron of the Ana ribozyme is mutated to... AA ATAA TT G CT (SEQ ID NO:75), in which the 5' intron P1 guide sequence 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 below. Figure 20 As shown.

[0196] 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:76, P1 is strictly complementary; SEQ ID NO:77, 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:78). A T7 promoter (SEQ ID NO:79) was added to the 5' end of these three nucleic acid chains, and then the molecules were cloned into the pUC19 plasmid from 5' to 3' through the EcoRI and XbaI restriction sites.

[0197] Based on the literature's partitioning of the HRV-B3-eIF4GIRES domain and RNAFold's prediction of the secondary structure of the HRV-B3-eIF4GIRES sequence (SEQ ID NO:80), such as Figure 17As shown, stem-loop structures in HRV-B3-eIF4G were screened. The loop position sequence CCAAGTAAC (SEQ ID NO:65) in the longer stem-loop structure of Domain I simultaneously met the splicing requirements of both T4td and Ana ribozymes. The bases at the loop position were split at the slash position (5'-CCAAGT / AAC-3'), yielding fragment HRV-B3-eIF4G IRES I (SEQ ID NO:66, identical to the sequence of HRV-B3 IRES I) including CCAAGT and its upstream sequence, and fragment HRV-B3-eIF4G IRES II (SEQ ID NO:81) including AAC bases and its downstream sequence. HRV-B3-eIF4G IRES II was linked to the 3' end of the 3' intron of the T4td ribozyme (SEQ ID NO:68). HRV-B3-eIF4G IRES I was linked to the 5' end of the loosely paired 5' intron of the T4td ribozyme (SEQ ID NO:71).

[0198] Linking the Fluc protein coding sequence from 5' to 3' between the downstream of HRV-B3-eIF4GIRES II and the upstream of HRV-B3-eIF4GIRES I yielded a nucleic acid molecule (SEQ ID NO:82) composed of T4td ribozyme 3' intron - HRV-B3-eIF4GIRES II - Fluc ORF - HRV-B3-eIF4GIRES 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:83) 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:79) 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.

[0199] 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.

[0200] 2. Preparation of circular RNA

[0201] The steps for plasmid linearization, in vitro transcription, in vitro circularization, and linear RNA digestion are as described in Example 1.

[0202] 3. RNA circularization identification

[0203] 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.

[0204] Figure 21 Electrophoresis showed that the nucleic acid molecule with the nucleotide sequence shown in SEQ ID NO:76 was successfully circularized, and sequencing results showed that it was circularized at the correct site. Quantitative analysis by ImageJ showed that the circularization efficiency was 98.3%. Figure 22 Electrophoresis showed that the nucleic acid molecule with the nucleotide sequence shown in SEQ ID NO:77 was successfully circularized, and sequencing results showed that it was circularized at the correct site. Quantitative analysis by ImageJ showed that the circularization efficiency was 96.4%. Figure 23 Electrophoresis showed that the nucleic acid molecule with the nucleotide sequence shown in SEQ ID NO:78 was successfully circularized, and sequencing results showed that it was circularized at the correct site. Quantitative analysis by ImageJ showed that the circularization efficiency was 97.5%. Figure 24 Electrophoresis showed that the nucleic acid molecule with the nucleotide sequence shown in SEQ ID NO:82 was successfully circularized, and sequencing results showed that it was circularized at the correct site. Quantitative analysis by ImageJ showed that the circularization efficiency was 97.2%. Figure 25 Electrophoresis showed that the nucleic acid molecule with the nucleotide sequence shown in SEQ ID NO:83 was successfully circularized, and sequencing results showed that it was circularized at the correct site. Quantitative analysis by ImageJ showed that the circularization efficiency was 97.4%.

[0205] Example 5: Evaluation of the potency of circular mRNA encoding tumor antigen peptides

[0206] 1. Isolation of peripheral blood mononuclear cells (PBMCs):

[0207] 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.

[0208] 2. Circular mRNA encoding tumor antigen peptides stimulates PBMCs

[0209] Take 100 μl (10 μL) of the separated PBMCs from each well. 5100 μl of coated LNP-LNP cells (containing 0.1 μg of circRNA, whose nucleotide sequence is shown in SEQ ID NO:76 or SEQ ID NO:77) 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.

[0210] 3. ELISpot color development

[0211] 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 1 μg / ml of biotin-labeled 7-B6-1 antibody 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 per well, and develop at room temperature in the dark for 5-30 min. Pour off the liquid in the wells, rinse with tap water to stop the chromogenic process, and allow to air dry. Analyze the spots using an ELISpot speckle analyzer. Results are as follows: Figure 26The results showed that the circular RNA formed by SEQ ID NO:76 (SEQ 76 in the figure) and SEQ ID NO:77 (SEQ 77 in the figure) stimulated PBMCs and produced significantly more IFN-γ points than the negative control group, indicating that the circular RNA formed by SEQ ID NO:76 and SEQ ID NO:77 can effectively express the encoded tumor antigen peptide in DC cells and activate T cells.

[0212] Example 6: Evaluation of the potency of circ mRNA encoding Fluc protein

[0213] According to Lipofectamine TM HEK 293T cells were transfected with the circular RNA formed by SEQ ID NO:82 using 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. Results are as follows: Figure 27 The results showed that the circular RNA formed by SEQ ID NO:82 expressed significantly higher levels of Fluc protein in HEK 293T cells than in the negative and positive controls.

[0214] Example 7: Preparation of scarless circular RNA based on stem-loop structure in HRV-B4 IRES

[0215] 1. Plasmid construction

[0216] Secondary structure prediction of the HRV-B4 IRES sequence (SEQ ID NO:60) was performed using RNAFold, and the positions of stem-loop structures in HRV-B4 IRES were screened as follows: Figure 16 As shown, the CCGTAAC in its loop position sequence (Table 3) contains a T base. Upstream of the T base are 3 free CCG bases, and downstream of the T base are 3 free AAC bases, which meets the requirements of Ana ribozyme splicing (N1N2N3TN4N5N6). The bases in its loop position are split at the slash position (5'-CCGT / AAC-3'), resulting in fragment HRV-B4 IRES I (nucleotide sequence as shown in SEQ ID NO:84) including CCGT and its upstream sequence, and fragment HRV-B4 IRES II (nucleotide sequence as shown in SEQ ID NO:85) including the AAC base and its downstream sequence.

[0217] HRV-B4 IRES II is linked to the 3' end of the 3' intron (SEQ ID NO:69) of the Ana ribozyme. HRV-B4 IRESI is linked to the 5' end of the 5' intron mutant of the Ana ribozyme (SEQ ID NO:86). The 5' intron mutant region of the Ana ribozyme is mutated to... AA ATAA TT G CG The 5' intron P1 guide sequence mutates to CG (opposite to the CG in the loop position), and the P10 guide sequence TT is opposite to the AA base in the loop position (e.g., Figure 28 ).

[0218] The antigen peptide protein-coding sequence (the sequence shown in 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 IRESI-Ana ribozyme 5' intron (its nucleotide sequence is shown in SEQ ID NO:87). 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 The T7 promoter (SEQ ID NO:79) was added to the 5' end of this nucleic acid strand, and then cloned into the pUC19 plasmid from the 5' to 3' direction via the EcoRI and XbaI restriction sites.

[0219] 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.

[0220] 2. Preparation of circular RNA

[0221] The steps for plasmid linearization, in vitro transcription, in vitro circularization, and linear RNA digestion are as described in Example 1.

[0222] 3. RNA circularization identification

[0223] 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.

[0224] Figure 28 Electrophoresis showed that the nucleic acid molecule with the nucleotide sequence shown in SEQ ID NO:87 was successfully circularized, and the circularization efficiency was 37.8% as determined by ImageJ quantitative analysis. Figure 32As shown in column B4, reverse transcription PCR was performed on the circularized RNA. Primers were designed on both sides of the adapter for PCR. Sanger sequencing results showed that the nucleic acid molecule with the nucleotide sequence shown in SEQ ID NO:87 was successfully circularized at the predetermined base positions.

[0225] Example 8: Preparation of scarless circular RNA based on stem-loop structure in Human XIAP IRES

[0226] 1. Plasmid construction

[0227] Secondary structure prediction of the Human XIAP IRES sequence (SEQ ID NO:64) was performed using RNAFold, and the positions of stem-loop structures in the Human XIAP IRES were screened as follows: Figure 16 As shown, 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 are split at the slash position (5'-AATT / AAT-3') to obtain fragment XIAP IRES I (nucleotide sequence as shown in SEQ ID NO:88) including AAT and its upstream sequence and fragment XIAP IRES II (nucleotide sequence as shown in SEQ ID NO:89) including AAT bases and its downstream sequence.

[0228] XIAP IRES II is linked to the 3' end of the 3' intron of the Ana ribozyme (SEQ ID NO:69). XIAP IRES I is linked to the 5' end of the 5' intron mutant of the Ana ribozyme (SEQ ID NO:90), and the 5' intron mutant region of the Ana ribozyme is mutated to... AA ATAA TT G AT The 5' intron P1 guide sequence is mutated to AT (opposite to the AT in the loop position), and the P10 guide sequence TT is opposite to the AA base in the loop position (e.g., Figure 29 ).

[0229] The antigen peptide protein-coding sequence (SEQ ID NO:14) was ligated between the downstream of XIAP IRES II and the upstream of XIAPIRES 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:91). 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 The T7 promoter (SEQ ID NO:79) was added to the 5' end of this nucleic acid strand, and then cloned into the pUC19 plasmid from the 5' to 3' direction via the EcoRI and XbaI restriction sites.

[0230] 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.

[0231] 2. Preparation of circular RNA

[0232] The steps for plasmid linearization, in vitro transcription, in vitro circularization, and linear RNA digestion are as described in Example 1.

[0233] 3. RNA circularization identification

[0234] 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.

[0235] Figure 29 Electrophoresis showed that the nucleic acid molecule with the nucleotide sequence shown in SEQ ID NO:91 was successfully circularized, and the circularization efficiency was 62.6% as determined by ImageJ quantitative analysis. Figure 32 As shown in the XIAP column, reverse transcription PCR was performed on the circularized RNA. Primers were designed on both sides of the adapter for PCR. Sanger sequencing results showed that the nucleic acid molecule with the nucleotide sequence shown in SEQ ID NO:91 was successfully circularized at the predetermined base positions.

[0236] Example 9: Preparation of scarless circular RNA based on the stem-loop structure in HRV-B97 IRES

[0237] 1. Plasmid construction

[0238] Secondary structure prediction of the HRV-B97 IRES sequence (SEQ ID NO:58) was performed using RNAFold, and the stem-loop structure positions in HRV-B97 IRES were screened as follows: Figure 16 As shown, in the CGTTAGA loop position sequence (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 in the loop position are split at the slash position (5'-CGTT / AGA-3'), resulting in fragment HRV-B97 IRES I (nucleotide sequence as shown in SEQ ID NO:92) including CGTT and its upstream sequence and fragment HRV-B97 IRES II (nucleotide sequence as shown in SEQ ID NO:93) including AGA bases and its downstream sequence.

[0239] HRV-B97 IRES II is linked to the 3' end of the 3' intron (SEQ ID NO: 69) of the Ana ribozyme. HRV-B97IRES I is linked to the 5' end of the 5' intron mutant (SEQ ID NO: 94) of the Ana ribozyme, and the 5' intron mutant region of the Ana ribozyme is mutated to... AG ATAA CT G AC The 5' intron P1 guide sequence is mutated to AC (reverse complementary to GT at the loop position), and the P10 guide sequence CT is reverse complementary to AG at the loop position (e.g., Figure 30 ).

[0240] 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-B97IRES I-Ana ribozyme 5' intron (its nucleotide sequence is shown in SEQ ID NO:95). 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 The T7 promoter (SEQ ID NO:79) was added to the 5' end of this nucleic acid strand, and then it was cloned into the pUC19 plasmid from the 5' to 3' direction via the EcoRI and XbaI restriction sites.

[0241] 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.

[0242] 2. Preparation of circular RNA

[0243] The steps for plasmid linearization, in vitro transcription, in vitro circularization, and linear RNA digestion are as described in Example 1.

[0244] 3. RNA circularization identification

[0245] 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.

[0246] Figure 30 Electrophoresis showed that the nucleic acid molecule with the nucleotide sequence shown in SEQ ID NO:95 was successfully circularized, and the circularization efficiency was 64.3% as determined by ImageJ quantitative analysis. Figure 32As shown in column B97, reverse transcription PCR was performed on the circularized RNA. Primers were designed on both sides of the adapter for PCR. Sanger sequencing results showed that the nucleic acid molecule with the nucleotide sequence shown in SEQ ID NO:95 was successfully circularized at the predetermined base positions.

[0247] Example 10: Preparation of scarless circular RNA based on stem-loop structure in ShuffledIRES#42IRES

[0248] 1. Plasmid construction

[0249] Secondary structure prediction was performed on the ShuffledIRES#42IRES sequence (SEQ ID NO:62) using RNAFold, and the positions of stem-loop structures in ShuffledIRES#42IRES were screened as follows: Figure 16 As shown, in the loop position sequence (Table 3), ATGTAAC has 3 free bases upstream of the second T base and 3 free bases downstream of the second T base, which meets the requirements of Ana ribozyme splicing (N1N2N3TN4N5N6). The bases in the loop position are split at the slash position (5'-ATGT / AAC-3'), resulting in the fragment ShuffledIRES#42IRES I (nucleotide sequence as shown in SEQ ID NO:96) including ATGT and its upstream sequence, and the fragment ShuffledIRES#42IRES II (nucleotide sequence as shown in SEQ ID NO:97) including the AGA base and its downstream sequence.

[0250] ShuffledIRES#42IRES II is linked to the 3' end of the 3' intron of the Ana ribozyme (SEQ ID NO:69). ShuffledIRES#42IRES I is linked to the 5' end of the 5' intron mutant of the Ana ribozyme (SEQ ID NO:98), and the 5' intron mutant region of the Ana ribozyme is mutated to... AA ATAA TT G CA The 5' intron P1 guide sequence mutates to CA (opposite to TG at the loop position), and the P10 guide sequence TT is opposite to AA at the loop position (e.g., Figure 31 ).

[0251] 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 Ana ribozyme 3' intron-ShuffledIRES#42IRESII-antigen peptide ORF-ShuffledIRES#42IRES I-Ana ribozyme 5' intron (its nucleotide sequence is shown in SEQ ID NO:99). 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 The T7 promoter (SEQ ID NO:79) was added to the 5' end of this nucleic acid strand, and then cloned into the pUC19 plasmid from the 5' to 3' direction via the EcoRI and XbaI restriction sites.

[0252] 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.

[0253] 2. Preparation of circular RNA

[0254] The steps for plasmid linearization, in vitro transcription, in vitro circularization, and linear RNA digestion are as described in Example 1.

[0255] 3. RNA circularization identification

[0256] 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.

[0257] Figure 31 Electrophoresis showed that the nucleic acid molecule with the nucleotide sequence shown in SEQ ID NO:99 was successfully circularized, and the circularization efficiency was 94.5% as determined by ImageJ quantitative analysis. Figure 32 As shown in column S42, reverse transcription PCR was performed on the circularized RNA. Primers were designed on both sides of the adapter for PCR. Sanger sequencing results showed that the nucleic acid molecule with the nucleotide sequence shown in SEQ ID NO:99 was successfully circularized at the predetermined base positions.

[0258] Example 11: Regular stem-loop structures are more conducive to scarless RNA circularization.

[0259] Based on the experimental results above, we can conclude that more regular stem-loop structures, such as HRV-B3 IRES and ShuffledIRES#42, have relatively higher circularization efficiency. Conversely, 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, have relatively lower circularization efficiency. To further verify that regular stem-loop structures are more conducive to scarless RNA circularization, we further selected four IRES with more regular stem-loop structures—HRV-B92 (SEQ ID NO:54), HRV-B37 (SEQ ID NO:55), shuffledIRES#01 (SEQ ID NO:56), and shuffledIRES#38 (SEQ ID NO:57)—to design and prepare scarless circular RNA.

[0260] 1. Plasmid construction

[0261] The four IRES ring position sequences HRV-B37, HRV-B92, shuffledIRES#01, and shuffledIRES#38 all contain CCAAGTAAC (SEQ ID NO:65). 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:100), HRV-B37 IRES II (SEQ ID NO:101), HRV-B92 IRES I (SEQ ID NO:102), HRV-B92 IRES II (SEQ ID NO:103), shuffledIRES#01IRES I (SEQ ID NO:104), shuffledIRES#01IRES II (SEQ ID NO:105), shuffledIRES#38IRES I (SEQ ID NO:106), and shuffledIRES#38IRES II (SEQ ID NO:107). The split IRES II sequences were ligated to the 3' end of the 3' intron of the Ana ribozyme (SEQ ID NO: 69), and IRES I sequences were ligated to the 5' end of the 5' intron mutant of the Ana ribozyme (SEQ ID NO: 72). 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: 108, SEQ ID NO: 109, SEQ ID NO: 110, and SEQ ID NO: 111.

[0262] The T7 promoter (SEQ ID NO:79) 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.

[0263] 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.

[0264] 2. Preparation of circular RNA

[0265] The steps for plasmid linearization, in vitro transcription, in vitro circularization, and linear RNA digestion are as described in Example 1.

[0266] 3. RNA circularization identification

[0267] The method for detecting circular RNA by formaldehyde denaturation electrophoresis is as described in Example 1. Figure 33 Electrophoresis results showed that circular RNAs designed using regular stem-loop structures from HRV-B37, HRV-B92, shuffledIRES#01, and shuffledIRES#38 all achieved good circularization. Specifically, the circularization efficiency was 91% for the HRV-B37 stem-loop structure, 87% for the HRV-B92 stem-loop structure, 90% for the shuffledIRES#01 stem-loop structure, and 79% for the shuffledIRES#38 stem-loop structure. This indicates that using more regular stem-loop structures to design and prepare scar-free circular RNA is more conducive to improving RNA circularization efficiency.

[0268] Example 12: Scarless circular RNA is difficult to prepare without the stem-loop structure rule in this invention.

[0269] 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 regularity in this invention during the design and preparation of scarless circular RNA, we took CVB3IRES (SEQ ID NO:112) as an example, selected sites in the random coil region of CVB3 IRES for truncation, spliced ​​it with Ana ribozyme introns, and mutated the intron guide sequence according to the mutation rules in this invention, and performed in vitro transcription circularization verification.

[0270] 1. Plasmid construction

[0271] CVB3 IRES has eight main structural domains: I, II, III, IV, V, VI, VII, and a variable region. Figure 34 ), using RNAFold to predict the secondary structure of the CVB3 IRES sequence, such as Figure 35In addition to the variable region, there is also an unstable random coil between the V and VI domains. First, two sites (S1 and S2) were selected in the variable region for truncation. The S1 site was truncated at the slash of the sequence CACTTAGCTT / AAAGAGGTTA, spliced ​​with the Ana ribozyme, and the Ana ribozyme intron 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:113 was obtained. The S2 site was truncated at the slash of the sequence GAGGTTACTT / AAAACATTAC, spliced ​​with the Ana ribozyme, and the Ana ribozyme intron 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:114 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 ribozyme intron 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:115 is obtained.

[0272] The T7 promoter (SEQ ID NO:79) 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 through the EcoRI and XbaI restriction sites.

[0273] 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.

[0274] 2. Preparation of circular RNA

[0275] The steps for plasmid linearization, in vitro transcription, in vitro circularization, and linear RNA digestion are as described in Example 1.

[0276] 3. RNA circularization identification

[0277] The method for detecting circular RNA by formaldehyde denaturation electrophoresis is as described in Example 1. Figure 36 Electrophoresis results showed that nucleic acid molecules with nucleotide sequences as shown in SEQ ID NO:113, SEQ ID NO:114, and SEQ ID NO:115 failed to complete circularization well and were degraded into diffuse bands after treatment with RNase R. This indicates that using the stem-loop structure rule designed in this invention to prepare scarless circular RNA can effectively improve the success rate of RNA circularization.

[0278] The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiments. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

[0279] Sequence information:

[0280] SEQ ID NO:1 (T4td ribozyme 3' intron, containing exon 21nt)

[0281] TGGTTCTACATAAATGCCTAACGACTATCCCTTTGGGGAGTAGGGTCAAGTGACTCGAAACGATAGACAACTTGCTTTAACAAGTTGGAGATATAGTCTGCTCTGCATGGTGACATGCAGCTGGATATAATTCCGGGGTAAGATTAACGACCTTATCTGAACATAATGCTACCGTTTAATATTGCGTCA

[0282] SEQ ID NO:2 (T4td ribozyme 5' intron, containing 16nt exon)

[0283] AGATGTTTTCTTGGGTTAATTGAGGCCTGAGTATAAGGTGACTTATACTTGTAATCTATCTAAACGGGGAACCTCTCTAGTAGACAATCCCGTGCTAAATTGTAGGACTGCCCTTTAAT

[0284] SEQ ID NO:3 (Ana ribozyme 3' intron, containing 51 nt exon)

[0285] TGACTTACAACTAATCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCAAGAGAATGAAAATCCGTTGACCTTAAACGGTCGTGTGGGTTCAAGTCCCTCCACCCCCA

[0286] SEQ ID NO:4 (Ana ribozyme 5' intron, containing 15nt exon)

[0287] AGACGCTACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTCAACAATAGATGSEQ ID NO:5(IRES MP,75 )

[0288] TTCGTTTGCTTTTTGTAGTATAATTAAATATTTGTCATATAAGAGATTGGTTAGAGATTTGTTCTTTGTTTGATCCACC

[0289] SEQ ID NO:6 Tumor Antigen Peptide MAGE-1

[0290] EADPTGHSY

[0291] SEQ ID NO:7 Tumor Antigen Peptide TRP-2

[0292] SVYDFFVWL

[0293] SEQ ID NO:8 Tumor Antigen Peptide gp100

[0294] IMDQVPFSV

[0295] SEQ ID NO:9 Tumor antigen peptide IL13Rα2

[0296] WLPFGFILI

[0297] SEQ ID NO:10 Tumor antigen peptide Melan A

[0298] EAAGIGILTV

[0299] SEQ ID NO:11 Tumor antigen peptide BST2

[0300] LLLGIGILVL

[0301] SEQ ID NO:12 Tumor Antigen Peptide IMP2

[0302] NLSALGIFST

[0303] SEQ ID NO:13

[0304] MEAAGIGILTVILGKLLLGILVLLIIFWLPFGFILILVIKEADPTGHSYVLVTIMDQVPFSVS

[0305] VSLNLSALGIFSTGLSCSVYDFFVWLHYYZ

[0306] SEQ ID NO:14

[0307] ATGGAAGCCGCTGGCATCGGCATCCTCACCGTGATCCTGGGCAAGCTGCTGCTGGGCATTGGAATCCTGGTGCTGCTGATCATCTTCTGGCTGCCTTTCGGCTTTATCCTGATCCTGGTCATCAAGGAGGCCGACCCCACCGGCCACAGCTATGTGCTGGTGACAATCATGGATCAGGTGCCTTTTAGCGTGTCTGTGTCCCTGAACCTGAGCGCCCTGGGAATCTTCAGCACCGGCCTGTCTTGCAGCGTGTACGACTTCTTCGTGTGGCTGCACTACTACTAA

[0308] SEQ ID NO:15

[0309] TGTAATACGACTCACTATAGGGTGGTTCTACATAAATGCCTAACGACTATCCCTTTGGGGAGTAGGGTCAAGTGACTCGAAACGATAGACAACTTGCTTTAACAAGTTGGAGATATAGTCTGCTCTGCATGGTGACATGCAGCTGGATATAATTCCGGGGTAAGATTAACGACCTTATCTGAACATAATGCTACCGTTTAATATTGCGTCATTCGTTTGCTTTTTGTAGTATAATTAAATATTTGTCATATAAGAGATTGGTTAGAGATTTGTTCTTTGTTTGATCCACCATGGAAGCCGCTGGCATCGGCATCCTCACCGTGATCCTGGGCAAGCTGCTGCTGGGCATTGGAATCCTGGTGCTGCTGATCATCTTCTGGCTGCCTTTCGGCTTTATCCTGATCCTGGTCATCAAGGAGGCCGACCCCACCGGCCACAGCTATGTGCTGGTGACAATCATGGATCAGGTGCCTTTTAGCGTGTCTGTGTCCCTGAACCTGAGCGCCCTGGGAATCTTCAGCACCGGCCTGTCTTGCAGCGTGTACGACTTCTTCGTGTGGCTGCACTACTACTAAAGATGTTTTCTTGGGTTAATTGAGGCCTGAGTATAAGGTGACTTATACTTGTAATCTATCTAAACGGGGAACCTCTCTAGTAGACAATCCCGTGCTAAATTGTAGGACTGCCCTTTAATTCTAGA

[0310] SEQ ID NO:16

[0311] TGTAATACGACTCACTATAGGGTGACTTACAACTAATCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCAAGAGAATGAAAATCCGTTGACCTTAAACGGTCGTGTGGGTTCAAGTCCCTCCACCCCCATTCGTTTGCTTTTTGTAGTATAATTAAATATTTGTCATATAAGAGATTGGTTAGAGATTTGTTCTTTGTTTGATCCACCATGGAAGCCGCTGGCATCGGCATCCTCACCGTGATCCTGGGCAAGCTGCTGCTGGGCATTGGAATCCTGGTGCTGCTGATCATCTTCTGGCTGCCTTTCGGCTTTATCCTGATCCTGGTCATCAAGGAGGCCGACCCCACCGGCCACAGCTATGTGCTGGTGACAATCATGGATCAGGTGCCTTTTAGCGTGTCTGTGTCCCTGAACCTGAGCGCCCTGGGAATCTTCAGCACCGGCCTGTCTTGCAGCGTGTACGACTTCTTCGTGTGGCTGCACTACTACTAAAGACGCTACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTCAACAATAGATGTCTAGA

[0312] SEQ ID NO:17

[0313] TGTAATACGACTCACTATAGGGTGGTTCTACATAAATGCCTAACGACTATCCCTTTGGGGAGTAGGGTCAAGTGACTCGAAACGATAGACAACTTGCTTTAACAAGTTGGAGATATAGTCTGCTCTGCATGGTGACATGCAGCTGGATATAATTCCGGGGTAAGATTAACGACCTTATCTGAACATAATGCTACCGTTTAATATTGCGTCAAAAACAAAACAAAAAACAAAAACACAAAACAAAAAACAAAAAAAAAAAACTTCGTTTGCTTTTTGTAGTATAATTAAATATTTGTCATATAAGAGATTGGTTAGAGATTTGTTCTTTGTTTGATCCACCATGGAAGCCGCTGGCATCGGCATCCTCACCGTGATCCTGGGCAAGCTGCTGCTGGGCATTGGAATCCTGGTGCTGCTGATCATCTTCTGGCTGCCTTTCGGCTTTATCCTGATCCTGGTCATCAAGGAGGCCGACCCCACCGGCCACAGCTATGTGCTGGTGACAATCATGGATCAGGTGCCTTTTAGCGTGTCTGTGTCCCTGAACCTGAGCGCCCTGGGAATCTTCAGCACCGGCCTGTCTTGCAGCGTGTACGACTTCTTCGTGTGGCTGCACTACTACTAAAAAAACAAAAACACAAAAAAACAAAAAAAACAAAAAAACAAAAACAAAAAAGATGTTTTCTTGGGTTAATTGAGGCCTGAGTATAAGGTGACTTATACTTGTAATCTATCTAAACGGGGAACCTCTCTAGTAGACAATCCCGTGCTAAATTGTAGGACTGCCCTTTAATTCTAGA

[0314] SEQ ID NO:18

[0315] TGTAATACGACTCACTATAGGGTGACTTACAACTAATCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCAAGAGAATGAAAATCCGTTGACCTTAAACGGTCGTGTGGGTTCAAGTCCCTCCACCCCCAAAAACAAAACAAAAAACAAAAACACAAAACAAAAAACAAAAAAAAAAAACTTCGTTTGCTTTTTGTAGTATAATTAAATATTTGTCATATAAGAGATTGGTTAGAGATTTGTTCTTTGTTTGATCCACCATGGAAGCCGCTGGCATCGGCATCCTCACCGTGATCCTGGGCAAGCTGCTGCTGGGCATTGGAATCCTGGTGCTGCTGATCATCTTCTGGCTGCCTTTCGGCTTTATCCTGATCCTGGTCATCAAGGAGGCCGACCCCACCGGCCACAGCTATGTGCTGGTGACAATCATGGATCAGGTGCCTTTTAGCGTGTCTGTGTCCCTGAACCTGAGCGCCCTGGGAATCTTCAGCACCGGCCTGTCTTGCAGCGTGTACGACTTCTTCGTGTGGCTGCACTACTACTAAAAAAACAAAAACACAAAAAAACAAAAAAAACAAAAAAACAAAAACAAAAAAGACGCTACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTCAACAATAGATGTCTAGA

[0316] SEQ ID NO:19

[0317] TGTAATACGACTCACTATAGGGTGGTTCTACATAAATGCCTAACGACTATCCCTTTGGGGAGTAGGGTCAAGTGACTCGAAACGATAGACAACTTGCTTTAACAAGTTGGAGATATAGTCTGCTCTGCATGGTGACATGCAGCTGGATATAATTCCGGGGTAAGATTAACGACCTTATCTGAACATAATGCTACCGTTTAATATTGCGTCATGCTGGTGGTTCGTTTGCTTTTTGTAGTATAATTAAATATTTGTCATATAAGAGATTGGTTAGAGATTTGTTCTTTGTTTGATCCACCATGGAAGCCGCTGGCATCGGCATCCTCACCGTGATCCTGGGCAAGCTGCTGCTGGGCATTGGAATCCTGGTGCTGCTGATCATCTTCTGGCTGCCTTTCGGCTTTATCCTGATCCTGGTCATCAAGGAGGCCGACCCCACCGGCCACAGCTATGTGCTGGTGACAATCATGGATCAGGTGCCTTTTAGCGTGTCTGTGTCCCTGAACCTGAGCGCCCTGGGAATCTTCAGCACCGGCCTGTCTTGCAGCGTGTACGACTTCTTCGTGTGGCTGCACTACTACTAACCACCAGTGAGATGTTTTCTTGGGTTAATTGAGGCCTGAGTATAAGGTGACTTATACTTGTAATCTATCTAAACGGGGAACCTCTCTAGTAGACAATCCCGTGCTAAATTGTAGGACTGCCCTTTAATTCTAGA

[0318] SEQ ID NO:20TGTAATACGACTCACTATAGGGTGACTTACAACTAATCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCAAGAGAATGAAAATCCGTTGACCTTAAACGGTCGTGTGGGTTCAAGTCCCTCCACCCCCATGGGCAGGCTGCTGGTGGTTCGTTTGCTTTTTGTAGTATAATTAAATATTTGTCATATAAGAGATTGGTTAGAGATTTGTTCTTTGTTTGATCCACCATGGAAGCCGCTGGCATCGGCATCCTCACCGTGATCCTGGGCAAGCTGCTGCTGGGCATTGGAATCCTGGTGCTGCTGATCATCTTCTGGCTGCCTTTCGGCTTTATCCTGATCCTGGTCATCAAGGAGGCCGACCCCACCGGCCACAGCTATGTGCTGGTGACAATCATGGATCAGGTGCCTTTTAGCGTGTCTGTGTCCCTGAACCTGAGCGCCCTGGGAATCTTCAGCACCGGCCTGTCTTGCAGCGTGTACGACTTCTTCGTGTGGCTGCACTACTACTAACCACCAGTGCAGGCTGCCTAAGACGCTACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTCAACAATAGATGTCTAGA

[0319] SEQ ID NO:21(CVB3 IRES)

[0320] TTAAAACAGCCTGTGGGTTGATCCCACCCACAGGCCCATTGGGCGCTAGCACTCTGGTATCACGGTACCTTTGTGCGCCTGTTTTATACCCCCTCCCCCAACTGTAACTTAGAAGTAACACACACCGATCAACAGTCAGCGTGGCACACCAGCCACGTTTTGATCAAGCACTTCTGTTACCCCGGACTGAGTATCAATAGACTGCTCACGCGGTTGAAGGAGAAAGCGTTCGTTATCCGGCCAACTACTTCGAAAAACCTAGTAACACCGTGGAAGTTGCAGAGTGTTTCGCTCAGCACTACCCCAGTGTAGATCAGGTCGATGAGTCACCGCATTCCCCACGGGCGACCGTGGCGGTGGCTGCGTTGGCGGCCTGCCCATGGGGAAACCCATGGGACGCTCTAATACAGACATGGTGCGAAGAGTCTATTGAGCTAGTTGGTAGTCCTCCGGCCCCTGAATGCGGCTAATCCTAACTGCGGAGCACACACCCTCAAGCCAGAGGGCAGTGTGTCGTAACGGGCAACTCTGCAGCGGAACCGACTACTTTGGGTGTCCGTGTTTCATTTTATTCCTATACTGGCTGCTTATGGTGACAATTGAGAGATCGTTACCATATAGCTATTGGATTGGCCATCCGGTGACTAATAGAGCTATTATATATCCCTTTGTTGGGTTTATACCACTTAGCTTGAAAGAGGTTAAAACATTACAATTCATTGTTAAGTTGAATACAGCAAAGCCACC

[0321] SEQ ID NO:22(FLUC DNA sequence)

[0322]

[0323] SEQ ID NO:23

[0324]

[0325] SEQ ID NO:24

[0326]

[0327] SEQ ID NO:25

[0328]

[0329] SEQ ID NO:26

[0330]

[0331] SEQ ID NO:27

[0332]

[0333] SEQ ID NO:28

[0334]

[0335] SEQ ID NO:29

[0336] TGTAATACGACTCACTATAGGGTGACTTACAACTAATCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCAAGAGAATGAAAATGGCAGGCTGCTGGTGGTTCGTTTGCTTTTTGTAGTATAATTAAATATTTGTCATATAAGAGATTGGTTAGAGATTTGTTCTTTGTTTGATCCACCATGGAAGCCGCTGGCATCGGCATCCTCACCGTGATCCTGGGCAAGCTGCTGCTGGGCATTGGAATCCTGGTGCTGCTGATCATCTTCTGGCTGCCTTTCGGCTTTATCCTGATCCTGGTCATCAAGGAGGCCGACCCCACCGGCCACAGCTATGTGCTGGTGACAATCATGGATCAGGTGCCTTTTAGCGTGTCTGTGTCCCTGAACCTGAGCGCCCTGGGAATCTTCAGCACCGGCCTGTCTTGCAGCGTGTACGACTTCTTCGTGTGGCTGCACTACTACTAACCACCAGTGCAGGCTGCCGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTCAACAATAGATGTCTAGA

[0337] SEQ ID NO:30

[0338] TGTAATACGACTCACTATAGGGTGACTTACAACTAATCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCAAGAGAATGAAAAGGCAGGCTGCTGGTGGTTCGTTTGCTTTTTGTAGTATAATTAAATATTTGTCATATAAGAGATTGGTTAGAGATTTGTTCTTTGTTTGATCCACCATGGAAGCCGCTGGCATCGGCATCCTCACCGTGATCCTGGGCAAGCTGCTGCTGGGCATTGGAATCCTGGTGCTGCTGATCATCTTCTGGCTGCCTTTCGGCTTTATCCTGATCCTGGTCATCAAGGAGGCCGACCCCACCGGCCACAGCTATGTGCTGGTGACAATCATGGATCAGGTGCCTTTTAGCGTGTCTGTGTCCCTGAACCTGAGCGCCCTGGGAATCTTCAGCACCGGCCTGTCTTGCAGCGTGTACGACTTCTTCGTGTGGCTGCACTACTACTAACCACCAGTGCAGGCTGCCACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTCAACAATAGATGTCTAGA

[0339] SEQ ID NO:31

[0340] TGTAATACGACTCACTATAGGGTGACTTACAACTAATCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCAAGAGAATGAAAGGCAGGCTGCTGGTGGTTCGTTTGCTTTTTGTAGTATAATTAAATATTTGTCATATAAGAGATTGGTTAGAGATTTGTTCTTTGTTTGATCCACCATGGAAGCCGCTGGCATCGGCATCCTCACCGTGATCCTGGGCAAGCTGCTGCTGGGCATTGGAATCCTGGTGCTGCTGATCATCTTCTGGCTGCCTTTCGGCTTTATCCTGATCCTGGTCATCAAGGAGGCCGACCCCACCGGCCACAGCTATGTGCTGGTGACAATCATGGATCAGGTGCCTTTTAGCGTGTCTGTGTCCCTGAACCTGAGCGCCCTGGGAATCTTCAGCACCGGCCTGTCTTGCAGCGTGTACGACTTCTTCGTGTGGCTGCACTACTACTAACCACCAGTGCAGGCTGCCCTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTCAACAATAGATGTCTAGA

[0341] SEQ ID NO:32

[0342] TGTAATACGACTCACTATAGGGTGACTTACAACTAATCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCAAGAGAATGAAGGCAGGCTGCTGGTGGTTCGTTTGCTTTTTGTAGTATAATTAAATATTTGTCATATAAGAGATTGGTTAGAGATTTGTTCTTTGTTTGATCCACCATGGAAGCCGCTGGCATCGGCATCCTCACCGTGATCCTGGGCAAGCTGCTGCTGGGCATTGGAATCCTGGTGCTGCTGATCATCTTCTGGCTGCCTTTCGGCTTTATCCTGATCCTGGTCATCAAGGAGGCCGACCCCACCGGCCACAGCTATGTGCTGGTGACAATCATGGATCAGGTGCCTTTTAGCGTGTCTGTGTCCCTGAACCTGAGCGCCCTGGGAATCTTCAGCACCGGCCTGTCTTGCAGCGTGTACGACTTCTTCGTGTGGCTGCACTACTACTAACCACCAGTGCAGGCTGCCTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTCAACAATAGATGTCTAGA

[0343] SEQ ID NO:33

[0344] TGTAATACGACTCACTATAGGGTGGTTCTACATAAATGCCTAACGACTATCCCTTTGGGGAGTAGGGTCAAGTGACTCGAAACGATAGACAACTTGCTTTAACAAGTTGGAGATATAGTCTGCTCTGCATGGTGACATGCAGCTGGATATAATTCCGGGGTAAGATTAACGACCTTATCTGAACATAATGCTACGCTGGTGGTTCGTTTGCTTTTTGTAGTATAATTAAATATTTGTCATATAAGAGATTGGTTAGAGATTTGTTCTTTGTTTGATCCACCATGGAAGCCGCTGGCATCGGCATCCTCACCGTGATCCTGGGCAAGCTGCTGCTGGGCATTGGAATCCTGGTGCTGCTGATCATCTTCTGGCTGCCTTTCGGCTTTATCCTGATCCTGGTCATCAAGGAGGCCGACCCCACCGGCCACAGCTATGTGCTGGTGACAATCATGGATCAGGTGCCTTTTAGCGTGTCTGTGTCCCTGAACCTGAGCGCCCTGGGAATCTTCAGCACCGGCCTGTCTTGCAGCGTGTACGACTTCTTCGTGTGGCTGCACTACTACTAACCACCAGCGCTTGGGTTAATTGAGGCCTGAGTATAAGGTGACTTATACTTGTAATCTATCTAAACGGGGAACCTCTCTAGTAGACAATCCCGTGCTAAATTGTAGGACTGCCCTTTAATTCTAGA

[0345] SEQ ID NO:34

[0346] TGTAATACGACTCACTATAGGGTGGTTCTACATAAATGCCTAACGACTATCCCTTTGGGGAGTAGGGTCAAGTGACTCGAAACGATAGACAACTTGCTTTAACAAGTTGGAGATATAGTCTGCTCTGCATGGTGACATGCAGCTGGATATAATTCCGGGGTAAGATTAACGACCTTATCTGAACATAATGCTCGCTGGTGGTTCGTTTGCTTTTTGTAGTATAATTAAATATTTGTCATATAAGAGATTGGTTAGAGATTTGTTCTTTGTTTGATCCACCATGGAAGCCGCTGGCATCGGCATCCTCACCGTGATCCTGGGCAAGCTGCTGCTGGGCATTGGAATCCTGGTGCTGCTGATCATCTTCTGGCTGCCTTTCGGCTTTATCCTGATCCTGGTCATCAAGGAGGCCGACCCCACCGGCCACAGCTATGTGCTGGTGACAATCATGGATCAGGTGCCTTTTAGCGTGTCTGTGTCCCTGAACCTGAGCGCCCTGGGAATCTTCAGCACCGGCCTGTCTTGCAGCGTGTACGACTTCTTCGTGTGGCTGCACTACTACTAACCACCAGCGTTGGGTTAATTGAGGCCTGAGTATAAGGTGACTTATACTTGTAATCTATCTAAACGGGGAACCTCTCTAGTAGACAATCCCGTGCTAAATTGTAGGACTGCCCTTTAATTCTAGA

[0347] SEQ ID NO:35

[0348] TGTAATACGACTCACTATAGGGTGGTTCTACATAAATGCCTAACGACTATCCCTTTGGGGAGTAGGGTCAAGTGACTCGAAACGATAGACAACTTGCTTTAACAAGTTGGAGATATAGTCTGCTCTGCATGGTGACATGCAGCTGGATATAATTCCGGGGTAAGATTAACGACCTTATCTGAACATAATGCCGCTGGTGGTTCGTTTGCTTTTTGTAGTATAATTAAATATTTGTCATATAAGAGATTGGTTAGAGATTTGTTCTTTGTTTGATCCACCATGGAAGCCGCTGGCATCGGCATCCTCACCGTGATCCTGGGCAAGCTGCTGCTGGGCATTGGAATCCTGGTGCTGCTGATCATCTTCTGGCTGCCTTTCGGCTTTATCCTGATCCTGGTCATCAAGGAGGCCGACCCCACCGGCCACAGCTATGTGCTGGTGACAATCATGGATCAGGTGCCTTTTAGCGTGTCTGTGTCCCTGAACCTGAGCGCCCTGGGAATCTTCAGCACCGGCCTGTCTTGCAGCGTGTACGACTTCTTCGTGTGGCTGCACTACTACTAACCACCAGCGTGGGTTAATTGAGGCCTGAGTATAAGGTGACTTATACTTGTAATCTATCTAAACGGGGAACCTCTCTAGTAGACAATCCCGTGCTAAATTGTAGGACTGCCCTTTAATTCTAGA

[0349] SEQ ID NO:36

[0350] TGTAATACGACTCACTATAGGGTGGTTCTACATAAATGCCTAACGACTATCCCTTTGGGGAGTAGGGTCAAGTGACTCGAAACGATAGACAACTTGCTTTAACAAGTTGGAGATATAGTCTGCTCTGCATGGTGACATGCAGCTGGATATAATTCCGGGGTAAGATTAACGACCTTATCTGAACATAATGCGCTGGTGGTTCGTTTGCTTTTTGTAGTATAATTAAATATTTGTCATATAAGAGATTGGTTAGAGATTTGTTCTTTGTTTGATCCACCATGGAAGCCGCTGGCATCGGCATCCTCACCGTGATCCTGGGCAAGCTGCTGCTGGGCATTGGAATCCTGGTGCTGCTGATCATCTTCTGGCTGCCTTTCGGCTTTATCCTGATCCTGGTCATCAAGGAGGCCGACCCCACCGGCCACAGCTATGTGCTGGTGACAATCATGGATCAGGTGCCTTTTAGCGTGTCTGTGTCCCTGAACCTGAGCGCCCTGGGAATCTTCAGCACCGGCCTGTCTTGCAGCGTGTACGACTTCTTCGTGTGGCTGCACTACTACTAACCACCAGCGGGGTTAATTGAGGCCTGAGTATAAGGTGACTTATACTTGTAATCTATCTAAACGGGGAACCTCTCTAGTAGACAATCCCGTGCTAAATTGTAGGACTGCCCTTTAATTCTAGA

[0351] SEQ ID NO:37

[0352]

[0353] SEQ ID NO:38

[0354]

[0355] SEQ ID NO:39

[0356]

[0357] SEQ ID NO:40

[0358]

[0359] SEQ ID NO:41(8nt50-1)

[0360]

[0361] SEQ ID NO:42(7nt-5 50-1)

[0362]

[0363] SEQ ID NO:43(7nt-3 50-1)

[0364]

[0365] SEQ ID NO:44(6nt50-1)

[0366]

[0367] SEQ ID NO:45(NoAa)

[0368] TGTAATACGACTCACTATAGGGTGACTTACAACTAATCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCAAGAGAATGAAAATTCGTTTGCTTTTTGTAGTATAATTAAATATTTGTCATATAAGAGATTGGTTAGAGATTTGTTCTTTGTTTGATCCACCATGGAAGCCGCTGGCATCGGCATCCTCACCGTGATCCTGGGCAAGCTGCTGCTGGGCATTGGAATCCTGGTGCTGCTGATCATCTTCTGGCTGCCTTTCGGCTTTATCCTGATCCTGGTCATCAAGGAGGCCGACCCCACCGGCCACAGCTATGTGCTGGTGACAATCATGGATCAGGTGCCTTTTAGCGTGTCTGTGTCCCTGAACCTGAGCGCCCTGGGAATCTTCAGCACCGGCCTGTCTTGCAGCGTGTACGACTTCTTCGTGTGGCTGCACTACTACTAAACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTCAACAATAGATG

[0369] SEQ ID NO:46(6ntAa)

[0370] TGTAATACGACTCACTATAGGGTGACTTACAACTAATCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCAAGAGAATGAAAAGCCGGGACTTCGTTTGCTTTTTGTAGTATAATTAAATATTTGTCATATAAGAGATTGGTTAGAGATTTGTTCTTTGTTTGATCCACCATGGAAGCCGCTGGCATCGGCATCCTCACCGTGATCCTGGGCAAGCTGCTGCTGGGCATTGGAATCCTGGTGCTGCTGATCATCTTCTGGCTGCCTTTCGGCTTTATCCTGATCCTGGTCATCAAGGAGGCCGACCCCACCGGCCACAGCTATGTGCTGGTGACAATCATGGATCAGGTGCCTTTTAGCGTGTCTGTGTCCCTGAACCTGAGCGCCCTGGGAATCTTCAGCACCGGCCTGTCTTGCAGCGTGTACGACTTCTTCGTGTGGCTGCACTACTACTAACCCGGCACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTCAACAATAGATG

[0371] SEQ ID NO:47(8ntAa)

[0372] TGTAATACGACTCACTATAGGGTGACTTACAACTAATCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCAAGAGAATGAAAACGCCGCGGACTTCGTTTGCTTTTTGTAGTATAATTAAATATTTGTCATATAAGAGATTGGTTAGAGATTTGTTCTTTGTTTGATCCACCATGGAAGCCGCTGGCATCGGCATCCTCACCGTGATCCTGGGCAAGCTGCTGCTGGGCATTGGAATCCTGGTGCTGCTGATCATCTTCTGGCTGCCTTTCGGCTTTATCCTGATCCTGGTCATCAAGGAGGCCGACCCCACCGGCCACAGCTATGTGCTGGTGACAATCATGGATCAGGTGCCTTTTAGCGTGTCTGTGTCCCTGAACCTGAGCGCCCTGGGAATCTTCAGCACCGGCCTGTCTTGCAGCGTGTACGACTTCTTCGTGTGGCTGCACTACTACTAACCGCGGCGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTCAACAATAGATG

[0373] SEQ ID NO:48(20ntAa)

[0374] TGTAATACGACTCACTATAGGGTGACTTACAACTAATCGGAAGGTGCAGAGACTCGACGGGAGCTAACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCAAGAGAATGAAAATGGGCAGGCTGCTGGTGGTTC TTTGCTTTTTGTAGTATAATTAAATATTTGTCATATAAGAGATTGGTTAGAGATTTGTTCTTTGTTTGATCCCACCATGGAAGCCGCTGGCATCGGCATCCTCACCGTGATCCTGGGCAAGCTGCTGCTGGGCATTGGAATCCTGGTGCTGCTGATCATCTTCTGGCTGCCT TTCGGCTTTATCCTGATCCTGGTCATCAAGGAGGCCGACCCCACCGGCCACAGCTATGTGCTGGTGACAATCATGGATCAGGTGCCTTTTTAGCGTGTCTGTGTCCCTGAACCTGAGCGCCCTGGGAATCTTCAGCACCGGCCTGTCTTGCAGCGTGTACGACTTCTTCGTG TGGCTGCACTACTACTAACCACCAGTGCAGGCTGCCTAACTTAAATAATTGAGCCTTAAAAGAAATTCTTTAAGTGGATGCTCTCCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTCAACAATAGATG

[0375] SEQ ID NO:49(NOAT)

[0376] TGTAATACGACTCACTATAGGGTGGTTCTACATAAATGCCTAACGACTATCCCTTTGGGGAGTAGGGTCAAGTGACTCGAAACGATAGACAACTTGCTTTAACAAGTTGGAGATATAGTCTGCTCTGCATGGTGACATGCAGCTGGATATAATTCCGGGGTAAGATTAACGACCTTATCTGAACATAATGCTTTCGTTTGCTTTTTGTAGTATAATTAAATATTTGTCATATAAGAGATTGGTTAGAGATTTGTTCTTTGTTTGATCCACCATGGAAGCCGCTGGCATCGGCATCCTCACCGTGATCCTGGGCAAGCTGCTGCTGGGCATTGGAATCCTGGTGCTGCTGATCATCTTCTGGCTGCCTTTCGGCTTTATCCTGATCCTGGTCATCAAGGAGGCCGACCCCACCGGCCACAGCTATGTGCTGGTGACAATCATGGATCAGGTGCCTTTTAGCGTGTCTGTGTCCCTGAACCTGAGCGCCCTGGGAATCTTCAGCACCGGCCTGTCTTGCAGCGTGTACGACTTCTTCGTGTGGCTGCACTACTACTAATTGGGTTAATTGAGGCCTGAGTATAAGGTGACTTATACTTGTAATCTATCTAAACGGGGAACCTCTCTAGTAGACAATCCCGTGCTAAATTGTAGGACTGCCCTTTAAT

[0377] SEQ ID NO:50(6ntAT)

[0378] TGTAATACGACTCACTATAGGGTGGTTCTACATAAATGCCTAACGACTATCCCTTTGGGGAGTAGGGTCAAGTGACTCGAAACGATAGACAACTTGCTTTAACAAGTTGGAGATATAGTCTGCTCTGCATGGTGACATGCAGCTGGATATAATTCCGGGGTAAGATTAACGACCTTATCTGAACATAATGCTCCCCCCACTTCGTTTGCTTTTTGTAGTATAATTAAATATTTGTCATATAAGAGATTGGTTAGAGATTTGTTCTTTGTTTGATCCACCATGGAAGCCGCTGGCATCGGCATCCTCACCGTGATCCTGGGCAAGCTGCTGCTGGGCATTGGAATCCTGGTGCTGCTGATCATCTTCTGGCTGCCTTTCGGCTTTATCCTGATCCTGGTCATCAAGGAGGCCGACCCCACCGGCCACAGCTATGTGCTGGTGACAATCATGGATCAGGTGCCTTTTAGCGTGTCTGTGTCCCTGAACCTGAGCGCCCTGGGAATCTTCAGCACCGGCCTGTCTTGCAGCGTGTACGACTTCTTCGTGTGGCTGCACTACTACTAAGGGGGGTTGGGTTAATTGAGGCCTGAGTATAAGGTGACTTATACTTGTAATCTATCTAAACGGGGAACCTCTCTAGTAGACAATCCCGTGCTAAATTGTAGGACTGCCCTTTAAT

[0379] SEQ ID NO:51(8ntAT)

[0380] TGTAATACGACTCACTATAGGGTGGTTCTACATAAATGCCTAACGACTATCCCTTTGGGGAGTAGGGTCAAGTGACTCGAAACGATAGACAACTTGCTTTAACAAGTTGGAGATATAGTCTGCTCTGCATGGTGACATGCAGCTGGATATAATTCCGGGGTAAGATTAACGACCTTATCTGAACATAATGCTCGCTGGTGTTCGTTTGCTTTTTGTAGTATAATTAAATATTTGTCATATAAGAGATTGGTTAGAGATTTGTTCTTTGTTTGATCCACCATGGAAGCCGCTGGCATCGGCATCCTCACCGTGATCCTGGGCAAGCTGCTGCTGGGCATTGGAATCCTGGTGCTGCTGATCATCTTCTGGCTGCCTTTCGGCTTTATCCTGATCCTGGTCATCAAGGAGGCCGACCCCACCGGCCACAGCTATGTGCTGGTGACAATCATGGATCAGGTGCCTTTTAGCGTGTCTGTGTCCCTGAACCTGAGCGCCCTGGGAATCTTCAGCACCGGCCTGTCTTGCAGCGTGTACGACTTCTTCGTGTGGCTGCACTACTACTAACACCAGCGTTGGGTTAATTGAGGCCTGAGTATAAGGTGACTTATACTTGTAATCTATCTAAACGGGGAACCTCTCTAGTAGACAATCCCGTGCTAAATTGTAGGACTGCCCTTTAAT

[0381] SEQ ID NO:52(9ntAT)

[0382] TGTAATACGACTCACTATAGGGTGGTTCTACATAAATGCCTAACGACTATCCCTTTGGGGAGTAGGGTCAAGTGACTCGAAACGATAGACAACTTGCTTTAACAAGTTGGAGATATAGTCTGCTCTGCATGGTGACATGCAGCTGGATATAATTCCGGGGTAAGATTAACGACCTTATCTGAACATAATGCTTGCTGGTGGTTCGTTTGCTTTTTGTAGTATAATTAAATATTTGTCATATAAGAGATTGGTTAGAGATTTGTTCTTTGTTTGATCCACCATGGAAGCCGCTGGCATCGGCATCCTCACCGTGATCCTGGGCAAGCTGCTGCTGGGCATTGGAATCCTGGTGCTGCTGATCATCTTCTGGCTGCCTTTCGGCTTTATCCTGATCCTGGTCATCAAGGAGGCCGACCCCACCGGCCACAGCTATGTGCTGGTGACAATCATGGATCAGGTGCCTTTTAGCGTGTCTGTGTCCCTGAACCTGAGCGCCCTGGGAATCTTCAGCACCGGCCTGTCTTGCAGCGTGTACGACTTCTTCGTGTGGCTGCACTACTACTAACCACCAGTGTTGGGTTAATTGAGGCCTGAGTATAAGGTGACTTATACTTGTAATCTATCTAAACGGGGAACCTCTCTAGTAGACAATCCCGTGCTAAATTGTAGGACTGCCCTTTAAT

[0383] SEQ ID NO:53(HRV-B3 IRES)

[0384] TTAAAACAGCGGATGGGTACCCCACCATCCGACCCACTGGGTGTAGTACTCTGGTACTTCGTACCTTTGTACGCCTGTTCTTCCCATTGTACCCTTCCTGAACTTCCAACCCAAGTAACGTTAGAAGCTCAACATTTAGTACAACAGGAAGCACCACATCCAGTGGTGTTTAGTACAAGCACTTCTGTTTCCCCGGAGCGAGGTATAGGCTGTACCCACTGCCAAAAACCTTTAACCGTTATCCGCCAACCAACTACGTAAAAGCTAGTAGTATTATGTTTTTAACTAGGCGTTCGATCAGGTGGATTTCCCCTCCACTAGTTTGGTCGATGAGGCTAGGAATTCCCCACGGGTGACCGTGTCCTAGCCTGCGTGGCGGCCAACCCAGCTTATGCTGGGACGCCTTTTTATAGACATGGTGTGAAGACTCGCATGTGCTTGGTTGTGATTCCTCCGGCCCCTGAATGCGGCTAACCTTAACCCTGGAGCCTTGTGTCACAAACCAGTGATGATAAGGTCGTAATGAGCAATTCCGGGACGGGACCGACTACTTTGGGTGTCCGTGTTTCTTATTTTTCTTATTATTGTCTTATGGTCACAGCATATATATAACATATACTGTGATC

[0385] SEQ ID NO:54(HRV-B92 IRES)

[0386] TTAAAACAGCGGATGGGTATCCCACCATCCGGCCCACTGGGTGTAGTACTCTGGTACATTGTACCTTTGTACGCCTGTTTTCCCCCTCTTGTACCCGCCCTTCAAGCTCCTTGCCCAAGTAACGTTAGAAGTTTGAACATTGGTACAATAGGAAGCATCACATCCAGTGGTGTACTGTACAAACACTTCTGTTGCCCCGGAGCGAGGTATAGATGGTCCCCACCGTCAAAAGCCTTTAACCGTTATCCGCCAATCAACTACGTAATGGCTAGTAGCACCTTGGATTTAAGTTGGCGTTCGATCAGGTGGTAACCCCCACTAGTTTGGTCGATGAGGCTAGGAATTCCCCACGGGTGACCGTGTCCTAGCCTGCGTGGCGGCCAACCCAGCATCCGCTGGGACGCCAATTTAATGACATGGTGTGAAGACCTGCATGTGCTTGATTGTGAGTCCTCCGGCCCCTGAATGCGGCTAACCCTAACCCCGGAGCCTTGCAGCACAATCCAGTGTTGTTAAGGTCGTAATGAGCAATTCTGGGATGGGACCGACTACTTTGGGTGTCCGTGTTTCTTATTTTTCTTGAATTTTTCTTATGGTCACAGCATATATACATTATATACTGTGATC

[0387] SEQ ID NO:55(HRV-B37 IRES)

[0388] TTTAAACAGCGGATGGGTATCCCACCATCCGACCCACTGGGTGTAGTACTCTGGTATTTTGTACCTTTGTACGCCTGTTGTTCCTAATGTACCCACCCTAAAACTTCCTACCCAAGTAACGTTAGAAGTTTCATCAACAAGTACAATAGGAAGCATCACATCCAGTGGTGTTTTGTACAAGCACTTCTGTTTCCCCGGAGCGAGGTATAGGCTGTACCCACTGCCGAAAGCCTTTAACCGTTATCCGCCAACCAACTACGTAAAAGCTAGTATCATCATGTTTTAAAATAGGCGTTCGATCAGGTGGTACCCCCCTCCACTAGTTTGGTCGATGAGGCTAGGAACTCCCCACGGGTGACCGTGTCCTAGCCTGCGTGGCGGCCAACCCAGCTTCTGCTGGGACGCCTTTTTATGGACATGGTGTGAAGACTCGCATGTGCTTGGTTGTGACTCCTCCGGCCCCTGAATGCGGCTAACCTTAACCCCGGAGCCCTGTGTTGCAATCCAGTAACATTAGGGTCGTAATGAGCAATTTCGGGACGGGACCGACTACTTTGGGTGTCCGTGTTTCTCATTTTTCTTATTATTGTCTTATGGTCACAGCATATATATAACGTATATACTGTGATC

[0389] SEQ ID NO:56(shuffledIRES#01)

[0390] TTAAAACAGCGGATGGGTACCCCACCATCCGACCCACTGGGTGTAGTACTCTGGTATTTTGTACCTTTGTACGCCTGTTGTTCCTAATGTACCCACCCTAAAACTTCCAACCCAAGTAACGTTAGAAGTTTGACATTAAAGTACAATAGGTTGCACTACATCCAGTGGTGTTTCGTACAAGCACTTCTGTTTCCCCGGAGCGGGGTATAGACGGTACCCACTGTCAAAAGCCCTTAACCGTTATCCGCCAATCAACTACGTAATGGCTAGTAACATCATGAAGGTGAGTTGACGTTCGATCAGGTGGTAACCCCCACTAGTTTGGTCGATGAGGCTAGGAATCCCCCACGGGTAACCGTGTCCTAGCCTGCGTGGCGGCCAACCCAGCGTTTGCTGGGACGCCAATTCACTGACATGGTGCGAATACTTGCATGTGCTTGATTGTGATTCCTCCGGCCCCTGAATGCGGCTAACCCTAACCCTGGAGCCTTGCACCACAATCCAGTGGTGTCTGGGTCGTAATGAGTAATTCTGGGATGGGACCGACTACTTTGGGTGTCCGTGTTTCTCATTTTTCTTATTATTGTCTTATGGTCACAGCATATAGTAATATATACTGTGATC

[0391] SEQ ID NO:57(shuffledIRES#38)

[0392] TTAAAACAGCGGATGGGTATCCCACCATCCGACCCACTGGGTGTAGTACTCTGGTACTTTGTACCTTTGTACGCCTGTTTTCCCCCTCTTGTACCCGCCCTTCAAGCTCCTTGCCCAAGTAACGTTAGAAGTTTGAACATTGGTACAATAGGAAGCATCACATCCAGTGGTGTACTGTACAAACACTTCTGTTGCCCCGGAGCGAGGTATAGATGGTCCCCACCGTCAAAAGCCTTTAACCGTTATCCGCCAACCAACTACGAGACAGCTAGTAATGCCCTGTTCTCCTGCAAGGCGTTCGATCAGACAGATTTCCCCTCTGTTAGTCTGGTCGATGAGGCTAGGAGCTCCCCACGGGCGACCGTGTCCTAGCCTGCGTGGCGGCCAGCCCAGCTTTTGCTGGGACGCCTTTTCAAAGACATGGTGTGAAGACCTGCATGTGCTTGGTTGTGAGTCCTCCGGCCCCTGAATGCGGCTAACCTTAACCCTGGAGCCTTATGCCACGAACCAGTGGTTGTAAGGTCGTAATGAGTAATTCTGGGATGGGACCGACTACTTTGGGTGTCCGTGTTTCCTTTATTCTTATCATTTGTGTTTCATGGTTACAAGCATTGTTTGTAATC

[0393] SEQ ID NO:58(HRV-B97)

[0394] TTAAAACAGCGGATGGGTTTCCCACCATCCGACCCACTGGGTGTAGTGCTCTGGTATTTTGTACCTTTGCACGCCTGTTTCCCCTTTGTACCCATCCTGAATTTCCTCCCTCTGCAACGTTAGAAGTTTGTGAAATTAAAAGTACAATAGGAAGCATCACATCCAGTGGTGTTCAGTACAAGCACTCCTGTTTCCCCGGAGCGAGGTATAGGTTGTACCCACGACCGAAAGCCTTTAACCGTTATCCGCCAATCAACTACATAACGGCTAGTATCATCATGTTTTTGATCTGGCGTTCGATCAGGTGGTTTCCCCCACTAGTCTGGTCGATGAGGCTAGGATTTCCCCACGGGCGACCGTGTCCTAGCCTGCGTGGCGGCCAGCCCAGCTTATGCTGGGACGCCTTTTTAAAGACATGGTGTGAAGACCTGCATGTGCTTGATTGTGAGTCCTCCGGCCCCTGAATGCGGCTAACCTTAACCCTGGAGCCCGACAGCATAATCCAATGTTGTTTGGGTCGTAATGAGCAATTCCGGGATGGGACCGACTACTTTGGGTGTCCGTGTTTCTTTTTATTCTTATATTGTCTTATGGTCACAGCATATATAGTATATATACTGTGATC

[0395] SEQ ID NO:59(shuffledIRES#03)

[0396] TTAAAACAGCGGATGGGTTTCCTACCATCCGACCCACTGGGTGTAGTGCTCTGGTATATTGTACCTTTGCACGCCTGTTTCCCCTTTGTACCCATCCTGAATTTCCTCCCTCTGCAACGTTAGAAGTTTGTGAAATTAAAAGTACAATAGGAAGCATCACATCCAGTGGTGTTCAGTACAAGCACTTCTGTTTCCCCGGAGCGAGGTATAGGCTGTACCCACTGCCGAAAGCCTTTAACCGTTATCCGCCAGCCAACTACGTAACGGCTAGTAGCACCTTGGATTTAAGTTGGCGTTCGATCAGGTGGTAACCCCCCACTAGTTTGGTCGATGAGGCTGAGAACTCCCCACGGGTAACCGTGTCTCAGCCTGCGTGGCGGCCAACCCAGCGTTTGCTGGGACGCCCTTTTATAGACATGGTGTGAAGACTCGCATGTGCTTGGTTGTGATTCCTCCGGCCCCTGAATGCGGCTAACCCTAACCCTGGAGCCTTGTGTTACAAACCAGTAATATTAAGGTCGTAATGAGCAATTCCGGGACGGGACCGACTACTTTGGGTGTCCGTGTTTCCTGTTTTTCTTTAAAATGTCTTATGGTCACAGCATATATACTGTATATACTGTGATC

[0397] SEQ ID NO:60(HRV-B4)

[0398] TTAAAACAGCGGATGGGTTTCCCACCATCCGACCCACTGGGTGTAGTGCTCTGGTATTTTGTACCTTTGCACGCCTGTTTCCCATTTGTACCCTTCCTTAATCTCCTTCCCCCGTAACGTTAGAAGTTTTGGAATTTTAAAGTACAATAGGAAGCGCCACATCCAGTGGTGTTGCGTACAAATACTTCTGTTTACCCGGAGCGAGGTATAGGTTGTACCCACGGCCAAAAGCCTTTAACCGTTATCCGCCAATCAACTACGTAACGGCTAGTATCATCTTGCTTTTGATTTGGTGTTCGATCAGGTGGTATCCCCCACTAGTCTGGTCGATGAGGCTAGGAATTCCCCACGGGCGACCGTGTCCTAGCCTGCGTGGCGGCCAGCCCAGCTTTTGCTGGGACGCCTTTTCAAAGACATGGTGTGAAGACCTGCATGTGCTTGGTTGTGAGTCCTCCGGCCCCTGAATGCGGCTAACCTTAACCCTGGAGCCCAGCAGCATAATCCAATGTTGTTTGGGTCGTAATGAGCAATTCCGGGACGGGACCGACTACTTTGGGTGTCCGTGTTTCCTTTTATTCTTACATTGTCTTATGGTCACAGCATATATATTATATATACTGTGATC

[0399] SEQ ID NO:61(HRV-C11)

[0400] TTAAAACTGGATACAGGTTGTTCCCACCTGTATCACCCAAGTGGTGTGGTGCTCTTGTATTTCGGTACGTTTGCACGCCAGTTTGCTACCCCTTCCCTTTTACGTAACTTAGAAGTTTACACAAAGACCAATAGGCGGTGGTAAATCCATACCACTAACGGTCAAATACTTCTGTTTCCCCGGCATGCGAGGAATAGGCTCCAAAAGGGCTGAAGCCACTAGTGTCGTTATCCGCATTGGTACTACGCAAAGCCTAGTATTACCTTGAAAATTTCTTGGCTGGTCGCTCCACCAGATACCCCACTGGTAGACCTGGCAGATGAGGCAGGACTTACCCCACTGGCGACAGTGGTCCTGCCTGCGTGGCTGCCTGCACACCCCTTACGGGGTGTGAAGCCCAGAAACAGACAAGGTGTGAAGAGCCCCGTGTGCTACTAGTGAGTCCTCCGGCCCCTGAATGCGGCTAATCTTACCCCACAGCTGTTGCACGCAAACCAGCGTGTATGCAGTCGTAATGAGCAATTGTGGGATGGAACCGACTACTTTGGGTGTCCGTGTTTCTTTTTATTCCTATTATTTGCTTATGGTGACAATATTGATATTATCAGTGTTGTCATC

[0401] SEQ ID NO:62(shuffledIRES#42)

[0402] TTAAAACAGTGGATGGGTTTCCTACCATCCGACCCACTGGGTGTAGTGCTCTGGTATATTGTACCTTTGCACGCCTGTTTTCCCATTGTACCCTTCCTTAAATTCCTCCCCATGTAACGTTAGAAGTTTAAGAACATAAATGTACAATAGGAAGCATCACATCCAGTGGTGTATGGTACAAGCACTTCTGTTTATCCGGAGCGAGGTATAAGCGGTACCCACTGCTGAAAGCCTTTAACCGTTACCCGCCAGCCAACTACGTAAAAGCTAGTAACATCTTGTTTGTAACTTGGCGTTCGATCAGGTGGATTCCCCCTCCACTAGTTTGGTCGATGAGGCTAGGAACTCCCCACGGGTGACCGTGTCCTAGCCTGCGTGGCGGCCAACCCAGCTCTTGCTGGGACGCCCTTTTATAGACATGGTGTGAAGACTCGCATGTGCTTGGTTGTGATTCCTCCGGCCCCTGAATGCGGCTAACCCTAACCCCGGAGCCGTTGCCCATAATCCAATGGGTTTGCGGTCGTAATGGGCAACTCTGGGATGGGACCAACTACTTTGGGTGTCCGTGTTTCCTGTTTTTCTTTAAAATGTCTTATGGTCACAGCATATAGTAATTATATACTGTGATC

[0403] SEQ ID NO:63(iPV2)

[0404] TTAAAACAGCTCTGGGGTTGTTCCCACCCCAGAGGCCCACGTGGCGGCCAGTACACTGGTATCGCGGTACCTTTGTACGCCTGTTTTATACTCCCTTCCCCCGTAACTTAGAAGCACAACGTCCAAGTTCAATAGAAGGGGGTACAAACCAGTACCACCACGAACAAGCACTTCTGTTCCCCCGGTGAGGCTGTATAGGCTGTTTCCACGGCTAAAAGCGGCTGATCCGTTATCCGCTCATGTACTTCGAGAAGCCTAGTATCACCTTGGAATCTTCGATGCGTTGCGCTCAACACTCAACCCCAGAGTGTAGCTTAGGTCGATGAGTCTGGACGTTCCTCACCGGCGACGGTGGTCCAGGCTGCGTTGGCGGCCTACCTGTGGCCCAAAGCCACAGGACGCTAGTTGTGAACAAGGTGTGAAGAGCCTATTGAGCTACCTGAGAGTCCTCCGGCCCCTGAATGCGGCTAATCCTAACCACGGAGCAGGCAGTGGCAATCCAGCGACCAGCCTGTCGTAACGCGCAAGTTCGTGGCGGAACCGACTACTTTGGGTGTCCGTGTTTCCTTTTATTTTTACAATGGCTGCTTATGGTGACAATTATTGATAGTTATCATAAAGCAAATTGGATTGGCCATCCGGTGAGAATTTGATTATTAAATTACTCTCTTGTTGGGATTGCTCCTTTGAAATCCTGTGCACTCACACCTATTGGAATTACCTCATTGTTGAGATATTATTACCACT

[0405] SEQ ID NO:64(Human XIAP)

[0406] CTTGTAAAAACAACTTTGATGCCTTGAATATATAATGATTCATTATAACAATTATGCATAGATTTTAATAATCTGCATATTTTATGCTTTCATGTTTTTCCTAATTAATGATTTGACATGGTTAATAATTATAATATATTCTGCATCACAGTTTACATATTTATGTAAAATAAGCATTTAAAAATTATTAGTTTTATTCTGCCTGCTTAAATATTACTTTCCTCAAAAAGAGAAAACAAAAATGCTAGATTTTACTTTATGACTTGAATGATGTGGTAATGTCGAACTCTAGTATTTAGAATTAGAATGTTTCTTAGCGGTCGTGTAGTTATTTTTATGTCATAAGTGGATAATTTGTTAGCTCCTATAACAAAAGTCTGTTGCTTGTGTTTCACATTTTGGATTTCCTAATATAATGTTCTCTTTTTAGAAAAGGTGGACAAGTCCTATTTTCAAGAGA

[0407] SEQ ID NO:65 (HRV-B3 IRES loop position sequence)

[0408] CCAAGTAAC

[0409] SEQ ID NO:66 (HRV-B3 IRES I)

[0410] TTAAAACAGCGGATGGGTACCCCACCATCCGACCCACTGGGTGTAGTACTCTGGTACTTCGTACCTTTGTACGCCTGTTCTTCCCATTGTACCCTTCCTGAACTTCCAACCCAAGT

[0411] SEQ ID NO:67 (HRV-B3 IRES II)

[0412] AACGTTAGAAGCTCAACATTTAGTACAACAGGAAGCACCACATCCAGTGGTGTTTAGTACAAGCACTTCTGTTTCCCCGGAGCGAGGTATAGGCTGTACCCACTGCCAAAAACCTTTAACCGTTATCCGCCAACCAACTACGTAAAAGCTAGTAGTATTATGTTTTTAACTAGGCGTTCGATCAGGTGGATTTCCCCTCCACTAGTTTGGTCGATGAGGCTAGGAATTCCCCACGGGTGACCGTGTCCTAGCCTGCGTGGCGGCCAACCCAGCTTATGCTGGGACGCCTTTTTATAGACATGGTGTGAAGACTCGCATGTGCTTGGTTGTGATTCCTCCGGCCCCTGAATGCGGCTAACCTTAACCCTGGAGCCTTGTGTCACAAACCAGTGATGATAAGGTCGTAATGAGCAATTCCGGGACGGGACCGACTACTTTGGGTGTCCGTGTTTCTTATTTTTCTTATTATTGTCTTATGGTCACAGCATATATATAACATATACTGTGATC

[0413] SEQ ID NO:68 (T4td ribozyme 3' intron)

[0414] TGGTTCTACATAAATGCCTAACGACTATCCCTTTGGGGAGTAGGGTCAAGTGACTCGAAACGATAGACAACTTGCTTTAACAAGTTGGAGATATAGTCTGCTCTGCATGGTGACATGCAGCTGGATATAATTCCGGGGTAAGATTAACGACCTTATCTGAACATAATG

[0415] SEQ ID NO:69 (Ana ribozyme 3' intron)

[0416] TGACTTACAACTAATCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCAAGAGAATG

[0417] SEQ ID NO:70 (T4td ribozyme 5' intron strict)

[0418] AAATTGTTGCTTGGGTATAAGGTGACTTATACTTGTAATCTATCTAAACGGGGAACCTCTCTAGTAGACAATCCCGTGCTAAATTGTAGGACTGCCCTTTAAT

[0419] SEQ ID NO:71 (T4td ribozyme 5' intron is not rigorous)

[0420] AAATTGTTGCTTGTGTATAAGGTGACTTATACTTGTAATCTATCTAAACGGGGAACCTCTCTAGTAGACAATCCCGTGCTAAATTGTAGGACTGCCCTTTAAT

[0421] SEQ ID NO:72 (Ana ribozyme 5' intron)

[0422] AAATAATTGTCCCTTAAAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTCAACAATAGATG

[0423] SEQ ID NO:73 (T4td ribozyme intron P1 and P10 region mutants - strict)

[0424] AAATTGTTGCTTGG

[0425] SEQ ID NO:74 (T4td ribozyme intron P1 and P10 region mutants are not rigorous)

[0426] AAATTGTTGCTTGT

[0427] SEQ ID NO:75 (Ana ribozyme intron P1 and P10 region mutant)

[0428] AAATAATTGCT

[0429] SEQ ID NO:76(P1 rigorous complement 44-7.13, T4td ribozyme 3' intron-HRV-B3 IRES II-antigen peptide ORF-HRV-B3 IRES I-T4td ribozyme 5' intron)

[0430]

[0431] SEQ ID NO:77(P1 non-rigid complement 44-7.12, T4td ribozyme 3' intron-HRV-B3 IRES II-antigen peptide ORF-HRV-B3 IRES I-T4td ribozyme 5' intron)

[0432]

[0433] SEQ ID NO:78(Ana 44-7.11, Ana ribozyme 3' intron-HRV-B3 IRES II-antigen peptide ORF-HRV-B3IRES I-Ana ribozyme 5' intron)

[0434]

[0435] SEQ ID NO:79(T7 Promoter)

[0436] TAATACGACTCACTATAGGG

[0437] SEQ ID NO:80(HRV-B3-eIF4G IRES)

[0438] TTAAAACAGCGGATGGGTACCCCACCATCCGACCCACTGGGTGTAGTACTCTGGTACTTCGTACCTTTGTACGCCTGTTCTTCCCATTGTACCCTTCCTGAACTTCCAACCCAAGTAACGTTAGAAGCTCAACATTTAGTACAACAGGAAGCACCACATCCAGTGGTGTTTAGTACAAGCACTTCTGTTTCCCCGGAGCGAGGTATAGGCTGTACCCACTGCCAAAAACCTTTAACCGTTATCCGCCAACCAACTACGTAAAAGCTAGTAGTATTATGTTTTTAACTAGGCGTTCGATCAGGTGGATTTCCCCTCCACTAGTTTGGTCGATGAGGCTAGGAATTCCCCACGGGTGACCGTGTCCTAGCCTGCGTGGCGGCCAACCCAGCCCACTCACTATTTGTTTTCGCGCCCAGTTGCAAAAAGTGTCGGGGCTGGGACGCCTTTTTATAGACATGGTGTGAAGACTCGCATGTGCTTGGTTGTGATTCCTCCGGCCCCTGAATGCGGCTAACCTTAACCCTGGAGCCTTGTGTCACAAACCAGTGATGATAAGGTCGTAATGAGCAATTCCGGGACGGGACCGACTACTTTGGGTGTCCGTGTTTCTTATTTTTCTTATTATTGTCTTATGGTCACAGCATATATATAACATATACTGTGATC

[0439] SEQ ID NO:81(HRV-B3-eIF4G IRES II)

[0440] AACGTTAGAAGCTCAACATTTAGTACAACAGGAAGCACCACATCCAGTGGTGTTTAGTACAAGCACTCTGTTTCCCCGGAGCGAGGTATAGCTGTACCCACTGCCAAAACTTTAACCGTTATCCGCCAACCAAA CTACGTAAAAGCTAGTAGTATTATGTTTTTAACTAGGCGTTCGATCAGGTGGATTTCCCCTCCACTAGTTTGGTCGATGAGCTAGGAATTCCCCACGGGTGACCGTCCTAGCGTGCGTGGCCAACCCAGCCCC ACTCACTATTTGTTTTCGCGCCCAGTTGCAAAAAGTGTCGGGGCTGGGACGCCTTTTTATAGATGGTGTGAAGACTCGCATGTGCTTGGTTGTGATTCCTCCGGCCCCTGAATGCGGCTAACCTTAACCCTGGAG CCTTGTGTCACAAACCAGTGATGATAAGGTCGTAATGAGCAATTCCGGGACGGGACCGACTACTTTGGGTGTCCGTGTTTCTTATTTTTTTATTGTCTTATGGTCAGCATATATAACATATACTGTGATC

[0441] SEQ ID NO:82(HRV-B3-eIF4G IRES FLUC 44-7.10,T4td-HRV-B3-eIF4GIRES II-Fluc ORF-HRV-B3-eIF4G IRES I-T4td-Fluc)

[0442]

[0443] SEQ ID NO:83(44-7.14HRVB3 FLUC Ana, Ana ribozyme 3' intron-HRV-B3 IRES II-Fluc ORF-HRV-B3 IRES I-Ana ribozyme 5' intron)

[0444]

[0445] SEQ ID NO:84 (HRV-B4 IRES I)

[0446] TTAAAACAGCGGATGGGTTTCCCACCATCCGACCCACTGGGTGTAGTGCTCTGGTATTTTGTACCTTTGCACGCCTGTTTCCCATTTGTACCCTTCCTTAATCTCCTTCCCCCGT

[0447] SEQ ID NO:85 (HRV-B4 IRES II)

[0448] AACGTTAGAAGTTTTGGAATTTTAAAGTACAATAGGAAGCGCCACATCCAGTGGTGTTGCGTACAAATACTTCTGTTTACCCGGAGCGAGGTATAGGTTGTACCCACGGCCAAAAGCCTTTAACCGTTATCCGCCAATCAACTACGTAACGGCTAGTATCATCTTGCTTTTGATTTGGTGTTCGATCAGGTGGTATCCCCCACTAGTCTGGTCGATGAGGCTAGGAATTCCCCACGGGCGACCGTGTCCTAGCCTGCGTGGCGGCCAGCCCAGCTTTTGCTGGGACGCCTTTTCAAAGACATGGTGTGAAGACCTGCATGTGCTTGGTTGTGAGTCCTCCGGCCCCTGAATGCGGCTAACCTTAACCCTGGAGCCCAGCAGCATAATCCAATGTTGTTTGGGTCGTAATGAGCAATTCCGGGACGGGACCGACTACTTTGGGTGTCCGTGTTTCCTTTTATTCTTACATTGTCTTATGGTCACAGCATATATATTATATATACTGTGATC

[0449] SEQ ID NO:86 (5' intron of Ana ribozyme)

[0450] AAATAATTGCGCCTTAAAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTCAACAATAGATG

[0451] SEQ ID NO:87 (Ana ribozyme HRVB4 polypeptide)

[0452]

[0453] SEQ ID NO:88 (Human XIAP IRES I)

[0454] CTTGTAAAAACAACTTTGATGCCTTGAATATATAATGATTCATTATAACAATTATGCATAGATTTTAATAATCTGCATATTTTATGCTTTCATGTTTTTCCTAATT

[0455] SEQ ID NO:89 (Human XIAP IRES II)

[0456] AATGATTTGACATGGTTAATAATTATAATATATTCTGCATCACAGTTTACATATTTATGTAAAATAAGCATTTAAAAATTATTAGTTTTATTCTGCCTGCTTAAATATTACTTTCCTCAAAAAGAGAAAACAAAAATGCTAGATTTTACTTTATGACTTGAATGATGTGGTAATGTCGAACTCTAGTATTTAGAATTAGAATGTTTCTTAGCGGTCGTGTAGTTATTTTTATGTCATAAGTGGATAATTTGTTAGCTCCTATAACAAAAGTCTGTTGCTTGTGTTTCACATTTTGGATTTCCTAATATAATGTTCTCTTTTTAGAAAAGGTGGACAAGTCCTATTTTCAAGAGA

[0457] SEQ ID NO:90 (5' intron mutant of Ana ribozyme)

[0458] AAATAATTGATCCTTAAAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTCAACAATAGATG

[0459] SEQ ID NO:91 (Ana ribozyme XIAP polypeptide)

[0460]

[0461] SEQ ID NO:92 (HRV - B97 IRES I)

[0462] TTAAAACAGCGGATGGGTTTCCCACCATCCGACCCACTGGGTGTAGTGCTCTGGTATTTTGTACCTTTGCACGCCTGTTTCCCCTTTGTACCCATCCTGAATTTCCTCCCTCTGCAACGTT

[0463] SEQ ID NO:93 (HRV - B97 IRES II)

[0464] AGAAGTTTGTGAAATTAAAAGTACAATAGGAAGCATCACATCCAGTGGTGTTCAGTACAAGCACTCCTGTTTCCCCGGAGCGAGGTATAGGTTGTACCCACGACCGAAAGCCTTTAACCGTTATCCGCCAATCAACTACATAACGGCTAGTATCATCATGTTTTTGATCTGGCGTTCGATCAGGTGGTTTCCCCCACTAGTCTGGTCGATGAGGCTAGGATTTCCCCACGGGCGACCGTGTCCTAGCCTGCGTGGCGGCCAGCCCAGCTTATGCTGGGACGCCTTTTTAAAGACATGGTGTGAAGACCTGCATGTGCTTGATTGTGAGTCCTCCGGCCCCTGAATGCGGCTAACCTTAACCCTGGAGCCCGACAGCATAATCCAATGTTGTTTGGGTCGTAATGAGCAATTCCGGGATGGGACCGACTACTTTGGGTGTCCGTGTTTCTTTTTATTCTTATATTGTCTTATGGTCACAGCATATATAGTATATATACTGTGATC

[0465] SEQ ID NO:94 (5' intron mutant of Ana ribozyme)

[0466] AGATAACTGACCCTTAAAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTCAACAATAGATG

[0467] SEQ ID NO:95(HRV-B97 Ana peptide)

[0468]

[0469] SEQ ID NO:96 (ShuffledIRES#42IRES I)

[0470] TTAAAACAGTGGATGGGTTTCCTACCATCCGACCCACTGGGTGTAGTGCTCTGGTATATTGTACCTTTGCACGCCTGTTTTCCCATTGTACCCTTCCTTAAATTCCTCCCCATGT

[0471] SEQ ID NO:97 (ShuffledIRES#42IRES II)

[0472] AACGTTAGAAGTTTAAGAACATAAATGTACAATAGGAAGCATCACATCCAGTGGTGTATGGTACAAGCACTTCTGTTTATCCGGAGCGAGGTATAAGCGGTACCCACTGCTGAAAGCCTTTAACCGTTACCCGCCAGCCAACTACGTAAAAGCTAGTAACATCTTGTTTGTAACTTGGCGTTCGATCAGGTGGATTCCCCCTCCACTAGTTTGGTCGATGAGGCTAGGAACTCCCCACGGGTGACCGTGTCCTAGCCTGCGTGGCGGCCAACCCAGCTCTTGCTGGGACGCCCTTTTATAGACATGGTGTGAAGACTCGCATGTGCTTGGTTGTGATTCCTCCGGCCCCTGAATGCGGCTAACCCTAACCCCGGAGCCGTTGCCCATAATCCAATGGGTTTGCGGTCGTAATGGGCAACTCTGGGATGGGACCAACTACTTTGGGTGTCCGTGTTTCCTGTTTTTCTTTAAAATGTCTTATGGTCACAGCATATAGTAATTATATACTGTGATC

[0473] SEQ ID NO:98 (5' intron mutant of Ana ribozyme)

[0474] AAATAATTGCACCTTAAAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTCAACAATAGATG

[0475] SEQ ID NO:99 (Ana ribozyme ShuffledIRES#42 IRES polypeptide)

[0476]

[0477] SEQ ID NO:100(HRV-B37 IRES I)

[0478] TTTAAACAGCGGATGGGTATCCCACCATCCGACCCACTGGGTGTAGTACTCTGGTATTTTGTACCTTTGTACGCCTGTTGTTCCTAATGTACCCACCCTAAAACTTCCTACCCAAGT

[0479] SEQ ID NO:101(HRV-B37 IRES II)

[0480] AACGTTAGAAGTTTCATCAACAAGTACAATAGGAAGCATCACATCCAGTGGTGTTTTGTACAAGCACTTCTGTTTCCCCGGAGCGAGGTATAGGCTGTACCCACTGCCGAAAGCCTTTAACCGTTATCCGCCAACCAACTACGTAAAAGCTAGTATCATCATGTTTTAAAATAGGCGTTCGATCAGGTGGTACCCCCCTCCACTAGTTTGGTCGATGAGGCTAGGAACTCCCCACGGGTGACCGTGTCCTAGCCTGCGTGGCGGCCAACCCAGCTTCTGCTGGGACGCCTTTTTATGGACATGGTGTGAAGACTCGCATGTGCTTGGTTGTGACTCCTCCGGCCCCTGAATGCGGCTAACCTTAACCCCGGAGCCCTGTGTTGCAATCCAGTAACATTAGGGTCGTAATGAGCAATTTCGGGACGGGACCGACTACTTTGGGTGTCCGTGTTTCTCATTTTTCTTATTATTGTCTTATGGTCACAGCATATATATAACGTATATACTGTGATC

[0481] SEQ ID NO:102(HRV-B92 IRES I)

[0482] TTAAAACAGCGGATGGGTATCCCACCATCCGGCCCACTGGGTGTAGTACTCTGGTACATTGTACCTTTGTACGCCTGTTTTCCCCCTCTTGTACCCGCCCTTCAAGCTCCTTGCCCAAGT

[0483] SEQ ID NO:103(HRV-B92 IRES II)

[0484] AACGTTAGAAGTTTGAACATTGGTACAATAGGAAGCATCACATCCAGTGGTGTACTGTACAAACACTTCTGTTGCCCCGGAGCGAGGTATAGATGGTCCCCACCGTCAAAAGCCTTTAACCGTTATCCGCCAATCAACTACGTAATGGCTAGTAGCACCTTGGATTTAAGTTGGCGTTCGATCAGGTGGTAACCCCCACTAGTTTGGTCGATGAGGCTAGGAATTCCCCACGGGTGACCGTGTCCTAGCCTGCGTGGCGGCCAACCCAGCATCCGCTGGGACGCCAATTTAATGACATGGTGTGAAGACCTGCATGTGCTTGATTGTGAGTCCTCCGGCCCCTGAATGCGGCTAACCCTAACCCCGGAGCCTTGCAGCACAATCCAGTGTTGTTAAGGTCGTAATGAGCAATTCTGGGATGGGACCGACTACTTTGGGTGTCCGTGTTTCTTATTTTTCTTGAATTTTTCTTATGGTCACAGCATATATACATTATATACTGTGATC

[0485] SEQ ID NO:104(shuffledIRES#01IRES I)

[0486] TTAAAACAGCGGATGGGTACCCCACCATCCGACCCACTGGGTGTAGTACTCTGGTATTTTGTACCTTTGTACGCCTGTTGTTCCTAATGTACCCACCCTAAAACTTCCAACCCAAGT

[0487] SEQ ID NO:105(shuffledIRES#01IRES II)

[0488] AACGTTAGAAGTTTGACATTAAAGTACAATAGGTTGCACTACATCCAGTGGTGTTTCGTACAAGCACTTCTGTTTCCCCGGAGCGGGGTATAGACGGTACCCACTGTCAAAAGCCCTTAACCGTTATCCGCCAATCAACTACGTAATGGCTAGTAACATCATGAAGGTGAGTTGACGTTCGATCAGGTGGTAACCCCCACTAGTTTGGTCGATGAGGCTAGGAATCCCCCACGGGTAACCGTGTCCTAGCCTGCGTGGCGGCCAACCCAGCGTTTGCTGGGACGCCAATTCACTGACATGGTGCGAATACTTGCATGTGCTTGATTGTGATTCCTCCGGCCCCTGAATGCGGCTAACCCTAACCCTGGAGCCTTGCACCACAATCCAGTGGTGTCTGGGTCGTAATGAGTAATTCTGGGATGGGACCGACTACTTTGGGTGTCCGTGTTTCTCATTTTTCTTATTATTGTCTTATGGTCACAGCATATAGTAATATATACTGTGATC

[0489] SEQ ID NO:106(shuffledIRES#38IRES I)

[0490] TTAAAACAGCGGATGGGTATCCCACCATCCGACCCACTGGGTGTAGTACTCTGGTACTTTGTACCTTTGTACGCCTGTTTTCCCCCTCTTGTACCCGCCCTTCAAGCTCCTTGCCCAAGT

[0491] SEQ ID NO:107(shuffledIRES#38IRES II)

[0492] AACGTTAGAAGTTTGAACATTGGTACAATAGGAAGCATCACATCCAGTGGTGTACTGTACAAACACTTCTGTTGCCCCGGAGCGAGGTATAGATGGTCCCCACCGTCAAAAGCCTTTAACCGTTATCCGCCAACCAACTACGAGACAGCTAGTAATGCCCTGTTCTCCTGCAAGGCGTTCGATCAGACAGATTTCCCCTCTGTTAGTCTGGTCGATGAGGCTAGGAGCTCCCCACGGGCGACCGTGTCCTAGCCTGCGTGGCGGCCAGCCCAGCTTTTGCTGGGACGCCTTTTCAAAGACATGGTGTGAAGACCTGCATGTGCTTGGTTGTGAGTCCTCCGGCCCCTGAATGCGGCTAACCTTAACCCTGGAGCCTTATGCCACGAACCAGTGGTTGTAAGGTCGTAATGAGTAATTCTGGGATGGGACCGACTACTTTGGGTGTCCGTGTTTCCTTTATTCTTATCATTTGTGTTTCATGGTTACAAGCATTGTTTGTAATC

[0493] SEQ ID NO:108 (B37 Ana polypeptide)

[0494]

[0495] SEQ ID NO:109(B92 Ana polypeptide)

[0496]

[0497] SEQ ID NO:110(S01 Ana polypeptide)

[0498]

[0499] SEQ ID NO:111(S38 Ana polypeptide)

[0500]

[0501] SEQ ID NO:112 (CVB3 IRES)

[0502] TTAAAACAGCCTGTGGGTTGATCCCACCCACAGGCCCATTGGGCGCTAGCACTCTGGTATCACGGTACCTTTGTGCGCCTGTTTTATACCCCCTCCCCCAACTGTAACTTAGAAGTAACACACACCGATCAACAGTCAGCGTGGCACACCAGCCACGTTTTGATCAAGCACTTCTGTTACCCCGGACTGAGTATCAATAGACTGCTCACGCGGTTGAAGGAGAAAGCGTTCGTTATCCGGCCAACTACTTCGAAAAACCTAGTAACACCGTGGAAGTTGCAGAGTGTTTCGCTCAGCACTACCCCAGTGTAGATCAGGTCGATGAGTCACCGCATTCCCCACGGGCGACCGTGGCGGTGGCTGCGTTGGCGGCCTGCCCATGGGGAAACCCATGGGACGCTCTAATACAGACATGGTGCGAAGAGTCTATTGAGCTAGTTGGTAGTCCTCCGGCCCCTGAATGCGGCTAATCCTAACTGCGGAGCACACACCCTCAAGCCAGAGGGCAGTGTGTCGTAACGGGCAACTCTGCAGCGGAACCGACTACTTTGGGTGTCCGTGTTTCATTTTATTCCTATACTGGCTGCTTATGGTGACAATTGAGAGATCGTTACCATATAGCTATTGGATTGGCCATCCGGTGACTAATAGAGCTATTATATATCCCTTTGTTGGGTTTATACCACTTAGCTTGAAAGAGGTTAAAACATTACAATTCATTGTTAAGTTGAATACAGCAAA

[0503] SEQ ID NO:113 (S1 loop RNA precursor)

[0504]

[0505] SEQ ID NO:114 (S2 circular RNA precursor)

[0506]

[0507] SEQ ID NO:115 (S3 circular RNA precursor)

[0508]

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 intron mutant fragment (intron fragment II), b. Unit I fragment II, whose 5' end includes the II ribozyme recognition fragment. c. Functional units, e. The first unit fragment I, whose 3' end includes the I-th ribozyme recognition fragment. f.5'I group intron mutant fragment (intron fragment I); The functional units include IRES, RNA aptamers, protein-binding sequences, protein-coding regions, non-coding regions, etc., or combinations thereof; The intron fragment II is located at the 3' end of the intron fragment I, that is, the complete group I intron mutant sequence includes: 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 bond structure, or a local hairpin structure, wherein the local stem structure, local double bond structure, or local hairpin structure is adjacent to or includes the cyclic 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 recognizes the region of the original fragment in the first unit 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', 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’ 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 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, 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: 53, 54, 55, 56, 57, 58, 60, 62, 64 and 80; Preferably, the nucleotide sequence of the translation initiation element fragment I is as shown in SEQ ID NO:66, and the nucleotide sequence of the translation initiation element fragment II is as shown in SEQ ID NO:67; Preferably, the nucleotide sequence of the translation initiation element fragment I is shown in SEQ ID NO:92, and the nucleotide sequence of the translation initiation element fragment II is shown in SEQ ID NO:93; Preferably, the nucleotide sequence of the translation initiation element fragment I is shown in SEQ ID NO:84, and the nucleotide sequence of the translation initiation element fragment II is shown in SEQ ID NO:85; Preferably, the nucleotide sequence of the translation initiation element fragment I is shown in SEQ ID NO:96, and the nucleotide sequence of the translation initiation element fragment II is shown in SEQ ID NO:97; Preferably, the nucleotide sequence of the translation initiation element fragment I is shown in SEQ ID NO:88, and the nucleotide sequence of the translation initiation element fragment II is shown in SEQ ID NO:89; Preferably, the nucleotide sequence of the translation initiation element fragment I is shown in SEQ ID NO:66, and the nucleotide sequence of the translation initiation element fragment II is shown in SEQ ID NO:

81. Preferably, the nucleotide sequence of the translation initiation element fragment I is shown in SEQ ID NO:100, and the nucleotide sequence of the translation initiation element fragment II is shown in SEQ ID NO:

101. 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:104, and the nucleotide sequence of the translation initiation element fragment II is shown in SEQ ID NO:

105. 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.

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: 70, 71, 72, 86, 90, 94 and 98, and the nucleotide sequence of intron fragment II is shown in SEQ ID NO: 68 or 69; Preferably, the nucleotide sequence of intron fragment I is as shown in 70 or 71, and the nucleotide sequence of intron fragment II is as shown in SEQ ID NO:68; Preferably, the nucleotide sequence of intron fragment I is shown in any one of 72, 86, 90, 94 and 98, and the nucleotide sequence of intron fragment II is shown in SEQ ID NO:

69.

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, 2A peptide, 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: 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.

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 truncated fragment II, the coding element, the translation initiation element truncated 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: 76, 77, 78, 82, 83, 87, 91, 95, 99, 108, 109, 110 and 111.

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 promoters at both ends 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:

79.

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 prepared by the method according to claim 16.

18. A host cell expressing the recombinant nucleic acid molecule of any one of claims 1-14, the recombinant expression vector of claim 15, 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, the method targeting the T4td ribozyme intron, 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 unit sequence is linked to the 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; 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, the loop position sequence being N1N2N3N4N5TN6N7 or N1N2N3N4N5UN6N7, N1-N7 being A, G, C, U, T or absent, and the intron ribozyme in group I being 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’ When N6 and N7 are complementary, and N6N7 are 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 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 a first unit sequence, wherein the method targets an Ana ribozyme intron, 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 is screened. The loop position contains at least 6 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 3 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 3' group I intron. (4) The 5' end of the functional unit sequence is linked to the 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) preferably 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.

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