A nucleic acid construct for circular RNA expression and its application in protein expression

Abstract

The present disclosure relates to a nucleic acid construct for circular RNA expression and its application in protein expression. The nucleic acid construct includes the following structures from 5' to 3' end: 3' intron, exon E2, 5' spacer, IRES element, target gene, 3' spacer, exon E1, 5' intron. Through a specific combination of elements, it has the effect of promoting circular RNA ring formation and improving the ring formation rate, and can also achieve protein expression of the target gene.

Description

Technical Field

[0001] The present invention belongs to the field of molecular biology and bioengineering technology, and specifically relates to a nucleic acid construct for circular RNA expression and its application in protein expression. Background Art

[0002] Circular RNA (circRNA) is a type of covalently closed single-stranded RNA molecule that is produced by alternative splicing of mRNA precursors and has no 5' end mRNA. 7 G cap and 3' end Poly A tail structure. The structure of circular RNA is not affected by nucleases, and is more stable in vivo than linear RNA molecules and is not easily degraded. Due to the low abundance of circular RNA expression and the limitations of detection technology, it was considered to be a non-functional byproduct of abnormal RNA splicing in the early days. With the development of high-throughput sequencing technology, a large number of RNAs in circular conformations have been discovered in eukaryotes. Circular RNA can regulate the gene expression function of host cells and is closely related to the pathogenesis of many human diseases. Studies have shown that it plays an important role in the occurrence of cancer, cardiovascular disease, neurological disease and autoimmune diseases. Compared with linear RNA, circular RNA has stronger stability. As a new type of nucleic acid drug treatment tool, it has great development prospects in the field of gene therapy.

[0003] Currently, circular RNA is prepared in vitro mainly through chemical methods, T4 ligase or ribozyme catalysis.

[0004] The chemical method uses condensation reagents such as cyanogen bromide (BrCN) or 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) to connect the two ends of the RNA chain into a ring, but the phosphate migration will lead to the formation of unnatural 2'-5' phosphate bonds, which is only suitable for the connection of small fragments of circular RNA. In addition, the chemical method requires the introduction of certain environmentally harmful and explosive chemical reagents. [1] .

[0005] The ligase method uses nucleic acid ligase (T4 DNA ligase, T4 RNA ligase I or T4 RNA ligase II) to catalyze the formation of new phosphodiester bonds between the 5' and 3' ends of linear RNA to obtain circular RNA molecules. The T4 ligase circularization method is an early application method, but it has problems such as low preparation efficiency, difficult purification process, easy generation of polymer byproducts, and difficulty in obtaining circular RNA with precise sequence. [2] .

[0006] The ribozyme method for RNA cyclization mainly utilizes the self-splicing of type I introns or type II introns that have the ability to specifically recognize and cleave RNA, catalyzing the circularization of linear RNA by cutting the phosphodiester bond of the substrate RNA, which is called the permuted intron-exon self-splicing system (PIE system). [3-5] The current main circularization strategy is to catalyze RNA circularization by self-cleavage of type I introns. Patent CN113278635B shows that different combinations of sequence elements will affect RNA circularization. It is necessary to perform combinatorial screening of sequence elements to further guide the construction and expression of RNA circularization vectors.

[0007] The circular RNA overexpression circularization vector DNA sequence disclosed in the prior art can realize the circularization expression of circular RNA. Although it can achieve the purpose of circularization expression, its circularization efficiency is relatively low. In order to meet the industrial production needs of in vitro protein expression, it is still necessary to develop a circular RNA expression vector with higher circularization efficiency and better protein expression persistence.

[0008] 1.Prisca Obi,et al.The design and synthesis of circular RNAs.Methods2021,196,85-103.

[0009] 2.Xinjie Chen, et al. Circular RNA: Biosynthesis in vitro. Front. Bioeng. Biotechnol. 2021, 9, 787881.

[0010] 3. R. Alexander Wesselhoeft, et al. RNA circularization diminishes immunogenicity and can extend translation duration in vivo. Molecular Cell2019,74,508-520.

[0011] 4. Liang Qu, et al. Circular RNA vaccines against SARS-CoV-2 and emerging variants. Cell 2022, 185, 1728-1744.

[0012] 5. Guangzhou Aiji Biotechnology Co., Ltd. A sequence combination for promoting circular RNA ring formation and its application [P]. Chinese patent: CN113278635B, 2023-05-19. Summary of the invention

[0013] In view of the deficiencies in the prior art, it is still necessary to develop an in vivo circular RNA expression vector with high cyclization efficiency and good expression persistence. To this end, the present disclosure mainly provides a nucleic acid construct for circular RNA expression and its application in protein expression. The nucleic acid construct for circular RNA expression includes the following structures from 5' to 3' end in sequence: 3' intron, exon E2, 5' spacer, internal ribosome entry site element (IRES element), target gene, 3' spacer, exon E1, 5' intron. It has the effect of promoting circular RNA into a ring and improving the ring rate through a specific combination of elements. At the same time, the circular RNA molecule formed by transcription contains a specific combination element, which has the effect of promoting circular RNA into a ring, and can achieve continuous and efficient expression of target protein in eukaryotic cells.

[0014] In one aspect, the present disclosure provides a nucleic acid construct for circular RNA expression, wherein the structure from 5' to 3' end includes: 3' intron, exon E2, 5' spacer, IRES element, target gene, 3' spacer, exon E1, 5' intron.

[0015] In some embodiments, the size of the 5' spacer region is 20 to 200 bp.

[0016] In some embodiments, the size of the 3' spacer region is 20 to 200 bp.

[0017] In some embodiments, the IRES element is selected from one of the following:

[0018] IRES IRES element content SEQ ID NO: HRV-B3 IRES element of rhinovirus B3 SEQ ID NO:6 EMCV IRES element of encephalomyocarditis virus SEQ ID NO:7 HCV Hepatitis C virus IRES element SEQ ID NO:8 HRV-B17 IRES element of rhinovirus B17 SEQ ID NO:9 HRV-A1 IRES element of rhinovirus A1 SEQ ID NO:10

[0019] In some embodiments, the IRES element is EMCV or HRV-B3. In some embodiments, the IRES element is EMCV.

[0020] In some embodiments, the 5' spacer is selected from one of the following:

[0021] 5' spacer 5' spacer content SEQ ID NO: HBA2 PR Sequence of hemoglobin α2 bound to the α complex SEQ ID NO:11 HBA2 PR M1 HBA2 PR mutation sequence: G8A, G19A, G21A, T28A SEQ ID NO:12 HBA2 PR M2 HBA2 PR mutation sequence: C3A, G8A, T25A, T33A SEQ ID NO:13 HBA2 PR M3 HBA2 PR mutation sequence: T6A, G19A, T28A, T33A SEQ ID NO:14

[0022] In some embodiments, the 5' spacer is HBA2 PR M2 or HBA2 PR M3. In some embodiments, the 5' spacer is HBA2 PR M2.

[0023] In some embodiments, the 3' spacer is selected from one of the following:

[0024] 3' spacer 3' spacer content SEQ ID NO: Human β-globin Human β-globin 3'UTR SEQ ID NO:15 Human β-globin M1 Human β-globin 3'UTR sequence: 19 nt truncated at the 3' end SEQ ID NO:16 Human β-globin M2 Human β-globin 3'UTR sequence: 5' end truncated 25nt SEQ ID NO:17

[0025] In some embodiments, the 3' spacer is human β-globin M2, human β-globin. In some embodiments, the 3' spacer is human β-globin M2.

[0026] In some embodiments, the 3' intron, exon E2, exon E1, and 5' intron are a PIE system.

[0027] In some embodiments, the PIE system is a T4 phage (Thymidylate Synthase of T4, T4td) PIE system and / or anabaena (Anabaena pre-tRNA) PIE system. In some embodiments, the PIE system is an Anabaena pre-tRNA PIE system.

[0028] In some embodiments, the components of the T4 td PIE system are:

[0029]

[0030] In some embodiments, the components of the Anabaena pre-tRNA PIE system are:

[0031]

[0032] In some embodiments, any one of the following groups is included:

[0033] (1) The IRES element is EMCV and the 5' spacer is HBA2 PR M2;

[0034] (2) the IRES element is EMCV and the 5' spacer is HBA2 PR M3;

[0035] (3) the IRES element is HRV-B3, and the 5' spacer is HBA2 PR M2;

[0036] (4) the IRES element is EMCV, and the 3' spacer is human β-globin M2;

[0037] (5) The IRES element is HRV-B3, and the 5' spacer is HBA2 PR M3.

[0038] In some embodiments, any one of the following groups is included:

[0039] (1) The IRES element is EMCV; the 3' spacer is human β-globin M2; and the 5' spacer is HBA2 PR M2;

[0040] (2) the IRES element is EMCV; the 3' spacer is human β-globin M2; and the 5' spacer is HBA2 PR M3;

[0041] (3) the IRES element is HRV-B3; the 3' spacer is human β-globin M2; and the 5' spacer is HBA2 PR M2;

[0042] (4) the IRES element is HRV-B3; the 3' spacer is human β-globin M2; and the 5' spacer is HBA2 PR M3;

[0043] (5) The Anabaena pre-tRNA PIE system, the IRES element is HRV-B3; the 5' spacer is HBA2 PRM2;

[0044] (6) The Anabaena pre-tRNA PIE system, the IRES element is EMCV; the 5' spacer is HBA2 PR M2;

[0045] (7) In the Anabaena pre-tRNA PIE system, the IRES element is EMCV and the 5' spacer is HBA2 PR M3.

[0046] In some embodiments, any one of the following groups is included:

[0047] (1) The Anabaena pre-tRNA PIE system, wherein the 5' spacer is HBA2 PR M2; the IRES element is EMCV; and the 3' spacer is human β-globin M2;

[0048] (2) The Anabaena pre-tRNA PIE system, wherein the 5' spacer is HBA2 PR M3; the IRES element is EMCV; and the 3' spacer is human β-globin M2;

[0049] (3) The Anabaena pre-tRNA PIE system, wherein the 5' spacer is HBA2 PR M2; the IRES element is HRV-B3; and the 3' spacer is human β-globin M2;

[0050] (4) The Anabaena pre-tRNA PIE system, wherein the 5' spacer is HBA2 PR M3; the IRES element is HRV-B3; and the 3' spacer is human β-globin M2.

[0051] In some embodiments, the target gene includes a gene encoding a human protein or a non-human protein.

[0052] In some embodiments, the size of the target gene is 100 to 6000 bp.

[0053] In some embodiments, the target gene encodes a target polypeptide. In some embodiments, the target polypeptide is a therapeutic polypeptide. In some embodiments, the therapeutic polypeptide is selected from the group consisting of hormones, neurotransmitters, growth factors, enzymes, cytokines, antibodies, Fc fusion proteins, anticoagulants, blood factors, bone morphogenetic proteins, interferons, interleukins, and thrombolytic agents.

[0054] In some embodiments, the enzyme is selected from the group consisting of an oxidoreductase, a metabolic enzyme, a mitochondrial enzyme, an oxygenase, a dehydrogenase, an ATP-independent enzyme, a lysosomal enzyme, a desaturase.

[0055] In another aspect, the present disclosure provides use of a nucleic acid construct for circular RNA expression to prepare circular RNA in vitro.

[0056] In another aspect, the present disclosure provides a method for preparing circular RNA in vitro using a nucleic acid construct for circular RNA expression, comprising the following steps:

[0057] 1) Transcription step: using the nucleic acid construct to transcribe and form a linear nucleic acid molecule;

[0058] 2) Circularization step: The linear nucleic acid molecule undergoes a circularization reaction to obtain a circular RNA.

[0059] In another aspect, the present disclosure provides circular RNA prepared by the method.

[0060] In another aspect, the present disclosure provides a circular RNA, the structure of which from the 5' end to the 3' end sequentially comprises: an exon, a 5' spacer, an IRES element, a target gene and a 3' spacer.

[0061] In some embodiments, the structure of the circular RNA from 5' to 3' end includes: exon E2, 5' spacer, IRES element, target gene, 3' spacer and exon E1.

[0062] In some embodiments, the exon, 5' spacer, IRES element, gene of interest and 3' spacer are defined as described above.

[0063] In another aspect, the present disclosure provides use of the circular RNA in expressing a target polypeptide.

[0064] Beneficial Effects

[0065] The nucleic acid construct for circular RNA expression provided by the present disclosure includes the following structures from 5' to 3' end: 3' intron, exon E2, 5' spacer, IRES element, target gene, 3' spacer, exon E1, 5' intron. It has the effect of promoting circular RNA ring formation and improving the ring formation rate through a specific combination of elements. At the same time, the circular RNA molecule formed by transcription contains a specific combination element, which has the effect of promoting circular RNA ring formation, and can achieve continuous and efficient expression of target proteins in eukaryotic cells.

[0066] 1. Effects of each component

[0067] 1. The 3' / 5' introns, 5' spacer, and 3' spacer elements that make up the circular RNA are the same. The circularization efficiency of the circular RNA with EMCV as the IRES element is the highest, followed by HRBV3 and then HCV.

[0068] The IRES element circularization efficiency from high to low is: (1-2)EMCV > (1-1)HRV-B3 > (1-3)HCV>(1-5)HRV-A1>(1-4)HRV-B17.

[0069] The circular RNA composition includes 3' / 5'T4 td intron, 5'HBA2 PR spacer, 3'human β-globin spacer, and IRES. The circular RNA circularization efficiency of EMCV is 65.7%; the circularization efficiency of HRV-B3 is 60.3%; the circularization efficiency of HCV is 56.8%; the circularization efficiency of HRV-A1 is 52.1%; and the circularization efficiency of HRV-B17 is 47.5%.

[0070] 2. The 3' / 5' intron, IRES, and 3' spacer elements that make up the circular RNA are the same, and the combination with the 5' spacer sequence of HBA2PR M2 has the best circularization effect, reaching 74.6%; followed by HBA2 PR M3, reaching 69.8%; and then HBA2 PR.

[0071] The looping efficiency of the 5' spacer sequence is from high to low:

[0072] HBA2 PR M2>HBA2 PR M3>HBA2 PR>HBA2 PR M1.

[0073] In the combination of 3' / 5' intron being T4 td, 3' spacer being human β-globin, and IRES being EMCV, HRV-B3 or HCV, the circularization efficiency of circular RNA with 5' spacer sequence being HBA2 PR M2 was 61.5-74.6%; the circularization efficiency of HBA2 PR M3 was 58.7-69.8%; the circularization efficiency of HBA2 PR was 54.1-65.7%; and the circularization efficiency of HBA2 PR M1 was 50.9-56.3%.

[0074] 3. Among the circular RNA compositions, the 3' / 5'T4 td intron, the 5' spacer region is HBA2 PR M2 or HBA2 PR M3, the IRES is EMCV or HRV-B3, and the 3' spacer region is human β-globin M2, with the highest circularization efficiency reaching 79.7%, followed by human β-globin, reaching 74.6%.

[0075] The looping efficiency of 3' spacer sequences from high to low is: human β-globin M2>human β-globin>human β-globin M1.

[0076] In the combination of 3' / 5' intron being T4 td, IRES being EMCV, HRV-B3 or HCV, and 5' spacer sequence being HBA2 PR M2, HBA2 PR M3 or HBA2 PR, the circularization efficiency of circular RNA with 3' spacer being human β-globin M2 was 67.8-79.9%; the circularization efficiency of human β-globin was 64.7-74.6%; and the circularization efficiency of human β-globin M1 was 59.4-64.8%.

[0077] 4. Among the circular RNA compositions, the circular RNA with 5' spacer of HBA2 PR M2 or HBA2 PR M3, IRES of EMCV or HRV-B3, 3' spacer of human β-globin M2 or human β-globin, and 3' / 5' intron of Anabaena pre-tRNA had a higher circularization efficiency than T4 td, reaching 83.5%.

[0078] The circularization efficiency of 3' / 5' intron sequences is from high to low: Anabaena pre-tRNA >T4 td.

[0079] In the combination where the 5' spacer sequence is HBA2 PR M2 or HBA2 PR M3, IRES is EMCV or HRV-B3, and the 3' spacer is human β-globin M2 or human β-globin, the circularization efficiency of circular RNA with 3' / 5' intron being Anabaena pre-tRNA is 69.7-83.5%; the circularization efficiency of T4 td is 67.8-79.7%.

[0080] 2. Unit Components

[0081] The circularization efficiency was 55.8-65.7% when any one of the 3' / 5' introns (Anabaena pre-tRNA), 5' spacer (HBA2 PR M2 or HBA2 PR M3), IRES (EMCV or HRV-B3) and 3' spacer (human β-globin M2) constituting the circular RNA was selected. Among them, the circularization efficiency of EMCV was the highest.

[0082] Compared with the circular RNA cyclization efficiency not containing the above elements, the circularization efficiency is increased by 8.9-18.2%, and the improvement effect is significant.

[0083] 3. Dual Components

[0084] The circularization efficiency of any two elements selected from the 3' / 5' intron (Anabaena pre-tRNA), 5' spacer (HBA2 PR M2 or HBA2 PR M3), IRES (EMCV or HRV-B3) and 3' spacer (human β-globin M2) constituting the circular RNA was 61.7-74.6%. Among them, the combination of HBA2 PR M2 and EMCV had the highest circularization efficiency.

[0085] Compared with the circular RNA cyclization efficiency not containing the above elements, the cyclization efficiency is increased by 17.8-27.1%, and the improvement effect is significant.

[0086] Four, three components

[0087] The circularization efficiency of any three elements selected from the 3' / 5' intron (Anabaena pre-tRNA), 5' spacer (HBA2 PR M2 or HBA2 PR M3), IRES (EMCV or HRV-B3) and 3' spacer (human β-globin M2) constituting the circular RNA was 69.3-79.7%. Among them, the combination of HBA2 PR M2, EMCV and human β-globin M2 had the highest circularization efficiency.

[0088] Compared with the circularization efficiency of circular RNA not containing the above elements, the circularization efficiency is increased by 22.9-32.2%, which is a significant improvement.

[0089] Five, four components

[0090] The 4 elements of the 3' / 5' intron (Anabaena pre-tRNA), 5' spacer (HBA2 PR M2 or HBA2 PR M3), IRES (EMCV or HRV-B3) and 3' spacer (human β-globin M2) that make up the circular RNA were selected, and the circularization efficiency was 71.4-83.5%. Among them, the combination of Anabaena pre-tRNA PIE system, HBA2 PR M2, EMCV and human β-globin M2 had the highest circularization efficiency of 83.5%.

[0091] Compared with the circularization efficiency of circular RNA not containing the above elements, the circularization efficiency is increased by 26.7-36%, which is the best improvement.

[0092] VI. Protein Expression

[0093] The nucleic acid construct for expressing circular RNA disclosed in the present invention comprises a circular RNA molecule formed by transcription, which has the function of promoting the circularization of circular RNA and can realize the continuous and efficient expression of target protein in eukaryotic cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0094] Figure 1 Shown are schematic diagrams of the construction of DNA vectors, linear RNA, and circular RNA;

[0095] Figure 2 It was identified by T4 td intron combined circular RNA sequencing;

[0096] Figure 3 It is a method for identifying circular RNA of T4 td intron combination SEQ ID NO:48; wherein, Lane 1: linear RNA-linear primer control; Lane 2: linear RNA-circular primer; Lane 3: circular RNA-linear primer control; Lane 4: circular RNA-circular primer;

[0097] Figure 4 Identification of Anabaena pre-tRNA intron combination circular RNA sequencing;

[0098] Figure 5 Identification of circular RNA of Anabaena pre-tRNA intron combination SEQ ID NO:55; wherein, Lane 1: linear RNA-linear primer control; Lane 2: linear RNA-circular primer; Lane 3: circular RNA-linear primer control; Lane 4: circular RNA-circular primer;

[0099] Figure 6 Expression results of NanoLuc circular RNA;

[0100] Figure 7 Expression results of EGFP circular RNA; A and B: linear EGFP mRNA expression; C and D: circular EGFP mRNA expression; scale bar is 100 μm. DETAILED DESCRIPTION

[0101] The present disclosure is further described below by way of examples. It should be understood that the examples of the present disclosure are only provided for illustrating the present disclosure, rather than limiting the present disclosure, and simple improvements to the present disclosure based on the technical solution of the present disclosure all fall within the protection scope of the present disclosure.

[0102] Main reagents

[0103] The experimental techniques and methods used in this example are conventional techniques unless otherwise specified, usually according to conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. The materials, reagents, etc. used in the examples, unless otherwise specified, can be obtained through regular commercial channels.

[0104] The DNA vector for constructing circular RNA disclosed in the present invention comprises: T7 promoter (SEQ ID NO: 1), 3' intron and exon E2, 5' spacer, IRES element, Nano Luciferase (NanoLuc) luciferase gene (i.e. coding region gene, target gene), 3' spacer, exon E1 sequence and 5' intron.

[0105] The circular RNA designed in the present disclosure includes: exon E2, 5' spacer, IRES element, Nano Luciferase (NanoLuc) luciferase gene (ie, coding region gene, target gene), 3' spacer, exon E1.

[0106] The schematic diagram of the composition of the expression vector construction is as follows Figure 1 shown.

[0107] Sequence Description:

[0108] The sequence shown in SEQ ID NO: 1 is the nucleotide sequence of T7 promoter;

[0109] The sequence shown in SEQ ID NO:2 is the nucleotide sequence of the 3' intron of the T4 td PIE system;

[0110] The sequence shown in SEQ ID NO:3 is the nucleotide sequence of exon E2 of the T4 td PIE system;

[0111] The sequence shown in SEQ ID NO:4 is the nucleotide sequence of exon E1 of the T4 td PIE system;

[0112] The sequence shown in SEQ ID NO:5 is the nucleotide sequence of the 5' intron of the T4 td PIE system;

[0113] The sequence shown in SEQ ID NO:6 is the nucleotide sequence of HRV-B3 IRES;

[0114] The sequence shown in SEQ ID NO:7 is the nucleotide sequence of EMCV IRES;

[0115] The sequence shown in SEQ ID NO:8 is the nucleotide sequence of HCV IRES;

[0116] The sequence shown in SEQ ID NO:9 is the nucleotide sequence of HRV-B17 IRES;

[0117] The sequence shown in SEQ ID NO: 10 is the nucleotide sequence of HRV-A1 IRES;

[0118] The sequence shown in SEQ ID NO: 11 is the nucleotide sequence of the 5' spacer region HBA2 PR;

[0119] The sequence shown in SEQ ID NO: 12 is the nucleotide sequence of the 5' spacer HBA2 PR M1;

[0120] The sequence shown in SEQ ID NO: 13 is the nucleotide sequence of the 5' spacer HBA2 PR M2;

[0121] The sequence shown in SEQ ID NO: 14 is the nucleotide sequence of the 5' spacer HBA2 PR M3;

[0122] The sequence shown in SEQ ID NO: 15 is the nucleotide sequence of the 3' spacer region of human β-globin;

[0123] The sequence shown in SEQ ID NO: 16 is the nucleotide sequence of the 3' spacer region of human β-globin M1;

[0124] The sequence shown in SEQ ID NO: 17 is the nucleotide sequence of the 3' spacer region of human β-globin M2;

[0125] The sequence shown in SEQ ID NO: 18 is the nucleotide sequence of the 3' intron of the Anabaena pre-tRNA PIE system;

[0126] The sequence shown in SEQ ID NO:19 is the nucleotide sequence of exon E2 of the Anabaena pre-tRNA PIE system;

[0127] The sequence shown in SEQ ID NO:20 is the nucleotide sequence of exon E1 of the Anabaena pre-tRNA PIE system;

[0128] The sequence shown in SEQ ID NO:21 is the nucleotide sequence of the 5' intron of the Anabaena pre-tRNA PIE system;

[0129] The sequence order shown in SEQ ID NO:22 is a linear RNA sequence combination of Anabaena pre-tRNA PIE system 3' intron, Anabaena pre-tRNA PIE system exon E2, 5' spacer HBA2 PR M2, IRES element EMCV, coding region gene NanoLuc, 3' spacer human β-globin M2, Anabaena pre-tRNA PIE system exon E1, and Anabaena PIE system 5' intron;

[0130] The sequence shown in SEQ ID NO:23 is the nucleotide sequence of NanoLuc encoding DNA;

[0131] The sequence shown in SEQ ID NO:24 is the NanoLuc amino acid sequence;

[0132] The sequence shown in SEQ ID NO:25 is T4 td PIE system exon E2, 5' spacer HBA2

[0133] PR, IRES element HRV-B3, coding region gene NanoLuc, 3′ spacer human β-globin, circular RNA sequence combination of exon E1 of T4 td PIE system;

[0134] The sequence shown in SEQ ID NO:26 is T4 td PIE system exon E2, 5' spacer HBA2

[0135] PR, IRES element EMCV, coding region gene NanoLuc, 3′ spacer human β-globin, circular RNA sequence combination of exon E1 of T4 td PIE system;

[0136] The sequence shown in SEQ ID NO:27 is T4 td PIE system exon E2, 5' spacer HBA2

[0137] PR, IRES element HCV, coding region gene NanoLuc, 3′ spacer human β-globin, circular RNA sequence combination of exon E1 of T4 td PIE system;

[0138] The sequence shown in SEQ ID NO:28 is T4 td PIE system exon E2, 5' spacer HBA2

[0139] PR, IRES element HRV-B17, coding region gene NanoLuc, 3′ spacer human β-globin, circular RNA sequence combination of exon E1 of T4 td PIE system;

[0140] The sequence shown in SEQ ID NO:29 is T4 td PIE system exon E2, 5' spacer HBA2

[0141] PR, IRES element HRV-A1, coding region gene NanoLuc, 3′ spacer human β-globin, circular RNA sequence combination of exon E1 of T4 td PIE system;

[0142] The sequence shown in SEQ ID NO:30 is the forward primer nucleotide sequence for amplifying linear RNA;

[0143] The sequence shown in SEQ ID NO:31 is the reverse primer nucleotide sequence for amplifying linear RNA;

[0144] The sequence shown in SEQ ID NO:32 is the forward primer nucleotide sequence for amplifying circular RNA (T4 td);

[0145] The sequence shown in SEQ ID NO:33 is the reverse primer nucleotide sequence for amplifying circular RNA (T4 td);

[0146] The sequence shown in SEQ ID NO:34 is the reverse primer nucleotide sequence for amplifying circular RNA (Anabaena pre-tRNA);

[0147] The sequence shown in SEQ ID NO:35 is T4 td PIE system exon E2, 5' spacer HBA2

[0148] Combination of circular RNA sequences of PR M3, IRES element EMCV, coding region gene NanoLuc, 3' spacer region human β-globin, and exon E1 of T4 td PIE system;

[0149] The sequence shown in SEQ ID NO:36 is T4 td PIE system exon E2, 5' spacer HBA2

[0150] Combination of circular RNA sequences of PR M1, IRES element EMCV, coding region gene NanoLuc, 3' spacer region human β-globin, and exon E1 of T4 td PIE system;

[0151] The sequence shown in SEQ ID NO:37 is T4 td PIE system exon E2, 5' spacer HBA2

[0152] Combination of circular RNA sequences of PR M2, IRES element EMCV, coding region gene NanoLuc, 3' spacer human β-globin, and exon E1 of T4 td PIE system;

[0153] The sequence shown in SEQ ID NO:38 is T4 td PIE system exon E2, 5' spacer HBA2

[0154] Combination of circular RNA sequences of PR M3, IRES element HCV, coding region gene NanoLuc, 3' spacer human β-globin, and exon E1 of T4 td PIE system;

[0155] The sequence shown in SEQ ID NO:39 is T4 td PIE system exon E2, 5' spacer HBA2

[0156] Combination of circular RNA sequences of PR M1, IRES element HCV, coding region gene NanoLuc, 3' spacer human β-globin, and exon E1 of T4 td PIE system;

[0157] The sequence shown in SEQ ID NO:40 is T4 td PIE system exon E2, 5' spacer HBA2

[0158] Combination of PR M2, IRES element HCV, coding region gene NanoLuc, 3' spacer human β-globin, circular RNA sequence of exon E1 of T4 td PIE system;

[0159] The sequence shown in SEQ ID NO:41 is T4 td PIE system exon E2, 5' spacer HBA2

[0160] Combination of PR M3, IRES element HRV-B3, coding region gene NanoLuc, 3' spacer region human β-globin, and circular RNA sequence of exon E1 of T4 td PIE system;

[0161] The sequence shown in SEQ ID NO:42 is T4 td PIE system exon E2, 5' spacer HBA2

[0162] Combination of circular RNA sequences of PR M1, IRES element HRV-B3, coding region gene NanoLuc, 3' spacer region human β-globin, and exon E1 of T4 td PIE system;

[0163] The sequence shown in SEQ ID NO:43 is T4 td PIE system exon E2, 5' spacer HBA2

[0164] Combination of circular RNA sequences of PR M2, IRES element HRV-B3, coding region gene NanoLuc, 3' spacer region human β-globin, and exon E1 of T4 td PIE system;

[0165] The sequence shown in SEQ ID NO:44 is T4 td PIE system exon E2, 5' spacer HBA2

[0166] PR, IRES element EMCV, coding region gene NanoLuc, 3′ spacer human β-globin M2, circular RNA sequence combination of exon E1 of T4 td PIE system;

[0167] The sequence shown in SEQ ID NO:45 is T4 td PIE system exon E2, 5' spacer HBA2

[0168] Combination of circular RNA sequences of PR M3, IRES element EMCV, coding region gene NanoLuc, 3' spacer region human β-globin M1, and exon E1 of T4 td PIE system;

[0169] The sequence shown in SEQ ID NO:46 is T4 td PIE system exon E2, 5' spacer HBA2

[0170] Combination of circular RNA sequences of PR M3, IRES element EMCV, coding region gene NanoLuc, 3' spacer human β-globin M2, and exon E1 of T4 td PIE system;

[0171] The sequence shown in SEQ ID NO:47 is T4 td PIE system exon E2, 5' spacer HBA2

[0172] Combination of circular RNA sequences of PR M2, IRES element EMCV, coding region gene NanoLuc, 3' spacer human β-globin M1, and exon E1 of T4 td PIE system;

[0173] The sequence shown in SEQ ID NO:48 is T4 td PIE system exon E2, 5' spacer HBA2

[0174] Combination of circular RNA sequences of PR M2, IRES element EMCV, coding region gene NanoLuc, 3' spacer human β-globin M2, and exon E1 of T4 td PIE system;

[0175] The sequence shown in SEQ ID NO:49 is T4 td PIE system exon E2, 5' spacer HBA2

[0176] Combination of circular RNA sequences of PR M3, IRES element HRV-B3, coding region gene NanoLuc, 3' spacer region human β-globin M1, and exon E1 of T4 td PIE system;

[0177] The sequence shown in SEQ ID NO:50 is T4 td PIE system exon E2, 5' spacer HBA2

[0178] Combination of circular RNA sequences of PR M3, IRES element HRV-B3, coding region gene NanoLuc, 3' spacer human β-globin M2, and exon E1 of T4 td PIE system;

[0179] The sequence shown in SEQ ID NO:51 is T4 td PIE system exon E2, 5' spacer HBA2

[0180] Combination of circular RNA sequences of PR M2, IRES element HRV-B3, coding region gene NanoLuc, 3' spacer region human β-globin M1, and exon E1 of T4 td PIE system;

[0181] The sequence shown in SEQ ID NO:52 is T4 td PIE system exon E2, 5' spacer HBA2

[0182] Combination of circular RNA sequences of PR M2, IRES element HRV-B3, coding region gene NanoLuc, 3' spacer human β-globin M2, and exon E1 of T4 td PIE system;

[0183] The sequence shown in SEQ ID NO:53 is T4 td PIE system exon E2, 5' spacer HBA2

[0184] PR, IRES element EMCV, coding region gene NanoLuc, 3′ spacer human β-globin M1, circular RNA sequence combination of exon E1 of T4 td PIE system;

[0185] The sequence order shown in SEQ ID NO:54 is a circular RNA sequence combination of exon E2 of the Anabaena pre-tRNA PIE system, 5' spacer HBA2 PR M3, IRES element EMCV, coding region gene NanoLuc, 3' spacer human β-globin M2, and exon E1 of the Anabaena pre-tRNA PIE system;

[0186] The sequence order shown in SEQ ID NO:55 is a circular RNA sequence combination of exon E2 of the Anabaena pre-tRNA PIE system, 5' spacer HBA2 PR M2, IRES element EMCV, coding region gene NanoLuc, 3' spacer human β-globin M2, and exon E1 of the Anabaena pre-tRNA PIE system;

[0187] The sequence order shown in SEQ ID NO:56 is a combination of the circular RNA sequence of exon E2 of the Anabaena pre-tRNA PIE system, 5' spacer HBA2 PR M3, IRES element HRV B3, coding region gene NanoLuc, 3' spacer human β-globin M2, and exon E1 of the Anabaena pre-tRNA PIE system;

[0188] The sequence order shown in SEQ ID NO:57 is a combination of the circular RNA sequence of exon E2 of the Anabaena pre-tRNA PIE system, 5' spacer HBA2 PRM2, IRES element HRV B3, coding region gene NanoLuc, 3' spacer human β-globin M2, and exon E1 of the Anabaena pre-tRNA PIE system;

[0189] The sequence order shown in SEQ ID NO:58 is a combination of the circular RNA sequence of exon E2 of the Anabaena pre-tRNA PIE system, 5' spacer HBA2 PR M3, IRES element EMCV, coding region gene NanoLuc, 3' spacer human β-globin, and exon E1 of the Anabaena pre-tRNAPIE system;

[0190] The sequence order shown in SEQ ID NO:59 is a combination of the circular RNA sequence of exon E2 of the Anabaena pre-tRNA PIE system, 5' spacer HBA2 PR M2, IRES element EMCV, coding region gene NanoLuc, 3' spacer human β-globin, and exon E1 of the Anabaena pre-tRNA PIE system;

[0191] The sequence order shown in SEQ ID NO:60 is a combination of the circular RNA sequence of exon E2 of the Anabaena pre-tRNA PIE system, 5' spacer region HBA2 PR M2, IRES element HRV-B3, coding region gene NanoLuc, 3' spacer region human β-globin, and exon E1 of the Anabaena pre-tRNA PIE system;

[0192] The sequence shown in SEQ ID NO:61 is the nucleotide sequence of EGFP encoding DNA;

[0193] The sequence shown in SEQ ID NO:62 is the EGFP amino acid sequence;

[0194] The sequence order shown in SEQ ID NO:63 is a combination of the circular RNA sequence of exon E2 of the Anabaena pre-tRNA PIE system, 5' spacer HBA2 PR M2, IRES element EMCV, coding region gene EGFP, 3' spacer human β-globin M2, and exon E1 of the Anabaena pre-tRNA PIE system;

[0195] The nucleotide sequence of the Bsa I restriction site is GGAGACC; the nucleotide sequence of the Xba I restriction site is TCTAGA.

[0196] Circular RNA Preparation Method

[0197] 1-1 Plasmid construction

[0198] A sequence containing the NanoLuc luciferase reporter gene and different elements was constructed. This step was entrusted to Nanjing GenScript Biotechnology Co., Ltd. for gene synthesis and cloning. The gene fragment was cloned into the pVAX1 vector (purchased from Thermo Fisher Scientific, catalog number V26020) between base 664 and base 785 to obtain a successfully constructed circular plasmid and glycerol bacteria.

[0199] The elements for constructing the circular RNA DNA vector are as follows:

[0200] IRES element, selected from Table 1,

[0201] 5' spacer, selected from Table 2,

[0202] Target gene (coding region gene): Nano Luciferase (NanoLuc) luciferase gene (SEQ ID NO: 23);

[0203] 3' spacer, selected from Table 3,

[0204] The intron and exon are selected from the T4 td PIE system or the Anabaena pre-tRNA PIE system (PIE system, Permuted intron-exon self-splicing system) in Table 4.

[0205] Table 1 IRES elements

[0206] IRES IRES element content SEQ ID NO: HRV-B3 IRES element of rhinovirus B3 SEQ ID NO:6 EMCV IRES element of encephalomyocarditis virus SEQ ID NO:7 HCV Hepatitis C virus IRES element SEQ ID NO:8 HRV-B17 IRES element of rhinovirus B17 SEQ ID NO:9 HRV-A1 IRES element of rhinovirus A1 SEQ ID NO:10

[0207] Table 2 5' spacer sequence

[0208] 5' spacer 5' spacer content SEQ ID NO: HBA2 PR Sequence of hemoglobin α2 bound to the α complex SEQ ID NO:11 HBA2 PR M1 HBA2 PR mutation sequence: G8A, G19A, G21A, T28A SEQ ID NO:12 HBA2 PR M2 HBA2 PR mutation sequence: C3A, G8A, T25A, T33A SEQ ID NO:13 HBA2 PR M3 HBA2 PR mutation sequence: T6A, G19A, T28A, T33A SEQ ID NO:14

[0209] Table 3 3' spacer sequence

[0210] 3' spacer content SEQ ID NO: Human β-globin Human β-globin 3'UTR SEQ ID NO:15 Human β-globin M1 Human β-globin 3'UTR sequence: 19 nt truncated at the 3' end SEQ ID NO:16 Human β-globin M2 Human β-globin 3'UTR sequence: 5' end truncated 25nt SEQ ID NO:17

[0211] Table 4 PIE system

[0212]

[0213]

[0214] 1-2 Preparation of linearized plasmid

[0215] 1) Plasmid extraction

[0216] The glycerol bacteria were expanded and cultured at 37°C, 220 rpm, overnight in LB medium containing 50 mg / mL kanamycin.

[0217] Collect the overnight culture medium and use the endotoxin-free plasmid extraction kit (purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.) to extract the plasmid. The detailed steps are as follows:

[0218] a) Add 2.5 mL of equilibration solution to the adsorption column (the adsorption column is placed in a 50 mL collection tube), centrifuge at 8000 rpm for 2 min, pour out the waste liquid in the collection tube, and put the adsorption column back into the collection tube.

[0219] b) Take 100 mL of overnight cultured bacterial solution and add it to a centrifuge tube. Centrifuge at 8000 rpm for 3 min at room temperature to collect the bacteria and remove the supernatant as much as possible.

[0220] c) Add 8 mL of solution P1 and vortex to thoroughly suspend the precipitate.

[0221] d) Add 8 mL of solution P2 to the centrifuge tube, immediately and gently invert the tube upside down 8 times to fully lyse the bacteria, and leave it at room temperature for 5 min.

[0222] e) Add 8 mL of solution P4 to the centrifuge tube, and immediately and gently turn it upside down 6-8 times to mix thoroughly until white dispersed flocculent precipitates appear in the solution. Then leave it at room temperature for about 10 minutes. Centrifuge at 8000 rpm for 5-10 minutes to allow the white precipitate to centrifuge to the bottom of the tube. Pour all the solution carefully into the filter, slowly push the push handle to filter, and collect the filtrate in a clean 50 mL tube.

[0223] f) Add 0.3 times the volume of isopropanol to the filtrate, mix by inversion, and transfer to the adsorption column. Centrifuge at 8000 rpm for 2 min at room temperature, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube.

[0224] g) Add 10 mL of rinse solution to the adsorption column, centrifuge at 8000 rpm for 2 min, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube.

[0225] h) Repeat step g).

[0226] i) Add 3 mL of anhydrous ethanol to the adsorption column, centrifuge at 8000 rpm for 2 min at room temperature, and discard the waste liquid.

[0227] j) Place the adsorption column back into the collection tube and centrifuge at 8000 rpm for 5 min to remove the remaining rinse solution in the adsorption column.

[0228] k) Place the adsorption column in a clean 50mL collection tube, add 1mL of enzyme-free water to the middle of the adsorption film, place at room temperature for 5 minutes, and then centrifuge at 8000rpm for 2 minutes. Transfer all the eluate in the 50mL centrifuge tube to a clean 1.5mL centrifuge tube and store at -20℃.

[0229] 2) Plasmid digestion

[0230] The plasmid containing the HCV IRES sequence was digested with Xba I (purchased from New England Biotechnology (NEB)), and the remaining plasmids were digested with Bsa I (purchased from Nanjing Novozyme Biotechnology Co., Ltd.) to obtain linear plasmids.

[0231] The enzyme digestion system is as follows:

[0232] Table 5 Plasmid linearization restriction enzyme system

[0233] Reagents Mass / Volume Plasmids 10 μg Restriction enzymes 5μL 10× Buffer 20μL Enzyme-free water Make up to 200 μL

[0234] Enzyme digestion conditions: 37℃ reaction for 3h.

[0235] The digestion product was purified using a DNA column purification kit (purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.), and the detailed steps are as follows:

[0236] a) Add 500 μL of equilibration solution to the adsorption column, centrifuge at 12,000 rpm for 1 min, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube.

[0237] b) Add 5 times the volume of the enzyme digestion reaction solution and mix thoroughly.

[0238] c) Add the solution obtained in the previous step to the adsorption column and place it at room temperature for 2 minutes. Centrifuge at 12000 rpm for 1 minute, pour out the waste liquid in the collection tube, and place the adsorption column in the collection tube.

[0239] d) Add 600 μL of rinsing solution to the adsorption column, centrifuge at 12,000 rpm for 1 min, pour out the waste liquid in the collection tube, and place the adsorption column in the collection tube.

[0240] e) Repeat step d).

[0241] f) Place the centrifugal adsorption column back into the collection tube and centrifuge at 12000 rpm for 2 minutes to remove as much rinsing solution as possible. Place the adsorption column at room temperature for several minutes to dry thoroughly.

[0242] g) Take out the adsorption column and place it in a clean centrifuge tube. Add 30 μL of enzyme-free water to the middle of the adsorption membrane. Leave it at room temperature for 2 minutes. Centrifuge at 12,000 rpm for 2 minutes to collect the DNA solution.

[0243] 1-3 Preparation of linear mRNA by in vitro transcription

[0244] 1) In vitro transcription synthesis of mRNA

[0245] The linear plasmid obtained in 1-2 was used as a template to synthesize mRNA using a T7 in vitro transcription kit (T7 High Yield RNA Transcription Kit, purchased from Yisheng Biotechnology (Shanghai) Co., Ltd.). The specific reaction system is as follows:

[0246] Table 6 In vitro transcription reaction system

[0247] Reagents Mass / Volume 10× Reaction buffer 2.0μL ATP(100mM) 2.0μL CTP (100 mM) 2.0μL GTP (100 mM) 2.0μL UTP(100mM) 2.0μL Linearized DNA template 1.0 μg T7 RNA Polymerase Mix 2.0μL Enzyme-free water Make up to 20 μL

[0248] Use a pipette to gently mix the components, collect them by brief centrifugation, and incubate them at 37°C for 2 hours; then add 1.0 μL DNase I, mix well, and incubate at 37°C for 20 minutes to digest the linear DNA template.

[0249] 2) Linear mRNA purification

[0250] The transcription product obtained above was purified using lithium chloride precipitation method.

[0251] a) Add 7.5 M lithium chloride solution (final concentration 2.8 M) to the transcription product and incubate at -20°C for 2 h.

[0252] b) Centrifuge at 12000 rpm for 15 min, remove the supernatant, add 500 μL 70% ethanol for washing, centrifuge at 12000 rpm for 5 min, and repeat this step once.

[0253] c) Thoroughly remove the 70% ethanol solution and air dry, then add 100 μL of enzyme-free water to dissolve the mRNA.

[0254] d) Determination of mRNA concentration and integrity.

[0255] 1-4 Linear mRNA circularization

[0256] RNA cyclization principle: in Mg 2+ Under the action of GTP, the base junction between the 5' intron and exon E1 is broken, and the broken exon E1 will attack the base junction between the 3' intron and exon E2, causing the junction to break, the 3' intron to dissociate, and exon E1 and exon E2 to form a covalent connection to form a circular RNA.

[0257] 1) cyclization reagents: GTP buffer: 100 mM Tris-HCl, 20 mM MgCl2, 2 mM DTT, pH 7.5.

[0258] 2) Cyclization system and conditions

[0259] Table 7 Cyclization system

[0260] Reagents volume mRNA 20 μg GTP (100 mM) 0.4μL GTP buffer 9.6μL Enzyme-free water Make up to 20 μL

[0261] The above solution was heated at 55°C for 15 min and then placed on ice for 3 min. The circularized RNA product was purified using the lithium chloride precipitation method, and the operation steps were consistent with the linear mRNA purification.

[0262] 1-5 Circular RNA Identification:

[0263] According to the principle of RNA cyclization, by amplifying and sequencing the junction sequence containing exon E1 and exon E2, it can be determined whether the RNA is circular.

[0264] cDNA is obtained by RNA reverse transcription reaction. Using cDNA as a template, PCR amplification is performed using specific primers. By sequencing the specific product, it is determined that the cyclized product is a connection sequence containing E1 and E2, indicating that the circular RNA has been successfully connected.

[0265] In this example, RT-qPCR reverse transcription and amplification were performed, and sequencing was performed to identify the sequence integrity and accuracy at the circular RNA looping site.

[0266] 1) Reverse transcription system and conditions

[0267] Table 8 RT-qPCR reverse transcription reaction system

[0268] Reagents Mass / Volume RNA 1 μg RT primer mix 4.0μL Primerscript RT Enzyme Mix 1.0μL 5xPrimerscript buffer 4.0μL Enzyme-free water Make up to 20 μL

[0269] The circularized mRNA and linear mRNA were prepared according to the above system, heated at 50°C for 15 min, heated at 85°C for 5 s, and stored at 4°C.

[0270] 2) PCR amplification system and conditions of reverse transcription products

[0271] The cDNA obtained by reverse transcription of linear and circular RNA was used as template for PCR amplification.

[0272] Table 9 Primer sequences

[0273]

[0274]

[0275] The PCR reaction system is as follows:

[0276] Table 10 QPCR reverse transcription reaction system

[0277] Reagents Volume (μL) Reverse transcription product 1.0 10× Buffer 2.0 dNTP 1.6 Upstream primer (10 μM) 1.0 Downstream primer (10 μM) 1.0 Polymerase 0.5 Enzyme-free water Top up to 20

[0278] Table 11 QPCR reaction procedure

[0279]

[0280] The amplified PCR product was sent to Qingke Biotechnology for sequencing. The sequencing results verified that the sequence contained the connected E1-E2 sequence, indicating that the RNA had been connected into a ring.

[0281] 3) Gel electrophoresis detection

[0282] The amplified product was subjected to gel electrophoresis, and the grayscale analysis of the electrophoresis bands was performed using software. The specific steps are as follows:

[0283] ① Weigh 1g agarose into a conical flask, add 100mL 1x TBE electrophoresis buffer, and heat in a microwave to dissolve.

[0284] ② When cooled to 60-70℃, add 10μL nucleic acid dye, pour the gel into the prepared gel plate, and let it stand at room temperature for about 30 minutes until the gel solidifies.

[0285] ③ Take the sample and add DNA loading buffer, mix well and then load the sample, perform electrophoresis at 120V / 40min, and observe the electrophoresis bands on the gel imaging system.

[0286] ④ Use the analysis software provided by Bio-Rad instrument to analyze the grayscale of the bands.

[0287] Example 1 Circular RNAs Formed by Different IRES Elements

[0288] In the above synthesis method, "1-1 plasmid construction" is as follows:

[0289] The DNA vector for constructing circular RNA includes: T7 promoter, 3'T4 td intron and exon E2 (SEQ ID NO: 2-3), 5'HBA2 PR spacer, IRES element, Nano Luciferase (NanoLuc) luciferase gene, 3'human β-globin spacer, 5'T4 td intron and exon E1 sequence (SEQ ID NO: 4-5).

[0290] The IRES element is selected from the following sequences:

[0291] Table 12 IRES elements

[0292] IRES IRES element content SEQ ID NO: HRV-B3 IRES element of rhinovirus B3 SEQ ID NO:6 EMCV IRES element of encephalomyocarditis virus SEQ ID NO:7 HCV Hepatitis C virus IRES element SEQ ID NO:8 HRV-B17 IRES element of rhinovirus B17 SEQ ID NO:9 HRV-A1 IRES element of rhinovirus A1 SEQ ID NO:10

[0293] The resulting NanoLuc-expressing circular RNA sequence is shown below:

[0294] Table 13 Different IRES element circular RNA

[0295] Serial number 3' / 5' intron 5' spacer IRES 3' spacer Coding region gene SEQ ID NO: 1-1 T4 td HBA2 PR HRV-B3 Human β-globin NanoLuc SEQ ID NO:25 1-2 T4 td HBA2 PR EMCV Human β-globin NanoLuc SEQ ID NO:26 1-3 T4 td HBA2 PR HCV Human β-globin NanoLuc SEQ ID NO:27 1-4 T4 td HBA2 PR HRV-B17 Human β-globin NanoLuc SEQ ID NO:28 1-5 T4 td HBA2 PR HRV-A1 Human β-globin NanoLuc SEQ ID NO:29

[0296] For other reagents and operation steps, refer to 1-1 to 1-5.

[0297] Example 2 Circular RNAs mediated by different 5' spacers

[0298] On the basis of the above Example 1, the same intron and exon elements as those in the above Example 1 were used, EMCV, HCV, and HRV-B3 were used as IRES elements of circular RNA, and different combinations of 5' spacer sequences in Example 1 were screened. The 5' spacer sequences were selected from HBA2 PR and corresponding mutant sequences, as follows:

[0299] Table 14 5' spacer sequence

[0300] 5' spacer 5' spacer content SEQ ID NO: HBA2 PR Sequence of hemoglobin α2 bound to the α complex SEQ ID NO:11 HBA2 PR M1 HBA2 PR mutation sequence: G8A, G19A, G21A, T28A SEQ ID NO:12 HBA2 PR M2 HBA2 PR mutation sequence: C3A, G8A, T25A, T33A SEQ ID NO:13 HBA2 PR M3 HBA2 PR mutation sequence: T6A, G19A, T28A, T33A SEQ ID NO:14

[0301] The resulting NanoLuc-expressing circular RNA sequence is shown below:

[0302] Table 15 Circular RNAs formed by different 5' spacer regions

[0303] Serial number 3' / 5' intron 5' spacer IRES 3' spacer Coding region gene SEQ ID NO: 2-1 T4 td HBA2 PR EMCV Human β-globin NanoLuc SEQ ID NO:26 2-2 T4 td HBA2 PR M3 EMCV Human β-globin NanoLuc SEQ ID NO:35 2-3 T4 td HBA2 PR M1 EMCV Human β-globin NanoLuc SEQ ID NO:36 2-4 T4 td HBA2 PR M2 EMCV Human β-globin NanoLuc SEQ ID NO:37 2-5 T4 td HBA2 PR HCV Human β-globin NanoLuc SEQ ID NO:27 2-6 T4 td HBA2 PR M3 HCV Human β-globin NanoLuc SEQ ID NO:38 2-7 T4 td HBA2 PR M1 HCV Human β-globin NanoLuc SEQ ID NO:39 2-8 T4 td HBA2 PR M2 HCV Human β-globin NanoLuc SEQ ID NO:40 2-9 T4 td HBA2 PR HRV-B3 Human β-globin NanoLuc SEQ ID NO:25 2-10 T4 td HBA2 PR M3 HRV-B3 Human β-globin NanoLuc SEQ ID NO:41 2-11 T4 td HBA2 PR M1 HRV-B3 Human β-globin NanoLuc SEQ ID NO:42 2-12 T4 td HBA2 PR M2 HRV-B3 Human β-globin NanoLuc SEQ ID NO:43

[0304] For other reagents and operation steps, refer to 1-1 to 1-5.

[0305] Example 3 Circular RNAs mediated by different 3' spacers

[0306] On the basis of the above Example 2, the same intron and exon elements as those in the above Example 2 were used, EMCV and HRV-B3 were used as IRES elements of circular RNA, HBA2 PR, HBA2 PR M2 and HBA2 PR M3 were used as 5' spacer sequence elements, and different combinations of 3' spacer sequences in Example 2 were screened. The 3' spacer sequence was selected from human β-globin and its truncated sequence, as follows:

[0307] Table 16 3' spacer sequence

[0308] 3' spacer 3' spacer content SEQ ID NO: Human β-globin Human β-globin 3'UTR SEQ ID NO:15 Human β-globin M1 Human β-globin 3'UTR sequence: 19 nt truncated at the 3' end SEQ ID NO:16 Human β-globin M2 Human β-globin 3'UTR sequence: 5' end truncated 25nt SEQ ID NO:17

[0309] The resulting NanoLuc-expressing circular RNA sequence is shown below:

[0310] Table 17 Circular RNAs formed by different 3' spacer regions

[0311]

[0312]

[0313] For other reagents and operation steps, refer to 1-1 to 1-5.

[0314] Example 4 Circular RNAs mediated by different type I introns

[0315] On the basis of the above Example 3, EMCV and HRV-B3 were used as the IRES sequence elements of circular RNA, HBA2 PR M2 and HBA2 PR M3 were used as 5' spacer sequence elements, human β-globin and human β-globin M2 were used as 3' spacer sequence elements of circular RNA, and the type I intron and exon sequences in Example 3 were replaced with the intron and exon sequences of the Anabaena pre-tRNA PIE system (SEQ ID NO: 18-21).

[0316] Table 18 PIE system

[0317]

[0318] The resulting NanoLuc-expressing circular RNA sequence is shown below:

[0319] Table 19 Circular RNAs formed by different 3' / 5' introns

[0320]

[0321]

[0322] For other reagents and operation steps, refer to 1-1 to 1-5.

[0323] Example 5 Detection of protein expression of circular RNA encoding NanoLuc in HEK293T cells

[0324] In this example, the vector for promoting circular RNA circularization prepared in Example 4 was used to perform circular RNA circularization in vitro, and the expression and circularization efficiency were detected.

[0325] Table 20 Circular RNA composed of different elements

[0326]

[0327] 5-1 Transfection of HEK293T / ACE2 cells with NanoLuc circular RNA

[0328] 1) Cell culture

[0329] HEK293T / ACE2 cells (Vazyme: DD1701-01) were inoculated in DMEM high-glucose medium containing 10% fetal bovine serum, 1% penicillin and streptomycin, and cultured in a 37°C, 5% CO2 incubator. The cells were subcultured every 2-3 days.

[0330] 2) Cell transfection

[0331] Before transfection, HEK293T / ACE2 cells were cultured at 2×10 4 Each well was inoculated in a 96-well plate and cultured in a 37°C, 5% CO2 incubator. After the cells reached 70-90% confluency, the cell culture medium was replaced with Opti-MEM I reduced blood culture medium (purchased from Thermo Fisher Scientific, Catalog No. 31985070). RNA was transfected into HEK293T / ACE2 cells at 100 ng / well using Lipofectamine MessengerMAX transfection reagent (purchased from Thermo Fisher Scientific, Catalog No. LMRNA003). The specific operation is as follows:

[0332] (1) Take 25 μL of Opti-MEM I blood-reduced medium, mix with 0.75 μL of Lipofectamine MessengerMAX, and incubate at room temperature for 10 min.

[0333] (2) Take 1 μg of mRNA and add Opti-MEM I blood reduction medium to 25 μL;

[0334] (3) Mix the diluted mRNA and Lipofectamine MessengerMAX (1:1) and incubate at room temperature for 5 min.

[0335] 10 μL of the above mixed solution was slowly added to a 96-well plate and incubated in a 37°C, 5% CO2 incubator.

[0336] 5-2NanoLuc luciferase expression detection

[0337] Prepare Nano-Glo Live Cell Reagent luciferase reaction substrate solution according to the NanoLuc luciferase detection kit (Nano-Glo Live Cell Assay System, purchased from Promega (Beijing) Biotechnology Co., Ltd.). After 24 hours of cell transfection, remove the cell culture medium and add 100 μL Opti-MEM medium to each well. After adding 25 μL Nano-Glo Live Cell Reagent, mix gently and detect the luminescence value with an ELISA reader.

[0338] Example 6 Detection of protein expression of circular RNA encoding EGFP in HEK293T cells

[0339] Based on the above Example 5, the same IRES element, 5' spacer element, 3' spacer element and type I intron sequence as in the above Example were used, and the NanoLuc sequence in Example 5 was replaced with the EGFP sequence (SEQ ID NO: 61).

[0340] Table 21 Different coding region genes

[0341] Example Coding region gene SEQ ID NO: 6 EGFP SEQ ID NO:61 5 NanoLuc SEQ ID NO:23

[0342] Table 22 Circular RNA element types

[0343]

[0344] Other reagents and preparation methods are consistent with those in Example 1.

[0345] The method of transfecting HEK293T cells with circular RNA encoding EGFP was the same as in Example 5.

[0346] EGFP protein expression detection: One day after transfection, HEK293T cells were observed under a 200× fluorescence microscope for EGFP expression.

[0347] Results Analysis 1

[0348] IRES is very important for the activity of initiating protein translation. It is mainly derived from the viral UTR sequence and does not require special chemical modification. There is a correlation between the activity and length of IRES, and IRES activity has different expression activities in different cell types.

[0349] According to the principle of RNA cyclization, in Mg 2+ Under the action of GTP, the base junction between the 5' intron and exon E1 is broken, and the broken exon E1 will attack the base junction between the 3' intron and exon E2, causing the junction to break, the 3' intron to dissociate, and exon E1 and exon E2 to form a covalent connection to form a circular RNA. Therefore, by amplifying and sequencing the junction sequence containing exon E1 and exon E2, it can be determined whether the RNA is circular.

[0350] cDNA was obtained by RNA reverse transcription reaction. Using cDNA as a template, PCR amplification was performed using specific primers. The experimental results showed that there was no obvious specific amplification band in the linear mRNA group, and specific amplification products were visible in the circular RNA group. By sequencing the specific product, it was determined that the circularized product was a connection sequence containing E1 and E2, indicating that the circular RNA had been successfully connected.

[0351] The gray value of the electrophoresis bands was analyzed, and the circularization efficiency was calculated by the ratio of the gray value of the circular RNA product amplification band to the linear RNA amplification band. The results are as follows:

[0352] Table 23 Circularization efficiency of NanoLuc with different IRES combinations

[0353]

[0354] in conclusion:

[0355] When the 3' / 5' intron of the circular RNA is T4 td, the 5' spacer is HBA2 PR, and the 3' spacer is human β-globin, the circular RNA with IRES as EMCV has the highest circularization efficiency of 65.7%, followed by HRV-B3, 60.3%, HCV, 56.8%, and HRV-A1 and HRV-B17, 52.1% and 47.5%, respectively.

[0356] summary:

[0357] Example 2 used EMCV, HRBV3 and HCV in combination with different 5' spacer elements to compare the circularization efficiency of circular RNA. The results showed that:

[0358] The 3' / 5' introns, 5' spacer, and 3' spacer elements that make up the circular RNA are the same. The circularization efficiency of the circular RNA with EMCV as the IRES element is the highest, followed by HRBV3 and then HCV.

[0359] The circular RNA composition includes 3' / 5'T4 td intron, 5'HBA2 PR spacer, 3'human β-globin spacer, and IRES. The circular RNA circularization efficiency of EMCV is 65.7%; the circularization efficiency of HRV-B3 is 60.3%; the circularization efficiency of HCV is 56.8%; the circularization efficiency of HRV-A1 is 52.1%; and the circularization efficiency of HRV-B17 is 47.5%.

[0360] The IRES element circularization efficiency from high to low is: (1-2)EMCV > (1-1)HRV-B3 > (1-3)HCV>(1-5)HRV-A1>(1-4)HRV-B17.

[0361] Results Analysis 2

[0362] Example 2 The circular RNA compositions all contain 3' / 5'T4 td introns and 3' human β-globin spacer, the difference being that the IRES and 5' spacer sequences used are different. IRES uses one of EMCV, HRBV3 and HCV combined with different 5' spacer sequences, and the circular RNA circularization efficiency of different combinations is tested. The results are shown in the following table:

[0363] Table 24 Circularization efficiency of NanoLuc with different 5' spacers

[0364]

[0365] Table 24, in the combination where the 3' / 5' intron is T4 td, the 3' spacer is human β-globin, and the IRES is EMCV, the cyclization efficiency of the combination with the 5' spacer sequence of HBA2 PR M2 is 74.6%, which is about 9% higher than the cyclization efficiency of the combination using HBA2 PR (65.7%); the cyclization efficiency of the combination with the 5' spacer sequence of HBA2 PR M3 reaches 69.8%, which is about 4% higher than that of HBA2 PR; the cyclization efficiency of the combination of HBA2 PR M1 is lower than that of HBA2 PR.

[0366] The looping efficiency of the 5' spacer sequence is from high to low: (2-4)HBA2 PR M2>(2-2)HBA2PR M3>(2-1)HBA2 PR>(2-3)HBA2 PR M1.

[0367] Table 25 Circularization efficiency of NanoLuc with different 5' spacers

[0368]

[0369]

[0370] Table 25, in the combination where the 3' / 5' intron is T4 td, the 3' spacer is human β-globin, and the IRES is HCV, the combination where the 5' spacer sequence is HBA2 PR M2 has a cyclization efficiency of 61.5%, which is about 7% higher than the cyclization efficiency of the combination using HBA2 PR (54.1%); the combination where the 5' spacer sequence is HBA2 PR M3 has a cyclization efficiency of 58.7%, which is 4% higher than HBA2 PR; the combination where the 5' spacer sequence is HBA2 PR M1 has a lower cyclization efficiency than HBA2 PR.

[0371] The looping efficiency of the 5' spacer sequence is from high to low: (2-8)HBA2 PR M2>(2-6)HBA2PR M3>(2-5)HBA2 PR>(2-7)HBA2 PR M1.

[0372] Table 26 Circularization efficiency of NanoLuc with different 5' spacers

[0373]

[0374] Table 26, in the combination where the 3' / 5' intron is T4 td, the 3' spacer is human β-globin, and the IRES is HRV-B3, the combination where the 5' spacer sequence is HBA2 PR M2 has a cyclization efficiency of 68.2%, which is about 8% higher than the cyclization efficiency of the combination using HBA2 PR (60.3%); the combination where the 5' spacer sequence is HBA2 PR M3 has a cyclization efficiency of 64.7%, which is about 4% higher than HBA2 PR; the combination where the 5' spacer sequence is HBA2 PR M1 has a lower cyclization efficiency than HBA2 PR.

[0375] The looping efficiency of the 5' spacer sequence is from high to low: (2-12)HBA2 PR M2>(2-10)HBA2PR M3>(2-9)HBA2 PR>(2-11)HBA2 PR M1.

[0376] in conclusion:

[0377] When the 3' / 5' introns are T4 td, the 3' spacer is human β-globin, and the IRES element is the same, the circular RNA with the 5' spacer being HBA2PR M2 has the highest circularization efficiency, followed by the circular RNA composed of HBA2 PR M3, then the HBA2 PR combination, and the HBA2 PR M1 combination is the worst.

[0378] summary:

[0379] The 3' / 5' intron, IRES, and 3' spacer elements that make up the circular RNA are the same, and the combination of HBA2 PRM2 with a 5' spacer sequence has the best circularization effect, reaching 74.6%; followed by HBA2 PR M3, reaching 69.8%; and then HBA2 PR.

[0380] In the combination of 3' / 5' intron being T4 td, 3' spacer being human β-globin, and IRES being EMCV, HRV-B3 or HCV, the circularization efficiency of circular RNA with 5' spacer sequence being HBA2 PR M2 was 61.5-74.6%; the circularization efficiency of HBA2 PR M3 was 58.7-69.8%; the circularization efficiency of HBA2 PR was 54.1-65.7%; and the circularization efficiency of HBA2 PR M1 was 50.9-56.3%, that is,

[0381] The looping efficiency of the 5' spacer sequence is from high to low:

[0382] HBA2 PR M2>HBA2 PR M3>HBA2 PR>HBA2 PR M1.

[0383] Results Analysis 3

[0384] As can be seen from Example 2, the circular RNA compositions all contain 3' / 5'T4 td introns and 3' human β-globin spacer, the IRES element is EMCV or HRV-B3, and the 5' spacer is HBA2 PR M2, HBA2 PR M3 or HBA2 PR, and the circularization efficiency exceeds 60%. Therefore, Example 3 uses the above IRES and 5' spacer elements and 3' / 5'T4 td introns, with different 5' spacer sequences to form circular RNAs, and detects their circularization efficiency. The results are as follows:

[0385] Table 27 Circular RNAs formed by different 3' spacer regions

[0386]

[0387] For other reagents and operation steps, refer to 1-1 to 1-5.

[0388] Table 28 Circularization efficiency of NanoLuc with different 3' spacers, 5' spacer is HBA2 PR, IRES is EMCV

[0389]

[0390]

[0391] Table 28, among the circular RNAs with T4 td as 3' / 5' intron, EMCV as IRES, and HBA2 PR as 5' spacer, the combination with human β-globin M2 as 3' spacer had a circularization efficiency of 67.8%, which was about 2% higher than the combination using human β-globin (65.7%); the combination using human β-globin M1 had a circularization efficiency of 60.5%, which was lower than the combination using human β-globin.

[0392] The looping efficiency of the 3' spacer sequence is from high to low: (3-3) human β-globin M2> (3-1) human β-globin> (3-2) human β-globin M1.

[0393] Table 29 Circularization efficiency of NanoLuc with different 3' spacers, 5' spacer is HBA2 PR M3, IRES is EMCV

[0394]

[0395] Table 29, among the circular RNAs with T4 td as 3' / 5' intron, EMCV as IRES, and HBA2 PR M3 as 5' spacer, the combination with human β-globin M2 as 3' spacer has a circularization efficiency of 73.5%, which is about 4% higher than the combination using human β-globin (68.8%); the combination using human β-globin M1 has a circularization efficiency of 62.5%, which is lower than the combination using human β-globin.

[0396] The looping efficiency of the 3' spacer sequence is from high to low: (3-6) human β-globin M2> (3-4) human β-globin> (3-5) human β-globin M1.

[0397] Table 30 Circularization efficiency of NanoLuc with different 3' spacers, 5' spacer of human β-globin M2 and IRES of EMCV

[0398]

[0399] Table 30, among the circular RNAs with 3' / 5' introns being T4 td, IRES being EMCV, and 5' spacer being HBA2 PR M2, the combination with 3' spacer being human β-globin M2 had a cyclization efficiency of 79.7%, which was about 5% higher than the combination using human β-globin (74.6%); the combination using human β-globin M1 had a cyclization efficiency of 64.8%, which was lower than the combination using human β-globin. cDNA was obtained by RNA reverse transcription reaction. PCR amplification was performed using specific primers using cDNA as a template. The experimental results showed that there was no obvious specific amplification band in the linear mRNA group of SEQ ID NO:48, while a specific amplification product was visible in the circular RNA group. By sequencing the specific product, it was determined that the cyclization product was a junction sequence containing E1 and E2, indicating that the circular RNA had been successfully connected ( Figure 2 The gray value of the electrophoresis bands was analyzed, and the circularization efficiency was calculated by the ratio of the gray value of the circular RNA product amplification band to the linear RNA amplification band ( Figure 3 ).

[0400] The looping efficiency of the 3' spacer sequence is from high to low: (3-9) human β-globin M2> (3-7) human β-globin> (3-8) human β-globin M1.

[0401] Table 31 Circularization efficiency of NanoLuc with different 3' spacers, 5' spacer is HBA2 PR M3, IRES is HRV-B3

[0402]

[0403] Table 31, among the circular RNAs with 3' / 5' introns being T4 td, IRES being HRV-B3, and 5' spacer being HBA2 PR M3, the combination with 3' spacer being human β-globin M2 had a circularization efficiency of 69.3%, which was about 5% higher than the combination using human β-globin (64.7%); the combination using human β-globin M1 had a circularization efficiency of 59.4%, which was lower than the combination using human β-globin.

[0404] The looping efficiency of the 3' spacer sequence is from high to low: (3-12) human β-globin M2>(3-10) human β-globin>(3-11) human β-globin M1.

[0405] Table 32 Circularization efficiency of NanoLuc with different 3' spacers, 5' spacer is HBA2 PR M2, IRES is HRV-B3

[0406]

[0407] Table 32, among the circular RNAs with T4 td as 3' / 5' intron, HRV-B3 as IRES, and HBA2 PR M2 as 5' spacer, the combination with human β-globin M2 as 3' spacer had a circularization efficiency of 72.5%, which was about 5% higher than the combination using human β-globin (67.8%); the combination using human β-globin M1 had a circularization efficiency of 61.7%, which was lower than the combination using human β-globin.

[0408] The looping efficiency of the 3' spacer sequence is from high to low: (3-15) human β-globin M2> (3-13) human β-globin> (3-14) human β-globin M1.

[0409] in conclusion:

[0410] When the 3' / 5' introns were T4 td, the 5' spacer and the IRES element were the same, the circularization efficiency of the circular RNA composed of human β-globin M2 in the 3' spacer was the best, followed by human β-globin, and human β-globin M1 was the worst.

[0411] summary:

[0412] The circular RNA composition consists of 3' / 5'T4 td intron, 5' spacer is HBA2 PR M2 or HBA2PR M3, IRES is EMCV or HRV-B3, and 3' spacer is human β-globin M2 with the highest circularization efficiency, reaching 79.7%, followed by human β-globin, reaching 74.6%.

[0413] In the combination of 3' / 5' intron being T4 td, IRES being EMCV, HRV-B3 or HCV, and 5' spacer sequence being HBA2 PR M2, HBA2 PR M3 or HBA2 PR, the circularization efficiency of 3' spacer human β-globin M2 circular RNA was 67.8-79.9%; the circularization efficiency of human β-globin was 64.7-74.6%; the circularization efficiency of human β-globin M1 was 59.4-64.8%, that is,

[0414] The looping efficiency of 3' spacer sequences from high to low is: human β-globin M2>human β-globin>human β-globin M1.

[0415] Results Analysis 4

[0416] As can be seen from Example 3, the circular RNA composition includes 3' / 5'T4 td intron, 5' spacer, IRES and 3' spacer are combined as follows: HBA2 PR M2 and EMCV and human β-globin M2, HBA2PR M3 and EMCV and human β-globin M2, HBA2PR M2 and HRV-B3 and human β-globin M2 and HBA2 PR M2 and EMCV and human β-globin, and the circularization efficiency exceeds 70%. Therefore, Example 4 is based on the above element combination, changes the 3' / 5' intron, replaces T4 td with Anabaena pre-tRNA, and compares its circularization efficiency. The results are as follows:

[0417] Table 33 Circularization efficiency of different 3' / 5' intron combinations with 5' spacer HBA2 PR M2, IRES EMCV, and 3' spacer human β-globin M2

[0418]

[0419]

[0420] Table 33, the circular RNA with 5' spacer region of HBA2 PR M2, IRES of EMCV, 3' spacer region of human β-globin M2, and 3' / 5' intron of Anabaena pre-tRNA has a circularization efficiency of 83.5%, which is about 4% higher than the combination of T4td (79.7%). cDNA was obtained by RNA reverse transcription reaction. PCR amplification was performed using specific primers using cDNA as a template. The experimental results showed that there was no obvious specific amplification band in the linear mRNA group of SEQ ID NO:55, and a specific amplification product was visible in the circular RNA group. By sequencing the specific product, it was determined that the circularized product was a junction sequence containing E1 and E2, indicating that the circular RNA had been successfully connected ( Figure 4 The gray value of the electrophoresis bands was analyzed, and the circularization efficiency was calculated by the ratio of the gray value of the circular RNA product amplification band to the linear RNA amplification band ( Figure 5 ).

[0421] The circularization efficiency of 3' / 5' intron sequences is from high to low: (4-9)Anabaena pre-tRNA> (4-2)T4td.

[0422] Table 34 Circularization efficiency of different 3' / 5' intron combinations with 5' spacer HBA2 PR M3, IRES EMCV, and 3' spacer human β-globin M2

[0423]

[0424] Table 34, the circular RNA circularization efficiency of the circular RNA with HBA2 PR M3 as the 5' spacer, EMCV as the IRES, human β-globin M2 as the 3' spacer, and Anabaena pre-tRNA as the 3' / 5' intron was 75.6%, which was about 2% higher than that of the combination of T4td (73.5%).

[0425] The circularization efficiency of 3' / 5' intron sequences is from high to low: (4-8)Anabaena pre-tRNA> (4-1)T4td.

[0426] Table 35 Circularization efficiency of different 3' / 5' intron combinations with 5' spacer HBA2 PR M2, IRES HRV-B3, and 3' spacer human β-globin M2

[0427]

[0428]

[0429] Table 35, the circular RNA circularization efficiency of the circular RNA with 5' spacer of HBA2 PR M2, IRES of HRV-B3, 3' spacer of human β-globin M2, and 3' / 5' intron of Anabaena pre-tRNA was 74.2%, which was about 2% higher than that of the combination of T4 td (72.5%).

[0430] The circularization efficiency of 3' / 5' intron sequences is from high to low: (4-11)Anabaena pre-tRNA>(4-4)T4 td.

[0431] Table 36 Circularization efficiency of different 3' / 5' intron combinations with 5' spacer HBA2 PR M2, IRES EMCV, and 3' spacer human β-globin

[0432]

[0433] Table 36, the circular RNA circularization efficiency of the circular RNA with HBA2 PR M2 as the 5' spacer, EMCV as the IRES, human β-globin as the 3' spacer, and Anabaena pre-tRNA as the 3' / 5' intron was 78.2%, which was about 1.6% higher than that of the combination of T4 td (74.6%).

[0434] The circularization efficiency of 3' / 5' intron sequences is from high to low: (4-13)Anabaena pre-tRNA >(4-6)T4td.

[0435] Table 37 Circularization efficiency of different 3' / 5' intron combinations with 5' spacer HBA2 PR M3, IRES HRV-B3, and 3' spacer human β-globin M2

[0436]

[0437] Table 37, the circular RNA circularization efficiency of the circular RNA with 5' spacer of HBA2 PR M3, IRES of HRV-B3, 3' spacer of human β-globin M2, and 3' / 5' intron of Anabaena pre-tRNA was 71.4%, which was about 2% higher than that of the combination of T4 td (69.3%).

[0438] The circularization efficiency of 3' / 5' intron sequences is from high to low: (4-10)Anabaena pre-tRNA>(4-3)T4 td.

[0439] Table 38 Circularization efficiency of different 3' / 5' intron combinations with 5' spacer HBA2 PR M3, IRES EMCV, and 3' spacer human β-globin

[0440]

[0441] Table 38, the circular RNA circularization efficiency of the circular RNA with HBA2 PR M3 as the 5' spacer, EMCV as the IRES, human β-globin as the 3' spacer, and Anabaena pre-tRNA as the 3' / 5' intron was 72.6%, which was about 4% higher than that of the combination of T4 td (68.8%).

[0442] The circularization efficiency of 3' / 5' intron sequences is from high to low: (4-12)Anabaena pre-tRNA>(4-5)T4 td.

[0443] Table 39 Circularization efficiency of different 3' / 5' intron combinations with 5' spacer HBA2 PR M2, IRES HRV-B3, and 3' spacer human β-globin

[0444]

[0445] Table 39, the circularization efficiency of the circular RNA with HBA2 PR M2 as the 5' spacer, HRV-B3 as the IRES, human β-globin as the 3' spacer, and Anabaena pre-tRNA as the 3' / 5' intron was 69.7%, which was about 2% higher than that of the combination of T4td (67.8%);

[0446] The circularization efficiency of 3' / 5' intron sequences is from high to low: (4-14)Anabaena pre-tRNA>(4-7)T4 td.

[0447] in conclusion:

[0448] When the 5' spacer, IRES, and 3' spacer elements are the same, circular RNAs composed of 3' / 5' introns of Anabaena type I are more efficient than those composed of T4 td type I introns.

[0449] summary:

[0450] The circular RNA composition, in which the 5' spacer is HBA2 PR M2 or HBA2 PR M3, IRES is EMCV or HRV-B3, 3' spacer is human β-globin M2 or human β-globin, and 3' / 5' intron is Anabaena pre-tRNA, has a higher circularization efficiency than T4 td, reaching 83.5%.

[0451] In the combination of 5' spacer sequence being HBA2 PR M2 or HBA2 PR M3, IRES being EMCV or HRV-B3, 3' spacer being human β-globin M2 or human β-globin, the circularization efficiency of circular RNA with 3' / 5' intron being Anabaena pre-tRNA is 69.7-83.5%; the circularization efficiency of T4 td is 67.8-79.7%, that is,

[0452] The circularization efficiency of 3' / 5' intron sequences is from high to low: Anabaena pre-tRNA>T4 td.

[0453] Results Analysis 1-4 Summary

[0454] The circular RNA composition includes: 3' / 5' introns are Anabaena pre-tRNA, 5' spacer is HBA2 PR M2 or HBA2 PR M3, IRES is EMCV or HRV-B3, 3' spacer is human β-globin M2, and the circularization efficiency is high;

[0455] Among them, the circular RNA with Anabaena pre-tRNA as 3' / 5' intron, HBA2 PR M2 as 5' spacer, EMCV as IRES, and human β-globin M2 as 3' spacer had the highest circularization efficiency, reaching 83.5%.

[0456] 1) Through the detection of circular RNA cyclization efficiency of different combinations of Examples 1 to 4, the cyclization efficiency of each element is obtained from high to low:

[0457] 3' / 5' intron: Anabaena pre-tRNA>T4 td;

[0458] 5′ spacer sequence: HBA2 PR M2>HBA2 PR M3>HBA2 PR>HBA2 PR M1;

[0459] IRES element: EMCV>HRV-B3>HCV>HRV-A1>HRV-B3;

[0460] 3' spacer sequence: human β-globin M2>human β-globin>human β-globin M1.

[0461] 2) Screen out the 3' / 5' introns, 5' spacer, IRES and 3' spacer that make up the circular RNA. The elements with high circularization efficiency are as follows:

[0462] Table 40 Elements that make up circular RNA

[0463]

[0464] 3) The circularization efficiency after optimizing the circular RNA combination using the elements in the table below is as follows:

[0465] Table 41 Circular RNA Circularization Efficiency Not Containing the Elements in Table 40

[0466]

[0467]

[0468] As can be seen from Table 41, the circular RNA composition does not contain the elements in Table 40, and the cyclization efficiency is 47.5-56.8%.

[0469] Table 42 Circular RNA Circularization Efficiency Containing 1 Element in Table 40

[0470]

[0471] As shown in Table 42, when the circular RNA composition contains any one of the elements in Table 40, the cyclization efficiency is 55.8-65.7%; among them, the combination of IRES and EMCV (sequence number 3-1) has the highest cyclization efficiency.

[0472] Compared with the circular RNA cyclization efficiency not containing the elements in Table 40 (sequence numbers 1-4, 1-5, and 1-3), the IRES is different, and the other elements are the same, and the circularization efficiency is increased by 8.9 to 18.2%. The second is the combination of the 5' spacer region of HBA2 PR M2 (sequence number 2-8). Compared with sequence number 2-7, the 5' spacer region is different, and the other elements are the same, and the circularization efficiency is increased by about 10%, which is a significant improvement.

[0473] Table 43 Circular RNA Circularization Efficiency Containing 2 Elements in Table 40

[0474]

[0475]

[0476] As shown in Table 43, when the circular RNA composition contains any two elements in Table 40, the circularization efficiency is 61.7-74.6%; among them, the combination of 5' spacer region of HBA2 PR M2 and IRES of EMCV (sequence number 3-7) has the highest circularization efficiency.

[0477] Compared with the circular RNA cyclization efficiency not containing the elements in Table 40 (No. 1-4, 2-7, 1-5, 1-3), the 5' spacer and IRES are different, and the other elements are the same, the circularization efficiency is increased by 17.8-27.1%, which is a significant improvement.

[0478] Table 44 Circular RNA Circularization Efficiency Containing 3 Elements in Table 40

[0479]

[0480] As shown in Table 44, when the circular RNA composition contains any three elements in Table 40, the circularization efficiency is 69.3-79.7%, among which the combination of HBA2 PR M2 as the 5' spacer, EMCV as the IRES and human β-globin M2 as the 3' spacer (sequences 4-9) has the highest circularization efficiency.

[0481] Compared with the circular RNA circularization efficiency not containing the elements in Table 40 (No. 1-4, 2-7, 1-5, 1-3), the 3' and 5' spacers and IRES are different, and the other elements are the same, the circularization efficiency is increased by 22.9-32.2%, which is a significant increase.

[0482] Table 45 Circular RNA Circularization Efficiency of the 4 Elements in Table 40

[0483]

[0484] The circular RNA composition contains four elements in Table 40, and the circularization efficiency is 71.4-83.5%. Among them, the combination of 3' and 5' introns as Anabaena pre-tRNA PIE system, 5' spacer as HBA2 PR M2, IRES as EMCV and 3' spacer as human β-globin M2 (sequence number 4-9) has the highest circularization efficiency (83.5%).

[0485] Compared with the circular RNA circularization efficiency not containing the elements in Table 40 (No. 1-4, 2-7, 1-5, 1-3), the 3' and 5' spacers, 3' / 5' introns and IRES are different, and the other elements are the same, the circularization efficiency is increased by 26.7-36%, which is the best improvement.

[0486] Results Analysis 5

[0487] The circular RNAs in the table below and their corresponding linear RNAs were transfected into HEK293T / ACE2 cells, and the expression of NanoLuc fluorescent protein was detected. The results are shown in Figure 6 .

[0488] Table 46

[0489]

[0490]

[0491] The fluorescence intensity quantitative detection results of the circular (SEQ ID NO: 55) and linear mRNA (SEQ ID NO: 22) composed of Anabaena pre-tRNA, HBA2 PR M2, EMCV and human β-globin M2 encoding NanoLuc are shown in Figure 2. Figure 6 As shown, compared with the linear product of SEQ ID NO:22, the expression of NanoLuc protein in cells transfected with the circularized product of SEQ ID NO:55 was stronger, about 7 times; the expression of NanoLuc protein in cells transfected with linear mRNA was close to zero on the third day, while NanoLuc in cells transfected with circular RNA was still highly expressed on the fifth day (fluorescence intensity of about 1700), and the expression time was longer. Moreover, the circularized product of SEQ ID NO:48, the circularized product of SEQ ID NO:50, and the circularized product of SEQ ID NO:56 were all expressed, verifying that the artificial circularized vector combination has the function of protein expression.

[0492] Results Analysis 6

[0493] The circular RNA encoding EGFP comprises a combination of Anabaena pre-tRNA, HBA2 PR M2, EMCV and human β-globin M2, and the circularization efficiency is 83%. The fluorescence signal intensity of the transiently transfected cells is analyzed, and the transiently transfected cells are observed under a fluorescence microscope. Figure 7 It can be seen that the transfected cells exhibited a typical post-transfection morphology. Under a fluorescence microscope, obvious EGFP green fluorescence could be observed in the cells transfected with the circularized product, while the EGFP green fluorescence in the cells transfected with the linear product was weak, indicating that the circular RNA had been successfully expressed.

[0494] Comparative Example 1:

[0495] Robert Chen, et al. Engineering circular RNA for enhanced protein production. 2022, Nat. Biotechnol. 41, 262-272.

[0496] In the present disclosure and comparative example 1, when different IRES elements were explored with different 5' spacers and 3' spacers, other elements used were as shown in the following table.

[0497] Table 47 Circularization efficiency of NanoLuc with different IRES combinations

[0498]

[0499] The results showed that the intensity of fluorescent protein expression of the IRES element in Comparative Example 1 (HEK293T cells) was as follows: HRV-B3>HRV-A1>HCV>HRV-B17>EMCV, and there were significant differences in the protein expression intensity detected in different cells, and the circularization efficiency was not explored and detected.

[0500] The circularization efficiency of the IRES elements disclosed in the present invention is from high to low: EMCV>HRV-B3>HCV>HRV-A1>HRV-B17.

[0501] From the comparison of the above data, it can be seen that ① compared with the present disclosure, the purpose of the determination is different. The present disclosure is to increase the circularization rate, and the comparative example 1 is to increase the protein expression intensity. The two are not comparable. ② The order of the influence of the IRES element on the circularization rate in comparative example 1 and the present disclosure is also different. The circularization efficiency of the IRES element of the present disclosure is from high to low: EMCV>HRV-B3>HCV>HRV-A1>HRV-B17. In comparative example 1, the intensity of fluorescent protein expressed by the IRES element (HEK293T cells) is from high to low: HRV-B3>HRV-A1>HCV>HRV-B17>EMCV.

[0502] Comparative Example 2:

[0503] R. Alexander Wesselhoeft, et al. Engineering circular RNA for potent and stable translation in eukaryotic cells. 2018, Nat. Commun. 9, 2629.

[0504] Table 48 Comparative Example 2 Cyclization efficiency of Gaussia luciferase with different element combinations

[0505]

[0506] The component combination of Comparative Example 2 was used to replace the coding region gene with the coding region gene NanoLuc disclosed in the present invention, and its circularization rate was measured to be only 75.1%.

[0507] The above experimental data show that even if the elements are the same, the looping rates for genes in different coding regions are different and have significant differences.

Claims

1. A nucleic acid construct for circular RNA expression, the structure from 5' to 3' end comprising: 3' intron, exon E2, 5' spacer, IRES element, target gene, 3' spacer, exon E1, 5' intron; the 5' spacer is HBA2PR M2, whose sequence is shown in SEQ ID NO: 13; the 3' spacer is human β-globin M2, whose sequence is shown in SEQ ID NO: 17; The 3' intron, exon E2, exon E1, and 5' intron are derived from the bacteriophage T4 td PIE system or the Anabaena pre-tRNA PIE system; and The IRES element is selected from one of the following: 。 2. The nucleic acid construct for circular RNA expression according to claim 1, wherein The IRES element is EMCV.

3. The nucleic acid construct for circular RNA expression according to claim 1, wherein: The components of the bacteriophage T4 tdPIE system are: 。 4. The nucleic acid construct for circular RNA expression according to claim 1, wherein: The components of the Anabaena pre-tRNAPIE system are: 。 5. The nucleic acid construct for circular RNA expression according to claim 1, wherein Any of the following groups: (1) The IRES element is EMCV and the 5' spacer is HBA2 PR M2; (2) The IRES element is HRV-B3, and the 5' spacer is HBA2 PR M2; (3) The IRES element is EMCV and the 3' spacer is human β-globin M2.

6. The nucleic acid construct for circular RNA expression according to claim 1, wherein: Any of the following groups: (1) The IRES element is EMCV; the 3' spacer is human β-globin M2; and the 5' spacer is HBA2 PR M2; (2) The IRES element is HRV-B3; the 3' spacer is human β-globin M2; and the 5' spacer is HBA2 PR M2; (3) The 3' intron, exon E2, exon E1, and 5' intron are the Anabaena pre-tRNA PIE system, the IRES element is HRV-B3; and the 5' spacer is HBA2 PR M2; (4) The 3' intron, exon E2, exon E1, and 5' intron are the Anabaena pre-tRNA PIE system, the IRES element is EMCV; and the 5' spacer is HBA2 PR M2.

7. The nucleic acid construct for circular RNA expression according to claim 1, wherein Any of the following groups: (1) The 3' intron, exon E2, exon E1, and 5' intron are the Anabaena pre-tRNA PIE system, the 5' spacer is HBA2 PR M2; the IRES element is EMCV; and the 3' spacer is human β-globin M2; (2) The 3' intron, exon E2, exon E1, and 5' intron are the Anabaena pre-tRNA PIE system, the 5' spacer is HBA2 PR M2; the IRES element is HRV-B3; and the 3' spacer is human β-globin M2.

8. The nucleic acid construct for circular RNA expression according to claim 1, wherein The target gene includes a gene encoding a human protein or a non-human protein.

9. The nucleic acid construct for circular RNA expression according to claim 1, wherein: The size of the target gene is 100 to 6000 bp.

10. The nucleic acid construct for circular RNA expression according to claim 1, wherein: The target gene encodes a target polypeptide, and the target polypeptide is a therapeutic polypeptide.

11. The nucleic acid construct for circular RNA expression according to claim 10, wherein: The therapeutic polypeptide is selected from the group consisting of hormones, neurotransmitters, growth factors, enzymes, cytokines, antibodies, Fc fusion proteins, anticoagulants, blood factors, bone morphogenetic proteins, interferons, interleukins, and thrombolytic agents.

12. The nucleic acid construct for circular RNA expression according to claim 11, wherein The enzyme is selected from the group consisting of oxidoreductases, metabolic enzymes, mitochondrial enzymes, oxygenases, dehydrogenases, ATP-independent enzymes, lysosomal enzymes, and desaturases.

13. Use of the nucleic acid construct for circular RNA expression according to any one of claims 1 to 12 in preparing circular RNA in vitro.

14. A method for preparing circular RNA in vitro using the nucleic acid construct for circular RNA expression according to any one of claims 1 to 12, comprising the following steps: 1) Transcription step: using the nucleic acid construct to transcribe and form a linear nucleic acid molecule; 2) Circularization step: The linear nucleic acid molecule undergoes a circularization reaction to obtain a circular RNA.

15. A circular RNA prepared by the method according to claim 14.

16. A circular RNA, the structure of which from the 5' to the 3' end comprises: exons, 5' spacer, IRES element, target gene, and 3' spacer; Its structure from 5' to 3' end includes: exon E2, 5' spacer, IRES element, target gene, 3' spacer and exon E1; The definitions of the exon, 5' spacer, IRES element, target gene and 3' spacer are as described in any one of claims 1-12.

17. Use of the circular RNA according to claim 15 or 16 in expressing a target polypeptide.

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