Sequence optimization method for improving stability of circular RNA, application and product

Abstract

The invention discloses a sequence optimization method for improving the stability of circular RNA, application and a product, and belongs to the technical field of biotechnology and molecular biology. In order to solve the problem that the existing circular RNA is easy to be specifically degraded by RNAseK and lysosome after entering a cell, synonymous mutation optimization is carried out on an open reading frame of a target gene of the circular RNA by identifying nucleic acid motifs (a first type of nucleic acid motifs and a second type of nucleic acid motifs) identified by two degradation mechanisms. The optimized circular RNA is prepared by means of in-vitro cyclization of an anabaena I-type intron self-splicing mediator, after cells are transfected, the intracellular RNA expression level of the circular RNA is remarkably improved compared with that of a wild type, and the target protein expression efficiency is also remarkably improved. The intracellular stability and expression efficiency of the circular RNA are remarkably enhanced, and the method can be widely applied to the scenes of gene expression regulation, protein synthesis, gene therapy, vaccine development and the like.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and molecular biology, and relates to the design, modification and application of circular RNA. Specifically, it relates to sequence optimization methods to improve the intracellular stability of circular RNA, optimized circular RNA products and their applications in gene expression regulation, protein synthesis, gene therapy or vaccine development. Background Technology

[0002] Circular RNA (circRNA) is a covalently closed circular RNA molecule formed by backsplicing of precursor mRNA. It lacks a 5' cap and a 3' polyadenylated tail. Its unique circular topology naturally resists RNA exonuclease degradation, resulting in a significantly longer half-life than linear RNA, allowing it to maintain its presence and functional activity more persistently within cells. By inserting an internal ribosome entry site (IRES) sequence into the circular RNA sequence, cap-independent protein translation can be achieved. This overcomes the traditional functional limitation of circular RNA as merely a regulatory molecule, transforming it into a novel biological tool capable of efficiently expressing target proteins, demonstrating enormous application potential in several cutting-edge fields such as gene therapy, vaccine development, and protein replacement therapy.

[0003] In recent years, the technology for preparing functional circular RNA in vitro has been continuously developing and iterating. Core optimization directions mainly focus on improving circularization efficiency, simplifying purification processes, reducing linear RNA residue, and expanding application scenarios. For example, by optimizing the source and sequence of self-splicing introns, adjusting the in vitro transcription reaction system, and improving affinity chromatography purification strategies, the large-scale preparation and purity improvement of circular RNA have been achieved. However, despite the increasingly mature in vitro preparation technology, circular RNA still faces the challenge of intracellular specific degradation pathways after entering cells. This problem directly limits its intracellular accumulation and the expression efficiency of target proteins, becoming a key bottleneck restricting its industrial application.

[0004] Related studies have confirmed that there are two core degradation mechanisms targeting circular RNA within cells: one is a protein-mediated degradation pathway, where proteins can recognize conserved nucleic acid motifs on circular RNA and initiate degradation reactions, significantly reducing the intracellular abundance of circular RNA; the other is a degradation pathway involving the lysosomal system, where various acidic nucleases within lysosomes can mediate RNA hydrolysis, further exacerbating the degradation of circular RNA. Currently, existing technologies have not been specifically optimized for these two key degradation mechanisms, resulting in insufficient intracellular stability of circular RNA and translation efficiency of target proteins that cannot meet the needs of clinical applications or industrial production, greatly limiting its translational applications in gene therapy, vaccine development, and other fields. Therefore, developing a technology that can circumvent intracellular specific degradation at the sequence level and significantly improve the intracellular stability and expression efficiency of circular RNA has significant theoretical and practical value. Summary of the Invention

[0005] The purpose of this invention is to provide: A functional circular RNA resistant to degradation, its preparation method, and its application are disclosed to address the technical problems of existing in vitro synthesized circular RNAs, such as easy recognition and degradation by RNAseK and lysosomes after entering cells, resulting in low RNA expression levels, insufficient translation efficiency of target proteins, and short duration of action in vivo, or a combination thereof.

[0006] Terminology Explanation: Unless otherwise defined, all technical terms in this document have the same meanings as commonly understood by one of ordinary skill in the art to which the subject matter of the claims pertains. Unless otherwise stated, all patents, patent inventions, and publications cited in this document are incorporated herein by reference in their entirety. If multiple definitions exist for terms in this document, the definitions in this chapter shall prevail.

[0007] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0008] The definition of the standard chemical terminology can be found in the reference "Molecular Cloning: A Laboratory Manual" (4th edition, by J. Sambrook).

[0009] Unless otherwise specified, conventional methods within the scope of the art, such as T7 in vitro transcription, RNA extraction, DNase I digestion, reverse transcription, real-time quantitative PCR, cell transfection, live cell fluorescence quantitative assay, dual-luciferase activity assay, and single-factor ANOVA, shall be used.

[0010] Unless specifically defined herein, the use of all commercially available products herein employs standard techniques. For example, it may be carried out using the manufacturer's instructions for use with the kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein can generally be implemented according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0011] The terms “optional / arbitrary” or “optionally / arbitrarily” mean that the event or situation described below may or may not occur, including both the occurrence and non-occurrence of the event or situation.

[0012] The term "circular RNA" as used in this article refers to a covalently closed circular RNA molecule formed by backsplicing of precursor mRNA. It lacks a 5' cap structure and a 3' polyadenylated tail and can achieve translational expression of the target protein by inserting into an internal ribosome entry site.

[0013] The term “RNAseK” used in this article refers to a membrane-integrated endoribonuclease widely found in eukaryotic cells that can specifically recognize specific nucleic acid motifs on circular RNA and mediate their degradation. Knocking down this protein can upregulate the expression of circular RNA containing the corresponding motif.

[0014] The term "lysosome" as used in this article refers to an organelle in eukaryotic cells that contains a variety of acidic hydrolases. Nucleases such as RNase 1 inside the lysosome can specifically recognize specific motifs on circular RNA and mediate their degradation. Inhibiting their activity can upregulate the expression level of circular RNA containing the corresponding motif.

[0015] The term "nucleic acid motif" as used in this article refers to a conserved segment of a nucleic acid sequence that has a specific function. In this article, it specifically refers to a specific sequence segment that can be recognized by RNAseK or nucleases in lysosomes, including AG-rich motifs and CU-rich motifs and / or motifs containing CA base pairs.

[0016] The term "synonymous mutation" as used in this article refers to a base substitution in a DNA sequence without altering the amino acid sequence encoded by the target gene or the structure and function of the protein, in order to circumvent the recognition of specific nucleic acid motifs by enzymes that are involved in their degradation.

[0017] The term "type I self-splicing intron" as used in this article refers to a class of introns with self-splicing activity, which can complete splicing through transesterification without the participation of proteins. They are often used to mediate the circularization of target RNA in vitro. In this article, it specifically refers to type I introns derived from pre-tRNA of the genus Anabaena.

[0018] The term “internal ribosome entry site (IRES)” used in this article refers to a nucleic acid sequence that enables ribosomes to initiate translation inside mRNA. It can initiate protein synthesis without the need for a 5' cap structure and is a key element for circular RNA to achieve the expression of target proteins.

[0019] The term "MEME software" as used in this article refers to a bioinformatics tool used to identify conserved nucleic acid motifs or amino acid motifs from biological sequences, which can predict statistically significant conserved motifs by analyzing multiple sequence alignment results.

[0020] The term "GFP" used in this article refers to Green Fluorescent Protein, whose encoding gene can be used as a reporter gene. The expression level of the target gene can be reflected by detecting the fluorescent signal produced by its expression.

[0021] The term "FLuc (Firefly Luciferase)" used in this article refers to firefly luciferase, whose encoding gene can be used as a reporter gene. The expression level of the target gene can be reflected by detecting its activity in catalyzing luciferin luminescence.

[0022] The term "mCherry" used in this article refers to a red fluorescent protein whose encoding gene can be used as an internal reference reporter gene to normalize the expression detection results of the target gene.

[0023] The term "RLuc (Renilla Luciferase)" used in this article refers to Renilla luciferase, whose encoding gene can be used as an internal reference reporter gene to normalize the activity detection results of firefly luciferase.

[0024] The term "T7 promoter" as used in this article refers to a DNA sequence that can be specifically recognized and transcribed by T7 RNA polymerase, and is commonly used to initiate RNA synthesis in in vitro transcription systems.

[0025] The term "polyA sequence" as used in this article refers to a nucleic acid sequence consisting of multiple adenine nucleotides, used here as a label for linear RNA to facilitate the separation and purification of circular RNA from linear RNA.

[0026] The term "pcDNA3.0 plasmid" as used in this article refers to a commonly used cloning vector that can be used to construct protein expression plasmids.

[0027] The term "pUC57 plasmid" as used in this article refers to a commonly used cloning vector containing restriction enzyme sites such as EcoRI and HindIII, which can be used to construct in vitro transcription template plasmids.

[0028] The term "in vitro transcription" as used in this article refers to the technique of synthesizing RNA using DNA as a template by RNA polymerase in vitro, simulating the in vivo transcription process. In this article, it is used for the synthesis of circular RNA precursors.

[0029] The term "real-time quantitative PCR (qPCR)" as used in this article refers to a technique that detects the intensity of fluorescence signals in real time during a PCR reaction, thereby enabling the quantitative analysis of target nucleic acid fragments. It is commonly used in 2... - Ct The relative expression level of the target gene is calculated using this method.

[0030] The term "Ct value" used in this article refers to the number of cycles in a real-time quantitative PCR reaction at which the fluorescence signal reaches a set threshold, and can be used to quantitatively analyze the initial concentration of the target nucleic acid.

[0031] The term "2" used in this article - Ct "Quantitative real-time PCR" refers to a method used for analyzing the results of quantitative real-time PCR, which quantifies the relative expression level of the target gene by calculating the fold increase of the target gene relative to the internal reference gene.

[0032] The term "dual luciferase reporter gene assay" used in this article refers to a method that uses firefly luciferase (FLuc) and kidney luciferase (RLuc) as reporter genes and measures the ratio of their activities to quantitatively analyze the expression or regulation efficiency of the target gene.

[0033] The term "DNase I" as used in this article refers to an endonuclease that degrades double-stranded or single-stranded DNA and is used to remove residual DNA impurities after RNA extraction.

[0034] The term "reverse transcription" as used in this article refers to the process of synthesizing cDNA using RNA as a template under the action of reverse transcriptase. It is often used to convert RNA templates into DNA templates for PCR amplification.

[0035] The term "Random primer" as used in this article refers to a random primer that can bind to any sequence of an RNA template to initiate a reverse transcription reaction to synthesize cDNA.

[0036] The term "one-way ANOVA test" used in this article refers to a statistical method used to analyze the significance of differences between multiple sample groups. In this article, it is used to test whether the differences in RNA expression levels, fluorescence intensity, or enzyme activity among different treatment groups are statistically significant.

[0037] As used in this article, the term "N" refers to any nucleotide base, including adenine (A), guanine (G), cytosine (C), and uracil (U, in RNA) or thymine (T, in DNA).

[0038] The term "sequence optimization" used in this article refers to codon modification techniques targeting specific nucleic acid motifs within the open reading frame of a target gene. By using synonymous mutations without altering the amino acid sequence of the target protein, conserved sequence fragments that can be recognized by specific nucleases are modified, thereby increasing the accumulation level of circular RNA transcribed from the gene in the cell and improving the translation efficiency of the target protein.

[0039] The term "HEK293F cell" used in this article refers to the human embryonic kidney cell line, which is commonly used in experimental studies such as gene expression and protein purification.

[0040] The term "HEK293T cell" used in this article refers to the human embryonic kidney cell line, which is commonly used in experimental studies such as gene expression and cell transfection.

[0041] The term "HeLa cell" used in this article refers to a human cervical cancer cell line, which is commonly used in molecular biology and cell biology-related experimental research.

[0042] The term "N2a cell" used in this article refers to the mouse neuroblastoma cell line, which is commonly used in experimental studies related to the nervous system.

[0043] The term "3T3-L1 cells" used in this article refers to the mouse embryonic fibroblast cell line, which is commonly used in experimental studies related to cell differentiation and metabolism.

[0044] The term "SEQ ID NO." used in this article refers to the unique number of each nucleic acid or amino acid sequence in the sequence listing, which is used to clearly distinguish different sequence fragments and facilitate the description and implementation of technical solutions.

[0045] The term “open reading frame (ORF)” as used in this article refers to a continuous nucleic acid sequence in a gene, from the start codon to the stop codon, that encodes a complete protein.

[0046] The term "primer" as used in this article refers to a short nucleic acid fragment complementary to the template nucleic acid sequence, which can initiate DNA synthesis under the action of DNA polymerase and is commonly used in techniques such as PCR amplification and real-time quantitative PCR.

[0047] The term "internal control" as used in this article refers to a reference indicator used in experiments to calibrate errors and normalize detection results. In this article, it specifically refers to circular RNA expressing mCherry or RLuc, used to correct cell transfection efficiency and detection system errors.

[0048] The term "WT" as used in this article refers to the wild type, specifically the original GFP or FLuc gene sequence that has not undergone synonymous mutations in its nucleic acid motif.

[0049] The term "Mut1" as used in this article refers to the gene sequence resulting from a synonymous mutation targeting only RNAseK-related nucleic acid motifs (type 1 nucleic acid motifs).

[0050] The term "Mut2" as used in this article refers to a gene sequence resulting from synonymous mutations targeting only lysosomal and RNase 1-related nucleic acid motifs (type II nucleic acid motifs).

[0051] The term "Dual-mut" as used in this article refers to a gene sequence resulting from simultaneous synonymous mutations of both RNAseK-related nucleic acid motifs (type I nucleic acid motifs) and lysosomal and RNase 1-related nucleic acid motifs (type II nucleic acid motifs).

[0052] The term "circGFP" as used in this article refers to a circular RNA containing a GFP coding sequence, which can initiate the translational expression of the GFP protein via an IRES sequence.

[0053] The term "circFLuc" used in this article refers to a circular RNA containing the FLuc coding sequence, which can initiate the translational expression of firefly luciferase via the IRES sequence.

[0054] The term "circmCherry" used in this article refers to a circular RNA containing the mCherry coding sequence, which was used as an internal control to normalize the GFP expression detection results.

[0055] The term "circRLuc" used in this article refers to a circular RNA containing the RLuc coding sequence, used as an internal control to normalize the activity assay results of FLuc.

[0056] In a first aspect, the present invention provides a sequence optimization method for improving the stability of circular RNA.

[0057] This method involves synonymous mutation or removal of one or more nucleic acid motifs to be optimized contained in the target gene of circular RNA, wherein the nucleic acid motifs to be optimized are Class I nucleic acid motifs and / or Class II nucleic acid motifs.

[0058] The first type of nucleic acid motif is an AG-rich motif that can be recognized and mediated to degrade by RNAseK.

[0059] The second type of nucleic acid motif is a CU-rich motif and / or containing CA base pairs that can be recognized and mediated to degrade by lysosomes or their internal RNase 1.

[0060] The first type of nucleic acid motif includes one or more selected from AGAGNAGNAG, AGAG, AGNAG, AGNNAG, and AGNAGNAG.

[0061] The preferred nucleic acid motifs for the first type are AGAGNAGNAG, AGAG, and AGNAG.

[0062] Among them, the first type of nucleic acid motif is further preferably AGAGNAGNAG.

[0063] The second type of nucleic acid motif includes one or more selected from CANNCUGCUG, CUGCUG, CANGCUG, CANCUGC, CANUGCU, and CANUGCA.

[0064] The preferred second type of nucleic acid motif is CANNCUGCUG, CUGCUG, or CANGCUG.

[0065] The second type of nucleic acid motif is further preferably CANNCUGCUG.

[0066] The synonymous mutation is a codon substitution that does not change the amino acid sequence of the target protein.

[0067] Based on a further solution to the technical problem of the present invention, a preferred embodiment of the technical solution provided in the first aspect of the present invention includes: The first preferred option: The first type of nucleic acid motif includes one or more selected from AGAGNAGNAG, AGAG, AGNAG, AGNNAG, and AGNAGNAG. This option, based on modifying AG-rich motifs to resist RNAseK degradation, further provides experimentally identified specific motif sequences that are efficiently recognized by RNAseK, making sequence optimization more targeted and effective.

[0068] The second preferred option: The second type of nucleic acid motif includes one or more selected from CANNCUGCUG, CUGGCUG, CANGCUG, CANCUGC, CANUGCU, and CANUGCA. This option, based on modifying CU-rich motifs to resist lysosomal degradation, further provides experimentally identified specific motif sequences that are efficiently recognized by lysosomal RNase 1, thereby achieving more precise evasion of the lysosomal degradation pathway.

[0069] The third preferred option is that the nucleic acid motif to be optimized includes both the first type of nucleic acid motif and the second type of nucleic acid motif, and both are subjected to synonymous mutations or removal. This option, while improving resistance to a single degradation pathway, further achieves a synergistic enhancement of circular RNA stability by simultaneously modifying the key recognition motifs of the two degradation mechanisms, resulting in better intracellular accumulation and protein expression effects compared to modifying only a single motif type.

[0070] Secondly, the present invention provides a template DNA molecule for preparing circular RNA.

[0071] The template DNA molecule optimizes the circular RNA sequence in the circular RNA template DNA using the sequence optimization method described in any one of the first aspects, and is used to generate the target circular RNA in vitro or in vivo.

[0072] Thirdly, the present invention provides a sequence-optimized circular RNA.

[0073] The circular RNA contains the target gene sequence optimized by the sequence optimization method described in any of the first aspects.

[0074] Fourthly, the present invention provides the application of the circular RNA described in the third aspect.

[0075] This application is selected from applications in gene expression regulation, protein synthesis, or vaccine development.

[0076] Fifthly, the present invention provides a pharmaceutical composition.

[0077] The pharmaceutical composition contains the circular RNA described in the third aspect.

[0078] The pharmaceutical composition further includes a pharmaceutical carrier and / or pharmaceutically acceptable excipients.

[0079] The pharmaceutical carrier is selected from one or more of lipid nanoparticle delivery systems, liposome carriers, viral carriers, polymer carriers, and inorganic nanocarriers.

[0080] The pharmaceutical carrier is preferably a lipid nanoparticle delivery system, a liposome carrier, or a viral carrier.

[0081] The pharmaceutical carrier is further preferably a lipid nanoparticle delivery system.

[0082] The pharmaceutically acceptable excipients are selected from one or more of the following: fillers, binders, disintegrants, lubricants, solubilizers, stabilizers, and preservatives.

[0083] The dosage form of the pharmaceutical composition is one of injection, gel, or cream.

[0084] The preferred dosage form is an injection.

[0085] Sixthly, the present invention provides a method for preparing a vaccine.

[0086] This method optimizes the gene encoding the antigen protein using any of the sequence optimization methods described in the first aspect, and then constructs a circular RNA vaccine.

[0087] In a seventh aspect, the present invention provides a vaccine.

[0088] The vaccine comprises the circular RNA, vaccine vector, and optional vaccine excipients described in the third aspect.

[0089] The vaccine carrier is selected from one or more of lipid nanoparticles, liposomes, polymer nanoparticles, virus-like particles, inorganic nanocarriers, or aluminum adjuvants.

[0090] The vaccine carrier is preferably a lipid nanoparticle.

[0091] The vaccine excipients are selected from one or more of adjuvants, buffer salts, sugar stabilizers, surfactants, acid-base regulators, or preservatives.

[0092] The adjuvant is selected from one or more of aluminum salt adjuvants, MF59, AS01, AS03, AS04, CpG oligonucleotides, Poly I:C, or cytokines.

[0093] The buffer salt is selected from phosphate buffer salt, Tris buffer salt, histidine buffer salt or citrate buffer salt.

[0094] The sugar stabilizer is selected from sucrose, trehalose, or mannitol.

[0095] The surfactant is selected from polysorbate 80, polysorbate 20, poloxamer 188 or polyethylene glycol-phospholipid.

[0096] The acid-base regulator is selected from sodium hydroxide, hydrochloric acid, citric acid, acetic acid, or a combination thereof.

[0097] The preservative is selected from phenol, m-cresol, benzyl alcohol, methylparaben, propylparaben, or thimerosal.

[0098] Eighthly, the present invention provides the use of the circular RNA described in the third aspect for the treatment or prevention of protein expression-related diseases.

[0099] More preferably, the protein expression-related disease refers to a disease that can be treated or prevented by expressing functional proteins in the patient's cells.

[0100] The protein expression-related diseases mentioned above are selected from: Protein dysfunction or defect diseases; or Infectious diseases that require the expression of exogenous antigens to trigger protective immunity.

[0101] The protein function loss or defect disease includes genetic diseases, certain metabolic diseases, or acquired diseases caused by a deficiency of a specific protein.

[0102] The hereditary disease is selected from hemophilia, α1-antitrypsin deficiency, cystic fibrosis, or Duchenne muscular dystrophy.

[0103] The infectious disease is caused by a virus or bacteria.

[0104] The present invention has at least the following beneficial effects: 1. Compared with existing technologies, this invention has better technical effects in terms of intracellular RNA expression level of circular RNA, expression efficiency of target protein, and duration of action in vivo. According to experimental tests, after transfecting HeLa cells with Dual-mut circGFP of this invention for 48 hours, the RNA expression level was more than 3 times higher than that of wild-type (WT), and the GFP fluorescence intensity was more than 1.8 times higher; after transfecting N2a cells with Dual-mut circFLuc for 48 hours, the RNA expression level was more than 2.5 times higher than that of WT, and the luciferase activity was more than 2 times higher.

[0105] 2. Compared with existing technologies, this invention provides a technical solution with a different technical concept, and its technical effect is significantly superior to existing technologies. The difference between the technical concept of this invention and existing technologies lies in the fact that existing technologies focus on the circularization efficiency of circular RNA or the optimization of delivery systems, without designing for intracellular specific degradation mechanisms; while this invention, by identifying specific motifs recognized by RNAseK and lysosomes, uses synonymous mutations for precise modification, fundamentally solving the degradation problem at the sequence level, making the technical concept more targeted and innovative. Attached Figure Description

[0106] Figure 1 The figures show the verification results of RNAseK and lysosomal RNase 1 protein in vitro degrading circular RNA; where A is the result of RNAseK in vitro degrading circular RNA in Example 1; and B is the result of lysosomal RNase 1 in vitro degrading circular RNA in Example 1.

[0107] Figure 2 The sequence characteristics of the circular RNA in Example 2 are shown below; where A is the nucleic acid sequence preferentially regulated by the RNAseK protein; and B is the nucleic acid sequence preferentially regulated by the lysosome.

[0108] Figure 3 The diagrams show the GFP or FLuc sequence mutation and in vitro circularization in Example 3; where A is a diagram of synonymous mutation of the DNA sequence of GFP or FLuc targeting the first type of nucleic acid motif and / or the second type of nucleic acid motif; and B is a diagram of circularization for in vitro synthesis of circGFP / circFLuc RNA.

[0109] Figure 4 In Example 3, gel images of the in vitro synthesized circGFP / circFLuc and their corresponding sequence-optimized circular RNAs, as well as the purified RNA recovered from their corresponding internal control circmCherry / circRLuc.

[0110] Figure 5 In Example 4, the expression levels of circGFP or circFLuc and their corresponding sequence-optimized circular RNAs were detected at the RNA level 48 hours after transfection into cells. Among them, A is the qPCR result of RNA expression level after transfection of human HeLa cells or mouse N2a cells with sequence-optimized circGFP; B is the qPCR result of RNA expression level after transfection of human HeLa cells or mouse N2a cells with sequence-optimized circFLuc.

[0111] Figure 6 The results of fluorescence intensity detection on the first to fifth days after transfecting human HeLa cells with circGFP and its corresponding sequence-optimized circular RNA in Example 4 are shown.

[0112] Figure 7 In Example 4, the fluorescence intensity decay kinetics and linear regression analysis were performed on the first to fifth days after transfecting human HeLa cells with circGFP and its corresponding sequence-optimized circular RNA.

[0113] Figure 8This is the quantitative result of GFP protein fluorescence intensity 48 hours after transfecting human HeLa cells or mouse N2a cells with circGFP and its corresponding sequence-optimized circular RNA, as shown in Example 4. The scale bar is 50 μm.

[0114] Figure 9 The results of luciferase activity detection in human HeLa cells or mouse N2a cells 48 hours after transfecting circFLuc and its corresponding sequence-optimized circular RNA in Example 4 are shown. Detailed Implementation

[0115] Unless otherwise specified, all raw materials and reagents used in this invention were purchased from commercial suppliers, and experiments were conducted in accordance with the operating instructions. Unless otherwise specified, all instruments, equipment, and apparatus used in this invention are conventional instruments, equipment, and apparatus, and experiments were conducted in accordance with the operating instructions and the accompanying reagents.

[0116] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Unless otherwise specified in the embodiments, conditions are performed under conventional conditions or conditions recommended by the manufacturer. All reagents or instruments without specified manufacturers are commercially available conventional products. Numerous specific details are provided in the following detailed embodiments to better illustrate the invention. The specific embodiments described herein are for illustrative purposes only and are not intended to constitute any limitation on the invention.

[0117] Data analysis and statistical analysis were performed using professional data processing software. One-way ANOVA was used for significance analysis, and P<0.05 was considered to indicate a significant difference.

[0118] Example 1: In vitro degradation of circular RNA by RNAseK and lysosomal RNase 1 1.1 Experimental Methods 1.1.1 Expression and purification of human FLAG-tagged RNAseK protein (1) Expression of RNAseK protein: Using pcDNA3.0 plasmid as backbone, plasmids were constructed to overexpress human RNAseK protein with 3×FLAG tag (WT RNAseK, SEQ ID NO.1) or RNAseK protein mutant (lysine at position 10 is mutated to alanine, K10A RNAseK, SEQ ID NO.2). The above plasmids were transfected into HEK293F cells. 10 mM sodium butyrate was added 12 hours after transfection, and the cells were cultured at 30℃ for 48 hours. The cells were then collected by centrifugation.

[0119] (2) Purification of RNAseK protein: Lysis buffer (50 mM Tris-HCl pH8.0, 150 mM NaCl, 20% glycerol, 1× protease inhibitor, 1% dodecyl-β-D-maltodextrin, 2% cholesterol succinate, 5 mM MgCl2 and 5 mM ATP) was added to the cell pellet, and the cells were lysed at 4°C for 2 hours. Then, the pellet was centrifuged at 45,000 rpm for 45 minutes and the pellet was discarded. The supernatant was incubated with a FLAG gel to enrich the FLAG-tagged RNAseK protein. The RNAseK protein was eluted with 3×FLAG peptides. The eluent was concentrated to 1 mL through a 10 kDa ultrafiltration tube and then separated by a Superdex® 200Increase 10 / 300 column (Cytiva) molecular sieve to obtain human WT or K10A RNAseK protein with 3×FLAG tag.

[0120] 1.1.2 In vitro circularization and purification of circular RNA Circular RNAs circIARS (SEQ ID NO.3) and circANKRD11 (SEQ ID NO.4), which are specifically regulated by the RNAseK protein in cells, were selected as substrates for the RNAseK in vitro reaction; circILKAP (SEQ ID NO.5) and circFANCA (SEQ ID NO.6), which are specifically regulated by the RNase 1 protein, were selected as substrates for the RNase 1 in vitro reaction. After PCR amplification and recovery, the linearized transcription templates of the above circular RNAs with T7 promoters and initiation in guanine were obtained. The linearized precursors of the circular RNAs were synthesized in vitro using a T7 transcription kit (Thermo Scientific, K0441).

[0121] Table 1 Mixture

[0122] After reacting the mixture in Table 1 at 37°C for 4 hours, DNase I was added to remove the template DNA, terminating the reaction. The transcribed RNA was then purified using TRIzol reagent. The linearized precursor of the circular RNA was obtained and circularized overnight at 16°C using T4 RNA ligase 1. The circular RNA and linear RNA were separated by urea denaturing gel electrophoresis, and the circular RNA was recovered by gel excision for subsequent experiments.

[0123] 1.1.3 In vitro experiments with RNAseK protein Human WT RNAseK and K10A RNAseK proteins were reacted in the reaction systems shown in Table 2: Table 2 Reaction System

[0124] The 10× Reaction Buffer contains: 500 mM Tris-HCl pH 8.0, and 1.5 M NaCl. Since RNAseK protein is known to be unaffected by human placental RNase inhibitors, an RNase inhibitor was added to the RNAseK protein in vitro reaction system to exclude the influence of other RNases.

[0125] After reacting the reaction system in Table 2 and the enzyme-free control at 37°C for 30, 60, or 90 minutes, an equal volume of 2×RNA loading was added and denatured in a 70°C metal bath for 10 minutes to terminate the reaction. Finally, the samples were subjected to gel electrophoresis and GelRed staining, photographed using a gel imaging system (Tanon 1600), and the digestion results were quantitatively analyzed using ImageJ software.

[0126] 1.1.4 In vitro experiments with RNase 1 protein The human RNase 1 protein (MCE, HY-P71089; SEQ ID NO.7) was reacted in the reaction system shown in Table 3: Table 3 Reaction System

[0127] The 10× Reaction Buffer contains: 500 mM Tris-HCl pH 8.0 and 1.5 M NaCl.

[0128] After reacting the reaction system in Table 3 and the enzyme-free control (No Enzyme) at 37℃ for 30, 60, or 90 minutes, an equal volume of 2×RNA loading was added and denatured in a 70℃ metal bath for 10 minutes to terminate the reaction. Finally, the samples were subjected to gel electrophoresis and GelRed staining, photographed using a gel imaging system (Tanon 1600), and the digestion results were quantitatively analyzed using ImageJ software.

[0129] 1.2 Experimental Results like Figure 1 As shown in Figure A, WT RNAseK protein effectively degraded circIARS and circANKRD11 in vitro, while K10A RNAseK protein lost some RNase activity and its ability to degrade circular RNA was significantly reduced, further supporting the effectiveness of WT RNAseK in degrading circular RNA.

[0130] like Figure 1As shown in B, lysosomal RNase 1 protein effectively degraded circFANCA and circILKAP in vitro.

[0131] Example 2: Characteristics of circular RNA sequences regulated by RNAseK and lysosomes 2.1 Experimental Methods Circular RNA sequence prediction: The RNA sequences of circular RNAs whose expression levels were upregulated after knocking down RNAseK protein or inhibiting lysosomal activity were obtained from human and mouse cell lines (human: HeLa and HEK293T; mouse: N2a and 3T3-L1) using high-throughput sequencing of circular RNAs. The nucleic acid sequences were then predicted using MEME software with default parameters.

[0132] 2.2 Experimental Results like Figure 2 As shown in A, in the four cell lines, the circular RNA sequences upregulated after knockdown of RNAseK contained AG-rich sequences, which are summarized as the first type of nucleic acid motif: referring to AG-rich nucleic acid motifs that can be specifically recognized by the ribonuclease RNAseK and mediate the degradation of circular RNA, corresponding to AGAGNAGNAG, AGAG, AGNAG, AGNNAG, and AGNAGNAG, with the core sequence being AGAGNAGNAG (N represents any base of A, U, G, or C). like Figure 2 As shown in B, among the four cell lines, the circular RNA sequences upregulated after inhibiting lysosomal activity contained sequences rich in CU. At the same time, based on the known preference of lysosomal RNase 1 for CA base pairs, it is summarized as a second type of nucleic acid motif: a nucleic acid motif rich in CU and containing CA base pairs that can be specifically recognized by lysosomes (or their internal RNase 1) and mediate the degradation of circular RNA, corresponding to CANNCUGCUG, CUGGCUG, CANGCUG, CANCUGC, CANUGCU, and CANUGCA, with the core sequence being CANNCUGCUG (N represents any of the bases A, U, G, and C).

[0133] Example 3: Optimization and in vitro circularization of GFP / Firefly Luciferase sequence 3.1 Experimental Methods 3.1.1 Optimization of GFP / Firefly Luciferase sequence (1) Within the open reading frame region of GFP, search for nucleic acid motifs representing the first and second classes of nucleic acid motifs, and perform synonymous mutations on them. Among them, the GFP sequence without mutation is WT GFP (SEQ ID NO.8), the GFP sequence with mutations only targeting RNAseK-related motifs (first class of nucleic acid motifs) is Mut1 GFP (SEQ ID NO.9), the GFP sequence with mutations only targeting lysosome and RNase 1-related motifs (second class of nucleic acid motifs) is Mut2 GFP (SEQ ID NO.10), and the GFP sequence with mutations targeting both classes of motifs is Dual-mut GFP (SEQ ID NO.11).

[0134] (2) Within the open reading frame region of firefly luciferase (FLuc), search for the first and second class nucleic acid motifs and perform synonymous mutations on them. Among them, the unmutated FLuc sequence is WT FLuc (SEQ ID NO.12), the FLuc sequence with mutations only targeting RNAseK-related motifs (first class nucleic acid motifs) is Mut1 FLuc (SEQ ID NO.13), the FLuc sequence with mutations only targeting lysosome and RNase 1-related motifs (second class nucleic acid motifs) is Mut2 FLuc (SEQ ID NO.14), and the FLuc sequence with mutations targeting both classes of motifs is Dual-mut FLuc (SEQ ID NO.15).

[0135] (3) The optimized gene sequence was obtained by chemical synthesis by the sequencing company.

[0136] 3.1.2 A method for circular RNA circularization mediated by introns of type I self-splicing in the genus Anabaena. (1) Construct template plasmids for in vitro circularization.

[0137] Following the order of T7 promoter, polyA sequence, Anabaena pre-tRNA-Leu type I intron and its exon 1 (Exon1) sequence, IRES sequence, conventional GFP / Firefly luciferase sequence or GFP / Firefly luciferase sequence synthesized according to 3.1.1, and Anabaena pre-tRNA-Leu exon 2 (Exon2) sequence, the above sequences are inserted between the EcoR I and Hind III restriction sites of the pUC57 plasmid to obtain a template plasmid for transcribing RNA that needs to be circularized.

[0138] In addition, this embodiment also uses the mCherry open reading frame sequence (SEQ ID NO.16) and the Renilla Luciferase (RLuc. SEQ ID NO.17) open reading frame sequence to construct a template plasmid for in vitro circularization, so as to generate IRES-initiated circular RNA expressing mCherry (circmCherry) and RLuc (circRLuc), which serves as an internal reference for cell transfection and subsequent experiments.

[0139] (2) In vitro synthesis of linear precursors of circular RNA The template plasmid was amplified and recovered by PCR, and the purified linearized transcription template was obtained. Circular RNA precursor RNA was synthesized in vitro using the T7 transcription kit (Thermo Scientific, K0441).

[0140] Table 4 Mixture

[0141] After reacting the mixture in Table 4 at 37°C for 4 hours, DNase I was added to remove the template DNA, thus terminating the reaction. The transcribed RNA was then purified using TRIzol reagent (Invitrogen, 15596018CN).

[0142] (3) In vitro cyclization reaction The transcribed linear RNA was placed in a cyclization buffer (10 mM MgCl2, 50 mM Tris-HCl, pH 7.5, 1 mM DTT) and reacted at 55°C for 15 minutes.

[0143] (4) Purification of cyclization reaction products After the cyclization reaction was complete, enzyme-free water was added to a final volume of 100 µL, and the product was transferred to a new EP tube. 0.5 times the volume of 7.5 M lithium chloride was added, and the tube was incubated at -20°C for at least 30 minutes to precipitate. Then, the tube was centrifuged at 12000 g for 15 minutes at 4°C, the supernatant was discarded, and 1 mL of 80% ethanol was added for inverted washing. The tube was then centrifuged again at 12000 g for 5 minutes at 4°C, the supernatant was discarded, and the tube was inverted and air-dried for 10 minutes before adding an appropriate amount of enzyme-free water to dissolve the RNA.

[0144] (5) Isolation and purification of circular RNA The circular RNA obtained from in vitro circularization needs to be further separated using a Sepax Monomix dT20 affinity resin column (Sepax, 2221509D0). After preparing the column according to the affinity resin column instructions, 1 mL of purified circularization product (containing 500 mM NaCl, 10 mM EDTA, pH 7.5) was injected into the column using a syringe. Since linear RNA other than circular RNA carries a polyA sequence, it will be captured by the dT20 affinity resin. Therefore, the circularization product can be preferentially eluted with elution buffer (100 mM Tris-HCl, 250 mM NaCl, 1 mM EDTA, pH 7.5), while the linear RNA will be eluted with enzyme-free water.

[0145] The elution fraction containing circular RNA was purified by lithium chloride precipitation and dissolved in enzyme-free water to obtain in vitro synthesized circular GFP (circGFP), circular mCherry (circmCherry), circular Fireflyluciferase (circFLuc), and circular Renilla luciferase (circRLuc) RNAs containing the IRES sequence, which were used for subsequent experiments.

[0146] 3.2 Experimental Results like Figure 3 As shown in A, the sequence involves searching for a Class I nucleotide motif, or a DNA sequence corresponding to a Class II nucleotide motif, within the GFP or Firefly Luciferase gene sequence, and then performing a synonymous mutation on it. WT represents the unmutated GFP or Firefly Luciferase sequence, Mut1 represents the sequence with a synonymous mutation targeting a Class I nucleotide motif, Mut2 represents the sequence with a synonymous mutation targeting a Class II nucleotide motif, and Dual-mut represents the sequence with synonymous mutations targeting both Class I and Class II nucleotide motifs.

[0147] like Figure 3 Figure B in the diagram illustrates a schematic of the cyclization method mediated by introns of the type I self-splicing of Anabaena.

[0148] like Figure 4 The schematic diagram shown in Figure A and the gel image of its cyclization product identification were obtained after in vitro cyclization and purification. Four circular RNAs expressing GFP protein or four circular RNAs expressing FLuc (WT, Mut1, Mut2, Dual-mut) were obtained.

[0149] like Figure 4 As shown in B, internal reference circular RNAs expressing mCherry and RLuc were obtained.

[0150] Example 4: Expression detection of circGFP / circFLuc in cells after sequence optimization 4.1 Experimental Methods 4.1.1 Transfection of HeLa cells or N2a cells Four types of circular RNA expressing GFP protein or four types expressing Luciferase (WT, Mut1, Mut2, Dual-mut) were transfected into HeLa cells (ATCC, CCL-2) or N2a cells (ATCC, CCL-131) using Lipofectamine 2000 reagent. Detailed steps are as follows: (1) Passage the cells one day before transfection and seed them into a six-well plate so that the cells are in the logarithmic phase and the density is about 60%-70% when transfected the next day.

[0151] (2) Take 5 µL of Lipofectamine 2000 and add it to 125 µL of OPTI-MEM. Mix well and let stand for 5 minutes.

[0152] (3) Take 3 nM circular RNA (1.5 nM each of circGFP and circmCherry; 1.5 nM each of circFLuc and circRLuc; calculated based on a final volume of 1 mL) and add it to 125 µL OPTI-MEM. After mixing, add it to the mixture containing Lipofectamine 2000 in step (2), mix gently, and let stand for 20 minutes.

[0153] (4) Remove the old culture medium from the cells to be transfected, and add 750 µL of fresh culture medium without streptomycin / penicillin and the mixture of RNA and Lipofectamine 2000 from step (3). Gently shake well and place in a 37°C cell culture incubator for further culture.

[0154] (5) After 6 hours, the culture medium in the transfected cells was replaced with a new complete culture medium and cultured for another 48 hours before proceeding with subsequent experiments.

[0155] 4.1.2 Total RNA extraction All RNA extraction procedures used RNase-free reagents and consumables, and the workbench was wiped with 3% hydrogen peroxide.

[0156] (1) Remove the culture medium from the cells, add 1 mL of PBS buffer to wash the cells twice, remove the PBS, and add 1 mL of TRIzol reagent.

[0157] (2) After the cells were fully lysed by pipetting, they were transferred to a 1.5 mL centrifuge tube and placed on a vertical mixer to continue lysis for 10 minutes.

[0158] (3) Add 200 µL of chloroform to the centrifuge tube, shake vigorously for 30 seconds to mix it, let it stand for 5 minutes, and then centrifuge at 12000 g for 15 minutes in a centrifuge at 4℃.

[0159] (4) Carefully aspirate the upper aqueous phase from the centrifuge tube into a new 1.5 mL centrifuge tube, add an equal volume of isopropanol, mix well, and precipitate at -20℃ for 30 minutes.

[0160] (5) Centrifuge at 12000 g for 15 minutes in a centrifuge at 4℃, discard the supernatant, add 1 mL of 80% ice ethanol and wash the RNA precipitate by inverting the centrifuge tube, centrifuge at 7500 g for 5 minutes in a centrifuge at 4℃, discard the supernatant again, and then invert the centrifuge tube to dry for 10 minutes or more.

[0161] (6) Add the DNA digestion system (39 µl enzyme-free water, 1 µL RNase inhibitor, 5 µL DNase I, 5 µL DNase Buffer) to the dried centrifuge tube, mix gently, and digest in a 37°C water bath for 30 minutes. After digestion, add 5 µL 0.5 M EDTA and place in a 70°C metal bath to terminate the digestion. Then add 45 µl enzyme-free water, 10 µL 3 M sodium acetate, and 250 µL anhydrous ethanol, mix well, and place in a -80°C environment to precipitate RNA again.

[0162] (7) Centrifuge at 12000 g for 15 minutes in a centrifuge at 4℃, discard the supernatant, add 1 mL of 80% ice ethanol and wash the RNA precipitate by inverting the centrifuge tube, centrifuge at 7500 g for 5 minutes in a centrifuge at 4℃, discard the supernatant again, invert the centrifuge tube and air dry for 10 minutes or more, then add an appropriate amount of enzyme-free water and place it on ice to completely dissolve the RNA.

[0163] 4.1.3 Obtaining cDNA via reverse transcription RNA reverse transcription was performed according to the instructions of the ABScript II cDNA First Strand Synthesis Kit (ABclonal, RK20400), using RNase-free reagents and consumables throughout the process, as detailed below: (1) Prepare the reaction system in Table 5 in a 200 µL centrifuge tube, mix well, and denature in a metal bath at 65℃ for 5 minutes.

[0164] Table 5 Reaction System

[0165] (2) After cooling on ice for 2 minutes, add 10 µL of Reaction buffer and 2 µL of Enzyme mix and mix well.

[0166] (3) In a PCR instrument, the reaction was carried out according to the following reaction program: 25℃ for 5 minutes, 42℃ for 1 hour, and 80℃ for 5 minutes. The cDNA product obtained was used for subsequent RNA expression level detection.

[0167] 4.1.4 Real-time quantitative PCR To detect the expression levels of circGFP and circLuciferase in cells, specific primers for quantitative real-time PCR were designed based on their sequences. The primers had a single melting curve, and the Ct value increased linearly with template gradient dilution.

[0168] The primer information involved is shown in Table 6: Table 6 Primer Information

[0169] (1) Prepare the reaction system in Table 7 according to the instructions and mix well.

[0170] Table 7 Reaction System

[0171] (2) Add the above reaction system to a 96-well QPCR plate, add 15 µL to each well, and make three replicates for each sample. After sealing with a sealing film and centrifuging, place the 96-well plate in a real-time PCR instrument and react according to the reaction procedure in Table 8.

[0172] Table 8 Reaction Procedure

[0173] (3) The Ct value obtained after the reaction is completed is used as 2 - Ct The relative expression level of the target gene was calculated using this method.

[0174] 4.1.5 Time-continuous live-cell fluorescence detection (1) Pass HeLa cells one day before transfection and seed them into 12-well plates so that the cells are in the logarithmic phase and the density is about 50%-60% when transfected the next day.

[0175] (2) Mix circGFP and circmCherry in equal proportions (1.5 nM: 1.5 nM, total 3 nM) and transfect the cells. At the same time, set up blank control wells without transfection.

[0176] (3) One day after transfection, wash the cells with 1 mL PBS and repeat twice.

[0177] (4) Using a microplate reader (Molecular Devices, SpectraMax iD5), fluorescence readings of cells were collected under excitation light of 485 nm or 587 nm, respectively, and GFP or mCherry fluorescence signals in five fields of view were collected in each sample well.

[0178] (5) After collection, replace PBS with cell culture medium in a sterile biosafety cabinet and continue to culture in a cell culture incubator. Repeat the above fluorescence reading collection operation every day for two to five days after transfection.

[0179] (6) Data Analysis: GFP fluorescence intensity value = (GFP fluorescence intensity F - blank control group F) / (mCherry fluorescence intensity R - blank control group R).

[0180] Statistical analysis of GFP fluorescence intensity data was performed using R software (version 4.3.1). GFP fluorescence intensity values ​​were first logarithmically transformed (ln value) to linearize the exponential decay process. A linear mixed-effects model was used to analyze the differences in degradation rates among different treatment groups. The degradation rate constant (k) was calculated from the linear regression slope of the logarithmically transformed data, and the half-life (t½) was calculated using the formula t½ = ln(2) / k.

[0181] 4.1.6 Quantitative PCR for live cells (1) Pass HeLa cells or N2a cells one day before transfection and seed them into six-well plates so that the cells are in the logarithmic phase and the density is about 60%-70% when transfected the next day.

[0182] (2) Transfect cells with a mixture of circGFP and circmCherry in equal proportions (1.5 nM: 1.5 nM, total 3 nM); (3) After 48 hours, wash the cells with 1 mL PBS and repeat twice.

[0183] (4) Using a Leica Thunder DMi8 microscope, live cells were photographed under excitation light of 488 nm or 546 nm, and GFP or mCherry fluorescence signals in multiple fields of view were collected in each sample well.

[0184] (5) The GFP or mCherry fluorescence signal in each acquired image was quantified using ImageJ software. Finally, the GFP signal value was normalized by dividing the mCherry signal value.

[0185] 4.1.7 Detection of luciferase activity in fireflies This experiment used a dual-luciferase reporter gene assay kit (Yeasen, 11402ES80) and was conducted in accordance with the instructions.

[0186] (1) Pass HeLa cells or N2a cells one day before transfection and seed them into six-well plates so that the cells are in the logarithmic phase and the density is about 60%-70% when transfected the next day.

[0187] (2) Mix circFLuc and circRLuc in equal proportions (1.5 nM: 1.5 nM, total 3 nM) and transfect cells. After 48 hours, discard the culture medium and wash the cells with ice-cold PBS.

[0188] (3) Add 300 µL of lysis buffer (taking a 12-well plate as an example), lyse on ice for 5 minutes, and then sonicate in an ice-water bath for 30 seconds (over 3 seconds, stop for 6 seconds).

[0189] (4) Set up experimental group, control group (no treatment after transfection and cell lysis) and blank control group (cell lysate without transfection). Take 20 µL of cell lysate into a 96 microplate, and set up 3 replicates for each sample.

[0190] (5) Add 100 µL of firefly luciferase substrate working solution (equilibrate to room temperature) to the experimental group and the blank control group, shake the plate to mix well, and quickly place it in the microplate reader (wavelength 350-700 nm) for detection to obtain experimental group F, control group F and blank control group F.

[0191] (6) Add 100 µL of Renin luciferase substrate working solution (equilibrate to room temperature) to the experimental group and the blank control group, shake the plate to mix well, and quickly place it in the microplate reader (wavelength 350-700 nm) to detect and obtain experimental group R, control group R and blank control group R.

[0192] (7) Data Analysis: Experimental group value = (Experimental group F - Blank control group F) / (Experimental group R - Blank control group R); Control group value = (Control group F - Blank control group F) / (Control group R - Blank control group R); Enzyme activity change factor = experimental group value / control group value.

[0193] 4.2 Experimental Results like Figure 5The qPCR results for A in the figure show that, in both human HeLa cells and mouse N2a cells, compared with WTcircGFP, Mut1circGFP or Mut2circGFP, which are sequence-optimized for the first or second nucleic acid motif according to the present invention, or Dual-mut circGFP, which is sequence-optimized for both nucleic acid motifs simultaneously, exhibit significantly increased RNA expression levels 48 hours after cell transfection, suggesting stronger stability. Furthermore, Dual-mut circGFP, which is sequence-optimized for both nucleic acid motifs simultaneously, showed the highest expression level among the four circular RNAs.

[0194] like Figure 5 The qPCR results for B in the study showed that, compared to WTcircFLuc, Mut1 circFLuc or Mut2 circFLuc, which were sequence-optimized for the first or second type of nucleic acid motifs according to the present invention, or Dual-mut circFLuc, which were sequence-optimized for both types of nucleic acid motifs simultaneously, exhibited significantly higher RNA expression levels 48 hours after cell transfection, indicating stronger stability, whether in human HeLa cells or mouse N2a cells. Furthermore, Dual-mut circFLuc, which was sequence-optimized for both types of nucleic acid motifs simultaneously, showed the highest expression level among the four circular RNAs.

[0195] like Figure 6 The results of live-cell GFP fluorescence showed that after sequence optimization, the expression level of circGFP in cells was significantly increased, and the expression level of Dual-mut circGFP was the highest.

[0196] like Figure 7 The GFP fluorescence intensity decay kinetics and linear regression analysis showed that after sequence optimization, the GFP fluorescence intensity decay rate expressed by circGFP was significantly slower.

[0197] like Figure 8 The results of live-cell GFP fluorescence showed that after sequence optimization, the expression level of circGFP in cells was significantly increased, and the expression level of Dual-mut circGFP was the highest.

[0198] like Figure 9 The results of dual-luciferase activity assays showed that after sequence optimization, the luciferase activity of circFLuc was significantly increased, and the Dual-mut group of fireflies had the highest luciferase activity.

[0199] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A sequence optimization method for improving stability of a circular RNA, characterized by, performing synonymous mutation or removing one or more nucleic acid motifs to be optimized contained in the gene of interest in the circular RNA, wherein the nucleic acid motif to be optimized is a first type of nucleic acid motif and / or a second type of nucleic acid motif; the first type of nucleic acid motif is an AG-rich motif that can be recognized and mediated to be degraded by RNAse K; the second type of nucleic acid motif is a CU-rich motif that can be recognized and mediated to be degraded by lysosome or RNase 1 inside the lysosome and / or contains CA base pair.

2. The sequence optimization method of claim 1, wherein, the first type of nucleic acid motif comprises one or more selected from AGAGNAGNAG, AGAG, AGNAG, AGNNAG and AGNAGNAG.

3. The sequence optimization method of claim 1, wherein, the second type of nucleic acid motif comprises one or more selected from CANNCUGCUG, CUGCUG, CANGCUG, CANCUGC, CANUGCU and CANUGCA.

4. The sequence optimization method of claim 1, wherein, the synonymous mutation is codon substitution that does not change the amino acid sequence of the protein of interest.

5. A template DNA molecule for preparing a circular RNA, characterized by, the sequence of the circular RNA in the circular RNA template DNA is optimized by the sequence optimization method according to any one of claims 1-4, and is used to produce the target circular RNA in vitro or in vivo.

6. A sequence-optimized circular RNA, characterized in that, comprises the sequence of the gene of interest optimized by the sequence optimization method according to any one of claims 1-4.

7. The circular RNA of claim 6 is used in gene expression regulation, protein synthesis or vaccine development.

8. A pharmaceutical composition, characterized by, comprises the circular RNA of claim 6.

9. The pharmaceutical composition of claim 8, wherein, the pharmaceutical composition further comprises a pharmaceutical carrier and / or a pharmaceutically acceptable excipient.

10. The pharmaceutical composition of claim 9, wherein, the pharmaceutical carrier is selected from one or more of a lipid nanoparticle delivery system, a liposome carrier, a viral carrier, a polymer carrier or an inorganic nanocarrier.

11. The pharmaceutical composition of claim 9, wherein, the pharmaceutically acceptable excipient is selected from one or more of a filler, a binder, a disintegrant, a lubricant, a solubilizer, a stabilizer or a preservative.

12. The pharmaceutical composition of claim 8, wherein, the dosage form of the pharmaceutical composition is an injection, a gel or a cream.

13. A method of preparing a vaccine, characterized by, the gene encoding the antigen protein is optimized by the sequence optimization method according to any one of claims 1-4, and is constructed into a circular RNA vaccine.

14. A vaccine comprising a polynucleotide of claim 1.

15. comprises the circular RNA of claim 6, a vaccine carrier and optionally a vaccine excipient.

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