Vector for preparing circular RNA (Ribonucleic Acid) and construction method

Abstract

According to the invention, a structural domain reaction substrate sequence consisting of Exon1 (E1), P1 and Exon2 (E2) sequences of ribozyme is mutated, and E1, P1 and E2 sequences are mutated under the condition of maintaining the structural stability, so that the Azoarcus group I intron ribozyme still has enzyme activity and can maintain the capability of forming circular RNA (Ribose Nucleic Acid). The invention discloses a flexible vector construction method for preparing circular RNA (Ribonucleic Acid) without limitation of a substrate sequence, which comprises the following steps: determining a target to-be-cyclized site sequence NNUNNNN, and segmenting the target to-be-cyclized site sequence NNUNNNN into E1: NNU and E2: NNNN; with E1 and E2 sequences as references, designing IGS sequences to respectively form complementary pairing with E1 and E2; and 5'and 3 'homologous arms, IRES, CDS and other elements are respectively added. The method provided by the invention can be used for preparing the circular RNA for any target sequence, has no residual sequence, and has relatively high cyclization efficiency. The FlexCirc cyclization system designed on the basis of Azoarcus group I intron ribozyme can form the circular RNA, the cyclization substrate sequence has the characteristic of flexible design, and the cyclization efficiency can realize a relatively high cyclization proportion.

Description

Technical Field

[0001] The present invention belongs to the technical fields of molecular biology and bioengineering, and specifically relates to recombinant nucleic acids and vectors for preparing circular RNAs, and methods for preparing circular RNAs. Background Art

[0002] Circular RNAs, as the name implies, are a class of RNA molecules with a circular closed morphological structure and are ubiquitous in nature. Circular RNAs can generally be formed in different organisms through a back-splicing mechanism. Compared with linear mRNAs, circular RNAs have no 5' and 3' ends, and thus can tolerate exonuclease hydrolysis and have relatively high stability. In 2018, an article titled "Engineering circular RNA for potent and stable translation in eukaryotic cells" published by Wesselhoeft R.A in Nature Communication initiated the application of circular RNAs for protein expression. The article utilized a type I ribozyme derived from microorganisms, namely Anabaena group I intron, which was split into a 5' half intron and a 3' half intron, and rearranged to form a PIE (permuted intron-exon) basic structure. Based on the PIE basic structure, homologous arms on both sides and an internal spacer sequence were added, thereby improving the circularization efficiency and the circularization load size of Anabaena group I intron PIE, and defining this circularization system as AnaPIE (Wesselhoeft, Kowalski et al. 2018). However, for the AnaPIE circularization system, ~186 bp of bases remain in the circular RNA to form a scar sequence, and this remaining scar sequence has been studied to have potential immunogenicity problems (Liu, Guo et al. 2022).

[0003] In 2021, Rausch's research on the Tetrahymena T4td ribozyme determined that the core sequence of the T4td ribozyme substrate is a 10-bp core sequence (Rausch, Heinz et al. 2021). To solve the problem of residual scars in circular RNAs, the published patent CN114574483A further reduced the core sequence of the substrate to 8 bp: TTGGGTCT based on the research of Rausch in 2021. In this invention, this sequence TTGGGTCT is hidden in the target sequence to be circularized, such as the CDS of the target protein or inside the IRES element, and a CleanPIE circularization system that hides the scar sequence is designed using the PIE structure. However, the circularization site of this CleanPIE system is still limited by the above-mentioned substrate core sequence TTGGGTCT and cannot be circularized at any site.

[0004] The patent KR102442946B1 discloses another STS circularization system designed using the Tetrahymena T4td ribozyme. The circularization site forms a G:U wobble base pair between the 5’ IGS and the 3’ target site, where G is inside the 5’ IGS and U is at the end of the 3’ target site region. This STS circularization system does not require the ribozyme to be split, and only needs to keep the 5’ IGS sequence and the 3’ target site sequence to form complementary pairing to form circular RNA. However, according to the circularization efficiency disclosed in the patent KR102442946B1, that is, the proportion of circular RNA is only 12%, which cannot be effectively used for production and application. Summary of the Invention

[0005] Basic Definitions

[0006] In the claims and / or the specification, when used in conjunction with the term "comprising", the words "a" or "an" can mean "one", but can also mean "one or more", "at least one", and "one or more than one".

[0007] As used in the claims and the specification, the words "comprising", "having", "including", or "containing" indicate the presence of the stated features, integers, steps, operations, elements, or components, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components, or groups thereof.

[0008] Throughout the application, the term "about" means: a value includes the standard deviation of the error of the device or method used to measure that value.

[0009] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein and refer to amino acid polymers of any length. The polymer can be linear or branched, it can contain modified amino acids, and it can be interrupted by non-amino acids. The term also includes amino acid polymers that have been modified (e.g., disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation or any other manipulation, such as conjugation with a labeled component).

[0010] Definition of professional terms

[0011] Homologous arms (5’arm 3’arm)

[0012] The 5' and 3' homologous arms (5’arm 3’arm) can be synthetic sequences, different from the internal homologous region but functionally similar. The length of the homologous arms can be, for example, about 5 - 55 nucleotides, about 9 - 19 nucleotides, e.g., a length of about 5, about 10, about 20, about 30, about 40 or about 50 nucleotides. In another embodiment, the length of the homologous arm can be 9 nucleotides. In another embodiment, the length of the homologous arm can be 19 nucleotides. In certain embodiments, the length of the homologous arm is at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19 nucleotides. In certain embodiments, the length of the homologous arm does not exceed 50, 45, 40, 35, 30, 25 or 20 nucleotides. In certain embodiments, the length of the homologous arm is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55 nucleotides.

[0013] Ribozyme

[0014] "Ribozyme", also known as ribozyme, is used to describe an RNA molecule with catalytic activity, usually having a specific three-dimensional structure. In some embodiments, the ribozyme recognition site in the present invention refers to the polynucleotide sequence in the spatial structure that can internally undergo phosphodiester bond cleavage and re-linkage when the RNA molecule forms a three-dimensional ribozyme molecule with catalytic function.

[0015] PIE system

[0016] Also known as permuted introns and exons, that is, type I introns are split at specific positions to form two parts, 5’ half intron and 3’ half intron. The two split introns are rearranged, and a target gene sequence is added in the middle of the rearranged introns. Using the self-splicing and ligation reactions of group I introns, a method for ligating the target gene sequence to form a circular RNA molecule.

[0017] Spacer

[0018] As described herein, a spacer can refer to a domain that provides space between other domains. Relative to a comparable engineered polynucleotide lacking a spacer domain, adding a spacer can provide improvements in the engineered polynucleotide's targeting of a target polynucleotide (such as increased specificity, enhanced editing efficiency, etc.). In some cases, a spacer domain can contain a filler sequence. In some cases, a filler sequence can contain a spacer domain.

[0019] In some cases as used herein, "spacer" refers to any continuous nucleotide sequence that: 1) is predicted to avoid interfering with proximal structures, such as from an IRES, coding or non-coding region, or intron; 2) is at least 7 nucleotides in length (optionally no more than 100 nucleotides); 3) is located downstream and adjacent to a 3' intron fragment and / or upstream and adjacent to a 5' intron fragment; and / or 4) contains one or more of the following: a) an unstructured region at least 5 nt in length; b) a region predicted to base pair with a distal (i.e., non-adjacent) sequence at least 5 nt in length, which includes another spacer; and / or c) a structured region at least 7 nt in length, which is limited to the sequence of the spacer.

[0020] In certain embodiments, the spacer sequence can be, for example, at least 10 nucleotides in length, at least 15 nucleotides in length, or at least 30 nucleotides in length. In certain embodiments, the spacer sequence is at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, or 30 nucleotides in length. In certain embodiments, the spacer sequence is no more than 100, 90, 80, 70, 60, 50, 45, 40, 35, or 30 nucleotides in length.

[0021] The spacer sequence can be a polyA sequence, a polyA-C sequence, a polyC sequence, or a poly-U sequence, or the spacer sequence can be specifically engineered according to the IRES. The spacer sequences described herein can have two functions: (1) promoting circularization and (2) promoting functionality by allowing the intron and IRES to fold correctly. More specifically, the spacer sequences described herein are designed to have three priorities: 1) being inert to the folding of the proximal intron and IRES structure; 2) sufficiently separating the intron and IRES secondary structures; and 3) containing a spacer-spacer complementary region to promote the formation of a "splicing bubble". In one embodiment, the vector is compatible with many possible IRESs and coding or non-coding regions, as well as two spacer sequences.

[0022] The term "Flexible circularization (FlexCirc) system" refers to a flexible circularization system, which can also be interpreted as a flexible or elastic circularization system. Compared with the disadvantage of the AnaPIE circularization system that residual scar sequences are formed in the final circular RNA product, no residual scar sequences are present in the final circular RNA product of the FlexCirc circularization system of the present invention. And compared with the CleanPIE circularization system that is limited by a fixed substrate sequence, the FlexCirc circularization system of the present invention is not limited by the substrate sequence. The FlexCirc circularization system can first determine the sequences of the sites E1 and E2 to be circularized, and form complementary pairing with E1 and E2 by mutating the IGS sequence to maintain the stability of the substrate domain and retain the ability of the circularization reaction. The E1 and E2 sequences finally retained in the circular RNA product can be flexibly variable and freely designed according to the target sequences of the sites to be circularized.

[0023] Definition of genetic engineering terms

[0024] The term "circular" as used in the context of a nucleic acid molecule generally refers to a nucleic acid molecule having a polynucleotide sequence that can be represented as a circular two-dimensional form of one nucleotide following another, wherein the polynucleotide represented is circular.

[0025] A promoter refers to a starting control region sequence that can be used to drive the expression of a relevant coding region in a desired host cell and is familiar to those skilled in the art. In fact, any promoter capable of driving these genetic elements is suitable for use in the present disclosure. The termination control region can also be derived from various genes that are native to the preferred host.

[0026] The term "coding region" refers to a nucleic acid molecule sequence that can be translated into amino acids and ultimately form a protein. More specifically, it is the nucleic acid molecule sequence in a messenger RNA molecule that can be translated into amino acids and ultimately form a protein.

[0027] The term "recombinant nucleic acid molecule" refers to a polynucleotide having sequences that are not linked together in nature. The recombinant polynucleotide can be included in a suitable vector, and the vector can be used for transformation into a suitable host cell. Then the polynucleotide is expressed in the recombinant host cell to produce, for example, "recombinant polypeptide", "recombinant protein", "fusion protein", etc.

[0028] The term "encoding" as applied to a polynucleotide can refer to such a polynucleotide that, if in its native state or when manipulated by methods well known to those skilled in the art, can be transcribed, translated, or transcribed and translated to produce mRNA of a polypeptide or a fragment thereof, then it can be said to "encode" the polypeptide. The antisense strand can be the complementary strand of such a nucleic acid, and the coding sequence can be deduced therefrom.

[0029] A "coding sequence" is a nucleic acid sequence that is transcribed into RNA (such as mRNA, rRNA, tRNA, snRNA, sense RNA, or antisense RNA). In other aspects, the RNA is not translated to produce a protein, but functions as an RNA molecule to regulate protein expression.

[0030] The term "vector" can refer to a nucleic acid construct designed for transfer between different hosts, including but not limited to plasmids, viruses, cosmids, phages, BACs, YACs, etc. In some embodiments, a "viral vector" is defined as a recombinantly produced virus or viral particle that contains a polynucleotide to be delivered in vivo, ex vivo, or in vitro to a host cell. In some embodiments, a plasmid vector can be prepared from commercially available vectors. In other embodiments, viral vectors can be produced from baculoviruses, retroviruses, adenoviruses, AAVs, etc., according to techniques known in the art. In one embodiment, the viral vector is a lentiviral vector. Examples of viral vectors include retroviral vectors, adenoviral vectors, adeno-associated viral vectors, alphavirus vectors, etc.

[0031] In some cases, the recombinant vector is a binary vector capable of functioning in multiple organisms. The organisms can be microorganisms, plants, or animals.

[0032] The term "host cell" refers to a cell into which an exogenous polynucleotide has been introduced, including progeny of such cells. Host cells include "transformants" and "transformed cells", which include primary transformed cells and progeny derived therefrom. A host cell is any type of cell system that can be used to produce the protein molecules of the present invention, including eukaryotic cells, such as mammalian cells, insect cells, yeast cells; and prokaryotic cells, such as Escherichia coli cells. Host cells include cultured cells, and also include cells within transgenic animals, transgenic plants, or cultured plant or animal tissues.

[0033] In this article, the term "recombinant cell" generally refers to a cell obtained by modifying or recombining the genetic material of a host cell using genetic engineering techniques or cell fusion techniques, which has unique traits with stable inheritance. It includes host cells that are different from the parental cells after introducing recombinant nucleic acid molecules, recombinant expression vectors, or circular RNAs, and can be eukaryotic cells or prokaryotic cells. The terms "transformed" or "transfected" as used herein refer to the introduction of nucleic acids (such as vectors) into cells by various techniques known in the art. Suitable host cells can be transformed or transfected with the nucleic acids of the present invention, such as DNA and / or RNA sequences, and can be used for the expression and / or secretion of target proteins. Examples of suitable host cells that can be used in the present invention include immortalized hybridoma cells, NS / 0 myeloma cells, HEK293T, HEK293, 293 cells, Chinese hamster ovary (CHO) cells, HeLa cells, Cap cells (human amniotic fluid-derived cells), CoS cells, 3T3, NS0, HT-1080, PERC6, CAP, HKB-11, Huh-7.

[0034] The terms "transformation, transfection, transduction" have the meanings commonly understood by those skilled in the art, which refer to the process of introducing exogenous nucleic acids (such as DNA, RNA, etc.) into a host. The methods of transformation, transfection, and transduction include any method of introducing nucleic acids into cells, and these methods include, but are not limited to, electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, and lithium acetate-DMSO method.

[0035] "Complementary" refers to the ability of nucleic acids to form hydrogen bonds through traditional Watson-Crick base pairing or other non-traditional types of base pairing. For the nucleic acid molecules of the present invention, the binding free energy between the nucleic acid molecule and its complementary sequence is sufficient to enable the relevant functions of the nucleic acid to be carried out, such as RNAi activity. The determination of the binding free energy of nucleic acid molecules is well known in the art (see, for example, Turner et al., 1987, CSH Symp. Quant. Biol., 1987, LII, pp. 123-133, Frier et al., P.N.A.S., 1986, 83, 9373-9377; Turner et al., J Am. Chem. Soc, 1987, 109, 3783-3785). The percentage of complementarity represents the nucleotides in a nucleic acid molecule that can base pair with a second nucleic acid sequence (for example, 5, 6, 7, 8, 9, or 10 nucleotides out of a total of 10 nucleotides, representing 50%, 60%, 70%, 80%, 90%, and 100% complementarity, respectively, in a first oligonucleotide that base pairs with a second nucleic acid sequence having 10 nucleotides). "Completely complementary" means that all consecutive residues of a nucleic acid sequence will form hydrogen bonds with the same number of consecutive residues in a second nucleic acid sequence.

[0036] Treatment-related Definitions

[0037] The terms "individual", "patient", or "subject" include (depending on the addition selected based on the presence of a therapeutic use) mammals. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats).

[0038] The term "treatment" means: The term "treatment" as used herein can be used to mean obtaining a desired pharmacological effect, physiological effect, or any combination thereof. In some cases, treatment can reverse side effects attributable to a disease or disorder. In some cases, treatment can stabilize a disease or disorder. In some cases, treatment can delay the progression of a disease or disorder. In some cases, treatment can cause the regression of a disease or disorder. In some cases, treatment can prevent the occurrence of a disease or disorder. In some embodiments, the treatment effect can be measured. In some cases, the measured values can be compared before and after administration of the composition. In some cases, including after contracting a disease, exposing (e.g., administering) a subject to the circular RNA, circularized precursor RNA, recombinant nucleic acid vector, composition, etc. of the present invention, such that the symptoms of the disease are alleviated compared to when not exposed, does not necessarily mean complete suppression of the symptoms of the disease. Contracting a disease means: The body has exhibited symptoms of the disease.

[0039] The term "prevention" means: Before contracting a disease, by exposing (e.g., administering) a subject to the circular RNA, recombinant nucleic acid vector, composition, etc. of the present invention, such that the symptoms after contracting the disease are alleviated compared to when not exposed, does not necessarily mean complete suppression of the disease.

[0040] The term "effective amount" refers to such an amount or dose of the recombinant nucleic acid molecule, recombinant expression vector, circularized precursor RNA, circular RNA, vaccine, or composition of the present invention that, when administered to a patient in a single or multiple doses, produces an expected effect in a patient in need of treatment or prevention. The effective amount can be readily determined by an attending physician, who is a person skilled in the art, by considering various factors such as: the species of the mammal; its size, age, and general health; the specific disease involved; the degree or severity of the disease; the response of the individual patient; the specific antibody administered; the mode of administration; the bioavailability characteristics of the administered formulation; the selected dosing regimen; and the use of any concomitant therapies.

[0041] Variant Verification

[0042] To further verify the technical effects, the inventors further verified multiple variants of exons, spacer sequences, introns, etc. on the basis of the sequences disclosed in the present invention. On the basis of identities of at least 66%, 67%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, for example, verification of various different types of anticodon stems, verification of introns from different sources, verification of spacers of different lengths, etc. On the basis of more than 66% homology ensured by truncation, mutation, segmentation, etc., by adopting the verification methods in the following invention content and specific implementation manners, first synthesizing recombinant nucleic acid molecules, then performing a cyclization reaction, and then designing PCR primers at specific sites to verify the cyclization effect, the inventors determined that the same technical effects can be achieved.

[0043] For the verification method of the variants, the percentage of sequence identity can be determined by comparing the new sequence with the query sequence. The commonly used standard methods for comparing the similarity of two nucleic acids or polypeptides and the amino acid positions can be used for this comparison. By using computer programs, such as BLAST or FASTA, for example, two polypeptides can be aligned to perform the best alignment of their respective amino acids (which can be along the full length of one or both sequences, or along a predetermined local part of one or both sequences). Such programs provide a "defect" opening penalty and a "defect" gap penalty, and a scoring matrix such as PAM250 (a standard scoring matrix; see Dayhoff et al., Atlas of Protein Sequence and Structure, Vol. 5, Supp. 3 (1978)) or a BLOSUM scoring matrix can be used in combination with the computer program. Then the percentage of identity is calculated.

[0044] To improve the technical effects of the coding region, the invention has made sequence improvements to the fluorescent protein in various structures, including but not limited to codon optimization technology, for enhancing the luminescence effect of the fluorescent protein.

[0045] The existing cyclization system based on type I ribozymes is limited by the substrate sequence restriction and cannot cyclize any sequence, which will form residues, leading to potential immunogenicity and other problems.

[0046] The present invention discloses a method for constructing a vector for preparing circular RNA, which can achieve the preparation of circular RNA for any sequence without being restricted by the substrate sequence, has no residual redundant sequences, and has a high cyclization efficiency.

[0047] To solve the above technical problems, the present invention adopts the following technical solutions.

[0048] The present invention discloses a Flexible circularization (FlexCirc) system, which comprises: a 5' homologous arm, an IGS sequence, a ribozyme, a substrate E2 sequence, a spacer sequence, an IRES element, a target protein CDS sequence, a substrate E1 sequence, and a 3' homologous arm.

[0049] Preferably, the circularization system is a recombinant nucleic acid molecule.

[0050] Preferably, the system sequentially comprises the following elements from the 5' end to the 3' end: a 5' homologous arm, an IGS sequence, a ribozyme, a substrate E2 sequence, a spacer sequence, an IRES element, a target protein CDS sequence, a substrate E1 sequence, and a 3' homologous arm.

[0051] Preferably, the system further comprises a 5' end T7 promoter sequence and a DNA linearization restriction site.

[0052] Preferably, the restriction site is EcoR I.

[0053] Preferably, the substrate E1 sequence is selected from SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:12; the substrate E2 sequence is selected from SEQ ID NO:13, SEQ ID NO:20 or SEQ ID NO:21.

[0054] Preferably, the sequence of the 5' homologous arm is as shown in SEQ ID NO:1.

[0055] Preferably, the IGS sequence is selected from SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:7.

[0056] Preferably, the ribozyme sequence is as shown in SEQ ID NO:22.

[0057] Preferably, the spacer sequence is selected from SEQ ID NO:23 or SEQ ID NO:26.

[0058] Preferably, the IRES element sequence is as shown in SEQ ID NO:24.

[0059] Preferably, the target protein CDS sequence is as shown in SEQ ID NO:25.

[0060] Preferably, the sequence of the 3' homologous arm is selected from SEQ ID NO:27, SEQ ID NO:28 or SEQ ID NO:29.

[0061] Preferably, the circularization system is a recombinant nucleic acid molecule, and the sequence of the recombinant nucleic acid molecule is shown in SEQ ID NO: 17, SEQ ID NO: 18 or SEQ ID NO: 19.

[0062] The present invention discloses a recombinant vector, and the recombinant vector includes the flexible circularization system described above.

[0063] Preferably, the recombinant vector is pBlueScript, but it is not used for limitation, and all recombinant vectors that can achieve the invention in the prior art are within the protection scope.

[0064] The present invention discloses a cell, and the cell includes the flexible circularization system or the recombinant vector described above; the cell can also be referred to as a recombinant cell.

[0065] The present invention discloses a method for preparing circular RNA, and the method includes transcribing a precursor RNA using the recombinant vector described above and further preparing it into circular RNA.

[0066] The present invention discloses a composition, which is characterized in that the composition includes at least one of the flexible circularization system, the recombinant vector according to the claims, and the cell described above.

[0067] The present invention discloses a design method for a flexible circularization system (Flexible circularization (FlexCirc) system), which is characterized by including the following steps:

[0068] 1) Determine the sequence of the site to be circularized NNUNNNN, and divide it into E1: NNU, E2: NNNN, where N is any nucleotide;

[0069] 2) Taking the E1 and E2 sequences as references, design IGS sequences to form base complementary pairs with E1 and E2 respectively;

[0070] 3) Further add 5' and 3' homologous arm sequences, ribozyme sequences, IRES, spacer sequences and the CDS sequence of the target protein respectively.

[0071] Preferably, the order of each element in step 3) from 5' to 3' is 5' homologous arm, IGS sequence, ribozyme, substrate E2 sequence, spacer sequence, IRES element, CDS sequence of the target protein, substrate E1 sequence, 3' homologous arm.

[0072] Preferably, it is characterized in that it further includes:

[0073] 4) Introduce a 5'-end T7 promoter sequence and a DNA template linearization restriction site into the flexible circularization system.

[0074] Preferably, the restriction site is EcoR I, but this is not intended to be limiting, and any restriction site capable of achieving restriction digestion function is within the protection scope.

[0075] The present invention discloses a recombinant nucleic acid molecule, characterized in that the recombinant nucleic acid molecule comprises: a 5' homologous arm, a first intron, a substrate enhancing element, a spacer sequence, a translation initiation element, a coding / non-coding region, a spacer sequence, a substrate enhancing element, a second intron, and a 3' homologous arm.

[0076] Preferably, the recombinant nucleic acid molecule sequentially comprises a 5' homologous arm, a first intron, a substrate enhancing element, a spacer sequence, a translation initiation element, a coding / non-coding region, a spacer sequence, a substrate enhancing element, a second intron, and a 3' homologous arm from the 5' end to the 3' end.

[0077] Preferably, the intron fragment is preferably a type I intron fragment, more preferably an Azoarcus group I intron;; further, the type I intron can be derived from any one of the following type I introns: the Azoarcus sp gene of nitrogen-fixing bacteria, the Td gene of T4 phage, the tRNALeu gene of the cyanobacterium Anabaena, TpaCOX2, Ptu, etc.

[0078] Preferably, the spacer sequence is selected from at least one of a polyA sequence, a polyA-C sequence, a polyC sequence, or a poly-U sequence.

[0079] Preferably, the spacer sequence can be 5 to 100 nucleotides.

[0080] Preferably, the translation initiation element is selected from at least one of an IRES sequence, a 5' UTR sequence, a Kozak sequence, a sequence containing m6A modification (N(6)-methyladenosine modification), a complementary sequence of ribosomal 18S rRNA, and an initiation sequence for rolling circle translation.

[0081] Preferably, the coding / non-coding region can encode at least one protein selected from Gluc (Gauss luciferase), Fluc (firefly luciferase), eGFP (enhanced green fluorescent protein), hEPO (human erythropoietin), or Cas9 endonuclease.

[0082] Preferably, the sequence of the recombinant nucleic acid molecule is selected from at least one of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:15, or SEQ ID NO:16.

[0083] The present invention discloses a recombinant vector comprising the recombinant nucleic acid molecule.

[0084] The present invention discloses a method for preparing circular RNA, including transcribing the recombinant vector to form precursor RNA, and further preparing it into circular RNA.

[0085] The present invention discloses a cell, which contains the recombinant vector described above.

[0086] The present invention discloses a composition, characterized in that the composition includes at least one of the recombinant nucleic acid molecule, the recombinant vector, and the cell described above.

[0087] Preferably, it further contains more than one pharmaceutically acceptable excipient, carrier, adjuvant, vehicle.

[0088] The present invention discloses a method for preventing, diagnosing, and / or treating a disease, and its use in preparing a drug for preventing and / or treating a disease, including administering to a subject at least one of the flexible cyclization system, the recombinant vector, the cell, the recombinant nucleic acid molecule, the recombinant vector, and the composition described above.

[0089] Beneficial effects

[0090] The present invention discloses a method for constructing a vector for preparing circular RNA that is flexible and not restricted by the substrate sequence. Based on previous research findings, by mutating the substrate sequence of the domain composed of the Exon1 (E1), P1, and Exon2 (E2) sequences of the Azoarcus group I intron ribozyme, it is found that when maintaining the structural stability, mutating the E1, P1, and E2 sequences, the Azoarcus group I intron ribozyme still has enzymatic activity and can maintain the ability to form circular RNA.

[0091] Based on the research and understanding of the substrate domain of Azoarcus group I intron, the present invention discloses a method for constructing a vector for preparing circular RNA that is flexible and not restricted by the substrate sequence, and the steps are as follows: Step 1: Determine the target sequence NNUNNNN to be cyclized, and divide it into E1: NNU, E2: NNNN. Step 2: Using the E1 and E2 sequences as references, design IGS sequences to form complementary pairs with E1 and E2 respectively. Step 3: Add 5' and 3' homologous arms respectively. The target sequence to be cyclized includes IRES and CDS, which are concatenated by E2 and E1. The present invention can prepare circular RNA for any target sequence without residual sequences and has a high cyclization efficiency, which is a technical effect that cannot be achieved by the prior art.

[0092] Based on the Azoarcus group I intron ribozyme adopting the PIE structure, various mutations were carried out on the substrates, and the circularized vectors after mutation could all form circular RNAs. The FlexCirc circularization system designed based on the Azoarcus group I intron ribozyme could all form circular RNAs, and the circularization substrate sequences had the characteristic of flexible design. The FlexCirc circularization system designed based on the Azoarcus group I intron ribozyme could achieve a relatively high circularization ratio in terms of circularization efficiency. Description of the Drawings

[0093] Figure 1 . Among them, a is the substrate domain of the Azoarcus group I intron ribozyme composed of the sequences of Exon1 (E1), P1, and Exon2 (E2). E1 is the 5'-end lowercase letter sequence cucau, E2 is the 3'-end lowercase letter sequence aauc, P1 is the domain formed by the ribozyme 1-11 nt and E1, and IGS is the intron 6-11 nt region in the P1 stem-loop domain;; b is a schematic diagram of the construct of the FlexCirc (Flexibility self-Circularization system) circularization substrate with flexible design of the present invention.

[0094] Figure 2 . Schematic diagram of the design of variable vector of different substrate sequences under the condition of maintaining the structural stability of the substrate domain formed by E1, P1, and E2 during the preparation of circular RNA by the Azoarcus group I intron ribozyme using the rearranged intron-exon circularization system (PIE).

[0095] Figure 3 . Schematic diagram of the vector structure of the circular RNA prepared by the Azoarcus group I intron ribozyme using the PIE system.

[0096] Figure 4 . Electrophoresis separation results of circular RNAs in the circularization products of circular RNA vectors with 5 different substrate sequences on a 2% E-gel electrophoresis.

[0097] Figure 5a . Electrophoresis verification results after digestion of circular RNA vectors with sequences 10 and 11 at different concentrations of Rnase R on a 1% agarose gel.

[0098] Figure 5b . Electrophoresis verification results after digestion of circular RNA vectors with sequences 14, 15, and 16 at different concentrations of Rnase R on a 1% agarose gel.

[0099] Figure 6. Sanger sequencing results of the first generation after RT-PCR amplification of the cyclization sites of different substrate cyclization carrier sequences 11, sequence 14, sequence 15, and sequence 16.

[0100] Figure 7 . Schematic diagram of the cyclization carrier design of different substrate sequences of FlexCirc: sequence 17, sequence 18, and sequence 19.

[0101] Figure 8 . Schematic diagram of FlexCirc circular RNA formation, where E1 - E2 can be set inside the GOI or IRES sequence.

[0102] Figure 9 . Results of 2% E-gel electrophoresis separation of the cyclization products of the carriers of different substrate sequences of FlexCirc: sequence 17, sequence 18, and sequence 19.

[0103] Figure 10 . Results of 1% agarose gel electrophoresis of the cyclization products of the carriers of different substrate sequences of FlexCirc: sequence 17, sequence 18, and sequence 19 after digestion with Rnase R at different concentrations.

[0104] Figure 11 . Results of 1% agarose gel electrophoresis of the RT-PCR amplification products of the cyclization sites of the cyclization products of the carriers of different substrate sequences of FlexCirc: sequence 17, sequence 18, and sequence 19.

[0105] Figure 12 . Sequencing verification results of the cyclization sites of the cyclization products of the carriers of different substrate sequences of FlexCirc: sequence 17, sequence 18, and sequence 19.

[0106] Figure 13a . Results of 4% denatured PAGE gel electrophoresis separation and purity evaluation of the cyclization products of the carriers of different substrate sequences of FlexCirc: sequence 17, sequence 18, and sequence 19 before and after digestion with Rnase R.

[0107] Figure 13b . Results of purity evaluation based on gray values after 4% denatured PAGE gel electrophoresis separation of the cyclization products of the carriers of different substrate sequences of FlexCirc: sequence 17, sequence 18, and sequence 19 before digestion with Rnase R.

[0108] Figure 14 . Results of verification of eGFP fluorescent protein expression on the 3rd day after transfection of the cyclization products of the carriers of different substrate sequences of FlexCirc: sequence 17, sequence 18, and sequence 19 into HEK293T cells. Detailed implementation methods

[0109] Example 1: Verifying the sequence diversity of the substrate core domain composed of the Exon1, P1, and Exon2 sequences of the Azoarcus group I intron ribozyme

[0110] Construction of the AzoPIE vector

[0111] In this example, for the Azoarcus group I intron ribozyme, the PIE strategy was adopted to split the ribozyme at the P6 loop to form the 5’ half intron and the 3’ half intron respectively, as Figure 3 shown in the construction of the self-cyclizing vector AzoPIE. For the substrate core domain composed of Exon1, P1, and Exon2 together, mutant vectors were constructed. Under the condition of maintaining base pairing, sequences in Exon1, P1, and Exon2 were respectively used to construct mutant vectors, as Figure 2 shown. The mutant vector sequences, including the 5’-end T7 promoter sequence and the DNA template linearization restriction enzyme site EcoR I sequence, were recombined into the pBlueScript plasmid, and the required plasmid was obtained by the direct synthesis method of a third-party plasmid.

[0112] In vitro transcription and cyclization verification

[0113] 1-2-1. Preparation of the DNA linearized template for in vitro transcription:

[0114] Each mutant vector plasmid synthesized by a third party was linearized by the restriction enzyme EcoR I to obtain the transcription template. Specifically, the plasmid qualified by PCR was linearized by the EcoR I restriction enzyme to prepare the IVT transcription template, and the enzyme digestion system is shown in Table 1. The purity of the linearized template was detected by HPLC method respectively to ensure that the prepared linearized template meets the requirements of subsequent IVT transcription.

[0115] Table 1 Recipe of the 500 μL EcoR I enzyme digestion system

[0116] Component Volume (μL) Plasmid 50 μg EcoR I (10 U / μL) 50 Tingo Buffer 50 Enzyme-free water Up to 500

[0117] 1-2-2. In vitro transcription and circularization verification: Prepare 20 μL reaction systems according to Table 2, and conduct 2 parallel experiments for each sequence template. Place the transcription system in a PCR instrument and react at 37 °C for 2 hours. After the reaction is completed, add 1 μL of DNase I and 2 μL of 10×DNase I buffer to each reaction system, and react at 37 °C for 20 minutes. Transfer each transcription system to a 1.5 mL centrifuge tube, add an RNA precipitation solution containing 0.5 times the volume of lithium chloride at a concentration of 7.5 M and 50 mM EDTA, mix well, and precipitate at -20 °C in the refrigerator for 1 h. After precipitation, centrifuge at 18360×g at 4 °C for 15 min, try to remove the supernatant as much as possible, add 500 μL of 70% ethanol solution, blow and wash the precipitate, centrifuge again under the same conditions, try to remove the supernatant as much as possible, and then evaporate the ethanol to dryness. Resuspend each experimental group with 100 μL of enzyme-free water. Sample and detect the RNA concentration, purity, and use 2% E-Gel electrophoresis to determine the circRNA synthesis effect.

[0118] Table 2 In vitro transcription system

[0119]

[0120] Experimental results, as Figure 4 shown, in the 2% E-gel electrophoresis separation results of 5 different mutant vector sequences, the introns dropped after the self-cleavage of ribozymes, as well as the circular RNA bands and nicked RNA bands after circularization, can be seen. Among them, the circular RNA has a slower migration rate in 2% E-gel.

[0121] Rnase R digestion verification of circular RNA

[0122] To further verify whether circular RNA exists in 5 different mutant vector sequences, the circularized products were verified by RnaseR digestion. RNase R can cleave and degrade RNA from the 3'-5' direction, and can digest almost all linear RNA molecules, but it is not easy to digest circular RNA, lariat structures, or double-stranded RNA molecules with a 3'-terminal overhang of less than 7 nt. In this example, Rnase R enzymes with different concentration units were used for digestion tolerance verification. After digestion at 37 °C for 30 minutes, 1% agarose gel electrophoresis (120 V, 30 minutes) was used to observe the tolerance of circular RNA to Rnase R digestion.

[0123] Experimental results, as Figure 5a, as shown in Figure 5b: Under the condition of no Rnase R digestion, the circularized products have two obvious bands in 1% agarose gel electrophoresis, namely the linear precursor and circular RNA product bands. Under the condition of Rnase R digestion, the linear precursor mRNA in the circularized products will be degraded, and with the increase of the concentration of R enzyme, the degradation is more obvious. Compared with the linear mRNA precursor, circular RNA can tolerate digestion by different concentrations of R enzyme and has good tolerance. The corresponding linear mRNA control samples are easily degraded under R enzyme digestion, especially completely degraded under the condition of a concentration greater than 1 U / L.

[0124] RT-PCR and Sanger sequencing verification of the circularization site of circular RNA

[0125] In this example, reverse transcription RT-PCR was used to amplify the circularization site, and the amplification products were sent to a third-party sequencing company for first-generation Sanger sequencing.

[0126] Table 3 Primer information for RT-PCR of circularization site

[0127]

[0128]

[0129] The sequencing results are as Figure 6 shown: All four different mutant vector designs can form circles, and the sequencing results of the circularization site sequences show that they can all circularize at the expected sites.

[0130] In this example, mutations were designed for the substrate domains Exon1, P1, and Exon2 of the Azoarcus group I intron. The PIE circularization design was used. Through circularization verification and RT-PCR sequencing verification of the circularization site, it was proved that different substrate mutant sequences can form circular RNA, and circularization linkage occurred at the expected designed sites. It is indicated that the Azoarcus group I intron ribozyme consists of Exon1, P1, and Exon2 to form the substrate domain, and has the characteristic of variable substrate sequence under the condition of maintaining structural stability.

[0131] Example 2: Design and verification of the FlexCirc circularization system

[0132] 2-1. Construction of the FlexCirc system vector

[0133] This second example is a creative invention of the Flexible circularization (FlexCirc) system based on the results and hints of the first example, that is, the variable substrate sequence of the Azoarcus group I intron.

[0134] The design method of the FlexCirc system of the present invention is as follows, as shown in the schematic Figure 1 b, Figure 7 shown:

[0135] Step 1: Determine the sequence of the site to be circularized NNUNNNN, and divide it into E1: NNU, E2: NNNN

[0136] Step 2: Using the E1 and E2 sequences as references, design IGS sequences to form base complementary pairs with E1 and E2 respectively

[0137] Step 3: Add 5' and 3' homologous arm sequences respectively, that is, the target sequence to be circularized, including IRES, spacer and the CDS sequence of the target protein.

[0138] The order of each element of the FlexCirc system circularization system is as shown in the appendix Figure 8 shown: 5' homologous arm, IGS sequence, ribozyme, substrate E2 sequence, spacer sequence spacer, IRES element, CDS sequence of the target protein, substrate E1 sequence, 3' homologous arm

[0139] The above FlexCirc system circularization system sequence, including the 5'-end T7 promoter sequence and the DNA template linearization restriction site EcoR I sequence, was recombined into the pBlueScript plasmid and obtained by the direct synthesis method of a third-party sequence synthesis company.

[0140] The 3 circularization substrate sequences tested in this example are:

[0141] Table 4 Information on 3 substrate sequences to be circularized tested in this example

[0142]

[0143]

[0144] 2-2. In vitro transcription and circularization verification

[0145] 2-2-1. Preparation of DNA linearized template: The mutant vector plasmid synthesized by a third party was linearized by the restriction enzyme EcoR I to obtain a transcription template. Specifically, the plasmid qualified by PCR was linearized by the EcoR I restriction enzyme to prepare the IVT transcription template, and the enzyme digestion system is shown in Table 1. The purity of the linearized template was detected by HPLC method respectively to ensure that the prepared linearized template meets the requirements of subsequent IVT transcription.

[0146] 2-2-2. In vitro transcription and circularization verification: Prepare 20 μL reaction systems according to Table 2, perform 2 parallel experiments for each sequence template, place the transcription system in a PCR instrument, and react at 37°C for 2 hours. After the reaction is completed, add 1 μL of DNase I and 2 μL of 10×DNase I buffer to each reaction system, and react at 37°C for 20 minutes. Transfer each transcription system to a 1.5 mL centrifuge tube, add 0.5 times the volume of 7.5 M lithium chloride and 50 mM EDTA RNA precipitation solution, mix well and precipitate in a -20°C refrigerator for 1 hour. After precipitation is completed, centrifuge at 18360×g for 15 minutes at 4°C, remove the supernatant as much as possible, add 500 μL of 70% ethanol solution, blow and wash the precipitate, centrifuge again under the same conditions, remove the supernatant as much as possible, and evaporate the ethanol. Resuspend each experimental group with 100 μL of enzyme-free water. Samples were taken to test RNA concentration and purity, and 2% E-Gel electrophoresis was used to determine the circRNA synthesis effect.

[0147] Experimental results, such as Figure 9 As shown, in 2% E-gel electrophoresis, sequence 17, sequence 18, and sequence 19 can form circular RNA, nicked RNA, and detached intron product bands. Among them, there is only one detached intron band, which is different from the two intron bands formed by the PIE ring-forming system, which is consistent with the design that the Azoarcus ribozyme is not split.

[0148] 2-3. Circular RNA RNase R digestion verification

[0149] To further verify whether there is circular RNA in the three different mutant vector sequences, the cyclized products were subjected to RNase R digestion verification. In this example, RNase R enzymes of different concentrations were used to verify the digestion resistance.

[0150] Experimental results, such as Figure 10 Display: Under the condition of no Rnase R digestion, the cyclized product has two obvious bands in 1% agarose gel electrophoresis, namely the linear precursor and the circular RNA product band. Under the condition of Rnase R digestion, the linear precursor mRNA in the cyclized product will be degraded, and as the concentration of R enzyme increases, the degradation becomes more obvious. Compared with the linear mRNA precursor, circular RNA can tolerate digestion by different concentrations of R enzyme and has better tolerance. The corresponding linear mRNA control sample is degraded under R digestion, especially when the concentration is greater than 1U / L, it can be completely degraded. It is suggested that sequence 17, sequence 18, and sequence 19 in this embodiment all produce circular RNA.

[0151] 2-4. Circular RNA cyclization site verification by Sanger sequencing

[0152] In this example, reverse transcription RT-PCR was used to amplify the circularization site, and the amplification product was sent to a third-party sequencing company for first-generation Sanger sequencing.

[0153] The primer information in Table 5 is as follows

[0154]

[0155] The sequencing results are as Figure 11 shown: Amplification products were generated for Sequence 17, Sequence 18, and Sequence 19, and the size of the electrophoresis band was approximately 400 bp. By performing first-generation Sanger sequencing on the amplification products, the sequencing results are as Figure 12 shown, indicating that Sequence 17, Sequence 18, and Sequence 19 were all circularized. Moreover, the circularization substrate sequences of Sequence 17 and Sequence 18 were different. This shows that the FlexCirc circularization system of the present invention can successfully prepare circular RNAs from different substrate sequences. Different mutant vector designs can all be circularized, and the sequencing results of the circularization site sequences show that they can all be circularized at the expected sites. 2-5. Evaluation of the circularization efficiency of circular RNAs

[0156] In this example, 4% polyacrylamide-7M urea denaturing PAGE gel electrophoresis was used to measure the circularization efficiency of FlexCirc circRNA

[0157] The FlexCirc circularization products before and after RNase R digestion were diluted to 80 ng / μL. Take 5 μL of the diluted sample, add 5 μL of ultrapure water and 10 μL of gel loading buffer II (a 1-2X solution composed of 95% formamide, 18 mM EDTA, 0.025% SDS, xylene cyanol, and bromophenol blue, Invitrogen Cat#AM8546G), mix them 1:1, pre-denature at 65°C (on a PCR instrument) for 5 min, and then immediately cool on ice. RNA Marker (3 μL of Marker plus 7 μL of ultrapure water and 10 μL of gel loading buffer II) was treated in the same way. Prepare 4% polyacrylamide-7M urea denaturing PAGE, and perform pre-electrophoresis at 120 V in 1% TBE buffer for 30 min. Use a 200 μL pipette to aspirate the buffer and rinse the loading wells 1-2 times to avoid the urea precipitated from the gel affecting the loading. Then load 15 μL (300 ng) per well (the loading amount can be adjusted appropriately, and the loading volume needs to be kept consistent), perform electrophoresis at 120 V at room temperature for 15 min, and then increase the voltage to 200 V and perform electrophoresis for another 70 min. Stain the gel with SYBRGreen nucleic acid dye (Thermo Fisher Scientific) diluted 1:10000 for about 10 min, and analyze it using the Tanon5200 chemiluminescence imaging system.

[0158] Experimental results, such as Figure 13a shown: In 4% polyacrylamide - 7M urea denaturing PAGE gel electrophoresis, the circular RNAs in the FlexCirc cyclization products are located at the upper end of the gel due to their slow migration rate. There are various different products in the products before Rnase R digestion, including nicked RNA, precursor RNA, and intron. After Rnase R digestion, the linear heterobands all disappear and are degraded, only circular RNA and nicked RNA are retained. The quantitative analysis results of the bands in each lane show that, as Figure 13b shown, the cyclization efficiencies of sequence 17, sequence 18, and sequence 19 in this example before digestion are 33.3%, 32.7%, and 31.0% respectively. The proportions of circular RNA after Rnase R digestion are 60.8%, 62.0%, and 59.6% respectively.

[0159] Among them, sequence 19 extends the number of bases complementary to IGS in E2 based on sequence 18, that is, E2 is extended from NNNN to NNNNNNN. Theoretically, it can improve the binding stability between E2 and IGS, thereby aiming to improve the cyclization efficiency. As the experimental results Figure 13a , 13b shown, extending the number of bases complementary to IGS in E2 fails to significantly improve the cyclization efficiency. The cyclization efficiency of sequence 19 is slightly lower than that of sequence 17 and sequence 18, suggesting that the extension of the complementary pairing between E2 sequence and IGS cannot improve the cyclization efficiency, that is, it is suggested that the E2 sequence of NNNN is the best.

[0160] 2 - 6. Verification of circular RNA protein expression

[0161] Cell culture

[0162] Take a bottle of HEK293T / 17 cells cultured in a T75 flask and plate them when the cell confluence reaches 80%-90%. Discard the culture medium, add PBS for washing, discard the PBS, add 0.05% trypsin to digest the cells. Wait until the cells become round, then discard the trypsin. Pipette the cells with 10 mL of 10% FBS cell medium without antibiotics to disperse them. Count the cells and adjust the cell concentration to 1×106 cells / mL ± 10%. Take 10 48-well plates and inoculate 0.2 mL of the cell suspension into each well. Then place the cell culture plates in an incubator at 37°C and 5% CO2 for 24 hours ± 4 hours. Dissolve 0.4 μg of circRNA in 25 μL of serum-free DMEM medium and mix gently. Let it stand at room temperature for 5 minutes. Dissolve 1 μL of Lipohigh transfection reagent in 25 μL of serum-free DMEM medium and mix gently. Let it stand at room temperature for 5 minutes. Add the medium containing LipoHigh from the previous step to the medium containing circRNA and mix gently. Let it stand at room temperature for 20 minutes. Take out the cell culture plates from the incubator and dropwise add the mixture into the inoculated cells. Mix gently and then place them back in the incubator at 37°C and 5% CO2 for culture. After 4-8 hours, discard the liquid and add 0.5 mL of medium containing 2% FBS for continued culture.

[0163] Cell culture and fluorescence detection

[0164] At 24 hours ± 4 hours after transfection, take one 48-well plate and observe and photograph the GFP fluorescence expression using a fluorescence microscope with a 10× eyepiece and a 10× objective. After photographing, measure the fluorescence intensity value using a multifunctional microplate reader. From the fourth day to the twelfth day, perform the detection according to the operation on the third day to monitor the process of fluorescence from non-existent to peak and then from peak to decrease during these days.

[0165] Experimental results, as Figure 14 shown, on the third day after transfection of HEK293T / 17 cells with the circular RNAs of sequence 17, sequence 18, and sequence 19 after digestion and purification with Rnase R, green fluorescence could be observed, indicating that the eGFP protein was normally expressed. This shows that the circular RNAs prepared by FlexCirc circRNA have the ability to express proteins.

Claims

1. A flexible circularization (FlexCirc) system, characterized in that, The system includes: 5' homologous arm, IGS sequence, ribozyme, substrate E2 sequence, spacer sequence, IRES element, target protein CDS sequence, substrate E1 sequence, 3' homologous arm.

2. The flexible cyclization system according to claim 1, wherein From the 5' end to the 3' end, the system sequentially includes the following elements: 5' homologous arm, IGS sequence, ribozyme, substrate E2 sequence, spacer sequence, IRES element, target protein CDS sequence, substrate E1 sequence, 3' homologous arm.

3. The flexible cyclization system according to any one of claims 1-2, characterized in that, The system further includes a 5' end T7 promoter sequence and a DNA linearization restriction site.

4. The flexible cyclization system according to claim 3, characterized in that, The restriction site is EcoR I.

5. The flexible cyclization system according to any one of claims 1-2, characterized in that, The substrate E1 sequence is selected from SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:12; the substrate E2 sequence is selected from SEQ ID NO:13, SEQ ID NO:20 or SEQ ID NO:

21.

6. The flexible cyclization system according to any one of claims 1-2, characterized in that, The sequence of the 5' homologous arm is as shown in SEQ ID NO:

1.

7. The flexible cyclization system according to any one of claims 1-2, characterized in that, The IGS sequence is selected from SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:

7.

8. The flexible cyclization system according to any one of claims 1-2, characterized in that, The ribozyme sequence is as shown in SEQ ID NO:

22.

9. The flexible cyclization system according to any one of claims 1-2, characterized in that, The spacer sequence is selected from SEQ ID NO:23 or SEQ ID NO:

26.

10. The flexible cyclization system according to any one of claims 1-2, characterized in that, The IRES element sequence is as shown in SEQ ID NO:

24.

11. The flexible cyclization system according to any one of claims 1-2, characterized in that, The target protein CDS sequence is as shown in SEQ ID NO:

25.

12. The flexible cyclization system according to any one of claims 1-2, characterized in that, The sequence of the 3' homologous arm is selected from SEQ ID NO:27, SEQ ID NO:28 or SEQ ID NO:

29.

13. The flexible cyclization system according to any one of claims 1-2, characterized in that, The circularization system is a recombinant nucleic acid molecule, and the sequence of the recombinant nucleic acid molecule is as shown in SEQ ID NO:17, SEQ ID NO:18 or SEQ ID NO:

19.

14. Recombinant vector, characterized in that, The recombinant vector includes the flexible circularization system according to any one of claims 1-13.

15. The recombinant vector according to claim 14, characterized in that, The recombinant vector is pBlueScript.

16. A cell, characterized in that, The cell includes the flexible circularization system according to any one of claims 1-13 or the recombinant vector according to any one of claims 14-15.

17. A method for preparing circular RNA, characterized in that, The method includes transcribing the recombinant vector according to any one of claims 14-15 to form precursor RNA, and further preparing it into circular RNA.

18. Composition, characterized in that, The composition includes at least one of the flexible circularization system according to any one of claims 1-13, the recombinant vector according to any one of claims 14-15, and the cell according to claim 16.

19. A design method for a Flexible circularization (FlexCirc) system, characterized in that, It includes the following steps: 1) Determine the sequence of the site to be circularized NNUNNNN, and divide it into E1: NNU, E2: NNNN, where N is any nucleotide; 2) Using the E1 and E2 sequences as references, design the IGS sequence to form base complementary pairing with E1 and E2 respectively; 3) Further add 5' and 3' homologous arm sequences, ribozyme sequence, IRES, spacer sequence and target protein CDS sequence respectively.

20. The method according to claim 19, wherein In step 3), the order of the elements from 5' to 3' is 5' homologous arm, IGS sequence, ribozyme, substrate E2 sequence, spacer sequence, IRES element, target protein CDS sequence, substrate E1 sequence, 3' homologous arm.

21. The method according to any one of claims 19-20, characterized in that, Further comprising: 4) Introduce a 5'-end T7 promoter sequence and a DNA template linearization restriction site into the flexible cyclization system.

22. The method according to claim 21, wherein The restriction site is EcoR I.

23. Recombinant nucleic acid molecule, characterized in that, The recombinant nucleic acid molecule comprises: 5' homologous arm, first intron, substrate enhancer element, spacer sequence, translation initiation element, coding / non-coding region, spacer sequence, substrate enhancer element, second intron, 3' homologous arm.

24. The recombinant nucleic acid molecule according to claim 23, wherein, The recombinant nucleic acid molecule sequentially comprises a 5' homologous arm, a first intron, a substrate enhancer element, a spacer sequence, a translation initiation element, a coding / non-coding region, a spacer sequence, a substrate enhancer element, a second intron, and a 3' homologous arm from the 5'-end to the 3'-end.

25. The recombinant nucleic acid molecule according to any one of claims 24, characterized in that, The intron fragment is preferably a type I intron fragment, more preferably an Azoarcus group I intron;; further, the type I intron can be derived from any one of the following type I introns: the Azoarcus sp gene of nitrogen-fixing bacteria, the Td gene of T4 phage, the tRNALeu gene of the cyanobacterium Anabaena, TpaCOX2, Ptu, etc.

26. The recombinant nucleic acid molecule according to any one of claims 23-24, characterized in that, The spacer sequence is selected from at least one of a polyA sequence, a polyA-C sequence, a polyC sequence, or a poly-U sequence.

27. The recombinant nucleic acid molecule according to claim 26, wherein, The spacer can be 5 to 100 nucleotides.

28. The recombinant nucleic acid molecule according to any one of claims 23-24, characterized in that, The translation initiation element is selected from at least one of an IRES sequence, a 5'UTR sequence, a Kozak sequence, a sequence containing m6A modification (N(6)-methyladenosine modification), a complementary sequence of ribosomal 18S rRNA, and an initiation sequence of rolling circle translation.

29. The recombinant nucleic acid molecule according to any one of claims 23-24, characterized in that, The coding / non-coding region can encode at least one protein selected from Gluc (Gauss luciferase), Fluc (firefly luciferase), eGFP (enhanced green fluorescent protein), hEPO (human erythropoietin), or Cas9 endonuclease.

30. The recombinant nucleic acid molecule according to any one of claims 23-24, characterized in that, The sequence of the recombinant nucleic acid molecule is selected from at least one of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:15, or SEQ ID NO:

16.

31. A recombinant vector comprising the recombinant nucleic acid molecule according to any one of claims 23-30.

32. A method for preparing circular RNA, comprising transcribing a precursor RNA using the recombinant vector according to claim 31 and further preparing it into circular RNA.

33. A cell comprising the recombinant vector according to claim 31.

34. A composition, characterized in that, The composition comprises at least one of the recombinant nucleic acid molecule according to any one of claims 23-30, the recombinant vector according to claim 31, and the cell according to claim 33.

35. The composition according to claim 34, further comprising one or more pharmaceutically acceptable excipients, carriers, adjuvants, vehicles.

36. A method for preventing, diagnosing, and / or treating a disease, comprising administering to a subject at least one of the flexible cyclization systems according to any one of claims 1-13, the recombinant vectors according to any one of claims 14-15, the cells according to claim 16, the recombinant nucleic acid molecules according to any one of claims 23-30, the recombinant vectors according to claim 31, the cells according to claim 33, and the compositions according to claims 34-35.

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