Vector for preparing circular RNA and construction method

Abstract

A flexible method for constructing a vector for preparing circular RNA without being limited by substrate sequence is provided. The method comprises the following steps: determining a target site sequence NNUNNNN to undergo cyclization, and dividing said sequence into E1: NNU and E2: NNNN; using the E1 and E2 sequences as references to design IGS sequences to form complementary pairings with E1 and E2, respectively; adding 5' and 3' homologous arms, as well as IRES and CDS and other elements, respectively. In the provided method, circular RNA can be prepared for any target sequence without residual sequences, and the described method has high cyclization efficiency. Also provided is a FlexCirc ring-forming system designed on the basis of an Azoarcus group I intron ribozyme, which can form circular RNA. The ring-forming substrate sequence has the characteristics of flexible design, and the ring-forming efficiency can achieve a high ring-forming ratio.

Description

A vector for preparing circular RNA and its construction method Technical Field

[0001] The present invention belongs to the field of molecular biology and bioengineering technology, and specifically relates to a recombinant nucleic acid and a vector for preparing circular RNA, as well as a method for preparing circular RNA. Background Art

[0002] Circular RNA, as the name suggests, is a type of RNA molecule with a circular closed structure, which is ubiquitous in nature. Circular RNA can generally be formed in different organisms through the mechanism of reverse shearing. Compared with linear mRNA, circular RNA has no 5' and 3' ends, so it can withstand hydrolysis by nucleases and has relatively high stability. In 2018, Wesselhoeft RA published an article entitled "Engineering circular RNA for potent and stable translation in eukaryotic cells" in Nature Communication, which pioneered the application of circular RNA for protein expression. The article uses a type I ribozyme from microorganisms, namely Anabaena group I intron, to split it into 5'half intron and 3'half intron, and rearranges it to form the PIE (permuted intron-exon) basic structure. Based on the PIE basic structure, the addition of flanking homology arms and an internal spacer sequence improves the looping efficiency and looping payload of the Anabaena group I intron PIE, thereby defining this looping system as AnaPIE (Wesselhoeft, Kowalski et al. 2018). However, the AnaPIE looping system leaves a ~186bp base residue within the circular RNA, forming a scar sequence. This residual scar sequence has been shown to have potential immunogenicity issues (Liu, Guo et al. 2022).

[0003] In 2021, Rausch conducted research on the Tetrahymena T4td ribozyme and determined that the core sequence of the T4td ribozyme substrate is a 10bp core sequence (Rausch, Heinz et al. 2021). In order to solve the problem of residual scar in circular RNA, the patent CN114574483A, based on the research of Rausch 2021, further reduced the substrate core sequence to 8bp: TTGGGT CT. The invention hides this sequence TTGGGT CT in the target sequence to be cyclized, such as the target protein CDS, or inside the IRES element, and adopts the PIE structure to design a CleanPIE cyclization system that hides the scar sequence. However, the cyclization site of the CleanPIE system is still limited by the above-mentioned substrate core sequence TTGGGT CT, and cannot be cyclized at any site.

[0004] Patent KR102442946B1 discloses another STS looping system designed using the Tetrahymena T4td ribozyme. The looping site forms a G:U wobble base pair between the 5' IGS and the 3' target site, with the G located within the 5' IGS and the U at the end of the 3' target site region. This STS looping system does not require segmentation of the ribozyme; it only requires maintaining a complementary pairing between the 5' IGS and 3' target site sequences to form circular RNA. However, the looping efficiency disclosed in patent KR102442946B1, i.e., the proportion of circular RNA, is only 12%, making it ineffective for production and application.

[0005] Summary of the Invention

[0006] Basic Definition

[0007] In the claims and / or description, when used in conjunction with the term "comprising", the word "a" or "an" can mean "one", but can also mean "one or more", "at least one" and "one or more than one".

[0008] As used in the claims and the specification, the words “comprises,” “has,” “includes,” or “contains” indicate the presence of stated features, integers, steps, operations, elements, or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, or groups thereof.

[0009] Throughout this application, the term "about" indicates that a value includes the standard deviation of error for the device or method being employed to determine the value.

[0010] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein and refer to amino acid polymers of any length. The polymer may be linear or branched, it may contain modified amino acids, and it may be interrupted by non-amino acids. The term also includes amino acid polymers that have been modified (e.g., by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component).

[0011] Definition of professional terms

[0012] Homology arms (5'arm 3'arm)

[0013] 5' and 3' homology arms (5'arm 3'arm) can be synthetic sequences, different from internal homology regions, but similar in function. The length of the homology arms can be, for example, about 5-55 nucleotides, about 9-19 nucleotides, for example, a length of about 5, about 10, about 20, about 30, about 40 or about 50 nucleotides. In another embodiment, the length of the homology arms can be 9 nucleotides. In another embodiment, the length of the homology arms can be 19 nucleotides. In certain embodiments, the length of the homology arms 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 homology arms is no more than 50, 45, 40, 35, 30, 25 or 20 nucleotides. In certain embodiments, the homology arms are 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 in length.

[0014] Ribozymes

[0015] "Ribozyme," also known as ribozyme, is a term used to describe catalytically active RNA molecules, typically possessing a specific three-dimensional structure. In some embodiments, the ribozyme recognition site of the present invention refers to a polynucleotide sequence within the spatial structure that enables the cleavage and reconnection of phosphodiester bonds within the RNA molecule when it forms a catalytically active three-dimensional ribozyme.

[0016] PIE system

[0017] Also known as permuted introns and exons, this method involves splitting the group I intron at a specific position to form a 5' half intron and a 3' half intron. The two introns formed by the split are then rearranged, and the target gene sequence is added between the rearranged introns. The target gene sequence is then connected to form a circular RNA molecule by utilizing the self-cleavage and ligation reaction of the group I intron (Group I intron).

[0018] spacer

[0019] As described herein, spacer can refer to the domain that provides space between other domains.Relative to the comparable engineered polynucleotide lacking a spacer domain, increasing a spacer can provide the improvement (such as increased specificity, enhanced editing efficiency, etc.) of engineered polynucleotide to the target polynucleotide.In some cases, the spacer domain can include a stuffing sequence.In some cases, the stuffing sequence can include a spacer domain.

[0020] As used herein, "spacer" in some cases refers to any contiguous nucleotide sequence that: 1) is predicted to avoid interfering with proximal structure, such as from an IRES, coding or noncoding 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' intronic segment and / or upstream and adjacent to a 5' intronic segment, and / or 4) comprises one or more of: a) an unstructured region at least 5 nt long b) a region predicted to base pair with a distal (i.e., non-adjacent) sequence at least 5 nt long that includes another spacer, and / or c) a structured region at least 7 nt long that is limited to the sequence of the spacer.

[0021] 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.

[0022] 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 modified according to the IRES. The spacer sequence described herein can have two functions: (1) promote circularization and (2) promote functionality by allowing the intron and IRES to fold correctly. More specifically, the spacer sequence described herein is designed to have three priorities: 1) be inert to the folding of the proximal intron and IRES structure; 2) fully separate the intron and IRES secondary structure; and 3) contain a spacer-spacer complementary region to promote the formation of a "splicing bubble". In one embodiment, the vector is compatible with many possible IRES and coding or non-coding regions and two spacer sequences.

[0023] The term "Flexible circularization (FlexCirc) system" refers to a flexible circularization system, which can also be interpreted as a flexible circularization system or an elastic circularization system. Compared with the disadvantage that the AnaPIE circularization system will form residual scar sequences in the final circular RNA product, the FlexCirc circularization system of the present invention does not leave any scar sequences in the final circular RNA product. And compared with the CleanPIE circularization system, which is restricted by a fixed substrate sequence, the FlexCirc circularization system of the present invention is not restricted 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 pairs with E1 and E2 by mutating the IGS sequence to maintain the stability of the substrate domain and the ability to maintain the circularization reaction. The E1 and E2 sequences finally retained in the circular RNA product can be flexible and freely designed according to the sequence of the target site to be circularized.

[0024] Definition of Genetic Engineering Terms

[0025] The term "circular" used in the context of nucleic acid molecules may generally refer to nucleic acid molecules that may represent a polynucleotide sequence in a circular 2-dimensional form, one nucleotide after another, wherein the represented polynucleotide is circular.

[0026] Promoters are initiation control region sequences that are used to drive expression of the associated coding region in the desired host cell and are 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. Termination control regions can also be derived from various genes native to the preferred host.

[0027] The term "coding region" refers to a nucleic acid sequence that can be translated into amino acids and ultimately form a protein. More specifically, it refers to a nucleic acid sequence in a messenger RNA molecule that can be translated into amino acids and ultimately form a protein.

[0028] 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 to transform into a suitable host cell. The polynucleotide is then expressed in the recombinant host cell to produce, for example, a "recombinant polypeptide," "recombinant protein," "fusion protein," and the like.

[0029] The term "encoding" as applied to a polynucleotide may refer to a polynucleotide that, in its native state or when manipulated by methods well known to those skilled in the art, can be transcribed, translated, or both transcribed and translated to produce mRNA for a polypeptide or fragment thereof. The antisense strand may be the complementary strand of such a nucleic acid, and the coding sequence may be deduced therefrom.

[0030] A "coding sequence" is a nucleic acid sequence that is transcribed into RNA (e.g., mRNA, rRNA, tRNA, snRNA, sense RNA, or antisense RNA). In other aspects, the RNA is not translated to produce a protein, but rather functions as an RNA molecule to regulate protein expression.

[0031] The term "vector" may 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 comprising a polynucleotide to be delivered to a host cell in vivo, in vitro, or in vitro. In some embodiments, a plasmid vector can be prepared by a commercially available vector. In other embodiments, the viral vector can be produced by baculovirus, retrovirus, adenovirus, AAV, 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, alphaviral vectors, etc.

[0032] In some cases, the recombinant vector is a binary vector that can function in a variety of organisms. The organism can be a microorganism, a plant, or an animal.

[0033] The term "host cell" refers to a cell into which an exogenous polynucleotide has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include primary transformed cells and progeny derived therefrom. Host cells are any type of cell system that can be used to produce the protein molecules of the present invention, including eukaryotic cells, for example, mammalian cells, insect cells, yeast cells; and prokaryotic cells, for example, Escherichia coli cells. Host cells include cultured cells, as well as cells within transgenic animals, transgenic plants, or cultured plant tissues or animal tissues.

[0034] In this article, the term "recombinant cell" generally refers to the use of genetic engineering technology or cell fusion technology to modify or reorganize the genetic material of the host cell to obtain a cell with a unique trait of stable inheritance, encompassing host cells that are different from the parent cell after the introduction of recombinant nucleic acid molecules, recombinant expression vectors, and circular RNA, and can be eukaryotic or prokaryotic cells. As used herein, the term "transformed" or "transfected" refers 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 expression and / or secretion of target proteins. Examples of suitable host cells that can be used for the present invention include immortalized hybridoma cells, NS / 0 myeloma cells, HEK293T, HEK293, 293 cells, Chinese hamster ovary (CHO) cells, HeLa cells, Cap cells (cells derived from human amniotic fluid), CoS cells, 3T3, NSO, HT-1080, PERC6, CAP, HKB-11, Huh-7.

[0035] The terms "transformation," "transfection," and "transduction" have the meaning generally understood by those skilled in the art, i.e., the process of introducing exogenous nucleic acid (DNA, RNA, etc.) into a host. Such methods of transformation, transfection, and transduction include any method for introducing nucleic acid into a cell, including, but not limited to, electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG), DEAE-dextran, cationic liposomes, and lithium acetate-DMSO.

[0036] "Complementarity" refers to the ability of a nucleic acid to form hydrogen bonds through traditional Watson-Crick base pairing or other non-traditional types of base pairing. With respect to the nucleic acid molecules of the present invention, the binding free energy of a nucleic acid molecule with its complementary sequence is sufficient to enable the relevant function of the nucleic acid, such as RNAi activity. 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., PNAS, 1986, 83, 9373-9377; Turner et al., J Am. Chem. Soc, 1987, 109, 3783-3785). Percent complementarity refers to the number of nucleotides in a nucleic acid molecule that can base pair with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, or 10 nucleotides out of a total of 10 nucleotides in a first oligonucleotide that base pairs with a second nucleic acid sequence of 10 nucleotides represents 50%, 60%, 70%, 80%, 90%, and 100% complementarity, respectively). "Perfectly 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.

[0037] Treatment-related definitions

[0038] The terms "individual," "patient," or "subject" include (optionally added if therapeutic use is present) 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).

[0039] The term "treatment" refers to: the term "treatment" can be used herein to mean obtaining a desired pharmacological effect, physiological effect, or any combination thereof. In some cases, treatment can reverse the side effects attributable to the disease or condition. In some cases, treatment can stabilize the disease or condition. In some cases, treatment can delay the progression of the disease or condition. In some cases, treatment can cause the regression of the disease or condition. In some cases, treatment can prevent the occurrence of the disease or condition. In some embodiments, the therapeutic 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 suffering from the disease, the subject is contacted with (e.g., administered) the circular RNA, circularized precursor RNA, and recombinant nucleic acid vector, composition, etc. of the present invention, thereby alleviating the symptoms of the disease compared to when not in contact, and does not mean that the symptoms of the disease must be completely suppressed. Suffering from a disease means that the body has symptoms of the disease.

[0040] The term "prevention" means that before a subject develops a disease, by exposing him or her to (e.g., administering) the circular RNA, recombinant nucleic acid vector, composition, etc. of the present invention, the symptoms after the disease is alleviated compared to when the subject has not been exposed to the disease, and does not necessarily mean that the disease must be completely suppressed.

[0041] The term "effective amount" refers to an amount or dosage of a recombinant nucleic acid molecule, recombinant expression vector, circularized precursor RNA, circular RNA, vaccine, or composition of the present invention that produces the desired effect in a patient in need of treatment or prevention after administration to the patient in a single or multiple doses. An effective amount can be readily determined by the attending physician, who is skilled in the art, by considering a variety of factors, such as the species of mammal; its size, age, and general health; the specific disease involved; the extent 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 dosing regimen selected; and the use of any concomitant therapy.

[0042] Variant Validation

[0043] To further verify the technical effect, the inventors further verified multiple variants of exons, spacer sequences, introns, etc. based on the disclosed sequences of the present invention. On the basis 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%, and 99.9% identity, for example, verification of multiple different types of anticodon stems, verification of introns from different sources, verification of spacers of different lengths, etc., and ensuring more than 66% homology by truncation, mutation, segmentation, etc., by adopting the verification method in the following invention summary and specific embodiments, first synthesizing recombinant nucleic acid molecules, then performing a cyclization reaction, and then performing PCR primer design for specific sites to verify the cyclization effect, the inventors determined that the same technical effect can be achieved.

[0044] The variants can be verified by comparing the new sequence with the query sequence to determine the percentage of sequence identity. Standard methods for comparing the similarity and amino acid position of two nucleic acids or polypeptides that are commonly used can be used to perform such comparisons. Using a computer program such as BLAST or FASTA, for example, two polypeptides can be aligned to perform optimal alignment of their respective amino acids (either along the full length of one or both sequences, or along a predetermined portion of one or both sequences). Such programs provide "defective" open penalties and "defective" gap penalties. 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 the BLOSUM scoring matrix can be used in conjunction with the computer program. The percentage of identity is then calculated.

[0045] In order to improve the technical effect of the coding region, the invention has made various structural sequence improvements to the fluorescent protein, including but not limited to codon optimization technology, to enhance the luminescence effect of the fluorescent protein.

[0046] The existing type I ribozyme-based cyclization system is limited by the substrate sequence restriction and cannot cyclize any sequence, which will form residues and lead to potential immunogenicity and other problems.

[0047] The present invention discloses a method for constructing a circular RNA preparation vector, which is not restricted by substrate sequence and can realize circular RNA preparation for any sequence without residual redundant sequences and has high circularization efficiency.

[0048] In order to solve the above technical problems, the present invention adopts the following technical solutions.

[0049] The present invention discloses a flexible circularization (FlexCirc) system, which comprises: a 5' homology 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' homology arm.

[0050] Preferably, the circularization system is a recombinant nucleic acid molecule.

[0051] Preferably, the system comprises the following elements from the 5' end to the 3' end: 5' homology arm, IGS sequence, ribozyme, substrate E2 sequence, spacer sequence, IRES element, target protein CDS sequence, substrate E1 sequence, 3' homology arm.

[0052] Preferably, the system further comprises a 5' end T7 promoter sequence and a DNA linearization restriction site.

[0053] Preferably, the restriction enzyme cleavage site is EcoR I.

[0054] 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.

[0055] Preferably, the sequence of the 5' homology arm is as shown in SEQ ID NO: 1.

[0056] Preferably, the IGS sequence is selected from SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:7.

[0057] Preferably, the ribozyme sequence is shown as SEQ ID NO: 22.

[0058] Preferably, the spacer sequence is selected from SEQ ID NO:23 or SEQ ID NO:26.

[0059] Preferably, the IRES element sequence is shown in SEQ ID NO: 24.

[0060] Preferably, the CDS sequence of the target protein is shown in SEQ ID NO: 25.

[0061] Preferably, the sequence of the 3' homology arm is selected from SEQ ID NO: 27, SEQ ID NO: 28 or SEQ ID NO: 29.

[0062] Preferably, the cyclization 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.

[0063] The present invention discloses a recombinant vector comprising the flexible cyclization system.

[0064] Preferably, the recombinant vector is pBlueScript, but this is not intended to be limiting. All recombinant vectors in the prior art that can realize the invention are within the scope of protection.

[0065] The present invention discloses a cell, wherein the cell comprises the flexible circularization system or the recombinant vector; the cell can also be called a recombinant cell.

[0066] The invention discloses a method for preparing circular RNA, which comprises adopting the recombinant vector to transcribe to form precursor RNA, and further preparing the circular RNA.

[0067] The present invention discloses a composition, characterized in that the composition comprises at least one of the flexible cyclization system, the recombinant vector according to the claim and the cell.

[0068] The present invention discloses a design method for a flexible circularization (FlexCirc) system, which is characterized by comprising the following steps:

[0069] 1) Determine the sequence of the site to be cyclized NNUNNNN and split it into E1:NNU, E2:NNNN, where N is any nucleotide;

[0070] 2) Using the E1 and E2 sequences as references, design IGS sequences to form base complementary pairs with E1 and E2, respectively;

[0071] 3) Further add 5' and 3' homology arm sequences, ribozyme sequence, IRES, spacer sequence and target protein CDS sequence respectively.

[0072] Preferably, the order of the elements in step 3) from 5' to 3' is 5' homology arm, IGS sequence, ribozyme, substrate E2 sequence, spacer sequence, IRES element, target protein CDS sequence, substrate E1 sequence, 3' homology arm.

[0073] Preferably, it is characterized in that it further comprises:

[0074] 4) Introducing a 5' end T7 promoter sequence and a DNA template linearization restriction site into the flexible circularization system.

[0075] Preferably, the enzyme cleavage site is EcoR I, but this is not intended to be limiting, and all enzyme cleavage sites that can achieve enzyme cleavage function are within the protection scope.

[0076] The present invention discloses a recombinant nucleic acid molecule, characterized in that the recombinant nucleic acid molecule comprises: a 5' homology arm, a first intron, a substrate enhancement element, a spacer sequence, a translation initiation element, a coding / non-coding region, a spacer sequence, a substrate enhancement element, a second intron, and a 3' homology arm.

[0077] Preferably, the recombinant nucleic acid molecule comprises, from the 5' end to the 3' end, a 5' homology 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' homology arm.

[0078] Preferably, the intron fragment is preferably a type I intron fragment, more preferably an Azoarcus group I intron; further, the type I intron may be derived from any of the following type I introns: nitrogen-fixing bacteria Azoarcus sp gene, T4 phage Td gene, cyanobacteria Anabaena tRNALeu gene, TpaCOX2, Ptu, etc.

[0079] 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.

[0080] Preferably, the spacer sequence may be 5 to 100 nucleotides.

[0081] 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 an m6A modification (N(6)methyladenosine modification), a complementary sequence of ribosomal 18S rRNA, and a rolling circle translation initiation sequence.

[0082] Preferably, the coding / non-coding region can encode at least one protein selected from Gaussia luciferase (Gluc), firefly luciferase (Fluc), enhanced green fluorescent protein (eGFP), human erythropoietin (hEPO) or Cas9 nuclease.

[0083] 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.

[0084] The invention discloses a recombinant vector comprising the recombinant nucleic acid molecule.

[0085] The invention discloses a method for preparing circular RNA, which comprises the steps of transcribing the recombinant vector to form a precursor RNA, and further preparing the circular RNA.

[0086] The invention discloses a cell comprising the recombinant vector.

[0087] The present invention discloses a composition, characterized in that the composition comprises at least one of the recombinant nucleic acid molecule, the recombinant vector and the cell.

[0088] Preferably, it further comprises one or more pharmaceutically acceptable excipients, carriers, auxiliary materials, and vehicles.

[0089] The present invention discloses a method for preventing, diagnosing and / or treating a disease, and use thereof in preparing a medicament for preventing and / or treating a disease, comprising 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. Beneficial effects

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

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

[0092] The FlexCirc cyclization system, designed based on the Azoarcus group I intron ribozyme, employs a PIE structure and employs various substrate mutations. All of the mutated cyclization vectors are capable of forming circular RNAs. The FlexCirc cyclization system, designed based on the Azoarcus group I intron ribozyme, is capable of forming circular RNAs, and the cyclization substrate sequence offers flexible design. The FlexCirc cyclization system, designed based on the Azoarcus group I intron ribozyme, achieves a high cyclization ratio. BRIEF DESCRIPTION OF THE DRAWINGS

[0093] Figure 1. In which, a is the substrate domain of the Azoarcus group I intron ribozyme consisting of Exon1 (E1), P1, and Exon2 (E2) sequences, E1 is the lowercase sequence cucau at the 5' end, E2 is the lowercase sequence aauc at the 3' end, P1 is the domain formed by ribozyme 1-11nt and E1, and IGS is the intron 6-11nt region in the P1 stem-loop domain; b is a schematic diagram of the self-circularization system construct of the FlexCirc (Flexibility self-Circularization system) of the present invention, in which the cyclization substrate can be flexibly designed.

[0094] Figure 2. Schematic diagram of the design of variable vectors with different substrate sequences while maintaining structural stability in the substrate domain formed by E1, P1, and E2 during the process of verifying the use of the rearranged intron-exon looping system (PIE) for the preparation of circular RNA by Azoarcus group I intron ribozyme.

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

[0096] Figure 4. Circular products of 5 circularized RNA vectors with different substrate sequences, and electrophoretic separation of circular RNA in a 2% E-gel.

[0097] Figure 5a. Circularized RNA vector sequence 10 and sequence 11 were digested with different concentrations of RNase R and then verified by electrophoresis on 1% agarose gel.

[0098] Figure 5b. Circularized RNA vector sequences 14, 15, and 16 were digested with different concentrations of RNase R and then verified by electrophoresis on a 1% agarose gel.

[0099] Figure 6. First-generation Sanger sequencing results after RT-PCR amplification of the circularization sites of different substrate circularization vector sequences 11, 14, 15, and 16.

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

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

[0102] Figure 9. Separation results of 2% E-gel electrophoresis of the cyclization products of FlexCirc substrates with different sequences: sequence 17, sequence 18, and sequence 19.

[0103] Figure 10. 1% agarose gel electrophoresis results of the circularized products of FlexCirc vectors with different substrate sequences: sequence 17, sequence 18, and sequence 19, after digestion with RNase R at different concentrations.

[0104] Figure 11. Circularization products of FlexCirc vectors with different substrate sequences: sequence 17, sequence 18, and sequence 19, and 1% agarose gel electrophoresis results of RT-PCR amplification products of the circularization sites.

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

[0106] Figure 13a. 4% denatured PAGE gel electrophoresis separation and purity assessment of the circularization products of FlexCirc vectors with different substrate sequences: sequence 17, sequence 18, and sequence 19 before and after RNase R digestion.

[0107] Figure 13b. Circularization products of FlexCirc vectors with different substrate sequences: sequence 17, sequence 18, and sequence 19 were separated by 4% denatured PAGE gel electrophoresis before RNase R digestion and then their purity was assessed based on grayscale values.

[0108] Figure 14. eGFP fluorescent protein expression verification results on day 3 after HEK293T cells were transfected with the circularized products of FlexCirc vectors with different substrate sequences: sequence 17, sequence 18, and sequence 19. DETAILED DESCRIPTION

[0109] Example 1: Verification of the Sequence Diversity of the Substrate Core Domain of the Azoarcus group I Intron Ribozyme Composed of Exon1, P1, and Exon2 Sequences

[0110] AzoPIE vector construction

[0111] In this example, the PIE strategy was used to split the Azoarcus group I intron ribozyme at the P6 loop, forming a 5' half intron and a 3' half intron, respectively. The self-circularized vector AzoPIE was constructed as shown in Figure 3. Mutation vectors were constructed for the substrate core domain composed of Exon 1, P1, and Exon 2. While maintaining base matching, mutation vectors were constructed for sequences in Exon 1, P1, and Exon 2, respectively, as shown in Figure 2. The mutation vector sequence, including the 5' end T7 promoter sequence and the DNA template linearization restriction site EcoRI sequence, was recombined into the pBlueScript plasmid, and the desired plasmid was obtained by third-party direct plasmid synthesis.

[0112] In vitro transcription and circularization verification

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

[0114] Each mutant vector plasmid synthesized by a third party was linearized with the restriction enzyme EcoRI to obtain a transcription template. Specifically, plasmids that passed PCR analysis were linearized and digested with EcoRI to prepare IVT transcription templates. The enzyme digestion system is shown in Table 1. The purity of the linearized templates was tested using HPLC to ensure that they met the requirements for subsequent IVT transcription.

[0115] Table 1 EcoR I enzyme 500 μL digestion system formula

[0116] 1-2-2. In vitro transcription and circularization verification: Prepare 20 μL of each reaction system according to Table 2. Perform two replicates for each sequence template. Place the transcription system in a PCR instrument and incubate at 37°C for 2 hours. After completion, add 1 μL of DNase I and 2 μL of 10× DNase I buffer to each reaction system and incubate at 37°C for 20 minutes. Transfer each transcription system to a 1.5 mL centrifuge tube, add 0.5 volumes of RNA precipitation solution containing 7.5 M lithium chloride and 50 mM EDTA, mix well, and precipitate at -20°C for 1 hour. After precipitation, centrifuge at 18,360 × g for 15 minutes at 4°C. Remove as much supernatant as possible, add 500 μL of 70% ethanol solution, and wash the pellet. Centrifuge again under the same conditions, remove as much supernatant as possible, evaporate the ethanol, and resuspend each experimental group in 100 μL of enzyme-free water. Samples are collected to test RNA concentration and purity, and the synthesis of the circRNA is confirmed by 2% E-Gel electrophoresis.

[0117] Table 2 In vitro transcription system

[0118] The experimental results, as shown in Figure 4, show that in the 2% E-gel electrophoresis separation results of the five different mutant vector sequences, introns dropped after spontaneous cleavage by the ribozyme, as well as circular RNA bands and nicked RNA bands after circularization can be seen. Among them, the migration rate of circular RNA in the 2% E-gel is slower.

[0119] Circular RNARnase R enzyme digestion verification

[0120] To further verify the presence of circular RNA in the five different mutant vector sequences, the circularized products were subjected to RNase R digestion verification. 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, lasso structures, or double-stranded RNA molecules with less than 7nt protruding at the 3' end. This example uses different concentrations of RNase R enzyme to verify the resistance to enzyme digestion. After enzyme digestion at 37°C for 30 minutes, 1% agarose gel electrophoresis (120V, 30 minutes) is used to observe the tolerance of circular RNA under RNase R enzyme digestion.

[0121] The experimental results, shown in Figures 5a and 5b, show that in the absence of RNase R digestion, the circularized product exhibits two distinct bands on 1% agarose gel electrophoresis: the linear precursor and the circular RNA product. However, in the presence of RNase R digestion, the linear precursor mRNA in the circularized product is degraded, with degradation increasing with increasing RNase R concentration. Compared to linear mRNA precursors, circular RNAs are more resistant to cleavage by varying RNase R concentrations. The corresponding linear mRNA control sample is readily degraded by RNase R digestion, and is completely degraded at concentrations exceeding 1 U / L.

[0122] RT-PCR and Sanger sequencing verification of circular RNA circularization sites

[0123] In this example, reverse transcription RT-PCR was used to amplify the circularization site, and the amplified product was sent to a third-party sequencing company for first-generation Sanger sequencing.

[0124] Table 3 RT-PCR primer information for cyclization sites

[0125] The sequencing results are shown in Figure 6: the four different mutation vector designs can all form circles, and the results of circle site sequencing show that they can all form circles at the expected sites.

[0126] This example employed mutational design in the substrate domains Exon 1, P1, and Exon 2 of the Azoarcus group I intron, employing a PIE looping design. Validation of looping and RT-PCR sequencing of looped sites demonstrated that different substrate mutation sequences all formed circular RNAs, with circularization occurring at the intended design sites. This suggests that the Azoarcus group I intron ribozyme, comprised of Exon 1, P1, and Exon 2, possesses the characteristic of substrate sequence variability while maintaining structural stability.

[0127] Example 2: Design and verification of the FlexCirc ring system

[0128] 2-1. FlexCirc system vector construction

[0129] This Example 2 is based on the results and suggestions of Example 1, that is, a flexible circularization (FlexCirc) system was creatively invented on the basis of the variable substrate sequence of Azoarcus group I intron.

[0130] The design method of the FlexCirc system of the present invention is as follows, as shown in schematic diagram 1b and FIG7 :

[0131] Step 1: Determine the sequence of the site to be cyclized NNUNNNN and split it into E1:NNU, E2:NNNN

[0132] Step 2: Using E1 and E2 sequences as reference, design IGS sequences to form base complementary pairs with E1 and E2 respectively

[0133] Step 3: Add 5' and 3' homology arm sequences respectively, that is, the target sequence to be circularized, including IRES, spacer and target protein CDS sequence.

[0134] The order of the components of the FlexCirc system is shown in Figure 8: 5' homology arm, IGS sequence, ribozyme, substrate E2 sequence, spacer sequence, IRES element, target protein CDS sequence, substrate E1 sequence, 3' homology arm

[0135] The FlexCirc system cyclization 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 the desired plasmid was obtained by direct synthesis by a third-party sequence synthesis company.

[0136] The three cyclization substrate sequences tested in this example are:

[0137] Table 4 Sequence information of 3 substrates to be cyclized tested in this example

[0138] 2-2. In vitro transcription and circularization verification

[0139] 2-2-1. Preparation of linearized DNA template: The mutant vector plasmid synthesized by a third party is linearized with the restriction endonuclease EcoRI to obtain a transcription template. Specifically, plasmids that have passed PCR verification are linearized and digested with EcoRI restriction endonuclease to prepare IVT transcription templates. The enzyme digestion system is shown in Table 1. The purity of the linearized templates is tested by HPLC to ensure that the prepared linearized templates meet the requirements for subsequent IVT transcription.

[0140] 2-2-2. In vitro transcription and circularization verification: Prepare 20 μL of each reaction system according to Table 2. Perform two parallel experiments for each sequence template. Place the transcription system in a PCR instrument and incubate at 37°C for 2 hours. After the reaction is complete, add 1 μL of DNase I and 2 μL of 10× DNase I buffer to each reaction system and incubate at 37°C for 20 minutes. Transfer each transcription system to a 1.5 mL centrifuge tube, add 0.5 volumes of RNA precipitation solution containing 7.5 M lithium chloride and 50 mM EDTA, mix well, and precipitate at -20°C for 1 hour. After precipitation, centrifuge at 18,360 × g for 15 minutes at 4°C. Remove as much supernatant as possible, add 500 μL of 70% ethanol solution, and wash the pellet. Centrifuge again under the same conditions, remove as much supernatant as possible, evaporate the ethanol, and resuspend each experimental group in 100 μL of enzyme-free water. Samples are collected to test RNA concentration and purity. CircRNA synthesis efficiency is confirmed by 2% E-Gel electrophoresis.

[0141] The experimental results, shown in Figure 9, show that sequences 17, 18, and 19 all formed circular RNA, nicked RNA, and shed intron product bands on a 2% E-gel electrophoresis. There was only one shed intron band, distinct from the two intron bands formed by the PIE looping system, consistent with the design of the Azoarcus ribozyme, which was not split.

[0142] 2-3. Circular RNA RNase R Enzyme Digestion Verification

[0143] To further verify the presence of circular RNA in the three different mutant vector sequences, the circularized products were subjected to RNase R digestion verification. In this example, different concentrations of RNase R were used to verify the resistance to digestion.

[0144] The experimental results, as shown in Figure 10, show that in the absence of RNase R digestion, the cyclized product has two distinct bands on 1% agarose gel electrophoresis: a linear precursor and a circular RNA product band. However, in the presence of RNase R digestion, the linear precursor mRNA in the cyclized product is degraded, and degradation becomes more pronounced as the concentration of the R enzyme increases. Compared to linear mRNA precursors, circular RNA can withstand digestion by different concentrations of R enzyme and has better tolerance. The corresponding linear mRNA control sample is degraded under R enzyme digestion, especially when the concentration is greater than 1 U / L, and can be completely degraded. This suggests that sequences 17, 18, and 19 in this example all produce circular RNA.

[0145] 2-4. Sanger sequencing verification of circular RNA cyclization sites (see Figures 10a, b)

[0146] In this example, reverse transcription RT-PCR was used to amplify the circularization site, and the amplified product was sent to a third-party sequencing company for first-generation Sanger sequencing.

[0147] Table 5 Primer information is as follows

[0148] The sequencing results are shown in Figure 11: Sequence 17, sequence 18, and sequence 19 all produce amplified products, and the electrophoresis band size is about 400bp. By performing first-generation Sanger sequencing on the amplified products, the sequencing results shown in Figure 12 show that sequence 17, sequence 18, and sequence 19 are all cyclized. And the cyclization substrate sequences of sequence 17 and sequence 18 are different. This shows that the FlexCirc cyclization system of the present invention can successfully prepare circular RNA for different substrate sequences. Different mutation vector designs can all form a circle, and the results of sequencing the cyclization site sequence show that they can all form a circle as expected. 2-5. Evaluation of circular RNA cyclization efficiency

[0149] In this example, 4% polyacrylamide-7M urea denaturing PAGE gel electrophoresis was used to measure the circularization efficiency of FlexCirc circRNA.

[0150] Samples of the FlexCirc circularized product were diluted to 80 ng / μL before and after RNase R digestion. 5 μL of this diluted sample was then added to 5 μL of ultrapure water and 10 μL of gel loading buffer II (a 1-2X solution consisting of 95% formamide, 18 mM EDTA, 0.025% SDS, xylene cyanol, and bromophenol blue, Invitrogen Cat# AM8546G) in a 1:1 ratio. The mixture was heated at 65°C (on a PCR instrument) for 5 minutes for pre-denaturation and then immediately cooled on ice. RNA marker (3 μL of marker, 7 μL of ultrapure water, and 10 μL of gel loading buffer II) was treated in the same manner. A 4% polyacrylamide-7 M urea denaturing PAGE was prepared and pre-electrophoresis was performed in 1% TBE buffer at 120 V for 30 minutes. Aspirate the buffer with a 200 μL pipette and rinse the sample wells 1-2 times to prevent urea from precipitating from the gel from interfering with sample loading. Then, load 15 μL (300 ng) of sample per well (the sample volume can be adjusted as needed, but the loading volume must remain consistent). Electrophoresis was performed at 120 V for 15 min at room temperature, then the voltage was increased to 200 V and continued for an additional 70 min. The gel was stained with SYBR Green nucleic acid dye (Thermo Fisher Scientific) at a dilution of 1:10,000 for approximately 10 min and analyzed using a Tanon 5200 chemiluminescence imaging system.

[0151] The experimental results, as shown in Figure 13a, show that in 4% polyacrylamide-7M urea denaturing PAGE gel electrophoresis, the circular RNA in the FlexCirc circularization product is located at the upper end of the gel due to its slow migration rate. The products before RNase R digestion contain a variety of different products, including nicked RNA, precursor RNA, and intron. After RNase R digestion, the linear miscellaneous bands disappear and degrade, leaving only the circular RNA and nicked RNA. Quantitative analysis of the bands in each lane shows, as shown in Figure 13b, that the circularization efficiencies of Sequences 17, 18, and 19 in this example before digestion were 33.3%, 32.7%, and 31.0%, respectively. After RNase R digestion, the proportions of circular RNA were 60.8%, 62.0%, and 59.6%, respectively.

[0152] Sequence 19, based on sequence 18, extends the number of bases in the complementary pairing between E2 and IGS, that is, E2 is extended from NNNN to NNNNNNN. This theoretically improves the stability of the binding between E2 and IGS, thereby increasing the efficiency of circularization. As shown in Figures 13a and 13b of the experimental results, extending the number of bases in the complementary pairing between E2 and IGS did not significantly improve the circularization efficiency. The circularization efficiency of sequence 19 was slightly lower than that of sequence 17 and sequence 18, indicating that extending the complementary pairing between the E2 sequence and IGS does not improve the circularization efficiency, suggesting that the optimal E2 sequence is NNNN.

[0153] 2-6. Circular RNA Protein Expression Verification

[0154] Cell culture

[0155] Take a bottle of HEK293T / 17 cells cultured in a T75 well and plate them at a confluence of 80%-90%. Discard the culture medium, wash with PBS, discard the PBS, and disintegrate the cells with 0.05% trypsin. Once the cells become rounded, discard the trypsin and disperse the cells by pipetting with 10 mL of 10% FBS cell culture medium without antibiotics. Count the cells and adjust the cell concentration to 1×106 cells / mL ± 10%. Inoculate 0.2 mL of the cell suspension in each of ten 48-well plates and incubate the plates at 37°C in a 5% CO2 incubator for 24 hours ± 4 hours. Dissolve 0.4 μg of circRNA in 25 μL of serum-free DMEM medium, mix gently, and incubate at room temperature for 5 minutes. Dissolve 1 μL of Lipohigh transfection reagent in 25 μL of serum-free DMEM medium, mix gently, and incubate at room temperature for 5 minutes. Add the culture medium containing LipoHigh from the previous step to the culture medium containing circRNA, mix gently, and let it stand at room temperature for 20 minutes. Remove the cell culture plate from the incubator, add dropwise to the seeded cells, mix gently, and place in a 37°C, 5% CO2 incubator for culture. After 4 to 8 hours, discard the solution and add 0.5 mL of culture medium containing 2% FBS to continue culturing.

[0156] Cell culture and fluorescence detection

[0157] 24 hours ± 4 hours after transfection, examine one 48-well plate and observe and photograph GFP fluorescence expression using a fluorescence microscope with a 10x eyepiece and a 10x objective. After imaging, measure fluorescence intensity using a multifunctional microplate reader. From day 4 to day 12, follow the same procedures as day 3, monitoring the progression of fluorescence from zero to peak and then from peak to decline.

[0158] As shown in Figure 14, the circular RNAs of sequences 17, 18, and 19 were digested and purified with RNase R and then transfected into HEK293T / 17 cells. Green fluorescence was observed on day 3, indicating normal expression of eGFP protein. This demonstrates that circular RNAs prepared using FlexCirc circRNA have the ability to express proteins.

Claims

1. A flexible circularization (FlexCirc) system, characterized in that the system 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.

2. The flexible circularization system of claim 1, characterized in that the system comprises following elements in sequence from 5’ end to 3’ end: the 5’ homologous arm, the IGS sequence, the ribozyme, the substrate E2 sequence, the spacer sequence, the IRES element, the target protein CDS sequence, the substrate E1 sequence, and the 3 ’ homologous arm.

3. The flexible circularization system of any one of claims 1-2, characterized in that the system further comprises a T7 promoter sequence at the 5’ end and a DNA linearization enzyme cleavage site.

4. The flexible circularization system of claim 3, characterized in that the enzyme cleavage site is EcoR I.

5. The flexible circularization system of 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 circularization system of any one of claims 1-2, characterized in that a sequence of the 5’ homologous arm is shown as SEQ ID NO: 1.

7. The flexible circularization system of 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 circularization system of any one of claims 1-2, characterized in that the ribozyme sequence is shown as SEQ ID NO: 22.

9. The flexible circularization system of 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 circularization system of any one of claims 1-2, characterized in that the IRES element sequence is shown as SEQ ID NO: 24.

11. The flexible circularization system of any one of claims 1-2, characterized in that the target protein CDS sequence is shown as SEQ ID NO: 25.

12. The flexible circularization system of any one of claims 1-2, characterized in that a 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 circularization system of any one of claims 1-2, characterized in that the circularization system is a recombinant nucleic acid molecule, and a sequence of the recombinant nucleic acid molecule is shown as SEQ ID NO: 17, SEQ ID NO: 18, or SEQ ID NO: 19.

14. A recombinant vector, characterized in that the recombinant vector comprises the flexible circularization system of any one of claims 1-13.

15. The recombinant vector of claim 14, characterized in that the recombinant vector is pBlueScript.

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

17. A method for preparing circular RNA, characterized in that the method comprises transcribing a precursor RNA using the recombinant vector of any one of claims 1415, and further preparing the circular RNA from the precursor RNA.

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

19. A design method for a flexible circularization (FlexCirc) system, characterized inthat the design method comprises the following steps:1) determining a site sequence NNUNNNN to be cyclized, dividing the site sequence into E1: NNU, and E2: NNNN, wherein N is any nucleotide;2) Using the E1 and E2 sequences as references to design an IGS sequence to form complementary base pairings with E1 and E2, respectively;3) further adding 5’ and 3’ homologous arm sequences, a ribozyme sequence, IRES, a spacer sequence and a target protein CDS sequence, respectively.

20. The method of claim 19, characterized in that, in step 3), the order of each element from 5’ to 3’ ends is the 5’ homologous arm, the IGS sequence, the ribozyme, the substrate E2 sequence, the spacer sequence, the IRES element, the target protein CDS sequence, the substrate E1 sequence, and the 3’ homologous arm.

21. The method of any one of claims 19-20, characterized in that, the method further comprises:4) introducing a T7 promoter sequence at the 5’ end and a DNA template linearization enzyme cleavage site into the flexible circularization system.

22. The method of claim 21, characterized in that, the enzyme cleavage site is EcoR I.

23. 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-codingregion, a spacer sequence, a substrate enhancing element, a second intron, and a 3’ homologous arm.

24. The recombinant nucleic acid molecule of claim 23, characterized in that, the recombinant nucleic acid molecule comprises the 5’ homologous arm, the first intron, the substrate enhancing element, the spacer sequence, the translation initiation element, the coding / non-coding region, the spacer sequence, the substrate enhancing element, the second intron, and the 3 ’ homologous arm in sequence from 5 ’ end to 3 ’ end.

25. The recombinant nucleic acid molecule of claim 24, characterized in that, a fragment of the intron is preferably a type I intron fragment, and more preferably, an Azoarcus group I intron; further, the type I intron is derivable from any of following type I introns: Azoarcus sp gene from azotobacteria, Td gene from T4 bacteriophage, tRNALeu gene from cyanobacterium Anabaena, TpaCOX2, Ptu, etc.

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

27. The recombinant nucleic acid molecule of claim 26, characterized in that, the spacer can be 5 to 100 nucleotides in length.

28. The recombinant nucleic acid molecule of any one of claims 23-24, characterizedin that, the translation initiation element is selected from at least one of a 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 of any one of claims 23-24, characterized in that, the coding / non-coding region can encode at least one protein selected from Gaussian luciferase (Gluc), fire fly luciferase (Fluc), enhanced green fluorescent protein (eGFP), human erythropoietin (hEPO), or Cas9 endonuclease.

30. The recombinant nucleic acid molecule of any one of claims 23-24, characterized in that, a 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 of any one of claims 23-30.

32. A method for preparing circular RNA, comprising transcribing a precursor RNA using the recombinant vector of claim 31, and further preparing the circular RNA from the precursor RNA.

33. A cell comprising the recombinant vector of claim 31.

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

535. The composition of claim 34, further comprising one or more pharmaceutically acceptable excipients, carriers, adjuvants, and vehicles.

36. A method for preventing, diagnosing, and / or treating diseases, comprising 10   administering to a subject at least one of the flexible circularization system of any oneof claims 1-13, the recombinant vector of any one of claims 14-15, the cell of claim 16, the recombinant nucleic acid molecule of any one of claims 23-30, the recombinant vector of claim 31, the cell of claim 33, and the composition of any one of claims 3435.15

Images