WO2025011502A1 - Method for preparing circular rna and nucleic acid sequence for method - Google Patents

Abstract

Provided is a method for preparing a circular RNA, which method comprises inserting purification tags, such as poly(A) tags or functional variants thereof into two terminal arms of a linear RNA precursor, and separating the circular RNA, which is obtained by means of the cyclization reaction of the linear RNA precursor, from other byproducts of the cyclization reaction as impurities using an affinity chromatographic medium capable of simultaneously binding to the tags. Further provided are a linear RNA precursor with a purification tag inserted in a specific region and an encoding DNA thereof. The insertion of the purification tag in the specific region does not affect the yield of linear RNA precursors produced by means of the in-vitro transcription (IVT) of the encoding DNA.

Description

Method for preparing circular RNA and nucleic acid sequence used in the method Technical Field

The present invention relates to methods and nucleic acid sequences for preparing and purifying circular RNA.

Background Art

In recent years, the development and application of mRNA vaccines or mRNA drugs have played a vital role in controlling the COVID-19 pandemic, and have also greatly accelerated the development of mRNA drugs in other disease treatment fields. It has been noted that linear mRNA drugs face major challenges in vivo, such as low stability and short half-life. Since circular RNA does not contain free ends, it can effectively avoid degradation by exonuclease in cells, thus having significantly higher stability than linear mRNA molecules (see non-patent literature 1), providing an effective candidate solution to the above challenges.

Circular RNA (circRNA) is a type of covalently closed circular RNA molecule. Since the genome of plant viroids was first discovered as a circular RNA molecule in 1976, hundreds of natural circular RNAs have been identified in viruses (such as hepatitis D virus HDV) and eukaryotic cells (such as mammalian cells), and it has been found that these molecules perform a variety of different biological functions in vivo, including but not limited to encoding proteins, regulating gene transcription, acting as microRNA sponges, protein scaffolds, etc. (see non-patent literature 2). Related molecular biology and functional research and analysis have promoted the in vitro synthesis technology of circular RNA, and chemical synthesis, ligase method (T4 DNA ligase, T4 RNA ligase 1, T4 RNA ligase 2), and ribozyme method (including type I intron ribozyme and type II intron ribozyme) have been reported (see non-patent literature 1).

Among them, the ribozyme method is one of the main ways to synthesize circular RNA in the current field of nucleic acid drugs. It uses the PIE system (Permuted Intron-Exon System) established by modifying type I or type II self-splicing introns to prepare circular RNA. It is reported that the splicing of type I introns does not depend on any protein. In the presence of Mg ²⁺ and guanosine, splicing can be achieved through a two-step transesterification reaction: in the first step, guanosine attacks the 5' splicing site (5'SS), and the 3' hydroxyl group of guanosine undergoes an ester exchange reaction at this site, causing splicing between the 5' exon and the 5'intron; in the second step, the 3'-OH of the intermediate formed after the first reaction attacks the 3' splicing site (3'SS), and a second ester exchange reaction is carried out at this site, resulting in the exons on both sides of the intron being directly connected, and the 5' end of the intron itself is cyclized with the 3' end and then sheared off. According to the above mechanism of action, the natural type I self-splicing intron is divided into two, and the arrangement order of "exon 1-intron fragment 1-intron fragment 2-exon 2" is artificially replaced with the arrangement of "intron fragment 2-exon 2-exon 1-intron fragment 1", and the circular RNA obtained by cyclization of the "exon 2-exon 1" fragment can be prepared in the presence of Mg ²⁺ and guanosine. For example, non-patent documents 3 and 4 respectively reported the PIE system established by transforming the type I self-splicing intron of Anabaena (fish algae) pre-Trna or T4 phage Td gene through the above replacement method. The above system can successfully cyclize RNAs of varying lengths of ~100nt or ~550nt. The PIE system based on these two type I self-splicing intron ribozymes is still widely used in circular RNA synthesis. Non-patent document 5 also reports the optimization of the PIE system based on Anabaena pre-tRNA type I intron ribozyme, which can significantly improve the in vitro cyclization efficiency by adding external homology arms, internal homology arms, etc., and shorten the length of the cyclizable RNA fragment to 1. The length of circular RNA was increased to ~5kb, laying a good foundation for the synthesis of circular RNA drugs.

The synthesis process of the above circular RNA will produce a variety of RNA byproducts, including but not limited to: introns on both sides that are cut off after the circularization reaction, linear RNA precursors that have not yet been circularized, and linear and circular high molecular weight polymerized RNA produced by polymerization of multiple linear precursors. The prior art has reported that the presence of these byproducts has various adverse effects on the final circular RNA biological products, which not only reduces the purity and titer of the products, but also significantly increases the immunogenicity in vivo (see non-patent document 2). Therefore, there is an urgent need for a method that can efficiently prepare high-purity circular RNA molecules, especially a preparation method that can meet the production level of industrialization.

The existing preparation methods for separating byproducts from circular RNA mainly utilize the difference in molecular weight between the two, or the difference in physical properties between linear and circular structures, such as size exclusion chromatography (SEC) or high performance liquid chromatography (HPLC) (see non-patent literature 2 to 6, patent literature 1). However, the above purification methods make it difficult to distinguish between the target circular RNA and linear RNA precursors with very similar molecular weights, as well as nicked RNA with exactly the same molecular weight. Therefore, the conventional practice in the industry is to perform RNase R digestion before SEC or HPLC to remove the above impurities. At the same time, SEC and HPLC themselves have common problems such as low loading capacity, high cost, complex process, and low resolution. After adding additional enzyme treatment steps, it is even more difficult to simultaneously achieve improvements in yield, efficiency, and purity, let alone industrial-level circular RNA production.

Patent document 2 reports a method for purifying linear mRNA with a poly(A) structure using column chromatography using oligo dT as an affinity ligand. Although patent document 2 also mentions that poly(A) sequences can be artificially added to circular RNA molecules, the patent method adds poly(A) to the circular region of the RNA molecule, so theoretically it is impossible to simultaneously separate all self-splicing byproducts and target circular RNA molecules in one column chromatography step, especially it is impossible to separate linear RNA precursors, nicked RNA and target circular RNA molecules that also have poly(A) sequences and have not yet formed a circle.

Patent document 3 reports another method for purifying circular RNA, which includes the steps of adding a poly(A) tail to linear RNA mixed with circular RNA, and then removing the RNA containing the poly(A) tail using oligo(dT)25-coupled magnetic beads. However, the main purpose of this method is to separate natural circular RNA and its corresponding linear transcript fragments, rather than various byproducts in the synthesis process of circular RNA.

Patent document 4 reports a method of incorporating at least one purification tag into the 3' or 5' end of a linear precursor RNA, thereby enabling the use of antisense oligo affinity purification of circular RNA, but Patent document 4 has clearly excluded the use of poly(A) and its variants as purification tags as a whole. Specifically, poly(A) is an essential element constituting the circular RNA of Patent document 4. If it is also inserted as a tag into the 3' or 5' end of the linear RNA precursor, it is obvious that according to the design principle of affinity purification, it will not be possible to distinguish between the desired isolated circular RNA and the uncircularized impurities. Patent document 4 also clearly states that "as a result of self-splicing, the corresponding circularized RNA no longer contains 3' and/or 5' end purification tags". Therefore, Patent document 4 actually provides technical teachings that are completely opposite to the present invention.

In addition, Patent Document 4 also describes the use of SEQ ID NO: 208 (tctttaccctcgtcttgacg) and 209 (tatgctgttatccgtcgatt) as oligos for affinity purification of circular RNA. However, FIG. 6 of the document shows that the circular RNA The enrichment effect occurs after the step of separating the target circular RNA from the impurities that are not completely circularized. In other words, in Patent Document 4, adding a purification tag to the 3' or 5' end does not directly improve the circularization reaction efficiency and circular RNA purity.

References:

Non-patent literature 1: Chen et al., Frontiers in Bioengineering and Biotechnology (2021). 9: 787881;

Non-patent literature 2: Liu et al., Molecular Cell (2022), 82: 1-15;

Non-patent literature 3: Been et al., Nucleic Acids Research (1992). 20: 5357-5364;

Non-patent literature 4: Ford et al., Proceedings of National Academy of Science (1994).91:3117-3121;

Non-patent literature 5: Wesselhoeft et al., Nature Communications (2018). 9: 2629;

Non-patent literature 6: Wesselhoeft et al., Molecular Cell (2019). 74: 508-520;

Patent document 1: WO2019/236673A1;

Patent document 2: CN114381454A;

Patent document 3: CN110283895A;

Patent document: 4: WO2023/073228A1.

Summary of the invention

The inventors have found through in-depth research that by inserting a tag sequence into the two end arms of the linear RNA precursor used to prepare circular RNA, the specific binding of the tag sequence to the corresponding affinity ligand can be utilized to separate the prepared target circular RNA molecule from various RNA byproducts generated during the preparation process using affinity chromatography. Compared with the traditional circular RNA preparation method, the method of the present invention can use an affinity chromatography process that is easy to operate and industrialized, and can achieve industrial production while reducing production costs. The inventors also surprisingly found that after inserting polyadenylic acid or its functional variants at both ends of the linear RNA precursor, the production of byproducts such as nicked RNA can be significantly reduced or even basically eliminated, thereby greatly reducing the amount of enzyme used when using RNase R treatment during the circular RNA purification process, and even omitting this enzyme treatment step, which not only simplifies the process flow and reduces costs, but also significantly improves the production efficiency, product purity and yield of circularized RNA. In addition, the method of the present invention does not need to insert a tag that is irrelevant to its function into the target circular RNA, which improves the safety of the circular RNA. At the same time, the tag sequence exists in all impurities, which greatly simplifies purity detection and quality control. The circular RNA prepared by the method of the present invention substantially does not contain linear RNA byproducts, providing excellent active pharmaceutical ingredients for the development of circular RNA drugs. The present invention also screened the linear RNA precursor and its encoding DNA for suitable insertion of purification tags. The results showed that after the purification tag was inserted into a specific region of the encoding DNA of the linear RNA precursor, the yield of the linear RNA precursor produced by in vitro transcription (IVT) of the encoding DNA would not be reduced.

Therefore, in one aspect, the present invention provides a method for preparing circular RNA, the method comprising:

Step A: generating a linear RNA precursor, wherein the linear precursor comprises a 5' end arm, a 3' self-splicing site, a circularization region, a 5' self-splicing site and a 3' end arm in a manner operably connected to each other in sequence from 5' to 3' direction, wherein a first tag is inserted into the 5' end arm and a second tag is inserted into the 3' end arm;

Step B: placing the linear RNA precursor under conditions suitable for self-splicing of the 3' self-splicing site and the 5' self-splicing site, to obtain a mixture comprising a linear RNA fragment with the first tag and/or the second tag and a circular RNA obtained by cyclization of the cyclization region;

Step C: contacting the mixture with an affinity chromatography medium capable of simultaneously binding the first tag and the second tag for a period of time sufficient to allow the affinity chromatography medium to bind to the linear RNA fragment containing at least one of the tags; and

Step D: separating the affinity chromatography medium and the mixture after contacting the affinity chromatography medium, collecting the supernatant, and obtaining the circular RNA.

Wherein, the first tag and/or the second tag are independently selected from a poly(A) tag consisting of 20 to 100, preferably 30 to 90, more preferably 40 to 80, further preferably 45 to 70, and most preferably 50 to 65 consecutive adenine nucleotides or a functional variant thereof, and the first tag and the second tag are not present in the cyclization region and the circular RNA.

In some embodiments, the functional variant of the poly(A) tag used in the method of the present invention is to insert one or more non-A bases into the poly(A) tag, preferably insert 1 to 20 non-A bases, and more preferably insert 1 to 10 non-A bases.

In some embodiments, functional variants of the poly(A) tag used in the methods of the present invention include the following:

(1) a single element a, at least one element b, and at least one element c,

(2) only one element a, at least one element b, and at least one element d; or

(3) a single element a, at least one element b, at least one element c and at least one element d,

wherein the element a is composed of more than 20 consecutive adenine nucleotides, the element b is composed of more than 3 and less than 20 consecutive adenine nucleotides, the element c is composed of a nucleotide selected from uracil nucleotides, cytosine nucleotides, and guanine nucleotides, the element d is composed of more than 2 and less than 20 nucleotides, the nucleotides are arbitrarily selected from adenine nucleotides, uracil nucleotides, cytosine nucleotides, and guanine nucleotides, and the element d does not contain more than 3 consecutive adenine nucleotides, and the 5' and 3' terminal nucleotides are not adenine nucleotides,

Wherein, when the Poly(A) tag contains two or more of the element b, the element c or the element d at the same time, the sequences of every two elements b may be the same or different, the sequences of every two elements c may be the same or different, and the sequences of every two elements d may be the same or different.

Furthermore, the element a and the element b, the element c and the element d, the elements b, the elements c, and the elements d are not adjacent to each other.

In some embodiments, element a used in the method of the present invention consists of more than 20 and less than 80 consecutive adenine nucleotides, preferably consists of 30 to 70, 35 to 65, 40 to 60, or 45 to 55 consecutive adenine nucleotides, and more preferably consists of 60 consecutive adenine nucleotides.

In some embodiments, the element b used in the method of the present invention consists of 3 to 10, 10 to 19, 12 to 15, 14 nt to 17, or 16 to 19, preferably 19 consecutive adenine nucleotides. In some embodiments, the number of element b used in the method of the present invention is 2 to 10, preferably 2 to 5, and more preferably 3.

In some embodiments, the element c used in the method of the present invention is a guanine nucleotide. In some embodiments, the number of the element c used in the method of the present invention is 2 to 10, 3 to 8, 4 to 6, or 2 to 5, preferably 2.

In some embodiments, the element d used in the method of the present invention consists of 3 to 18, 5 to 16, 4 to 10, or 6 to 12 nucleotides, preferably 6 nucleotides. In a preferred embodiment, the element d used in the method of the present invention can be independently selected from any one of GAUAUC, GUAUAC, GAAUCU, GCAUAUGACU or GAUAUCGUAUAC. In some embodiments, the number of elements d used in the method of the present invention is 0 to 5, preferably 1 to 3, and more preferably 1.

In some embodiments, when element c and element d are present at the same time, the total number of element c and element d used in the method of the present invention is 2 to 15, preferably 3 to 5, and more preferably 3.

In a preferred embodiment, the functional variant of the poly(A) tag used in the method of the present invention has any one structure selected from the following structures: element a-element c-element b-element c-element b-element c-element b-element c-element b, element b-element c-element b-element c-element a-element d-element b-element c-element b-element c-element b, element b-element c-element b-element c-element b-element d-element a-element c, element a-element d-element b-element c-element b-element c-element b-element b, or, element b-element c-element b-element c-element b-element d-element a.

In an optional embodiment, the functional variant of the poly (A) tag used in the method of the present invention may further comprise an element e, wherein the element e is composed of one or two consecutive adenine nucleotides, wherein the element e is located at the 3' end of the Poly (A) tag sequence and is adjacent to the element d or the element c.

In some embodiments, the linear RNA precursor in the method of the present invention comprises a 5' external homology arm and a 3' intron fragment in the 5' end arm in the 5' to 3' direction, and the first tag is inserted in the 5' external homology arm, or inserted in the 5' terminal region of the 3' intron fragment near the 5' external homology arm, and the 5' terminal region is preferably 20 nucleotides at the 5' end of the 3' intron fragment, more preferably 15 nucleotides, and further preferably 10 nucleotides, or inserted at the 5' end of the 5' external homology arm. Upstream, and the linear RNA precursor in the method of the present invention also comprises a 5' intron fragment and a 3' external homologous arm in the 3' end arm in the 5' to 3' direction, and the second tag is inserted in the 3' external homologous arm, or inserted in the 3' terminal region of the 5' intron fragment close to the 3' external homologous arm, the 3' terminal region is preferably 20 nucleotides at the 3' end of the 5' intron fragment, more preferably 15 nucleotides, further preferably 10 nucleotides, or inserted downstream of the 3' end of the 3' external homologous arm.

In a preferred embodiment, the first tag is inserted at any position in the 5' external homology arm, and the second tag is inserted downstream of the 3' end of the 3' external homology arm (for example, the position immediately adjacent to the last nucleotide residue at the 3' end of the 3' external homology arm). In other preferred embodiments, the first tag is inserted at any position in the 5' external homology arm, and the second tag is inserted at any position in the 3' external homology arm. In other preferred embodiments, the first tag is inserted at any position in the 5' external homology arm, and the second tag is inserted at any position in the 3' terminal region of the 5' intron fragment. In a more preferred embodiment, the first tag is inserted at any position in the 5' external homology arm except the first nucleotide residue at the 5' end. In some preferred embodiments, the first tag is inserted at any position in the 5' external homology arm except the first nucleotide residue at the 5' end, and the second tag is inserted at any position in the 3' external homology arm. In some preferred embodiments, the first tag is inserted at any position in the 5' external homology arm except the first nucleotide residue at the 5' end, and the second tag is inserted at any position in the 3' external homology arm. In some preferred embodiments, the first tag is inserted at any position in the 5' external homology arm except the first nucleotide residue at the 5' end, and the second tag is inserted at any position in the 3' terminal region of the 5' intron fragment.

In some embodiments, the first tag is inserted at the 5' outer homology arm from the first nucleotide residue at the 5' end, in the 5' to 3' direction, at the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, or 20th residue position. In a preferred embodiment, the first tag is inserted at the 5' outer homology arm from the first nucleotide residue at the 5' end, at the 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, or 20th residue position. In a more preferred embodiment, the first tag is inserted at the 5' outer homology arm from the first nucleotide residue at the 5' end, at the 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, or 11th residue position.

Illustratively, the term "inserted at position n" or "inserted at residue position n" means that the 5'-most and 3'-most nucleotide residues of the described insertion sequence are covalently linked to the residues at positions n and n+1, respectively, before insertion. For example, "inserted at position 1" means that the inserted sequence is located between the original residues at positions 1 and 2.

In some embodiments, the second tag is inserted in the 3' outer homology arm from the last nucleotide residue at the 3' end, in the 3' to 5' direction at the 1st, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20th residue position. In other more preferred embodiments, the second tag is inserted in the 3' outer homology arm from the last nucleotide residue at the 3' end, in the 3' to 5' direction at the 1st, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11th residue position. In some embodiments, the second tag is inserted in the 5' intron fragment from the last nucleotide residue at the 3' end, in the 3' to 5' direction at the 1st, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20th residue position. In other more preferred embodiments, the second tag is inserted into the 5' intron fragment at the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th or 11th residue position in the 3' to 5' direction starting from the last nucleotide residue at the 3' end.

In some embodiments, the circularization region comprises a 3' coding region fragment, a translation initiation element, and a 5' coding region fragment in a manner that is operably connected to each other in the 5' to 3' direction. In other embodiments, the circularization region comprises a 3' exon fragment, a 5' internal homology arm, an insert fragment, a 3' internal homology arm, and a 5' exon fragment in a manner that is operably connected to each other in the 5' to 3' direction. In optional embodiments, the circularization region comprises a first spacer between the insert fragment and the 5' internal homology arm, and a second spacer between the insert fragment and the 3' internal homology arm. In other embodiments, the insert fragment comprises a translation initiation element, or comprises a translation initiation element and a coding region. In some embodiments, the translation initiation element is an IRES sequence. In a preferred embodiment, the IRES sequence can be selected from, but not limited to, the following IRES sequences: Taura syndrome virus, blood-sucking assassin bug virus, Theile's encephalomyelitis virus, simian virus 40, fire ant virus 1, cereal aphid virus, reticuloendotheliosis virus, Forman polio virus 1, soybean looper virus, Kashmir bee virus, human rhinovirus 2, glass leafhopper virus-1, human immunodeficiency virus type 1, glass leafhopper virus-1, lice P virus, hepatitis C virus, hepatitis A virus, GB hepatitis virus, foot-and-mouth disease virus, human enterovirus 71, equine rhinovirus, tea looper virus Like virus, encephalomyocarditis virus (EMCV), Drosophila C virus, crucifer tobacco virus, cricket paralysis virus, bovine viral diarrhea virus 1, black queen cell virus, aphid lethal paralysis virus, avian encephalomyelitis virus, acute bee paralysis virus, hibiscus yellow ringspot virus, swine fever virus, human FGF2, human SFTPA1, human AML1/RUNX1, Drosophila antennae, human AQP4, human AT1R, human BAG-1, human BCL2, human BiP, human c-IAPl, human c-myc, human eIF4G, mouse NDST4L, human LEF1, mouse HIF1α, Human n.myc, mouse Gtx, human p27kipl, human PDGF2/c-sis, human p53, human Pim-1, mouse Rbm3, Drosophila reaper, canine Scamper, Drosophila Ubx, human UNR, mouse UtrA, human VEGF-A, human XIAP, Drosophila hairless, Saccharomyces cerevisiae TFIID, Saccharomyces cerevisiae YAP1, human c-src, human FGF-1, simian picornavirus, turnip shrunken virus, aptamer of eIF4G, coxsackievirus B3 (CVB3) or coxsackievirus A (CVB1/2). In some embodiments, the IRES sequence is a wild-type IRES sequence or a modified IRES sequence. In certain embodiments, the IRES sequence is about 50 nucleotides in length.

In some embodiments, the inserted fragment comprises the coding sequence of a structural gene or its functional fragment or the sequence of a non-coding RNA or its complementary sequence, wherein the structural gene is selected from a polypeptide, a protein subunit, a protein active center, a protein or a protein hybrid of a non-natural catalytic group, a recombinant protein active subunit or active center/, a recombinant artificial enzyme or other biological effector units mainly composed of amino acids, and the non-coding RNA is selected from microRNA (miRNA), small interfering RNA (siRNA), PIWI protein-interacting RNA (piRNA), transfer RNA-derived small RNA (tsRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), long non-coding RNA (lncRNA), pseudogene, ceRNA (competing endogenous RNAs), microRNA sponge or other types of non-mRNA RNA.

In an optional embodiment, the length of the 5' and 3' outer homology arms is each independently greater than 5 nt, greater than 10 nt, greater than 15 nt, greater than 20 nt, greater than 25 nt, greater than 30 nt, greater than 40 nt, greater than 50 nt, greater than 60 nt, less than 5 nt, less than 10 nt, less than 15 nt, less than 20 nt, less than 25 nt, less than 30 nt, less than 40 nt, less than 50 nt, less than 60 nt, 5-60 nt, 10-55 nt, 15-50 nt, 20-45 nt, 25-40 nt, 30-35 nt, 10 nt, 15 nt, 20 nt, 25 nt, 30 nt, 35 nt, 40 nt, 45 nt, or 50 nt. In an optional embodiment, the length of the 5' and 3' intron fragments is each independently greater than 5 nt, greater than 10 nt, greater than 15 nt, greater than 20 nt, greater than 25 nt, greater than 30 nt, greater than 40 nt, greater than 50 nt, greater than 60 nt, less than 5 nt, less than 10 nt, less than 15 nt, less than 20 nt, less than 25 nt, less than 30 nt, less than 40 nt, less than 50 nt, less than 60 nt, less than 70 nt, less than 80 nt, less than 90 nt, less than 100 nt, less than 150 nt, less than 20 nt, less than 25 nt, less than 30 nt, less than 40 nt, less than 50 nt, less than 60 nt, 0nt, less than 90nt, less than 100nt, less than 150nt, less than 200nt, 5-200nt, 10-150nt, 50-200nt, 50-150nt, 5-60nt, 10-55nt, 15-50nt, 20-45nt, 25-40nt, 30-35nt, 10nt, 15nt, 20nt, 25nt, 30nt, 35nt, 40nt, 45nt, or 50nt. In an optional embodiment, the length of the 5' and 3' exonic fragments is each independently greater than 5 nt, greater than 10 nt, greater than 15 nt, greater than 20 nt, greater than 25 nt, greater than 30 nt, greater than 40 nt, greater than 50 nt, greater than 60 nt, less than 5 nt, less than 10 nt, less than 15 nt, less than 20 nt, less than 25 nt, less than 30 nt, less than 40 nt, less than 50 nt, less than 60 nt, 5-60 nt, 10-55 nt, 15-50 nt, 20-45 nt, 25-40 nt, 30-35 nt, 10 nt, 15 nt, 20 nt, 25 nt, 30 nt, 35 nt, 40 nt, 45 nt, or 50 nt. In optional embodiments, the lengths of the 5' and 3' internal homology arms are each independently greater than 5 nt, greater than 10 nt, greater than 15 nt, greater than 20 nt, greater than 25 nt, greater than 30 nt, greater than 40 nt, greater than 50 nt, greater than 60 nt, less than 5 nt, less than 10 nt, less than 15 nt, less than 20 nt, less than 25 nt, less than 30 nt, less than 40 nt, Less than 50nt, less than 60nt, 5-60nt, 10-55nt, 15-50nt, 20-45nt, 25-40nt, 30-35nt, 10nt, 15nt, 20nt, 25nt, 30nt, 35nt, 40nt, 45nt, or 50nt. In an optional embodiment, the length of the insert is greater than 50 nt, greater than 100 nt, greater than 150 nt, greater than 200 nt, greater than 250 nt, greater than 300 nt, greater than 400 nt, greater than 500 nt, greater than 600 nt, greater than 1k nt, greater than 1.5k nt, greater than 2k nt, greater than 3k nt, less than 50 nt, less than 100 nt, less than 150 nt, less than 200 nt, less than 250 nt, less than 300 nt, less than 400 nt, less than 500 nt, less than 600 nt, less than 600 nt, less than 1k nt, less than 1.5k nt, less than 2k nt, less than 3k nt, 50-5k nt, 50-5k nt, 50-4k nt, 50-3k nt, 50-2k nt, 50-1.5k nt, 50-1k nt, 50~600nt, 100~550nt, 150~500nt, 200~450nt, 250~400nt, 300~350nt.

In a preferred embodiment, the 5' external homology arm has a sequence as shown in SEQ ID NO: 1 or 56, and the 3' external homology arm has a sequence as shown in SEQ ID NO: 2 or 57. In a preferred embodiment, the 3' intron fragment and the 5' intron fragment are both from type I introns, preferably from the cyanobacterium Anabaena pre-tRNA gene or the T4 phage Td gene. In a more preferred embodiment, the 3' intron fragment has a sequence as shown in SEQ ID NO: 3, and the 5' intron fragment has a sequence as shown in SEQ ID NO: 4. In a preferred embodiment, the 3' intron fragment and the 5' intron fragment are both from type II introns, preferably from type II introns of Clostridium such as Clostridium tetani, or type II introns of Bacillus such as Bacillus thuringiensis. In a more preferred embodiment, the 3' intron fragment and the 5' intron fragment are from the type II intron contained in the nucleotide sequence shown in SEQ ID NO:5 or 6. In a preferred embodiment, the 3' exon fragment and the 5' exon fragment are from the 3' terminal region and the 5' terminal region of the natural exon, respectively, preferably from the cyanobacterium Anabaena pre-tRNA gene or the T4 phage Td gene. In a more preferred embodiment, the 3' exon fragment has a sequence as shown in SEQ ID NO:7, and the 5' exon fragment has a sequence as shown in SEQ ID NO:8. In a preferred embodiment, the 5' internal homology arm has a sequence as shown in SEQ ID NO:9, and the 3' internal homology arm has a sequence as shown in SEQ ID NO:10. In some embodiments, the first spacer is the same as or different from the second spacer.

In some embodiments, the affinity chromatography medium used in the method of the present invention is operably linked to an affinity ligand capable of specifically binding to the first tag and/or the second tag. In a preferred embodiment, the affinity ligand is selected from the group consisting of poly X1, poly _X1 - _X2 , poly _X1 - _X2 - _X3 , poly _{X1- X2} - _X3 - _X4 , wherein _X1 , _X2 , _X3 , _X4 are independently any one of A, G, C, T, U. In a more preferred embodiment, the affinity ligand is selected from any one of the group consisting of Oligo dT, Oligo dC, Oligo dG, Oligo dU.

In some embodiments, the affinity chromatography medium used in the method of the present invention is selected from any one of the group consisting of magnetic beads, dextran molecules, polyacrylamide macromolecules, macromolecular cellulose molecules, chitosan materials, modified polylactic acid materials, PET materials, inorganic silicate materials or other high molecular polymers.

In some embodiments, the methods of the present invention do not include the step of adding RNase R to the mixture comprising circular RNA to remove linear RNA.

In other embodiments, when the method of the present invention comprises adding RNase R to a mixture comprising circular RNA In the step of removing linear RNA, the amount of RNase R added is reduced to 30-50%, preferably 40% or 50%, of the amount used for purification without the poly(A) tag or its functional variant.

In another aspect, the present invention provides a circular RNA prepared using the preparation method of the present invention, which substantially does not contain non-circular RNA molecules. In a preferred embodiment, the circular RNA prepared using the preparation method of the present invention has a purity of greater than 90%, greater than 91%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, greater than 99%, greater than 99.5%, greater than 99.9% or higher. In other preferred embodiments, the circular RNA prepared using the method of the present invention has a purity of more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99%, more than 99.5%, more than 99.9% or more. In other preferred embodiments, the content of non-ideal nucleic acids (e.g., various intermediate complexes that are not completely cyclized but still retain elements such as ribozymes, etc.) in the circular RNA prepared using the method of the present invention is no more than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1% or less of the total RNA.

In another aspect, the present invention provides a linear RNA precursor for use in the methods of the present invention.

In another aspect, the present invention provides a nucleic acid sequence that can be transcribed to produce the linear RNA precursor of the present invention under conditions suitable for transcription. In a preferred embodiment, the nucleic acid sequence of the present invention also comprises a regulatory sequence necessary for transcription to produce the linear RNA precursor in an operably linked manner, including but not limited to a promoter, a terminator, a transcription factor binding site, an untranslated region (UTR), an enhancer, a palindrome, a cis-acting element, a trans-acting element, a TATA box, a CAAT box, an operator, and a transposon.

In another aspect, the present invention provides a vector comprising the nucleic acid sequence of the present invention. In some embodiments, the vector of the present invention is a linear DNA, a plasmid, a viral nucleic acid fragment or a cell genomic DNA fragment.

In another aspect, the present invention provides an engineered cell comprising the linear RNA precursor, circular RNA, nucleic acid sequence, or vector of the present invention.

In another aspect, the present invention provides a composition comprising the linear RNA precursor, circular RNA, nucleic acid sequence, vector, or engineered cell of the present invention.

In another aspect, the present invention also provides the use of the linear RNA precursor, nucleic acid sequence, vector, or engineered cell of the present invention to prepare circular RNA.

In another aspect, the present invention also provides the use of the linear RNA precursor, circular RNA, nucleic acid sequence, vector, or engineered cell of the present invention for preparing a drug, a cytotoxic agent, or an immunomodulatory preparation. In some embodiments, the therapeutic drug, cytotoxic agent, or immunomodulatory preparation is selected from viruses, pluripotent or multipotent stem cells, iPS, engineered immune cells, antibodies or antibody fragments, antibodies or antibody fragments conjugated to drugs, chemotherapeutic agents, immunosuppressive or modulatory agents, anti-infective drugs, anticancer agents, hypoglycemic drugs, cardiovascular and cerebrovascular disease therapeutic drugs, degenerative neurological disease drugs, obesity therapeutic drugs, hematological disease therapeutic drugs, respiratory disease therapeutic drugs, or retroviral disease therapeutic drugs.

In another aspect, the present invention also provides a method for administering circular RNA, which comprises administering an effective amount of the circular RNA of the present invention to an organism in need thereof, or administering an effective amount of the circular RNA of the present invention using the nucleic acid sequence, vector, engineered cell or the like. The circular RNA is prepared from cells or compositions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 schematically shows a nucleic acid sequence of a linear RNA precursor for preparing circular RNA according to the present application, wherein the following elements are sequentially included in the 5′ to 3′ direction: a 5′ external homology arm, a 3′ intron fragment, a 3′ exon fragment, a 5′ internal homology arm, a first spacer, a miRNA binding region (micro RNA binding sites), a second spacer, a 3′ internal homology arm, a 5′ exon fragment, a 5′ intron fragment, and a 3′ external homology arm, wherein the locations of two self-splicing sites are schematically shown by vertical arrows;

FIG2 shows a schematic diagram of a linear RNA precursor for preparing circular RNA according to the present application;

FIG3 shows a schematic diagram of another linear RNA precursor for preparing circular RNA according to the present application;

FIG4 shows a schematic diagram of another linear RNA precursor for preparing circular RNA according to the present application;

Figures 5A and 5B show gel electrophoresis images of the cyclization reaction products of the linear RNA precursor according to some embodiments of the present invention and the products after RNase R digestion. In the figure, M represents the Riboruler low molecular weight RNA reference; CK represents the comparative example 1 without poly (A) insertion;

FIG6 shows the relative IVT yields of the cyclization reaction products of the linear RNA precursors according to Preparation Examples 6 to 24 of the present application. In the figure, the IVT yield of each preparation example is expressed as a multiple of the control IVT yield and subjected to One-way ANOVA statistical analysis. **** indicates P<0.0001;

FIG7 shows the relative IVT yields of the cyclization reaction products of the linear RNA precursors according to Preparation Examples 25 to 29 of the present application. In the figure, the IVT yield of each preparation example is expressed as a multiple of the control IVT yield and subjected to One-way ANOVA statistical analysis. *** indicates P < 0.001, **** indicates P < 0.0001;

Figures 8A to 8D show HPLC graphs of the cyclization reaction products of the linear RNA precursor according to the present application before and after RNase R enzymatic cleavage treatment.

DETAILED DESCRIPTION

definition

Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure will have the meanings commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear, however, in the event of any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definitions.

As used herein, the term "comprising" or "including" means that the sequence, composition and method include the described components or steps, but do not exclude other components or steps. "Mainly composed of...", when used to define sequences, compositions and methods, should mean excluding any other components or other steps that are obviously important for the technical effect it should achieve. "Composed of..." should mean excluding other components and steps not mentioned.

As used herein, the term "linear RNA precursor" or "linear precursor" is used interchangeably and refers to an RNA precursor that is not covalently closed in a circle but can generate circular RNA during the circularization process, which is generally transcribed from a template DNA. In this article, the linear RNA precursor comprises a complete circular RNA sequence that has not yet been circularized, and a self-splicing sequence (e.g., a self-splicing site, an intron ribozyme fragment, and a homology arm, etc.) required for the circularization of the RNA sequence. These self-splicing sequences are removed from the linear RNA precursor during the circularization process to produce circular RNA and linear RNA fragments as by-products. In some embodiments, the linear RNA precursor of the present invention can be used to produce circular RNA by incubation in the presence of magnesium ions and guanosine nucleotides or nucleosides at a temperature (e.g., between 20° C. and 60° C.) at which RNA circularization occurs.

In a preferred embodiment, the linear RNA precursor includes the following elements: 5' external homology arm, first tag, 3' intron fragment, 3' self-splicing site, 3' exon fragment, 5' internal homology arm, inserted fragment, 3' internal homology arm, 5' exon fragment, 5' self-splicing site, 5' intron fragment, second tag and 3' external homology arm. In some embodiments, the 3' intron fragment, 3' self-splicing site, 3' exon fragment located 5' upstream of the inserted fragment, and the 5' exon fragment, 5' self-splicing site, 5' intron fragment located 3' downstream of the inserted fragment play a key role in the process of forming circular RNA by splicing of linear RNA precursor. For example, by utilizing the ribozyme properties of the 3' intron fragment, the 3' self-splicing site connecting the 3' intron fragment and the 3' exon fragment is broken under GTP initiation, and the break site can further trigger the break of the 5' self-splicing site connecting the 5' intron fragment and the 5' exon fragment, so that the 5' exon fragment and the 3' exon fragment are connected at the self-splicing site to form a circular RNA. The presence of the external homology arm and the internal homology arm in pairs can improve the self-splicing efficiency and the cyclization efficiency in the above-mentioned cyclization reaction.

The nucleotides in the linear RNA precursor of the present invention may be unmodified natural nucleotides or partially or completely modified non-natural nucleotides. In some embodiments, the linear RNA precursor of the present invention comprises only naturally occurring nucleotides. In other embodiments, the linear RNA precursor of the present invention comprises one or more modifications that can increase stability, such as 2'-O-methyl, fluorine or O-methoxyethyl conjugates, thiophosphate backbones or 2',4'-cyclic 2'-O-ethyl modifications (Holdt et al., Front Physiol., 9: 1262 (2018); Krutzfeldt et al., Nature, 438 (7068): 685-9 (2005); Crooke et al., Cell Metab 27 (4): 714-739 (2018)), and/or one or more modifications that can reduce the innate immunogenicity of the circular RNA molecule in the host, such as at least one N6-methyladenosine (m ⁶ A)

As used herein, the term "linear RNA fragment" refers to all RNA products derived from the linear RNA precursor after the linear RNA precursor undergoes a cyclization reaction, except for the target circular RNA molecule produced thereby, including but not limited to: the self-splicing sequences on both sides that are sheared off after the cyclization reaction, and the various splicing intermediate sequences produced during the cyclization reaction. Since the linear RNA precursor molecules may not all undergo a cyclization reaction, the "linear RNA fragment" may also include the linear RNA precursor sequence remaining in the cyclization reaction product. In addition, the length of the "linear RNA fragment" is not necessarily less than the linear RNA precursor from which it is derived, because the term also covers linear and circular high molecular weight polymerized RNA produced by polymerization of multiple identical or different sequences mentioned above. In this article, the term "linear RNA fragment" has substantially the same meaning as the "byproduct" or "impurity" produced by the cyclization reaction.

As used herein, the terms "circular RNA" or "circRNA" are used interchangeably and refer to polyribonucleotides that form a closed circular structure through covalent bonds. It has been reported that circular RNA is a 3-5' covalently closed RNA ring without a 5' end cap and a 3' end poly (A) tail. Due to the lack of free ends required for exonuclease-mediated degradation, it has the property of resisting RNA enzyme degradation and has a longer life or half-life than ordinary linear RNA products (e.g., mature mRNA). Circular RNA can be produced by a splicing process, and cyclization mainly occurs at the annotated exon boundaries using conventional splicing sites (Starke et al., 2015; Szabo et al., 2015). In a preferred embodiment, such a circular RNA is a single-stranded RNA molecule. Unless otherwise explicitly stated, the circular RNA molecule herein can have any structure suitable for circular RNA known in the art, but does not contain the same sequence as the first tag and the second tag present in the circular RNA precursor.

RNA nicking refers to the phenomenon of single-point breakage of the RNA chain. During the preparation of circular RNA, the presence of metal ions (especially Mg2+) in the reaction system causes random breakage of circular RNA, thereby generating nicked RNA, which has the same molecular weight as circular RNA and is easily digested by RNase.

As used herein, the term "intron fragment" refers to, for example, a sequence having 75% or higher similarity to a natural type I or type II intron ribozyme (Ribozyme or intronribozyme) or its main active fragment, or a small ribozyme (e.g., satellite RNA, mainly including hammerhead ribozymes, hairpin ribozymes, hepatitis D virus (HDV) RNA, Varkud satellite (VS) ribozyme, and GlmS riboswitch). For example, an exemplary type I intron ribozyme can be a natural intron self-cleaving ribozyme sequence of Anabaena (whose accession number is included in the GenBank database (GenBank: AY768517), with a total sequence length of 313 bases), or a main active fragment of the sequence (e.g., a sequence as described in SEQ ID NO.2 in CN115786374A, with a total sequence length of 246 bases), or an equivalent of the above ribozyme with various base substitutions or truncations. Exemplary type II ribozymes can be derived from yeast mitochondrial DNA (as described in the following references: Zimmerly et al., Mob DNA, 2015), the Ll.LtrB intron of Lactococcus lactis, the TeI3c/4c type II intron of Thermosynechococcus elongatus (as described in the following references: Monat et al., PloS One, 2020; Costa et al., Sscience, 2016), type II introns of Clostridium such as Clostridium tetani, type II introns of Bacillus such as Bacillus thuringiensis, or the commercial ribozyme tool Targetron. As used herein, the term "3' intron fragment" refers to a sequence having 75% or higher similarity to the 3' terminal region of the Anabaena ribozyme, and the term "5' intron fragment" refers to a sequence having 75% or higher similarity to the 5' terminal region of the Anabaena ribozyme, as long as the two intron fragments can be close to each other in the spatial structure of the RNA to form a complex with complete ribozyme activity (as described in the following reference: Wesselhoeft et al., Nature Communication, 2018). A person skilled in the art can determine the sequences that can serve as the "3' intron fragment" and the "5' intron fragment" in the natural intron ribozyme.

As used herein, the term "exon fragment" may refer to an exon recognized and spliced by an intron ribozyme in the intron-exon (PIE) system of a ribozyme, or a fragment of a signal sequence recognized by an intron ribozyme. For example, a "3' exon fragment" may refer to a fragment of a signal sequence recognized by an intron ribozyme. To represent a sequence having 75% or higher similarity with the 3' end region of the exon recognized and cleaved by the Anabaena ribozyme, a "5' exon fragment" can represent a sequence having 75% or higher similarity with the 5' end region of the exon recognized and cleaved by the Anabaena ribozyme, as long as the two exon fragments are reversed in direction, can be recognized by the ribozyme and undergo self-splicing (as described in the following reference: Wesselhoeft et al., Nature Communication, 2018). A person skilled in the art can determine the sequences in the exons of natural genes that can serve as "3' exon fragments" and "5' exon fragments".

As used herein, the term "self-splicing site" or "splice site" refers to the position between dinucleotides where cleavage occurs during the RNA circularization reaction and generates free ends for circularization.

As used herein, the term "circularization region" refers to a region that is included in the formed circular RNA but not in the sheared RNA after linear RNA is prepared into circular RNA in vitro or circular RNA is formed in vivo by splint-mediated method, permuted intron-exon method, RNA ligase-mediated method or other methods, and is irrelevant to the RNA circularization method, the structure and function of the circularization intermediate.

As used herein, the term "tag" refers to a sequence that can be used to bind to a complementary oligonucleotide (which can be referred to as an "affinity ligand") on an affinity chromatography matrix, so that molecules containing the tag are specifically adsorbed to the affinity chromatography matrix and separated from molecules that do not contain the tag. The nucleotides constituting the tag can be naturally occurring nucleotides or artificially modified non-natural nucleotides.

It should be noted that the expressions "first", "second" or similar expressions used here and in the text of this article are only intended to distinguish the two elements in a specific category, and do not indicate the importance, order, etc. of the elements; the "first" and "second" elements can be the same or different referents/concepts. For example, in some cases, the "first tag" and the "second tag" can represent the same tag (e.g., polyA) or different tags (e.g., polyA or its functional variants).

As used herein, the term "poly (A) tag" has a meaning recognized and understood by a person of ordinary skill in the art, for example, it refers to a sequence composed of polyadenine nucleotides, which is generally located near the 3' terminal region or the 3' end (also commonly referred to as the poly (A) tail) in a linear messenger RNA molecule. It is generally believed that the poly (A) sequence at the 3' end generally protects the mRNA from 3' end degradation in a linear mRNA molecule and plays an important role in cap-dependent protein translation. Patent document 4 also reports that the poly (A) sequence in circular RNA has the effect of reducing its immunogenicity. The poly (A) tag of the present invention is generally composed of about 20 to up to about 100 adenine nucleotides, preferably 30 to 90, more preferably 40 to 80, further preferably 45 to 70, and most preferably 50 to 65 consecutive adenine nucleotides. In some embodiments, the poly (A) tag of the invention is composed of 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, 56, 57, 58 , 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 consecutive adenine nucleotides.

As used herein, the term "Poly(A) tag" may also refer to a class of poly(A) tags comprising at least one non-A base, including but not limited to PCT application PCT/CN2023/079037, Chinese patent application CN112805386A, U.S. patent application Various poly (A) sequences described in US10717982B2. The above application or patent text is hereby incorporated in its entirety. Such poly (A) sequences are also referred to herein as "functional variants of poly (A)", meaning that the above sequence consisting of continuous adenine nucleotides is interrupted by individual non-A nucleotides, but still has substantially the same functional activity (such as the effect on 3' terminal stability and/or translation activity) as the sequence consisting of continuous adenine nucleotides.

Specifically, the "functional variant of poly(A)" in the present invention includes the following different situations:

(1) a functional variant comprising only one element a, at least one element b, and at least one element c,

(2) a functional variant comprising only element a, at least one element b, and at least one element d; or

(3) a functional variant comprising only one element a, at least one element b, and at least one element c and at least one element d,

Among them, element a is composed of multiple consecutive adenine (A) nucleotides, and the length range is ≥20nt; element b is composed of multiple consecutive A nucleotides, and in some embodiments, the length range of element b is 3nt≤b＜20nt; element c is composed of a non-A nucleotide, and the nucleotide is selected from thymine (T), cytosine (C), and guanine (G) nucleotides; element d is composed of any two or more consecutive nucleotides, and the nucleotides are selected from A, T, C, and G nucleotides, wherein the nucleotides at the 5' and 3' ends of element d are not A nucleotides, and element d does not contain more than 3 consecutive A nucleotides, and the length range of element d is 2nt≤d≤20nt; element e is composed of one or two consecutive A, and when it exists, it is located and can only be located at the 3' end of the Poly (A) coding sequence, and is adjacent to element d or element c.

When a Poly(A) contains two or more "element b", "element c", and "element d" at the same time, the sequences of every two elements b can be the same or different, the sequences of every two elements c can be the same or different, and the sequences of every two elements d can be the same or different, as long as they each meet the above definitions of elements a, b, c and d.

In some embodiments, element a and element b in the poly(A) tag are not adjacent, element c and element d are not adjacent, elements b are not adjacent to each other, elements c are not adjacent to each other, and elements d are not adjacent to each other.

In some embodiments, the Poly(A) tag further comprises a unique element e, which consists of one or two consecutive A's and is located at the 3' end of the Poly(A) tag and adjacent to element d or element c.

In some embodiments, Poly(A) and its functional variants can be a segment of RNA or a hybrid molecule of DNA and RNA.

In some embodiments, the sequence structure of the Poly(A) tag is selected from:

Element a-element c-element b-element c-element b-element c-element b-element c-element b;

Element b-element c-element b-element c-element a-element d-element b-element c-element b-element c-element b;

Element b-element c-element b-element c-element b-element d-element a-element c;

element a-element d-element b-element c-element b-element c-element b; or

Element b-element c-element b-element c-element b-element d-element a.

As used herein, the term "binding" refers to the reversible complementary pairing between nucleotide bases through intermolecular interaction forces (e.g., hydrogen bonds), or complementary pairing through chemical bonds, so that nucleic acid molecules can be reversibly paired under specific conditions or reactions. A certain affinity can be achieved between nucleic acids and matrix materials (e.g., organic polymer materials or magnetic beads) carrying nucleic acids, or between matrix materials carrying nucleic acids. For example, a certain length (e.g., 50 nt) of polyA oligosingle-stranded RNA or a long single-stranded RNA containing the oligosingle-stranded polyA can "bind" to a dextran matrix carrying oligo dT under certain conditions by means of complementary base pairing, and optionally, the hydrogen bond between polyA and oligo dT can be opened by changing environmental conditions (e.g., temperature change, strong ion conditions, or strong acid-base conditions), thereby making the "binding" reversible, and further making the oligosingle-stranded RNA or the long single-stranded RNA containing the oligosingle-stranded polyA detached from the dextran matrix; in addition, optionally, an irreversible chemical bond can be formed between polyA and oligo dT under certain conditions, thereby making the RNA irreversibly bound to the dextran matrix.

As used herein, the term "affinity chromatography medium" refers to materials with oligonucleotides complementary to purification tags and affinity chromatography matrices. Ideal oligonucleotides can be designed according to specific needs. For example, in the case where the tag is poly(A), the ideal oligonucleotide on the medium can be oligo dT. Affinity chromatography matrices can be selected from stable materials that are unreactive with nucleotides or inert with nucleotide molecules, and there is no obvious mutual repulsion or mutual attraction between the molecular surface and the nucleotide molecules. For example, the matrix can be dextran molecules, polyacrylamide macromolecules, macromolecular cellulose molecules, chitosan materials, modified polylactic acid materials, PET materials, inorganic silicate materials, metal materials with coating layers, etc. Generally, the oligonucleotides and affinity chromatography matrices are cross-linked by covalent bonds. In addition, the oligonucleotides on the medium can also be replaced by other molecules, as long as such molecules have a preferred intermolecular force with the nucleotides. For example, in the case where the tag is an oligonucleotide composed of at least one of U, C and A, hypoxanthine can be cross-linked to the matrix to form a desired affinity chromatography medium.

As used herein, the term "separation" refers to the separation of circular RNA products from linear RNA precursors, linear RNA fragments, various intermediate complexes formed by self-splicing processes, etc., thereby simply obtaining the effect of purification. Techniques for purifying target polynucleotides and polypeptides are well known in the art and include, for example, ion exchange chromatography, affinity chromatography, and sedimentation according to density. Generally, a substance is purified when it is present in a sample in an amount greater than its naturally occurring amount relative to other components of the sample. In a preferred embodiment herein, the circular RNA separated by affinity chromatography or affinity adsorption has a purity of greater than 90%, greater than 91%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, greater than 99%, greater than 99.5%, greater than 99.9% or higher, or has a purity of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or higher, wherein the content of non-ideal nucleic acids (e.g., various intermediate complexes that are not completely circularized and still retain elements such as ribozymes, etc.) therein is no more than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1% or less.

Linear nucleic acid molecules are said to have a "5'-terminus" (5' end) and a "3'-terminus" (3' end) because nucleic acid phosphodiester bonds are present at the 5' carbon and 3' carbon of the sugar portion of the substituted mononucleotide. In some examples, the 5' terminal nucleotide of the nucleic acid molecule is a nucleotide that can form a new phosphodiester bond with its ribose 5' carbon, and correspondingly, the 3' terminal nucleotide is a nucleotide that can form a new phosphodiester bond with its ribose 3' carbon.

As used herein, the term "5'upstream" refers to the portion of a nucleic acid fragment that precedes the fragment in the 5' to 3' direction and is not within the fragment. The term "3' end downstream" refers to the position of a specific nucleic acid fragment in the 5' to 3' direction, but not in the fragment. For example, in a 1kb nucleic acid sequence, the "5' end upstream" of a specific nucleic acid fragment located at the 100bp to 199bp may include any position from the 0bp to the 99bp, and its "3' end downstream" may include any position from the 200bp to the 1000bp. Based on a similar concept, the term "5' terminal region" refers to a number of consecutive nucleotide residues in the 5' to 3' direction from the 5' terminal nucleotide of a specific nucleic acid fragment, and the term "3' terminal region" refers to a number of consecutive nucleotide residues in the 3' to 5' direction from the 3' terminal nucleotide of a specific nucleic acid fragment. Those skilled in the art can easily determine the 5' and 3' terminal nucleotides of the specific fragment by comparing with the complete nucleic acid molecule. For example, in a 1kb nucleic acid sequence, the "5' terminal region" of a specific nucleic acid fragment located at 100bp to 199bp can represent 100bp to 119bp, 100bp to 114bp, 100bp to 109bp, and its "3' terminal region" can represent 170bp to 199bp, 175bp to 199bp, 180bp to 199bp. It should be noted that the specific nucleic acid lengths and positions listed in the explanations here are exemplary, and these terms can be supplemented, extended, expanded, replaced, synonymous, and equivalent without violating the concept and spirit of the present disclosure.

As used herein, the term "structural gene" refers to a gene that can encode various polypeptides or proteins, and "functional fragments of structural genes" may refer to fragments of broken structural genes that can ultimately be expressed as polypeptides in whole or in part. Non-limiting examples of functional fragments of structural genes include protein subunits, protein active centers, protein hybrids of proteins or non-natural catalytic groups, recombinant protein active subunits or active centers, recombinant artificial enzymes or other biological effect units mainly composed of amino acids, etc.; "non-coding RNA" may refer to RNA that does not encode polypeptide or protein products, including rRNA, tRNA, snRNA, snoRNA, microRNA, micronRNA sponge, miRNA, lncRNA, circRNA, piRNA and other RNAs with known functions, and also include RNAs with unknown functions.

As used herein, the term "spacer" refers to a nucleic acid sequence between a functional sequence (e.g., a promoter or a ribosome entry sequence) and another functional sequence (e.g., a structural gene or a fragment thereof, or a non-coding RNA or a DNA transcribed from a non-coding RNA), which is used to space the binding factors of the two functional sequences at a certain distance, or to prevent the two functional sequences from affecting each other's spatial structure, or to space the binding factors of a functional sequence at a certain distance from the spatial structure of the other functional sequence itself, which does not encode a polypeptide, protein or non-coding RNA with a biological effect, or the transcription product or translation product at least does not degrade the biological activity of the polypeptide, protein or non-coding RNA. For example, in some embodiments, the spacer can be a scrambled, mononucleotide repeated, or polynucleotide repeated sequence of a specific length (e.g., 9nt).

As used herein, the term "RNase R" (Ribonuclease R, RNase R) is a ribonuclease exonuclease derived from the Escherichia coli RNR superfamily, which can cut and degrade linear RNA molecules from the 3' to 5' direction, but basically does not digest circular RNA, lasso structure or double-linked RNA molecules with 7nt missing at the 3' protruding end. For specific examples of RNase R, please refer to the following reference: Cheng ZF, Biol Chem, 2002. It can be understood by those skilled in the art that the term "RNase R" can include polypeptides, hybrid enzymes, polypeptide analogs, etc. with the same enzyme activity that are artificially modified or recombinantly produced based on RNase R.

As used herein, the term "immunogenicity" refers to the potential to induce an immune response to a substance. When an organism's immune system or a certain type of immune cell is exposed to an immunogenic substance, an immune response may be induced.

As used herein, the term "cyclization efficiency" refers to a measure of the resulting circular polyribonucleotide compared to its linear starting material.

As used herein, the term "translation efficiency" refers to the rate or amount of protein or peptide produced from ribonucleotide transcripts. In some embodiments, translation efficiency can be expressed as the amount of protein or peptide produced per a given amount of transcript encoding a protein or peptide.

The term "nucleotide" refers to a ribonucleotide, a deoxyribonucleotide, a modified form thereof, or an analog thereof. Nucleotides include substances including purines (e.g., adenine, hypoxanthine, guanine, and derivatives and analogs thereof) and pyrimidines (e.g., cytosine, uracil, thymine, and derivatives and analogs thereof). Nucleotide analogs include nucleotides having modifications in the chemical structure of bases, sugars, and/or phosphates, including but not limited to, 5'-position pyrimidine modifications, 8'-position purine modifications, modifications at the cytosine exocyclic amine, and substitutions of 5-bromo-uracil; and 2'-position sugar modifications, including but not limited to sugar-modified ribonucleotides, wherein 2'-OH is substituted by a group such as H, OR, R, halo, SH, SR, NH2, NHR, NR2, or CN, wherein R is an alkyl moiety as defined herein. Nucleotide analogs are also intended to include nucleotides having bases such as inosine, quercetin, xanthine; sugars such as 2'-methylribose; non-natural phosphodiester linkages such as methylphosphonate, phosphorothioate, and peptide linkages. Nucleotide analogs include 5-methoxyuridine, 1-methylpseudouridine, and 6-methyladenosine.

The terms "nucleic acid" and "polynucleotide" are used interchangeably herein to describe a polymer of any length (e.g., greater than about 2 bases, greater than about 10 bases, greater than about 100 bases, greater than about 500 bases, greater than 1000 bases, or up to about 10,000 or more bases), composed of nucleotides (e.g., deoxyribonucleotides or ribonucleotides), and can be produced enzymatically or synthetically (e.g., as described in U.S. Pat. No. 5,948,902 and references cited therein), which can hybridize with naturally occurring nucleic acids in a sequence-specific manner similar to two naturally occurring nucleic acids, for example, can participate in Watson-Crick base pairing interactions. Naturally occurring nucleic acids are composed of nucleotides, including guanine, cytosine, adenine, thymine, and uracil (G, C, A, T, and U, respectively).

As used herein, the terms "ribonucleic acid" and "RNA" refer to a polymer composed of ribonucleotides.

As used herein, the terms "deoxyribonucleic acid" and "DNA" refer to a polymer composed of deoxyribonucleotides.

As used herein, when two "homology arms" or "homologous regions" have a sufficient level of sequence identity with each other's reverse complementary sequences to serve as substrates for hybridization reactions, the two regions are complementary to each other or complementary. As used herein, a polynucleotide sequence has "homology" when it is identical or shares sequence identity with a reverse complementary sequence or "complementary" sequence. The percentage of sequence identity between a homologous region and the reverse complementary sequence of the corresponding homologous region can be any percentage of sequence identity that allows hybridization to occur. In some embodiments, the internal duplex forming region of the polynucleotide of the present invention is capable of forming a duplex with another internal duplex forming region and does not form a duplex with an external duplex forming region.

"Transcription" refers to the formation or synthesis of RNA molecules by RNA polymerase using a DNA molecule as a template. The present invention is not limited to the RNA polymerase used for transcription. For example, in some embodiments, a T7-type RNA polymerase may be used. Enzymes. "Translation" refers to the formation of polypeptide molecules by ribosomes based on RNA templates.

It should be understood that the terms used herein are for the purpose of describing specific embodiments only and are not intended to be limiting. As used in this specification and the appended claims, unless the content clearly indicates otherwise, the singular forms "a/an" and "the" include plural referents. Thus, for example, reference to "a cell" includes a combination of two or more cells, or the entire culture of cells; reference to "polynucleotides" actually includes many copies of the polynucleotides. Unless expressly provided or obvious from the context, as used herein, the term "or" is understood to be inclusive. Unless otherwise defined herein or and in the remainder of the specification below, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which the invention belongs.

Unless expressly provided or obvious from the context, as used herein, the term "about" should be understood to be within the normal tolerance range of the field, such as within 2 standard deviations of the mean value. "About" can be understood to be within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02% or 0.01% of the value. Unless otherwise obvious from the context, all numerical values provided herein are modified by the term "about".

As used herein, the term "encoding" refers broadly to any process in which information in a polymeric macromolecule is used to direct the production of a second molecule that is different from the first molecule. The second molecule may have a chemical structure that is different in chemical nature from the first molecule.

"Combination" or "co-administration" refers to the co-administration of a therapeutic agent provided herein and one or more additional therapeutic agents sufficiently close in time that the therapeutic agent provided herein can enhance the effect of the one or more additional therapeutic agents, or vice versa.

As used herein, the terms "treat" and "prevent" and words derived therefrom do not necessarily mean 100% or complete treatment or prevention. Rather, there are varying degrees of treatment or prevention that one of ordinary skill in the art recognizes as having potential benefits or therapeutic effects. The treatment or prevention provided by the methods disclosed herein may include treating or preventing one or more disorders or symptoms of a disease. In addition, for purposes herein, "prevention" may include delaying the onset of a disease or a symptom or disorder thereof.

As used herein, the term "expressed sequence" may refer to a nucleic acid sequence encoding a product such as a peptide or polypeptide, a regulatory nucleic acid, or a non-coding nucleic acid. An exemplary expressed sequence encoding a peptide or polypeptide may comprise a plurality of nucleotide triplets, each of which may encode an amino acid and is referred to as a "codon".

The term "antibody" (Ab) includes, but is not limited to, glycoprotein immunoglobulins that specifically bind to an antigen. In general, an antibody may comprise at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen-binding molecule thereof. Each H chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region comprises three constant domains, CH1, CH2, and CH3. Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region comprises one constant domain, CL. The VH and VL regions can be further subdivided into hypervariable regions, called complementary determining regions (CDRs), interspersed with more conservative regions, called framework regions (FRs). Each VH and VL comprises three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system. Antibodies may include, for example, monoclonal antibodies. Clonal antibodies, recombinantly produced antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, engineered antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies comprising two heavy chains and two light chain molecules, antibody light chain monomers, antibody heavy chain monomers, antibody light chain dimers, antibody heavy chain dimers, antibody light chain-antibody heavy chain pairs, intrabodies, antibody fusions (sometimes referred to herein as "antibody conjugates"), heteroconjugate antibodies, single domain antibodies, monovalent antibodies, single chain antibodies or single chain Fv (scFv), camelized antibodies, affibodies, Fab fragments, F(ab')2 fragments, disulfide-linked Fv (sdFv), anti-idiotype antibodies (including, for example, anti-anti-Id antibodies), miniantibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as "antibody mimetics"), and antigen-binding fragments of any of the above. In some embodiments, the antibodies described herein refer to polyclonal antibody populations.

Immunoglobulins can be derived from any known isotype, including but not limited to IgA, secretory IgA, IgG and IgM. IgG subclasses are also well known to those skilled in the art, including but not limited to human IgG1, IgG2, IgG3 and IgG4. "Isotype" refers to the Ab class or subclass (e.g., IgM or IgG1) encoded by the heavy chain constant region gene. For example, the term "antibody" includes naturally occurring and non-naturally occurring antibodies; monoclonal and polyclonal antibodies; chimeric and humanized antibodies; human or non-human antibodies; fully synthetic antibodies; and single-chain antibodies. Non-human antibodies can be humanized by recombinant methods to reduce their immunogenicity in humans. In the absence of explicit instructions and unless otherwise indicated in the context, the term "antibody" also includes an antigen-binding fragment or antigen-binding portion of any of the above-mentioned immunoglobulins, and includes monovalent and divalent fragments or portions, as well as single-chain antibodies.

"Antigen binding molecule", "antigen binding portion" or "antibody fragment" refers to any molecule that comprises an antigen binding portion (e.g., CDR) of an antibody from which the molecule is derived. Antigen binding molecules may include antigen complementary determining regions (CDRs). Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2 and Fv fragments, dAbs, linear antibodies, scFv antibodies, and multispecific antibodies formed by antigen binding molecules. Peptibodies (i.e., Fc fusion molecules comprising peptide binding domains) are another example of suitable antigen binding molecules. In some embodiments, the antigen binding molecules bind to antigens on tumor cells. In some embodiments, the antigen binding molecules bind to antigens on cells involved in hyperproliferative diseases or to viral or bacterial antigens. In some embodiments, the antigen binding molecules bind to BCMA. In other embodiments, the antigen binding molecules are antibody fragments that specifically bind to antigens, including one or more complementary determining regions (CDRs) thereof. In other embodiments, the antigen binding molecules are single-chain variable fragments (scFv).

"Cancer" or "cancer" refers to a broad variety of diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and growth lead to the formation of malignant tumors that invade adjacent tissues and may also metastasize to distant parts of the body via the lymphatic system or bloodstream. "Cancer" or "cancerous tissue" may include tumors. Examples of cancers that may be treated by the methods disclosed herein include, but are not limited to, cancers of the immune system, including lymphomas, leukemias, myelomas, and other white blood cell malignancies. In some embodiments, the methods disclosed herein can be used to reduce the size of tumors originating from, for example, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, testicular cancer, uterine cancer, multiple myeloma, Hodgkin's disease, non-Hodgkin's lymphoma (NHL), primary mediastinal large B-cell lymphoma (PMBC), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), transformed follicular lymphoma, splenic marginal zone lymphoma (SMZL), esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer adenocarcinoma, adrenal cancer, urethral cancer, penile cancer, chronic or acute leukemia, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia (ALL) (including non-T cell ALL), chronic lymphocytic leukemia (CLL), solid tumors in children, lymphocytic lymphoma, bladder cancer, kidney cancer or ureteral cancer, central nervous system (CNS) tumors, primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brain stem glioma, pituitary adenoma, epidermoid carcinoma, squamous cell carcinoma, T cell lymphoma, environmentally induced cancers including those induced by asbestos, other B cell malignancies, and combinations of the described cancers. In some embodiments, the methods disclosed herein can be used to reduce the size of tumors derived from, for example, sarcomas and carcinomas, fibrosarcomas, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, Kaposi's sarcoma, soft tissue sarcomas and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, lung cancer, colorectal cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma (e.g., pancreatic, colon, ovarian, lung, breast, stomach, prostate, cervical cancer, or esophageal adenocarcinoma), sweat gland cancer, sebaceous gland cancer, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocarcinoma, bile duct cancer, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumors, bladder cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, renal pelvis cancer, CNS tumors (such as glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma and retinoblastoma). Specific cancers may respond to chemotherapy or radiation therapy, or the cancer may be refractory. Refractory cancer refers to cancer that is not suitable for surgical intervention and the cancer initially does not respond to chemotherapy or radiation therapy, or the cancer becomes unresponsive over time.

As used herein, the term "immune cell" or "lymphocyte" includes natural killer (NK) cells, T cells or B cells. NK cells are a type of cytotoxic (cytotoxic) lymphocytes that represent the main components of the innate immune system. NK cells repel tumors and cells infected by viruses. It works through the process of apoptosis or programmed cell death. They are called "natural killers" because they do not need to be activated to kill cells. T cells play a major role in cell-mediated immunity (without antibody involvement).

The term "genetic engineering" or "engineering" refers to a method of modifying the genome of a cell, including but not limited to deleting a coding or non-coding region or a portion thereof or inserting a coding region or a portion thereof. In some embodiments, the modified cell is a lymphocyte, such as a T cell, which can be obtained from a patient or a donor. The cell can be modified to express an exogenous construct incorporated into the cell genome, such as a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

As used herein, the expression "sequence identity" or, for example, "a sequence that is 50% identical to ... " refers to the degree to which the sequences are identical on a nucleotide-by-nucleotide or amino acid-by-amino acid basis over the comparison window. Therefore, the "percentage of sequence identity" can be calculated by comparing two optimally aligned sequences over the comparison window, determining the number of positions where the same nucleic acid base (e.g., A, T, C, G, I) or the same amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) appears in the two sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to obtain the percentage of sequence identity. Including sequences that have at least about 50%, 55%, 60%, 65%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, 500%, 510%, 520%, 530%, 540%, 550%, 5 Nucleotides and polypeptides with 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, typically wherein the polypeptide variant retains at least one biological activity of the reference polypeptide.

As used herein, the term "treatment" refers to both therapeutic treatment and prophylactic or preventive or preventive measures, wherein the purpose is to prevent or slow down (mitigate) an undesirable pathological change or condition. For purposes of the present invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction in disease severity, delay or slowing of disease progression, improvement or alleviation of the disease state, and remission (whether partial or complete), whether detectable or undetectable.

As used herein, the term "therapeutically effective amount" refers to an amount of a compound of the invention that can: (i) treat or prevent a disease or condition described herein, (ii) ameliorate or eliminate one or more diseases or conditions described herein, or (iii) prevent or delay the onset of one or more symptoms of a disease or condition described herein.

As used herein, the term "non-coding RNA" or "ncRNA" mainly includes microRNA (miRNA), small interfering RNA (siRNA), PIWI-interacting RNA (piRNA), tRNA-derived small RNA (tsRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), long non-coding RNA (lncRNA), circular RNA (circRNA), pseudogenes, etc. miRNA is a short ncRNA of about 22 to 23 nucleotides. Its coding gene is transcribed by RNA polymerase II and regulates the expression of mRNA by binding to the 3′ untranslated region (3′ UTR) of mRNA. More than 60% of coding genes are potential regulatory targets of miRNA. lncRNA is a non-coding RNA with a length of more than 200 nucleotides. Its biogenesis process is similar to that of mRNA. lncRNA plays an important role in a variety of biological processes such as cell cycle regulation, chromatin modification, and mRNA translation. CircRNA belongs to lncRNA, which is mainly produced by exon or intron sequences. It is a single-stranded circular RNA molecule that can bind to miRNA as a competitive endogenous RNA to regulate transcription or affect parental gene expression. piRNA is a small RNA with a length of about 21 to 35 nucleotides, which is processed from long single-stranded transcripts. The genomic sites of these transcripts are aggregated throughout the genome and transcribed by RNA polymerase II. There are about 20,000 piRNAs in the human genome, which are mainly expressed in gonadal cells. tsRNA is derived from the cleavage of tRNA by nucleases. It is usually 18 to 40 nucleotides in length and plays an important role in regulating translation, maintaining the stability of mRNA, gene silencing, reverse transcription, etc.

As used herein, the term "genome" or "genomic DNA" refers to the heritable information of a host organism. The genomic DNA includes all genetic material of a cell or organism, including nuclear DNA (chromosomal DNA), extrachromosomal DNA, and organelle (e.g., mitochondrial) DNA. Preferably, the term "genome" or genomic "DNA" refers to the chromosomal DNA of the nucleus.

As used herein, where the term "recombinant" is used to describe an organism or cell (eg, a microorganism), it is used to convey that the organism or cell contains at least one "transgene," "transgenic," or "recombinant" polynucleotide, typically described later.

As used herein, a polynucleotide that is "exogenous" with respect to an individual organism is a polynucleotide that has been introduced into that organism by any means other than sexual hybridization.

As used herein, the term "promoter" or "RNase binding site" is a polynucleotide region that initiates transcription of a coding sequence. The promoter is located near the transcription start site of a gene, on the same strand of DNA and upstream (towards the 5' region of the sense strand). Some Promoters are constitutive in that they are active under all circumstances in the cell, whereas other promoters are regulated to become active in response to a specific stimulus (e.g., inducible promoters). As used herein, the term "promoter activity" and its grammatical equivalents refer to the extent of expression of a nucleotide sequence operably linked to the promoter whose activity is being measured. Promoter activity can be measured directly by determining the amount of RNA transcript produced, such as by Northern blot analysis, or indirectly by determining the amount of product encoded by a linked nucleic acid sequence, such as a reporter nucleic acid sequence linked to the promoter.

As used herein, the term "plasmid" refers to an extrachromosomal element that often carries genes that are not part of the core metabolic machinery of the cell, and is usually in the form of a circular double-stranded DNA molecule. These elements can be autonomous replication sequences, genome integration sequences, phage or nucleotide sequences of any origin, linear, circular or supercoiled single-stranded or double-stranded DNA or RNA. Typically, a plasmid contains a replication origin that is functional in a host cell (e.g., E. coli), and a selectable marker for detecting host cells containing the plasmid.

As used herein, the term "transformation" refers to the introduction of one or more exogenous polynucleotides into a host cell by using physical or chemical methods.

As used herein, the term "expression" is the process of revealing the information encoded within a gene. If the gene encodes a protein, expression includes transcribing the DNA into mRNA, processing the mRNA (if necessary) into a mature mRNA product, and translating the mature mRNA into a protein.

The present invention is further described below by way of specific embodiments, but this is not intended to limit the present invention. Those skilled in the art may make various modifications or adjustments based on the teachings of the present invention without departing from the spirit and scope of the present invention.

Example

The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials, reagents, instruments, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources. The quantitative tests in the following examples, unless otherwise specified, are the average values of three repeated experiments. In the following examples, unless otherwise specified, the nucleotide sequences in the sequence table are written from left to right in the order of 5' to 3' end, and the amino acid sequences are written from left to right in the order of amino terminal to carboxyl terminal.

Reagents, instruments, and cell lines used

Unless otherwise specified, the reagents, instruments and cell lines used in the present invention are commercially available.

Important reagents are listed below:

BspQ I (Cat. No. ON-123, Shanghai Zhaowei Technology Development Co., Ltd.);

High Yield T7 RNA Synthesis Kit (Cat. No. ON-040, Shanghai Zhaowei Technology Development Co., Ltd.);

Monarch RNA Cleanup Kit (Cat. No. T2040L, NEB);

RNase R (Cat. No. E224, nearshore protein);

Oligo d(T)25 magnetic beads (Cat. No. S1419S, NEB).

The important instruments are listed below:

Agilent 5200 Fragment Analyzer System (Agilent, U.S.),

Agarose gel electrophoresis system (model DYY-6C, Beijing Liuyi Biotechnology Co., Ltd.),

Nanodrop spectrophotometer (model Nanodrop ONE ^c , Thermo Fisher Scientific),

Centrifuge (model Sorvall Legend Micro 17R, Thermo Fisher Scientific),

Metal bath (Model HWS-12, Shanghai Yiheng Scientific Instrument Co., Ltd.).

Example 1: Preparation of linear RNA precursor

Suzhou Jinweizhi Biotechnology Co., Ltd. was commissioned to synthesize a DNA template double strand for preparing a linear RNA precursor. The template double strand was incorporated into the pUC57 vector (company name, product number) by gene synthesis, and placed downstream of the 3' end of the T7 promoter sequence (TAATACGACTCACTATA) to obtain the template plasmid pUC-PIE-miR21 for synthesizing a linear RNA precursor

A 20 μl system (1X reaction buffer, 7.5 mM each of ATP, GTP, CTP and UTP, 1 μl of T7 RNA polymerase mix, 1 μg of linear template plasmid pUC-PIE-miR21) was used for in vitro transcription at 37°C for 4 h to prepare the linear RNA precursor shown in SEQ ID NO: 11. The precursor contains the elements listed in Table 1 in the 5' to 3' direction.

The underlines in SEQ ID NO: 11 indicate the 5' outer homology arm near the 5' end and the 3' outer homology arm near the 3' end, respectively. Homology arm. For ease of description, the first cytosine (C) in the 5' outer homology arm is numbered as position 1, and the guanine (G) adjacent to it is the transcription start point of the T7 promoter (TAATACGACTCACTATA), which is called position 0. The last nucleotide in SEQ ID NO:11 in the 5' to 3' direction is called position 807, and the 3' end nucleotide adjacent to it can be called position 808 accordingly.

In SEQ ID NO:11, the symbols "#" and "*" respectively indicate the positions of the chemical bonds targeted by the two-step transesterification reaction of the linear precursor during the cyclization. The "UG" and "AA" shown in bold are the dinucleotides that determine the self-splicing sites in the 3' intron fragment and the 5' intron fragment, respectively. For this specific sequence, the nucleotide residues 1 to 151 located 5' upstream of "#" constitute its 5' end arm, and the nucleotide residues 673 to 807 located 3' downstream of "*" constitute its 3' end arm; the circular RNA formed by it will be cyclized by the sequence represented by the nucleotides 152 to 672 located between "#" and "*", that is, the cyclized region specifically includes: 3' exon fragment (positions 152 to 202), 5' internal homology arm (positions 203 to 227), insert fragment (positions 228 to 635), 3' internal homology arm (positions 636 to 657), 5' exon fragment (positions 658 to 672).

FIG1 schematically shows the linear RNA precursor, and shows the name and start and end position numbers of each element above the corresponding position.

Table 1: PIE system sequence

Example 2: Linear RNA precursors with purification tags in one or both end arms and IVT yield and circularization efficiency test

Using the same method as in Example 1, linear RNA precursors having nucleotide sequences as shown in SEQ ID NO: 26 to SEQ ID NO: 49 were prepared as Preparation Examples 1 to 24, respectively. As shown in Table 2 below, compared with the nucleotide sequence shown in SEQ ID NO: 11, the linear RNA precursors of Preparation Examples 1 to 24 respectively contained a poly (A) with a length of 50 nt as a purification tag at different positions of SEQ ID NO: 11. At the same time, SEQ ID NO: 11 prepared in Example 1 was used as a comparative example (CK).

For the insertion position, an exemplary explanation is as follows: when the insertion position is 1, poly A is located at the 3' end of the first nucleotide residue of the linear RNA precursor and is directly connected to the first nucleotide residue.

Table 2:

Using known methods, Preparation Examples 1 to 5, 7, 11, 24 and Comparative Example 1 were subjected to cyclization reaction under the same conditions. For example, after incubating the in vitro transcription system of Examples 1 and 2 at 37°C for 4 hours, 2 mm GTP was added and transferred to 55°C for incubation for 15 minutes, and then the incubated mixture was purified with RNA Cleanup Kit according to the manufacturer's instructions to obtain the cyclization reaction product before RNase R treatment (-). The RNA content of the above cyclization reaction product was determined using the Nanodrop method. 20 U RNase R and 2 μg RNA were added to a 100 μl reaction system, incubated at 37°C for 15 minutes, and the cyclization reaction product (+) after RNase R treatment was obtained. The above two reaction products were detected by 1.0% agarose gel under the same conditions. Among them, the electrophoresis results of the product with a poly (A) tag on the single-side end arm are shown in Figure 5A, and the electrophoresis results of the product with a poly (A) tag on the double-side end arm are shown in Figure 5B.

RNase R is a 3’-5’ exonuclease that can only degrade linear RNA, not circular RNA. Figure 5 shows that after RNase R digestion, obvious bands corresponding to circular RNA can be observed in Preparation Examples 1 to 5, 7, 11, 24 and Comparative Example 1 (CK), indicating that the insertion of poly(A) did not affect the cyclization of these linear RNA precursors. However, in the lanes corresponding to Preparation Examples 1 to 5, 7, 11, and 24, no obvious secondary bands were observed except for the main band, whether before (-) or after (+) RNase R digestion. In contrast, in the lane corresponding to Comparative Example 1 as a control, two separate obvious bands can be observed before RNase R digestion (-), among which the band with high mobility disappears after RNase R digestion (+), indicating that the band mainly corresponds to Nicked RNA impurities generated by random breakage of circular RNA.

In summary, this example shows that, without being constrained by any theoretical mechanism, inserting a purification tag into the two end arms of a linear RNA precursor (especially the region of the 5' external homology arm, the 5' intron or the 3' external homology arm) generally does not significantly interfere with the cyclization reaction, but can surprisingly improve the cyclization reaction efficiency of the linear RNA precursor, significantly increase the proportion of the desired complete cyclization product in the reaction products, and significantly reduce or even eliminate undesirable by-products such as nicked RNA, thereby significantly improving the purity of the crude product of the cyclization reaction, reducing or even eliminating the dependence of the circular RNA purification method on the RNase R enzyme treatment step, and ultimately greatly simplifying the subsequent purification process while not affecting or even significantly improving the quality of the cyclization product.

Example 3: Linear RNA precursor with purification tags in both side arms and IVT yield and circularization efficiency test

Using the same method as Example 2, a linear RNA precursor having a nucleotide sequence as shown in SEQ ID NO:50 was prepared as Comparative Example 2. Except for the 5' external homology arm and the 3' external homology arm shown in Table 3, the remaining element sequences in the linear RNA precursor were the same as those in Table 1.

Table 3

Using the same method, linear RNA precursors having nucleotide sequences as shown in SEQ ID NO: 51 to 55 were prepared as Preparation Examples 25 to 29, respectively. As shown in Table 4 below, compared with Comparative Example 2, the linear RNA precursors of Preparation Examples 25 to 29 respectively contained poly (A) with a length of 50 nt as a purification tag at different positions of SEQ ID NO: 50.

Table 4:

Using the same method as Example 2, the relative yields of Preparation Examples 25 to 29 compared with Comparative Example 2 (CK) under the same conditions were measured, and the results are shown in FIG. 7 .

This example still shows that linear RNA precursors with purification tags can produce the desired cyclized products through in vitro cyclization reactions. Changing the specific sequence of the linear RNA precursor, including changing the end arm sequence (such as changing the 5' and/or 3' external homology arms), does not affect the in vitro cyclization of the precursor molecule with the purification tag. However, when the purification tag is inserted at a certain position in the linear RNA precursor, the IVT yield will be significantly reduced, specifically, the residue position of the linear RNA precursor immediately upstream of the 5' end of the 5' external homology arm (position 0), and the first residue position (position 1) at the end of the 5' end of the 5' external homology arm. However, a reduction in the amount of non-target products produced can be observed in all cases.

Example 4: Other exemplary linear RNA precursors

According to the same method as in Example 1, a linear RNA precursor having the element structure shown in Figures 2 to 4 is prepared. The external homology arms, intron fragments, coding region fragments, translation initiation elements, exon fragments, internal homology arms, spacer sequences, and insertion sequences shown in the figures can all use elements known in the art to have corresponding functions, or elements that can be reasonably inferred to have corresponding functions, including but not limited to various functional elements described in CN112399860A and CN115404240A. The above-mentioned application or patent text is hereby incorporated herein in its entirety.

Example 5: Purification efficiency of affinity chromatography cyclization reaction products

The purification efficiency of linear RNA precursors with poly(A) inserted into both end arms was measured by affinity chromatography relative to a control without poly(A).

The final reaction solution containing 5 μg of RNA was taken from the cyclization reaction products of Preparation Example 24 and Comparative Example 1 for purification. Without adding RNase R, the final solution was incubated with 200 μl of Oligo d(T)25 magnetic beads for 20 minutes to allow the magnetic beads to fully contact and adsorb the RNA with the poly(A) label. After 20 minutes, the sample was placed on a magnetic stand and allowed to stand for 10 seconds to separate the magnetic beads from the supernatant, the magnetic beads were removed, and the supernatant was recovered using the RNA Cleanup Kit according to the manufacturer's instructions. The purity of the cyclization reaction product before RNase R treatment (-) and the cyclization reaction product after RNase R treatment (+) was detected using an Agilent 5200 fragment analyzer under default settings. The results are shown in Figures 8A to 8D.

FIG8 shows that before incubation with Oligo d(T)25 magnetic beads, the purity of circular RNA in the samples of Comparative Example 1 and Preparation Example 24 was 81.3% and 92.7%, respectively. After incubation with magnetic beads, the purity of circular RNA in Comparative Example 1 was determined to be 78.1%, and the purity of Preparation Example 24 was increased to 100%. The above results show that after inserting poly(A) into the two end arms of the linear RNA precursor, various impurities coexisting with the target circular RNA in the cyclization reaction system can be removed by affinity chromatography media (such as Oligo d(T)25 magnetic beads), and high-purity circular RNA can be obtained.

It is noteworthy that even before incubation with Oligo d(T)25 magnetic beads, a significant difference in the purity of the cyclization reaction products of Preparation Example 24 and Comparative Example 1 can be observed (92.7% vs 81.3%). This result is consistent with the electrophoresis results observed in Example 3, indicating that the poly(A) inserted into the end arm can directly improve the purity of the cyclization reaction product independently of the affinity purification mechanism.

The sequences used in the above examples of the present application are shown in the sequence table. It should be understood that these sequences are only exemplary sequences of the present application's embodiments, rather than any restriction to the present application's scheme. Although what is indicated in the electronic sequence table is a DNA sequence, the nucleotide sequence in the present application's sequence table can represent both a DNA sequence and an RNA sequence, and when it represents an RNA sequence, "T" therein represents uridine.

Claims (66)

A method for preparing circular RNA, the method comprising: Step A: generating a linear RNA precursor, wherein the linear precursor comprises a 5' terminal arm, a 3' self-splicing site, a circularization region, a 5' self-splicing site and a 3' terminal arm in a manner operably connected to each other in sequence from 5' to 3' direction, wherein a first tag is inserted into the 5' terminal arm, and a second tag is inserted into the 3' terminal arm; Step B: placing the linear RNA precursor under conditions suitable for self-splicing of the 3' self-splicing site and the 5' self-splicing site, to obtain a mixture comprising a linear RNA fragment with the first tag and/or the second tag and a circular RNA obtained by cyclization of the cyclization region; Step C: contacting the mixture with an affinity chromatography medium capable of simultaneously binding the first tag and the second tag for a period of time sufficient to allow the affinity chromatography medium to bind to the linear RNA fragment containing at least one of the tags; and Step D: separating the affinity chromatography medium and the mixture after contacting the affinity chromatography medium, collecting the supernatant, and obtaining the circular RNA. Wherein, the first tag and/or the second tag are independently selected from a poly(A) tag consisting of 20 to 100, preferably 30 to 90, more preferably 40 to 80, further preferably 45 to 70, and most preferably 50 to 65 consecutive adenine nucleotides or a functional variant thereof, and the first tag and the second tag are not present in the cyclization region and the circular RNA. The method according to claim 1, wherein the functional variant of the poly(A) tag is to insert one or more non-A bases into the poly(A) tag, preferably insert 1 to 20 non-A bases, and more preferably insert 1 to 10 non-A bases. The method according to any of the preceding claims, wherein the functional variant of the poly(A) tag comprises (1) a single element a, at least one element b, and at least one element c, (2) only one element a, at least one element b, and at least one element d; or (3) a single element a, at least one element b, at least one element c and at least one element d, wherein the element a is composed of more than 20 consecutive adenine nucleotides, the element b is composed of more than 3 and less than 20 consecutive adenine nucleotides, the element c is composed of a nucleotide selected from uracil nucleotides, cytosine nucleotides, and guanine nucleotides, the element d is composed of more than 2 and less than 20 nucleotides, the nucleotides are arbitrarily selected from adenine nucleotides, uracil nucleotides, cytosine nucleotides, and guanine nucleotides, and the element d does not contain more than 3 consecutive adenine nucleotides, and the 5' and 3' terminal nucleotides are not adenine nucleotides, Wherein, when the Poly(A) tag contains two or more of the element b, the element c or the element d at the same time, the sequences of every two elements b may be the same or different, the sequences of every two elements c may be the same or different, and the sequences of every two elements d may be the same or different. Furthermore, the element a and the element b, the element c and the element d, the elements b, the elements c, and the elements d are not adjacent to each other. The method according to any of the preceding claims, wherein the element a consists of more than 20 and less than 80 consecutive adenine nucleotides, preferably 30 to 70, 35 to 65, 40 to 60, or 45 to 55 consecutive adenine nucleotides. The nucleotide sequence is preferably composed of 60 consecutive adenine nucleotides. A method according to any preceding claim, wherein the element b consists of 3 to 10, 10 to 19, 12 to 15, 14 to 17, or 16 to 19, preferably 19 consecutive adenine nucleotides. The method according to any one of the preceding claims, wherein the number of the elements b is 2 to 10, preferably 2 to 5, and more preferably 3. A method according to any preceding claim, wherein the element c is a guanine nucleotide. The method according to any of the preceding claims, wherein the number of the elements c is 2 to 10, 3 to 8, 4 to 6, or 2 to 5, preferably 2. The method according to any of the preceding claims, wherein the element d consists of 3 to 18, 5 to 16, 4 to 10, or 6 to 12 nucleotides, preferably consists of 6 nucleotides. A method according to any preceding claim, wherein the element d is selected from any of GAUAUC, GUAUAC, GAAUCU, GCAUAUGACU or GAUAUCGUAUAC. The method according to any one of the preceding claims, wherein the number of the elements d is 0 to 5, preferably 1 to 3, more preferably 1. The method according to any of the preceding claims, wherein when element c and element d are present at the same time, the total number of the elements c and d is 2 to 15, preferably 3 to 5, more preferably 3. The method according to any of the preceding claims, wherein the functional variant of the poly(A) tag has any one structure selected from the following structures: component a-component c-component b-component c-component b-component c-component b-component c-component b, Element b-element c-element b-element c-element a-element d-element b-element c-element b-element c-element b, Element b-element c-element b-element c-element b-element d-element a-element c, element a-element d-element b-element c-element b-element c-element b, or Element b-element c-element b-element c-element b-element d-element a. The method according to any of the preceding claims, wherein the 5' end arm comprises a 5' external homology arm and a 3' intron fragment in the 5' to 3' direction, and the first tag is inserted in the 5' external homology arm, or inserted in the 5' terminal region of the 3' intron fragment close to the 5' external homology arm, the 5' terminal region is preferably 20 nucleotides at the 5' end of the 3' intron fragment, more preferably 15 nucleotides, and further preferably 10 nucleotides, or inserted upstream of the 5' end of the 5' external homology arm, The 3' end arm comprises a 5' intron fragment and a 3' external homology arm in the 5' to 3' direction, and the second tag is inserted in the 3' external homology arm, or inserted in the 3' terminal region of the 5' intron fragment close to the 3' external homology arm, the 3' terminal region is preferably 20 nucleotides at the 3' end of the 5' intron fragment, more preferably 15 nucleotides, and further preferably 10 nucleotides, or inserted downstream of the 3' end of the 3' external homology arm, Preferably, The first tag is inserted into the 5' outer homology arm, and the second tag is inserted downstream of the 3' end of the 3' outer homology arm; The first tag is inserted in the 5' outer homology arm and the second tag is inserted in the 3' outer homology arm; or The first tag is inserted in the 5' external homology arm, and the second tag is inserted in the 3' terminal region of the 5' intron fragment; More preferably, the first tag is inserted at any position in the 5' external homology arm except the first nucleotide residue at the 5' end, and the second tag is inserted downstream of the 3' end of the 3' external homology arm; The first tag is inserted in any position of the 5' outer homology arm except the first nucleotide residue at the 5' end, and the second tag is inserted in the 3' outer homology arm; or The first tag is inserted at any position in the 5' external homology arm except the first nucleotide residue at the 5' end, and the second tag is inserted at the 3' terminal region of the 5' intron fragment. The method according to any of the preceding claims, wherein the circularization region comprises a 3' coding region fragment, a translation initiation element, and a 5' coding region fragment in a manner operably linked to each other in sequence from 5' to 3' direction. A method according to any of the preceding claims, wherein the circularization region comprises a 3' exon fragment, a 5' internal homology arm, an insert fragment, a 3' internal homology arm and a 5' exon fragment in sequence in a manner operably connected to each other from 5' to 3' direction, and optionally, the circularization region comprises a first spacer region between the insert fragment and the 5' internal homology arm, and a second spacer region between the insert fragment and the 3' internal homology arm. The method according to claim 16, wherein the inserted fragment comprises a translation initiation element, or comprises a translation initiation element and a coding region, wherein the translation initiation element is preferably an IRES sequence. The method according to any of the preceding claims, wherein the inserted fragment comprises a structural gene or a functional fragment thereof or a sequence of a non-coding RNA or its complementary sequence, wherein the structural gene encodes a polypeptide, a protein subunit, a protein active center, a protein or a protein hybrid of a non-natural catalytic group, a recombinant protein active subunit or active center, a recombinant artificial enzyme or other biological effect units mainly composed of amino acids, and the non-coding RNA is selected from microRNA (miRNA), small interfering RNA (siRNA), PIWI protein-interacting RNA (piRNA), transfer RNA-derived small RNA (tsRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), long non-coding RNA (lncRNA), pseudogene, ceRNA (competing endogenous RNAs), microRNA sponge or other types of non-mRNA RNA. The method according to any of the preceding claims, wherein the lengths of the 5' and 3' outer homology arms are each independently greater than 5 nt, greater than 10 nt, greater than 15 nt, greater than 20 nt, greater than 25 nt, greater than 30 nt, greater than 40 nt, greater than 50 nt, greater than 60 nt, less than 5 nt, less than 10 nt, less than 15 nt, less than 20 nt, less than 25 nt, less than 30 nt, less than 40 nt, less than 50 nt, less than 60 nt, 5-60 nt, 10-55 nt, 15-50 nt, 20-45 nt, 25-40 nt, 30-35 nt, 10 nt, 15 nt, 20 nt, 25 nt, 30 nt, 35 nt, 40 nt, 45 nt, or 50 nt, Optionally, the lengths of the 5' and 3' intron fragments are each independently greater than 5 nt, greater than 10 nt, greater than 15 nt, greater than 20 nt, greater than 25 nt, greater than 30 nt, greater than 40 nt, greater than 50 nt, greater than 60 nt, less than 5 nt, less than 10 nt, less than 15 nt, less than 20 nt, less than 25 nt, less than 30 nt, less than 40 nt, less than 50 nt, less than 60 nt, less than 70 nt, less than 80 nt. , less than 90nt, less than 100nt, less than 150nt, less than 200nt, 5-200nt, 10-150nt, 50-200nt, 50-150nt, 5-60nt, 10-55nt, 15-50nt, 20-45nt, 25-40nt, 30-35nt, 10nt, 15nt, 20nt, 25nt, 30nt, 35nt, 40nt, 45nt, or 50nt, Optionally, the length of the 5' and 3' exon fragments are each independently greater than 5 nt, greater than 10 nt, greater than 15 nt, greater than 20 nt, greater than 25 nt, greater than 30 nt, greater than 40 nt, greater than 50 nt, greater than 60 nt, less than 5 nt, less than 10 nt, less than 15 nt, less than 20 nt, less than 25 nt, less than 30 nt, less than 40 nt, less than 50 nt, less than 60 nt, 5-60 nt, 10-55 nt, 15-50 nt, 20-45 nt, 25-40 nt, 30-35 nt, 10 nt, 15 nt, 20 nt, 25 nt, 30 nt, 35 nt, 40 nt, 45 nt, or 50 nt, Optionally, the lengths of the 5' and 3' internal homology arms are each independently greater than 5 nt, greater than 10 nt, greater than 15 nt, greater than 20 nt, greater than 25 nt, greater than 30 nt, greater than 40 nt, greater than 50 nt, greater than 60 nt, less than 5 nt, less than 10 nt, less than 15 nt, less than 20 nt, less than 25 nt, less than 30 nt, less than 40 nt, less than 50 nt, less than 60 nt, 5-60 nt, 10-55 nt, 15-50 nt, 20-45 nt, 25-40 nt, 30-35 nt, 10 nt, 15 nt, 20 nt, 25 nt, 30 nt, 35 nt, 40 nt, 45 nt, or 50 nt, The length of the inserted fragment is optionally greater than 50 nt, greater than 100 nt, greater than 150 nt, greater than 200 nt, greater than 250 nt, greater than 300 nt, greater than 400 nt, greater than 500 nt, greater than 600 nt, greater than 1k nt, greater than 1.5k nt, greater than 2k nt, greater than 3k nt, less than 50 nt, less than 100 nt, less than 150 nt, less than 200 nt, less than 250 nt, less than 300 nt, less than 400 nt, less than 50 0nt, less than 600nt, less than 600nt, less than 1k nt, less than 1.5k nt, less than 2k nt, less than 3k nt, 50~5knt, 50~5k nt, 50~4k nt, 50~3k nt, 50~2k nt, 50~1.5k nt, 50~1k nt, 50~600nt, 100~550nt, 150~500nt, 200~450nt, 250~400nt, 300~350nt. A method according to any of the preceding claims, wherein the 5’ external homology arm has a sequence as shown in SEQ ID NO:1 or 56, and the 3’ external homology arm has a sequence as shown in SEQ ID NO:2 or 57. The method according to any of the preceding claims, wherein the 3' intron fragment and the 5' intron fragment are derived from type I introns, preferably from the cyanobacterium Anabaena pre-tRNA gene or the T4 phage Td gene, more preferably the 3' intron fragment has a sequence as shown in SEQ ID NO:3, and the 5' intron fragment has a sequence as shown in SEQ ID NO:4. The method according to any of the preceding claims, wherein the 3' intron fragment and the 5' intron fragment are derived from type II introns, preferably type II introns from Clostridium such as Clostridium tetani, or type II introns from Bacillus such as Bacillus thuringiensis, more preferably the type II intron is a type II intron contained in the nucleotide sequence shown in SEQ ID NO: 5 or 6. The method according to any of the preceding claims, wherein the 3' exon fragment and the 5' exon fragment The 3' terminal region and the 5' terminal region are respectively derived from natural exons, preferably from the cyanobacterium Anabaena pre-tRNA gene or the T4 phage Td gene, more preferably the 3' exon fragment has the sequence shown in SEQ ID NO:7, and the 5' exon fragment has the sequence shown in SEQ ID NO:8. A method according to any of the preceding claims, wherein preferably the 5’ internal homology arm has a sequence as shown in SEQ ID NO:9, and the 3’ internal homology arm has a sequence as shown in SEQ ID NO:10. A method according to any preceding claim, wherein the first spacer is the same as or different from the second spacer. The method according to any of the preceding claims, wherein an affinity ligand capable of specifically binding to the first tag and/or the second tag is operably linked to the affinity chromatography medium, preferably the affinity ligand is selected from the group consisting of polymer X1, polymer _X1 - _X2 , polymer _X1 - _X2 - _X3 , polymer _X1 - _X2 - _{X3-X4 ,} wherein _X1 , _X2 , _X3 , _X4 are independently any one of A, G, C, T, U, more preferably any one of the group consisting of Oligo dT, Oligo dC, Oligo dG, Oligo dU. The method according to any of the preceding claims, wherein the affinity chromatography medium is selected from any one of the group consisting of magnetic beads, dextran molecules, polyacrylamide macromolecules, macromolecular cellulose molecules, chitosan materials, modified polylactic acid materials, PET materials, inorganic silicate materials or other high molecular polymers. A method according to any preceding claim, which does not include the step of adding RNase R to the mixture to remove linear RNA. The method according to any of the preceding claims, when it includes the step of adding RNase R to the mixture to remove linear RNA, the amount of RNase R added is reduced to 30-50%, preferably 40% or 50% of the amount of enzyme used for purification when the poly (A) tag or its functional variant is not inserted. The circular RNA prepared by the method according to any one of claims 1 to 29, which substantially does not contain non-circular RNA molecules. A linear RNA precursor for use in the method according to any one of claims 1 to 29. A linear RNA precursor, characterized in that the linear RNA precursor comprises a 5' end arm, a 3' self-splicing site, a cyclization region, a 5' self-splicing site and a 3' end arm in a manner operably connected to each other in sequence from 5' to 3' direction, wherein a first tag is inserted into the 5' end arm and a second tag is inserted into the 3' end arm; Optionally, the first tag and/or the second tag are independently selected from a poly(A) tag consisting of 20 to 100, preferably 30 to 90, more preferably 40 to 80, further preferably 45 to 70, and most preferably 50 to 65 consecutive adenine nucleotides or a functional variant thereof, and the first tag and the second tag are not present in the cyclization region and the circular RNA. The linear RNA precursor according to claim 32, wherein the functional variant of the poly (A) tag is an insertion of one or more non-A bases into the poly (A) tag, preferably an insertion of 1 to 20 non-A bases, and more preferably an insertion of 1 to 10 non-A bases. The linear RNA precursor according to claim 32 or 33, wherein the functional variant of the poly (A) tag comprises (1) a single element a, at least one element b, and at least one element c, (2) only one element a, at least one element b, and at least one element d; or (3) a single element a, at least one element b, at least one element c and at least one element d, wherein the element a is composed of more than 20 consecutive adenine nucleotides, the element b is composed of more than 3 and less than 20 consecutive adenine nucleotides, the element c is composed of a nucleotide selected from uracil nucleotides, cytosine nucleotides, and guanine nucleotides, the element d is composed of more than 2 and less than 20 nucleotides, the nucleotides are arbitrarily selected from adenine nucleotides, uracil nucleotides, cytosine nucleotides, and guanine nucleotides, and the element d does not contain more than 3 consecutive adenine nucleotides, and the 5' and 3' terminal nucleotides are not adenine nucleotides, Wherein, when the Poly(A) tag contains two or more of the element b, the element c or the element d at the same time, the sequences of every two elements b may be the same or different, the sequences of every two elements c may be the same or different, and the sequences of every two elements d may be the same or different. Furthermore, the element a and the element b, the element c and the element d, the elements b, the elements c, and the elements d are not adjacent to each other. The linear RNA precursor according to any one of claims 32 to 34, wherein the element a consists of more than 20 and less than 80 consecutive adenine nucleotides, preferably consists of 30 to 70, 35 to 65, 40 to 60, or 45 to 55 consecutive adenine nucleotides, more preferably consists of 60 consecutive adenine nucleotides. The linear RNA precursor according to any one of claims 32 to 35, wherein the element b consists of 3 to 10, 10 to 19, 12 to 15, 14 to 17, or 16 to 19, preferably 19 consecutive adenine nucleotides. The linear RNA precursor according to any one of claims 32 to 36, wherein the number of the element b is 2 to 10, preferably 2 to 5, and more preferably 3. The linear RNA precursor according to any one of claims 32 to 37, wherein the element c is a guanine nucleotide. The linear RNA precursor according to any one of claims 32 to 38, wherein the number of the elements c is 2 to 10, 3 to 8, 4 to 6, or 2 to 5, preferably 2. The linear RNA precursor according to any one of claims 32 to 39, wherein the element d consists of 3 to 18, 5 to 16, 4 to 10, or 6 to 12 nucleotides, preferably consists of 6 nucleotides. The linear RNA precursor according to any one of claims 32 to 40, wherein the element d is selected from any one of GAUAUC, GUAUAC, GAAUCU, GCAUAUGACU or GAUAUCGUAUAC. The linear RNA precursor according to any one of claims 32 to 41, wherein the number of the element d is 0 to 5, preferably 1 to 3, and more preferably 1. The linear RNA precursor according to any one of claims 32 to 42, wherein when element c and element d exist at the same time, the total number of element c and element d is 2 to 15, preferably 3 to 5, and more preferably 3. The linear RNA precursor according to any one of claims 32 to 43, wherein the functional variant of the poly (A) tag has any one structure selected from the following structures, component a-component c-component b-component c-component b-component c-component b-component c-component b, Element b-element c-element b-element c-element a-element d-element b-element c-element b-element c-element b, Element b-element c-element b-element c-element b-element d-element a-element c, element a-element d-element b-element c-element b-element c-element b, or Element b-element c-element b-element c-element b-element d-element a; Preferably, the functional variant of the poly(A) tag has a nucleotide sequence selected from any one of SEQ ID NO:12 to 25. The linear RNA precursor according to any one of claims 32 to 44, wherein the 5' end arm comprises a 5' external homology arm and a 3' intron fragment in the 5' to 3' direction, and the first tag is inserted in the 5' external homology arm, or inserted in the 5' terminal region of the 3' intron fragment close to the 5' external homology arm, the 5' terminal region is preferably 20 nucleotides at the 5' end of the 3' intron fragment, more preferably 15 nucleotides, and further preferably 10 nucleotides, or inserted upstream of the 5' end of the 5' external homology arm, The 3' end arm comprises a 5' intron fragment and a 3' external homology arm in the 5' to 3' direction, and the second tag is inserted in the 3' external homology arm, or inserted in the 3' terminal region of the 5' intron fragment close to the 3' external homology arm, the 3' terminal region is preferably 20 nucleotides at the 3' end of the 5' intron fragment, more preferably 15 nucleotides, and further preferably 10 nucleotides, or inserted downstream of the 3' end of the 3' external homology arm, Preferably, The first tag is inserted into the 5' outer homology arm, and the second tag is inserted downstream of the 3' end of the 3' outer homology arm; The first tag is inserted in the 5' outer homology arm and the second tag is inserted in the 3' outer homology arm; or The first tag is inserted in the 5' external homology arm, and the second tag is inserted in the 3' terminal region of the 5' intron fragment; More preferably, the first tag is inserted at any position in the 5' external homology arm except the first nucleotide residue at the 5' end, and the second tag is inserted downstream of the 3' end of the 3' external homology arm; The first tag is inserted in any position of the 5' outer homology arm except the first nucleotide residue at the 5' end, and the second tag is inserted in the 3' outer homology arm; or The first tag is inserted at any position in the 5' external homology arm except the first nucleotide residue at the 5' end, and the second tag is inserted at the 3' terminal region of the 5' intron fragment. The linear RNA precursor according to any one of claims 32 to 45, wherein the cyclization region comprises a 3' coding region fragment, a translation initiation element, and a 5' coding region fragment in a manner operably linked to each other in sequence from 5' to 3' direction. The linear RNA precursor according to any one of claims 32 to 46, wherein the cyclization region comprises a 3' exon fragment, a 5' internal homology arm, an insert fragment, a 3' internal homology arm and a 5' exon fragment in a manner operably connected to each other in sequence from 5' to 3' direction, and optionally, the cyclization region comprises a first spacer region between the insert fragment and the 5' internal homology arm, and a second spacer region between the insert fragment and the 3' internal homology arm. The linear RNA precursor according to claim 47, wherein the inserted fragment comprises a translation initiation element, or comprises a translation initiation element and a coding region, wherein the translation initiation element is preferably an IRES sequence. The linear RNA precursor according to any one of claims 32 to 48, wherein the inserted fragment comprises the coding sequence of a structural gene or its functional fragment or the sequence of a non-coding RNA or its complementary sequence, the structural gene is selected from a polypeptide, a protein subunit, a protein active center, a protein or a protein hybrid of a non-natural catalytic group, a recombinant protein active subunit or active center/, a recombinant artificial enzyme or other biological effect units mainly composed of amino acids, and the non-coding RNA is selected from microRNA (miRNA), small interfering RNA (siRNA), PIWI protein-interacting RNA (piRNA), transfer RNA-derived small RNA (tsRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), long non-coding RNA (lncRNA), pseudogene, ceRNA (competing endogenous RNAs), microRNA sponge or other types of non-mRNA RNA. The linear RNA precursor according to any one of claims 32 to 49, wherein the lengths of the 5' and 3' outer homology arms are each independently greater than 5 nt, greater than 10 nt, greater than 15 nt, greater than 20 nt, greater than 25 nt, greater than 30 nt, greater than 40 nt, greater than 50 nt, greater than 60 nt, less than 5 nt, less than 10 nt, less than 15 nt, less than 20 nt, less than 25 nt, less than 30 nt, less than 40 nt, less than 50 nt, less than 60 nt, 5-60 nt, 10-55 nt, 15-50 nt, 20-45 nt, 25-40 nt, 30-35 nt, 10 nt, 15 nt, 20 nt, 25 nt, 30 nt, 35 nt, 40 nt, 45 nt, or 50 nt, The lengths of the 5' and 3' intron fragments are each independently greater than 5 nt, greater than 10 nt, greater than 15 nt, greater than 20 nt, greater than 25 nt, greater than 30 nt, greater than 40 nt, greater than 50 nt, greater than 60 nt, less than 70 nt, less than 80 nt, less than 90 nt, less than 100 nt, less than 150 nt, less than 200 nt, 5-200 nt, 10-150 nt, 50-200 nt, 50-1 50nt, less than 5nt, less than 10nt, less than 15nt, less than 20nt, less than 25nt, less than 30nt, less than 40nt, less than 50nt, less than 60nt, 5-60nt, 10-55nt, 15-50nt, 20-45nt, 25-40nt, 30-35nt, 10nt, 15nt, 20nt, 25nt, 30nt, 35nt, 40nt, 45nt, or 50nt, Optionally, the length of the 5' and 3' exon fragments are each independently greater than 5 nt, greater than 10 nt, greater than 15 nt, greater than 20 nt, greater than 25 nt, greater than 30 nt, greater than 40 nt, greater than 50 nt, greater than 60 nt, less than 5 nt, less than 10 nt, less than 15 nt, less than 20 nt, less than 25 nt, less than 30 nt, less than 40 nt, less than 50 nt, less than 60 nt, 5-60 nt, 10-55 nt, 15-50 nt, 20-45 nt, 25-40 nt, 30-35 nt, 10 nt, 15 nt, 20 nt, 25 nt, 30 nt, 35 nt, 40 nt, 45 nt, or 50 nt, Optionally, the lengths of the 5' and 3' internal homology arms are each independently greater than 5 nt, greater than 10 nt, greater than 15 nt, greater than 20 nt, greater than 25 nt, greater than 30 nt, greater than 40 nt, greater than 50 nt, greater than 60 nt, less than 5 nt, less than 10 nt, less than 15 nt, less than 20 nt, less than 25 nt, less than 30 nt, less than 40 nt, less than 50 nt, less than 60 nt, 5 to 60 nt, 10 to 55 nt, 15-50 nt, 20-45 nt, 25-40 nt, 30-35 nt, 10 nt, 15 nt, 20 nt, 25 nt, 30 nt, 35 nt, 40 nt, 45 nt, or 50 nt, The length of the inserted fragment is optionally greater than 50 nt, greater than 100 nt, greater than 150 nt, greater than 200 nt, greater than 250 nt, greater than 300 nt, greater than 400 nt, greater than 500 nt, greater than 600 nt, greater than 1k nt, greater than 1.5k nt, greater than 2k nt, greater than 3k nt, less than 50 nt, less than 100 nt, less than 150 nt, less than 200 nt, less than 250 nt, less than 300 nt, less than 400 nt, less than 50 0nt, less than 600nt, less than 600nt, less than 1k nt, less than 1.5k nt, less than 2k nt, less than 3k nt, 50~5knt, 50~5k nt, 50~4k nt, 50~3k nt, 50~2k nt, 50~1.5k nt, 50~1k nt, 50~600nt, 100~550nt, 150~500nt, 200~450nt, 250~400nt, 300~350nt. A linear RNA precursor according to any one of claims 32 to 50, wherein the 5' external homologous arm has a sequence as shown in SEQ ID NO:1 or 56, and the 3' external homologous arm has a sequence as shown in SEQ ID NO:2 or 57. The linear RNA precursor according to any one of claims 32 to 51, wherein the 3' intron fragment and the 5' intron fragment are derived from type I introns, preferably from the cyanobacterium Anabaena pre-tRNA gene or the T4 phage Td gene, more preferably the 3' intron fragment has a sequence as shown in SEQ ID NO:3, and the 5' intron fragment has a sequence as shown in SEQ ID NO:4. The linear RNA precursor according to any one of claims 32 to 52, wherein the 3' intron fragment and the 5' intron fragment are derived from type II introns, preferably type II introns from Clostridium such as Clostridium tetani, or type II introns from Bacillus such as Bacillus thuringiensis, more preferably the type II intron is a type II intron contained in the nucleotide sequence shown in SEQ ID NO: 5 or 6. The linear RNA precursor according to any one of claims 32 to 53, wherein the 3' exon fragment and the 5' exon fragment are respectively derived from the 3' terminal region and the 5' terminal region of the natural exon, preferably from the cyanobacterium Anabaena pre-tRNA gene or the T4 phage Td gene, more preferably the 3' exon fragment has a sequence as shown in SEQ ID NO:7, and the 5' exon fragment has a sequence as shown in SEQ ID NO:8. The linear RNA precursor according to any one of claims 32 to 54, wherein the 5' internal homology arm preferably has a sequence as shown in SEQ ID NO:9, and the 3' internal homology arm has a sequence as shown in SEQ ID NO:10. The linear RNA precursor according to any one of claims 32 to 55, wherein the first spacer is the same as or different from the second spacer. The linear RNA precursor according to any one of claims 32 to 56, which has a nucleotide sequence selected from any one of SEQ ID NO: 26 to 49 and 51 to 55. A nucleic acid sequence capable of being transcribed to produce a linear RNA precursor for use in the method of any one of claims 1 to 29 or a linear RNA precursor of any one of claims 31 to 57, which preferably further comprises in an operably linked manner a regulatory sequence necessary for transcription to produce the linear RNA precursor, such as a promoter, a terminator, a transcription factor binding site ... Zygote site, untranslated region (UTR), enhancer, palindromic sequence, cis-acting element, trans-acting element, TATA box, CAAT box, operator, transposon. A vector comprising the nucleic acid sequence of claim 58. The vector according to claim 59, wherein the vector is a linear DNA, a plasmid, a viral nucleic acid fragment or a cell genomic DNA fragment. An engineered cell comprising the linear RNA precursor used in the method of any one of claims 1 to 29, the circular RNA of claim 30, the linear RNA precursor of any one of claims 31 to 57, the nucleic acid sequence of claim 58, or the vector of claim 59 or 60. A composition comprising a linear RNA precursor used in the method of any one of claims 1 to 29, a circular RNA according to claim 30, a linear RNA precursor according to any one of claims 31 to 57, a nucleic acid sequence according to claim 58, a vector according to claim 59 or 60, or an engineered cell according to claim 61. Use of a linear RNA precursor used in the method of any one of claims 1 to 29, a circular RNA according to claim 30, a linear RNA precursor according to any one of claims 31 to 57, a nucleic acid sequence according to claim 58, a vector according to claim 59 or 60, or an engineered cell according to claim 61 for preparing circular RNA. Use of a linear RNA precursor used in the method of any one of claims 1 to 29, a circular RNA according to claim 30, a linear RNA precursor according to any one of claims 31 to 57, a nucleic acid sequence according to claim 58, a vector according to claim 59 or 60, or an engineered cell according to claim 61 for preparing a drug, a cytotoxic agent or an immunomodulatory preparation. According to the use of claim 64, the therapeutic drug, cytotoxic agent or immunomodulatory preparation is selected from viruses, pluripotent or multipotent stem cells, iPS, engineered immune cells, antibodies or antibody fragments, drug-conjugated antibodies or antibody fragments, chemotherapeutic agents, immunosuppressive or modulatory agents, anti-infective drugs, anticancer agents, hypoglycemic drugs, cardiovascular and cerebrovascular disease therapeutic drugs, degenerative neurological disease drugs, obesity therapeutic drugs, hematological disease therapeutic drugs, respiratory disease therapeutic drugs, or retroviral disease therapeutic drugs. A method for administering circular RNA, comprising administering to an organism in need thereof an effective amount of a linear RNA precursor used in the method of any one of claims 1 to 29, a circular RNA according to claim 30, a linear RNA precursor according to any one of claims 31 to 57, a nucleic acid sequence according to claim 58, a vector according to claim 59 or 60, an engineered cell according to claim 61, or a circular RNA prepared using the composition according to claim 62.