Linear RNA cyclization component and use thereof

Abstract

A linear RNA cyclization component and a method for RNA chain cyclization. Ring forming primitives are linked to two ends of an RNA sequence to be undergone ring forming, and depending on the base complementary pairing interaction between the ring forming primitives, the 3' and 5' ends of the RNA sequence are extremely close in space, and are linked by means of an RNA ligase to complete cyclization of a target RNA. The cyclized RNA prepared by the method has higher stability than a linear RNA.

Description

Circularization components of linear RNA and their applications Technical Field

[0001] The present application belongs to the field of biomedicine technology, and specifically relates to a method for preparing single-stranded circular RNA. Background Art

[0002] Circular RNA (circRNA) is a type of single-stranded RNA characterized by a covalently closed topological structure, resulting in the absence of free ends. Compared to linear RNA, circular RNA is less susceptible to exonuclease digestion and exhibits greater stability, thus offering numerous promising applications both in vivo and in vitro. For example, it can serve as a small RNA (microRNA, miRNA) sponge, a protein-binding scaffold, an RNA expression vector, and a protein regulator. Consequently, the artificial synthesis of circular RNA has attracted widespread attention.

[0003] The currently commonly used methods for in vitro synthesis of circular RNA mainly include chemical ligation, intron-exon arrangement (PIE) and enzyme ligation. First, the chemical ligation method mainly uses chemical reagents such as cyanogen bromide (BrCN) and morpholino derivatives such as 2-(N-morpholino)-ethanesulfonic acid (MES) to connect the hydroxyl and phosphate groups at both ends of the linear RNA chain, thereby achieving the cyclization of the linear RNA chain. However, this type of method is generally only applicable to the cyclization of very small linear RNA chains (<100nt), and due to the presence of 2'hydroxyl groups in the RNA chain, side reactions such as the formation of 2'-5' phosphodiester bonds are very likely to occur. In addition, due to the introduction of a large number of chemical reagents, the biosafety of the prepared circular RNA is questionable, further limiting its scope of application. The PIE rule is achieved by utilizing two ester exchange reactions that determine the splicing site in the intron self-splicing reaction. However, a key limitation of existing PIE methods is that the circular RNA products based on the PIE method retain some exons of natural group I intron genes, and a large number of splicing intermediate byproducts are also produced in the reaction system. Moreover, since this method has very high requirements for the secondary structure of introns, it greatly increases the difficulty of sequence design. The enzyme ligation method mainly utilizes a series of DNA and RNA ligases derived from TA phage (T4 DNA ligase, T4 RNA ligase 1, T4 RNA ligase 2). These enzymes require linear RNA substrates to contain 5′ monophosphate for circularization. However, because the ligases are heavily dependent on the secondary structure of linear RNA, previous RNA circularization methods based on DNA and RNA ligases generally require the addition of additional splint chains, which increases the complexity of the system and the difficulty of post-processing. In addition, side reactions of intermolecular ligation of RNA chains are also prone to occur. How to achieve RNA molecule circularization efficiently, universally and conveniently is very important for the application of circular RNA molecules, which is difficult to achieve with current RNA circularization methods.

[0004] Summary of the Invention

[0005] To address the shortcomings of currently used RNA cyclization methods, the present invention provides a method for achieving cyclization using an RNA molecule linked to a specific sequence, which itself serves as a template. In this RNA cyclization method, the target RNA sequence relies on complementary base pairing between the cyclizing motifs, bringing the 3' and 5' ends of the target RNA into close spatial proximity. By adding RNA ligase, the RNA ends are covalently linked, achieving cyclization of the target RNA sequence.

[0006] In one aspect, the present invention provides a linear RNA for circularization. In another aspect, the present invention provides a circularization component for circularizing a linear RNA. In another aspect, the present invention provides a method for preparing a circular RNA. In another aspect, the present invention provides an expression vector for expressing a precursor linear RNA for circularization in vitro. In another aspect, the present invention provides uses of the circularization component for circularizing a linear RNA, the linear RNA for circularization, or the expression vector in treating, preventing, or diagnosing a disease, or in preparing a medicament for treating or preventing a disease, or a diagnostic or detection reagent.

[0007] The present invention provides a universal and simple RNA cyclization method and components, which can efficiently cyclize RNA chains of different lengths and sequences. This method uses the RNA itself as a template, eliminating the need for additional splint chains and generating no byproducts. Furthermore, this method simplifies sequence design and eliminates the need to consider the secondary structure of the sequence to be cyclized. Circular RNA prepared using this method has significantly improved stability compared to linear precursors, does not affect the original function of the RNA sequence, and can reduce the generation of polymer impurities.

[0008] In one aspect, the present invention provides a linear RNA for cyclization, wherein the cyclized linear RNA comprises the following structure from the 5' end to the 3' end:

[0009] In some embodiments, the circulation promoter sequence has the following structure from the 5' end to the 3' end:

[0010] GGGA-non-complementary region-X1X2X3X 4- ,

[0011] The GGGA- is completely complementary or partially complementary to the -X1X2X3X4-, so that the circular promoter forms a stem-loop structure.

[0012] In some embodiments, the lock unit is completely complementary to the key unit, so that the 5′ end of the circularized promoter sequence and the 3′ end of the key unit sequence form a cyclase action nick (Nick).

[0013] In some embodiments, GGGA- and -X1X2X3X4- are fully complementary or have at least 3 complementary pairs, preferably with only a mismatch between G and X3.

[0014] In some embodiments, X1 is selected from the base U, A, C or G, preferably the base U; X2, X3, X4 are each independently selected from the base G or C.

[0015] In some embodiments, the non-complementary region of the circular promoter is 3-15 nt in length. In some embodiments, the non-complementary region sequence is not complementary to GGGA-, -X1X2X3X4-, and the target RNA. In some embodiments, the ratio of bases C and A in the non-complementary region is 66%-100%. In some embodiments, the non-complementary region sequence in the circular promoter is -CCAAC- from the 5' end to the 3' end. In some embodiments, the circular promoter is GGGACCAACUCUC (SEQ ID NO: 1) from the 5' end to the 3' end. In some embodiments, the circular promoter can be replaced with GGGACCAACUCCC (SEQ ID NO: 2).

[0016] In some embodiments, the 5' end of the linear RNA used for circularization is triphosphorylated or monophosphorylated.

[0017] In some embodiments, the lock motif complements the key motif to form a pairing structure of at least 3 basepairs. In some embodiments, the lock motif complements the key motif to form a pairing structure of 3-15 bp, 3-9 bp, or more specifically, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 bp.

[0018] In some embodiments, the GC ratio in the paired structure formed by the key motif and the key motif is between 20% and 70%.

[0019] In some embodiments, the structure of the lock motif from the 5' end to the 3' end is -(UAG) m -, the sequence of the key motif from 5' to 3' end is -(CUA) n wherein m=n and m and n are selected from 1, 2, 3, 4 or 5.

[0020] In some embodiments, the lock motif has a structure of UAG from the 5' end to the 3' end. In some embodiments, the key motif has a sequence of CUA from the 5' end to the 3' end. In some embodiments, the lock motif has a sequence of UAGUAG from the 5' end to the 3' end, and the key motif has a sequence of CUACUA from the 5' end to the 3' end.

[0021] In some embodiments, the circularized promoter sequence, the lock motif sequence, and the key motif sequence form the following continuous primary structure after circularization:- (CUA) m -GGGACCAACUCUC-(UAG) n -, wherein m=n and m and n are selected from 1, 2, 3, 4 or 5. In some embodiments, GGGACCAACUCCC (SEQ ID NO: 2) can be substituted in the circular promoter.

[0022] In some embodiments, the linear RNA used for circularization has GGGACCAACUCUC-(UAG) from the 5' end to the 3' end. m -Target RNA-(CUA) n wherein m=n and m and n are selected from 1, 2, 3, 4 or 5. In some embodiments, GGGACCAACUCCC (SEQ ID NO: 2) can be substituted in the circular promoter.

[0023] In some embodiments, the circularization promoter sequence, the lock motif sequence, and the key motif sequence in the linear RNA used for circularization form the following structural fragments after circularization:

[0024] In some embodiments, structures are formed that are at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% homologous to the above structural fragments.

[0025] In some embodiments, the length of the target RNA sequence (i.e., the RNA sequence to be circularized) is 50 nt or more, preferably 100 nt to 10,000 nt, or greater than 200 nt, 300 nt, 400 nt, 500 nt, 600 nt, 700 nt, 800 nt, 900 nt, 1000 nt, 1500 nt, 2000 nt, 3000 nt, 4000 nt, 5000 nt, 6000 nt, 7000 nt, 8000 nt, 9000 nt, or 10,000 nt.

[0026] In some embodiments, the target RNA sequence regulates the target nucleic acid in a subject, or directly encodes and expresses a target polypeptide or protein. In some embodiments, the target RNA sequence regulates the function of pre-mRNA, mRNA, miRNA, lncRNA, circRNA, and tRNA in a subject, or expresses a polypeptide or protein encoded by a single-stranded circular RNA.

[0027] In some embodiments, the linear RNA used for cyclization and any elements therein comprise unmodified, partially modified, or fully modified nucleosides. The modifications include, but are 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. Nucleotide analogs are also intended to include nucleotides having bases such as inosine, quercetin, and xanthines; sugars such as 2'-methyl ribose; and non-natural phosphodiester linkages such as methylphosphonates, phosphorothioates, and peptide linkages. Nucleotide analogs include 5-methoxyuridine, 1-methylpseudouridine, and 6-methyladenosine.

[0028] In another aspect, the present invention provides a circularization component for circularizing linear RNA, comprising: a circularization promoter, a lock motif, and a key motif.

[0029] In some embodiments, the circularized promoter, lock motif, and key motif form a linear structure with the target RNA as follows:

[0030] In some embodiments, the circular promoter has the following structure from the 5' end to the 3' end:

[0031] GGGA-non-complementary region-X1X2X3X 4- ,

[0032] The GGGA- is fully complementary or partially complementary to the -X1X2X3X4-, so that the cyclic promoter forms a stem-loop structure. The lock motif is selected from a sequence that is fully complementary to the key motif from the 3' end to the 5' end.

[0033] In some embodiments, the circularized promoter, lock motif, and key motif have the same definitions as described above for circularized linear RNA.

[0034] In some embodiments, the circular promoter, lock motif, and key motif in the circularization assembly of the circularized linear RNA form a circular intermediate RNA precursor chain having the following structure after hybridization: A cyclase action nick is formed between the circular promoter and the key motif.

[0035] In another aspect, the present invention provides a method for preparing circular RNA, comprising the following steps:

[0036] Step 1. The circular promoter, lock motif, target RNA sequence, and key motif of the present invention are sequentially connected to obtain a DNA template chain connected with the circular components;

[0037] Step 2: performing in vitro transcription on the DNA template chain to obtain the linear RNA chain for circularization of the present invention;

[0038] Step 3: Convert the 5′-terminal triphosphate of the RNA sequence obtained in step 2 into a monophosphate to obtain a circular RNA precursor chain;

[0039] Step 4: placing the circular RNA precursor chain in a buffer solution and annealing it to form an RNA precursor chain in a circular intermediate state;

[0040] Step 5: The RNA precursor chain in the circular intermediate state described in step 4 is treated with RNA ligase to connect the interfaces at the 5′ end and the 3′ end to obtain a covalently closed circular RNA.

[0041] In some embodiments, PCR can be used as step 1. In some embodiments, solid phase synthesis can be used to replace steps 1 and 2 to obtain the linear RNA chain for circularization.

[0042] In some embodiments, in step 3, the linear RNA chain is treated with Apyrase enzyme to convert the 5′-terminal triphosphate of the RNA sequence obtained in step 2 into a monophosphate to obtain a circular RNA precursor chain.

[0043] In some embodiments, step 5 is to obtain the RNA precursor chain in the circular intermediate state by annealing after step 4.

[0044] In some embodiments, the RNA ligase is selected from T4 RNA ligase 1 or T4 RNA ligase 2.

[0045] In some embodiments, the amount of T4 RNA ligase used in step 5 is 5-50 U / μM circular intermediate RNA precursor chain. The amount of T4 RNA ligase used can be selected from 5, 10, 20, 30, 40, 50 U / μM circular intermediate RNA precursor chain.

[0046] In some embodiments, the method of preparing circular RNA further comprises a purification step 6:

[0047] A. Enzyme digestion step: Use RNase R to treat the connected circular RNA system to completely remove the unreacted linear long single strands;

[0048] B. Product extraction step: Purify and extract circular RNA using one or more of the following methods: RNA purification kit method, ethanol precipitation ultrafiltration method, or polyacrylamide gel electrophoresis method.

[0049] In some embodiments, in the RNA purification kit method, the enzymatically purified circular RNA is extracted using an RNA Clean & Concentrator Kit (RCC) RNA purification kit. In the ethanol precipitation and ultrafiltration method, the circular RNA is precipitated with ethanol and then enriched by ultrafiltration for desalination. In the polyacrylamide gel electrophoresis method, the circular RNA is extracted after gel excision and purification using polyacrylamide gel electrophoresis.

[0050] In another aspect, the present invention provides an expression vector for expressing a linear precursor RNA in vitro for circularization. In some embodiments, the expression vector is selected from a linear DNA fragment, a plasmid vector, a viral vector, a bacterial artificial chromosome, or a yeast artificial chromosome. The linear DNA fragment is preferably a PCR product or a linear plasmid fragment.

[0051] In another aspect, the present invention provides the aforementioned linear RNA for circularization, circularization component for circularizing linear RNA, or expression vector, and their use in preparing circularized RNA.

[0052] On the other hand, the present invention provides the aforementioned linear RNA for circularization, the circularization component or expression vector for circularizing linear RNA, and its use in treating, preventing or diagnosing diseases or in preparing drugs or diagnostic or detection reagents for treating or preventing diseases.

[0053] definition

[0054] Listed below are definitions of various terms used to describe the nucleic acid combinations and compositions disclosed herein. These definitions apply to the terms used throughout this specification and claims, except where otherwise limited in specific cases, and these terms are used either individually or as part of a larger group.

[0055] As used herein, the terms "optional" and "optionally" include both selection and non-selection. For example, "optionally modified" includes both modification and non-modification.

[0056] As used herein, the terms "a," "an," and "the" are generally construed to cover both the singular and the plural.

[0057] The term "include" as used herein means and can be used interchangeably with the phrase "including (but not limited to)". The term "comprising" as used herein means and can be used interchangeably with the phrase "including (but not limited to)". The technical solutions using "include" or "comprising" in this patent can be further limited to "consisting of" or "composed of".

[0058] Throughout this specification, reference to "one embodiment," "some embodiments," "an embodiment," "a specific embodiment," "a related embodiment," "an embodiment," "an additional embodiment," or "a further embodiment," or combinations thereof, means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the appearances of the aforementioned phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0059] As used herein, the terms "circRNA" or "cyclic polyribonucleotide" or "circular RNA" are used interchangeably and refer to polyribonucleotides that form a circular structure through covalent bonds.

[0060] As used herein, the term "cyclization efficiency" refers to a measure of the resulting circular polyribonucleotide compared to its linear starting material.

[0061] As used herein, 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 the base, sugar, and / or phosphate, 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. Nucleotide analogs are also intended to include nucleotides having bases such as inosine, quercetin, xanthine; sugars such as 2'-methyl ribose; non-natural phosphodiester linkages such as methylphosphonate, phosphorothioate, and peptide linkages. Nucleotide analogs include 5-methoxyuridine, 1-methylpseudouridine, and 6-methyladenosine. In some embodiments, modified ribonucleosides include 5-methylcytidine, 5-methoxyuridine, 1-methyl-pseudouridine, N6-methyladenosine and / or pseudouridine. In some embodiments, such modified nucleosides provide additional stability and resistance to immune activation.

[0062] As used herein, the term "complementary" refers to the ability to pair between two sequences comprising naturally occurring or non-natural bases or their analogs by base stacking and specific hydrogen bonding. For example, if a base at a position of a nucleic acid can form a hydrogen bond with a base at the corresponding position of a target, the bases are considered to be complementary to each other at that position. Nucleic acids can include universal bases or inert abasic spacers that do not provide positive or negative contributions to hydrogen bonding. Base pairing can include canonical Watson-Crick base pairing and non-Watson-Crick base pairing (e.g., G:U Wobble base pairing and Hoogsteen base pairing). It should be understood that for complementary base pairing, adenosine bases (A) are complementary to thymidine bases (T) or uracil bases (U), cytosine bases (C) are complementary to guanosine bases (G), and universal bases such as 3-nitropyrroles or 5-nitroindole can hybridize with any A, C, U or T and are considered to be complementary thereto. Nichols et al., Nature, 1994; 369: 492 493 and Loakes et al., Nucleic Acids Res., 1994; 22: 4039 4043. Inosine (I) is also considered a universal base in the art and is considered complementary to any A, C, U, or T. See Watkins and SantaLucia, Nucl. Acids Research, 2005; 33(19): 6258-6267.

[0063] Those skilled in the art will understand that, unless otherwise indicated, the polynucleotide sequences described in this application will list "T" in representative DNA sequences, but when the sequence represents RNA, "T" will be replaced with "U".

[0064] Compared to the target RNA comprising the same expressed sequence, the circular RNA provided herein has higher functional stability. In some embodiments, compared to an mRNA comprising the same expressed sequence, 5moU modified, optimized UTR, cap and / or polyA tail, the circular RNA provided herein has higher functional stability.

[0065] The prior art cited in this specification is hereby incorporated by reference in its entirety and for all purposes. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] FIG1 shows a schematic diagram of RNA chain self-circularization.

[0067] FIG2 shows the polyacrylamide gel electrophoresis characterization of the 215 nt circular RNA prepared based on the present self-circularization strategy and the corresponding circularization efficiency.

[0068] FIG3 shows the RNase R and RNase H enzyme digestion verification of the 215nt circular RNA prepared based on the self-circularization strategy.

[0069] FIG4 shows the interface sequencing of the 215 nt circular RNA prepared based on the present self-circularization strategy.

[0070] Figure 5 shows a schematic diagram of the RNA precursor chain in the circular intermediate state described in step 4 of Example 1, where components 1, 2, and 3 correspond to the circularized promoter, component 4 corresponds to the lock motif, and component 5 corresponds to the key motif.

[0071] FIG6 shows the polymerization degree verification of circular RNA prepared based on the present self-circularization strategy.

[0072] FIG7 shows the polyacrylamide gel electrophoresis characterization of circular RNAs of different sizes prepared based on the present self-circularization strategy.

[0073] FIG8 shows the circularization efficiency of circular RNAs of different sizes prepared based on the present self-circularization strategy.

[0074] Figure 9 shows the polyacrylamide gel electrophoresis characterization of circular RNA prepared at different enzyme connection times in the 4M2 group without the "key motif-lock motif" sequence and the 4P group containing the "key motif-lock motif" sequence.

[0075] FIG10 shows the circularization efficiency of circular RNA prepared at different enzyme connection times in the 4M2 group without the "key motif-lock motif" sequence and the 4P group containing the "key motif-lock motif" sequence. Beneficial effects

[0076] The present invention provides a universal and simple RNA cyclization method that can efficiently cyclize RNA chains of varying lengths and sequences. This method uses the RNA itself as a template, eliminating the need for additional splinting chains and generating no byproducts. Furthermore, this method simplifies sequence design and eliminates the need to consider the secondary structure of the sequence to be cyclized. Circular RNAs prepared using this method exhibit significantly improved stability compared to linear precursors without compromising the original function of the RNA sequence. DETAILED DESCRIPTION

[0077] The embodiments of the present disclosure will be described in detail below with reference to the examples. However, those skilled in the art will appreciate that the following examples are intended only to illustrate the present disclosure and should not be construed as limiting the scope of the present disclosure. Where specific conditions are not specified in the examples, the experiments were performed under conventional conditions or the conditions recommended by the manufacturer. Where the manufacturer of the reagents or instruments used is not specified, they are all commercially available conventional products.

[0078] The experimental techniques and methods used in this example are conventional unless otherwise specified. For example, in the following examples, where specific conditions are not specified, conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989) or the conditions recommended by the manufacturer are generally followed. Materials and reagents used in the examples were obtained through commercial channels unless otherwise specified.

[0079] Example 1: Preparation of 215nt Circular RNA of Target RNA

[0080] Step 1: dsDNA template construction: PCR amplification was performed by adding 2.5 μL of forward primer (10 μM), 2.5 μL of reverse primer (10 μM), 1 μL of DNA template (1 ng), 19 μL of ddH2O, and 19 μL of 2× Phusion Plus mixer to a 50 μL PCR reaction system. The cyclization components, including the circularization promoter, lock motif, and key motif described in Table 1, were added. The resulting double-stranded DNA was separated and purified by agarose gel electrophoresis, verified by TA cloning, and Sanger sequencing. Finally, a PCR reaction was performed using the sequence-verified plasmid (TOPO-TA / Blunt Vector) as a template to obtain the final dsDNA template.

[0081] Table 1

[0082] Step 2: In vitro transcription of 5' triphosphorylated linear ssRNA using dsDNA as a template. The reaction system (20 μL) contains 500 ng linear dsDNA template, 4 μL T7 transcription 5× buffer, 8 μL rNTP (25 mM ATP, CTP, UTP and 3 mM GTP), 2 μL enzyme mixture (T7) and nuclease-free water. Incubate the reaction system at 37°C for 4 hours. Add 1 μL RNase-Free DNase to the reaction system and continue incubating at 37°C for 30 minutes to remove the template dsDNA. Finally, the reaction system is purified using the RNA Clean & Concentrator Kit (RCC) to obtain 5' triphosphorylated linear precursor ssRNA.

[0083] Step 3: Convert 5' triphosphorylated linear ssRNA to monophosphorylated form: The reaction system (10 μL) contains 2 μM 5' triphosphorylated linear ssRNA, 0.1 U Apyrase, 20 U RiboLock RNase inhibitor and DEPC water. The solution used in the reaction is 1× Apyrase reaction buffer (20 mM 4-morpholinoethanesulfonic acid (MES), 50 mM NaCl, 5 mM CaCl2, 1 mM dl-dithiothreitol (DTT) and 0.05% 20, (pH 7.5)). The reaction system was incubated at 37°C for 2 hours and inactivated at 65°C for 20 minutes to obtain the monophosphorylated linear ssRNA.

[0084] Step 4: Annealing of linear ssRNA: The reaction system (40 μL) contained 0.5 μM monophosphorylated linear ssRNA in 1×TE·Mg 2+ Buffer (10 mM Tris-HCl, 0.1 mM EDTA, 5 mM Mg 2+ The reaction system was incubated at 75°C for 5 minutes and cooled to 25°C at a rate of 1°C / min to obtain an RNA precursor chain in a circular intermediate state.

[0085] Step 5: T4 Rnl2 enzymatically ligates the circular intermediate RNA precursor to obtain circular ssRNA: The reaction system (80 μL) contains 0.25 μM of the circular intermediate RNA precursor, 10 U of T4 Rnl2, 40 U of RiboLock RNase Inhibitor, and DEPC water in 1× T4 Rnl2 reaction buffer (50 mM Tris-HCl, 2 mM MgCl2, 1 mM dl-dithiothreitol (DTT), and 400 μM adenosine triphosphate (ATP), pH 7.5). The reaction system is incubated at 37°C for 2 hours and inactivated with proteinase K to obtain the crude circular ssRNA product.

[0086] Step 6: Purification of circular ssRNA: The reaction system (20 μL) consists of 500 ng of crude circular ssRNA, 2 U of RNase R, and DEPC water. The reaction solution is 1× RNase R reaction buffer (20 mM Tris-HCl, 100 mM KCl, and 0.1 mM MgCl2, pH 8.0). The reaction system is incubated at 37°C for 30 minutes and at 70°C for 5 minutes. Finally, the reaction system is purified using the RNA Clean & Concentrator Kit (RCC) to obtain circular ssRNA.

[0087] Step 7, denaturing PAGE analysis and yield assessment: The samples were electrophoresed on a denaturing polyacrylamide gel containing 8M urea (denaturing PAGE). Depending on the length of the precursor RNA, the gel range was 4% to 10%, and the ratio of acrylamide to bisacrylamide was 19:1 to 39:1. The gel was analyzed by Sybr Gold TM Post-staining was performed and then imaged using a UV gel imager from BIO-RAD (Hercules, USA). Quantitative data were obtained using Image Lab software.

[0088] The yield of circular RNA was calculated using the following equation:

[0089] Here, I (环状RNA) Indicates the band intensity of circular RNA, I (tot) The band intensity of all bands in the lane (composed of circular RNA, linear precursor RNA, and polymerized byproducts) is shown in Figure 2. To confirm that the primary product of the circularization reaction is circRNA, RNase R and RNase H were used to identify the product. The results are shown in Figure 3. ○ represents circular RNA, - represents linear RNA precursor, and -- represents linear RNA dimer.

[0090] Example 2: Preparation of other circular RNAs of different nt lengths

[0091] In step 1, the target RNA of the same length was used to replace the target RNA in step 1 of Example 1, TAGTAG (SEQ ID NO: 5) was used to replace the lock motif in Example 1, and CTACTA (SEQ ID NO: 6) was used to replace the key motif in Example 1. The same method as in step 1 of Example 1 was used to prepare dsDNA full sequences of 173nt, 240nt, 375nt, 525nt, 675nt, 895nt and 1125nt in length as described in Table 2 (the sequences marked with _ in Table 2 correspond to target RNAs of 148nt, 215nt, 350nt, 500nt, 650nt, 870nt, and 1100nt, respectively), and the corresponding circular RNA was prepared using the methods of steps 2 to 6 of Example 1. The yield of circularized RNA was determined using the method of step 7 of Example 1, and the test results are shown in Figures 7-8. The results show that the cyclization method of the present invention is suitable for the cyclization of linear RNAs of different lengths.

[0092] Table 2

[0093] Example 3: Effect of enzyme connection time on cyclization efficiency

[0094] The same method as steps 1-7 of Example 1 was used to prepare circular RNAs of 256 nt in length (corresponding to the 4P group in Figures 9-10, containing a "key motif-lock motif" sequence), as well as circular RNAs with a lock motif and a key motif deleted (corresponding to the 4M2 group in Figures 9-10, not containing a "key motif-lock motif" sequence). In step 5, the cells were incubated for 0-60 min, and the yield of circularized RNA at different time points within the incubation period of 0-60 min was calculated according to the method of step 7 of the embodiment to evaluate the effect of the incubation time of the enzyme ligation reaction in step 5 on the circularization efficiency. The test results are shown in Figures 9-10. The results show that, compared to the 4M2 group without the "key motif-lock motif" sequence, the 4P group containing the "key motif-lock motif" sequence significantly increased the yield of circular RNA within the enzyme ligation time of 0-60 min; compared to the enzyme ligation reaction of 0-10 min, the circularization efficiency was better when the enzyme ligation reaction was 30 min or longer.

[0095] Table 3

Claims

1. A linear RNA for cyclization, characterized in that From the 5' end to the 3' end, it contains the following structure: The cyclized promoter has the following structure from the 5' end to the 3' end: GGGA-non-complementary region-X1X2X3X 4- , The GGGA- is completely complementary or partially complementary to the -X1X2X3X4-, so that the circularized promoter forms a stem-loop structure.

2. The linear RNA for cyclization according to claim 1, wherein the lock motif is completely complementary to the key motif, so that the 5' end of the cyclization promoter sequence and the 3' end of the key motif sequence form a cyclase action nick.

3. The linear RNA for cyclization according to claim 1 or 2, wherein the GGGA- is completely complementary to the -X1X2X3X4- or has at least 3 complementary pairs, and preferably there is only a mismatch between G and X3.

4. The linear RNA for cyclization according to claim 3, wherein X1 is selected from the group consisting of bases U, A, C or G; preferably base U; X2, X3, and X4 are each independently selected from the base G or C.

5. The linear RNA for cyclization according to any one of claims 1 to 4, wherein the non-complementary region of the cyclization promoter is 3-15 nt in length; and the non-complementary region sequence is not complementary to GGGA-, -X1X2X3X4-, and the target RNA. The linear RNA for cyclization according to any one of claims 1 to 5, wherein the ratio of bases C and A in the non-complementary region of the cyclized promoter is 66%-100%. The linear RNA for cyclization according to any one of claims 1 to 6, wherein the non-complementary region sequence in the cyclization promoter is -CCAAC- from the 5' end to the 3' end.

8. the linear RNA for cyclization described in any one in claim 1 to 7, wherein said lock motif and key motif are complementary to form the paired structure of at least 3 bp, preferably the paired structure of 3-15 bp, 3-9 bp.

9. The linear RNA for cyclization of any one of claims 1 to 8, wherein the lock motif is a UAG unit repeated 1-5 times from 5' to 3', and the key motif that is fully complementary to the lock motif is a CUA unit repeated 1-5 times from 5' to 3'; preferably, the lock motif is a sequence of UAGUAG from 5' to 3', and the key motif is a sequence of CUACUA from 5' to 3'.

10. The linear RNA for cyclization according to any one of claims 1 to 9, wherein the GC content of the key motif and the key motif is 20%-70%. The linear RNA for circularization according to any one of claims 1 to 10, wherein the circularization promoter has the sequence of SEQ ID NO. 1 or SEQ ID NO. 2 from the 5' end to the 3' end.

12. The linear RNA for cyclization according to any one of claims 1 to 11, wherein the cyclization promoter sequence, the lock motif sequence and the key motif sequence form the following structural fragments after cyclization:

13. The linear RNA for cyclization according to any one of claims 1 to 12, comprising one or more target RNAs, wherein the length of the target RNA sequence is independently more than 50 nt, preferably 100 nt-10000 nt.

14. The linear RNA for circularization of any one of claims 1 to 13, wherein one or more spacer regions (linker regions) are optionally inserted between one or more target RNAs, lock motifs, and key motifs.

15. A circularization assembly for circularizing linear RNA, comprising: A cyclization promoter, a lock motif and a key motif, wherein the cyclization promoter, the lock motif and the key motif have the definitions as described in any one of claims 1 to 14; the cyclization promoter, the lock motif and the key motif form the following linear structure with the target RNA, 16. The cyclization assembly for cyclizing linear RNA as claimed in claim 15, wherein the cyclization promoter, the lock motif and the key motif form a circular intermediate RNA precursor chain with the following structure after hybridization, forming an action incision of a cyclase between AGGG and the key motif, 17. The cyclization assembly for cyclizing linear RNA as claimed in claim 15 or 16, wherein the cyclization promoter sequence, the lock motif sequence and the key motif sequence form the following continuous primary structure after cyclization: -(CUA) m -GGGACCAACUCUC-(UAG) n -, or -(CUA) m -GGGACCAACUCCC-(UAG) n -; wherein m=n and m and n are selected from 1, 2, 3, 4 or 5.

18. A method for preparing circular RNA, characterized in that: The following steps are involved: Step 1. sequentially connect the circularized promoter, lock motif, target RNA sequence and key motif according to any one of claims 1 to 14 to obtain a DNA template chain connected with a circular component; Step 2: performing in vitro transcription on the DNA template chain to obtain the linear RNA chain for circularization according to any one of claims 1 to 14; Optionally, using solid phase synthesis to replace step 1 and step 2 to obtain the linear RNA chain for cyclization according to any one of claims 1 to 14; Step 3: convert the 5′-terminal triphosphate of the RNA sequence obtained in step 2 into a monophosphate to obtain a circular RNA precursor chain; Step 4: placing the circular RNA precursor chain in a buffer solution and annealing to form an RNA precursor chain in a circular intermediate state; Step 5: The circular intermediate RNA precursor chain described in step 4 is treated with RNA ligase to connect the interfaces of the 5′ end and the 3′ end to obtain a covalently closed circular RNA.

19. The method for preparing circular RNA according to claim 18, characterized in that: The RNA ligase is selected from T4 RNA ligase 1 or T4 RNA ligase 2.

20. The method for preparing circular RNA according to claims 18-19, characterized in that: The dosage of T4 RNA ligase in step 5 is 5-50 U / μM circular intermediate RNA precursor chain.

21. The method for preparing circular RNA according to claims 18-20, further comprising a purification step 6: A. Enzyme digestion step: Use RNase R to treat the connected circular RNA system to completely remove the unreacted linear long single strands; B. Product extraction step: use one or more of the RNA purification kit method, ethanol precipitation ultrafiltration method or polyacrylamide gel electrophoresis method to purify and extract circular RNA.

22. The method for preparing circular RNA according to claim 21, wherein: In the RNA purification kit method, the RNA Clean & Concentrator Kit (RCC) RNA purification kit is used to extract the enzymatically purified circular RNA; In the ethanol precipitation and ultrafiltration method, the circular RNA is precipitated by ethanol, and then the circular RNA is enriched by ultrafiltration and desalting; In the polyacrylamide gel electrophoresis, circular RNA is extracted after gel excision and purification using polyacrylamide gel electrophoresis.

23. An expression vector, which expresses the precursor linear RNA for circularization according to any one of claims 1 to 14 in vitro.

24. The expression vector of claim 23, wherein the expression vector is selected from a linear DNA fragment, a plasmid vector, a viral vector, a bacterial artificial chromosome, and a yeast artificial chromosome; the linear DNA fragment is preferably a PCR product or a linear plasmid fragment.

25. Use of the linear RNA for circularization according to any one of claims 1 to 14, the circularization component for circularizing linear RNA according to any one of claims 15 to 17, or the expression vector according to claims 23 to 24 in preparing circularized RNA.

26. The linear RNA for circularization according to any one of claims 1 to 14, the circularization component for circularizing linear RNA according to any one of claims 15 to 17, or the expression vector according to claims 23 to 24, and its use in treating, preventing or diagnosing a disease or in preparing a drug for treating or preventing a disease or a diagnostic or detection reagent.

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