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

Cyclization components for linear RNA and applications thereof TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and specifically relates to a method for preparing single-stranded circular RNA. BACKGROUND

[0002] Circular RNA is a kind of single-stranded RNA characterized by a covalently closed topological structure, and therefore does not have free ends. Compared with linear RNA, circular RNA is not easily digested by exonuclease, and exhibits higher stability, and therefore has many practical application prospects in vivo and in vitro. For example, it can be used as a microRNA (miRNA) sponge, a protein binding framework, an RNA expression vector, and a protein regulator, etc. Therefore, how to artificially synthesize circular RNA has attracted widespread attention.

[0003] The in vitro synthesis method of circular RNA commonly used at present mainly includes chemical ligation method, intron-exon arrangement method (PIE) and enzyme ligation method. 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, so as to realize the circularization of the linear RNA chain. However, such a method is generally only suitable for the circularization of very small linear RNA chains (<100 nt), and due to the presence of 2' hydroxyl in the RNA chain, a side reaction of generating 2'-5' phosphodiester bond is prone to occur. In addition, due to the introduction of a large amount of chemical reagents, the biosafety of the prepared circular RNA is questionable, which further limits its application range. The PIE method is to realize the two ester exchange reactions based on the determined splicing sites in the intron self-splicing reaction. However, a key limitation of the existing PIE method is that the circular RNA product based on the PIE method will retain part of the exons of the natural group I intron gene, and a large amount of splicing intermediate by-products will be produced in the reaction system, and due to the very high requirement of the method on the secondary structure of the intron, the difficulty of sequence design is greatly increased. The enzyme ligation method mainly uses a series of DNA and RNA ligases (T4 DNA ligase, T4 RNA ligase 1, T4 RNA ligase 2) derived from the T4 bacteriophage. These enzymes need the linear RNA substrate to contain 5' monophosphate for circularization. However, due to the serious dependence of the ligase on the secondary structure of the linear RNA, the previous RNA ring-forming method based on DNA and RNA ligase generally needs to additionally add a splint strand, which increases the complexity of the system and the difficulty of post-processing. In addition, side reactions of intermolecular connection of RNA chains are also prone to occur. How to efficiently, universally and conveniently realize the circularization of RNA molecules is very important for the application of circular RNA molecules, which is difficult to achieve by the current RNA ring-forming method.

[0004] SUMMARY

[0005] In view of the disadvantages of the commonly used RNA circularization methods, the present application provides a method for circularizing RNA molecules with specific sequences, which uses itself as a template. In the RNA circularization method of the present application, the 3' end and 5' end of the RNA sequence to be circularized are extremely close in space due to the base complementary pairing between the circularization motifs, so that the covalent connection of the two ends of the RNA is achieved by adding RNA ligase, thereby realizing the circularization of the target RNA sequence.

[0006] In one aspect, the present application provides a linear RNA for circularization. In another aspect, the present application provides a circularization assembly for circularizing a linear RNA. In another aspect, the present application provides a method for preparing a circular RNA. In another aspect, the present application provides an expression vector for expressing a precursor linear RNA for circularization in vitro. In another aspect, the present application provides the use of the circularization assembly for circularizing a linear RNA, the linear RNA for circularization, or the expression vector in the treatment, prevention or diagnosis of diseases, or in the preparation of a drug for treating or preventing diseases or a diagnostic or detection reagent.

[0007] The universal and simple RNA circularization method and assembly provided by the present application can efficiently circularize RNA chains of different lengths and sequences. The method uses itself as a template, does not require the additional introduction of a clamp strand, and does not generate by-products. In addition, the method has a simple sequence design and does not require consideration of the secondary structure of the sequence to be circularized. The circular RNA prepared by this method has greater stability than the linear precursor, does not affect the original function of the RNA sequence, and can reduce the generation of polymer impurities.

[0008] In one aspect, the present application provides a linear RNA for circularization, which comprises the following structure from 5' end to 3' end:

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

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

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

[0012] In some embodiments, the lock unit and the key unit are fully complementary, such that the 5' end of the circularization initiator sequence and the 3' end of the key unit sequence form a circularase action nick (Nick).

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

[0014] In some embodiments, X1is selected from base U, A, C or G, preferably base U; each of X2, X3, X4is independently selected from base G or C.

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

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

[0017] In some embodiments, the lock unit and the key unit are complementary to form a paired structure of at least 3 bp (base pairs). In some embodiments, the lock unit and the key unit are complementary to form a paired structure of 3-15 bp, 3-9 bp. Specifically, a paired structure of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 bp is formed.

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

[0019] In some embodiments, the structure of the lock unit from 5' end to 3' end is -(UAG) m , and the sequence of the key unit from 5' end 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 the structure UAG from 5' end to 3' end. In some embodiments, the key motif has the sequence CUA from 5' end to 3' end. In some embodiments, the lock motif has the sequence UAGUAG from 5' end to 3' end and the key motif has the sequence CUACUA from 5' end to 3' end.

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

[0022] In some embodiments, the linear RNA for circularization has the structure GGGACCAACUCUC-(UAG) m -target RNA-(CUA) n wherein m = n and m and n are selected from 1, 2, 3, 4, or 5. In some embodiments, the circularization initiator can be replaced with GGGACCAACUCCC (SEQ ID NO: 2).

[0023] In some embodiments, the circularization initiator sequence, the lock motif sequence, and the key motif sequence in the linear RNA for circularization, after circularization, form the following structure fragment:

[0024] In some embodiments, a structure is formed that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% homologous to the above structure fragment.

[0025] In some embodiments, the target RNA sequence (i.e., the RNA sequence to be circularized) has a length of 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, 10,000 nt.

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

[0027] In some embodiments, the linear RNA for circularization and any element therein comprises unmodified, partially modified or fully modified nucleosides. The modifications include, but are not limited to, 5'-position pyrimidine modification, 8'-position purine modification, modification at the exocyclic amine of cytosine, and substitution of 5-bromo-uracil; and 2'-position sugar modification, including but not limited to sugar-modified ribonucleotides, nucleotide analogs also intended to include nucleotides with bases such as inosine, queuosine, 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.

[0028] In another aspect, the present application provides a circularization assembly for circularizing a linear RNA, comprising: a circularization initiator, a lock motif and a key motif.

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

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

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

[0032] The GGGA- is fully or partially complementary to the -X1X2X3X4- to form a stem-loop structure for the circularization initiator. The lock motif is selected from a sequence fully complementary to the key motif from 3' end to 5' end.

[0033] In some embodiments, the circularization initiator, the lock motif and the key motif have the same definition as the aforementioned linear RNA for circularization.

[0034] In some embodiments, the circularization initiator, the lock motif and the key motif in the circularization assembly for circularizing a linear RNA form a circular intermediate RNA precursor strand with the following structure after hybridization. A circularization enzyme action cut is formed between the circularization initiator and the key motif.

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

[0036] Step 1. sequentially connecting the cyclization initiator, the lock motif, the target RNA sequence and the key motif of the application to obtain a DNA template strand with a cyclization component connected;

[0037] Step 2: in vitro transcription of the DNA template strand to obtain a linear RNA strand for cyclization of the application;

[0038] Step 3: converting the 5' end triphosphate of the RNA sequence obtained in step 2 into monophosphate to obtain a circular RNA precursor strand;

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

[0040] Step 5: using RNA ligase to treat the RNA precursor strand in a circular intermediate state in step 4 to connect the interface of the 5' end and the 3' end to obtain a covalently closed circular RNA.

[0041] In some embodiments, step 1 can select PCR method. In some embodiments, solid phase synthesis method can be used to replace step 1 and step 2 to obtain the linear RNA strand for cyclization.

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

[0043] In some embodiments, step 5 obtains the RNA precursor strand in a circular intermediate state by annealing after step 4.

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

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

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

[0047] A, enzyme digestion step: using RNase R to treat the circular RNA system after ligation to completely remove unreacted linear long single strands;

[0048] B. Product extraction step: Purification and extraction of circular RNA using one or several of RNA purification kit method, ethanol precipitation ultrafiltration method or polyacrylamide gel electrophoresis method.

[0049] In some embodiments, in the RNA purification kit method, the circular RNA after enzyme digestion purification is extracted by using RNA Clean & Concentrator kit (RCC) RNA purification kit. In the ethanol precipitation and ultrafiltration method, the circular RNA is enriched by ethanol precipitation and then salt-removing by ultrafiltration. In the polyacrylamide gel electrophoresis, the circular RNA is extracted after gel purification by polyacrylamide gel electrophoresis.

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

[0051] In another aspect, the present application provides the use of the aforementioned linear RNA for circularization, the circularization assembly for circularizing linear RNA or the expression vector in the preparation of a circularized RNA.

[0052] In another aspect, the present application provides the use of the aforementioned linear RNA for circularization, the circularization assembly for circularizing linear RNA or the expression vector in the preparation of a circularized RNA.

[0053] Definitions

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

[0055] As used herein, the term "optionally" "optionally" includes both the scenario of selection and the scenario of non-selection. For example, "optionally modified" includes both the scenario of being modified and the scenario of not being modified.

[0056] As used herein, the terms "a" and "the" are generally interpreted to encompass both the singular and the plural forms.

[0057] As used herein, the term "comprise" means "comprising, but not limited to," and can be used interchangeably with the phrase "include (but not limited to)." As used herein, the term "include" means "include, but not limited to," and can be used interchangeably with the phrase "comprise (but not limited to)." The technical solutions using "comprise" or "include" in the patent can be further limited to "consist" or "constitute."

[0058] Throughout this specification, reference can be made to "one embodiment," "some embodiments," "an embodiment," "certain embodiments," "related embodiments," "a certain embodiment," "further embodiments," or "additional embodiments" or combinations thereof. It is understood that such references mean that a particular feature, structure, or characteristic being described is included in at least one embodiment of the application. Therefore, appearances of the foregoing phrases in various places throughout this specification are not necessarily intended to refer to the same embodiment. Furthermore, the particular features, structures, or characteristics can be combined in any suitable manner in one or more embodiments.

[0059] As used herein, the term "circRNA" or "circular polyribonucleotide" or "circular RNA" are used interchangeably and refer to a polyribonucleotide that forms a circular structure through a covalent bond.

[0060] As used herein, the term "circularization 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 that include 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 modified chemical structures at the base, sugar, and / or phosphate, including but not limited to, 5'-position pyrimidine modifications, 8'-position purine modifications, modifications at the exocyclic amines of cytosine, and substitution 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, queosine, xanthine; sugars such as 2'-methylribose; non-natural phosphodiester linkages such as methylphosphonate, phosphorothioate, and peptide linkages. Nucleotide analogs include 5-methoxyuridine, 1-methyl-pseudouridine, and 6-methyladenosine. In some embodiments, the 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 of two sequences comprising naturally or non-naturally occurring bases or analogs thereof to pair by base stacking and specific hydrogen bonding. For example, a base at a position of a nucleic acid is considered to be complementary to a base at a corresponding position of a target if the base is capable of forming a hydrogen bond with the base. Nucleic acids can comprise universal bases or inert no-base spacers that do not contribute positively or negatively 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 will be appreciated that for complementary base pairing, an adenosine-type base (A) is complementary to a thymidine-type base (T) or a uracil-type base (U), a cytosine-type base (C) is complementary to a guanosine-type base (G), and a universal base such as 3-nitropyrrole or 5-nitroindole can be hybridized to and considered complementary to any A, C, U, or T. 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] It will be appreciated by those skilled in the art that the polynucleotide sequences described in this application will recite "T" in representative DNA sequences, unless otherwise specified, but that "T" will be replaced with "U" when the sequence represents RNA.

[0064] The circular RNAs provided herein have higher functional stability compared to target RNAs comprising the same expression sequence. In some embodiments, the circular RNAs provided herein have higher functional stability compared to mRNAs comprising the same expression sequence, 5moU modifications, optimized UTRs, caps, and / or polyA tails.

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

[0066] Figure 1 shows a schematic of RNA strand self-circularization.

[0067] Figure 2 shows polyacrylamide gel electrophoresis characterization of 215 nt circular RNAs prepared based on the self-circularization strategy and the corresponding circularization efficiency.

[0068] Figure 3 shows RNase R and RNase H enzyme digestion verification of 215 nt circular RNA prepared based on the self-circularization strategy.

[0069] Figure 4 shows interface sequencing of 215 nt circular RNA prepared based on the self-circularization strategy.

[0070] Figure 5 shows a schematic diagram of the RNA precursor strand of the circular intermediate state described in Example 1, Step 4. Component 1, Component 2 and Component 3 correspond to the circularization promoter, Component 4 corresponds to the lock motif, and Component 5 corresponds to the key motif.

[0071] Figure 6 shows the verification of the polymerization degree of circular RNA prepared based on the self-circularization strategy.

[0072] Figure 7 shows the polyacrylamide gel electrophoresis characterization of circular RNA of different sizes prepared based on the self-circularization strategy.

[0073] Figure 8 shows the circularization efficiency of circular RNA of different sizes prepared based on the self-circularization strategy.

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

[0075] Figure 10 shows the circularization efficiency of circular RNA prepared at different enzyme ligation times for the 4M2 group without the "key motif-lock motif" sequence and the 4P group with the "key motif-lock motif" sequence. Advantages

[0076] The present application provides a universal and simple RNA circularization method, which can efficiently circularize RNA strands of different lengths and different sequences. The method uses itself as a template, does not require the additional introduction of a clamp strand, and does not generate byproducts. In addition, the method has a simple sequence design and does not need to consider the secondary structure of the sequence to be circularized. The stability of the circular RNA prepared by this method is greatly improved compared to linear precursors, and the original function of the RNA sequence is not affected. DETAILED DESCRIPTION

[0077] The embodiments of the present disclosure will be described in detail below with reference to the examples, but those skilled in the art will understand that the following examples are only for illustration of the present disclosure and should not be considered as limiting the scope of the present disclosure. The specific conditions not specified in the examples are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used are not specified by the manufacturer, and are all conventional products that can be obtained by market purchase.

[0078] The experimental techniques and experimental methods used in this example are conventional technical methods, for example, the experimental methods in the following examples without specific conditions are usually carried out according to the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. The materials, reagents, etc. used in the examples, unless otherwise specified, can be obtained through regular commercial channels.

[0079] Example 1: Preparation of circular RNA of 215 nt target RNA

[0080] Step one, construction of dsDNA template: 2.5 μL forward primer (10 μM), 2.5 μL reverse primer (10 μM), 1 μL DNA template (1 ng), 19 μL ddH2O and 19 μL 2x Phusion plus mixer were added in a 50 μL PCR reaction system for PCR amplification and addition of circularization components, the circularization components included the circularization promoters, lock motifs and key motifs described in Table 1, the obtained double-stranded DNA was separated and purified by agarose gel electrophoresis, and was verified by TA cloning and Sanger sequencing. Finally, the plasmid (TOPO-TA / Blunt Vector) verified by sequencing was used as a template to obtain the final dsDNA template by PCR reaction.

[0081] Table 1

[0082] Step two, in vitro transcription of 5' triphosphorylated linear ssRNA with dsDNA as template. The reaction system (20 μL) contained 500 ng linear dsDNA template, 4 μL T7 transcription 5x buffer, 8 μL rNTP (25 mM ATP, CTP, UTP and 3 mM GTP), 2 μL enzyme mixture (T7) and nuclease-free water. The reaction system was incubated at 37°C for 4 hours. 1 μL RNase-Free DNase was added to the reaction system, and incubation was continued at 37°C for 30 minutes to remove the template dsDNA. Finally, the reaction system was purified using RNA Clean & Concentrator kit (RCC) to obtain 5' triphosphorylated linear precursor ssRNA.

[0083] Step three, conversion of 5' triphosphorylated linear ssRNA to monophosphorylated form: reaction mixture (10 μΐ^) contains 2 μΜ 5' triphosphorylated linear ssRNA, 0.1 U Apyrase, 20 U RiboLock RNase inhibitor and DEPC water, reaction solution is 1 x Apyrase reaction buffer (20 mM 4-morpholineethanesulfonic acid (MES), 50 mM NaCl, 5 mM CaCl2, 1 mM dl-dithiothreitol (DTT) and 0.05% 20, (pH 7.5)). Reaction mixture is incubated at 37 °C for 2 hours and inactivated at 65 °C for 20 minutes to obtain monophosphorylated linear ssRNA.

[0084] Step four, annealing of linear ssRNA: reaction mixture (40 μΐ^) contains 0.5 μΜ monophosphorylated linear ssRNA, reaction solution is 1 x TE-Mg 2+ buffer (10 mM Tris-HCl, 0.1 mM EDTA, 5 mM Mg 2+ , pH 7.6). Reaction mixture is incubated at 75 °C for 5 minutes and cooled to 25 °C at a rate of 1 °C / min to obtain RNA precursor strand in a circular intermediate state.

[0085] Step five, T4 Rnl2 enzyme ligation of RNA precursor strand in a circular intermediate state to obtain circular ssRNA: reaction mixture (80 μΐ^) contains 0.25 μΜ RNA precursor strand in a circular intermediate state, 10 U T4 Rnl2, 40 U RiboLock RNase inhibitor and DEPC water, reaction solution is in 1 x T4 Rnl2 reaction buffer (50 mM Tris-HCl, 2 mM MgCl2, 1 mM dl-dithiothreitol (DTT) and 400 μΜ adenosine triphosphate (ATP), pH 7.5). Reaction mixture is incubated at 37 °C for 2 hours and inactivated with proteinase K to obtain crude product of circular ssRNA.

[0086] Step six, purification of circular ssRNA: reaction mixture (20 μΐ^) contains 500 ng crude product of circular ssRNA, 2 U RNase R and DEPC water, reaction solution is 1 x RNase R reaction buffer (20 mM Tris-HCl, 100 mM KCl and 0.1 mM MgCl2, pH 8.0). Reaction mixture is incubated at 37 °C for 30 minutes and at 70 °C for 5 minutes. Finally, reaction mixture is purified with RNA Clean & Concentrator kit (RCC) to obtain circular ssRNA.

[0087] Step seven, denaturing PAGE analysis and yield evaluation: samples were subjected to electrophoresis on denaturing polyacrylamide gel (denaturing PAGE) with 8M urea. The gel ranged from 4% to 10% according to the length of the precursor RNA, with the ratio of acrylamide to bisacrylamide being 19:1 to 39:1. The gel was post-stained by Sybr Gold TM and then imaged using a UV gel imager from BIO-RAD (Hercules, USA). Quantitative data was obtained using Image Lab software.

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

[0089] Here, I (环状RNA) represents the band intensity of circular RNA, I (tot) represents the band intensity of all bands in the lane (consisting of circular RNA, linear precursor RNA and polymerization byproducts). The results are shown in Figure 2. To confirm that the main product of the circularization reaction is circRNA, the product was identified using two enzymes, RNase R and RNase H, and the results are shown in Figure 3. Wherein o represents circular RNA, - represents linear RNA precursor, and -- represents linear RNA dimer.

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

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

[0092] Table 2

[0093] Example 3: Effect of enzyme incubation time on circularization efficiency

[0094] The same method as steps 1-7 of Example 1 was used to prepare 256nt long circular RNA (corresponding to 4P group in FIG. 9-10, containing the“key-motif-lock motif” sequence), and circular RNA without lock motif and key motif (corresponding to 4M2 group in FIG. 9-10, not containing the“key-motif-lock motif” sequence), respectively. The incubation time of 0-60min in step 5 was used to evaluate the effect of incubation time of step 5 enzyme ligation reaction on the circularization efficiency. The yield of circularized RNA at different time points within 0-60min incubation was calculated according to the method of Example step seven, respectively. The test results are shown in FIG. 9-10. The results show that, compared with the 4M2 group without the“key-motif-lock motif” sequence, the 4P group containing the“key-motif-lock motif” sequence significantly provides the yield of circular RNA within 0-60min enzyme ligation time; the circularization efficiency is better at 0-10min, 30min and above enzyme ligation reaction.

[0095] Table 3

Claims

1. A linear RNA for circularization, characterized in that, comprises the following structure from the 5' end to the 3' end: The circularization promoter has the following structure from 5' end to 3' end: GGGA-non-complementary region-X1X2X3X 4- , The GGGA- and the -X1X2X3X4- are fully complementary or partially complementary, so that the circularization promoter forms a stem-loop structure.

2. The linear RNA for circularization of claim 1, wherein the lock motif and the key motif are fully complementary, so that the 5' end of the circularization promoter sequence and the 3' end of the key motif sequence form a circularizer action cut.

3. The linear RNA for circularization of claim 1 or 2, wherein the GGGA- and the -X1X2X3X4- are fully complementary or at least 3 complementary pairs, preferably only a mismatch between G and X3.

4. The linear RNA for circularization of claim 3, wherein X1 is selected from base U, A, C or G; preferably base U; X2, X3, X4 are each independently selected from base G or C.

5. The linear RNA for circularization of any one of claims 1 to 4, wherein the non-complementary region of the circularization promoter is 3-15 nt in length; the non-complementary region sequence is non-complementary to GGGA-, -X1X2X3X4- and the target RNA.

6. The linear RNA for circularization of any one of claims 1 to 5, wherein the proportion of base C and A in the non-complementary region of the circularization promoter is 66%-100%.

7. The linear RNA for circularization of any one of claims 1 to 6, wherein the non-complementary region sequence in the circularization promoter from 5' end to 3' end is -CCAAC-.

8. The linear RNA for circularization of any one of claims 1 to 7, wherein the lock motif and the key motif are complementary to form a paired structure of at least 3 bp, preferably a paired structure of 3-15 bp, 3-9 bp.

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

10. The linear RNA for circularization of any one of claims 1 to 9, wherein the GC proportion of the key motif and the key motif is 20%-70%.

11. The linear RNA for circularization of any one of claims 1 to 10, wherein the circularization promoter from 5' end to 3' end has the sequence described in SEQ ID NO. 1 or SEQ ID NO.

2.

12. The linear RNA for circularization of any one of claims 1 to 11, wherein the circularization initiator sequence, the lock motif sequence, and the key motif sequence form the following structural fragment upon circularization:

13. The linear RNA for circularization of any one of claims 1 to 12, wherein one or more target RNAs are contained, and the length of each of the target RNA sequences is independently 50 nt or more, preferably 100 nt-10000 nt.

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

15. A circularization assembly for circularizing a linear RNA, comprising: a circularization initiator, a lock motif and a key motif, having the definition as in any one of claims 1 to 14; said circularization initiator, lock motif and key motif forming with the target RNA the following linear structure, 16. The circularization assembly for circularizing a linear RNA of claim 15, wherein the circularization initiator, the lock motif and the key motif form, upon hybridization, a circular intermediate state of the RNA precursor strand having the structure, upon hybridization, a circular intermediate state of the RNA precursor strand having the structure, wherein the cleavage nick for the circulase is formed between AGGG and the key motif.

17. The circularization assembly for circularizing linear RNA as described in claim 15 or 16, wherein the circularization promoter sequence, lock motif sequence, and key motif sequence form the following sequential primary structure after circularization: -(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 of preparing a circular RNA, characterized by, comprising the following steps: Step 1. sequentially ligate the circularization promoter, the lock motif, the target RNA sequence and the key motif according to any one of claims 1-14 to obtain a DNA template strand with a circularization component ligated; Step 2: in vitro transcribe the DNA template strand to obtain a linear RNA strand for circularization according to any one of claims 1-14; Optionally, replace the step 1 and step 2 with solid phase synthesis to obtain a linear RNA strand for circularization according to any one of claims 1-14; Step 3: convert the 5' end triphosphate of the RNA sequence obtained in step 2 to monophosphate to obtain a circular RNA precursor strand; Step 4: place the circular RNA precursor strand in a buffer solution to anneal to form an RNA precursor strand in a circular intermediate state; Step 5: use RNA ligase to treat the circular intermediate state RNA precursor strand in step 4 to connect the interface of the 5' end and the 3' end to obtain a covalently closed circular RNA.

19. The method of claim 18, wherein the method of producing a circular RNA is characterized by, The RNA ligase is selected from T4 RNA ligase 1 (T4 RNA ligase 1) or T4 RNA ligase 2 (T4 RNA Ligase 2).

20. The method of claim 18-19 for preparing a circular RNA, characterized in that, The amount of T4 RNA ligase used in step 5 is 5-50 U / μM of circular intermediate state RNA precursor strand.

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

22. The method for preparing a circular RNA according to claim 21, wherein, In the RNA purification kit method, use the RNA Clean & Concentrator kit (RCC) RNA purification kit to extract the circular RNA after enzyme digestion and purification; In the ethanol precipitation and ultrafiltration method, the circular RNA is enriched by ethanol precipitation and then salt-free ultrafiltration; In the polyacrylamide gel electrophoresis, the circular RNA is extracted after gel purification by polyacrylamide gel electrophoresis.

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

24. The expression vector according to claim 23, wherein the expression vector is selected from a linear DNA fragment, a plasmid vector, a viral vector, a bacterial artificial chromosome, a yeast artificial chromosome; and 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 1-14, the circularization component for circularizing a linear RNA according to any one of claims 15-17 or the expression vector according to claims 23-24 for the preparation of a circular RNA.

26. Use of the linear RNA for circularization according to any one of claims 1 to 14 or the circularization assembly for circularizing a linear RNA according to any one of claims 15 to 17 or the expression vector according to claims 23 to 24 for the treatment, prevention or diagnosis of a disease or for the preparation of a medicament for the treatment, prevention of a disease or a diagnostic or detection reagent.

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