Purification method for circular RNA

Abstract

The present invention provides a novel purification method for circular RNA that makes it possible to separate and purify the circular RNA by means of reversed-phase HPLC. The purification method comprises a purification step for subjecting a sample containing circular RNA to reversed-phase HPLC using a mobile phase containing a quaternary salt.

Description

Method for purifying circular RNA

[0001] This invention relates to a method for purifying circular RNA, etc.

[0002] Circular RNA (ribonucleic acid) is known to have higher biological stability, longer protein expression duration, and the ability to suppress excessive immune responses compared to linear RNA (see, for example, Non-Patent Document 1).

[0003] Molecular Cell 2019, 74(3), 508-520.

[0004] Since circular RNA is produced by cyclizing its precursor linear RNA in solution, the reaction solution contains impurities other than circular RNA, such as linear RNA and its degradation products. When circular RNA is used as a pharmaceutical, the concentration of circular RNA in the pharmaceutical composition must be strictly controlled, so these impurities must be clearly separated from and removed from the circular RNA.

[0005] In particular, linear RNA has drawbacks such as being easily degraded in vivo and triggering a strong immune response even with small amounts of contamination. Therefore, in order to apply circular RNA as a pharmaceutical, there is a need to develop purification methods that can reduce the contamination of linear RNA and improve the purity of circular RNA.

[0006] Typically, triethylammonium, a tertiary salt, is most commonly used in separation methods using reverse-phase HPLC (high-performance liquid chromatography). However, conventional purification methods using tertiary salts have made it difficult to separate circular RNA from impurities in solution (particularly separating circular RNA and linear RNA of similar size).

[0007] This invention has been made in view of the current state of the prior art described above, and its main objective is to provide a novel method for purifying circular RNA that can separate and purify circular RNA by reverse-phase HPLC.

[0008] The inventors have diligently conducted research to achieve the above-mentioned objectives. As a result, surprisingly, the inventors have discovered that by using a quaternary salt instead of the conventionally used tertiary salt as the mobile phase of reversed-phase HPLC, circular RNA can be separated and purified with higher purity and in a simple manner. Based on this finding, the inventors have conducted further research and completed the present invention. That is, the present invention encompasses the following components.

[0009] Item 1. A method for purifying circular RNA, comprising a purification step of subjecting a sample containing circular RNA to reverse-phase HPLC using a mobile phase containing a quaternary salt. Item 2. The method according to Item 1, wherein the quaternary salt comprises at least one salt selected from the group consisting of ammonium salts, imidazolium salts, pyrrolidinium salts, pyridinium salts, piperidinium salts, and phosphonium salts. Item 3. The method according to Item 1 or 2, wherein the quaternary salt comprises an ammonium salt. Item 4. The method according to any one of Items 1 to 3, wherein the quaternary salt has four identical or different hydrocarbon groups having 1 to 10 carbon atoms on a nitrogen atom or a phosphorus atom. Item 5-1. The method according to any one of Items 1 to 4, wherein the concentration of the quaternary salt in the mobile phase is 1 mM to 1000 mM. Item 5-2. The method according to any one of Items 1 to 5-1, wherein the circular RNA in the mobile phase has a random coil structure. Item 6. The method according to any one of claims 1 to 5-2, wherein the sample further comprises linear RNA. Claim 7. The method according to claim 6, wherein the sample further comprises the circular RNA and the linear RNA, and at least one substance selected from the group consisting of plasmid DNA, deoxyoligonucleotides, deoxynucleoside monophosphate, ribonucleoside triphosphate, and protein. Claim 8. The method according to claim 6 or 7, wherein the resolution (Rs) of the peaks of the circular RNA and the linear RNA in the chromatogram obtained in the purification step is 0.5 or higher. Claim 9. The method according to any one of claims 1 to 8, wherein the circular RNA is single-stranded RNA. Claim 10. The method according to any one of claims 1 to 9, wherein the base length of the circular RNA is 20 to 15000. Claim 11. The method according to any one of claims 1 to 10, wherein the reverse-phase HPLC uses at least one stationary phase selected from the group consisting of organic silica hybrid substrates and macroporous spherical polystyrene. Claim 12. A method for producing circular RNA, comprising: a cyclization step of obtaining circular RNA by cyclizing linear RNA; and a purification step of subjecting a sample containing the circular RNA to reverse-phase HPLC using a mobile phase containing a quaternary salt. Item 13. A circular RNA composition comprising circular RNA and a quaternary salt, and substantially free of linear RNA.Item 14. A mobile phase for reversed-phase HPLC for purifying circular RNA, comprising a quaternary salt.

[0010] According to the present invention, a novel method for purifying circular RNA that enables separation and purification of circular RNA by reversed-phase HPLC can be provided.

[0011] These are the results of reversed-phase HPLC of Example 1-1; the results of denaturing PAGE of Example 1-1; the results of reversed-phase HPLC of Example 2-1; the results of denaturing PAGE of Example 2-1; the results of reversed-phase HPLC of Example 2-2; the results of denaturing PAGE of Example 2-2; the results of reversed-phase HPLC of Example 2-3; the results of denaturing PAGE of Example 2-3; the results of reversed-phase HPLC of Example 2-4; the results of denaturing PAGE of Example 2-4; the results of reversed-phase HPLC of Example 2-5; the results of denaturing PAGE of Example 2-5; the results of reversed-phase HPLC of Comparative Example 1; the results of denaturing PAGE of Comparative Example 1; the results of reversed-phase HPLC of Comparative Example 2; the results of denaturing PAGE of Comparative Example 2; the results of reversed-phase HPLC of Example 3; the results of denaturing PAGE of Example 3 and Comparative Example 3; the results of HS-AFM measurement of Example 3; the results of reversed-phase HPLC of Comparative Example 3; the results of HS-AFM measurement of Comparative Example 3; the results of HS-AFM measurement of Example 4 (when the quaternary salt is replaced with a tertiary salt); and the results of HS-AFM measurement of Example 4 (when the tertiary salt is replaced with a quaternary salt).

[0012] As used herein, the term "comprising" is a concept that encompasses all of "comprising (comprise)", "consisting essentially of (consist essentially of)", and "consisting of (consist of)".

[0013] In addition, as used herein, the notation of a numerical range "A to B" means "not less than A and not more than B".

[0014] 1. Method for Purifying Circular RNA The present invention provides a method for purifying circular RNA (hereinafter sometimes simply referred to as the "purification method"), comprising a purification step of subjecting a sample containing circular RNA to reverse-phase HPLC using a mobile phase containing a quaternary salt. The purification method of the present invention allows for the separation and purification of circular RNA with higher purity by using a mobile phase containing a quaternary salt in reverse-phase HPLC. The purification method of the present invention is particularly advantageous over the prior art in that it can separate and purify circular RNA with high purity from a solution containing a mixture of circular RNA and linear RNA of similar size. However, since the purification method of the present invention can suppress the aggregation of circular RNA by using a mobile phase containing a quaternary salt, it is possible to purify circular RNA not only by separating and removing linear RNA, but also by separating circular RNAs from each other or from other substances, and preferably by purifying circular RNA with higher purity. The purification method of the present invention can be used not only for the purification of circular RNA but also for the purification of circular DNA.

[0015] Furthermore, the purification method of the present invention is advantageous compared to conventional purification methods that purify circular RNA by electrophoresis in that it can efficiently purify a sample containing a larger amount of circular RNA in a single, simple operation. In addition, the purification method of the present invention can achieve highly efficient purification even for large circular RNAs that are unsuitable for electrophoresis.

[0016] Furthermore, in conventional purification methods that degrade impurities other than circular RNA (especially linear RNA) with exonucleases, nicks are introduced into the circular RNA, or parts of it are also degraded. In contrast, the purification method of the present invention does not involve deterioration or degradation of the quality of circular RNA, thus enabling the efficient purification of higher-quality circular RNA.

[0017] The present invention will be described in detail below.

[0018] 1-1. Sample The sample used in the purification method of the present invention is not particularly limited as long as it contains circular RNA.

[0019] The sample may be, for example, a sample obtained when producing circular RNA by in vitro transcription, in vivo (cell system) transcription, or chemosynthesis.

[0020] The sample may be in solution or in a dry state. From the viewpoint of the solubility of circular RNA, it is preferable that the sample be in solution during purification. Furthermore, from the viewpoint of the stability of circular RNA, it is preferable that the sample be stored frozen before purification.

[0021] The sample is preferably a crude sample obtained after the synthesis of circular RNA or a purified sample thereof.

[0022] The sample is preferably subjected to nucleic acid extraction before being subjected to the purification method of the present invention, if necessary. Examples of nucleic acid extraction methods include enzymatic treatment, surfactant treatment, sonication, and the like.

[0023] Furthermore, it is preferable to pre-purify the sample before using the purification method of the present invention. Examples of pre-purification include LiCl precipitation, alcohol precipitation, phenol / chloroform extraction, simple purification by spin column, and ultrafiltration.

[0024] 1-1-1. Circular RNA The sample used in the purification method of the present invention contains circular RNA.

[0025] Circular RNA is not particularly limited as long as it is a circular RNA without terminal structures, but it is preferably mRNA that codes for a protein. Circular RNA may also be non-coding RNA (ncRNA). mRNA may be modified or unmodified mRNA. Examples of ncRNA may include tRNA, rRNA, snRNA, snoRNA, miRNA, piRNA, tsRNA, and lncRNA.

[0026] The circular RNA may be partially or entirely single-stranded RNA, or partially or entirely double-stranded RNA. In one embodiment, the sample used in the purification method of the present invention contains single-stranded RNA.

[0027] Circular RNA may have branched structures (branched chains) as long as it includes circular RNA without terminal structures, and such branched structures may have terminal structures. The production method of the present invention allows for the separation and purification of circular RNA by reverse-phase HPLC even if the circular RNA has branched structures.

[0028] From the viewpoint of purification efficiency, the base length of the circular RNA is preferably 20 to 15,000, more preferably 100 to 5,000, and even more preferably 100 to 3,000. The base length may or may not be a multiple of 3. When the base length of the circular RNA is a multiple of 3, the reading frame does not shift during translation.

[0029] Circular RNA may contain not only typical bases such as adenine (A), uracil (U), guanine (G), and cytosine (C), but also other bases such as hypoxanthine (I) and modified bases. Examples of modified bases include pseudouracil (e.g., N). １ -Pseudouracil), 3-methyluracil, dihydrouracil, 5-alkylcytosine (e.g., 5-methylcytosine), 5-alkyluracil (e.g., 5-ethyluracil), 5-halouracil (5-bromouracil), 6-azapyrimidine, 6-alkylpyrimidine (6-methyluracil), 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, 1-methyladenine, 1-methylhypoxanthine, Examples include 2,2-dimethylguanine, 3-methylcytosine, 2-methyladenine, 2-methylguanine, N6-methyladenine, 7-methylguanine, 5-methoxyaminomethyl-2-thiouracil, 5-methylaminomethyluracil, 5-methylcarbonylmethyluracil, 5-methyloxyuracil, 5-methyl-2-thiouracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid, 2-thiocytosine, purines, 2-aminopurines, isoguanine, indole, imidazole, xanthine, etc.

[0030] Preferably, circular RNA contains only phosphodiester bonds between nucleosides, but may also contain phosphoramidate bonds, phosphorothioate bonds, phosphorodithioate bonds, or methylphosphonate bonds between nucleosides.

[0031] In circular RNA, a hydroxyl group may be substituted with -OR (R is, for example, CH ３ (2'-O-Me), CH ２ CH ２ OCH ３ (2'-O-MOE), CH ２ CH ２ NHC(NH)NH ２ , CH ２ CONHCH ３ , CH ２ CH ２ CN, etc.) may include a sugar (ribose).

[0032] Circular RNA may include, for example, a phosphate moiety or hydroxyl moiety modified with biotin, an amino group, a lower alkylamine group, an acetyl group, or the like. Further, in circular RNA, the conformation of the sugar moiety may be fixed to N-type (LNA) by crosslinking the 2' oxygen and 4' carbon of the sugar moiety.

[0033] Circular RNA may or may not have a start codon and / or a stop codon. If the circular RNA does not have a termination codon in the same reading frame as the start codon for expressing a desired protein, protein repeats of theoretically infinite chain length can be synthesized.

[0034] It is also preferable that the circular RNA has a kozak sequence upstream of the start codon. The specific base sequence of the kozak sequence can be appropriately determined in consideration of the base sequence of the translation region, the species of the organism of the translation system, and the like.

[0035] Circular RNA can be subjected to reverse-phase HPLC singly in one kind or in combination of two or more kinds.

[0036] In the mobile phase described later, circular RNA preferably has a random coil structure. In this specification, "having a random coil structure" means a state in which the higher-order structure of the nucleic acid molecule is unfolded, or in other words, a state in which the nucleic acid chains are not aggregated. The presence or absence of higher-order structure and / or aggregation can be evaluated, for example, by high-speed atomic force microscopy (high-speed AFM). For example, in AFM measurement, aggregated nucleic acid molecules preferably have a thickness of about 1.0 nm or more, and monodisperse nucleic acid molecules preferably have a thickness of less than about 1.0 nm (e.g., 0.2 nm to 0.5 nm).

[0037] Circular RNA may be a natural product, a commercially available product, or one that has been synthesized in-house. Examples of methods for synthesizing circular RNA include the circular RNA production method (cyclization step) described later. Linear RNA, which is a precursor to circular RNA, can be synthesized, for example, using an automated nucleic acid synthesizer or a polynucleotide synthase.

[0038] In the sample, the amount of crude purified circular RNA before purification is preferably 0.005 mg to 10 mg, more preferably 0.01 mg to 5 mg, and even more preferably 0.1 mg to 1 mg, from the viewpoint of purification efficiency.

[0039] 1-1-2. Impurities The sample used in the purification method of the present invention is generally assumed to contain impurities other than circular RNA. In this specification, "impurities" refers to substances other than circular RNA, such as various reagents and nucleic acids (vectors, polynucleotides, etc.) and linear RNA, that are added to the reaction system in the production of circular RNA. In the purification method of the present invention, by using a mobile phase containing a quaternary salt, circular RNA can be separated and purified with higher purity in reverse-phase HPLC.

[0040] In one embodiment, the sample may further contain linear RNA. Linear RNA is an uncyclized RNA such as a precursor of circular RNA, a fragment thereof, or a dimer, and is an unwanted substance in the cyclization reaction solution.

[0041] Linear RNA is not particularly limited as long as it is linear RNA with terminal structures. Examples of linear RNA may also include aptamers and ribozymes.

[0042] Linear RNA is preferably mRNA that codes for proteins, but may also be non-protein-coding RNA (ncRNA). mRNA may be modified or unmodified. Examples of ncRNA may include tRNA, rRNA, snRNA, snoRNA, miRNA, piRNA, tsRNA, and lncRNA.

[0043] Linear RNA is preferably single-stranded RNA, but part of it or its entire length may be double-stranded RNA.

[0044] Linear RNA may have branched structures, and such branched structures may also have terminal structures, as long as they are linear RNA having terminal structures. The production method of the present invention can separate and remove circular RNA by reverse-phase HPLC, even if it further contains linear RNA having branched structures.

[0045] From the viewpoint of purification efficiency, the base length of linear RNA is preferably 20 to 15,000, more preferably 20 to 10,000, and even more preferably 20 to 8,000.

[0046] Furthermore, it is preferable that the base length of the linear RNA is substantially the same as that of the circular RNA. Even if the production method of the present invention further contains linear RNA having substantially the same base length, it can be separated from the circular RNA by reverse-phase HPLC and removed. It should be noted that the base lengths of the circular RNA and linear RNA being substantially the same means that there is no difference between them beyond the size change due to cyclization or ring opening, and specifically, the ratio of the base length of the circular RNA to the base length of the linear RNA (circular / linear) is preferably 0.8 to 1.2, more preferably 0.9 to 1.1, and even more preferably 1.0.

[0047] Furthermore, it is preferable that the sequence of the linear RNA is substantially identical to that of the circular RNA. Even if the production method of the present invention further contains linear RNA having substantially identical sequences, it can be separated from the circular RNA by reverse-phase HPLC and removed. It should be noted that the sequences of the circular RNA and linear RNA being substantially identical means that there are no differences between them beyond sequence changes due to cyclization or ring opening, and specifically, the identity or homology between the sequences of the circular RNA and linear RNA is preferably 80% to 100%, more preferably 90% to 100%, and even more preferably 99% to 100%.

[0048] In the sample, the amount of crude purified linear RNA before purification is preferably 0.005 mg to 1000 mg, more preferably 0.005 mg to 500 mg, and even more preferably 0.1 mg to 10 mg, from the viewpoint of purification efficiency.

[0049] The sample may include biological samples for use in the synthesis of circular RNA (e.g., components of plants, bacteria, and viruses (e.g., nucleic acids and proteins)), linear RNA, plasmid DNA, deoxyoligonucleotides, deoxynucleoside monophosphate, ribonucleoside triphosphate, and enzymes (proteins) for use in the reaction. In one embodiment, the sample further contains linear RNA in addition to circular RNA. In another embodiment, the sample further contains circular RNA and the above linear RNA, and at least one substance selected from the group consisting of plasmid DNA, deoxyoligonucleotides, deoxynucleoside monophosphate, ribonucleoside triphosphate, and proteins.

[0050] 1-2. Mobile Phase The mobile phase used in the purification method of the present invention contains a quaternary salt. A mobile phase containing a quaternary salt is useful as a mobile phase for reverse-phase HPLC for the purification of circular RNA. In this specification, "mobile phase" refers to the solution that passes through the column in reverse-phase HPLC. Furthermore, when "mobile phase A" or "mobile phase B" is mentioned, it refers to the solutions mixed to prepare the mobile phase, respectively. In some cases, "mobile phase A" and "mobile phase B" together are referred to as "mobile phase".

[0051] 1-2-1. Quaternary Salts Quaternary salts are not particularly limited as long as they are compounds having a quaternary nitrogen atom or a quaternary phosphorus atom. In conventional purification methods using reversed-phase HPLC, quaternary salts, which are difficult to remove due to their low volatility, are not used, and triethylammonium, a tertiary salt, is the most commonly used. As mentioned above, the inventors have found that the purification efficiency of cyclic RNA in reversed-phase HPLC is improved by using a mobile phase containing a quaternary salt compared to a tertiary salt.

[0052] From the viewpoint of purification efficiency, ammonium salts, imidazolium salts, pyrrolidinium salts, pyridinium salts, piperidinium salts, and / or phosphonium salts may be used as quaternary salts. In one embodiment, the quaternary salt used in the method for purifying circular RNA of the present invention includes at least one salt selected from the group consisting of ammonium salts, imidazolium salts, pyrrolidinium salts, pyridinium salts, piperidinium salts, and phosphonium salts. In one embodiment, the quaternary salt used in the method for purifying circular RNA of the present invention includes an ammonium salt.

[0053] Quaternary salts are given by general formula (1): XR ４ ＋ A － (1) Preferably the compound is represented by the formula [wherein X represents a nitrogen atom or a phosphorus atom. The four Rs are the same or different, representing a substituted or unsubstituted hydrocarbon group. Alternatively, two or more of the four Rs may be linked together with adjacent nitrogen or phosphorus atoms to form one or more substituted or unsubstituted rings. A represents a counter anion.]

[0054] In general formula (1), X is preferably a nitrogen atom from the viewpoint of purification efficiency. Although not particularly limited, typically X is a nitrogen atom when the quaternary salt is an ammonium salt, imidazolium salt, pyrrolidinium salt, pyridinium salt, or piperidinium salt, and X is a phosphorus atom when the quaternary salt is a phosphonium salt.

[0055] In general formula (1), examples of hydrocarbon groups represented by R include alkyl groups, alkenyl groups, alkynyl groups, cycloalkyl groups, cycloalkenyl groups, cycloalkadienyl groups, aryl groups, aralkyl groups, and the like. From the viewpoint of solubility and purification efficiency, the number of carbon atoms in the hydrocarbon group is preferably 1 to 10, more preferably 2 to 6, and even more preferably 2 to 4. The hydrocarbon group may be linear or branched. The hydrocarbon groups represented by R may be different groups, but it is preferable that they are all the same group.

[0056] In general formula (1), examples of substituents that the hydrocarbon group represented by R may have include hydroxyl groups, halogen atoms (fluorine atoms, chlorine atoms, bromine atoms, iodine atoms, etc.), alkyl groups, aryl groups, heteroaryl groups, alkoxy groups (methoxy groups, ethoxy groups, propoxy groups, etc.), carboxyl groups, amino groups, and the like.

[0057] In general formula (1), examples of rings that R can form include imidazole rings, pyrrolidine rings, pyridine rings, and piperidine rings.

[0058] From the viewpoint of purification efficiency, the quaternary salt preferably has four identical or different hydrocarbon groups having 1 to 10 carbon atoms on a nitrogen atom or phosphorus atom, preferably four hydrocarbon groups having 2 to 6 carbon atoms, and more preferably four hydrocarbon groups having 2 to 4 carbon atoms.

[0059] In general formula (1), the counter anion represented by A is preferably the conjugate base of the Brønsted acid from the viewpoint of purification efficiency. Examples of Brønsted acids include inorganic and organic acids. Examples of inorganic acids include hydrofluoric acid, hydrochloric acid, perchloric acid, sulfuric acid, persulfuric acid, fluorosulfuric acid, chlorosulfuric acid, phosphoric acid, boric acid, nitric acid, and HBF. ４ HB ２ F ７ HBCl ４ HBCl ３ F, HBBr ３ F, HPF ６ HAsF ６ , HSbF ４ , HSbF ６ , HSbCl ６ , HSbCl５ F, HSb ２ F １１ , HAlF ４ , HAlCl ４ , HAlCl ３ F, and HAlF ３ It may include Cl. Examples of organic acids may include carboxylic acids such as acetic acid, trifluoroacetic acid, trichloroacetic acid, and pentafluoropropionic acid, and sulfonic acids such as methanesulfonic acid, trifluoromethanesulfonic acid, trichloromethanesulfonic acid, ethanesulfonic acid, perfluoroethanesulfonic acid, propanesulfonic acid, perfluoropropanesulfonic acid, butanesulfonic acid, perfluorobutanesulfonic acid, benzenesulfonic acid, toluenesulfonic acid, and nitrobenzenesulfonic acid; alkyl sulfuric acids such as methylsulfonic acid; and sulfonylimidic acids such as bis(methanesulfonyl)imide acid and bis(trifluoromethanesulfonyl)imide acid.

[0060] Quaternary salts can be used in the mobile phase either individually or in combination of two or more types.

[0061] In the mobile phase, the concentration of the quaternary salt is preferably 1 mM to 1000 mM, more preferably 5 mM to 500 mM, and even more preferably 10 mM to 100 mM, from the viewpoint of purification efficiency. In one embodiment, the concentration of the quaternary salt contained in the mobile phase used in the purification method of the present invention is 1 mM to 1000 mM. In one embodiment, the concentration of the quaternary salt contained in the mobile phase used in the purification method of the present invention is 5 mM to 500 mM. In one embodiment, the concentration of the quaternary salt contained in the mobile phase used in the purification method of the present invention is 10 mM to 100 mM.

[0062] 1-2-2. The solvent used in the mobile phase is not particularly limited as long as it is a solvent in which the quaternary salt dissolves, but it is preferable that it contains a polar solvent, and preferably water.

[0063] Examples of polar solvents other than water (polar organic solvents) may include nitrile solvents such as acetonitrile; alcohol solvents such as methanol, ethanol, and 1-propanol; and ketone solvents such as acetone. Among these, acetonitrile is preferred as the polar organic solvent from the viewpoint of solubility and purification efficiency of cyclic RNA. The mobile phase may be a solution consisting of a single concentration of polar solvent, or a solution prepared by mixing two polar solvents of different concentrations.

[0064] From the viewpoint of purification efficiency, the content (volume ratio) of the polar organic solvent is preferably 0 to 100 volume%, more preferably 0 to 75 volume%, and even more preferably 0 to 50 volume%, relative to the total amount of solvent. In one embodiment, the solvent used in the purification method of the present invention contains 5 to 90 volume% of the polar organic solvent.

[0065] The solvent may include a low-polarity solvent, as long as it does not impair the effects of the present invention. Examples of non-polar solvents include chloroform, benzene, methylene chloride, and toluene.

[0066] The solvent can be used as the mobile phase either alone or in combination of two or more solvents.

[0067] The mobile phase may or may not contain a denaturing agent such as formamide or urea. According to the mobile phase of the present invention, by using a mobile phase containing a quaternary salt, the purification efficiency of circular RNA can be improved without using conventionally known denaturing agents.

[0068] The pH of the mobile phase is preferably 6.0 to 8.0, more preferably 6.5 to 7.5, and even more preferably 7.0.

[0069] The mobile phase can be prepared, for example, by mixing an aqueous solution of a quaternary salt with a polar organic solvent. The mixing ratio is not particularly limited and can be appropriately determined depending on the type of circular RNA and quaternary salt.

[0070] 1-3. Stationary Phase The stationary phase used in the purification method of the present invention is not particularly limited, as long as it is a reversed phase containing a nonpolar compound.

[0071] The stationary phase is not particularly limited, and a wide range of known reversed phases can be used. In particular, from the viewpoint of purification efficiency, it is preferable to use at least one stationary phase selected from the group consisting of organic silica hybrid substrates and macroporous spherical polystyrene. In this specification, "organic silica hybrid substrate" refers to a material in which organic and inorganic materials are composited at the nanoscale by utilizing weak interactions between organic polymers and silica or organic-inorganic covalent bonds, and is used as a packing material for columns used in HPLC. As an HPLC column containing an organic silica hybrid substrate, any column such as Shimadzu Corporation's Shim-pack Scepter series or YMC's Triart series can be used. In one embodiment, the HPLC column used in the purification method of the present invention is a column selected from the group consisting of YMC-Triart Bio C4, YMC-Triart Bio C18, and Agilent PLRP-S. In one embodiment, the HPCL column used in the purification method of the present invention is a YMC-Triart C4 column.

[0072] The organic groups in the stationary phase are not particularly limited and can be appropriately determined depending on the type of circular RNA and quaternary salt. Specific examples of organic groups in the stationary phase include octadecyl (C18), octyl (C8), butyl (C4), ethyl (C2), and methyl (C1). Among these, octadecyl (C18) and butyl (C4) are preferred as organic groups in the stationary phase.

[0073] The stationary phase is preferably packed into the column in the form of porous beads or blocks.

[0074] From the viewpoint of purification efficiency, the pore size of the stationary phase is preferably 150 Å to 4000 Å, more preferably 300 Å to 4000 Å, and even more preferably 300 Å to 1000 Å.

[0075] From the viewpoint of purification efficiency, the particle size of the stationary phase is preferably 1.9 μm to 100 μm, more preferably 1.9 μm to 50 μm, and even more preferably 5 μm to 50 μm.

[0076] 1-4. Purification Process (Reverse-Phase HPLC) In the purification process, the sample containing circular RNA is subjected to reverse-phase HPLC using a mobile phase containing a quaternary salt. Specifically, it is preferable to subject the sample containing circular RNA to a reverse-phase HPLC column in the presence of a mobile phase containing a quaternary salt. The injection of the sample into the reverse-phase HPLC and the operation of the HPLC instrument can be easily performed by those skilled in the art according to conventional methods.

[0077] In the purification method of the present invention, isocratic separation or gradient separation in reverse-phase HPLC can be used.

[0078] In one embodiment, the purification method of the present invention is gradient separation. In this case, the composition of the mobile phase is determined by a gradient program. The mixing ratio (volume ratio) of polar organic solvents to the total amount of solvent is preferably increased in the gradient program during gradient separation. For example, conditions can be adopted in which the mixing ratio (volume ratio) of each solvent increases by 0 to 100 vol%, 10 to 90 vol%, 20 to 80 vol%, and 10 to 50 vol% during gradient separation.

[0079] In a gradient program, it is preferable that the multiple mobile phases have the same quaternary salt concentration and pH.

[0080] An example of a gradient program is as follows in one embodiment: Mobile phase A: 1 mM to 1000 mM tetrabutylammonium salt (pH = 7.0), 5% to 50% by volume of polar organic solvent Mobile phase B: 1 mM to 1000 mM tetrabutylammonium salt (pH = 7.0), 5% to 90% by volume of polar organic solvent Composition of mobile phase: At the start, mobile phase B is 50% or more by volume, after a predetermined time has elapsed, mobile phase B is 75% or more by volume, and after another predetermined time has elapsed, mobile phase B is 100% by volume

[0081] An example of a gradient program in another embodiment is as follows: Mobile phase A: 1 mM to 1000 mM tetraethylammonium salt (pH = 7.0), 5% to 50% by volume of polar organic solvent Mobile phase B: 1 mM to 1000 mM tetraethylammonium salt (pH = 7.0), 5% to 50% by volume of polar organic solvent Composition of mobile phase: At the start, mobile phase B is 5% by volume or more, after a predetermined time elapsed, mobile phase B is 10% or more, and after another predetermined time elapsed, mobile phase B is 100% by volume

[0082] The flow rate of the mobile phase is not particularly limited and can be appropriately determined depending on the type of circular RNA and quaternary salt. The flow rate can be controlled, for example, by a pump. The flow rate is, for example, 0.3 mL / min to 50 mL / min.

[0083] The column temperature is not particularly limited and can be appropriately determined depending on the type of circular RNA and quaternary salt. In particular, from the viewpoint of purification efficiency, the column temperature is preferably 10°C to 90°C, more preferably 20°C to 80°C, even more preferably 30°C to 70°C, and especially preferably 40°C to 65°C. The column temperature can be controlled, for example, by a heater, an incubator, or a column oven.

[0084] The detection method is not particularly limited as long as circular RNA can be detected reliably and effectively, and can be appropriately determined depending on the type of circular RNA. For example, detection is preferably performed using a UV detector based on absorbance at a wavelength of 250 to 260 nm.

[0085] When a sample subjected to reverse-phase HPLC contains linear RNA, the resolution (Rs) of the circular RNA peak and the linear RNA peak in the chromatogram obtained in the purification step is preferably 0.5 or higher, more preferably 0.8 or higher, even more preferably 1.0 or higher, and particularly preferably 1.5 or higher (complete separation). Here, the resolution (Rs) can be calculated by the following formula.

[0086] t ｌｉｎｅａｒ : Retention time of the peak of linear RNA t ｃｉｒｃｕｌａｒ : Retention time of the peak of circular RNA W １／２ｌｉｎｅａｒ : Peak width at half maximum of linear RNA W１／２ｃｉｒｃｕｌａｒ : Peak width at half maximum of circular RNA

[0087] In the purification method of the present invention, it is preferable to recover a fraction mainly containing circular RNA. It is also preferable to recover a fraction mainly containing impurities. Recovery is preferably carried out using a fraction collector, for example. In this way, a high-purity circular RNA (composition) can be obtained in the purification method of the present invention. It is preferable that the high-purity circular RNA (composition) is substantially free of linear RNA. Here, "substantially free of linear RNA" means that the linear RNA content is typically 10% by mass or less, preferably 5% by mass or less, more preferably 3% by mass or less, even more preferably 1% by mass or less, and particularly preferably 0.1% by mass or less, based on 100% by mass of the total sum of the circular RNA and linear RNA content.

[0088] The recovered circular RNA can be concentrated to further improve its purity. Concentration methods are not limited to those mentioned above and include, for example, alcohol precipitation, centrifugal evaporator, and ultrafiltration.

[0089] When a sample subjected to reverse-phase HPLC contains linear RNA, the amount of circular RNA that can be determined by electrophoresis is preferably 70% or more, more preferably 90% or more, and even more preferably 95% or more, in terms of band intensity ratio (signal intensity ratio) relative to the total amount of circular RNA and linear RNA.

[0090] 2. Method for Producing Circular RNA The present invention also provides a method for producing circular RNA. The method for producing circular RNA of the present invention comprises a cyclization step of obtaining circular RNA by cyclizing linear RNA, and a purification step of subjecting a sample containing the circular RNA to reverse-phase HPLC using a mobile phase containing a quaternary salt. That is, the method for producing circular RNA of the present invention comprises a cyclization step before the purification step. By including the purification step, high-purity circular RNA can be obtained.

[0091] The cyclization step is not particularly limited, and a wide range of known cyclization reactions used for circular RNA synthesis can be employed. Among these, the method of obtaining circular RNA by making 5' phosphorylated RNA circular is preferred. For example, in the cyclization step, circular RNA can be synthesized by synthesizing linear RNA (single-stranded RNA) by a known chemical synthesis reaction and then ligating it with a ligase. Alternatively, circular RNA can be synthesized by synthesizing multiple linear RNAs and then ligating them in an appropriate order with a ligase. During the ligase reaction, the ligase reaction can be carried out efficiently by using DNA probes that hybridize to both ends of the two single-stranded RNAs to be ligated. Alternatively, it may be prepared using a splicing reaction.

[0092] Furthermore, circular RNA can also be synthesized using polymerase-mediated transcription. First, a double-stranded DNA template, with a base sequence complementary to the target circular RNA positioned downstream of the promoter sequence, is used as a template. Polymerase-mediated transcription is then performed in a cell-free system to synthesize linear RNA (single-stranded RNA). The desired circular RNA can then be synthesized by ligating the 5' and 3' ends of the synthesized linear RNA.

[0093] In the method for producing circular RNA of the present invention, the purification step can be the purification step exemplified in "1. Method for purifying circular RNA".

[0094] 3. Circular RNA Composition The circular RNA composition purified or produced by the purification method or production method of the present invention contains circular RNA and quaternary salts, and substantially does not contain linear RNA.

[0095] Here, "substantially free of linear RNA" means that the linear RNA content is typically 10% by mass or less, preferably 5% by mass or less, more preferably 3% by mass or less, even more preferably 1% by mass or less, particularly preferably 0.1% by mass or less, and most preferably below the detection limit (i.e., the linear RNA content itself is below the detection limit).

[0096] In a circular RNA composition, the concentration of circular RNA is preferably 10 nM or higher, more preferably 50 nM to 1 M, and even more preferably 100 nM to 1 mM. The present invention can provide a composition containing a high concentration of circular RNA. The concentration of circular RNA can be determined by absorbance method or calibration curve method.

[0097] In a circular RNA composition, the lower the concentration of linear RNA, the better. While there is no particular lower limit, it is preferably less than 100 nM, more preferably less than 10 nM, and even more preferably less than 1 nM. The concentration of linear RNA can be determined by absorbance or calibration curve.

[0098] In the circular RNA composition, the concentration of the quaternary salt is preferably 1 mM to 1000 mM, more preferably 5 mM to 500 mM, and even more preferably 10 mM to 100 mM. If necessary, it is preferable to remove the quaternary salt from the circular RNA composition. Examples of methods for removing the quaternary salt may include alcohol precipitation, desalting with a reversed-phase column, and ultrafiltration.

[0099] The circular RNA composition may or may not contain the solvent (e.g., water, polar organic solvent, etc.) of the sample or mobile phase used in the purification process. If solvent remains, it can be removed by standard methods.

[0100] Because circular RNA compositions substantially do not contain linear RNA that induces an excessive immune response, they are useful as components in pharmaceutical compositions for use as vaccines when immunizing a subject against various diseases. These compositions typically contain RNA and a pharmaceutically acceptable carrier. The pharmaceutical compositions of the present invention may also contain RNA carriers such as liposomes, oil-in-water emulsions, and microparticles.

[0101] The following examples and comparative examples will be shown to further clarify the features of the present invention, but the present invention is not limited to the following examples.

[0102] To simplify the description, the following abbreviations may be used in the examples.

[0103]

[0104] The following equipment was used in each test.

[0105]

[0106] For the reverse-phase HPLC, the following equipment manufactured by Shimadzu Corporation was used.

[0107]

[0108] In each test, reagents and solvents were commercially available first-grade or special-grade products unless otherwise specified. Sterile water was prepared according to the Operating Instructions for the Thermo Scientific Barnstend Pacific-T II Water Purification System (Thermo SCIENTIFIC). The DNA oligonucleotides (PCR primers, sprint DNA) used in the experiments were purchased from Eurofins Genomics in OPC grade and used without purification unless otherwise specified.

[0109] The following buffer solutions were used in each test.

[0110]

[0111] The following size markers were used in each test.

[0112]

[0113] In each test, LiCl precipitation and alcohol precipitation were performed using the following methods.

[0114] (LiCl precipitation) Half the volume of LiCl aqueous solution (7.5 M) was added to the nucleic acid aqueous solution and mixed well, then allowed to stand at -30°C for 30 minutes. After that, the mixture was centrifuged at the maximum speed of the tube for 30 minutes to form a pellet. The supernatant was removed, 80% ethanol aqueous solution was added, and the mixture was stretched at the maximum speed for 2 minutes. After removing the supernatant, the mixture was air-dried to remove the ethanol. The pellet was redissolved in MQ and used in subsequent experiments.

[0115] (Alcohol Precipitation) To an aqueous solution of nucleic acid, one-tenth the volume of NaOAc (3M, pH=5.2) and an equal volume of 2-propanol were added, and the mixture was allowed to stand at -30°C for 1 hour. Then, the mixture was centrifuged at the maximum speed of the tube for 30 minutes to form a pellet. The supernatant was removed, an 80% ethanol aqueous solution was added, and the mixture was stretched at the maximum speed for 2 minutes. After removing the supernatant, the mixture was air-dried to remove the ethanol. The pellet was redissolved in MQ and used in subsequent experiments.

[0116] [Preparation Example 1: Preparation of Mobile Phase] Preparation Example 1-1: Tetrabutylammonium salt (NBu ４ ＋ A 40% aqueous solution of tetrabutylammonium hydroxide (TCI) was diluted with MQ water, and the pH was adjusted to 7.0 using HCl to prepare a 200 mM tetrabutylammonium hydroxide solution. A mobile phase containing 10 mM tetrabutylammonium salt was prepared using the prepared solution, HPLC-grade acetonitrile (Kanto Chemical), and MQ water. Mobile phase A (hereinafter, "10 mM NBu4+ (pH 7.0), 5% MeCN") contained 5% acetonitrile, and mobile phase B (hereinafter, "10 mM NBu4+ (pH 7.0), 90% MeCN") contained 90% acetonitrile. The prepared mobile phases were subjected to suction filtration using an Advantec VH050P disposable vacuum filter unit, followed by degassing, and then used in HPLC.

[0117] Manufacturing Example 1-2: Tetraethylammonium salt (NET ４ ＋ ) In the same manner as in Production Example 1-1, a 35% aqueous solution of tetraethylammonium hydroxide (TCI) was prepared, and a mobile phase containing 10 mM tetraethylammonium salt was prepared. Mobile phase A (hereinafter, "10 mM NEt4+ (pH 7.0), 5% MeCN") contains 5% acetonitrile, and mobile phase B (hereinafter, "10 mM NEt4+ (pH 7.0), 50% MeCN") contains 50% acetonitrile.

[0118] Manufacturing Examples 1-3: Triethylamine acetate (TEAA; HNET) ３ ＋A 2M acetic acid-2M triethylamine solution with pH 7.0 was prepared using acetic acid (Kanto Chemical) and triethylamine (Kanto Chemical). The prepared solution was diluted to 100 mM with HPLC-grade acetonitrile and MQ water to prepare the mobile phase. Mobile phase A (hereinafter referred to as "100 mM HNEt3") + (pH 7.0), 5% MeCN) contains 5% acetonitrile, and mobile phase B (hereinafter referred to as "100 mM HNEt3") + The mobile phase (pH 7.0, 30% MeCN) contains 30% acetonitrile. The prepared mobile phase was degassed after suction filtration using an Advantec VH050P disposable vacuum filter unit and then used in HPLC.

[0119] Preparation Example 1-4: Hexylammonium Acetate (HAA) A mobile phase containing 50 mM hexylammonium was prepared using hexylammonium (TCI), a primary salt, in the same manner as in Preparation Example 1-3. Mobile phase A (hereinafter, "50 mM HAA (pH 7.0), 5% MeCN") contains 5% acetonitrile, and mobile phase B (hereinafter, "50 mM HAA (pH 7.0), 75% MeCN") contains 75% acetonitrile.

[0120] [Production Example 2: Preparation of Circular mRNA] In each production example, circular RNA having the following sequence was prepared.

[0121]

[0122] Preparation Example 2-1: EmGFP (SEQ ID NO: 1) A circular EmGFP-mRNA (SEQ ID NO: 1) containing the nucleotide sequence encoding GFP was prepared by the following method. The plasmid containing the EmGFP coding sequence was prepared by replacing the Nluc coding sequence of the Nluc vector pNL1.1TK[Nluc / TK]Vector (Promega) with the EmGFP coding sequence by restriction enzyme digestion and Gibson assembly. The sequences of the obtained circular RNA and the various polynucleotides (oligonucleotides) used are shown in Table 7.

[0123]

[0124] (Step 1: Restriction enzyme cleavage of plasmid vector) pNL1.1TK[Nluc / TK]Vector (Promega) was used as the plasmid vector. A reaction solution was prepared (3.6 μg of plasmid vector, 15 U of Hind III (Takara, 15 U / μL), 15 U of XbaI (Takara, 15 U / μL), 1× Mbuffer, and made up to 100 μL with MQ water), and incubated at 37°C for 3 hours to cleave the plasmid vector with restriction enzymes. The reaction solution was subjected to electrophoresis using a 1% agarose gel, and the cleaved plasmid vector was purified using Wizard SV Gel and PCR Clean-Up System (Promega).

[0125] (Step 2: Insert preparation) Polynucleotides encoding EmGFP were prepared by PCR. PCR reaction mixture (1×PCR buffer for KOD plus Neo, MgSO4) ４ A solution was prepared using (1.5 mM), NTPs (0.2 mM), forward primer (0.3 μM), reverse primer (0.3 μM), KOD plus neo (Toyobo; 0.02 U / μL), plasmid DNA (Cloning vector pMSW107; 1 ng / μL), and MQ water (to make up). PCR conditions were 95°C for 2 minutes / (95°C for 15 seconds, 50°C for 15 seconds, 68°C for 30 seconds) × 30 cycles / 12°C. After PCR, the sample was purified using Wizard SV Gel and PCR Clean-Up System (Promega). The absorbance of the nucleic acid aqueous solution at 260 nm was measured to determine the concentration. PCR products were electrophoresed on an agarose gel (1%, 1×TBE, 100V, 30min), stained with EtBr, and confirmed by ChemiDoc.

[0126] (Step 3: Preparation of EmGFP Expression Vector) Using the plasmid vector and insert prepared in Steps 1 and 2, an EmGFP expression vector (hereinafter referred to as "pNL1.1TK-EmGFP vector") was prepared by Gibson assembly according to a standard procedure. Specifically, a reaction solution prepared using 1×fusion mix (Takara) with a molar ratio of vector to insert of 1:2 was used. Jm109 (Nippon Gene, 313-02413) was used as the competent cell. The resulting colonies were confirmed by colony PCR, and after purifying the plasmid, the sequence was confirmed by capillary sequencing (ABI PRISM 3500XL Genetic Analyzer) to prepare the pNL1.1TK-EmGFP vector.

[0127] (Step 4: Preparation of transcription template DNA by PCR) Template DNA for use in the transcription reaction in Step 5 was prepared by PCR. Specifically, the PCR reaction solution was 1×PCR buffer for KOD plus Neo, MgSO ４ A solution was prepared by suspending (1.5 mM), NTPs (0.2 mM), forward primer (0.15 μM), reverse primer (0.15 μM), KOD plus neo (Toyobo; 0.02 U / μL), and plasmid DNA (1 ng / μL) in MQ water. PCR conditions were 95°C for 2 minutes / (95°C for 15 seconds, 50°C for 15 seconds, 68°C for 30 seconds) × 30 cycles / 12°C. After PCR, the sample was purified using Wizard SV Gel and PCR Clean-Up System (Promega). The concentration of the nucleic acid aqueous solution was determined by measuring the absorbance at 260 nm. PCR products were electrophoresed on an agarose gel (1%, 1×TAE, 100V, 30min), stained with EtBr, and confirmed by ChemiDoc.

[0128] (Step 5: In vitro transcription reaction (IVT)) EmGFP mRNA was prepared using the IVT method. Specifically, the transcription reaction mixture consisted of template DNA (2.5 ng / μL), 1×T7 RNA Polymerase buffer (Tris-HCl (40 mM, pH=8.0), MgCl)２ A mixed solution of (8 mM), spermidine (2 mM), T7 RNA Polymerase (2.5 U / μL, house-made), DTT (5 mM), ATP (2 mM), UTP (2 mM), GTP (2 mM), CTP (2 mM), GMP (10 mM), and Pyrophosphate (1 mU / μL, house-made) was prepared and incubated at 37°C for 2 hours. Recombinant DNase I (RNase-free) (Takara) was added to the reaction mixture to a concentration of 0.1 U / μL, and the template DNA was degraded by incubation at 37°C for 15 minutes. Linear EmGFP-mRNA (hereinafter referred to as "linear mRNA") was purified by precipitation with LiCl. Subsequently, the concentration was determined by measuring the absorbance of the nucleic acid aqueous solution at 260 nm. The linear mRNA obtained by the transcription reaction was subjected to electrophoresis using 5% denatured PAGE, stained with SYBR Green II, and confirmed by ChemiDoc.

[0129] (Step 6: HPLC purification) Linear mRNA prepared by the transcription reaction was purified by HPLC. The conditions used were as follows:

[0130]

[0131] (Step 7: Cyclization reaction) A cyclization reaction of mRNA was carried out using the linear mRNA and sprint DNA purified in Step 6. Specifically, the reaction solution consisted of linear mRNA (final concentration 0.5 μM), sprint DNA (1.0 μM), and 1×T4 RNA ligase 2 reaction buffer (Tris-HCl (50 mM, pH=7.5), MgCl). ２A mixed solution of (2 mM), DTT (2 mM), and ATP (400 μM) was used. The mixture was heated in an incubator at 90°C for 3 minutes and then gradually cooled to room temperature. Reagents such as PEG8000 (final concentration 10%) and T4 RNA ligase 2 (final concentration 25 ng / μL, house-made) were added to the reaction solution and incubated at 37°C for 1 hour. The reaction solution was purified by phenol / chloroform extraction and alcohol precipitation. The crude circular mRNA was subjected to electrophoresis using 5% denatured PAGE, stained with SYBR Green II, and confirmed by ChemiDoc. A crude sample containing circular EmGFP-mRNA was obtained by the above method.

[0132] Manufacturing Example 2-2: Nluc (Sequence ID 2) Circular Nluc-mRNA (Sequence ID 2) was prepared using the same method as in steps 4-7 of Manufacturing Example 2-1. pNL1.1TK[Nluc / TK]Vector (Promega) was used as the plasmid containing the coding sequence for Nluc. The sequences of the various polynucleotides (oligonucleotides) used are shown in Table 9.

[0133]

[0134] Manufacturing Example 2-3: GFP-mcherry (Self-Splifying Method; Sequence ID No. 3) Circular GFP-mcherry-mRNA (Sequence ID No. 3) was prepared in the same manner as steps 1-7 of Manufacturing Example 2-1. Specifically, cyclization was performed by self-splicing. The circular RNA obtained by this method is called circular GFP-mcherry (SS). The sequences of the various polynucleotides (oligonucleotides) used are shown in Table 10.

[0135]

[0136] Production Example 2-4: GFP-mcherry (enzyme ligation method; Sequence ID No. 4) A circular mRNA (Sequence ID No. 4) was prepared in the same manner as steps 1-7 of Production Example 2-1, except that cyclization was performed by enzyme ligation. Specifically, cyclization was performed by enzyme ligation. The sequences of the various polynucleotides (oligonucleotides) used are shown in Table 11.

[0137]

[0138] Manufacturing Example 2-5: Capped Branched Nluc (SEQ ID NO: 22) Linear mRNA (SEQ ID NO: 27) was prepared and purified in the same manner as in steps 4-6 of Production Example 2-1, and sprint DNA 1 (SEQ ID NO: 28), sprint DNA 2 (SEQ ID NO: 29), and branched chemosynthetic RNA (SEQ ID NO: 30; 5'm) were prepared. ７ G-ppp-N ｎ Circular mRNA (SEQ ID NO: 22) was produced by cyclization using an enzymatic linkage method (see below for structure). Specifically, the reaction solution consisted of 1.0 μM mRNA, 4.0 μM sprint DNA, 2.0 μM branched chemosynthetic RNA, and 1 × T4 RNA ligase 2 reaction buffer (50 mM Tris-HCl (pH = 7.5), 2 mM MgCl). ２ A reaction solution (2 mM DTT, 400 μM ATP) was prepared. It was heated in an incubator at 90°C for 3 minutes and then gradually cooled to room temperature. Reagents were added to the reaction solution to a final concentration of 10% PEG8000, 25 ng / μL T4 RNA ligase 2 (house made), and incubated at 37°C for 1 hour. The reaction solution was purified by phenol / chloroform extraction and alcohol precipitation. The purified circular mRNA was subjected to electrophoresis using 5% denatured PAGE, stained with SYBR Green II, and confirmed by ChemiDoc. The sequences of the various polynucleotides (oligonucleotides) used are shown in Table 12.

[0139] Furthermore, branched chemosynthetic RNA (SEQ ID NO: 30; 5'm) ７ G-ppp-N ｎ Specifically, as shown below, m ７ It has a structure capped by part G.

[0140]

[0141]

[0142] [Example 1: Quaternary salt (NBu ４ ＋ [Circular RNA Purification Using )] Example 1-1: EmGFP (Purification and Separation by Reverse-Phase HPLC) A crude sample containing circular EmGFP-mRNA (SEQ ID NO: 1; Production Example 2-1) was subjected to reverse-phase HPLC using a mobile phase containing tetrabutylammonium salt (Production Example 1-1) to purify circular EmGFP-RNA from the crude sample. The crude sample is presumed to contain not only circular EmGFP-mRNA, but also linear EmGFP-mRNA, dimerized EmGFP-mRNA or its degradation products (short linear RNA), or polynucleotides in the reaction solution. Reverse-phase HPLC was performed under the conditions shown in Table 13.

[0143]

[0144] Figure 1 shows the chromatogram obtained by reverse-phase HPLC. In Figure 1, the difference in retention time between the peak corresponding to circular EmGFP-mRNA and the peak corresponding to linear EmGFP-mRNA was approximately 8 minutes. In Figure 1, the peak for circular EmGFP-mRNA was sufficiently separated not only from the peaks corresponding to linear EmGFP-mRNA, but also from the peaks corresponding to short linear EmGFP-mRNA and dimerized linear EmGFP-mRNA. This result indicates that we successfully separated circular EmGFP-mRNA from other substances using reverse-phase HPLC with a mobile phase containing quaternary salts. Each peak was isolated and used for the following analysis.

[0145] Table 14 shows the resolution (Rs) of the circular RNA peak and the linear RNA peak in the chromatogram. Here, the resolution (Rs) can be calculated using the following formula.

[0146] t ｌｉｎｅａｒ : Retention time of the peak of linear RNA t ｃｉｒｃｕｌａｒ : Retention time of the peak of circular RNA W １／２ｌｉｎｅａｒ : Peak width at half maximum of linear RNA W １／２ｃｉｒｃｕｌａｒ : Peak width at half maximum of circular RNA

[0147]

[0148] (Analysis of fractions by denatured PAGE) A 5% denatured polyacrylamide gel (acrylamide:bisacrylamide = 19:1) was prepared and electrophoresis was performed under constant current of 26 mA, 60 min, and 1×TBE conditions. Each sample was prepared so that the amount of nucleic acid applied was 50 ng. After mixing with 2× loading buffer, it was heated at 90°C for 3 minutes and immediately cooled rapidly on ice. The gel was stained with SYBR Green II and imaged with ChemiDoc.

[0149] The results of the denatured PAGE are shown in Figure 2. In Figure 2, the fraction collected between 13 and 32 minutes showed the highest purity of circular RNA. In this fraction, no bands other than those corresponding to circular RNA were detected.

[0150] Table 15 shows the fluorescence intensities of the bands corresponding to circular RNA, linear RNA, short linear RNA, and dimerized linear RNA contained in the sample. The purity of circular RNA, as determined by the ratio of circular RNA to the total amount of nucleic acid (signal intensity ratio), was 95.4%. These results indicate that high-purity circular RNA was successfully produced by purification using reverse-phase HPLC with a mobile phase containing quaternary salts.

[0151]

[0152] [Example 2: Quaternary salt (NET) ４ ＋ [Circular mRNA purification using )] Example 2-1: Except that EmGFP quaternary salt was replaced with tetraethylammonium salt (Production Example 1-2) and reverse-phase HPLC was performed under the conditions shown in Table 16, circular RNA was purified, separated, and denatured by PAGE in the same manner as in Example 1-1.

[0153]

[0154] The results of reverse-phase HPLC are shown in Figure 3, and the results of denatured PAGE are shown in Figure 4. Table 17 shows the resolution (Rs) of the circular RNA peaks and linear RNA peaks obtained from the chromatograms.

[0155]

[0156] Table 18 also shows the fluorescence intensities of the bands corresponding to circular RNA, linear RNA, short linear RNA, and dimerized linear RNA contained in the sample. The purity of circular RNA, determined by the ratio of circular RNA to the total amount of nucleic acid (signal intensity ratio), was 93.3%.

[0157]

[0158] Example 2-2: The circular RNA was purified, separated, and denatured by PAGE in the same manner as in Example 2-1, except that circular Nluc-mRNA (SEQ ID NO: 2; Production Example 2-2) was replaced with circular Nluc-mRNA, and reverse-phase HPLC was performed under the conditions shown in Table 19.

[0159]

[0160] The results of reverse-phase HPLC are shown in Figure 5, and the results of denatured PAGE are shown in Figure 6. Table 20 shows the resolution (Rs) of the circular RNA peaks and linear RNA peaks obtained from the chromatograms.

[0161]

[0162] Table 21 also shows the fluorescence intensities of the bands corresponding to circular RNA, linear RNA, short linear RNA, and dimerized linear RNA contained in the sample. The purity of circular RNA, determined by the ratio of circular RNA to the total amount of nucleic acid (signal intensity ratio), was 90.1%.

[0163]

[0164] Example 2-3: GFP-mCherry (self-splicing) circular RNA was purified, separated, and denatured by PAGE in the same manner as in Example 2-1, except that circular GFP-mCherry-mRNA (SEQ ID NO: 3; Production Example 2-3) was used instead of circular RNA, and reverse-phase HPLC was performed under the conditions shown in Table 22.

[0165]

[0166] The results of reverse-phase HPLC are shown in Figure 7, and the results of denatured PAGE are shown in Figure 8. Table 23 shows the resolution (Rs) of the circular RNA peaks and linear RNA peaks obtained from the chromatograms.

[0167]

[0168] Table 24 also shows the fluorescence intensities of the bands corresponding to circular RNA, linear RNA, nicked linear RNA, intron 1, and intron 2 contained in the sample. The purity of circular RNA, determined by the ratio of circular RNA to the total amount of nucleic acid (signal intensity ratio), was 93.7%.

[0169]

[0170] Example 2-4: Purification, separation, and denaturation of circular RNA were performed in the same manner as in Example 2-1, except that circular GFP-mCherry-mRNA (SEQ ID NO: 4; Production Example 2-4) was used instead of circular GFP-mCherry-mRNA, and reverse-phase HPLC was performed under the conditions shown in Table 25.

[0171]

[0172] The results of reverse-phase HPLC are shown in Figure 9, and the results of denatured PAGE are shown in Figure 10. Table 26 shows the resolution (Rs) of the circular RNA peaks and linear RNA peaks obtained from the chromatograms.

[0173]

[0174] Table 27 shows the fluorescence intensities of the bands corresponding to circular RNA, linear RNA, and short linear RNA contained in the sample. The purity of circular RNA, determined by the ratio of circular RNA to the total amount of nucleic acid (signal intensity ratio), was 96.9%.

[0175]

[0176] Example 2-5: Nluc (capped branched) circular RNA was replaced with capped branched circular Nluc-mRNA (SEQ ID NO: 22; Production Example 2-5), and reverse-phase HPLC was performed under the conditions shown in Table 28. The circular RNA was purified, separated, and denatured by PAGE in the same manner as in Example 2-1.

[0177]

[0178] The results of reverse-phase HPLC are shown in Figure 11, and the results of denatured PAGE are shown in Figure 12. Table 29 shows the resolution (Rs) of the circular RNA peaks and linear RNA peaks obtained from the chromatograms.

[0179]

[0180] Table 30 also shows the fluorescence intensities of the bands corresponding to circular RNA, nicked linear RNA, and short linear RNA contained in the sample. The purity of circular RNA, determined by the ratio of circular RNA to the total amount of nucleic acid (signal intensity ratio), was 90.7861%.

[0181]

[0182] [Comparative Example 1: Tertiary Salt (HNET ３ ＋ [Circular mRNA purification using )] Except that the quaternary salt was replaced with the tertiary salt triethylamine acetate (Production Example 1-3) and reverse-phase HPLC was performed under the conditions shown in Table 31, the circular RNA was purified, separated, and denatured by PAGE in the same manner as in Example 2-1.

[0183]

[0184] The results of reverse-phase HPLC are shown in Figure 13, and the results of denatured PAGE are shown in Figure 14. Table 32 shows the resolution (Rs) of the circular RNA peaks and linear RNA peaks obtained from the chromatograms.

[0185]

[0186] In reverse-phase HPLC using a mobile phase containing tertiary salts, a tendency for circular RNA to elute first was observed, but it was not possible to separate the peak corresponding to circular RNA from the linear RNA peak, and therefore high-purity circular RNA could not be produced.

[0187] [Comparative Example 2: Purification of circular mRNA using primary salt (HAA)] Except that the quaternary salt was replaced with the primary salt hexylammonium acetate (Production Example 1-4), and reverse-phase HPLC was performed under the conditions shown in Table 33, the circular RNA was purified, separated, and denatured by PAGE in the same manner as in Comparative Example 1.

[0188]

[0189] The results of reverse-phase HPLC are shown in Figure 15, and the results of denatured PAGE are shown in Figure 16. Table 34 shows the resolution (Rs) of the circular RNA peaks and linear RNA peaks obtained from the chromatograms.

[0190]

[0191] In reverse-phase HPLC using a mobile phase containing primary salts, a tendency for circular RNA to elute first was observed, but it was not possible to separate the peak corresponding to circular RNA from the linear RNA peak, and therefore high-purity circular RNA could not be produced.

[0192] [Example 3: High-speed atomic force microscopy (HS-AFM) measurement in a mobile phase containing a quaternary salt] (Reverse-phase HPLC and denaturation PAGE) The circular RNA was purified, separated, and denatured by PAGE in the same manner as in Example 2-1, except that reverse-phase HPLC was performed on a crude sample (Preparation Example 2-1) containing circular EmGFP-mRNA under the conditions shown in Table 35.

[0193]

[0194] The results of reverse-phase HPLC are shown in Figure 17, and the results of denatured PAGE are shown in Figure 18. For the HS-AFM measurement, the sample solutions used were obtained by separating the fractions shown in the chromatograms Fr. 66-69, Fr. 83-86, and Fr. 98-101, and then concentrating them in a centrifugal evaporator (room temperature, 60 minutes). In each of the concentrated fractions, the nucleic acid concentrations determined from the absorbance at 260 nm using Nanodrop were: Fr. 66-69: 203.5 nM, Fr. 83-86: 223.7 nM, and Fr. 98-101: 29.3 nM.

[0195] (HS-AFM Measurement) HS-AFM imaging was performed in tapping mode using a laboratory-fabricated apparatus. A small microcantilever (Olympus: BL-AC7, length 6 μm-7 μm, width 2 μm, thickness 90 nm) with a spring constant of approximately 0.1 N / m, a Q value of approximately 1, and a resonant frequency of approximately 800 kHz in solution was used. The free vibration amplitude of the cantilever was approximately 2 nm, and the feedback was set to a vibration amplitude of 1.5 nm. A lipid membrane (DPPC:DPTAP = 7:3) was used as the solid substrate. First, a 5 mM magnesium chloride aqueous solution was dropped onto mica immediately after cutting. After incubation for 10 minutes, the substrate was thoroughly washed with Milli-Q water. Next, the sample solution was dropped onto the substrate and incubated for 5 minutes, and then tetraethylammonium salt aqueous solution (10 mM NET) was added as the observation buffer. ４ ＋ The samples were washed (as described in Manufacturing Examples 1-2). Then, the samples were immersed in observation buffer and AFM imaging was performed. For observation, each sample was diluted 100-fold with observation buffer before use.

[0196] The results of HS-AFM measurements are shown in Figure 19. Circular RNA (Figure 19 left), linear RNA (Figure 19 center), and linear ligatures such as dimers of linear RNA (Figure 19 right) all showed unraveling of their higher-order structures and exposure of single-stranded regions in the mobile phase containing quaternary salts. In other words, circular RNA exhibited a random coil structure in the mobile phase containing quaternary salts.

[0197] [Comparative Example 3: High-speed atomic force microscopy (HS-AFM) measurement in a mobile phase containing a tertiary salt] (Reverse-phase HPLC and denaturation PAGE) Except for performing reverse-phase HPLC under the conditions shown in Table 36, circular RNA was purified, separated, and denatured PAGE was performed in the same manner as in Example 3.

[0198]

[0199] The results of reverse-phase HPLC are shown in Figure 20, and the results of denatured PAGE are shown in Figure 18. For the HS-AFM measurement, the sample solutions used were obtained by separating the fractions shown in the chromatograms Fr. 21-23, Fr. 24-26, and Fr. 37-40, and concentrating them in a centrifugal evaporator (room temperature, 60 minutes). In each of the concentrated fractions, the nucleic acid concentrations determined from the absorbance at 260 nm using Nanodrop were: Fr. 21-23: 469.6 nM, Fr. 24-26: 516.8 nM, and Fr. 37-40: 35.0 nM.

[0200] (HS-AFM measurement) Triethylamine acetate aqueous solution (100 mM HNET) was used as the observation buffer. ３ ＋ HS-AFM imaging was performed in the same manner as in Example 3, except that the preparations from Production Examples 1-3) were used. When observing, each sample was diluted 50-fold, 200-fold, or 800-fold with observation buffer before use.

[0201] The results of the HS-AFM measurements are shown in Figure 21. Fr. 21-23 (Figure 21 left), Fr. 24-26 (Figure 21 center), and Fr. 37-40 (Figure 21 right) all showed aggregation in the mobile phase containing the tertiary salt, with the nucleic acid chains adopting higher-order structures, resulting in an overall compact folded structure.

[0202] [Example 4: Effect of Salt Exchange] To confirm the effect of changes in the mobile phase, in the AFM measurement of Fr. 66-69 in Example 3, the tetraethylammonium salt aqueous solution (Production Example 1-2), which was the observation buffer, was replaced with a triethylamine acetate aqueous solution (Production Example 1-3). The results of the AFM measurements in each aqueous solution are shown in Figure 22.

[0203] Similarly, in the AFM measurement of Fr. 21-23 in Comparative Example 3, the observation buffer solution, triethylamine acetate aqueous solution (Production Examples 1-3), was replaced with tetraethylammonium salt aqueous solution (Production Example 1-2). The results of the AFM measurements in each aqueous solution are shown in Figure 23.

[0204] The results shown in Figures 22 and 23 indicate that in the presence of a quaternary salt, nucleic acid molecules were monodisperse, with one strand exposed (i.e., possessing a random coil structure), while in the presence of a tertiary salt, nucleic acid molecules aggregated. These results confirm that nucleic acid molecules can reversibly change their (higher-order) structure through salt exchange.

Claims

1. A method for purifying circular RNA, comprising a purification step of subjecting a sample containing circular RNA to reverse-phase HPLC using a mobile phase containing a quaternary salt.

2. The method according to claim 1, wherein the quaternary salt comprises at least one salt selected from the group consisting of ammonium salts, imidazolium salts, pyrrolidinium salts, pyridinium salts, piperidinium salts, and phosphonium salts.

3. The method according to claim 2, wherein the quaternary salt includes an ammonium salt.

4. The method according to any one of claims 1 to 3, wherein the quaternary salt has four identical or different hydrocarbon groups having 1 to 10 carbon atoms on a nitrogen atom or a phosphorus atom.

5. The method according to any one of claims 1 to 3, wherein the concentration of the quaternary salt in the mobile phase is 1 mM to 1000 mM.

6. The method according to any one of claims 1 to 3, wherein the sample further comprises linear RNA.

7. The method according to claim 6, wherein the sample further comprises the cyclic RNA and the linear RNA, and at least one substance selected from the group consisting of plasmid DNA, deoxyoligonucleotides, deoxynucleoside monophosphates, ribonucleoside triphosphates, and proteins.

8. The method according to claim 6, wherein the resolution (Rs) of the circular RNA peak and the linear RNA peak in the chromatogram obtained in the purification step is 0.5 or higher.

9. The method according to any one of claims 1 to 3, wherein the circular RNA is a single-stranded RNA.

10. The method according to any one of claims 1 to 3, wherein the base length of the circular RNA is 20 to 15,000.

11. The method according to any one of claims 1 to 3, wherein the reversed-phase HPLC uses at least one stationary phase selected from the group consisting of an organic silica hybrid substrate and a macroporous spherical polystyrene.

12. A method for producing circular RNA, comprising: a cyclization step of obtaining circular RNA by cyclizing linear RNA; and a purification step of subjecting a sample containing the circular RNA to reverse-phase HPLC using a mobile phase containing a quaternary salt.

13. A circular RNA composition comprising circular RNA and a quaternary salt, and substantially free of linear RNA.

14. Mobile phase for reverse-phase HPLC for the purification of circular RNA containing quaternary salts.

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