RNA-capping enzyme derived from faustovirus s17

Abstract

The present invention provides a composition that includes (i) an RNA-capping enzyme derived from Faustovirus S17, (ii) uncapped target RNA, (iii) guanosine triphosphate (GTP), (iv) a methyl group donor, and (v) a buffer. The present invention also provides a target RNA capping method that includes a step for incubating a composition that includes (i) an RNA-capping enzyme derived from Faustovirus S17, (ii) uncapped target RNA, (iii) guanosine triphosphate (GTP), (iv) a methyl group donor, and (v) a buffer.

Description

RNA capping enzyme derived from Faustovirus strain S17

[0001] The present invention relates to a composition comprising an RNA capping enzyme derived from the Faustovirus strain S17 (hereinafter sometimes referred to as "FCE(S17)").

[0002] mRNA medicines are attracting attention as a new medical modality. mRNA medicines contain mRNA, which encodes a protein, as an active ingredient. By producing the protein in the body after administration, the medicine exerts medical effects based on the function of that protein. During the global spread of SARS-CoV-2 that occurred in 2020, mRNA vaccines, one type of mRNA medicine, were put into practical use and administered to many people, making a significant contribution to preventing infection. mRNA medicines are superior to other medicines in that vaccines can be designed in a short period of time based on the genetic information of the virus, and different types of vaccines can be mass-produced using the same manufacturing process.

[0003] The 5' end of eukaryotic mRNA contains a modification called a cap. The cap not only contributes to mRNA stabilization, but is also said to enhance translation efficiency by being recognized by translation initiation factors in eukaryotic cells. Therefore, adding a cap to synthetic RNA is highly desirable in mRNA medicine.

[0004] Currently, two methods are used for capping RNA. In the first method (hereinafter referred to as "enzymatic RNA capping"), target RNA is converted into capped RNA using an RNA capping enzyme. In the second method (hereinafter referred to as "co-transcriptional capping"), a cap analog, such as an anti-reverse cap analog (ARCA), is added to the in vitro transcription reaction. In the co-transcriptional capping method, the cap analog is co-transcriptionally incorporated into the RNA molecule during in vitro transcription.

[0005] Compared with the co-transcriptional capping method, the enzymatic RNA capping method provides a higher yield of capped RNA. However, the enzymatic RNA capping method has problems such as the need to use a large amount of enzyme and the need to purify the capped RNA from the reaction solution after capping. Furthermore, there is also the problem that a portion of the RNA is decomposed at the optimum temperature (around 37°C) of conventional RNA capping enzymes.

[0006] Vaccinia virus RNA capping enzyme (hereinafter referred to as "VCE") has been widely used in enzymatic RNA capping methods. However, VCE is a heterodimeric enzyme, which is difficult to produce and has unstable enzymatic activity. To avoid these problems, a "single-stranded RNA capping enzyme" consisting of a single polypeptide chain has recently been reported.

[0007] For example, WO2021 / 041260 (Patent Document 1) discloses single-stranded RNA capping enzymes derived from giant viruses such as Faustovirus, Mimivirus, and Muumuvirus. More specifically, as Faustovirus RNA capping enzymes (hereinafter referred to as "FCEs"), FCEs derived from the Faustovirus D5b strain, the Faustovirus E12 strain, the Faustovirus ST1 strain, and the Faustovirus LC9 strain are disclosed.

[0008] Furthermore, RNA. 2023 Nov;29(11):1803-1817 (Non-Patent Document 1) showed that the RNA capping enzyme derived from the Faustovirus D5b strain exhibits advantageous properties for the production of synthetic mRNA compared to VCE.

[0009] Furthermore, WO 2022 / 164428 (Patent Document 2) discloses an FCE mutant having an amino acid substitution at a specific position in the RNA capping enzyme derived from the Faustovirus D5b strain, in which asparagine (N), an N-linked glycosylation site, is substituted with glutamine (Q), resulting in reduced or no glycosylation.

[0010] International Publication No. WO2021 / 041260 International Publication No. WO2022 / 164428

[0011] RNA. 2023 Nov;29(11):1803-1817

[0012] In order to perform enzymatic RNA capping more efficiently, a single-stranded RNA capping enzyme that has higher enzymatic activity and retains high activity even at low temperatures is required.

[0013] As a result of intensive efforts to solve the above problems, the inventors discovered that the RNA capping enzyme derived from the Faustovirus S17 strain has high enzymatic activity and retains high activity even at low temperatures, thereby completing the present invention.

[0014] That is, the present invention can be summarized as follows: [1] A composition comprising (i) an RNA capping enzyme derived from the Faustovirus strain S17, (ii) an uncapped target RNA, (iii) guanosine triphosphate (GTP), (iv) a methyl group donor, and (v) a buffer; [2] The composition according to [1], which further comprises a nonionic surfactant; [3] The composition according to [2], wherein the nonionic surfactant is a polysorbate; [4] The composition according to any one of [1] to [3], wherein the RNA capping enzyme derived from the Faustovirus strain S17 is an RNA capping enzyme comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 1; [5] The composition according to any one of [1] to [3], wherein the RNA capping enzyme derived from the Faustovirus strain S17 is an RNA capping enzyme comprising an amino acid sequence that is 100% identical to SEQ ID NO: 1; [6] A method for capping a target RNA, comprising a step of incubating a composition comprising (i) an RNA capping enzyme derived from the Faustovirus strain S17, (ii) an uncapped target RNA, (iii) guanosine triphosphate (GTP), (iv) a methyl group donor, and (v) a buffer; [7] The method according to [6], wherein the incubating step is carried out at a temperature of 15°C to 35°C; [8] The method according to [6], wherein the incubating step is carried out at a temperature of 20°C to 30°C; [9] The method according to any one of [6] to [8], wherein the composition further comprises a nonionic surfactant;

[10] The method according to [9], wherein the nonionic surfactant is a polysorbate;

[11] The method according to any one of [6] to

[10] , wherein the RNA capping enzyme derived from the Faustovirus strain S17 is an RNA capping enzyme comprising an amino acid sequence at least 90% identical to SEQ ID NO: 1;

[12] The method according to any one of [6] to

[10] , wherein the RNA capping enzyme derived from the Faustovirus strain S17 is an RNA capping enzyme comprising an amino acid sequence 100% identical to SEQ ID NO: 1.

[0015] The present invention provides compositions suitable for enzymatic RNA capping methods.

[0016] FIG. 1 shows the RNA capping activity ratios of FCE (S17), FCE (NEB), and VCE at each reaction temperature.

[0017] (Definition of Terms) As used herein, the term "RNA capping enzyme" refers to an enzyme that has the activity of adding a cap to RNA. More specifically, the term "RNA capping enzyme" refers to a protein that generates a Cap-0 structure through three enzymatic activities: RNA triphosphatase activity, guanylyltransferase activity, and guanine methyltransferase activity.

[0018] As used herein, the term "single-stranded RNA capping enzyme" refers to an RNA capping enzyme consisting of a single polypeptide chain that has RNA triphosphatase activity, guanylyltransferase activity, and guanine methyltransferase activity. The RNA capping enzymes of Faustovirus, Mimivirus, and Muumuuvirus are examples of single-stranded RNA capping enzymes. On the other hand, VCE is a heterodimer and is not a single-stranded RNA capping enzyme.

[0019] (1) Composition of the Present Invention The composition of the present invention is characterized by comprising (i) an RNA capping enzyme derived from Faustovirus strain S17, (ii) an uncapped target RNA, (iii) guanosine triphosphate (GTP), (iv) a methyl group donor, and (v) a buffering agent.

[0020] Faustovirus is a giant virus that was first isolated in 2015. It is approximately 0.2 micrometers in diameter and contains a 466-kb double-stranded DNA genome. Faustovirus infects amoebas. The amino acid sequence of the RNA capping enzyme (hereinafter sometimes referred to as "FCE(S17)") derived from the Faustovirus S17 strain is disclosed in Gene Bank ID: QJX72631.1 (SEQ ID NO: 1).

[0021] The RNA capping enzyme used in the present invention may be an enzyme derived from the wild-type Faustovirus S17 strain (SEQ ID NO: 1). Furthermore, the RNA capping enzyme used in the present invention may contain an amino acid sequence having identity to the amino acid sequence of SEQ ID NO: 1, so long as it is functionally equivalent. Here, the function refers to the activity of the RNA capping enzyme derived from the Faustovirus S17 strain (the activity of capping RNA). Although not particularly limited, the RNA capping enzyme used in the present invention may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% amino acid sequence identity to SEQ ID NO: 1.

[0022] As long as the RNA capping enzyme used in the present invention is functionally equivalent, it may contain an amino acid sequence in which one or several amino acids have been substituted, deleted, inserted or added in the amino acid sequence of SEQ ID NO: 1. As used herein, "one or several" is not particularly limited, but refers to a range of 1 to 15, preferably a range of 1 to 10, more preferably a range of 1 to 5, and particularly preferably a range of 1 to 3.

[0023] In one embodiment of the present invention, the RNA capping enzyme used in the present invention may have another peptide attached thereto. Examples of such peptides include, but are not limited to, a signal peptide required for the secretory expression of the enzyme and an affinity tag useful for purifying the enzyme. The signal peptide may be selected from known secretory proteins or modified versions thereof depending on the host used for production, and is usually attached to the N-terminus of the RNA capping enzyme to be produced. Furthermore, the affinity tag may be selected from known affinity tags as long as the ligand that can be used for capturing the affinity tag is known. Examples of affinity tags include histidine (His) tags, HN tags, HAT tags, glutathione S-transferase (GST) tags, maltose binding protein (MBP) tags, C-Myc tags, and Strep (II) tags consisting of eight amino acid residues (Trp-Ser-His-Pro-Gln-Phe-Glu-Lys). These tags may be added to either the N-terminus or C-terminus of the RNA capping enzyme to be produced. Furthermore, affinity tags can be removed from the RNA capping enzyme after purification. For this purpose, an affinity tag is used in which a protease recognition sequence, such as that for Factor Xa, PreScission Protease, thrombin, enterokinase, or TEV protease (Tobacco Etch Virus Protease), can be inserted between the RNA capping enzyme and the affinity tag. The RNA capping enzyme used in the present invention is not particularly limited, and an example thereof is FCE(S17) (SEQ ID NO: 3) with an 8x histidine tag added to the C-terminus.

[0024] The RNA capping enzyme used in the present invention can be produced using known recombinant DNA technology. The following description will use FCE (S17) as an example. Known hosts and vectors can be used. For example, bacteria (such as Escherichia coli and Bacillus subtilis), yeast, filamentous fungi, insect cells, or animal cells (such as mammalian cells, including human cells) can be used as hosts, and a vector suitable for each host can be used. The vector carries a nucleic acid encoding FCE (S17). The nucleic acid may be codon-optimized as necessary. FCE (S17) expressed in a host into which a vector carrying the nucleic acid has been introduced, or secreted into the culture supernatant of the host, can be purified to the desired purity using known protein purification methods.

[0025] "Cap" refers to a structure found in mRNA of eukaryotic cells, in which a nucleoside is attached to the 5' end of RNA with three phosphate groups between them. In "capped RNA," the nucleoside is 7-methylguanosine (N7-methylguanosine, sometimes called standard cap), and the ribose moiety is bound to a phosphate group at the 5' position. The structure of RNA with a cap is, for example, m ７ GpppN １ (pN) Ｘ Here, m ７ G represents 7-methylguanosine, ppp represents a triphosphate bridge between the 5' carbon of 7-methylguanosine and the first nucleotide of the RNA produced by transcription, and N １ (pN) Ｘ represents the RNA produced by transcription. １ is the nucleotide located at the 5' end of the RNA produced by transcription. "p" represents a phosphate group, "G" represents a guanosine nucleoside, and "m ７" represents a methyl group at position 7 of guanine. The cap can have a Cap-0 structure, a Cap-1 structure, or a Cap-2 structure (reviewed in Ramanathan, Nucleic Acids Res., 2016, 44:7511-7526), ​​depending on the enzyme used in the capping reaction and / or whether a SAM is present. RNA having any cap structure is referred to herein as a "capped RNA."

[0026] As used herein, "capping" refers to the addition of a cap to the 5' end of RNA. The cap can be added to the 5' end of RNA (e.g., an uncapped RNA transcript) chemically, enzymatically, or co-transcriptionally, independent of transcription, to produce a 5'-capped RNA. In the present invention, the cap is added by an enzymatic reaction independent of transcription.

[0027] As used herein, "uncapped" refers to RNA that does not have a cap structure at the 5' end. Uncapped RNA typically has a triphosphoryl, diphosphoryl, monophosphoryl, or hydroxyl group at the 5' end.

[0028] As used herein, "target RNA" refers to a polyribonucleotide of interest. The polyribonucleotide may be, but is not limited to, a therapeutic RNA or a precursor thereof. The target RNA may result from intracellular or in vitro transcription. The target RNA may be present in a mixture, such as an in vitro transcription reaction mixture, a cell, or a cell lysate. The target RNA may be uncapped.

[0029] In some embodiments, the uncapped target RNA may be, but is not limited to, 100 nucleotides in length or more and may be RNA encoding a polypeptide such as a therapeutic protein or a therapeutic vaccine. Target RNA with secondary structure, including therapeutic RNA, may be more efficiently capped using the methods of the present invention.

[0030] Uncapped target RNA can be produced by solid-phase synthesis or by transcribing a DNA template using an RNA polymerase in an in vitro transcription reaction. In some embodiments, the RNA produced in an in vitro transcription reaction can be purified prior to capping.

[0031] In vitro refers to events that occur not within a multicellular organism but rather in an artificial environment such as a test tube or reaction vessel, cell culture, etc.

[0032] The compositions of the present invention include guanosine triphosphate (GTP). As used herein, the term guanosine triphosphate (GTP) also encompasses modified GTP. Examples of modified GTP include those having a methyl group at the N7 position of the guanine ring, those having a label attached to the 2nd or 3rd position of the ribose, and the like. In some embodiments, the label attached to the modified GTP may be an oligonucleotide, a detection label such as a fluorophore, or a capture moiety such as biotin or desthiobiotin, in which case the label may optionally be linked to the ribose of the nucleotide, for example, by a linker. See, for example, WO2015 / 085142.

[0033] The concentration of guanosine triphosphate (GTP) can be set with reference to the composition of a known reaction solution. The concentration of guanosine triphosphate (GTP) contained in the composition of the present invention is not particularly limited, but is, for example, 0.01 mM to 10 mM, preferably 0.05 mM to 5 mM, and more preferably 0.1 mM to 1 mM.

[0034] The composition of the present invention includes a methyl group donor, such as S-adenosylmethionine (hereinafter referred to as "SAM").

[0035] The concentration of the methyl group donor can be set with reference to the composition of a known reaction solution. The concentration of the methyl group donor contained in the composition of the present invention is not particularly limited, but in the case of SAM, it is exemplified as 0.001 mM to 10 mM, preferably 0.005 mM to 5 mM, and more preferably 0.01 mM to 0.5 mM.

[0036] As used herein, the term "buffer" refers to an agent that suppresses changes in the pH of a solution when an acid or alkali is added to the solution. Examples of buffers contained in the composition of the present invention include Tris, HEPES, TAPS, MOPS, tricine, and MES. Tris is a preferred example. The pH of the composition of the present invention is usually adjusted to a range of 7.0 to 9.0.

[0037] The composition of the present invention may further contain various components such as salts (NaCl, KCl, etc.), metal ions (magnesium ions, etc.), reducing agents (dithiothreitol, etc.), surfactants, RNase inhibitors, etc. The concentrations of these components can be set with reference to the compositions of known reaction solutions.

[0038] Examples of surfactants that may be contained in the composition of the present invention include nonionic surfactants such as polysorbates (polyoxyethylene sorbitan fatty acid esters), Triton X-100 (Polyoxyethylene(10) octylphenyl ether), Poloxamer 188, and Nonidet P-40 (Octylphenyl-polyethylene glycol), anionic surfactants such as poly(ethylene glycol) 4-nonylphenyl 3-sulfopropyl ether (PNSE), cationic surfactants such as distearyldimethylammonium chloride, and amphoteric surfactants such as cocamidopropyl betaine. Polysorbates include polysorbate 20, polysorbate 21, polysorbate 40, polysorbate 60, polysorbate 61, polysorbate 65, polysorbate 80, polysorbate 81, polysorbate 85, etc. The surfactant contained in the composition of the present invention is not particularly limited, but preferred examples include nonionic surfactants, more preferred examples include polysorbates, and even more preferred examples include polysorbate 20. Polysorbate 20 is also called "Tween 20" or "Polyoxyethylene Sorbitan Monolaurate."

[0039] The concentration of the surfactant contained in the composition of the present invention is not particularly limited, but examples thereof include 0.001% by volume to 1% by volume, preferably 0.005% by volume to 0.5% by volume, more preferably 0.01% by volume to 0.1% by volume, and even more preferably 0.02% by volume.

[0040] More efficient capping of RNA substrates means adding less enzyme to the capping composition to perform the capping, using the same amount of enzyme to produce more capped RNA (as a percentage of RNA in the reaction), terminating the reaction earlier and / or more efficiently capping RNA with secondary structure at the 5' end.

[0041] The present invention provides a kit for capping uncapped target RNA. The kit of the present invention is characterized by comprising an RNA capping enzyme derived from the Faustovirus S17 strain, guanosine triphosphate (GTP), a methyl group donor, and a buffer. The capping enzyme, GTP, methyl group donor, and buffer described above for the composition of the present invention can each be used. These components may be individually packaged so that they can be mixed at the time of use to prepare a reaction solution, or multiple components may be packaged as a mixture to facilitate the preparation of a reaction solution. Examples of the kit of the present invention include a kit containing a premix solution that can be completed by simply adding uncapped target RNA and water (e.g., sterile water) at the time of use, and a kit containing the premix in a dry state. Other components that can be included in the kit include salts (e.g., NaCl, KCl), metal ions (e.g., magnesium ions), reducing agents (e.g., dithiothreitol), surfactants, RNase inhibitors, etc.

[0042] The composition of the present invention can be used to cap target RNA. The composition of the present invention can be used, for example, to cap target RNA at low temperatures. As used herein, "low temperature" refers to a temperature lower than the optimum temperature (around 37°C) of conventional RNA capping enzymes, and includes, but is not limited to, temperatures ranging from 10°C to less than 35°C, temperatures ranging from 10°C to 30°C, or temperatures ranging from 15°C to 25°C.

[0043] (2) The method for capping a target RNA of the present invention The method for capping a target RNA of the present invention is characterized by comprising a step of incubating a composition comprising (i) an RNA capping enzyme derived from the Faustovirus S17 strain, (ii) an uncapped target RNA, (iii) guanosine triphosphate (GTP), (iv) a methyl group donor, and (v) a buffer.

[0044] The method for capping a target RNA of the present invention can also be achieved by incubating the composition of the present invention.

[0045] The incubating step is carried out at a temperature and for a time sufficient to form capped target RNA.

[0046] The temperature of the incubation step can range from 10°C to 99°C. However, there was a problem that the optimum temperature (around 37°C) of conventional RNA capping enzymes resulted in partial decomposition of the RNA. FCE(S17) has the characteristic of retaining significant capping activity even at low temperatures. As used herein, "low temperature" refers to a temperature lower than the optimum temperature (around 37°C) of conventional RNA capping enzymes, and includes, but is not limited to, temperatures in the range of 10°C or higher and lower than 35°C, temperatures in the range of 10°C or higher and 30°C or lower, or temperatures in the range of 15°C or higher and 25°C or lower. Therefore, the temperature of the incubation step is not particularly limited, and examples include, for example, 10°C to 55°C, 15°C to 55°C, 10°C to 35°C, 15°C to 35°C, 20°C to 30°C, or 15°C to 25°C. The temperature of the incubation step may be constant or may vary.

[0047] The incubation time may range from a few seconds to a few hours, and is not particularly limited, but examples include 1 minute to 24 hours, 3 minutes to 12 hours, and 5 minutes to 5 hours.

[0048] The target RNA capping method of the present invention can further include monitoring the appearance of capped target RNA. For example, capped RNA can be monitored / detected by denaturing urea polyacrylamide gel electrophoresis, radiometric assay, capillary electrophoresis, or mass spectrometry-based methods.

[0049] During the capping reaction, target RNA is methylated using a methyl group donor as a starting material. When SAM is used as the methyl group donor, S-adenosylhomocysteine ​​(SAH) is generated as the product. The capping activity of the enzyme can be calculated by measuring and analyzing the amount of SAH produced using a commercially available methyltransferase activity assay kit.

[0050] The methods of the present invention produce capped target RNA.

[0051] The capped target RNA can be used for various purposes either directly or in a processed form, for example, encapsulated in lipid nanoparticles, etc., and may be formulated into a form suitable for delivery to a subject (e.g., a human). Formulations may include liquid formulations (solutions, suspensions, dispersions), topical formulations (gels, ointments, drops, creams), liposomal formulations, etc.

[0052] The formulation may include pharmaceutically acceptable additives, binders, buffers, coatings, colorants, release-controlling agents, delivery agents, diluents, disintegrants, dyes, excipients, fillers, lipids, lubricants, salts, and adsorbents.

[0053] The capped target RNA can be delivered to eukaryotes. The capped target RNA can be delivered to animals or animal cells. Examples of animals include humans and non-human animals (e.g., mice, rats, rabbits, dogs, cats, cows, pigs, sheep, horses, or primates). The capped target RNA can also be delivered to plants or plant cells to confer or enhance resistance or tolerance to environmental conditions (e.g., drought, salinity), and / or to prevent, alleviate, or treat herbivory, pathogen infection, or their effects. Furthermore, the capped target RNA can also be delivered to yeast cells.

[0054] The capped target RNA, as well as formulations containing the RNA, can be administered to a subject by any suitable route of administration, including intracellular (e.g., muscle or organ), intravenous, subcutaneous, transdermal, oral, enteral, parenteral, ocular, otic, transmucosal, sublingual, and pulmonary routes (e.g., by nebulization and / or inhalation), and combinations thereof.

[0055] The capped target RNA can be introduced into cells in vitro or ex vivo, more specifically, the cells can be part of a mammal, part of tissue removed from a mammal, or isolated from the blood of a mammal.

[0056] The present invention will be described below with reference to examples, but the present invention is not particularly limited to these examples.

[0057] Example 1 Preparation of FCE (S17) The amino acid sequence of the RNA capping enzyme derived from the fast virus strain S17 (hereinafter referred to as "FCE (S17)") is disclosed in Gene Bank ID: QJX72631.1 (SEQ ID NO: 1). Using this amino acid sequence, a gene encoding FCE (S17) tagged with 8X histidine at the C-terminus was chemically synthesized (SEQ ID NO: 2). The resulting synthetic gene was introduced into plasmid pCold IV (Takara Bio) in which the ampicillin resistance gene had been replaced with a kanamycin resistance gene, using In-Fusion (registered trademark) HD ​​Cloning Kit (Takara Bio). The amino acid sequence of FCE (S17) tagged with 8X histidine at the C-terminus is shown in SEQ ID NO: 3.

[0058] Next, Escherichia coli strain BL21 (Takara Bio Inc.) was transformed with the plasmid and statically cultured overnight at 37°C on a 1.5% agarose LB plate containing 50 μg / mL kanamycin. A single colony was selected from this plate, spread onto a 1.5% agarose LB plate containing 50 μg / mL kanamycin, and streaked at 37°C for 16 to 24 hours. The grown E. coli was inoculated into LB medium containing 50 μg / mL kanamycin (hereinafter referred to as "LB-Km medium") and cultured with shaking at 37°C until the OD660 value reached 0.45. A portion of the culture medium was taken from the culture with shaking, and 60% glycerol was added and mixed to a final glycerol concentration of 15%, to prepare a glycerol stock (hereinafter referred to as "RCB"). 1 mL of RCB was inoculated into 100 mL of LB-Km medium for pre-preculture and cultured overnight with shaking at 37°C. 2 mL of this culture was inoculated into 200 mL of LB-Km medium for preculture and cultured with shaking at 37°C. When the OD600 value reached 0.5, the culture was inoculated into 2000 mL of LB-Km medium for main culture and cultured with shaking at 37°C. When the OD600 value reached 1.5, IPTG was added to the culture to a final concentration of 1 mM, and induction culture was further carried out at 20°C for 18 to 22 hours, after which the bacterial cells were collected.

[0059] The obtained bacterial cells were suspended in a solution containing 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 0.1% Tween 20, 1 mM PMSF, and 5% glycerol in an amount 5 times the bacterial weight. The bacterial cells in the suspension were then disrupted using a high-pressure homogenizer. To remove debris, the suspension was clarified using an MF membrane (0.05 μm, manufactured by Repligen). The clarified solution was then filtered through a sterile filtration filter (0.2 μm, manufactured by Sartorius) to obtain a filtrate. A 1 / 24 volume of a solution containing 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 0.1% Tween 20, 500 mM imidazole, and 5% glycerol was added to the filtrate, mixed, and then filtered through a bottle-top filter (0.22 μm, manufactured by Corning). The filtrate was applied to a Nuvia IMAC Resin (BIO RAD) column to adsorb the capping enzyme. After washing the column, the adsorbed material was eluted with a buffer containing imidazole. To the resulting eluate, a two-fold volume of a solution containing 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 0.1% Tween 20, and 5% glycerol was added and mixed, and then filtered through a bottle-top filter (0.22 μm, Corning). The filtrate was applied to a Nuvia IMAC Resin (BIO RAD) column to adsorb the capping enzyme. After washing the column, the adsorbed material was eluted with a buffer containing imidazole. To the resulting eluate, a 9-fold volume of a solution containing 50 mM potassium phosphate buffer (pH 6.5), 0.1 mM EDTA, 1 mM DTT, and 5% glycerol was added and mixed, followed by filtration through a bottle-top filter (0.22 μm, Corning). The filtrate was applied to a Cellufine MAX CM (JNC) column to adsorb the capping enzyme. After washing the column, the adsorbed material was eluted with a buffer containing sodium chloride. The resulting eluate was used as FCE (S17) in the following tests.

[0060] SEQ ID NO: 1: MSRRLQRCRDVDQVCEYYNAKGAIGELELRFDKLTPDLFAHVFDKLKPDGEISTTMRVSNSDGTAREITFGGGVKTGETFVRKQNICVFDVVDIFSYKVAVSTEETLVDKPAMEKDASVRFKIRMSVEGAVPNWRIDLTAVKTAELGKIAQHTASLVLQTFPPNLLKMSGAEVAKLANNSYELELEYIGKTPATKERVDAAAKYAVDLLAGIKNANSAVGAV LGESISDICRVAKVIHTPDYATVVCRNPSFKMLLPQVISLTKSSYYGGIYPPEGMYVAGKTDGVRALVLCEDGVAKVITAESVDITTGTCAGTTILDCELSTGKSGATLHV FDIIMHNSKPIHTQPFSTRIATDISDVKIPEYKIAIKPFVKIQATALEAAFKEVYKAPHNEGLILIMDGNDYAMTKTYKWKPLSHNTIDFLIKACPKQLLNIDPYKPRPGH KLWLLFTTISLDQQRELGIEFIPAWKMLFTDINLFGSRVPIQFMPAINPLAYICYLPEAATCATGDAINDGDIVEMRAVDGFDTVPKWEPIRVRSDRKDEPGFYGNNYKIA SDIYLNYIDIFQFEDLYKYNPGYFEKNKSDIYVAPNKYRRFLIKNIFSKYLKNAKWVIDAAAGRGADLHLYKAECVENLLAIDIDPTAISELVRRRNEITGYNRGHRGHRG GSMRAHMGASHHGAQNCAKSTTLHALVADLRTDPDVLIPKIIQSRPPERGYDAIVINFAIHYLCDTDEHIRDFLITVSRLLAPNGIFMFTTMDGESIVKLLETHKVKSGES WTVHTGADDPEAGVVKYSIRRLYDSDKLTKTGQQIAVLLPMSGEMKTEPLCNIKNIISIARKMGLDLVESADFSVMYEAFARAYPEISARLTPDDKLYNDLHSYAVFKRKK

[0061]

[0062] SEQ ID NO: 3: MSRRLQRCRDVDQVCEYYNAKGAIGELELRFDKLTPDLFAHVFDKLKPDGEISTTMRVSNSDGTAREITFGGGVKTGETFVRKQNICVFDVVDIFSYKVAVSTEETLVDKPAMEKDASVRFKIRMSVEGAVPNWRIDLTAVKTAELGKIAQHTASLVLQTFPPNLLKMSGAEVAKLANNSYELELEYIGKTPATKERVDAAAKYAVDLLAGIKNANSAVGAVLG ESISDICRVAKVIHTPDYATVVCRNPSFKMLLPQVISLTKSSYYGGIYPPEGMYVAGKTDGVRALVLCEDGVAKVITAESVDITTGTCAGTTILDCELSTGKSGATLHVFDI IMHNSKPIHTQPFSTRIATDISDVKIPEYKIAIKPFVKIQATALEAAFKEVYKAPHNEGLILIMDGNDYAMTKTYKWKPLSHNTIDFLIKACPKQLLNIDPYKPRPGHKLWL LFTTISLDQQRELGIEFIPAWKMLFTDINLGFGSRVPIQFMPAINPLAYICYLPEAATCATGDAINDGDIVEMRAVDGFDTVPKWEPIRVRSDRKDEPGFYGNNYKIASDIYL NYIDIFQFEDLYKYNPGYFEKNKSDIYVAPNKYRRFLIKNIFSKYLKNAKWVIDAAAGRGADLHLYKAECVENLLAIDIDPTAISELVRRRNEITGYNRGHRGHRGGSMRAH MGASHHGAQNCAKSTTLHALVADLRTDPDVLIPKIIQSRPPERGYDAIVINFAIHYLCDTDEHIRDFLITVSRLLAPNGIFMFTTMDGESIVKLLETHKVKSGESWTVHTGA DDPEAGVVKYSIRRLYDSDKLTKTGQQIAVLLPMSGEMKTEPLCNIKNIISIARKMGLDLVESADFSVMYEAFARAYPEISARLTPDDKLYNDLHSYAVFKRKKHHHHHHHH

[0063] Example 2 Effect of Capping Buffer Composition on FCE Enzyme Activity 2 μL of an 80-fold diluted solution of FCE (S17) obtained in Example 1 or 3.1 U of commercially available FCE RNA capping enzyme (New England Biolabs, model number: M2081L) was mixed with 1 μg of 100-base RNA, 0.1 mM GTP, and 0.02 mM SAM in 20 μL of RNA capping buffer (composition shown in Table 1), and the mixture was incubated at 37° C. for 30 minutes. The commercially available FCE [FCE (NEB)] was used based on the activity listed on the product label.

[0064]

[0065] The nucleotide sequence of 100 base RNA is shown in SEQ ID NO: 4. During the capping reaction, 100 base RNA is methylated using SAM as a starting material, producing SAH as the product. The amount of SAH produced was measured and analyzed using a methyltransferase activity measurement kit, MTase-Glo Methyltransferase Assay (Promega, model number: V7601), to calculate the capping activity of each enzyme. Furthermore, the enzyme activity ratio was calculated, assuming that the activity of each enzyme in Buffer No. 1 was 1. The results are shown in Table 2.

[0066]

[0067] Both FCE(S17) and FCE(NEB) showed 4.5-fold higher activity in the presence of Tween 20 (Buffers 3 and 4) than in the absence of Tween 20 (Buffer 1). This activity also exceeded the activity in the presence of Poloxamer 188 (Buffer 2). This result indicates that the addition of Tween 20 to the capping buffer particularly enhances the capping activity of FCE.

[0068] SEQ ID NO: 4: GGGAACCTGTGGCATTTGTGCTGCCGGGAACGGCGTTTCGTGTCTCTGCCGGTGTGGCAGCCGAAATGACAGAGCGCGGCCTGGCCAGAATGCAAAAGCT

[0069] Example 3 Enzyme activity of FCE (S17) over a wide temperature range Ten microliters of a 400-fold diluted solution of FCE (S17) obtained in Example 1, 3.1 U equivalent of FCE (NEB) used in Example 2, or 2.5 U equivalent of a commercially available RNA capping enzyme derived from vaccinia virus (VCE; Takara Bio Inc., model number: 2460A) was dissolved in 20 μL of RNA capping buffer (50 mM Tris-HCl, 5 mM KCl, 1 mM MgCl ２ After mixing with 1 μg of 100 base RNA, 0.1 mM GTP, and 0.02 mM SAM in a buffer containing 1 mM DTT and 0.02% Tween 20, the mixture was incubated at temperatures ranging from 15°C to 55°C for 30 minutes. FCE (NEB) and VCE were used based on the activity listed in the product specifications.

[0070] The nucleotide sequence of the 100 base RNA is shown in SEQ ID NO: 4. The capping activity of the enzyme was calculated using the method described in Example 2. Furthermore, the activity ratio of each enzyme at 15°C to 55°C was calculated, assuming that the activity at 35°C was 100%. The results are shown in Figure 1.

[0071] While FCE(NEB) and VCE showed the highest capping activity at 35°C, FCE(S17) showed capping activity at 25°C equal to or greater than that at 35°C. Furthermore, FCE(S17) maintained higher capping activity than FCE(NEB) and VCE in the low temperature range (15°C to 25°C).

[0072] The present invention provides compositions suitable for enzymatic RNA capping methods.

[0073] SEQ ID NO:1: Faustovirus 17S RNA capping enzyme Genbank accession No. QJX72631.1 SEQ ID NO:2: Synthetic gene encoding Faustovirus 17S RNA capping enzyme with 8X histidine tag SEQ ID NO:3: Faustovirus 17S RNA capping enzyme with 8X histidine tag SEQ ID NO:4: 100 base RNA

Claims

1. A composition comprising: (i) an RNA capping enzyme derived from Faustovirus strain S17; (ii) an uncapped target RNA; (iii) guanosine triphosphate (GTP); (iv) a methyl group donor; and (v) a buffer.

2. The composition of claim 1, further comprising a nonionic surfactant.

3. The composition according to claim 2, wherein the nonionic surfactant is a polysorbate.

4. The composition according to any one of claims 1 to 3, wherein the RNA capping enzyme derived from Faustovirus strain S17 is an RNA capping enzyme comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:

1.

5. A composition described in any one of claims 1 to 3, wherein the RNA capping enzyme derived from Faustovirus strain S17 is an RNA capping enzyme comprising an amino acid sequence 100% identical to SEQ ID NO:

1.

6. A method for capping a target RNA, comprising the step of incubating a composition comprising: (i) an RNA capping enzyme derived from Faustovirus strain S17; (ii) uncapped target RNA; (iii) guanosine triphosphate (GTP); (iv) a methyl group donor; and (v) a buffer.

7. The method of claim 6, wherein the incubating step is carried out at a temperature between 15°C and 35°C.

8. The method of claim 6, wherein the incubating step is carried out at a temperature of 20°C to 30°C.

9. The method of any one of claims 6 to 8, wherein the composition further comprises a nonionic surfactant.

10. The method of claim 9, wherein the nonionic surfactant is a polysorbate.

11. The method according to any one of claims 6 to 10, wherein the RNA capping enzyme derived from Faustovirus strain S17 is an RNA capping enzyme comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:

1.

12. The method according to any one of claims 6 to 10, wherein the RNA capping enzyme derived from Faustovirus strain S17 is an RNA capping enzyme comprising an amino acid sequence 100% identical to SEQ ID NO: 1.

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