Methyltransferase for methylating hydroxyl group of ribose moiety in third nucleotide from 5'-end of RNA or hydroxyl groups of ribose moieties respectively in third and fourth nucleotides from 5'-end of RNA, methylation method, use of methyltransferase, kit, and nucleic acid therapeutics

Abstract

Provided is a methyltransferase for methylating a hydroxyl group of a ribose moiety in the third nucleotide from the 5'-end of RNA or hydroxyl groups of ribose moieties respectively in the third and fourth nucleotides from the 5'-end of RNA.

Description

Methyltransferase for methylating the hydroxyl group of ribose at the third or third and fourth nucleotides from the 5' end of RNA, methylation method, use of methyltransferase, kit, and nucleic acid medicine

[0001] The present invention relates to a methyltransferase for methylating the hydroxyl group of ribose in the third nucleotide or the third and fourth nucleotides from the 5' end of RNA, a methylation method, use of the methyltransferase, a kit, and a nucleic acid medicine.

[0002] 7-methylguanylic acid (m) is attached to the 5' end of RNA such as mRNA (messenger RNA). 7 It is known that the 5' cap structure to which 7-methylguanylic acid (G) is attached contributes to functions such as translation activity and stability. It has been reported that such RNA functions can be improved by methylating the ribose hydroxyl group (2'-hydroxyl group) of the nucleotide that is linked to 7-methylguanylic acid via triphosphate, particularly the ribose hydroxyl groups of the first and second nucleotides from the 5' end (Non-Patent Document 1).

[0003] On the other hand, although methylation of the ribose hydroxyl group in the third and fourth nucleotides from the 5' end of RNA is known, it is still in the research stage and there is little knowledge about its effects (Non-Patent Document 2). Therefore, if methylation of the ribose hydroxyl group in the third and fourth nucleotides from the 5' end of RNA could be facilitated, it could potentially advance the development of nucleic acid medicines such as mRNA medicines. A structure in which the hydroxyl group of ribose in the first nucleotide from the 5' end of RNA is not methylated is called a Cap0-type structure, a structure in which the hydroxyl group (2'-hydroxyl group) of ribose in the first nucleotide from the 5' end of RNA is methylated is called a Cap1-type structure, a structure in which the hydroxyl groups of ribose in the first and second nucleotides from the 5' end of RNA are methylated is called a Cap2-type structure, a structure in which the hydroxyl groups of ribose in the first to third nucleotides from the 5' end of RNA are methylated is called a Cap3-type structure, and a structure in which the hydroxyl groups of ribose in the first to fourth nucleotides from the 5' end of RNA are methylated is called a Cap4-type structure.

[0004] Magdalena Byszewska and 3 others, "RNA methyltransferases involved in 5' cap biosynthesis", RNA Biology, vol. 11(12), pp. 1597-1607, 2014 DecemberJesse R. Zamudio and 6 others, "Complete Cap 4 Formation Is Not Required for Viability in Trypanosoma brucei", Eukaryotic Cell, vol. 5(6), pp. 905-915, June 2005

[0005] The present invention has been made to solve the above-mentioned problems, and aims to provide a methyltransferase capable of methylating the hydroxyl group of ribose in the third nucleotide or the third and fourth nucleotides from the 5' end of RNA, a methylation method, use of the methyltransferase, and a kit. Another aim of the present invention is to provide a nucleic acid medicine having RNA with excellent translation activity.

[0006] The present inventors have conducted extensive research to solve the above-mentioned problems, and as a result have discovered a methyltransferase that can methylate the hydroxyl group of ribose at the third nucleotide or the third and fourth nucleotides from the 5' end of RNA, thereby completing the present invention. That is, the present invention is exemplified as follows.

[0007] <1> A methyltransferase for methylating the hydroxyl group of ribose in the third nucleotide or the third and fourth nucleotides from the 5' end of RNA.

[0008] <2> The methyltransferase according to <1>, which is (a), (b), or (c) below: (a) a protein consisting of the amino acid sequence of SEQ ID NO: 1; (b) a protein consisting of the amino acid sequence of SEQ ID NO: 1 in which one or several amino acids have been deleted, substituted, inserted, and / or added; or (c) a protein consisting of an amino acid sequence that has a homology of 80% or more to the protein consisting of the amino acid sequence of (a).

[0009] <3> A method for methylating the hydroxyl group of ribose at the third nucleotide or the third and fourth nucleotides from the 5' end of RNA, the method comprising carrying out a methylation reaction of RNA in the presence of a methyl group donor and the methyltransferase according to <1> or <2>.

[0010] <4> The method according to <3>, wherein the RNA used for the methylation is at least one selected from Cap0-type RNA, Cap1-type RNA, Cap2-type RNA, RNA having a cap structure at the 5'-end and only the hydroxyl group of the ribose at the second nucleotide from the 5'-end being methylated, Cap3-type RNA, RNA having a cap structure at the 5'-end and only the hydroxyl group of the ribose at the third nucleotide from the 5'-end being methylated, RNA having a cap structure at the 5'-end and only the hydroxyl groups of the ribose at the second and third nucleotides from the 5'-end being methylated, and RNA having a cap structure at the 5'-end and only the hydroxyl groups of the ribose at the first and third nucleotides from the 5'-end being methylated.

[0011] <5> The method according to <3>, wherein a nucleoside triphosphate is further present in the methylation reaction, and the 5'-end of the RNA is simultaneously capped.

[0012] <6> Use of the methyltransferase according to <1> or <2> for methylating the hydroxyl group of ribose in the third nucleotide or the third and fourth nucleotides from the 5' end of RNA.

[0013] <7> A kit used for methylating the hydroxyl group of ribose in the third nucleotide or the third and fourth nucleotides from the 5' end of RNA, the kit comprising the methyltransferase according to <1> or <2>.

[0014] <8> The kit according to <7>, further comprising a methyl group donor.

[0015] <9> A nucleic acid drug comprising a Cap3-type RNA having a 5'-cap structure and in which the hydroxyl groups of the ribose in the first to third nucleotides from the 5'-end are methylated.

[0016] <10> A nucleic acid drug comprising a Cap4-type RNA having a 5'-cap structure and in which the hydroxyl groups of the ribose in the first to fourth nucleotides from the 5'-end are methylated.

[0017] The present invention provides a methyltransferase capable of methylating the hydroxyl group of ribose at the third nucleotide or the third and fourth nucleotides from the 5' end of RNA, a methylation method, use of the methyltransferase, and a kit. The present invention also provides a nucleic acid medicine having RNA with excellent translation activity.

[0018] 1 shows the results of LC-MS analysis (quantitative analysis) when 5'pppGAAA was used as RNA in an example. 2 shows the results of LC-MS analysis (quantitative analysis) when 5'pppGUUU was used as RNA in an example. 3 shows the results of LC-MS analysis (qualitative analysis) when 5'pppGAAA was used as RNA in an example. 4 shows the results of LC-MS analysis (qualitative analysis) when 5'pppGUUU was used as RNA in an example. 5'pppGUUU was used as RNA in an example. 7 1 shows the results of LC-MS analysis (qualitative analysis) when GpppGAAAA was used. 7 1 shows the results of sequence analysis by Confirm Sequence in LC-MS / MS analysis when GpppGAAAA was used. 2 shows the base sequence of Cap0-type mRNA used in the examples. 3 shows graphs illustrating the results of evaluation of translation activity in the examples.

[0019] The following is a detailed description of the embodiments of the present invention. The present invention is not limited to the following embodiments, and it should be understood that modifications and improvements made to the following embodiments based on the ordinary knowledge of those skilled in the art without departing from the spirit of the present invention are also within the scope of the present invention.

[0020] (1. Methyltransferase) The methyltransferase according to an embodiment of the present invention is an enzyme used to methylate the hydroxyl group of ribose at the third nucleotide from the 5' end of RNA, or at the third and fourth nucleotides from the 5' end. Note that, herein, methylation of the hydroxyl group of ribose at the third nucleotide from the 5' end of RNA is sometimes referred to as Cap3 formation, and methylation of the hydroxyl group of ribose at the fourth nucleotide from the 5' end of RNA is sometimes referred to as Cap4 formation. The methyltransferase according to an embodiment of the present invention can perform Cap3 formation, or both Cap3 formation and Cap4 formation, by controlling the reaction time. Specifically, Cap3 formation can be performed by shortening the reaction time, and Cap4 formation in addition to Cap3 formation can be performed by extending the reaction time. The reaction time varies depending on the type of RNA (e.g., the number of nucleotides bound to 7-methylguanylic acid via triphosphate), and can be appropriately adjusted depending on the type of RNA used.

[0021] The methyltransferase according to the embodiment of the present invention is preferably the following (a), (b), or (c): (a) a protein consisting of the amino acid sequence of SEQ ID NO: 1; (b) a protein consisting of the amino acid sequence of SEQ ID NO: 1 in which one or several amino acids have been deleted, substituted, inserted, and / or added; or (c) a protein consisting of an amino acid sequence that is 80% or more homologous to the protein consisting of the amino acid sequence of (a).

[0022] Here, the protein consisting of the amino acid sequence of SEQ ID NO: 1 is derived from Acanthamoeba castellanii medusavirus. (SEQ ID NO: 1) MSSTKRDRSCLDGELGAVRPERNKRPRYDERDYASRRRRSYEDELHAKRREEEDEDRRRRRSHHHHRSHESSRCESHRSHHDNPHVAQCDAPPTNIPFSRILAPD ATRMPYRRRRGEDKSVIHWGQRKLLMSEIEFLTRYGHTSSSRTVVYAGAAPGTHTNWLADIFPDLKFVLVDPNPFVAQPTDRVEIVKDYFTDETAQKYAGQSVLFIS DVRTANWREQDENAVEKHVMHDMLAQQRWVEIMKPSMSMLKFRLPYPDREGCQGSTEYLDGEVFLPVWGPQTTTETRLVTPGTAKRQWDHTVYEQQMFFFNTQVRVG LYDHDVRAVGLDRCYDCTSEVRVLREYLDKYGLESLPWARDMLRHRHRREPEDEYAADVGDEDDKRFVIGRLSEYISRICSPKGRTLQSAPVQGFNFDNTIRNHPLL

[0023] The methyltransferase according to the embodiment of the present invention can methylate the hydroxyl group of ribose at the third nucleotide or the third and fourth nucleotides from the 5' end of RNA. This facilitates the production of RNA in which the hydroxyl groups of ribose at the third nucleotide or the third and fourth nucleotides from the 5' end are methylated, which may advance the development of nucleic acid medicines such as mRNA medicines.

[0024] In the protein (b) above, the number of amino acids that may be deleted, substituted, inserted, and / or added is not particularly limited, and may be 1 to 10, 1 to 8, 1 to 5, 1 to 4, 1 to 3, or 1 to 2. Site-directed mutagenesis kits for site-directed mutagenesis, such as deletion, substitution, insertion, and addition, can be used, for example, the GeneTailor® Site-Directed Mutagenesis System (Invitrogen Corporation) and the TakaRa Site-Directed Mutagenesis System (Prime STAR® Mutagenesis Basal kit, Mutan®-Super Express Km, etc.: Takara Bio Inc.). Whether or not mutations such as deletion, substitution, insertion, and addition have been introduced can be confirmed using various amino acid sequencing methods, as well as structural analysis methods such as X-ray and NMR.

[0025] The homology (identity) of the protein (c) to the protein consisting of the amino acid sequence (a) is not particularly limited as long as it is 80% or more, but is preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more. Here, the homology to the protein consisting of the amino acid sequence (a) can be determined using an alignment program such as BLAST (Basic Local Alignment Search Tool). For example, the homology to the protein consisting of the amino acid sequence (a) can be calculated using the algorithm blastp provided by NCBI (National Center for Biotechnology Information) with default parameters.

[0026] The protein used in the methyltransferase according to the embodiment of the present invention can be obtained by growing Escherichia coli or the like from a vector carrying the corresponding gene and inducing protein expression. The method for producing a vector carrying the corresponding gene is not particularly limited, and various techniques for synthesizing artificial genes may be used as appropriate. Commercially available Escherichia coli can be used as appropriate, and examples include E. coli BL21 manufactured by Takara Bio Inc. The growth of Escherichia coli and the induction of protein expression can also be performed according to known techniques. When purifying the methyltransferase according to the embodiment of the present invention, the Escherichia coli can be disrupted and centrifuged to remove bacterial debris, and the resulting product can be purified based on the tag (His-Tag) derived from the vector.

[0027] (2. Methylation Method) A methylation method according to an embodiment of the present invention is a method for methylating the hydroxyl group of ribose at the third nucleotide or the third and fourth nucleotides from the 5' end of RNA. This methylation method comprises carrying out a methylation reaction of RNA in the presence of a methyl group donor and the above-described methyltransferase. The above-described methyltransferase can methylate the hydroxyl group of ribose at the third nucleotide or the third and fourth nucleotides from the 5' end of RNA, thereby facilitating the production of Cap3-type RNA and the like.

[0028] The methyl group donor is not particularly limited, and S-adenosylmethionine (SAM) or the like can be used.

[0029] The conditions for the methylation reaction are not particularly limited and can be carried out according to general conditions. For example, a methyl group donor, the above-mentioned methyltransferase, and RNA can be added to an appropriate buffer solution and reacted at 30 to 45°C. The reaction time is also not particularly limited, but may be, for example, 30 minutes to 50 hours. The position of the hydroxyl group to be methylated can be controlled by the reaction time. For example, if Cap3 formation (methylation of the hydroxyl group of ribose at the third nucleotide from the 5' end of RNA) is performed, the reaction time can be shortened. If Cap4 formation (methylation of the hydroxyl group of ribose at the fourth nucleotide from the 5' end of RNA) is performed in addition to Cap3 formation, the reaction time can be lengthened. The reaction time can be adjusted appropriately depending on the type of RNA used. Furthermore, the concentrations of the methyl group donor, the above-mentioned methyltransferase, and RNA in the reaction solution for the methylation reaction can be set appropriately depending on their types.

[0030] Examples of RNA that can be used for methylation include Cap0-type RNA, Cap1-type RNA, Cap2-type RNA, RNA having a cap structure at the 5'-end and only the ribose hydroxyl group at the second nucleotide from the 5'-end being methylated, Cap3-type RNA, RNA having a cap structure at the 5'-end and only the ribose hydroxyl group at the third nucleotide from the 5'-end being methylated, RNA having a cap structure at the 5'-end and only the ribose hydroxyl groups at the second and third nucleotides from the 5'-end being methylated, and RNA having a cap structure at the 5'-end and only the ribose hydroxyl groups at the first and third nucleotides from the 5'-end being methylated. By using such mRNA, the ribose hydroxyl group at the third nucleotide from the 5'-end or the third and fourth nucleotides from the 5'-end can be methylated.

[0031] Nucleoside triphosphates may also be added to the methylation reaction to simultaneously cap the 5'-end of RNA. This allows capping of the 5'-end of RNA and methylation of the ribose hydroxyl groups at the third or third and fourth nucleotides from the 5'-end to be achieved in a single reaction step, significantly reducing the time and cost required to produce a given RNA. When nucleoside triphosphates are added, the concentration of nucleoside triphosphates in the reaction solution may be appropriately determined depending on the types of other components. Furthermore, the reaction conditions are not particularly limited and can be carried out according to general conditions.

[0032] (3. Use of Methyltransferase) The use of a methyltransferase according to an embodiment of the present invention is for methylating the hydroxyl group of ribose at the third nucleotide or the third and fourth nucleotides from the 5' end of RNA. For this use of a methyltransferase, the above-described methyltransferase is used. The above-described methyltransferase can methylate the hydroxyl group of ribose at the third nucleotide or the third and fourth nucleotides from the 5' end of RNA, facilitating the production of Cap3-type RNA and the like.

[0033] (4. Kit) A kit according to an embodiment of the present invention is used for methylating the hydroxyl group of ribose at the third nucleotide or the third and fourth nucleotides from the 5' end of RNA. This kit includes the above-described methyltransferase. The above-described methyltransferase can methylate the hydroxyl group of ribose at the third nucleotide or the third and fourth nucleotides from the 5' end of RNA, facilitating the production of Cap3-type RNA and the like. Therefore, this kit is useful for methylating the hydroxyl group of ribose at the third nucleotide or the third and fourth nucleotides from the 5' end of RNA.

[0034] The kit according to an embodiment of the present invention may further comprise a methyl donor in addition to the above-described methyltransferase. Because the methyl donor is a necessary component for the methylation reaction, this kit is useful for methylating the hydroxyl group of ribose at the third nucleotide or the third and fourth nucleotides from the 5' end of RNA.

[0035] (5. Nucleic Acid Drugs) Nucleic acid drugs according to embodiments of the present invention include Cap3-type RNAs having a 5'-cap structure and in which the hydroxyl groups of the ribose in the first to third nucleotides from the 5'-end are methylated. Cap3-type RNAs have excellent translation activity and can be used in the development of various nucleic acid drugs, particularly mRNA drugs and vaccines. mRNA drugs and vaccines can express therapeutic proteins and antigens administered to living organisms. Furthermore, nucleic acid drugs according to embodiments of the present invention include Cap4-type RNAs having a 5'-cap structure and in which the hydroxyl groups of the ribose in the first to fourth nucleotides from the 5'-end are methylated. While the efficacy of Cap4-type RNA is unclear, it has excellent translation activity similar to Cap4-type RNA and may be used in the development of various nucleic acid drugs, particularly mRNA drugs and vaccines. In addition to the 5'-cap structure, Cap3-type RNAs and Cap4-type RNAs have a 5'-untranslated region (5'UTR), an open reading frame region (ORF), a 3'-untranslated region (3'UTR), and a poly(A) tail region. These regions are not particularly limited and may be selected appropriately depending on the type of nucleic acid drug.

[0036] Cap3-type RNA can be produced according to the method described above in (2. Methylation Method). For example, Cap3-type RNA can be produced by methylating Cap2-type RNA in the presence of a methyl group donor and a methyltransferase according to an embodiment of the present invention. Cap3-type RNA can also be produced by methylating Cap1-type RNA in the presence of a methyl group donor, a Cap2 transferase, and a methyltransferase according to an embodiment of the present invention. Cap3-type RNA can also be produced by methylating Cap0-type RNA in the presence of a methyl group donor, a Cap1 transferase, a Cap2 transferase, and a methyltransferase according to an embodiment of the present invention. Furthermore, Cap3-type RNA can be produced by simultaneously capping and methylating the 5'-end of a specific RNA that is not capped at the 5' end in the presence of a methyl group donor, a specific capping enzyme, and a nucleoside triphosphate.

[0037] Cap4-type RNA can be obtained by producing a mixture of Cap3-type RNA and Cap4-type RNA according to the above-mentioned (2. Methylation Method), and then isolating Cap4-type RNA from the mixture. For example, a mixture of Cap3-type RNA and Cap4-type RNA can be produced by methylating Cap3-type RNA in the presence of a methyl group donor and a methyltransferase according to an embodiment of the present invention. Alternatively, a mixture of Cap3-type RNA and Cap4-type RNA can be produced by methylating Cap1-type RNA in the presence of a methyl group donor, Cap2-transferase, and a methyltransferase according to an embodiment of the present invention. Alternatively, a mixture of Cap3-type RNA and Cap4-type RNA can be produced by methylating Cap0-type RNA in the presence of a methyl group donor, Cap1-transferase, Cap2-transferase, and a methyltransferase according to an embodiment of the present invention. Furthermore, a mixture of Cap3-type RNA and Cap4-type RNA can also be produced by using a specific RNA whose 5' end is not capped, and simultaneously capping and methylating the 5' end of the RNA in the presence of a methyl group donor, a specific capping enzyme, and a nucleoside triphosphate. The method for isolating Cap4-type RNA from a mixture of Cap3-type RNA and Cap4-type RNA is not particularly limited, and can be carried out using a known method.

[0038] The present invention will be described in detail below with reference to examples, but the present invention should not be construed as being limited to these examples.

[0039] Preparation of a methyltransferase (protein consisting of the amino acid sequence of SEQ ID NO: 1) according to an embodiment of the present invention: 1 μL of a plasmid vector solution for expressing a protein consisting of the amino acid sequence of SEQ ID NO: 1 was added to 25 μL of Escherichia coli strain BL21, and the mixture was incubated on ice for 10 minutes, at 42°C for 30 seconds, and then on ice for 3 minutes. Next, 1 mL of LB medium was added, and the mixture was incubated at 37°C for 1 hour and at room temperature for 3 minutes. After centrifugation, the mixture was plated on LB agar medium (1.0% (w / v) peptone, 0.5% (w / v) yeast extract, 0.5% (w / v) sodium chloride) containing kanamycin and cultured overnight (16 hours) at 37°C. After confirming colony growth, a single colony was aseptically inoculated into LB liquid medium containing kanamycin and cultured with shaking at 37°C for 16 hours to obtain a preculture solution. One mL of the resulting preculture was inoculated into 50 mL of LBE medium (2.0% (w / v) potato peptone, 1.0% (w / v) yeast extract, 0.5% (w / v) sodium chloride) containing 50 μg / mL kanamycin, and cultured at 37°C with shaking at 120 rpm. Isopropyl-β-thiogalactopyranoside (IPTG) was added to a final concentration of 0.4 mmol / L at an OD600 of approximately 0.8, and the culture temperature was then set to 17°C, followed by overnight culture (16 hours). After culture was completed, the cells were collected by centrifugation (7,000 × g, 10 minutes), suspended in 5 mL of buffer (50 mmol / L HEPES aqueous solution, pH 8.0), and disrupted by sonication. The cell debris was further removed by centrifugation (12,000 × g, 10 minutes), and the resulting supernatant was used as a crude enzyme solution.

[0040] The protein was crudely purified from the obtained crude enzyme solution using Talon (registered trademark) resin (manufactured by Clontech), a resin for His-Tag purification. Specifically, 400 μL of Talon (registered trademark) resin was taken and loaded into an empty column, and 5 mL of buffer (50 mmol / L HEPES aqueous solution pH 8.0) was added. The operation of draining the liquid under its own weight was repeated twice. Then, the crude enzyme solution was added and waited for the liquid to drain completely. As a washing operation, 5 mL of equilibration buffer was added and the operation of draining the liquid was repeated twice, and then 5 mL of washing buffer (50 mmol / L HEPES aqueous solution pH 8.0 + 50 mmol / L imidazole) was added and the operation of draining the liquid was repeated twice. The target protein was then eluted with 500 μL of elution buffer (50 mmol / L HEPES aqueous solution pH 8.0 + 500 mmol / L imidazole) and mixed with 500 μL of 100% glycerol to prepare a crude enzyme solution. The molecular weight of the protein obtained above was determined by SDS-PAGE. Specifically, an SDS-PAGE gel was prepared, electrophoresed, and the electrophoresed gel was stained with CBB staining solution. The position of each protein band was confirmed based on the mobility of the molecular weight marker, and the molecular weight was then determined. As a result, the molecular weight of the protein was 29.8 kDa. The obtained protein (enzyme) was recovered from the crude enzyme solution by centrifugation using an ultrafiltration membrane, stored at -20 °C, and used in the following evaluation.

[0041] <Evaluation 1 of Methylation Reaction> First, 7.5 μL of a nucleic acid solution containing 0.5 mmol / L of RNA (5′pppGAAA or 5′pppGUUU), 5 μL of a capping buffer (containing 50 mmol / L of Tris-HCl, 5 mmol / L of KCl, 1 mmol / L of MgCl, and 1 mmol / L of dithiothreitol (DTT), with a pH of 8.0 at 25° C.), 2.5 μL of a 10 mmol / L solution of S-adenosylmethionine (SAM) diluted in NFW, 2.5 μL of a 10 mmol / L solution of guanosine triphosphate (GTP) diluted in NFW, 27.5 μL of water (NFW), 2.5 μL of ribonuclease inhibitor, and VCE (Vaccinia Capping Enzyme). A reaction solution was prepared by mixing 2.5 μL of mRNA Cap 2′-O-methyltransferase (Takara Bio Inc.), 2.5 μL of 2′-O-MTase (mRNA Cap 2′-O-methyltransferase; Takara Bio Inc.), and 2.5 μL of an NFW solution of Cap2 transferase (protein of SEQ ID NO: 2 below), and the reaction was carried out at 37° C. for 1.5 hours.

[0042] <SEQ ID NO: 2: Amino acid sequence of a protein derived from Leishmania major strain Friedlin> MFFDQRRVLTENAPRRPYQNNASTASFECPLLKGLHYGQRKLMLSEVEFLVQISMHIKRRCSGAAPKRVLVVYAGAACGLHLPFLFSLFPALDFVLIDPAPFCPRVKEIASKEGSCVLELIEDLCTAELCLRIRRTYHETHDIFLVSDIRSGEPTGMSLNQEHTDMIQQDNNAQREWCFSLEVEAAMLKFHPPYPAAKDSDPANYKAQDATVEEYTYLD GTQLLGVWAPKSSSEVRLIVVGPFKRGYAAPTRRYYCTVHEEQCYAYNTDNRYEKDCTAERLILEAYLSAFPGAYQSVEPLSKELSEQLEYPLFCPLEPGFTEQHARWV TLLYSTRKPAALQYFDLLKNDMSVEQVAGLVTKYKDCCEIPAGVKVGDIELTPHFWSVFAAGDFAVVYCFPFVRWEWKRHAHRRQPDKRRDDGRKNHRRFAGKKRTANTA

[0043] Next, 5 μL of the reaction mixture was diluted with 95 μL of eluent A [20 mmol / L hexafluoroisopropanol (HFIP), 20 mmol / L triethylamine acetate (TEA-A) aqueous solution (pH 7.0)], filtered through a 0.45 μm pore size filter, placed in an HPLC vial, and subjected to LC-MS analysis. Next, 2.5 μL of a 10 mmol / L NFW diluted solution of SAM and 2 μL of a 0.3 mg / mL NFW solution of methyltransferase (a protein consisting of the amino acid sequence of SEQ ID NO: 1) were added to the reaction mixture, and the mixture was allowed to react at 37°C for 1 hour (total reaction time: 2.5 hours). 5 μL was collected from the reaction solution, diluted with 95 μL of eluent A [20 mmol / L hexafluoroisopropanol (HFIP), 20 mmol / L triethylamine acetate (TEA-A) aqueous solution (pH 7.0)], filtered through a 0.45 μm pore size filter, placed in an HPLC vial, and subjected to LC-MS analysis. Next, the reaction solution was allowed to react for an additional 14.5 hours (total reaction time: 17 hours). 5 μL was collected from the reaction solution, diluted with 95 μL of eluent A [20 mmol / L hexafluoroisopropanol (HFIP), 20 mmol / L triethylamine acetate (TEA-A) aqueous solution (pH 7.0)], filtered through a 0.45 μm pore size filter, placed in an HPLC vial, and subjected to LC-MS analysis.

[0044] LC-MS analysis was carried out under the following conditions. System: Acquity UPLC system (Waters Corporation) Column: Biozen (registered trademark) Oligo, particle size 1.7 μm, inner diameter 2.1 mm × length 150 mm (Phenomenex Corporation) Injection volume: 10 μL Temperature: 35°C Flow rate: 0.2 mL / min Eluent A: 20 mmol / L hexafluoroisopropanol (HFIP), 20 mmol / L triethylamine acetate (TEA-A) aqueous solution (pH 7.0) Eluent B: 20 ​​mmol / L HFIP, 20 mmol / L TEA-A aqueous solution (pH 7.0), 40% methanol (MeOH) Elution mode: Gradient elution

[0045] Graphs showing the results of LC-MS analysis (quantitative analysis results) of the above methylation reaction are shown in Figures 1 and 2. Figure 1 shows the results when 5'pppGAAA was used as the RNA, and Figure 2 shows the results when 5'pppGUUU was used as the RNA. Graphs showing the results of LC-MS analysis (qualitative analysis results) of the above methylation reaction are shown in Figures 3 and 4. Figure 3 shows the results when 5'pppGAAA was used as the RNA, and Figure 4 shows the results when 5'pppGUUU was used as the RNA. In Figures 1 to 4, substrate RNA is represented as "5'ppp," Cap0-type RNA as "Cap0," Cap0-type RNA in which the hydroxyl group of the ribose at the first nucleotide from the 5' end is methylated (Cap1-type RNA) as "Cap01," Cap0-type RNA in which the hydroxyl group of the ribose at the second nucleotide from the 5' end is methylated as "Cap02," Cap0-type RNA in which the hydroxyl groups of the ribose at both the first and second nucleotides from the 5' end are methylated (Cap2-type RNA) as "Cap012," and Cap0-type RNA in which the hydroxyl groups of the ribose at both the first to third nucleotides from the 5' end are methylated (Cap3-type RNA) as "Cap0123." The numbers in parentheses in Figures 3 and 4 represent measured mass values.

[0046] As shown in Figures 1 to 4, Cap3-type RNA began to be produced after 1 hour of methylation reaction (total reaction time: 2.5 hours) following the addition of methyltransferase (a protein consisting of the amino acid sequence of SEQ ID NO: 1), and most of the RNA became Cap3-type RNA after 14.5 hours of methylation reaction (total reaction time: 17 hours).

[0047] <Evaluation of methylation reaction 2> 0.3 mmol / L (635.5 ng / μL) of RNA (m 7A reaction solution was prepared by mixing 10 μL of a nucleic acid solution containing Gppp-GAAAA), 10 μL of capping buffer (containing 50 mmol / L Tris-HCl, 5 mmol / L KCl, 1 mmol / L MgCl, and 1 mmol / L dithiothreitol (DTT), with a pH of 8 at 25°C), 5 μL of a 10 mmol / L NFW diluted solution of S-adenosylmethionine (SAM), 68 μL of water (NFW), 2 μL of ribonuclease inhibitor, and 5 μL of a NFW solution of methyltransferase (a protein consisting of the amino acid sequence of SEQ ID NO: 1), and the mixture was reacted at 37°C for 2.5 or 15 hours. After the reaction, 100 μL of eluent A was added to the reaction solution, which was then filtered through a 0.45 μm pore filter and placed in an HPLC vial for LC-MS and LC-MS / MS analysis.

[0048] Using the data obtained from the LC-MS / MS analysis, sequence analysis was performed to identify the site of methyl group transfer using the Confirm Sequence program in the UNIFY (Waters) software. The analysis using this program was performed under the following conditions: mass error 20 ppm, intensity cutoff: 500, isotope intensity ratio cutoff (%): 70, isotope similarity cutoff (%): 70. The results of the LC-MS analysis are shown in Figure 5, and the results of the sequence analysis using Confirm Sequence are shown in Figure 6. The action of methyltransferase converts the substrate m 7As the reaction progressed, the peak of GpppGAAAA (measured mass 2118.2976 Da) receded on the LC chromatogram and a mass increase of one methyl group (approximately 14 Da) was detected. Sequence analysis of the reactants confirmed that Reactant-1 had methylated the hydroxyl group of the ribose at the third nucleotide from the 5' end of the RNA (OMEA in Figure 6), while Reactant-2 had methylated the hydroxyl groups of the ribose at the third and fourth nucleotides from the 5' end of the RNA (OMEA in Figure 6). This demonstrates that methyltransferase can also express Cap4 activity after Cap3 activity. Furthermore, since there is a large difference in the reaction time required for the Cap3 conversion activity and the subsequent Cap4 conversion activity, by controlling the reaction time, it is possible to synthesize RNA in which the hydroxyl group of the ribose in the third nucleotide from the 5' end is methylated, or RNA in which the hydroxyl groups of the ribose in the third and fourth nucleotides from the 5' end are methylated.

[0049] <Synthesis of mRNA and evaluation of its translation activity> A 3075 nt mRNA encoding LacZ (β-galactosidase) was synthesized by in vitro transcription and purified using NucleoSpin (registered trademark) RNA (Takara Bio Inc.). This mRNA was capped with an m-terminus at the 5' end using Vaccinia Capping Enzyme (Takara Bio Inc.). 7 A G Cap structure was added and purified again using NucleoSpin® RNA to obtain Cap0 type mRNA (SEQ ID NO: 3: FIG. 7).

[0050] Next, a reaction solution was prepared by mixing 5 μL of a nucleic acid solution containing 406.32 ng / μL of Cap0-type mRNA, 2 μL of capping buffer (NEB), 2 μL of a 10 mmol / L NFW diluted solution of S-adenosylmethionine (SAM), 5 μL of water (NFW), 2 μL of RNase inhibitor, and 1 μL of a 0.3 mg / mL NFW solution of Cap1 transferase (mRNA Cap2'-O-Methyltransferase, Takara Bio Inc.). The reaction solution was then incubated at 37°C for 30 minutes, and then purified using NucleoSpin® RNA to obtain Cap1-type mRNA.

[0051] Next, a reaction solution was prepared by mixing 5 μL of a nucleic acid solution containing 406.32 ng / μL of Cap1-type mRNA, 2 μL of capping buffer (NEB), 2 μL of a 10 mmol / L NFW diluted solution of S-adenosylmethionine (SAM), 5 μL of water (NFW), 2 μL of RNase inhibitor, and 1 μL of a 0.3 mg / mL NFW solution of each Cap2-conjugating enzyme (proteins of SEQ ID NOs: 2 and 4 to 6). The reaction solution was then reacted at 37°C for 30 minutes and then purified using NucleoSpin (registered trademark) RNA to obtain Cap2-type mRNA.

[0052] <SEQ ID NO: 4: Amino acid sequence of protein derived from Trypanosoma cruzi> MKFPASRLLTDKFPRRAYVPSRADSLENVVVKGLHFGQRKLLLSEIEFLSAYLESQKKSAKPLLVVYAGAARGTHLPFLFKLFEKVKFVLIDPAPFCASVQELAKDEKGPILELLEKYCTDELCLRLLRSYCSNYDIILISDIRSGEPVNQSNKENTLMIMRDNEMQRSWCWSLKAKAALLKFHPPYPRCKDTASRYYDASDDTPD SVDYFDGNRLFGVWAPKSSSELRLCVAGPFIPGAKVSMRKYDCTVHEEQCYFYNTEDRYARDCEAEKEILERYLKLNYGTFSGDVVSLSNEISKFLNFPLFAP LEPSFTEN DARWLTLIYSTRDPKCEEWFEPLRGLMTLEVVQNLIKTYRNESSVPLNVTVGPTTLTRDFWKVMCTGNLVEAYGLPRPHWRFFSQIVSRKSKRTSL

[0053] <SEQ ID NO: 5: Amino acid sequence of protein derived from Trypanosoma cruzi marinkellei> MKFPASRLLTDESPRRAYVPSRAASLENFLVKGLHFGQRKLLLSEIEFLSAYLESQKKSAKPLLVVYAGAARGTHLPFLFKLFERVKFVLIDPAPFCTSVQELAKDGKGGPILELLEEYCTDELCLRLSRSYRSNYNIILISDIRSGEPVNQSNKENTVMIMRDNEMQRSWCWSLKAEAALLKFHPPYPRCKDTASRYYDASDDTPD SVEYFDGNRLFGVWAPKSSSEVRLCVAGPFIPGAKVSMRKYDCTVHEEQCYFYNTEGRYARDCEAEKEILERYLKLNSGAYSGGVVSLSNDISKFLNFPLFAP LEPSFTEN DARWLALIYSTRDPKCEELFEPLRGLMTLDVQNLVKTYRNESSVPLNVTVGPTTLTRDFWKVMCTGNLVEAYGLPRLPWRFASQIVSRKSKRTSL

[0054] <SEQ ID NO: 6: Amino acid sequence of protein derived from Phytomonas sp. EM1> MYFDSSRILDSRCPRTPYKPTSETLSLDSISVKGLHFGQRKLLLSEIEFLTAIFDKRKCDIIESDHKQRPVLVVYAGAANGSHLPILFRLFAEARFILIDPAPFCEEVEVIAEDVQGPIIELINDYCTDELCLRLSRMYGTQYDLYLISDIRTGVPKKMQKNIEHTEMMIRDNAAQRSWCWSLKAVAAMLKFHPPYPPSTDSSGQKCEDDDNTPDEIEY LDGIQLFGVWAPKSSSEVRLVVQGPFCHGYNAKMRKYNCKEHEEQCYHYNTSNRYLKDCAAERIIVDRYLKCIPNEYKNDITIVSNQISKDLKFPLFCPLENDFTEDHAR WVYLLYSSRRHDIMHLYEPFKDVMRFDTVKDLIKRYGQSEKVPEDAQVSGKPLSIDFWKAFCRCDFESAYSLPRIQWKFYPLLDPKRPHRRENKGAESSRNRTQFARKES

[0055] A reaction solution was prepared by mixing 5 μL of a nucleic acid solution containing 406.32 ng / μL of Cap0 mRNA, 2 μL of capping buffer (NEB), 2 μL of a 10 mmol / L NFW diluted solution of S-adenosylmethionine (SAM), 5 μL of water (NFW), 2 μL of RNase inhibitor, and 1 μL of a 0.3 mg / mL NFW solution of each Cap2 conjugation enzyme (proteins of SEQ ID NOs: 2 and 4-6). The reaction solution was then incubated at 37°C for 30 minutes and then purified using NucleoSpin (registered trademark) RNA to obtain mRNA in which the hydroxyl group of the ribose in the second nucleotide from the 5' end of the Cap0 mRNA was methylated.

[0056] Next, a reaction solution was prepared by mixing 5 μL of a nucleic acid solution containing 406.32 ng / μL of Cap2-type mRNA, 2 μL of capping buffer (NEB), 2 μL of a 10 mmol / L NFW diluted solution of S-adenosylmethionine (SAM), 5 μL of water (NFW), 2 μL of RNase inhibitor, and 1 μL of a 0.3 mg / mL NFW solution of methyltransferase (protein of SEQ ID NO: 1). The reaction solution was then incubated at 37°C for 30 minutes and then purified using NucleoSpin® RNA to obtain Cap3-type mRNA.

[0057] A reaction solution was prepared by mixing 5 μL of nucleic acid solution containing 406.32 ng / μL Cap0 mRNA, 2 μL of capping buffer (NEB), 2 μL of 10 mmol / L S-adenosylmethionine (SAM) diluted in NFW, 5 μL of water (NFW), 2 μL of RNase inhibitor, and 1 μL of 0.3 mg / mL methyltransferase (protein of SEQ ID NO: 1) in NFW. The reaction solution was then incubated at 37°C for 30 minutes and then purified using NucleoSpin® RNA to obtain mRNA in which the ribose hydroxyl group at the third nucleotide from the 5' end of the Cap0 mRNA was methylated. The purity of each mRNA prepared above was confirmed by bioanalyzer analysis.

[0058] Reagent 1 was obtained by mixing 1 mL of Opti-MEM (registered trademark) medium and 80 μL of Lipofectamine (registered trademark) LTX in an Eppendorf tube. Furthermore, 751 μL of Opti-MEM (registered trademark) medium and 15 μL of PLUS (registered trademark) reagent were mixed in an Eppendorf tube to obtain Reagent 2. Next, 31.9 μL of Reagent 2 was dispensed into 20 Eppendorf tubes. 12.5 μL and 2.5 μL of each 50 ng / μL mRNA were added to these Eppendorf tubes and mixed. 31.3 μL of Reagent 1 was added to these Eppendorf tubes and mixed. The mixture was then incubated at room temperature for 5 minutes, and 10 μL of each was introduced into cells (RAW264.7).

[0059] Next, the translation activity was evaluated by quantifying LacZ (β-galactosidase) using the Beta-GLO (registered trademark) assay system manufactured by Promega Corp. The results are shown in FIG.

[0060] In Figure 8, Cap0-type mRNA is represented as "Cap0," Cap1-type mRNA as "Cap01," Cap2-type mRNA as "Cap012," Cap0-type mRNA in which the hydroxyl group of the ribose at the second nucleotide from the 5' end is methylated as "Cap02," Cap3-type mRNA as "Cap0123," and Cap0-type mRNA in which the hydroxyl group of the ribose at the third nucleotide from the 5' end is methylated as "Cap03." The results for Cap02, Cap012, and Cap0123 were the average of the results for each mRNA prepared using the proteins of SEQ ID NOs: 2 and 4-6. As shown in Figure 8, Cap3-type mRNA had superior translation activity compared to the other mRNAs.

[0061] As can be seen from the above results, the present invention can provide a methyltransferase capable of methylating the hydroxyl group of ribose at the third nucleotide or the third and fourth nucleotides from the 5' end of RNA, a methylation method, use of the methyltransferase, and a kit.

Claims

1. Methyltransferases for methylating the hydroxyl group of ribose in the third or third and fourth nucleotides from the 5' end of RNA.

2. The methyltransferase according to claim 1, which is one of the following (a), (b), or (c): (a) a protein consisting of the amino acid sequence of SEQ ID NO: 1; (b) a protein consisting of the amino acid sequence of SEQ ID NO: 1 in which one or more amino acids have been deleted, substituted, inserted, and / or added; or (c) a protein consisting of an amino acid sequence that is 80% or more identical to the protein consisting of the amino acid sequence of (a).

3. A method for methylating the hydroxyl group of ribose in the third nucleotide or the third and fourth nucleotides from the 5' end of RNA, comprising carrying out a methylation reaction of RNA in the presence of a methyl group donor and the methyltransferase according to claim 1 or 2.

4. The method of claim 3, wherein the RNA used for the methylation is at least one selected from Cap0-type RNA, Cap1-type RNA, Cap2-type RNA, RNA having a cap structure at the 5'-end and only the hydroxyl group of the ribose at the second nucleotide from the 5'-end being methylated, Cap3-type RNA, RNA having a cap structure at the 5'-end and only the hydroxyl group of the ribose at the third nucleotide from the 5'-end being methylated, RNA having a cap structure at the 5'-end and only the hydroxyl groups of the ribose at the second and third nucleotides from the 5'-end being methylated, and RNA having a cap structure at the 5'-end and only the hydroxyl groups of the ribose at the first and third nucleotides from the 5'-end being methylated.

5. The method according to claim 3, wherein a nucleoside triphosphate is further present in the methylation reaction, and the 5' end of the RNA is simultaneously capped.

6. Use of the methyltransferase according to claim 1 or 2 for methylating the hydroxyl group of ribose in the third or third and fourth nucleotides from the 5' end of RNA.

7. A kit used for methylating the hydroxyl group of ribose in the third nucleotide or the third and fourth nucleotides from the 5' end of RNA, comprising the methyltransferase according to claim 1 or 2.

8. The kit of claim 7, further comprising a methyl group donor.

9. A nucleic acid drug comprising Cap3-type RNA having a 5' cap structure and in which the hydroxyl groups of the ribose in the first to third nucleotides from the 5' end are methylated.

10. A nucleic acid drug comprising Cap4-type RNA having a 5' cap structure and in which the hydroxyl groups of the ribose in the first to fourth nucleotides from the 5' end are methylated.

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