Methyltransferase for methylating hydroxyl group of ribose moiety in second nucleotide from 5'-end of RNA, methylation method, use of methyltransferase, and kit

Abstract

Provided is a methyltransferase for methylating a hydroxyl group of a ribose moiety in the second nucleotide from the 5'-end of RNA. This methyltransferase comprises a protein which has a product of a methyl group transfer activity ratio and an expression amount ratio with respect to a protein comprising the amino acid sequence represented by SEQ ID NO: 1 of 1.20 or more.

Description

Methyltransferase for methylating the hydroxyl group of ribose in the penultimate nucleotide of the 5' end of RNA, methylation method, use of methyltransferase, and kit

[0001] The present invention relates to a methyltransferase, a methylation method, the use of the methyltransferase, and a kit for methylating the hydroxyl group of ribose in the penultimate 5' nucleotide of RNA.

[0002] 7-methylguanylic acid (m) is attached to the 5' end of RNA such as mRNA (messenger RNA). 7 The 5'-cap structure to which 7-methylguanylic acid (G) is attached is known to contribute to functions such as translation activity and stability. It has been reported that such RNA functions are improved by methylating the ribose hydroxyl group (2'-hydroxyl group) of the nucleotide linked via 7-methylguanylic acid and triphosphate, particularly the ribose hydroxyl group of the second nucleotide from the 5' end (Non-Patent Documents 1 to 4). A structure in which the ribose hydroxyl group of the first nucleotide from the 5' end of RNA is not methylated is called a Cap0-type structure, a structure in which the ribose hydroxyl group (2'-hydroxyl group) of the first nucleotide from the 5' end of RNA is methylated is called a Cap1-type structure, and a structure in which the ribose hydroxyl groups of the first and second nucleotides from the 5' end of RNA are methylated is called a Cap2-type structure.

[0003] Magdalena Byszewska and 3 others, “RNA methyltransferases involved in 5' cap biosynthesis”, RNA Biology, vol. 11(12), pp. 1597-1607, 2014 DecemberVladimir Despic and 1 other person, “mRNA aging shapes the Cap2 methylome in mammalian mRNA”, Nature, vol. 614, pp. 358-366, 2023Megan P. Hall and 1 other, "Functional characterization of a 48 kDa Trypanosoma brucei cap 2 RNA methyltransferase", Nucleic Acids Research, vol. 34, No. 19, pp. 5594-5602, 2006Maria Werner and 9 others, "2'-O-ribose methylation of cap2 in human: function and evolution in a horizontally mobile family”, Nucleic Acids Research, Vol, 39, No.11, pp.4756-4768, 2011

[0004] A method using a methyltransferase from Trypanosoma brucei or the like is known as a method for methylating the hydroxyl group of ribose in the second nucleotide from the 5' end of RNA. However, the expression level and methyltransfer activity of conventional methyltransferases are low, and therefore, they are not at a level suitable for industrial use.

[0005] The present invention has been made to solve the above-mentioned problems, and aims to provide a methyltransferase that has high expression level and / or methyltransfer activity and can be used industrially, a methylation method, use of the methyltransferase, and a kit.

[0006] The present inventors have conducted extensive research to solve the above-mentioned problems and have found that a specific protein can methylate the hydroxyl group of ribose in the second nucleotide from the 5' end of RNA, has high expression level and / or high methyl group transfer activity, and is suitable for industrial use, thereby completing the present invention. Specifically, the present invention is exemplified as follows.

[0007] <1> A methyltransferase for methylating the hydroxyl group of ribose in the second nucleotide from the 5' end of RNA, the methyltransferase being composed of a protein having a product of the methyltransfer activity ratio and the expression level ratio relative to a protein consisting of the amino acid sequence of SEQ ID NO: 1 of 1.20 or more.

[0008] <2> The methyltransferase according to <1>, wherein the methyltransferase is selected from proteins consisting of the amino acid sequences of SEQ ID NOs: 2 to 22 and proteins consisting of the amino acid sequences of SEQ ID NOs: 2 to 22 in which one or several amino acids have been deleted, substituted, inserted, and / or added.

[0009] <3> The methyltransferase according to <1>, wherein the expression level ratio of the protein to a protein consisting of the amino acid sequence of SEQ ID NO: 1 is 5.00 or more.

[0010] <4> The methyltransferase according to <3>, wherein the methyltransferase is selected from proteins consisting of the amino acid sequences of SEQ ID NOs: 2, 3, 5 to 8, 11 to 14, 16, 17, and 20, and proteins consisting of the amino acid sequences of SEQ ID NOs: 2, 3, 5 to 8, 11 to 14, 16, 17, and 20 in which one or several amino acids have been deleted, substituted, inserted, and / or added.

[0011] <5> The methyltransferase according to <1>, wherein the protein comprises at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 23 to 25.

[0012] <6> The methyltransferase according to <5>, wherein the methyltransferase is selected from proteins consisting of the amino acid sequences of SEQ ID NOs: 2, 4, 5, 7 to 10, 12, 14, and 18, and proteins consisting of the amino acid sequences of SEQ ID NOs: 2, 4, 5, 7 to 10, 12, 14, and 18 in which one or several amino acids have been deleted, substituted, inserted, and / or added.

[0013] <7> The methyltransferase according to <1>, wherein the methyltransferase activity ratio of the protein to a protein consisting of the amino acid sequence of SEQ ID NO: 1 is 1.50 or more.

[0014] <8> The methyltransferase according to <7>, wherein the methyltransferase is selected from proteins consisting of the amino acid sequences of SEQ ID NOs: 2 to 7, 10 to 13, 15, and 16, and proteins consisting of the amino acid sequences of SEQ ID NOs: 2 to 7, 10 to 13, 15, and 16 in which one or several amino acids have been deleted, substituted, inserted, and / or added.

[0015] <9> The methyltransferase according to <1>, which has a methyltransferase activity on RNA having three nucleotides linked from the 5'-end.

[0016] <10> The methyltransferase according to <9>, wherein the methyltransferase is selected from proteins consisting of the amino acid sequences of SEQ ID NOs: 3, 7, 8, 11 to 14, 16, and 17, and proteins consisting of the amino acid sequences of SEQ ID NOs: 3, 7, 8, 11 to 14, 16, and 17 in which one or several amino acids have been deleted, substituted, inserted, and / or added.

[0017] <11> A method for methylating the hydroxyl group of ribose in the second nucleotide 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 a methyltransferase, wherein the methyltransferase is the methyltransferase according to any one of <1> to <10>.

[0018] <12> The method according to <11>, wherein the RNA used for the methylation has a Cap0 type structure and / or a Cap1 type structure.

[0019] <13> The method according to <11>, wherein a nucleoside triphosphate is further present in the methylation reaction, and the 5'-end of the RNA is simultaneously capped.

[0020] <14> Use of the methyltransferase according to any one of <1> to <10> for methylating the hydroxyl group of ribose in the second nucleotide from the 5' end of RNA.

[0021] <15> A kit used for methylating the hydroxyl group of ribose in the second nucleotide from the 5' end of RNA, the kit comprising the methyltransferase according to any one of <1> to <10>.

[0022] <16> The kit according to <15>, further comprising a methyl group donor.

[0023] According to the present invention, it is possible to provide a methyltransferase that has a high expression level and / or high methyltransferase activity and can be used industrially, a methylation method, use of the methyltransferase, and a kit.

[0024] 1 is a diagram showing the base sequence of Cap0-type mRNA used in Examples. 2 is a graph showing the results of evaluation of translation activity in Examples.

[0025] Hereinafter, embodiments of the present invention will be described in detail. The present invention is not limited to the following embodiments, and it should be understood that modifications and improvements to the following embodiments, as appropriate, based on the common knowledge of those skilled in the art, fall within the scope of the present invention, as long as they do not deviate from the spirit of the present invention. With regard to numerical ranges described in stages in this specification, the upper limit of a certain numerical range may be replaced with the upper limit of another numerical range described in stages or a value shown in an example. Similarly, with regard to numerical ranges described in stages in this specification, the lower limit of a certain numerical range may be replaced with the lower limit of another numerical range described in stages or a value shown in an example. Furthermore, with regard to numerical ranges described in this specification, a range may be created by combining the upper and lower limits of a certain numerical range.

[0026] (1. Methyltransferase) The methyltransferase according to an embodiment of the present invention is an enzyme used to methylate the hydroxyl group of ribose in the second nucleotide from the 5' end of RNA. The RNA is not particularly limited, but is preferably mRNA. The methyltransferase according to an embodiment of the present invention is a protein having a product of the methyltransferase activity ratio and the expression level ratio relative to a protein consisting of the amino acid sequence of SEQ ID NO: 1 (hereinafter abbreviated as "product of methyltransferase activity ratio and expression level ratio") of 1.20 or more. Such a characteristic allows for a higher expression level and / or methyltransferase activity than known methyltransferases, facilitating industrial use. From the viewpoint of increasing the level of industrial use, the product of the methyltransferase activity ratio and the expression level ratio is preferably 3.00 or more, more preferably 5.00 or more, even more preferably 10.00 or more, and particularly preferably 20.00 or more. The upper limit of the product of the methyltransferase activity ratio and the expression level ratio is not particularly limited, but may be, for example, 200.00, 150.00, or 120.00.

[0027] Here, the protein consisting of the amino acid sequence of SEQ ID NO: 1 below is derived from Trypanosoma brucei, has a molecular weight of 48 kDa, and is known as a methyltransferase (Non-Patent Documents 1, 3, and 4). <SEQ ID NO: 1: Amino acid sequence of the protein derived from Trypanosoma brucei> PGDNFPDSRVLSDEFPRRKYKPSSATRLEDIVLKGLHYGQRKLLLSEIEFLSAYLEGRQAGAKPTLVVYAGAANGSHLPFLFQLFEAVKFVLIDPAPFCDAVRE ISLNKQGPILDLVQGFCTDELCKQLSSSYGSTYDILLVSDIRSGVPEKQSNRENTLMIMRDNDDMQRSWWTLKAEAALLKFHPPYPPCRDRNSRHYDEADDTPES IEYLDGVRLFGVWAPKSSSELRLCVQGPFLQGTSFPMRRYDCTTTHEEQCYFYNTDNRYTRDCAAESGILERYLQLFPSAPYSDPTALSMTISNFLGFPLFLPLDS SFSESDARWVTLLYSTRIPTCLELFTTLRGRVTHTVMKQLAEEWQSATTVPKGVSIDSVELTSEFWKAVCAGDLTEAYSFPNIRWRFANLLISRRRRKRSASDRAP

[0028] As used herein, the "transmethylation activity ratio relative to a protein consisting of the amino acid sequence of SEQ ID NO: 1" refers to the ratio of the transmethylation activity of a target protein to the transmethylation activity of a protein consisting of the amino acid sequence of SEQ ID NO: 1. The transmethylation activity can be evaluated by quantifying the amount of S-adenosylhomocysteine ​​(SAH) produced after a methylation reaction using a 60-mer Cap1 RNA as a substrate in the presence of a methyltransferase (protein) and S-adenosylmethionine (SAM) as a methyl donor. The transmethylation activity is evaluated as 1 U, which is the activity that produces 1 μm of SAH per hour at 37°C. The conditions for measuring the transmethylation activity are as described in the Examples section below.

[0029] As used herein, the expression level ratio relative to a protein consisting of the amino acid sequence of SEQ ID NO: 1 refers to the ratio of the expression level of a target protein in an expression system using E. coli to the expression level of a protein consisting of the amino acid sequence of SEQ ID NO: 1 in an expression system using E. coli. The expression level in an expression system using E. coli can be evaluated by expressing a His-tagged (histidine-tagged) protein in E. coli, crudely purifying it by immobilized metal affinity chromatography (IMAC), and analyzing the protein contained in the crude solution by polyacrylamide gel electrophoresis (SDS-PAGE). The conditions for measuring the expression level are as described in the Examples below.

[0030] The protein used in the methyltransferase according to the embodiment of the present invention preferably has a query coverage of 50% or more and a sequence identity (Per Ident) of 25% or more relative to the protein consisting of the amino acid sequence of SEQ ID NO: 1. Controlling the query coverage and sequence identity within these ranges facilitates improving the expression level and / or methyltransferase activity of the methyltransferase. The query coverage may be 60% or more, 80% or more, or 90% or more. The sequence identity may be 30% or more, 40% or more, or 50% or more. The upper limit of the query coverage is not particularly limited, but may be, for example, 100% or 99%. The upper limit of the sequence identity is also not particularly limited, but may be, for example, 90%, 80%, or 70%. The query coverage and sequence identity of the protein used in the methyltransferase according to the embodiment of the present invention relative to the protein consisting of the amino acid sequence of SEQ ID NO: 1 can be determined using an alignment program such as BLAST (Basic Local Alignment Search Tool). For example, the query coverage of a protein used in a methyltransferase according to an embodiment of the present invention for a protein consisting of the amino acid sequence of SEQ ID NO: 1 can be calculated using the algorithm blastp provided by NCBI (National Center for Biotechnology Information) with default parameters.

[0031] Examples of methyltransferases having the above-described characteristics include proteins consisting of the amino acid sequences of SEQ ID NOs: 2 to 22, and proteins consisting of the amino acid sequences of SEQ ID NOs: 2 to 22 in which one or more amino acids have been deleted, substituted, inserted, and / or added. These may be used alone or in combination of two or more types.

[0032] <SEQ ID NO: 2: Amino acid sequence of protein derived from Trypanosoma cruzi> MKFPASRLLTDKFPRRAYVPSRADSLENVVVKGLHFGQRKLLLSEIEFLSAYLESQKKSAKPLLVVYAGAARGTHLPFLFKLFEKVKFVLIDPAPFCASVQELAKDEKGPILELLEKYCTDELCLRLLRSYCSNYDIILISDIRSGEPVNQSNKENTLMIMMRDNEMQRSWCWSLKAKAALLKFHPPYPRCKDTASRYYDASDDTPD SVDYFDGNRLFGVWAPKSSSELRLCVAGPFIPGAKVSMRKYDCTVHEEQCYFYNTEDRYARDCEAEKEILERYLKLNYGTFSGDVVSLSNEISKFLNFPLFAP LEPSFTEN DARWLTLIYSTRDPKCEEWFEPLRGLMTLEVVQNLIKTYRNESSVPLNVTVGPTTLTRDFWKVMCTGNLVEAYGLPRPHWRFFSQIVSRKSKRTSL

[0033] <SEQ ID NO: 3: Amino acid sequence of a protein derived from Leishmania major strain Friedlin> MFFDQRRVLTENAPRRPYQNNASTASFECPLLKGLHYGQRKLMLSEVEFLVQISMHIKRRCSGAAPKRVLVVYAGAACGLHLPFLFSLFPALDFVLIDPAPFCPRVKEIASKEGSCVLELIEDLCTAELCLRIRRTYHETHDIFLVSDIRSGEPTGMSLNQEHTDMIQQDNNAQREWCFSLEVEAAMLKFHPPYPAAKDSDPANYKAQDATVEEYTYLD GTQLLGVWAPKSSSEVRLIVVGPFKRGYAAPTRRYYCTVHEEQCYAYNTDNRYEKDCTAERLILEAYLSAFPGAYQSVEPLSKELSEQLEYPLFCPLEPGFTEQHARWV TLLYSTRKPAALQYFDLLKNDMSVEQVAGLVTKYKDCCEIPAGVKVGDIELTPHFWSVFAAGDFAVVYCFPFVRWEWKRHAHRRQPDKRRDDGRKNHRRFAGKKRTANTA

[0034] <SEQ ID NO: 4: Amino acid sequence of protein derived from Trypanosoma theileri> MKFPAVRVLTDDFPRRTYKPSNLATIDDIVVKGLHYGQRKLLLSEIEFLSAYIETSRTPKKPLLVVYAGAANGSHLPFLFNLFEDVKFVLIDPAPFCTAVQDIAKRSKKHILELVEGYCSDELCIRLSRSYHSDYNILFVSDIRSGEPKKQSNKENTAMIMRDNEMQRSWCCSLKAMAAMLKFHPPYPRCKDPKSRSYDENDDTPDSIEYMDGN RLFGIWAPKSSSELRLCVQGPFTPGKKIAMRRYDCTLHEEQCYFYNTSDRYSRDCEAEKNVLQRYLKLFPMAFSADVVKLSNAISEFLKFPLFRPLDPSFTESDARW VALLYSTRDAESLVLFKPLRGLITLSVIKELIKTWRDATSVPENVKIGSVTLSREFWKVMCTGDLVETYNLPRTRWGFAHEIISRKLKRSHSECVPSKKKVTKRNKK

[0035] <SEQ ID NO: 5: Amino acid sequence of protein derived from Trypanosoma melophagium> MKFPLDRVLTDDFPRRTYKPSNLATTDNIVVKGLHYGQRKLLLSEIEFLSAYLETPRTTKQPLLVVYAGAANGSHLPFLFDLFKDVKFVLIDPAPFCAAVREIAQLSKGPIVELVEGYCTDELCLRLSRSYHLDYNILFISDIRSGEPKRQSNKENTAMIMRDNEMQRSWCWSLKANAALLKFHPPYPRCKDPNSRSYDENDNTPDDSVEYMDGN RLFGIWAPKSSELRLCVQGPFTTGTKIAMRIYDCSLHEEQCYFYNTSDRYTRDCEAEKNVLQRYLTLFPVAFSADVVKLSNAISEFLKFPLFRPLDPSFTEKDARW VTLLYSTRDSESLVLFDHLRGLVTLGVIQDLIKMWRDAKSVPENVTIGSVTLSREFWKVMCTGDLVGAYGLPRMRWGFAHEILSRKSGRSSNEWTAPQQKKMMKRNKK

[0036] <SEQ ID NO: 6: Amino acid sequence of protein derived from Trypanosoma rangeli SC58> MKFSPLRLLTDDFPRRAYVPSHSATLESVTVKGLHFGQRKLLLSEIEFLSAYLEAPKASDKPLLVVYAGAANGSHLPFLFGLFEKVKFVLIDPASFCAKVREIAKDGEGPVLELVEGYCTDELCLRLSRLYGSVYDILLVSDIRSGEPLTQSNKENTAMIMRDNEMQRSWCSSLKARAALLKFHPPYPRCKDATSRYYDSADDTPDSDVEYMDGSRL FGVWAPKSSSEVRLCVEGPFLSSAKVPMRQYDCTVHEEQCYFYNTEDRYARDCVAERDILSRYLKANPGAYPDGVVSFSNDISKFLQFPLFVPLEPSFTETEARWVTL LYSTRDPKCLEWVEPLRGLMTLDVIRDLAKRYHDERSVPSDVTVGAVALSRDFWRVMCAGDLTEAYGLPRIRWRFAPQIVSHGGKRPRSLRVSRCLTPSLQNVNALKE

[0037] <SEQ ID NO: 7: Amino acid sequence of protein derived from Trypanosoma conorhini> MKFSPFRLLTDDFPRRAYVPSHSATLENVVVKGLHFGQRKLLLSEVEFLSAYMEAPRASNKALLVVYAGAANGSHLPFLFGLFATIKFILIDPAPFCAEVREIAKDRKGSVLELVEGYCTDELCLRLSRAYSSEYDILLVSDIRSGEPVTQSNKENTAMIMRDNEMQRSWCWSLKAEAALLKFHPPYPRCKDAASRYYDAADDTPD SVEYMDGSRLFGVWAPKSSSEVRLCVEGPFVPGATVPMRQYDCTLHEEQCYFYNTEDRYARDCAAERDILTRYLKANPDAYPGGVVSLSNAISKFLQFPLFVP LDPSFTESEARWVTLAYSARDPKCLEWIEPLRGLMTLDVIRDLVKRHRNDSAVPSEATVGPVTLSREFWRVMCAGDLAGAYGLPPIRWRFAAQILSRRPKRTRP

[0038] <SEQ ID NO: 8: Amino acid sequence of protein derived from Trypanosoma cruzi marinkellei> MKFPASRLLTDESPRRAYVPSRAASLENFLVKGLHFGQRKLLLSEIEFLSAYLESQKKSAKPLLVVYAGAARGTHLPFLFKLFERVKFVLIDPAPFCTSVQELAKDGKGGPILELLEEYCTDELCLRLSRSYRSNYNIILISDIRSGEPVNQSNKENTVMIMRDNEMQRSWCWSLKAEAALLKFHPPYPRCKDTASRYYDASDDTPD SVEYFDGNRLFGVWAPKSSSEVRLCVAGPFIPGAKVSMRKYDCTVHEEQCYFYNTEGRYARDCEAEKEILERYLKLNSGAYSGGVVSLSNDISKFLNFPLFAP LEPSFTEN DARWLALIYSTRDPKCEELFEPLRGLMTLDVQNLVKTYRNESSVPLNVTVGPTTLTRDFWKVMCTGNLVEAYGLPRLPWRFASQIVSRKSKRTSL

[0039] <SEQ ID NO: 9: Amino acid sequence of protein derived from Trypanosoma vivax Y486> MFISVAFLHFFLLDVCTCHPSRILMKFSASRLLTDESPRRVYKPSSSSSSLHDVVVKGLHYGQRKLLLSEIEFLSAYHEADSSESPPLVVYAGAANGSHLPFIFRLFPGVRF VLIDPAPFCAQVRDIAQCSDGPIVERVEGYCTDELCRRLAHSHSSSYKILLISDIRSGVPAKLSNKENTAMIMRDNKMQQSWCWSLGAEAALLKFHPPYPRCNDPDSAHYDA SDDTPNSVEYLDGERLFGVWAPKSSSEVRLCVRGPFSEDVEPRLRHYDCVVHEEQCYYYNTDGRYARDCKAEREILNRYLELFPGVFSGDVVALSSAISEFLRFPMFRPLD PSFSEDDARWVTLLYSVGDQACLDLFDTLRGHVSLKFLQEQLAKWHTSVSVPEDVTAGSVKLTRDFWRVICTGSLSSEAYGIPPVRCNFAHVFVSRKRRRAISLRAVGLSKER

[0040] <SEQ ID NO: 10: Amino acid sequence of protein derived from Trypanosoma grayi> MQKENAKPLLVVYAGAANGSHLPFLFDLFKTIKFVLIDPAPFCPAVREIAQRADGPIKEMVEGYCTDELCLRLSRTYRSDYDILLISDIRSGEPKKQSNKDNTAMIMRDNEMQRSWCWSLRRAEAALLKFHPPYPPCRDTKSRHYDAADDTPESVEYMDGIRLFGVWAPKSSSELRLCVK GPFVPGAKIAMRHYDCSMHEEQCAFYNTEGRYARDCEAEKNILARYLKLFPSAYSGDVVALSHAVSAFLKFPLFRPLESTFTEDDARWV TLLYSTRDVENLAWFERLRGLMTSKVVQDLVKMWRDSTCVPVDATIGSIALSKEFWTIMCTGNLVEAYSLPRIWWRFAPQIISRKTKRSS

[0041] <SEQ ID NO: 11: Amino acid sequence of protein derived from Leishmania donovani> MFFDKRRVLTEEAPRRPYQNNASTASFECPLLKGLHYGQRKLLLSEVEFLVQISMHVKCRGSGASPKKVLVVYAGAAYGLHLPLIFSLFPALDFVLIDPAPFCSRVKEIASKEGSCVLELIDDFCTAELCLRIRRSYQETHDIFLVSDIRSGQPTGMSMNQEHTDMIQQDNKAQREWCFSLEVEAAMLKFHPPYPAAKDSDPANYKAQDATVEEYTYLD GTQLLGVWAPKSSSEVRLIVVGPFKRGYAAPTRRYYCTVHEEQCYAYNTDNRYEKDCAAERLILETYLSAFPGASKSVEQLSKTLSEQLEYPLFCPLEPGFTEQHARWV TLLYSTRNPAALQYFDLLKNYMSVEQVAGLVTKYKDCCEIPTGVKVGDIELTPHFWSVFAAGDFAVVYCFPAVRWGWKQHAHRRQPNKQEDDDRENHRRFAGKKRRANAA

[0042] <SEQ ID NO: 12: Amino acid sequence of protein derived from Leishmania martiniquensis> MLFDQRRVLTEDAPRRPYQSNASTASFEQRLLKGLHYGQRKLILSEVEFFLQIAVYTKRHESVASPKKALIVYAGAACGFHLPFLFSLFPTLEFVLIDPAPFCSQVKEIASKEKSCVLELIEDLCTPELCLRIRRTYVDSHDIFLVSDIRSGRPTEMSLNQEHTDMILQDNDAQREWCFSLEAAAAAMLKFHPPYPAAKGSNAEKYKTHDTTMEEYMYLD GTQLFGVWAPKSSSEIRLVVVGPFKQGYEPPTRRYYCTAHEEQCYAYNVDKRYDKDCAAERLILESYLSAFPGVYESVEQLSKTISGKLEYPLFCPLEPGFTEQHARWV TLLYSTRKPSALQYFDLLKNVMSVDQVAALVMKYKDSREIPTGVKVGDIELTPHFWSVFSAGDFAAVYCFPLVRWGWKQHNYRRQANSQQCHGSQNHRRCAGRKRAFNAA

[0043] <SEQ ID NO: 13: Amino acid sequence of protein derived from Leishmania enriettii> MLFDQRRVLTEDAPRRPYQNNASTASFEHRLLKGLHYGQRKLILSEIEFFLQIAMYTERRGSTASPKKVLIVYAGAACGFHLPFLFSLFPMLEFVLIDPAPMCPQVKAIASEEESCVLELIEDICTPELCLRIRRTYFETHDIFLVSDIRSGQPTRMSLNQEHTDMIVHDNNAQREWCFSLEVEAAMLKFHPPYPAAKDSNSGQDNAEDTTMEEYMYLD GTQLLGVWAPKSSSEVRLVVVGPFKRGYEPPTRRYYCTAHEEQCYAYNMDNRYDKDCAAERLILESYLSAFPGVYESVEQLSKTLSEKLEYSLFCPLEPGFTEQHARWV TLLYSTKKPSALQYFEILKNDMSVDQVAGLVTKYKDCKEIPTDVKVGSIELTSHFWSVLAAGDFAAVYCFPLVRWGWKQHSYRRQSNNQQCYGNHSRRRCAGRKRASSAA

[0044] <SEQ ID NO: 14: Amino acid sequence of protein derived from Phytomonas sp. EM1> MYFDSSRILDSRCPRTPYKPTSETLSLDSISVKGLHFGQRKLLLSEIEFLTAIFDKRKCDIIESDHKQRPVLVVYAGAANGSHLPILFRLFAEARFILIDPAPFCEEVEVIAEDVQGPIIELINDYCTDELCLRLSRMYGTQYDLYLISDIRTGVPKKMQKNIEHTEMMIRDNAAQRSWCWSLKAVAAMLKFHPPYPPSTDSSGQKCEDDDNTPDEIEY LDGIQLFGVWAPKSSSEVRLVVQGPFCHGYNAKMRKYNCKEHEEQCYHYNTSNRYLKDCAAERIIVDRYLKCIPNEYKNDITIVSNQISKDLKFPLFCPLENDFTEDHAR WVYLLYSSRRHDIMHLYEPFKDVMRFDTVKDLIKRYGQSEKVPEDAQVSGKPLSIDFWKAFCRCDFESAYSLPRIQWKFYPLLDPKRPHRRENKGAESSRNRTQFARKES

[0045] <SEQ ID NO: 15: Amino acid sequence of protein derived from Phytomonas sp. Hart1> MYFDSSRILDSRCPRTPYKPTSETLSLVNISVKGLHFGQRKLLLSEIEFLTAIFDKQKSDSVESSREQRPVLVVYPGAANGSHLPILFCLFKDVRFILIDPAPFCKELEVIAEDEDGPVIELINDYCTDELCLRISRMYGTQYDLYIVSDIRTGVPKKMQKNIEHTEMILHDNAIQRSWCWSLKAIAAMLKFHPPYPPSTDLSVQKCDLDDNTPNEIEYF DGIQLLGVWAPKSSSEVRLVVEGPFSHGHIAKTRKYNCRTHEEQCYHYNISNRYAKDCAAERVIMDRYLKCVSNKYGNNITALSNQISKDLRFPLFCPLEKDFTEDNARW LYLLYSSRRNNIIHLYEPFKNIMKFDTVKSLIKQYGESEKVPENAQVSGKQLSIDFWKDFCRCDFESAYSLPRIQWKFYGLLESRCTSYKREKRGSTSSFCNQVKFPRKEY

[0046] <SEQ ID NO: 16: Amino acid sequence of protein derived from Angomonas deanei> MFDKSRILSDESPPKVYEPSEASDWKKLTVKGLHYGQRKLLLSEVEVLTVLNSSPSLGGRPILVVYAGAAPGLHIPFLCTMFPQIKMVLIDPAPFSGNLQKLSEDSNGPILELINDVCTDELCIRLSRDYGETYYILFISDIRTGNPSGMKTNVEHTKMMEMDNNLQKSWCFSIRADAALLKFHPPYPAVTDKSSPAYDENDTTPSS ITYLDGVLLWGVWAPKSSSEIRLFVNQPFSATADVKERSYNCSEHEQKCYYYNTDSRYAKDCEAERLILQRYIDANLGGTWSCASLSEKISEELGFALFTPLA EGFSEDNARWVSLLYSTRNPANLSMFEQLRSVMTFKAVYHLVSTFKDSDSIPKNEKTDGVELGADFWKVFCRGNFAEAYSFPKFRWNFYSQLFGANKSVGKRRR

[0047] <SEQ ID NO: 17: Amino acid sequence of protein derived from Leptomonas seymouri> MEFTEGRVLADDAPRSTYQNNAATSSFEQPLLKGLHYGQRKLMLSEVEFFVAILKRIRAQGERAGDKKVLIVYAGAACGLHLPFLFSLFEDMQFILIDPAPFCAQVRDAAAKEGSCVLELIEDICTPELCLRRIRRSYCETHQLYLVSDIRSGAPTEMNQNQEHTEMIQRDNAAQREWCFSLEVEAAMLKFHPPYPTAQDPKSAKYNSADTTAENYVYLS GTELLGVWAPKSSSEVRLVVVGPFTKGYRAPSRTYSCTKHEEQCYAYNIDNRYEKDCNAERCILQSYLDLFSDKYSSVEQLSRTLSERLEYPLFCPLEPGFTEQHARWV SLLYSTGKPAALTFFAPLKEEMSVQRVAKLVVDYKDSSEIPTGVQIGGVELTRSFWVALAEADFGAVYCFPFFRWGWKRRLQQQKKAGGGRQRVAGTKRHAYECSQEGGL

[0048] <SEQ ID NO: 18: Amino acid sequence of protein derived from Bodo saltans> MDFPSFRQLVASSERRVYKPSRGSTFDGTVDVVKGLHYGQRKLILSEIEFLTAVLQAETDSTKPILVVYAGAANGSHLPHLFQLFPQVKFVLIDPAPFCEAVRRISQTDGPIVEIIEGYYCTDELCMRLKRSHQGEYRIVLVSDIRSGAPNRSTNKEHTEMI MRDNAWQRGWYSCLNAESAMLKFHPPYPKVTDPASPKYEPEDDTPNIMPYLEGKLLWGVWAPKSSSEVRLIVQGELKERL YDAVEFEEQCYFYNTTDRFVRDVEAERAILKSYVAYVKPDADDVDVLSKALSEFLGFPKFLPLQQSEDEARIISLLYLSKTR

[0049] <SEQ ID NO: 19: Amino acid sequence of protein derived from Acanthamoeba castellanii medusavirus> MSSTKRDRSCLDGELGAVRPERNKRPRYDERDYASRRRRSYEDELHAKRREEEDEDRRRRRRSHHHHRSHESSRCESRSHRHHDNPHVAQCDAPPTNIPFSRILAPDATRMPYRRRRRGEDKSVIHWGQRKLLMSEIEFLTRYGHTSSSRTVVYAGAAPGTHTNWLADIFPDLKFVLVDPNPFVAQPTDRVEIVKDYFTDETAQKYAGQSVLFIS DVRTANWREQDENAVEKHVMHDMLAQQRWVEIMKPSMSMLKFRLPYPDREGCQGSTEYLDGEVFLPVWGPQTTTETRLVTPGTAKRQWDHTVYEQQMFFFNTQVRVG LYDHDVRAVGLDRCYDCTSEVRVLREYLDKYGLESLPWARDMLRHRHRREPEDEYAADVGDEDDKRFVIGRLSEYISRICSPKGRTLQSAPVQGFNFDNTIRNHPLL

[0050] <SEQ ID NO: 20: Amino acid sequence of protein derived from Barrevirus sp.> VPINFIDLPTNFPYRENAKPNNIHIGQRKLLLNEVYFLSRFGNLSNTVVYAGAAPGTHISLLAELFPNHKFILYDPSRFNIKETDNIKIFTGEDEGYFTGPIAKQYANQNVLFISDIRSVKGITDFKEFESRVIID NELQKEWVTIMKPAKASLKFRIPFTIKEPYEYFDGLIEVQAWAPKTSSETRLITDGLKMKKYDPIAYE NKMFYFNNYIRPNKTYGNLSWDTAYEIFIWQQYFKKTNIDNQFIRDKIKDIMNKVTQVLNRDIRGKYL

[0051] <SEQ ID NO: 21: Amino acid sequence of protein derived from Brachionus calyciflorus> MSSADSIHTFQNKNSEYSKNFFRILTDDFPEEKYKPKEKDQDRSSVHYGQRKLFLSEVEFLTNVCCQVPKEKLFKKIVIIYAGAAPGIHIDFLNQLFPFMYFVLVDPAKMAVKTSEKIRIHQEYFTDETAQSLKKEFQHDLILFISDIRRFGPGAKLTDSEIEEIVEDMRNQKNWFKILNPFRALLKFRLPYVENRT NAQRFLEYLDGDVYFQIWPPNSSSETRLYVKENAGLKEYDCIKYENQLYRFNNNERVMCYEHEYLVDGLDHCYDCRAELYVFENYLENFSKIEALNRKV KITKLKVEDYVKQLNHHLEADKKFLRFSYNKQEYQIKFTEIKYGKKVEDLFIDENLAKNKFNKWEKRTFHFDHQKTKIPKNDMCLLKKRLNSTVIKRLNK

[0052] <SEQ ID NO: 22: Amino acid sequence of protein derived from Mimivirus LCMiAC01> MTNKRYLCTIDAKLYNTLKHNPHTIQFFCGVKNIYADSQKEYNEAFHDDKTLLYYYKRDRVPHALKKELLVNKPPYKNNKLPRKLEDIHTSIKYKGRQQIPETVLHWGQLKLLMGEMDFFNRYLNPKKKYTIVYAGAARGDHVILSLELYKNLNFVLVDPAKFNKDLYNKKRVRIINDYFTNDLARKLKKEYDNIIYISDIRTSTKEEDVKENHKMQK KWYEIMNPDYTLLKFRLPWDQDTFDYLDGEIYLQSFPPRTSTETRLVKKSAKIKTYDSKEYENKLFYYNLYDRARYYDHNYKVDDLYMDHCCDCTSTLNIIDDYLKSD FNEAFKSFSVEKLMKRIINLLAKYRKRGDILKIHYLELINKLSIRGSKHDSKFLIKSHSFDLYEHKYHKYKQKYLRLKYIHENLYKKKYLGGEASRLIKISNDVTKKEIV

[0053] 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 that utilize site-specific mutagenesis techniques for deletion, substitution, insertion, addition, and the like, such as 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.), can be used. Furthermore, 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.

[0054] Although the reason why the expression level and / or transmethylation activity of the proteins having the amino acid sequences of SEQ ID NOs: 2 to 22 are improved compared to the protein having the amino acid sequence of SEQ ID NO: 1 is unclear, it is highly likely that at least one common partial amino acid sequence selected from SEQ ID NO: 23 (KGLHYGQRKLLLSEIEF), SEQ ID NO: 24 (KFVLIDPAPFC), and SEQ ID NO: 25 (LFGVWAPKSSSE) in the amino acid sequences influences this effect. Proteins having a common amino acid sequence include proteins having the amino acid sequences of SEQ ID NOs: 2, 4, 5, 7 to 10, 12, 14, and 18, and proteins having the amino acid sequences of SEQ ID NOs: 2, 4, 5, 7 to 10, 12, 14, and 18 in which one or several amino acids have been deleted, substituted, inserted, and / or added.

[0055] The protein used in the methyltransferase according to the embodiment of the present invention preferably has an expression level ratio of 2.00 or more relative to the protein consisting of the amino acid sequence of SEQ ID NO: 1. In particular, an expression level ratio of 5.00 or more is superior to the protein consisting of the amino acid sequence of SEQ ID NO: 1 in mass productivity and is therefore easier to use industrially. Examples of proteins having an expression level ratio of 5.00 or more include proteins consisting of the amino acid sequences of SEQ ID NOs: 2, 3, 5 to 8, 11 to 14, 16, 17, and 20, and proteins consisting of the amino acid sequences of SEQ ID NOs: 2, 3, 5 to 8, 11 to 14, 16, 17, and 20 in which one or several amino acids have been deleted, substituted, inserted, and / or added.

[0056] The protein used in the methyltransferase according to the embodiment of the present invention preferably has a transmethylation activity ratio of 1.50 or more, more preferably 2.00 or more, relative to the protein consisting of the amino acid sequence of SEQ ID NO: 1. A transmethylation activity ratio of 1.50 or more is superior to the protein consisting of the amino acid sequence of SEQ ID NO: 1 in specific activity, making it suitable for industrial use. Examples of proteins having a transmethylation activity ratio of 1.50 or more include proteins consisting of the amino acid sequences of SEQ ID NOs: 2 to 7, 10 to 13, 15, and 16, as well as proteins consisting of the amino acid sequences of SEQ ID NOs: 2 to 7, 10 to 13, 15, and 16 in which one or several amino acids have been deleted, substituted, inserted, and / or added. The upper limit of the transmethylation activity ratio is not particularly limited, but may be, for example, 10.00, 8.00, or 6.00.

[0057] The methyltransferase according to the embodiment of the present invention preferably has methyl transfer activity toward RNA having three nucleotides linked from the 5' end (3-mer RNA). Conventional methyltransferases consisting of proteins with the amino acid sequence of SEQ ID NO: 1 were able to methylate the ribose hydroxyl group at the second nucleotide from the 5' end of RNA having at least 35 nucleotides linked from the 5' end (RNA of 35-mer or longer), but were unable to methylate RNA with a short chain length. In contrast, a methyltransferase selected from proteins with the amino acid sequences of SEQ ID NOs: 3, 7, 8, 11-14, 16, and 17, and proteins with the amino acid sequences of SEQ ID NOs: 3, 7, 8, 11-14, 16, and 17 in which one or more amino acids have been deleted, substituted, inserted, and / or added, can also methylate RNA having three nucleotides linked from the 5' end (3-mer RNA).

[0058] 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.

[0059] (2. Methylation Method) A methylation method according to an embodiment of the present invention is a method for methylating the hydroxyl group of ribose in the second nucleotide 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 a methyltransferase. The methyltransferase comprises the above-described protein. The methyltransferase comprising the above-described protein has higher expression level and / or methyltransfer activity than conventional methyltransferases, and is therefore capable of methylating the hydroxyl group of ribose in the second nucleotide from the 5' end of RNA at an industrial level.

[0060] The methyl group donor is not particularly limited, and S-adenosylmethionine (SAM) or the like can be used.

[0061] 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, a 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 can be, for example, 30 minutes to 50 hours. The concentrations of the methyl group donor, methyltransferase, and RNA in the reaction solution for the methylation reaction can be appropriately set depending on their types.

[0062] The RNA used for methylation can have a Cap0-type structure and / or a Cap1-type structure. By using RNA with a Cap0-type structure, the hydroxyl group of ribose at the second nucleotide from the 5'-end of RNA with a Cap0-type structure can be methylated. In this case, RNA with a Cap2-type structure can be produced by allowing an existing Cap1-transferase to coexist. Furthermore, by using RNA with a Cap1-type structure, RNA with a Cap2-type structure can be produced by methylating the hydroxyl group of ribose at the second nucleotide from the 5'-end of RNA with a Cap1-type structure.

[0063] The RNA used for methylation does not need to have a 5'-cap structure. In this case, by further adding a nucleoside triphosphate to the methylation reaction, capping of the 5'-end of the RNA can be simultaneously performed. This allows the 5'-end capping and methylation of the RNA to be performed in a single reaction step, significantly reducing the time and cost required to produce the desired RNA. When adding a nucleoside triphosphate, the concentration of the nucleoside triphosphate in the reaction solution can be appropriately determined depending on the types of other components. Furthermore, the reaction conditions are not particularly limited and can be performed according to general conditions.

[0064] (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 in the second nucleotide from the 5' end of RNA. For this use of a methyltransferase, a methyltransferase consisting of the above protein is used. The methyltransferase consisting of the above protein has a higher expression level and / or a higher methyltransfer activity than conventional methyltransferases, and is therefore capable of methylating the hydroxyl group of ribose in the second nucleotide from the 5' end of RNA at an industrial level.

[0065] (4. Kit) A kit according to an embodiment of the present invention is used for methylating the hydroxyl group of ribose in the second nucleotide from the 5' end of RNA. This kit includes a methyltransferase consisting of the above-described protein. The methyltransferase consisting of the above-described protein has a higher expression level and / or a higher methyltransfer activity than conventional methyltransferases, and is therefore capable of methylating the hydroxyl group of ribose in the second nucleotide from the 5' end of RNA on an industrial level. Therefore, this kit is useful for methylating the hydroxyl group of ribose in the second nucleotide from the 5' end of RNA.

[0066] The kit according to the embodiment of the present invention may further comprise a methyl donor in addition to the methyltransferase. Since the methyl donor is a necessary component for the methylation reaction, the kit is useful for methylating the hydroxyl group of ribose in the penultimate nucleotide from the 5' end of RNA.

[0067] 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.

[0068] Expression and crude purification of proteins consisting of the amino acid sequences of SEQ ID NOS: 1 to 22, and evaluation of expression levels. 1 μL of each protein expression plasmid vector solution was added to 25 μL of E. 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.

[0069] The target 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. This operation of allowing the liquid to flow completely under its own weight was repeated twice. Then, the crude enzyme solution was added and the column was allowed to flow completely. As a washing operation, 5 mL of equilibration buffer was added and the operation of allowing the liquid to flow completely 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 allowing the liquid to flow completely 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 protein concentration in the resulting crude enzyme solution was measured using the Bradford method (Bio-Rad). The enzyme (protein) expression level ratio was determined by calculating the ratio of the protein concentration in the crude enzyme solution containing the protein consisting of the amino acid sequence of SEQ ID NO: 1 to that of the standard.

[0070] The molecular weight of the protein obtained above was determined by SDS-PAGE. Specifically, an SDS-PAGE gel was prepared and run, 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. Furthermore, the query coverage and sequence identity (Per Ident) of the proteins consisting of the amino acid sequences of SEQ ID NOs: 2 to 22 relative to the protein consisting of the amino acid sequence of SEQ ID NO: 1 were determined according to the above method. The obtained protein (enzyme) was collected from the crude enzyme solution by centrifugation using an ultrafiltration membrane, stored at -20°C, and used in the following evaluations.

[0071] <Evaluation 1 of the methyl transfer activity of proteins consisting of the amino acid sequences of SEQ ID NOs: 1 to 22> 60mer Cap1 type mRNA (m 7 GpppAmGACCAUCAUCAUCAUCAUCAUCUACUACAUCUCGAGGAUCCUCUAGAGUCGACCUG: SEQ ID NO: 26, m 7 where m represents the methyl group attached to the 7th position of the guanosine base, and m represents the O-methyl group at the 2'-position of ribose. Thermal denaturation was performed on 1 μL of a nucleic acid water (NFW) solution containing 0.3 mmol / L of SAM. The solution was heated at 65°C for 10 minutes and then cooled to 8°C. Next, an enzyme water (NFW) solution containing 0.3 mg / mL of each protein was prepared, and 2.5 μL of the enzyme solution was added to the nucleic acid solution. Next, 0.75 μL of a 0.5 mmol / L NFW solution of S-adenosylmethionine (SAM), 0.75 μL of a 50 mmol / L NFW solution of Tris-HCl (pH 7.5), 0.5 μL of ribonuclease inhibitor, and 4.5 μL of NFW were further added to the nucleic acid solution, mixed, and then reacted at 37°C for 15 hours. Next, 40 μL of eluent A [20 mmol / L hexafluoroisopropanol (HFIP) and 20 mmol / L triethylamine acetate (TEA-A) aqueous solution (pH 7.0)] was added to 10 μL of the reaction mixture, and the mixture was centrifuged at 4°C and 15,000 rpm (22,400 × g centrifugal force) for 10 minutes. The supernatant was then collected and subjected to LC analysis. In the LC analysis, SAH (S-adenosylhomocysteine: methyl group donor) produced by the methylation reaction was quantified. The transmethylation activity ratio was determined by calculating the ratio of the transmethylation activity of the protein consisting of the amino acid sequence of SEQ ID NO: 1 to that of the standard.

[0072] The LC 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

[0073] <Evaluation 2 of the methyl transfer activity of proteins consisting of the amino acid sequences of SEQ ID NOS: 1 to 22> 3mer Cap0 type mRNA (m 7 Using GpppGAA as a substrate, we investigated whether methylation of the ribose hydroxyl group in the second nucleotide from the 5' end proceeds. First, a reaction solution was prepared by mixing 2 μL of a nucleic acid solution containing 0.3 mmol / L Cap0 mRNA, 1 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 8.0 at 25°C), 1 μL of a 10 mmol / L NFW diluted solution of S-adenosylmethionine (SAM), 5 μL of water (NFW), and 1 μL of a 0.3 mg / mL NFW solution of each protein. The reaction solution was then incubated at 37°C for 14 hours. Next, 40 μL of [eluent A: 20 mmol / L hexafluoroisopropanol (HFIP) and 20 mmol / L triethylamine acetate (TEA-A) aqueous solution (pH 7.0)] was added to the reaction solution, and the mixture was filtered through a filter with a pore size of 0.45 μm and placed in an HPLC vial, followed by liquid chromatography (LC) analysis.

[0074] In the LC analysis, a peak of mRNA in which the hydroxyl group of the ribose in the second nucleotide from the 5' end was methylated was confirmed, and an x ​​was confirmed when such a peak was not confirmed.

[0075] The results of the above evaluations are shown in Table 1.

[0076]

[0077] As shown in Table 1, it was confirmed that the proteins consisting of the amino acid sequences of SEQ ID NOs: 2 to 22 have higher expression level ratios and / or methyl group transfer activity ratios than the protein consisting of the amino acid sequence of SEQ ID NO: 1, and are easier to use industrially.

[0078] <Evaluation of translation activity> A 3075 nt mRNA encoding LacZ (β-galactosidase) was synthesized by in vitro transcription and purified using NucleoSpin (registered trademark) RNA (manufactured by Takara Bio Inc.). This mRNA was capped with an m-terminus at the 5' end using Vaccinia Capping Enzyme (manufactured by Takara Bio Inc.). ７ A G Cap structure (Cap0) was added, and the resulting mixture was purified again using NucleoSpin (registered trademark) RNA to obtain Cap0-type mRNA (SEQ ID NO: 27: Figure 1). 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 an RNase inhibitor, and 1 μL of a 0.3 mg / mL NFW solution of the protein of SEQ ID NO: 2, 3, 8, or 14. Next, the reaction solution was 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 was methylated (hereinafter referred to as "Cap02-type mRNA"). For comparison, Cap1-type mRNA was obtained under the same conditions as above, except that a Cap1 transferase (mRNA Cap2'-O-Methyltransferase manufactured by Takara Bio Inc.) was used. For comparison, the Cap0-type mRNA obtained above was also prepared.

[0079] 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 50 ng / μL of each mRNA (Cap0 mRNA, Cap1 mRNA, and Cap02 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). Furthermore, translation activity was evaluated by quantifying LacZ (β-galactosidase) using the Beta-GLO (registered trademark) assay system manufactured by Promega. The results are shown in Figure 2. The translation activity results for Cap02-type mRNA are the average values ​​of the translation activity results for each Cap02-type mRNA synthesized using the protein of SEQ ID NO: 2, 3, 8, or 14. As shown in Figure 2, Cap02-type mRNA had superior translation activity to Cap0-type mRNA and Cap1-type mRNA.

[0080] As can be seen from the above results, the present invention can provide a methyltransferase, a methylation method, use of the methyltransferase, and a kit that have high expression levels and / or methyltransferase activity and can be used industrially.

Claims

1. A methyltransferase for methylating the hydroxyl group of ribose in the second nucleotide from the 5' end of RNA, wherein the product of the methyltransfer activity ratio and the expression level ratio relative to a protein consisting of the amino acid sequence of SEQ ID NO: 1 is 1.20 or more.

2. The methyltransferase according to claim 1, wherein the methyltransferase is selected from proteins consisting of the amino acid sequence of SEQ ID NOs: 2 to 22 and proteins consisting of the amino acid sequence of SEQ ID NOs: 2 to 22 in which one or several amino acids have been deleted, substituted, inserted and / or added.

3. The methyltransferase according to claim 1, wherein the expression level ratio of said protein to a protein consisting of the amino acid sequence of SEQ ID NO: 1 is 5.00 or more.

4. The methyltransferase according to claim 3, wherein the methyltransferase is selected from proteins consisting of the amino acid sequences of SEQ ID NOs: 2, 3, 5 to 8, 11 to 14, 16, 17, and 20, and proteins consisting of the amino acid sequences of SEQ ID NOs: 2, 3, 5 to 8, 11 to 14, 16, 17, and 20 in which one or several amino acids have been deleted, substituted, inserted, and / or added.

5. The methyltransferase of claim 1, wherein the protein comprises at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 23-25.

6. The methyltransferase according to claim 5, wherein the methyltransferase is selected from proteins consisting of the amino acid sequences of SEQ ID NOs: 2, 4, 5, 7 to 10, 12, 14 and 18, and proteins consisting of the amino acid sequences of SEQ ID NOs: 2, 4, 5, 7 to 10, 12, 14 and 18 in which one or several amino acids have been deleted, substituted, inserted and / or added.

7. The methyltransferase according to claim 1, wherein the methyltransferase activity ratio of said protein to a protein consisting of the amino acid sequence of SEQ ID NO: 1 is 1.50 or more.

8. The methyltransferase according to claim 7, wherein the methyltransferase is selected from proteins consisting of the amino acid sequences of SEQ ID NOs: 2 to 7, 10 to 13, 15 and 16, and proteins consisting of the amino acid sequences of SEQ ID NOs: 2 to 7, 10 to 13, 15 and 16 in which one or several amino acids have been deleted, substituted, inserted and / or added.

9. The methyltransferase according to claim 1, which has methyltransfer activity on RNA having three nucleotides linked from the 5' end.

10. The methyltransferase according to claim 9, wherein the methyltransferase is selected from proteins consisting of the amino acid sequences of SEQ ID NOs: 3, 7, 8, 11 to 14, 16 and 17, and proteins consisting of the amino acid sequences of SEQ ID NOs: 3, 7, 8, 11 to 14, 16 and 17 in which one or several amino acids have been deleted, substituted, inserted and / or added.

11. A method for methylating the hydroxyl group of ribose in the second nucleotide 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 a methyltransferase, wherein the methyltransferase is the methyltransferase according to any one of claims 1 to 10.

12. The method of claim 11, wherein the RNA used for the methylation has a Cap0 type structure and / or a Cap1 type structure.

13. The method according to claim 11, wherein a nucleoside triphosphate is further present in the methylation reaction, and the 5' end of the RNA is simultaneously capped.

14. Use of the methyltransferase according to any one of claims 1 to 10 for methylating the hydroxyl group of ribose in the second nucleotide from the 5' end of RNA.

15. A kit used for methylating the hydroxyl group of ribose in the second nucleotide from the 5' end of RNA, the kit comprising the methyltransferase according to any one of claims 1 to 10.

16. The kit of claim 15, further comprising a methyl group donor.

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