Methyltransferase for methylation of ribose hydroxyl group in 1st and 2nd nucleotides from 5' end of RNA, methylation method, use of methyltransferase, and kit

Abstract

The present invention provides a methyltransferase for methylation of a ribose hydroxyl group in the 1st and 2nd nucleotides from the 5' end of RNA.

Description

Methyltransferase for methylating the hydroxyl group of ribose in the first and second nucleotides from 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 first and second nucleotides from the 5' end 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] Although methyltransferases that methylate the hydroxyl group of ribose in the first nucleotide from the 5' end of RNA (hereinafter sometimes referred to as "Cap1 transferase") and methyltransferases that methylate the hydroxyl group of ribose in the second nucleotide from the 5' end of RNA (hereinafter sometimes referred to as "Cap2 transferase") are known, no methyltransferases that methylate the hydroxyl groups of ribose in both the first and second nucleotides from the 5' end of RNA are known.

[0005] The present invention has been made to solve the above-mentioned problems, and aims to provide a methyltransferase capable of methylating the hydroxyl groups of ribose in both the first and second nucleotides from the 5' end of RNA, 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 as a result have discovered a methyltransferase that can methylate the hydroxyl groups of ribose in both the first and second 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 groups of ribose in the first and second 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 groups of ribose in the first and second 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 has a Cap0 structure.

[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 groups of ribose in the first and second nucleotides from the 5' end of RNA.

[0013] <7> A kit used for methylating the hydroxyl groups of ribose in the first and second 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] According to the present invention, it is possible to provide a methyltransferase capable of methylating the hydroxyl group of ribose in both the first and second nucleotides from the 5' end of RNA, a methylation method, use of the methyltransferase, and a kit.

[0016] LC analysis results in an example. The top row shows the LC analysis results when a methylation reaction was performed using a protein consisting of the amino acid sequence of SEQ ID NO: 1, the middle row shows the LC analysis results when a methylation reaction was performed using a protein consisting of the amino acid sequence of SEQ ID NO: 2, and the bottom row shows the LC analysis results of Cap0-type mRNA that was not subjected to a methylation reaction. LC analysis results in an example where a methylation reaction was performed using a protein consisting of the amino acid sequence of SEQ ID NO: 1. LC analysis results in an example where a methylation reaction was performed using a protein consisting of the amino acid sequence of SEQ ID NO: 2.

[0017] 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.

[0018] (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 first and second nucleotides 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 preferably (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 is 80% or more homologous to the protein consisting of the amino acid sequence of (a).

[0019] Here, the protein consisting of the amino acid sequence of SEQ ID NO: 1 is derived from Barrevirus sp. (SEQ ID NO: 1) VPINFIDLPTNFPYRENAKPNNIHIGQRKLLLNEVYFLSRFGNLSNTVVYAGAAPGTHISLLAELFPNHKFILYDPSRFNIKETDNIKIFTGEDEGYFTGPIAKQYANQNVLFISDIRSVKGITDFKEFESRVIID NELQKEWVTIMKPAKASLKFRIPFTIKEPYEYFDGLIEVQAWAPKTSSETRLITDGLKMKKYDPIAYE NKMFYFNNYIRPNKTYGNLSWDTAYEIFIWQQYFKKTNIDNQFIRDKIKDIMNKVTQVLNRDIRGKYL

[0020] The methyltransferase according to the embodiment of the present invention can methylate the hydroxyl groups of ribose in both the first and second nucleotides from the 5' end of RNA. In particular, whereas conventional methods required the production of Cap1-type RNA using a Cap1-transferase and then the production of Cap2-type RNA using a Cap2-transferase, the use of the methyltransferase according to the embodiment of the present invention makes it possible to produce Cap2-type RNA, thereby eliminating the need to produce Cap1-type RNA using a Cap1-transferase. Therefore, the time and cost required for producing Cap2-type RNA can be significantly reduced.

[0021] 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.

[0022] 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.

[0023] 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.

[0024] (2. Methylation Method) A methylation method according to an embodiment of the present invention is a method for methylating the hydroxyl groups of ribose in the first and second nucleotides from the 5' end of RNA. This methylation method comprises carrying out a methylation reaction of RNA in the presence of a methyl donor and the above-described methyltransferase. The above-described methyltransferase can methylate the hydroxyl groups of ribose in both the first and second nucleotides from the 5' end of RNA, thereby enabling the production of Cap2-type RNA in a one-step reaction. Therefore, the time and cost required for producing Cap2-type RNA can be significantly reduced.

[0025] The methyl group donor is not particularly limited, and S-adenosylmethionine (SAM) or the like can be used.

[0026] 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.

[0027] The RNA used for methylation can have a Cap0-type structure. By using the Cap0-type structure, a Cap2-type structure can be produced in a one-step reaction.

[0028] Nucleoside triphosphates may also be added to the methylation reaction to simultaneously cap the 5'-end of the RNA. This allows for capping of the 5'-end of the RNA and Cap2 formation in a single reaction step, significantly reducing the time and cost required to produce Cap2-type 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 the reaction may be carried out under general conditions.

[0029] (3. Use of Methyltransferase) The use of a methyltransferase according to an embodiment of the present invention is for methylating the hydroxyl groups of ribose in the first and second 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 groups of ribose in both the first and second nucleotides from the 5' end of RNA, and therefore Cap2-type RNA can be produced in a one-step reaction. Therefore, the time and cost required for producing Cap2-type RNA can be significantly reduced.

[0030] (4. Kit) A kit according to an embodiment of the present invention is used for methylating the hydroxyl groups of ribose in the first and second nucleotides from the 5' end of RNA. This kit includes the above-described methyltransferase. The above-described methyltransferase can methylate the hydroxyl groups of ribose in both the first and second nucleotides from the 5' end of RNA, thereby enabling the production of Cap2-type RNA in a one-step reaction. Therefore, this kit is useful for methylating the hydroxyl groups of ribose in both the first and second nucleotides from the 5' end of RNA.

[0031] The kit according to the embodiment of the present invention may further comprise a methyl donor in addition to the methyltransferase. Because the methyl donor is a necessary component for the methylation reaction, the kit is useful for methylating the hydroxyl groups of ribose at both the first and second nucleotides from the 5' end of RNA.

[0032] 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.

[0033] Preparation of Protein Comprising the Amino Acid Sequence of SEQ ID NO: 1: 1 μL of a plasmid vector solution for expressing a protein comprising 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.

[0034] 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 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 32.9 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.

[0035] <Preparation of a protein consisting of the amino acid sequence of SEQ ID NO: 2> A protein consisting of the amino acid sequence of SEQ ID NO: 2, derived from Trypanosoma brucei, was prepared as a known methyltransferase. (SEQ ID NO: 2: Amino acid sequence of the protein derived from Trypanosoma brucei) PGDNFPDSRVLSDEFPRRKYKPSSATRLEDIVLKGLHYGQRKLLLSEIEFLSAYLEGRQAGAKPTLVVYAGAANGSHLPFLFQLFEAVKFVLIDPAPFCDAVRE ISLNKQGPILDLVQGFCTDELCKQLSSSYGSTYDILLVSDIRSGVPEKQSNRENTLMIMRDNDDMQRSWWTLKAEAALLKFHPPYPPCRDRNSRHYDEADDTPES IEYLDGVRLFGVWAPKSSSELRLCVQGPFLQGTSFPMRRYDCTTHEEQCYFYNTDNRYTRDCAAESGILERYLQLFPSAPYSDPTALSMTISNFLGFPLFLPLDSSFSESDARWVTLLYSTRIPTCLELFTTLRGRVTHTVMKQLAEEWQSATTVPKGVSIDSVELTSEFWKAVCAGDLTEAYSFPNIRWRFANLLISRRRKRSASDRAP The protein was prepared in the same manner as above, except that the type of plasmid vector solution for protein expression was changed. The molecular weight of the protein obtained in the same manner as above was 48 kDa.

[0036] <Evaluation of Methylation Reaction 1> Using 5mer RNA (5'pppGAAAA) as a substrate, we investigated whether methylation of the ribose hydroxyl groups at the first and second nucleotides from the 5' end proceeded. First, 10 μL of a nucleic acid solution containing 0.5 mmol / L RNA, 5 μL of a 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), 4.5 μL of a 10 mmol / L NFW diluted solution of S-adenosylmethionine (SAM), 2.5 μL of a 10 mmol / L NFW diluted solution of guanosine triphosphate, 24 μL of water (NFW), and 4 μL of VCE (Vaccinia Capping Enzyme; Takara Bio Inc.) were mixed to prepare a reaction solution, and the reaction solution was allowed to react at 37 °C for 4 hours. Next, 5 μL of the reaction solution was dispensed into 8 tubes, and 2 μL of a 0.3 mg / mL NFW solution of each protein was added thereto and allowed to react overnight at 37° C. Next, 93 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 0.45 μm pore filter, placed in an HPLC vial, and then subjected to liquid chromatography (LC) analysis.

[0037] 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

[0038] A graph showing the results of the above LC analysis is shown in Figure 1. In Figure 1, the upper row shows the results of LC analysis when a methylation reaction was performed using a protein consisting of the amino acid sequence of SEQ ID NO: 1, the middle row shows the results of LC analysis when a methylation reaction was performed using a protein consisting of the amino acid sequence of SEQ ID NO: 2, and the lower row shows the results of LC analysis of Cap0-type mRNA that was not subjected to a methylation reaction. In Figure 1 and other figures, the peak of Cap0-type mRNA is referred to as "Cap0," the peak of Cap0-type mRNA in which the hydroxyl group of the ribose at the first nucleotide from the 5' end is methylated (Cap1-type mRNA) is referred to as "Cap01," the peak of Cap0-type mRNA in which the hydroxyl group of the ribose at the second nucleotide from the 5' end is methylated is referred to as "Cap02," and the peak of Cap0-type mRNA in which the hydroxyl groups of the ribose at both the first and second nucleotides from the 5' end are methylated (Cap2-type mRNA) is referred to as "Cap012" (the same applies hereinafter).

[0039] As shown in Figure 1, when the methylation reaction was performed using a protein consisting of the amino acid sequence of SEQ ID NO: 1, the hydroxyl groups of the ribose at both the first and second nucleotides from the 5' end were methylated (Cap2-type mRNA was obtained), whereas when the methylation reaction was performed using a protein consisting of the amino acid sequence of SEQ ID NO: 2, only the hydroxyl group of the ribose at the second nucleotide from the 5' end was methylated.

[0040] <Evaluation of methylation reaction 2> 5mer Cap0 mRNA (m 7The progress of the methylation reaction using proteins (methyltransferases) consisting of the amino acid sequences of SEQ ID NOS: 1 and 2 was investigated using a substrate (GpppGAAAA). First, 5 μL of a nucleic acid solution containing 0.3 mmol / L Cap0 mRNA, 5 μL of capping buffer (NEB), 5 μL of a 10 mmol / L NFW diluted solution of S-adenosylmethionine (SAM), 27 μL of water (NFW), 3 μL of ribonuclease inhibitor, and 5 μL of each protein (methyltransferase) were mixed and reacted at 37°C. LC analysis was performed at 0, 25, 60, 120, 180, 240, 1230, and 1600 minutes. For LC analysis, 5 μL of the mixture (reaction solution) was taken, mixed with 45 μL of eluent A [20 mmol / L hexafluoroisopropanol (HFIP) and 20 mmol / L triethylamine acetate (TEA-A) aqueous solution (pH 7.0)], filtered through a filter with a pore size of 0.45 μm, and placed in an HPLC vial. The LC analysis conditions were the same as those described above.

[0041] Graphs showing the results of LC analysis are shown in Figures 2 and 3. Figure 2 shows the results of LC analysis when a methylation reaction was carried out using a protein consisting of the amino acid sequence of SEQ ID NO: 1, and Figure 3 shows the results of LC analysis when a methylation reaction was carried out using a protein consisting of the amino acid sequence of SEQ ID NO: 2.

[0042] As shown in Figure 2, in the methylation reaction using the protein consisting of the amino acid sequence of SEQ ID NO: 1, Cap01 was produced in large amounts in the early stages, but the production of Cap012 increased over time. In contrast, in the methylation reaction using the protein consisting of the amino acid sequence of SEQ ID NO: 2, as shown in Figure 3, no change was observed in the amount of Cap02 produced in the early stages, and the production of Cap012 was not confirmed even over time.

[0043] As can be seen from the above results, the present invention provides a methyltransferase capable of methylating the hydroxyl groups of ribose in both the first and second nucleotides from the 5' end of RNA, a methylation method, use of the methyltransferase, and a kit.

Claims

1. A methyltransferase that methylates the hydroxyl groups of ribose in the first and second 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 groups of ribose in the first and second 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 has a Cap0 type structure.

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 groups of ribose in the first and second nucleotides from the 5' end of RNA.

7. A kit used for methylating the hydroxyl groups of ribose in the first and second 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.

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