5'-Capped Oligonucleotides for RNA Synthesis

Abstract

The present invention relates to a novel oligonucleotide primer for use in synthesizing 5'-capped RNA. In the present invention, the novel oligonucleotide primer for RNA capping provided by Chemical Formula 1 can be useful in the field of nucleic acid therapeutics or vaccines.

Description

[Technical field]

[0001] Technical Field The present invention relates to oligonucleotides for the synthesis of 5'-capped RNA.

[0002] Background technology 5'-capping of vRNA is the first step in pre-mRNA processing in eukaryotic cells, in which a cap is placed on the 5' end of the transcribed RNA. The 5' end of eukaryotic mRNA molecules contains the 7-methylguanosine (mGU) residue. 7 A 5' cap (cap-0) is generated in which a 5' cap is linked to a 5' ribose sugar (G) via a 5' triphosphate bond, which is mediated in vivo by methyltransferase enzymes. In eukaryotes, mRNA can have further modifications at the 5' end, including methylation of the 2' hydroxyl group of the first ribose sugar (cap-1) and methylation of the 2' hydroxyl group of the second ribose sugar (cap-2). Through this process of 5' capping of mRNA, the mRNA acquires resistance to degradative enzymes such as 5' exonucleases when it leaves the nucleus and enters the cytoplasm.

[0003] For medical or research purposes, capped mRNA encoding a specific gene can be transfected into eukaryotes or microinjected into cells or embryos to artificially express said gene. However, if uncapped RNA is used in this process, not only will the RNA be rapidly degraded, but it will also be immunogenic and the efficiency of protein translation will be rapidly reduced. Therefore, it is very important that 5' capping is properly performed in order to synthesize mRNA in vitro.

[0004] The process of synthesizing mRNA by first transcribing RNA with a 5'-triphosphate group in vitro and then 5'-capping it with a capping enzyme has proven to be costly and inefficient. This has led to the development of a synthetic method in which an oligonucleotide with a 5'-capping structure is produced and used as a primer to initiate in vitro transcription. For example, International Publication No. WO2008 / 016473 and International Publication No. WO2013 / 059475 disclose dinucleotide mRNA cap analogs for synthesizing 5'-capped RNA, and International Publication No. WO2017 / 053297 discloses trinucleotide mRNA cap analogs for synthesizing 5'-capped mRNA.

[0005] Since capped RNA transcripts can be applied to therapeutic and / or preventive fields requiring protein synthesis, such as nucleic acid therapeutics and vaccines, it is necessary to develop oligonucleotide primers for 5'-capping to efficiently prepare them. For example, there is a pressing need for the development of RNA cap analogs that have simpler and more cost-effective manufacturing processes than conventional methods, efficiently advance the desired transcription reaction, improve the yield of RNA production, reduce the production of unwanted heterogeneous products, do not require additional enzymatic reactions, or enable large-scale synthesis of RNA. Furthermore, if a cap structure is provided that can maintain mRNA stability in vivo, improve translation efficiency, or reduce side effects such as immunogenicity when applied as an actual RNA therapeutic or vaccine, it should be possible to achieve universal usefulness in the RNA therapeutic or vaccine field.

[0006] Summary of the Invention Problem to be solved by the invention An object of the present invention is to provide a novel oligonucleotide primer for RNA capping, a method for producing the same, and uses thereof. Another object of the present invention is to provide an RNA molecule produced using the oligonucleotide primer for RNA capping, and uses thereof.

[0007] Means for solving the problem In order to achieve the above object, the present inventors provide a novel oligonucleotide primer for RNA capping, its manufacturing method and its use method. Unless otherwise specified, all terms used in this application have the same meaning as commonly understood by those of ordinary skill in the art to which this invention belongs. All patent and non-patent literature referenced throughout the disclosure of this application is incorporated by reference in its entirety.

[0008] Novel RNA capping oligonucleotide primers The present invention provides a compound represented by the following chemical formula 1, a stereoisomer thereof, or a salt thereof: [ka] In the above chemical formula, B1 and B2 are each independently a natural, non-natural or modified nucleoside base; X1 is -OH, -O(C 1-4 -alkyl), or -halo; X2 is -H or combines with X1 to form an LNA ring, where one or more H of the LNA ring is -(C 1-4 alkyl), -OH, or -O(C 1-4 alkyl) may be substituted}; Y1 is -O(C 1-4 alkyl), -O(C 1-4 Alkyl)O(C 1-4 alkyl), -CH2O(C 1-4 -alkyl), or -halo; Y2 is -H or combines with Y1 to form an LNA ring, where one or more H of the LNA ring is -(C 1-4 alkyl), -OH, or -O(C 1-4 alkyl) may be substituted}; Z1 and Z2 each independently represent -OH, -O(C 1-4 alkyl), -O(C1-4 Alkyl)O(C 1-4 alkyl), -CH2O(C 1-4 -alkyl), or -halo; Z3 is -H or combines with Z1 to form an LNA ring, where one or more H of the LNA ring is -(C 1-4 alkyl), -OH, or -O(C 1-4 alkyl) may be substituted}; n is an integer from 0 to 3; m is an integer of 1 to 10 {wherein, when m is 2 or more, each of B2, Z1 and Z2 may be different from each other}; R1 and R2 are each independently -H or -(C 1-4 alkyl); R3 is -(C 1-4 alkyl).

[0009] In the present invention, the nucleoside base may be not only a natural nucleoside base, but also a non-natural or modified nucleoside base.

[0010] In the present invention, the most common base rings in naturally occurring nucleosides are the purine and pyrimidine rings. Naturally occurring purine rings include, for example: [ka] Naturally occurring pyrimidine rings include, for example: [ka] Includes.

[0011] According to an embodiment of the invention, the non-natural or modified nucleoside base is an isomer of the natural nucleoside base or one or more -H of the natural nucleoside base is -(C 1-4 alkyl), -O(C 1-4and optionally substituted with one or more selected from the group consisting of -alkyl) or -halo, one or more =CH- are replaced with =N-, and one or more =O are replaced with =S.

[0012] In the present invention, the unnatural or modified nucleoside base may also be a base derived from a nucleoside analogue with an artificial base that can be recognized by RNA polymerase as a substitute for either the natural NTP (e.g., ATP, UTP, CTP and GTP) or other specific NTP (see literature [Loakes, D., Nucleic Acids Res., 29: 2437-2447 (2001); Crey-Desbiolles, C., et al., Nucleic Acids Res., 33: 1532-1543 (2005); Kincaid, K., et al., Nucleic Acids Res., 33: 2620-2628 (2005); Preparata, FP, Oliver, JS, J. Comput. Biol. 753-765 (2004); and Hill, F., et al., Proc Natl. 1999, 14: 1111-1115 (2004)). Acad. Sci. USA, 95:4258-4263 (1998)] etc.

[0013] In addition, in the present invention, the non-natural or modified nucleoside base is a halogen-substituted purine (e.g., 6-fluoropurine), a halogen-substituted pyrimidine, N 6 -Ethyladenine, N 4 The base may be derived from, but is not limited to, -(alkyl)-cytosine, 5-ethylcytosine, and the like.

[0014] In the present invention, other examples of the unnatural or modified nucleoside base include 8-azaguanosine, pseudouridine (ψ), 5-methyl-cytidine (m 5 C), 1-methyl-pseudouridine (m 1 ψ), 1-methyl-pseudouridine (m 1 ψ) and 5-methyl-cytidine (m 5 C), 2-thiouridine (s 2 U), 2-thiouridine (s2 U) and 5-methyl-cytidine (m 5 C), 5-methoxy-uridine (mo 5 U), 5-methoxy-uridine (mo 5 U) and 5-methyl-cytidine (m 5 C), 2'-O-methyluridine, 2'-O-methyluridine and 5-methyl-cytidine (m 5 C), N 6 -Methyl-adenosine (m 6 A) or N 6 -Methyl-adenosine (m 6 A) and 5-methyl-cytidine (m 5 C), but is not limited to these.

[0015] In addition, examples of natural, unnatural or modified nucleoside bases that can be used in the present invention can be found in International Publication WO2018 / 144775 or International Publication WO2018 / 144082.

[0016] According to an embodiment of the present invention, B1 and B2 are each independently [ka] may be also possible.

[0017] According to a further embodiment of the present invention, X1 is -OH; X2 may be -H.

[0018] According to a further embodiment of the present invention, Y1 is -O(C 1-4 alkyl) or -O(C 1-4 Alkyl)O(C 1-4 -alkyl), or -halo; Y2 may be -H or may combine with Y1 to form an LNA ring, where one or more H of the LNA ring is -(C 1-4 alkyl).

[0019] According to a further embodiment of the present invention, Z1 is -OH or -O(C 1-4 alkyl); Z2 is -OH; Z3 may be -H.

[0020] Also, according to an embodiment of the present invention, n may be 0 or 1.

[0021] Also, according to an embodiment of the present invention, m may be 1.

[0022] According to a further embodiment of the present invention, R1 and R2 are each independently -H; R3 is -(C 1-4 It may be alkyl.

[0023] Specifically, the compound represented by Chemical Formula 1 may be any one selected from the group consisting of the following compounds: [ka] JPEG0007644229000006.jpg190161 JPEG0007644229000007.jpg177161 JPEG0007644229000008.jpg170161

[0024] According to an embodiment of the present invention, the compound represented by Formula 1 may be one or more selected from the group consisting of the following compounds: 2-amino-9-((2R,3R,4S,5R)-5-((((((((2R,3R,4R,5R)-3-(((((2R,3S,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phospho R(6-amino-9H-purin-9-yl)-4-fluorotetrahydrofuran-2-yl)methoxy)(hydroxy)phospho R (oxy)(hydroxy)phospho R (oxy)(hydroxy)phospho R (oxy)methyl)-3,4-dihydroxytetrahydrofuran-2-yl)-7-methyl-6-oxo-6,9-dihydro-1H-purin-7-ium (oligonucleotide 1) 2-amino-9-((2R,3R,4S,5R)-5-((((((((2R,3R,4R,5R)-3-(((((2R,3S,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-5-(6-amino-9H-purin-9-yl)-4-(2-methoxyethoxy)tetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)methyl)-3,4-dihydroxytetrahydrofuran-2-yl)-7-methyl-6-oxo-6,9-dihydro-1H-purin-7-ium (oligonucleotide 2); 2-Amino-9-(( Round 2, Round 3, Round 4, Round 5 )-5-(((((((((1S,3R,4R,6S,7S)-7-(((((2R,3S,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-3-(6-amino-9H-purin-9-yl)-6-methyl-2,5-dioxabicyclo[2.2.1]heptan-1-yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)methyl)-3,4-dihydroxytetrahydrofuran-2-yl)-7-methyl-6-oxo-6,9-dihydro-1H-purin-7-ium (oligonucleotide 3); 2-Amino-9-(( Round 2, Round 3, Round 4, Round 5)-5-(((((((((2R,3R,4R,5R)-3-(((((2R,3S,4R,5R)-5-(5-amino-7-oxo-6,7-dihydro-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-5-(6-amino-9H-purin-9-yl)-4-methoxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy) ( Hydroxy)phosphoryl)oxy)methyl)-3,4-dihydroxytetrahydrofuran-2-yl)-7-methyl-6-oxo-6,9-dihydro-1H-purin-7-ium (oligonucleotide 4); 2-Amino-9-(( Round 2, Round 3, Round 4, Round 5 )-5-(((((((((1R,3R,4R,7S)-7-(((((2R,3S,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-3-(6-amino-9H-purin-9-yl)-2,5-dioxabicyclo[2.2.1]heptan-1-yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy) ( Hydroxy)phosphoryl)oxy)methyl)-3,4-dihydroxytetrahydrofuran-2-yl)-7-methyl-6-oxo-6,9-dihydro-1H-purin-7-ium (oligonucleotide 5); 2-Amino-9-(( Round 2, Round 3, Round 4, Round 5 )-5-(((((((((2R,3R,4R,5R)-3-(((((2R,3S,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-5-(6-amino-9H-purin-9-yl)-4-fluorotetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy) (Hydroxy)phosphoryl)oxy)methyl)-3-hydroxy-4-methoxytetrahydrofuran-2-yl)-7-methyl-6-oxo-6,9-dihydro-1H-purin-7-ium (oligonucleotide 6); 2-Amino-9-(( Round 2, Round 3, Round 4, Round 5 )-5-(((((((((2R,3R,4R,5R)-3-(((((2R,3S,4R,5R)-5-(5-amino-7-oxo-6,7-dihydro-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-5-(6-amino-9H-purin-9-yl)-4-fluorotetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy) ( Hydroxy)phosphoryl)oxy)methyl)-3-hydroxy-4-methoxytetrahydrofuran-2-yl)-7-methyl-6-oxo-6,9-dihydro-1H-purin-7-ium (Oligonucleotide 7); 2-Amino-9-(( Round 2, Round 3, Round 4, Round 5 )-5-(((((((((2R,3R,4R,5R)-3-(((((2R,3S,4R,5R)-5-(5-amino-7-oxo-6,7-dihydro-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-5-(6-amino-9H-purin-9-yl)-4-fluorotetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy) ( Hydroxy)phosphoryl)oxy)methyl)-3,4-dihydroxytetrahydrofuran-2-yl)-7-methyl-6-oxo-6,9-dihydro-1H-purin-7-ium (oligonucleotide 8); 2-Amino-9-(( Round 2, Round 3, Round 4, Round 5)-5-(((((((((2R,3R,4R,5R)-5-(6-amino-9H-purin-9-yl)-3-(((((2R,3S,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-4-fluorotetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy) ( Hydroxy)phosphoryl)oxy)methyl)-3,4-dihydroxytetrahydrofuran-2-yl)-7-methyl-6-oxo-6,9-dihydro-1H-purin-7-ium (oligonucleotide 9); 2-Amino-9-(( Round 2, Round 3, Round 4, Round 5 )-5-(((((((((2R,3R,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-3-(((((2R,3S,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-4-fluorotetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy) ( Hydroxy)phosphoryl)oxy)methyl)-3-hydroxy-4-methoxytetrahydrofuran-2-yl)-7-methyl-6-oxo-6,9-dihydro-1H-purin-7-ium (oligonucleotide 10); 2-Amino-9-(( Round 2, Round 3, Round 4, Round 5 )-5-(((((((((2R,3R,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-3-(((((2R,3S,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-4-fluorotetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy) (Hydroxy)phosphoryl)oxy)methyl)-3,4-dihydroxytetrahydrofuran-2-yl)-7-methyl-6-oxo-6,9-dihydro-1H-purin-7-ium (oligonucleotide 11); and 2-Amino-9-(( Round 2, Round 3, Round 4, Round 5 )-5-(((((((2R,3R,4R,5R)-3-(((((2R,3S,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-5-(6-amino-9H-purin-9-yl)-4-fluorotetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)methyl)-3,4-dihydroxytetrahydrofuran-2-yl)-7-methyl-6-oxo-6,9-dihydro-1H-purin-7-ium (oligonucleotide 12).

[0025] According to an embodiment of the present invention, the compound represented by Chemical Formula 1 may be an oligonucleotide primer for RNA capping.

[0026] In the present invention, the term "stereoisomer" refers to a compound of the present invention or a salt thereof that has a similar chemical or molecular formula but is sterically different. Each such stereoisomer and mixtures thereof are also included within the scope of the present invention. In addition, unless otherwise specified, a solid bond (-) attached to an asymmetric carbon atom is not necessarily a wedge-shaped bond representing the absolute configuration of the stereocenter. [ka] or wedge-shaped dashed bond [ka] may include.

[0027] In the present invention, the term "salt" refers to a salt commonly used in the pharmaceutical industry. Specifically, it may be a base addition salt. For example, inorganic ion salts made with calcium, potassium, sodium, magnesium, etc.; amino acid salts made with arginine, lysine, histidine, etc.; and amine salts made with trimethylamine, triethylamine, ammonia, pyridine, picoline, etc. are included, but the types of salts meant in the present invention are not limited to these listed salts.

[0028] RNA capped with a novel oligonucleotide primer The above-mentioned oligonucleotide primer for RNA capping can be used to prepare an RNA molecule comprising the same. Thus, the present invention provides an RNA molecule comprising an oligonucleotide primer for RNA capping represented by Chemical Formula 1.

[0029] The oligonucleotide primer for RNA capping according to the present invention can be extended by RNA polymerase by incorporating NTP at the 3'-end. Transcription can be initiated in vitro under the control of a promoter in a transcription system containing essential components such as a DNA template (e.g., a DNA plasmid), RNA polymerase, nucleoside 5'-triphosphates and a suitable buffer. Here, the oligonucleotide primer is complementary to the DNA template at the initiation site.

[0030] In the present invention, promoter refers to a specific region of a dsDNA template that induces and controls the initiation of transcription of a specific DNA sequence (e.g., gene). Promoter is located on the same strand and upstream (towards the 5' region of the sense strand) on DNA. Promoter is usually located immediately adjacent to the DNA sequence to be transcribed or partially overlaps with such sequence. Nucleotide position in promoter is designed based on the transcription start site where transcription of DNA starts (position +1). Initiating oligonucleotide primer is complementary to the initiation site of promoter sequence (which in certain embodiments is at position +1 and +2, and in the case of the initiating tetramer, is at position +1, +2 and +3).

[0031] In one embodiment, the RNA capping oligonucleotide primer is attached to the 5' end of an RNA molecule to form a 5'-capped RNA, such as, but not limited to, mRNA, small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), small Cajal body-specific RNA (scaRNA).

[0032] In one embodiment, the RNA molecule is an mRNA and can include one or more coding sequences (CDS). In addition, the RNA molecule can include a polyA sequence and / or a polyadenylation signal. The polyA sequence can be entirely or mostly composed of adenine nucleotides, analogs, or derivatives thereof. The polyA sequence can be a tail located adjacent to the 3' untranslated region of the nucleic acid.

[0033] The present invention also provides a method for synthesizing a 5'-capped RNA molecule, the method comprising: (S-1) mixing a DNA template, an RNA capping oligonucleotide primer represented by the above-mentioned chemical formula 1, and an RNA polymerase; and (S-2) incubating the mixture and transcribing the polynucleotide template.

[0034] Meanwhile, methods for DNA-templated and promoter-controlled synthesis of RNA using an oligonucleotide primer, RNA polymerase and nucleoside 5'-triphosphate (NTP) are well known in the art to which the present invention pertains. Thus, an RNA molecule containing the oligonucleotide primer for RNA capping represented by Chemical Formula 1 provided in the present invention can be synthesized.

[0035] According to embodiments of the present invention, the DNA template may be a double-stranded linear DNA, a partially double-stranded linear DNA, a circular double-stranded DNA, a DNA plasmid, a PCR amplicon, or any modified nucleic acid capable of reacting appropriately with an RNA polymerase.

[0036] According to a specific embodiment of the invention, the synthesis of the RNA molecule is performed in vitro. For large-scale transcription in vitro, single subunit phage polymerases from T7, T3, SP6, K1-5, K1E, K1F or K11 bacteriophages can be used. Polymerases of this family have simple minimal promoter sequences of ∼17 nucleotides with no auxiliary proteins and minimal constraints on the initiation nucleotide sequence. One example of an RNA polymerase that can be used in the present invention is T7 RNA polymerase (T7 RNAP), although one of ordinary skill in the relevant art will understand that the present invention can be practiced with other RNA polymerases. Unlike DNA polymerases, T7 RNAP initiates RNA synthesis in the absence of a primer. The first step in initiation is called de novo RNA synthesis, in which the RNA polymerase recognizes a specific sequence in the DNA template, selects a first pair of nucleotide triphosphates complementary to the template residues at positions +1 and +2, and catalyzes the formation of phosphodiester bonds to form a dinucleotide.

[0037] Medicinal uses of capped RNA The 5'-capped RNA molecule of the present invention as described above can be usefully utilized for pharmaceutical purposes.

[0038] According to an embodiment of the present invention, the 5'-capped RNA molecule produced according to the present invention can be utilized as a nucleic acid therapeutic or vaccine. For example, the nucleic acid therapeutic or vaccine can be used as an RNA vaccine (a vaccine for preventing cancer or infectious diseases). The nucleic acid therapeutic or vaccine can be administered to a subject and translated in vivo to produce a peptide of interest.

[0039] According to an embodiment of the invention, the 5'-capped RNA molecule can be introduced into a cell to produce a protein capable of treating a medical condition of the cell or exerting a therapeutic effect on the cell.

[0040] According to an embodiment of the present invention, the nucleic acid therapeutic agent or vaccine may further comprise, together with the RNA molecule, a carrier capable of introducing the RNA molecule into a target cell.

[0041] According to an embodiment of the invention, the invention provides a peptide translated from the 5'-capped RNA molecule described above.

[0042] According to a specific embodiment of the present invention, there is provided a cell into which the above-mentioned 5'-capped RNA molecule has been introduced, said cell may be a somatic cell of a subject or a cell line that can be cultured in vitro.

[0043] According to a further embodiment of the present invention, there is provided a peptide produced from a cell into which a 5'-capped RNA molecule has been introduced.

[0044] In the present invention, pharmaceutical compositions for RNA therapeutics or vaccines can be formulated for administration by injection or other suitable routes known to those of ordinary skill in the relevant art to treat a particular condition. Injectable compositions for parenteral administration usually contain the active compound in a compatible solution and / or pharmaceutical carrier, such as sterile physiological saline. The composition can also be formulated as a suspension in lipids or phospholipids, a liposomal suspension, or an aqueous emulsion.

[0045] Methods for preparing compositions and / or dosage forms for RNA therapeutics or vaccines are known to those of ordinary skill in the relevant art (see Remington's Pharmaceutical Sciences (1999)). th (See, e.g., Williams & Wilkins, Ed., 1995). The composition to be administered contains a specific amount of a selected compound in a pharma- ceutical , safe and effective amount to increase expression of a protein of interest in a target cell or tissue.

[0046] In some embodiments, the pharmaceutical composition contains at least 0.1% (w / v) of the compound as described above, in some embodiments, the pharmaceutical composition contains more than 0.1%, in some embodiments, the pharmaceutical composition contains about 10% or less, in some embodiments, the pharmaceutical composition contains about 5% or less, and in some embodiments, the pharmaceutical composition contains about 1% or less (w / v). The selection of the appropriate concentration depends on factors such as the desired dose, frequency, and method of delivery of the active agent.

[0047] When treating a subject, e.g., a mammal or human, the dosage is determined based on factors such as the subject's weight and general health, the condition being treated, the severity of the condition, and the like. The dosage and concentration are determined to provide the desired benefit while avoiding any undesirable side effects. Typical dosages of the subject compounds are in the range of about 0.0005-500 mg / day for human patients, and in some embodiments, in the range of about 1-100 mg / day. For example, higher dosage regimens include, e.g., 50-100, 75-100, or 50-75 mg / day, and lower dosage regimens include, e.g., 1-50, 25-50, or 1-25 mg / day.

[0048] Effect of the Invention The novel oligonucleotide according to the present invention can be used in the synthesis of 5'-capped RNA, and can not only improve the RNA production process (e.g., synthesis yield, synthesis scale, purity, etc.), but also improve the efficacy (e.g., RNA stability and / or protein expression efficiency) and reduce side effects (e.g., reduced immunogenicity) of nucleic acid therapeutics or vaccines using the same. Therefore, the present invention can be usefully applied in the field of nucleic acid therapeutics or vaccines. [Brief description of the drawings]

[0049] [Figure 1] FIG. 1 shows the results of electrophoresis for in vitro transcribed mRNA. [Diagram 2] FIG. 2 shows the results of GFP mRNA expression through a fluorescent microscope.

[0050] Best Mode for Carrying Out the Invention The present invention will be described in more detail with reference to the following examples and experimental examples. These examples and experimental examples are merely for the purpose of illustrating the present invention, and the scope of the present invention is not limited to these examples and experimental examples.

[0051] Preparation Example 1: Preparation of ((2R,3R,4R,5R)-3-(((((2R,3S,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-5-(6-amino-9H-purin-9-yl)-4-fluorotetrahydrofuran-2-yl) dihydrogen methyl phosphate, 2-triethylamine salt Step 1. Preparation of (2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-fluoro-2-(hydroxymethyl)tetrahydrofuran-3-yl(2-cyanoethyl)(((3aR,4R,6R,6aR)-6-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl)phosphate N-(9-((3aR,4R,6R,6aR)-6-(hydroxymethyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide (4 g, 10.17 mmol) was dissolved in acetonitrile. After cooling to 0-5 °C, (2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-fluorotetrahydrofuran-3-yl(2-cyanoethyl)diisopropylphosphoramidite (8.91 g, 10.17 mmol) and 1H-tetrazole (2.85 g, 40.7 mmol) were added in sequence. After 10 minutes, the temperature was slowly raised to room temperature and stirred for 1 hour. The reaction solution was cooled to 0-5°C, and then I2 (0.1M) (7.74g, 30.5mmol) in THF / distilled water / pyridine (66:33:1) (101.6mL) was added. The temperature was slowly raised to room temperature and stirred for 1 hour. After the reaction was completed, the mixture was diluted with DCM, and a solution of 10% sodium thiosulfate pentahydrate was added, and the organic layer and the aqueous layer were separated. The organic layer was dehydrated with sodium sulfate and concentrated under reduced pressure. The concentrated residue was dissolved in DCM (200 mL). After cooling to 0-5°C, 10.17 mL of TFA was diluted in 817 mL of DCM and slowly added, and stirred for 10 minutes. The reaction solution was slowly warmed to room temperature and stirred for 10 minutes. After cooling to 0-5°C, saturated NaHCO3(aq) solution was slowly added to adjust the pH to between 8 and 9. The organic layer and the aqueous layer were separated, and the organic layer was dehydrated with sodium sulfate and filtered. After concentration under reduced pressure, separation was performed by column chromatography to obtain the title compound (5.62 g) as a yellow solid. LC-MS (ESI, m / z) = 882.1 (M+H + ).

[0052] Step 2. Preparation of (2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-2-(((bis(2-cyanoethoxy)phosphoryl)oxy)methyl)-4-fluorotetrahydrofuran-3-yl(2-cyanoethyl)(((3aR,4R,6R,6aR)-6-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl)phosphate (2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-fluoro-2-(hydroxymethyl)tetrahydrofuran-3-yl(2-cyanoethyl)(((3aR,4R,6R,6aR)-6-(2-isobutylamido-6-oxo-1,6-dihydro-9H-purin-9-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl)phosphate (5.6 g, 6.35 mmol) obtained in step 1 was dissolved in DCM (21.2 mL). After cooling to 0-5 ° C., bis(2-cyanoethyl)diisopropylphosphoramidite (3.32 mL, 12.70 mmol) and 1H-tetrazole (0.890 g, 12.70 mmol) were added in sequence. After 10 minutes, the temperature was slowly raised to room temperature and stirred for 1 hour. After cooling the reaction solution to 0-5°C, I2 (0.1M) (4.84g, 19.05mmol) in THF / distilled water / pyridine (66:33:1) (63.6mL) was added. The temperature was slowly raised to room temperature and stirred for 1 hour. After the reaction was completed, the mixture was diluted with DCM, and a solution of 10% sodium thiosulfate pentahydrate was added, and the organic layer and the aqueous layer were separated. The organic layer was dehydrated with sodium sulfate, concentrated under reduced pressure, and then separated by column chromatography to obtain the title compound (5.78g) as a yellow solid. LC-MS (ESI, m / z) = 1086.1 (M+H + ).

[0053] Step 3. Preparation of ((2R,3R,4R,5R)-3-(((((2R,3S,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-5-(6-amino-9H-purin-9-yl)-4-fluorotetrahydrofuran-2-yl)phosphoric acid dihydrogen methyl 2 triethylamine salt (2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-2-(((bis(2-cyanoethoxy)phosphoryl)oxy)methyl)-4-fluorotetrahydrofuran-3-yl(2-cyanoethyl)(((3aR,4R,6R,6aR)-6-(2-isobutylamido-6-oxo-1,6-dihydro-9H-purin-9-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl)phosphate (2.89g, 2.71mmol) obtained in the above step 2 was dissolved in 1M HCl aqueous solution (162mL, 162mmol) and stirred at room temperature for 16 hours. After cooling the reaction solution to 0-5°C, ammonia water (42.2mL, 1083mmol) was slowly added. After slowly warming to room temperature, the mixture was heated for 23 hours at 50 to 55° C. The reaction mixture was washed with DCM (150 mL x 2), and the aqueous layer was purified with DEAE Sephadex resin to obtain the title compound (1.98 g) as a white solid. LC-MS (ESI, m / z) = 695.1 (M+H + ).

[0054] Preparation Example 2: Preparation of 2-amino-9-((2R,3R,4S,5R)-3,4-dihydroxy-5-(((hydroxy((hydroxy(1H-imidazol-1-yl)phosphoryl)oxy)phosphoryl)oxy)methyl)tetrahydrofuran-2-yl)-7-methyl-6-oxo-6,9-dihydro-1H-purin-7-ium sodium salt Step 1. Preparation of 2-amino-9-((2R,3R,4S,5R)-3,4-dihydroxy-5-(((hydroxy(phosphonooxy)phosphoryl)oxy)methyl)tetrahydrofuran-2-yl)-7-methyl-6-oxo-6,9-dihydro-1H-purin-7-ium triethylamine salt ((2R,3S,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl) diphosphate trihydrogen methyl.2 triethylamine salt (5g, 7.74mmol) was dissolved in purified water (155mL) and the pH of the solution was adjusted to 4.5 with acetic acid. Dimethyl sulfate (5.18mL, 54.2mmol) was slowly added for 1 minute, and the mixture was stirred at room temperature for 1 hour. At this time, the pH of the solution was adjusted to 4-4.5 with 5N aqueous sodium hydroxide solution. After the reaction was completed, the mixture was washed with DCM (200mL x 3 times), and the aqueous layer was adjusted to pH 5.5-6 with 1M TEAB. The product was purified with DEAE Sephadex resin to obtain the title compound (3.03g) as a white solid. LC-MS(ESI,m / z)=456.0(MH + ).

[0055] Step 2. Preparation of 2-amino-9-((2R,3R,4S,5R)-3,4-dihydroxy-5-(((hydroxy((hydroxy(1H-imidazol-1-yl)phosphoryl)oxy)phosphoryl)oxy)methyl)tetrahydrofuran-2-yl)-7-methyl-6-oxo-6,9-dihydro-1H-purin-7-ium sodium salt 2-Amino-9-((2R,3R,4S,5R)-3,4-dihydroxy-5-(((hydroxy(phosphonooxy)phosphoryl)oxy)methyl)tetrahydrofuran-2-yl)-7-methyl-6-oxo-6,9-dihydro-1H-purin-7-ium. triethylamine salt (3.03 g, 5.42 mmol) obtained in the above step 1, imidazole (3.69 g, 54.2 mmol) were dissolved in DMF (38.7 mL). 2,2'-dithiodipyridine (5.97 g, 27.1 mmol) was added and stirred at room temperature for 10 hours. After the reaction was completed, the reaction solution was cooled to -10 to -20 ° C., and 0.1 M sodium percolate in acetone (108 mL, 10.83 mmol) was added dropwise. The resulting solid was filtered and dried to give the title compound (2.33 g) as a white solid. LC-MS(ESI,m / z)=507.0(MH + ).

[0056] Preparation Example 3: Preparation of 2-amino-9-((2R,3R,4S,5R)-3-hydroxy-5-(((hydroxy((hydroxy(1H-imidazol-1-yl)phosphoryl)oxy)phosphoryl)oxy)methyl)-4-methoxytetrahydrofuran-2-yl)-7-methyl-6-oxo-6,9-dihydro-1H-purin-7-ium sodium salt ((2R,3S,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-4-hydroxy-3-methoxytetrahydrofuran-2-yl) diphosphate trihydrogen methyl salt The reaction was carried out in the same manner as in Preparation Example 2 using 2-triethylamine salt (190 mg, 0.331 mmol) to give the title compound (159 mg) as a white solid. LC-MS (ESI, m / z) = 522.0 (M+H + ).

[0057] Example 1: 2-amino-9-((2R,3R,4S,5R)-5-((((((((2R,3R,4R,5R)-3-(((((2R,3S,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phospho R (6-amino-9H-purin-9-yl)-4-fluorotetrahydrofuran-2-yl)methoxy)(hydroxy)phospho R (oxy)(hydroxy)phospho R (oxy)(hydroxy)phospho R Preparation of (3,4-dihydroxytetrahydrofuran-2-yl)-7-methyl-6-oxo-6,9-dihydro-1H-purin-7-ium.3 triethylamine salt (oligonucleotide 1) The compound obtained in Production Example 1 (20 mg, 0.029 mmol) and the compound obtained in Production Example 2 (23.18 mg, 0.041 mmol) were dissolved in DMSO (0.29 mL), MgCl2 (3.84 mg, 0.041 mmol) was added, and the mixture was stirred at room temperature for 7 hours. After the reaction was completed, the mixture was diluted with 1.44 mL of 0.25 mM EDTA 2Na salt solution (aq). After confirming that the mixture was completely dissolved, the mixture was diluted with 144 mL of purified water and purified with DEAE Sephadex resin to obtain the title compound (22.4 mg) as a white solid. LC-MS(ESI,m / z)=1132.6(MH + ).

[0058] Example 2: Preparation of 2-amino-9-((2R,3R,4S,5R)-5-((((((((2R,3R,4R,5R)-3-(((((2R,3S,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-5-(6-amino-9H-purin-9-yl)-4-(2-methoxyethoxy)tetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)methyl)-3,4-dihydroxytetrahydrofuran-2-yl)-7-methyl-6-oxo-6,9-dihydro-1H-purin-7-ium.3 triethylamine salt (oligonucleotide 2) ((2R,3R,4R,5R)-3-(((((2R,3S,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-5-(6-amino-9H-purin-9-yl)-4-(2-methoxyethoxy)tetrahydrofuran-2-yl)phosphoric acid dihydrogen methyl.2 triethylamine salt (92 mg, 0.123 mmol) obtained by synthesis in a similar manner to that of Production Example 1 and the compound obtained in Production Example 2 (91.3 mg, 0.172 mmol) were reacted in a similar manner to that of Example 1 to obtain the title compound (73 mg) as a white solid. LC-MS (ESI, m / z) = 1187.7 (M-3H + )

[0059] Example 3: 2-Amino-9-(( Round 2, Round 3, Round 4, Round 5 Preparation of )-5-(((((((((1S,3R,4R,6S,7S)-7-(((((2R,3S,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-3-(6-amino-9H-purin-9-yl)-6-methyl-2,5-dioxabicyclo[2.2.1]heptan-1-yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)methyl)-3,4-dihydroxytetrahydrofuran-2-yl)-7-methyl-6-oxo-6,9-dihydro-1H-purin-7-ium.3 triethylamine salt (oligonucleotide 3) ((1S,3R,4R,6S,7S)-7-(((((2R,3S,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-3-(6-amino-9H-purin-9-yl)-6-methyl-2,5-dioxabicyclo[2.2.1]heptan-1-yl)phosphoric acid dihydrogen methyl.2 triethylamine salt (30 mg, 0.034 mmol) obtained by synthesis in the same manner as in Production Example 1 and the compound obtained in Production Example 2 (35 mg, 0.069 mmol) were reacted in the same manner as in Example 1 to obtain the title compound (6 mg) as a white solid. LC-MS(ESI,m / z)=1156.1(MH + )

[0060] Example 4: 2-amino-9-(( Round 2, Round 3, Round 4, Round 5 )-5-(((((((((2R,3R,4R,5R)-3-(((((2R,3S,4R,5R)-5-(5-amino-7-oxo-6,7-dihydro-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-5-(6-amino-9H-purin-9-yl)-4-methoxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy) ( Preparation of Hydroxy)phosphoryl)oxy)methyl)-3,4-dihydroxytetrahydrofuran-2-yl)-7-methyl-6-oxo-6,9-dihydro-1H-purin-7-ium.3 triethylamine salt (oligonucleotide 4) ((2R,3R,4R,5R)-3-(((((2R,3S,4R,5R)-5-(5-amino-7-oxo-6,7-dihydro-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-5-(6-amino-9H-purin-9-yl)-4-methoxytetrahydrofuran-2-yl)phosphoric acid dihydrogen methyl.2 triethylamine salt (17 mg, 0.024 mmol) obtained by synthesis in a similar manner to that of Production Example 1 and the compound obtained in Production Example 2 (24.43 mg, 0.048 mmol) were reacted in a similar manner to that of Example 1 to obtain the title compound (3.8 mg) as a white solid. LC-MS (ESI, m / z) = 1145.8 (M-2H + )

[0061] Example 5: 2-Amino-9-(( Round 2, Round 3, Round 4, Round 5 )-5-(((((((((1R,3R,4R,7S)-7-(((((2R,3S,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-3-(6-amino-9H-purin-9-yl)-2,5-dioxabicyclo[2.2.1]heptan-1-yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy) ( Preparation of Hydroxy)phosphoryl)oxy)methyl)-3,4-dihydroxytetrahydrofuran-2-yl)-7-methyl-6-oxo-6,9-dihydro-1H-purin-7-ium.3 triethylamine salt (Oligonucleotide 5) ((1R,3R,4R,7S)-7-(((((2R,3S,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-3-(6-amino-9H-purin-9-yl)-2,5-dioxabicyclo[2.2.1]heptan-1-yl)phosphoric acid dihydrogen methyl.2 triethylamine salt (50 mg, 0.055 mmol) obtained by synthesis in a similar manner to that of Production Example 1 and the compound obtained in Production Example 2 (112.0 mg, 0.221 mmol) were reacted in a similar manner to that of Example 1 to obtain the title compound (8 mg) as a white solid. LC-MS(ESI,m / z)=1143.0(MH + )

[0062] Example 6: 2-Amino-9-(( Round 2, Round 3, Round 4, Round 5 )-5-(((((((((2R,3R,4R,5R)-3-(((((2R,3S,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-5-(6-amino-9H-purin-9-yl)-4-fluorotetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy) ( Preparation of Hydroxy)phosphoryl)oxy)methyl)-3-hydroxy-4-methoxytetrahydrofuran-2-yl)-7-methyl-6-oxo-6,9-dihydro-1H-purin-7-ium.3 triethylamine salt (Oligonucleotide 6) The compound obtained in Production Example 1 (30 mg, 0.03 mmol) and the compound obtained in Production Example 3 (32.7 mg, 0.06 mmol) were reacted in the same manner as in Example 1 to obtain the title compound (9 mg) as a white solid. LC-MS (ESI, m / z) = 1149.1 (M+H + )

[0063] Example 7: 2-amino-9-(( Round 2, Round 3, Round 4, Round 5)-5-(((((((((2R,3R,4R,5R)-3-(((((2R,3S,4R,5R)-5-(5-amino-7-oxo-6,7-dihydro-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-5-(6-amino-9H-purin-9-yl)-4-fluorotetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy) ( Preparation of Hydroxy)phosphoryl)oxy)methyl)-3-hydroxy-4-methoxytetrahydrofuran-2-yl)-7-methyl-6-oxo-6,9-dihydro-1H-purin-7-ium.3 triethylamine salt (Oligonucleotide 7) ((2R,3R,4R,5R)-3-(((((2R,3S,4R,5R)-5-(5-amino-7-oxo-6,7-dihydro-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-5-(6-amino-9H-purin-9-yl)-4-fluorotetrahydrofuran-2-yl)phosphoric acid dihydrogen methyl.2 triethylamine salt (20 mg, 0.029 mmol) obtained by synthesis in a similar manner to that of Production Example 1 and the compound obtained in Production Example 3 (31.3 mg, 0.058 mmol) were reacted in a similar manner to that of Example 1 to obtain the title compound (16 mg) as a white solid. LC-MS (ESI, m / z) = 1147.1 (M-2H + )

[0064] Example 8: 2-amino-9-(( Round 2, Round 3, Round 4, Round 5)-5-(((((((((2R,3R,4R,5R)-3-(((((2R,3S,4R,5R)-5-(5-amino-7-oxo-6,7-dihydro-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-5-(6-amino-9H-purin-9-yl)-4-fluorotetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy) ( Preparation of hydroxy)phosphoryl)oxy)methyl)-3,4-dihydroxytetrahydrofuran-2-yl)-7-methyl-6-oxo-6,9-dihydro-1H-purin-7-ium.3 triethylamine salt (oligonucleotide 8) ((2R,3R,4R,5R)-3-(((((2R,3S,4R,5R)-5-(5-amino-7-oxo-6,7-dihydro-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-5-(6-amino-9H-purin-9-yl)-4-fluorotetrahydrofuran-2-yl)phosphoric acid dihydrogen methyl.2 triethylamine salt (20 mg, 0.029 mmol) obtained by synthesis in a similar manner to that of Production Example 1 and the compound obtained in Production Example 2 (30.5 mg, 0.058 mmol) were reacted in a similar manner to that of Example 1 to obtain the title compound (14.5 mg) as a white solid. LC-MS (ESI, m / z) = 1133.1 (M-2H + )

[0065] Example 9: 2-amino-9-(( Round 2, Round 3, Round 4, Round 5)-5-(((((((((2R,3R,4R,5R)-5-(6-amino-9H-purin-9-yl)-3-(((((2R,3S,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-4-fluorotetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy) ( Preparation of Hydroxy)phosphoryl)oxy)methyl)-3,4-dihydroxytetrahydrofuran-2-yl)-7-methyl-6-oxo-6,9-dihydro-1H-purin-7-ium.3 triethylamine salt (Oligonucleotide 9) ((2R,3R,4R,5R)-5-(6-amino-9H-purin-9-yl)-3-(((((2R,3S,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-4-fluorotetrahydrofuran-2-yl)phosphoric acid dihydrogen methyl.2 triethylamine salt (50 mg, 0.058 mmol) obtained by synthesis in the same manner as in Production Example 1 and the compound obtained in Production Example 2 (61.8 mg, 0.117 mmol) were reacted in the same manner as in Example 1 to obtain the title compound (34.6 mg) as a white solid. LC-MS (ESI, m / z) = 1092.8 (M-2H + )

[0066] Example 10: 2-amino-9-(( Round 2, Round 3, Round 4, Round 5 )-5-(((((((((2R,3R,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-3-(((((2R,3S,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-4-fluorotetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy) (Preparation of Hydroxy)phosphoryl)oxy)methyl)-3-hydroxy-4-methoxytetrahydrofuran-2-yl)-7-methyl-6-oxo-6,9-dihydro-1H-purin-7-ium.3 triethylamine salt (oligonucleotide 10) ((2R,3R,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-3-(((((2R,3S,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-4-fluorotetrahydrofuran-2-yl)phosphoric acid dihydrogen methyl.2 triethylamine salt (20 mg, 0.02 mmol) obtained by synthesis in a similar manner to that of Production Example 1 and the compound obtained in Production Example 3 (21.47 mg, 0.039 mmol) were reacted in a similar manner to that of Example 1 to obtain the title compound (15.2 mg) as a white solid. LC-MS (ESI, m / z) = 1165.0 (M+H + )

[0067] Example 11: 2-amino-9-(( Round 2, Round 3, Round 4, Round 5 )-5-(((((((((2R,3R,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-3-(((((2R,3S,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-4-fluorotetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy) ( Preparation of Hydroxy)phosphoryl)oxy)methyl)-3,4-dihydroxytetrahydrofuran-2-yl)-7-methyl-6-oxo-6,9-dihydro-1H-purin-7-ium.3 triethylamine salt (oligonucleotide 11) ((2R,3R,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-3-(((((2R,3S,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-4-fluorotetrahydrofuran-2-yl)phosphoric acid dihydrogen methyl.2 triethylamine salt (50 mg, 0.049 mmol) obtained by synthesis in the same manner as in Production Example 1 and the compound obtained in Production Example 2 (52.3 mg, 0.099 mmol) were reacted in the same manner as in Example 1 to obtain the title compound (26.3 mg) as a white solid. LC-MS (ESI, m / z) = 1147.9 (M-2H + )

[0068] Example 12: 2-amino-9-(( Round 2, Round 3, Round 4, Round 5 Preparation of )-5-(((((((2R,3R,4R,5R)-3-(((((2R,3S,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-5-(6-amino-9H-purin-9-yl)-4-fluorotetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)methyl)-3,4-dihydroxytetrahydrofuran-2-yl)-7-methyl-6-oxo-6,9-dihydro-1H-purin-7-ium.3 triethylamine salt (oligonucleotide 12) The compound obtained in Production Example 1 (40 mg, 0.047 mmol) and 2- amino 9-(( Round 2, Round 3, Round 4, Round 5 )-3,4-dihydroxy-5-(((hydroxy(1H-imidazol-1-yl)phosphoryl)oxy)methyl)tetrahydrofuran-2-yl)-7-methyl-6-oxo-6,9-dihydro-1H- PPhosphorus-7-ium sodium salt (42.6 mg, 0.095 mmol) was reacted in the same manner as in Example 1 to obtain the title compound (6.5 mg) as a white solid. LC-MS (ESI, m / z) = 1052.1 (M-2H + )

[0069] Experimental Example 1: Preparation of GFP DNA template To obtain a DNA template for mRNA synthesis, a plasmid gene with a T7 RNA promoter, 5'UTR, EGFP, 3'UTR, and poly A sequence was synthesized from Bionix Co., Ltd. Then, in order to change the promoter sequence according to the capping material, the plasmid gene was mixed with primeSTAR HS premix (Takara catalog #R040A), 2 μmol of forward primer (sequence number 1), and 2 μmol of reverse primer (sequence number 2), and PCR (polymerase chain reaction) was performed, after which a plasmid gene was produced through cloning using XbaI (Takara catalog #1093A) and BamHI (Takara catalog #1010A) restriction enzymes in the conventional plasmid gene. The produced plasmid gene was linearized with SamI (Takara catalog #1085A) restriction enzyme, and then the GFP DNA transcription template (sequence number 3) was secured using the AccuPrep PCR purification kit (Bionia catalog #K-3037).

[0070] Experimental example 2: In vitro transcription of GFP mRNA 2-1: In vitro transcription of non-capped GFP mRNA For the synthesis of mRNA without a 5'-capped structure, 40 mM Tris·HCl (pH 7.9), 25 mM magnesium chloride, 2 mM spermidine, 2 mM ATP, 2 mM UTP, 2 mM CTP, 2 mM GTP, 5% DMSO (Sigma-Aldrich catalog #472301-500ML), 5 mg / mL GFP DNA transcription template, 800 U / mL recombinant RNase inhibitor protein (Takara catalog #2316A), 2 U / mL yeast inorganic pyrophosphatase (Thermo Scientific catalog #EF0221), and 2500 U / mL T7 RNA polymerase (Biodyne catalog #dy1670) were mixed.

[0071] The transcription reaction mixture was incubated for 5 hours at 37°C. The reaction was supplemented with 40 mM Tris·HCl (pH 7.5), 8 mM magnesium chloride, 5 mM DTT, and 5000 U / mL recombinant DNAse I (Takara catalog #2270A) and incubated for 1 hour at 37°C. The generated mRNA was purified using Zymo Research RNAclean & concentrator-25 kit (catalog #1017) according to the manufacturer's instructions.

[0072] The synthesis of mRNA was confirmed by electrophoresis using 1% agarose gel containing Lonza GelStarTM Nucleic Acid gel Stain (catalog #50535) (Figure 1). UV analysis of the synthesized mRNA was measured using a Thermo Scientific Nanodrop one UV-vis spectrometer. As a result, it was confirmed that GFP mRNA containing approximately 1066 nt of poly A(120) was produced.

[0073] 2-2: In vitro transcription of GFP mRNA through co-transcriptional capping with ARCA cap analogues For the ARCA-capped mRNA synthesis reaction, 40 mM Tris·HCl (pH 7.9), 25 mM magnesium chloride, 2 mM spermidine, 2 mM ATP, 2 mM UTP, 2 mM CTP, 0.4 mM GTP, 1.6 mM ARCA (Trilink, catalog #N-7003), 5% DMSO (Sigma-Aldrich catalog #472301-500ML), 5 mg / mL GFP DNA transcription template, 800 U / mL recombinant RNase inhibitor protein (Takara catalog #2316A), 2 U / mL yeast inorganic pyrophosphatase (Thermo Scientific catalog #EF0221), and 2500 U / mL T7 RNA polymerase (Biodyne catalog #dy1670) were mixed.

[0074] The transcription reaction mixture was incubated for 5 hours at 37°C. The reaction was supplemented with 40 mM Tris·HCl (pH 7.5), 8 mM magnesium chloride, 5 mM DTT, and 5000 U / mL recombinant DNAse I (Takara catalog #2270A) and incubated for 1 hour at 37°C. The generated mRNA was purified using Zymo Research RNAclean & concentrator-25 kit (catalog #1017) according to the manufacturer's instructions.

[0075] The synthesis of mRNA was confirmed by electrophoresis using 1% agarose gel containing Lonza GelStarTM Nucleic Acid gel Stain (catalog #50535) (Figure 1). UV analysis of the synthesized mRNA was measured using a Thermo Scientific Nanodrop one UV-vis spectrometer. As a result, it was confirmed that dimeric ARCA capping GFP mRNA containing approximately 1067 nt of poly A(120) was produced.

[0076] 2-3: In vitro transcription of GFP mRNA through co-transcriptional capping in accordance with the embodiment For the mRNA synthesis reaction using the example compounds of the present invention, 40 mM Tris·HCl (pH 7.9), 25 mM magnesium chloride, 2 mM spermidine, 2 mM ATP, 2 mM UTP, 2 mM CTP, 2 mM GTP, 3.2 mM example compound, 5% DMSO (Sigma-Aldrich catalog #472301-500ML), 5 mg / mL GFP DNA transcription template, 800 U / mL recombinant RNase inhibitor protein (Takara catalog #2316A), 2 U / mL yeast inorganic pyrophosphatase (Thermo Scientific catalog #EF0221), and 2500 U / mL T7 RNA polymerase (Biodyne catalog #dy1670) were mixed.

[0077] The transcription reaction mixture was incubated for 5 hours at 37°C. The reaction was supplemented with 40 mM Tris·HCl (pH 7.5), 8 mM magnesium chloride, 5 mM DTT, and 5000 U / mL recombinant DNAse I (Takara catalog #2270A) and incubated for 1 hour at 37°C. The generated mRNA was purified using Zymo Research RNAclean & concentrator-25 kit (catalog #1017) according to the manufacturer's instructions.

[0078] The synthesis of mRNA was confirmed by electrophoresis using 1% agarose gel containing Lonza GelStar™ Nucleic Acid gel Stain (catalog #50535) (Figure 1). UV analysis of the synthesized mRNA was measured using a Thermo Scientific Nanodrop one UV-vis spectrometer. As a result, it was confirmed that the example capping GFP mRNA containing approximately 1067 nt of poly A(120) was produced.

[0079] Experimental Example 3: Translation of mRNA in HeLa cells The translation activity of mRNA generated by in vitro transcription was evaluated in a human cervix cancer cell line (Hela). Hela cells were cultured in DMEM supplemented with 10% FBS and 1% penicillin / streptomycin at 37°C under an atmosphere of 5% carbon dioxide. 6 Hela cells were plated at 100 / well in a 6-well plate. The next day, the cells were transfected using a transfection reagent (messengerMAX lipofectamine; Invitrogen catalog #LMRNA003): Tube A was prepared by diluting 7.5 μL of the transfection reagent in 125 μL of complex medium (Opti-MEM; Life technologies) and incubating at room temperature for 10 min according to the transfection reagent manufacturer's instructions, and Tube B was prepared by diluting 5 μg of the prepared mRNA in 125 μL of Opti-MEM. The solutions in Tube A and Tube B were mixed and then incubated at room temperature for 5 min. During the incubation time, the cell culture medium was replaced with DMEM containing 10% FBS without penicillin / streptomycin. The incubated mixed solution was then used to transfect the cells. After 3–4 h of incubation at 37°C under a 5% carbon dioxide atmosphere, the cell culture medium was replaced with DMEM supplemented with 10% FBS and 1% penicillin / streptomycin. Fluorescent protein expression was confirmed using a fluorescence microscope 96 h after transfection.

[0080] As a result, the mRNA produced using the example compounds showed excellent fluorescence activity. On the other hand, the control group, mRNA without cap, showed no fluorescence activity at all. In addition, compared with the mRNA synthesized using the ARCA cap, the mRNA produced using the example compounds all showed excellent protein expression rates (Figure 2).

[0081] Experimental Example 4: Western Blot Analysis After the transfection experiment, the culture medium was removed and washed once with PBS. After treating with 200 μL of cell lysis buffer (RIPA + phosphatase inhibitor + PMSF), the cells were lysed at 4 °C for 10 minutes. The cell lysate was collected and centrifuged at 12000 rpm, 4 °C, for 10 minutes. The supernatant was then collected, mixed with 5 × SDS-sample loading buffer, and boiled at 100 °C for 5 to 10 minutes. A 10% SDS PAGE-gel was run at 120 V for 10 minutes and 170 V for 1 hour. Transfer was performed at 30 V for 1 hour using Xcell 2 blot Module (Invitrogen). Blocking was performed at room temperature for 1 hour using 5% BSA-PBST. Rabbit anti-GFP antibody was diluted 1:1000 in 5% BSA-PBST and reacted overnight at 4 °C. Then, it was washed three times with PBST for 10 minutes. HRP-conjugated rabbit secondary antibody was diluted 1:10000 in 2.5% BSA-PBST and incubated at room temperature for 1 hour. Then, the cells were washed three times for 10 minutes with PBST. After ECL treatment, the cells were examined by chemiluminescence and analyzed for β-actin versus GFP expression levels (Table 1). [Table 1]

[0082] Experimental Example 5: FACS analysis After the transfection experiment, the culture medium was removed and washed once with PBS. After trypsin-EDTA treatment, the cells were incubated at 37°C for about 2 to 3 minutes until they began to fall off. After neutralization with culture medium (HG DMEM + 10% FBS + 1% penicillin / streptomycin), the cells were centrifuged at 1200 rpm for 2 minutes at room temperature. After scooping out the supernatant, 1 mL of PBS was dispensed, and the aggregated cells were released by pipetting. GFP signals (FITC) were measured using FACS (BD FACSDiva 8.0.3) (FSC, SSC confirmation, Table 2). [Table 2]

[0083] Although the specific parts of the present invention have been described in detail above, it is obvious to those skilled in the art that such specific descriptions are merely preferred embodiments and experimental examples, and are not intended to limit the scope of the present invention. Therefore, the true scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. The following chemical formula 1: 【Chemistry 8】 An oligonucleotide primer compound for RNA capping represented by the formula: In the above Chemical Formula 1, B １ and B ２ are each independently a natural, non-natural or modified nucleoside base; X １ is -OH; X ２ is -H; Y １ is -halo; Y ２ is -H or Y １ and form an LNA ring, wherein at least one H of the LNA ring is -(C １－４ alkyl), -OH, or -O(C １－４ alkyl)}; Z １ and Z ２ are each independently -OH or -O(C １－４ alkyl); Z ３ is -H; n is 1; m is 1; R １ and R ２ are each independently -H or -(C １－４ alkyl); and R ３ is -(C １－４ alkyl), An oligonucleotide primer compound for RNA capping, a stereoisomer thereof, or a salt thereof.

2. The non-natural or modified nucleoside base is an isomer of the natural nucleoside base or at least one -H of the natural nucleoside base is -(C １－４ alkyl), -O(C １－４ -alkyl) or -halo, at least one ═CH— is replaced by ═N—, and at least one ═O is replaced by ═S; The oligonucleotide primer compound for RNA capping according to claim 1, its stereoisomer, or its salt.

3. B １ and B ２ are each independently 【Chemistry 9】 is; The oligonucleotide primer compound for RNA capping according to claim 1, its stereoisomer, or its salt.

4. Y １ is -halo; and Y ２ is -H or Y １ and form an LNA ring, wherein at least one H of the LNA ring is -(C １－４ alkyl)}; The oligonucleotide primer compound for RNA capping according to claim 1, its stereoisomer, or its salt.

5. Z １ is -OH or -O(C １－４ alkyl); Z ２ is —OH; and Z ３ is -H; The oligonucleotide primer compound for RNA capping according to claim 1, its stereoisomer, or its salt.

6. R １ and R ２ are each independently —H; and R ３ is -(C １－４ alkyl); The oligonucleotide primer compound for RNA capping according to claim 1, its stereoisomer, or its salt.

7. The following compound: 【Chemistry 10】 【change】 【change】 The oligonucleotide primer compound for RNA capping according to claim 1, its stereoisomer, or a salt thereof, selected from the group consisting of:

8. An RNA molecule comprising an oligonucleotide primer for RNA capping according to any one of claims 1 to 7.

9. The RNA molecule of claim 8, wherein the RNA capping oligonucleotide primer is attached to the 5' top end of the RNA molecule.

10. The RNA molecule of claim 8, wherein the RNA molecule is an mRNA comprising at least one coding sequence (CDS).

11. (S-1) mixing a DNA template, an oligonucleotide primer for RNA capping according to any one of claims 1 to 7, and an RNA polymerase; and (S-2) incubating the mixture to effect transcription of the polynucleotide template; 9. A method for synthesizing an RNA molecule according to claim 8, comprising:

12. The method for synthesizing an RNA molecule according to claim 11, wherein the synthesis of the RNA molecule is carried out in vitro.

13. A cell into which the RNA molecule of claim 8 has been introduced.

14. A nucleic acid therapeutic comprising the RNA molecule of claim 8.

15. The nucleic acid therapeutic agent of claim 14, comprising a carrier capable of introducing the RNA molecule into a target cell.

16. A vaccine comprising the RNA molecule of claim 8.

17. The vaccine of claim 16, comprising a vehicle capable of introducing the RNA molecule into a target cell.

18. 17. The vaccine of claim 16 for preventing cancer or infectious disease.

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