Polynucleotides and Pharmaceutical Compositions

Abstract

The present invention addresses the problem of providing a polynucleotide that has modified sites in a translation region but yet sustains the translation activity. This problem can be solved by a polynucleotide that contains a translation region from an imitation codon to a termination codon, wherein: the translation region comprises n codons; n is a positive integer of 2 or greater; the n codons each comprise the first, second and third nucleotides; and, in at least two codons among the n codons, the first nucleotide is a sugar-modified nucleotide.

Description

[Technical Field]

[0001] The present invention relates to polynucleotides and pharmaceutical compositions comprising said polynucleotides. [Background technology]

[0002] Genetic information in cells is transmitted by the transcription of messenger RNA (hereinafter sometimes referred to as "mRNA") by RNA polymerase using DNA as a template, and then ribosomes bind to the transcribed single-stranded mRNA and synthesize proteins through translation. This mode of transmission is called the "central dogma" in molecular biology and is a fundamental principle common to both prokaryotes and eukaryotes. mRNA, an intermediate in genetic information transmission, has the base sequence information and structure required for direct recognition by ribosomes and translation into proteins.

[0003] Nucleic acid medicines have been gaining increasing attention as next-generation medicines in recent years. Polynucleotides in the form of mRNA (hereinafter referred to as "artificial mRNA") can be used as nucleic acid medicines for protein replacement therapy through enhanced or increased expression, or as nucleic acid medicines for vaccine therapy through peptide expression. However, it is known that when artificial mRNA composed only of natural bases is introduced into cells from the outside, it binds to intracellular Toll-like receptors (TLR3, TLR7, TLR8, RIG-I, etc.) and rapidly induces an immune response, resulting in an inflammatory reaction and a decrease in protein translation (Non-Patent Document 1). To express proteins intracellularly, some method must be devised to reduce the immune reactivity of the artificial mRNA itself without reducing the translation yield. Furthermore, because RNA composed only of natural bases is vulnerable to nucleases, the introduction of modified nucleotides is also necessary to enhance stability (Non-Patent Document 2). Among modified nucleotides, polynucleotides containing sugar-modified nucleotides, such as 2'-O-methylated RNA, 2'-F-modified RNA, and cross-linked nucleic acids, have been shown to be effective in both reducing the immune reactivity of nucleic acid drugs and conferring resistance to nucleases (Non-Patent Document 3).

[0004] In recent years, there has been a vigorous movement to use mRNA artificially synthesized in a test tube by in vitro transcription (hereinafter referred to as "IVT") as a pharmaceutical (Non-Patent Document 4). For example, Non-Patent Document 5 reports that in a clinical trial of an artificial mRNA cancer vaccine targeting melanoma patients, the incidence of metastasis significantly decreased after the administration of the cancer vaccine began, and certain results are beginning to be reported. However, these artificial mRNAs used in clinical applications are produced by IVT. Artificial mRNAs produced by IVT have the following two problems. First, the introduction position of modified nucleotides, which are introduced for the purposes of reducing immune reactivity or imparting stability to nucleases, cannot be controlled. Patent Document 1 discloses cases in which artificial mRNAs into which 2'-F-modified RNAs have been introduced by IVT have exhibited reduced or lost peptide translation ability. Second, modified nucleotides other than those recognized as substrates by the RNA polymerase used in IVT cannot be introduced. Patent Document 1 also discloses that it is difficult to prepare artificial mRNAs containing 2'-O-methylated modified RNAs by IVT reactions using general RNA polymerases. Therefore, it is difficult to say that the position and type of modified nucleotides have been thoroughly considered in artificial mRNAs prepared by introducing modified nucleotides by IVT.

[0005] A method for artificially synthesizing mRNA using a technique for chemically linking multiple RNAs has been reported (Non-Patent Documents 6 and 7). Using this method, it is possible to introduce modified nucleotides containing sugar modifications into any position of an artificial mRNA, including the coding sequence (hereinafter sometimes referred to as "CDS"). In fact, Non-Patent Documents 6 and 7 disclose that artificial mRNAs were prepared in which 2'-O-methylated modified RNA was introduced into a single position in the CDS of mRNA, and their peptide translation ability was confirmed. However, it has also been disclosed that peptide translation ability can be significantly attenuated depending on the position at which the sugar-modified nucleotide is introduced. Therefore, further knowledge regarding the modification rate, position, and type of modified nucleotides is needed to achieve sufficiently low immunoreactivity and high stability as nucleic acid drugs. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] International Publication No. 2014 / 093574 [Non-patent literature]

[0007] [Non-Patent Document 1] Nature Reviews Drug Discovery, Vol. 13, pp. 759-780 (2014) [Non-patent document 2] Nature Biotechnology, Vol. 35, No. 3, pp. 238-248 (2017) [Non-patent document 3] Drug Discovery Today, Vol. 13, No. 19 / 20, pp. 842-855 (2008) [Non-patent document 4] Nature Biotechnology, Vol. 35, No. 3, pp. 193-197 (2017) [Non-Patent Document 5] Nature, Vol. 547, No. 7662, pp. 222-226 (2017) [Non-patent document 6] Nucleic Acids Research, Vol. 44, No. 2, pp. 852-862 (2015) [Non-Patent Document 7] Genes, Vol. 10, No. 2, p. 84 (2019) Summary of the Invention [Problem to be solved by the invention]

[0008] An object of the present invention is to provide a polynucleotide that maintains translation activity while having a modified site in the translation region. [Means for solving the problem]

[0009] As a result of intensive research, the inventors have found that translation activity is maintained even if the sugar moiety of the first nucleotide, among the first, second and third nucleotides contained in each of the multiple codons that make up the translation region, is modified.

[0010] The present invention includes the following embodiments. [1] It includes the translation region from the start codon to the stop codon, the translation region contains n codons, where n is a positive integer of 2 or more; each of the n codons comprises a first nucleotide, a second nucleotide, and a third nucleotide; the first nucleotide in at least two of the n codons is a sugar-modified nucleotide; Polynucleotide. [2] The polynucleotide according to [1], wherein the sugar-modified nucleotide comprises a sugar moiety modified at least at the 2' position. [3] The sugar moiety modified at least at the 2' position is selected from the following: [ka] [2] The polynucleotide according to [2]. [4] the sugar-modified nucleotide comprises a base moiety corresponding to a base selected from the group consisting of adenine, guanine, cytosine, and uracil; The polynucleotide according to any one of [1] to [3], wherein the types of bases are at least two. [5] The polynucleotide according to any one of [1] to [4], wherein at least one of the sugar-modified nucleotides contains a modified base moiety. [6] The polynucleotide according to any one of [1] to [5], wherein the first nucleotide in all of the n codons is a sugar-modified nucleotide. [6-1] The polynucleotide according to any one of [1] to [5], wherein the proportion of the first nucleotide in the n codons that is a sugar-modified nucleotide is 5% or more. [6-2] The polynucleotide according to any one of [1] to [5], wherein the proportion of the first nucleotide in the n codons that is a sugar-modified nucleotide is 10% or more. [6-3] The polynucleotide according to any one of [1] to [5], wherein the proportion of the first nucleotide in the n codons that is a sugar-modified nucleotide is 15% or more. [6-4] The polynucleotide according to any one of [1] to [5], wherein the proportion of the first nucleotide in the n codons that is a sugar-modified nucleotide is 20% or more. [6-5] The polynucleotide according to any one of [1] to [5], wherein the proportion of the first nucleotide in the n codons that is a sugar-modified nucleotide is 25% or more. [6-6] The polynucleotide according to any one of [1] to [5], wherein the proportion of the first nucleotide in the n codons that is a sugar-modified nucleotide is 30% or more. [6-7] The polynucleotide according to any one of [1] to [5], wherein the proportion of the first nucleotide in the n codons that is a sugar-modified nucleotide is 35% or more. [6-8] The polynucleotide according to any one of [1] to [5], wherein the proportion of the first nucleotide in the n codons that is a sugar-modified nucleotide is 40% or more. [6-9] The polynucleotide according to any one of [1] to [5], wherein the proportion of the first nucleotide in the n codons that is a sugar-modified nucleotide is 45% or more. [6-10] The polynucleotide according to any one of [1] to [5], wherein the proportion of the first nucleotide in the n codons that is a sugar-modified nucleotide is 50% or more. [6-11] The polynucleotide according to any one of [1] to [5], wherein the proportion of the first nucleotide in the n codons that is a sugar-modified nucleotide is 55% or more. [6-12] The polynucleotide according to any one of [1] to [5], wherein the proportion of the first nucleotide in the n codons that is a sugar-modified nucleotide is 60% or more. [6-13] The polynucleotide according to any one of [1] to [5], wherein the proportion of the first nucleotide in the n codons that is a sugar-modified nucleotide is 65% or more. [6-14] The polynucleotide according to any one of [1] to [5], wherein the proportion of the first nucleotide in the n codons that is a sugar-modified nucleotide is 70% or more. [6-15] The polynucleotide according to any one of [1] to [5], wherein the proportion of the first nucleotide in the n codons that is a sugar-modified nucleotide is 75% or more. [6-16] The polynucleotide according to any one of [1] to [5], wherein the proportion of the first nucleotide in the n codons that is a sugar-modified nucleotide is 80% or more. [6-17] The polynucleotide according to any one of [1] to [5], wherein the proportion of the first nucleotide in the n codons that is a sugar-modified nucleotide is 90% or more. [6-18] The polynucleotide according to any one of [1] to [5], wherein the proportion of the first nucleotide in the n codons that is a sugar-modified nucleotide is 95% or more. [7] The polynucleotide according to any one of [1] to [6-18], wherein the first, second and third nucleotides of the stop codon are sugar-modified nucleotides. [8] The polynucleotide according to any one of [1] to [7], wherein the first, second and third nucleotides of the initiation codon are sugar-modified nucleotides. [9] The polynucleotide according to any one of [1] to [8], wherein the second nucleotide of at least one of the n codons is a sugar-modified nucleotide. [9-1] The polynucleotide according to any one of [1] to [9], wherein the proportion of the second nucleotide in the n codons that is a sugar-modified nucleotide is 50% or less. [9-2] The polynucleotide according to any one of [1] to [9], wherein the proportion of the second nucleotide in the n codons that is a sugar-modified nucleotide is 45% or less. [9-3] The polynucleotide according to any one of [1] to [9], wherein the proportion of the second nucleotide in the n codons that is a sugar-modified nucleotide is 40% or less. [9-4] The polynucleotide according to any one of [1] to [9], wherein the proportion of the second nucleotide in the n codons that is a sugar-modified nucleotide is 35% or less. [9-5] The polynucleotide according to any one of [1] to [9], wherein the proportion of the second nucleotide in the n codons that is a sugar-modified nucleotide is 30% or less. [9-6] The polynucleotide according to any one of [1] to [9], wherein the proportion of the second nucleotide in the n codons that is a sugar-modified nucleotide is 25% or less. [9-7] The polynucleotide according to any one of [1] to [9], wherein the proportion of the second nucleotide in the n codons that is a sugar-modified nucleotide is 20% or less. [9-8] The polynucleotide according to any one of [1] to [9], wherein the proportion of the second nucleotide in the n codons that is a sugar-modified nucleotide is 15% or less. [9-9] The polynucleotide according to any one of [1] to [9], wherein the proportion of the second nucleotide in the n codons that is a sugar-modified nucleotide is 10% or less. [9-10] The polynucleotide according to any one of [1] to [9], wherein the proportion of the second nucleotide in the n codons that is a sugar-modified nucleotide is 5% or less. [9-11] The polynucleotide according to any one of [1] to [6-18], wherein the proportion of the second nucleotide in the n codons that is a sugar-modified nucleotide is 0%.

[10] The polynucleotide according to any one of [1] to [9-11], wherein the third nucleotide in at least one of the n codons is a sugar-modified nucleotide. [10-1] The polynucleotide according to any one of [1] to

[10] , wherein the proportion of the third nucleotide in the n codons that is a sugar-modified nucleotide is 100%. [10-2] The polynucleotide according to any one of [1] to

[10] , wherein the proportion of the third nucleotide in the n codons that is a sugar-modified nucleotide is 90% or less. [10-3] The polynucleotide according to any one of [1] to

[10] , wherein the proportion of the third nucleotide in the n codons that is a sugar-modified nucleotide is 80% or less. [10-4] The polynucleotide according to any one of [1] to

[10] , wherein the proportion of the third nucleotide in the n codons that is a sugar-modified nucleotide is 70% or less. [10-5] The polynucleotide according to any one of [1] to

[10] , wherein the proportion of the third nucleotide in the n codons that is a sugar-modified nucleotide is 60% or less. [10-6] The polynucleotide according to any one of [1] to

[10] , wherein the proportion of the third nucleotide in the n codons that is a sugar-modified nucleotide is 50% or less. [10-7] The polynucleotide according to any one of [1] to

[10] , wherein the proportion of the third nucleotide in the n codons that is a sugar-modified nucleotide is 45% or less. [10-8] The polynucleotide according to any one of [1] to

[10] , wherein the proportion of the third nucleotide in the n codons that is a sugar-modified nucleotide is 40% or less. [10-9] The polynucleotide according to any one of [1] to

[10] , wherein the proportion of the third nucleotide in the n codons that is a sugar-modified nucleotide is 35% or less. [10-10] The polynucleotide according to any one of [1] to

[10] , wherein the proportion of the third nucleotide in the n codons that is a sugar-modified nucleotide is 30% or less. [10-11] The polynucleotide according to any one of [1] to

[10] , wherein the proportion of the third nucleotide in the n codons that is a sugar-modified nucleotide is 25% or less. [10-12] The polynucleotide according to any one of [1] to

[10] , wherein the proportion of the third nucleotide in the n codons that is a sugar-modified nucleotide is 20% or less. [10-13] The polynucleotide according to any one of [1] to

[10] , wherein the proportion of the third nucleotide in the n codons that is a sugar-modified nucleotide is 15% or less. [10-14] The polynucleotide according to any one of [1] to

[10] , wherein the proportion of the third nucleotide in the n codons that is a sugar-modified nucleotide is 10% or less. [10-15] The polynucleotide according to any one of [1] to

[10] , wherein the proportion of the third nucleotide in the n codons that is a sugar-modified nucleotide is 5% or less. [10-16] The polynucleotide according to any one of [1] to [6-18] and [9] to [9-11], wherein the proportion of the third nucleotide in the n codons that is a sugar-modified nucleotide is 0%.

[11] The polynucleotide according to any one of [1] to [10-16], wherein n is an integer of 2 to 2000. [11-1] The polynucleotide according to any one of [1] to [10-16], wherein n is an integer of 2 to 1500. [11-2] The polynucleotide according to any one of [1] to [10-16], wherein n is an integer of 2 to 1000. [11-3] The polynucleotide according to any one of [1] to [10-16], wherein n is an integer of 2 to 500. [11-4] The polynucleotide according to any one of [1] to [10-16], wherein n is an integer of 5 to 2000. [11-5] The polynucleotide according to any one of [1] to [10-16], wherein n is an integer of 5 to 1500. [11-6] The polynucleotide according to any one of [1] to [10-16], wherein n is an integer of 5 to 1000. [11-7] The polynucleotide according to any one of [1] to [10-16], wherein n is an integer of 5 to 500. [11-8] The polynucleotide according to any one of [1] to [10-16], wherein n is an integer of 10 to 2000. [11-9] The polynucleotide according to any one of [1] to [10-16], wherein n is an integer of 10 to 1500. [11-10] The polynucleotide according to any one of [1] to [10-16], wherein n is an integer of 10 to 1000. [11-11] The polynucleotide according to any one of [1] to [10-16], wherein n is an integer of 10 to 500. [11-12] The polynucleotide according to any one of [1] to [10-16], wherein n is an integer of 50 to 2000. [11-13] The polynucleotide according to any one of [1] to [10-16], wherein n is an integer of 50 to 1500. [11-14] The polynucleotide according to any one of [1] to [10-16], wherein n is an integer of 50 to 1000. [11-15] The polynucleotide according to any one of [1] to [10-16], wherein n is an integer of 50 to 500. [11-16] The polynucleotide according to any one of [1] to [10-16], wherein n is an integer of 100 to 2000. [11-17] The polynucleotide according to any one of [1] to [10-16], wherein the n is an integer of 100 to 1500. [11-18] The polynucleotide according to any one of [1] to [10-16], wherein n is an integer of 100 to 1000. [11-19] The polynucleotide according to any one of [1] to [10-16], wherein n is an integer of 100 to 500. [11-20] The polynucleotide according to any one of [1] to [10-16], wherein n is an integer of 200 to 2000. [11-21] The polynucleotide according to any one of [1] to [10-16], wherein n is an integer of 200 to 1500. [11-22] The polynucleotide according to any one of [1] to [10-16], wherein n is an integer of 200 to 1000. [11-23] The polynucleotide according to any one of [1] to [10-16], wherein n is an integer of 200 to 500.

[12] The polynucleotide according to any one of [1] to [11-23], further comprising a 5'-side untranslated region.

[13] the 5'-side untranslated region contains a base-modified nucleotide containing the following base: [ka] [wherein R is an alkyl group having 1 to 6 carbon atoms]

[12] The polynucleotide according to

[12] .

[14] The polynucleotide according to

[12] or

[13] , wherein the first, second, and third nucleotides from the 5' end of the 5' untranslated region are sugar-modified nucleotides.

[15] The polynucleotide according to any one of

[12] to

[14] , further comprising a 5' cap structure.

[16] The polynucleotide according to any one of [1] to

[15] , further comprising a 3' untranslated region.

[17] The polynucleotide according to

[16] , wherein the 3' untranslated region comprises a poly(A) tail.

[18] The polynucleotide according to

[16] or

[17] , wherein the first, second, and third nucleotides from the 3' end of the 3' untranslated region are sugar-modified nucleotides.

[19] The polynucleotide according to any one of

[12] to

[18] , wherein the 5' untranslated region and / or the 3' untranslated region contains sugar-modified nucleotides.

[20] It includes the following structure: [ka] [In the formula, R 1 and R 2 each independently represents H, OH, F, or OCH3; B 1 and B 2 each independently represents a base moiety, X 1 represents O, S or NH, X 2 represents O, S, NH or the following structure: [ka] X 3 represents OH, SH or a salt thereof; However, X 1 and X 2 is not simultaneously O] A polynucleotide according to any one of claims [1] to

[19] . [twenty one] The polynucleotide according to any one of claims [1] to

[20] , comprising a phosphorothioate structure. [twenty two] The polynucleotide according to any one of [1] to

[21] , wherein the first and second nucleotides in at least one of the n codons are linked by a phosphorothioate. [twenty three] The polynucleotide according to any one of [1] to

[22] , wherein the first and second nucleotides, the first to third nucleotides, the first to fourth nucleotides, or the first to fifth nucleotides from the 5' end of the 5' untranslated region are linked by phosphorothioate. [twenty four] The polynucleotide according to any one of [1] to

[23] , wherein the first and second nucleotides, the first to third nucleotides, the first to fourth nucleotides, or the first to fifth nucleotides from the 3' end of the 3' untranslated region are linked by phosphorothioate. [twenty five] A pharmaceutical composition comprising the polynucleotide according to any one of claims [1] to

[24] .

[0011] The present invention further includes the following embodiments. [1A] The polynucleotide according to any one of [1] to

[24] or the pharmaceutical composition according to

[25] , for use in the treatment of a disease. [1B] A method for treating a disease, comprising administering to a patient in need thereof a therapeutically effective amount of the polynucleotide described in any one of [1] to

[24] or the pharmaceutical composition described in

[25] . [1C] Use of the polynucleotide according to any one of [1] to

[24] or the pharmaceutical composition according to

[25] for treating a disease. [1D] Use of the polynucleotide according to any one of [1] to

[24] in the manufacture of a medicament for treating a disease. [1E] The polynucleotide according to any one of [1] to

[24] , for use in the manufacture of a medicament for treating a disease. [1F] A kit for use in treating a disease, comprising the polynucleotide according to any one of [1] to

[24] or the pharmaceutical composition according to

[25] and instructions for use.

[0012] The present invention further includes the following embodiments. [2A] It includes a translated region and a 5' untranslated region, A polynucleotide, wherein the first, second, and third nucleotides from the 5' end of the 5' untranslated region are sugar-modified nucleotides. [2B] a translated region and a 3' untranslated region, A polynucleotide in which the first, second, and third nucleotides from the 3' end of the 3' untranslated region are sugar-modified nucleotides. [2C] a translated region, a 5' untranslated region, and a 3' untranslated region; the first, second, and third nucleotides from the 5' end of the 5' untranslated region are sugar-modified nucleotides; A polynucleotide in which the first, second, and third nucleotides from the 3' end of the 3' untranslated region are sugar-modified nucleotides. [2D] It includes a translated region and a 5' untranslated region, A polynucleotide in which the first to third nucleotides, the first to fourth nucleotides, or the first to fifth nucleotides from the 5' end of the 5' untranslated region are linked by phosphorothioate. [2E] a translated region and a 3' untranslated region, A polynucleotide in which the first to third nucleotides, the first to fourth nucleotides, or the first to fifth nucleotides from the 3' end of the 3' untranslated region are linked by phosphorothioate. [2F] a translated region, a 5' untranslated region, and a 3' untranslated region; the first to third nucleotides, the first to fourth nucleotides, or the first to fifth nucleotides from the 5' end of the 5' untranslated region are linked by phosphorothioate; A polynucleotide in which the first to third nucleotides, the first to fourth nucleotides, or the first to fifth nucleotides from the 3' end of the 3' untranslated region are linked by phosphorothioate. [2G] It includes a translated region and a 5' untranslated region, the first, second, and third nucleotides from the 5' end of the 5' untranslated region are sugar-modified nucleotides; A polynucleotide in which the first to third nucleotides, the first to fourth nucleotides, or the first to fifth nucleotides from the 5' end of the 5' untranslated region are linked by phosphorothioate. [2H] a translated region and a 3' untranslated region, the first, second, and third nucleotides from the 3' end of the 3' untranslated region are sugar-modified nucleotides; A polynucleotide in which the first to third nucleotides, the first to fourth nucleotides, or the first to fifth nucleotides from the 3' end of the 3' untranslated region are linked by phosphorothioate. [2I] a translated region, a 5' untranslated region, and a 3' untranslated region; the first, second, and third nucleotides from the 5' end of the 5' untranslated region are sugar-modified nucleotides; the first to third nucleotides, the first to fourth nucleotides, or the first to fifth nucleotides from the 5' end of the 5' untranslated region are linked by phosphorothioate; the first, second, and third nucleotides from the 3' end of the 3' untranslated region are sugar-modified nucleotides; A polynucleotide in which the first to third nucleotides, the first to fourth nucleotides, or the first to fifth nucleotides from the 3' end of the 3' untranslated region are linked by phosphorothioate. [Effects of the Invention]

[0013] According to the present invention, it is possible to provide a polynucleotide that maintains translation activity while having a modification site in the translation region. [Brief explanation of the drawings]

[0014] [Figure 1] Schematic of a coding region where the first nucleotide of two codons is a sugar-modified nucleotide. [Figure 2] Schematic of an open reading frame where the first nucleotide in every codon is a sugar-modified nucleotide. [Figure 3] A schematic diagram of the translated region in which the first nucleotide of every codon and all three nucleotides in the stop codon are sugar-modified nucleotides. [Figure 4] This figure shows the results of Western blot analysis of translation reactions performed using PURExpress® with compound R1 and compound R17 as substrates. Lanes are as follows: 1-3: compound R17 (respectively at concentrations of 1, 3, and 5 μM in the reaction solution), 4-6: compound R1 (respectively at concentrations of 1, 3, and 5 μM in the reaction solution), 7: no RNA, M: protein size marker (Precision Plus Protein Dual Extra Standards (BIORAD)). The numbers on the left of the figure indicate the molecular weight of the protein, and the arrows indicate the translation products produced. [Figure 5] The figure shows the results of Western blot analysis of translation reactions performed using RRL and compound R2 and compound R18 as substrates. Lanes are as follows: 1: no RNA, 2: compound R18 (5 μg), 3: compound R2 (5 μg), M: protein size marker (Precision Plus Protein Dual Extra Standards (BIORAD)). The numbers on the left of the figure indicate the molecular weight of the protein, and the arrows indicate the translation products produced. DETAILED DESCRIPTION OF THE INVENTION

[0015] <Polynucleotide> One embodiment of the present invention relates to a polynucleotide comprising a translation region from a start codon to a stop codon, the translation region comprising n codons, where n is a positive integer greater than or equal to 2, the n codons each comprising a first, second, and third nucleotide, and the first nucleotide of at least two of the n codons is a sugar-modified nucleotide.

[0016] FIG. 1 shows a schematic diagram of a coding region in which the first nucleotide of any two codons is a sugar-modified nucleotide.

[0017] Since translation activity is maintained even when the sugar moiety of the first nucleotide in multiple codons constituting the translation region is modified, the polynucleotide of this embodiment maintains translation activity even though it has a modification site in the translation region. As used herein, "translation activity" refers to the activity of mRNA being translated to synthesize a polypeptide ("polypeptide" as used herein encompasses proteins). The polynucleotide of this embodiment also has excellent stability against enzymes (e.g., nucleases).

[0018] As used herein, "maintaining translation activity" refers to a polynucleotide in which the sugar moieties of the first nucleotides in multiple codons are modified, having 60% or more of the translation activity of an unmodified polynucleotide. The translation activity of the modified polynucleotide is preferably 70% or more, 80% or more, 90% or more, or 100% or more of the translation activity of an unmodified polynucleotide.

[0019] The polynucleotide of this embodiment is understood to be a polynucleotide that has a function equivalent to, for example, mRNA, small open reading frame (smORF), non-canonical open reading frame, long noncoding RNA (lncRNA), or pri-microRNA (pri-miRNA), in that the translation region is translated into a polypeptide.

[0020] (Translation area) The polynucleotide of this embodiment includes a translation region. The translation region is also called a coding sequence (CDS). One polynucleotide may include multiple translation regions. The translation region is composed of multiple codons from an initiation codon to a termination codon (also called a stop codon), and is a region where a polypeptide is synthesized through translation. A codon is a unit that codes for each amino acid that constitutes a polypeptide, and each unit is composed of three nucleotides.

[0021] Based on the natural codon table, an example of the start codon is, but is not limited to, AUG, which encodes methionine. Non-canonical start codons other than AUG include CUG, GUG, UUG, ACG, AUC, AUU, AAG, AUA, and AGG. Examples of stop codons include UAA, UAG, and UGA. The type of codons constituting the translation region is not particularly limited and can be selected appropriately depending on the polypeptide of interest.

[0022] The number of codons (n) constituting the translation region is preferably an integer of 2 to 2000, more preferably an integer of 2 to 1500, even more preferably an integer of 2 to 1000, and most preferably an integer of 2 to 500. The lower limit of the above numerical range may be changed to 5, 10, 50, 100, 200, etc. When the lower limit is changed, the number of codons (n) constituting the translation region is preferably an integer of 5 to 2000, 10 to 2000, 50 to 2000, 100 to 2000, or 200 to 2000, more preferably an integer of 5 to 1500, 10 to 1500, 50 to 1500, 100 to 1500, or 200 to 1500, even more preferably an integer of 5 to 1000, 10 to 1000, 50 to 1000, 100 to 1000, or 200 to 1000, and most preferably an integer of 5 to 500, 10 to 500, 50 to 500, 100 to 500, or 200 to 500.

[0023] Each codon contains a first, second, and third nucleotide. For example, for the start codon (AUG), the first nucleotide is A, the second nucleotide is U, and the third nucleotide is G.

[0024] A nucleotide typically contains a sugar moiety, a base moiety, and a phosphate moiety. The sugar moiety corresponds to the sugar moiety in a nucleotide, the base moiety corresponds to the base moiety in a nucleotide, and the phosphate moiety corresponds to the phosphate moiety in a nucleotide.

[0025] As used herein, a nucleotide with a modified sugar moiety is referred to as a "sugar-modified nucleotide," a nucleotide with a modified base moiety is referred to as a "base-modified nucleotide," and a nucleotide with a modified phosphate moiety is referred to as a "phosphate-modified nucleotide." As used herein, "modification" refers to changing the structure of the sugar moiety, base moiety, or phosphate moiety. The change in structure due to modification is not particularly limited. Examples of modifications include substitution of any position with any substituent.

[0026] As used herein, sugar moiety modifications do not include substitution of H for the OH bonded to the 2' carbon of the sugar moiety (i.e., substitution of a ribose moiety with a 2'-deoxyribose moiety), nor substitution of H for OH bonded to the 2' carbon of the sugar moiety (i.e., substitution of a 2'-deoxyribose moiety with a ribose moiety). Therefore, when a ribonucleotide is used as the reference, a 2'-deoxyribonucleotide corresponding to a ribonucleotide is not a "sugar moiety-modified nucleotide." Furthermore, when a 2'-deoxyribonucleotide is used as the reference, a ribonucleotide corresponding to a 2'-deoxyribonucleotide is not a "sugar moiety-modified nucleotide."

[0027] The unmodified sugar moiety is preferably a sugar moiety corresponding to ribose or 2'-deoxyribose, more preferably a sugar moiety corresponding to ribose. That is, in the polynucleotide of this embodiment, nucleotides other than sugar-modified nucleotides preferably contain a sugar moiety corresponding to ribose or 2'-deoxyribose, more preferably a sugar moiety corresponding to ribose.

[0028] [Sugar-modified nucleotides] In the polynucleotide of this embodiment, at least two of the first nucleotides contained in the multiple codons constituting the translated region are sugar-modified nucleotides. The position of the codon containing the sugar-modified nucleotide is not particularly limited. The percentage of first nucleotides that are sugar-modified nucleotides is preferably 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 100%. A percentage of 100% means that all first nucleotides are sugar-modified nucleotides. A higher percentage tends to result in better stability against enzymes. Figure 2 shows a schematic diagram of a translated region in which all first nucleotides are sugar-modified nucleotides. Although not particularly limited, when the first nucleotide is a sugar-modified nucleotide, the substituent at the 2'-position of the sugar moiety of the first nucleotide is preferably fluorine.

[0029] In the polynucleotide of this embodiment, at least one of the second nucleotides contained in the multiple codons constituting the translation region may be a sugar-modified nucleotide, but the sugar moiety of the second nucleotide may be unmodified. The proportion of sugar-modified nucleotides at the second nucleotide may be 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, or 0%. A proportion of 0% means that none of the second nucleotides are sugar-modified nucleotides. Although not particularly limited, when the second nucleotide is a sugar-modified nucleotide, the substituent at the 2'-position of the sugar moiety of the second nucleotide is preferably fluorine.

[0030] In the polynucleotide of this embodiment, at least one of the third nucleotides contained in the multiple codons constituting the translation region may be a sugar-modified nucleotide. The proportion of sugar-modified third nucleotides may be 100%, 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, or 0%.

[0031] In the polynucleotide of this embodiment, the first, second, and third nucleotides of the stop codon may be sugar-modified nucleotides in order to improve translation activity. Figure 3 shows a schematic diagram of a translation region in which all first nucleotides and all nucleotides of the stop codon are sugar-modified nucleotides.

[0032] In the polynucleotide of this embodiment, the first, second, and third nucleotides of the start codon may be sugar-modified nucleotides in order to improve stability against nucleases. Although not particularly limited, it is preferred that the substituents at the 2'-positions of the sugar moieties of the first, second, and third nucleotides of the start codon are all fluorine.

[0033] The sugar-modified nucleotide is not particularly limited as long as the sugar moiety of the nucleotide is modified, but preferably contains a sugar moiety modified at least at the 2'-position. Modification at the 2'-position can improve stability against enzymes. The sugar moiety modified at least at the 2'-position may be a sugar moiety in which the 2'- and 4'-positions are cross-linked.

[0034] Examples of modified sugar moieties include the following: [ka] [In the formula, M is R 1 , OR 1 , R 2 OR 1 , S.H., S.R. 1 , NH2, NHR 1 , N.R. 1 2, N3, CN, F, Cl, Br or I; R 1 are each independently an alkyl or an aryl, preferably an alkyl having 1 to 6 carbon atoms, more preferably an alkyl having 1 to 3 carbon atoms; R 2 is alkylene, preferably alkylene having 1 to 6 carbon atoms.

[0035] In this specification, examples of alkyl having 1 to 6 carbon atoms include linear or branched alkyl having 1 to 6 carbon atoms. Examples of linear alkyl having 1 to 6 carbon atoms include methyl, ethyl, propyl, butyl, pentyl, and hexyl. Examples of branched alkyl having 1 to 6 carbon atoms include isopropyl, isobutyl, sec-butyl, tert-butyl, and pentyl substituted with methyl. Examples of the alkyl having 1 to 3 carbon atoms include methyl, ethyl, propyl, and isopropyl.

[0036] As used herein, aryl includes, for example, optionally substituted phenyl and optionally substituted naphthyl.

[0037] In this specification, alkylene having 1 to 6 carbon atoms is a group in which one hydrogen atom bonded to a carbon atom of alkyl having 1 to 6 carbon atoms has been removed.

[0038] As used herein, the term "modified sugar moiety" refers to a modified sugar structure contained in a sugar-modified nucleotide. The modified sugar moiety M can also include 2-(methoxy)ethoxy, 3-aminopropoxy, 2-[(N,N-dimethylamino)oxy]ethoxy, 3-(N,N-dimethylamino)propoxy, 2-[2-(N,N-dimethylamino)ethoxy]ethoxy, 2-(methylamino)-2-oxoethoxy, 2-(N-methylcarbamoyl)ethoxy, and 2-cyanoethoxy.

[0039] Modified sugar moieties further include the sugar moieties of the following nucleic acids: ·Locked Nucleic Acid (LNA) [Tetrahedron Letters, 38, 8735 (1997) and Tetrahedron, 54, 3607 (1998)]; ·Ethylene bridged nucleic acid (ENA) [Nucleic Acids Research, 32, e175 (2004)]; ·Constrained Ethyl (cEt) [The Journal of Organic Chemistry 75, 1569 (2010)]; ·Amido-Bridged Nucleic Acid (AmNA) [Chem Bio Chem 13, 2513 (2012)]; ·2'-O,4'-c-Spirocyclopropylene bridged nucleic acid (scpBNA) [Chem. Commun., 51, 9737 (2015)]; ·tricycloDNA (tcDNA) [Nat. Biotechnol., 35, 238 (2017)]; ·Unlocked Nucleic Acid (UNA) [Mol. Ther. Nucleic Acids 2, e103 (2013)]; 3'-Fluorohexitol nucleic acid (FHNA) [Nat. Biotechnol., 35, 238 (2017)]; Peptide nucleic acid (PNA) [Acc. Chem. Res., 32, 624 (1999)]; Oxypeptide nucleic acid (OPNA) [J. Am. Chem. Soc., 123, 4653 (2001)]; Peptide ribonucleic acid (PRNA) [J. Am. Chem. Soc., 122, 6900 (2000)].

[0040] The modified sugar moiety is not particularly limited, but is preferably selected from the following: [ka]

[0041] The sugar-modified nucleotide preferably contains a base moiety corresponding to a base selected from the group consisting of adenine (A), guanine (G), cytosine (C), and uracil (U), and preferably contains at least two types of bases. Here, "at least two types of bases" means, for example, that one sugar-modified nucleotide contains a base moiety corresponding to adenine, and another sugar-modified nucleotide contains a base moiety corresponding to guanine.

[0042] The sugar-modified nucleotide may be a base-modified nucleotide and / or a phosphate-modified nucleotide (in other words, the sugar-modified nucleotide may further comprise a modified base moiety and / or a modified phosphate moiety). At least one of the sugar-modified nucleotides may comprise a modified base moiety.

[0043] [Base-modified nucleotides] The translated region may contain a base-modified nucleotide. The position of the base-modified nucleotide in the translated region is not particularly limited. The base-modified nucleotide may be a sugar-modified nucleotide and / or a phosphate-modified nucleotide (in other words, the base-modified nucleotide may further contain a modified sugar moiety and / or a modified phosphate moiety).

[0044] The base-modified nucleotide is not particularly limited as long as the base of the nucleotide is modified. Examples of unmodified bases include bases corresponding to adenine, guanine, cytosine, and uracil. Examples of modified bases include bases in which the oxygen of the unmodified base is replaced with sulfur, bases in which the hydrogen of the unmodified base is replaced with alkyl having 1 to 6 carbon atoms, halogen, or the like, bases in which the methyl of the unmodified base is replaced with hydrogen, hydroxymethyl, alkyl having 2 to 6 carbon atoms, or the like, and bases in which the amino of the unmodified base is replaced with alkyl having 1 to 6 carbon atoms, alkanoyl having 1 to 6 carbon atoms, oxo, hydroxy, or the like.

[0045] Specific examples of base moiety-modified nucleotides include 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyladenine, 6-methylguanine, 2-propyladenine, 2-propylguanine, 2-thiouracil, 2-thiothymine, 2-thiocytosine, 5-propynyluracil, 5-propynylcytosine, 6-azouracil, 6-azocytosine, 6-azothymine, 5-pseudouracil, 4-thiouracil, 8-haloadenine, 8-haloguanine, 8-aminoadenine, 8-aminoguanine, 8-mercaptoadenine, 8-mercaptoguanine, 8-alkylthioadenine, 8- Examples include alkylthioguanine, 8-hydroxyadenine, 8-hydroxyguanine, 5-bromouracil, 5-bromocytosine, 5-trifluoromethyluracil, 5-trifluoromethyluracil, 7-methylguanine, 7-methyladenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 3-deazaguanine, 7-deazaadenine, 3-deazaadenine, pyrazolo[3,4-d]pyrimidine, imidazo[1,5-a]1,3,5 triazinone, 9-deazapurine, imidazo[4,5-d]pyrazine, thiazolo[4,5-d]pyrimidine, pyrazin-2-one, 1,2,4-triazine, pyridazine, and 1,3,5-triazine.

[0046] [Phosphate-modified nucleotides] The translated region may contain a phosphate-modified nucleotide. The position in the translated region where the phosphate-modified nucleotide is present is not particularly limited. The phosphate-modified nucleotide may be a sugar-modified nucleotide and / or a base-modified nucleotide (in other words, the phosphate-modified nucleotide may further contain a modified sugar moiety and / or a modified base moiety).

[0047] The phosphate-modified nucleotide is not particularly limited as long as the phosphate moiety (phosphodiester bond) of the nucleotide is modified. Examples of modified phosphate moieties include phosphorothioate bond, phosphorodithioate bond, alkylphosphonate bond, and phosphoramidate bond.

[0048] The translation region may contain phosphate-modified nucleotides in which the modified phosphate moiety is an optical isomer (Rp, Sp). Methods for selectively synthesizing optical isomers of phosphorothioate bonds are disclosed, for example, in J. Am. Chem. Soc., 124, 4962 (2002), Nucleic Acids Research, 42, 13546 (2014), and Science 361, 1234 (2018).

[0049] (5' untranslated region) The polynucleotide of this embodiment may further comprise a 5' untranslated region (5' UTR). The 5' untranslated region is located upstream of the translated region (towards the 5' end) and is a region where translation for polypeptide synthesis does not occur. The number of nucleotides constituting the 5' untranslated region is preferably an integer of 1 to 1,000, more preferably an integer of 1 to 500, even more preferably an integer of 1 to 250, and particularly preferably an integer of 1 to 100.

[0050] The 5'-untranslated region may contain sugar-modified nucleotides. The positions of the sugar-modified nucleotides are not particularly limited, but from the viewpoint of improving translation activity, the first, second, and third nucleotides from the 5'-end may be sugar-modified nucleotides.

[0051] Furthermore, all nucleotides in the 5'-untranslated region may be sugar-modified nucleotides.

[0052] Specific examples of modified sugar moieties of sugar-modified nucleotides include those described above in the section [Sugar-modified nucleotides] (translation region).

[0053] The 5'-untranslated region may contain a base-modified nucleotide. The position of the base-modified nucleotide in the 5'-untranslated region is not particularly limited. The base-modified nucleotide may be a sugar-modified nucleotide and / or a phosphate-modified nucleotide (in other words, the base-modified nucleotide may further contain a modified sugar moiety and / or a modified phosphate moiety).

[0054] Specific examples of the modified base moiety of the base-modified nucleotide include those described in the section "Base-modified nucleotide" above (translated region). From the viewpoint of improving translation activity, the 5'-untranslated region preferably contains, but is not limited to, the following modified base moiety: [ka] [wherein R is an alkyl group having 1 to 6 carbon atoms] The alkyl group R of the modified base moiety is preferably methyl or ethyl.

[0055] Specific examples of alkyl having 1 to 6 carbon atoms include those described above in the section [Sugar-modified nucleotides] in (translation region).

[0056] The 5'-untranslated region may contain a phosphate-modified nucleotide. The position of the phosphate-modified nucleotide in the 5'-untranslated region is not particularly limited. The phosphate-modified nucleotide may be a sugar-modified nucleotide and / or a base-modified nucleotide (in other words, the phosphate-modified nucleotide may further contain a modified sugar moiety and / or a modified base moiety).

[0057] Specific examples of the modified phosphate moiety of the phosphate moiety-modified nucleotide include those described above in the section [Phosphate moiety-modified nucleotide] (translated region).

[0058] (5' cap structure) The polynucleotide of this embodiment may further comprise a 5' cap structure. The 5' cap structure is located upstream of the 5' untranslated region. The inclusion of a 5' cap structure tends to improve translation activity.

[0059] (3' untranslated region) The polynucleotide of this embodiment may further comprise a 3' untranslated region (3'UTR). The 3' untranslated region is located downstream of the translated region and is not translated for polypeptide synthesis.

[0060] The number of nucleotides constituting the 3' untranslated region is preferably an integer of 1 to 6,000, more preferably an integer of 1 to 3,000, even more preferably an integer of 1 to 1,000, and particularly preferably an integer of 1 to 500.

[0061] The 3' untranslated region may contain a poly(A) tail. The 3' untranslated region may contain both a poly(A) tail and a polynucleotide other than a poly(A) tail, or may contain only one of them. The inclusion of a poly(A) tail tends to improve translation activity.

[0062] The length of the poly(A) tail is preferably 1 to 500 bases, more preferably 1 to 200 bases, and even more preferably 1 to 40 bases.

[0063] The 3'-untranslated region may contain sugar-modified nucleotides. The position of the sugar-modified nucleotides is not particularly limited. The sugar-modified nucleotides may be contained in both the polynucleotide other than the polyA strand and the polyA strand, or in only one of them. From the viewpoint of improving translation activity, the first, second, and third nucleotides from the 3'-end of the 3'-untranslated region may be sugar-modified nucleotides. Although not particularly limited, it is preferred that the substituents at the 2'-position of the sugar moieties of the first, second, and third nucleotides from the 3'-end are all 2'-O-methoxyethyl (MOE) groups.

[0064] Specific examples of modified sugar moieties of sugar-modified nucleotides include those described above in the section [Sugar-modified nucleotides] (translation region).

[0065] The 3'-untranslated region may contain a base-modified nucleotide. The position of the base-modified nucleotide in the 3'-untranslated region is not particularly limited. The base-modified nucleotide may be a sugar-modified nucleotide and / or a phosphate-modified nucleotide (in other words, the base-modified nucleotide may further contain a modified sugar moiety and / or a modified phosphate moiety).

[0066] Specific examples of the modified base moiety of the base-modified nucleotide include those described above in the section [Base-modified nucleotide] in (translation region).

[0067] The 3'-untranslated region may contain a phosphate-modified nucleotide. The position of the phosphate-modified nucleotide in the 3'-untranslated region is not particularly limited. The phosphate-modified nucleotide may be a sugar-modified nucleotide and / or a base-modified nucleotide (in other words, the phosphate-modified nucleotide may contain a modified sugar moiety and / or a modified base moiety).

[0068] Specific examples of the modified phosphate moiety of the phosphate moiety-modified nucleotide include those described above in the section [Phosphate moiety-modified nucleotide] (translated region).

[0069] Since phosphate-modified nucleotides can confer stability against endonucleases, which are a type of nuclease, it is preferable that two or more consecutive phosphate-modified nucleotides are included from the 5'-end and / or 3'-end of the polynucleotide of the present invention.

[0070] (Connection part) The polynucleotide of this embodiment may include the following linkages: [ka] [In the formula, R 1 and R 2 each independently represents H, OH, F, or OCH3; B 1 and B 2 each independently represents a base moiety, X 1 represents O, S or NH, X 2 represents O, S, NH or the following structure: [ka] X 3 represents OH, SH or a salt thereof (X 3 OH and SH are respectively O - and S - may also be written as However, X 1 and X 2 is not simultaneously O]

[0071] The left and right nucleotides in the linking portion are the two nucleotides that constitute the polynucleotide of this embodiment. Even if the linking portion is included, translation activity can be maintained. Nucleotide A on the right side (5'-end) of the linking portion and nucleotide B on the left side (3'-end) of the linking portion, as well as nucleotide C on the 3'-end adjacent to nucleotide B and nucleotide D on the 3'-end adjacent to nucleotide C, may not be modified.

[0072] X of the connecting part 3 Examples of OH and SH salts include pharmaceutically acceptable salts. Examples of pharmaceutically acceptable salts include alkali metal salts, alkaline earth metal salts, ammonium salts, organic amine salts, and amino acid salts. Examples of alkali metal salts include sodium salts, lithium salts, and potassium salts. Examples of alkaline earth metal salts include calcium salts and magnesium salts.

[0073] Specific examples of the connecting portion include the following. [ka] [In the formula, R 1 , R 2 , B 1 , B 2 , and X 3 is as above]

[0074] The position of the linking portion is not particularly limited. The linking portion may be present in any of the translated region, the 5' untranslated region, and the 3' untranslated region, but when a linking portion is present, it is preferably present at least in the translated region.

[0075] The number of linkages is not particularly limited and can be appropriately selected depending on the length of the polynucleotide, and examples of the number of linkages include 1 to 200, 1 to 100, 1 to 50, 1 to 20, 1 to 10, 1 to 8, 1 to 6, 1 to 4, 1 to 3, or 1 or 2.

[0076] In the polynucleotide of this embodiment, the first and second nucleotides in at least one of the codons constituting the translation region may be linked by a phosphorothioate bond. The number of phosphorothioate bonds is not particularly limited and can be selected appropriately depending on the length of the polynucleotide. Examples of the number of phosphorothioate bonds include 1 to 200, 1 to 100, 1 to 50, 1 to 20, 1 to 10, 1 to 8, 1 to 6, 1 to 4, 1 to 3, or 1 or 2.

[0077] From the viewpoint of improving translation activity, the first and second nucleotides, the first to third nucleotides, the first to fourth nucleotides, or the first to fifth nucleotides from the 5' end of the 5'-untranslated region may be linked by phosphorothioate. For example, "the first to third nucleotides are linked by phosphorothioate" means that the first and second nucleotides are linked by phosphorothioate, and the second and third nucleotides are linked by phosphorothioate.

[0078] From the viewpoint of improving translation activity, the first and second nucleotides, the first to third nucleotides, the first to fourth nucleotides, or the first to fifth nucleotides from the 3' end of the 3' untranslated region may be linked by phosphorothioate.

[0079] Another embodiment of the invention comprises a translated region and a 5' untranslated region, The polynucleotide relates to a polynucleotide in which the first, second, and third nucleotides from the 5' end of the 5' untranslated region are sugar-modified nucleotides.

[0080] Another embodiment of the invention is a nucleic acid sequence comprising a translated region and a 3' untranslated region, The polynucleotide relates to a polynucleotide in which the first, second, and third nucleotides from the 3' end of the 3' untranslated region are sugar-modified nucleotides.

[0081] Another embodiment of the invention is a nucleic acid sequence comprising a translated region, a 5' untranslated region, and a 3' untranslated region, the first, second, and third nucleotides from the 5' end of the 5' untranslated region are sugar-modified nucleotides; The polynucleotide relates to a polynucleotide in which the first, second, and third nucleotides from the 3' end of the 3' untranslated region are sugar-modified nucleotides.

[0082] Another embodiment of the invention comprises a translated region and a 5' untranslated region, The present invention relates to a polynucleotide in which the first to third nucleotides, the first to fourth nucleotides, or the first to fifth nucleotides from the 5' end of the 5' untranslated region are linked by phosphorothioate.

[0083] Another embodiment of the invention is a nucleic acid sequence comprising a translated region and a 3' untranslated region, The present invention relates to a polynucleotide in which the first to third nucleotides, the first to fourth nucleotides, or the first to fifth nucleotides from the 3' end of the 3' untranslated region are linked by phosphorothioate.

[0084] Another embodiment of the invention is a nucleic acid sequence comprising a translated region, a 5' untranslated region, and a 3' untranslated region, the first to third nucleotides, the first to fourth nucleotides, or the first to fifth nucleotides from the 5' end of the 5' untranslated region are linked by phosphorothioate; The present invention relates to a polynucleotide in which the first to third nucleotides, the first to fourth nucleotides, or the first to fifth nucleotides from the 3' end of the 3' untranslated region are linked by phosphorothioate.

[0085] Another embodiment of the invention comprises a translated region and a 5' untranslated region, the first, second, and third nucleotides from the 5' end of the 5' untranslated region are sugar-modified nucleotides; The present invention relates to a polynucleotide in which the first to third nucleotides, the first to fourth nucleotides, or the first to fifth nucleotides from the 5' end of the 5' untranslated region are linked by phosphorothioate.

[0086] Another embodiment of the invention is a nucleic acid sequence comprising a translated region and a 3' untranslated region, the first, second, and third nucleotides from the 3' end of the 3' untranslated region are sugar-modified nucleotides; The present invention relates to a polynucleotide in which the first to third nucleotides, the first to fourth nucleotides, or the first to fifth nucleotides from the 3' end of the 3' untranslated region are linked by phosphorothioate.

[0087] Another embodiment of the invention is a nucleic acid sequence comprising a translated region, a 5' untranslated region, and a 3' untranslated region, the first, second, and third nucleotides from the 5' end of the 5' untranslated region are sugar-modified nucleotides; the first to third nucleotides, the first to fourth nucleotides, or the first to fifth nucleotides from the 5' end of the 5' untranslated region are linked by phosphorothioate; the first, second, and third nucleotides from the 3' end of the 3' untranslated region are sugar-modified nucleotides; The present invention relates to a polynucleotide in which the first to third nucleotides, the first to fourth nucleotides, or the first to fifth nucleotides from the 3' end of the 3' untranslated region are linked by phosphorothioate.

[0088] (Other sequences) The polynucleotide of this embodiment may further comprise a Kozak sequence and / or a Ribosome Binding sequence (RBS).

[0089] <Method of producing polynucleotides> The polynucleotide of this embodiment can be produced, for example, by chemical synthesis. Specifically, the polynucleotide of this embodiment can be produced by using a known chemical synthesis method to introduce predetermined sugar-modified nucleotides at predetermined positions while extending the polynucleotide chain. Known chemical synthesis methods include, for example, the phosphoramidite method, the phosphorothioate method, the phosphotriester method, and the CEM method (see Nucleic Acids Research, 35, 3287 (2007)). Alternatively, an ABI3900 high-throughput nucleic acid synthesizer (manufactured by Applied Biosystems) can also be used.

[0090] More specifically, known chemical synthesis methods include those described in the following literature: Tetrahedron, Vol. 48, No. 12, pp. 2223-2311 (1992); Current Protocols in Nucleic Acids Chemistry, John Wiley & Sons (2000); Protocols for Oligonucleotides and Analogs, Human Press (1993); Chemistry and Biology of Artificial Nucleic Acids, Wiley-VCH (2012); Genome Chemistry: A Scientific Approach Utilizing Artificial Nucleic Acids, Kodansha (2003); New Trends in Nucleic Acid Chemistry, Kagaku Dojin (2011).

[0091] A phosphoramidite that is not commercially available can be chemically synthesized and used as a starting material to produce the polynucleotide of this embodiment. The method for synthesizing phosphoramidite (f), which is the raw material for base-modified nucleotides, is shown below. [ka] [In the synthesis scheme, Ra represents a hydrogen atom, F, OMe, or OCH2CH2OMe; Rb represents a protecting group that can be removed with a fluoride ion, such as di-tert-butylsilyl; Rc represents an alkyl group having 1 to 6 carbon atoms; and Rd represents a protecting group used in solid-phase nucleic acid synthesis, such as a p,p'-dimethoxytrityl group.]

[0092] (Process A) Compound (b) can be produced by reacting compound (a) with, for example, a corresponding silylating agent in a solvent in the presence of a base at a temperature between 0°C and 80°C for 10 minutes to 3 days. Examples of the solvent include DMF, DMA, and NMP, which may be used alone or in combination. Examples of the base include imidazole, triethylamine, diisopropylethylamine and the like. Examples of the silylating agent include di-tert-butylsilyl bis(trifluoromethanesulfonate).

[0093] (Process B) Compound (c) can be produced by reacting compound (b) with a corresponding alkylating agent in a solvent in the presence of a base at a temperature between 0° C. and 150° C. for 10 minutes to 3 days. The reaction can be accelerated by the use of an appropriate additive. Examples of the solvent include DMF, pyridine, dichloromethane, THF, ethyl acetate, 1,4-dioxane, and NMP, which may be used alone or in combination. Examples of the base include aqueous sodium hydroxide solution, potassium carbonate, pyridine, triethylamine, N-ethyl-N,N-diisopropylamine, and the like. Examples of the alkylating agent include methyl iodide, ethyl iodide, and methyl bromide. An example of the additive is tetrabutylammonium bromide.

[0094] (Process C) Compound (d) can be produced by reacting compound (c) with a fluorine reagent in a solvent at a temperature between -80°C and 200°C for 10 seconds to 72 hours. A base may also be added at this time. Examples of fluorine reagents include hydrogen fluoride, triethylamine hydrofluoric acid, and tetrabutylammonium fluoride (TBAF). Examples of the base include triethylamine, N,N-diisopropylethylamine, and the like. Examples of the solvent include dichloromethane, chloroform, acetonitrile, toluene, ethyl acetate, THF, 1,4-dioxane, DMF, N,N-dimethylacetamide (DMA), NMP, and dimethyl sulfoxide (DMSO).

[0095] (Process D) Compound (e) can be produced by reacting compound (d) with a corresponding alkylating agent in a solvent in the presence of a base at a temperature between 0° C. and 150° C. for 10 minutes to 3 days. The reaction can also be accelerated by the use of an appropriate activating agent. Examples of the solvent include DMF, pyridine, dichloromethane, THF, ethyl acetate, 1,4-dioxane, and NMP, which may be used alone or in combination. Examples of the base include pyridine, triethylamine, N-ethyl-N,N-diisopropylamine, 2,6-lutidine, and the like. Examples of the alkylating agent include trityl chloride, p,p'-dimethoxytrityl chloride, and the like. The activating agent may, for example, be 4-dimethylaminopyridine.

[0096] (Process E) Compound (f) can be produced by reacting compound (e) with compound (g) in a solvent in the presence of a base at a temperature between 0°C and 100°C for 10 seconds to 24 hours. Examples of the solvent include dichloromethane, acetonitrile, toluene, ethyl acetate, THF, 1,4-dioxane, DMF, and NMP, which may be used alone or in combination. Examples of the base include triethylamine, N,N-diisopropylethylamine, pyridine, etc., which may be used alone or in combination.

[0097] The 5'-cap structure can be introduced using known methods (e.g., enzymatic methods and chemical synthesis methods), such as those described in Top. Curr. Chem. (Z) (2017) 375:16 and Beilstein J. Org. Chem. 2017, 13, 2819-2832.

[0098] When the polynucleotide of this embodiment has a long base length, a plurality of polynucleotide units may be linked together. The linking method is not particularly limited, and examples thereof include enzymatic methods and chemical synthesis methods.

[0099] Enzymatic ligation can be achieved, for example, by ligation using a ligase. Examples of ligases include T4 DNA Ligase, T4 RNA Ligase 1, T4 RNA Ligase 2, T4 RNA Ligase 2, truncated T4 RNA Ligase 2, truncated KQ, E. coli DNA Ligase, and Taq DNA Ligase. These can be used alone or in combination. In enzymatic ligation, it is generally preferred that nucleotide A at the 3' end of the polynucleotide unit constituting the 5' end of the polynucleotide (hereinafter referred to as the "5' end polynucleotide unit"), nucleotide B at the 5' end of the polynucleotide unit constituting the 3' end of the polynucleotide (hereinafter referred to as the "3' end polynucleotide unit") (nucleotides A and B are adjacent to each other in the ligated polynucleotide), nucleotide C adjacent to nucleotide B, and nucleotide D adjacent to nucleotide C are unmodified. On the other hand, when T4 RNA ligase 2 described in Molecular Cell, Vol. 16, 211-221, October 22, 2004, or the like is used, the nucleotides A to D may be modified.

[0100] In enzymatic linkage, polydisperse polyethylene glycol (PEG) may be used to promote the linkage reaction by the molecular crowding effect. Examples of polydisperse PEG include PEG4000, PEG6000, PEG8000, and PEG10000, which may be used alone or in combination.

[0101] An example of a chemical synthesis method (also referred to as "chemical ligation") is a method in which the 3' end of the 5'-end polynucleotide unit (on the right side below) and the 5' end of the 3'-end polynucleotide unit (on the left side below) are condensed in the presence of a condensing agent, as shown below. [ka] [In the formula, R 1 , R 2 , B 1 , B 2 , X 1 , X 2 , and X 3 is as above]

[0102] Examples of condensing agents include 1,3-dicyclohexanecarbodiimide (DCC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), carbonyldiimidazole, benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate, (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate, O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), and 2-chloro-1-methylpyridinium iodide.

[0103] The condensation reaction is preferably carried out in the presence of template DNA containing a nucleotide chain complementary to the 3'-terminal nucleotide chain of the 5'-terminal polynucleotide unit and the 5'-terminal nucleotide chain of the 3'-terminal polynucleotide unit. The template DNA is preferably a nucleotide chain that is preferably 2 to 50 bases long, more preferably 5 to 40 bases long, from the 3'-terminal of the 5'-terminal polynucleotide unit, and a nucleotide chain that is complementary to the nucleotide chain that is preferably 2 to 50 bases long, more preferably 5 to 40 bases long, from the 5'-terminal of the 3'-terminal polynucleotide unit. Here, "complementary" means that the base sequence identity is, for example, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 100%.

[0104] In the condensation reaction, an additive may be added, such as 1-hydroxybenzotriazole (HOBt) and 4-dimethylaminopyridine (DMAP).

[0105] The condensation reaction may be carried out in the presence of a buffer solution, such as an acetate buffer solution, a Tris buffer solution, a citrate buffer solution, a phosphate buffer solution, or water.

[0106] The temperature for the condensation reaction is not particularly limited, but may be, for example, room temperature to 200° C. The time for the condensation reaction is not particularly limited, but may be, for example, 5 minutes to 100 hours.

[0107] Specific examples of the condensation reaction between the 3' end of the 5' end polynucleotide unit (right side below) and the 5' end of the 3' end polynucleotide unit (left side below) include the following. [ka] [In the formula, R 1 , R 2 , B 1 , B 2 , and X 3 is as described above, and X 4 is a leaving group.]

[0108] Specific examples of the leaving group include a chloro group, a bromo group, an iodo group, a methanesulfonyl group, a p-toluenesulfonyl group, and a trifluoromethanesulfonyl group. The leaving group is not particularly limited, but is preferably a chloro group or a bromo group.

[0109] The polynucleotide units may be linked multiple times depending on the length of the desired polynucleotide. The number of times of linkage is not particularly limited, and may be, for example, 1 to 200 times, 1 to 100 times, 1 to 50 times, 1 to 20 times, 1 to 10 times, 1 to 8 times, 1 to 6 times, 1 to 4 times, 1 to 3 times, or 1 or 2 times.

[0110] The methods for producing Compound (M) and Compound (N), which are the 5'-terminal polynucleotide units used for ligation, are shown below. [ka] [In the formula, B p represents a base which may be protected with a protecting group, B represents a base, and Polymer represents a solid support. 4 represents a selectively deprotectable protecting group, such as a tert-butyldimethylsilyl group or a triethylsilyl group; R 3 is a protecting group used in solid-phase nucleic acid synthesis, for example, p,p'-dimethoxytrityl group, and X a represents a nucleic acid sequence, and Y a and Y b are each independently a leaving group, for example, a halogen, preferably a chlorine atom or a bromine atom. In this specification, a nucleic acid sequence is a partial structure in a nucleic acid that forms a nucleic acid together with a compound to which it is bound. When there are multiple Bs in a molecule, each B may be the same or different.

[0111] (Process 1) Compound (B) can be produced by reacting compound (A) in a solvent at a temperature between 60° C. and the boiling point of the solvent used for 10 seconds to 3 days. Examples of solvents include toluene, xylene, 1,2-dichloroethane, 1,4-dioxane, N,N-dimethylformamide (DMF), N-methylpyrrolidone (NMP), 1,2-dichlorobenzene, and water, and these can be used alone or in combination. Compound (A) can be produced, for example, by the method described in J. Am. Chem. Soc. (1999), 121, 5661-5665.

[0112] B in compound (A) p is not particularly limited, but preferably has one of the following structures:

[0113] [ka] R 6 is a group constituting a part of a protecting group for a base, and represents, for example, a methyl group, an isopropyl group, an optionally substituted phenyl group, etc. Examples of the substituent in the optionally substituted phenyl group include a methyl group, an isopropyl group, and a tert-butyl group.

[0114] (Process 2) Compound (C) can be produced by reacting compound (B) with, preferably, 1 to 100 equivalents of an additive in a solvent in the presence of 1 to 100 equivalents of an oxidizing agent at a temperature between 0°C and the boiling point of the solvent used for 10 seconds to 3 days. Examples of the solvent include aprotic solvents such as chloroform and dichloromethane, which can be used alone or in combination. Examples of the oxidizing agent include organic oxidizing agents such as Jones reagent, chromic acid, pyridinium dichromate, ruthenium tetroxide, sodium chlorite, and Dess-Martin reagent, and inorganic oxidizing agents such as pyridinium chlorochromate, and these can be used alone or in combination. Examples of additives include pyridine, triethylamine, N,N-diisopropylethylamine, etc., which can be used alone or in combination.

[0115] (Step 3) Compound (D) can be produced by reacting compound (C) in the presence of hydroxylamine hydrochloride in a solvent such as pyridine at a temperature between 0°C and the boiling point of the solvent used for 10 seconds to 3 days.

[0116] (Step 4) Compound (E) can be produced by reacting compound (D) in a solvent in the presence of 1 to 100,000 equivalents of a deprotecting agent at a temperature between 0°C and the boiling point of the solvent used for 10 seconds to 3 days. Examples of the solvent include toluene, xylene, water, etc., and these can be used alone or in combination. Examples of deprotecting agents include trifluoroacetic acid, trichloroacetic acid, acetic acid, and hydrochloric acid, and these can be used alone or in combination.

[0117] (Step 5) Compound (F) can be produced by reacting compound (E) in a solvent in the presence of a reducing agent at a temperature between 0°C and the boiling point of the solvent used for 10 seconds to 3 days. Examples of the solvent include trifluoroacetic acid, trichloroacetic acid, acetic acid, hydrochloric acid, toluene, xylene, tetrahydrofuran, methanol, ethanol, 1,4-dioxane, and water, and these can be used alone or in combination. Examples of the reducing agent include sodium borohydride, sodium cyanoborohydride, lithium borohydride, and sodium triacetoxyborohydride.

[0118] (Step 6) Compound (G) can be produced by reacting compound (F) in a solvent in the presence of a catalyst under a hydrogen atmosphere at a temperature between 0°C and the boiling point of the solvent used for 10 seconds to 3 days. Examples of the solvent include trifluoroacetic acid, acetic acid, diluted hydrochloric acid, methanol, ethanol, isopropanol, and water, and these can be used alone or in combination. Examples of the catalyst include palladium on carbon and ruthenium on carbon. Compound (G) can also be produced, for example, by the method described in WO 2017 / 123669.

[0119] (Step 7) Compound (H) can be produced by reacting compound (G) with, preferably 1 to 1,000 equivalents of a base in a solvent in the presence of 1 to 100 equivalents of compound (G') and a base at a temperature between 0°C and the boiling point of the solvent used for 10 seconds to 3 days. Examples of solvents include methanol, ethanol, isopropanol, dichloromethane, acetonitrile, toluene, ethyl acetate, tetrahydrofuran (THF), 1,4-dioxane, N,N-dimethylformamide (DMF), N-methylpyrrolidone (NMP), and water, and these can be used alone or in combination. Examples of the base include pyridine, triethylamine, N-ethyl-N,N-diisopropylamine, 2,6-lutidine, etc., which can be used alone or in combination. Compound (G') may be a commercially available product.

[0120] (Step 8) Compound (I) can be produced by reacting compound (H) with p,p'-dimethoxytrityl chloride in a solvent such as pyridine, optionally in the presence of a co-solvent, at a temperature between 0°C and 100°C for 5 minutes to 100 hours. Examples of co-solvents include methanol, ethanol, dichloromethane, chloroform, 1,2-dichloroethane, toluene, ethyl acetate, acetonitrile, diethyl ether, tetrahydrofuran, 1,2-dimethoxyethane, dioxane, N,N-dimethylformamide (DMF), N,N-dimethylacetamide, N-methylpyrrolidone, triethylamine, N,N-diisopropylethylamine, and water, and these can be used alone or in combination.

[0121] (Step 9) Compound (J) can be produced by reacting compound (I) with 1 to 10 equivalents of an additive in a solvent at a temperature between 0°C and the boiling point of the solvent used for 10 minutes to 10 days. Examples of the solvent include dichloromethane, acetonitrile, toluene, ethyl acetate, THF, 1,4-dioxane, DMF, DMA, and NMP, and these can be used alone or in combination. Examples of additives include tetrabutylammonium fluoride, triethylamine trihydrofluoride, etc., which may be used alone or in combination.

[0122] (Step 10) Compound (K) can be produced by reacting compound (J) with succinic anhydride in a solvent in the presence of 1 to 30 equivalents of a base at a temperature between room temperature and 200°C for 5 minutes to 100 hours. Examples of solvents include methanol, ethanol, dichloromethane, chloroform, 1,2-dichloroethane, toluene, ethyl acetate, acetonitrile, diethyl ether, tetrahydrofuran, 1,2-dimethoxyethane, dioxane, N,N-dimethylformamide (DMF), N,N-dimethylacetamide, N-methylpyrrolidone, pyridine, and water, and these can be used alone or in combination. Examples of the base include cesium carbonate, potassium carbonate, potassium hydroxide, sodium hydroxide, sodium methoxide, potassium tert-butoxide, triethylamine, diisopropylethylamine, N-methylmorpholine, pyridine, 1,8-diazabicyclo[5.4.0]-7-undecene (DBU), and N,N-dimethyl-4-aminopyridine (DMAP), and these can be used alone or in combination.

[0123] (Step 11) Compound (L) can be produced by reacting compound (K) with a terminally aminated solid support, without a solvent or in a solvent, in the presence of 1 to 30 equivalents of a base, a condensing agent, and, if necessary, 0.01 to 30 equivalents of an additive, at a temperature between room temperature and 200°C for 5 minutes to 100 hours, and then reacting in an acetic anhydride / pyridine solution at a temperature between room temperature and 200°C for 5 minutes to 100 hours. Examples of the solvent include those exemplified in Step 4. Examples of the base include cesium carbonate, potassium carbonate, potassium hydroxide, sodium hydroxide, sodium methoxide, potassium tert-butoxide, triethylamine, diisopropylethylamine, N-methylmorpholine, pyridine, 1,8-diazabicyclo[5.4.0]-7-undecene (DBU), and N,N-dimethyl-4-aminopyridine (DMAP), and these can be used alone or in combination. Examples of condensing agents include 1,3-dicyclohexanecarbodiimide (DCC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), carbonyldiimidazole, benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate, (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate, O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), and 2-chloro-1-methylpyridinium iodide. Examples of additives include 1-hydroxybenzotriazole (HOBt), 4-dimethylaminopyridine (DMAP), etc., which can be used alone or in combination. The solid phase support is not particularly limited as long as a known aminated solid phase support is used in solid phase synthesis, and examples thereof include solid phase supports such as controlled pore glass (CPG) and polystyrene resin (PS) modified with long-chain alkylamino groups. For example, a commercially available long-chain alkylamine controlled pore glass (LCAA-CPG) can be used.

[0124] (Step 12) Compound (M) can be produced by using compound (L) to elongate the corresponding nucleotide chain by a known oligonucleotide chemical synthesis method, followed by detachment from the solid phase, deprotection of the protecting group, and purification. The removal and deprotection from the solid phase can be achieved by treating the oligonucleotide after chemical synthesis with a base in a solvent or without a solvent at a temperature between −80° C. and 200° C. for 10 seconds to 72 hours. Examples of bases include ammonia, methylamine, dimethylamine, ethylamine, diethylamine, isopropylamine, diisopropylamine, piperidine, triethylamine, ethylenediamine, 1,8-diazabicyclo[5.4.0]-7-undecene (DBU), potassium carbonate, etc., and these can be used alone or in combination. Examples of the solvent include water, methanol, ethanol, THF, etc., which can be used alone or in combination. Oligonucleotides can be purified using a C18 reversed-phase column or an anion exchange column, or preferably by combining the two methods. The purity of the nucleic acid complex after purification is preferably 90% or more, more preferably 95% or more.

[0125] (Step 13) Compound (N) can be produced by reacting compound (M) in a buffer solution in the presence of 1 to 1,000 equivalents of compound (O) at a temperature between room temperature and 100°C for 5 minutes to 100 hours. Examples of the buffer solution include acetate buffer, Tris buffer, citrate buffer, phosphate buffer, water, etc., and these can be used alone or in combination. Compound (O) may be a commercially available product.

[0126] The method for producing compound (W), which is the 3'-terminal polynucleotide unit used for linking, is shown below. [ka] [In the formula, B p represents a base which may be protected by a protecting group, B represents a base, R 7 represents a protecting group, such as a tert-butyldimethylsilyl group or a triethylsilyl group; Yc represents, for example, a chlorine atom, a bromine atom or a tosylate group; X b represents a nucleic acid sequence. When multiple Bs are present in a molecule, each B may be the same or different.]

[0127] (Step 14) Compound (Q) can be produced by reacting compound (P) in a solvent in the presence of an additive and a base at a temperature between 0°C and the boiling point of the solvent used for 10 seconds to 3 days. Examples of the solvent include dichloromethane, acetonitrile, toluene, ethyl acetate, THF, 1,4-dioxane, DMF, DMA, and NMP, and these can be used alone or in combination. Examples of additives include tosylic anhydride, tosyl chloride, thionyl chloride, oxalyl chloride, etc., which may be used alone or in combination. Examples of the base include pyridine, triethylamine, N-ethyl-N,N-diisopropylamine, potassium carbonate, etc., and these can be used alone or in combination. Compound (P) can be a commercially available product.

[0128] (Step 15) Compound (R) can be produced by reacting compound (Q) in a solvent in the presence of an azidating agent and, if necessary, a base, at a temperature between room temperature and the boiling point of the solvent used for 10 seconds to 3 days. Examples of the solvent include dichloromethane, acetonitrile, toluene, ethyl acetate, THF, 1,4-dioxane, DMF, DMA, and NMP, and these can be used alone or in combination. Examples of the azidating agent include sodium azide. Examples of the base include pyridine, triethylamine, N-ethyl-N,N-diisopropylamine, potassium carbonate, etc., and these can be used alone or in combination.

[0129] (Step 16) Compound (S) can be produced by reacting compound (R) in a solvent in the presence of a silylating agent and a base at a temperature between room temperature and the boiling point of the solvent used for 10 seconds to 3 days. Examples of the solvent include dichloromethane, acetonitrile, toluene, ethyl acetate, THF, 1,4-dioxane, DMF, DMA, and NMP, and these can be used alone or in combination. Examples of the silylating agent include tert-butyldimethylsilyl chloride, tert-butyldimethylsilyl triflate, and triethylsilyl chloride. Examples of the base include pyridine, triethylamine, N-ethyl-N,N-diisopropylamine, potassium carbonate, potassium hydroxide, sodium hydroxide, sodium methoxide, potassium tert-butoxide, triethylamine, diisopropylethylamine, N-methylmorpholine, pyridine, 1,8-diazabicyclo[5.4.0]-7-undecene (DBU), and N,N-dimethyl-4-aminopyridine (DMAP), and these can be used alone or in combination.

[0130] (Step 17) Compound (T) can be produced by adding a reducing agent to compound (S) in a solvent and reacting the mixture at a temperature between room temperature and the boiling point of the solvent used for 10 seconds to 3 days. Examples of solvents include methanol, ethanol, dichloromethane, chloroform, 1,2-dichloroethane, toluene, ethyl acetate, acetonitrile, diethyl ether, tetrahydrofuran, 1,2-dimethoxyethane, dioxane, N,N-dimethylformamide (DMF), N,N-dimethylacetamide, N-methylpyrrolidone, triethylamine, N,N-diisopropylethylamine, acetic acid, and water, and these can be used alone or in combination. Examples of the reducing agent include sodium borohydride, sodium cyanoborohydride, lithium borohydride, sodium triacetoxyborohydride, palladium on carbon under a hydrogen atmosphere, and the like.

[0131] (Step 18) Compound (U) can be produced in the same manner as in Step 7 using compound (T).

[0132] (Step 19) Compound (V) can be produced by reacting compound (U) with compound (AA) in a solvent in the presence of a base at a temperature between 0°C and 100°C for 10 seconds to 24 hours. Examples of the solvent include dichloromethane, acetonitrile, toluene, ethyl acetate, THF, 1,4-dioxane, DMF, and NMP, and these can be used alone or in combination. Examples of the base include triethylamine, N,N-diisopropylethylamine, pyridine, etc., which can be used alone or in combination. Compound (AA) may be a commercially available product.

[0133] (Step 20) Compound (W) can be produced in the same manner as in Step 12 using compound (V).

[0134] When the polynucleotide of this embodiment is produced by linking multiple polynucleotide units, it may contain some polynucleotide units produced by IVT. The method for linking polynucleotides produced by IVT is not particularly limited, and examples include the above-mentioned enzymatic method and chemical synthesis method. An example of a method for producing polynucleotide units using IVT is a method in which RNA is transcribed from a template DNA having a promoter sequence using RNA polymerase. More specifically, examples of known IVT include the methods described in the following literature. RNA, Methods in Molecular Biology (Methods and Protocols), Volume 703, Chapter 3 (2011); Cardiac Gene Therapy: Methods in Moleculer Biology (Methods and Protocols), Vol. 1521, Chapter 8 (2016); Journal of Molecular Biology, Vol. 249, pp. 398-408 (1995)

[0135] Template DNA used in IVT includes, for example, those produced by chemical synthesis, those produced by polymerase chain reaction, plasmid DNA, and those produced by linearizing plasmid DNA with a restriction enzyme, and these can be used alone or in combination. RNA polymerases include T3 RNA polymerase, T7 RNA polymerase, SP6 RNA polymerase, etc., and these can be used alone or in combination. The ribonucleoside triphosphate used during transcription may be modified, and multiple types of ribonucleoside triphosphates can also be used in combination. As described in Cardiac Gene Therapy: Methods in Molecular Biology (Methods and Protocols), Vol. 1521, Chapter 8 (2016), a 5' Cap structure can be added using compounds such as m7G(5')ppp(5')G (Trilink, Catalog No. S1404) or Anti Reverse Cap Analog, 3'-O-Me-m7G(5')ppp(5')G (Trilink, Catalog No. N-7003). As described in Journal of Molecular Biology, Vol. 249, pp. 398-408 (1995), inserting a sequence such as the Hepatitis delta virus (HDV) ribozyme into template DNA allows for cleavage of the 5' or 3' end of RNA after transcription.

[0136] <Pharmaceutical Composition> One embodiment of the present invention relates to a pharmaceutical composition comprising the polynucleotide. By administering the pharmaceutical composition of this embodiment to a patient with a disease, the polynucleotide is translated, a polypeptide encoded by the polynucleotide is synthesized, and the disease is treated.

[0137] Although not particularly limited, the present invention provides a method for treating diseases characterized by the loss or abnormality of the function or activity of a specific protein by compensating for that function or activity with a polypeptide translated from the polynucleotide. Alternatively, the present invention also provides a therapeutic method for artificially controlling immune responses by expressing a foreign antigen peptide or its analog in vivo using a polypeptide translated from the polynucleotide. Furthermore, by expressing in vivo a specific protein in the body, such as a transcription factor, or a polypeptide not naturally occurring in the body, using a polypeptide translated from the polynucleotide, it is also possible to artificially control or modify the function, differentiation, proliferation, etc. of cells, thereby providing a therapeutic method for restoring the function of tissues or cells in diseases characterized by damaged tissues or cells or reduced or abnormal function or activity.

[0138] Diseases include, but are not limited to, cancer and proliferative diseases, infectious and parasitic diseases, blood and hematopoietic diseases, autoimmune diseases, endocrine, nutritional and metabolic diseases (including congenital metabolic disorders), mental and nervous system diseases, skin and subcutaneous tissue diseases, eye diseases, ear diseases, respiratory system diseases, digestive system diseases, renal, urinary and reproductive system diseases, cardiovascular diseases, cerebrovascular diseases, musculoskeletal and connective tissue diseases, miscarriage, perinatal diseases, congenital malformations, acquired injuries, and poisoning.

[0139] The pharmaceutical composition may be administered in a predetermined formulation. Examples of the formulation include liquid dosage forms for oral or parenteral administration, such as pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents (e.g., water or other solvents), solubilizers, and emulsifiers commonly used in the art (e.g., ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (especially cottonseed, peanut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol, and fatty acid esters of sorbitan, and mixtures thereof). Formulations for oral administration may include adjuvants (e.g., wetting agents, emulsifying and suspending agents), sweeteners, flavoring agents, and / or perfuming agents. Formulations for parenteral administration may include solubilizing agents (e.g., Cremophor®, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and combinations thereof).

[0140] Methods for administering the pharmaceutical composition include, for example, local administration into lymph nodes, local administration into tumors, intramuscular administration, intradermal administration, subcutaneous administration, intratracheal administration, intrathecal administration, intraventricular administration, intraocular administration, intratympanic administration, catheter administration into coronary arteries, catheter administration into hepatic portal vein, catheter administration into myocardium, transurethral catheter administration, and intravenous administration.

[0141] In addition to the polynucleotide, the pharmaceutical composition may contain optional ingredients, such as one or more pharmaceutically acceptable additives selected from solvents, aqueous solvents, non-aqueous solvents, dispersion media, diluents, dispersions, suspending aids, surfactants, isotonicity agents, thickeners, emulsifiers, preservatives, lipids, lipidoid liposomes, lipid nanoparticles, core-shell nanoparticles, polymers, lipoplexes, peptides, proteins, cells, hyaluronidase, and mixtures thereof. [Example]

[0142] The present invention will be described in more detail below using examples and reference examples, but the technical scope of the present invention is not limited to these.

[0143] Reagents used in the synthesis of compounds were purchased from Sigma-Aldrich, Tokyo Chemical Industry Co., Ltd., Wako Pure Chemical Industries, Ltd., and Kanto Chemical Co., Ltd. and were used without further purification. Anhydrous solvents were prepared by drying over activated molecular sieves 4A for 12 hours, or commercially available anhydrous-grade solvents were used. Reactions were monitored by thin-layer silica gel chromatography (silica gel 70F254 TLC plate - Wako, Wako Pure Chemical Industries, Ltd.). Compounds were purified using flash chromatography silica gel 60N (spherical, neutral, particle size 40–50 μm) (Kanto Chemical Co., Ltd.). NMR was measured using a JEOL ECS 400 MHz (JEOL Ltd.) with deuterated solvents (CDCl3, CD3OD, DMSO-d6) (Kanto Chemical Co., Ltd.). The acquired NMR data was analyzed using JEOL Delta software (JEOL Ltd.), and chemical shift values ​​were corrected using residual signals in deuterated solvents (CDCl3: 7.26, CD3OD: 3.31, DMSO-d6: 2.50) (Organometallics 2010, 29, 2176-2179). 1H NMR data are reported as chemical shift (δ), integral (H), signal splitting mode, and coupling constant (Hz) (s: Singlet, d: Doublet, t: Triplet, sept.: Septet, m: Multiplet, br.: Broad). High-resolution mass spectrometry was performed using a microTOF-QII ESI (Bruker Daltonics), and accurate mass correction was performed using ESI TUNING MIX (Agilent Technologies) as an internal standard.

[0144] Compound 12, which is a starting material for polynucleotides, was synthesized according to the following scheme. [ka]

[0145] Step 1: Synthesis of Compound 4 N-(9-((2R,3S,4S,5R)-3-(tert-butyldimethylsilyloxy)-5-((tert-butyldimethylsilyloxy)methyl)-4-hydroxy-tetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide Compound 3, obtained by a literature method (J. Am. Chem. Soc., 1999, 121, 5661-5665), was dissolved in 1,2-dichlorobenzene (2.0 mL) and stirred in an oil bath (160°C) for 4 hours. After returning to room temperature, the reaction mixture was purified by flash column chromatography (neutral silica gel, dichloromethane / methanol = 40:1) without concentration to give compound 4 as a white solid (0.31 g, 53% yield). 11H NMR (400 MHz, CDCl3) δ 12.01 (1H, s), 8.50 (1H, s), 8.07 (1H, s), 5.86 (1H, d, J = 6.0 Hz), 4.47 (1H, s), 4.24 - 4.23 (1H, m), 4.22 - 4.21 (1H, m), 3.93 (1H, dd, J = 11.6, 2.0 Hz), 3.82 (1H, dd, J = 11.6, 2.0 Hz), 2.66 (1H, sept., J = 6.8 Hz), 1.27 (3H, d, J = 6.8 Hz), 1.25 (3H, d, J = 6.8 Hz), 0.93 (9H, s), 0.82 (9H, s), 0.13 (3H, s), 0.12 (3H, s), -0.07 (3H, s), -0.20 (3H, s) 13 13C NMR (100 MHz, CDCl3) δ 179.0, 155.7, 148.5, 148.7, 147.8, 136.8, 121.0, 87.4, 85.4, 77.6, 71.8, 63.6, 36.2, 25.9, 25.4, 19.1, 18.7, 18.3, 17.8, -5.3, -5.4, -5.5, -5.6 ESI-HRMS: calcd for C[[ID=�]] 26 H 48 N5O6Si2582.31 [M+H] + , found : 582.31 [M+H]<0̇000067>

[0146] Engineering 2 Synthesis of Compound 5 N-(9-((2R,3S,5S)-3-(tert-butyldimethylsilyloxy)-5-((tert-butyldimethylsilyloxy)methyl)-4-(hydroxyimino)-tetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide To a solution of chromic acid (129 mg, 1.29 mmol) in anhydrous dichloromethane (2.0 mL), molecular sieves 3A (powder) (258 mg) was added and cooled in an ice bath. To this solution, anhydrous pyridine (207 μL, 1.29 mmol) was added dropwise under stirring, and the mixture was stirred in an ice bath. After 30 minutes, acetic anhydride (122 μL, 1.29 mmol) was added dropwise, and the mixture was stirred in an ice bath. After 30 minutes, a solution of compound 4 (250 mg, 0.43 mmol) in dichloromethane (1.3 mL) was added dropwise, and the mixture was stirred at room temperature for 2 hours. After confirming the disappearance of the starting materials by thin-layer chromatography, the reaction mixture was diluted with ethyl acetate, filtered through a silica pad (2 cm thick), and the filtrate was concentrated under reduced pressure to give a colorless solid. This crude product 4' was used directly in the next reaction. Hydroxylamine hydrochloride (299 mg, 4.30 mmol) was added to a solution of crude product 4' (0.43 mmol) in pyridine (4 mL) and stirred at room temperature. After 24 hours, the reaction mixture was concentrated under reduced pressure, and water was added to the residue, followed by extraction with ethyl acetate. The organic layer was washed with saturated brine and dried over anhydrous sodium sulfate. The organic layer was concentrated under reduced pressure, and the residue was purified by flash column chromatography (neutral silica gel, dichloromethane / methanol = 40:1) to give compound 5 as a white solid (255 mg, 68% yield for 2 steps). 1 H NMR (400 MHz, CDCl3) δ 12.14 (1H, s), 9.27 (1H, s), 8.78 (1H, s), 8.11 (1H, s), 5.78 (1H, d, J = 7.6 Hz), 5.09 (1H, s), 4.92 (1H, d, J = 7.2 Hz), 4.14 (1H, d, J = 11.4 Hz), 3.92 (1H, d, J = 11.4 Hz), 2.79 - 2.74 (1H, m), 1.27 - 1.21 (6H, m), 0.91 (9H, s), 0.71 (9H, s), 0.10 (3H, s), 0.07 (3H, s), -0.10 (3H, s), -0.23 (3H, s) 13C NMR (100 MHz, CDCl3) δ 178.9, 157.8, 155.6, 148.7, 147.8, 136.8, 120.8, 87.5, 86.5, 62.2, 36.3, 25.9, 25.5, 25.2, 19.1, 18.8, 18.3, 18.0, -5.0, -5.5, -5.6, -5.7 ESI-HRMS: calculation for C 26 H 47 N6O6Si2595.31[M+H] + , found: 595.31[M+H] +

[0147] Step 3: Synthesis of Compound 6 N-(9-((2R,3S,5S)-3-(tert-butyldimethylsilyloxy)-4-(hydroxyimino)-5-(hydroxymethyl)-tetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide Compound 5 (129 mg, 0.22 mmol) was added to ice-cooled 90% aqueous trifluoroacetic acid (1.0 mL) and stirred in an ice bath for 30 minutes. The reaction mixture was concentrated under reduced pressure, and the resulting residue was azeotroped three times with toluene and water (1:1, v / v) under reduced pressure. The resulting residue was purified by flash column chromatography (neutral silica gel, dichloromethane / methanol = 50:1 to 40:1) to give compound 6 as a white solid (96 mg, 92% yield). 1H NMR (400 MHz, CD3OD) δ 8.36 (1H, s), 5.87 (1H, d, J = 7.6 Hz), 5.18 (1H, dd, J = 7.6, 2.0 Hz), 5.02 (1H, d, J = 2.0 Hz), 4.11 (1H, dd, J = 2.0 Hz), 2.0 Hz), 3.92 (1H, d, J = 12.0, 2.0 Hz), 2.71 (1H, Sept., J = 7.2 Hz), 1.21 (6H, d, J = 7.2 Hz), 0.72 (9H, s), 0.00 (3H, s), -0.16 (3H, s); 13 C NMR (100 MHz, CD3OD) δ 181.8, 157.4, 156.8, 151.0, 150.0, 139.8, 121.3, 88.4, 79.7, 76.5, 61.6, 36.9, 25.9, 19.4, 19.2,0. -4.5, -5 ESI-HRMS : calcd forC 20 H 32 N6NaO6Si 503.21[M+Na] + , found : 503.20[M+Na] +

[0148] Scene 4 in Scene 7th N-(9-((2R,3S,4S,5S)-4-amino-3-(tert-butyldimethylsilyloxy)-5-(hydroxymethyl)-tetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide To a solution of compound 6 (93 mg, 0.19 mmol) in acetic acid (1.9 mL), sodium borohydride (15 mg, 0.38 mmol) was added and the mixture was stirred at room temperature for 1 hour. After confirming the disappearance of the starting materials by thin-layer chromatography, the reaction mixture was concentrated under reduced pressure. The residue was dissolved in ethyl acetate, washed with saturated brine, and dried over anhydrous sodium sulfate. The organic layer was concentrated under reduced pressure, and the resulting residue was purified by flash column chromatography (neutral silica gel, dichloromethane / methanol = 20:1) to give compound 7 as a white solid (51 mg, 55% yield). 1 H NMR (400 MHz, CD3OD) δ 8.34 (1H, s), 6.06 (1H, d, J = 6.0 Hz), 4.75 (1H, t, J = 6.4 Hz), 4.27 (1H, d, J = 2.8 Hz), 3.86 (1H, dd, J = 12.4, 2.0 Hz), 3.73 (1H, d, J = 12.4, 2.0 Hz), 3.62 - 3.60 (1H, m), 2.71 (1H, sept., J = 6.8 Hz), 1.21 (6H, d, J = 6.8 Hz), 0.82 (9H, s), -0.02 (3H, s), -0.23 (3H, s) 13 C NMR (100 MHz, CD3OD) δ 181.8, 157.4, 150.8, 149.8, 139.6, 139.4, 121.1, 89.7, 84.5, 77.7, 65.4, 65.2, 36.9, 26.0, -5.2, -5.3 ESI-HRMS: calculation for C 20 H 35 N6O6Si 483.24[M+H] + , found: 483.23[M+H] +

[0149] Step 5 Synthesis of Compound 8 N-(9-((2R,3S,4S,5S)-4-amino-3-(tert-butyldimethylsilyloxy)-5-(hydroxymethyl)-tetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide To a solution of compound 7 (50 mg, 0.10 mmol) in 90% aqueous acetic acid (1.5 mL), 10% palladium-carbon (20 mg) was added and the mixture was stirred at room temperature under a hydrogen atmosphere for 18 hours. After confirming the disappearance of the starting materials by thin-layer chromatography, the reaction mixture was diluted with methanol and the palladium-carbon was removed by filtration through Celite. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by flash column chromatography (neutral silica gel, dichloromethane / methanol = 15:1 to 10:1) to give compound 8 as a white solid (41 mg as acetate, 75% yield). 1 H NMR (400 MHz, CD3OD) δ 8.32 (1H, s), 5.99 (1H, s), 4.60 (1H, s), 3.95 - 3.68 (4H, m), 2.73 (1H, br. s), 1.22 (6H, br. s), 0.05 (3H, s), -0.06 (3H, s) ESI-HRMS: calculation for C 20 H 34 N6NaO5Si 489.2258[M+Na] + , found: 489.2231[M+Na] +

[0150] Step 6 Synthesis of Compound 9 N-(9-((2R,3S,4R,5S)-3-(tert-butyldimethylsilyloxy)-5-(hydroxymethyl)-4-(2,2,2-trifluoroacetamido)-tetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide To a solution of compound 8 (40 mg, 0.076 mmol) from the literature (WO2017 / 123669) and triethylamine (45 L, 0.38 mmol) in methanol (0.76 mL), ethyl trifluoroacetate (0.76 mL) was added and stirred at room temperature for 24 hours. After confirming the disappearance of the raw materials by thin-layer chromatography, the reaction mixture was concentrated under reduced pressure, and the resulting residue was purified by flash column chromatography (neutral silica gel, dichloromethane / methanol = 20:1 to 12:1) to obtain compound 9 as a white solid (12 mg, 28% yield). 1 H NMR (400 MHz, CDCl3) δ 12.26 (1H, s), 10.11 (1H, s), 7.76 (1H, s), 7.26 (1H, d, J = 3.6 Hz), 5.71 (1H, d, J = 3.6 Hz), 4.98 (1H, dd, J = 6.8 Hz), 4.78 (1H, dd, J = 6.8, 3.6 Hz), 4.21 (1H, d, J = 6.8 Hz), 4.03 (1H, dd, J =11.2 Hz), 3.82 (1H, dd, J =11.2 Hz), 2.79 (1H, sept., J = 6.8 Hz), 1.26 (3H, d, J = 6.8 Hz), 1.24 (3H, d, J = 6.8 Hz), 0.85 (9H, s), -0.01 (3H, s), -0.11 (3H, s) 13 C NMR (100 MHz, CDCl3) δ 179.8, 158.0, 157.7, 157.3, 156.9, 155.2, 148.3, 147.3, 138.6, 122.0, 120.0, 117.1, 114.2, 111.3, 91.6, 83.7, 74.5, 61.3, 51.0, 36.1, 25.2, 18.9, 17.7, -5.0, -5.4 ESI-HRMS: calculation for C 22 H 33 F3N6NaO6Si 585.21[M+Na] +, found: 585.21[M+Na] +

[0151] Step 7 Synthesis of Compound 10 N-(9-((2R,3S,4R,5S)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-(tert-butyldimethylsilyloxy)-4-(2,2,2-trifluoroacetamido)-tetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide To a solution of compound 9 (10 mg, 0.017 mmol) in anhydrous pyridine (1 mL) was added dimethoxytrityl chloride (18 mg, 0.053 mmol) and the mixture was stirred at room temperature for 1.5 hours. Subsequently, dimethoxytrityl chloride (18 mg, 0.053 mmol) was added, and the mixture was stirred at room temperature for 30 minutes. After confirming the disappearance of the starting materials by thin-layer chromatography, methanol (1 mL) was added to the reaction mixture and the mixture was concentrated under reduced pressure. The residue was dissolved in ethyl acetate and washed with water and then with saturated brine. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The resulting residue was purified by flash column chromatography (neutral silica gel, hexane / ethyl acetate = 5:1 to 2:1) to give compound 10 as a white solid (15.2 mg, 99% yield). 1H NMR (400 MHz, CDCl3) δ 11.99 (1H, s), 10.11 (1H, s), 8.07 (1H, s), 7.81 (1H, s), 7.45 (2H, dd, J = 8.2, 2.0 Hz), 7.32 (4H, dd, J = 9.2, 3.6 Hz), 7.24 - 7.29 (3H, m), 7.01 (1H, d, J = 7.2 Hz), 6.76 (4H, J = 9.2, 3.6 Hz), 5.71 (1H, d, J = 4.2 Hz), 5.16 (1H, dd, J = 6.4, 4.2 Hz), 4.20 - 4.17 (1H, m), 3.76 (3H, s), 3.75 (3H, s), 3.56 (1H, dd, J = 11.2, 2.8 Hz), 3.22 (1H, dd, J = 11.2, 2.8 Hz), 1.82 (1H, d, J = 6.8 Hz), 0.97 (3H, d, J = 6.8 Hz), 0.68 (9H, s), 0.79 (3H, d, J = 6.8 Hz), 0.04 (3H, s), -0.06 (3H, s) 13 C NMR (100 MHz, CDCl3) δ 171.2, 158.7, 158.0, 157.6, 157.2, 156.8, 155.4, 147.6, 147.2, 144.8, 139.2, 135.9, 135.4, 130.0, 127.9, 127.1, 122.6, 120.0, 117.0, 114.1, 111.2, 90.1, 86.3, 81.7, 73.4, 62.4, 60.4, 55.2, 51.4, 36.1, 25.4, 18.4, 17.8, -5.0, -5.3 ESI-HRMS: calcd for C 43 H 52 F3N6O8Si 865.36[M+H] + Found: 865.35 [M+H] +

[0152] Project 8 Synthesis of Compound 11 N-(9-((2R,3S,4S,5S)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-hydroxy-4-(2,2,2-trifluoroacetamido)-tetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide To a solution of compound 10 (14 mg, 0.016 mmol) in tetrahydrofuran (1 mL), tetrabutylammonium fluoride (1 M tetrahydrofuran solution, 19 μL, 0.019 mmol) was added and stirred at room temperature for 1 hour. After confirming the disappearance of the starting materials by thin-layer chromatography, the reaction mixture was concentrated under reduced pressure. The resulting residue was purified by flash column chromatography (neutral silica gel, dichloromethane / methanol = 30:1 to 15:1) to give compound 11 as a white solid (10.8 mg, 83% yield). 1 H NMR (400 MHz, CDCl3) δ 12.12 (1H, br. s), 8.76 (1H, br. s), 7.74 (1H, s), 7.81 (1H, s), 7.68 (1H, d, J = 5.4 Hz), 7.48 (2H, d, J = 7.6 Hz), 7.37 (2H, d, J = 9.2 Hz), 7.34 (2H, d, J = 9.2 Hz), 7.25 - 7.21 (2H, m), 7.17 (1H, t, J = 7.2 Hz), 6.81 (2H, d, J = 9.2 Hz), 6.78 (2H, d, J = 9.2 Hz), 5.80 (1H, d, J = 4.0 Hz), 5.35 (1H, br. s), 5.08 (1H, dd, J = 12.4, 6.4 Hz), 4.30 - 4.29 (1H, m), 3.76 (3H, s), 3.74 (3H, s), 3.57 - 3.53 (1H, m), 3.29 - 3.26 (1H, m), 1.84 - 1.57 (1H, m), 0.94 (3H, d, J = 6.8 Hz), 0.68 (3H, d, J = 6.8 Hz) 13 C NMR (100 MHz, CDCl3) δ 179.4, 158.6, 158.4, 158.0, 157.6, 157.3, 147.8, 147.2, 144.8, 139.4, 136.3, 135.7, 130.1, 129.9, 128.1, 128.0, 127.0, 121.0, 120.0, 117.1, 114.2, 111.3, 91.2, 86.1, 82.5, 71.5, 62.7, 55.1, 51.2, 35.9, 18.5, 18.2 ESI-HRMS: calculation for C 37 H 38 F3N6O8751.27[M+H] + , found: 751.27[M+H] +

[0153] Step 9 Synthesis of Compound 12 To a solution of compound 11 (0.90 g, 1.20 mmol) and triethylamine (0.42 mL, 3.0 mmol) in acetonitrile (12 mL), succinic anhydride (0.24 g, 2.40 mmol) and dimethylaminopyridine (29 mg, 0.24 mmol) were added and stirred at room temperature for 1 hour. After confirming the disappearance of the starting materials by thin-layer chromatography, the reaction mixture was concentrated under reduced pressure. The residue was dissolved in ethyl acetate and washed twice with saturated aqueous sodium bicarbonate and then with saturated brine. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was azeotropically evaporated with a dichloromethane / methanol solution (1:1, v / v) to give a white foamy solid (1.11 g, 97% as the triethylamine salt). This compound 12 was used directly in the next reaction.

[0154] Compound 12 can also be synthesized from the starting material 13 shown below to obtain intermediate 6. [ka]

[0155] Step 10 Synthesis of compound 14 N-(9-((2R,3R,5S)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-((tert-butyldimethylsilyl)oxy)-4-(hydroxyimino)tetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide Under an argon atmosphere, compound 13 (ChemGenes, 5.0 g, 6.5 mmol) was dissolved in anhydrous dichloromethane (50 mL) and stirred in an ice bath. While the reaction mixture was cooled, sodium bicarbonate (8.2 g, 97.3 mmol) and nor-AZADO (36 mg, 0.260 mmol) were added. Iodobenzene diacetate (3.14 g, 9.73 mmol) was added in portions, carefully monitoring the internal temperature. The mixture was then stirred for 21 hours and 10 minutes while warming to room temperature. After confirming the disappearance of the starting materials, isopropyl alcohol (7.5 mL) was added to the reaction mixture and stirred for 4 hours (to quench excess oxidant). The reaction mixture was poured into ice water, and chloroform was added to separate the layers. The aqueous layer was then extracted again with chloroform. The combined organic layers were washed once with water and once with saturated brine, and then dehydrated over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated to obtain a crude product (9.01 g, containing a compound in which the DMTr group had been partially deprotected) as an orange solid. Under an argon atmosphere, the crude product (9.01 g) was dissolved in anhydrous pyridine (40 mL) and stirred while cooling in an ice bath. While the reaction mixture was cooled, hydroxylamine hydrochloride (4.06 g, 58.7 mmol) was added, and the mixture was stirred for 17 hours and 25 minutes while warming to room temperature. After confirming the disappearance of the starting materials, the reaction mixture was transferred to a recovery flask while washing with chloroform (containing 1% triethylamine) and concentrated. The residue was added to saturated aqueous sodium bicarbonate and stirred for 15 minutes, then extracted twice with chloroform. The organic layers were combined, washed once with saturated brine, and dehydrated over anhydrous sodium sulfate. The drying agent was removed by filtration, and the filtrate was concentrated to give compound 14 (4.13 g, diastereomeric mixture, 81% yield for two steps) as an orange foam. 1 H NMR (400MHz, CDCl3) δ: 12.04 (1H, d, J = 23.3 Hz), 9.23 (1H, s), 8.49 (1H, s), 7.89 (1H, s), 7.79 (1H, s), 7.66-7.58 (2H, m), 7.49-7.39 (4H, m), 7.31-7.14 (5H, m), 6.81-6.76 (2H, m), 6.73-6.68 (2H, m), 5.92 (1H, dd, J = 8.0, 1.6 Hz), 5.83 (1H, d, J = 3.7 Hz), 5.64 (1H, d, J = 8.2 Hz), 5.54 (1H, dd. d, J = 6.9 Hz), 0.86-0.76 (9H, m), 0.47 (2H, m), 0.11 (1H, s), 0.02--0.02 (3H, m), -0.07 (2H, s)[diastereomer mixture] ESI-HRMS: calculation for C 41 H 50 N6O8Si 781.97[MH] -, found: 781.84[MH] -

[0156] Step 11: Synthesis of Compound 6 from Compound 14 Compound 14 (3.80 g) obtained in Step 10 was used in the same manner as in Step 3 to give Compound 6 (2.12 g, 4.41 mmol, 91% yield). Detailed information on Compound 6 is as described in Step 3.

[0157] Synthesis of compound 15 [ka] A solution of compound 12 (380 mg, 0.50 mmol) in N,N-dimethylformamide (2.5 mL) was added to Native amino lcaa CPG (1000A, ChemGenes) (84 μmol / g, 1.20 g, 0.10 mmol), followed by a solution of HOBt (136 mg, 1.01 mmol) and EDC-HCl (193 mg, 1.01 mmol) in DMF (2.5 mL) and the mixture was shaken at room temperature. After 20 h, the reaction mixture was discarded, and the solid support was washed with N,N-dimethylformamide (5 mL, 4 times) and then with dichloromethane (5 mL, 4 times). Unreacted amino groups on the solid support were capped with 10% acetic anhydride / pyridine solution (5 mL) at room temperature for 16 h with shaking. The reaction mixture was discarded, and the solid support was washed with pyridine (5 mL, once) and then with dichloromethane (5 mL, four times), and then dried under vacuum to obtain Compound 15 (1.20 g), in which Compound 12 was supported on the solid support. The amount of compound 12 loaded on the solid phase was calculated using the following method. A specified amount of the resulting solid support was taken, and the color development of the 4,4'-dimethoxytrityl cation produced by the addition of a deblocking reagent (3 w / v% trichloroacetic acid / dichloromethane solution) was measured by UV-visible spectrophotometry (quartz cell, cell length: 10 mm). The amount of compound 12 loaded on the solid phase was calculated using the Lambert-Beer equation from the absorbance at 504 nm and the molar extinction coefficient of the 4,4'-dimethoxytrityl cation (wavelength 504 nm: 76,000). Specifically, the resulting solid support (2.0 mg) was weighed into a 2 mL volumetric flask, and the deblocking reagent was added to make a total volume of 2 mL. This was then mixed by inversion to prepare the measurement sample. A blank measurement was performed using a 3 w / v% trichloroacetic acid / dichloromethane solution, followed by measurement using the measurement sample. Absorbance at 504 nm: 0.377, loading: 24.8 μmol / g)

[0158] Compound 24 was synthesized according to the following scheme. [ka]

[0159] Step 12 Synthesis of compound 17 N-(9-((3aR,4R,6R,6aR)-6-(hydroxymethyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-9H-purin-6-yl)benzamide Under an argon atmosphere, commercially available N6-Benzyladenosine (compound 16) (100 g, 269 mmol, 1.0 eq.), acetone (2.70 L), and dimethoxypropane (166 mL, 1.35 mol, 5.0 eq.) were sequentially added to a 10 L four-neck flask. Concentrated sulfuric acid (1.44 mL, 26.9 mmol, 0.10 eq.) was added to the reaction solution and stirred at room temperature for 15 hours. After confirming that the starting materials remained, additional concentrated sulfuric acid (1.44 mL, 26.9 mmol, 0.10 eq.) was added and the mixture was stirred for 24 hours. After confirming that the starting materials remained, concentrated sulfuric acid (1.44 mL, 26.9 mmol, 0.10 eq.) was added and the mixture was stirred for 1 hour and 30 minutes. After that, concentrated sulfuric acid (2.87 mL, 53.8 mmol, 0.20 eq.) was added and the mixture was stirred for 4 hours. After confirming the progress of the reaction by LC / MS, the reaction mixture was cooled in an ice bath, and saturated aqueous sodium bicarbonate (400 mL) was added dropwise over 5 minutes to maintain the internal temperature at 3-5°C until the solution became neutral. The reaction mixture was concentrated under reduced pressure, and distilled water (2.0 L) was added to the residue. The solution was extracted three times with chloroform (1.0 L), and the organic layer was dehydrated over anhydrous sodium sulfate. After filtration, the solvent was evaporated under reduced pressure to give compound 17 (222 g). The obtained compound 17 was used in the next step without further purification.

[0160] Step 13 Synthesis of compound 18 ((3aR,4R,6R,6aR)-6-(6-benzamido-9H-purin-9-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl methanesulfonate Under an argon atmosphere, compound 17 (222 g) obtained in step 12 and pyridine (520 mL) were placed in a 2 L four-neck flask, the reaction solution was cooled in an ice bath, methanesulfonyl chloride (25.0 mL, 321 mmol, 1.2 eq.) was added dropwise over 15 minutes so that the internal temperature was 4°C to 9°C, and the mixture was stirred for 2 hours. After confirming the progress of the reaction by LC / MS, distilled water (500 mL) was added to the reaction mixture, and the solution was extracted three times with ethyl acetate (1.0 L). The organic layer was washed sequentially with 1N hydrochloric acid (1.0 L x 1, 500 mL x 2), saturated aqueous sodium bicarbonate (500 mL x 2), and saturated brine (500 mL x 2), and then dried over anhydrous sodium sulfate. After filtration, the solvent was removed under reduced pressure, and the resulting residue was azeotroped with toluene to give compound 18 (150 g, containing 17.6 wt % toluene). The resulting compound 18 was used in the next step without further purification.

[0161] Step 14 Synthesis of compound 19 N-(9-((3aR,4R,6R,6aR)-6-(azidomethyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-9H-purin-6-yl)benzamide Under an argon atmosphere, compound 18 (150 g) obtained in step 13 and dehydrated DMF (1.26 L) were placed in a 3 L four-neck flask. Sodium azide (82.8 g, 1.26 mol, 5.0 eq.) was added to the reaction solution, and the temperature was raised to 60°C over 30 minutes and the mixture was stirred at 60°C for 3 hours and 30 minutes. After confirming the progress of the reaction by LC / MS, the reaction mixture was gradually cooled to room temperature, and distilled water (1.0 L) and ethyl acetate (600 mL) were added. Distilled water (3.0 L) was added to the resulting solution, and the aqueous layer was extracted six times with ethyl acetate (500 mL). The organic layer was washed twice with distilled water (800 mL) and twice with saturated brine (800 mL), and then dehydrated over anhydrous sodium sulfate. After filtration, the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (SiO2 700 g, ethyl acetate) to give compound 19 (55.7 g, 128 mmol, 48% yield (3 steps from compound 16)).

[0162] Step 15 Synthesis of Compound 20 N-(9-((3aR,4R,6R,6aR)-2,2-dimethyl-6-((2,2,2-trifluoroacetamido)methyl) tetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-9H-purin-6-yl)benzamide Under an argon atmosphere, compound 19 (55.7 g, 128 mmol, 1.0 eq.) obtained in step 14 and methanol (1.28 L) were placed in a 3 L four-neck flask. 10% Pd / C (76.8 g, 21.2 mmol, 0.17 eq.) was added to the reaction solution, the atmosphere in the reaction solution was replaced with hydrogen, and the mixture was stirred at room temperature for 16 hours. After confirming the progress of the reaction by LC / MS, the reaction mixture was purged with argon gas and filtered through Celite. The filtrate was concentrated under reduced pressure, and the resulting residue was dissolved in methanol (985 mL) and transferred to a 3 L four-neck flask. The solution was cooled in an ice bath, and 1-(trifluoroacetyl)imidazole (17.0 mL, 149 mmol, 1.2 eq.) was added dropwise over 15 minutes to maintain the internal temperature at 2-4 °C. The mixture was stirred at 4 °C for 2 hours. After confirming the progress of the reaction by LC / MS, the reaction mixture was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (SiO2 800 g, heptane / ethyl acetate = 1:4) to give compound 20 (21.4 g, 42.2 mmol, 33% yield).

[0163] Step 16 Synthesis of compound 21 N-(9-((2R,3R,4S,5R)-3,4-dihydroxy-5-((2,2,2-trifluoroacetamido)methyl) tetrahydrofuran-2-yl)-9H-purin-6-yl)benzamide Compound 20 (10.0 g, 19.8 mmol, 10 eq.) obtained in step 15 and distilled water (50.0 mL) were added to a 1-L recovery flask, and the solution was cooled in an ice bath. Trifluoroacetic acid (50.0 mL, 640 mmol, 32.4 eq.) was added dropwise over 5 minutes under ice cooling, and the reaction mixture was warmed to room temperature and stirred for 4 hours and 30 minutes. After confirming the progress of the reaction by LC / MS, the reaction mixture was concentrated under reduced pressure, and the residue was azeotropically distilled with toluene. Isopropyl ether was added to the resulting residue to precipitate a solid, which was then filtered off. The resulting solid was dried under reduced pressure at room temperature to give compound 21 (8.86 g, 19.0 mmol, yield 96%).

[0164] Step 17 Synthesis of compound 22 N-(9-((2R,3R,4R,5R)-3-((tert-butyldimethylsilyl)oxy)-4-hydroxy-5-((2,2,2-trifluoroacetamido)methyl)tetrahydrofuran-2-yl)-9H-purin-6-yl)benzamide Under an argon atmosphere, compound 21 (15.6 g, 33.6 mmol, 1.0 eq.) obtained in step 16 and dehydrated DMF (111 mL) were placed in a 500 mL recovery flask, and the solution was cooled in an ice bath. Under ice cooling, imidazole (9.16 g, 134 mmol, 4.0 eq.) and t-butyldimethylsilyl chloride (15.2 g, 101 mmol, 3.0 eq.) were added so that the internal temperature was less than 6°C, and the mixture was stirred at the same temperature for 30 minutes. After confirming the progress of the reaction by LC / MS, ice water was added to the reaction mixture. The aqueous layer was extracted three times with ethyl acetate, washed with saturated brine, and dried over anhydrous sodium sulfate. After filtration, the solvent was evaporated under reduced pressure, and the resulting residue was purified by silica gel column chromatography (SiO2 800 g, chloroform / 2-butanone = 100:0 to 85:15) to give a mixture of compound 22 and compound 23 (10.9 g). The resulting mixture of compound 22 and compound 23 was used in the next step without further purification.

[0165] Step 18 Synthesis of compound 24 (2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-((tert-butyldimethylsilyl)oxy)-2-((2,2,2-trifluoroacetamido)methyl)tetrahydrofuran-3-yl (2-cyanoethyl) diisopropylphosphoramidite Under an argon atmosphere, a mixture of Compound 22 and Compound 23 (1.19 g, 2.05 mmol, Compound 22:Compound 23 = 9:1) obtained in Step 17 and dehydrated dichloromethane (6.83 mL) were placed in a 200 mL recovery flask, and the solution was cooled in an ice bath. Under ice cooling, a mixed solution of diisopropylethylamine (0.537 mL, 3.07 mmol, 1.5 eq.) and 3-((chloro(diisopropylamino)phosphanyl)oxy)propanenitrile (0.857 mL, 3.07 mmol, 1.5 eq.) in dichloromethane was added dropwise, and the reaction mixture was warmed to room temperature and stirred at room temperature for 2 hours. After confirming the disappearance of the raw materials by TLC, distilled water was added to the reaction mixture, which was then extracted twice with chloroform (50 mL). The organic layer was dried over anhydrous sodium sulfate. After filtration, the solvent was removed under reduced pressure, and the resulting residue was purified multiple times by silica gel column chromatography (heptane / ethyl acetate = 50:50 to 30:70, containing 0.5% triethylamine) to give the desired compound 24 (608 mg, 0.779 mmol, 38% yield) as a pale yellow amorphous substance. 1H NMR (400 MHz, CDCl3) δ: 9.82 (1H, d, J = 8.8 Hz), 9.07 (1H, s), 8.83-8.80 (1H, m), 8.06-8.01 (3H, m), 7.66-7.52 (3H, m), 5.86 (1H, d, J = 7.8 Hz), 4.88 (1H, dd, J = 7.8, 5.2 Hz), 4.50 (1H, br s), 4.39-4.29 (1H, m), 4.21-3.88 (3H, m), 3.74-3.63 (1H, m), 3.48-3.35 (1H, m), 2.73-2.66 (2H, m), 1.28-1.23 (12H, m), 1.08-1.04 (1H, m), 0.72-0.68 (9H, m), -0.17 (3H, s), -0.45 (3H, s). 31 P NMR(CDCl3) δ: 149.95

[0166] Compound 6a, which is a raw material for polynucleotides, was synthesized according to the following scheme. [ka]

[0167] Step 1: Synthesis of Compound 2a N-(9-((3aR,5R,6R,6aS)-2,2-di-tert-butyl-6-methoxytetrahydrofuro[2,3-d][1,3,2]dioxasilol-5-yl)-9H-purin-6-yl)benzamide To a solution of commercially available compound 1a (30.0 g, 78.0 mmol) in DMF (300 mL), di-t-butylsilylbis(trifluoromethanesulfonate) (68.6 g, 156 mmol) was slowly added under ice cooling. After stirring for 1 hour under ice cooling, the reaction mixture was added to saturated aqueous sodium bicarbonate and extracted twice with a mixed solvent of heptane / ethyl acetate. The organic layer was washed twice with water and once with saturated brine and then dried over anhydrous sodium sulfate. After filtration, the concentrated residue was purified by silica gel column chromatography (heptane / ethyl acetate = 7 / 3 → 3 / 7) to give compound 2a (38.7 g, 73.7 mmol) as a colorless solid (yield 95%). ESI-MS: Calculated: 524.23 [MH] - , Actual measurement: 524.5 [MH] - 1 H-NMR (CDCl3, 400 MHz) δ: 9.32 (s, 1H), 8.76 (s, 1H), 8.05 (s, 1H), 8.03 (t, J = 6.6 Hz, 2H), 7.60 (t, J = 7.3 Hz, 1H), 7.51 (t, J = 7.8 Hz, 2H), 6.01 (s, 1H), 4.66 (dd, J = 9.6, 5.0 Hz, 1H), 4.48 (dd, J = 8.9, 4.8 Hz, 1H), 4.31 (d, J = 4.6 Hz, 1H), 4.20 (ddd, J = 10.1, 5.0, 5.0 Hz, 1H), 4.03 (t, J = 9.8 Hz, 1H), 3.70 (s, 3H), 1.10 (s, 9H), 1.06 (s, 9H).

[0168] Step 2 Synthesis of compound 3a N-(9-((3aR,5R,6R,6aS)-2,2-di-tert-butyl-6-methoxytetrahydrofuro[2,3-d][1,3,2]dioxasilol-5-yl)-9H-purin-6-yl)-N-methylbenzamide Compound 2a (10.0 g, 19.0 mmol) was dissolved in dichloromethane (50 mL), and tetrabutylammonium bromide (9.20 g, 28.5 mmol) and 1 M aqueous sodium hydroxide solution (50 mL) were added. Methyl iodide (4.76 mL, 76.0 mmol) was slowly added dropwise. The mixture was then stirred at room temperature for 1 hour and 10 minutes. After confirming the disappearance of the starting materials, the reaction mixture was quenched by adding it to ice-cold water / chloroform = 1 / 1. The organic layer was washed twice with water and then dehydrated over anhydrous sodium sulfate. The desiccant was removed by filtration, and the filtrate was concentrated. The concentrated residue was purified by silica gel column chromatography (heptane / ethyl acetate = 90 / 10 → 50 / 50) to give compound 3a (6.25 g, 11.6 mmol) as a colorless amorphous solid (61% yield). ESI-MS: Calculated value: 540.26 [M+H] + , Measured value: 540.4 [M+H] + 1 H-NMR (CDCl3, 400 MHz) δ: 8.56 (s, 1H), 7.94 (s, 1H), 7.49-7.46 (m, 2H), 7.34-7.29 (m, 1H), 7.21 (t, J = 7.6 Hz, 2H), 5.94 (s, 1H), 4.61 (dd, J = 9.6, 5.0 Hz, 1H), 4.46 (dd, J = 9.2, 5.0 Hz, 1H), 4.22 (d, J = 4.6 Hz, 1H), 4.17 (ddd, J = 10.0, 5.2, 5.0 Hz, 1H), 4.00 (dd, J = 10.5, 9.2 Hz, 1H), 3.79 (s, 3H), 3.67 (s, 3H), 1.08 (s, 9H), 1.05 (s, 9H).

[0169] Step 3: Synthesis of compound 4a N-(9-((2R,3R,4S,5S)-4,5-dihydroxy-3-methoxytetrahydrofuran-2-yl)-9H-purin-6-yl)-N-methylbenzamide Compound 3a (6.25 g, 11.6 mmol) was dissolved in tetrahydrofuran (63 mL) and cooled in an ice bath. Triethylamine (8.07 mL, 57.9 mmol) and triethylamine trihydrofluoride (1.89 mL, 11.6 mmol) were added, and the mixture was stirred for 1 hour and 5 minutes while cooling in an ice bath. After confirming the disappearance of the starting materials, the reaction was quenched by adding triethylamine (10 mL, 76.0 mmol). The reaction mixture was diluted with chloroform and concentrated. The concentrated residue was purified by silica gel column chromatography (chloroform / methanol = 100 / 0 → 90 / 10) to obtain compound 4a (4.25 g, 10.6 mmol) as a colorless amorphous solid. (Yield: quant.) ESI-MS: Calculated value: 400.16 [M+H] + , Measured value: 400.3 [M+H] + 1 H-NMR (CDCl3, 400 MHz) δ: 8.56 (s, 1H), 7.97 (s, 1H), 7.50-7.48 (m, 2H), 7.35-7.31 (m, 1H), 7.22 (t, J = 7.8 Hz, 2H), 5.87 (d, J = 7.3 Hz, 1H), 5.86 (dd, J = 11.4, 2.3 Hz, 1H), 4.63 (dd, J = 7.3, 4.6 Hz, 1H), 4.57-4.56 (m, 1H), 4.36-4.34 (m, 1H), 3.99-3.94 (m, 1H), 3.81 (s, 3H), 3.77 (td, J = 12.3, 1.7 Hz, 1H), 3.31 (s, 3H), 2.77 (d, J = 1.4 Hz, 1H).

[0170] Step 4 Synthesis of compound 5a N-(9-((2R,3R,4S,5S)-5-(bis(4-methoxyphenyl)(phenyl)methoxy)-4-hydroxy-3-methoxytetrahydrofuran-2-yl)-9H-purin-6-yl)-N-methylbenzamide Compound 4a (4.25 g, 10.6 mmol) was dissolved in pyridine (43 mL) and stirred in an ice bath. 4,4'-Dimethoxytrityl chloride (5.41 g, 20.0 mmol) was added to the reaction mixture, and the mixture was stirred at room temperature for 2 hours and 25 minutes. After confirming the disappearance of the starting materials, the reaction mixture was quenched by adding it to ice-cooled sodium bicarbonate water and extracted with ethyl acetate. The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated. The concentrated residue was purified by silica gel column chromatography (heptane / ethyl acetate (containing 1% triethylamine) = 70 / 30 → 50 / 50) to obtain compound 5a (5.35 g, 7.62 mmol) as a colorless amorphous solid (71% yield). ESI-MS: Calculated value: 702.29 [M+H] + , Measured value: 702.6 [M+H] + 1 H-NMR (CDCl3, 400 MHz) δ: 8.50 (s, 1H), 8.14 (s, 1H), 7.45-7.40 (m, 4H), 7.33-7.22 (m, 8H), 7.16 (t, J = 7.6 Hz, 2H), 6.81 (dd, J = 8.9, 1.1 Hz, 4H), 6.15 (d, J = 3.7 Hz, 1H), 4.48 (dd, J = 11.9, 5.0 Hz, 1H), 4.35 (dd, J = 5.3, 3.9 Hz, 1H), 4.21-4.19 (m, 1H), 3.80 (s, 3H), 3.79 (s, 6H), 3.53 (s, 3H), 3.50 (dd, J = 10.8, 3.0 Hz, 1H), 3.40 (dd, J = 10.8, 4.4 Hz, 1H), 2.66 (d, J = 6.4 Hz, 1H).

[0171] Step 5: Synthesis of amidite 6a (2S,3S,4R,5R)-2-(bis(4-methoxyphenyl)(phenyl)methoxy)-4-methoxy-5-(6-(N-methylbenzamido)-9H-purin-9-yl)tetrahydrofuran-3-yl (2-cyanoethyl) diisopropylphosphoramidite Compound 5a (5.30 g, 7.55 mmol) was dissolved in dichloromethane (48 mL), and diisopropylethylamine (2.64 mL, 15.1 mmol) was added. The mixture was cooled in an ice bath. 2-Cyanoethyldiisopropylchlorophosphoramidite (2.68 g, 11.3 mmol) dissolved in dichloromethane (5 mL) was added dropwise over 5 minutes. The mixture was then stirred for 1 hour and 10 minutes while warming to room temperature. After confirming the disappearance of the starting materials, the reaction mixture was quenched by adding it to ice-cold saturated aqueous sodium bicarbonate. Extraction was performed with ethyl acetate. The organic layer was washed with saturated brine and dried over anhydrous sodium sulfate. The drying agent was removed by filtration, and the filtrate was concentrated. The concentrated residue was purified by silica gel column chromatography (heptane / ethyl acetate (containing 1% triethylamine) = 70 / 30 → 50 / 50) to give amidite 6a (6.22 g, 6.90 mmol) as a colorless amorphous solid (yield 91%). ESI-MS: Calculated value: 902.40 [M+H] + , Measured value: 902.5 [M+H] + 1H-NMR (CDCl3, 400 MHz) δ: 8.47 (s, 0.35H), 8.47 (s, 0.65H), 8.14 (s, 0.35H), 8.09 (s, 0.65H), 7.44-7.39 (m, 4H), 7.33-7.21 (m, 8H), 7.16-7.12 (m, 2H), 6.93-6.78 (m, 4H), 6.12 (d, J = 5.5, 0.65H), 6.10 (d, J = 5.0, 0.35H), 4.66-4.53 (m, 2H), 4.41-4.38 (m, 0.35H), 4.34-4.32 (m, 0.65H), 3.97-3.78 (m, 10H), 3.70-3.44 (m, 7H), 3.36-3.30 (m, 1H), 2.64 (t, J = 6.2 Hz, 1.3H), 2.38 (t, J = 6.4 Hz, 0.70H), 1.22-1.17 (m, 8H), 1.06 (d, J = 6.9 Hz, 4H). 31 P-NMR(CDCl3, 162 MHz) δ: 150.70, 150.94.

[0172] Compound 6b, which is a raw material for polynucleotides, was synthesized according to the following scheme. [ka]

[0173] Step 1: Synthesis of Compound 2b N-(9-((4aR,6R,7R,7aS)-2,2-di-tert-butyl-7-fluorotetrahydro-4H-furo[3,2-d][1,3,2]dioxasilin-6-yl)-9H-purin-6-yl)benzamide To a solution of commercially available compound 1b (30.0 g, 80.4 mmol) in DMF (300 mL), di-t-butylsilylbis(trifluoromethanesulfonate) (70.8 g, 161 mmol) was slowly added under ice cooling. After stirring for 1 hour under ice cooling, the reaction mixture was added to saturated aqueous sodium bicarbonate and extracted twice with a mixed solvent of heptane / ethyl acetate. The organic layer was washed twice with water and once with saturated brine and then dried over anhydrous sodium sulfate. After filtration, the concentrated residue was purified by slurrying with heptane / ethyl acetate = 9 / 1 to give compound 2b (38.7 g, 75.4 mmol) as a colorless solid (yield 94%). ESI-MS: Calculated value: 514.23 [M+H] + , Measured value: 514.5 [M+H] + 1 H-NMR (DMSO-d6, 400 MHz) δ: 11.27 (s, 1H), 8.74 (s, 1H), 8.65 (s, 1H), 8.04 (d, J = 8.7 Hz, 2H), 7.65 (t, J = 7.5 Hz, 1H), 7.55 (t, J = 7.5 Hz, 2H), 6.45 (d, J = 23 Hz, 1H), 5.71 (dd, J = 54.5, 4.1 Hz, 1H), 5.03 (m, 1H), 4.44 (q, J = 3.7 Hz, 1H), 4.09 (m, 2H), 1.11 (s, 9H), 1.02 (s, 9H).

[0174] Step 2: Synthesis of Compound 3b N-(9-((4aR,6R,7R,7aS)-2,2-di-tert-butyl-7-fluorotetrahydro-4H-furo[3,2-d][1,3,2]dioxasilin-6-yl)-9H-purin-6-yl)-N-methylbenzamide Compound 2b (10.0 g, 19.5 mmol) was dissolved in dichloromethane (50 mL), and tetrabutylammonium bromide (9.41 g, 29.2 mmol) and 1 M aqueous sodium hydroxide solution (50 mL) were added. Methyl iodide (1.83 mL, 29.2 mmol) was slowly added dropwise. The mixture was then stirred at room temperature for 1 hour. After confirming the disappearance of the starting materials, the reaction mixture was quenched by adding it to ice-cold water / chloroform = 1 / 1. The organic layer was washed twice with water and then dehydrated over anhydrous sodium sulfate. The desiccant was removed by filtration, and the filtrate was concentrated. The concentrated residue was purified by silica gel column chromatography (heptane / ethyl acetate = 90 / 10 → 50 / 50) to give compound 3b (6.86 g, 12.8 mmol) as a colorless amorphous solid (65% yield). ESI-MS: Calculated value: 528.24 [M+H] + , Measured value: 538.6 [M+H] + 1 H-NMR (CDCl3, 400 MHz) δ: 8.54 (s, 1H), 7.94 (s, 1H), 7.47 (d, J = 8.1 Hz, 2H), 7.32 (t, J = 7.3 Hz, 2H), 7.21 (t, J = 7.6 Hz, 2H), 6.10 (d, J = 22.0 Hz, 1H), 5.46 (dd, J = 54.5, 4.1 Hz, 1H), 4.86 (ddd, J = 27.2, 9.8, 4.1 Hz, 1H), 4.47 (dd, J = 9.2, 5.0 Hz, 1H), 4.14 (m, 1H), 4.03 (t, J = 9.8 Hz, 1H), 3.78 (s, 3H), 1.11 (s, 9H), 1.05 (s, 9H).

[0175] Step 3: Synthesis of compound 4b N-(9-((2R,3R,4R,5R)-3-fluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-9H-purin-6-yl)-N-methylbenzamide Compound 3b (6.67 g, 12.6 mmol) was dissolved in tetrahydrofuran (66 mL) and cooled in an ice bath. Triethylamine (8.81 mL, 63.2 mmol) and triethylamine trihydrofluoride (2.05 mL, 12.6 mmol) were added, and the mixture was stirred for 1 hour and 5 minutes while cooling in an ice bath. After confirming the disappearance of the starting materials, the reaction was quenched by adding triethylamine (10.6 mL, 76.0 mmol). The reaction mixture was diluted with chloroform and concentrated. The concentrated residue was purified by silica gel column chromatography (chloroform / methanol = 100 / 0 → 90 / 10) to obtain compound 4b (4.98 g, 12.9 mmol) as a colorless amorphous solid. (Yield: quant.) ESI-MS: Calculated value: 388.14 [M+H] + , Measured value: 388.4 [M+H] + 1 H-NMR (DMSO-d6, 400 MHz) δ: 8.70 (s, 1H), 8.58 (s, 1H), 7.30 (m, 5H), 6.31 (dd, J = 16.9, 2.3 Hz, 1H), 5.75 (d, J = 6.4 Hz, 1H), 5.41 (m, 1H), 5.15 (t, J = 5.3 Hz, 1H), 4.46 (m, 1H), 3.98 (m, 1H), 3.75 (dq, J = 12.4, 2.6 Hz, 1H), 3.67 (s, 3H), 3.61 - 3.56 (m, 1H).

[0176] Step 4 Synthesis of compound 5b N-(9-((2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-fluoro-4-hydroxytetrahydrofuran-2-yl)-9H-purin-6-yl)-N-methylbenzamide Compound 4b (4.93 g, 12.7 mmol) was dissolved in pyridine (49 mL) and stirred in an ice bath. 4,4'-Dimethoxytrityl chloride (6.47 g, 29.2 mmol) was added to the reaction mixture, and the mixture was stirred at room temperature for 1 hour and 20 minutes. After confirming the disappearance of the starting materials, the reaction mixture was quenched by adding it to ice-cooled sodium bicarbonate water and extracted with ethyl acetate. The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated. The concentrated residue was purified by silica gel column chromatography (heptane / ethyl acetate (containing 1% triethylamine) = 70 / 30 → 50 / 50) to obtain compound 5b (8.34 g, 12.1 mmol) as a colorless amorphous solid (yield 95%). ESI-MS: Calculated value: 690.27 [M+H] + , Measured value: 690.7 [M+H] + 1 H-NMR (CDCl3, 400 MHz) δ: 8.51 (s, 1H), 8.10 (s, 1H), 7.43 (dd, J = 8.2, 1.4 Hz, 2H), 7.37 (dd, 8.2, 1.4 Hz, 2H), 7.28-7.20 (m, 8H), 7.12 (t, J = 7.5 Hz, 2H), 6.79 (d, J = 8.7 Hz), 6.23 (dd, J = 17.1, 2.5 Hz, 1H), 5.58 (dq, J = 52.9, 2.3 Hz, 1H), 4.78 (m, 1H), 4.19 (m, 1H), 3.78 (s, 6H), 3.47 (ddd, J = 57.9, 10.6, 3.5 Hz, 2H), 2.44 (dd, J = 7.5, 2.5 Hz, 1H).

[0177] Step 5: Synthesis of amidite 6b (2R,3R,4R,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-fluoro-5-(6-(N-methylbenzamido)-9H-purin-9-yl)tetrahydrofuran-3-yl (2-cyanoethyl) diisopropylphosphoramidite Compound 5b (10.0 g, 14.6 mmol) was dissolved in dichloromethane (80 mL), and diisopropylethylamine (5.08 mL, 29.1 mmol) was added. The mixture was cooled in an ice bath. 2-Cyanoethyldiisopropylchlorophosphoramidite (4.18 g, 21.8 mmol) dissolved in dichloromethane (15 mL) was added dropwise over 5 minutes. The mixture was then stirred for 1 hour while warming to room temperature. After confirming the disappearance of the starting materials, the reaction mixture was quenched by adding it to ice-cold saturated aqueous sodium bicarbonate. Extraction was performed with ethyl acetate. The organic layer was washed with saturated brine and dried over anhydrous sodium sulfate. The drying agent was removed by filtration, and the filtrate was concentrated. The concentrated residue was purified by silica gel column chromatography (heptane / ethyl acetate (containing 1% triethylamine) = 70 / 30 → 50 / 50) to give amidite 6b (12.1 g, 13.6 mmol) as a colorless amorphous solid (yield 93%). ESI-MS: Calculated value: 890.38 [M+H] + , Measured value: 890.8 [M+H] + 1H-NMR (CDCl3, 400 MHz) δ: 8.53 (s, 0.49H), 8.50 (s, 0.51H), 8.14 (s, 1H), 7.43-7.39 (m, 2H), 7.37-7.33 (m, 2H), 7.27-7.20 (m, 8H), 7.09-7.04 (m, 2H), 6.77 (t, J = 9.1 Hz, 4H), 6.28-6.19 (m, 1H), 5.74 (dq, J = 18.5, 2.2 Hz, 0.50H), 5.61 (dq, J = 19.2, 2.3 Hz, 0.50H), 5.10-5.00 (m, 0.47H), 4.94-4.85 (m, 0.53H), 4.31 (m, 1H), 3.97-3.82 (m, 1H), 3.79 (s, 3H), 3.79 (s, 3H), 3.63-3.53 (m, 4H), 3.31-3.27 (m, 1H), 2.59 (t, J = 6.2 Hz, 1H), 2.41 (t, J = 6.4 Hz, 1H), 1.20-1.15 (m, 9H), 1.04 (d, J = 6.4 Hz, 3H). 31 P-NMR(CDCl3, 162 MHz) δ: 151.97, 151.92, 151.19, 151.11.

[0178] Compound 6c, which is a starting material for polynucleotides, was synthesized according to the following scheme. [ka]

[0179] Step 1: Synthesis of compound 3c N-(9-((4aR,6R,7R,7aS)-2,2-di-tert-butyl-7-methoxytetrahydro-4H-furo[3,2-d][1,3,2]dioxasilin-6-yl)-9H-purin-6-yl)-N-ethylbenzamide Compound 2a (11.7 g, 22.3 mmol) was dissolved in dichloromethane (58.5 mL), and tetrabutylammonium bromide (10.8 g, 33.4 mmol) and 1 M aqueous sodium hydroxide solution (58.5 mL) were added. Ethyl iodide (10.8 mL, 134 mmol) was slowly added dropwise. The mixture was then stirred at room temperature for 2 hours. After confirming the disappearance of the starting materials, the reaction mixture was quenched by adding it to ice-cooled water / chloroform = 1 / 1. The organic layer was washed twice with water and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated. The concentrated residue was purified by slurry with toluene, and the filtrate was concentrated again. The concentrated residue was purified by silica gel column chromatography (heptane / ethyl acetate = 90 / 10 → 70 / 30) to give compound 3c (6.14 g, 11.1 mmol) as a colorless amorphous solid (49% yield). ESI-MS: Calculated value: 554.28 [M+H] + , Measured value: 554.6 [M+H] + 1 H-NMR (CDCl3, 400 MHz) δ: 8.56 (s, 1H), 7.91 (s, 1H), 7.47-7.45 (m, 2H), 7.31-7.27 (m, 1H), 7.19 (t, J = 7.5 Hz, 2H), 5.94 (s, 1H), 4.61 (dd, J = 9.6, 4.6 Hz, 1H), 4.46 (dd, J = 9.1, 5.0 Hz, 1H), 4.40 (q, J = 7.0 Hz, 2H), 4.22 (d, J = 4.6 Hz, 1H), 4.16 (ddd, J = 10.1, 4.9, 4.8 Hz, 1H), 4.00 (dd, J = 10.5, 9.6 Hz, 1H), 3.67 (s, 3H), 1.34 (t, J = 7.1 Hz, 3H), 1.09 (s, 9H), 1.05 (s, 9H).

[0180] Step 2: Synthesis of compound 4c N-ethyl-N-(9-((2R,3R,4R,5R)-4-hydroxy-5-(hydroxymethyl)-3-methoxytetrahydrofuran-2-yl)-9H-purin-6-yl)benzamide Compound 3c (6.14 g, 11.1 mmol) was dissolved in tetrahydrofuran (61.4 mL) and cooled in an ice bath. Triethylamine (7.73 mL, 55.4 mmol) and triethylamine trihydrofluoride (1.81 mL, 11.1 mmol) were added, and the mixture was stirred for 2 hours while cooling in an ice bath. After confirming the disappearance of the starting materials, the reaction was quenched by adding triethylamine (10 mL, 76.0 mmol). The reaction mixture was diluted with chloroform and concentrated. The concentrated residue was purified by silica gel column chromatography (chloroform / methanol = 100 / 0 → 90 / 10) to obtain compound 4c (4.60 g, 11.1 mmol) as a colorless amorphous solid. (Yield: quant.) ESI-MS: Calculated value: 414.18 [M+H] + , Measured value: 414.3 [M+H] + 1 H-NMR (CDCl3, 400 MHz) δ: 8.56 (s, 1H), 7.92 (s, 1H), 7.50-7.46 (m, 2H), 7.32-7.28 (m, 1H), 7.20 (t, J = 7.5 Hz, 2H), 5.89 (dd, J = 11.6, 2.1 Hz, 1H), 5.85 (d, J = 7.3 Hz, 1H), 4.62 (dd, J = 7.3, 4.6 Hz, 1H), 4.57-4.56 (m, 1H), 4.45-4.39 (m, 2H), 4.36-4.34 (m, 1H), 3.96 (dt, J = 12.9, 1.9, 1H), 3.80-3.73 (m, 1H), 3.29 (s, 3H), 2.70 (d, J = 1.4 Hz, 1H), 1.37 (t, J = 7.1 Hz, 3H).

[0181] Step 3: Synthesis of compound 5c N-(9-((2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxy-3-methoxytetrahydrofuran-2-yl)-9H-purin-6-yl)-N-ethylbenzamide Compound 4c (4.58 g, 11.1 mmol) was dissolved in pyridine (46 mL) and stirred in an ice bath. 4,4'-Dimethoxytrityl chloride (5.63 g, 16.6 mmol) was added to the reaction mixture and stirred at room temperature for 2 hours. After confirming the disappearance of the starting materials, the reaction mixture was quenched by adding it to ice-cooled sodium bicarbonate water and extracted with ethyl acetate. The organic layer was washed with saturated brine and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated. The concentrated residue was purified by silica gel column chromatography (heptane / ethyl acetate (containing 1% triethylamine) = 70 / 30 → 50 / 50) to obtain compound 5c (7.55 g, 10.6 mmol) as a colorless amorphous solid (yield 95%). ESI-MS: Calculated value: 716.31 [M+H] + , Measured value: 716.2 [M+H] + 11H-NMR (CDCl3, 400 MHz) δ: 8.51 (s, 1H), 8.11 (s, 1H), 7.43-7.40 (m, 4H), 7.32-7.22 (m, 8H), 7.13 (t, J = 7.5 Hz, 2H), 6.81 (dd, J = 9.1, 1.4 Hz, 4H), 6.14 (d, J = 3.7 Hz, 1H), 4.47 (dd, J = 5.7, 5.6 Hz, 1H), 4.41 (q, J = 7 Hz, 2H), 4.34 (dd, J = 8.4, 4.1 Hz, 1H), 4.21-4.17 (m, 1H), 3.79 (s, 6H), 3.53 (s, 3H), 3.50 (dd, J = 10.5, 3.2 Hz, 1H), 3.39 (dd, J = 10.7, 4.3 Hz, 1H), 2.64 (d, J = 6.4 Hz, 1H), 1.34 (t, J = 7.1 Hz, 3H).

[0182] Synthesis of Engineering 4 Amidite 6c (2R,3R,4R,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-(6-(N-ethylbenzamido)-9H-purin-9-yl)-4-methoxytetrahydrofuran-3-yl (2-cyanoethyl) diisopropylphosphoramidite Compound 5c (8.83 g, 12.3 mmol) was dissolved in dichloromethane (74 mL), and diisopropylethylamine (4.31 mL, 24.7 mmol) was added. The mixture was cooled in an ice bath. 2-Cyanoethyldiisopropylchlorophosphoramidite (4.40 g, 18.6 mmol) dissolved in dehydrated dichloromethane (14 mL) was added dropwise over 9 minutes. The mixture was then stirred for 1 hour while warming to room temperature. After confirming the disappearance of the starting materials, the reaction mixture was quenched by adding it to ice-cold saturated aqueous sodium bicarbonate. Ethyl acetate was added for extraction. The organic layer was washed with saturated brine and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated. The concentrated residue was purified by silica gel column chromatography (heptane / ethyl acetate (containing 1% triethylamine) = 70 / 30 → 50 / 50) to give amidite 6c (10.4 g, 11.3 mmol) as a colorless amorphous solid (yield 92%). ESI-MS: Calculated value: 916.42 [M+H] + , Measured value: 917.3 [M+H] + 1 H-NMR (CDCl3, 400 MHz) δ: 8.49 (s, 0.37H), 8.48 (s, 0.63H), 8.11 (s, 0.34H), 8.05 (s, 0.66H), 7.43-7.38 (m, 4H), 7.32-7.21 (m, 8H), 7.11 (t, J = 7.8 Hz, 2H), 6.80 (m, 4H), 6.10 (m, 1H), 4.64-4.52 (m, 2H), 4.43-4.32 (m, 3H), 3.94-3.83 (m, 1H), 3.79-3.78 (m, 6H), 3.67 -3.46 (m, 4H), 3.35-3.29 (m, 1H), 2.64 (t, J = 6.4 Hz, 1.3H), 2.37 (t, J = 6.4 Hz, 0.70H), 1.33 (t, J = 7.1 Hz, 3H), 1.18 (m, 8H), 1.06 (d, J = 6.9 Hz, 4H). 31P-NMR(CDCl3, 162 MHz) δ: 151.67, 150.92.

[0183] Compound 6d, which is a starting material for polynucleotides, was synthesized according to the following scheme. [ka]

[0184] Step 1: Synthesis of compound 3d N-(9-((4aR,6R,7R,7aS)-2,2-di-tert-butyl-7-fluorotetrahydro-4H-furo[3,2-d][1,3,2]dioxasilin-6-yl)-9H-purin-6-yl)-N-ethylbenzamide Compound 2b (1.00 g, 1.95 mmol) was dissolved in dichloromethane (5.0 mL), and tetrabutylammonium bromide (0.942 g, 2.92 mmol) and 1 M aqueous sodium hydroxide solution (5.0 mL) were added. Methyl iodide (0.942 mL, 11.7 mmol) was slowly added dropwise. The mixture was then stirred at room temperature for 2 hours. After confirming the disappearance of the starting materials, the reaction mixture was quenched by adding it to ice-cold water / chloroform = 1 / 1. The organic layer was washed twice with water and then dehydrated over anhydrous sodium sulfate. The desiccant was removed by filtration, and the filtrate was concentrated. The concentrated residue was purified by silica gel column chromatography (heptane / ethyl acetate = 80 / 20 → 70 / 30) to give compound 3d (629 mg, 1.16 mmol) as a colorless amorphous solid (60% yield). ESI-MS: Calculated value: 542.26 [M+H] + , Measured value: 542.6 [M+H] + 1H-NMR (CDCl3, 400 MHz) δ: 8.55 (s, 1H), 7.91 (s, 1H), 7.46 (d, J = 7.3 Hz, 2H), 7.30 (t, J = 7.1 Hz, 1H), 7.19 (t, J = 7.8 hz, 2H), 6.09 (d, J = 22.4 Hz, 1H), 5.45 (dd, J = 54.1, 3.9 Hz, 1H), 4.86 (ddd, J = 27.2, 9.8, 4.1 Hz, 1H), 4.48 (dd, J = 9.1, 5.0, 1H), 4.40 (q, J = 7.2 Hz, 2H), 4.04 (t, J = 9.8 Hz, 1H), 1.34 (t, J = 7.1 Hz, 3H), 1.11 (s, 9H), 1.05 (s, 9H).

[0185] Step 2: Synthesis of compound 4d N-ethyl-N-(9-((2R,3R,4R,5R)-3-fluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-9H-purin-6-yl)benzamide Compound 4d was obtained in the same manner as in the process for obtaining compound 4c. ESI-MS: Calculated value: 401.40 [M+H] + , Measured value: 402.1 [M+H] +

[0186] Step 3: Synthesis of compound 5d N-(9-((2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-fluoro-4-hydroxytetrahydrofuran-2-yl)-9H-purin-6-yl)-N-ethylbenzamide Compound 5d was obtained in the same manner as in the step for obtaining compound 5c. ESI-MS: Calculated value: 738.25 [M+Cl] - , Measured value: 738.7 [M+Cl] - 1 1H-NMR (CDCl3, 400 MHz) δ: 8.52 (s, 1H), 8.07 (s, 1H), 7.43 - 7.41 (m, 2H), 7.38 - 7.35 (m, 2H), 7.30 - 7.20 (m, 8H), 7.10 (t, J = 7.8 Hz, 2H), 6.79 (d, J = 8.2 Hz, 4H), 6.22 (dd, J = 17.4, 2.3 Hz, 1H), 5.58 (ddd, J = 53.0, 2.4, 1.2 Hz, 1H), 4.93 - 4.74 (m, 1H), 4.40 (q, J = 7.2 Hz, 2H), 4.19 - 4.16 (m, 1H), 3.78 (s, 6H), 3.54 (dd, J = 11.0, 3.2 Hz, 1H), 3.40 (dd, J = 10.5, 3.1 Hz, 1H), 2.23 (dd, J = 6.9, 2.3 Hz, 1H), 1.33 (t, J = 7.1 Hz, 3H).

[0187] Synthesis of Engineering 4 Amidite 6d (2R,3R,4R,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-(6-(N-ethylbenzamido)-9H-purin-9-yl)-4-fluorotetrahydrofuran-3-yl (2-cyanoethyl) diisopropylphosphoramidite Compound 5d (1.93 g, 2.74 mmol) was dissolved in dichloromethane (16 mL), and diisopropylethylamine (0.958 mL, 5.48 mmol) was added. The mixture was cooled in an ice bath. 2-Cyanoethyldiisopropylchlorophosphoramidite (970 mg, 4.11 mmol) dissolved in dehydrated dichloromethane (3.8 mL) was added dropwise. The mixture was then stirred for 1 hour while warming to room temperature. After confirming the disappearance of the starting materials, the reaction mixture was quenched by adding it to ice-cold saturated aqueous sodium bicarbonate. Ethyl acetate was added for extraction. The organic layer was washed with saturated brine and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated. The concentrated residue was purified by silica gel column chromatography (heptane / ethyl acetate (containing 1% triethylamine) = 90 / 10 → 60 / 40) to obtain amidite 6d (2.29 g, 2.53 mmol) as a colorless amorphous solid. (92% yield) ESI-MS: Calculated value: 938.36 [M+Cl] - , Measured value: 938.7 [M+Cl] - 1 H-NMR (CDCl3, 400 MHz) δ: 8.53 (s, 0.5H), 8.51 (s, 0.5H), 8.11 (s, 1H), 7.41-7.33 (m, 4H), 7.27-7.16 (m, 8H), 7.03 (t, J = 7.8 Hz, 2H), 6.79-6.75 (m, 4H), 6.27-6.18 (m, 1H), 5.78-5.58 (m, 1H), 5.10-4.85 (m, 1H), 4.42-4.38 (m, 2H), 4.31-4.30 (m, 1H), 3.96-3.72 (m, 7H), 3.68-3.51 (m, 4H), 3.29-3.27 (m, 1H), 2.59 (t, J = 6.2 Hz, 1H), 2.40 (t, J = 6.4 Hz, 1H), 1.33-1.31 (m, 3H), 1.19-1.15 (m, 9H), 1.04 (d, J = 6.9 Hz, 3H). 31P-NMR(CDCl3, 162 MHz) δ: 151.94, 151.89, 151.20, 151.11.

[0188] RNA oligonucleotides were synthesized using 2'-TOM (triisopropylsilyloxymethyl)-protected β-cyanoethyl phosphoramidites (DMT-2'-O-TOM-rA(Ac), DMT-2'-O-TOM-rG(Ac), DMT-2'-O-TOM-rC(Ac), and DMT-2'-O-TOM-rU) (Glen Research and ChemGenes, respectively). DNA oligonucleotides were synthesized using β-cyanoethyl phosphoramidites (DMT-dA(Bz), DMT-dG(iBu), DMT-dC(Ac), and DMT-T). Each phosphoramidite monomer was prepared in 0.05 mol / L acetonitrile and synthesized using 0.2 μmol or 0.8 μmol of solid support on a DNA / RNA solid-phase synthesizer (NTS M-2-MX, Nippon Techno Service Co., Ltd.). The DNA oligonucleotides were prepared using CPG 1000A (dA-CPG, dG-CPG, Ac-dC-CPG, dT-CPG) (Glen Research) as a solid support, and the condensation time was 2 minutes. For RNA with a phosphate group at the 5' end (5'-monophosphate RNA), Universal UnyLinker Support 2000A (ChemGenes) was used as the solid support. The condensation time for the first base was 15 minutes, and for each subsequent base was 3 minutes. The hydroxyl group at the 5' end was phosphorylated using a chemical phosphorylation reagent (0.05 mol / L acetonitrile solution) (Glen Research or ChemGenes). Solid-phase synthesis of an RNA oligonucleotide having a 3'-aminoguanosine monomer introduced at the 3' end was carried out using compound 15. The condensation time for the first base was 15 minutes, and each of the subsequent bases was 3 minutes.

[0189] The following reagents were used in the solid-phase synthesis system. The dimethoxytrityl group at the 5'-terminus hydroxyl group was removed using a commercially available deblocking reagent (Deblocking Solution-1, 3 w / v% trichloroacetic acid / dichloromethane solution) (Wako Pure Chemical Industries, Ltd.) and allowed to react for 10 seconds. A commercially available activator solution (Activator Solution 3) (Wako Pure Chemical Industries, Ltd.) was used as the phosphoramidite activator. Unreacted 5'-terminus hydroxyl groups were capped using commercially available capping solutions (Cap A Solution-2 and Cap B Solution-2) (Wako Pure Chemical Industries, Ltd.) and allowed to react for 10 seconds. The oxidizing agent used to produce the phosphate ester was a solution containing pyridine, THF, water, and iodine (Oxidizer, 0.01 M iodine, 29.2% water, 6.3% pyridine, 64.5% acetonitrile, Honeywell), and allowed to react for 10 seconds. After solid-phase synthesis, the dimethoxytrityl group at the 5'-terminus of the RNA oligonucleotide was deprotected on the solid support. The synthesized DNA and RNA oligonucleotides were deprotected using standard methods (concentrated aqueous ammonia, 55°C, 12 hours). DNA oligonucleotides were purified using a cartridge column (MicroPure II Column, LGC Biosearch Technology) according to the manufacturer's protocol. The solution obtained by deprotection was concentrated to dryness using a centrifugal evaporator. The TOM protecting group at the 2'-hydroxyl group of the RNA oligonucleotide was then removed using 1 mL of tetrabutylammonium fluoride (1 M tetrahydrofuran solution) (50°C for 10 minutes, followed by 12 hours at room temperature, or 50°C for 10 minutes, followed by 6 hours at 35°C). Tris-HCl buffer (1 M, pH 7.4) (1 mL) was added to the solution, mixed, and the tetrahydrofuran was removed by concentration using a centrifugal evaporator. The resulting solution was treated using a gel filtration column (NAP-25, GE Healthcare) equilibrated with ultrapure water according to the manufacturer's protocol.The resulting fraction containing the RNA oligonucleotide was concentrated using a centrifugal evaporator and then purified using denaturing polyacrylamide gel (hereinafter referred to as dPAGE).

[0190] (Purification of RNA fragments using dPAGE) Acrylamide gel solution (containing 7M urea as a denaturant) was prepared by adding an aqueous solution of ammonium persulfate (APS) and N,N,N',N'-tetramethylethylenediamine (TEMED) as a polymerization agent, followed by solidification at room temperature for 6–12 hours. The RNA sample was mixed with gel loading buffer (80% formamide, TBE), heated at 90°C for 3 minutes, and then loaded onto the gel. After electrophoresis, RNA bands were detected by UV light (254 nm) and excised from the gel with a razor blade. The excised gel pieces were finely crushed and extracted with ultrapure water at room temperature for 6–12 hours with shaking. The RNA extract was desalted and concentrated using an Amicon Ultra 10K (Millipore) and ethanol precipitated with 0.3M sodium acetate (pH 5.2) / 70% ethanol to obtain an RNA pellet. The RNA pellet was rinsed with 80% ethanol and air-dried at room temperature for 1 hour. The resulting RNA pellet was dissolved in ultrapure water and diluted to the appropriate concentration. The absorbance at 260 nm was measured using UV-visible spectrophotometry (NanoDrop, Thermo Scientific). The concentration was determined from the molar extinction coefficient of each RNA sequence (the following values ​​were used for the molar extinction coefficients of each base: A = 15300, G = 11800, C = 7400, T = 9300, U = 9900). The structure of the purified oligonucleotide was determined by mass spectrometry using MALDI-TOF MS (Ultraflex III, Bruker Daltonics) (matrix: 3-hydroxypicolinic acid) or by analysis using denaturing polyacrylamide gel electrophoresis.

[0191] (Analysis of chemical ligation reactions using dPAGE) For analysis of chemical ligation reactions, the reaction mixture was appropriately diluted with ultrapure water and used as a sample. The diluted sample was mixed with gel loading buffer (80% formamide / TBE), heated at 90°C for 3 minutes, and then loaded onto a gel. After electrophoresis, the gel was stained (room temperature, 15 minutes) with SYBR® Green II Nucleic Acid Stain (Lonza) diluted 10,000-fold with ultrapure water to detect RNA bands (equipment used: ChemiDoc, BIORAD). The yield of the chemical ligation reaction was calculated by comparing the band intensity of the RNA ligation product with that of the isolated and purified ligation product by dPAGE, using it as a standard substance.

[0192] (Purification of chemical ligation products using dPAGE) The RNA ligation product obtained by the chemical ligation reaction was recovered from the reaction mixture as an RNA pellet by ethanol precipitation (0.3 M sodium acetate (pH 5.2) / 70% ethanol), and then purified by dPAGE.

[0193] The sequence information of the compounds (polynucleotides) used in Examples 1 to 4 is shown below. In Tables 1 and 2, each nucleotide N represents RNA, N(M) represents 2'-O-methyl modified RNA, N(F) represents 2'-F modified RNA, and dN represents DNA. p indicates that the 3' or 5' end is phosphorylated. The underlined "AUG" represents the start codon, and the underlined "UGA" represents the stop codon. A diagonal line ( / ) in a sequence indicates that polynucleotides are linked at that position. [Table 1] [Table 2]

[0194] Example 1 Three batches of ultrapure aqueous solutions (200 μL, final nucleic acid concentration: 50 μM) containing the chemically synthesized RNA fragment E1-1 (10 nmol) obtained as sequence 2, the 5'-phosphate RNA fragment E1-2 (10 nmol) obtained as sequence 3, and template DNA1 (10 nmol) were prepared. Each solution was mixed with 100 μL of T4 RNA Ligase 2 Reaction Buffer (10X) (New England BioLabs) and 440 μL of ultrapure water, heated at 90°C for 5 minutes, and then cooled to room temperature over 30 minutes. A 60% PEG 6000 aqueous solution was added to each solution to a final concentration of 15%. After mixing, 10 μL of T4 RNA Ligase 2 (New England BioLabs) (10 units / μL) was added and the mixture was placed on a temperature-controlled heating block at 37°C for 16 hours. An equal volume of chloroform was added to the reaction mixture, mixed by vortexing, and centrifuged. The upper layer was separated and subjected to alcohol precipitation (0.3 M sodium acetate (pH 5.2) / 70% ethanol) to obtain an RNA pellet. The RNA obtained from each batch was pooled and purified on a 7.5% denaturing polyacrylamide gel to obtain the RNA ligation product E1 (9.7 nmol, 32% yield).

[0195] Example 2 Using the RNA fragment E2-1 obtained by chemical synthesis as sequence 6, the 5'-phosphate RNA fragment E2-2 obtained as sequence 7, and template DNA1, the RNA ligation product E2 (10.9 nmol, yield 36%) was obtained in the same manner as in Example 1.

[0196] Example 3 The RNA ligation product E3 (13.4 nmol, yield 45%) was obtained in the same manner as in Example 1 using the chemically synthesized RNA fragment E3-1 obtained as sequence 9, the 5'-phosphate RNA fragment E3-2 obtained as sequence 10, and template DNA1.

[0197] Example 4 The RNA ligation product E4 (6.8 nmol, yield 34%) was obtained in the same manner as in Example 1 using the chemically synthesized RNA fragment E4-1 obtained as sequence 12, the 5'-phosphate RNA fragment E4-2 obtained as sequence 13, and template DNA1.

[0198] The sequence information of the compounds (polynucleotides) used in Reference Examples 1 to 18 is shown below. In Tables 3 to 8, N represents RNA, N(M) represents 2'-O-methyl-modified RNA, N(F) represents 2'-F-modified RNA, N(L) represents LNA, N(MOE) represents 2'-O-methoxyethyl-modified RNA, and dN represents DNA. A(m6) represents N6-methyladenine as the base. p represents phosphorylation at the 3' or 5' end, and p(S) represents phosphorothioation at the 3' or 5' end. NH2 represents substitution of the hydroxyl group at the 3' or 5' end with an amino group. -N / P- and -P / N- represent substitution of a phosphate bond with a phosphoamide bond in the nucleotide, and -NHAc / S- represents substitution of a phosphate bond with -NHC(O)-CH2-SP(O)(OH)-O- in the nucleotide. The underlined "AUG" represents the start codon, and the underlined "UGA" represents the stop codon. A slash ( / ) in the sequence indicates that a polynucleotide has been ligated at that point. [Table 3] [Table 4] [Table 5] [Table 6] [Table 7] [Table 8]

[0199] Reference example 1 An ultrapure aqueous solution of the chemically synthesized 3'-amino RNA fragment R1-1 (200 μM), obtained as sequence 15, the 5'-phosphate RNA fragment R1-2 (200 μM), obtained as sequence 16, and template DNA2 (200 μM) was heated at 90°C for 3 minutes and then allowed to cool to room temperature over 30 minutes. The ligation reaction was initiated by adding an equal volume of a separately prepared condensation agent buffer solution (1M EDC-HCl / 1M HOBt, 500 mM HEPES-NaOH (pH 8.5), 200 mM NaCl, 200 mM MgCl2) and mixing. The reaction mixture (nucleic acid concentration: 100 μM, 0.5M EDC-HCl / 0.5M HOBt, 250 mM HEPES-NaOH (pH 8.5), 100 mM NaCl, 100 mM MgCl2) was placed on a temperature-controlled heat block (25°C) overnight. After the reaction, RNA pellets were obtained by alcohol precipitation (0.3 M aqueous sodium acetate solution (pH 5.2) / 70% ethanol). This RNA was analyzed on a 7.5% denaturing polyacrylamide gel, and the reaction yield (52%) was calculated.

[0200] Reference example 2 Using R2-1 and R2-2 obtained by chemical synthesis and template DNA1, the production yield (73%) of the RNA ligation product R2 was calculated in the same manner as in Reference Example 1.

[0201] Reference example 3 Using chemically synthesized R3-1 (100 μM), R3-2 (100 μM), and template DNA3 (100 μM), the RNA ligation product R3 (7.7 nmol, yield 26%) was obtained in the same manner as in Reference Example 1.

[0202] Reference example 4 Using chemically synthesized R4-1 (100 μM), R4-2 (100 μM), R4-3 (100 μM), template DNA1 (100 μM), and template DNA4 (100 μM), the RNA ligation product R4 (2.4 nmol, yield 5.6%) was obtained in the same manner as in Reference Example 1.

[0203] Reference example 5 Chemically synthesized R5-1 (100 μM) was mixed with phosphate buffer (50 mM), MiliQ water, and a DMF solution of iodoacetic acid NHS ester (5 mM) and incubated at 30°C for 2 hours. Subsequently, a pellet of iodoacetylated R5-1 was obtained by alcohol precipitation (0.3 M sodium acetate aqueous solution (pH 5.2) / 70% ethanol). Chemically synthesized R5-2 (50 μM), template DNA1 (50 μM), sodium chloride aqueous solution (100 mM), and phosphate buffer (20 mM, pH 7.5) were mixed and heated to 90°C for 3 minutes, then allowed to cool to room temperature for at least 30 minutes. The iodoacetylated R5-1 (50 μM) was added to this solution and incubated at 30°C for 6 hours. After the reaction, a pellet of RNA was obtained by alcohol precipitation (0.3 M sodium acetate aqueous solution (pH 5.2) / 70% ethanol). This RNA was purified through a 7.5% denaturing polyacrylamide gel to obtain the RNA ligation product R5 (2.32 nmol, yield 7.3%).

[0204] Reference example 6 Using chemically synthesized R6-1 and R6-2 and template DNA1, an RNA ligation product R6 (11.1 nmol, yield 37%) was obtained in the same manner as in Example 1.

[0205] Reference example 7 Using chemically synthesized R7-1 and R7-2 and template DNA1, an RNA ligation product R7 (3.1 nmol, yield 10%) was obtained in the same manner as in Example 1.

[0206] Reference example 8 Using chemically synthesized R8-1 and R8-2 and template DNA1, an RNA ligation product R8 (2.6 nmol, yield 9%) was obtained in the same manner as in Example 1.

[0207] Reference example 9 Using chemically synthesized R9-1 and R9-2 and template DNA1, an RNA ligation product R9 (5.9 nmol, yield 20%) was obtained in the same manner as in Example 1.

[0208] Reference example 10 Using chemically synthesized R10-1 and R10-2 and template DNA1, an RNA ligation product R10 (2.1 nmol, yield 9%) was obtained in the same manner as in Example 1.

[0209] Reference example 11 An ultrapure aqueous solution of the chemically synthesized RNA fragment R11-1 (200 μM), sequence 49, the 5'-amino RNA fragment R11-2 (200 μM), sequence 50, and template DNA1 (200 μM) was heated at 90°C for 3 minutes and then cooled to room temperature over 30 minutes. An equal volume of a separately prepared condensation agent buffer solution (1M EDC-HCl / 1M HOBt, 500 mM HEPES-NaOH (pH 8.5), 200 mM NaCl, 200 mM MgCl2) was added to this solution and mixed to initiate the ligation reaction. The reaction mixture (nucleic acid concentration: 100 μM, 0.5M EDC-HCl / 0.5M HOBt, 250 mM HEPES-NaOH (pH 8.5), 100 mM NaCl, 100 mM MgCl2) was placed on a temperature-controlled heat block (25°C) for 7 hours. After the reaction, the RNA was pelleted by alcohol precipitation (0.3 M sodium acetate aqueous solution (pH 5.2) / 70% ethanol), and purified on a 7.5% denaturing polyacrylamide gel to obtain the RNA ligation product R11 (1.2 nmol, 15% yield).

[0210] Reference example 12 A solution of chemically synthesized R12-1 (10 μM), R12-2 (10 μM), and template DNA1 (10 μM) in 1x T4 DNA ligase buffer (66 mM Tris-HCl (pH 7.6), 6.6 mM MgCl2, 10 mM DTT, 0.1 mM ATP) (Takara Bio) (total 2.1 mL) was heated at 90 °C for 5 min and then slowly cooled to room temperature. 60% PEG6000 was added to this solution to a final concentration of 15%. T4 DNA ligase (Takara Bio) (350 units / μL) (135 μL) was added to this solution, mixed, and then placed on a temperature-controlled heat block (25 °C, 16 h). An equal volume of chloroform was added to the reaction mixture, mixed by vortexing, and centrifuged. The upper layer was separated and subjected to alcohol precipitation (0.3 M sodium acetate (pH 5.2) / 70% ethanol) to obtain an RNA pellet. This RNA was purified on a 7.5% denaturing polyacrylamide gel to obtain the RNA ligation product R12 (1.4 nmol, 27% yield).

[0211] Reference example 13 Using chemically synthesized R13-1 and R13-2 and template DNA1, an RNA ligation product R13 (1.3 nmol, yield 26%) was obtained in the same manner as in Reference Example 12.

[0212] Reference example 14 Using chemically synthesized R14-1 and R14-2 and template DNA1, the RNA ligation product R14 (9.4 nmol, yield 31%) was obtained in the same manner as in Example 1.

[0213] Reference example 15 Using chemically synthesized R15-1 and R15-2 and template DNA1, the RNA ligation product R15 (8.6 nmol, yield 29%) was obtained in the same manner as in Example 1.

[0214] Reference example 16 Using chemically synthesized R16-1 (10 μM), R16-2 (10 μM), and template DNA1 (10 μM), the RNA ligation product R16 (1.1 nmol, yield 37%) was obtained in the same manner as in Reference Example 12.

[0215] Reference example 17 A solution of chemically synthesized R17-1 (50 μM), R17-2 (50 μM), and template DNA5 (100 μM) in 1x T4 DNA ligase buffer (66 mM Tris-HCl (pH 7.6), 6.6 mM MgCl2, 10 mM DTT, 0.1 mM ATP) (Takara Bio) (total 2.1 mL) was heated at 90 °C for 5 minutes and then slowly cooled to room temperature. 50% PEG 6000 was added to this solution to a final concentration of 10%. T4 DNA ligase (Takara Bio) (350 units / μL) (135 μL) was added to this solution, mixed, and then placed on a temperature-controlled heat block (25 °C, 16 hours). An equal volume of chloroform was added to the reaction mixture, mixed by vortexing, and centrifuged. The upper layer was separated and subjected to alcohol precipitation (0.3 M sodium acetate (pH 5.2) / 70% ethanol) to obtain an RNA pellet. This RNA was purified on a 7.5% denaturing polyacrylamide gel to obtain the RNA ligation product L84(PO) (30 nmol, 22% yield). A total of 80 μL of L84(PO) (50 μM, 20 μL), R17-3 (160 μM, 12.5 μL), and template DNA6 (160 μM, 12.5 μL) in 1x T4 DNA ligase buffer (66 mM Tris-HCl (pH 7.6), 6.6 mM MgCl2, 10 mM DTT, 0.1 mM ATP) (Takara Bio) was heated at 90°C for 5 minutes and then slowly cooled to room temperature. 50% PEG 6000 was added to this solution to a final concentration of 10%. 5 μL of T4 DNA ligase (Takara Bio) (350 units / μL) was added to this solution, mixed, and then placed on a temperature-controlled heat block (25°C, 16 hours). An equal volume of chloroform was added to the reaction mixture, mixed by vortexing, and centrifuged. The upper layer was separated and subjected to alcohol precipitation (0.3 M sodium acetate (pH 5.2) / 70% ethanol) to obtain an RNA pellet. This RNA was purified on a 7.5% denaturing polyacrylamide gel to obtain the RNA ligation product R17 (360 pmol, 36% yield).

[0216] Reference example 18 Using chemically synthesized R18-1 and R18-2 and template DNA1, an RNA ligation product R18 (11.9 nmol, yield 40%) was obtained in the same manner as in Example 1.

[0217] Test Example 1 (translation reaction of mRNA sample) The translation reaction was carried out using a commercially available translation kit according to the manufacturer's protocol. The translation reaction in a prokaryotic cell system was carried out using Compound R1 and Compound R17 as substrate RNAs and PURExpress (registered trademark) (New England BioLabs) as a reagent. The translation reaction in the eukaryotic cell system was carried out using Compound R2 and Compound R18 as substrate RNAs and Rabbit Reticulocyte Lysate System, Nuclease Treated (hereinafter, RRL) (Promega) as a reagent.

[0218] (Sample preparation for translation reaction) Preparations were made according to the recommended protocols for each kit. The reaction mixture, containing all materials except the substrate RNA, served as the translation reaction mixture. The translation reaction mixture was added to a tube containing an RNA sample that had been dried using a centrifugal evaporator, mixed, and then placed on a heat block at the appropriate temperature to initiate the translation reaction. The translation product was detected by Western blotting using an anti-FLAG antibody. The primary antibody used was an anti-FLAG antibody (F1804, Sigma), and the secondary antibody was an anti-mouse IgG antibody (anti-mouse IgG-HRP) (A9044, Sigma).

[0219] (translation reaction in prokaryotic cells) The translation reaction mixture was prepared by mixing Solution A (2 μL), Solution B (1.5 μL), RNase inhibitor (Murine RNase Inhibitor, New England BioLabs) (0.1 μL), and ultrapure water (1.4 μL) included in the PURExpress® kit. This solution was added to the tube containing the RNA sample and mixed, allowing the translation reaction to proceed (37°C, 2 hours).

[0220] (translation reaction in eukaryotic cells) The translation reaction mixture was prepared by mixing Rabbit Reticulocyte Lysate (14.0 μL) provided with the Rabbit Reticulocyte Lysate System Kit, Amino Acid Mixture - Met (0.20 μL), Amino Acid Mixture - Leu (0.20 μL), Murine RNase Inhibitor (New England BioLabs) (0.4 μL), and ultrapure water (5.2 μL). This solution was added to the tube containing the RNA sample and mixed, allowing the translation reaction to proceed (30°C, 2 hours).

[0221] (SDS-PAGE analysis of translation products) A discontinuous buffer system was used for SDS-PAGE. The gel composition is shown below. The upper layer was a 5% polyacrylamide (acrylamide:bisacrylamide = 29:1) (0.125 M Tris-HCl (pH 6.8), 0.1% SDS) concentrating gel (approximately 1.5 cm). The lower layer was a 15% polyacrylamide (acrylamide:bisacrylamide = 29:1) (0.375 M Tris-HCl (pH 8.8), 0.1% SDS) separating gel. A solution of APS and TEMED were added as polymerization agents, and the gel was allowed to solidify (room temperature, 30 minutes). The translation reaction mixture was mixed with 2x SDS-PAGE loading buffer (125 mM Tris-HCl (pH 6.8), 30 (v / v)% glycerol, 4% sodium dodecyl sulfate (SDS), 0.03% bromophenol blue (BPB)) and heated at 90°C for 3 minutes to prepare the sample for SDS-PAGE analysis. The sample for SDS-PAGE analysis was immediately subjected to electrophoresis on an SDS-PAGE gel (using 25 mM Tris, 192 mM glycine, 0.1% SDS as the running buffer).

[0222] (Detection of translation products by Western blotting) After electrophoresis, the translation products on the gel were transferred to a membrane for Western blotting (Immobilon®-P) (IPVH00010, Millipore) using the semi-dry method (the membrane was pre-treated with methanol to make it hydrophilic, and then immersed in blotting buffer (25 mM Tris, 192 mM Glycine, 20% MeOH)). A constant current condition (current application time: 1 hour) was used for the transfer, and the current value used was determined based on the size of the membrane. That is, the membrane area (cm 2 The current value (mA) was set so that the value was 2. The 5% ECL Prime solution in TBS-T used in the following procedures was prepared by mixing Amersham ECL Prime (GE Healthcare) with TBS-T (0.05 M Tris-HCl (pH 7.4), 150 mM NaCl, 0.05% Tween 20). The membrane onto which the translation product was transferred was blocked (5% ECL Prime in TBS-T, room temperature, 1 hour with shaking), then treated with primary antibody (4,000-fold diluted, 0.5% ECL Prime in TBS-T, 4°C, 12 hours with shaking), washed (TBS-T, 5 minutes x 5 times with shaking), treated with secondary antibody (50,000-fold diluted, 0.5% ECL Prime in TBS-T, room temperature, 1 hour with shaking), and washed (TBS-T, 5 minutes x 5 times). After antibody treatment, the translation products on the membrane were detected using a chemiluminescent reagent (SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific)) according to the manufacturer's recommended protocol. After treatment with the chemiluminescent reagent, the translation product signals were detected using a ChemiDoc (BIORAD) (detection mode: chemiluminescence, exposure time: 90-300 seconds). The results are shown in Figures 4 and 5.

[0223] The results of the Western blot analysis of the translation reaction products using PURExpress® with compounds R1 and R17 as substrates, as well as the Western blot analysis of the translation reaction products using RRL with compounds R2 and R18 as substrates, demonstrate that the amounts of translation products from compounds R1 and R2, which contain non-natural linking moieties, are comparable to those from natural compounds R17 and R18, in both prokaryotic and eukaryotic systems.

[0224] Test Example 2 (Translation reaction in eukaryotic cells; Translation reaction test using rabbit erythrocyte lysate) The translation activity in a eukaryotic cell system was evaluated using a Rabbit-Reticulocyte-Lysate-System-Nuclease-Treated kit (Promega, catalog number L4960) for each compound obtained in Examples 1 to 3 and Reference Examples 6 to 10, 14, 15, and 18. First, each mRNA sample was diluted with THE RNA storage solution (Thermo Fisher Scientific, catalog number AM7001) to a final concentration of 0.3 μM, and 2 μL of the diluted sample was dispensed into a 96-well PCR plate (AS ONE). Next, a master mix was prepared by mixing 7.0 μL of reticulocyte lysate, nuclease treated per reaction, 0.1 μL of amino acid mixture minus leucine per reaction, 0.1 μL of amino acid mixture minus methionine per reaction, 0.4 μL of RNase inhibitor, murine (New England BioLabs, catalog number M0314) per reaction, and 0.4 μL of purified water per reaction. 8 μL of this mixture was dispensed into the PCR plate containing the mRNA samples, and after addition and mixing, the mixture was allowed to stand at 37°C for 1 hour to carry out the translation reaction. The translation product in the reaction solution after the translation reaction was detected by the sandwich ELISA method described below. First, 6*His, His-Tag antibody (Proteintech, catalog no. 66005-1-Ig) was diluted to 3 μg / mL in 0.1 M carbonate buffer (pH 9.4), and 50 μL of the solution was dispensed into each well of a 96-well ELISA plate (Nunc). The plate was then left to stand overnight at 4°C to prepare an antibody-immobilized plate. The plate was then washed with Tris-Buffered Saline with Tween 20 (Santa Cruz, catalog no. sc-24953) diluted 1x with purified water (hereafter referred to as the washing solution). Then, 200 μL of a washing solution (hereafter referred to as the blocking solution) prepared by diluting bovine serum albumin (Wako Pure Chemical Industries, catalog no. 017-22231) to a final concentration of 3% was dispensed into each well and left to stand at room temperature for 1 hour. After washing the plate with washing solution, 50 μL of the translation reaction solution diluted 100-fold with blocking solution was dispensed into each well and incubated at room temperature for 1 hour. At this time, translation product polypeptide preparations (Scrum) were also diluted to various concentrations in blocking solution and dispensed into the plate. After washing the plate with washing solution, 50 μL of Monoclonal Anti-FLAG M2-Peroxidase (HRP) Ab produced in mouse (Sigma, catalog antibody A8592-1MG) diluted 10,000-fold with blocking solution was dispensed into each well and incubated at room temperature for 1 hour. After washing the plate with washing solution, 50 μL of 1-Step Ultra TMB-ELISA (Thermo Fisher Scientific, catalog number 34028) was dispensed into each well and incubated at room temperature for several minutes. The reaction was then stopped by dispensing 50 μL of 0.5 M sulfuric acid (Wako Pure Chemical Industries, Ltd.) per well, and the absorbance was measured using a spectrophotometer (Bio-Rad) at a measurement wavelength of 450 nm and a reference wavelength of 570 nm. The translation product concentration (μM) in each translation reaction solution and the relative translation product amount, relative to the amount of R18 with no sugar modification set at 1, were quantified using a calibration curve prepared based on the absorbance of the polypeptide preparation. These are shown in Tables 9 to 11.

[0225] [Table 9]

[0226] [Table 10]

[0227] [Table 11]

[0228] As is clear from Tables 9 to 11, after each compound was added to rabbit erythrocyte lysate, the polypeptide encoded by the gene sequence was produced by the translation system of the eukaryotic cells.

[0229] (Translation reaction test using HeLa cell line lysate of mRNA sample) The translation activity in a human cell line was evaluated using a 1-Step Human Coupled IVT Kit (Thermo Fisher Scientific, catalog number 88882) for each compound obtained in Examples 1 to 4 and Reference Examples 6, 10, 14, 15, and 18. First, each compound was diluted with THE RNA storage solution (Thermo Fisher Scientific, catalog number AM7001) to a final concentration of 0.3 μM, and 1 μL of the diluted solution was dispensed into a 96-well PCR plate (AS ONE). Next, a master mix was prepared by mixing 5.0 μL of Hela Lysate per reaction, 1.0 μL of Accessory Proteins per reaction, 2.0 μL of Reaction Mix per reaction, 0.2 μL of RNase Inhibitor Murine (New England BioLabs, catalog number M0314) per reaction, and 0.8 μL of purified water per reaction. 9 μL of this mixture was dispensed into the PCR plate containing the mRNA samples, and after addition and mixing, the mixture was allowed to stand at 37°C for 45 minutes to allow the translation reaction to proceed. The translation product in the reaction solution after the translation reaction was measured by the sandwich ELISA method described in Test Example 2 (Translation reaction in a eukaryotic cell system; translation reaction test using rabbit erythrocyte lysate) in exactly the same manner, except that the translation reaction solution was diluted 20-fold with blocking solution and added to the plate. The measurement results were quantified using a calibration curve prepared based on the absorbance of the polypeptide preparation. The translation product concentration (μM) in each translation reaction solution and the relative amount of translation product when the amount of compound R18 without sugar moieties was set to 1 are shown in Tables 12 to 14.

[0230] [Table 12]

[0231] [Table 13]

[0232] [Table 14]

[0233] As is clear from Tables 12 to 14, after each compound was added to HeLa cell lysate, the polypeptide encoded by the gene sequence was produced by the translation system of the human cells.

[0234] (In vitro translation reaction test using HeLa cell line with mRNA sample) The in vitro translation activity of each compound obtained in Examples 2 and 3 and Reference Examples 6 and 15 was evaluated using a Hela cell line. First, Hela cells suspended in RPMI medium (Nacalai Tesque) containing 10% fetal bovine serum were seeded onto a 96-well culture plate for adherent cells at a cell density of 10,000 cells / 100 μL per well, and cultured overnight at 37°C and 5% CO2. After overnight cell culture, the culture supernatant was removed from the cells, and 40 μL of RPMI medium containing 10% fetal bovine serum was added per well. Each compound was diluted with Lipofectamin MessengerMAX Transfection Reagent (Thermo Fisher Scientific, catalog number: LMRNA008) in Optimem (Thermo Fisher Scientific, catalog number: 31985-070) to a final concentration of 0.3 μM. The mixture was added to each well at 10 μL per well and cultured at 37°C under 5% CO2 for 6 hours. After 6 hours of culture, the culture supernatant was removed from the cells, washed once with ice-cold D-PBS(-) (Nacalai Tesque), and then 20 μL of NP-40 (Invitrogen, FNN0021) containing 2% protease inhibitor cocktail (for animal cell extracts) was added per well. Cells were lysed by vigorous shaking for 5 minutes. The translation products in the obtained cell lysates were analyzed by sandwich ELISA in exactly the same manner as in Test Example 2 (Translation reaction in a eukaryotic cell system; translation reaction test using rabbit erythrocyte lysate), except that the cell lysates were diluted 10-fold with blocking solution and added to the plate. The measurement results were quantified using a calibration curve prepared based on the absorbance of the polypeptide preparation. The translation product concentrations (nM) in each translation reaction solution and the relative amounts of the translation products, when the amount of compound R6 without sugar modification was taken as 1, are shown in Table 15.

[0235] [Table 15]

[0236] As is clear from Table 15, when each compound was added to HeLa cells, it produced the polypeptide encoded by the gene sequence.

[0237] Test Example 3 (Stability test of each compound using phosphodiesterase I) The stability of each compound (RNA solution) prepared in Example 1 and Reference Examples 10 and 18 against phosphodiesterase I (SVPD) was evaluated by the following method. 10 μL of MQ water, 4 μL of 5 μM RNA solution, 4 μL of 5x reaction buffer (0.1 M Tris-HCl pH 8.0, 0.5 M NaCl, 0.075 M MgCl2), and 4 μL of 5.5 U / μL SVPD (Warthington, catalog number 3926) were added and incubated at 37°C. 6 μL aliquots were taken at 15, 30, and 60 minutes after the start of the reaction. (RNA degradation assessment by PAGE) The entire sample was applied to a 5% dPAGE (5% acrylamide, 7M urea, small gel, 8 cm x 8 cm) and electrophoresed at 300 V constant for 18 min. The gel was stained with SYBR Green II, photographed, and quantified. The remaining amount of each compound in each sample was calculated as the relative amount remaining compared to the unreacted amount (0 min) and is shown in Table 16.

[0238] [Table 16]

[0239] As is clear from Table 16, the compounds with sugar modifications had improved resistance to phosphodiesterase I compared with compound R18, which did not have a sugar modification.

[0240] Test Example 4 (Stability test of mRNA samples using phosphodiesterase I) The enzyme stability of each compound obtained in Examples 1 to 4 and Reference Examples 6, 14, 15, and 18 was evaluated using phosphodiesterase I (Warthington, catalog number 3926). First, phosphodiesterase I was prepared in SVPD stock solution (110 mM Tris-HCl (Nippon Gene, catalog number 314-90401), 110 mM NaCl (Ambion, catalog number AM9759), 15 mM MgCl (Nacalai Tesque, catalog number 20942-34)) to a final concentration of 55 U / mL, and this was further diluted 10-fold with 5x SVPD reaction buffer (100 mM Tris-HCl (Ambion, catalog number AM9855G), 500 mM NaCl, 75 mM MgCl) to prepare a 5.5 U / mL SVPD enzyme solution. Each compound was diluted with THE RNA storage solution (Thermo Fisher Scientific, catalog number AM7001) to a final concentration of 5 μM. To prepare the enzyme reaction premix, 30 μL of distilled water, 12 μL of 5 μM mRNA, and 12 μL of 5x SVPD reaction buffer were added to a 96-well PCR plate. For the enzyme unreacted (0 min) run, 9 μL of the enzyme reaction premix was dispensed into another 96-well PCR plate, to which 3.5 μL of a mixture of 1 μL of SVPD solution and 2.5 μL of 25 mM EDTA (Ambion, catalog number AM9260G) was added and stored at -30°C. For the enzyme reaction run, 40.5 μL of the enzyme reaction premix was dispensed into another 96-well PCR plate, to which 4.5 μL of 5.5 U / mL SVPD enzyme solution was added and mixed well. This was dispensed in 10 μL aliquots into four new 96-well PCR plates and reacted at 37°C for the specified time (15 min, 30 min, 60 min) on each plate. After that, 2.5 μL of 25 mM EDTA was added and the plates were stored at -30°C until measurement. The amount of residual mRNA in the reaction solution after the enzymatic reaction was detected using the RT-qPCR method described below. A standard curve was created using compound E4. A dilution series was prepared using the RNA storage solution, with 11 concentrations starting from 4 μM and diluted 4-fold. 2.5 μL of the standard curve and post-enzymatic reaction samples were diluted 1071-fold with distilled water containing Ribonuclease Inhibitor (Takara Bio, catalog no. 2311B) at a final concentration of 0.2 U / mL. Reverse transcription cDNA was prepared using 5 μL of this diluted sample and 2 μL of 2 μM RT primer (Sigma-Aldrich) using the iScript Select cDNA Synthesis Kit (BIO-RAD, catalog no. 1708897). The reaction temperatures were 25°C for 5 min, 42°C for 30 min, and 85°C for 5 min. qPCR was performed by mixing 2 μL of cDNA, 10 μL of TaqMan Gene Expression Master Mix, 0.28 μL of Fw primer (Sigma-Aldrich), 0.33 μL of Rv primer (Sigma-Aldrich), 0.38 μL of TaqMan MGB Probe (Thermo Fisher Scientific, catalog number 4316033), and 7.01 μL of distilled water. The Quantstudio12K Flex (Applied Biosystems) was used. The DNA sequences of the primers and TaqMan MGB Probe used are as follows. Based on the C values ​​of the standard samples, the concentration of each compound in each sample was quantified using a calibration curve. The relative remaining amount compared to the unreacted enzyme (0 min) is shown in Table 17. RT primer: 5'-TCAGTGGTGGTGGTGGTGGTGTTTG-3' (SEQ ID NO: 75) Fw primer: 5'-ATCTTGTCGTCGTCGTCCTT-3' (SEQ ID NO: 76) Rv primer: 5'-GAATACAAGCTACTTGTTCTTTT-3' (SEQ ID NO: 77) Taqman MGB Probe: 5'-CAGCCACCATG-3' (SEQ ID NO: 78)

[0241] [Table 17]

[0242] As is clear from Table 17, E1, E4, R14 and R15, which have sugar moiety modifications, have improved resistance to phosphodiesterase I compared with compound R18, which does not have sugar moiety modifications. Similarly, E2 and E3, which have sugar modifications, have improved resistance to phosphodiesterase I compared with compound R6, which does not have a sugar modification.

[0243] Test Example 5 (translation reaction in eukaryotic cells) The translation reaction of each compound (RNA) obtained in Reference Examples 3-5, 11-13, 16, and 18 in a eukaryotic cell system was evaluated using the following method. A solution containing Rabbit-Reticulocyte-Lysate-System_-Nuclease-Treated (Promega L4960), RNase inhibitor Murine (New England Biolabs, M0314S), and RNA were mixed in the following composition: RRL 7.0 μL, 1 mM AA-Leu 0.1 μL, 1 mM AA-Met 0.1 μL, RNase Inhibitor 0.4 μL, 5 μM RNA 2.0 μL, and MQ 0.4 μL. This solution was incubated at 37°C for 1 hour. After incubation, 5 μL of the solution was diluted 100-fold with 495 μL of blocking buffer (3% BSA / TBST). 100 μL of the diluted translation solution was used for ELISA.

[0244] (Detection of translation products by sandwich ELISA) Anti-Histag solution (Proteintech, catalog number 66005-1-Ig) (3 μg / mL in 0.1 M carbonate buffer, pH 9.4) was added to a 96-well plate at 100 μL / well. The plate was covered with parafilm and incubated at 4°C for 12 hours. The solution in the plate was discarded, and the plate was washed three times with 200 μL / well of TBST. Blocking buffer (3% BSA / TBST) was added at 200 μL / well, and the plate was incubated at room temperature for 1 hour. The solution in the plate was discarded, and the plate was washed three times with 200 μL / well of TBST. Translation reaction solution was added at 100 μL / well, and the plate was incubated at room temperature for 1 hour. The translation reaction solution in the plate was discarded, and the plate was washed three times with 200 μL / well of TBST. 100 μL / well of Anti-Flag solution (Cell Signaling Technology, Catalog No. 2368) (1:1000 in blocking buffer) was added. The plate was covered with parafilm to prevent drying and condensation, and incubated overnight at 4°C. The solution in the plate was discarded, and the plate was washed three times with 200 μL / well of TBST. 100 μL / well of Anti-rabbit IgG HRP solution (Sigma-Aldrich) (1:10,000 in blocking buffer) was added, and the plate was incubated at room temperature for 1 hour. The solution in the plate was discarded, and the plate was washed four times with 200 μL / well of TBST. 100 μL / well of TMB substrate solution was added, and the plate was incubated at room temperature for 5 minutes. 100 μL / well of 2M H2SO4 solution was added to the wells containing the TBM substrate solution. The translation efficiency was evaluated by measuring absorbance at 450 nm using a plate reader (Mithras LB940 (Berthold)). The measurement results were quantified using a calibration curve prepared based on the absorbance of the polypeptide preparation. The translation product concentration (μM) in each translation reaction solution and the relative amount of translation product when the amount of R18, which has no sugar modification, is set to 1, are shown in Tables 18 to 20.

[0245] [Table 18]

[0246] [Table 19]

[0247] [Table 20]

[0248] As is clear from Tables 18 to 20, after each compound was added to rabbit erythrocyte lysate, the polypeptide encoded by the gene sequence was produced by the translation system of eukaryotic cells.

[0249] The sequence information of the example compounds (polynucleotides) is shown below. In the table below, each nucleotide N (uppercase) represents RNA, each nucleotide n (lowercase) represents DNA, N(M) represents 2'-O-methyl modified RNA, N(F) represents 2'-F modified RNA, N(L) represents LNA, and N(MOE) represents 2'-O-methoxyethyl modified RNA. Am6 indicates that the base is N6-methyladenine, and Ae6 indicates that the base is N6-ethyladenine. p indicates that the 3' or 5' end is phosphorylated. ^ indicates that the phosphate group connecting the sugar moieties is phosphorothioate. N(B) represents 2',4'-BNA containing the sugar moiety shown below. NC (Me). [ka] BDBD represents an artificial dangling end having the following structure: [ka]

[0250] [Table 21] [Table 22] [Table 23] Table 24 Table 25 Table 26 Table 27 Table 28 Table 29 Table 30 Table 31 Table 32 Table 33 Table 34 Table 35 Table 36 Table 37 Table 38 Table 39 Table 40 Table 41 Table 42 Table 43 Table 44 Table 45 Table 46 Table 47 Table 48 Table 49 Table 50 Table 51 Table 52 Table 53 Table 54 Table 55 Table 56 Table 57 Table 58 [Table 59] [Table 60] [Table 61] [Table 62] [Table 63] [Table 64] [Table 65] [Table 66] [Table 67]

[0251] Test Example 6 (Translation reaction test using HeLa cell line lysate of mRNA sample) The translation activity of each compound shown in Tables 21 to 56 in a human cell line was evaluated using a 1-Step Human Coupled IVT Kit (Thermo Fisher Scientific, catalog number 88882). The translation reaction was carried out under the same conditions as in Test Example 2 (translation reaction test using HeLa cell line lysate as an mRNA sample). The translation product in the reaction solution after the translation reaction was detected by the sandwich ELISA method described below. First, 6*His, His-Tag antibody (Proteintech, catalog no. 66005-1-Ig) was diluted to 3 μg / mL in 0.1 M carbonate buffer (pH 9.4), and 50 μL of the solution was dispensed into each well of a 96-well ELISA plate (Nunc). The plate was then left to stand overnight at 4°C to prepare an antibody-immobilized plate. The plate was then washed with Tris-Buffered Saline with Tween 20 (Santa Cruz, catalog no. sc-24953) diluted 1x with purified water (hereafter referred to as the washing solution). Then, 200 μL of a washing solution (hereafter referred to as the blocking solution) prepared by diluting bovine serum albumin (Wako Pure Chemical Industries, catalog no. 017-22231) to a final concentration of 3% was dispensed into each well and left to stand at room temperature for 1 hour. After washing the plate with washing solution, 50 μL of the translation reaction solution diluted with blocking solution was dispensed into each well and allowed to stand at room temperature for 1 hour. The translation product polypeptide preparation shown in SEQ ID NO: 539 (Cosmo Bio) was also diluted to various concentrations in blocking solution and dispensed into the plate. After washing the plate with washing solution, 50 μL of Monoclonal Anti-FLAG M2-Peroxidase (HRP) Ab produced in mouse (Sigma, catalog antibody A8592-1MG) diluted 1:10,000 in blocking solution was dispensed into each well and allowed to stand at room temperature for 1 hour. After washing the plate with washing solution, 50 μL of 1-Step Ultra TMB-ELISA (Thermo Fisher Scientific, catalog number 34028) was dispensed into each well and allowed to stand at room temperature for several minutes. Then, 50 μL of 0.5 M sulfuric acid (manufactured by Wako Pure Chemical Industries, Ltd.) was dispensed per well to stop the reaction, and the absorbance was measured using an absorption spectrophotometer (manufactured by Bio-Rad) at a measurement wavelength of 450 nm and a reference wavelength of 570 nm.The translation product concentration (nM) in each translation reaction solution, quantified using a calibration curve prepared based on the absorbance of a polypeptide preparation, and the relative amount of translation product when the amount of R18, which has no sugar modification, is set to 1, are shown in the table below. Translation product polypeptide preparation: NH2-MDYKDDDDKIIDYKDDDDKGGDYKDDDDKHHHHHH-COOH (SEQ ID NO: 539)

[0252] [Table 68] [Table 69] [Table 70] [Table 71] [Table 72] [Table 73] [Table 74] [Table 75] [Table 76] [Table 77] [Table 78] [Table 79] [Table 80] [Table 81] Table 82 Table 83 Table 84 Table 85 Table 86 Table 87 Table 88 Table 89 Table 90 Table 91 Table 92 Table 93 Table 94 Table 95 Table 96 Table 97 Table 98 Table 99 [Table 100] [Table 101] [Table 102]

[0253] As is clear from the above test results, when each compound having a sugar moiety modification was added to HeLa cell lysate, the polypeptide encoded by the gene sequence was produced by the translation system of the eukaryotic cells.

[0254] Test Example 7 (In vitro translation reaction test using HeLa cell line with mRNA sample) The in vitro translation activity of each compound listed in Tables 21 to 56 was evaluated using a HeLa cell line. First, HeLa cells suspended in RPMI medium (Nacalai Tesque) containing 10% fetal bovine serum were seeded onto a 96-well culture plate for adherent cells at a cell density of 10,000 cells / 100 μL per well and cultured overnight at 37°C and 5% CO2. After overnight cell culture, the culture supernatant was removed from the cells, and 40 μL of RPMI medium containing 10% fetal bovine serum was added per well. Each compound was diluted with Optimem (Thermo Fisher Scientific, catalog number: LMRNA008) at a final concentration of 0.3% to a final concentration of 0.1 μM, and mixed. 10 μL of the mixture was added to each culture plate per well and cultured at 37°C and 5% CO2 for 5 hours. After 5 hours of incubation, the culture supernatant was removed from the cells and washed once with ice-cold D-PBS(-) (Nacalai Tesque). For Table 103, 20 μL of NP-40 (Invitrogen, FNN0021) containing 2% protease inhibitor cocktail (for animal cell extracts) was added per well and vigorously shaken for 30 seconds to lyse the cells. For Tables 104 and 105, 20 μL of iScript RT-qPCR Sample Preparation Reagent (Bio-Rad, 1708898) containing 2% protease inhibitor cocktail (for animal cell extracts) was added per well and vigorously shaken for 30 seconds to lyse the cells. The translation products in the obtained cell lysates were analyzed by the same sandwich ELISA method as described in Test Example 6 (translation reaction test using HeLa cell line lysate as an mRNA sample). As a result of the measurement, the translation product concentrations (nM) in each translation reaction solution were quantified using a calibration curve prepared based on the absorbance of the polypeptide preparation, and are shown in the table below.

[0255] [Table 103] [Table 104] [Table 105] [Table 106]

[0256] As is clear from the above test results, after addition of each mRNA with sugar modifications to HeLa cells, it produced the polypeptide encoded by the gene sequence, and its activity was equal to or greater than that of mRNA without sugar modifications.

[0257] Test Example 8 (In vitro translation reaction test of mRNA samples using mouse primary hepatocytes) The in vitro translation activity of each compound listed in Tables 21 to 56 was evaluated using mouse primary hepatocytes (Thermo Fisher Scientific, Catalog No. MSCP10). First, mouse primary hepatocytes suspended in William's E Medium, no phenol red (Thermo Fisher Scientific, Catalog No. A1217601) containing Primary Hepatocyte Thawing and Plating Supplements (Thermo Fisher Scientific, Catalog No. CM3000) were seeded onto a 96-well collagen I-coated culture plate (Corning, Catalog No. 356407) at a cell density of 10,000 cells / 100 μL per well and cultured at 37°C and 5% CO for 5 hours. After incubation, the culture supernatant was removed from the cells, and 100 μL of William's E Medium, no phenol red containing Primary Hepatocyte Maintenance Supplements (Thermo Fisher Scientific, catalog number CM4000) was added per well. The cells were further incubated at 37°C for 5 hours under 5% CO2. After overnight incubation, the culture supernatant was removed from the cells, and 40 μL of William's E Medium, no phenol red containing Primary Hepatocyte Maintenance Supplements was added per well. Each compound was diluted with Optimem and mixed with 0.3% Lipofectamin MessengerMAX Transfection Reagent to a final concentration of 0.1 μM. The mixture was added to each culture plate at 10 μL per well and incubated at 37°C for 5 hours under 5% CO2. After 5 hours of culture, the culture supernatant was removed from the cells, and 100 μL of William's E Medium, no phenol red, containing Primary Hepatocyte Maintenance Supplements was added per well, followed by further culture at 37°C and 5% CO2 for 1 hour.After culturing for 1 hour, the culture supernatant was removed from the cells and washed once with ice-cold D-PBS(-) (Nacalai Tesque). 20 μL of NP-40 (Invitrogen, FNN0021) containing 2% protease inhibitor cocktail (for animal cell extracts) was then added per well, and the cells were lysed by vigorously shaking for 30 seconds. The translation products in the obtained cell lysates were analyzed by the same sandwich ELISA method as described in Test Example 6 (translation reaction test using HeLa cell line lysate as an mRNA sample). As a result of the measurement, the translation product concentrations (nM) in each translation reaction solution were quantified using a calibration curve prepared based on the absorbance of the polypeptide preparation, and are shown in the table below.

[0258] [Table 107] [Table 108] [Table 109]

[0259] As is clear from the above test results, after addition of each mRNA with sugar modifications to mouse hepatocytes, it produced the polypeptide encoded by the gene sequence, and its activity was equal to or greater than that of mRNA without sugar modifications.

[0260] Test Example 9 (In vitro translation reaction test using HeLa cell line with mRNA sample) The in vitro translation activity of each compound listed in Tables 21 to 56 was evaluated using a HeLa cell line. First, each compound was diluted to 20 μM in THE RNA Storage Solution (Thermo Fisher Scientific, Catalog No. AM7000). HeLa cells were suspended at 200,000 cells / 19 μL in a mixture of SE Cell Line Nucleofector Solution and Supplement 1 included in the SE Cell Line 96-well Nucleofector Kit (Lonza, Catalog No. V4SC-1096). The prepared nucleic acid solution and HeLa cell suspension were mixed at a volume ratio of 1:19, and electroporation was performed using the Nucleofector™ 96-well Shuttl System (Lonza) under pulse conditions FF-137. Ten minutes after electroporation, cells were suspended in RPMI medium (Nacalai Tesque) containing 10% fetal bovine serum and seeded onto a 96-well culture plate for adherent cells at 50,000 cells / 145 μL per well. The cells were cultured at 37°C for 3 hours under 5% CO2 conditions. After 3 hours of culture, the culture supernatant was removed and the cells were washed once with ice-cold D-PBS(-) (Nacalai Tesque). Then, 20 μL of iScript RT-qPCR Sample Preparation Reagent (Bio-Rad, 1708898) containing 2% protease inhibitor cocktail (for animal cell extracts) was added per well and lysed by vigorously shaking for 30 seconds. The translation products in the obtained cell lysates were analyzed by the same sandwich ELISA method as described in Test Example 6 (translation reaction test using HeLa cell line lysate as an mRNA sample). As a result of the measurement, the translation product concentrations (nM) in each translation reaction solution were quantified using a calibration curve prepared based on the absorbance of the polypeptide preparation, and are shown in the table below.

[0261] [Table 110]

[0262] As is clear from the above test results, mRNA with sugar modifications produced the polypeptide encoded by the gene sequence after electroporation into HeLa cells, and its activity was superior to that of mRNA without sugar modifications in the translation region.

[0263] Test Example 10 (Translation reaction test using HeLa cell line lysate of mRNA sample) The translation activity of each compound listed in Tables 21 to 56 in a human cell line was evaluated using a 1-Step Human Coupled IVT Kit (Thermo Fisher Scientific, catalog number 88882). The translation reaction was carried out in the same manner as in Test Example 2 (translation reaction test using HeLa cell line lysate as an mRNA sample), with the nucleic acid concentration at a final concentration of 1 μM. The translation product in the reaction solution after the translation reaction was measured in the same manner as in Test Example 6 (translation reaction test using HeLa cell line lysate as an mRNA sample) using the sandwich ELISA method, except that the peptide shown in SEQ ID NO: 540 (manufactured by Cosmo Bio) was used as the translation product polypeptide preparation. The translation product concentration (nM) in each translation reaction solution, quantified using a calibration curve prepared based on the absorbance of the polypeptide preparation, and the relative translation product amount, when each compound in the table that does not have a sugar modification in the translation region is set to 1, are shown in the table below. Translation product polypeptide preparation: NH2-MDYKDDDDKGGHHHHHH-COOH (SEQ ID NO: 540)

[0264] [Table 111] [Table 112] [Table 113] [Table 114] [Table 115] [Table 116] [Table 117] [Table 118] [Table 119] [Table 120] [Table 121] [Table 122]

[0265] As is clear from the above test results, after each mRNA compound was added to HeLa cell lysate, the polypeptide encoded by the gene sequence was produced by the translation system of the eukaryotic cells.

[0266] Test Example 11 (In vitro translation reaction test using HeLa cell line with mRNA sample) The in vitro translation activity of each compound listed in Tables 21 to 56 was evaluated using a HeLa cell line. Transfection of each compound into the cell line and preparation of cell lysates were performed in the same manner as in Test Example 7 (in vitro translation reaction test using a HeLa cell line for mRNA samples), and cells were lysed using iScript RT-qPCR Sample Preparation Reagent containing 2% protease inhibitor cocktail (for animal cell extracts). The translation products in the obtained cell lysates were analyzed by the same sandwich ELISA method as described in Test Example 10 (translation reaction test using HeLa cell line lysate as an mRNA sample). As a result of the measurement, the translation product concentrations (nM) in each translation reaction solution were quantified using a calibration curve prepared based on the absorbance of the polypeptide preparation, and are shown in the table below.

[0267] [Table 123]

[0268] As is clear from the test results, after addition of each mRNA with sugar modifications to HeLa cells, it produced the polypeptide encoded by the gene sequence, and its activity was superior to that of mRNA without sugar modifications in the translation region.

[0269] Test Example 12 (Translation reaction test using HeLa cell line lysate of mRNA sample) The translation activity of each compound listed in Tables 21 to 56 in a human cell line was evaluated using a 1-Step Human Coupled IVT Kit (Thermo Fisher Scientific, catalog number 88882). The translation reaction was carried out under the same conditions as in Test Example 2 (translation reaction test using HeLa cell line lysate as an mRNA sample), with the nucleic acid concentration at a final concentration of 1 μM. The translation product in the reaction solution after the translation reaction was measured in the same manner as in Test Example 6 (translation reaction test using HeLa cell line lysate of mRNA sample) using the sandwich ELISA method, except that the peptide shown in SEQ ID NO: 541 (manufactured by Cosmo Bio) was used as the translation product polypeptide preparation. The translation product concentration (nM) in each translation reaction solution, quantified using a calibration curve prepared based on the absorbance of the polypeptide preparation, and the relative translation product amount when E82, which has no sugar modification in the translation region, is set to 1, are shown in Tables 124 to 133. Translation product polypeptide preparation: NH2-MDYKDDDDKGGDYKDDDDKHHHHHH-COOH (SEQ ID NO: 541)

[0270] [Table 124] [Table 125] [Table 126] [Table 127] [Table 128] [Table 129] [Table 130] [Table 131] [Table 132] [Table 133]

[0271] As is clear from the above test results, after each mRNA compound having a sugar moiety modification was added to HeLa cell lysate, the polypeptide encoded by the gene sequence was produced by the translation system of the eukaryotic cell.

[0272] Test Example 13 (In vitro translation reaction test using HeLa cell line with mRNA sample) The in vitro translation activity of each compound listed in Tables 21 to 56 was evaluated using a HeLa cell line. Transfection of each compound into the cell line and preparation of cell lysates were performed in the same manner as in Test Example 7 (in vitro translation reaction test using a HeLa cell line for mRNA samples), and cells were lysed using iScript RT-qPCR Sample Preparation Reagent containing 2% protease inhibitor cocktail (for animal cell extracts). The translation products in the obtained cell lysates were analyzed by the same sandwich ELISA method as described in Test Example 12 (translation reaction test using HeLa cell line lysate as an mRNA sample). As a result of the measurement, the translation product concentrations (nM) in each translation reaction solution were quantified using a calibration curve prepared based on the absorbance of the polypeptide preparation, and are shown in the table below.

[0273] [Table 134]

[0274] As is clear from the above test results, after addition of each mRNA with sugar modifications to HeLa cells, it produced the polypeptide encoded by the gene sequence, and its activity was superior to that of mRNA without sugar modifications.

[0275] Test Example 14 (Translation reaction in eukaryotic cells; Translation reaction test using rabbit erythrocyte lysate) The translation activity of each compound listed in Tables 21 to 56 in a eukaryotic cell system was evaluated using a Rabbit-Reticulocyte-Lysate-System-Nuclease-Treated kit. The translation reaction was carried out in the same manner as in Test Example 2 (translation reaction in a eukaryotic cell system; translation reaction test using rabbit erythrocyte lysate). The translation product in the reaction solution after the translation reaction was detected by the sandwich ELISA method described below. First, anti-human EGF antibody (Peprotech, catalog number 500-P45) was diluted to 3 μg / mL with 0.1 M carbonate buffer (pH 9.4), and 50 μL of the solution was dispensed into each well of a 96-well ELISA plate (Nunc). The plate was then left to stand overnight at 4°C to prepare an antibody-immobilized plate. The plate was then washed with Tris-Buffered Saline with Tween 20 diluted 1x with purified water (hereinafter referred to as the washing solution). Then, 200 μL of a washing solution containing bovine serum albumin diluted to a final concentration of 3% (hereinafter referred to as the blocking solution) was dispensed into each well and left to stand at room temperature for 1 hour. After washing the plate with washing solution, 50 μL of translation reaction solution diluted 100-fold with blocking solution was dispensed per well and allowed to stand at room temperature for 1 hour. After washing the plate with washing solution, 50 μL of Monoclonal ANTI-FLAG M2-Peroxidase (HRP) Ab produced in mouse (SIGMA, catalog antibody A8592-1MG) diluted 10,000-fold with blocking solution was dispensed per well and allowed to stand at room temperature for 1 hour. After washing the plate with washing solution, 50 μL of 1-Step Ultra TMB-ELISA (Thermo Fisher Scientific, catalog number 34028) was dispensed per well and allowed to stand at room temperature for several minutes. The reaction was then stopped by dispensing 50 μL of 0.5 M sulfuric acid (Wako Pure Chemical Industries, Ltd.) into each well, and the absorbance was measured at a measurement wavelength of 450 nm and a reference wavelength of 570 nm using an absorption spectrophotometer (Bio-Rad Laboratories). The absorbance (absorbance at the measurement wavelength minus absorbance at the reference wavelength) measured for each translation reaction solution is shown in the table below.

[0276] [Table 135]

[0277] As is clear from the above test results, after each mRNA compound having sugar modifications was added to rabbit erythrocyte lysate, the FLAG-EGF peptide encoded in the gene sequence was produced by the translation system of eukaryotic cells.

[0278] Test Example 15 (Translation reaction test using HeLa cell line lysate of mRNA sample) The translation activity of each compound listed in Tables 21 to 56 in a human cell line was evaluated using a 1-Step Human Coupled IVT Kit (Thermo Fisher Scientific, catalog number 88882). The translation reaction was carried out under the same conditions as in Test Example 2 (translation reaction test using HeLa cell line lysate as an mRNA sample), with the nucleic acid concentration at a final concentration of 1 μM. The translation product in the reaction solution after the translation reaction was analyzed in the same manner as in Test Example 14 (Translation reaction in a eukaryotic cell system; translation reaction test using rabbit erythrocyte lysate), except that the translation reaction solution was diluted 20-fold with blocking solution. The absorbance (absorbance at the measurement wavelength minus absorbance at the reference wavelength) measured for each translation reaction solution is shown in the table below.

[0279] [Table 136]

[0280] As is clear from the above test results, after each mRNA compound having sugar modifications was added to HeLa cell lysate, the FLAG-EGF peptide encoded in the gene sequence was produced by the translation system of the eukaryotic cells.

[0281] Test Example 16 (In vitro translation reaction test using HeLa cell line with mRNA sample) The in vitro translation activity of each compound listed in Tables 21 to 56 was evaluated using a HeLa cell line. Transfection of each compound into the cell line and preparation of cell lysates were performed in the same manner as in Test Example 7 (in vitro translation reaction test using a HeLa cell line for mRNA samples), and cells were lysed using iScript RT-qPCR Sample Preparation Reagent containing 2% protease inhibitor cocktail (for animal cell extracts). The translation products in the obtained cell lysates were analyzed by the sandwich ELISA method described in Test Example 14 (Translation reaction in a eukaryotic cell system; translation reaction test using rabbit erythrocyte lysate), except that the cell lysates were diluted 30-fold with blocking solution. The absorbance (absorbance at the measurement wavelength minus absorbance at the reference wavelength) measured for each cell lysate is shown in the table below.

[0282] [Table 137]

[0283] As is clear from the above test results, after addition of each mRNA compound with sugar modifications to HeLa cells, the eukaryotic translation system produced the FLAG-EGF peptide encoded in the gene sequence, and its activity was superior to that of mRNA without sugar modifications.

[0284] Test Example 17 (Translation reaction in eukaryotic cells; Translation reaction test using rabbit erythrocyte lysate) The translation activity of each compound listed in Tables 21 to 56 in a eukaryotic cell system was evaluated using a Rabbit-Reticulocyte-Lysate-System-Nuclease-Treated kit. The translation reaction was carried out in the same manner as in Test Example 2 (translation reaction in a eukaryotic cell system; translation reaction test using rabbit erythrocyte lysate). The translation product in the reaction solution after the translation reaction was measured in the same manner as in Test Example 6 (translation reaction test using HeLa cell line lysate as an mRNA sample) using the sandwich ELISA method, except that the peptide shown in SEQ ID NO: 542 (manufactured by Cosmo Bio) was used as the translation product polypeptide standard. The translation product concentration (nM) in each translation reaction solution, quantified using a calibration curve prepared based on the absorbance of the polypeptide standard, and the relative translation product amount when E6, which has no sugar modification in the translation region, is set to 1, are shown in the table below. Translation product polypeptide preparation: NH2-MDYKDDDDKIIDYKDDDDKGGDYKDDDDKSIINFEKLHHHHHH-COOH (SEQ ID NO: 542)

[0285] [Table 138]

[0286] As is clear from the above test results, after each mRNA compound having a sugar moiety modification was added to rabbit erythrocyte lysate, the peptide encoded by the gene sequence was produced by the translation system of eukaryotic cells.

[0287] Test Example 18 (Translation reaction test using HeLa cell line lysate of mRNA sample) The translation activity of each compound listed in Tables 21 to 56 in a human cell line was evaluated using a 1-Step Human Coupled IVT Kit (Thermo Fisher Scientific, catalog number 88882). The translation reaction was carried out under the same conditions as in Test Example 2 (translation reaction test using HeLa cell line lysate as an mRNA sample), with the nucleic acid concentration at a final concentration of 1 μM. The translation product in the reaction solution after the translation reaction was assayed in the same manner as in Test Example 17 (Translation reaction in a eukaryotic cell system; translation reaction test using rabbit erythrocyte lysate), except that the translation reaction solution was diluted 10-fold with blocking solution. The translation product concentration (nM) in each translation reaction solution, quantified using a calibration curve prepared based on the absorbance of the polypeptide preparation, and the relative amount of translation product, when E6, which has no sugar modification in the translated region, is set to 1, are shown in the table below.

[0288] [Table 139]

[0289] As is clear from the above test results, when each mRNA compound having sugar moieties was added to HeLa cell lysate, the peptide encoded by the gene sequence was produced by the translation system of the eukaryotic cells.

[0290] Test Example 19 (Serum stability test of mRNA samples) For each compound listed in Tables 21-56, nucleic acid stability in serum was evaluated using commercially available mouse serum (Takara Bio, catalog number 2311B, lot number AJ10759A). First, mouse serum was diluted 15-fold with UltraPure™ DNase / RNase-Free Distilled Water (DW) (Invitrogen, catalog number 10977-015) to prepare a diluted serum solution. Each compound was diluted with THE RNA storage solution (Thermo Fisher Scientific, catalog number AM7001) to a final concentration of 5 μM. For the enzyme-unreacted (0 min) run, 10.5 μL of a mixture of 8 μL of diluted serum solution, 2.5 μL of 6 U / μL Ribonuclease Inhibitor (Takara Bio, catalog no. 2311B), and 2 μL of 5 μM mRNA was added to another 96-well PCR plate and stored at -30°C. For the enzyme reaction, 28 μL of diluted serum solution and 7 μL of 5 μM mRNA were added to another 96-well PCR plate and mixed well. Ten μL of this mixture was dispensed into four new 96-well PCR plates, and each plate was incubated at 37°C for the specified time (5, 15, 30, and 60 min). After adding 2.5 μL of 6 U / μL Rnase inhibitor, the plates were stored at -30°C until analysis. The amount of residual mRNA in the reaction solution after the enzymatic reaction was detected using the RT-qPCR method described below. For the standard curve, compound R18 was used for Table 140, and the corresponding compound for Table 141. A dilution series was prepared using the RNA storage solution, with 11 concentrations starting from 4 μM and diluted 4-fold. 2.5 μL of the standard curve and post-enzymatic reaction samples were diluted 1071-fold with DW containing ribonuclease inhibitor at a final concentration of 0.2 U / mL. 5 μL of this diluted sample and 1 μL of 2 μM RT primer (Sigma-Aldrich) were used to prepare reverse-transcribed cDNA using the TaqMan Micro RNA RT kit (Thermo Fisher Scientific, catalog number 4366597). The reaction temperatures were 16°C for 30 min, 42°C for 30 min, and 85°C for 5 min. qPCR was performed by mixing 5 μL of cDNA, 10 μL of TaqMan Gene Expression Master Mix, 0.28 μL of Fw primer (Sigma-Aldrich), 0.33 μL of Rv primer (Sigma-Aldrich), 0.38 μL of TaqMan MGB Probe (Thermo Fisher Scientific, catalog number 4316033), and 4.01 μL of distilled water. The Quantstudio12K Flex (Applied Biosystems) was used. The DNA sequences of the primers and TaqMan MGB Probe used are as follows: Based on the C values ​​of the standard samples, the concentration of each compound in each sample was quantified using a calibration curve. The relative remaining amount compared to the unreacted enzyme (0 minutes) is shown in the table below. RT primer: 5'-TCAGTGGTGGTGGTGGTGGTGTTTG-3' (SEQ ID NO: 75) Fw primer: 5'-ATCTTGTCGTCGTCGTCCTT-3' (SEQ ID NO: 76) Rv primer: 5'-GAATACAAGCTACTTGTTCTTTT-3' (SEQ ID NO: 77) Taqman MGB Probe: 5'-CAGCCACCATG-3' (SEQ ID NO: 78) [Table 140] [Table 141]

[0291] As is clear from the above test results, mRNA with sugar modifications had improved resistance to degradation in serum compared to mRNA without sugar modifications.

[0292] Test Example 20 (Translation reaction of VEGF protein) <Synthesis of comparative compound RN> The sequence information of the materials (polynucleotides) used in the synthesis of the comparative compound RN, which is translated into VEGF protein, is shown below. In the table, each nucleotide N represents RNA, N(M) represents 2'-O-methyl modified RNA, N(F) represents 2'-F-modified RNA, N(MOE) represents 2'-O-methoxyethyl modified RNA, and dN represents DNA.

[0293] [Table 142]

[0294] Comparative compound RN was obtained by the following series of operations. (Preparation of double-stranded DNA by PCR reaction and preparation of 3'-end polynucleotide fragments by in vitro transfection) The plasmid DNA used was one in which the artificial synthetic gene sequence GN shown in SEQ ID NO: 546 was inserted into the Sma I site of a commercially available pUC19 vector (manufactured by FASMAC Co., Ltd.). PCR was performed using plasmid DNA, FW primers and RV primers. The final concentrations of the PCR solution were as follows: template DNA 0.2 ng / μL, dNTP 250 μM, primer 0.3 μM, PrimeSTAR HS DNA Polymerase (Takara) 0.025 U / μL, PrimeSTAR Buffer (Mg 2+ The temperature cycle was as follows: 94°C for 5 minutes, [98°C for 10 seconds, 55°C for 30 seconds, 72°C for 60 seconds] x 30 cycles. Crude purification was performed by phenol-chloroform extraction and isopropanol precipitation. Subsequently, the template plasmid was digested and removed by DpnI treatment. The final concentrations of the reaction mixture were PCR product 3.43 μg / μL, Dpn I (Takara) 1 U / μL, and T buffer (Takara) 1x. The reaction was incubated at 37°C for 1 hour. Crude purification was performed by phenol-chloroform extraction and isopropanol precipitation.

[0295] Next, transcription reaction was performed using T7 RNA polymerase. The final concentrations of the reaction solution were as follows: DNA 10 ng / μL, DTT 5 mM, ATP, CTP, and UTP 2 mM each, GMP 2 mM, GTP 0.5 mM, T7 RNA polymerase (Takara) 2.5 U / μL, T7 RNA Polymerase buffer (Takara) 1x, RNase Inhibitor, Murine (NEB) 0.2 U / μL. The mixture was incubated at 37°C for 2 hours. DNase (recombinant, RNase-free, Takara) was then added to a final concentration of 0.1 U / μL, and the mixture was incubated at 37°C for 30 minutes. The 3'-terminal polynucleotide fragment RN-2 was obtained by crude purification via phenol-chloroform extraction and isopropanol precipitation.

[0296] (Preparation of RNA ligation products by RNA ligation) A ligation reaction using RNA ligase 2 was performed using the 3'-terminal polynucleotide fragment RN-1 obtained by chemical synthesis according to standard methods, the RNA fragment RN-2 obtained by in vitro transcription, and template DNA-A. The final concentrations were RN-1 2 μM, RN-2 2 μM, DNAN 4 μM, PEG8000 10% (v / v), T4 RNA ligase 2 (NEB) 1 U / μL, T4 RNA ligase 2 buffer (NEB) 1x, and the RNase inhibitor Murine (NEB) 1 U / μL. After adding the substrates and reagents (except for PEG8000, T4 RNA ligase 2, and RNase inhibitor) to the tube, the mixture was heated to 90°C for 3 minutes and then allowed to cool to room temperature. The remaining reagents and enzymes were added and incubated at 45°C for 1 hour. DNase was added to a final concentration of 0.33 U / μL, and the mixture was incubated at 37°C for 15 minutes. The ligated RNA was crudely purified by phenol-chloroform extraction and isopropanol precipitation. The reaction sample was analyzed by dPAGE (5% acrylamide, 7M urea). Gel purification was performed as follows: After electrophoresis on dPAGE (6% acrylamide, 7.5M urea, 25% formamide), the corresponding band was excised and extracted with MQ water for 12 hours. The gel extract was obtained by Amicon 10K (Merck Millipore) treatment and isopropanol precipitation.

[0297] (Sequence analysis of the junction of RNA ligation products) The sequence of the ligation site of the RNA ligation product RN was analyzed using the Smarter RACE 5' 3' Kit (Takara Bio, catalog number 634859) by the following series of steps. First, 1 μL of RN prepared at 100 ng / μL was taken and mixed with 1 μL of 5' CDS Primer A and 9 μL of nuclease-free water. The mixture was heated at 72°C for 4 minutes and cooled at 4°C for 2 minutes. The reaction mixture was then mixed with 1 μL of SMARTER II A Oligo, 4 μL of 5x First Strand Buffer, 0.5 μL of 100 mM DTT, 1 μL of 20 mM dNTPs, 0.5 μL of 40 U / μL RNase Inhibitor, and 2 μL of SMARTScribe Reverse Trancriptase. The mixture was heated at 42°C for 90 minutes, 72°C for 10 minutes, cooled at 4°C, and then diluted with 240 μL of TE. The resulting template cDNA was synthesized by adding buffer and mixing. Subsequently, PCR reactions were performed using the resulting template cDNA as follows. Specifically, 2.5 μL of template cDNA, 5 μL of 10x UPM, 0.1 μL of 100 μM RV Primer 2 (SEQ ID NO: 548), 25 μL of SeqAmp Buffer, 1 μL of SeqAmp DNA polymerase, and 16.4 μL of nuclease-free water were mixed and heated to 94°C for 30 seconds using a thermal cycler, followed by 20 cycles of heating at 68°C for 30 seconds and 72°C for 60 seconds, and then cooled to 4°C. The resulting PCR product was electrophoresed on a 1.5% agarose-TAE gel, and the corresponding band was excised and purified using a QIAquick Gel Extraction Kit (QIAGEN, catalog number 28704). 1 μL of the resulting PCR product was extracted with the Zero Blunt TOPO PCR Cloning Kit for Sequencing. The mixture was mixed with 0.5 μL of TOPO vector contained in (Invitrogen, Catalog No. 45-0031), 0.5 μL of salt solution, and 1 μL of nuclease-free water, and the mixture was allowed to react at room temperature for 5 minutes.The resulting reaction product was transformed into Competent Quick DH5α (Toyobo Co., Ltd., catalog number DNA-913), plated on an LB agar plate containing 100 μg / mL ampicillin, and cultured overnight at 37°C to obtain a single colony. The single colony was inoculated into LB liquid medium containing 100 μg / mL ampicillin and cultured overnight at 37°C, and plasmid DNA was extracted from the resulting E. coli using the PureYield Plasmid Miniprep System (Promega Corp., catalog number A1222).

[0298] 500 ng of the resulting plasmid DNA was mixed with 6.4 pmol of a sequencing primer (M13FW or M13RV, SEQ ID NOs: 549 and 550, respectively), and the mixture was adjusted to 14 μL with nuclease-free water. Capillary sequencing analysis based on the Sanger method was performed by FASMAC. Sequence analysis of three independent single colonies confirmed that the sequence of the ligation site of the RNA ligation product RN matched the corresponding site in the gene sequence shown in SEQ ID NO: 546. RV primer 2: 5'- GATTACGCCAAGCTTTGG CTTGAAGATGTA CTCGATCTCATCAGG-3' SEQ ID NO: 548 Sequencing primer M13FW: 5'-CTCGATCTCATCAGG-3' SEQ ID NO: 549 Sequencing primer M13RV: 5'-CAGGAAACAGCTATGAC-3' SEQ ID NO: 550

[0299] <Synthesis of comparative compound RN'> The sequence information of the materials (polynucleotides) used in the synthesis of the comparative compound RN', which is translated into VEGF protein, is shown below. In the table, each nucleotide N represents RNA, N(M) represents 2'-O-methyl modified RNA, N(F) represents 2'-F-modified RNA, N(MOE) represents 2'-O-methoxyethyl modified RNA, and dN represents DNA. [Table 143]

[0300] A comparative compound RN' was obtained by the following series of operations. (Preparation of linearized plasmid DNA and preparation of RNA fragments by in vitro transcension) The plasmid DNA used was a commercially available pUC19 vector with the synthetic gene sequence GN' (SEQ ID NO: 552) inserted into the EcoRV and XbaI sites (GeneWiz Co., Ltd.). The plasmid DNA was linearized using the restriction enzyme XbaI. The final concentrations of the reaction solution were 20 ng / μL of plasmid DNA, 0.75 U / μL of XbaI (Takara), 1x M buffer (Takara), and 0.01% BSA, and the mixture was incubated at 37°C for 1 hour. Crude purification was performed by phenol-chloroform extraction and isopropanol precipitation. The resulting linearized DNA was used for transcription reactions. The final concentrations of the reaction mixture were as follows: DNA 10 ng / μL, DTT 5 mM, ATP, CTP, and UTP 2 mM each, GMP 2 mM, GTP 0.5 mM, T7 RNA polymerase (Takara) 2.5 U / μL, T7 RNA Polymerase buffer (Takara) 1x, RNase Inhibitor, Murine (NEB) 0.2 U / μL. The mixture was incubated at 37°C for 2 hours. Subsequently, DNase (recombinant, RNase-free, Takara) was added to a final concentration of 0.1 U / μL, and the mixture was incubated at 37°C for 30 minutes. The 3'-terminal polynucleotide fragment, RN'-2, was crudely purified by phenol-chloroform extraction and isopropanol precipitation.

[0301] (Preparation of RNA ligation products by RNA ligation) A ligation reaction using RNA ligase 2 was performed using the 5'-terminal polynucleotide fragment RN'-1 obtained by chemical synthesis according to standard methods, the 3'-terminal polynucleotide fragment RN'-2 obtained by in vitro transcription, and template DNA-B. The final concentrations were RN-1 2 μM, RN-2 2 μM, DNAN 4 μM, PEG8000 10% (v / v), T4 RNA ligase 2 (NEB) 1 U / μL, T4 RNA ligase 2 buffer (NEB) 1x, and the RNase inhibitor Murine (NEB) 1 U / μL. After adding the substrates and reagents (except for PEG8000, T4 RNA ligase 2, and RNase inhibitor) to the tube, the mixture was heated to 90°C for 3 minutes and then allowed to cool to room temperature. The remaining reagents and enzymes were added and incubated at 45°C for 1 hour. DNase was added to a final concentration of 0.33 U / μL, and the mixture was incubated at 37°C for 15 minutes. The ligated RNA was crudely purified by phenol-chloroform extraction and isopropanol precipitation. The reaction sample was analyzed by dPAGE (5% acrylamide, 7M urea). Gel purification was performed as follows: After electrophoresis on dPAGE (6% acrylamide, 7.5M urea, 25% formamide), the corresponding band was excised and extracted with MQ water for 12 hours. The gel extract was obtained by Amicon 10K (Merck Millipore) treatment and isopropanol precipitation.

[0302] <Synthesis of EN, a compound with sugar moiety modification> The sequence information of the materials (polynucleotides) used in the synthesis of compound EN, which is translated into VEGF protein, is shown below. In the table, each nucleotide N represents RNA, N(M) represents 2'-O-methyl modified RNA, N(F) represents 2'-F-modified RNA, N(MOE) represents 2'-O-methoxyethyl modified RNA, and dN represents DNA.

[0303] [Table 144]

[0304] The sugar-modified compound EN was obtained by the following series of operations. (Preparation of linearized plasmid DNA and preparation of RNA fragments by in vitro transcension) The 3' fragment was prepared in the same manner as in the above RN'-2 preparation method.

[0305] (Preparation of RNA ligation products by RNA ligation) A ligation reaction using RNA ligase 2 was performed using chemically synthesized RNA fragments EN-1 and RN'-2 and template DNA-C. The final concentrations were EN-1 2 μM, RN'-2 2 μM, template DNA 4 μM, PEG8000 10% (v / v), T4 RNA ligase 2 (NEB) 1 U / μL, T4 RNA ligase 2 buffer (NEB) 1x, and RNase inhibitor Murine (NEB) 1 U / μL. After adding the substrates and reagents (except for PEG8000, T4 RNA ligase 2, and RNase inhibitor) to the tube, the mixture was heated to 90°C for 3 minutes and allowed to cool to room temperature. The remaining reagents and enzymes were added and incubated at 45°C for 1 hour. DNase (recombinant, RNase-free, Takara) was added to a final concentration of 0.33 U / μL, and the mixture was incubated at 37°C for 15 minutes. The ligated RNA was crudely purified by phenol-chloroform extraction and isopropanol precipitation. The reaction sample was analyzed by dPAGE (5% acrylamide, 7M urea). Gel purification was performed as follows: After electrophoresis on dPAGE (6% acrylamide, 7.5M urea, 25% formamide), the corresponding band was excised and extracted with MQ water for 12 hours. The gel extract was obtained by Amicon 10K (Merck Millipore) treatment and isopropanol precipitation.

[0306] (translation reaction of mRNA sample) The sequence information of RN, RN', and EN obtained above is shown in Table 145. In Table 145, each nucleotide N (uppercase) represents RNA, each nucleotide N(F) represents 2'-F modified RNA, and N(MOE) represents 2'-O-methoxyethyl modified RNA. [Table 145]

[0307] The translational activity of RN, RN', and EN was evaluated in vitro using HeLa cells. First, HeLa cells suspended in RPMI medium (Nacalai Tesque) containing 10% fetal bovine serum were seeded onto a 96-well culture plate at 10,000 cells / 100 μL per well and cultured overnight at 37°C in 5% CO2. After overnight culture, the culture supernatant was removed from the cells, and 40 μL of RPMI medium containing 10% fetal bovine serum was added per well. Each compound was diluted with Optimem (Thermo Fisher Scientific, catalog number LMRNA008) at a final concentration of 0.3% to give final compound concentrations of 0.3, 1, 3, and 10 nM. The mixture was then added to each culture plate at a volume of 10 μL per well and cultured at 37°C under 5% CO2 for 4 hours. After 4 hours of culture, the culture supernatant was collected, and 50 μL of RPMI medium containing 10% fetal bovine serum was added per well. The cells were further cultured overnight at 37°C under 5% CO2. The culture supernatant was then collected after 24 hours of culture.

[0308] The amount of VEGF protein in the culture supernatant was measured using Human VEGE Quantikine ELISA (R&D, catalog number DVE00) according to the manual provided with the kit. The VEGF protein concentration (ng / mL) in each culture supernatant is shown in the table below.

[0309] [Table 146]

[0310] As is clear from the above evaluation results, after addition of EN with glycosylation to HeLa cells, it produced the VEGF protein encoded by the gene sequence, and the efficiency was superior to that of RN and RN' without glycosylation. [Sequence List Free Text]

[0311] SEQ ID NO: 1: Compound E1 SEQ ID NO: 2: Compound E1-1 SEQ ID NO: 3: Compound E1-2 SEQ ID NO: 4: Template DNA1 SEQ ID NO: 5: Compound E2 SEQ ID NO: 6: Compound E2-1 SEQ ID NO: 7: Compound E2-2 SEQ ID NO: 8: Compound E3 SEQ ID NO: 9: Compound E3-1 SEQ ID NO: 10: Compound E3-2 SEQ ID NO: 11: Compound E4 SEQ ID NO: 12: Compound E4-1 SEQ ID NO: 13: Compound E4-2 SEQ ID NO: 14: Compound R1 SEQ ID NO: 15: Compound R1-1 SEQ ID NO: 16: Compound R1-2 SEQ ID NO: 17: Template DNA2 SEQ ID NO: 18: Compound R2 SEQ ID NO: 19: Compound R2-1 SEQ ID NO: 20: Compound R2-2 SEQ ID NO: 21: Compound R3 SEQ ID NO: 22: Compound R3-1 SEQ ID NO: 23: Compound R3-2 SEQ ID NO: 24: Template DNA3 SEQ ID NO: 25: Compound R4 SEQ ID NO: 26: Compound R4-1 SEQ ID NO: 27: Compound R4-2 SEQ ID NO: 28: Compound R4-3 SEQ ID NO: 29: Template DNA4 SEQ ID NO: 30: Compound R5 SEQ ID NO: 31: Compound R5-1 SEQ ID NO: 32: Compound R5-2 SEQ ID NO: 33: Compound R6 SEQ ID NO: 34: Compound R6-1 SEQ ID NO: 35: Compound R6-2 SEQ ID NO: 36: Compound R7 SEQ ID NO: 37: Compound R7-1 SEQ ID NO: 38: Compound R7-2 SEQ ID NO: 39: Compound R8 SEQ ID NO: 40: Compound R8-1 SEQ ID NO: 41: Compound R8-2 SEQ ID NO: 42: Compound R9 SEQ ID NO: 43: Compound R9-1 SEQ ID NO: 44: Compound R9-2 SEQ ID NO: 45: Compound R10 SEQ ID NO: 46: Compound R10-1 SEQ ID NO: 47: Compound R10-2 SEQ ID NO: 48: Compound R11 SEQ ID NO: 49: Compound R11-1 SEQ ID NO: 50: Compound R11-2 SEQ ID NO: 51: Compound R12 SEQ ID NO: 52: Compound R12-1 SEQ ID NO: 53: Compound R12-2 SEQ ID NO: 54: Compound R13 SEQ ID NO: 55: Compound R13-1 SEQ ID NO: 56: Compound R13-2 SEQ ID NO: 57: Compound R14 SEQ ID NO: 58: Compound R14-1 SEQ ID NO: 59: Compound R14-2 SEQ ID NO: 60: Compound R15 SEQ ID NO: 61: Compound R15-1 SEQ ID NO: 62: Compound R15-2 SEQ ID NO: 63: Compound R16 SEQ ID NO: 64: Compound R16-1 SEQ ID NO: 65: Compound R16-2 SEQ ID NO: 66: Compound R17 SEQ ID NO: 67: Compound R17-1 SEQ ID NO: 68: Compound R17-2 SEQ ID NO: 69: Compound R17-3 SEQ ID NO: 70: Template DNA5 SEQ ID NO: 71: Template DNA6 SEQ ID NO: 72: Compound R18 SEQ ID NO: 73: Compound R18-1 SEQ ID NO: 74: Compound R18-2 SEQ ID NO: 75: RT primer SEQ ID NO: 76: Fw primer SEQ ID NO: 77: Rv primer SEQ ID NO: 78: Taqman MGB Probe SEQ ID NOs: 79 to 538: Compounds E5 to E248-1 SEQ ID NOs: 539 to 542: Translation product polypeptide preparation SEQ ID NO: 543: FW primer SEQ ID NO: 544: RV primer 1 SEQ ID NO: 545: 5' end polynucleotide sequence RN-1 SEQ ID NO: 546: Artificial synthetic gene sequence GN SEQ ID NO: 547: Template DNA-A SEQ ID NO: 548: RV primer 2 SEQ ID NO: 549: Sequencing primer M13FW SEQ ID NO: 550: Sequencing primer M13RV SEQ ID NO: 551: 5' end polynucleotide sequence RN'-1 SEQ ID NO: 552: Artificial synthetic gene sequence GN' SEQ ID NO: 553: Template DNA-B SEQ ID NO: 554: EN-1 SEQ ID NO: 555: Template DNA-C SEQ ID NO: 556: Compounds RN and RN' SEQ ID NO: 557: Compound EN

Claims

1. It includes the translation region from the start codon to the stop codon, the translation region comprises n codons, n is a positive integer of 2 or more, each of the n codons comprises a first, second, and third nucleotide; The first nucleotide of at least two of the n codons is a nucleotide of the following formula: 【Chemistry 1】 The sugar-modified nucleotide is represented by the first, second, and third nucleotides of the stop codon are sugar-modified nucleotides; Polynucleotide.

2. 2. The polynucleotide of claim 1, wherein the sugar-modified nucleotides in the three nucleotides of the stop codon include a sugar moiety modified at at least the 2' position.

3. The sugar moieties modified at the 2'-positions of the three nucleotides of the stop codon are selected from the following: 【Chemistry 2】 The polynucleotide of claim 2.

4. the sugar-modified nucleotide comprises a base moiety corresponding to a base selected from the group consisting of adenine, guanine, cytosine, and uracil; The polynucleotide according to any one of claims 1 to 3, wherein the types of bases are at least two.

5. The polynucleotide of any one of claims 1 to 4, wherein at least one of the sugar-modified nucleotides comprises a modified base moiety.

6. The polynucleotide according to any one of claims 1 to 5, wherein the first nucleotide in all of the n codons is a sugar-modified nucleotide.

7. The polynucleotide according to any one of claims 1 to 6, wherein the first, second and third nucleotides of the initiation codon are sugar-modified nucleotides.

8. The polynucleotide according to any one of claims 1 to 7, wherein the second nucleotide of at least one codon other than a stop codon among the n codons is a sugar-modified nucleotide.

9. The polynucleotide according to any one of claims 1 to 8, wherein the third nucleotide of at least one codon other than a stop codon among the n codons is a sugar-modified nucleotide.

10. The polynucleotide according to any one of claims 1 to 9, wherein n is an integer from 2 to 2000.

11. The polynucleotide according to any one of claims 1 to 10, further comprising a 5' untranslated region.

12. the 5'-side untranslated region contains a base-modified nucleotide containing the following base: 【Transformation 3】 [wherein R is an alkyl group having 1 to 6 carbon atoms] The polynucleotide of claim 11.

13. The polynucleotide according to claim 11 or 12, wherein the first, second, and third nucleotides from the 5' end of the 5' untranslated region are sugar-modified nucleotides.

14. The polynucleotide of any one of claims 11 to 13, further comprising a 5' cap structure.

15. The polynucleotide according to any one of claims 1 to 14, further comprising a 3' untranslated region.

16. The polynucleotide of claim 15, wherein the 3' untranslated region comprises a polyA tail.

17. The polynucleotide according to claim 15 or 16, wherein the first, second, and third nucleotides from the 3' end of the 3' untranslated region are sugar-modified nucleotides.

18. The polynucleotide according to any one of claims 11 to 17, wherein the 5' untranslated region and / or the 3' untranslated region comprises sugar-modified nucleotides.

19. It includes the following structure: 【Chemistry 4】 [In the formula, R １ and R ２ are each independently H, OH, F, or OCH ３ represents B １ and B ２ each independently represents a base moiety, X １ represents O, S or NH, X ２ represents O, S, NH or the following structure: 【Transformation 5】 X ３ represents OH, SH or a salt thereof; However, X １ and X ２ is not O at the same time. A polynucleotide according to any one of claims 1 to 18.

20. The polynucleotide according to any one of claims 1 to 19, comprising a phosphorothioate structure.

21. 21. The polynucleotide of any one of claims 1 to 20, wherein the first and second nucleotides in at least one of the n codons are linked by a phosphorothioate.

22. The polynucleotide according to any one of claims 1 to 21, wherein the first to second nucleotides, the first to third nucleotides, the first to fourth nucleotides, or the first to fifth nucleotides from the 5' end of the 5' untranslated region are linked by a phosphorothioate.

23. The polynucleotide according to any one of claims 1 to 22, wherein the first to second nucleotides, the first to third nucleotides, the first to fourth nucleotides, or the first to fifth nucleotides from the 3' end of the 3' untranslated region are linked by phosphorothioate.

24. A pharmaceutical composition comprising the polynucleotide of any one of claims 1 to 23.

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