Single-stranded polynucleotide production method and modified polynucleotide

Abstract

The main objective of the inventors of the present invention was to provide new technology for purifying a single-stranded polynucleotide. The present inventors have discovered that a single-stranded polynucleotide can be purified by adding a hydrophobic tag having a specific structure to the polynucleotide. Further additional improvements have been made to arrive at the present disclosure.

Description

Method for producing single-stranded polynucleotides and modified polynucleotides

[0001] This disclosure relates to a method for producing single-stranded polynucleotides, a method for purifying single-stranded polynucleotides, modified polynucleotides, PCR primers, and compositions for producing single-stranded polynucleotides.

[0002] (α) Single-stranded polynucleotides are used in a wide range of fields, including molecular biology, diagnostic technologies, gene therapy, and nanotechnology. For example, single-stranded DNA is used as a template for homologous recombination repair in RNA ligation and gene knock-in using the CRISPR-Cas system, and is also used in the creation of DNA origami (DNA nanostructures). Furthermore, single-stranded DNA or single-stranded RNA can be used as nucleic acid aptamers.

[0003] (β) In recent years, nucleic acid drugs have attracted attention as the next generation of medicine following antibody drugs and small molecule drugs. In particular, therapies using messenger RNA (mRNA) are expected to be responsive and highly effective, and the development of mRNA drugs is progressing rapidly in the fields of mRNA vaccines and gene therapy. mRNA drugs consist of a cap structure (5' cap), a 5' untranslated region (5' UTR), a coding region (Coding Sequence, CDS), a 3' untranslated region (3' UTR), and a poly(A) tail, in order from the 5' end. In order to use mRNA as a pharmaceutical, it is important to obtain high-purity mRNA. Furthermore, structural characteristics of mRNA, such as the efficiency of capping and the length of the poly(A) tail, affect in vivo stability and translation efficiency, so there is a need for technology to purify mRNA with a desired structure to high purity. For example, Patent Document 2 describes a technology for purifying nucleotides with a desired 5' end structure by introducing a hydrophobic protecting group to the 5' end of the nucleotides.

[0004] Japanese Patent Publication No. 2023-000571, International Publication No. 2023 / 282245

[0005] Inagaki, M., Abe, N., Li, Z. et al. Cap analogs with a hydrophobic photocleavable tag enable facile purification of fully capped mRNA with various cap structures. Nat Commun 14, 2657(2023). https: / / doi.org / 10.1038 / s41467-023-38244-8

[0006] (α) Conventionally, single-stranded polynucleotides have been purified by methods such as the streptavidin-biotin method and the λ exonuclease treatment method. In the streptavidin-biotin method, double-stranded DNA is generated by PCR using biotin-labeled primers and unmodified primers, and then only the biotin-labeled strand is captured using streptavidin-immobilized beads and separated from the unmodified strand. The streptavidin-biotin method has high specificity and can produce relatively high-purity single-stranded DNA, but it requires biotin-labeled primers and streptavidin beads, making it costly and resulting in low yield. In the λ exonuclease treatment method, double-stranded DNA is generated by PCR using phosphorylated and unphosphorylated primers, and then treated with λ exonuclease, an enzyme that specifically degrades DNA strands with phosphorylated 5' ends, thereby selectively leaving the unphosphorylated strand and obtaining single-stranded DNA. In the λ exonuclease treatment method, it is necessary to remove the exonuclease after the reaction.

[0007] The inventors' primary objective was to provide a novel technology for purifying single-stranded polynucleotides.

[0008] (β) In addition, Non-Patent Document 1 uses high-performance liquid chromatography (HPLC) as a method for purifying nucleic acids. However, the chromatography supports commonly used in HPLC have small particle sizes of about 5 μm, making it difficult for the mobile phase to flow through the column. For this reason, while HPLC has high separation ability, it is time-consuming and unsuitable for processing many samples. Furthermore, HPLC equipment is expensive and large, requiring a large space for installation.

[0009] The inventors attempted to purify a desired polynucleotide using a chromatography support with a larger particle size than those commonly used in HPLC. However, the technique described in Patent Document 2 failed to purify the target polynucleotide when a chromatography support with a relatively large particle size was used.

[0010] The present inventors primarily aimed to provide a technique for purifying a target polynucleotide using a chromatography support with a relatively large particle size, as an additional or different problem from the above problem (α).

[0011] (α) The inventors have found that single-stranded polynucleotides can be purified by attaching a hydrophobic tag having a specific structure to the polynucleotide.

[0012] (β) Furthermore, the inventors have found that by attaching a hydrophobic tag having an alkyl or alkoxy group with approximately 8 to 30 carbon atoms and a specific structure to the polynucleotide, it is possible to purify the target polynucleotide even with a chromatography support having a relatively large particle size. Through further improvements, they have completed this disclosure.

[0013] This disclosure includes, for example, the following subject matter: Section 1. General formula (I): [k represents an integer greater than or equal to 1; R Y R represents a k-valent group obtained by removing k atoms or groups from a polynucleotide; R is the same or different, and the general formula is (II): (R 1 and R 2 are the same or different and each represent a hydrogen atom, an alkyl group, or an alkoxy group; R 3 to R 7 are the same or different and each represent a hydrogen atom, an alkyl group, an alkoxy group, or a nitro group (provided that at least one of R 1 to R 7 is an alkyl group having 3 to 30 carbon atoms or an alkoxy group); and R X represents a linker)], which comprises subjecting the modified polynucleotide represented by said formula to liquid chromatography. Item 2. The production method according to Item 1, further comprising a step of performing a polynucleotide synthesis reaction using the modified polynucleotide represented by General Formula (I) as a primer. Item 3. The production method according to Item 1, further comprising: a step of cleaving a plasmid with a restriction enzyme; and a step of binding a nucleotide derivative or polynucleotide derivative to which a hydrophobic tag represented by the following General Formula (VIII) is added to an exposed base at the end cleaved by said restriction enzyme, to obtain said modified polynucleotide: 1 to R 7 , wherein at least one of R Y is an alkyl group having 8 to 30 carbon atoms or an alkoxy group. The production method according to any one of Items 1 to 3, wherein: X are the same or different and each represent a single bond, -(CH2) n -, -O-(CH2) n -, or -R 8 - [wherein n represents an integer of 1 or greater; R 8 is represented by General Formula (VI): (X 1 and X 2A method of manufacture according to any one of items 1 to 6, wherein R is the same or different and represents a divalent group represented by general formula (III): (R 1 and R 2 R represents the same or different hydrogen atom, alkyl group, or alkoxy group; 4 ~R 7 These are the same or different, representing a hydrogen atom, an alkyl group, or an alkoxy group (however, R 1 , R 2 , and R 4 ~R 7 (At least one of these is an alkyl or alkoxy group having 3 to 30 carbon atoms); R X (where indicates a linker) or general formula (VII): (R 1 and R 2 R represents the same or different hydrogen atom, alkyl group, or alkoxy group; 3 , R 4 , R 6 , and R 7 These are the same or different, representing a hydrogen atom, an alkyl group, or an alkoxy group (however, R 3 , R 4 , R 6 , and R 7 (At least one of these is an alkyl or alkoxy group having 3 to 30 carbon atoms); R X The method according to any one of items 1 to 7, wherein (where represents a linker). Item 9. The method according to any one of items 1 to 8, wherein the pH of the sample subjected to the liquid chromatography is 8.0 or higher, and / or the pH of the mobile phase used in the liquid chromatography is 8.0 or higher. Item 10. The method according to any one of items 1 to 9, wherein the liquid chromatography is reversed-phase chromatography. Item 11. The method according to any one of items 1 to 10, wherein the liquid chromatography is solid-phase extraction and purification. Item 12. The method according to any one of items 1 to 11, wherein the single-stranded polynucleotide is single-stranded DNA. Item 13. General formula (I): [k represents an integer greater than or equal to 1; RY R represents a k-valent group obtained by removing k atoms or groups from a polynucleotide; R is the same or different, and the general formula is (II): (R 1 and R 2 R represents the same or different hydrogen atom, alkyl group, or alkoxy group; 3 ~R 7 These are the same or different, representing a hydrogen atom, alkyl group, alkoxy group, or nitro group (however, R 1 ~R 7 (At least one of these is an alkyl or alkoxy group having 3 to 30 carbon atoms); R X (where indicates a linker) is represented as, where R is R Y A modified polynucleotide to which at least one nucleotide located 1 to 10 bases from the end is attached. Item 14. In the modified polynucleotide, R X They are the same or different, single bond, -(CH2) n -, -O-(CH2) n -, or -R 8 -[In the formula, n represents an integer greater than or equal to 1; R 8 The general formula is (VI): (X 1 and X 2 A modified polynucleotide as described in item 13, where R is the same or different, and represents a divalent group represented by O or S. Item 15. R is the same or different, and represents general formula (III): (R 1 and R 2 R represents the same or different hydrogen atom, alkyl group, or alkoxy group; 4 ~R 7 These are the same or different, representing a hydrogen atom, an alkyl group, or an alkoxy group (however, R 1 , R 2 , and R 4 ~R 7 (At least one of these is an alkyl or alkoxy group having 3 to 30 carbon atoms); R X (where indicates a linker) or general formula (VII): (R 1 and R 2R represents the same or different hydrogen atom, alkyl group, or alkoxy group; 3 , R 4 , R 6 , and R 7 These are the same or different, representing a hydrogen atom, an alkyl group, or an alkoxy group (however, R 3 , R 4 , R 6 , and R 7 (At least one of these is an alkyl or alkoxy group having 3 to 30 carbon atoms); R X A modified polynucleotide as described in item 13 or 14, wherein (where is a linker). Item 16. A primer comprising a modified polynucleotide as described in any of items 13 to 15. Item 17. A composition for the production of a single-stranded polynucleotide comprising the primer described in item 16. Item 18. The composition according to item 17, wherein the single-stranded polynucleotide is single-stranded DNA. Item 19. General formula (I): [k represents an integer greater than or equal to 1; R Y R represents a k-valent group obtained by removing k atoms or groups from a polynucleotide; R is the same or different, and the general formula is (II): (R 1 and R 2 R represents the same or different hydrogen atom, alkyl group, or alkoxy group; 3 ~R 7 These are the same or different, representing a hydrogen atom, alkyl group, alkoxy group, or nitro group (however, R 1 ~R 7 (At least one of these is an alkyl or alkoxy group having 3 to 30 carbon atoms); R X A method for purifying a single-stranded polynucleotide, comprising the step of subjecting a modified polynucleotide represented by [where R represents a linker] to liquid chromatography. Item 20. The purification method according to item 19, further comprising the step of carrying out a polynucleotide synthesis reaction using the modified polynucleotide represented by the general formula (I) as a primer. Item 21. In the modified polynucleotide, R is R Y Purification method according to item 19 or 20, wherein at least one nucleotide located 1 to 30 bases from the end of is bound. Item 22. In the modified polynucleotide, RX are the same or different, and each is a single bond, -(CH2) n -, -O-(CH2) n -, or -R 8 -[wherein n represents an integer of 1 or greater; R 8 is a divalent group represented by general formula (VI): (X 1 and X 2 are the same or different, and each is O or S), the purification method according to any one of Items 19 to 21. Item 23. In the modified polynucleotide, R are the same or different, and each is general formula (III): (R 1 and R 2 are the same or different, and each represents a hydrogen atom, an alkyl group, or an alkoxy group; R 4 to R 7 are the same or different, and each represents a hydrogen atom, an alkyl group, or an alkoxy group (provided that at least one of R 1 , R 2 , and R 4 to R 7 is an alkyl group having 3 to 30 carbon atoms or an alkoxy group having 3 to 30 carbon atoms); R X represents a linker), or is general formula (VII): (R 1 and R 2 are the same or different, and each represents a hydrogen atom, an alkyl group, or an alkoxy group; R 3 , R 4 , R 6 , and R 7 are the same or different, and each represents a hydrogen atom, an alkyl group, or an alkoxy group (provided that at least one of R 3 , R 4 , R 6 , and R 7 is an alkyl group having 3 to 30 carbon atoms or an alkoxy group having 3 to 30 carbon atoms); R XA purification method according to any one of items 19 to 22, wherein (where represents a linker). Item 24. A purification method according to any one of items 19 to 23, wherein the pH of the sample subjected to the liquid chromatography is 8.0 or higher, and / or the pH of the mobile phase used in the liquid chromatography is 8.0 or higher. Item 25. A purification method according to any one of items 19 to 24, wherein the liquid chromatography is reversed-phase chromatography. Item 26. A purification method according to any one of items 19 to 25, wherein the liquid chromatography is solid-phase extraction purification. Item 27. A purification method according to any one of items 19 to 26, wherein the single-stranded polynucleotide is single-stranded DNA. Item 28. General formula (I): [k represents an integer greater than or equal to 1; R Y R represents a k-valent group obtained by removing k atoms or groups from a polynucleotide; R is the same or different, and the general formula is (II): (R 1 and R 2 R represents the same or different hydrogen atom, alkyl group, or alkoxy group; 3 ~R 7 These are the same or different, representing a hydrogen atom, alkyl group, alkoxy group, or nitro group (however, R 1 ~R 7 (At least one of these is an alkyl or alkoxy group having 8 to 30 carbon atoms); R X A composition comprising a modified polynucleotide represented by (where represents a linker), used to adsorb the modified polynucleotide onto a chromatography support with a particle size of 10 to 150 μm. Item 29. In the modified polynucleotide, R is R Y The composition according to claim 28, wherein at least one nucleotide located 1 to 30 bases from the end of is bonded. Claim 30. In the modified polynucleotide, R X They are the same or different, single bond, -O-(CH2) n -, or - (CH2) n- (where n is an integer greater than or equal to 1) The composition according to item 28 or 29. Item 31. The composition according to any one of items 28 to 30, wherein the chromatography support is a reversed-phase chromatography support. Item 32. The composition according to any one of items 28 to 31, wherein the polynucleotide is mRNA. Item 33. General formula (I): [k represents an integer greater than or equal to 1; R Y R represents a k-valent group obtained by removing k atoms or groups from a polynucleotide; R is the same or different, and the general formula is (II): (R 1 and R 2 R represents the same or different hydrogen atom, alkyl group, or alkoxy group; 3 ~R 7 These are the same or different, representing a hydrogen atom, alkyl group, alkoxy group, or nitro group (however, R 1 ~R 7 (At least one of these is an alkyl or alkoxy group having 8 to 30 carbon atoms); R X A complex comprising a modified polynucleotide represented by (where represents a linker) and a chromatography support with a particle size of 10 to 150 μm, wherein the modified polynucleotide is adsorbed onto the chromatography support. Item 34. In the modified polynucleotide, R is R Y The complex according to item 33, wherein at least one nucleotide located 1 to 30 bases from the end of is bound. Item 35. In the modified polynucleotide, R X They are the same or different, single bond, -O-(CH2) n -, or - (CH2) n - (where n is an integer greater than or equal to 1), the complex according to item 33 or 34. Item 36. The complex according to any one of items 33 to 35, wherein the chromatography support is a reversed-phase chromatography support. Item 37. The complex according to any one of items 33 to 36, wherein the polynucleotide is mRNA. Item 38. General formula (I): [k represents an integer greater than or equal to 1; R Y R represents a k-valent group obtained by removing k atoms or groups from a polynucleotide; R is the same or different, and the general formula is (II): (R 1 and R 2 R represents the same or different hydrogen atom, alkyl group, or alkoxy group; 3 ~R 7 These are the same or different, representing a hydrogen atom, alkyl group, alkoxy group, or nitro group (however, R 1 ~R 7 (At least one of these is an alkyl or alkoxy group having 8 to 30 carbon atoms); R X A method for producing a polynucleotide, comprising the step of adsorbing a modified polynucleotide represented by (where represents a linker) onto a chromatography support with a particle size of 10 to 150 μm. Item 39. In the modified polynucleotide, R is R Y The method for producing the modified polynucleotide described in item 38, wherein at least one nucleotide located 1 to 30 bases from the end of is bound to R X They are the same or different, single bond, -O-(CH2) n -, or - (CH2) n A method for producing a product according to item 38 or 39, wherein n is an integer greater than or equal to 1. Item 41. A method for producing a product according to any one of items 38 to 40, wherein the chromatography support is a reversed-phase chromatography support. Item 42. A method for producing a product according to any one of items 38 to 41, further comprising the step of performing solid-phase extraction. Item 43. A method for producing a product according to any one of items 38 to 42, wherein the polynucleotide is mRNA.

[0014] This disclosure provides a novel technology for purifying single-stranded polynucleotides.

[0015] Example A1-1: A schematic diagram of the prepared forward primer is shown. R represents a t-butyl group. Examples A1-1 and A1-2-1: The sequences of the prepared primers are shown. * in the forward primer represents a hydrophobic tag enclosed in a rectangle in Figure 1 or Figure 17. Example A1-2: The sequences of the sense strand and antisense strand of the double-stranded DNA amplified by PCR are shown. Example A1-2: The results of analysis of the PCR product by agarose gel electrophoresis are shown. From left to right, a 500 bp DNA ladder marker, the PCR product, and a sample of the PCR product irradiated with ultraviolet light were applied, respectively. Example A1-3: The results of HPLC are shown. Example A1-3: The results of agarose gel electrophoresis are shown. From left to right, the lanes are: a 500 bp DNA ladder marker, double-stranded DNA (dsDNA) prepared in steps 1-2, single-stranded DNA (ssDNA) prepared separately from this test, a sample prepared by isolating the peak with a retention time of 13.629 minutes by HPLC, and a sample prepared by isolating the peak with a retention time of 12.880 minutes by HPLC. Example A1-4: HPLC results are shown. Example A1-4: Agarose gel electrophoresis results are shown. From left to right, the lanes are: a 500 bp DNA ladder marker, double-stranded DNA (dsDNA) prepared in steps 1-2, single-stranded DNA (ssDNA) prepared separately from this test, a sample prepared by isolating the peak with a retention time of 13.156 minutes by HPLC, and a sample prepared by isolating the peak with a retention time of 13.621 minutes by HPLC. Example A1-5: HPLC results are shown. Example A1-5: Agarose gel electrophoresis results are shown. From left to right, the lanes are: a 500 bp DNA ladder marker, double-stranded DNA (dsDNA) prepared in steps 1-2, single-stranded DNA (ssDNA) prepared separately from this test, a sample prepared by isolating the peak with a retention time of 13.134 minutes by HPLC, and a sample prepared by isolating the peak with a retention time of 13.612 minutes by HPLC. Example A1-6: Shows the HPLC results. Example A1-7: Shows the agarose gel electrophoresis results. In the figure, Exonuclease I(-) represents a sample that was not treated with exonuclease I, and Exonuclease I(+) represents a sample that was treated with exonuclease I.Example A1-8-1: HPLC results are shown. Example A1-8-1: Agarose gel electrophoresis results are shown. From left to right, the lanes are: 500 bp DNA ladder marker, double-stranded DNA (dsDNA) prepared in 1-2, single-stranded DNA (ssDNA) prepared separately from this test, a sample prepared by isolating the peak with a retention time of 13.301 minutes by HPLC, and a sample prepared by isolating the peak with a retention time of 13.420 minutes by HPLC. Example A1-8-2: HPLC results are shown. Example A1-8-2: Agarose gel electrophoresis results are shown. From left to right, the lanes are: 500 bp DNA ladder marker, double-stranded DNA (dsDNA) prepared in 1-2, single-stranded DNA (ssDNA) prepared separately from this test, and a sample prepared by isolating the peak with a retention time of 14.297 minutes by HPLC. Example A2-1: A schematic diagram of the prepared forward primer is shown. In the diagram, R represents a linear alkyl group with 19 carbon atoms (C. 19 H 39) represents. Example A2-2: Results of agarose gel electrophoresis are shown. From left to right, a 500 bp DNA ladder marker, a PCR product, and a sample of the PCR product irradiated with ultraviolet light were applied. Example A2-3: Results of agarose gel electrophoresis are shown. A 500 bp DNA ladder marker was applied to lane 1, double-stranded DNA with a hydrophobic tag (dsDNA) prepared in 2-2 was applied to lane 2, single-stranded DNA with a hydrophobic tag (ssDNA) prepared separately from this test was applied to lane 3, single-stranded DNA from which the hydrophobic tag was removed by ultraviolet light irradiation was applied to lane 4, and double-stranded DNA from which the hydrophobic tag was removed by ultraviolet light irradiation was applied to lane 5. The flow-through fraction (FT) was applied to lane 6. For lanes 7-16, "wash" represents the washing buffer, "Elu" represents each elution buffer, the "-1" at the end represents the fraction eluted when the buffer was loaded into the column for the first time, and "-2" represents the fraction eluted when the buffer was loaded into the column for the second time. Example A2-3: Shows the HPLC results. Example A3-1: Shows (A) Decyl-dATP and (B) Nb-dATP as examples of the prepared dNTPs. R1 is a linear alkyl group with 10 carbon atoms (C 10 H 21 ), R2 is a linear alkyl group with 11 carbon atoms (C 11 H 23(This shows) Example A3-2: This shows the insertion results using various DNA polymerases in primer extension experiments. (A) shows the complex of a primer labeled with fluorescein (FAM) at 5' and a DNA template, and the incorporation of Decyl-dATP and NB-dATP into the Counter base indicated by N was confirmed. (B) shows the incorporation of Decyl-dATP or Nb-dATP into the various Counter bases T, A, C, and G using two types of DNA polymerase (Klenow Fragment 3'->5' exo-, Taq polymerase 5'->3' exo-). Example A3-3: (A) Overview of Decyl-dATP incorporation into plasmid digests, and (B) Confirmation of BsaI digestion by agarose gel electrophoresis. Example A3-4: HPLC analysis results. Each chart shows, from top to bottom, plasmid digest, 30-minute extension product, 2-hour extension product, and 12-hour extension product, with the percentage of products incorporating Decyl-dATP shown in red. Example B2-1: UPLC analysis results for each reaction product are shown. The top shows the UPLC analysis results of the reaction product when using the tBuNb modified chemical capping reagent. The bottom shows the UPLC analysis results of the reaction product when using the C11Nb modified chemical capping reagent. Example B2-3-1: Results of LC-MS analysis of the concentrated sample eluted with 20% acetonitrile / 0.1M TEAA buffer in Example B2-2-1 are shown. The top shows the obtained UPLC profile. The bottom shows the MS analysis results of each UPLC peak. Example B2-3-2: The results of LC-MS analysis of samples eluted with 20%, 40%, and 60% acetonitrile / 0.1M TEAA buffer and concentrated in Example B2-2-2 are shown. From top to bottom on the left, the ULC profiles of the samples eluted with 20%, 40%, and 60% acetonitrile / 0.1M TEAA buffer and concentrated are shown. On the right, the MS analysis results of the ULC peak derived from C11Nb-modified capped mRNA eluted with 40% acetonitrile / 0.1M TEAA buffer are shown. Example B2-4: An overview of this test is shown.Example B2-4: The translational activity of HiBiT A, HiBiT B, and HiBiT C was evaluated using a luciferase assay with the Nano-Glo® Luciferase Assay System (Promega). Example B3-1: The structures of each chemically capped mRNA are shown. Example B3-2: The results of denatured polyacrylamide electrophoresis (dPAGE) are shown. The numbers at the top of the lanes correspond to the 11 types of mRNA shown in Figure 30. Example B3-2: The HiBiT-Cap2 RNA samples obtained in Example B3-1 were eluted with 20, 40, and 60% acetonitrile / 0.1M TEAA buffer in cartridge purification, and the concentrated samples were analyzed by LCMS. Example B3-2: The HiBiT-Cap2-r-fm RNA sample obtained in Example B3-1 was eluted with 20, 40, and 60% acetonitrile / 0.1M TEAA buffer in cartridge purification, and the concentrated sample was analyzed by LCMS. The results of LCMS analysis of the concentrated sample are shown. Example B3-2: The HiBiT-Cap2-fr-r RNA sample obtained in Example B3-1 was eluted with 20, 40, and 60% acetonitrile / 0.1M TEAA buffer in cartridge purification, and the concentrated sample was analyzed by LCMS. The results of LCMS analysis of the concentrated sample are shown. Example B3-2: The HiBiT-Cap2-fr-m RNA sample obtained in Example B3-1 was eluted with 20, 40, and 60% acetonitrile / 0.1M TEAA buffer in cartridge purification, and the concentrated sample was analyzed by LCMS. Example B3-2: The HiBiT-Cap2-fr-fm RNA sample obtained in Example B3-1 was eluted with 20, 40, and 60% acetonitrile / 0.1M TEAA buffer in cartridge purification, and the concentrated sample was analyzed by LCMS. The results of LCMS analysis of the concentrated sample are shown.Example B3-2: The HiBiT-Cap2-fm-m RNA sample obtained in Example B3-1 was eluted with 20, 40, and 60% acetonitrile / 0.1M TEAA buffer in cartridge purification, and the concentrated sample was analyzed by LCMS. The results of LCMS analysis of the concentrated sample are shown. Example B3-2: The HiBiT-Cap2-fm-fm RNA sample obtained in Example B3-1 was eluted with 20, 40, and 60% acetonitrile / 0.1M TEAA buffer in cartridge purification, and the concentrated sample was analyzed by LCMS. The results of LCMS analysis of the concentrated sample are shown. Example B3-2: The HiBiT-mr RNA sample obtained in Example B3-1 was eluted with 20, 40, and 60% acetonitrile / 0.1M TEAA buffer in cartridge purification, and the concentrated sample was analyzed by LCMS. Example B3-2: The HiBiT-mm RNA sample obtained in Example B3-1 was eluted with 20, 40, and 60% acetonitrile / 0.1M TEAA buffer in cartridge purification, and the concentrated sample was analyzed by LCMS. Example B3-2: The HiBiT-m-fm RNA sample obtained in Example B3-1 was eluted with 20, 40, and 60% acetonitrile / 0.1M TEAA buffer in cartridge purification, and the concentrated sample was analyzed by LCMS. Example B5-2: The fraction obtained from the eluate of 20% acetonitrile in cartridge purification of the reaction product of 5'-phosphorylated RNA and 2'-O-4-n-butylbenzyl-N7-methylguanosine 5'-O-diphosphate imidazolide was analyzed using liquid-phase chromatography. Example B5-2: This shows the results of analyzing fractions obtained by eluting 20%, 40%, and 60% acetonitrile during cartridge purification of the reaction product of 5'-phosphorylated RNA and 2'-O-(4-(dodecyloxymethyl)benzyl)-N7-methylguanosine 5'-O-diphosphate imidazolide using liquid-phase chromatography. Example B5-3: This shows an overview of the test.Example B5-3: The translational activity of RNA without a cap structure (Cap(-)) and synthetic mRNA with Cap2 and C12OM-Bn modifications was evaluated using a luciferase assay with the Nano-Glo® Luciferase Assay System (Promega). Example B6-3: The results of LC-MS analysis of samples eluted with 20%, 40%, and 60% ACN / TEAA and concentrated are shown. From top to bottom on the left, the UPLC profiles of samples eluted with 20%, 40%, and 60% ACN / TEAA and concentrated are shown. On the right, the MS analysis results of the UPLC peaks of samples eluted with 40% and 60% ACN / TEAA are shown. Example B6-3: The dPAGE results are shown. Lane 1 was loaded with a sample eluted with 20% ACN / TEAA, lane 2 with a sample eluted with 40% ACN / TEAA, and lane 3 with a sample eluted with 60% ACN / TEAA. Example B6-4: The results of LC-MS analysis of samples eluted with 20%, 40%, and 60% ACN / TEAA and concentrated are shown. From top to bottom on the left, the UPLC profiles of the samples eluted with 20%, 40%, and 60% ACN / TEAA and concentrated are shown. On the right, the MS analysis results of the UPLC peaks of the samples eluted with 40% and 60% ACN / TEAA are shown. Example B6-4: The dPAGE results are shown. The sample eluted with 40% ACN / TEAA was loaded into lane 1, and the sample eluted with 60% ACN / TEAA was loaded into lane 2. Example B7-8: The results of LC-MS analysis of the capping reaction product obtained in 7-7 are shown. Example B7-8: The results of LC-MS analysis of each eluted fraction after cartridge purification are shown.

[0016] The embodiments included in this disclosure will be described in more detail below. This disclosure preferably includes, but is not limited to, methods for producing single-stranded polynucleotides, modified polynucleotides with hydrophobic tags, PCR primers, compositions for producing single-stranded polynucleotides, etc., and encompasses everything disclosed herein and recognizable to those skilled in the art. This disclosure also preferably includes modified polynucleotides with hydrophobic tags, compositions containing such modified polynucleotides, and methods for producing such modified polynucleotides, etc.

[0017] 1. Definitions In this disclosure, “polynucleotide” and “nucleic acid” are used interchangeably and refer to polymers of two or more nucleotides of any length. The term “polynucleotide” also includes “polynucleotide derivatives.” That is, the term “polynucleotide” in this disclosure includes polynucleotides containing nucleotide derivatives; polynucleotides having unusual nucleotide bonding; and polynucleotides containing nucleotide derivatives and having unusual nucleotide bonding. In this disclosure, polynucleotides may be linear, branched, or cyclic.

[0018] Specific examples of polynucleotide derivatives include those that have undergone arbitrary chemical modifications. To prevent degradation by hydrolytic enzymes such as nucleases, the phosphate residues of each nucleotide can be replaced with chemically modified phosphate residues such as phosphorothioates (PS), methylphosphonates, or phosphorodithionates. The hydroxyl group at position 2 of the sugar (ribose) may also be replaced with an -OR (where R represents, for example, CH3 (2'-O-Me), CH2CH2OCH3 (2'-O-MOE), CH2CH2NHC(NH)NH2, CH2CONHCH3, CH2CH2CN, etc.). Furthermore, the base portion (pyrimidine, purine) may be chemically modified, for example, by introducing a methyl group or cationic functional group at position 5 of the pyrimidine base, or by substituting the carbonyl group at position 2 with a thiocarbonyl group. In addition, the phosphate or hydroxyl portion may be modified with, for example, biotin, amino groups, lower alkylamine groups, acetyl groups, etc., but is not limited to these examples. Furthermore, BNA (LNA), in which the conformation of the sugar portion of a nucleotide is fixed to the N-type by cross-linking the 2' oxygen and 4' carbon atoms of the sugar portion, can also be used.

[0019] Specific examples of nucleic acid bases that make up nucleotides include not only typical bases in DNA and RNA (adenine (A), uracil (U), guanine (G), cytosine (C), thymine (T), etc.), but also other bases, such as hypoxanthine (I) and modified bases. Modified bases include, for example, pseudouracil, 3-methyluracil, dihydrouracil, 5-alkylcytosine (e.g., 5-methylcytosine), 5-alkyluracil (e.g., 5-ethyluracil), 5-halouracil (5-bromouracil), 6-azapyrimidine, 6-alkylpyrimidine (6-methyluracil), 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, 1-methyladenine, 1-methylhypoxanthine Examples include santhine, 2,2-dimethylguanine, 3-methylcytosine, 2-methyladenine, 2-methylguanine, N6-methyladenine, 7-methylguanine, 5-methoxyaminomethyl-2-thiouracil, 5-methylaminomethyluracil, 5-methylcarbonylmethyluracil, 5-methyloxyuracil, 5-methyl-2-thiouracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid, 2-thiocytosine, purines, 2-aminopurines, isoguanine, indole, imidazole, xanthine, etc.

[0020] The polynucleotide may be either DNA or RNA, but is preferably DNA. The polynucleotide may be single-stranded or double-stranded, but is preferably single-stranded. While not particularly limited, in this disclosure, the polynucleotide is particularly preferably single-stranded DNA.

[0021] Alternatively, although not particularly limited, in one embodiment, the polynucleotide is preferably RNA, and particularly preferably mRNA, from the viewpoint of the high need for the technology of this disclosure and the ability to obtain a product with superior functionality through the technology of this disclosure. In this disclosure, mRNA means RNA that includes, in order from the 5' end, a cap structure (5' cap), a 5' untranslated region (5' UTR), a coding region (Coding Sequence, CDS), a 3' untranslated region (3' UTR), and a polyA chain.

[0022] Natural mRNA typically contains 7-methylguanosine (m 7 G) has a 5' cap (m) attached to the 5' terminal base (N) via three phosphate groups (p). 7 It has GpppN, Cap 0). In the art of this disclosure, in addition to Cap 0, in addition to the structure of Cap 0, the first base (N) is 2'-O-methylated Cap 1 (m 7 In addition to the structure of Cap 1 (GpppNm), Cap 2 (m) also has a 2'-O-methylated second base (N). 7 Other natural 5' caps such as GpppNmNm, and ARCA (Anti-Reverse Cap Analog, 3'-O-Me-m) in which the 3'-O position of guanosine is methylated. 7 Synthetic capped analogs such as GpppG are also included in the 5' cap category.

[0023] The method for preparing the polynucleotides used in this disclosure is not particularly limited, and conventionally known methods or methods easily conceivable from conventionally known methods can be employed. For example, they may be chemically synthesized using a commercially available automated nucleic acid synthesizer, enzymatically synthesized using a template polynucleotide encoding a desired sequence and a polynucleotide synthase, or multiple polynucleotides may be linked together. Alternatively, a hydrophobic tag may be attached to a nucleotide triphosphate, which is a substrate of polymerase, and the tag may be incorporated into the polynucleotide. Examples of polynucleotide synthases include DNA polymerase and RNA polymerase. Furthermore, (mono or poly)nucleotide derivatives may be used as raw materials or substrates in the synthesis, and modifications may be performed after the polynucleotide has been synthesized.

[0024] 2. Modified Polynucleotides in this Disclosure The modified polynucleotides included in this disclosure are defined by general formula (I): [k represents an integer greater than or equal to 1; R Y R represents a k-valent group obtained by removing k atoms or groups from a polynucleotide; R is the same or different, and the general formula is (II): (R 1 and R 2 R represents the same or different hydrogen atom, alkyl group, or alkoxy group; 3 ~R 7 These are the same or different, representing a hydrogen atom, alkyl group, alkoxy group, or nitro group (however, R 1 ~R 7 (At least one of these is an alkyl or alkoxy group having 3 to 30 carbon atoms); R X It has a structure represented by (where represents a linker). Hereinafter, the modified polynucleotide included in this disclosure may be referred to as the "modified polynucleotide of this disclosure". Furthermore, the structure of the above general formula (II) excluding the linker may be referred to as the "hydrophobic tag" in this disclosure. That is, the following general formula (VIII): The structure represented by this may be referred to as a "hydrophobic tag" in this disclosure.

[0025] There are no particular restrictions, but R may be the same or different, as in general formula (III): (R 1 and R 2 R represents the same or different hydrogen atom, alkyl group, or alkoxy group; 4 ~R 7 These are the same or different, representing a hydrogen atom, an alkyl group, or an alkoxy group (however, R 1 , R 2 , and R 4 ~R 7 (At least one of these is an alkyl or alkoxy group having 3 to 30 carbon atoms); R X A structure represented by (where indicates a linker), or general formula (VII): (R 1 and R 2 R represents the same or different hydrogen atom, alkyl group, or alkoxy group; 3 , R 4 , R 6 , and R 7 These are the same or different, representing a hydrogen atom, an alkyl group, or an alkoxy group (however, R 3 , R 4 , R 6 , and R 7 (At least one of these is an alkyl or alkoxy group having 3 to 30 carbon atoms); R X It is preferable to have a structure represented by (where indicates a linker).

[0026] In this disclosure, k is an integer of 1 or more and is not particularly limited insofar as the effects of this disclosure are achieved. k may be between 1 and 100, between 1 and 50, preferably between 1 and 20, more preferably between 1 and 10, and particularly preferably between 1 and 5. The upper or lower limit of the range may be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100.

[0027] The alkyl group may be linear or branched. The number of carbon atoms in the alkyl group is not particularly limited; for example, it may be 1 to 30 carbon atoms, preferably 1 to 20, more preferably 1 to 10, and particularly preferably 1 to 5. The upper or lower limit of the above range may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30.

[0028] Examples of alkyl groups include methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, tert-butyl group, sec-butyl group, n-pentyl group, neopentyl group, n-hexyl group, 3-methylpentyl group, n-heptyl group, n-octyl group, 2-ethylhexyl group, n-nonyl group, n-decyl group, n-undecyl group, n-dodecyl group, n-tridecyl group, n-tetradecyl group, n-pentadecyl group, n-hexadecyl group, n-heptadecyl group, n-octadecyl group, n-nonadecyl group, and n-eicosanyl group.

[0029] The alkoxy group may be linear or branched. The number of carbon atoms in the alkoxy group is not particularly limited; for example, it may be 1 to 30 carbon atoms, preferably 1 to 20, more preferably 1 to 10, and particularly preferably 1 to 5. The upper or lower limit of the above range may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30.

[0030] Examples of alkoxy groups include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, n-pentyloxy, neopentyloxy, n-hexyloxy, 2-ethylhexyloxy, n-heptyloxy, n-octyloxy, n-nonyloxy, n-decyloxy, n-undecyloxy, n-dodecyloxy, n-tridecyloxy, n-tetradecyloxy, n-pentadecyloxy, n-hexadecyloxy, n-heptadecyloxy, n-octadecyloxy, n-nonadecyloxy, and n-icosanyloxy.

[0031] However, R 1 ~R 7 at least one of (preferably R 1 or R 2 ) is an alkyl or alkoxy group having 3 to 30 carbon atoms, preferably an alkyl or alkoxy group having 3 to 25 carbon atoms, more preferably an alkyl or alkoxy group having 3 to 20 carbon atoms, even more preferably an alkyl or alkoxy group having 4 to 19 carbon atoms, and particularly preferably an alkyl group having 4 to 19 carbon atoms. The upper or lower limit of the above range may be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30.

[0032] Also, although not particularly limited, in one manner, R 1 ~R 7 At least one of these groups may be an alkyl or alkoxy group having 8 to 30 carbon atoms, preferably an alkyl or alkoxy group having 9 to 25 carbon atoms, more preferably an alkyl or alkoxy group having 10 to 20 carbon atoms, and even more preferably an alkyl or alkoxy group having 11 to 19 carbon atoms. The upper or lower limit of the range may be 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30.

[0033] While not particularly limited, the technology of this disclosure is R 3 ~R 7 R 3 or R 5 It is preferable that the first is a nitro group, and the others are hydrogen atoms, alkyl groups, or alkoxy groups, R 3 It is more preferable that the first is a nitro group, and the others are hydrogen atoms, alkyl groups, or alkoxy groups. 3 It is even more preferable that one of the atoms is a nitro group and the others are hydrogen atoms. 3 If the group is a nitro group, the hydrophobic tag has an o-nitrobenzyl skeleton. 5 When the group is a nitro group, the hydrophobic tag has a p-nitrobenzyl skeleton. In this disclosure, the structure represented by the following general formula (IV) is referred to as the o-nitrobenzyl skeleton, and the structure represented by the general formula (V) is referred to as the p-nitrobenzyl skeleton. In this disclosure, the part having the o-nitrobenzyl skeleton or the p-nitrobenzyl skeleton may be referred to as the "nitrobenzyl part".

[0034] General formula (IV): General formula (V): [In the formula, R' represents any substituent other than a nitro group, or a hydrogen atom.]

[0035] In the technology of this disclosure, if the hydrophobic tag has an o-nitrobenzyl skeleton, the hydrophobic tag can be detached by light irradiation or reduction treatment. On the other hand, if the hydrophobic tag has a p-nitrobenzyl skeleton, the hydrophobic tag will not be detached by light irradiation, but it can be detached by reduction treatment.

[0036] R X R represents a linker, and its structure is not particularly limited as long as the desired effect is obtained. X For example, the structure is a single bond, -(CH2) n -, -O-CH2-, -O-(CH2) n -, -OC(=O)-, -C(=O)-, -R 8 -, -O-(CH2) n -R 8 -, -OC (=O)- (CH2) n -R 8-[In the formula, n represents an integer greater than or equal to 1; R 8 The general formula is (VI): (X 1 and X 2 Examples include: R, which represents a divalent group (which is the same or different, and is either O or S). X This is a single bond, -(CH2) n -, -O-(CH2) n -, or -R 8 - is preferred, -R 8 - is more preferable, -R 8 - and X 1 and X 2 It is even more preferable that n is O. n may be, for example, 1 to 10, preferably 1 to 5, more preferably 1 to 3, and even more preferably 1 or 2.

[0037] R Y R represents a k-valent group obtained by removing k atoms or groups from a polynucleotide. As stated above, the term "polynucleotide" in this disclosure includes "polynucleotide derivatives". Therefore, R Y This may be a k-valent group obtained by removing k atoms or groups from a polynucleotide derivative. Examples of the polynucleotide derivative include those that have been alkylated, such as by methylation.

[0038] Examples of atoms or groups removed from polynucleotides include phosphate groups, hydroxyl groups or hydrogen atoms constituting phosphate groups, hydroxyl groups or hydrogen atoms in the sugar portion, and amino groups or hydrogen atoms in nucleic acid bases.

[0039] There are no particular restrictions, but R is R Y It is preferable that it is bound to at least one nucleotide located 1 to 30 bases from the end. In other words, in the art of the present disclosure, it is preferable that the modified polynucleotide has at least one hydrophobic tag located 1 to 30 bases from the end. The end may be a 5' end or a 3' end, but it is preferable that it be a 5' end. From the viewpoint of improving the purification efficiency of single-stranded polynucleotides, in the modified polynucleotide of the present disclosure, R is R YIt is more preferable that the molecule is bonded to at least one nucleotide located 1 to 20 bases from the end (preferably the 5' end), even more preferable that it is bonded to at least one nucleotide located 1 to 10 bases, and particularly preferable that it is bonded to at least one nucleotide located 1 to 5 bases. The upper or lower limit of the range may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30.

[0040] In this disclosure, the length of the modified polynucleotide is not particularly limited insofar as the effects of this disclosure are achieved. For example, it may be 5 to 1,000,000 base pairs long, or 10 to 500,000 base pairs long, preferably 20 to 100,000 base pairs long, more preferably 50 to 50,000 base pairs long, and particularly preferably 100 to 10,000 base pairs long. The upper or lower limits of the above range are 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 1 The base lengths may be 0000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 200000, 300000, 400000, 500000, 600000, 700000, 800000, 900000, or 1000000 bases.

[0041] 3. Uses The modified polynucleotides of this disclosure may be used to produce single-stranded polynucleotides. A composition containing the modified polynucleotides of this disclosure and used to produce single-stranded polynucleotides may be referred to as the “Composition of this Disclosure.” While not particularly limited, the single-stranded polynucleotide is preferably single-stranded DNA.

[0042] While not particularly limited, the modified polynucleotides of this disclosure can also be used as primers. In this disclosure, a primer is a polynucleotide that serves as the starting point for a polynucleotide synthesis reaction by a nucleic acid polymerase (e.g., DNA polymerase, RNA polymerase, etc.), and is not particularly limited to that extent. PCR primers are particularly preferred as primers. The length of the primer is not particularly limited as long as the desired effect is achieved, and may be 10 to 300 nucleotides long, preferably 10 to 200 nucleotides long, more preferably 15 to 150 nucleotides long, even more preferably 20 to 100 nucleotides long, and particularly preferably 20 to 55 nucleotides long. The upper or lower limits of the above range may be 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 90, 100, 150, 200, 250, or 300 nucleotides long.

[0043] Polynucleotide synthesis reaction products (e.g., PCR products) obtained using the modified polynucleotides of this disclosure as primers also have the structure of the modified polynucleotides of this disclosure. That is, the compositions of this disclosure include both compositions containing primers having the structure of the modified polynucleotides of this disclosure, and compositions containing polynucleotide synthesis reaction products (e.g., PCR products) having the structure of the modified polynucleotides of this disclosure. The polynucleotide synthesis reaction product may be single-stranded or double-stranded. Specifically, immediately after the polynucleotide synthesis reaction, the polynucleotide synthesis reaction product is usually a double-stranded modified polynucleotide having the structure of the general formula (I) above. However, if a step is performed to dissociate the double-stranded polynucleotide into a single-stranded polynucleotide after the polynucleotide synthesis reaction, the polynucleotide synthesis reaction product may be a single-stranded modified polynucleotide having the structure of the general formula (I) above. Polynucleotide synthesis reaction products having the structure of the modified polynucleotides of this disclosure can be subjected to liquid chromatography as described later.

[0044] 4. Liquid Chromatography In the art of this disclosure, the modified polynucleotides of this disclosure may be subjected to liquid chromatography. Liquid chromatography is a type of chromatography in which the mobile phase is a liquid. In the art of this disclosure, single-stranded polynucleotides having a desired sequence can be separated by liquid chromatography. The modified polynucleotides of this disclosure subjected to liquid chromatography may be single-stranded or double-stranded.

[0045] Although not particularly limited, liquid chromatography in the techniques of this disclosure may be solid-phase extraction purification. Solid-phase extraction purification is a method of purification in which a target substance in a sample is selectively adsorbed onto a solid phase, and then eluted using a suitable solvent.

[0046] The modified polynucleotides of this disclosure, subjected to liquid chromatography, may be adsorbed onto a chromatography support, although this is not particularly limited. Here, "chromatography support" means a solid-phase material used to adsorb and separate target substances in chromatography. Specific examples of chromatography supports include column packing materials. "Adsorption onto a chromatography support" means that a substance contained in the mobile phase interacts with binding sites on the surface of the chromatography support and is retained on the support. Examples of such interactions include hydrophobic interactions, ionic bonds, hydrogen bonds, and specific ligand binding.

[0047] In the technology disclosed herein, the material of the chromatography support is not particularly limited, and for example, porous silica gel, polymer resin, ceramic, etc. may be used. Furthermore, functional groups such as acidic, basic, hydrophilic, or hydrophobic groups may be introduced to the surface of the chromatography support, or ligands or metal ions may be immobilized on it.

[0048] While not particularly limited, in the technology of this disclosure, liquid chromatography is preferably reversed-phase chromatography (i.e., the chromatography support is a reversed-phase chromatography support). Reverse-phase chromatography is a separation method that uses a hydrophobic stationary phase (support) and a highly polar mobile phase. Specific examples of reversed-phase chromatography supports include styrenedivinylbenzene-based supports and silica-based supports in which ethyl groups (C2), butyl groups (C4), octyl groups (C8), octadecyl groups (C18), triacontyl groups (C30), phenyl groups, or cyano groups are bonded to the surface of silica gel. While not particularly limited, in the technology of this disclosure, the reversed-phase chromatography support is preferably a styrenedivinylbenzene-based support or a silica-based support in which octyl groups (C8), octadecyl groups (C18), or triacontyl groups (C30) are introduced to the surface of silica gel particles, and more preferably a styrenedivinylbenzene-based support or octadecylsilyl group-modified silica gel.

[0049] In the technology of this disclosure, the particle size of the chromatography support is not particularly limited and may be, for example, 1 to 150 μm. Here, particle size means the average of the maximum diameters of individual particles. Generally, particle size can be measured by laser diffraction scattering, dynamic light scattering, scanning electron microscopy (SEM) image analysis, or sieving tests. In particular, in this disclosure, particle size means the average (i.e., number mean) of the maximum diameters of individual particles measured by sieving tests for 100 randomly selected particles.

[0050] The particle size of the chromatography support of this disclosure may be, for example, 1 to 150 μm, preferably 5 to 150 μm, more preferably 10 to 120 μm, even more preferably 15 to 100 μm, particularly preferably 20 to 80 μm, and most preferably 25 to 50 μm. The upper or lower limit of the above range may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 μm.

[0051] Alternatively, the particle size of the chromatography carrier of this disclosure is preferably 1 to 20 μm, more preferably 1 to 15 μm, and even more preferably 5 to 10 μm. The upper or lower limit of the range may be 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 μm.

[0052] Alternatively, although not particularly limited, in one embodiment the particle size of the chromatography support may be 10 to 150 μm, preferably 15 to 120 μm, more preferably 20 to 100 μm, even more preferably 25 to 80 μm, and particularly preferably 30 to 50 μm. The upper or lower limits of the above range may be 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 μm.

[0053] Generally, the particle size of chromatography supports used in HPLC is around 3 to 8 μm. The particle size of chromatography supports used in ultra-high-performance liquid chromatography (ULC) is around 2 μm or less. Chromatography supports with a particle size of around 10 to 150 μm are used in solid-phase extraction and purification, such as cartridge purification using disposable columns.

[0054] The specific liquid chromatography method used in the technology of this disclosure is not particularly limited and may include, for example, HPLC, ULC, flash chromatography, and solid-phase extraction purification. However, while HPLC and ULC have high separation capabilities, the mobile phase does not flow easily, resulting in longer processing times and making them not necessarily suitable for processing multiple samples. Therefore, when the particle size of the chromatography support in this disclosure is about 10 to 150 μm, solid-phase extraction purification, such as cartridge purification using a disposable column, is preferred from the viewpoint of efficiently processing multiple samples.

[0055] While not particularly limited, in the technology of this disclosure, the chromatography support preferably consists of particles with a particle size of 10 to 150 μm, accounting for 80% or more (number ratio) of the total when filled in a container; more preferably consisting of particles with a particle size of 10 to 150 μm, even more preferably consisting of particles with a particle size of 10 to 150 μm, and particularly preferably consisting of particles with a particle size of 10 to 150 μm, accounting for 90% or more (number ratio).

[0056] In the art of the present disclosure, the pore size of the chromatography support is not particularly limited as long as the desired effect is obtained. For example, it may be 5 to 100 nm, preferably 8 to 80 nm, and more preferably 10 to 50 nm. Alternatively, in one embodiment, the pore size of the chromatography support may be 5 to 50 nm, preferably 8 to 30 nm, and more preferably 10 to 15 nm. The upper or lower limit of the above range may be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 nm. The present disclosure includes a composition comprising the modified polynucleotide of the present disclosure, used for adsorbing the modified polynucleotide onto the above-described chromatography support (preferably a chromatography support with a particle size of 10 to 150 μm). Furthermore, this disclosure includes a complex comprising the modified polynucleotide of this disclosure and the above-mentioned chromatography carrier (preferably a chromatography carrier with a particle size of 10 to 150 μm), wherein the modified polynucleotide is adsorbed onto the chromatography carrier.

[0057] In the techniques of this disclosure, the method for adsorbing the modified polynucleotide onto a chromatography support is not particularly limited, as long as the desired effect is obtained. For example, the modified polynucleotide may be adsorbed onto the chromatography support by flowing or adding a sample solution containing the modified polynucleotide to a column packed with the chromatography support (column method). Alternatively, the modified polynucleotide may be adsorbed onto the chromatography support by mixing a sample solution containing the modified polynucleotide with the chromatography support and allowing it to stand or be stirred for a certain period of time (batch method).

[0058] The modified polynucleotides of this disclosure can be adsorbed onto a chromatography support by interacting with functional groups on the surface of the chromatography support via a hydrophobic tag. It is preferable that the modified polynucleotides of this disclosure are adsorbed onto a chromatography support by interacting with hydrophobic functional groups on the surface of the chromatography support via a hydrophobic tag. It is more preferable that the modified polynucleotides of this disclosure are adsorbed onto a chromatography support by interacting with alkyl groups on the surface of alkyl (e.g., butyl, octyl, or octadecyl)silyl group-modified silica gel via a hydrophobic tag. It is even more preferable that the modified polynucleotides of this disclosure are adsorbed onto a chromatography support by interacting with octadecyl groups on the surface of octadecylsilyl group-modified silica gel via a hydrophobic tag.

[0059] In the technology of this disclosure, the chromatography support can be packed into a container of any shape. Specific examples of container shapes include cylindrical, syringe, spin column, disk, and well plate types. While not particularly limited, the container in which the chromatography support is packed in the technology of this disclosure is preferably cylindrical, syringe, or spin column type. These containers come in various capacities, and by selecting a container with a larger capacity than a typical HPLC or UPLC column, a larger quantity of single-stranded polynucleotides can be purified more efficiently. Also, while not particularly limited, the container in which the chromatography support is packed in the technology of this disclosure may be made of metal, glass, or resin, and is preferably made of resin.

[0060] In the technology disclosed herein, the method of liquid passage is not particularly limited. For example, liquid passage can be performed using any of the following methods: pressurization, suction, centrifugal, or gravity-feed. Pressurization refers to a method in which a sample solution or eluate is passed into a container by applying external pressure to the upstream side of the container filled with the support. Specifically, this can be done by manually applying pressure using a syringe or by using a pump. Note that HPLC and ULC also use pressurization to pass liquid, but when using chromatography supports with a particle size of about 10 to 150 μm, liquid can be passed at a much lower pressure (for example, 10 bar or less, preferably 5 bar or less) than HPLC and ULC. Suction refers to a method in which a sample solution or eluate is passed through a container filled with the support by applying negative pressure to the downstream side of the container. Specifically, this can be done by using a suction manifold and applying negative pressure to the downstream side of the column with a vacuum pump. The centrifugal method involves setting a container filled with a carrier (e.g., a spin column) in a centrifuge and using centrifugal force to pass the sample solution or eluent through it. The gravity-fed method, on the other hand, uses gravity to allow the sample solution or eluent to pass through it naturally.

[0061] While not particularly limited, a washing step may be optionally performed in the technology of this disclosure. Here, the washing step refers to the step of removing impurities present in a container such as a column after the target substance has been adsorbed onto the chromatography support. The composition, pH, etc., of the solution used in the washing step are not particularly limited as long as the desired effect is obtained, and can be appropriately selected by those skilled in the art. Specific washing solutions include, for example, water, a buffer, or any aqueous solution (preferably an aqueous solution of tetraethylammonium hydroxide (TEA-OH)) containing 0-10% organic solvent (preferably acetonitrile). The concentration of the organic solvent is preferably 2-8%, more preferably 3-7%, and even more preferably 4-6%. In this disclosure, unless otherwise specified, % in the context of a solution means v / v% of the entire solution.

[0062] The pH of the washing solution may be, for example, pH 3.0 to 14.0, preferably pH 8.0 to 14.0, more preferably pH 9.0 to 14.0, even more preferably pH 10.0 to 13.0, and particularly preferably pH 11.0 to 13.0. The upper or lower limit of the above range may be 3.0, 4.0, 5.0, 6.0, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, or 14.0.

[0063] In the technology disclosed herein, the elution method, the composition of the elution solution, pH, etc., are not particularly limited as long as the desired effect is obtained, and can be appropriately selected by those skilled in the art. For example, at least one organic solvent selected from the group consisting of acetonitrile, methanol, ethanol, isopropanol, n-hexane, toluene, ethyl acetate, dichloromethane, and tetrahydrofuran, a mixed solution of at least one organic solvent selected from the group and water (water-acetonitrile, water-methanol, etc.), or a mixed solution of at least one organic solvent selected from the group and any buffer or aqueous solution may be used for elution. Furthermore, the elution operation may be performed in multiple steps, using an elution solution of a different composition each time. In addition, gradient elution may be performed by continuously changing the water / organic solvent ratio. Specific examples of elution solutions include water, a buffer, or any aqueous solution (preferably an aqueous solution of tetraethylammonium hydroxide (TEA-OH)) containing 5-90% organic solvent (preferably acetonitrile). The concentration of the organic solvent is preferably 5 to 70%, more preferably 10 to 50%, even more preferably 15 to 40%, and particularly preferably 15 to 30%.

[0064] In the technology of this disclosure, although not particularly limited, the pH of the elution solution is preferably 8.0 or higher, more preferably 9.0 or higher, even more preferably 10.0 or higher, and particularly preferably 11.0 or higher. The pH of the elution solution may also be, for example, pH 3.0 to 14.0, preferably pH 8.0 to 14.0, more preferably pH 9.0 to 14.0, even more preferably pH 10.0 to 13.0, and particularly preferably pH 11.0 to 13.0. The upper or lower limit of the above range may be 3.0, 4.0, 5.0, 6.0, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, or 14.0. A higher pH of the elution solution allows for more stable dissociation of single-stranded polynucleotides while efficiently producing them.

[0065] In the technologies of this disclosure, although not particularly limited, the pH of the chromatographic mobile phase is preferably 8.0 or higher, more preferably 9.0 or higher, even more preferably 10.0 or higher, and particularly preferably 11.0 or higher. The pH of the mobile phase may also be, for example, pH 3.0 to 14.0, preferably pH 8.0 to 14.0, more preferably pH 9.0 to 14.0, even more preferably pH 10.0 to 13.0, and particularly preferably pH 11.0 to 13.0. The upper or lower limits of the above range may be 3.0, 4.0, 5.0, 6.0, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, or 14.0. A higher pH of the mobile phase in chromatography allows for more stable dissociation of single-stranded polynucleotides while efficiently producing them.

[0066] While not particularly limited, the chromatographic mobile phase in the art of this disclosure may contain a denaturing agent. Specific examples of denaturing agents include formamide and urea. When the chromatographic mobile phase contains a denaturing agent, single-stranded polynucleotides can be produced efficiently while maintaining a more stable dissociated state of the single-stranded polynucleotides.

[0067] In the art of this disclosure, although not particularly limited, the pH of the sample subjected to chromatography is preferably 8.0 or higher, more preferably 9.0 or higher, even more preferably 10.0 or higher, and particularly preferably 11.0 or higher. Furthermore, the pH of the sample subjected to chromatography may be, for example, pH 3.0 to 14.0, preferably pH 8.0 to 14.0, more preferably pH 9.0 to 14.0, even more preferably pH 10.0 to 13.0, and particularly preferably pH 11.0 to 13.0. The upper or lower limits of the above range may be 3.0, 4.0, 5.0, 6.0, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, or 14.0. A higher pH of the sample subjected to chromatography allows for more stable dissociation of single-stranded polynucleotides while efficiently producing them.

[0068] 5. Detachment of Hydrophobic Tags While not particularly limited, the technology of this disclosure may include a step for detaching the hydrophobic tag. As described above, in the technology of this disclosure, if the hydrophobic tag has an o-nitrobenzyl skeleton, the hydrophobic tag can be detached by light irradiation or reduction treatment. On the other hand, if the hydrophobic tag has a p-nitrobenzyl skeleton, the hydrophobic tag will not be detached by light irradiation, but it can be detached by reduction treatment.

[0069] The conditions for light irradiation are not particularly limited as long as the hydrophobic tag can be detached, and can be set appropriately according to the properties of the hydrophobic tag, etc. For example, light with a wavelength of 300 nm to 400 nm at 0.5 to 10 mW / cm². 2 You may irradiate it with this light intensity for about 1 to 60 minutes.

[0070] The conditions for the reduction treatment are not particularly limited as long as the hydrophobic tag can be removed, and can be set appropriately according to the properties of the hydrophobic tag. For example, incubation may be performed at 20 to 45°C in a 1 to 1000 mM sodium dithionite solution for 10 to 60 minutes.

[0071] Furthermore, modified polynucleotides from which the hydrophobic tag has been removed exhibit decreased hydrophobicity (i.e., increased hydrophilicity), and this can be used to confirm the removal of the hydrophobic tag. For example, if the retention time in reverse-phase HPLC is shorter for a sample after light irradiation or reduction treatment than for a sample before such treatment, it can be concluded that the hydrophobic tag has been removed.

[0072] 6. Method of Production of the Disclosure The Disclosure also includes a method of production of single-stranded polynucleotides, which includes the step of subjecting the modified polynucleotides of the Disclosure to liquid chromatography. This method of production may be referred to as the "Method of Production of the Disclosure." The matters described with respect to the modified polynucleotides of the Disclosure and the compositions of the Disclosure may be appropriately incorporated into the Method of Production of the Disclosure. Furthermore, the matters described with respect to the Method of Production of the Disclosure may be appropriately incorporated into the modified polynucleotides of the Disclosure and the compositions of the Disclosure.

[0073] While not particularly limited, the manufacturing method of the Disclosure may further include a step of carrying out a polynucleotide synthesis reaction using the modified polynucleotide of the Disclosure as a primer. PCR is particularly preferred as the polynucleotide synthesis reaction. The polynucleotide synthesis reaction product (e.g., PCR product) obtained by the polynucleotide synthesis reaction step is preferably subjected to liquid chromatography.

[0074] Furthermore, both the primer and the polynucleotide synthesis reaction product may have the structure of the modified polynucleotide of this disclosure. Specifically, the primer may be a single-stranded modified polynucleotide having the structure of general formula (I) and a base length sufficient to function as a primer. The polynucleotide synthesis reaction product may, immediately after the polynucleotide synthesis reaction, be a double-stranded modified polynucleotide having the structure of general formula (I). However, if a step is taken to dissociate the double-stranded polynucleotide into a single-stranded polynucleotide after the polynucleotide synthesis reaction, the polynucleotide synthesis reaction product may be a single-stranded modified polynucleotide having the structure of general formula (I).

[0075] The manufacturing method of the present disclosure is not particularly limited, but includes the steps of cleaving a plasmid with a restriction enzyme, and applying the following general formula (VIII) to the exposed bases at the cleavage ends by the restriction enzyme: The process may further include the step of attaching a nucleotide derivative or polynucleotide derivative to which a hydrophobic tag represented by is attached, in order to obtain the modified polynucleotide.

[0076] Specific examples of nucleotide derivatives to which the hydrophobic tag is attached include, for example, nucleoside triphosphates (NTPs) or deoxynucleoside triphosphates (dNTPs) to which the hydrophobic tag is attached. Here, the base (N) contained in the NTP or dNTP may be, for example, A, T, G, or C. Furthermore, the position to which the hydrophobic tag is attached in the NTP or dNTP is not particularly limited, but it is preferably the hydroxyl group at the 3' position of the sugar moiety. Specific examples of polynucleotide derivatives to which the hydrophobic tag is attached include, for example, polynucleotides containing a nucleotide to which the hydrophobic tag is attached at the terminal or internally (preferably at the terminal) (for example, polynucleotides into which a hydrophobic tag-attached NTP or dNTP is incorporated).

[0077] The method for preparing the plasmid to be used in the cleavage step is not particularly limited. For example, it may be prepared by transforming host cells such as E. coli into plasmids, culturing them, and then extracting and purifying them using the alkaline SDS method or a commercially available plasmid purification kit.

[0078] The type of restriction enzyme is not particularly limited as long as the desired effect is obtained, and can be appropriately selected by those skilled in the art depending on the sequence of the target modified polynucleotide.

[0079] The method for attaching a hydrophobic tagged nucleotide derivative or polynucleotide derivative is not particularly limited. For example, a method can be employed in which the nucleotide derivative is covalently bonded to the bases (overhanging or blunt ends) exposed at the cleavage ends of a polynucleotide cleaved by restriction enzymes using an enzymatic reaction. Specifically, (i) a method in which a hydrophobic tagged nucleotide derivative or polynucleotide derivative is incorporated using a DNA polymerase (e.g., Klenow fragment, Taq DNA polymerase, etc.) using the cleavage ends as a template, and the ends are extended; (ii) a method in which the ends of the derivative are designed to contain a base sequence complementary to the sequence of the cleavage ends, and after hybridizing the two by annealing, a phosphodiester bond is formed using a DNA ligase (e.g., T4 DNA ligase); or (iii) a method in which a hydrophobic tagged nucleotide derivative or polynucleotide derivative is attached to the 3' end using a terminal transferase.

[0080] While not particularly limited, the manufacturing method of the present disclosure may further include a step of dissociating double-stranded polynucleotides into single-stranded polynucleotides. The specific method for dissociating double-stranded polynucleotides into single-stranded polynucleotides is not particularly limited as long as the desired effect is obtained. For example, double-stranded polynucleotides may be dissociated into single-stranded polynucleotides by at least one method selected from the group consisting of thermal denaturation, alkaline denaturation, chemical denaturation, and enzymatic treatment. While not particularly limited, in the art of the present disclosure, it is preferable to dissociate double-stranded polynucleotides into single-stranded polynucleotides by alkaline denaturation or chemical denaturation.

[0081] In addition, in thermal denaturation, double-stranded polynucleotides can be dissociated into single-stranded polynucleotides by heating them to a high temperature (for example, 80°C or higher, preferably 85°C or higher, more preferably 90°C or higher, and even more preferably 95°C or higher). In alkaline denaturation, double-stranded polynucleotides can be dissociated into single-stranded polynucleotides by exposing them to a high pH (for example, pH 8.0 or higher, preferably pH 9.0 or higher, more preferably pH 10.0 or higher, even more preferably pH 11.0 or higher, and particularly preferably pH 12.0 or higher). The type of alkali used to raise the pH is not particularly limited, and for example, sodium hydroxide or potassium hydroxide can be used. In chemical denaturation, double-stranded polynucleotides can be dissociated into single-stranded polynucleotides by treating them with a denaturing agent such as formamide or urea. In enzymatic treatment, double-stranded polynucleotides can be dissociated into single-stranded polynucleotides by treating them with an enzyme such as λ exonuclease or DNA helicase.

[0082] The step of dissociating double-stranded polynucleotides into single-stranded polynucleotides can be performed at any time as long as the desired effect is obtained. For example, it may be performed before subjecting the modified polynucleotide of this disclosure to liquid chromatography, or it may be performed in parallel with the liquid chromatography. In other words, the modified polynucleotide of this disclosure subjected to liquid chromatography may be single-stranded or double-stranded. If the step of dissociating double-stranded polynucleotides into single-stranded polynucleotides is performed before subjecting the modified polynucleotide of this disclosure to liquid chromatography, for example, an alkali (preferably sodium hydroxide and / or potassium hydroxide) and / or a denaturant (preferably formamide) may be mixed with the sample containing the modified polynucleotide of this disclosure. If the step of dissociating double-stranded polynucleotides into single-stranded polynucleotides is performed in parallel with the liquid chromatography, for example, a mobile phase containing an alkali and / or a denaturant may be used in the liquid chromatography.

[0083] 7. Purification Method of the Disclosure The Disclosure also includes a method for purifying single-stranded polynucleotides, which includes the step of subjecting the modified polynucleotides of the Disclosure to liquid chromatography. This manufacturing method may be referred to as the "Purification Method of the Disclosure." The information described with respect to the modified polynucleotides of the Disclosure, the compositions of the Disclosure, and the manufacturing methods of the Disclosure shall be incorporated into the Purification Method of the Disclosure as appropriate.

[0084] In this specification, the term “comprising” includes not only “containing” but also “essentially consisting of” and “consisting of.” Furthermore, this disclosure encompasses all combinations of the constituent elements described herein.

[0085] Furthermore, the various characteristics (properties, numerical values, structure, function, etc.) described for each embodiment of this disclosure described above may be combined in any way to identify the subject matter covered by this disclosure. In other words, this disclosure covers all subject matter consisting of any combination of the combinable characteristics described herein.

[0086] The embodiments of this disclosure will be described in more detail below with examples, but the embodiments of this disclosure are not limited to the examples below.

[0087] <Example A>

[0088] 1. Purification of single-stranded DNA using hydrophobic tags with t-butyl groups 1-1. Preparation of PCR primers A schematic diagram of the prepared forward primer is shown in Figure 1. In Figure 1, R represents a t-butyl group. The sequences of the prepared primers are shown in Figure 2. In the forward primer in Figure 2, * represents the hydrophobic tag enclosed in a rectangle in Figure 1.

[0089] Primers were synthesized using the DNA / RNA synthesizer NR-2A_7MX or NRs-4A_10R7NP (Nippon Techno Service). DNA amidite and CPG (Chemgenes; deoxyguanosine (n-ibu) 3'-ICAa CPG, 1000) ÅA TV (47.3 μmol / g) was used. In the case of forward primers with hydrophobic tags, the DNA was further coupled with a chemical phosphorylation reagent (CPR) containing the hydrophobic tag after DNA synthesis.

[0090] Amidite and CPR were dissolved in dehydrated acetonitrile and prepared to 50 mM and 100 mM, respectively. The reagents used in the synthesis apparatus were as follows: 10 (w / v)% dichloromethane (Wako, 135-02441) for deprotection, 0.25 M 5-benzylthio-1H-tetrazole in acetonitrile (Wako) for coupling, a mixture of acetic anhydride / tetrahydrofuran / pyridine (1:8:1, Wako) and 10 (v / v)% 1-methylimidazole in tetrahydrofuran (Wako) for capping, and 0.01 M iodine (Honeywell) in 64% acetonitrile, 6% pyridine, and 30% water for oxidation.

[0091] After DNA synthesis, the DNA was cleaved from the support and deprotected overnight at room temperature with a 1:1 mixture (1 mL) of 40% methylamine aqueous solution and 33% methylamine ethanol solution, or allowed to stand for 30 minutes at 65 °C with a 1:1 mixture (1 mL) of 40% methylamine aqueous solution and 30% ammonia water. The supernatant was filtered and dried under reduced pressure. The DNA was purified by reverse-phase HPLC. The purification conditions were as follows: Column: YMC Triart Bio C4, 250 × 10 mm ID, S-5 μm, 30 nm; Solution A: 50 mM TEAA (pH 7.0 + 5% acetonitrile); Solution B: 100% acetonitrile; Gradient of Solution B: 0-100%; Column temperature: 50 °C; Flow rate: 1 mL / min; Detection wavelength: 260 nm.

[0092] After purification, the sample was added to 3 M sodium acetate (pH 5.2) and 2-propanol and allowed to stand at -30 °C for 1 hour. Then, it was centrifuged at 15000 rpm for 30 minutes, the supernatant was removed, and the pellet was washed with 80% ethanol. The pellet was then dissolved in Milli-Q water, and the DNA concentration was measured using NanoDrop2000 (Thermo Fisher Scientific). The DNA concentration was determined from the absorbance at 260 nm. Purity was confirmed by denatured polyacrylamide gel electrophoresis (PAGE) using 7.5 M urea as the denaturant. The ratio of acrylamide to N,N'-methylenebisacrylamide was 19:1. After electrophoresis, the gel was stained with SYBR Green II Nucleic Acid Gel Stain (Lonza) and examined using the ChemiDoc XRS Plus system (Bio-Rad).

[0093] 1-2. Preparation of double-stranded DNA with t-butyl hydrophobic tag Double-stranded DNA was prepared by PCR using the Nano luc plasmid (pNL1.1TK, promega) as a template and PCR primers prepared in 1-1.

[0094] The PCR reaction mixture was prepared with the following composition: 0.3 μM each of primers, 1 ng / μL of pNL1.pNL1.1TK vector, 0.2 mM of dNTPs, 1.5 mM of MgSO4, 1× PCR buffer for KOD-Plus-Neo, and 0.02 units / μL of KOD-Plus-Neo (Toyobo). The reaction was carried out under the following thermal cycling conditions: 94 °C, 2 min – (98 °C, 10 sec – 55 °C, 30 sec – 72 °C, 36 sec) × 40 cycles – 72 °C, 3 min. The sequences of the sense and antisense strands of the double-stranded DNA amplified by this PCR are shown in Figure 3. The results of agarose gel electrophoresis analysis of the PCR product are shown in Figure 4. In Figure 4, from left to right, the 500 bp DNA ladder marker, the PCR product, and the PCR product irradiated with ultraviolet light were applied, respectively.

[0095] 1-3. Purification of single-stranded DNA using reversed-phase HPLC (Triart Bio C18 column) An equal volume of 1 M sodium hydroxide aqueous solution was added to the double-stranded DNA sample with hydrophobic tags prepared in 1-2, and the mixture was gently mixed. The mixed sample was analyzed and the peak portion was separated using reversed-phase HPLC. The analytical conditions are shown below. Note that the YMC Triart Bio C18 is a column packed with an organosilica hybrid support having the functional group C18.

[0096] <Analysis Conditions> Column used: YMC Triart Bio C18 (Pore size: 30 nm, Particle size: 5 μm, Column internal dimensions: 250 × 4.6 mm I.D.) Analysis conditions: 0-60%, 1 mL / min, 20 minutes Analysis temperature: 30 °C Mobile phase A: 10 mM NaOH (pH 12) Mobile phase B: 10 mM NaOH (pH 12) + 50% acetonitrile

[0097] Each sample was neutralized with 1 M hydrochloric acid. Then, an equal volume of isopropanol and 1 / 10 volume of 3 M sodium acetate aqueous solution were added, and the mixture was allowed to stand at -20 °C for 1 hour. The samples were then centrifuged at 15,000 rpm for 30 minutes. After centrifugation, the supernatant was removed, and 1.5 equivalents of 80% ethanol were added by volume, and the mixture was centrifuged again at 15,000 rpm for 5 minutes. The supernatant was removed, and the mixture was air-dried to remove the ethanol. Milli-Q water was added to dissolve the pellet, and then agarose gel electrophoresis was performed.

[0098] The HPLC results are shown in Figure 5. Peaks were observed at 12.880 minutes and 13.629 minutes, respectively. The agarose gel electrophoresis results are shown in Figure 6. From left to right in Figure 6, the lanes are: 500 bp DNA ladder marker, double-stranded DNA (dsDNA) prepared in steps 1-2, single-stranded DNA (ssDNA) prepared separately from this test, a sample prepared by isolating the peak at 13.629 minutes by HPLC, and a sample prepared by isolating the peak at 12.880 minutes by HPLC. Single-stranded DNA has approximately half the molecular weight of double-stranded DNA, but comparing the bands in the second and third lanes from the left, the band for single-stranded DNA is slightly higher. This is thought to be because not only molecular weight but also DNA structure affected electrophoretic mobility.

[0099] As shown in Figure 6, both the sample with a retention time of 12.880 minutes and the sample with a retention time of 13.629 minutes were suggested to be single-stranded DNA. Furthermore, since a hydrophobic tag was attached only to the forward primer during PCR, the peak with the longer retention time of 13.629 minutes in reverse-phase HPLC is presumed to be single-stranded DNA of the sense strand, which has a more hydrophobic feel and to which the hydrophobic tag has been attached.

[0100] 1-4. Purification of single-stranded DNA using reversed-phase HPLC (Agilent PLRP-S) An equal volume of 1 M sodium hydroxide aqueous solution was added to the double-stranded DNA sample with hydrophobic tags prepared in 1-2, and the mixture was gently mixed. The mixed sample was analyzed and the peak portion was separated using reversed-phase HPLC. The analytical conditions are shown below. Note that Agilent PLRP-S is a column packed with a styrene-divinylbenzene-based support.

[0101] <Analysis Conditions> Column used: Agilent PLRP-S (Pore size: 4000 Å, Particle size: 8 μm, Column internal dimensions: 150 × 4.6 mm I.D.) Analysis conditions: 0-30%, 1 mL / min, 20 minutes Analysis temperature: 30 °C Mobile phase A: 10 mM TEA-OH (pH 12) Mobile phase B: 10 mM TEA-OH (pH 12) + 80% acetonitrile

[0102] Each sample was neutralized with 1 M hydrochloric acid. Then, an equal volume of isopropanol and 1 / 10 of a 3 M sodium acetate aqueous solution were added, and the mixture was allowed to stand at -20 °C for 1 hour. The samples were then centrifuged at 15,000 rpm for 30 minutes. After centrifugation, the supernatant was removed, and 1.5 equivalents of 80% ethanol were added by volume, and the mixture was centrifuged again at 15,000 rpm for 5 minutes. The supernatant was removed, and the mixture was air-dried to remove the ethanol. Milli-Q water was added to dissolve the pellet, and then agarose gel electrophoresis was performed.

[0103] The HPLC results are shown in Figure 7. Peaks were observed at 13.156 minutes and 13.621 minutes, respectively. The agarose gel electrophoresis results are shown in Figure 8. From left to right in Figure 8, the lanes are: a 500 bp DNA ladder marker, double-stranded DNA (dsDNA) prepared in steps 1-2, single-stranded DNA (ssDNA) prepared separately from this test, a sample prepared by isolating the peak at 13.156 minutes by HPLC, and a sample prepared by isolating the peak at 13.621 minutes by HPLC. As shown in Figure 8, both the sample with a retention time of 13.156 minutes and the sample with a retention time of 13.621 minutes were suggested to be single-stranded DNA. The peak at 13.621 minutes, which has a longer retention time in reverse-phase HPLC, is presumed to be single-stranded DNA of the sense strand to which a hydrophobic tag has been attached.

[0104] 1-5. Purification of single-stranded DNA using reverse-phase HPLC (Agilent PLRP-S) The double-stranded DNA sample with hydrophobic tags prepared in 1-2 was analyzed and peak regions were separated using reverse-phase HPLC. The analytical conditions are shown below. Unlike in 1-4, in this test, the step of adding NaOH to the sample before chromatography was not performed.

[0105] <Analysis Conditions> Column used: Agilent PLRP-S (Pore size: 4000 Å, Particle size: 8 μm, Column internal dimensions: 150 × 4.6 mm I.D.) Analysis conditions: 0-30%, 1 mL / min, 20 minutes Analysis temperature: 30 °C Mobile phase A: 10 mM TEA-OH (pH 12) Mobile phase B: 10 mM TEA-OH (pH 12) + 80% acetonitrile

[0106] Each sample was neutralized with 1 M hydrochloric acid. Then, an equal volume of isopropanol and 1 / 10 of a 3 M sodium acetate aqueous solution were added, and the mixture was allowed to stand at -20 °C for 1 hour. The samples were then centrifuged at 15,000 rpm for 30 minutes. After centrifugation, the supernatant was removed, and 1.5 equivalents of 80% ethanol were added by volume, and the mixture was centrifuged again at 15,000 rpm for 5 minutes. The supernatant was removed, and the mixture was air-dried to remove the ethanol. Milli-Q water was added to dissolve the pellet, and then agarose gel electrophoresis was performed.

[0107] The HPLC results are shown in Figure 9. Peaks were observed at 13.134 minutes and 13.612 minutes, respectively. The agarose gel electrophoresis results are shown in Figure 10. From left to right in Figure 10, the lanes are: a 500 bp DNA ladder marker, double-stranded DNA (dsDNA) prepared in steps 1-2, single-stranded DNA (ssDNA) prepared separately from this test, a sample prepared by isolating the peak at 13.134 minutes by HPLC, and a sample prepared by isolating the peak at 13.612 minutes by HPLC. As shown in Figure 10, both the sample with a retention time of 13.134 minutes and the sample with a retention time of 13.612 minutes were suggested to be single-stranded DNA. The peak at 13.612 minutes, which had a longer retention time in reverse-phase HPLC, is presumed to be single-stranded DNA of the sense strand to which a hydrophobic tag has been attached.

[0108] 1-6. Removal of hydrophobic tags by ultraviolet light irradiation A portion of the sample collected in 1-4 was irradiated with 4 mW / cm² 2 The samples were irradiated with ultraviolet light at 365 nm for 10 minutes and analyzed by reverse-phase HPLC. The analytical conditions are as follows.

[0109] <Analysis Conditions> Column used: Agilent PLRP-S (Pore size: 4000 Å, Particle size: 8 μm, Column internal dimensions: 150 × 4.6 mm I.D.) Analysis conditions: 0-30%, 1 mL / min, 20 minutes Analysis temperature: 30 °C Mobile phase A: 10 mM TEA-OH (pH 12) Mobile phase B: 10 mM TEA-OH (pH 12) + 80% acetonitrile

[0110] The results are shown in Figure 11. The sample prepared by extracting the peak with a retention time of 13.156 minutes in 1-4 had a retention time of 13.025 minutes before UV irradiation and a retention time of 13.007 minutes after UV irradiation. In other words, the retention time remained almost unchanged before and after UV irradiation. On the other hand, the sample prepared by extracting the peak with a retention time of 13.612 minutes in 1-4 had a retention time of 13.479 minutes before UV irradiation and a retention time of 13.002 minutes after UV irradiation. In other words, the retention time was significantly shortened by UV irradiation.

[0111] These results suggest that single-stranded DNA with a hydrophobic tag has a longer retention time in reverse-phase chromatography than single-stranded DNA without a hydrophobic tag, allowing it to be separated from the other strand, and that the hydrophobic tag can be removed by ultraviolet light irradiation after purification of the single-stranded DNA.

[0112] 1-7. Exonuclease I Treatment Double-stranded DNA (dsDNA) prepared in 1-2, single-stranded DNA (ssDNA) prepared separately from this test, a sample prepared by isolating the peak with a retention time of 13.156 minutes in 1-4, and a sample prepared by isolating the peak with a retention time of 13.612 minutes in 1-4 and then irradiating with ultraviolet light were each treated with exonuclease I. Specifically, 50 ng DNA, 10× Exonuclease I Reaction Buffer, and Exonuclease I were mixed and reacted at 37°C for 15 minutes to perform exonuclease I treatment. After the reaction, the enzyme was inactivated by heating at 80°C for 15 minutes. Subsequently, each sample was analyzed by agarose gel electrophoresis.

[0113] The results are shown in Figure 12. In the figure, Exonuclease I(-) represents samples that were not treated with exonuclease I, and Exonuclease I(+) represents samples that were treated with exonuclease I. Since the band that was present in Exonuclease I(-) disappeared in Exonuclease I(+), it was confirmed that both the sample with a retention time of 13.156 minutes in 1-4 and the sample with a retention time of 13.612 minutes in 1-4 were single-stranded DNA.

[0114] 1-8. Purification of single-stranded DNA under neutral conditions. Unlike the reverse-phase HPLC performed in 1-3 to 1-6, reverse-phase HPLC was performed using a neutral mobile phase. The analytical conditions are shown below.

[0115] <Analysis Conditions> Column used: Agilent PLRP-S (Pore size: 4000 Å, Particle size: 8 μm, Column internal dimensions: 150 × 4.6 mm I.D.) Analysis conditions: 0-30%, 1 mL / min, 20 minutes Analysis temperature: 30 °C Mobile phase A: 10 mM TEA-OH (pH 7) Mobile phase B: 10 mM TEA-OH (pH 7) + 80% acetonitrile

[0116] 1-8-1. Alkali treatment: An equal volume of 1 M sodium hydroxide aqueous solution was added to the double-stranded DNA sample with hydrophobic tags prepared in 1-2, and the mixture was gently mixed. The mixed sample was analyzed by reverse-phase HPLC under the above conditions, and the peak regions were separated. An equal volume of isopropanol and 1 / 10 the volume of 3 M sodium acetate aqueous solution were added to each separated sample, and the mixture was allowed to stand at -20 °C for 1 hour. The samples were then centrifuged at 15000 rpm for 30 minutes. After centrifugation, the supernatant was removed, and 1.5 equivalents of 80% ethanol were added by volume, and the mixture was centrifuged again at 15000 rpm for 5 minutes. The supernatant was removed, and the mixture was air-dried to remove the ethanol. Milli-Q water was added to dissolve the pellet, and then agarose gel electrophoresis was performed.

[0117] The HPLC results are shown in Figure 13. Peaks were observed at 13.301 minutes and 13.420 minutes, respectively. The agarose gel electrophoresis results are shown in Figure 14. From left to right in Figure 14, the lanes are: a 500 bp DNA ladder marker, double-stranded DNA (dsDNA) prepared in steps 1-2, single-stranded DNA (ssDNA) prepared separately from this test, a sample prepared by isolating the peak at 13.301 minutes by HPLC, and a sample prepared by isolating the peak at 13.420 minutes by HPLC. As shown in Figure 14, both the sample with a retention time of 13.301 minutes and the sample with a retention time of 13.420 minutes were suggested to be single-stranded DNA. The peak at 13.420 minutes, which has a longer retention time in reverse-phase HPLC, is presumed to be single-stranded DNA of the sense strand to which a hydrophobic tag has been attached.

[0118] 1-8-2. No Alkali Treatment Double-stranded DNA samples with hydrophobic tags prepared in 1-2 were analyzed by reverse-phase HPLC under the above conditions without alkali treatment, and the peak regions were separated. To each separated sample, an equal volume of isopropanol and 1 / 10 volume of 3 M sodium acetate aqueous solution were added, and the samples were allowed to stand at -20 °C for 1 hour. The samples were then centrifuged at 15000 rpm for 30 minutes. After centrifugation, the supernatant was removed, and 1.5 equivalents of 80% ethanol were added by volume, and the samples were centrifuged again at 15000 rpm for 5 minutes. The supernatant was removed, and the samples were air-dried to remove the ethanol. Milli-Q water was added to dissolve the pellet, and then agarose gel electrophoresis was performed.

[0119] The HPLC results are shown in Figure 15. A single peak was observed at 14.297 minutes. The agarose gel electrophoresis results are shown in Figure 16. From left to right in Figure 16, the lanes are: a 500 bp DNA ladder marker, double-stranded DNA (dsDNA) prepared in steps 1-2, single-stranded DNA (ssDNA) prepared separately from this test, and a sample prepared by isolating the peak with a retention time of 14.297 minutes from the HPLC. As shown in Figure 16, the sample with a retention time of 14.297 minutes was suggested to be double-stranded DNA.

[0120] 2. Purification of single-stranded DNA using hydrophobic tags with C19 alkyl groups 2-1. Preparation of PCR primers A schematic diagram of the prepared forward primer is shown in Figure 17. In Figure 17, R is a linear alkyl group with 19 carbon atoms (C19 alkyl group). 19 H 39 This represents the hydrophobic tag shown in Figure 17. The sequence of the fabricated primers is as shown in Figure 2, and the asterisks in the forward primer in Figure 2 represent the hydrophobic tag shown enclosed in a rectangle in Figure 17. In other words, the primers fabricated in 1-1 and the primers fabricated in 1-2 are identical except for the structure of the hydrophobic tag in the forward primer.

[0121] Primers were synthesized using the DNA / RNA synthesizer NR-2A_7MX or NRs-4A_10R7NP (Nippon Techno Service). DNA amidite and CPG (Chemgenes; deoxyguanosine (n-ibu) 3'-ICAa CPG, 1000) Å A TV (47.3 μmol / g) was used. In the case of forward primers with hydrophobic tags, the DNA was further coupled with a chemical phosphorylation reagent (CPR) containing the hydrophobic tag after DNA synthesis. Amidite and CPR were dissolved in anhydrous acetonitrile and prepared to 50 mM and 100 mM, respectively. The reagents used in the synthesis apparatus were as follows: 10 (w / v)% dichloromethane (Wako, 135-02441) for deprotection, 0.25 M 5-benzylthio-1H-tetrazole in acetonitrile (Wako) for coupling, a mixture of acetic anhydride / tetrahydrofuran / pyridine (1:8:1, Wako) and 10 (v / v)% 1-methylimidazole in tetrahydrofuran (Wako) for capping, and 0.01 M iodine (Honeywell) in 64% acetonitrile, 6% pyridine, and 30% water for oxidation.

[0122] After DNA synthesis, the DNA was cleaved from the support and deprotected overnight at room temperature with a 1:1 mixture (1 mL) of 40% methylamine aqueous solution and 33% methylamine ethanol solution, or allowed to stand for 30 minutes at 65 °C with a 1:1 mixture (1 mL) of 40% methylamine aqueous solution and 30% ammonia water. The supernatant was filtered and dried under reduced pressure. The DNA was purified by reverse-phase HPLC. The purification conditions were as follows: Column: YMC Triart Bio C4, 250 × 10 mm ID, S-5 μm, 30 nm; Solution A: 50 mM TEAA (pH 7.0 + 5% acetonitrile); Solution B: 100% acetonitrile; Gradient of Solution B: 0-100%; Column temperature: 50 °C; Flow rate: 1 mL / min; Detection wavelength: 260 nm.

[0123] After purification, the sample was added to 3 M sodium acetate (pH 5.2) and 2-propanol and allowed to stand at -30 °C for 1 hour. Then, it was centrifuged at 15000 rpm for 30 minutes to remove the residue, and the pellet was washed with 80% ethanol. The pellet was then dissolved in Milli-Q water, and the DNA concentration was measured using NanoDrop2000 (Thermo Fisher Scientific). The DNA concentration was determined from the absorbance at 260 nm. Purity was confirmed by denatured polyacrylamide gel electrophoresis (PAGE) using 7.5 M urea as the denaturant. The ratio of acrylamide to N,N'-methylenebisacrylamide was 19:1. After electrophoresis, the gel was stained with SYBR Green II Nucleic Acid Gel Stain (Lonza) and examined using the ChemiDoc XRS Plus system (Bio-Rad).

[0124] 2-2. Preparation of double-stranded DNA with a C19 hydrophobic tag Double-stranded DNA was prepared by PCR using the Nano luc plasmid (pNL1.1TK, promega) as a template and PCR primers prepared in 2-1.

[0125] The PCR reaction mixture was prepared with the following composition: 0.3 μM each of primers, 1 ng / μL of pNL1.1TK vector, 0.2 mM of dNTPs, 1.5 mM of MgSO4, 1× PCR buffer for KOD-Plus-Neo, and 0.02 units / μL of KOD-Plus-Neo (Toyobo). The reaction was carried out under the following thermal cycling conditions: 94 °C, 2 min – (98 °C, 10 sec – 55 °C, 30 sec – 72 °C, 36 sec) × 40 cycles – 72 °C, 3 min. The sequences of the sense and antisense strands of the double-stranded DNA amplified by this PCR are shown in Figure 3. The results of agarose gel electrophoresis analysis of the PCR product are shown in Figure 18. In Figure 18, from left to right, the 500 bp DNA ladder marker, the PCR product, and the PCR product irradiated with ultraviolet light were applied, respectively.

[0126] 2-3. Purification of single-stranded DNA using a disposable column First, a disposable cartridge column, Presep DNA / RNA type A (85 mg / 1 mL) column (Wako), was washed and equilibrated by passing 1 mL of 100% acetonitrile twice and 1 mL of 100 mM TEA-OH (pH 12) three times. Next, the double-stranded DNA sample with a hydrophobic tag prepared in 2-2 was mixed with an equal volume of 1 M sodium hydroxide aqueous solution and gently combined. The mixed sample was immediately loaded onto the Presep DNA / RNA type A (85 mg / 1 mL) column. Subsequently, the solution was washed with 5% acetonitrile / 10 mM TEA-OH (pH 12), and then sequentially eluted with 20% acetonitrile / 10 mM TEA-OH (pH 12), 40% acetonitrile / 10 mM TEA-OH (pH 12), 60% acetonitrile / 10 mM TEA-OH (pH 12), and 80% acetonitrile / 10 mM TEA-OH (pH 12). The eluates were collected in 1 mL portions. The specific cartridge purification conditions are described below.

[0127]

[0128] Each recovered fraction was analyzed by agarose gel electrophoresis. The results are shown in Figure 19. In Figure 19, lane 1 contains a 500 bp DNA ladder marker, lane 2 contains double-stranded DNA (dsDNA) with a hydrophobic tag prepared in 2-2, lane 3 contains single-stranded DNA (ssDNA) with a hydrophobic tag prepared separately from this test, lane 4 contains single-stranded DNA from which the hydrophobic tag was removed by UV irradiation, and lane 5 contains double-stranded DNA from which the hydrophobic tag was removed by UV irradiation. Lane 6 contains the flow-through fraction (FT). For lanes 7-16, "wash" refers to the washing buffer in the table above, and "Elu" refers to each elution buffer in the table above. The "-1" at the end indicates the fraction eluted when the buffer was loaded onto the column for the first time, and "-2" indicates the fraction eluted when the buffer was loaded onto the column for the second time.

[0129] Of the samples fractionated using a disposable column, wash-1, Elution 1-1, and Elution 2-1 were analyzed by reverse-phase HPLC. The analytical conditions are as follows.

[0130] <Analysis Conditions> Column used: YMC Triart Bio C4 (Pore size: 30 nm, Particle size: 5 μm, Column internal dimensions: 250 × 4.6 mm I.D) Analysis conditions: 0-100%, 1 mL / min, 20 minutes Analysis temperature: 30 °C Mobile phase A: 50 mM TEAA (pH 7) + 5% acetonitrile Mobile phase B: 100% acetonitrile

[0131] The results are shown in Figure 20. The peaks with a retention time of approximately 12 minutes observed in Wash-1 and Elution 1-1 are thought to originate from single-stranded DNA without hydrophobic tags, while the peaks with a retention time of approximately 15 minutes observed in Elution 1-1 and Elution 2-1 are thought to originate from single-stranded DNA with hydrophobic tags. In other words, it is thought that only single-stranded DNA of the sense strand with a hydrophobic tag was eluted in Elution 2-1. These results suggest that the technology described herein may allow for the simple purification of desired single-stranded DNA using a disposable cartridge column.

[0132] 3. Purification of single-stranded DNA using dNTPs containing hydrophobic tags 3-1. Synthesis of Decyl-dATP and Nb-dATP

[0133] Dmf group deprotection 2'-Deoxyadenosine anhydrous (2.00 g, 7.96 mmol) was suspended in dry methanol (92.2 mL), and N,N-dimethylformamide dimethyl acetal (4.84 g, 5.33 mL, 39.8 mmol) was added. After stirring at 50 °C for 3.5 hours, the reaction mixture was concentrated using a rotary evaporator. Dichloromethane was added to the residue to suspend it, and the mixture was diluted with hexane (3 times the volume). The resulting precipitate was filtered by suction and washed with hexane. The obtained solid was dried under reduced pressure to obtain the target compound as a white powder (2.45 g, quantitative yield). 1H NMR (600 MHz, CD3OD) δ 8.93 (s, 1H, CHN(CH3)2), 8.44 (s, 1H, H-2), 8.43 (s, 1H, H-8), 6.48 (dd, J = 8.0, 5.9 Hz, 1H, H-1'), 4.59 (dt, J = 5.4, ​​2.8 Hz, 1H, H-3'), 4.07 (q, J = 3.2 Hz, 1H, H-4'), 3.85 (dd, J = 12.2, 3.1 Hz, 1H, one of H-5'), 3.75 (dd, J = 12.2, 3.5 Hz, 1H, one of H-5'), 3.25 (s, 3H, NCH3), 3.24 (s, 3H, NCH3), 2.86 - 2.81 (m, 1H, one of H-2'), 2.43 (ddd, J = 13.5, 6.1, 2.9 Hz, 1H, one of H-2') ppm. Spectral data were consistent with literature values. (J. Ju et al., PNAS 2006, 103(52), 19635-19640)

[0134] TBS protection To a solution of dmf-protected 2'-deoxyadenosine (2.00 g, 6.53 mmol) and imidazole (1.07 g, 15.7 mmol) in dry DMF, tert-butyldimethylsilyl chloride (1.14 g, 7.57 mmol) was added. After stirring at room temperature for 4 hours, the reaction mixture was diluted with ethyl acetate and washed sequentially with saturated sodium bicarbonate aqueous solution, Milli-Q water, and saturated brine. The organic layer was dried over sodium sulfate and concentrated using a rotary evaporator. The resulting crude product was purified by silica gel column chromatography eluting with 2.4 → 9.1% methanol / dichloromethane to obtain the target compound as a white foamy solid (1.68 g, yield 61.1%). 1H NMR (400 MHz, CD3OD) δ 8.88 (s, 1H, CHN(CH3)), 8.42 (2s, 2H, H-2 and H8), 6.48 (t, J = 6.5 Hz, 1H, H-1'), 4.58 (dt, J = 5.9, 3.7 Hz, 1H, H-3'), 4.03 (q, J = 3.6 Hz, 1H, H-4'), 3.92 (dd, J = 11.3, 3.7 Hz, 1H, one of H-5'), 3.83 (dd, J = 11.3, 3.7 Hz, 1H, , one of H-5'), 3.24 (d, J = 1.8 Hz, 6H, NCH3), 2.75 (dt, J = 13.4, 6.2 Hz, 1H, one of H-2'), 2.51 (ddd, J = 13.4, 6.3, 4.0 Hz, 1H, one of H-2'), 0.88 (s, 9H, C(CH3)3), 0.06 (d, J = 1.6 Hz, 6H, SiCH3 x2) ppm. The spectral data were consistent with the literature values. (J. Ju et al., PNAS 2006, 103(52), 19635-19640).

[0135] 2'-O-alkylation To a solution of 2'-deoxyadenosine (1.09 g, 2.59 mmol) dissolved in dichloromethane (80.2 mL), tetrabutylammonium bromide (419 mg, 1.30 mmol) and 1-bromodecane (2.88 g, 13.0 mmol) were added. A 40% aqueous sodium hydroxide solution (w / w, 40.1 mL) was added, and the mixture was vigorously stirred at room temperature for 1 hour. Then, 1-bromodecane (2.88 g, 13.0 mmol) was added, and the mixture was stirred at room temperature for 5 hours. The reaction mixture was diluted with saturated aqueous sodium bicarbonate and extracted twice with ethyl acetate. The organic layer was washed with saturated brine, dried over sodium sulfate, and concentrated using a rotary evaporator. The resulting crude product was purified by silica gel column chromatography under 0→6.3% methanol / dichloromethane elution conditions to obtain the target compound as a yellow oil (222 mg, yield 15.3%). 1H NMR (600 MHz, CD3OD) δ 8.89 (s, 1H, CHN(CH3)2), 8.42 (s, 1H, H2), 8.39 (s, 1H, H8), 6.46 - 6.39 (m, 1H, H1’), 4.27 (dt, J = 6.6, 3.2 Hz, 1H, H3’), 4.14 - 4.11 (m, 1H, H4’), 3.90 - 3.86 (m, 1H, one of H5’), 3.80 (dd, J = 11.0, 3.7 Hz, 1H, one of H5’), 3.54 - 3.49 (m, 2H, 3’-O-CH2-), 3.23 (2s, 6H, CHN(CH3)2), 2.76 (ddd, J = 13.3, 7.4, 5.9 Hz, 1H, one of H2’), 2.57 (ddd, J = 13.5, 6.2, 3.1 Hz, 1H, one of H2’), 1.62 - 1.57 (m, 2H, -CH2- of 3’-O-alkyl), 1.41 - 1.37 (m, 2H, -CH2- of 3’-O-alkyl), 1.31 - 1.27 (m, 12H, -CH2- of 3’-O-alkyl), 0.89 (d, J = 5.8 Hz, 12H, Si-C(CH3)3 and -CH3 of 3’-O-alkyl), 0.08 (s, 6H, Si-(CH3)2) ppm. 13C NMR (151 MHz, CD3OD) δ 161.05 (C6), 160.07 (CHN(CH3)2), 153.43 (C2), 152.12 (C4), 142.19 (C8), 126.82 (C5), 86.81 (C4’), 86.02 (C1’), 80.71 (C3’), 70.55 (3’-O-CH2-), 64.58 (C5’), 41.50 (one of CHN(CH3)2), 38.70 (C2’), 35.24 (one of CHN(CH3)2), 33.08 (-CH2- of 3’-O-Alkyl), 30.91 (-CH2- of 3’-O-Alkyl), 30.78 (-CH2- of 3’-O-Alkyl), 30.73 (-CH2- of 3’-O-Alkyl), 30.58 (-CH2- of 3’-O-Alkyl), 30.48 (-CH2- of 3’-O-Alkyl), 27.32 (-CH2- of 3’-O-Alkyl), 26.48 (Si-C(CH3)3), 23.75 (-CH2- of 3’-O-Alkyl), 19.21 (Si-C(CH3)3), 14.52 (-(CH2)9CH3), -5.22 (Si(CH3)2) ppm. HR-ESI-TOF-MS m / z calcd. for C 29 H 53 N6O3Si [M + H] + 561.3943; found 561.3946

[0136] 2’-O-Decyl-dA 3'-O-decyl-protected deoxyadenosine (222 mg, 396 μmol) was dissolved in THF (9.38 mL), and 1 M tetrabutylammonium fluoride (436 μL, 436 μmol) in THF was added to the solution. The mixture was stirred at room temperature for 2.5 hours, and then concentrated using a rotary evaporator. The resulting residue was dissolved in 7 M ammonia-methanol solution (7.10 mL) and stirred at 50 °C for 6 hours. After concentrating the reaction mixture, it was purified by silica gel column chromatography eluting with 2.4 → 6.3% methanol / dichloromethane to obtain the target compound as a white solid (142 mg, yield 91.6%). 1 H NMR (600 MHz, CDCl3) δ 8.23 ​​(s, 1H, H2), 7.82 (s, 1H, H8), 6.76 (s, 2H, 6-NH2), 6.62 (s, 1H, 5'-OH), 6.22 (dd, J = 9.4, 5.6 Hz, 1H, H1'), 4.29 (d, J = 5.4 Hz, 1H, H3'), 4.24 (d, J = 1.3 Hz, 1H, H4'), 3.95 (dd, J = 12.7, 1.8 Hz, 1H, H5'), 3.72 (d, J = 13.1 Hz, 1H, H5'), 3.43 (tt, J = 6.6, 3.2 Hz, 2H, 3'-O-CH2-), 2.91 (ddd, J = 13.3, 9.4, 5.4 Hz, 1H, H2'), 2.36 (dd, J = 12.8, 5.8 Hz, 1H, H2'), 1.58 - 1.52 (m, 2H, -CH2-of 3'-O-Alkyl), 1.33 - 1.21 (m, 14H, -CH2-of 3'-O-Alkyl), 0.83 (t, J = 7.0 Hz, 3H, -(CH2)9CH3) ppm. 13C NMR (151 MHz, CDCl3) δ 156.34 (C6), 152.39 (C2), 148.57 (C4), 139.98 (C8), 121.03 (C5), 87.63 (C1’), 87.11 (C4’), 80.76 (C3’), 69.44 (3’-O-CH2-), 63.78 (C5’), 37.93 (C2’), 31.90 (-CH2- of 3’-O-Alkyl), 29.83 (-CH2- of 3’-O-Alkyl), 29.61 (-CH2- of 3’-O-Alkyl), 29.58 (-CH2- of 3’-O-Alkyl), 29.48 (-CH2- of 3’-O-Alkyl), 29.33 (-CH2- of 3’-O-Alkyl), 26.21 (-CH2- of 3’-O-Alkyl), 22.69 (-CH2- of 3’-O-Alkyl), 14.15 (-(CH2)9CH3) ppm. HR-ESI-TOF-MS m / z calcd. for C 20 H 33 N5NaO3 [M + Na] + 414.2476; found 414.2487.

[0137] 2’-O-Decyl-dATP A solution of 3'-O-decyl 2'-deoxyadenosine (134 mg, 342 μmol) dissolved in trimethyl phosphate (1.71 mL) and tetrahydrofuran (400 μL) was cooled in an ice bath, and phosphoryl chloride (57.7 mg, 35.2 μL, 376 μmol) was added. After stirring at 0 °C for 2 hours, a mixture of 375 mM bis-tributylammonium pyrophosphate (2.28 mL, 855 μmol) and tributylamine (380 mg, 488 μL, 2.05 mmol) in acetonitrile was added to the reaction mixture in an ice bath. After stirring at room temperature for 41 minutes, the reaction was stopped by adding 0.1 M TEAB buffer (pH 7.9, 5 mL), and the mixture was stirred at room temperature for a further 44 minutes. The resulting mixture was concentrated using a rotary evaporator and then heated in a DEAE Sephadex A-25 (φ = 4.5 cm, h = 8.4 cm, 140 cm). 3 The solution was purified by ion-exchange chromatography (12 mL / min) using a linear gradient of 0-1.1 M TEAB buffer (pH 7.9) and 0-7% acetonitrile (270 minutes). The fraction containing the target product was collected and concentrated. The residue was dissolved in methanol (5.00 mL), and then suspended in 600 mM sodium perchlorate / acetone (40.0 mL). The mixture was centrifuged at 3,500 rpm for 10 minutes. The supernatant was removed, and the precipitate was resuspended in acetone. The same suspension and centrifugation procedure was repeated four additional times. The resulting precipitate was dried under reduced pressure to obtain the target triphosphate as a white solid (173 mg, yield 70.2%) as the sodium salt. 1 H NMR (600 MHz, D2O) δ 8.46 (s, 1H), 8.15 (s, 1H), 6.36 (d, J = 6.1 Hz, 1H), 4.44 (s, 1H), 4.37 (s, 1H), 4.15 (s, 2H), 3.66 - 3.53 (m, 2H), 2.70 (s, 1H), 2.57 (d, J = 9.1 Hz, 1H), 1.62 - 1.49 (m, 2H), 1.32 - 1.12 (m, 14H), 0.75 (t, J = 7.0 Hz, 3H) ppm.13 C NMR (151 MHz, D2O) δ 154.09, 151.07, 148.54, 140.13, 118.12, 84.08, 83.56, 79.80, 69.43, 66.29, 37.14, 31.78, 29.54, 29.39, 29.24, 25.99, 22.51, 13.85 ppm. 31 P NMR (162 MHz, D2O) δ -9.62 (d, J = 21.5 Hz, 1P), -10.73 (d, J = 21.5 Hz, 1P), -22.82 (t, J = 21.5 Hz, 1P) ppm. HR-ESI-TOF-MS m / z calcd. for C 20 H 35 N5O 12 P3 [M - H] - 630.1501; found 630.1908.

[0138] 2'-デオキシアデノシンのprotection A mixture of 2'-deoxyadenosine (653 mg, 1.55 mmol) and a methylthioacetal derivative (651 mg, 1.77 mmol) was azeotropically removed three times with benzene (10 mL). A 3 Å molecular sieve (650 mg) was added to the resulting mixture and suspended in tetrahydrofuran (11.6 mL). N-iodosuccinimide (398 mg, 1.77 mmol) was then added, and the reaction mixture was cooled to -40 °C. After stirring at -40 °C for 20 minutes, trifluoromethanesulfonic acid (266 mg, 157 μL, 1.77 mmol) was added, and the mixture was stirred for a further 2 hours at -40 °C. Triethylamine (5.80 mL) was added to the reaction mixture to stop the reaction, and the mixture was diluted with ethyl acetate (100 mL). The mixture was sequentially washed with saturated sodium bicarbonate aqueous solution (100 mL × 2), saturated sodium thiosulfate aqueous solution (100 mL × 2), and saturated saline solution (100 mL × 1). The organic layer was dried over sodium sulfate and concentrated. The residue was purified by silica gel column chromatography (eluent: 1.00-4.8% methanol / dichloromethane) to obtain the target compound as a brown oily substance (319 mg, yield 27.7%). 1H NMR (600 MHz, CDCl3) δ 8.89 (s, 1H), 8.49 (d, J = 10.1 Hz, 1H), 8.12 (d, J = 3.1 Hz, 1H), 7.90 - 7.82 (m, 1H), 7.68 (ddd, J = 7.9, 3.4, 1.5 Hz, 1H), 7.62 - 7.52 (m, 1H), 7.41 - 7.31 (m, 1H), 6.38 (dd, J = 8.0, 5.7 Hz, 0.2H), 6.32 - 6.29 (m, 0.5H), 5.55 (d, J = 4.6 Hz, 0.3H), 5.19 - 5.14 (m, 1H), 4.69 (d, J = 7.2 Hz, 0.4H), 4.55 (dd, J = 14.9, 7.2 Hz, 1H), 4.44 - 4.43 (m, 0.7H), 4.31 - 4.27 (m, 0.3H), 4.24 (ddd, J = 5.1, 3.8, 1.3 Hz, 0.3H), 4.11 (q, J = 1.4 Hz, 0.5H), 3.96 - 3.93 (m, 0.3H), 3.84 (d, J = 7.1 Hz, 0.5H), 3.77 (d, J = 3.2 Hz, 0.3H), 3.75 - 3.71 (m, 0.7H), 3.64 (dd, J = 10.9, 3.7 Hz, 0.4H), 3.54 (dd, J = 11.0, 3.4 Hz, 0.2H), 3.46 (dd, J = 10.8, 5.2 Hz, 0.3H), 3.22 (s, 3H), 3.16 (s, 3H), 2.67 - 2.62 (m, 0.2H), 2.53 (ddd, J = 13.4, 5.7, 2.4 Hz, 0.3H), 2.35 (ddd, J = 13.6, 8.0, 5.8 Hz, 0.6H), 2.21 - 2.18 (m, 0.6H), 2.08 (ddd, J = 13.4, 5.6, 4.6 Hz, 0.4H), 1.79 - 1.69 (m, 2H), 1.53 - 1.45 (m, 1H), 1.38 (dd, J = 9.4, 5.3 Hz, 1H), 1.20 (d, J = 13.0 Hz, 16H), 0.89 (s, 4H), 0.86 (d, J = 0.5 Hz, 3H), 0.83 - 0.82 (m, 5H), 0.06 (s, 2H), 0.03 (d, J = 6.0 Hz, 2H), -0.00 (d, J = 4.7 Hz, 2H) ppm. . 13 C NMR (151 MHz, CDCl3) δ 159.60, 158.17, 152.64, 152.60, 151.37, 151.30, 148.52, 148.43, 140.03, 140.00, 138.65, 133.25, 128.51, 128.48, 128.13, 126.36, 126.30, 124.30, 99.48, 99.08, 92.45, 87.74, 86.63, 85.62, 85.31, 84.54, 84.33, 76.79, 74.57, 74.24, 73.37, 72.92, 64.54, 63.85, 63.63, 63.49, 62.05, 44.40, 41.51, 41.32, 38.58, 38.08, 37.89, 35.21, 31.94, 29.68, 29.65, 29.55, 29.49, 29.38, 28.27, 26.09, 26.03, 25.99, 25.91, 22.72, 18.41, 18.36, 18.32, 14.17, -5.29, -5.36, -5.44, -5.50 ppm. HR-ESI-TOF-MS m / z calcd. for C 38 H 62 N7O6Si [M + H] + 740.4525; found 740.4493

[0139] 3'-O-nitrobenzyl-modified 2'-deoxyadenosine Protective 2'-deoxyadenosine (319 mg, 431 μmol) was dissolved in THF (10.2 mL) and 1 M tetrabutylammonium fluoride (474 ​​μL, 474 μmol) in THF was added. The mixture was stirred at room temperature for 12 hours and then concentrated using a rotary evaporator. The resulting residue was dissolved in 7 M ammonia / methanol solution (7.72 mL) and stirred at 50 °C for 4 hours. After concentrating the reaction mixture, it was purified by silica gel column chromatography using 2.0 → 3.2% methanol / dichloromethane as the eluent to obtain the target compound as a yellow amorphous solid (144 mg, yield 58.5%). 1 H NMR (600 MHz, CD3OD) δ 8.26 - 8.18 (m, 2H), 7.85 (dt, J = 8.4, 1.7 Hz, 0.5H), 7.79 (dt, J = 8.2, 1.7 Hz, 0.5H), 7.71 - 7.66 (m, 2H), 7.46 (dddt, J = 15.1, 10.1, 6.6, 1.7 Hz, 1H), 6.50 - 6.34 (m, 1H), 5.02 (ddt, J = 8.2, 6.1, 3.1 Hz, 1H), 4.71 (dd, J = 7.4, 3.3 Hz, 1H), 4.62 - 4.54 (m, 1H), 4.42 - 4.35 (m, 1.5H), 4.22 (td, J = 4.0, 1.9 Hz, 0.5H), 3.63 (t, J = 3.8 Hz, 1H), 3.52 (dt, J = 11.9, 3.7 Hz, 0.5H), 3.48 - 3.40 (m, 0.5H), 2.88 - 2.77 (m, 0.5H), 2.62 - 2.51 (m, 1H), 2.03 (dd, J = 14.9, 1.6 Hz, 0.5H), 1.77 - 1.70 (m, 2H), 1.24 (d, J = 11.0 Hz, 18H), 0.89 - 0.87 (m, 3H) ppm. 13C NMR (151 MHz, CD3OD) δ 157.23, 153.72, 153.68, 150.36, 150.15, 150.07, 140.88, 139.27, 138.84, 134.32, 129.72, 129.60, 129.47, 124.95, 124.90, 120.08, 94.77, 94.48, 89.03, 88.88, 86.55, 86.32, 79.17, 78.93, 76.04, 75.33, 69.21, 68.57, 63.43, 63.37, 59.50, 40.19, 39.35, 38.77, 38.69, 33.06, 30.72, 30.65, 30.55, 30.46, 30.40, 30.35, 26.99, 26.84, 24.79, 23.74, 20.71, 19.53, 14.46, 13.94 ppm. HR-ESI-TOF-MS m / z calcd. for C 29 H 42 N6NaO6 [M + Na] + 593.3058; found 593.3052.

[0140] Nb-dATP 3'-O-nitrobenzyloxymethyl-2'-deoxyadenosine (144 mg, 252 μmol) was dissolved in trimethyl phosphate (1.26 mL) and cooled in an ice bath. Phosphoryl chloride (42.5 mg, 25.9 μL, 277 μmol) was added to this mixture and stirred in an ice bath for 1 hour and 25 minutes. Subsequently, phosphoryl chloride (19.3 mg, 11.8 μL, 126 μmol) was added to the reaction mixture. After stirring at 0 °C for 1 hour, a mixture of 375 mM bis-tributylaminium pyrophosphate (1.68 mL, 630 μmol) and tributylamine (280 mg, 359 μL, 1.51 mmol) in acetonitrile was added to the reaction mixture in an ice bath. The mixture was then stirred at room temperature for 46 minutes, and 0.1 M TEAB buffer (pH 7.9, 5 mL) was added to stop the reaction. The mixture was then stirred overnight at room temperature. The resulting mixture was concentrated using a rotary evaporator and then evaporated in a DEAE Sephadex A-25 (φ = 4.5 cm, h = 8.4 cm, 140 cm). 3 The solution was purified by ion-exchange chromatography (12 mL / min) using a linear gradient of 0-1.5 M TEAB buffer (pH 7.9) and 0-10% acetonitrile (270 minutes). The fraction containing the target compound was collected and concentrated, and the residue was dissolved in methanol (5.00 mL). 600 mM sodium perchlorate / acetone (40.0 mL) was added to this to suspend the solution, and the mixture was centrifuged at 3,500 rpm for 10 minutes. The supernatant was removed, and the precipitate was resuspended in acetone. This suspension and centrifugation procedure was repeated three more times. The final precipitate was dried under reduced pressure to obtain the target triphosphate as a white solid (116 mg, yield 46.0%) as the sodium salt. 1H NMR (400 MHz, D2O) δ 8.53 - 8.25 (m, 1H), 8.06 (s, 1H), 7.85 - 7.59 (m, 3H), 7.54 - 7.32 (m, 1H), 6.29 - 5.93 (m, 1H), 5.08 (s, 1H), 4.75 (m, 0.5H), 4.63 (m, 0.5H), 4.51 - 3.64 (m, 4H), 3.25 - 2.95 (m, 1H), 2.90 - 2.29 (m, 1.5H), 1.90 (m, 0.5H), 1.66 (br, 2H), 0.99 (br, 18H), 0.60 (br, 3H) ppm. 13 C NMR (151 MHz, D2O) δ 151.89, 148.16, 137.90, 133.80, 128.57, 124.01, 117.88, 94.69, 84.36, 78.42, 75.66, 66.18, 41.16, 37.60, 37.17, 31.85, 30.34, 29.72, 29.37, 25.85, 25.49, 22.53, 13.74 ppm. 31 P NMR (163 MHz, D2O) δ -9.33 (1P), -10.38 (1P), -21.33 (1P) ppm. HR-ESI-TOF-MS m / z calcd. for C 29 H 44 N6O 15 P3 [M - H] - 809.2083; found 809.2346.

[0141] 3-2. Incorporation of Decyl-dATP and Nb-dATP by DNA Polymerase First, we evaluated suitable DNA polymerases that can utilize Decyl-dATP and Nb-dATP as substrates. We used a 20 nt DNA primer labeled with 5' fluorescein (FAM) and a 34 nt DNA template containing a counterbase indicated by N (T, A, C, G), as shown in Figure 21(A). Two types of DNA polymerases were used: Klenow Fragment (3'->5' exo-) (NewEnglandBiolabs) and Taq DNA polymerase (5'->3' exo-) (NewEnglandBiolabs, HemKlenTaq). A mixture of 1x ThermoPol Reaction buffer (NewEnglandBiolabs), 0.1 μM DNA primer, 0.15 μM DNA template, 0.2 mM Decyl-dATP or Nb-dATP, and 0.125 units / µl DNA polymerase was mixed and reacted at 37 °C for 30 minutes. An equal volume of 2x RNA Loading Dye (NewEnglandBiolabs) was added, and the mixture was treated at 90 °C for 5 minutes. This mixture was then added to a 1x TBE, 8 M urea, 15% acrylamide gel and electrophoresed at 600 V for 150 minutes. After electrophoresis, fluorescein fluorescence was observed as a band using a ChemiDoc (BioRad) gel imaging system. Figure 21(B) shows the gel electrophoresis results. In the uptake using Klenow fragments, Decyl-dATP and Nb-dATP were quantitatively incorporated into the T counterpart. However, Nb-dATP was also incorporated into the C counterpart. Taq DNA polymerase quantitatively incorporated Nb-dATP into the T molecule, while hardly incorporating Decyl-dATP at all.

[0142] 3-3. Incorporation of Decyl-dATP into Plasmid Digestion Products and Purification of Single-Stranded DNA As a method for large-scale preparation of single-stranded DNA, target DNA is prepared as a plasmid, digested with restriction enzymes, one strand is labeled with hydrophobic-tagged dNTPs, and purified by HPLC. Target sequences of Type IIS restriction enzymes with different sequence recognition sites and digestion sites are placed at both ends of the target DNA sequence. This introduces arbitrary adherent ends at both ends of the target DNA sequence. At this time, the sequence is designed so that hydrophobic-tagged dNTPs are incorporated into only one strand. An overview is shown in Figure 22(A). In this experiment, a plasmid with BsaI recognition sequences at both ends of a 600 bp target DNA sequence was prepared and digested by treatment at 37 °C for 12 hours with a composition of 1x CutSmart Buffer (NewEnglandBiolabs), 500 ng / ul plasmid, and 0.05 units / ul BsaI-HF (NewEnglandBiolabs). Figure 22(B) shows the gel electrophoresis results. To this digest product, 1 / 10 equivalent of 3 M sodium acetate (pH 5.2) and 1 equivalent of 2-propanol were added and the mixture was allowed to stand at -30 °C for 1 hour. After that, the mixture was centrifuged at 15000 rpm for 30 minutes to remove the residue, and then the pellet was washed with 80% ethanol. The pellet was then dissolved in Milli-Q water, and the DNA concentration was measured using NanoDrop2000. The DNA concentration was determined from the absorbance at 260 nm. This digest product has a 5'-dTTTT-3' overhang at the 5' end of the sense strand and a 5'-dCGCC-3' overhang at the 5' end of the antisense strand. Therefore, by incorporating Decyl-dATP, a hydrophobic tag can be selectively introduced into the antisense strand. A mixture of 1x ThermoPol Reaction buffer, 100 ng / µl plasmid digest, 0.2 mM Decyl-dATP, and 0.08 units / µl Klenow Fragment (3'->5' exo-) was incubated at 37 °C for 0.5, 2, and 12 hours.

[0143] 3-4. Purification of Decyl-dATP-incorporated DNA using reversed-phase HPLC (Agilent PLRP-S) The hydrophobic tagged double-stranded DNA sample prepared in 3-3 was analyzed. <Analysis conditions> Column used: Agilent PLRP-S (pore size: 4000 Å, particle size: 8 μm, column internal dimensions: 150 × 4.6 mm I.D.) Analysis conditions: 0-100%, 1 mL / min, 20 minutes Analysis temperature: 30 °C Mobile phase A: 100 mM TEAA (pH 7.0) Mobile phase B: 100% acetonitrile

[0144] The results obtained from HPLC analysis are shown in Figure 23. The plasmid digest product contains two types of DNA strands, and peaks were observed at the 10.331 minute mark. Unreacted decyl-dATP was found at the 13.056 minute mark. A new peak appeared at 12.260 minutes due to the extension reaction, and it was confirmed that its proportion increased as the extension time was extended. This confirmed the introduction of a hydrophobic tag.

[0145] <Example B>

[0146] 1. Synthesis of chemical capping reagents 1

[0147] Chemical capping reagent (5) was synthesized according to the above scheme. Each step of the synthesis is described in detail below. The synthesis of compound 1 was carried out according to M. Inagaki et al., 2023 (Non-Patent Literature 1).

[0148] 1-1. Synthesis of Compound 2 A suspension of 2'-C11Nb-N7-methylguanosine (1) (100 mg, 117 μmol) was suspended in trimethyl phosphate (585 μL) and cooled to -10 °C. Subsequently, 2,6-Lutidine (28.8 mg, 31.3 μL, 269 μmol) and phosphoryl chloride (41.2 mg, 25.1 μL, 269 μmol) were added sequentially. After stirring at -10 °C for 6 hours, the reaction was stopped by adding 0.2 M TEAB buffer (pH 7.9, 1.00 mL) at -10 °C. The mixture was then raised to room temperature and diluted with methanol (18.0 mL). Further dilute with 50% methanol / water (approx. 100 mL) and test on a CHRMATREX C8 A31 SMB-100-20 / 45 column (column size: φ = 3.0 cm, h = 10 cm, 70 cm). 3 The compound was purified using a linear gradient (7 mL / min), solvent A: 50 mM TEAA buffer (pH 6.0) + 0.5% CH3CN, solvent B: CH3CN, 10-100% B / 60 min. The fraction containing the target compound was collected, concentrated, and lyophilized to obtain the target monophosphate compound (2) as a white solid (50.3 mg, yield 61.7%). Spectral data for this compound is reported in Non-Patent Document 1.

[0149] 1-2. Synthesis of Compound 3 2'-Nb-7mGMP(2) (238 mg, 0.342 mmol), mimidazole (187 mg, 2.74 mmol), and 2,2'-dithiodipyridine (227 mg, 1.03 mmol) were dissolved in N,N-dimethylformamide (4.07 mL). Triethylamine (69.2 mg, 95.6 μL, 0.684 mmol) was added, followed by triphenylphosphine (270 mg, 1.03 mmol). After stirring at room temperature for 5.5 hours, a solution of sodium perchlorate (502 mg, 4.10 mmol) dissolved in 4% triethylamine / dry acetone (41.0 mL) was added dropwise under stirring. The mixture was centrifuged at 4,000 rpm for 7 minutes, the supernatant was removed, and the precipitate was resuspended in acetone. This suspension and centrifugation procedure was repeated three more times. Finally, the precipitate was dried under reduced pressure to obtain 2'-Nb-7mGMP-Imidazolide (3) (164 mg, yield 62.4%) as a yellow solid.

[0150] 1 ​1H NMR (600 MHz, DMSO-D6) δ 9.55 - 9.26 (m, 1H), 7.92 - 7.81 (m, 1H), 7.74 - 7.60 (m, 3H), 7.54 - 7.43 (m, 1H), 7.11 (dd, J = 4.8, 1.3 Hz, 1H), 6.86 (dd, J = 5.5, 1.2 Hz, 1H), 5.95 (d, J = 5.2 Hz, 0.7H), 5.80 (d, J = 4.2 Hz, 0.3H), 5.42 (d, J = 5.8 Hz, 0.3H), 5.35 (d, J = 4.9 Hz, 0.7H), 5.09 - 5.02 (m, 0.3H), 4.89 - 4.80 (m, 1H), 4.71 - 4.66 (m, 1H), 4.61 (d, J = 7.1 Hz, 0.3H), 4.47 - 4.42 (m, 1H), 4.18 - 4.13 (m, 0.4H), 4.07 - 3.96 (m, 6H), 3.74 - 3.69 (m, 1H), 1.66 - 1.49 (m, 2H), 1.23 - 1.11 (m, 18H), 0.83 (m, 3H) ppm. 13 13C NMR (151 MHz, DMSO-D6) δ 150.28, 148.90, 148.19, 139.69, 137.04, 134.11, 129.23, 129.02, 128.95, 124.50, 124.32, 120.41, 108.56, 92.54, 87.33, 85.38, 78.59, 73.69, 69.36, 64.32, 40.60, 37.33, 35.84, 31.82, 29.54, 29.36, 29.27, 29.24, 25.42, 22.62, 14.48 ppm. 31 31P NMR (243 MHz, DMSO-D6) δ -9.57 ppm. C 33 H 46 N8O 10 P (M - H) - calcd. m / z 745.3080, found m / z 745.3158.

[0151] 1-3. Synthesis of Compound 4 2'-Nb-7mGMP-imidazolide (3) (164 mg, 0.213 mmol) was dissolved in DMF (4.27 mL), and tris(triethylammonium)phosphate (683 mg, 1.70 mmol) and zinc chloride (232 mg, 1.70 mmol) were added sequentially. After stirring at room temperature for 10 hours, the reaction was stopped by adding 500 mM EDTA-NaOH aqueous solution (pH 8.0, 34.0 mL, EDTA: 17.0 mmol). The mixture was then transmitted to a CHRMATREX C8 A31 SMB-100-20 / 45 column (column size: φ = 3.0 cm, h = 10 cm, 70 cm). 3 The solution was purified using a linear gradient (7 mL / min), solvent A: 50 mM TEAA buffer (pH 6.0) + 0.5% CH3CN, solvent B: CH3CN, 10-100% B / 60 min. The fraction containing the target compound was collected, concentrated, and lyophilized to obtain the target diphosphate compound (4) (161 mg, 164 μmol, yield 77.0%) as a white solid.

[0152] 1 ​H NMR(600 MHz, MeOD)δ 9.59 - 9.42(m, 0.5H), 7.86 - 7.80(m, 1H), 7.75 - 7.63(m, 2H), 7.49 - 7.41(m, 1H), 7.29(s, 0.5H), 6.10(d, J = 2.5 Hz, 0.8H), 5.90(d, J = 2.3 Hz, 0.2H), 5.25(dd, J = 7.6, 4.5 Hz, 0.4H), 5.20(dd, J = 7.6, 4.5 Hz, 1H), 5.04(d, J = 7.1 Hz, 0.6H), 4.98(s, 1H), 4.77(dd, J = 11.5, 7.1 Hz, 1H), 4.62 - 4.57(m, 1H), 4.52(dd, J = 6.8, 4.6 Hz, 1H), 4.42 - 4.34(m, 1H), 4.24 - 4.16(m, 2H), 4.14 - 4.06(m, 4H), 3.18(q, J= 7.3 Hz, 12H), 1.71(tt, J = 13.4, 7.4 Hz, 2H), 1.43 - 1.36(m, 1H), 1.30(t, J = 7.3 Hz, 18H), 1.23 - 1.16(m, 17H), 0.84(td, J = 7.1, 1.7 Hz, 3H)ppm. 13C NMR(151 MHz, MeOD)δ 175.39, 156.39, 156.30, 153.95, 153.92, 149.40, 149.28, 148.72, 148.35, 137.83, 137.53, 137.24, 137.04, 133.28, 133.13, 128.61, 128.51, 128.33, 128.15, 128.04, 123.88, 123.78, 107.61, 107.48, 93.76, 93.52, 88.64, 88.61, 84.50, 84.34, 84.28, 80.25, 80.02, 74.62, 74.32, 67.98, 63.39, 63.23, 46.03, 37.44, 37.38, 35.56, 35.54, 31.75, 29.47, 29.44, 29.41, 29.27, 29.16, 25.39, 25.31, 22.43, 20.61, 13.22, 7.84 ppm. 31 P NMR(243 MHz, MeOD)δ -9.28(1P), -10.69(1P)ppm. ESI-TOF-MS C 30 H 45 N6O 14 P2(M - H) - calcd. m / z 775.2475, found m / z 775.2741.

[0153] 1-4. Compound 5: Synthesis of C11Nb-modified chemical capping reagent 2'-Nb-7mGDP (4) (70.0 mg, 71.5 μmol), mimidazole (38.9 mg, 572 μmol), and 2,2'-dithiodipyridine (47.4 mg, 215 μmol) were dissolved in N,N-dimethylformamide (851 μL). Triethylamine (14.5 mg, 20.0 μL, 143 μmol) was added, followed by triphenylphosphine (56.4 mg, 215 μmol). After stirring at room temperature for 6 hours, a solution of sodium perchlorate (105 mg, 858 μmol) dissolved in 4% triethylamine / dry acetone (8.58 mL) was added dropwise under stirring. The mixture was centrifuged at 4,000 rpm for 7 minutes, the supernatant was removed, and the precipitate was resuspended in acetone. This suspension and centrifugation procedure was repeated three more times. Finally, the precipitate was dried under reduced pressure to obtain 2'-Nb-7mGDP-Imidazolide (5) (78.3 mg, quant.) as a white powder. In the following examples, compound 5 may be referred to as the "C11Nb-modified chemical capping reagent".

[0154] 1 ​H NMR(600 MHz, DMSO-D6)δ 9.71 - 9.43(m, 1H), 7.87(dt, J = 8.2, 1.7 Hz, 0.7H), 7.78(d, J = 7.9 Hz, 0.3H), 7.73 - 7.70(m, 1H), 7.68 - 7.63(m, 1H), 7.51 - 7.38(m, 1H), 7.17(d, J = 7.4 Hz, 1H), 6.78(d, J = 3.9 Hz, 1H), 5.93(d, J = 4.4 Hz, 0.7H), 5.75(d, J = 4.0 Hz, 0.3H), 5.69(d, J = 5.3 Hz, 0.3H), 5.61(d, J = 5.3 Hz, 0.7H), 5.10 - 4.91(m, 1H), 4.88 - 4.74(m, 1H), 4.70 - 4.55(m, 1H), 4.53 - 4.40(m, 1H), 4.34 - 4.16(m, 1H), 4.04 - 3.93(m, 4H), 3.89(d, J = 11.7 Hz, 1H), 3.82 - 3.76(m, 1H), 1.65 - 1.44(m, 2H), 1.23 - 1.05(m, 18H), 0.80(td, J = 7.1, 4.7 Hz, 3H)ppm. 13 C NMR(151 MHz, DMSO-D6)δ 150.19, 149.84, 148.77, 139.79, 137.37, 134.22, 129.15, 129.02, 128.32, 124.49, 120.71, 108.22, 93.16, 87.28, 85.35, 79.63, 74.00, 69.34, 63.66, 40.60, 37.39, 35.89, 31.82, 31.24, 29.53, 29.37, 29.30, 29.26, 29.23, 25.45, 22.62, 14.48 ppm. 31 P NMR(243 MHz, DMSO-D6)δ -10.62(1P), -19.05(1P)ppm. ESI-TOF-MS C 33 H 47 N8O 13 P2(M - H) -calcd. m / z 825.2743, found m / z 825.2775.

[0155] 1-5. Synthesis of tBuNb-modified chemical capping reagent Compound 5: A tBuNb-modified chemical capping reagent was synthesized using a method similar to the synthesis method for C11Nb-modified chemical capping reagent, in which a tert-butyl group was added instead of a C11 alkyl group.

[0156] 2. Chemical Capping Reaction and Cartridge Purification 2-1. Chemical Capping Reaction

[0157] 68-base 5' phosphorylated RNA (7mGpppA) encoding the HiBiT peptide m G mAGCCACCAUGGUGAGCGGCUGGCGGCUGUUCAAGAAGAUUAGCUGAAAAAAAAAAAAAAAAAAAAA: Sequence ID No. 5 was synthesized using an automated nucleic acid synthesizer. An aqueous solution of the 5' phosphorylated RNA (5 nmol) was measured out, 100 mM calcium chloride aqueous solution (50 μL) was added, and it was freeze-dried for 2.5 hours. The resulting solid was dissolved in DMSO (117 μL), and then 15 mM C11Nb modification chemical capping reagent (5) (333 μL, DMSO solution) or tBuNb modification chemical capping reagent (333 μL, DMSO solution) was added. Next, 100 mM 2-nitroimidazole (50 μL, DMSO solution) was added, and the mixture was treated at 55°C for 3-4 hours. The reaction solution (500 μL) was diluted with Milli Q water (500 μL), and then 3 M sodium acetate aqueous solution (125 μL) was added. Isopropanol (1.25 mL) was added, followed by 20 mg / mL glycogen aqueous solution, and the mixture was cooled at -80°C for 40 minutes. The RNA was precipitated by centrifugation (14,000 rpm, 4°C, 10 minutes). After removing the supernatant, 80% ethanol aqueous solution (1.00 mL) was added to the obtained RNA pellet, and the mixture was further centrifuged (14,000 rpm, 4°C, 7 minutes). After removing the supernatant, the RNA pellet was dried under reduced pressure for 20-30 minutes to obtain a crude product. The obtained RNA was dissolved in Milli-Q water (500 μL), and then 1 M TEAA buffer (pH 7) (500 μL) was added to obtain an RNA sample.

[0158] Figure 25 shows the UPLC analysis results for each reaction product. The upper part of Figure 25 shows the UPLC analysis results for the reaction product when using the tBuNb-modified chemical capping reagent. The peak around a retention time of 4.8 minutes represents unreacted material without a cap structure, and the peak around a retention time of 4.9 minutes represents tBuNb-modified capped mRNA. The lower part of Figure 25 shows the UPLC analysis results for the reaction product when using the C11Nb-modified chemical capping reagent. The peak around a retention time of 4.8 minutes represents unreacted material without a cap structure, and the peak around a retention time of 6.0 minutes represents C11Nb-modified capped mRNA. By introducing C11Nb modification, which is more hydrophobic than tBuNb, a significant delay of more than 1 minute in retention time was observed compared to uncapped RNA, resulting in clear separation.

[0159] The conditions for LCMS analysis are as follows: UPLC System: Agilent 1290 Infinity II, MS System: Agilent 6530 LC / Q-TOF, Method file: C18_0-90p_20min_60dG_300uL_woref, Column No. 2, ACQUITY UPLC Oligonucleotide BEH C18 Column, 130A, 1.7 um, 2.1 mm x 50 mm Part No., 186003949; Serial No., 04373306818442, Sol A; 100 mM HIFP (pH 8.3), 8.6 mM TEA Sol B; 100% MeOH, Column Temp.: 60 deg., Detection: 260 nm, Flow rate: 0.3 mL / min, Gradient Program: 0-90%B (0-12 minutes), 90%B (12.0-15 minutes), 90-0%B (15-15.1 minutes), 0%B (15.1-20 minutes)

[0160] 2-2. Cartridge Purification The RNA sample obtained in 2-1 was loaded onto a cartridge column MP-1602-10 MicroPure II column (LGC) and purified by cartridge. This column is packed with cross-linked vinylbenzene support with a particle size of 50–100 μm.

[0161] 2-2-1. RNA samples reacted using tBuNb-modified chemical capping reagent

[0162]

[0163] First, the column was conditioned with acetonitrile followed by 1M TEAA buffer. Then, the RNA sample reacted with the tBuNb modification chemical capping reagent obtained in 2-1 was loaded onto the column. Next, the column was washed with 5% acetonitrile / 0.1M TEAA buffer, and then eluted with 20% acetonitrile / 0.1M TEAA buffer, 30% acetonitrile / 0.1M TEAA buffer, 40% acetonitrile / 0.1M TEAA buffer, and 60% acetonitrile / 0.1M TEAA buffer.

[0164] Each eluate (Elution 1-4) was concentrated by centrifugation to a volume of 2 mL. Analysis of each eluate using a NanoDrop micro-spectrophotometer (Thermo Fisher Scientific) revealed the absorbance around 260 nm (Abs). 260 The elution values ​​were 1.585 for Elution 1 (20% acetonitrile / 0.1M TEAA buffer), 0.125 for Elution 2 (30% acetonitrile / 0.1M TEAA buffer), 0.030 for Elution 3 (40% acetonitrile / 0.1M TEAA buffer), and 0.036 for Elution 4 (60% acetonitrile / 0.1M TEAA buffer). These results suggest that most of the RNA adsorbed to the carrier was eluted with Elution 1 (20% acetonitrile / 0.1M TEAA buffer). Therefore, the samples eluted with 20% acetonitrile / 0.1M TEAA buffer and concentrated were subjected to purity confirmation by LCMS analysis, as described later.

[0165] 2-2-2. RNA samples reacted using C11Nb-modified chemical capping reagent

[0166]

[0167] First, the column was conditioned with acetonitrile followed by 1M TEAA buffer. Then, the RNA sample reacted with the C11Nb modification chemical capping reagent obtained in 2-1 was loaded onto the column. Next, the column was washed with 5% acetonitrile / 0.1M TEAA buffer, and then eluted with 20% acetonitrile / 0.1M TEAA buffer, 40% acetonitrile / 0.1M TEAA buffer, 60% acetonitrile / 0.1M TEAA buffer, and 80% acetonitrile / 0.1M TEAA buffer.

[0168] Each eluate (Elution 1-4) obtained by loading RNA samples reacted with a C11Nb-modified chemical capping reagent was concentrated by centrifugation to a volume of 300 μL. Analysis of each eluate using a NanoDrop micro-spectrophotometer revealed the absorbance around 260 nm (Abs). 260 The elution values ​​were 2.187 for Elution 1 (20% acetonitrile / 0.1M TEAA buffer), 3.417 for Elution 2 (40% acetonitrile / 0.1M TEAA buffer), 0.345 for Elution 3 (60% acetonitrile / 0.1M TEAA buffer), and 0.061 for Elution 4 (80% acetonitrile / 0.1M TEAA buffer). These results suggest that RNA adsorbed to the carrier was eluted with Elutions 1-3 (20%, 40%, 60% acetonitrile / 0.1M TEAA buffer). Therefore, samples eluted with 20%, 40%, and 60% acetonitrile / 0.1M TEAA buffer and concentrated were subjected to purity confirmation by LCMS analysis, as described later.

[0169] 2-3. Purity Confirmation by LC-MS Analysis 2-3-1. tBuNb-Modified Chemically Capped mRNA Samples eluted with 20% acetonitrile / 0.1M TEAA buffer in 2-2-1 and concentrated were analyzed by LC-MS. The results are shown in Figure 26. The obtained UPLC profile is shown at the top of Figure 26. A mixture of tBuNb-modified capped and uncapped mRNA was detected. The results of MS analysis of each UPLC peak are shown at the bottom of Figure 26. An MS peak of 22,257.770 Da was observed from the peak around a UPLC retention time of 4.7 minutes, and it was identified as unreacted material without a cap structure (calcd. 22,256.669 Da). An MS peak of 22,918.010 Da was observed from the peak around a UPLC retention time of 4.9 minutes, and it was identified as tBuNb-modified capped mRNA (calcd. 22,917.138 Da).

[0170] These results indicate that RNA samples reacted with a tBuNb-modified chemical capping reagent cannot be adequately separated into tBuNb-modified and uncapped RNAs by cartridge purification.

[0171] The conditions for LCMS analysis are as follows: UPLC System: Agilent 1290 Infinity II, MS System: Agilent 6530 LC / Q-TOF, Method file: C18_0-90p_20min_60dG_300uL_woref, Column No. 2, ACQUITY UPLC Oligonucleotide BEH C18 Column, 130A, 1.7 um, 2.1 mm x 50 mm Part No., 186003949; Serial No., 04373306818442, Sol A; 100 mM HIFP (pH 8.3), 8.6 mM TEA Sol B; 100% MeOH, Column Temp.: 60 deg., Detection: 260 nm, Flow rate: 0.3 mL / min, Gradient Program: 0-90%B (0-12 minutes), 90%B (12.0-15 minutes), 90-0%B (15-15.1 minutes), 0%B (15.1-20 minutes)

[0172] 2-3-2. C11Nb-modified chemically capped mRNA Samples eluted with 20%, 40%, and 60% acetonitrile / 0.1M TEAA buffer in 2-2-2 and concentrated were analyzed by LC-MS. The results are shown in Figure 27. From top to bottom left of Figure 27, the ULC profiles of samples eluted with 20%, 40%, and 60% acetonitrile / 0.1M TEAA buffer and concentrated are shown. In the ULC profile of the sample eluted with 20% acetonitrile / 0.1M TEAA buffer and concentrated, a peak originating from unreacted material without a cap structure was observed (top panel). In the ULC profile of the sample eluted with 40% acetonitrile / 0.1M TEAA buffer and concentrated, a peak originating from C11Nb-modified capped mRNA was detected (middle panel). UPLC profiles of concentrated samples eluted with 60% acetonitrile / 0.1M TEAA buffer showed peaks originating from multiple impurities, but no peaks originating from the target C11Nb-modified capped mRNA were observed (bottom panel).

[0173] Figure 27 shows the MS analysis results of the UPLC peak derived from C11Nb-modified capped mRNA eluted with 40% acetonitrile / 0.1M TEAA buffer. A peak at 23,1016.819 Da was observed, derived from C11Nb-modified capped mRNA (calcd. 23,015.328 Da). This result indicates that the desired C11Nb-modified capped mRNA was obtained in high purity by chemical capping with C11Nb modification and cartridge purification.

[0174] The conditions for LCMS analysis are as follows: UPLC System: Agilent 1290 Infinity II, MS System: Agilent 6530 LC / Q-TOF, Method file: C18_0-90p_20min_60dG_300uL_woref, Column No. 2, ACQUITY UPLC Oligonucleotide BEH C18 Column, 130A, 1.7 um, 2.1 mm x 50 mm Part No., 186003949; Serial No., 04373306818442, Sol A; 100 mM HIFP (pH 8.3), 8.6 mM TEA Sol B; 100% MeOH, Column Temp.: 60 deg., Detection: 260 nm, Flow rate: 0.3 mL / min, Gradient Program: 0-90%B (0-12 minutes), 90%B (12.0-15 minutes), 90-0%B (15-15.1 minutes), 0%B (15.1-20 minutes)

[0175] 2-4. Evaluation of Translation Efficiency The effect of C11Nb modification on translation efficiency was evaluated. An overview of this study is shown in Figure 28. Specifically, mRNA from which C11Nb modification was removed by light irradiation (i.e., unmodified mRNA) (HiBiT A in Figure 29), tBuNb-modified capped mRNA (HiBiT B in Figure 29), and C11Nb-modified capped mRNA (HiBiT C in Figure 29) were transfected into HeLa cells using Lipofectamine® Messenger MAX (Thermo Fisher Scientific). 24 hours after transfection, a luciferase assay was performed using the Nano-Glo® Luciferase Assay System (Promega) to evaluate translational activity. The results are shown in Figure 29.

[0176] As shown in Figure 29, no significant differences in translational activity were observed among HiBiT A, HiBiT B, and HiBiT C. This suggests that C11Nb modification has little effect on mRNA translation efficiency.

[0177] 3. Demonstration of simultaneous chemical capping and parallel purification of multiple samples 3-1. Chemical capping reaction Eleven types of mRNA (encoding HiBiT peptide) with different chemical modifications were designed and solid-phase synthesis (200 nmol scale synthesis) was performed using a MerMade 48X synthesizer to synthesize RNA with a 5'-terminal monophosphate structure. The synthesized RNA with a 5'-terminal monophosphate structure was purified using a Presep DNA / RNA type A (255 mg / 3 mL) column (Fujifilm Wako Pure Chemical Industries), and then chemical capping was performed using C11Nb-modified 7mGDP-imidazolide (compound 5 of Example A1). The structure of each mRNA after chemical capping is shown in Figure 30. Note that although these mRNAs have different chemical modifications, their base sequences are identical (SEQ ID NO: 5). In Figure 30, X m X represents a 2'-O-methylated nucleotide. f indicates a 2'-O-deoxyfluoronucleotide. ^ indicates a phosphorothioate modification.

[0178] Analysis of each chemical capping reaction product by LC-MS revealed that each sample contained both the target C11Nb-capped mRNA and uncapped mRNA.

[0179] 3-2. Cartridge Purification The RNA sample obtained in 3-1 was loaded into a cartridge column Presep DNA / RNA type A (255 mg / 3 mL) (Fujifilm Wako Pure Chemical Industries) and purified by cartridge. In this test, a solid-phase extraction vacuum manifold (GL Sciences) was used, and the solution was passed through by suction. Details are described below.

[0180]

[0181] First, the column was conditioned with acetonitrile followed by 1M TEAA buffer, after which each RNA sample was loaded onto the column. Next, the samples were washed with 5% acetonitrile / 0.1M TEAA buffer, and then sequentially eluted with 20% acetonitrile / 0.1M TEAA buffer, 40% acetonitrile / 0.1M TEAA buffer, 60% acetonitrile / 0.1M TEAA buffer, and 80% acetonitrile / 0.1M TEAA buffer.

[0182] Each eluate (Elution 1-4) was concentrated by centrifugation, and each eluate was analyzed using a NanoDrop micro-spectrophotometer. Elutions showing absorption around 260 nm were subjected to denatured polyacrylamide electrophoresis (dPAGE). The results are shown in Figure 31. The numbers at the top of the lanes correspond to the 11 types of mRNA shown in Figure 30. As shown in Figure 31, a band originating from the 68-base + cap structure of the target length was observed for all mRNAs.

[0183] Samples were eluted with 20%, 40%, and 60% acetonitrile / 0.1M TEAA buffer, concentrated, and subjected to LC-MS analysis. The results are shown in Figures 32-42. As shown in Figures 32-42, in all mRNA samples, RNA without a cap structure was eluted with 20% acetonitrile / 0.1M TEAA buffer, the target C11Nb-modified capped mRNA was eluted with 40% acetonitrile / 0.1M TEAA buffer, and unreacted capping reagent was eluted with 60% acetonitrile / 0.1M TEAA buffer. These results demonstrate that the target C11Nb-modified capped mRNA can be separated from RNA without a cap structure and unreacted capping reagent by cartridge purification.

[0184] The conditions for LCMS analysis are as follows: UPLC System: Agilent 1290 Infinity II, MS System: Agilent 6530 LC / Q-TOF, Method file: C18_0-90p_20min_60dG_300uL_woref, Column No.: 2, ACQUITY UPLC Oligonucleotide BEH C18 Column, 130A, 1.7 um, 2.1 mm x 50 mm Part No.: 186003949; Serial No.: 0413320191832; Lot No.: 0413320191, Sol A: 100 mM HIFP (pH 8.3), 8.6 mM TEA Sol B: 100% MeOH, Column Temp.: 60 deg., Detection: 260 nm, Flow rate: 0.3 mL / min, Gradient Program: 0-60%B (0-12 minutes), 60-90%B (12-12.1 minutes), 90%B (12.1-15 minutes), 90-0%B (15-15.1 minutes), 0%B (15.1-20 minutes), Injection volume and solution condition: 10 μL (Milli Q water), Injection amount: 10 pmol

[0185] 4. Synthesis of chemical capping reagents 2

[0186] The chemical capping reagent was synthesized according to the above scheme. Each step of the synthesis is described in detail below.

[0187] 4-1. Synthesis of 4-(dodecyloxymethyl)benzyl bromide

[0188] 1-dodecanol (1.86 g, 10 mmol, 1.0 equiv) was dissolved in THF (25 mL), and sodium hydride (60% oil dispersion, 600 mg, 15 mmol, 1.5 equiv), washed with hexane, was added at 0°C. After stirring at the same temperature for 1 hour, α,α'-dibromo-p-xylene (5.28 g, 20 mmol, 2.0 equiv) was added to the reaction solution along with THF (15 mL). After stirring at room temperature for 24 hours, the resulting solid was filtered off using celite. The filtrate was concentrated under reduced pressure, and hexane was added to recover the excess α,α'-dibromo-p-xylene as yellow crystals (2.37 g, 8.98 mmol). The residue was purified by column chromatography using silica gel (hexane / DCM 0-20%) to obtain 4-(dodecyloxymethyl)benzyl bromide as a white solid (2.04 g, 5.52 mmol, 55%).

[0189] 1 H NMR (400 MHz, CDCl3): δ 7.37 (d, J = 8.1 Hz, 2H), 7.31 (d, J = 8.3 Hz, 2H), 4.50 (s, 2H), 4.49 (s, 2H), 3.46 (t, J = 6.6 Hz, 2H), 1.61 (quint, J = 7.0 Hz, 2H), 1.40-1.20 (m, 18H), 0.88 (t, J = 6.9 Hz, 3H) ppm.

[0190] 4-2. Synthesis of 2'-O-(4-(dodecyloxymethyl)benzyl)-N2-isobutyrylguanosine

[0191] ​N2-isobutyrylguanosine (1.06 g, 3.0 mmol, 1.0 equiv) was dissolved in DMF (9 mL) and stirred at 0°C. Sodium hydride (60% oil dispersion, 288 mg, 7.2 mmol, 2.4 equiv), washed with hexane, was added to this solution, and the generation of hydrogen gas was confirmed. After 40 minutes, it was confirmed that the generation of hydrogen gas had stopped, and 4-(dodecyloxymethyl)benzyl bromide (1.66, 4.5 mmol, 1.5 equiv) was added to the resulting white suspension at 0°C. After continuing stirring for a further 5 hours, the reaction was quenched with ethanol (2 mL) and neutralized with 1N hydrochloric acid (7.2 mL). The reaction vessel was removed from the ice bath and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (DCM / MeOH 5%) to obtain 2'-O-(4-(dodecyloxymethyl)benzyl)-N2-isobutyrylguanosine (977 mg, 1.52 mmol, 51%) as a white solid.

[0192] 1 H NMR (400 MHz, DMSO-d6): δ 12.1 (s, 1H), 11.6 (s, 1H), 8.20 (s, 1H), 7.20-7.15 (m, 4H), 5.95 (d, J = 6.3 Hz, 1H), 5.32 (d, J = 4.9 Hz, 1H), 5.07 (t, J = 5.4 Hz, 1H), 4.68 (d, J = 12.1 Hz, 1H), 4.49-4.40 (m, 2H), 4.39-4.30 (m, 3H), 3.93 (dd, J = 7.3, 4.2 Hz, 1H), 3.67-3.51 (m, 2H), 3.36 (t, J = 6.5 Hz, 2H), 2.77 (sept, J = 6.8 Hz, 1H), 1.54-1.45 (m, 2H), 1.33-1.17 (m, 18H), 1.12 (d, J = 6.7 Hz, 6H), 0.85 (t, J = 6.8 Hz, 3H) ppm.

[0193] ​4-3. Synthesis of 2'-O-(4-(dodecyloxymethyl)benzyl)guanosine

[0194] 2'-O-(4-(dodecyloxymethyl)benzyl)-N2-isobutyrylguanosine (963 mg, 1.5 mmol, 1.0 equiv) was dissolved in acetonitrile (12 mL), and aqueous ammonia (28%, 30 mL) was added and the mixture was stirred at 55°C for 6 hours. The reaction solution was then concentrated under reduced pressure and lyophilized to obtain 2'-O-(4-(dodecyloxymethyl)benzyl)guanosine (736 mg, 1.29 mmol, 86%) as a white solid.

[0195] 1 H NMR (400 MHz, DMSO-d6): δ 7.90 (s, 1H), 7.23-7.19 (m, 4H), 6.47-6.40 (brs, 2H), 5.87 (d, J = 6.0 Hz, 1H), 5.26 (d, J = 5.2 Hz, 1H), 5.07 (t, J = 5.5 Hz, 1H), 4.66 (d, J = 12.0 Hz, 1H), 4.49 (d, J = 12.1 Hz, 1H), 4.39 (s, 2H), 4.35-4.26 (m, 2H), 3.94 (q, J = 3.6 Hz, 1H), 3.65-3.49 (m, 2H), 3.37 (t, J = 6.3 Hz, 2H), 1.55-1.46 (m, 2H), 1.33-1.17 (m, 18H), 0.85 (t, J = 6.8 Hz, 3H) ppm.

[0196] ​4-4. Synthesis of 2'-O-(4-(dodecyloxymethyl)benzyl)-N7-methylguanosine 2'-O-(4-(dodecyloxymethyl)benzyl)guanosine (686 g, 1.2 mmol, 1.0 equiv) was dissolved in DMF (5 mL), and methyl iodide (598 μL, 9.6 mmol, 8.0 equiv) was added dropwise at room temperature. After stirring for 24 hours, the reaction solution was concentrated under reduced pressure to obtain 2'-O-(4-(dodecyloxymethyl)benzyl)-N7-methylguanosine (795 g, 1.11 mmol, 93%) as a pale yellow solid.

[0197] 1 H NMR (400 MHz, DMSO-d6): δ 11.70 (s, 1H), 9.22 (s, 1H), 7.33 (d, J = 8.1 Hz, 2H), 7.24 (d, J = 8.3 Hz, 2H), 6.00 (d, J = 3.3 Hz, 1H), 5.45-5.32 (brs, 1H), 5.25-5.06 (brs, 1H), 4.76 (d, J = 12.1 Hz, 1H), 4.65 (d, J = 12.1 Hz, 1H), 4.41 (s, 2H), 4.32-4.26 (m, 2H), 4.08-4.03(m, 1H), 3.97 (s, 3H), 3.74 (dd, J = 12.4, 3.2 Hz, 1H), 3.63 (dd, J = 12.1, 3.6 Hz, 1H), 3.39 (t, J = 6.5 Hz, 2H), 1.52 (quint, J = 6.8 Hz, 2H), 1.34-1.12 (m, 18H), 0.85 (t, J = 6.8 Hz, 1H) ppm.

[0198] 4-5. Synthesis of 2'-O-(4-(dodecyloxymethyl)benzyl)-N7-methylguanosine 5'-O-monophosphate

[0199] ​2'-O-(4-(dodecyloxymethyl)benzyl)-N7-methylguanosine (214 mg, 0.30 mmol, 1.0 equiv) was dissolved in trimethyl phosphate (1.5 mL) and stirred at -10°C. 2,6-lutidine (86.4 μL, 0.75 mmol, 2.5 equiv) and trichloride phosphate (70.1 μL, 0.75 mmol, 2.5 equiv) were added to the reaction solution, and stirring was continued at the same temperature for 6 hours. The reaction was then quenched with TBAB buffer solution (0.2 M, 3 mL) and diluted with methanol / Milli Q water (25 / 25 mL). The reaction mixture was purified by reverse-phase column chromatography (C8, A: 50 mM TBAA buffer with 0.5% ACN, B: ACN 10-90% linear gradient). The fraction containing the target product was concentrated and lyophilized to obtain 2'-O-(4-(dodecyloxymethyl)benzyl)-N7-methylguanosine 5'-O-monophosphate (33.5 mg, 0.0503 mmol, 17%) as a white solid.

[0200] 1 H NMR (600 MHz, CD3OD): δ 7.41 (d, J = 7.8 Hz, 2H), 7.30 (d, J = 7.8 Hz, 2H), 6.16 (d, J = 3.7 Hz, 1H), 4.78 (d, J = 12.2 Hz, 1H), 4.55-4.48 (m, 4H), 4.34-4.25 (m, 2H), 4.16-4.10 (m, 4H), 3.69-3.66 (m, 2H), 3.52 (t, J = 6.6 Hz, 2H), 1.65 (quint, J = 6.9 Hz, 2H), 1.45-1.29(m, 18H), 0.94 (t, J = 6.9 Hz, 3H) ppm. 31 P NMR (243 MHz, CD3OD): δ 1.39 ppm.

[0201] ​4-6. Synthesis of 2'-O-(4-(dodecyloxymethyl)benzyl)-N7-methylguanosine 5'-O-monophosphate imidazolide

[0202] 2'-O-(4-(dodecyloxymethyl)benzyl)-N7-methylguanosine 5'-O-monophosphate (33.5 mg, 0.050 mmol, 1.0 equiv), imidazole (27.2 mg, 0.40 mmol, 8.0 equiv), 2,2'-dithiodipyridine (33.0 mg, 0.15 mmol, 3.0 equiv), and DMF (1 mL) were added and the mixture was stirred at room temperature. Triethylamine (13.9 μL, 0.10 mmol, 2.0 equiv) and triphenylphosphine (39.3 mg, 0.15 mmol, 3.0 equiv) were added, and the mixture was stirred for 6 hours. Sodium hypochlorite solution (0.2 M in acetone with 4% NEt3, 2 mL, 0.40 mmol, 8 equiv) was added dropwise to the reaction mixture to obtain a white precipitate. This precipitate was collected by centrifugation, washed with acetone, and then dried under vacuum to obtain 2'-O-(4-(dodecyloxymethyl)benzyl)-N7-methylguanosine 5'-O-monophosphate imidazolide (21.3 mg, 0.0289 mmol, 58%).

[0203] 1 ​H NMR(600 MHz, DMSO-d6): δ 9.15(s, 1H), 7.65-7.64(m, 1H), 7.31(d, J = 8.2 Hz, 2H), 7.22(d, J = 8.2 Hz, 2H), 7.10-7.09(m, 1H), 6.87(s, 1H), 5.99(d, J = 3.7 Hz, 1H), 5.63(s, 1H), 5.40-5.36(brs, 1H), 4.73(d, J = 12.2 Hz, 1H), 4.62(d, J = 12.2 Hz, 1H)4.41(s, 2H), 4.27(t, J = 4.3 Hz, 1H), 4.14-4.10(m, 1H), 4.06-4.01(m, 2H), 3.98(s, 3H), 3.39(t, J = 6.6 Hz, 2H), 1.51(quint, J = 7.0 Hz, 2H), 1.32-1.18(m, 18H), 0.85(t, J = 7.0 Hz, 3H)ppm. 31 P NMR(243 MHz, DMSO-d6): δ -9.49 ppm.

[0204] 4-7. Synthetic phosphoric acid of 2'-O-(4-(dodecyloxymethyl)benzyl)-N7-methylguanosine 5'-O-diphosphate (12.1 μL, 0.232 mmol) and triethylamine (32.2 μL, 1.39 mmol) were stirred in acetonitrile solvent (1 mL) at room temperature for 3 hours, and tris(triethylammonium)phosphate was prepared by removing volatile substances. 2'-O-(4-(dodecyloxymethyl)benzyl)-N7-methylguanosine 5'-O-monophosphate imidazolide (21.3 mg, 0.0289 mmol, 1.0 equiv), the prepared tris(triethylammonium)phosphate (0.232 mmol, 8.0 equiv), and DMSO (1 mL) were packed and stirred at room temperature. Zinc chloride (31.6 mg, 0.232 mmol, 8.0 equiv) was added to the mixture and stirring was continued for 24 hours. After the reaction was complete, the mixed solution was cooled to 0°C and EDTA buffer (pH = 8.0, 3 mL) was added. The resulting reaction mixture was purified by reverse-phase column chromatography (C8 column, eluate A: 50 mM TEAA buffer + 0.5% acetonitrile, eluate B: acetonitrile, B 10 - 90%). The resulting fraction was concentrated and lyophilized to obtain 2'-O-(4-(dodecyloxymethyl)benzyl)-N7-methylguanosine 5'-O-diphosphate (7.6 mg, 8.02 μmol, 28%).

[0205] 1 ​H NMR (600 MHz, CD3OD-d6): δ 7.45 (d, J = 8.2 Hz, 2H), 7.30 (d, J = 7.8 Hz, 2H), 6.16 (d, J = 3.1 Hz, 1H), 4.68-4.70 (m, 1H), 4.54-4.52 (m, 1H), 4.51 (s, 2H), 4.34-4.29 (m, 2H), 4.16 (s, 3H), 3.51 (t, J = 6.6 Hz, 2H), 1.68-1.61 (m, 2H), 1.49-1.28 (m, 18H), 0.95 (t, J = 7.1 Hz, 3H) ppm.

[0206] 4-8. Synthesis of 2'-O-(4-(dodecyloxymethyl)benzyl)-N7-methylguanosine 5'-O-diphosphate imidazolide

[0207] 2'-O-(4-(dodecyloxymethyl)benzyl)-N7-methylguanosine 5'-O-diphosphate (7.6 mg, 8.0 μmol, 1.0 equiv), imidazole (4.4 mg, 64 μmol, 8.0 equiv), 2,2'-dithiodipyridine (5.3 mg, 24 μmol, 3.0 equiv), and DMF (200 μL) were added to a 15 mL Falcon tube and stirred at room temperature. Triethylamine (2.2 μL, 16 μmol, 2.0 equiv) and triphenylphosphine (6.3 mg, 24 μmol, 3.0 equiv) were added, and the mixture was stirred for 7 hours. Sodium hypochlorite solution (0.2 M in acetone with 4% NEt3, 1 mL) was added dropwise to the reaction mixture to obtain a white precipitate. This precipitate was collected by centrifugation, washed with acetone, and then dried under vacuum to obtain 2'-O-(4-(dodecyloxymethyl)benzyl)-N7-methylguanosine 5'-O-diphosphate imidazolide.

[0208] 1 ​H NMR (600 MHz, DMSO-d6): δ 7.71 (s, 1H), 7.36 (d, J = 7.8 Hz, 2H), 7.23 (d, J = 7.8 Hz, 2H), 7.20 (s, 1H), 6.78 (s, 1H), 6.01 (d, J = 3.1 Hz, 1H), 5.75 (s, 1H), 5.62-5.55 (brs, 1H), 4.78 (d, J = 12.6 Hz, 1H), 4.68 (J = 12.2 Hz, 1H), 4.43-4.36 (m, 4H), 4.05-3.96 (m, 5H), 3.94-3.91 (m, 1H), 3.42-3.39 (m, 2H), 1.54-1.49 (m, 2H), 1.31-1.18 (m, 18H), 0.85 (t, J = 6.8 Hz, 3H) ppm.

[0209] 5. Synthesis and Cartridge Purification of Non-Photo-Less Hydrophobic Tagged mRNA 5-1. Synthesis of Non-Photo-Less Hydrophobic Tagged mRNA p-mAmGAGCCACCATGGTGAGCGGCTGGCGGCTGTTCAAGAAGATTAGCTGAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 6) was synthesized using an automated nucleic acid synthesizer (Amidite: 2'-O-TOM nucleic acid amidite 50 mM acetonitrile solution, Oxidizing agent: Iodine 50 mM pyridine / water (9:1) solution, Activator: 5-benzylthio-1H-tetrazole 25 mM acetonitrile solution, Cap A: Anhydrous acetic acid 10% THF solution, Cap B: 1-methylimidazole 10% THF / pyridine (8:1) solution).

[0210] To the resin obtained by an automated nucleic acid synthesizer, ammonia water (28%, 500 μL) and methylamine (40%, 500 μL) were added, and the mixture was reacted at 60°C for 30 minutes to cleave the oligonucleotides from the resin and deprotect the acyl groups. The reaction solution was dried under reduced pressure, and the resulting residue was mixed with TBAF THF solution (1 M, 1 mL) and allowed to stand at room temperature for 15 hours to deprotect the TOM groups. Subsequently, Tris-HCl buffer solution (1 M, pH = 7.5, 1 mL) was added to neutralize the mixture. After removing the THF from the solution under reduced pressure to reduce the volume to approximately 1 mL, the solution was passed through a NAP-25 column for desalting. Sodium acetate aqueous solution and isopropyl alcohol were added to the resulting solution, and the mixture was allowed to stand at -30°C for 1 hour to obtain a precipitate. This precipitate was collected by centrifugation, washed with 80% ethanol, and dried to obtain an aqueous solution of 5'-phosphorylated RNA (110 μM, 300 μL, 33 nmol, 17%).

[0211] Synthesized 5'-phosphorylated RNA (5 nmol) was mixed with an aqueous calcium chloride solution (100 mM, 50 μL, 5 μmol) and lyophilized. The resulting solid was mixed with DMSO (117 μL), and 2'-O-(4-(dodecyloxymethyl)benzyl)-N7-methylguanosine 5'-O-diphosphate imidazolide or 2'-O-4-n-butylbenzyl-N7-methylguanosine 5'-O-diphosphate imidazolide (15 mM in DMSO, 333 μL, 5 μmol) or 2-nitroimidazole (100 mM in DMSO, 50 μL, 5 μmol) obtained in step 4-8 was added, and the mixture was incubated at 55°C for 3 hours. After the reaction was complete, Milli Q water (500 μL), sodium acetate aqueous solution (3 M, pH = 5.2, 125 μL), and isopropyl alcohol (1250 μL) were added, and vortexing was performed. The mixture was then allowed to stand at -30°C for 1 hour. The resulting precipitate was collected by centrifugation, washed with 80% ethanol, and then dried. The structures of the reaction products of 5'-phosphorylated RNA and 2'-O-(4-(dodecyloxymethyl)benzyl)-N7-methylguanosine 5'-O-diphosphate imidazolide (Cap2, C12OM-Bn modified HiBiT mRNA) and 5'-phosphorylated RNA and 2'-O-4-n-butylbenzyl-N7-methylguanosine 5'-O-diphosphate imidazolide (Cap2, 4nBuBn modified HiBiT mRNA) are shown below.

[0212] 5-2. Cartridge Purification Each reaction product obtained in 5-1 was purified using a cartridge column Presep DNA / RNA type A (255 mg / 3 mL) (Fujifilm Wako Pure Chemical Industries) (equilibrium: 100% acetonitrile 2 mL x 2, 1 M TEAA 3 mL x 2; washing: 5% acetonitrile w / 0.1 M TEAA; eluate 1: 20% acetonitrile w / 0.1 M TEAA 2 mL x 2; eluate 2: 40% acetonitrile w / 0.1 M TEAA 2 mL x 2; eluate 3: 60% acetonitrile w / 0.1 M TEAA 2 mL x 2). The column was packed with octadecylsilyl-modified silica gel with a particle size of 40 μm. Acetonitrile was removed from the obtained fraction under reduced pressure, and precipitation was performed using isopropyl alcohol as described above.

[0213] In cartridge purification of the reaction product of 5'-phosphorylated RNA and 2'-O-4-n-butylbenzyl-N7-methylguanosine 5'-O-diphosphate imidazolide, analysis of the fraction obtained by eluting with 20% acetonitrile using liquid-phase chromatography revealed that both capped and uncapped products were eluted simultaneously (Figure 43). This result suggests that cartridge purification cannot separate capped and uncapped products using the 4nBuBn modification tag.

[0214] In cartridge purification of the reaction product of 5'-phosphorylated RNA and 2'-O-(4-(dodecyloxymethyl)benzyl)-N7-methylguanosine 5'-O-diphosphate imidazolide, analysis of the fraction obtained from the eluate with 20% acetonitrile using liquid-phase chromatography revealed that only the uncapped product eluted. Analysis of the fraction obtained from the eluate with 40% acetonitrile using liquid-phase chromatography also revealed the elution of the capped product via detection MS (peak top: 6.09 min, Calcd, 22983.9, Found, 22985.8030). Analysis of the fraction obtained from the eluate with 60% acetonitrile showed no elution of capped RNA (Figure 44). These results suggest that the capped and uncapped products can be separated by cartridge purification if the C12OM-Bn modification tag is used.

[0215] 5-3. Evaluation of Intracellular Translational Activity An overview of this study is shown in Figure 45. Specifically, HeLa cells (RIKEN Cell Bank) were first cultured in Dulbecco's modified Eagle's medium (DMEM; Fujifilm Wako Pure Chemical Industries) supplemented with 10% fetal bovine serum (FBS; Invitrogen) (37 °C, 5% CO2). The day before transfection, HeLa cells were seeded into a 96-well plate (1.0 × 10⁶). 4(cell / well). The next day, the medium was removed and replaced with 100 μL / well Opti-MEM (Thermo Fisher Scientific). 0.15 μL Lipofectamine MessengerMAX, 25, and 50 ng RNA were diluted in 10 μL Opti-MEM and introduced into the cells. After 2 hours, the medium was replaced with Dulbecco's modified Eagle's medium (DMEM; Fujifilm Wako Pure Chemical Industries) supplemented with 10% fetal bovine serum (FBS; Invitrogen). After incubation at 37 °C for 6 and 24 hours, luminescence was measured using the Nano Glo HiBiT Lytic Detection System (Promega). The results are shown in Figure 46. As shown in Figure 46, synthetic mRNA with Cap2 and C12OM-Bn modifications showed higher translational activity compared to RNA without a cap structure (Cap(-)).

[0216] 6. Cartridge purification of 5' monophosphorylated nucleic acids 6-1. Synthesis of 5' monophosphorylation reagents

[0217] A stirred solution of compound 19 (0.100 g, 1.0 equivalent, 0.24 mmol) dissolved in DCM (4.0 mL) was cooled to 0 °C, and DIPEA (0.062 mL, 0.286 mmol, 1.5 equivalents) was added, followed by CEP-Cl (0.064 mL, 0.286 mmol, 1.2 equivalents). After the total amount was added, the reaction mixture was gradually heated to room temperature and stirred for 2.0 hours. The reaction was then stopped by adding saturated NaHCO3 aqueous solution. The aqueous layer was extracted three times with DCM, and the resulting organic layer was washed with saline solution, dried over anhydrous Na2SO4, filtered, and concentrated. The resulting pale yellow oily substance was purified by flash column chromatography to obtain the target compound 20 as a pale yellow semi-solid.

[0218] 1 ​H-NMR(400 MHz, CHLOROFORM-D)δ 7.86(dd, J = 8.2, 1.3 Hz, 1H), 7.81(dd, J = 8.0, 1.2 Hz, 1H), 7.61-7.57(m, 1H), 7.39-7.35(m, 1H), 5.42-5.36(m, 1H), 3.93-3.79(m, 2H), 3.50-3.41(m, 2H), 2.69-2.60(m, 2H), 1.83-1.72(m, 2H), 1.50-1.34(m, 2H), 1.30-1.24(m, 32H), 1.12(d, J = 6.8 Hz, 6H), 0.88-0.84(m, 9H)ppm. 13 C-NMR(101 MHz, CHLOROFORM-D)δ 147.5, 139.7, 133.2, 129.2, 127.9, 124.1, 117.6, 77.4, 77.1, 76.8, 70.6, 70.4, 58.9, 58.8, 43.3, 43.2, 39.3, 39.3, 32.0, 29.8, 29.7, 29.7, 29.6, 29.4, 29.3, 25.7, 24.8, 24.7, 24.1, 24.1, 22.8, 20.5, 20.4, 14.2 ppm. 31 P-NMR(162 MHz, CHLOROFORM-D)δ 148.8 ppm.

[0219] 6-2. Solid-phase DNA synthesis 5'-Phosphate ODN4 DNA (pTAATACGACTCACTATAGG, SEQ ID NO: 7) was synthesized on a 1 μmol scale using C19 phosphoramidite (20) with a DNA / RNA synthesizer NR-2A_7MX or NRs-4A_10R7NP (Nihon Techno Service). DNA phosphoramidite and CPG (Chemgenes; deoxyguanosine (n-ibu) 3'-lcaa CPG, 1000A TV 47.3 μmol / g) were used. For DNA synthesis, a 50 mM DNA phosphoramidite and 100 mM C19-Phosphoramidite solution (acetonitrile / DCM = 1:1) was used. The reagents used for the synthesis apparatus were as follows. For deprotection, a 3 w / v % trichloroacetic acid / dichloromethane solution was used; for coupling, a 0.25 M 5-benzylthio-1H-tetrazole / acetonitrile solution (Fujifilm Wako Pure Chemical Industries) was used; for capping, an anhydride / tetrahydrofuran / pyridine (1:8:1, Fujifilm Wako Pure Chemical Industries) and a 10 (v / v) % 1-methylimidazole / tetrahydrofuran solution (Fujifilm Wako Pure Chemical Industries) were used; and for oxidation, a 0.01 M iodine / 64% acetonitrile, 6% pyridine, 30% aqueous solution (Honeywell) was used.

[0220] After synthesis, the DNA was excised from the support and deprotected in a 40% methylamine hydroxide-28% ammonium hydroxide (1:1) solution at 65 °C for 30 minutes. The solution was dried using a vacuum concentrator and then dissolved in 500 μL of water. The structure of the target reaction product is shown below.

[0221] 6-3. Cartridge Purification The reaction product obtained in 6-2 was purified using a Presep DNA / RNA type A (255 mg / 3 mL) column (Fujifilm Wako Pure Chemical Industries). The column was pre-washed with 2 × 3 mL of acetonitrile (ACN), and then washed with 2 × 3 mL of 1 M triethylammonium acetate (TEAA) buffer (pH 7.0). The sample was loaded onto the column and sequentially washed with 2 × 2 mL of 5% ACN / TEAA (pH 7.0), followed by elution with 2 × 3 mL each of 20%, 40%, 60%, and 80% ACN / TEAA (pH 7.0). All eluted fractions were collected individually, concentrated, and precipitated with 3 M sodium acetate (pH 5.2) and 2-propanol.

[0222] The DNA concentration was determined by measuring the absorbance at 260 nm using a NanoDrop micro-spectrophotometer and using the extinction coefficient calculated with Oligo-Analyzer software (IDT). The mass of the target DNA was then confirmed by LC-MS. The results are shown in Figure 47. Elution of the target reaction product was confirmed in the 40% and 60% ACN / TEAA (pH 7.0) elution fractions.

[0223] DNA purity was evaluated by denatured polyacrylamide gel electrophoresis (dPAGE) in the presence of 7.5 M urea. The gel was stained with SYBR Green II and visualized using the ChemiDoc XRS Plus system (Bio-Rad). The results are shown in Figure 48. As shown in Figure 48, bands believed to be the target reaction product were confirmed in the 40% and 60% ACN / TEAA (pH 7.0) eluted fractions, consistent with the LCMS results. These results suggest that 5' monophosphorylated DNA can also be purified by cartridge using the technique described herein.

[0224] 6-4. Solid-phase RNA synthesis and cartridge purification 5'-Phosphate a12 LW RNA (pAmGmAGCCACCAUGGAGAGCAUCCUGGACGAGCACCUGCAGAGAGUGUGGUGAAAAAAAAAAAAAAAAAAAAA, SEQ ID NO: 8) was synthesized on a 0.2 μmol scale using C19 phosphoramidite in the same manner as the DNA described above. After excision from the support and deprotection, the RNA was treated overnight at room temperature with 1 M tetrabutylammonium fluoride / THF solution (1.0 mL). The reaction mixture was stopped by adding 1 M Tris-HCl buffer (pH 7.5), and the mixture was concentrated to remove THF.

[0225] Subsequently, the DNA / RNA was purified using a Presep DNA / RNA type A (255 mg / 3 mL) column (Fujifilm Wako Pure Chemical Industries). The column was pre-washed with 2 × 3 mL of acetonitrile (ACN), followed by washing with 2 × 3 mL of 1 M triethylammonium acetate (TEAA) buffer (pH 7.0). The sample was loaded onto the column and sequentially washed with 2 × 2 mL of 5% ACN / TEAA (pH 7.0), followed by elution with 2 × 3 mL each of 20%, 40%, 60%, and 80% ACN / TEAA (pH 7.0). All eluted fractions were collected individually, concentrated, and precipitated with 3 M sodium acetate (pH 5.2) and 2-propanol.

[0226] The RNA concentration was determined by measuring the absorbance at 260 nm using a NanoDrop micro-spectrophotometer and calculating the extinction coefficient using Oligo-Analyzer software (IDT). The mass of the target RNA was then confirmed by LC-MS. The results are shown in Figure 49. Elution of the target reaction product was confirmed in the 40% and 60% ACN / TEAA (pH 7.0) elution fractions.

[0227] RNA purity was evaluated by denatured polyacrylamide gel electrophoresis (dPAGE) in the presence of 7.5 M urea. The gel was stained with SYBR Green II and visualized using the ChemiDoc XRS Plus system (Bio-Rad). The results are shown in Figure 50. As shown in Figure 50, bands believed to be the target reaction product were confirmed in the 40% and 60% ACN / TEAA (pH 7.0) eluted fractions, consistent with the LCMS results. These results suggest that 5' monophosphorylated RNA can also be purified by cartridge using the technique described herein.

[0228] 7. HiBit nC 18 - Synthesis of capped mRNA and cartridge purification

[0229] According to the above scheme, HiBit nC 18 -Capped mRNA was synthesized. Each step of the synthesis is described in detail below.

[0230] 7-1. 2'-C 18 H 37 - Synthesis of Guanosine (2)

[0231] NaH (4.52 g, 169.8 mmol, 6.0 equiv.), nC 18 H 37 Compound I (10.23 g, 26.88 mmol, 0.95 equivalents) and guanosine isobutyrate 1 (10.0 g, 28.3 mmol, 1.0 equivalent) were dissolved in anhydrous DMF (100.0 mL) and treated in a two-necked round-bottom flask at 0 °C. The reaction mixture was gradually warmed to room temperature while stirring was continued. After 24 hours, EtOH:H2O (1:1) was added and the mixture was stirred for a further 16 hours at 50 °C. Subsequently, the reaction mixture was concentrated directly under reduced pressure and purified by flash column chromatography. This yielded compound 2 (7.37 g, 13.7 mmol, yield 51%) as a pale yellow viscous solid.

[0232] 1 ​1H-NMR (400 MHz, DMSO-D6) δ 10.59 (s, 1H), 7.89 (d, J = 9.5 Hz, 1H), 6.44 (d, J = 30.1 Hz, 2H), 5.73 (t, J = 6.7 Hz, 1H), 5.06-4.96 (m, 2H), 4.22-4.14 (m, 2H), 3.89-3.80 (m, 1H), 3.59-3.40 (m, 3H), 3.30-3.25 (m, 1H), 1.48-0.94 (m, 32H), 0.84-0.77 (m, 3H); 13 13C-NMR (100 MHz, DMSO-D6) δ 157.3, 154.2, 151.8, 135.9, 117.1, 86.5, 84.9, 81.6, 70.2, 69.4, 61.9, 40.6, 40.4, 40.2, 40.0, 39.8, 39.6, 39.4, 31.8, 29.6, 29.3, 29.2, 25.8, 22.6, 14.5; HRMS (ESI-MS): (M-H + )(C 28 H 48 N5O5-: 534.3660) (calculated); (M-H + )( C 28 H 48 N5O5-): 534.3672 (found).

[0233] 7-2. Synthesis of 2'-n-C 18 H 37 -7m-Guanosine (3)

[0234] Compound 2 (1.86 mmol, 1.0 eq) and CH3I (1.84 g, 13.02 mmol, 7.0 eq) were dissolved in DMF (10.0 mL), and the mixture was stirred in a round-bottom flask at room temperature for 7.0 hours. After 7.0 hours, water was added to the reaction mixture to quench the reaction. The aqueous layer was extracted with DCM (3×50 mL), the combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated. This afforded compound 3 (3.62 mmol, yield 97%) as a pale yellow solid.

[0235] 11H-NMR (400 MHz, DMSO-D6) δ 9.24 (s, 1H), 6.91-7.23 (1H), 5.87 (d, J = 2.7 Hz, 1H), 5.15 (dd, J = 23.0, 5.5 Hz, 2H), 4.22-4.17 (m, 2H), 3.97 (s, 4H), 3.68-3.48 (m, 4H), 1.48-1.44 (m, 2H), 1.19 (s, 32H), 0.83-0.80 (m, 3H); 13 13C-NMR (100 MHz, DMSO-D6) δ 191.6, 156.1, 153.9, 149.6, 136.7, 108.1, 87.4, 86.7, 82.1, 70.7, 68.7, 60.8, 40.7, 40.5, 40.3, 40.1, 39.8, 39.6, 39.4, 36.4, 35.0, 31.8, 29.7, 29.5, 29.4, 29.2, 26.7, 25.9, 22.6, 14.5. HRMS (ESI-MS): (M-H + ) (C 29 H 51 N5O5−: 548.3817) (calculated); (M-H + ) ( C 29 H 51 N5O5−): 548.3804 (found).

[0236] 7-3. Synthesis of 2'-n-C 18 H 37 -7m-Guanosine monophosphate (4)

[0237] Compound 3 (1.0 g, 1.82 mmol, 1.0 equivalent) was dissolved in 2,6-lutidine (0.53 mL, 4.54 mmol, 2.5 equivalents) and trimethyl phosphate (10 mL). POCl3 (0.43 mL, 2.5 equivalents, 4.54 mmol) was added to the stirred solution cooled to -10 °C, and stirring was continued at the same temperature. After 6 hours, 0.5 M TEAB (pH=8, 10 mL) buffer was added to the reaction mixture at -10 °C, and after stirring at room temperature, water (10 mL) was added for further dilution. After 2.0 hours, the resulting solid was collected by filtration and dried under reduced pressure. This yielded the target compound as a pale yellow solid (yield: 1.01 g, 1.61 mmol, 89%).

[0238] 1 H-NMR (600MHz, METHANOL-D4) δ 9.33 (s, 1H), 6.05 (d, J = 2.4 Hz, 1H), 4.44 (t, J = 5.4 Hz, 1H), 4.28-4.20 (m, 4H), 4.10-4.05 (m, 5H), 3.22-3.16 (m, 4H), 1.62 (q, J = 6.6 Hz, 2H), 1.26 (s, 31H), 0.87 (t, J = 7.1 Hz, 3H); 13 C-NMR (101 MHz, METHANOL-D4) δ 204.6, 204.3, 156.1, 153.9, 149.6, 136.5, 108.0, 88.3, ​​84.8, 84.7, 82.7, 71.0, 68.3, 68.2, 62.7, 62.6, 49.0, 48.7, 48.5, 48.3, 48.1, 47.9, 47.7, 47.5, 47.2, 47.0, 46.5, 35.3, 31.7, 29.6, 29.5, 29.3, 29.1, 25.8, 22.4, 13.1, 7.9; 31 P-NMR (241 MHz, METHANOL-d4) δ 1.313; HRMS (ESI-MS): (M-H+) (C 29 H 51 N5O8P - ):628.3480(calculated);(MH + ) (C​29 H 51 N5O8P - ): 628.3466 (found).

[0239] 7-4. 2'-nC 18 H 37 Synthesis of -7m-Guanosine-monophosphate-imidazolide (5)

[0240] Compound 4 (0.5 g, 0.796 mmol, 1.0 equivalent) was dissolved in anhydrous DMF (10.0 mL), and dithiodipyridine (0.526 g, 3.0 equivalents, 2.39 mmol), imidazole (0.433 g, 8.0 equivalents, 6.36 mmol), triethylamine (0.22 mL, 2.0 equivalents, 1.59 mmol), and PPh3 (0.418 g, 2.0 equivalents, 1.59 mmol) were sequentially added while stirring at room temperature. After 16.0 hours, the reaction mixture was poured into a NaClO4 stirred solution in a 4% Et3N-acetone mixture, and stirring was continued for 1.0 hour. Subsequently, the target solid compound 5 (0.3 g, 0.427 mmol, yield 54%) was isolated by centrifugation.

[0241] 31 P-NMR (241 MHz, DMSO-d6) δ 9.59; HRMS (ESI-MS): (MH + ) (C 32 H 53 N7O7P-): 678.3749(calculated.);(MH + ) (C 32 H 53 N7O7P-): 678.3752 (found).

[0242] 7-5. Synthesis of 2′-Octadecyl-7m-GDP(6)

[0243] ​Compound 5 (0.2 g, 0.284 mmol, 1.0 equivalent) was dissolved in anhydrous DMF (5.0 mL), and tris(triethylammonium) phosphate (1.14 g, 10.0 equivalents, 2.83 mmol) and ZnCl2 (0.39 g, 10.0 equivalents, 2.84 mmol) were sequentially added while stirring at room temperature. After 16 hours, the reaction was stopped by adding a 500 mM EDTA-NaOH aqueous solution (pH 8.0, 5.6 mL, EDTA: 2.84 mmol) to the reaction mixture, and the mixture was diluted with water (10 mL). The resulting insoluble substance was recovered by filtration, washed with water, and then freeze-dried. This yielded target compound 6 (0.1 g, 0.141 mmol, yield 50%) as an amorphous white solid.

[0244] 31 P-NMR (241 MHz, DMSO-d6) δ 0.393, -0.325; HRMS (ESI-MS): (MH + ) (C 29 H 52 N5O 11 P2-):708.3144(calculated);(MH + ) (C 29 H 52 N5O 11 P2-): 708.314.

[0245] 7-6. Synthesis of 2′-Octadecyl-7m-GDP-Im(7)

[0246] ​Compound 6 (0.1 g, 0.03 mmol, 1.0 equivalent) was dissolved in anhydrous DMF (1.0 mL), and while stirring at room temperature, dithiodipyridine (0.002 g, 3.0 equivalents, 0.01 mmol), imidazole (0.002 g, 8.0 equivalents, 0.024 mmol), triethylamine (0.001 mL, 2.0 equivalents, 0.006 mmol), and PPh3 (0.002 g, 2.0 equivalents, 0.006 mmol) were added sequentially. After 24 hours, the reaction mixture was poured into a NaClO4 stirred solution in a 4% Et3N-acetone mixture, and stirring was continued for 1.0 hour. Subsequently, the target solid compound 7 was isolated by centrifugation.

[0247] HRMS (ESI-MS): (MH + ) (C 32 H 54 N7O 10 P2-): 758.3412(calculated);(MH + ) (C 32 H 54 N7O 10 P2-:758.3405) (found).

[0248] 7-7. Chemical capping reaction using C18-modified capping reagent

[0249] HiBiT mRNA (pA m G mAGCCACCATGGTGAGCGGCTGGCGGCTGTTCAAGAAGATTAGCTGAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 9) and CaCl2 were placed in a 2 mL centrifuge tube, mixed by vortexing, and lyophilized to form a CaCl2-mRNA complex. After 1 hour, this complex was dissolved in anhydrous DMSO, and 2-NO2-imidazolide and 2'-GDP-Im were added sequentially, and the mixture was heated at 55 °C. After 3 hours, water was added to the reaction mixture at room temperature to stop the reaction. To purify the target compound, isopropanol was added, followed by 3M NaOAc solution, and the reaction mixture was stored in a freezer at -30 °C. After 1 hour, the reaction mixture was centrifuged at 4 °C for 15 minutes, the supernatant was decanted, the solid was collected, and washed with 80% ethanol. The solid was evaporated by centrifugation at 40 °C to dry it, and an aqueous solution was prepared.

[0250] 7-8. LC-MS and Cartridge Purification The capping reaction product obtained in 7-7 was analyzed by LC-MS. The results are shown in Figure 51. The peak around retention time 4.6 minutes represents unreacted material without a cap structure, and the peak around retention time 7.2 minutes represents C18-modified capped mRNA. In other words, the capping reaction product contained both capped and uncapped mRNA.

[0251] Cartridge purification was performed using the method described in 2-2-2, and LC-MS analysis was performed on each eluted fraction. The results are shown in Figure 52. From top to bottom on the left of Figure 52, the ULC profiles of samples eluted with 20% and 40% acetonitrile / 0.1M TEAA buffer and concentrated are shown. In the ULC profile of the sample eluted with 20% acetonitrile / 0.1M TEAA buffer and concentrated, a peak originating from unreacted material without a cap structure was observed (upper panel). In the ULC profile of the sample eluted with 40% acetonitrile / 0.1M TEAA buffer and concentrated, a peak originating from unreacted material without a cap structure and a peak originating from the target C18-modified capped mRNA were detected (lower panel). The MS analysis results of each ULC peak are shown on the right of Figure 52.

Claims

1. General formula (I): [k represents an integer greater than or equal to 1; R Y R represents a k-valent group obtained by removing k atoms or groups from a polynucleotide; R is the same or different, and general formula (II): (R 1 and R 2 R represents the same or different hydrogen atom, alkyl group, or alkoxy group; 3 ~R 7 These are the same or different, representing a hydrogen atom, alkyl group, alkoxy group, or nitro group (however, R 1 ~R 7 (At least one of these is an alkyl or alkoxy group having 3 to 30 carbon atoms); R X A method for producing a single-stranded polynucleotide, comprising the step of subjecting a modified polynucleotide represented by [where represents a linker] to liquid chromatography.

2. The manufacturing method according to claim 1, further comprising the step of carrying out a polynucleotide synthesis reaction using a modified polynucleotide represented by the general formula (I) as a primer.

3. A step of cutting the plasmid with a restriction enzyme, and applying the following general formula (VIII) to the exposed bases at the cut ends by the restriction enzyme: The manufacturing method according to claim 1, further comprising the step of attaching a nucleotide derivative or polynucleotide derivative to which a hydrophobic tag represented by is attached to obtain the modified polynucleotide.

4. The process according to any one of claims 1 to 3, wherein at least one of R 1 to R 7 in the general formula (II) is an alkyl group having 8 to 30 carbon atoms or an alkoxy group.

5. The manufacturing method according to claim 4, wherein the liquid chromatography includes the step of adsorbing the modified polynucleotide onto a chromatography support having a particle size of 10 to 150 μm.

6. In the modified polynucleotide, R is R Y A method for producing a product according to any one of claims 1 to 3, wherein at least one nucleotide located 1 to 30 bases from the end is bonded to it.

7. In the modified polynucleotide, R X They are the same or different, single bond, -(CH2) n -, -O-(CH2) n -, or -R 8 -[In the formula, n represents an integer greater than or equal to 1; R 8 The general formula is (VI): (X 1 and X 2 The manufacturing method according to any one of claims 1 to 3, wherein the group represents a divalent group (which is the same or different and is O or S).

8. In the modified polynucleotides, if R is the same or different, the general formula (III): (R 1 and R 2 R represents the same or different hydrogen atom, alkyl group, or alkoxy group; 4 ~R 7 These are the same or different, representing a hydrogen atom, an alkyl group, or an alkoxy group (however, R 1 , R 2 , and R 4 ~R 7 (At least one of these is an alkyl or alkoxy group having 3 to 30 carbon atoms); R X (where indicates a linker) or general formula (VII): (R 1 and R 2 R represents the same or different hydrogen atom, alkyl group, or alkoxy group; 3 , R 4 , R 6 , and R 7 These are the same or different, representing a hydrogen atom, an alkyl group, or an alkoxy group (however, R 3 , R 4 , R 6 , and R 7 (At least one of these is an alkyl or alkoxy group having 3 to 30 carbon atoms); R X The manufacturing method according to any one of claims 1 to 3, wherein (where indicates a linker).

9. The manufacturing method according to any one of claims 1 to 3, wherein the pH of the sample subjected to the liquid chromatography is 8.0 or higher, and / or the pH of the mobile phase used in the liquid chromatography is 8.0 or higher.

10. The manufacturing method according to any one of claims 1 to 3, wherein the liquid chromatography is reversed-phase chromatography.

11. The manufacturing method according to any one of claims 1 to 3, wherein the liquid chromatography is solid-phase extraction purification.

12. The manufacturing method according to any one of claims 1 to 3, wherein the single-stranded polynucleotide is single-stranded DNA.

13. General formula (I): [k represents an integer greater than or equal to 1; R Y R represents a k-valent group obtained by removing k atoms or groups from a polynucleotide; R is the same or different, and general formula (II): (R 1 and R 2 R represents the same or different hydrogen atom, alkyl group, or alkoxy group; 3 ~R 7 These are the same or different, representing a hydrogen atom, alkyl group, alkoxy group, or nitro group (however, R 1 ~R 7 (At least one of these is an alkyl or alkoxy group having 3 to 30 carbon atoms); R X (where indicates a linker) is represented as, where R is R Y A modified polynucleotide that is attached to at least one nucleotide located 1 to 10 bases from the end.

14. In the modified polynucleotide, R X They are the same or different, single bond, -(CH2) n -, -O-(CH2) n -, or -R 8 -[In the formula, n represents an integer greater than or equal to 1; R 8 The general formula is (VI): (X 1 and X 2 The modified polynucleotide according to claim 13, wherein the group represents a divalent group (which is the same or different and is O or S).

15. If R is the same or different, then general formula (III): (R 1 and R 2 R represents the same or different hydrogen atom, alkyl group, or alkoxy group; 4 ~R 7 These are the same or different, representing a hydrogen atom, an alkyl group, or an alkoxy group (however, R 1 , R 2 , and R 4 ~R 7 (At least one of these is an alkyl or alkoxy group having 3 to 30 carbon atoms); R X (where indicates a linker) or general formula (VII): (R 1 and R 2 R represents the same or different hydrogen atom, alkyl group, or alkoxy group; 3 , R 4 , R 6 , and R 7 These are the same or different, representing a hydrogen atom, an alkyl group, or an alkoxy group (however, R 3 , R 4 , R 6 , and R 7 (At least one of these is an alkyl or alkoxy group having 3 to 30 carbon atoms); R X The modified polynucleotide according to claim 13 or 14, wherein (indicates a linker).

16. A primer comprising the modified polynucleotide described in claim 13 or 14.

17. A composition for producing single-stranded polynucleotides, comprising the primer described in claim 16.

18. The composition according to claim 17, wherein the single-stranded polynucleotide is single-stranded DNA.

19. General formula (I): [k represents an integer greater than or equal to 1; R Y R represents a k-valent group obtained by removing k atoms or groups from a polynucleotide; R is the same or different, and general formula (II): (R 1 and R 2 R represents the same or different hydrogen atom, alkyl group, or alkoxy group; 3 ~R 7 These are the same or different, representing a hydrogen atom, alkyl group, alkoxy group, or nitro group (however, R 1 ~R 7 (At least one of these is an alkyl or alkoxy group having 8 to 30 carbon atoms); R X A composition comprising a modified polynucleotide represented by (where represents a linker), used to adsorb the modified polynucleotide onto a chromatography support with a particle size of 10 to 150 μm.

Images