In vitro transcription method and compounds therefor

Abstract

The present application provides a method for in vitro transcription of a DNA template into RNA, the method comprising providing a mixture comprising a buffer, ribonucleoside triphosphates (NTPs), one or more magnesium salts at a concentration of about 2 mM to about 60 mM, the DNA template, a recombinant RNA polymerase, and a cap analog comprising a structure having formula (I), (II), or (III); and incubating the reaction mixture at about 15° C. to about 35° C., optionally about 18° C. to about 31° C., for about 1 hour to about 12 hours, thereby producing the RNA.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is an international (PCT) application claiming priority to U.S. Provisional Patent Application No. 63 / 482,688, filed February 1, 2023, the contents of which are specifically incorporated by reference in their entirety for all purposes. Reference to sequence listing submitted in compatible XML file (.xml)

[0002] In accordance with the EFS-Web legal framework and 37 C.F.R. § 1.821-825 (see MPEP § 2442.03(a)), a sequence listing in a compatible XML file format (titled "3000076-010977Seq Listing ST26.xml," created on January 31, 2024, and 36,110 bytes in size) has been submitted contemporaneously with the present application, and the entire contents of said sequence listing are incorporated herein by reference. [Background technology]

[0003] The present disclosure relates generally to methods for synthesizing capped mRNA analogs and methods and compositions for in vitro transcription.

[0004] mRNA is a well-defined molecule whose structure includes a 5' cap, a 5' untranslated region (UTR), an open reading frame sequence encoding one or more genes of interest, a 3' UTR, and a poly(A) tail. The preparation of capped mRNA by in vitro synthesis may be of great significance for both basic science research and the development of new therapeutics. Several factors facilitate the production of mRNA that can have high expression levels, stability, and functionality.

[0005] An mRNA molecule may be flanked by 5'- and 3'-untranslated regions (UTRs). The 5'-UTR serves as the entry site for ribosomes to initiate translation, and the 3'-UTR plays an important role in translation termination and post-transcriptional modification, which may affect mRNA expression and half-life. The poly(A) tail of an mRNA can make the RNA molecule more stable and prevent mRNA degradation. The poly(A) tail can also allow the mature messenger RNA to be exported from the cell nucleus and translated into protein by ribosomes in the cytoplasm. See, for example, Sachs A and Wahle E, "Poly(A) tail metabolism and function in eucaryotes," J Biol Chem, (1993, November 5); 268(31):22955-8, incorporated herein by reference in its entirety.

[0006] The mRNA cap is a highly methylated modification at the 5' end of mRNA that protects mRNA from degradation, recruits complexes involved in mRNA processing, and can mark cellular mRNA to avoid recognition by the immune system. In mammals, the predominant 5' cap structure is an inverted 7-methylguanosine nucleotide linked to the first transcribed nucleotide via a 5'-5' triphosphate bond. 7-methylguanosine is methylated at its seventh nitrogen position, and m7 G or 7m The cap structure may be referred to as 5' G. m7 GpppN1(pN) x where N is any nucleotide and x is 0 or any number. In the Cap-0 structure, the first nucleotide has a 2' hydroxy group on its ribose, while in the Cap-1 structure, the first nucleotide has a 2'-o-methyl modification on the ribose, and the structure has a 2'-o-methyl modification on the ribose from the 5' end to the 3' end. m7 G 5’ pppN1 2’-OMe (pN) xwhere N is any nucleotide and x is any integer. m7 The 2' hydroxy groups of the first and second riboses up to G are methylated. The structure is m7 G 5’ pppN1 2’-OMe pN2 2’-OMe (pN) x where N is any nucleotide and x is any integer. See, e.g., Perry RP, "RNA processing comes of age," J Cell Biol. (1981 December);91(3 Pt 2):28s-38s, which is incorporated herein by reference in its entirety.

[0007] Capping can improve properties of mRNA, such as, but not limited to, its stability and its translation efficiency. See, e.g., Banerjee AK, "5'-terminal cap structure in eucaryotic messenger ribonucleic acids," Microbiol Rev. (June 1980); 44(2):175-205, incorporated herein by reference in its entirety. In vivo, each capping process can be carried out enzymatically. See, e.g., Perry. These processes can be time-consuming, inefficient, and expensive when performed in vitro.

[0008] The preparation of capped mRNA by in vitro synthesis may be of great significance to both basic science research, pharmacological development, and therapeutic development. Several factors may facilitate the production of mRNA that can have high expression levels, stability, and functionality. There is a need for effective in vitro transcription methods that allow for more efficient production of capped mRNA that can have high expression levels, stability, functionality, or a combination thereof. Summary of the Invention

[0009] In one aspect, embodiments of the present disclosure may include methods for in vitro transcription of a DNA template into RNA, the methods comprising: providing a mixture; and producing RNA by incubating the reaction mixture at about 15° C. to about 35° C., optionally at about 18° C. to about 31° C., for about 1 hour to about 12 hours; the mixture comprising a buffer, ribonucleoside triphosphates (NTPs), one or more magnesium salts at a concentration of about 2 mM to about 60 mM, the DNA template, a recombinant RNA polymerase, and a cap analog, wherein the cap analog comprises a structure having formula (I), (II), or (III): [ka] where R is H or CH3 and B1 is A or N6-methyl-adenine (m 6 A) and B2 is A, U, G or C; [ka] where: R1 is OCH3 and R2 is OH or H, or R1 is OH and R2 is H, or R1 is H and R2 is H or OCH3, or R1 and R2 are OCH3, B1 is A or N6-methyl-adenine (m 6 A) and B2 is A, U, G or C; [ka] where R is H or CH3 and B1 is A or N6-methyl-adenine (m 6 A) and B2 is A, U, G or C.

[0010] In another embodiment, the buffer may be Tris base, HEPES, or Tris-HCl.

[0011] In another embodiment, the concentration of the buffering agent is from about 1 mM to about 100 mM, from about 1 mM to about 90 mM, from about 1 mM to about 80 mM, from about 1 mM to about 70 mM, from about 1 mM to about 60 mM, from about 1 mM to about 50 mM, from about 1 mM to about 40 mM, from about 1 mM to about 30 mM, from about 1 mM to about 20 mM, from about 1 mM to about 10 mM, from about 1 mM to about 5 mM, from about 10 mM to about 20 mM, from about 10 mM to about 30 mM, from about 1 mM to about 100 mM, It may be 0 mM to about 40 mM, about 10 mM to about 50 mM, about 20 mM to about 50 mM, about 30 mM to about 50 mM, about 35 mM to about 45 mM, about 35 mM to about 40 mM, about 40 mM to about 50 mM, about 45 mM to about 50 mM, about 45 mM to about 55 mM, about 15 mM to about 45 mM, about 15 mM to about 35 mM, about 15 mM to about 30 mM, or about 15 mM to about 25 mM.

[0012] In another embodiment, the concentration of NTP is from about 1 mM to about 50 mM, from about 1 mM to about 40 mM, from about 1 mM to about 30 mM, from about 1 mM to about 20 mM, from about 1 mM to about 10 mM, from about 1 mM to about 5 mM, from about 2 mM to about 10 mM, from about 3 mM to about 10 mM, from about 3 mM to about 9 mM, from about 3 mM to about 8 mM, from about 3 mM to about 7 mM, from about 3 mM to about 6 mM, from about 3 mM to about 5 mM, from about 3 mM to about 4 mM, The concentration may be about 4 mM to about 10 mM, about 5 mM to about 10 mM, about 6 mM to about 10 mM, about 7 mM to about 10 mM, about 8 mM to about 10 mM, about 9 mM to about 10 mM, about 10 mM to about 50 mM, about 20 mM to about 50 mM, about 25 mM to about 50 mM, about 25 mM to about 45 mM, about 25 mM to about 40 mM, about 25 mM to about 35 mM, or about 20 mM to about 30 mM.

[0013] In another embodiment, the concentration of the one or more magnesium salts can be from about 2 mM to about 50 mM, from about 2 mM to about 40 mM, from about 2 mM to about 30 mM, from about 2 mM to about 40 mM, from about 2 mM to about 30 mM, from about 2 mM to about 20 mM, from about 2 mM to about 10 mM, from about 2 mM to about 5 mM, from about 5 mM to about 50 mM, from about 10 mM to about 45 mM, from about 15 mM to about 40 mM, from about 20 mM to about 35 mM, from about 20 mM to about 30 mM, from about 20 mM to about 25 mM, from about 22 mM to about 28 mM, or from about 25 mM to about 30 mM.

[0014] In another embodiment, the concentration of the DNA template is from about 0.001 μg / μl to about 2 μg / μl, from about 0.001 μg / μl to about 1.5 μg / μl, from about 0.001 μg / μl to about 1 μg / μl, from about 0.01 μg / μl to about 2 μg / μl, from about 0.01 μg / μl to about 1.5 μg / μl, from about 0.01 μg / μl to about 1 μg / μl, from about 0.01 μg / μl to about 0.5 μg / μl, from about 0.01 μg / μl to about 0.1 μg / μl, from about 0.01 μg / μl to about 0.05 μg / μl 1, about 0.02 μg / μl to about 0.04 μg / μl, about 0.02 μg / μl to about 0.1 μg / μl, about 0.03 μg / μl to about 0.1 μg / μl, about 0.04 μg / μl to about 0.1 μg / μl, about 0.05 μg / μl to about 0.1 μg / μl, about 0.06 μg / μl to about 0.1 μg / μl, about 0.07 μg / μl to about 0.1 μg / μl, about 0.08 μg / μl to about 0.1 μg / μl, or about 0.09 μg / μl to about 0.1 μg / μl.

[0015] In another embodiment, the concentration of the recombinant RNA polymerase may be from about 0.1 U / μl to about 2 U / μl, from about 0.5 U / μl to about 2 U / μl, from about 1 U / μl to about 2 U / μl, from about 1 U / μl to about 1.5 U / μl, or from about 1.5 U / μl to about 2 U / μl.

[0016] In another embodiment, the mixture may further comprise an antioxidant.

[0017] In another embodiment, the antioxidant can be dithiothreitol (DTT) and the concentration of dithiothreitol is from about 1 mM to about 50 mM, from about 2 mM to about 50 mM, from about 3 mM to about 50 mM, from about 4 mM to about 50 mM, from about 5 mM to about 50 mM, from about 6 mM to about 50 mM, from about 7 mM to about 50 mM, from about 8 mM to about 50 mM, from about 9 mM to about 50 mM, from about 10 mM to about 50 mM, from about 10 mM to about 40 mM, from about 15 mM to about 30 mM, from about 15 mM to about 25 mM, from about 15 mM to about 20 mM, from about 20 mM to about 50 mM, from about 30 mM to about 50 mM, or from about 40 mM to about 50 mM.

[0018] In another embodiment, the mixture may further comprise an RNase inhibitor, wherein the concentration of the RNase inhibitor is from about 0.001 U / μl to about 5 U / μl, from about 0.001 U / μl to about 4 U / μl, from about 0.001 U / μl to about 3 U / μl, from about 0.001 U / μl to about 2 U / μl, from about 0.001 U / μl to about 1 U / μl, from about 0.01 U / μl to about 5 U / μl, from about 0.01 U / μl to about 4 U / μl, from about 0.01 U / μl to about 3 ... / μl to about 2U / μl, about 0.01U / μl to about 1U / μl, about 0.01U / μl to about 0.5U / μl, about 0.01U / μl to about 0.1U / μl, about 0.01U / μl to about 0.05U / μl, about 0.01U / μl to about 0.04U / μl, about 0.01U / μl to about 0.03U / μl, about 0.01U / μl to about 0.02U / μl, about 0.1U / μl to about 5U / μl, about 0.1U / μl to about 4U / μl, about 0.1U / μl to about 3U / μl / μl, about 0.1U / μl to about 2U / μl, about 0.1U / μl to about 1U / μl, about 0.5U / μl to about 5U / μl, about 0.5U / μl to about 4U / μl, about 0.5U / μl to about 3U / μl, about 0.5U / μl to about 2U / μl, about 0.5U / μl to about 1U / μl, about 1U / μl to about 5U / μl, about 2U / μl to about 5U / μl, about 3U / μl to about 5U / μl, about 4U / μl to about 5U / μl, about 0.001U / μl to about 0.005 U / μl, about 0.001 U / μl to about 0.01 U / μl, about 0.005 U / μl to about 0.01 U / μl, about 0.01 U / μl to about 0.05 U / μl, about 0.01 U / μl to about 0.04 U / μl, about 0.01 U / μl to about 0.03 U / μl, about 0.01 U / μl to about 0.02 U / μl, about 0.02 U / μl to about 0.03 U / μl, about 0.02 U / μl to about 0.04 U / μl, or about 0.02 U / μl to about 0.05 U / μl.

[0019] In another embodiment, the concentration of the cap analog is from about 0.5 mM to about 50 mM, from about 0.5 mM to about 40 mM, from about 0.5 mM to about 30 mM, from about 0.5 mM to about 20 mM, from about 0.5 mM to about 10 mM, from about 0.5 mM to about 5 mM, from about 1 mM to about 10 mM, from about 2 mM to about 10 mM, from about 3 mM to about 10 mM, from about 3 mM to about 9 mM, from about 3 mM to about 8 mM, from about 3 mM to about 7 mM, from about 3 mM to about 9 mM, from about 3 mM to about 9 mM, from about 3 mM to about 10 ... to about 6 mM, about 3 mM to about 5 mM, about 3 mM to about 4 mM, about 4 mM to about 10 mM, about 5 mM to about 10 mM, about 6 mM to about 10 mM, about 6 mM to about 9 mM, about 6 mM to about 8 mM, about 6 mM to about 7 mM, about 7 mM to about 8 mM, about 7 mM to about 9 mM, about 7 mM to about 10 mM, about 8 mM to about 9 mM, about 8 mM to about 10 mM, or about 9 mM to about 10 mM.

[0020] In another embodiment, the mixture may further comprise a polyamine.

[0021] In another embodiment, the polyamine can be spermine, spermidine, or a combination thereof.

[0022] In another embodiment, the concentration of the polyamine is from about 0.1 mM to about 5 mM, from about 0.2 mM to about 4.9 mM, from about 0.2 mM to about 4.8 mM, from about 0.2 mM to about 4.7 mM, from about 0.2 mM to about 4.6 mM, from about 0.2 mM to about 4.5 mM, from about 0.2 mM to about 4.4 mM, from about 0.2 mM to about 4.3 mM, from about 0.2 mM to about 4.2 mM, from about 0.2 mM to about 4.1 mM, from about 0.2 mM to about 4 mM, from about 0.2 mM to about 3.5 mM, from about 0.2 mM to about 3 mM, from about 0.2 mM to about 2.5 mM, from about 0.5 mM to about 2.5 mM, from about 1.0 ... The concentration may be from about 1 mM to about 2.5 mM, from about 1.5 mM to about 2.5 mM, from about 0.2 mM to about 2 mM, from about 0.2 mM to about 1.5 mM, from about 0.2 mM to about 1 mM, from about 0.2 mM to about 0.9 mM, from about 0.2 mM to about 0.8 mM, from about 0.2 mM to about 0.7 mM, from about 0.2 mM to about 0.6 mM, from about 0.2 mM to about 0.5 mM, from about 0.2 mM to about 0.4 mM, from about 0.2 mM to about 0.3 mM, from about 1 to about 25 mM, from about 1 to about 20 mM, from about 1 to about 15 mM, from about 1 to about 10 mM, from about 1 to about 5 mM, or from about 1 to about 2.5 mM.

[0023] In another embodiment, the mixture may further comprise a pyrophosphatase, wherein the concentration of the pyrophosphatase is from about 0.01 mU / μl to about 2 mU / μl, from about 0.01 mU / μl to about 1.5 mU / μl, from about 0.01 mU / μl to about 1 mU / μl, from about 0.1 mU / μl to about 2 mU / μl, from about 0.1 mU / μl to about 1.5 mU / μl, from about 0.1 mU / μl to about 1 mU / μl, The concentration may be from about 0.9 mU / μl, from about 0.1 mU / μl to about 0.8 mU / μl, from about 0.1 mU / μl to about 0.7 mU / μl, from about 0.1 mU / μl to about 0.6 mU / μl, from about 0.1 mU / μl to about 0.5 mU / μl, from about 0.1 mU / μl to about 0.4 mU / μl, from about 0.1 mU / μl to about 0.3 mU / μl, or from about 0.1 mU / μl to about 0.2 mU / μl.

[0024] In another embodiment, incubating the reaction mixture is at about 15°C to about 35°C, about 16°C to about 35°C, about 17°C to about 35°C, about 18°C ​​to about 35°C, about 18°C ​​to about 34°C, about 18°C ​​to about 33°C, about 18°C ​​to about 32°C, about 18°C ​​to about 31°C, about 18°C ​​to about 30°C, about 18°C ​​to about 29°C, about 18°C ​​to about 28°C, about 18°C ​​to about 27°C, about 18°C ​​to about 26°C, about 18°C ​​to about 25°C, about 18°C ​​to about 24°C, about 18°C ​​to about 23°C, about 18°C ​​to about 22°C , about 18°C ​​to about 21°C, about 18°C ​​to about 20°C, about 18°C ​​to about 19°C, about 25°C to about 26°C, about 25°C to about 27°C, about 25°C to about 28°C, about 25°C to about 29°C, about 25°C to about 30°C, about 25°C to about 31°C, about 21°C to about 22°C, about 21°C to about 23°C, about 21°C to about 24°C, about 21°C to about 25°C, about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, about 30°C, about 31°C, about 32°C, about 33°C, about 34°C, or about 35°C.

[0025] In another embodiment, incubating the reaction mixture may be for about 1 hour to about 12 hours, about 1 hour to about 11 hours, about 1 hour to about 10 hours, about 1 hour to about 9 hours, about 1 hour to about 8 hours, about 1 hour to about 7 hours, about 1 hour to about 6 hours, about 1 hour to about 5 hours, about 1 hour to about 4 hours, about 1 hour to about 3 hours, about 1 hour to about 2 hours, about 2 hours to about 12 hours, about 3 hours to about 12 hours, about 4 hours to about 12 hours, about 5 hours to about 12 hours, about 6 hours to about 12 hours, about 7 hours to about 12 hours, about 8 hours to about 12 hours, about 9 hours to about 12 hours, about 10 hours to about 12 hours, about 11 hours to about 12 hours, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, or about 10 hours.

[0026] In another embodiment, incubating the reaction mixture may occur at about 31° C. for about 1 hour to about 5 hours, about 1 hour to about 4.5 hours, about 1 hour to about 4 hours, about 1 hour to about 3.5 hours, about 1 hour to about 3 hours, about 1 hour to about 2.5 hours, about 1 hour to about 2 hours, about 1 hour to about 1.5 hours, about 0.5 hours to about 1 hour, about 0.5 hours to about 1.5 hours, about 1 hour, about 2 hours, about 3 hours, about 4 hours, or about 5 hours.

[0027] In another aspect, the DNA template may comprise a promoter operably linked to a nucleic acid, the nucleic acid comprising a 5' untranslated region (5' UTR), an open reading frame (ORF) encoding an RNA of interest, a 3' UTR, and a polyA region, wherein the promoter comprises the sequence TAATACGACTCACTATAX1X2X3 (SEQ ID NO: 16), where X1 is A or G, X2 is A or G, and X3 is A, T, G, or C, wherein the 5' UTR is selected from SEQ ID NO: 1, 3, 5, or 9, and the 3' UTR is selected from SEQ ID NO: 2, 4, 6, or 8, and wherein the polyA region comprises at least 60 adenine bases (As).

[0028] In another embodiment, the promoter may comprise a sequence selected from SEQ ID NOs: 10-15.

[0029] In another embodiment, the 5'UTR and 3'UTR may be SEQ ID NOs: 1 and 2, 1 and 4, 1 and 6, 3 and 2, 3 and 4, 3 and 6, 3 and 8, 5 and 2, 5 and 4, 5 and 6, 7 and 2, 7 and 4, 7 and 6, 7 and 8, 9 and 2, 9 and 4, 9 and 6, or 9 and 8, respectively.

[0030] In another embodiment, the 5'UTR and 3'UTR may be SEQ ID NOs: 1 and 2, 1 and 4, 3 and 2, 1 and 6, 7 and 4, 9 and 2, or 3 and 6, respectively.

[0031] In another embodiment, the poly A region is from about 60 to about 200 A, from about 60 to about 190 A, from about 60 to about 180 A, from about 60 to about 170 A, from about 60 to about 160 A, from about 60 to about 150 A, from about 60 to about 140 A, from about 60 to about 130 A, from about 60 to about 120 A, from about 60 to about 110 A, from about 60 to about 100 A, from about 70 to about 150 A, from about 60 to about 160 A, from about 60 to about 170 A, from about 60 to about 180 A, from about 60 to about 190 A, from about 60 to about 200 A, from about 60 to about 210 A, from about 60 to about 220 A, from about 60 to about 230 A, from about 60 to about 240 A, from about 60 to about 250 A, from about 60 to about 260 A, from about 60 to about 270 A, from about 60 to about 280 A, from about 60 to about 290 A, from about 60 to about 300 A, from about 60 to about 310 A, from about 60 to about 320 A, from about 60 to about 330 A, from about 60 to about 340 A, from about 60 to about 350 A, from about 60 to about 360 A, from about 60 to about 370 A, from about 60 to about 380 A, from about 60 to about 390 A, from about 70 to about 800 A, from about 70 to about 810 A, from about 70 to about The amino acid sequence may comprise from about 1 to about 190 A, from about 80 to about 180 A, from about 90 to about 170 A, from about 100 to about 160 A, from about 100 to about 150 A, from about 100 to about 140 A, from about 100 to about 130 A, from about 100 to about 120 A, about 100 A, about 110 A, about 120 A, about 130 A, about 140 A, or about 150 A.

[0032] In another embodiment, the recombinant RNA polymerase may be selected from wild-type T7 RNA polymerase or a mutant thereof.

[0033] In another aspect, the cap analog is m 7 GpppA 2’Ome pA 2’Ome , m 7 GpppA 2’Ome pU 2’Ome , m 7 GpppA 2’Ome p.g. 2’Ome , m 7 GpppA 2’Ome PC 2’Ome , m 7 Gpppm 6 A 2’Ome pA 2’Ome , m 7 Gpppm 6 A 2’Ome pU 2’Ome , m 7 Gpppm 6 A 2’Ome p.g. 2’Ome , m 7 Gpppm 6 A 2’OmepC 2’Ome 、 m 7 G 3’Ome pppA 2’Ome pA 2’Ome 、 m 7 G 3’Ome pppA 2’Ome pU 2’Ome 、 m 7 G 3’Ome pppA 2’Ome pG 2’Ome 、 m 7 G 3’Ome pppA 2’Ome pC 2’Ome 、 m 7 G 3’Ome pppm 6 A 2’Ome pA 2’Ome 、 m 7 G 3’Ome pppm 6 A 2’Ome pU 2’Ome 、 m 7 G 3’Ome pppm 6 A 2’Ome pG 2’Ome 、 m 7 G 3’Ome pppm 6 A 2’Ome pC 2’Ome 、 m 7 G 2’Ome pppA 2’Ome pA 2’Ome 、 m 7 G 2’Ome pppA 2’Ome pU 2’Ome 、 m 7 G 2’Ome pppA 2’Ome pG 2’Ome 、 m 7 G 2’Ome pppA 2’Ome pC 2’Ome 、 m 7 G 2’Omepppm 6 A 2’Ome pA 2’Ome 、 m 7 G 2’Ome pppm 6 A 2’Ome pU 2’Ome 、 m 7 G 2’Ome pppm 6 A 2’Ome pG 2’Ome 、 m 7 G 2’Ome pppm 6 A 2’Ome pC 2’Ome 、 m 7 G 3’H pppA 2’Ome pA 2’Ome 、 m 7 G 3’H pppA 2’Ome pU 2’Ome 、 m 7 G 3’H pppA 2’Ome pG 2’Ome 、 m 7 G 3’H pppA 2’Ome pC 2’Ome 、 m 7 G 3’H pppm 6 A 2’Ome pA 2’Ome 、 m 7 G 3’H pppm 6 A 2’Ome pU 2’Ome 、 m 7 G 3’H pppm 6 A 2’Ome pG 2’Ome 、 m 7 G 3’H pppm 6 A 2’Ome pC 2’Ome 、 m 7 Gpp SpA 2’Ome pA 2’Ome 、 m 7 Gpp S pA 2’Ome pU 2’Ome 、 m 7 Gpp S pA 2’Ome pG 2’Ome 、 m 7 Gpp S pA 2’Ome pC 2’Ome 、 m 7 Gpp S pm 6 A 2’Ome pA 2’Ome 、 m 7 Gpp S pm 6 A 2’Ome pU 2’Ome 、 m 7 Gpp S pm 6 A 2’Ome pG 2’Ome 、 m 7 Gpp S pm 6 A 2’Ome pC 2’Ome 、 m 7 G 3’Ome pp S pA 2’Ome pA 2’Ome 、 m 7 G 3’Ome pp S pA 2’Ome pU 2’Ome 、 m 7 G 3’Ome pp S pA 2’Ome pG 2’Ome 、 m 7 G 3’Ome pp S pA 2’Ome pC 2’Ome 、 m 7 G 3’Ome ppS pm 6 A 2’Ome pA 2’Ome , m 7 G 3’Ome pp S pm 6 A 2’Ome pU 2’Ome , m 7 G 3’Ome pp S pm 6 A 2’Ome p.g. 2’Ome , and m 7 G 3’Ome pp S pm 6 A 2’Ome PC 2’Ome It may also be a cap analog selected from the group consisting of:

[0034] In another embodiment, the cap analog can be attached to the −1 and / or +1 nucleotide of the promoter.

[0035] In one aspect, an embodiment of the present disclosure may include a reaction mixture for in vitro transcription of a DNA template into RNA, the reaction mixture comprising a buffer substance at a concentration of about 45 mM to about 55 mM, an RNase inhibitor at a concentration of about 0.01 U / μl to about 0.03 U / μl, an NTP at a concentration of about 3 mM to about 5 mM, a cap analog at a concentration of about 6 mM to about 8 mM, and one or more ribonucleotides at a concentration of about 20 mM to about 30 mM. a magnesium salt; a polyamine at a concentration of about 1.5 mM to about 2.5 mM; a DNA template at a concentration of about 0.01 μg / μl to about 0.05 μg / μl; a pyrophosphatase at a concentration of about 0.1 mU / μl to about 0.5 mU / μl; and an RNA polymerase at a concentration of about 0.01 μg / μl to about 0.05 μg / μl, wherein the cap analog comprises a structure having formula (I), (II), or (III): [ka] where R is H or CH3 and B1 is A or N6-methyl-adenine (m 6 A) and B2 is A, U, G or C; [ka] where: R1 is OCH3 and R2 is OH or H, or R1 is OH and R2 is H, or R1 is H and R2 is H or OCH3, or R1 and R2 are each OCH3; B1 is A or N6-methyl-adenine (m 6 A) and B2 is A, U, G or C; [ka] where R is H or CH3 and B1 is A or N6-methyl-adenine (m 6 A) and B2 is A, U, G or C.

[0036] In another embodiment, incubating the reaction mixture may occur at about 15° C. to about 35° C. for about 1 hour to about 12 hours.

[0037] In another embodiment, incubating may comprise incubating the reaction mixture at about 18°C ​​to about 31°C.

[0038] In another embodiment, incubating may comprise incubating the reaction mixture at about 31° C. for about 3 hours.

[0039] In one aspect, embodiments of the present disclosure may include methods for in vitro transcribing a DNA template into RNA, the methods including: (1) providing a DNA template comprising a promoter operably linked to a nucleic acid comprising a 5' untranslated region (5' UTR), an open reading frame (ORF) encoding an RNA of interest, a 3' UTR, and a polyA region; and (2) providing a cap analog; incubating the DNA template and the cap analog in a reaction mixture; wherein the cap analog comprises a structure having formula (I), (II), or (III): [ka] where R is H or CH3 and B1 is A or N6-methyl-adenine (m 6 A) and B2 is A, U, G or C; [ka] where: R1 is OCH3 and R2 is OH or H, or R1 is OH and R2 is H, or R1 is H and R2 is H or OCH3, or R1 and R2 are each OCH3; B1 is A or N6-methyl-adenine (m 6 A) and B2 is A, U, G or C; wherein the promoter comprises the sequence TAATACGACTCACTATAX1X2X3 (SEQ ID NO: 16); wherein A at position 17 is the −1 nucleotide and X1 at position 18 is the +1 nucleotide; When X1 is G, and X2 and X3 are each A, T, G, or C, B1 is A and B2 is G; When X1 is A, and X2 and X3 are each A, T, G, or C, B1 is A and B2 is A; When X1 is C, and X2 and X3 are each A, T, G, or C, then B1 is A and B2 is C; and when X1 is T, and X2 and X3 are each A, T, G, or C, then B1 is A and B2 is U; [ka] where R is H or CH3 and B1 is A or N6-methyl-adenine (m 6 A) and B2 is A, U, G or C; wherein the cap analog is attached to the -1 and +1 nucleotides of the promoter, and wherein the incubating produces the RNA by incubating the reaction mixture at about 15°C to about 35°C for about 1 hour to about 12 hours.

[0040] In another embodiment, the promoter may comprise a sequence selected from SEQ ID NOs: 10, 11, 12, 13 and 14.

[0041] In another embodiment, the reaction mixture may include a buffer substance at a concentration of about 35 mM to about 45 mM, an RNase inhibitor at a concentration of about 0.01 U / μl to about 0.03 U / μl, an NTP at a concentration of about 20 mM to about 40 mM, a cap analog at a concentration of about 6 mM to about 8 mM, one or more magnesium salts at a concentration of about 20 mM to about 30 mM, a polyamine at a concentration of about 1.5 mM to about 2.5 mM, a DNA template at a concentration of about 0.01 μg / μl to about 0.05 μg / μl, a pyrophosphatase at a concentration of about 0.1 mU / μl to about 0.5 mU / μl, and an RNA polymerase at a concentration of about 1 U / μl to about 2 U / μl.

[0042] In one aspect, embodiments of the present disclosure may include a reaction mixture for in vitro transcription of a DNA template into RNA, the reaction mixture comprising the DNA template and a cap analog, wherein the cap analog comprises a structure having formula (I), (II), or (III): [ka] where R is H or CH3 and B1 is A or N6-methyl-adenine (m 6 A) and B2 is A, U, G or C; [ka] where: R1 is OCH3 and R2 is OH or H, or R1 is OH and R2 is H, or R1 is H and R2 is H or OCH3, or R1 and R2 are each OCH3; B1 is A or N6-methyl-adenine (m 6 A) and B2 is A, U, G or C; [ka] where R is H or CH3 and B1 is A or N6-methyl-adenine (m 6 A) and B2 is A, U, G or C; wherein the DNA template comprises a promoter, and wherein the cap analog binds to at least the −1 and +1 nucleotides of the promoter, or binds to at least the +1 and +2 nucleotides of the promoter.

[0043] In one aspect, embodiments of the present disclosure may include methods for synthesizing cap analogs, the methods comprising: (a) 5'-DMT-2'-O-methylguanosine (n-ibu) was reacted with acetic anhydride / pyridine, and the resulting product was then reacted with aqueous acetic acid to give compound (3): [ka] and (b) reacting compound (3) with 5'-dimethoxytrityl-N-benzoyl-adenosine, 2'-O-methyl, 3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite and tetrazole, followed by a first oxidizing agent and then an aqueous solution of acetic acid to give compound (4): [ka] and (c) reacting compound (4) with a chemical phosphorylation phosphoramidite reagent and tetrazole, followed by a second oxidizing agent and then ammonium hydroxide to give compound (5): [ka] and (d) reacting compound (5) with compound (2a) or (2b) in the presence of a divalent metal salt: [ka] to produce cap analogs (1a) or (1b): [ka] and obtaining the

[0044] In one aspect, embodiments of the present disclosure may include methods for synthesizing cap analogs, the methods comprising: (a) Compound 3: [ka] is reacted with 5'-DMT-2'-O-methyl-N6-methyl-adenosine 3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite and tetrazole, followed by a first oxidizing agent and then an aqueous solution of acetic acid to give compound (6): [ka] and (b) reacting compound (6) with a chemical phosphorylation phosphoramidite reagent and tetrazole, followed by a second oxidizing agent and then ammonium hydroxide to give compound (7): [ka] and (c) reacting compound (7) with compound (2b) in the presence of a divalent metal salt: [ka] to give the cap analog compound (8): [ka] and obtaining the

[0045] In another embodiment, the first oxidizing agent and the second oxidizing agent may be (1S)-(+)-(camphorsulfonyl)oxaziridine (CSO) or iodine, respectively.

[0046] In another embodiment, the divalent metal salt may be MnCl2 or ZnCl2.

[0047] In one aspect, embodiments of the present disclosure may include cap analogs, which include the formula (I): [ka] where R is H or CH3 and B1 is A or N6-methyl-adenine (m 6 A) and B2 is A, U, G or C.

[0048] In another embodiment, R is H, B1 is A, and B2 is A, U, G, or C, wherein the cap analog having formula (I) is m 7 GpppA 2’Ome pA 2’Ome , m 7 GpppA 2’OmepU 2’Ome , m 7 GpppA 2’Ome p.g. 2’Ome , and m 7 GpppA 2’Ome PC 2’Ome may be selected from the group consisting of:

[0049] In another embodiment, R is H and B1 is m 6 A and B2 is A, U, G or C, wherein the cap analog having formula (I) is m 7 Gpppm 6 A 2’Ome pA 2’Ome , m 7 Gpppm 6 A 2’Ome pU 2’Ome , m 7 Gpppm 6 A 2’Ome p.g. 2’Ome , and m 7 Gpppm 6 A 2’Ome PC 2’Ome may be selected from the group consisting of:

[0050] In another embodiment, R is CH3, B1 is A, and B2 is A, U, G, or C, wherein the cap analog having formula (I) is m 7 G 3’Ome pppA 2’Ome pA 2’Ome , m 7 G 3’Ome pppA 2’Ome pU 2’Ome , m 7 G 3’Ome pppA 2’Ome p.g. 2’Ome , and m 7 G 3’Ome pppA 2’Ome PC 2’Omemay be selected from the group consisting of:

[0051] In another embodiment, R is CH3 and B1 is m 6 A and B2 is A, U, G or C, wherein the cap analog having formula (I) is m 7 G 3’Ome pppm 6 A 2’Ome pA 2’Ome , m 7 G 3’Ome pppm 6 A 2’Ome pU 2’Ome , m 7 G 3’Ome pppm 6 A 2’Ome p.g. 2’Ome , and m 7 G 3’Ome pppm 6 A 2’Ome PC 2’Ome may be selected from the group consisting of:

[0052] In one aspect, embodiments of the present disclosure may include cap analogs, which include formula (II): [ka] where: R1 is OCH3 and R2 is OH or H, or R1 is OH and R2 is H, or R1 is H and R2 is H or OCH3, or R1 and R2 are each OCH3; B1 is A or N6-methyl-adenine (m 6 A) and B2 is A, U, G or C.

[0053] In another embodiment, R1 is OCH3 and R2 is OH, B1 is A and B2 is A, U, G or C, wherein the cap analog having formula (II) is m 7 G 2’Ome pppA 2’Ome pA 2’Ome , m 7 G 2’Ome pppA 2’Ome pU 2’Ome , m 7 G 2’Ome pppA 2’Ome p.g. 2’Ome , and m 7 G 2’Ome pppA 2’Ome PC 2’Ome may be selected from the group consisting of:

[0054] In another embodiment, R1 is OCH3 and R2 is OH, and B1 is m 6 A and B2 is A, U, G or C, wherein the cap analog having formula (II) is m 7 G 2’Ome pppm 6 A 2’Ome pA 2’Ome , m 7 G 2’Ome pppm 6 A 2’Ome pU 2’Ome , m 7 G 2’Ome pppm 6 A 2’Ome p.g. 2’Ome , and m 7 G 2’Ome pppm 6 A 2’Ome PC 2’Ome may be selected from the group consisting of:

[0055] In another embodiment, R1 is OH and R2 is H, B1 is A and B2 is A, U, G or C, wherein the cap analog having formula (II) is m 7 G 3’H pppA 2’Ome pA 2’Ome , m 7 G 3’H pppA 2’Ome pU 2’Ome , m 7 G 3’H pppA 2’Ome p.g. 2’Ome , and m 7 G 3’H pppA 2’Ome PC 2’Ome may be selected from the group consisting of:

[0056] In another embodiment, R1 is OH and R2 is H, and B1 is m 6 A and B2 is A, U, G or C, wherein the cap analog having formula (II) is m 7 G 3’H pppm 6 A 2’Ome pA 2’Ome , m 7 G 3’H pppm 6 A 2’Ome pU 2’Ome , m 7 G 3’H pppm 6 A 2’Ome p.g. 2’Ome , and m 7 G 3’H pppm 6 A 2’Ome PC 2’Ome may be selected from the group consisting of:

[0057] In one aspect, embodiments of the present disclosure may include a cap analog having formula (III): [ka] where R is H or CH3 and B1 is A or N6-methyl-adenine (m 6 A) and B2 is A, U, G or C.

[0058] In another embodiment, R is H, B1 is A, and B2 is A, U, G, or C, wherein the cap analog having formula (III) is m 7 Gpp S pA 2’Ome pA 2’Ome , m 7 Gpp S pA 2’Ome pU 2’Ome , m 7 Gpp S pA 2’Ome p.g. 2’Ome , and m 7 Gpp S pA 2’Ome PC 2’Ome may be selected from the group consisting of:

[0059] In another embodiment, R is H and B1 is m 6 A and B2 is A, U, G or C, wherein the cap analog having formula (III) is m 7 Gpp S pm 6 A 2’Ome pA 2’Ome , m 7 Gpp S pm 6 A 2’Ome pU 2’Ome , m 7 Gpp S pm 6 A 2’Ome p.g. 2’Ome , and m 7 Gpp S pm 6 A 2’Ome PC 2’Omemay be selected from the group consisting of:

[0060] In another embodiment, R is CH3, B1 is A, and B2 is A, U, G, or C, wherein the cap analog having formula (III) is m 7 G 3’Ome pp S pA 2’Ome pA 2’Ome , m 7 G 3’Ome pp S pA 2’Ome pU 2’Ome , m 7 G 3’Ome pp S pA 2’Ome p.g. 2’Ome , and m 7 G 3’Ome pp S pA 2’Ome PC 2’Ome may be selected from the group consisting of:

[0061] In another embodiment, R is CH3 and B1 is m 6 A and B2 is A, U, G or C, wherein the cap analog having formula (III) is m 7 G 3’Ome pp S pm 6 A 2’Ome pA 2’Ome , m 7 G 3’Ome pp S pm 6 A 2’Ome pU 2’Ome , m 7 G 3’Ome pp S pm 6 A 2’Ome p.g. 2’Ome , and m 7 G 3’Ome pp S pm 6 A 2’Ome PC2’Ome may be selected from the group consisting of:

[0062] In another aspect, embodiments of the present disclosure may include methods for synthesizing cap analogs, the methods comprising: (a) Compound (12) [ka] is reacted with dimethyl sulfate to give compound (13) [ka] and (b) reacting compound (13) with imidazole, triphenylphosphine, and 2,2'-dithiopyridine, followed by reaction with MnCl and bis-triethylammonium thiophosphate to give compound (14). [ka] and (c) Compound (5) [ka] with triphenylphosphine, imidazole and 2,2'-dithiopyridine to give compound (15). [ka] and (d) Compound (15) and Compound (14) [ka] in the presence of dimethylformamide and ZnCl to give compound (16). [ka] and obtaining the [Brief explanation of the drawings]

[0063] [Figure 1] 1 shows the use of a cap analog to initiate in vitro transcription at the −1 position, according to one embodiment of the present disclosure. [Figure 2] 1 illustrates a method for preparing a Cap-2 analog synthetic intermediate according to one embodiment of the present disclosure. [Figure 3] 1 shows a method for preparing a Cap-2 analog according to another embodiment of the present disclosure. [Figure 4] 1 shows a method for preparing another Cap-2 analog according to another embodiment of the present disclosure. [Figure 5] 1 shows the expression levels of mRNA prepared with various cap analogs, according to another embodiment of the present disclosure. [Figure 6] 1 shows a method for preparing another Cap-2 analog according to another embodiment of the present disclosure. [Figure 7] 1 shows a method for preparing another Cap-2 analog according to another embodiment of the present disclosure. [Figure 8] 1 shows a method for preparing another Cap-2 analog according to another embodiment of the present disclosure. [Figure 9] 1 shows the in vivo expression levels of mRNA prepared with various cap analogs, according to another embodiment of the present disclosure. [Figure 10] 1 shows the biodistribution of mRNA prepared with various cap analogs, according to one embodiment of the present disclosure. [Figure 11] 1 shows the effect of mRNA prepared with various cap analogs on mouse body weight, according to one embodiment of the present disclosure. DETAILED DESCRIPTION OF THE INVENTION

[0064] The present specification provides methods and compositions for in vitro mRNA synthesis. Also provided are methods and compositions for in vitro capped mRNA synthesis. Aspects of the disclosed methods and compositions can be used alone or in any combination. The disclosed methods and compositions can improve the efficiency of mRNA synthesis and provide mRNA with improved properties.

[0065] Regardless of the type of cap modification tested so far (e.g., cap-0 and cap-1), transcripts can be affected by the activity of DCP2, the major cellular decapping enzyme, which cooperates with its cellular partner, DCP1, to decap transcripts. 7 Another protein, DXO (decapping exoribonuclease), functions as a quality control factor to remove abnormally capped transcripts and can function as a decapping enzyme. Compared to DCP2, DXO can remove the entire cap structure by hydrolyzing the phosphodiester bond between the first and second transcribed nucleotides, resulting in the release of GDP and a monophosphorylated RNA strand. 7 This triggers the release of GpppN and monophosphorylated RNA, and as an exoribonuclease, it can subsequently degrade RNA in the 5'-3' direction. Interestingly, DXO was shown to have decapping activity that is inhibited by Cap-1 2'-O-methylation, indicating its potential role in distinguishing between "self" and "non-self" RNA.

[0066] A recent study by Drazkowska et al. demonstrated that 2'-O-methylation of the second transcribed nucleotide regulates protein biosynthesis in a cell-dependent manner (Drazkowska K, Tomecki R, Warminski M, Baran N, Cysewski D, Depaix A, Kasprzyk R, Kowalska J, Jemielity J, Sikorski PJ. 2'-O-Methylation of the second transcribed nucleotide within the mRNA 5' cap impacts the protein production level in a cell-specific manner and contributes to RNA immune evasion. Nucleic Acids Res. 2022 Sep 9;50(16):9051-9071, incorporated herein by reference in its entirety). Furthermore, RNA capped with Cap-2 was observed to be resistant to DXO-mediated decapping and degradation. Additionally, 2'-O-methylation of the second transcribed nucleotide and N6-methylation of adenosine as the first transcribed nucleotide serve as determinants that define the transcript as "self" and aid in immune evasion of the transcript.

[0067] Large-scale, efficient in vitro preparation of cap-2 mRNA may be of considerable importance for translating these findings into biomedical applications.

[0068] The disclosed methods and compositions can be used alone or in any combination to effect the in vitro synthesis of mRNAs of different sizes, for example, from about 100b to about 20Kb, from about 200b to about 19Kb, from about 300b to about 18Kb, from about 400b to about 17Kb, from about 500b to about 16Kb, from about 600b to about 15Kb, from about 700b to about 14Kb, from about 800b to about 13Kb, from about 900b to about 12Kb, from about 1Kb to about 11Kb, from about 1Kb to about 10Kb, from about 1Kb to about 9Kb, from about 1Kb to about 8Kb, from about 1Kb to about The mRNA may be in the range of, but is not limited to, 7 Kb, about 1 Kb to about 6 Kb, about 1 Kb to about 5 Kb, about 1 Kb to about 4 Kb, about 1 Kb to about 3 Kb, about 1 Kb to about 2 Kb, about 50 b to about 200 b, about 60 b to about 190 b, about 70 b to about 180 b, about 80 b to about 160 b, about 90 b to about 100 b, about 90 b to about 110 b, about 90 b to about 120 b, about 90 b to about 130 b, about 90 b to about 140 b, about 90 b to about 150 b, about 100 b to about 140 b, about 110 b to about 130 b, or about 110 b to about 120 b.

[0069] The mRNA synthesized using the disclosed compositions and / or methods can have many applications, including, but not limited to, in basic science research, pharmacological development, diagnostic development, therapeutic development, pharmaceutical applications, diagnostic applications, therapeutic applications, or any combination thereof.

[0070] Methods for RNA in vitro transcription are known in the art (see, e.g., Geall et al. (2013) Semin. Immunol. 25(2):152-159; Brunelle et al. (2013) Methods Enzymol. 530:101-14). The reagents used in the methods include a linear DNA template bearing a promoter sequence with high binding affinity for the corresponding RNA polymerase, ribonucleoside triphosphates (NTPs) of the four bases (adenine, cytosine, guanine, and uracil), cap analogs, other modified nucleotides, a DNA-dependent RNA polymerase (e.g., T7, T3, or SP6 RNA polymerase), a ribonuclease (RNase) inhibitor to inactivate any contaminating RNases, a pyrophosphatase to degrade transcription-inhibiting pyrophosphates, and Mg 2+ The solution may also contain MgCl and / or Mg(OAc) to provide as cofactors for RNA polymerase, an antioxidant (e.g., DTT), a polyamine such as spermidine, and a buffer to maintain an appropriate pH value.

[0071] A common buffer system used for RNA in vitro transcription may contain 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and tris(hydroxymethyl)aminomethane (Tris). The pH value of the buffer can generally be adjusted to be between 6 and 8.5. Some common transcription buffers may contain 80 mM HEPES / KOH (pH 7.5) and 40 mM Tris / HCl (pH 7.5).

[0072] The transfer buffer may further contain magnesium salts, such as MgCl2 and / or Mg(OAc)2, typically in the range of 5 mM to 50 mM. 2+ Magnesium ions (Mg) can function as a cofactor in the catalytic center of RNA polymerase and may be important for the RNA polymerization reaction. 2+) may be an essential component in RNA in vitro transcription buffer systems. In diffusion binding, fully hydrated Mg ions can also interact with RNA products via nonspecific long-range electrostatic interactions.

[0073] RNA in vitro transcription reactions can be performed in a batch reaction, where all components are mixed and incubated to allow the synthesis of RNA molecules until the reaction is complete. Furthermore, fed-batch reactions have been developed to improve the efficiency of RNA in vitro transcription reactions (Kern et al. (1997) Biotechnol. Prog. 13:747-756, Kern et al. (1999) Biotechnol. Prog. 15:174-184). In a fed-batch system, all components are combined, and different amounts of some reagents (e.g., NTPs, MgCl2, and / or Mg(OAc)2) are added over time to maintain constant reaction conditions.

[0074] For clarity and readability, the following definitions are provided. Any technical features mentioned in these definitions can be read in the context of the embodiments of the present invention. In particular, other definitions and interpretations can be provided in the context of these embodiments.

[0075] In vitro transcription: The terms "in vitro transcription" or "RNA in vitro transcription" may refer to a process in which RNA is synthesized in a cell-free system (in vitro). DNA, particularly plasmid DNA, is used as a template to produce RNA transcripts. RNA can be obtained by DNA-dependent in vitro transcription of a suitable DNA template, which, according to the present disclosure, may preferably be a linearized plasmid DNA template. The promoter for controlling in vitro transcription may be any promoter for any DNA-dependent RNA polymerase. Specific, non-limiting examples of DNA-dependent RNA polymerases are T7, T3, and SP6 RNA polymerases. A DNA template for in vitro RNA transcription can be obtained, for example, by cloning a nucleic acid (particularly a cDNA corresponding to the RNA of interest to be in vitro transcribed) and introducing it into a suitable vector for in vitro transcription, e.g., plasmid DNA. In a preferred embodiment of the present disclosure, the DNA template may be linearized with an appropriate restriction enzyme before in vitro transcription. cDNA can be obtained by reverse transcription of mRNA or chemical synthesis. Alternatively, a DNA template for in vitro RNA synthesis can also be obtained by gene synthesis.

[0076] For example, reagents used in in vitro transcription include: 1) a linearized DNA template having a promoter sequence with high binding affinity for the corresponding RNA polymerase (e.g., a phage-encoded RNA polymerase); 2) ribonucleoside triphosphates (NTPs) of the four bases (adenine, cytosine, guanine, and uracil); 3) optionally a cap analogue defined as follows: 4) a DNA-dependent RNA polymerase (e.g., T7, T3, or SP6 RNA polymerase) capable of binding to a promoter sequence within a linearized DNA template; 5) an optional ribonuclease (RNase) inhibitor to inactivate any contaminating RNases; and 6) an optional pyrophosphatase to degrade pyrophosphates that can inhibit transcription; and 7) Mg 2+ MgCl and / or magnesium acetate (Mg(CHO)) (Mg(OAc)), which provide ions as cofactors for the polymerase; 8) A buffer for maintaining an appropriate pH value, which may further contain optimal concentrations of antioxidants (e.g., DTT), amines (e.g., betaine), and / or polyamines (e.g., spermidine).

[0077] In the examples, the methods of RNA in vitro transcription according to the present disclosure do not use the following reagents, which are only necessary for in vitro translation of transcribed RNA into proteins, but are not necessary for RNA in vitro transcription. In particular, the mixture for RNA in vitro transcription may not contain any proteinogenic amino acids or tRNA. Furthermore, the mixture may not contain any proteinogenic amino acids, tRNA, or ribosome-containing cell extracts.

[0078] As used herein, the term "co-transcription" refers to the preparation of capped mRNA through a one-step in vitro transcription reaction using an RNA polymerase (e.g., T7 RNA polymerase). In contrast, one or more conventional post-transcriptional capping methods may require in vitro transcription (IVT) in the presence of an RNA polymerase to prepare uncapped RNA, followed by addition of a cap using a capping enzyme (e.g., vaccinia capping enzyme) with the aid of a 2'-O-methyltransferase to add methylation to the +1 base of the mRNA.

[0079] Nucleic Acid: The term "nucleic acid" refers to any DNA or RNA molecule and is used synonymously with polynucleotide. However, modifications or derivatives of nucleic acids as defined herein are expressly included in the general term "nucleic acid." For example, peptide nucleic acids (PNAs) are also included in the term "nucleic acid."

[0080] Nucleic acid template: The nucleic acid template provides a nucleic acid sequence that is transcribed into RNA by an in vitro transcription process and thus contains a nucleic acid sequence that is complementary to the RNA sequence transcribed therefrom. In addition to the nucleic acid sequence that is transcribed into RNA, the nucleic acid template further contains a promoter, and the RNA polymerase used in the in vitro transcription process binds to the promoter with high affinity.

[0081] Preferably, the nucleic acid template may be a linearized plasmid DNA template. Linear template DNA can be obtained by contacting plasmid DNA with a restriction enzyme under appropriate conditions, so that the restriction enzyme cleaves the plasmid DNA at one or more of its recognition sites, disrupting the circular plasmid structure. Preferably, the plasmid DNA is cleaved immediately after the end of the sequence to be transcribed into RNA. Therefore, the linear template DNA contains a free 5' end and a free 3' end that are not linked to each other. If the plasmid DNA contains only one recognition site for the restriction enzyme, the linear template DNA has the same number of nucleotides as the plasmid DNA. If the plasmid DNA contains multiple recognition sites for the restriction enzyme, the linear template DNA has fewer nucleotides than the plasmid DNA. Thus, the linear template DNA is a fragment of plasmid DNA that contains elements necessary for in vitro transcription, including promoter and template DNA elements for RNA transcription. According to base-pairing rules, the open reading frame (ORF) of the linear template DNA can determine the sequence of the transcribed RNA.

[0082] In other examples, the nucleic acid template may be selected from a synthetic double-stranded DNA construct, a single-stranded DNA template having a double-stranded DNA region containing a promoter bound by RNA polymerase, a circular double-stranded DNA template having a promoter and terminator sequence, or a linear DNA template amplified by PCR or isothermal amplification.

[0083] According to preferred embodiments of the present disclosure, the concentration of nucleic acid template in the in vitro transcription mixtures described herein may be within the range of about 1 to about 200 nM, about 10 nM to about 150 nM, about 20 nM to about 140 nM, about 30 nM to about 130 nM, about 40 nM to about 120 nM, about 50 nM to about 110 nM, about 60 nM to about 100 nM, about 65 nM to about 90 nM, about 65 nM to about 80 nM, about 65 nM to about 75 nM, about 65 nM to about 70 nM, about 70 nM to about 75 nM, about 1 to about 40 nM, about 1 to about 30 nM, about 1 to about 20 nM, or about 1 to about 10 nM. Even more preferably, the concentration of nucleic acid template may be about 10 to about 30 nM. Most preferably, the concentration of nucleic acid template may be about 40, 50, 60, 70, 80, 90 or 100 nM.

[0084] RNA, mRNA: RNA is a common abbreviation for ribose nucleic acid. It is a nucleic acid molecule, i.e., a polymer composed of nucleotide monomers. These nucleotides are generally adenosine monophosphate (AMP), uridine monophosphate (UMP), guanosine monophosphate (GMP), and cytidine monophosphate (CMP) monomers or their analogs linked together along a so-called backbone. The backbone is formed by a phosphodiester bond between the sugar of a first monomer (i.e., ribose) and the phosphate moiety of a second adjacent monomer. The specific order of the monomers (i.e., the order of the bases linked to the sugar / phosphate backbone) is called the RNA sequence. Generally, RNA can be obtained (e.g., intracellularly) by transcription of a DNA sequence. In eukaryotic cells, transcription generally occurs in the cell nucleus or mitochondria. In vivo, DNA transcription generally produces so-called premature RNA, which must be processed into so-called messenger RNA (commonly abbreviated as mRNA). For example, the processing of immature RNA in eukaryotic organisms involves several different post-transcriptional modifications, such as splicing, 5'-capping, polyadenylation, and export from the cell nucleus or mitochondria. The sum of these processes is also called RNA maturation. Mature messenger RNA generally provides a nucleotide sequence that can be translated into the amino acid sequence of a specific peptide or protein. Typically, mature mRNA contains a 5'-cap, optionally a 5'UTR, an open reading frame, optionally a 3'UTR, and a poly(A) sequence.

[0085] In addition to messenger RNA, there are also several non-coding types of RNA, which may be involved in regulating transcription and / or translation and immune stimulation. The term "RNA" further covers RNA molecules such as viral RNA, retroviral RNA and replicon RNA, small interfering RNA (siRNA), antisense RNA, CRISPR / Cas9 guide RNA, ribozyme, aptamer, ribose switch, immune stimulatory RNA, transfer RNA (tRNA), ribosomal RNA (rRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), microRNA (miRNA), and Piwi-interacting RNA (piRNA).

[0086] Dicarboxylic acids or their salts: Dicarboxylic acids are organic acids with two carboxyl groups (-COOH). The term refers to the general formula HO2C-(CH2) n These include linear saturated dicarboxylic acids with -COH, such as oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, pimelic acid, suberic acid, azelaic acid, and sebacic acid. They also include unsaturated dicarboxylic acids with at least one double bond (e.g., maleic acid and fumaric acid), and substituted dicarboxylic acids with at least one additional functional group (e.g., malic acid, tartaric acid, chicoric acid, and dimercaptosuccinic acid). Salts of dicarboxylic acids are obtained by combining a dicarboxylic acid anion with a suitable cation, such as Na. + , K. + , Ca 2+ or Mg 2+ Includes:

[0087] Tricarboxylic acids or their salts: Tricarboxylic acids are organic acids with three carboxyl groups (-COOH). Examples of tricarboxylic acids include citric acid, isocitric acid, aconitic acid, trimesic acid, nitrilotriacetic acid, and propane-1,2,3-tricarboxylic acid. In the buffer system and method of the present invention, citric acid (3-carboxy-3-hydroxypentane-1,5-dioic acid) is preferably used. Salts of tricarboxylic acids are formed by combining a tricarboxylic acid anion with a suitable cation, such as Na. + , K.+ , Ca 2+ or Mg 2+ Preferably, sodium citrate or magnesium citrate is used. When magnesium citrate is added to an RNA in vitro transcription reaction, the magnesium ions in the magnesium citrate can act as a cofactor for RNA polymerase, so it may not be necessary to add a magnesium salt to the reaction. Therefore, in such cases, the reaction mixture for RNA in vitro transcription contains magnesium citrate, a buffer substance, ribonucleoside triphosphates, a nucleic acid template, and an RNA polymerase.

[0088] Buffer: A buffer is a weak acid or base used to maintain the acidity (pH) of a solution near a selected value after the addition of another acid or base. Therefore, the function of a buffer is to prevent a sudden change in pH when an acid or base is added to a solution. Suitable buffers for use in the present invention include Tris (2-amino-2-hydroxymethyl-propane-1,3-diol) and HEPES (2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid). The buffer may further contain an acid or base to adjust the pH, such as HCl in the case of Tris (Tris-HCl) and KOH in the case of HEPES (HEPES-KOH). In a preferred embodiment of the present invention, citric acid is used to adjust the pH of the buffer (preferably Tris base), thereby avoiding the addition of other acids. In an alternative embodiment, the pH of the buffer is adjusted using an acid or base (e.g., HCl and KOH), and in addition to the pH-adjusted buffer, a salt of a dicarboxylic or tricarboxylic acid, preferably a citrate salt, is also present in the reaction mixture.

[0089] The concentration of the buffer in the mixture for in vitro transcription described herein may be about 10 to about 100 mM, about 10 to about 80 mM, about 10 to about 50 mM, about 10 to about 40 mM, about 10 to about 30 mM, or about 10 to about 20 mM. Preferably, the concentration of the buffer is 40 mM.

[0090] Preferably, the buffer has a pH value of about 6 to about 8.5, about 6.5 to about 8.0, about 7.0 to about 7.5, and even more preferably about 7.5 or about 8.0.

[0091] Ribonucleoside triphosphates: Ribonucleoside triphosphates (NTPs) (i.e., GTP, ATP, CTP, and UTP) are monomers polymerized in the in vitro transcription process. They can be provided with monovalent or divalent cations as counterions. Preferably, the monovalent cation is Li + , Na + , K. + , NH4 + or tris(hydroxymethyl)-aminomethane (Tris). Preferably, the divalent cation is selected from the group consisting of Mg 2+ , Ba 2+ and Mn 2+ More preferably, the monovalent cation is selected from the group consisting of Na + or tris(hydroxymethyl)-aminomethane (Tris).

[0092] The NTP concentration may be from about 1 mM to about 50 mM, from about 1 mM to about 40 mM, from about 1 mM to about 30 mM, from about 1 mM to about 20 mM, from about 1 mM to about 10 mM, from about 1 mM to about 5 mM, from about 2 mM to about 10 mM, from about 3 mM to about 10 mM, from about 3 mM to about 9 mM, from about 3 mM to about 8 mM, from about 3 mM to about 7 mM, from about 3 mM to about 6 mM, from about 3 mM to about 5 mM, from about 3 mM to about 4 mM, It may be 4 mM to about 10 mM, about 5 mM to about 10 mM, about 6 mM to about 10 mM, about 7 mM to about 10 mM, about 8 mM to about 10 mM, about 9 mM to about 10 mM, about 10 mM to about 50 mM, about 20 mM to about 50 mM, about 25 mM to about 50 mM, about 25 mM to about 45 mM, about 25 mM to about 40 mM, about 25 mM to about 35 mM, or about 20 mM to about 30 mM.

[0093] According to a preferred embodiment of the present invention, some or all of at least one ribonucleoside triphosphate in an in vitro transcription reaction mixture is replaced with a modified nucleoside triphosphate, as defined below.

[0094] Modified nucleoside triphosphate: As used herein, the term "modified nucleoside triphosphate" refers to chemical modifications, including backbone modifications and sugar or base modifications. These modified nucleoside triphosphates are also referred to herein as (nucleotide) analogs.

[0095] In this context, a modified nucleoside triphosphate as defined herein is a nucleotide analog / modification, such as a backbone modification, sugar modification, or base modification. A backbone modification according to the present invention is a modification in which the phosphate in the backbone of the nucleotide is chemically modified. A sugar modification according to the present invention is a chemical modification of the sugar in the nucleotide. Note that a base modification according to the present invention is a chemical modification of the base moiety of the nucleotide. In this context, the nucleotide analog or modification is preferably selected from nucleotide analogs applicable to transcription and / or translation. sugar modification

[0096] Modified nucleosides and nucleotides usable in the context of the present invention can be modified in the sugar moiety. For example, the 2' hydroxy group (OH) can be modified or replaced with several different "oxy" or "deoxy" substituents. Examples of "oxy"-2' hydroxy groups include alkoxy or aryloxy groups (-OR, e.g., R=H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugars), and polyethylene glycol (PEG), -O(CHCHO). nThese include, but are not limited to, CH2CH2OR, "locked" nucleic acids (LNAs) in which the 2' hydroxy group is linked to the 4' carbon of the same ribose, for example via a methylene bridge, and amino groups (-O-amino groups, where the amino group (e.g., NRR) can be an alkylamino group, dialkylamino group, heterocyclyl group, arylamino group, diarylamino group, heteroarylamino group, or diheteroarylamino group, ethylenediamine, polyamino group) or aminoalkoxy groups.

[0097] A "deoxy" modification can contain a hydrogen, an amino group (e.g., NH, alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid), or the amino group can be attached to the sugar via a linker, where the linker contains one or more of C, N, and O atoms.

[0098] The sugar group may contain one or more additional carbons that have the opposite stereochemical configuration to the corresponding carbons in ribose, so modified nucleotides may include, for example, nucleotides that contain arabinose as the sugar. skeletal modifications

[0099] The phosphate backbone may be further modified in modified nucleosides and nucleotides. The backbone phosphate group can be modified by replacing one or more oxygen atoms with different substituents. Additionally, modified nucleosides and nucleotides may include the complete replacement of the unmodified phosphate moiety with a modified phosphate as described herein. Examples of modified phosphate groups include, but are not limited to, thiophosphates, selenium phosphates, boranophosphates, boranophosphate esters, hydrogen phosphate esters, phosphoric acid amides, alkyl or aryl phosphonates, and phosphoric acid triesters. Dithiophosphates have two non-linked oxygens replaced with sulfur. The phosphate linker can be further modified by replacing the linking oxygen with nitrogen (bridged phosphoric acid amides), sulfur (bridged thiophosphates), and carbon (bridged methylene-phosphonates). Base Modification

[0100] The modified nucleosides and nucleotides usable in the present disclosure may be further modified in the nucleobase moiety. Examples of nucleobases found in RNA include, but are not limited to, adenine, guanine, cytosine, and uracil. For example, the nucleosides and nucleotides described herein may be chemically modified on the major groove surface. In some embodiments, the major groove chemical modification may include an amino group, a thiol group, an alkyl group, or a halo group.

[0101] In a particularly preferred embodiment of the present invention, the nucleotide analogue / modification may be selected from base modifications, which are preferably 2-amino-6-chloropurine nucleoside-5'-triphosphate, 2-aminopurine-nucleoside-5'-triphosphate, 2-aminoadenosine-5'-triphosphate, 2'-amino-2'-deoxycytidine-triphosphate, 2-thiocytidine-5'-triphosphate, 2-thiouridine-5'-triphosphate, 2'-fluorothymidine-5'-triphosphate, 2'-O-methylinosine-5'-triphosphate, 4-thiouridine-5'-triphosphate, 5-aminoallylcytidine-5'-triphosphate, 5-aminoallyluridine-5'-triphosphate, 5-bromocytidine-5'-triphosphate, 5-bromouridine-5'-triphosphate, 5-bromo-2'-deoxycytidine-5'-triphosphate, 5-bromo-2'-deoxyuridine-5'-triphosphate, 5-iodocytidine-5'-triphosphate, 5-iodo-2'-deoxycytidine-5'-triphosphate, 5- Iodouridine-5'-triphosphate, 5-iodo-2'-deoxyuridine-5'-triphosphate, 5-methylcytidine-5'-triphosphate, 5-methyluridine-5'-triphosphate, 5-propynyl-2'-deoxycytidine-5'-triphosphate, 5-propynyl-2'-deoxyuridine-5'-triphosphate, 6-azacytidine-5'-triphosphate, 6-azauridine-5'-triphosphate, 6-chloropurine nucleoside-5'-triphosphate, 7-deazaadenosine-5'-triphosphate, 7-deaza The base-modified nucleotides are selected from the group consisting of 5-methylcytidine-5'-triphosphate, 7-deazaguanosine-5'-triphosphate, 5-bromocytidine-5'-triphosphate, and pseudouridine-5'-triphosphate.

[0102] In some embodiments, the modified nucleoside is pyridine-4-ketoribose nucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinemethyluridine, 1-taurinemethyl-pseudouridine, 5-taurinemethyl-2-thio-uridine, 1-taurinemethyl- The pseudouridine may include 1-methyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine and 4-methoxy-2-thio-pseudouridine.

[0103] In some embodiments, the modified nucleoside is 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine May include isocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine and 4-methoxy-1-methyl-pseudoisocytidine.

[0104] In other examples, the modified nucleoside is 2-aminopurine, 2,6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6 These may include -(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, N6-glycylcarbamoyladenosine, N6-threoninoylcarbamoyladenosine, 2-methylthio-N6-threoninoylcarbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, and 2-methoxy-adenine.

[0105] In other examples, modified nucleosides may include inosine, 1-methyl-inosine, uiosine, uibutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine.

[0106] In some examples, the nucleotide may be modified on the major groove face and may include replacing the hydrogen on C-5 of uracil with a methyl or halo group.

[0107] In specific embodiments, the modified nucleoside is 5'-O-(1-thiophosphate)-adenosine, 5'-O-(1-thiophosphate)-cytidine, 5'-O-(1-thiophosphate)-guanosine, 5'-O-(1-thiophosphate)-uridine, or 5'-O-(1-thiophosphate)-pseudouridine.

[0108] In another specific embodiment, the modified nucleotide is 6-aza-cytidine, 2-thio-cytidine, α-thio-cytidine, pseudoisocytidine, 5-aminoallyl-uridine, 5-iodo-uridine, N1-methyl-pseudouridine, 5,6-dihydrouridine, α-thio-uridine, 4-thio-uridine, 6-aza-uridine, 5-hydroxy-uridine, deoxy-thymidine, 5-methyl-uridine, pyrrolo-cytidine, ino The nucleoside modifications may be selected from cytidine, α-thio-guanosine, 6-methyl-guanosine, 5-methyl-cytidine, 8-oxo-guanosine, 7-deaza-guanosine, N1-methyl-adenosine, 2-amino-6-chloro-purine, N6-methyl-2-amino-purine, pseudoisocytidine, 6-chloro-purine, N6-methyl-adenosine, α-thio-adenosine, 8-azidoadenosine, 7-deaza-adenosine.

[0109] Magnesium salt: A magnesium salt comprises a magnesium cation and a suitable anion (e.g., chloride or acetate). Preferably, the magnesium salt is magnesium chloride. In one or more in vitro transcription mixtures described herein, preferably, the initial free Mg 2+ The concentration may be about 1 to about 100 mM, about 1 to about 75 mM, about 1 to about 50 mM, about 1 to about 25 mM, or about 1 to about 10 mM. 2+ The concentration is about 5 to about 50 mM, about 10 to about 45 mM, about 15 to about 40 mM, or about 16 to about 37 mM, e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, or 37 mM. 2+The choice of concentration may be influenced by the initial total NTP concentration, as using a higher total NTP concentration in the in vitro transcription mixture may result in a higher Mg 2+ In some embodiments, the concentration of the magnesium salt may be from about 2 mM to about 50 mM, from about 2 mM to about 40 mM, from about 2 mM to about 30 mM, from about 2 mM to about 40 mM, from about 2 mM to about 30 mM, from about 2 mM to about 20 mM, from about 2 mM to about 10 mM, from about 2 mM to about 5 mM, from about 5 mM to about 50 mM, from about 10 mM to about 45 mM, from about 15 mM to about 40 mM, from about 20 mM to about 35 mM, from about 20 mM to about 30 mM, from about 20 mM to about 25 mM, from about 22 mM to about 28 mM, or from about 25 mM to about 30 mM.

[0110] RNA polymerase: An RNA polymerase is an enzyme that catalyzes the transcription of a DNA template into RNA. Suitable RNA polymerases for use in the present disclosure may include T7, T3, SP6, and E. coli RNA polymerase. Preferably, T7 RNA polymerase may be used. More preferably, the RNA polymerase for use in the present disclosure may be a recombinant RNA polymerase, meaning that it is added to the RNA in vitro transcription reaction as a single component, rather than as part of a cell extract containing other components in addition to the RNA polymerase. As will be understood by those skilled in the art, the choice of RNA polymerase depends on the promoter present in the DNA template, which must be bound to the appropriate RNA polymerase. Preferably, the concentration of RNA polymerase in one or more of the in vitro transcription mixtures described herein is from about 0.001 μg / μl to about 2 μg / μl, from about 0.001 μg / μl to about 1.5 μg / μl, from about 0.001 μg / μl to about 1 μg / μl, from about 0.01 μg / μl to about 1 μg / μl, from about 0.01 μg / μl to about 0.5 μg / μl, from about 0.01 μg / μl to about 0.5 μg / μl, from about 0.01 μg / μl to about 0.1 μg / μl, or from about 0.01 μg / μl to about 0.5 μg / μl. The concentration may be from about 0.01 μg / μl to about 0.05 μg / μl, from about 0.1 μg / μl to about 1 μg / μl, from about 0.1 μg / μl to about 0.9 μg / μl, from about 0.1 μg / μl to about 0.8 μg / μl, from about 0.1 μg / μl to about 0.7 μg / μl, from about 0.1 μg / μl to about 0.6 μg / μl, from about 0.1 μg / μl to about 0.5 μg / μl, from about 0.1 μg / μl to about 0.4 μg / μl, from about 0.1 μg / μl to about 0.3 μg / μl, or from about 0.1 μg / μl to about 0.2 μg / μl. Optionally, in one or more in vitro transcription mixtures described herein, the concentration of RNA polymerase can be from about 0.1 U / μl to about 2 U / μl, from about 0.5 U / μl to about 2 U / μl, from about 1 U / μl to about 2 U / μl, from about 1 U / μl to about 1.5 U / μl, or from about 1.5 U / μl to about 2 U / μl. As one of skill in the art will appreciate, the choice of RNA polymerase concentration can be influenced by the DNA template concentration.

[0111] Pyrophosphatase: Pyrophosphatase is an anhydride hydrolase that hydrolyzes diphosphate bonds. In in vitro transcription reactions, it is used to hydrolyze the bond within the diphosphate that is released after ribonucleoside triphosphates are incorporated into the nascent RNA strand. Preferably, in one or more in vitro transcription mixtures described herein, the concentration of pyrophosphatase is from about 0.01 mU / μl to about 2 mU / μl, from about 0.01 mU / μl to about 1.5 mU / μl, from about 0.01 mU / μl to about 1 mU / μl, from about 0.1 mU / μl to about 2 mU / μl, from about 0.1 mU / μl to about 1.5 mU / μl, from about 0.1 mU / μl to about 1 mU / μl, or from about 0. The concentration of pyrophosphatase may be from 1 mU / μl to about 0.9 mU / μl, from about 0.1 mU / μl to about 0.8 mU / μl, from about 0.1 mU / μl to about 0.7 mU / μl, from about 0.1 mU / μl to about 0.6 mU / μl, from about 0.1 mU / μl to about 0.5 mU / μl, from about 0.1 mU / μl to about 0.4 mU / μl, from about 0.1 mU / μl to about 0.3 mU / μl, or from about 0.1 mU / μl to about 0.2 mU / μl. Even more preferably, the concentration of pyrophosphatase may be about 0.3 mU / μl or about 0.5 mU / μl.

[0112] 5'-cap structure: A 5'-cap is typically a modified nucleotide, particularly a guanine nucleotide, added to the 5' end of an RNA molecule. Preferably, the 5'-cap can be added using a 5'-5'-triphosphate linkage. The 5'-cap may be methylated, e.g., m7GpppN, where N is the terminal 5' nucleotide of the 5'-capped nucleic acid, typically at the 5' end of the RNA. Naturally occurring 5'-caps may include m7GpppN.

[0113] The 5' cap structure may be formed from a cap analog.

[0114] Cap analog: A cap analog refers to a non-extendable dinucleotide or trinucleotide with capping function, which means that when incorporated at the 5' end of an RNA molecule, it promotes translation or localization and / or prevents degradation of the RNA molecule. Capped mRNA without a 5'-terminal triphosphate structure reduces its immunogenic side effects. Non-extendable means that the cap analog is incorporated only at the 5' end, does not have a 5' triphosphate, and therefore cannot be extended in the 3' direction by template-dependent RNA polymerase.

[0115] In some embodiments, the cap analog may include a cap-2 analog having the structure of formula (I): [ka] where R is H or CH3 and B1 is A or N6-methyl-adenine (m 6 A) and B2 is A, U, G or C.

[0116] When R=H, the cap-2 analog is m 7 GpppA 2’Ome pA 2’Ome , m 7 GpppA 2’Ome pU 2’Ome , m 7 GpppA 2’Ome p.g. 2’Ome , m 7 GpppA 2’Ome PC 2’Ome , m 7 Gpppm 6 A 2’Ome pA 2’Ome , m 7 Gpppm 6 A 2’Ome pU 2’Ome , m 7 Gpppm 6 A 2’Ome p.g.2’Ome , and m 7 Gpppm 6 A 2’Ome PC 2’Ome may include:

[0117] When R=CH3, the cap-2 analog is m 7 G 3’Ome pppA 2’Ome pA 2’Ome , m 7 G 3’Ome pppA 2’Ome pU 2’Ome , m 7 G 3’Ome pppA 2’Ome p.g. 2’Ome , m 7 G 3’Ome pppA 2’Ome PC 2’Ome , m 7 G 3’Ome pppm 6 A 2’Ome pA 2’Ome , m 7 G 3’Ome pppm 6 A 2’Ome pU 2’Ome , m 7 G 3’Ome pppm 6 A 2’Ome p.g. 2’Ome , and m 7 G 3’Ome pppm 6 A 2’Ome PC 2’Ome may include:

[0118] In some embodiments, the cap analog may include a cap-2 analog having the structure of formula (II): [ka] where: R1 is OCH3 and R2 is OH or H, or R1 is OH and R2 is H, or R1 is H and R2 is H or OCH3, or R1 and R2 are each OCH3; B1 is A or N6-methyl-adenine (m 6 A) and B2 is A, U, G or C.

[0119] When R1 is OCH3 and R2 is OH, the Cap-2 analog is m 7 G 2’Ome pppA 2’Ome pA 2’Ome , m 7 G 2’Ome pppA 2’Ome pU 2’Ome , m 7 G 2’Ome pppA 2’Ome p.g. 2’Ome , m 7 G 2’Ome pppA 2’Ome PC 2’Ome , m 7 G 2’Ome pppm 6 A 2’Ome pA 2’Ome , m 7 G 2’Ome pppm 6 A 2’Ome pU 2’Ome , m 7 G 2’Ome pppm 6 A 2’Ome p.g. 2’Ome , and m 7 G 2’Ome pppm 6 A 2’Ome PC 2’Ome may include:

[0120] When R1 is OH and R2 is H, the Cap-2 analog is m 7 G 3’H pppA 2’Ome pA 2’Ome , m 7 G 3’H pppA 2’Ome pU 2’Ome , m 7 G 3’H pppA 2’Ome p.g. 2’Ome , m 7 G 3’H pppA 2’Ome PC 2’Ome , m 7 G 3’H pppm 6 A 2’Ome pA 2’Ome , m 7 G 3’H pppm 6 A 2’Ome pU 2’Ome , m 7 G 3’H pppm 6 A 2’Ome p.g. 2’Ome , and m 7 G 3’H pppm 6 A 2’Ome PC 2’Ome may include:

[0121] Preferably, the cap analog may be added at an initial concentration ranging from about 1 to about 20 mM, about 1 to about 17.5 mM, about 1 to about 15 mM, about 1 to about 12.5 mM, about 1 to about 10 mM, about 1 to about 7.5 mM, about 1 to about 5 mM, or about 1 to about 2.5 mM, and even more preferably, from about 5 to about 20 mM, about 7.5 to about 20 mM, about 10 to about 20 mM, or about 12.5 to about 20 mM. In some embodiments, the concentration of the cap analog is from about 0.5 mM to about 50 mM, from about 0.5 mM to about 40 mM, from about 0.5 mM to about 30 mM, from about 0.5 mM to about 20 mM, from about 0.5 mM to about 10 mM, from about 0.5 mM to about 5 mM, from about 1 mM to about 10 mM, from about 2 mM to about 10 mM, from about 3 mM to about 10 mM, from about 3 mM to about 9 mM, from about 3 mM to about 8 mM, from about 3 mM to about 7 mM, It may be 3 mM to about 6 mM, about 3 mM to about 5 mM, about 3 mM to about 4 mM, about 4 mM to about 10 mM, about 5 mM to about 10 mM, about 6 mM to about 10 mM, about 6 mM to about 9 mM, about 6 mM to about 8 mM, about 6 mM to about 7 mM, about 7 mM to about 8 mM, about 7 mM to about 9 mM, about 7 mM to about 10 mM, about 8 mM to about 9 mM, about 8 mM to about 10 mM, or about 9 mM to about 10 mM.

[0122] Ribonuclease inhibitors: Ribonuclease inhibitors inhibit the action of ribonucleases, which degrade RNA. Preferably, in one or more in vitro transcription mixtures described herein, the concentration of the ribonuclease inhibitor is from about 0.001 U / μl to about 5 U / μl, from about 0.001 U / μl to about 4 U / μl, from about 0.001 U / μl to about 3 U / μl, from about 0.001 U / μl to about 2 U / μl, from about 0.001 U / μl to about 1 U / μl, from about 0.01 U / μl to about 5 U / μl, from about 0.01 U / μl to about 4 U / μl, from about 0.01 U / μl to about 3 ... 0.01U / μl to about 2U / μl, about 0.01U / μl to about 1U / μl, about 0.01U / μl to about 0.5U / μl, about 0.01U / μl to about 0.1U / μl, about 0.01U / μl to about 0.05U / μl, about 0.01U / μl to about 0.04U / μl, about 0.01U / μl to about 0.03U / μl, about 0.01U / μl to about 0.02U / μl, about 0.1U / μl to about 5U / μl, about 0.1U / μl to about 4U / μl, about 0.1U / μl to about 3U / μl, about 0.1U / μl to about 2U / μl, about 0.1U / μl to about 1U / μl, about 0.5U / μl to about 5U / μl, about 0.5U / μl to about 4U / μl, about 0.5U / μl to about 3U / μl, about 0.5U / μl to about 2U / μl, about 0.5U / μl to about 1U / μl, about 1U / μl to about 5U / μl, about 2U / μl to about 5U / μl, about 3U / μl to about 5U / μl, about 4U / μl to about 5U / μl, about 0.001U / μl to about 0.005U / μl, about 0.001 U / μl to about 0.01 U / μl, about 0.005 U / μl to about 0.01 U / μl, about 0.01 U / μl to about 0.05 U / μl, about 0.01 U / μl to about 0.04 U / μl, about 0.01 U / μl to about 0.03 U / μl, about 0.01 U / μl to about 0.02 U / μl, about 0.02 U / μl to about 0.03 U / μl, about 0.02 U / μl to about 0.04 U / μl, or about 0.02 U / μl to about 0.05 U / μl.

[0123] Antioxidants: Antioxidants inhibit the oxidation of other molecules. Suitable antioxidants for use in the present disclosure may include, but are not limited to, DTT (dithiothreitol), TCEP (tris(2-carboxyethyl)phosphine), NAC (N-acetylcysteine), β-mercaptoethanol, glutathione, cysteine, and cystine. Preferably, DTT can be used in the in vitro transcription reaction.

[0124] The concentration of antioxidant, preferably DTT, in one or more in vitro transcription mixtures described herein may be about 1 to about 50 mM, about 5 to about 48 mM, about 8 to about 47 mM, about 10 to about 46 mM, about 15 to about 45 mM, about 18 to about 44 mM, about 20 to about 43 mM, about 23 to about 42 mM, about 25 to about 41 mM, or about 28 to about 40 mM. Preferably, the concentration may be about 20 mM.

[0125] Amine: Preferably, the amine used in the present invention may be betaine (trimethylglycine). The concentration of the amine (preferably betaine) may be about 10 mM to about 2 M, preferably about 0.7 M to about 1.3 M.

[0126] Polyamine: Preferably, the polyamine may be selected from the group consisting of spermine and spermidine. Preferably, the concentration of the polyamine may be about 1 to about 25 mM, about 1 to about 20 mM, about 1 to about 15 mM, about 1 to about 10 mM, about 1 to about 5 mM, or about 1 to about 2.5 mM. Even more preferably, the concentration of the polyamine may be about 2 mM. Most preferably, the concentration of the polyamine may be about 2 mM spermidine.

[0127] DNase: DNase is an enzyme that hydrolyzes DNA by catalyzing the hydrolytic cleavage of phosphodiester linkages in the DNA backbone. Suitable DNases can be isolated from bovine pancreatic glands and are available from different suppliers (e.g., Sigma-Aldrich, New England Biolabs, Qiagen, and ThermoFisher). Preferably, DNase does not have any RNase activity. In the method of the present disclosure, DNase treatment can be performed after the RNA in vitro transcription reaction by adding DNase to the reaction mixture for RNA in vitro transcription. Preferably, an appropriate amount of calcium chloride can be added to the RNA in vitro transcription mixture together with DNase. The appropriate amount of CaCl2 can be about 1 to about 5 mM, preferably about 2 to about 4 mM, and more preferably about 3 mM. DNA can be treated with DNase for about 1 to about 5 hours, preferably about 1.5 to about 3 hours, and more preferably about 2 hours. DNase treatment can be performed at a temperature of about 37°C. In one example, about 3 mM CaCl2 and about 200 U / ml DNase I may be added to the RNA in vitro transcription mixture, and the resulting mixture may be incubated at about 37°C for about two hours. In another example, about 3 mM CaCl2 and about 400 U / ml DNase I may be added to the RNA in vitro transcription mixture, and the resulting mixture may be incubated at about 37°C for about two hours. In another example, DNase may be added to the RNA in vitro transcription mixture, and the resulting mixture may be incubated at about 31°C for about 30 minutes. DNase treatment may be stopped by adding EDTA or another chelating agent. Preferably, DNase treatment may be stopped by adding EDTA to a final concentration of about 25 mM.

[0128] Examples of the present disclosure can provide a combined solution for in vitro synthesis of mRNAs of different sizes, for example, but not limited to, about 1 Kb to 20 Kb, such as 1 Kb to 15 Kb or 1 Kb to 10 Kb, in a time-efficient manner with high capping efficiency, uniform polyA tails, high yield, and integrity. Examples of the present disclosure can include transcribing capped mRNAs in vitro using T7 RNA polymerase by designing a T7 promoter sequence in a DNA template to provide high affinity for the cap analog. Such a DNA template promoter design can include a T7 Φ6.5 promoter followed by the sequence GG. Such a design ensures efficient initiation of transcription, allowing for the preparation of capped mRNAs with high fidelity at the 5' end in a one-step process. 5'-end capping of mRNA

[0129] One of the important factors determining mRNA translation efficiency may be its 5'-end capping. Typically, capping is performed using a capping enzyme, and although the capping efficiency can be high, the process is time-consuming and expensive. Therefore, an efficient co-transcription method that allows for more rapid production of more efficient capped mRNA is needed. Ishikawa M., Ishikawa et al., "Preparation of eukaryotic mRNA having differentially methylated adenosine at the 5'-terminus and the effect of the methyl group in translation," Nucleic Acids Symposium Series, Vol. 53, Issue 1, September-October 2009, pp. 129-130, addressed this need. Ishikawa demonstrated the use of a trinucleotide cap analog with the structure m7GpppA*pG (where A* is adenosine or a methylated adenosine derivative) to prepare capped mRNA in one-step in vitro transcription. Using these molecules, Ishikawa demonstrated the ability to synthesize A, Am , m6 A or m6 A m We obtained reporter 5'-capped mRNAs with a cap analog (as the first transcribed nucleotide) and studied their translational properties in the rabbit reticulocyte system. Another study also demonstrated co-transcriptional capping of mRNAs using cap analogs. Sikorski, PJ et al., 2020, "The identity and methylation status of the first transcribed nucleotide in eukaryotic mRNA 5' cap modulates protein expression in living cells," Nucleic Acids Research, 48(4), pp. 1607-1626.

[0130] In embodiments, methods and / or compositions can be provided that increase capping efficiency, such as greater than about 50%, greater than about 55%, greater than about 60%, greater than about 65%, greater than about 70%, greater than about 75%, greater than about 80%, greater than about 85%, greater than about 90%, greater than about 95%, greater than about 96%, greater than about 96.5%, greater than about 97%, greater than about 97.5%, greater than about 98%, greater than about 98.5%, greater than about 99%, or greater than about 99.5%, as measured, for example, by cleaving mRNA with RNase H followed by measuring capping efficiency by LC-MS.

[0131] In an embodiment, a cap analog can initiate in vitro transcription to synthesize capped mRNA in a one-pot reaction. In an embodiment, the first base, methyl-A, after the reverse G cap can bind to the -1 position of a DNA template, and the second base, G, can bind to the +1 position of the DNA template, forming a complex with RNA polymerase to recruit the next ribonucleoside triphosphate (NTP), thereby elongating the RNA during the transcription process. For example, as shown in Figure 1, during the transcription process, a cap-2 analog (m ) can be used in an IVT. 7 GpppA 2’-Ome G 2’-Ome ), the first base of the methyl-A after the reverse G cap binds to the -1 position of the DNA template, and the second base of the G binds to the +1 position of the DNA template, thereby forming a complex with T7 RNA polymerase to recruit the next NTP and extend the RNA.

[0132] In embodiments, compositions are provided that include one or more cap analogs as described herein. In embodiments, methods are provided that use one or more cap analogs as described herein. In embodiments, cap analogs as described herein may be used in combination with compositions and / or methods described herein and / or may be used in combination with conventional compositions and / or methods. In examples, the methods and / or compositions described herein can increase mRNA capping efficiency to greater than about 50% efficiency, greater than about 55% efficiency, greater than about 60% efficiency, greater than about 65% efficiency, greater than about 70% efficiency, greater than about 75% efficiency, greater than about 80% efficiency, greater than about 85% efficiency, greater than about 90% efficiency, greater than about 95% efficiency, greater than about 96% efficiency, greater than about 96.5% efficiency, greater than about 97% efficiency, greater than about 97.5% efficiency, greater than about 98% efficiency, greater than about 98.5% efficiency, greater than about 99% efficiency, greater than about 99.5% efficiency, or at most about 100% efficiency. promoter

[0133] The promoter design in the DNA template can be important for initiation of in vitro transcription by DNA-dependent RNA polymerase. In an embodiment where T7 RNA polymerase (a monosubunit polymerase derived from T7 phage) can be used, the DNA template promoter design can include the T7 Φ6.5 promoter, followed by the sequence GG, GA, or AGG. In an embodiment, such a design can efficiently initiate transcription and produce mRNA with high fidelity at the 5' end in a one-step process.

[0134] In an embodiment, the promoter may have the sequence TAATACGACTCACTATAX1X2X3 (SEQ ID NO:16), where X1 is A or G, X2 is A or G, and X3 is A, T, G, or C.

[0135] In embodiments where T7 RNA polymerase can be used, in vitro transcription may be initiated using at least one of the promoter sequences in Table 1. Promoters can be added to a plasmid vector by gene synthesis or subcloning.

[0136] [Table 1]

[0137] In embodiments, at least one of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:15 may be used to prime in vitro transcription.

[0138] In examples, compositions are provided comprising one or more promoters as described herein. In examples, vectors are provided comprising one or more promoters as described herein. In examples, methods of using one or more promoters as described herein are provided. In examples, compositions are provided comprising mRNA, the mRNA comprising one or more promoters as described herein. In examples, vectors are provided comprising mRNA, the mRNA comprising one or more promoters as described herein. In examples, methods of using mRNA, the mRNA comprising one or more promoters as described herein are provided.

[0139] In some embodiments, in vitro transcription may be initiated at the -1 position, which helps form a more favorable complex with T7 RNA polymerase and produce RNA of greater length. Using the -1 and +1 positions of the initiation site for in vitro transcription allows for more flexibility in selecting the first mRNA base (not including the cap base) and leaves the +2 position open for custom sequences, such as those listed in Table 1, to produce mRNA. Currently, common practice for incorporating cap molecules during in vitro transcription is to use the +1 position to initiate mRNA synthesis, which requires the template to have the exact sequence AG or AT after the T7 promoter TATA box sequence. Examples of the present disclosure may include a method using a DNA template with a generic T7 promoter sequence (which has GG after the T7 promoter TATA box), which eliminates the need for specific mutagenesis of the DNA template when preparing cap-1 mRNA by co-transcription.

[0140] In some embodiments, a method for in vitro transcribing a DNA template into RNA includes: (1) a DNA template comprising a promoter operably linked to a nucleic acid comprising a 5' untranslated region (5' UTR), an open reading frame (ORF) encoding an RNA of interest, a 3' UTR, and a polyA region; and (2) a cap analog, wherein the cap analog comprises a structure having formula (I) or (II). [ka] where R is H or CH3 and B1 is A or N6-methyl-adenine (m 6 A) and B2 is A, U, G or C; [ka] where: R1 is OCH3 and R2 is OH or H, or R1 is OH and R2 is H, or R1 is H and R2 is H or OCH3, or R1 and R2 are each OCH3; B1 is A or N6-methyl-adenine (m 6 A) and B2 is A, U, G or C; wherein the promoter may contain the sequence TAATACGACTCACTATAX1X2X3 (SEQ ID NO: 16); wherein A at position 17 is the −1 nucleotide and X1 at position 18 is the +1 nucleotide; When X1 is G, and X2 and X3 are A, T, G or C, respectively, B1 is A or m 6 A and B 2 is G, When X1 is A, and X2 and X3 are A, T, G or C, respectively, B1 is A or m 6 A and B 2 is A, When X1 is C, and X2 and X3 are A, T, G or C, respectively, B1 is A or m6 A and B 2 is C, and When X1 is T, and X2 and X3 are A, T, G or C, respectively, B1 is A or m 6 A and B 2 is U, Here, the method may include providing a cap analog, wherein the cap analog is attached to the -1 and +1 nucleotides of the promoter, and incubating the DNA template and the cap analog in a reaction mixture, wherein the incubation is performed at about 15°C to about 35°C for about 1 hour to about 12 hours to produce RNA.

[0141] The promoter may contain a sequence selected from SEQ ID NOs: 10, 11, 12, 13 and 14.

[0142] In some embodiments, a method for in vitro transcribing a DNA template into RNA may include providing (1) a DNA template comprising a promoter operably linked to a nucleic acid comprising a 5' untranslated region (5' UTR), an open reading frame (ORF) for encoding an RNA of interest, a 3' UTR, and a polyA region, and (2) a cap analog attached to the -1 and +1 nucleotides of the promoter, and incubating the DNA template and the cap analog in a reaction mixture, wherein the incubation is at about 15°C to about 35°C, preferably about 18°C ​​to about 31°C, for a suitable period of time, preferably about 1 hour to about 12 hours, to produce the RNA.

[0143] In some embodiments, a method for in vitro transcribing a DNA template into RNA may include providing (1) a DNA template comprising a promoter operably linked to a nucleic acid comprising a 5' untranslated region (5' UTR), an open reading frame (ORF) encoding an RNA of interest, a 3' UTR, and a polyA region, and (2) a cap analog attached to the -1 and +1 nucleotides of the promoter, and producing the RNA by incubating the DNA template and the cap analog in a reaction mixture.

[0144] In some embodiments, the reaction mixture comprises NTPs and an RNA polymerase. In some embodiments, the reaction mixture may further comprise one or more of a buffer, an RNase inhibitor, a magnesium salt, a polyamine, and a pyrophosphatase.

[0145] In some embodiments, the reaction mixture comprises a buffer substance at a concentration of about 45 mM to about 55 mM, an RNase inhibitor at a concentration of about 0.01 U / μl to about 0.03 U / μl, an NTP at a concentration of about 1 mM to about 10 mM, a cap analog at a concentration of about 6 mM to about 8 mM, one or more magnesium salts at a concentration of about 20 mM to about 30 mM, a polyamine at a concentration of about 1.5 mM to about 2.5 mM, a DNA template at a concentration of about 0.01 μg / μl to about 0.05 μg / μl, a pyrophosphatase at a concentration of about 0.1 mU / μl to about 0.5 mU / μl, and an RNA polymerase at a concentration of about 0.01 μg / μl to about 0.05 μg / μl. 5'UTR and 3'UTR

[0146] The mRNA molecule may be flanked by 5'- and 3'-untranslated regions (UTRs). The 5'-UTR can be recognized by ribosomes to allow translation initiation, and the 3'-UTR may contain regulatory sequences that can affect the expression and half-life of the mRNA. When mRNA is expressed in mammalian cells, several different combinations of 5' and 3' UTRs can achieve high mRNA expression efficiency.

[0147] Cao et al. (Cao et al., "High-throughput 5'UTR engineering for enhanced protein production in non-viral gene therapies," Nature Communications, (2021) 12:4138, pp. 1-10, incorporated herein by reference in its entirety) reported a method for producing artificial 5'UTRs through a high-throughput screening process.

[0148] In an embodiment, a combination of an artificially selected 5' UTR and a human hemoglobin or mouse hemoglobin 3' UTR can be used to generate a construct for efficiently expressing an mRNA sequence. The combination can be selected and used to construct a vector for in vitro transcription (IVT). The combination can be used to produce an mRNA with efficient protein expression capabilities. In an embodiment, reference is made to one or more UTRs shown in Table 2 (see Example 1 below). In an embodiment, one or more UTRs shown in Table 2 can be used in any combination. In an embodiment, UTRs can be used in pairs, for example, as shown in Table 2, where the paired members are located in the same row. In embodiments, SEQ ID NO:1 and SEQ ID NO:2 are paired, SEQ ID NO:3 and SEQ ID NO:2 are paired, SEQ ID NO:1 and SEQ ID NO:4 are paired, SEQ ID NO:1 and SEQ ID NO:6 are paired, SEQ ID NO:3 and SEQ ID NO:6 are paired, and / or SEQ ID NO:9 and SEQ ID NO:2 are paired. In embodiments, one or more UTRs listed in Table 5 can achieve high expression efficiency when mRNA is expressed in mammalian cells. In embodiments, one or more pairs of UTRs listed in Table 5 can be used to achieve high expression efficiency when mRNA is expressed in mammalian cells.

[0149] Different 5'UTR and 3'UTR pairs can be cloned into the vector in the following orientation, from 5' to 3': promoter (e.g., T7 promoter (SEQ ID NO:10)), 5'UTR, Kozak sequence (GCCACC), eGFP coding sequence, and 3'UTR. The vector can then be subcloned into the pVAX vector to produce a plasmid for mRNA preparation. Examples of 5'UTR and 3'UTR pairs are shown in Table 2.

[0150] [Table 2-1] [Table 2-2]

[0151] In examples, a T7 promoter sequence, UTRs can be added to an open reading frame coding sequence by gene synthesis, which can then be subcloned into a plasmid vector to produce large-scale plasmid DNA for in vitro transcription applications. Compositions are provided that include one or more UTRs as described herein. In examples, vectors are provided that include one or more UTRs as described herein. In examples, methods are provided that use one or more UTRs as described herein. In examples, compositions are provided that include mRNA, the mRNA including one or more UTRs as described herein. In examples, vectors are provided that include mRNA, the mRNA including one or more UTRs as described herein. In examples, methods are provided that use mRNA, the mRNA including one or more UTRs as described herein. PolyA tail

[0152] The quality of the poly(A) tail (e.g., length and uniformity of length and distribution) can directly affect mRNA expression efficiency. Traditional methods for adding poly(A) tails to mRNA products in vitro use poly(A) polymerase. See, for example, Cao, GJ, and Sarkar, N., "Identification of the gene for an Escherichia coli poly(A) polymerase," Proc. Natl. Acad. Sci. USA, (1992) 89(21), 10380-10384, incorporated herein by reference in its entirety. However, such methods typically produce products with a wide distribution of poly(A) tail lengths, with only about 70% of the mRNA being tailed.

[0153] In addition to the length and distribution of capping and polyA tail, mRNA purity and integrity can be important factors affecting mRNA properties, such as, but not limited to, its stability and / or expression efficiency. Purification, promoter sequence, one or more 5' and / or 3' UTR sequences, and / or transcription conditions can aid in the production of high-quality mRNA.

[0154] Methods for adding polyA tails to mRNA products in vitro use polyA polymerase, as described, for example, by Cao et al. (Proc. Natl. Acad. Sci. USA. 89, 10380-10384). However, such methods can produce polyA products with a wide distribution of polyA tail lengths, for example, where only about 70% of the mRNA is polyadenylated.

[0155] In embodiments, the methods and / or compositions may include providing a capped analog, a method for providing and / or improving the length and / or distribution of a polyA tail, providing an effective promoter, providing an effective UTR (e.g., a UTR pair), providing effective transcription conditions, providing an effective transcription system, providing effective purification, or any combination thereof.

[0156] In embodiments, methods and / or compositions can be provided that increase the uniformity of poly-A tail length and / or distribution in transcribed mRNA molecules. In embodiments, the methods and / or compositions can produce an mRNA population, wherein greater than about 70% are tailed, wherein at least about 71% are tailed, wherein at least about 72% are tailed, wherein at least about 73% are tailed, wherein at least about 74% are tailed, wherein at least about 75% are tailed, wherein at least about 76% are tailed, wherein at least about 77% are tailed, wherein at least about 78% are tailed, wherein at least about 79% are tailed, wherein at least about 80% are tailed, wherein at least about 85% are tailed, wherein at least about 90% are tailed, wherein at least about 95% are tailed, and wherein at least about 99% are tailed.

[0157] In embodiments, the methods and / or compositions can produce a population of mRNA wherein the length (number of adenines) of the poly-A tail varies between mRNA molecules by at most about 70 to about 130 adenines, varies between mRNA molecules by at most about 60 to about 120 adenines, varies between mRNA molecules by at most about 50 to about 100 adenines, varies between mRNA molecules by at most about 40 to about 90 adenines, varies between mRNA molecules by at most about 50 to about 80 adenines, varies between mRNA molecules by at most about 40 to about 70 adenines, varies between mRNA molecules by at most about 30 to about 50 adenines, or varies between mRNA molecules by at most about 20 to about 40 adenines. The length of the polyA tail of mRNA can be measured by digesting the mRNA with RNase T1, followed by purifying and recovering the polyA fragment with oligo-dT magnetic beads, and detecting the length of the polyA fragment by capillary gel electrophoresis on a bioanalyzer.

[0158] In some embodiments, a polyA tail is added to a DNA template before transcription. In some embodiments, a method for adding a polyA tail to a DNA template before transcription can be provided. In some embodiments, this is a novel method for adding a polyA tail compared to the traditional method of adding a polyA tail to a DNA template by polymerase chain reaction (PCR) and then inserting the DNA template into a plasmid vector by gene synthesis. In some embodiments, adding a polyA tail to a DNA template can result in the production of more uniform polyA-tailed mRNA products, products with longer polyA tails, or both, either or both of which can result in more efficient mRNA expression.

[0159] In examples, compositions are provided that include mRNA, the mRNA comprising a polyA tail as described herein added by PCR. In examples, vectors are provided that include a polyA tail as described herein, the polyA tail being added to the vector by PCR. In examples, pVAX1 or pUC57 vectors are provided that include mRNA containing an ampicillin resistance gene, a T7 promoter sequence, 5'UTR and 3'UTR sequences, and a polyA tail (e.g., 100A) as described herein added by PCR.

[0160] In embodiments, the disclosed methods and / or compositions can produce a population of mRNA, wherein greater than about 70% is tailed, wherein at least about 71% is tailed, wherein at least about 72% is tailed, wherein at least about 73% is tailed, wherein at least about 74% is tailed, wherein at least about 75% is tailed, wherein at least about 76% is tailed, wherein at least about 77% is tailed, wherein at least about 78% is tailed, wherein at least about 79% is tailed, wherein at least about 80% is tailed, wherein at least about 85% is tailed, wherein at least about 90% is tailed, wherein at least about 95% is tailed, wherein at least about 99% is tailed, and wherein about 100% is tailed.

[0161] In embodiments, the disclosed methods and / or compositions can produce a population of mRNA wherein the length (number of adenines) of the poly-A tail varies between mRNA molecules by at most about 70 to about 130 adenines, varies between mRNA molecules by at most about 60 to about 120 adenines, varies between mRNA molecules by at most about 50 to about 100 adenines, varies between mRNA molecules by at most about 40 to about 90 adenines, varies between mRNA molecules by at most about 50 to about 80 adenines, varies between mRNA molecules by at most about 40 to about 70 adenines, varies between mRNA molecules by at most about 30 to about 50 adenines, or varies between mRNA molecules by at most about 20 to about 40 adenines.

[0162] Bacterial studies have demonstrated that repeat sequences (e.g., CTG, CAG) and the mode and level of plasmid replication and transcription can function in the amplification and deletion of repeat sequences. The deletion frequency of cloned repeat sequences can increase by up to 20-fold after inducing the lacZ promoter, which drives transcription of the insert in pUC19 (Bowater et al., 1997. Transcription increases the deletion frequency of long CTG·CAG triplet repeats from plasmids in Escherichia coli. Nucleic Acids Res 25:2861-2868, the contents of which are incorporated herein by reference in their entirety). Common vectors are typically maintained at high copy numbers, and inducing the transcription and translation of antibiotic resistance genes, indicator genes (e.g., blue / white screening genes), and inserts can cause instability for some classes of DNA sequences. As extremely repeat sequences and sequences with extremely low GC ratios, poly(A) sequences may be easily lost during the plasmid cloning and replication process. The presence of such sequences can lead to unexpected transcription and translation, influenced by the inducing activity of upstream and downstream promoters, further increasing the instability of the polyA sequence. To avoid the initiation activity of similar promoters in the vector for the inserted sequence, common strategies include fragmenting the inserted gene (if the inserted gene has obvious cytotoxicity) or directional cloning of the ORF in the "reverse" direction transcribed from the vector's promoter. However, these two strategies may not be applicable to gene cloning containing polyA sequences. While fragmentation can eliminate genotoxicity after expression of the cloned gene, such strategies may not solve the problem of polyA sequence instability because the transcription process is not yet eliminated and reverse insertion can only eliminate the effects of the promoter in one direction.Therefore, embodiments of the present disclosure may include modifying vectors by inserting transcription terminators upstream and downstream of the polyclonal region, which effectively disrupts the influence of promoters upstream and downstream of the polyclonal region on the inserted gene. This strategy effectively improves the stability of poly(A) sequences during the plasmid cloning and replication process, particularly for highly unstable poly(A)-containing cassettes. Results showed that adding transcription terminators upstream and downstream of the polyclonal region increased the clonal positive rate to 25%-50% (almost zero before adding the terminators), and the number of A bases in the poly(A) tail increased from 70-110 to approximately 120. Furthermore, adding terminators upstream and downstream of the inserted cassette also increased the plasmid yield by 32.5%. Without being limited to a particular theory, this increase may be due to the restoration of replication origin activity by the insertion of a transcription terminator (Stueber et al., 1982. Transcription from efficient promoters can interfere with plasmid replication and diminish expression of plasmid-specified genes. EMBO J 1:1399-1404, the contents of which are incorporated herein by reference in their entirety). The use of transcription terminators placed upstream and downstream of the polyclonal site to facilitate the cloning of cytotoxic or repeat sequences (e.g., polyA tails) has not yet been reported in the art.

[0163] A transcription termination sequence may be any nucleotide sequence that, when placed downstream of a nucleotide sequence encoding an open reading frame by transcription, causes transcription termination of the open reading frame. Such sequences are known in the art and may be derived from prokaryotic, eukaryotic, or phage genes. Examples of terminator sequences include, but are not limited to, the PTH terminator, pET-T7 terminator, T3-Tφ terminator, pBR322-P4 terminator, vesicular stomatitis virus terminator, rrnB-T1 terminator, rrnB-T2 terminator, λt0 terminator, rrnC terminator, Ttadc transcription terminator, and yeast-recognized termination sequences (e.g., Matα (α-factor) transcription terminator, native α-factor transcription termination sequence, ADR1 transcription termination sequence, ADH2 transcription termination sequence, and GAPD transcription termination sequence). A non-exhaustive list of transcription terminator sequences can be found in the iGEM Registry at: partsregistry.org / Terminators / Catalog. A series of 2, 3, 4, 5, 6, 7, or more first transcription terminator sequences may be placed directly 3' of the final nucleotide of the gene of interest (or open reading frame) or may be placed directly 3' of the final nucleotide of the gene of interest (or open reading frame) at least 1-5, 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, 45-50, 50-100, 100-150, 150-200, 200-300, 300-400, 400-500, 500-1,000, or more nucleotides away. The number of nucleotides between the tandem transcription terminator sequences can vary, for example, the transcription terminator sequences are separated by 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, 45-50 or more nucleotides. vector

[0164] In embodiments, a vector is provided, the vector comprising one or more of: (i) a polyA tail as described herein; (ii) one or more UTRs as described herein; (iii) one or more promoters as described herein; or (iv) a combination thereof. In embodiments, the vector may have a variety of uses, including, but not limited to, being used for inserting a target template nucleotide sequence. In embodiments, such a vector having a target template nucleotide sequence can be used (as a non-limiting example) for cloning or transcribing the target template nucleotide sequence. In embodiments, transcription may comprise in vitro transcription. In embodiments, transcription may be performed using T7 RNA polymerase. In embodiments, the vector may be a plasmid or a viral vector, such as, but not limited to, pVAX1 and / or pUC57. mRNA purity

[0165] mRNA integrity can affect cellular expression, so it can be important to initiate translation with mRNA of high integrity. Transcription conditions can affect the quality of the mRNA produced. Some transcription conditions can result in higher cleavage products.

[0166] Because mRNA integrity can be one of the key factors affecting cellular expression, starting with mRNA with high integrity can be very important. mRNA integrity is twofold. Typically, RNA tends to degrade faster than DNA due to its chemical instability, so storage conditions affect RNA quality. Also, transcription conditions can lead to higher cleavage products, so optimal buffer conditions can be empirically determined to achieve higher integrity and higher yields across a wide range of mRNA sizes.

[0167] The conditions for purification can affect the quality of the mRNA produced. For example, the presence of a trinucleotide cap analog in the final mRNA product. m7 GpppA* pG residues can inhibit mRNA translation efficiency in cells by competing with capped mRNA for ribosome recruitment. Purification after in vitro transcription can be a crucial step to obtain a final purified mRNA product.

[0168] The present disclosure further provides a solution for obtaining a pure mRNA product, which has minimal contaminants such as cap analogs, free NTPs, and other proteins that may interfere with or impair the performance of the mRNA product. mRNA purity is the ratio of full-length mRNA species in the crude transcribed mRNA product, which may be quantified by the full-length peak ratio in capillary gel electrophoresis.

[0169] In embodiments, purification as described herein can produce highly pure mRNA products with minimal contaminant analogs, free NTPs, and / or other proteins that may interfere with and / or damage the stability and / or translation efficiency of the mRNA product. In embodiments, the purification method includes binding the nucleic acid to a silica membrane column and washing with about 60% to about 80% ethanol in water, preferably about 70% to about 80% ethanol in water, followed by elution in water.

[0170] Other common purification methods, such as LiCl precipitation or affinity-based magnetic bead purification, can be used for the same purpose. In embodiments, purification methods such as those described herein may be used in combination with compositions and / or methods described herein and / or in combination with conventional compositions and / or methods.

[0171] In embodiments, methods and / or compositions for increasing the purity of transcribed mRNA can be provided. In embodiments, mRNA having a purity of about 79% or higher, mRNA having a purity of about 79.5% or higher, mRNA having a purity of about 80% or higher, mRNA having a purity of about 80.5% or higher, mRNA having a purity of about 81% or higher, mRNA having a purity of about 81.5% or higher, mRNA having a purity of about 82% or higher, mRNA having a purity of about 82.5% or higher, mRNA having a purity of about 83% or higher, mRNA having a purity of about 83.5% or higher, mRNA having a purity of about 84% or higher, mRNA having a purity of about 84.5% or higher, mRNA having a purity of about 85% or higher, mRNA having a purity of about 85.5% or higher, mRNA having a purity of about 86% or higher, mRNA having a purity of about 86.5% or higher, mRNA having a purity of about 87% or higher. The present invention provides methods and / or compositions for obtaining mRNA with a purity of about 87.5% or higher, about 88% or higher, about 88.5% or higher, about 89% or higher, about 89.5% or higher, about 90% or higher, about 91% or higher, about 92% or higher, about 93% or higher, about 94% or higher, about 95% or higher, about 96% or higher, about 97% or higher, about 98% or higher, about 99% or higher, or about 100% pure. mRNA purity may be measured by capillary gel electrophoresis using a bioanalyzer instrument, and the target peak area ratio (target length ± 15%) can be calculated for the purity measurement.

[0172] In embodiments, methods and / or compositions can be provided that increase the purity or integrity of transcribed mRNA. Obtaining high-quality long mRNA from in vitro transcription is a huge challenge due to the tendency of long mRNA to be easily degraded.

[0173] In some embodiments, the IVT is from about 15°C to about 35°C, from about 16°C to about 35°C, from about 17°C to about 35°C, from about 18°C ​​to about 35°C, from about 18°C ​​to about 34°C, from about 18°C ​​to about 33°C, from about 18°C ​​to about 32°C, from about 18°C ​​to about 31°C, from about 18°C ​​to about 30°C, from about 18°C ​​to about 29°C, from about 18°C ​​to about 28°C, from about 18°C ​​to about 27°C, from about 18°C ​​to about 26°C, from about 18°C ​​to about 25°C, from about 18°C ​​to about 24°C, from about 18°C ​​to about 23°C, from about 18°C ​​to about 2 ... to about 21°C, about 18°C ​​to about 20°C, about 18°C ​​to about 19°C, about 25°C to about 26°C, about 25°C to about 27°C, about 25°C to about 28°C, about 25°C to about 29°C, about 25°C to about 30°C, about 25°C to about 31°C, about 21°C to about 22°C, about 21°C to about 23°C, about 21°C to about 24°C, about 21°C to about 25°C, about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, about 30°C, about 31°C, about 32°C, about 33°C, about 34°C, or about 35°C.

[0174] In some embodiments, IVT may be administered for about 1 hour to about 12 hours, about 1 hour to about 11 hours, about 1 hour to about 10 hours, about 1 hour to about 9 hours, about 1 hour to about 8 hours, about 1 hour to about 7 hours, about 1 hour to about 6 hours, about 1 hour to about 5 hours, about 1 hour to about 4 hours, about 1 hour to about 3 hours, about 1 hour to about 2 hours, about 2 hours to about 12 hours, about 3 hours to about 12 hours, about 4 hours to about 12 hours, about 5 hours to about 12 hours, about 6 hours to about 12 hours, about 7 hours to about 12 hours, about 8 hours to about 12 hours, about 9 hours to about 12 hours, about 10 hours to about 12 hours, about 11 hours to about 12 hours, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, or about 10 hours.

[0175] In some examples, IVT may be performed at about 31° C. for about 1 hour to about 5 hours, about 1 hour to about 4.5 hours, about 1 hour to about 4 hours, about 1 hour to about 3.5 hours, about 1 hour to about 3 hours, about 1 hour to about 2.5 hours, about 1 hour to about 2 hours, about 1 hour to about 1.5 hours, about 0.5 hours to about 1 hour, about 0.5 hours to about 1.5 hours, about 1 hour, about 2 hours, about 3 hours, about 4 hours, or about 5 hours. Transcription mediated by T7 RNA polymerase

[0176] In eukaryotes, transcription of messenger RNA (mRNA) is completed by RNA polymerase II, a complex, multisubunit enzyme under intricate regulation. To perform large-scale in vitro transcription, researchers typically use monosubunit phage polymerases derived from T7, T3, SP6, K1-5, K1E, K1F, or K11 bacteriophages. This polymerase family uses a simple, minimal promoter sequence of approximately 17 nucleotides, which does not require auxiliary proteins and may have minimal restrictions on the initiating nucleotide sequence. While this application focuses on T7 RNA polymerase (T7 RNAP), those skilled in the art will understand that the present disclosure can be practiced with other RNA polymerases.

[0177] T7 RNA polymerase (RNAP) exists in at least two protein states. The first, called the "abortive complex," may be involved in transcription initiation. The second, which assumes a highly persistent conformation, is called the "elongation complex." In vitro transcription can be divided into six steps: 1) binding of RNA polymerase to promoter sequences; 2) initiation of transcription; 3) non-persistent elongation, called abortive transcription, during which the polymerase frequently releases the DNA template and short abortive transcripts; 4) conversion of the open complex to a closed complex; 5) persistent elongation; and 6) transcription termination. The large amount of RNA produced during transcription may include short abortive fragments approximately 2-8 nucleotides in length (Biochemistry 19:3245-3253 (1980); Nucleic Acids Res. 9:31-45 (1981); Nucleic Acids Res. 15:8783-8798 (1987); Biochemistry 27:3966-3974 (1988), each of which is incorporated herein by reference in its entirety). After synthesizing approximately 10-14 bases, the RNA polymerase exits the abortive cycle, loses specific contacts with the promoter DNA sequence, and can form a persistent elongation complex in which the RNA strand can elongate in a sequence-independent manner (J. Mol. Biol. 183:165-177 (1985); Proc. Natl. Acad. Sci. USA 83:3614-3618 (1986); Mol. Cell Biol. 7:3371-3379 (1987); each of which is incorporated herein by reference in its entirety).

[0178] [Table 3]

[0179] The shared sequence of the most active class III T7 promoters can cover 17 bp upstream and 6 bp downstream of the transcription start site (Cell 16:815-25. (1979), incorporated herein by reference in its entirety). The position of the first transcribed nucleotide is generally referred to as the +1 transcript nucleotide of the RNA, the second transcribed nucleotide as the +2 transcript nucleotide, and so on (Table 3). During transcription, the two strands can melt to form a transcription bubble, and the bottom strand of the duplex (shown as 3' to 5' in Table 3) is the transcription template. From transcript nucleotide +3 onwards, the template strand can define the identity of the transcribed nucleotide through Watson-Crick base pairing interactions. Here, the nucleotide encoding the first RNA transcript nucleotide is defined as the +1 nucleotide of the template. In the example shown in Table 3, the +1 transcript nucleotide is G and the +1 template nucleotide is C. Similarly, the +4 transcript nucleotide is an A and the +4 template nucleotide is a T.

[0180] T7 RNAP is known to be capable of initiating with short oligonucleotide primers. For example, 13 promoters in the T7 genome are known to be capable of initiating with pppGpG (J. Mol. Biol. 370:256-268 (2007), incorporated herein by reference in its entirety). It has been shown that T7 RNAP can initiate from dinucleotide primers (Biochemistry 24:5716-5723 (1985), incorporated herein by reference in its entirety). Axelrod et al. showed that an uncapped GpA dinucleotide can initiate from the +1 and +2 template nucleotides, 2'-deoxycytidine and 2'-deoxythymidine, respectively ("CT" template). The reaction conditions are 200 micromolar (μM) dimers and 100 μM ATP, CTP, GTP, and UTP. The reaction conditions further contained 100 μM 3'dATP, 3'dCTP, 3'dUTP, or 50 μM 3'dGTP. Only GpA-initiated RNA was observed; a mixture of GpA-initiated RNA and GTP-initiated 5'-triphosphate RNA was not observed. This may be due to the reaction conditions used. 100 μM GTP is much lower than the 2 mM Kd of T7 polymerase for the first initiating guanosine (J. Mol. Biol. (2007) 370, 256-268, incorporated herein by reference in its entirety). Because GTP competes with the initiator oligonucleotide for initiation, using a low GTP concentration favors GpA initiation but may result in low transcription yields (estimated maximum calculated yields of <150 μg / mL reaction). When ApG, CpG, UpG, or GpG was used to initiate transcription on the "CT" template, the formation of RNA transcripts with an additional non-templated 5' nucleotide (A, C, U, or G, respectively) was observed.

[0181] According to Ishikawa et al. m7 GpppApG, m7 Gppp m6 ApG, m7 GpppA 2’Ome pG or m7 Gppp m6 A 2’OmeA capped initiator oligonucleotide trimer of the pG structure can initiate transcription with 2'-deoxycytidine residues at template positions +1 and +2 on the template ("CC" template, Nucleic Acids Symposium Series, Vol. 53, No. 129 (2009), incorporated herein by reference in its entirety). m7 The results differed when G5'pppG was used. m7 pointed out that this may be due to base pairing between another adenosine (N1) in G5'pppN1pG and the 2'-deoxythymidine at the -1 position in the T7 promoter. This method is distinct from the method described in this disclosure, in which the +1 and +2 nucleotides of the initiating capped oligonucleotide trimer paired with the +1 and +2 nucleotides of the template nucleotide. Ishikawa et al. used 6 mM initiating oligonucleotide trimer, 0.9 mM GTP, and 7.5 mM each of ATP, CTP, and UTP. The authors used a capped initiating oligonucleotide primer (the most expensive nucleotide component in the transcription reaction) in a more than six-fold excess over competitive GTP to drive the transcription reaction toward capped RNA rather than pppRNA, which increased the overall cost of synthesizing RNA. In another embodiment, a low concentration of GTP (0.9 mM) limited the total yield of RNA in the transcription reaction (less than the theoretical 1.4 mg / mL). Conversely, the methods described herein can achieve efficient RNA capping and higher RNA yields (approximately 2 to 10 mg / mL) without limiting NTP concentrations, thereby allowing the production of high-quality mRNA at a commercially viable cost. T7 RNA polymerase

[0182] In some embodiments, the at least one modification of the T7 RNA polymerase may be selected from the group consisting of P266L, P270L, P270S, P270A, P270Y, Q744L, Q744P, Q744R, Y639F, H784A, E593G, Y639V, V685A, H784G, S430P, N433T, S633P, F849I, and F880Y. In some embodiments, the at least one modification comprises Y639F and H784A. In some embodiments, the at least one modification comprises E593G, Y639V, V685A, and H784G. In some embodiments, the at least one modification comprises S430P, N433T, S633P, F849I, and F880Y. In some embodiments, the at least one modification comprises S430P, N433T, S633P, F849I, F880Y, and P266L. In some embodiments, the at least one modification comprises S430P, N433T, S633P, F849I, F880Y, Y639F, and H784A. In some embodiments, the at least one modification comprises S430P, N433T, S633P, F849I, F880Y, P266L, Y639F, and H784A. In some embodiments, the at least one modification comprises S430P, N433T, S633P, F849I, F880Y, E593G, Y639V, V685A, and H784G. In some embodiments, the at least one modification comprises S430P, N433T, S633P, F849I, F880Y, P266L, E593G, Y639V, V685A, and H784G.

[0183] In some embodiments, at least one modification of the T7 RNA polymerase promotes initiation-elongation transition. In some embodiments, at least one modification increases the rate of promoter removal. In some embodiments, at least one modification increases the stability and / or activity of the polymerase. In some embodiments, at least one modification increases the thermostability of the polymerase. Magnesium ions in in vitro transcription

[0184] Magnesium ions (Mg 2+ ) is an essential component in RNA in vitro transcription buffer systems and is used to initiate transcription using cap analogs rather than GTP. Common buffer systems for RNA in vitro transcription (e.g., HEPES buffer, Tris-HCl buffer) may have high concentrations of free magnesium ions, which ensures high activity of RNA polymerase. 2+ In vitro transcription processes may require Mg ions. 2+ and NTPs complex during the reaction, and excess free Mg 2+ The absence of ions in the buffer system is important to ensure high capping efficiency and high integrity of the transcribed mRNA. Therefore, a high concentration of Mg 2+ This can be problematic, especially for high-yield / industrial-scale RNA production. 2+ Some ion-related problems can include magnesium-driven precipitation, which occurs when free Mg 2+ This can cause a decrease in the activity of the IVT reaction, thereby depleting magnesium ions from the RNA polymerase reaction center. As a result, the efficiency of RNA in vitro transcription is low. To evaluate the effect of Mg concentration on mRNA IVT yield and integrity, IVT was performed in the presence of increasing Mg concentrations. By maintaining the final Mg concentrations in the IVT reaction at 16.5 mM, 21 mM, 29 mM, and 37 mM, respectively, the free Mg in the IVT reaction system after complexing with NTPs and cap analogs was significantly reduced. 2+ The concentrations of Mg were set to -12 mM, -8 mM, 0 mM, and +8 mM, respectively. 2+ When the concentration is increased, the mRNA yield decreases and 2+ As the concentration increases, the integrity of the mRNA also decreases. example Example 1 Synthesis of cap analogues overview.

[0185] 5'-DMT-2'-O-methyl-guanosine (n-ibu) and 5'-DMT-2'-O-methyl-N6-methyl-adenosine 3'-phosphoramidite were purchased from ChemGenes, Inc. (Wilmington, MA). Chemical phosphorylation reagent [5'-phosphoramidite (O-DMT-2,2'-sulfonyldiethanol)] was purchased from Biosearch Technologies. Aluminum-coated silica gel 60 F (purchased from Sigma-Aldrich) was used. 254 The plate was used for TLC. 1 H and 31 P NMR spectra were collected on a Bruker 300 MHz spectrometer. HPLC chromatograms were obtained on an Agilent 1290 Infinity II system using a HALO 90A C18 column (4.6 × 150 mm) eluted with a gradient of 50 mM TEAB buffer (pH 7.8) / acetonitrile. Reaction completion is typically monitored using TLC and / or HPLC analysis. UV spectral data were obtained on a VWR UV-3100PL spectrophotometer. Buffer solutions were prepared using water dispensed from a Mill-Q EQ 7000 water purification system. The reported compounds were prepared as previously described: 2a: Sawai et al. (Synthesis and Reactions of Nucleoside 5'-Diphosphate Imidazolide. A Non-enzymatic Capping Agent for 5'-Monophosphorylated Oligo Ribonucleotides in Aqueous Solution, J. Org. Chem., 1999, 64, 5836-5840), 2b and 9: Jemielity et al. (Novel "anti-reverse" cap analogues with superior translational properties, RNA, 2003, 9, 1108-1122), the contents of which are incorporated herein by reference in their entirety. Synthesis of Cap-2 analogues

[0186] Preparation of intermediate 3 (Figure 2).

[0187] To a solution of 5'-DMT-2'-O-methylguanosine (n-ibu) (1.0 g, 1.49 mmol) in 20 mL of anhydrous pyridine was added 3.4 mL of a 9:1 solution of acetic anhydride / anhydrous THF, and the resulting solution was stirred under an argon atmosphere for 22 hours, after which the solution was evaporated to dryness.

[0188] The residue was then stirred in 10 mL of 80% aqueous acetic acid for 2 hours, the solvent was back-extracted under reduced pressure, and the residue was purified by silica gel chromatography (eluted with a gradient of 0 to 10% methanol in ethyl acetate). The pure product was partially combined and evaporated to give a white foam: 590 mg (96%) yield, TLC (ethyl acetate / methanol [9:1]) Rf = 0.48, 'H NMR (dimethyl-dsulfoxide) δ 12.10 (1H, br s), 11.68 (1H br s), 8.33 (1H, s), 5.88 (1H, d, J = 9.0 Hz), 5.44 (1H, d, J = 6.0 Hz), 5.31 (1H, t, J = 6.0 Hz), 4.56 (1H, m), 4.14 (1H, br t), 3.63 (2H, t, J = 3.0 Hz), 3.26 (3H, s), 2.77 (1H, m), 2.13 (3H, s), 1.14, and 1.11 (6H, 2 x s), chromatography (ESI+) m / z 410.16(M+H+).

[0189] Preparation of intermediate 4 (Figure 3).

[0190] Intermediate 3 (585 mg, 1.43 mmol) and 5'-dimethoxytrityl-N-benzoyl-adenosine, 2'-O-methyl, 3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (1.27 g, 1.48 mmol) were dissolved in 40 mL of anhydrous acetonitrile. Tetrazole (6.0 mL of a 0.45 M solution in anhydrous acetonitrile) was added, and the resulting solution was stirred for 18 h under an argon atmosphere. (1S)-(+)-(camphorsulfonyl)oxaziridine (CSO) oxidant (6.0 mL of a 0.50 M solution in acetonitrile) was added to the solution, and the resulting solution was stirred for 16 h. The solvent was back-extracted, and the residue was stirred in 10 mL of 80% aqueous acetic acid for 16 h. The solution was evaporated to dryness under reduced pressure, and the residue was purified by silica gel chromatography (eluting with a mobile phase gradient of 0 to 15% methanol in ethyl acetate). The pure fractions were combined and evaporated to give an amorphous solid: 865 mg (67%) yield, TLC (ethyl acetate / methanol [9:1]) R f =0.21, 31P NMR (dimethyl-d6 sulfoxide) δ -2.11 and -2.21 (1P,2 xs, diastereomeric pair), chromatography (ESI+) m / z 910.42 (M+H+).

[0191] Preparation of intermediate 5 (Figure 3).

[0192] Intermediate 4 (124 mg, 0.136 mmol) and "chemical phosphorylation reagent" (197 mg, 0.30 mmol) were stirred in 3.0 mL of 0.45 M tetrazole in anhydrous acetonitrile for 4.0 hours at room temperature under an argon atmosphere. HPLC analysis indicated that the coupling reaction was complete, and 3.0 mL of CSO oxidant solution (0.50 M in anhydrous acetonitrile) was added directly to the reaction solution. Stirring continued for 1.0 hour, and the solvent was back-extracted under reduced pressure.

[0193] To the residue was added 20 ml of 1 / 1 concentrated ammonium hydroxide / methanol. The reaction solution was sealed and allowed to stand at 37°C for 2 days. The solvent was back-extracted, and the residue was mixed with 20 ml of water and filtered. The filtrate was applied to a DEAE-Sephadex A25 column and eluted with a gradient of 0 to 0.70 M TEAB buffer (pH 7.8). The pure product was partially combined and evaporated to dryness. The TEAB salt was removed from the water by multiple evaporations. The final residue was dissolved in a minimum volume of 10% DMF in water, and the triethylammonium salt of the product was precipitated by adding acetonitrile: 57 mg (45%) yield. H NMR (DO) δ 8.31 (1H, s), 8.02 (1H, s), 7.43 (1H, s), 5.96 (1H d, J = 5.1 Hz), 5.8 (1H, d, J = 5.1 Hz), 4.83 (1H, m), 4.53 (1H, t, J = 4.4 Hz), 4.35 (3H, m), 4.22 (1H, m), 4.10 (2H, m), 3.97 (2H, m), 3.37 (3H, s), 3.34 (3H, s), 3.08 plus 1.16 (q and t, triethylammonium counterion); 31P NMR (DO) δ 0.26 (1P, s), -0.78 (1P, s); chromatographic (ESI) m / z 720.2.

[0194] Preparation of intermediate 1a (Figure 3).

[0195] A mixture of intermediate 5 (12.8 mg, 0.014 mmol), 2a (16.7 mg, 0.027 mmol), and MnCl2 (2.5 mg, 0.020 mmol) was stirred in anhydrous DMSO (257 μL) at room temperature for 18 hours. EDTA (75 μL of a 0.5 M solution in water) was added, and the solution was diluted to 2.0 mL with water. The solution was applied to a DEAE-Sephadex A25 column and eluted with a gradient of 0 to 0.70 M TEAB buffer (pH 7.8). The product was partially combined and evaporated to dryness under reduced pressure. The TEAB buffer salts were removed from the water by multiple evaporations. The residue was further purified by reverse-phase (C18) high-performance liquid chromatography: 4.7 μmol (34%) yield, chromatographic (ESI) m / z 1,159.8.

[0196] Preparation of intermediate 1b (Figure 3).

[0197] A mixture of intermediate 5 (7.0 mg, 0.076 mmol), 2a (7.0 mg, 0.011 mmol), and MnCl2 (2.5 mg, 0.016 mmol) was stirred in anhydrous DMSO (200 μL) for 18 h at room temperature. EDTA (50 μL of a 0.5 M solution in water) was added, the solution was diluted to 5.0 mL with water, and lyophilized to dryness. The crude product was purified by reverse-phase (C18) high-performance liquid chromatography: 5.9 μmol (78%) yield, chromatographic (ESI) m / z 1,173.2.

[0198] WO2022036858 disclosed a method for preparing Cap-2 analogs using enzymes, including phosphohydrolase, guanosine transferase, and T4 RNA ligase 1. In contrast, the present disclosure provides an embodiment in which the preparation of Cap-2 analogs does not involve enzymes. A comparison between the method disclosed in WO2022036858 and the method disclosed in the present disclosure is shown below.

[0199] [Table 4-1] [Table 4-2]

[0200] N6-methyl-adenine (m 6 A) Synthesis of Cap-2 Analogues Preparation of intermediate 7 (Figure 4).

[0201] Intermediate 3 (51 mg, 0.125 mmol) and 5'-DMT-2'-O-methyl-N6-methyl-adenosine 3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (100 mg, 0.125 mmol) were dissolved in tetrazole solution (1.0 mL of a 0.45 M solution in anhydrous acetonitrile) and stirred under argon for 2 h. CSO oxidant (1.0 mL of a 0.50 M solution in acetonitrile) was added to the solution, and the resulting solution was stirred for 20 min. The solvent was back-extracted, and the residue was stirred in 1.0 mL of 80% aqueous acetic acid at room temperature for 16 h. The solution was evaporated to dryness under reduced pressure, and the residue was purified by silica gel chromatography (eluted with a mobile phase gradient of 0 to 20% methanol in ethyl acetate). Pure portions of Intermediate 6 were combined and evaporated to give an amorphous solid: 80 mg (87%) yield.

[0202] Intermediate 6 (80 mg, 0.109 mmol) and chemical phosphorylation reagent (100 mg, 0.152 mmol) were dissolved in tetrazole solution (1.0 mL of a 0.45 M solution in anhydrous acetonitrile) and stirred for 2 h under an argon atmosphere. CSO oxidant (1.0 mL of a 0.50 M solution in acetonitrile) was added to the solution, and the resulting solution was stirred for 20 min, followed by back-extraction of the solvent. The residue was redissolved in a 50 / 50 mixture of methanol / ammonium hydroxide, and the sealed reaction vessel was incubated at 37 °C for 2 days. The reaction solution was then evaporated to dryness, dissolved in 12 mL of water, and applied to a DEAE Sephedex A25 column, eluting with a gradient of 0 to 0.60 M TEAB buffer (pH 7). The pure product was partially combined and evaporated to dryness. Excess TEAB buffer salts were removed from the water by multiple evaporations. The triethylammonium salt form of intermediate 7 was obtained as an amorphous solid: 28 mg yield, TLC (n-butanol / water / acetic acid, [5:3:1]) R f =0.13, chromatography (ESI) m / z 734.2.

[0203] Preparation of capping reagent analog 8 (Figure 4).

[0204] A mixture of the triethylammonium salt form of intermediate 7 (6.9 mg, 0.007 mmol), the triethylammonium salt form of 2b (10.0 mg, 0.016 mmol), and MnCl (2.0 mg, 0.016 mmol) in anhydrous DMSO (200 μL) was stirred at room temperature for 40 h. EDTA (100 μL of a 0.5 M solution in water) was added, and the solution was diluted to 5.0 mL with water and lyophilized to dryness. The residue was purified by reverse-phase (C18) high-performance liquid chromatography: 6.0 mg (54%) yield, chromatographic (ESI) m / z 1,187.6. Example 2 In vitro transcription

[0205] Both linearized DNA plasmids and PCR products could be used as DNA templates to prepare mRNA by in vitro transcription. To test in vitro transcription conditions, a plasmid vector containing a T7 promoter (SEQ ID NO: 10 or 12), 5'-UTR (SEQ ID NO: 9), eGFP coding sequence, 3'-UTR (SEQ ID NO: 2), and poly(A) tail (100 A) was linearized with BspQ1 or Bbs1 restriction enzyme and purified with magnetic beads. The linearized plasmid was transcribed to prepare capped mRNA using T7 RNA polymerase (wild-type), a cap analog (Cap-2), 10x transcription buffer, NTPs, and RNase inhibitors.

[0206] The in vitro transcription mixture and conditions are as described in Tables 4 to 6. More specifically, a 10x buffer solution containing HEPES buffer was prepared by adding magnesium acetate, spermidine, and DTT and stored at -20°C for use. For the in vitro transcription reaction, buffer, DNase-free and RNase-free water, NTPs (at concentrations specified in the tables), and cap analogs were added to the reaction tube, followed by the DNA template, T7 polymerase, RNase inhibitor, and inorganic pyrophosphatase. The reaction was maintained at the specified temperature range of 20°C to 40°C and transcription was carried out for 1 to 6 hours. The DNA template was then removed by digestion with RNase-free DNase 1 enzyme.

[0207] The cap analog may be any capped analog described herein. In the IVT reactions described in Tables 4-6, the cap-2 analog m 7 GpppA 2’Ome p.g. 2’Ome and m 7 G 3’Ome pppA 2’Ome p.g. 2’Ome In these examples, m 7 GpppA 2’Ome p.g. 2’Ome is referred to here as Cap-2. m 7 G 3’Ome pppA 2’Ome p.g. 2’OmeIn

[1999] , the 3'-OH group closer to m7G was replaced with methoxy (-OCH3). This modification is similar to that in the anti-reverse cap analog (ARCA: 3'-O-Me-m7GpppG). According to Stepinski et al. (Stepinski J, Waddell C, Stolarski R, Darzynkiewicz E, Rhoads RE (2001) Synthesis and properties of mRNAs containing the novel "anti-reverse" cap analogues 7-methyl(3'-O-methyl)GpppG and 7-methyl(3'deoxy)GpppG. RNA 7:1486-1495, the contents of which are incorporated herein by reference in their entirety), this 3'-O modification in ARCA prevents the production of mRNAs capped in the reverse orientation. 7 G 3’Ome pppA 2’Ome p.g. 2’Ome ARCA-Cap-2 was used as an analog of ARCA-Cap-1.

[0208] The HEPES buffer is 400 mM HEPES in water, pH 7.5. The RNA polymerase may be wild-type T7 RNA polymerase or a mutant T7 RNA polymerase with enhanced stability and / or the ability to incorporate a cap-2 analog. In vitro transcription may be performed in DNase-free, RNase-free plastic tubes ranging from 0.2 mL to 15 mL, at a defined temperature, with or without shaking.

[0209] Three IVT conditions (IVT1, IVT2, and IVT3) were tested as specified in Tables 4-6. Table 4 shows IVT1 conditions, in which IVT was performed with equal concentrations of NTP. Table 5 shows IVT2 conditions, in which the GTP concentration was reduced as indicated. Table 6 shows IVT3 conditions, in which the ATP and GTP concentrations were reduced as indicated.

[0210] [Table 5]

[0211] [Table 6]

[0212] [Table 7]

[0213] The mRNA was purified from the IVT reaction using a silica membrane column. Briefly, the mRNA from the in vitro transcription was mixed with buffer and ethanol, applied to the silica membrane column, followed by washing with 70% ethanol and elution with water or other mRNA storage buffer.

[0214] The amount of mRNA purified from the silica column was determined by Nanodrop to calculate the total yield of the IVT reaction, which is expressed as the amount (micrograms) of mRNA obtained per microliter of IVT reaction.

[0215] The integrity of the mRNA prepared by the disclosed method was analyzed using an Agilent Bioanalyzer to assess purity based on size. The Agilent RNA Nano 6000 kit was used. The purity of the analyzed mRNA was analyzed by smear analysis, and the proportion of the mRNA population with a target length ± 10% was calculated.

[0216] The capping efficiency of mRNA was examined by RNase H digestion and LC-MS, as described in Beverly et al. ("Label-free analysis of mRNA capping efficiency using RNase H probes and LC-MS." Anal Bioanal Chem. July 2016;408(18):5021-30, the contents of which are incorporated herein by reference in their entirety). Briefly, a short biotinylated RNA / DNA hybrid probe was annealed to the mRNA and subjected to RNase H digestion. The duplex containing the probe and the 5' end of the mRNA was then purified and subjected to LC-MS analysis.

[0217] The resulting eGFP mRNA bearing promoter SEQ ID NO: 10 or 12 was used in an expression efficiency assay in A549 cells. The day before, A549 cells were seeded into 96-well clear-bottom black plates. For eGFP mRNA samples, 200 ng / well of mRNA was added to 0.4 μl of lipofectamine MessengerMax and transfected in triplicate using OptiMEM. Cells were incubated with the mRNA overnight, and expression efficiency was assessed based on the relative fluorescence intensity of eGFP mRNA-treated cells using a microblade reader. Values ​​were normalized by relative cell number, as tested by Cyquant XTT cell viability assay. The normalized values ​​for the prepared mRNA bearing the Cap-2 cap analog were compared with those for the prepared mRNA bearing the AG Cap-1 cap analog. result

[0218] Using the conditions and procedures described above, mRNA was produced with a plasmid template encoding eGFP. The mRNA yields of the IVT reactions are shown in Table 7. Cap-2: [ka] ARCA-Cap 2: [ka]

[0219] Under IVT1 conditions and with promoter #2 (SEQ ID NO:10), mRNA yields using Cap-2 (8.07 μg / μl) or ARCA-Cap-2 (7.65 μg / μl) were similar to those using AG-Cap-1 (6.99 μg / μl). Using Cap-2 (2.12 μg / μl) and ARCA-Cap-2 (2.09 μg / μl), mRNA yields using promoter #1 (SEQ ID NO:12) were lower than those using promoter #2 (SEQ ID NO:10).

[0220] Using IVT2 conditions, the mRNA yield using Cap-2 and Promoter #1 (SEQ ID NO:12) is 2.14 μg / μl, and the mRNA yield using Promoter #2 (SEQ ID NO:10) is 2.56 μg / μl. Using Promoter #1 (SEQ ID NO:12), the mRNA yield using ARCA-Cap-2 is 2.08 μg / μl, and the mRNA yield using Promoter #2 (SEQ ID NO:10) is 2.06 μg / μl.

[0221] Using IVT3 conditions, the mRNA yield using Cap-2 and Promoter #1 (SEQ ID NO:12) is 1.98 μg / μl, and the mRNA yield using Promoter #2 (SEQ ID NO:10) is 2.62 μg / μl. The mRNA yield using ARCA-Cap-2 and Promoter #1 (SEQ ID NO:12) is 1.8 μg / μl, and the mRNA yield using Promoter #2 (SEQ ID NO:10) is 2.85 μg / μl.

[0222] Table 7. Yields of IVT reactions using modified IVT conditions.

[0223] [Table 8]

[0224] The integrity of IVT mRNA capped with cap-2 or ARCA-cap-2 was compared to that capped with AG-cap-1 using an Agilent bioanalyzer with an RNA Nano 6000 kit. The results are shown in Table 8. When using the promoter #1 (SEQ ID NO:12) template, the AG-cap-1 mRNA prepared had a purity of 71%-78% within the 900-1150 nt range. In contrast, the cap-2 and ARCA-cap-2 mRNAs prepared had purities of 82%-83% and 80%-84% within the same range. When using the promoter #2 (SEQ ID NO:10) template, the AG-cap-1 mRNA prepared had a purity of 85%-93% within the 900-1150 nt range. In contrast, the cap-2 and ARCA-cap-2 mRNAs prepared had purities of 89%-91% and 89%, respectively.

[0225] [Table 9]

[0226] As described above, mRNA capping efficiency was examined by RNase H digestion and LC-MS. The AG Cap-1 cap analog could be effectively cotranscribed and incorporated into IVT mRNA using IVT1 conditions, with a capping efficiency of >80%. Using the same IVT1 conditions, the efficiency of cotranscriptional capping with Cap-2 was lower. As shown in Table 9, using IVT1 conditions and promoter #1 (SEQ ID NO:12), the capping efficiencies of Cap-2 and ARCA-Cap-2 were 34.6% and 14.4%, respectively. Compared with the capping efficiency using promoter #1 (SEQ ID NO:12), the capping efficiency using promoter #1 (SEQ ID NO:10) and Cap-2 (44.4%) was higher, but the capping efficiency of ARCA-Cap II (2.3%) was lower. The efficiency of cotranscriptional capping of mRNA was improved when the modified IVT conditions were used. When using promoter #1 (SEQ ID NO:12) and Cap-2, the capping efficiencies under IVT2 and IVT3 conditions were 69.6% and 63.0%, respectively. When using promoter #2 (SEQ ID NO:10), further improvements were observed, e.g., 71.2% and 67.5% under IVT2 and IVT3 conditions, respectively. Similarly, when using ARCA-Cap-2 and promoter #2 (SEQ ID NO:12), the efficiencies were 62.6% (IVT2) and 67.9% (IVT3). Compared with the capping efficiency under IVT1 (2.30%), the capping efficiency of ARCA-Cap-2 using promoter #1 (SEQ ID NO:10) under the modified IVT2 (20.5%) and IVT3 (17.45%) conditions was improved, but the efficiency was lower than that using Cap-2 under IVT2 (71.2%) and IVT3 (67.5%).

[0227] [Table 10]

[0228] Furthermore, the mRNA was transfected into A549 cells to confirm its translatability. A549 cells were inoculated the day before and examined 24 hours later using a microblade reader. As shown in Figure 5, consistent with its low capping efficiency, mRNA produced using promoter #1 (SEQ ID NO:12) or promoter #2 (SEQ ID NO:10) under Cap-2, ARCA-Cap-2, and IVT1 conditions showed poorer expression than mRNA produced with AG Cap-1. For example, the expression level using promoter #1 (SEQ ID NO:12) was less than 30% of that using AG Cap-1, and the expression level using promoter #2 (SEQ ID NO:10) was approximately 10% of that using AG Cap-1. Using promoter #2 (SEQ ID NO:12) in the modified IVT conditions (IVT2 and IVT3) resulted in a significant increase in mRNA expression, consistent with their improved capping efficiency. For example, compared with AGCap-1 mRNA, >75% mRNA expression levels were obtained using Cap-2 and ARCA-CapII under IVT2 conditions. Approximately 60% and 80% mRNA expression levels were obtained using Cap-2 and ARCA-CapII, respectively. Lower levels of improved expression were also observed using promoter #2 (SEQ ID NO:10) under IVT2 and IVT3 conditions. For example, compared with AGCap-1 mRNA, >40% mRNA expression levels were observed using Cap-2 and ARCA-CapII under IVT2 conditions, and >20% mRNA expression levels were observed using Cap-2 and ARCA-CapII under IVT3 conditions. Example 3

[0229] A. Cap Reagent Analog 11(m 7 G 2’Ome pppA 2’Ome p.g. 2’Ome Preparation of (Figure 6) Intermediate 9 (16 mg, 0.028 mmol) was combined with triphenylphosphine (28 mg, 0.11 mmol), imidazole (13 mg, 0.19 mmol), and 2,2'-dithiopyridine (25 mg, 0.11 mmol). The mixture was dissolved in anhydrous DMSO (146 μl), and triethylamine (19 μl) was added. The resulting solution was stirred at room temperature for 45 minutes and then diluted with 1.8 mL of ice-cold 0.2 M sodium perchlorate in acetone. The reaction solution was diluted to a total of 1.8 mL with an ice-cold solution of 0.2 M sodium perchlorate in acetone, and the precipitate that formed was spun down into a crumb. The supernatant was discarded, and the crumb was washed with 1.8 mL of ice-cold acetone and spun down again into a crumb. The acetone washing cycle was repeated four more times, and the product crumb was dried under high vacuum to give Intermediate 10, an off-white solid. The solid was combined with intermediate 5 (9.0 mg, 0.0098 mmol) and MgCl2 (3.2 mg, 0.025 mmol). The mixture was dissolved in anhydrous DMSO (160 μl), and the resulting solution was incubated at 37°C for 18 hours. EDTA (400 μl of a 0.5 M solution) was added, and the resulting solution was diluted to 5.0 mL with water and lyophilized to dryness. The crude reaction product was dissolved in water and purified by reverse-phase (C18) high-performance liquid chromatography: 5.3 μmol (54%) yield of 11, chromatographic (ESI) m / z 1,172. Bm 7 G 3’Ome pp S pA 2’Ome p.g. 2’Ome Preparation of (16) B-1. Preparation of Intermediates 13 and 14 (Figure 7)

[0230] 3'-O-methylguanosine-5'-phosphate (12) was prepared according to Jemielity et al., RNA, 2003, 9, 1108-1122. The triethylammonium salt of intermediate 12 (132 mg, 0.28 mmol) was dissolved in 4.0 mL of water, and the solution was adjusted to pH 4.0 with acetic acid. Dimethyl sulfate (0.38 mL) was added dropwise over 10 minutes, and the reaction solution was stirred for 4 hours. Sodium hydroxide solution was added to maintain the pH at 3.75-4.25. After the 4-hour reaction period, the reaction solution was extracted with dichloromethane (3 × 16 mL), and the crude product contained in the aqueous phase was purified by reverse-phase (C18) HPLC using a gradient of TEAB (pH 7.8)-acetonitrile. The pure product was partially lyophilized to dryness to give a white solid: yield 110 mg (81%) of intermediate 13 as the triethylammonium salt.

[0231] Intermediate 13 (104 mg, 0.18 mmol) was combined with imidazole (85 mg, 1.25 mmol), triphenylphosphine (188 mg, 0.74 mmol), and 2,2′-dithiopyridine (164 mg, 0.72 mmol). The mixture was dissolved in 2.0 mL of dry DMSO containing triethylamine (130 μL). The resulting solution was stirred at room temperature for 4.0 hours. The reaction solution was then diluted to 25 mL with ice-cold 0.2 M sodium perchlorate / acetone solution. The precipitate formed was spun down to a crumb, and the supernatant was discarded. The crumb was suspended in 25 mL of ice-cold acetone, vortexed, and centrifuged to form a crumb. The supernatant was again discarded, and the process was repeated three more times. The final crumb was dried under high vacuum to give the 135′-phosphoryl imidazole ester analog as a white solid: yield: 101 mg.

[0232] The 5'-phosphoimidazole ester analog of 13 (101 mg) was combined with MnCl (3.0 mg, 0.024 mmol) and stirred at room temperature for 1.0 h in 4.0 mL of 0.25 M bis-triethylammonium thiophosphate in dimethylformamide. The reaction solution was then diluted to 50 mL with water. The crude product was purified by ion exchange chromatography (DEAE Sephedex A25 resin) using a gradient of 0 to 0.70 M TEAB (pH 8.3) buffer. The pure product was partially combined and evaporated from water several times to remove volatile salt species. The purified product (bistriethylammonium salt form of 14) was lyophilized to give a white solid: 63 mg (87% yield), chromatographic (ESI) m / z 486. B-2. Preparation of Intermediate 15 (Figure 8)

[0233] Intermediate 5 (28 mg, 0.030 mmol) was combined with triphenylphosphine (49 mg, 0.19 mmol), imidazole (22 mg, 0.32 mmol), and 2,2'-dithiopyridine (42 mg, 0.18 mmol). The mixture was dissolved in anhydrous DMSO (250 μl) containing triethylamine (33 μl). The reaction was stirred at room temperature for 30 minutes and then diluted with 15 ml of ice-cold 0.2 M sodium perchlorate / acetone solution. The precipitate that formed was spun down into a crumb, and the supernatant was discarded. The crumb was washed with 15 ml of ice-cold acetone and spun down again into a crumb. The supernatant was discarded, and the process was repeated three more times. Finally, the crumb was dried under high vacuum to give a white solid: yield 20 mg (82%), Intermediate 15 in the disodium salt form. B-3. ​​Preparation of capping reagent analog 16 (Figure 8)

[0234] Intermediate 15 (20 mg, 0.025 mmol) and intermediate 14 (9.0 mg, 0.015 mmol) were combined and stirred at 37°C for 18 hours in anhydrous dimethylformamide (145 μl) containing anhydrous tin chloride (22 mg). EDTA (400 μl of a 0.5 M solution) was added, and the solution was diluted to 5.0 mL with water and lyophilized to dryness. The powder was dissolved in water, and the two forms of capped analog 16 (diastereomers) were separated together by reverse-phase (C18) HPLC using a gradient of 50 mM TEAB (pH 8.0)-acetonitrile. The pure products were partially combined and lyophilized to dryness to give a white solid: 2.0 μmol yield (absorption coefficient = 30,540, determined spectrophotometrically at 255 nm), chromatographic (ESI) m / z 1188. Example 4

[0235] To test the in vivo protein expression of mRNA produced with a Cap-2 analog by IVT, mice were injected with firefly luciferase (Fluc) mRNA produced with a Cap-2 analog by IVT (Cap2-Fluc) or a Cap-1 analog by IVT (Cap1-Fluc), both encapsulated in the ALC0315-LNP formulation. The Cap-1 and Cap-2 analogs used in this example are shown below: Cap 1 Analogue (Cap 1-AG): [ka] Cap 2 Analog (Arca-Cap 2-AG, where R=CH3, B1=A, B2=G): [ka]

[0236] Mice injected with PBS served as a negative control. Ninety-six hours after injection, Fluc protein expression was examined by IVIS in vivo imaging. Figure 9 shows that the expression level of Cap2-Fluc mRNA was similar to that of Cap1-Fluc mRNA. Ninety-six hours later, biodistribution was further measured by bioluminescence. Figure 10 shows that the expression of Cap1-Fluc mRNA and Cap2-Fluc mRNA was primarily distributed in the liver. Figure 11 shows that injection of Cap1-Fluc mRNA or Cap2-Fluc mRNA did not affect the body weight of mice compared with that of PBS-injected mice.

[0237] Advantages of the present disclosure may include: (1) in vitro transcription reaction mixtures and conditions that can increase mRNA yield, integrity, and purity; (2) DNA templates and cap analogs that produce more full-length mRNA by binding to the -1 and / or +1 nucleotide of a promoter for in vitro transcription, thereby allowing greater flexibility in the selection of the first mRNA base and providing the +2 position open for custom sequences; and (3) cap-2 analogs that have 2'-O-methylation of the second transcribed nucleotide and N6-methylation of adenosine as the first transcribed nucleotide, which serve as a determinant defining the transcript as "self," thereby aiding in transcript immune evasion.

[0238] All references cited herein are incorporated by reference as if each reference were specifically and individually indicated to be incorporated by reference herein. Any reference is referenced as though disclosed prior to the filing date and should not be construed as an admission that such reference is not entitled to antedate the present disclosure by virtue of prior invention.

[0239] It should be understood that each one, two, or more of the above elements may find useful application in other types of methods different from those described above. Without further analysis, the above content sufficiently reveals the gist of the present disclosure so that others, by applying their current knowledge, can readily employ them in various applications without omitting features that, in the light of the prior art, fairly constitute essential features of the general or specific aspects of the present disclosure as set forth in the appended claims. The above embodiments are presented by way of example only, and the scope of the present disclosure is limited by the following claims.

Claims

1. 1. A method for in vitro transcription of a DNA template into RNA, said method comprising: providing a mixture; and producing the RNA by incubating the reaction mixture at about 15° C. to about 35° C., optionally at about 18° C. to about 31° C., for about 1 hour to about 12 hours, wherein the mixture comprises: A buffer substance; ribonucleoside triphosphates (NTPs); one or more magnesium salts at a concentration of about 2 mM to about 60 mM; the DNA template; a recombinant RNA polymerase and a cap analog, comprising a structure having formula (I), (II) or (III): 【Chemistry 1】 where R is H or CH ３ and B １ is A or N6-methyl-adenine (m ６ A) and B ２ is A, U, G or C, 【Chemistry 2】 where: R １ is OCH ３ and R ２ is OH or H, or R １ is OH and R ２ is H, or R １ is H and R ２ is H or OCH ３ or R １ and R ２ are OCH ３ and B1 is A or N6-methyl-adenine (m ６ A) and B2 is A, U, G or C; 【Transformation 3】 where R is H or CH ３ and B １ is A or N6-methyl-adenine (m ６ A) and B ２ is A, U, G or C.

2. 2. The method of claim 1, wherein the buffer is Tris base, HEPES, or Tris-HCl.

3. The concentration of the buffer substance is from about 1 mM to about 100 mM, from about 1 mM to about 90 mM, from about 1 mM to about 80 mM, from about 1 mM to about 70 mM, from about 1 mM to about 60 mM, from about 1 mM to about 50 mM, from about 1 mM to about 40 mM, from about 1 mM to about 30 mM, from about 1 mM to about 20 mM, from about 1 mM to about 10 mM, from about 1 mM to about 5 mM, from about 10 mM to about 20 mM, from about 10 mM to about 30 mM, from about 10 mM to about 40 mM, 3. The method of claim 1 or 2, wherein the ATP concentration is 0 mM, about 10 mM to about 50 mM, about 20 mM to about 50 mM, about 30 mM to about 50 mM, about 35 mM to about 45 mM, about 35 mM to about 40 mM, about 40 mM to about 50 mM, about 45 mM to about 50 mM, about 45 mM to about 55 mM, about 15 mM to about 45 mM, about 15 mM to about 35 mM, about 15 mM to about 30 mM, or about 15 mM to about 25 mM.

4. The concentration of the NTP is from about 1 mM to about 50 mM, from about 1 mM to about 40 mM, from about 1 mM to about 30 mM, from about 1 mM to about 20 mM, from about 1 mM to about 10 mM, from about 1 mM to about 5 mM, from about 2 mM to about 10 mM, from about 3 mM to about 10 mM, from about 3 mM to about 9 mM, from about 3 mM to about 8 mM, from about 3 mM to about 7 mM, from about 3 mM to about 6 mM, from about 3 mM to about 5 mM, from about 3 mM to about 4 mM, from about 4 mM to about 10 mM, 4. The method of claim 1, wherein the ATP concentration is 0 mM, about 5 mM to about 10 mM, about 6 mM to about 10 mM, about 7 mM to about 10 mM, about 8 mM to about 10 mM, about 9 mM to about 10 mM, about 10 mM to about 50 mM, about 20 mM to about 50 mM, about 25 mM to about 50 mM, about 25 mM to about 45 mM, about 25 mM to about 40 mM, about 25 mM to about 35 mM, or about 20 mM to about 30 mM.

5. 5. The method of any one of claims 1 to 4, wherein the concentration of the one or more magnesium salts is from about 2 mM to about 50 mM, from about 2 mM to about 40 mM, from about 2 mM to about 30 mM, from about 2 mM to about 40 mM, from about 2 mM to about 30 mM, from about 2 mM to about 20 mM, from about 2 mM to about 10 mM, from about 2 mM to about 5 mM, from about 5 mM to about 50 mM, from about 10 mM to about 45 mM, from about 15 mM to about 40 mM, from about 20 mM to about 35 mM, from about 20 mM to about 30 mM, from about 20 mM to about 25 mM, from about 22 mM to about 28 mM, or from about 25 mM to about 30 mM.

6. The concentration of the DNA template is from about 0.001 μg / μl to about 2 μg / μl, from about 0.001 μg / μl to about 1.5 μg / μl, from about 0.001 μg / μl to about 1 μg / μl, from about 0.01 μg / μl to about 2 μg / μl, from about 0.01 μg / μl to about 1.5 μg / μl, from about 0.01 μg / μl to about 1 μg / μl, from about 0.01 μg / μl to about 0.5 μg / μl, from about 0.01 μg / μl to about 0.1 μg / μl, from about 0.01 μg / μl to about 0.05 μg / μl, or about 0.02 μg / μl.

6. The method of claim 1, wherein the concentration of the IgG antibody is from about 0.01 μg / μl to about 0.04 μg / μl, from about 0.02 μg / μl to about 0.1 μg / μl, from about 0.03 μg / μl to about 0.1 μg / μl, from about 0.04 μg / μl to about 0.1 μg / μl, from about 0.05 μg / μl to about 0.1 μg / μl, from about 0.06 μg / μl to about 0.1 μg / μl, from about 0.07 μg / μl to about 0.1 μg / μl, from about 0.08 μg / μl to about 0.1 μg / μl, or from about 0.09 μg / μl to about 0.1 μg / μl.

7. 7. The method of any one of claims 1 to 6, wherein the concentration of the recombinant RNA polymerase is from about 0.1 U / μl to about 2 U / μl, from about 0.5 U / μl to about 2 U / μl, from about 1 U / μl to about 2 U / μl, from about 1 U / μl to about 1.5 U / μl, or from about 1.5 U / μl to about 2 U / μl.

8. The method of any one of claims 1 to 7, wherein the mixture further comprises an antioxidant.

9. 9. The method of claim 8, wherein the antioxidant is dithiothreitol (DTT) and the concentration of the dithiothreitol is from about 1 mM to about 50 mM, from about 2 mM to about 50 mM, from about 3 mM to about 50 mM, from about 4 mM to about 50 mM, from about 5 mM to about 50 mM, from about 6 mM to about 50 mM, from about 7 mM to about 50 mM, from about 8 mM to about 50 mM, from about 9 mM to about 50 mM, from about 10 mM to about 50 mM, from about 10 mM to about 40 mM, from about 15 mM to about 30 mM, from about 15 mM to about 25 mM, from about 15 mM to about 20 mM, from about 20 mM to about 50 mM, from about 30 mM to about 50 mM, or from about 40 mM to about 50 mM.

10. The mixture further comprises an RNase inhibitor, wherein the concentration of the RNase inhibitor is from about 0.001 U / μl to about 5 U / μl, from about 0.001 U / μl to about 4 U / μl, from about 0.001 U / μl to about 3 U / μl, from about 0.001 U / μl to about 2 U / μl, from about 0.001 U / μl to about 1 U / μl, from about 0.01 U / μl to about 5 U / μl, from about 0.01 U / μl to about 4 U / μl, from about 0.01 U / μl to about 3 U / μl, or from about 0.01 U / μl to about 2 U / μl. , about 0.01 U / μl to about 1 U / μl, about 0.01 U / μl to about 0.5 U / μl, about 0.01 U / μl to about 0.1 U / μl, about 0.01 U / μl to about 0.05 U / μl, about 0.01 U / μl to about 0.04 U / μl, about 0.01 U / μl to about 0.03 U / μl, about 0.01 U / μl to about 0.02 U / μl, about 0.1 U / μl to about 5 U / μl, about 0.1 U / μl to about 4 U / μl, about 0.1 U / μl to about 3 U / μl, about 0.1 U / μl to about 5 U / μl About 2 U / μl, about 0.1 U / μl to about 1 U / μl, about 0.5 U / μl to about 5 U / μl, about 0.5 U / μl to about 4 U / μl, about 0.5 U / μl to about 3 U / μl, about 0.5 U / μl to about 2 U / μl, about 0.5 U / μl to about 1 U / μl, about 1 U / μl to about 5 U / μl, about 2 U / μl to about 5 U / μl, about 3 U / μl to about 5 U / μl, about 4 U / μl to about 5 U / μl, about 0.001 U / μl to about 0.005 U / μl, about 0.001 U / μl to about 0.005 U / μl, 10. The method of any one of claims 1 to 9, wherein the concentration is about 0.01 U / μl, about 0.005 U / μl to about 0.01 U / μl, about 0.01 U / μl to about 0.05 U / μl, about 0.01 U / μl to about 0.04 U / μl, about 0.01 U / μl to about 0.03 U / μl, about 0.01 U / μl to about 0.02 U / μl, about 0.02 U / μl to about 0.03 U / μl, about 0.02 U / μl to about 0.04 U / μl, or about 0.02 U / μl to about 0.05 U / μl.

11. The concentration of the cap analog is from about 0.5 mM to about 50 mM, from about 0.5 mM to about 40 mM, from about 0.5 mM to about 30 mM, from about 0.5 mM to about 20 mM, from about 0.5 mM to about 10 mM, from about 0.5 mM to about 5 mM, from about 1 mM to about 10 mM, from about 2 mM to about 10 mM, from about 3 mM to about 10 mM, from about 3 mM to about 9 mM, from about 3 mM to about 8 mM, from about 3 mM to about 7 mM, from about 3 mM to about 6 mM, from about 3 mM to about 9 mM, 11. The method of any one of claims 1 to 10, wherein the HCl concentration is from about 5 mM to about 5 mM, from about 3 mM to about 4 mM, from about 4 mM to about 10 mM, from about 5 mM to about 10 mM, from about 6 mM to about 10 mM, from about 6 mM to about 9 mM, from about 6 mM to about 8 mM, from about 6 mM to about 7 mM, from about 7 mM to about 8 mM, from about 7 mM to about 9 mM, from about 7 mM to about 10 mM, from about 8 mM to about 9 mM, from about 8 mM to about 10 mM, or from about 9 mM to about 10 mM.

12. The method of any one of claims 1 to 11, wherein the mixture further comprises a polyamine.

13. 13. The method of claim 12, wherein the polyamine is spermine, spermidine, or a combination thereof.

14. The concentration of the polyamine is from about 0.1 mM to about 5 mM, from about 0.2 mM to about 4.9 mM, from about 0.2 mM to about 4.8 mM, from about 0.2 mM to about 4.7 mM, from about 0.2 mM to about 4.6 mM, from about 0.2 mM to about 4.5 mM, from about 0.2 mM to about 4.4 mM, from about 0.2 mM to about 4.3 mM, from about 0.2 mM to about 4.2 mM, from about 0.2 mM to about 4.1 mM, from about 0.2 mM to about 4 mM, from about 0.2 mM to about 3.5 mM, from about 0.2 mM to about 3 mM, from about 0.2 mM to about 2.5 mM, from about 0.5 mM to about 2.5 mM, from about 1.0 mM to about 2.5 mM 14. The method of claim 12 or 13, wherein the ATP concentration is about 1.5 mM to about 2.5 mM, about 0.2 mM to about 2 mM, about 0.2 mM to about 1.5 mM, about 0.2 mM to about 1 mM, about 0.2 mM to about 0.9 mM, about 0.2 mM to about 0.8 mM, about 0.2 mM to about 0.7 mM, about 0.2 mM to about 0.6 mM, about 0.2 mM to about 0.5 mM, about 0.2 mM to about 0.4 mM, about 0.2 mM to about 0.3 mM, about 1 to about 25 mM, about 1 to about 20 mM, about 1 to about 15 mM, about 1 to about 10 mM, about 1 to about 5 mM, or about 1 to about 2.5 mM.

15. The mixture further comprises a pyrophosphatase, wherein the concentration of the pyrophosphatase is from about 0.01 mU / μl to about 2 mU / μl, from about 0.01 mU / μl to about 1.5 mU / μl, from about 0.01 mU / μl to about 1 mU / μl, from about 0.1 mU / μl to about 2 mU / μl, from about 0.1 mU / μl to about 1.5 mU / μl, from about 0.1 mU / μl to about 1 mU / μl, or from about 0.1 mU / μl to about 0.9 mU / μl.

15. The method of any one of claims 1 to 14, wherein the concentration of ribonucleotides in the blood is about 0.1 mU / μl to about 0.8 mU / μl, about 0.1 mU / μl to about 0.7 mU / μl, about 0.1 mU / μl to about 0.6 mU / μl, about 0.1 mU / μl to about 0.5 mU / μl, about 0.1 mU / μl to about 0.4 mU / μl, about 0.1 mU / μl to about 0.3 mU / μl, or about 0.1 mU / μl to about 0.2 mU / μl.

16. Incubating the reaction mixture may be at a temperature ranging from about 15°C to about 35°C, about 16°C to about 35°C, about 17°C to about 35°C, about 18°C ​​to about 35°C, about 18°C ​​to about 34°C, about 18°C ​​to about 33°C, about 18°C ​​to about 32°C, about 18°C ​​to about 31°C, about 18°C ​​to about 30°C, about 18°C ​​to about 29°C, about 18°C ​​to about 28°C, about 18°C ​​to about 27°C, about 18°C ​​to about 26°C, about 18°C ​​to about 25°C, about 18°C ​​to about 24°C, about 18°C ​​to about 23°C, about 18°C ​​to about 22°C, about 18°C ​​to about 21°C, or about 18°C ​​to about 25°C.

16. The method of any one of claims 1 to 15, wherein the method is carried out at a temperature of about 18°C ​​to about 20°C, about 18°C ​​to about 19°C, about 25°C to about 26°C, about 25°C to about 27°C, about 25°C to about 28°C, about 25°C to about 29°C, about 25°C to about 30°C, about 25°C to about 31°C, about 21°C to about 22°C, about 21°C to about 23°C, about 21°C to about 24°C, about 21°C to about 25°C, about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, about 30°C, about 31°C, about 32°C, about 33°C, about 34°C, or about 35°C.

17. 17. The method of any one of claims 1 to 16, wherein the incubating the reaction mixture is performed for about 1 hour to about 12 hours, about 1 hour to about 11 hours, about 1 hour to about 10 hours, about 1 hour to about 9 hours, about 1 hour to about 8 hours, about 1 hour to about 7 hours, about 1 hour to about 6 hours, about 1 hour to about 5 hours, about 1 hour to about 4 hours, about 1 hour to about 3 hours, about 1 hour to about 2 hours, about 2 hours to about 12 hours, about 3 hours to about 12 hours, about 4 hours to about 12 hours, about 5 hours to about 12 hours, about 6 hours to about 12 hours, about 7 hours to about 12 hours, about 8 hours to about 12 hours, about 9 hours to about 12 hours, about 10 hours to about 12 hours, about 11 hours to about 12 hours, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, or about 10 hours.

18. 18. The method of any one of claims 1-17, wherein the incubating the reaction mixture is carried out at about 31°C for about 1 hour to about 5 hours, about 1 hour to about 4.5 hours, about 1 hour to about 4 hours, about 1 hour to about 3.5 hours, about 1 hour to about 3 hours, about 1 hour to about 2.5 hours, about 1 hour to about 2 hours, about 1 hour to about 1.5 hours, about 0.5 hours to about 1 hour, about 0.5 hours to about 1.5 hours, about 1 hour, about 2 hours, about 3 hours, about 4 hours, or about 5 hours.

19. the DNA template comprises a promoter operably linked to a nucleic acid, the nucleic acid comprising a 5' untranslated region (5' UTR), an open reading frame (ORF) encoding an RNA of interest, a 3' UTR, and a polyA region; wherein the promoter is TAATACGACTCACTATAX １ X ２ X ３ (SEQ ID NO: 16), wherein X １ is A or G, and X ２ is A or G, and X ３ is A, T, G or C, wherein the 5'UTR is selected from SEQ ID NO: 1, 3, 5 or 9, and the 3'UTR is selected from SEQ ID NO: 2, 4, 6 or 8; The method of any one of claims 1 to 18, wherein the poly A region contains at least 60 adenine bases (A).

20. 20. The method of claim 19, wherein the promoter comprises a sequence selected from SEQ ID NOs: 10-15.

21. 21. The method of claim 19 or 20, wherein the 5'UTR and the 3'UTR are SEQ ID NOs: 1 and 2, 1 and 4, 1 and 6, 3 and 2, 3 and 4, 3 and 6, 3 and 8, 5 and 2, 5 and 4, 5 and 6, 7 and 2, 7 and 4, 7 and 6, 7 and 8, 9 and 2, 9 and 4, 9 and 6, or 9 and 8, respectively.

22. 22. The method of claim 21, wherein the 5'UTR and the 3'UTR are SEQ ID NOs: 1 and 2, 1 and 4, 3 and 2, 1 and 6, 7 and 4, 9 and 2, or 3 and 6, respectively.

23. The poly A region may be from about 60 to about 200 A, from about 60 to about 190 A, from about 60 to about 180 A, from about 60 to about 170 A, from about 60 to about 160 A, from about 60 to about 150 A, from about 60 to about 140 A, from about 60 to about 130 A, from about 60 to about 120 A, from about 60 to about 110 A, from about 60 to about 100 A, from about 70 to about 190 A, from about 80 23. The method of any one of claims 19-22, comprising from about 180 A, from about 90 to about 170 A, from about 100 to about 160 A, from about 100 to about 150 A, from about 100 to about 140 A, from about 100 to about 130 A, from about 100 to about 120 A, about 100 A, about 110 A, about 120 A, about 130 A, about 140 A, or about 150 A.

24. The method of any one of claims 1 to 23, wherein the recombinant RNA polymerase is selected from wild-type T7 RNA polymerase or a mutant thereof.

25. The cap analog is m ７ GpppA ２’Ｏｍｅ pA ２’Ｏｍｅ 、 m ７ GpppA ２’Ｏｍｅ pU ２’Ｏｍｅ 、 m ７ Gp ２’Ｏｍｅ pG ２’Ｏｍｅ 、 m ７ GpppA ２’Ｏｍｅ pC ２’Ｏｍｅ 、 m ７ Gpppm ６ A ２’Ｏｍｅ pA ２’Ｏｍｅ 、 m ７ Gpppm ６ A ２’Ｏｍｅ pU ２’Ｏｍｅ 、 m ７ Gpppm ６ A ２’Ｏｍｅ pG ２’Ｏｍｅ 、 m ７ Gpppm ６ A ２’Ｏｍｅ pC ２’Ｏｍｅ 、 m ７ G ３’Ｏｍｅ AA0A ２’Ｏｍｅ AA ２’Ｏｍｅ 、 m ７ G ３’Ｏｍｅ AA0A ２’Ｏｍｅ UU ２’Ｏｍｅ 、 m ７ G ３’Ｏｍｅ pppA ２’Ｏｍｅ pG ２’Ｏｍｅ 、 m ７ G ３’Ｏｍｅ pppA ２’Ｏｍｅ pC ２’Ｏｍｅ 、 m ７ G ３’Ｏｍｅ pppm ６ A ２’Ｏｍｅ pA ２’Ｏｍｅ 、 m ７ G ３’Ｏｍｅ pppm ６ A ２’Ｏｍｅ pU ２’Ｏｍｅ 、 m ７ G ３’Ｏｍｅ pppm ６ A ２’Ｏｍｅ pG ２’Ｏｍｅ 、 m ７ G ３’Ｏｍｅ pppm ６ A ２’Ｏｍｅ pC ２’Ｏｍｅ 、 m ７ G ２’Ｏｍｅ AA0A ２’Ｏｍｅ AA ２’Ｏｍｅ 、 m ７ G ２’Ｏｍｅ AA0A ２’Ｏｍｅ UU ２’Ｏｍｅ 、 m ７ G ２’Ｏｍｅ pppA ２’Ｏｍｅ pG ２’Ｏｍｅ 、 m ７ G ２’Ｏｍｅ pppA ２’Ｏｍｅ pC ２’Ｏｍｅ 、 m ７ G ２’Ｏｍｅ pppm ６ A ２’Ｏｍｅ pA ２’Ｏｍｅ 、 m ７ G ２’Ｏｍｅ pppm ６ A ２’Ｏｍｅ pU ２’Ｏｍｅ 、 m ７ G ２’Ｏｍｅ pppm ６ A ２’Ｏｍｅ pG ２’Ｏｍｅ 、 m ７ G ２’Ｏｍｅ pppm ６ A ２’Ｏｍｅ pC ２’Ｏｍｅ 、 m ７ G ３’Ｈ AA0A ２’Ｏｍｅ AA ２’Ｏｍｅ 、 m ７ G ３’Ｈ AA0A ２’Ｏｍｅ UU ２’Ｏｍｅ 、 m ７ G ３’Ｈ pppA ２’Ｏｍｅ pG ２’Ｏｍｅ 、 m ７ G ３’Ｈ pppA ２’Ｏｍｅ pC ２’Ｏｍｅ 、 m ７ G ３’Ｈ pppm ６ A ２’Ｏｍｅ pA ２’Ｏｍｅ 、 m ７ G ３’Ｈ pppm ６ A ２’Ｏｍｅ pU ２’Ｏｍｅ 、 m ７ G ３’Ｈ pppm ６ A ２’Ｏｍｅ pG ２’Ｏｍｅ 、 m ７ G ３’Ｈ pppm ６ A ２’Ｏｍｅ pC ２’Ｏｍｅ 、 m ７ Gpp Ｓ p｡ ２’Ｏｍｅ p｡ ２’Ｏｍｅ 、 m ７ GA0 Ｓ AA ２’Ｏｍｅ UU ２’Ｏｍｅ 、 m ７ Gpp Ｓ pA ２’Ｏｍｅ pG ２’Ｏｍｅ 、 m ７ Gpp Ｓ pA ２’Ｏｍｅ pC ２’Ｏｍｅ 、 m ７ Gpp Ｓ pm ６ A ２’Ｏｍｅ pA ２’Ｏｍｅ 、 m ７ Gpp Ｓ pm ６ A ２’Ｏｍｅ pU ２’Ｏｍｅ 、 m ７ Gpp Ｓ pm ６ A ２’Ｏｍｅ pG ２’Ｏｍｅ 、 m ７ Gpp Ｓ pm ６ A ２’Ｏｍｅ pC ２’Ｏｍｅ 、 m ７ G ３’Ｏｍｅ pp Ｓ p｡ ２’Ｏｍｅ p｡ ２’Ｏｍｅ 、 m ７ G ３’Ｏｍｅ @@ Ｓ AA ２’Ｏｍｅ UU ２’Ｏｍｅ 、 m ７ G ３’Ｏｍｅ pp Ｓ pA ２’Ｏｍｅ pG ２’Ｏｍｅ 、 m ７ G ３’Ｏｍｅ pp Ｓ pA ２’Ｏｍｅ pC ２’Ｏｍｅ 、 m ７ G ３’Ｏｍｅ pp Ｓ pm ６ A ２’Ｏｍｅ pA ２’Ｏｍｅ 、 m ７ G ３’Ｏｍｅ pp Ｓ pm ６ A ２’Ｏｍｅ pU ２’Ｏｍｅ 、 m ７ G ３’Ｏｍｅ p.p. Ｓ pm ６ A ２’Ｏｍｅ p.g. ２’Ｏｍｅ , and m ７ G ３’Ｏｍｅ p.p. Ｓ pm ６ A ２’Ｏｍｅ PC ２’Ｏｍｅ The method of any one of claims 1 to 24, wherein the cap analog is selected from the group consisting of:

26. 26. The method of any one of claims 19 to 25, wherein the cap analog is attached to the -1 and / or +1 nucleotide of the promoter.

27. 1. A reaction mixture for in vitro transcription of a DNA template into RNA, said reaction mixture comprising: a buffering substance having a concentration of about 45 mM to about 55 mM; an RNase inhibitor at a concentration of about 0.01 U / μl to about 0.03 U / μl; NTP at a concentration of about 3 mM to about 5 mM; a cap analog at a concentration of about 6 mM to about 8 mM; one or more magnesium salts at a concentration of about 20 mM to about 30 mM; a polyamine at a concentration of about 1.5 mM to about 2.5 mM; a DNA template at a concentration of about 0.01 μg / μl to about 0.05 μg / μl; pyrophosphatase at a concentration of about 0.1 mU / μl to about 0.5 mU / μl; an RNA polymerase at a concentration of about 0.01 μg / μl to about 0.05 μg / μl; wherein the cap analog comprises a structure having formula (I), (II), or (III): 【Chemistry 4】 where R is H or CH ３ and B １ is A or N6-methyl-adenine (m ６ A) and B ２ is A, U, G or C, 【Transformation 5】 where: R １ is OCH ３ and R ２ is OH or H, or R １ is OH and R ２ is H, or R １ is H and R ２ is H or OCH ３ or R １ and R ２ are OCH ３ and B1 is A or N6-methyl-adenine (m ６ A) and B2 is A, U, G or C; 【Transformation 6】 where R is H or CH ３ and B １ is A or N6-methyl-adenine (m ６ A) and B ２ is A, U, G or C.

28. 28. The reaction mixture of claim 27, wherein the buffer is Tris base, HEPES, or Tris-HCl.

29. 29. The reaction mixture of claim 27 or 28, wherein the reaction mixture further comprises an antioxidant.

30. The cap analog is m ７ GpppA ２’Ｏｍｅ pA ２’Ｏｍｅ 、 m ７ GpppA ２’Ｏｍｅ pU ２’Ｏｍｅ 、 m ７ Gp ２’Ｏｍｅ pG ２’Ｏｍｅ 、 m ７ GpppA ２’Ｏｍｅ pC ２’Ｏｍｅ 、 m ７ Gpppm ６ A ２’Ｏｍｅ pA ２’Ｏｍｅ 、 m ７ Gpppm ６ A ２’Ｏｍｅ pU ２’Ｏｍｅ 、 m ７ Gpppm ６ A ２’Ｏｍｅ pG ２’Ｏｍｅ 、 m ７ Gpppm ６ A ２’Ｏｍｅ pC ２’Ｏｍｅ 、 m ７ G ３’Ｏｍｅ AA0A ２’Ｏｍｅ AA ２’Ｏｍｅ 、 m ７ G ３’Ｏｍｅ AA0A ２’Ｏｍｅ UU ２’Ｏｍｅ 、 m ７ G ３’Ｏｍｅ pppA ２’Ｏｍｅ pG ２’Ｏｍｅ 、 m ７ G ３’Ｏｍｅ pppA ２’Ｏｍｅ pC ２’Ｏｍｅ 、 m ７ G ３’Ｏｍｅ pppm ６ A ２’Ｏｍｅ pA ２’Ｏｍｅ 、 m ７ G ３’Ｏｍｅ pppm ６ A ２’Ｏｍｅ pU ２’Ｏｍｅ 、 m ７ G ３’Ｏｍｅ pppm ６ A ２’Ｏｍｅ pG ２’Ｏｍｅ 、 m ７ G ３’Ｏｍｅ pppm ６ A ２’Ｏｍｅ pC ２’Ｏｍｅ 、 m ７ G ２’Ｏｍｅ AA0A ２’Ｏｍｅ AA ２’Ｏｍｅ 、 m ７ G ２’Ｏｍｅ AA0A ２’Ｏｍｅ UU ２’Ｏｍｅ 、 m ７ G ２’Ｏｍｅ pppA ２’Ｏｍｅ pG ２’Ｏｍｅ 、 m ７ G ２’Ｏｍｅ pppA ２’Ｏｍｅ pC ２’Ｏｍｅ 、 m ７ G ２’Ｏｍｅ pppm ６ A ２’Ｏｍｅ pA ２’Ｏｍｅ 、 m ７ G ２’Ｏｍｅ pppm ６ A ２’Ｏｍｅ pU ２’Ｏｍｅ 、 m ７ G ２’Ｏｍｅ pppm ６ A ２’Ｏｍｅ pG ２’Ｏｍｅ 、 m ７ G ２’Ｏｍｅ pppm ６ A ２’Ｏｍｅ pC ２’Ｏｍｅ 、 m ７ G ３’Ｈ AA0A ２’Ｏｍｅ AA ２’Ｏｍｅ 、 m ７ G ３’Ｈ AA0A ２’Ｏｍｅ UU ２’Ｏｍｅ 、 m ７ G ３’Ｈ pppA ２’Ｏｍｅ pG ２’Ｏｍｅ 、 m ７ G ３’Ｈ pppA ２’Ｏｍｅ pC ２’Ｏｍｅ 、 m ７ G ３’Ｈ pppm ６ A ２’Ｏｍｅ pA ２’Ｏｍｅ 、 m ７ G ３’Ｈ pppm ６ A ２’Ｏｍｅ pU ２’Ｏｍｅ 、 m ７ G ３’Ｈ pppm ６ A ２’Ｏｍｅ pG ２’Ｏｍｅ 、 m ７ G ３’Ｈ pppm ６ A ２’Ｏｍｅ pC ２’Ｏｍｅ 、 m ７ Gpp Ｓ p｡ ２’Ｏｍｅ p｡ ２’Ｏｍｅ 、 m ７ GA0 Ｓ AA ２’Ｏｍｅ UU ２’Ｏｍｅ 、 m ７ Gpp Ｓ pA ２’Ｏｍｅ pG ２’Ｏｍｅ 、 m ７ Gpp Ｓ pA ２’Ｏｍｅ pC ２’Ｏｍｅ 、 m ７ Gpp Ｓ pm ６ A ２’Ｏｍｅ pA ２’Ｏｍｅ 、 m ７ Gpp Ｓ pm ６ A ２’Ｏｍｅ pU ２’Ｏｍｅ 、 m ７ Gpp Ｓ pm ６ A ２’Ｏｍｅ pG ２’Ｏｍｅ 、 m ７ Gpp Ｓ pm ６ A ２’Ｏｍｅ pC ２’Ｏｍｅ 、 m ７ G ３’Ｏｍｅ pp Ｓ p｡ ２’Ｏｍｅ p｡ ２’Ｏｍｅ 、 m ７ G ３’Ｏｍｅ @@ Ｓ AA ２’Ｏｍｅ UU ２’Ｏｍｅ 、 m ７ G ３’Ｏｍｅ pp Ｓ pA ２’Ｏｍｅ pG ２’Ｏｍｅ 、 m ７ G ３’Ｏｍｅ pp Ｓ pA ２’Ｏｍｅ pC ２’Ｏｍｅ 、 m ７ G ３’Ｏｍｅ pp Ｓ pm ６ A ２’Ｏｍｅ pA ２’Ｏｍｅ 、 m ７ G ３’Ｏｍｅ pp Ｓ pm ６ A ２’Ｏｍｅ pU ２’Ｏｍｅ 、 m ７ G ３’Ｏｍｅ p.p. Ｓ pm ６ A ２’Ｏｍｅ p.g. ２’Ｏｍｅ , and m ７ G ３’Ｏｍｅ p.p. Ｓ pm ６ A ２’Ｏｍｅ PC ２’Ｏｍｅ 30. The reaction mixture of any one of claims 27 to 29, wherein the cap analog is selected from the group consisting of:

31. The one or more magnesium salts may be MgCl ２ and / or magnesium acetate (Mg(C ２ H ３ O ２ ) ２ )(Mg(OAc) ２ 31. The reaction mixture according to any one of claims 27 to 30, wherein

32. 32. The reaction mixture of any one of claims 27 to 31, wherein the polyamine is spermine, spermidine, or a combination thereof.

33. the DNA template comprises a promoter operably linked to a nucleic acid, the nucleic acid comprising a 5' untranslated region (5' UTR), an open reading frame (ORF) encoding an RNA of interest, a 3' UTR, and a polyA region; wherein the promoter is TAATACGACTCACTATAX １ X ２ X ３ (SEQ ID NO: 16), wherein X １ is A or G, and X ２ is A or G, and X ３ is A, T, G or C, wherein the 5'UTR is selected from SEQ ID NO: 1, 3, 5 or 9, and the 3'UTR is selected from SEQ ID NO: 2, 4, 6 or 8; 33. The reaction mixture of any one of claims 27 to 32, wherein the polyA region comprises at least 60 adenine bases (A).

34. 34. The reaction mixture of claim 33, wherein the promoter comprises a sequence selected from SEQ ID NOs: 10-15.

35. 35. The reaction mixture of claim 33 or 34, wherein the 5'UTR and the 3'UTR are SEQ ID NOs: 1 and 2, 1 and 4, 1 and 6, 3 and 2, 3 and 4, 3 and 6, 3 and 8, 5 and 2, 5 and 4, 5 and 6, 7 and 2, 7 and 4, 7 and 6, 7 and 8, 9 and 2, 9 and 4, 9 and 6, or 9 and 8, respectively.

36. 36. The reaction mixture of claim 35, wherein the 5'UTR and the 3'UTR are SEQ ID NOs: 1 and 2, 1 and 4, 3 and 2, 1 and 6, 7 and 4, 9 and 2, or 3 and 6, respectively.

37. The poly A region may be from about 60 to about 200 A, from about 60 to about 190 A, from about 60 to about 180 A, from about 60 to about 170 A, from about 60 to about 160 A, from about 60 to about 150 A, from about 60 to about 140 A, from about 60 to about 130 A, from about 60 to about 120 A, from about 60 to about 110 A, from about 60 to about 100 A, from about 70 to about 190 A, from about 80 to about 160 A, from about 180 to about 190 A, from about 190 to about 200 A, from about 200 to about 210 A, from about 210 to about 220 A, from about 220 to about 230 A, from about 230 to about 240 A, from about 240 to about 250 A, from about 250 to about 260 A, from about 260 to about 270 A, from about 270 to about 280 A, from about 280 to about 300 A, from about 300 to about 310 A, from about 310 to about 320 A, from about 320 to about 330 A, from about 330 to about 340 A, from about 340 to about 350 A, from about 350 to about 360 A, from about 360 to about 370 A, from about 370 to about 380 A, from about 380 to about 400 A, from about 410 to about 420 A, from about 410 to about 430 A, from about 420 to about 440 A, from about 430 to about 450 A, 37. The reaction mixture of any one of claims 33-36, comprising from about 180 A, from about 90 to about 170 A, from about 100 to about 160 A, from about 100 to about 150 A, from about 100 to about 140 A, from about 100 to about 130 A, from about 100 to about 120 A, about 100 A, about 110 A, about 120 A, about 130 A, about 140 A, or about 150 A.

38. 38. The reaction mixture of any one of claims 27 to 37, wherein the recombinant RNA polymerase is selected from wild-type T7 RNA polymerase or a mutant thereof.

39. 1. A method for in vitro transcription of a DNA template into RNA, comprising: Providing a reaction mixture according to any one of claims 27 to 38; and incubating the reaction mixture at about 15°C to about 35°C for about 1 hour to about 12 hours to produce the RNA.

40. 40. The method of claim 39, wherein said incubating comprises incubating said reaction mixture at about 18°C ​​to about 31°C.

41. 40. The method of claim 39, wherein said incubating comprises incubating said reaction mixture at about 31°C for about 3 hours.

42. 1. A method for in vitro transcription of a DNA template into RNA, said method comprising: (1) providing a DNA template comprising a promoter operably linked to a nucleic acid comprising a 5' untranslated region (5'UTR), an open reading frame (ORF) encoding an RNA of interest, a 3'UTR, and a polyA region; and (2) a cap analog; incubating the DNA template and the cap analog in a reaction mixture; wherein the cap analog comprises a structure having formula (I), (II), or (III): 【Transformation 7】 where R is H or CH ３ and B １ is A or N6-methyl-adenine (m ６ A) and B ２ is A, U, G or C, 【Transformation 8】 where: R １ is OCH ３ and R ２ is OH or H, or R １ is OH and R ２ is H, or R １ is H and R ２ is H or OCH ３ or R １ and R ２ are OCH ３ and B1 is A or N6-methyl-adenine (m6A) and B2 is A, U, G, or C; The promoter is TAATACGACTCACTATAX １ X ２ X ３ (SEQ ID NO: 16), A at position 17 is the -1 nucleotide and X at position 18 is １ is the +1 nucleotide, X １ is G and X ２ and X ３ are A, T, G, or C, respectively, then B １ is A and B ２ is G, X １ is A and X ２ and X ３ are A, T, G, or C, respectively, then B １ is A and B ２ is A, X １ is C and X ２ and X ３ are A, T, G, or C, respectively, then B １ is A and B ２ is C, and X １ is T and X ２ and X ３ are A, T, G, or C, respectively, then B １ is A and B ２ is U, 【Chemistry 9】 R is H or CH ３ and B １ is A or N6-methyl-adenine (m6A), and B ２ is A, U, G or C, the cap analog is attached to the −1 and +1 nucleotides of the promoter; said incubating comprising incubating said reaction mixture at about 15°C to about 35°C for about 1 hour to about 12 hours to produce said RNA.

43. 43. The method of claim 42, wherein the promoter comprises a sequence selected from SEQ ID NOs: 10, 11, 12, 13 and 14.

44. 44. The method of claim 42 or 43, wherein the 5'UTR and the 3'UTR are SEQ ID NOs: 1 and 2, 1 and 4, 1 and 6, 3 and 2, 3 and 4, 3 and 6, 3 and 8, 5 and 2, 5 and 4, 5 and 6, 7 and 2, 7 and 4, 7 and 6, 7 and 8, 9 and 2, 9 and 4, 9 and 6, or 9 and 8, respectively.

45. 45. The method of claim 44, wherein the 5'UTR and the 3'UTR are SEQ ID NOs: 1 and 2, 1 and 4, 3 and 2, 1 and 6, 7 and 4, 9 and 2, or 3 and 6, respectively.

46. The poly A region may be from about 60 to about 200 A, from about 60 to about 190 A, from about 60 to about 180 A, from about 60 to about 170 A, from about 60 to about 160 A, from about 60 to about 150 A, from about 60 to about 140 A, from about 60 to about 130 A, from about 60 to about 120 A, from about 60 to about 110 A, from about 60 to about 100 A, from about 70 to about 190 A, from about 80 46. ​​The method of any one of claims 42-45, comprising from about 180 A, from about 90 to about 170 A, from about 100 to about 160 A, from about 100 to about 150 A, from about 100 to about 140 A, from about 100 to about 130 A, from about 100 to about 120 A, about 100 A, about 110 A, about 120 A, about 130 A, about 140 A, or about 150 A.

47. The cap analog is m ７ GpppA ２’Ｏｍｅ pA ２’Ｏｍｅ 、 m ７ GpppA ２’Ｏｍｅ pU ２’Ｏｍｅ 、 m ７ Gp ２’Ｏｍｅ pG ２’Ｏｍｅ 、 m ７ GpppA ２’Ｏｍｅ pC ２’Ｏｍｅ 、 m ７ Gpppm ６ A ２’Ｏｍｅ pA ２’Ｏｍｅ 、 m ７ Gpppm ６ A ２’Ｏｍｅ pU ２’Ｏｍｅ 、 m ７ Gpppm ６ A ２’Ｏｍｅ pG ２’Ｏｍｅ 、 m ７ Gpppm ６ A ２’Ｏｍｅ pC ２’Ｏｍｅ 、 m ７ G ３’Ｏｍｅ AA0A ２’Ｏｍｅ AA ２’Ｏｍｅ 、 m ７ G ３’Ｏｍｅ AA0A ２’Ｏｍｅ UU ２’Ｏｍｅ 、 m ７ G ３’Ｏｍｅ pppA ２’Ｏｍｅ pG ２’Ｏｍｅ 、 m ７ G ３’Ｏｍｅ pppA ２’Ｏｍｅ pC ２’Ｏｍｅ 、 m ７ G ３’Ｏｍｅ pppm ６ A ２’Ｏｍｅ pA ２’Ｏｍｅ 、 m ７ G ３’Ｏｍｅ pppm ６ A ２’Ｏｍｅ pU ２’Ｏｍｅ 、 m ７ G ３’Ｏｍｅ pppm ６ A ２’Ｏｍｅ pG ２’Ｏｍｅ 、 m ７ G ３’Ｏｍｅ pppm ６ A ２’Ｏｍｅ pC ２’Ｏｍｅ 、 m ７ G ２’Ｏｍｅ AA0A ２’Ｏｍｅ AA ２’Ｏｍｅ 、 m ７ G ２’Ｏｍｅ AA0A ２’Ｏｍｅ UU ２’Ｏｍｅ 、 m ７ G ２’Ｏｍｅ pppA ２’Ｏｍｅ pG ２’Ｏｍｅ 、 m ７ G ２’Ｏｍｅ pppA ２’Ｏｍｅ pC ２’Ｏｍｅ 、 m ７ G ２’Ｏｍｅ pppm ６ A ２’Ｏｍｅ pA ２’Ｏｍｅ 、 m ７ G ２’Ｏｍｅ pppm ６ A ２’Ｏｍｅ pU ２’Ｏｍｅ 、 m ７ G ２’Ｏｍｅ pppm ６ A ２’Ｏｍｅ pG ２’Ｏｍｅ 、 m ７ G ２’Ｏｍｅ pppm ６ A ２’Ｏｍｅ pC ２’Ｏｍｅ 、 m ７ G ３’Ｈ AA0A ２’Ｏｍｅ AA ２’Ｏｍｅ 、 m ７ G ３’Ｈ AA0A ２’Ｏｍｅ UU ２’Ｏｍｅ 、 m ７ G ３’Ｈ pppA ２’Ｏｍｅ pG ２’Ｏｍｅ 、 m ７ G ３’Ｈ pppA ２’Ｏｍｅ pC ２’Ｏｍｅ 、 m ７ G ３’Ｈ pppm ６ A ２’Ｏｍｅ pA ２’Ｏｍｅ 、 m ７ G ３’Ｈ pppm ６ A ２’Ｏｍｅ pU ２’Ｏｍｅ 、 m ７ G ３’Ｈ pppm ６ A ２’Ｏｍｅ pG ２’Ｏｍｅ 、 m ７ G ３’Ｈ pppm ６ A ２’Ｏｍｅ pC ２’Ｏｍｅ 、 m ７ Gpp Ｓ p｡ ２’Ｏｍｅ p｡ ２’Ｏｍｅ 、 m ７ GA0 Ｓ AA ２’Ｏｍｅ UU ２’Ｏｍｅ 、 m ７ Gpp Ｓ pA ２’Ｏｍｅ pG ２’Ｏｍｅ 、 m ７ Gpp Ｓ pA ２’Ｏｍｅ pC ２’Ｏｍｅ 、 m ７ Gpp Ｓ pm ６ A ２’Ｏｍｅ pA ２’Ｏｍｅ 、 m ７ Gpp Ｓ pm ６ A ２’Ｏｍｅ pU ２’Ｏｍｅ 、 m ７ Gpp Ｓ pm ６ A ２’Ｏｍｅ pG ２’Ｏｍｅ 、 m ７ Gpp Ｓ pm ６ A ２’Ｏｍｅ pC ２’Ｏｍｅ 、 m ７ G ３’Ｏｍｅ pp Ｓ p｡ ２’Ｏｍｅ p｡ ２’Ｏｍｅ 、 m ７ G ３’Ｏｍｅ @@ Ｓ AA ２’Ｏｍｅ UU ２’Ｏｍｅ 、 m ７ G ３’Ｏｍｅ pp Ｓ pA ２’Ｏｍｅ pG ２’Ｏｍｅ 、 m ７ G ３’Ｏｍｅ pp Ｓ pA ２’Ｏｍｅ pC ２’Ｏｍｅ 、 m ７ G ３’Ｏｍｅ pp Ｓ pm ６ A ２’Ｏｍｅ pA ２’Ｏｍｅ 、 m ７ G ３’Ｏｍｅ pp Ｓ pm ６ A ２’Ｏｍｅ pU ２’Ｏｍｅ 、 m ７ G ３’Ｏｍｅ p.p. Ｓ pm ６ A ２’Ｏｍｅ p.g. ２’Ｏｍｅ , and m ７ G ３’Ｏｍｅ p.p. Ｓ pm ６ A ２’Ｏｍｅ PC ２’Ｏｍｅ The method of any one of claims 42 to 46, selected from the group consisting of:

48. The reaction mixture comprises: a buffering substance having a concentration of about 35 mM to about 45 mM; an RNase inhibitor at a concentration of about 0.01 U / μl to about 0.03 U / μl; NTP at a concentration of about 20 mM to about 40 mM; the cap analog at a concentration of about 6 mM to about 8 mM; one or more magnesium salts at a concentration of about 20 mM to about 30 mM; a polyamine at a concentration of about 1.5 mM to about 2.5 mM; the DNA template at a concentration of about 0.01 μg / μl to about 0.05 μg / μl; pyrophosphatase at a concentration of about 0.1 mU / μl to about 0.5 mU / μl; 48. The method of any one of claims 42 to 47, comprising an RNA polymerase at a concentration of about 1 U / μl to about 2 U / μl.

49. 49. The method of claim 48, wherein the RNA polymerase is selected from wild-type T7 RNA polymerase or a mutant thereof.

50. 43. The method of claim 42, wherein said incubating comprises incubating said reaction mixture at about 18°C ​​to about 31°C.

51. 51. The method of claim 50, wherein said incubating comprises incubating said reaction mixture at about 31°C for about 3 hours.

52. 52. The method of any one of claims 19 to 26 and 42 to 51, wherein the DNA template further comprises at least one transcription terminator located upstream and / or downstream of the open reading frame (ORF).

53. A reaction mixture for in vitro transcription of a DNA template into RNA, the reaction mixture comprising the DNA template and a cap analog, wherein the cap analog comprises a structure having formula (I), (II), or (III): 【Chemistry 10】 where R is H or CH ３ and B １ is A or N6-methyl-adenine (m6A), and B ２ is A, U, G or C, 【Chemistry 11】 where: R １ is OCH ３ and R ２ is OH or H, or R １ is OH and R ２ is H, or R １ is H and R ２ is H or OCH ３ or R １ and R ２ are OCH ３ and B1 is A or N6-methyl-adenine (m6A) and B2 is A, U, G, or C; 【Chemistry 12】 where R is H or CH ３ and B １ is A or N6-methyl-adenine (m6A), and B ２ is A, U, G or C, wherein the DNA template comprises a promoter, and the cap analog is attached to at least the −1 and +1 nucleotides of the promoter, or to at least the +1 and +2 nucleotides of the promoter.

54. 1. A method for synthesizing a cap analog, the method comprising: (a) 5'-DMT-2'-O-methylguanosine (n-ibu) was reacted with acetic anhydride / pyridine, and the resulting product was then reacted with aqueous acetic acid to obtain compound (3): 【Chemistry 13】 and (b) reacting the compound (3) with 5'-dimethoxytrityl-N-benzoyl-adenosine, 2'-O-methyl, 3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite and tetrazole, followed by a first oxidizing agent, and then an aqueous acetic acid solution to obtain the compound (4): 【Chemistry 14】 and (c) reacting compound (4) with a chemical phosphorylation phosphoramidite reagent and tetrazole, followed by a second oxidizing agent, and then reacting with ammonium hydroxide to give compound (5): 【Chemistry 15】 and (d) reacting the compound (5) with the compound (2a) or (2b) in the presence of a divalent metal salt: 【Chemistry 16】 to produce the cap analog (1a) or (1b): 【Chemistry 17】 and obtaining a

55. 1. A method for synthesizing a cap analog, the method comprising: (a) Compound 3: [Chemistry 18] is reacted with 5'-DMT-2'-O-methyl-N6-methyl-adenosine 3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite and tetrazole, followed by a first oxidizing agent and then an aqueous acetic acid solution to give compound (6): 【Chemistry 19】 and (b) reacting compound (6) with a chemical phosphorylation phosphoramidite reagent and tetrazole, followed by a second oxidizing agent, and then reacting with ammonium hydroxide to give compound (7): 【Chemistry 20】 and (c) reacting the compound (7) with the compound (2b): 【Chemistry 21】 to give the cap analog compound (8): 【Chemistry 22】 and obtaining a

56. 56. The method of claim 54 or 55, wherein the first oxidizing agent and the second oxidizing agent are (1S)-(+)-(camphorsulfonyl)oxaziridine (CSO) or iodine, respectively.

57. The divalent metal salt is MnCl ２ or ZnCl ２ The method according to any one of claims 54 to 56, wherein

58. A cap analog, said cap analog comprising formula (I): 【Chemistry 23】 where R is H or CH ３ and B １ is A or N6-methyl-adenine (m ６ A) and B ２ is A, U, G or C, a cap analog.

59. R is H and B １ is A and B ２ is A, U, G or C.

60. The cap analog is m ７ GpppA ２’Ｏｍｅ pA ２’Ｏｍｅ 、 m ７ GpppA ２’Ｏｍｅ pU ２’Ｏｍｅ 、 m ７ GpppA ２’Ｏｍｅ p.g. ２’Ｏｍｅ , and m ７ GpppA ２’Ｏｍｅ PC ２’Ｏｍｅ 60. The cap analog of claim 59, selected from the group consisting of:

61. R is H and B １ is m ６ A and B ２ is A, U, G or C.

62. The cap analog is m ７ Gpppm ６ A ２’Ｏｍｅ pA ２’Ｏｍｅ 、 m ７ Gpppm ６ A ２’Ｏｍｅ pU ２’Ｏｍｅ 、 m ７ Gppm ６ A ２’Ｏｍｅ p.g. ２’Ｏｍｅ , and m ７ Gppm ６ A ２’Ｏｍｅ PC ２’Ｏｍｅ 62. The cap analog of claim 61, selected from the group consisting of:

63. R is CH ３ and B １ is A and B ２ is A, U, G or C.

64. The cap analog is m ７ G ３’Ｏｍｅ AA0A ２’Ｏｍｅ AA ２’Ｏｍｅ 、 m ７ G ３’Ｏｍｅ AA0A ２’Ｏｍｅ UU ２’Ｏｍｅ 、 m ７ G ３’Ｏｍｅ pppA ２’Ｏｍｅ p.g. ２’Ｏｍｅ , and m ７ G ３’Ｏｍｅ pppA ２’Ｏｍｅ PC ２’Ｏｍｅ 64. The cap analog of claim 63, selected from the group consisting of:

65. R is CH ３ and B １ is m ６ A and B ２ is A, U, G or C.

66. The cap analog is m ７ G ３’Ｏｍｅ pppm ６ A ２’Ｏｍｅ pA ２’Ｏｍｅ 、 m ７ G ３’Ｏｍｅ pppm ６ A ２’Ｏｍｅ pU ２’Ｏｍｅ 、 m ７ G ３’Ｏｍｅ ppm ６ A ２’Ｏｍｅ p.g. ２’Ｏｍｅ , and m ７ G ３’Ｏｍｅ ppm ６ A ２’Ｏｍｅ PC ２’Ｏｍｅ 66. The cap analog of claim 65, selected from the group consisting of:

67. A cap analog, said cap analog comprising formula (II): 【Chemistry 24】 where: R １ is OCH ３ and R ２ is OH or H, or R １ is OH and R ２ is H, or R １ is H and R ２ is H or OCH ３ or R １ and R ２ are OCH ３ and B1 is A or N6-methyl-adenine (m ６ A) and B2 is A, U, G, or C.

68. R １ is OCH ３ and R ２ is OH, and B １ is A and B ２ is A, U, G or C.

69. The cap analog is m ７ G ２’Ｏｍｅ AA0A ２’Ｏｍｅ AA ２’Ｏｍｅ 、 m ７ G ２’Ｏｍｅ AA0A ２’Ｏｍｅ UU ２’Ｏｍｅ 、 m ７ G ２’Ｏｍｅ pppA ２’Ｏｍｅ p.g. ２’Ｏｍｅ , and m ７ G ２’Ｏｍｅ pppA ２’Ｏｍｅ PC ２’Ｏｍｅ 69. The cap analog of claim 68, selected from the group consisting of:

70. R １ is OCH ３ and R ２ is OH, and B １ is m ６ A and B ２ is A, U, G or C.

71. The cap analog is m ７ G ２’Ｏｍｅ pppm ６ A ２’Ｏｍｅ pA ２’Ｏｍｅ 、 m ７ G ２’Ｏｍｅ pppm ６ A ２’Ｏｍｅ pU ２’Ｏｍｅ 、 m ７ G ２’Ｏｍｅ ppm ６ A ２’Ｏｍｅ p.g. ２’Ｏｍｅ , and m ７ G ２’Ｏｍｅ ppm ６ A ２’Ｏｍｅ PC ２’Ｏｍｅ 71. The cap analog of claim 70, selected from the group consisting of:

72. R １ is OH and R ２ is H and B １ is A and B ２ is A, U, G or C.

73. The cap analog is m ７ G ３’Ｈ AA0A ２’Ｏｍｅ AA ２’Ｏｍｅ 、 m ７ G ３’Ｈ AA0A ２’Ｏｍｅ UU ２’Ｏｍｅ 、 m ７ G ３’Ｈ pppA ２’Ｏｍｅ p.g. ２’Ｏｍｅ , and m ７ G ３’Ｈ pppA ２’Ｏｍｅ PC ２’Ｏｍｅ 73. The cap analog of claim 72, selected from the group consisting of:

74. R １ is OH and R ２ is H and B １ is m ６ A and B ２ is A, U, G or C.

75. The cap analog is m ７ G ３’Ｈ pppm ６ A ２’Ｏｍｅ pA ２’Ｏｍｅ 、 m ７ G ３’Ｈ pppm ６ A ２’Ｏｍｅ pU ２’Ｏｍｅ 、 m ７ G ３’Ｈ ppm ６ A ２’Ｏｍｅ p.g. ２’Ｏｍｅ , and m ７ G ３’Ｈ ppm ６ A ２’Ｏｍｅ PC ２’Ｏｍｅ 75. The cap analog of claim 74, selected from the group consisting of:

76. A cap analog, said cap analog comprising formula (III): 【Chemistry 25】 where R is H or CH ３ and B １ is A or N6-methyl-adenine (m ６ A) and B ２ is A, U, G or C, a cap analog.

77. R is H and B １ is A and B ２ is A, U, G or C.

78. The cap analog is m ７ Gpp Ｓ p｡ ２’Ｏｍｅ p｡ ２’Ｏｍｅ 、 m ７ GA0 Ｓ AA ２’Ｏｍｅ UU ２’Ｏｍｅ 、 m ７ Gpp Ｓ pA ２’Ｏｍｅ p.g. ２’Ｏｍｅ , and m ７ Gpp Ｓ pA ２’Ｏｍｅ PC ２’Ｏｍｅ 78. The cap analog of claim 77, selected from the group consisting of:

79. R is H and B １ is m ６ A and B ２ is A, U, G or C.

80. The cap analog is m ７ Gpp Ｓ pm ６ A ２’Ｏｍｅ pA ２’Ｏｍｅ 、 m ７ Gpp Ｓ pm ６ A ２’Ｏｍｅ pU ２’Ｏｍｅ 、 m ７ Gpp Ｓ pm ６ A ２’Ｏｍｅ p.g. ２’Ｏｍｅ , and m ７ Gpp Ｓ pm ６ A ２’Ｏｍｅ PC ２’Ｏｍｅ 79. The cap analog of claim 78, selected from the group consisting of:

81. R is CH ３ and B １ is A and B ２ is A, U, G or C.

82. The cap analog is m ７ G ３’Ｏｍｅ pp Ｓ p｡ ２’Ｏｍｅ p｡ ２’Ｏｍｅ 、 m ７ G ３’Ｏｍｅ @@ Ｓ AA ２’Ｏｍｅ UU ２’Ｏｍｅ 、 m ７ G ３’Ｏｍｅ p.p. Ｓ pA ２’Ｏｍｅ p.g. ２’Ｏｍｅ , and m ７ G ３’Ｏｍｅ p.p. Ｓ pA ２’Ｏｍｅ PC ２’Ｏｍｅ 82. The cap analog of claim 81, selected from the group consisting of:

83. R is CH ３ and B １ is m ６ A and B ２ is A, U, G or C.

84. The cap analog is m ７ G ３’Ｏｍｅ pp Ｓ pm ６ A ２’Ｏｍｅ pA ２’Ｏｍｅ 、 m ７ G ３’Ｏｍｅ pp Ｓ pm ６ A ２’Ｏｍｅ pU ２’Ｏｍｅ 、 m ７ G ３’Ｏｍｅ p.p. Ｓ pm ６ A ２’Ｏｍｅ p.g. ２’Ｏｍｅ , and m ７ G ３’Ｏｍｅ p.p. Ｓ pm ６ A ２’Ｏｍｅ PC ２’Ｏｍｅ 84. The cap analog of claim 83, selected from the group consisting of:

85. 1. A method for synthesizing a cap analog, the method comprising: (a) Compound (12) 【Chemistry 26】 is reacted with dimethyl sulfate to give compound (13). 【Chemistry 27】 and (b) reacting the compound (13) with imidazole, triphenylphosphine, and 2,2'-dithiopyridine, followed by addition of MnCl ２ and bis-triethylammonium thiophosphate to give compound (14). 【Chemistry 28】 and (c) Compound (5) 【Chemistry 29】 is reacted with triphenylphosphine, imidazole and 2,2'-dithiopyridine to give compound (15). 【Transformation 30】 and (d) Compound (15) and Compound (14) 【Chemistry 31】 in the presence of dimethylformamide and ZnCl to give compound (16). 【Chemistry 32】 and obtaining a

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