Compositions and methods for synthesizing 5'-capped rnas

Abstract

To provide an initiating capped oligonucleotide primer, an RNA molecule comprising the primer, a pharmaceutical composition comprising an RNA molecule, and a method for synthesizing an RNA molecule.SOLUTION: An initiating capped oligonucleotide primer is, for example, m7GpppApA, m7GpppAp (N6- methyladenine), m7GpppA2'OMepA or the like. A method for synthesizing RNA molecules includes the steps of: (a) introducing the initiating capped oligonucleotide primer represented by the formula m7GpppA2'OMepG into a mixture comprising an RNA polymerase under conditions conducive to transcription by the RNA polymerase of the polynucleotide template, where A and G in the formula are complementary to bases on the polynucleotide template at positions +1 and +2; and (b) incubating the mixture for a time sufficient to allow for the transcription.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation of U.S. Provisional Patent Application No. 62 / 221,2 filed on September 21, 2015. No. 48, the contents of which are incorporated herein by reference in their entirety. Sequence Listing This application contains a Sequence Listing, which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII code created on November 16, 2016 is incorporated herein by reference. The file is named 095109-000500PC-1022543_SL.txt , which is 785 bytes in size.

[0002] The present invention relates to methods and compositions for synthesizing 5'-capped RNA. In an embodiment, the present invention provides a method for the preparation of natural or modified 5'-cap 0, cap 1, cap A novel capping agent with trimethylguanosine-cap (TMG-cap) structure In a further aspect, the present invention relates to a 5-stranded initiation oligonucleotide primer. The present invention relates to a method for efficiently producing 5'-capped RNA and using the same. Methods for preparing the labeled RNA are provided. [Background technology]

[0003] The following explanation is provided to aid the reader's understanding. None of the references are admitted to be prior art in this invention.

[0004] Messenger RNAs that code for proteins of physiological importance for therapeutic applications ( mRNA) is a DNA-based plasmid and viral vector for delivering genetic material. The most important of these advantages are: be: (i) High level of safety (no chance of genome damage from viral or plasmid integration) (reduce (ii) mRNA delivery results in immediate protein expression (when using plasmids) (different from the delayed response that typically occurs in (iii) mRNA allows for robust, dose-dependent control over protein expression , and (iv) Large-scale synthesis of mRNA compared to plasmid and viral vector production. Simplicity.

[0005] The messenger RNA can code for virtually any known protein, Delivery to specific tissues and organs can be achieved by a variety of methods known to those skilled in the art. Once delivered, these mRNAs are used to express ribosomal proteins in targeted tissues. It directs the expression of various proteins, resulting in the production of hundreds of proteins per mRNA molecule.

[0006] present in each active mRNA molecule for efficient translation of the encoded protein Several structural elements are utilized to facilitate the synthesis of mRNA. A cap structure on the 5' end of A, which is found in all eukaryotes (and some The naturally occurring cap structure is a methyl group at the N7 position of the guanine base. This 7-methylguanosine ( 7m G) is mR The NA molecule is linked via a 5'-to-5'-triphosphate chain at the 5' end. , 7m and m7 are used synonymously and have the same meaning. 7m Gpp The presence of the p fragment is essential for mRNA maturation. Protects mRNA from exonucleolytic degradation Promotes transport of mRNA from the nucleus to the cytoplasm, It plays an important role in the assembly of the translation initiation complex (Cell 9:645-6 53, (1976); Nature vol. 266:235, (1977); Fede ration of Experimental Biologists Societ y Letters 96:1-11 (1978); Cell 40:223-2 4, (1985); Prog. Nuc. Acid Res. 35:173-20 7, (1988);Ann.Rev.Biochem.Volume 68:913~963 , (1999); J. Biol. Chem. Vol. 274: 30337-3040 (1 999)).

[0007] In cap-dependent translation, only mRNAs that retain the cap structure are active; "Decap" of mRNA terminates almost completely its template activity for protein synthesis. (Nature, vol. 255:33-37, (1975); J. Bi ol. Chem., vol. 253: 5228-5231, (1978); and Proc. c. Natl. Acad. Sci. USA, vol. 72: 1189-1193, (197 5 years)).

[0008] Another element in eukaryotic mRNAs is the presence of several In the case of The presence of 2'-O-methyl groups in mRNA is a key factor in the in vivo translation of mRNA. This is necessary for higher efficacy (Proc. Natl. Acad. Sci. USA, 77:3952-3956 (1980)), nuclease of 5' capped mRNA mRNA with cap 1 (and cap 2) further improves enzyme stability. This allows the cytoplasmic repressor to recognize the authentic 5' end of the mRNA and, in some cases, to recognize the infectious gene. Nucleic Acid Receptor arch 43:482-492 (2015).

[0009] Primary mRNA transcripts are RNA sequences that are initiated in vivo with NTPs (usually GTP). It retains the 5'-triphosphate group (5'-pppmRNA) that results from the initiation of mRNA transcription. The conversion of the 5'-triphosphorylated end of a transcript to a cap structure (Cap 0) is achieved by several enzymes. (J. Biol. Chem. 250:9322 (1975) 271:11936 (1996);J.Bio Chem. 267:16430 (1992). These enzymatic reaction steps The following groups are available: Step 1: RNA triphosphate enzyme converts the 5'-triphosphate of mRNA to 5'-diphosphate Convert to pppN1(pN) x →ppN1(pN) x + inorganic phosphate, Step 2: RNA guanyltransferase uses GTP to attach a GMP residue Transfer to the 5'-diphosphate of mRNA, ppN1 (pN) x +GTP→G(5')ppp(5 ')N1(pN) x + inorganic pyrophosphate, and Step 3: Guanine-7-methyltransferase reacts with S-adenosine as a cofactor. AdoMet is used to convert AdoMet to the 7-nitrogen of the guanine base. Transferring a methyl group, G(5')ppp(5')N1(pN) x +AdoMet→ 7m G(5 ')ppp(5')N1(pN) x +AdoHyc). The RNA resulting from these enzymatic activities is called "5'-capped RNA" or "capped RNA". The enzymes involved in this process, which result in the formation of "capped RNA," are called "capped RNAs." The combination of enzymes is called a "capping enzyme." Capping enzymes, including cleaved forms, have been identified and purified from a number of sources. and are well known in the art (Prog. Nucleic Acid Res. ol.Biol.Vol.66:1~40, (2001);Prog.Nucleic Acid Res. Mol. Biol. 50:101-129, (1995); and Microbiol. Rev. 44:175, (1980). Capsids are formed by the addition of cap nucleotides to the 5' end of primary RNA by a capping enzyme. The capped RNA is called a capped RNA with a "Cap 0 structure" (JB iol. Chem. 269:14974-14981, (1994); J. Biol. Vol. Chem. 271: 11936-11944 (1996). Cap type The enzyme is used to synthesize capped RNA with a cap 0 structure in vitro. (J. Biol. Chem. vol. 255: 11588, (1980 ); Proc. Natl. Acad. Sci. USA Vol. 94: 9573, (1997 267:16430, (1992);J.Bi ol. Chem. 269:14974, (1994); and J. Biol. hem. vol. 271:11936, (1996)).

[0010] Capped RNA with a 5'-Cap 0 structure is a (nucleoside-2'-O-) methyl This is further converted in vivo to the “cap 1” structure by the action of methyltransferase. (J. Biol. Chem. vol. 269: 14974-14981, (19 1994); J. Biol. Chem. Vol. 271: pp. 11936-11944, (199 6; and EMBO 21:2757-2768, (2002). For example, Vaccinia mRNA (nucleoside-2'-O) methyltransferase is a nucleotide analogue of The reaction results in the penultimate nucleotide of the 5' end of the 5' capped RNA with a Cap 0 structure. Can catalyze the methylation of the 2'-hydroxyl group of nucleotides: 7m G(5')ppp(5')N1pN2(pN) x +AdoMet→ 7m G(5') ppp(5')N1 2’-OMe pN2(pN) x +AdoHyc.

[0011] Dimethylated capped RNA with a cap 1 structure is more efficient than capped RNA with a cap 0 structure. It has been reported that RNA is translated more efficiently than RNA with a nucleic acid sequence similar to that of the nucleic acid sequence ... Res. 26:3208 (1998). Eukaryotic cells use another -2'-O) methyltransferase (e.g., hMTR2(Nuclease) in human cells) Acids Res. 39:4756 (2011)) The reaction results in the 2'-hydroxylation of the second transcribed nucleotide of the 5'-capped RNA. Catalyzes the methylation of the silyl group to convert the cap 1 structure to the cap 2 structure: 7m G(5')ppp(5')N1 2’-OMe pN2(pN) x +AdoMet→ 7 m G(5')ppp(5')N1 2’-OMe pN2 2’-OMe (pN) x +AdoH yc. Approximately 50% of eukaryotic mRNAs contain the Cap2 structure.

[0012] In the mid-1980s, in order to generate long functional RNA for various biological studies, In 2011, a method for the in vitro enzymatic synthesis of primary RNA was developed (Methods Enzymatic Synthesis). zymol. Vol. 180: pp. 51-62, (1989); Nucl. Acids Re s., vol. 10: 6353-6362, (1982); Meth. Enzymol. , Vol. 180: pp. 42-50 (1989); Nucl. Acids Res., No. 12 Volume: 7035-7056, (1984) and Nucleic Acid Research arch 15:8783-8798, (1987).

[0013] After in vitro transcription, the primary mRNA transcripts retaining the 5'-triphosphate group were capped However, in vitro enzymatic capping is not possible. 5'-capping is expensive, laborious, inefficient and difficult to control. It is difficult to do so.

[0014] Considering these disadvantages, chemically synthesized dinucleotides 7m G(5')ppp of capped mRNA, which initiates transcription using (5')G (also called mCAP) An alternative method for in vitro synthesis has been developed (RNA 1:957-967 , (1995)). The mCAP dinucleotide is the 5'-cap structure of mature mRNA. contains 2'-O-methyl nucleosides characteristic of the cap 1 and cap 2 structures. It does not have a

[0015] However, due to the initiation of in vitro transcription using synthetic mCAP dinucleotide There are two main disadvantages to using ppp-peptide synthesis. pppG is a strong competitor for pppG. When pppG is used to initiate mRNA, the resulting p-mRNA is translationally and immunogenically inactive due to the presence of 5'-triphosphate. Correspondingly, when mRNA is primed with mCAP, the resulting 5' cap The attached mRNA is translationally active but immunogenically inactive.

[0016] 5'-capped or 5'-uncapped (or 5'-triphosphorylated; pppm To improve the proportion of 5'-capped mRNA transcripts (up to 8 0-90%), the excess over pppG 7m GpppG(4 A downside to this approach is that transcription Rapid depletion of GTP supplies and the need for large amounts of synthetic mCAP dimers, which can be expensive This results in a significant reduction in the overall yield of mRNA. The crude mixture containing both A and 5'ppmRNA was purified with alkaline phosphatase. Further processing can be used to convert pppmRNA to cis-peptides to reduce the immunogenicity of the synthesized mRNA. This is necessary to remove the uncapped 5'-triphosphate group. The uncapped 5'-OH form of the mRNA is inactive and does not participate in the translation process. do not have.

[0017] Another disadvantage occurs when using asymmetric mCAP dinucleotides: bidirectional initiation. It is possible. 7m G in GpppG or 7m of any 3'-hydroxyl group of the G moiety, They tend to serve as initiation sites for transcription elongation with roughly equal probability. Depending on the type, approximately equal proportions 7m G(5')pppG(pN) n and G(5 ')ppp 7m G(pN) n This leads to the synthesis of two isomeric RNAs (RNA Vol. 1:9 pp. 57-967, (1995)).

[0018] To eliminate bidirectional initiation of mRNA synthesis when using mCAP dinucleotide , 7m A novel modified mCAP analogue in which the 3'-OH group of the G residue was replaced by OCH3 ("OM e"): 7m G(3'-O-Me)pppG (Anti-reverse cap analog) -Reverse Cap Analog (ARCA) was developed. ARCA initiates mRNA synthesis only in the correct forward orientation (RNA vol. 7: Several types of ARCA analogs are known in the art. (See, for example, U.S. Patent No. 7,074,596). To ensure that most mRNA transcripts have a 5'-cap structure, ppp A large molar excess of ARCA over G is still required. 1 structure of mRNA is 7m GpppG 2’-OMe Cap dimer (RNA vol. 1 957, (1995)) or its ARCA analogues. That is the thing.

[0019] Currently, the only known pathway for the generation of active long mRNAs containing the Cap1 structure is the enzyme Enzymatic 2'O-methylation of elementary capping and 5'-triphosphorylated mRNA transcripts is the enzymatic 2'-O-methylation of mCAP- or ARCA-capped pre-mRNA. Nucleosides, Nucleotides, and Nucleic A cids, vol. 25: 337-340, (2006) and Nucleosides ,Nucleotides,and Nucleic Acids Vol. 25(No. 3):3 Both approaches are quite labor intensive. are difficult to control, and even with substantial optimization, both approaches However, it is not possible to guarantee a high yield of capped and methylated pre-mRNA (J. Gen. Virol., 91:112-121, (2010). Furthermore, the cap 2 structure The methods for preparing mRNA with the desired structure are much more difficult and the results are less predictable. The enzyme for converting cap 1 to cap 2 is not currently commercially available.

[0020] Another important complication of in vitro synthesis of mRNA, especially for large-scale production, is the inactivation All uncapped mRNA forms are virulent and, in some cases, immunogenic. Therefore, it is necessary to isolate and purify active mRNA molecules that retain the cap. Unfortunately, these methods are not trivial and require extensive purification of capped RNA transcripts. Modified IgG antibodies conjugated with an affinity tag moiety that allows for easier isolation and purification. Synthesis of mCAP analogs with affinity as a reporter / affinity moiety is often required. Methods for synthesizing tagged mCAP analogs and isolating capped RNA from transcription reaction mixtures Novel protocols for the isolation of A are known in the art (e.g., U.S. Pat. No. 8,333,636). (See, e.g., 344,118.) Although these approaches are effective, they This requires the use of many more expensive mCAP analogs, and mRNAs containing only the Cap0 structure are This allows for the preparation and isolation of A.

[0021] In vitro synthesis of natural and modified RNA has been used to create ribozymes, antisense and biological It is used in a variety of applications, including physical and biochemical research. RNA transcripts were used in in vivo expression experiments (microinjection, transfection, etc.) tion and infection), in vitro translation experiments and assays, and treatments Various applications in drug, diagnostic, and vaccine development, labeling and detection abo-thib protein synthesis It is used for applications requiring

[0022] As a result, (a) it is less labor intensive than traditional methods, and (b) there is no need for double transcription. (c) eliminate or reduce directional initiation; (d) at a reduced cost compared to current methods; (e) generation of heterogeneous products with different 5' sequences, resulting in higher yields of RNA; (f) to incorporate cap 1 and cap 2 structures into the synthesized mRNA; Compositions and methods for large-scale synthesis of mRNA without the need for additional enzymatic reactions There is a need in the art for methods to provide specific modifications and modifications at or near the 5' end of the molecule. and / or affinity tags, such as fluorescent dyes, radioisotopes, mass tags, and / or biotin Modified and / or non-natural nucleic acids that retain one partner of a molecular binding pair, such as a nucleic acid The synthesis of various mRNAs containing nucleotides is also required. Summary of the Invention

[0023] Provided herein are methods and compositions for synthesizing 5' capped RNA. In one aspect of the invention, the initial capped oligonucleotide primer has the formula I: [ka] [In the formula, B1 to B 10 each is independently a natural, modified or non-natural nucleoside base; M is 0 or 1; L is 0 or 1; q1 is 1, Each of q2 to q9 is independently 0 or 1; R1 is H or methyl; R2 and R3 are independently H, OH, alkyl, O-alkyl, halogen, amine, or a a linker or a detectable marker; X1 to X 13 each is independently O or S; Y1 to Y 13 each independently represents OH, SH, BH3, aryl, alkyl, O-aryl alkyl or O-aryl; Z1 to Z 22 each independently represents O, S, NH, CH2, C(halogen)2 or CH (halogen), R4 to R 12 each independently being H, OH, OMe, a linker or a detectable marker. [is] It includes the general structure shown below.

[0024] In another aspect of the invention, a cap-containing initial oligonucleotide primer of formula I is RNA molecules, pharmaceutical compositions comprising such RNA, cells containing such RNA and and cells containing proteins or peptides translated from such RNA. do.

[0025] In yet another aspect of the invention, transcription of a polynucleotide template by an RNA polymerase Under conditions that promote synthesis of an RNA fragment containing a cap-containing initiation oligonucleotide primer of formula I, The nucleic acid is synthesized in a mixture containing a polynucleotide template and an RNA polymerase, and then Incubating the resulting mixture for a time sufficient to allow transcription of said template. is provided. [Brief description of the drawings]

[0026] [Figure 1] FIG. 1 shows an exemplary method for preparing 7-methylguanosine 5-diphosphate (pp7 mG) from guanosine 5′-diphosphate. [Diagram 2] FIG. 1 shows an exemplary method for preparing 7-methylguanosine 5′-diphosphate imidazolide (Im-pp7mG) from pp7mG. [Diagram 3] FIG. 1 shows an exemplary method for preparing 3′-O-methylguanosine 5′-phosphate (pG3′Ome) from 3′-O-methylguanosine. [Figure 4] FIG. 1 shows an exemplary method for preparing 3′-O-methylguanosine 5′-phosphorylimidazolide (Im-pG3′Ome) from pG3′Ome. [Diagram 5] FIG. 1 shows an exemplary method for preparing 3′-O-methylguanosine 5′-diphosphate (ppG3′Ome) from Im-pG3′Ome. [Figure 6] FIG. 1 shows an exemplary method for preparing 7-methyl-3′-O-methylguanosine 5-diphosphate (pp7mG3′Ome) from ppG3′Ome. [Figure 7] FIG. 1 shows an exemplary method for preparing 7-methyl-3′-O-methylguanosine 5-diphosphate imidazolide (Im-pp7mG3′Ome) from pp7mG3′Ome. [Figure 8] FIG. 1 shows a general procedure for the preparation of pN2′-OR1pN oligonucleotides (R1=H or Me). [Figure 9] FIG. 1 shows a general procedure for the synthesis of an initial oligonucleotide having a cap0, cap1 or cap2 structure. [Figure 10A] FIG. 1 shows the structure of an initial capped oligonucleotide primer used in the examples according to formula I, where B1 is guanine, M is 0, L is 1, q1 to q9 are 0, R1 is H, R2 is H, R3 is O-methyl, X1 is O, X2 is O, X13 is O, Y13 is OH, Z0 is O, Z1 is O, Z2 is O, and Z22 is O. [Figure 10B]FIG. 1 shows the structure of an initial capped oligonucleotide primer used in the examples according to formula I, where B1 is guanine, B10 is guanine, M is 0, L is 1, q1 is 1, q2 to q9 are 0, R1 is H, R2 is H, R3 is H, X1 is O, X2 is O, X4 is O, X13 is O, Y1 is OH, Y2 is OH, Y4 is OH, Y13 is OH, Z0 is O, Z1 is O, Z2 is O, Z4 is O, Z5 is O, Z22 is O, and R4 is O-methyl. [Figure 10C] FIG. 1 shows the structure of an initial capped oligonucleotide primer used in the examples according to formula I, where B1 is guanine, B10 is guanine, M is 0, L is 1, q1 is 1, q2 to q9 are 0, R1 is H, R2 is H, R3 is O-methyl, X1 is O, X2 is O, X4 is O, X13 is O, Y1 is OH, Y2 is OH, Y4 is OH, Y13 is OH, Z0 is O, Z1 is O, Z2 is O, Z4 is O, Z5 is O, Z22 is O, and R4 is O-methyl. [Figure 10D] FIG. 1 shows the structure of an initial capped oligonucleotide primer used in the examples according to formula I, where B1 is adenine, B10 is guanine, M is 0, L is 1, q1 is 1, q2 to q9 are 0, R1 is H, R2 is H, R3 is H, X1 is O, X2 is O, X4 is O, X13 is O, Y1 is OH, Y2 is OH, Y4 is OH, Y13 is OH, Z0 is O, Z1 is O, Z2 is O, Z4 is O, Z5 is O, Z22 is O, and R4 is O-methyl. [Figure 10E]FIG. 1 shows the structure of an initial capped oligonucleotide primer used in the examples according to formula I, where B1 is adenine, B10 is guanine, M is 0, L is 1, q1 is 1, q2 to q9 are 0, R1 is H, R2 is H, R3 is O-methyl, X1 is O, X2 is O, X4 is O, X13 is O, Y1 is OH, Y2 is OH, Y4 is OH, Y13 is OH, Z0 is O, Z1 is O, Z2 is O, Z4 is O, Z5 is O, Z22 is O, and R4 is O-methyl. [Figure 10F] FIG. 1 shows the structure of an initial capped oligonucleotide primer used in the examples according to formula I, where B1 is cytosine, B10 is guanine, M is 0, L is 1, q1 is 1, q2 to q9 are 0, R1 is H, R2 is H, R3 is H, X1 is O, X2 is O, X4 is O, X13 is O, Y1 is OH, Y2 is OH, Y4 is OH, Y13 is OH, Z0 is O, Z1 is O, Z2 is O, Z4 is O, Z5 is O, Z22 is O, and R4 is O-methyl. [Figure 10G] FIG. 1 shows the structure of an initial capped oligonucleotide primer used in the examples according to formula I, where B1 is cytosine, B10 is guanine, M is 0, L is 1, q1 is 1, q2 to q9 are 0, R1 is H, R2 is H, R3 is O-methyl, X1 is O, X2 is O, X4 is O, X13 is O, Y1 is OH, Y2 is OH, Y4 is OH, Y13 is OH, Z0 is O, Z1 is O, Z2 is O, Z4 is O, Z5 is O, Z22 is O, and R4 is O-methyl. [Figure 10H]FIG. 1 shows the structure of an initial capped oligonucleotide primer used in the examples according to formula I, where B1 is adenine, B2 is guanine, B1 0 is guanine, M is 0, L is 1, q1 is 1, q2 is 1, q3 to q9 are 0, R1 is H, R2 is H, R3 is O-methyl, X1 is O, X2 is O, X4 is O, X5 is O, X13 is O, Y1 is OH, Y2 is OH, Y4 is OH, Y5 is OH, Y13 is OH, Z0 is O, Z1 is O, Z2 is O, Z4 is O, Z5 is O, Z6 is O, Z22 is O, R4 is O-methyl, and R5 is O-methyl. [Figure 11] FIG. 1 shows luciferase activity of capped mRNA by mRNA co-transcription. [Fig. 12A-12H] 11A) with ARCA, 12B) with m7GpppG2'OmepG, 12C) with m7G3'OmepppG2'OmepG, 12D) with m7GpppA2'OmepG, 12E) with m7G3'OmepppA2'-OMepG, 12F) with m7GpppC2'OmepG, 12G) with m7G3' OmepppC2'OmepG, and 12H) with m7G3'OmepppA2'OmepG2'OmepG. Capping efficiencies in Figures 11B-11G are comparable to or significantly exceed those observed in Figure 11A. [Figure 13A-13D]13A illustrates the capping efficiency and fidelity of mRNA initiation capped by co-transcription with m7GpppA2'OmepG on the transcription template; FIG. 13A illustrates the use of 2'-deoxythymidine and 2'-deoxycytidine residues at template positions +1 and +2 using primer / NTP formulation 2; FIG. 13B illustrates the use of 2'-deoxythymidine and 2'-deoxycytidine residues at template positions +1 and +2 using primer / NTP formulation 3; FIG. 13C illustrates the use of 2'-deoxycytidine residues at template positions +1 and +2 using primer / NTP formulation 2; and FIG. 13D illustrates the use of 2'-deoxycytidine residues at template positions +1 and +2 using primer / NTP formulation 3. [Figure 14A-14B] FIG. 11 summarizes a comparison of translation in differentiated THP-1 cells of mRNAs generated with the m7GpppA2'OmepG initiator capped oligonucleotide on transcription templates with 2'-deoxythymidine and 2'-deoxycytidine residues at template positions +1 and +2 versus transcription templates with cytidine residues at template positions +1 and +2. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] Unless otherwise specified, all terms used herein are intended to be within the scope of the present invention. It has the same meaning as commonly understood by a person skilled in the art. All patents, patent applications and publications mentioned are incorporated by reference in their entirety. In the event that there are a plurality of definitions for terms herein, those in this section prevail. do.

[0028] As used herein, in connection with a numerical value, the term "approximately" or "about" refers to the stated value. Includes all values ​​within the defined range. Means plus or minus 30% of the value shown. Taste.

[0029] As used herein, the term "nucleic acid," "nucleotide sequence," or "nucleic acid sequence" refers to an A oligonucleotide, polynucleotide or any fragment thereof, any ribo- or deoxyribonucleotide, Ribonucleic acid derivatives and natural and / or modified nucleotide residues and internucleotide linkages These terms also refer to single-stranded, naturally occurring or synthetic molecules that contain a bond. It may be double-stranded, triple-stranded or quadruple-stranded and may represent the sense or antisense strand. DNA or RNA, or any DNA-like molecule, of natural (e.g., genomic) or synthetic origin. or RNA-like material. In reference to DNA sequences, "RNA equivalents" refer to All or most occurrences of are replaced with uracil and the sugar backbone is 2'-deoxyribonucleotide. A sequence that is identical to the reference DNA sequence except that it consists of ribose instead of ribose. Suitable for the methods and compositions provided herein. Alternative nucleic acid backbones include, but are not limited to, phosphorothioates, phosphorose Rhenoate, alkyl phosphotriester, aryl phosphotriester, alkyl phosphat Phosphonates, aryl phosphonates, phosphoboronates, morpholino nucleic acids (MNA), Examples include LNA and peptide nucleic acid (PNA).

[0030] As used herein, the term "primer" or "oligonucleotide primer" means , ribo- or deoxyribo- or chimeric ribo / deoxyribo-oligonucleotides A single strand may be naturally occurring or synthetic and typically consists of about two or more strands. between about 10 nucleotides, about 3 to about 8 nucleotides, or about 3 to about 5 nucleotides The oligonucleotide primer may contain one or more modification groups. Oligonucleotide primers may be RNA, DNA and / or other modified nucleotides. Those skilled in the art will recognize that oligonucleotides suitable for transcription of a DNA template sequence may include oligonucleotides. Otide primers can be designed and prepared.

[0031] As used herein, the term "initial capped oligonucleotide analog" or "initial capped oligonucleotide analog" refers to a "Capped oligonucleotide primer" refers to a primer having a cap at the 5' end. Initiating oligonucleotide containing a cap1, cap2 or TMG-cap structure A capped primer is a primer that has an unmodified or open 3'-OH group and is The primer can be extended by RNA polymerase by incorporation of NTPs at its 3' end. Components required: DNA template (e.g., DNA plasmid), RNA polymerase, nucleic acid In a transcription system containing riboside 5'-triphosphate and a suitable buffer, In vitro transcription can be initiated under the control of The "initiation primer" or "initiation oligonucleotide primer" used in The oligonucleotides bearing a terminal 3'-OH group, which is a valid substrate for NA polymerase, were In certain embodiments, the initiating oligonucleotide primer is It is a substrate for the primer and can be extended by incorporation of NTPs at the 3' end of the primer. The oligonucleotide primer is complementary to the DNA template at the initiation site.

[0032] As used herein, the relationship between the initiating capped oligonucleotide primer and NTPs In the context, the term "unsubstituted" or "unmodified" refers to an initial capped oligonucleotide that is not modified. Refers to nucleotide primers and NTPs.

[0033] As used herein, the term "modified initial capped oligonucleotide primer" refers to an initial capped oligonucleotide primer containing one or more additional modification groups. He points to Reimer.

[0034] As used herein, the term "modifying group" refers to, but is not limited to, a sugar, a nucleoside, At positions containing bases, triphosphate bridges and / or internucleotide phosphates, the initiating primer It refers to any chemical moiety that can be attached (see, for example, U.S. Patent Application No. 20070281308). The modification group of the initiating capped oligonucleotide primer is compatible with the process of transcription. The group may be of any nature.

[0035] As used herein, the term "internucleotide linkage" refers to an oligonucleotide primer or the bond or bonds that join two nucleosides of a nucleic acid; It may be a diester linkage or a modified linkage.

[0036] As used herein, the term "label" or "detectable label" refers to a molecule that can detect the label. or It refers to a compound or combination of compounds that can otherwise be associated with a molecule. isotopes (e.g., carbon, phosphorus, iodine, indium, sulfur, tritium, etc.) ), mass isotopes (e.g., H 2 , C 13 or N 15 ), dyes or fluorophores (e.g. cyanine, fluorescein or coumarin), hapten (e.g. biotin) or can be any other agent that can be detected directly or indirectly.

[0037] As used herein, the terms "hybridize" or "specifically hybridize" " means that the initiating capped oligonucleotide primer is properly stripped during the transcription reaction. This refers to the process of annealing to a DNA template under sensitive conditions. In certain embodiments, the 3-10 nucleotide polynucleotide containing a 5'-5' inverted cap structure is The sequence is carried out using an initiating capped oligonucleotide primer that is the length of the nucleic acid fragment. Nucleic acid hybridization techniques are well known in the art (see, e.g., Sambro ok et al., Molecular Cloning: A Laboratory Manual l, Second Edition, Cold Spring Harbor Pres. s, Plainview, NY (1989); Ausubel, FM et al., Cur. rent Protocols in Molecular Biology, John Wiley & Sons, Secaucus, N.J. (1994)).

[0038] As used herein, the term "initial capped oligonucleotide primer and DNA template" refers to a In the context of a complex, the terms "complement," "complementary," or "complementarity" refer to the standard Watson method. / Click base pairing rules. For example, the sequence "5'-AGTC-3'" It is complementary to the sequence "3'-TCAG-5'". Certain non-natural or synthetic nucleosides The nucleotides may be included in the nucleic acids described herein and, as such, are including, but not limited to, inosine, 7-deazaguanosine, 2'-O-methylguanosine, 2' -Fluoro-2'-deoxycytidine, pseudouridine, locked nucleic acid (LNA) and Base and sugar modified nucleosides, nucleotides and peptide nucleic acids (PNAs) Complementarity does not have to be perfect; a duplex can contain mismatched nucleic acids. The oligonucleotides may contain base pairs, degenerates, or non-matching nucleotides. The length of the nucleotide, the base composition and sequence of the oligonucleotide, and the occurrence of mismatched base pairs Several conditions, including the rate, ionic strength, composition of the hybridization buffer and reaction conditions, are Duplex stability can be determined empirically, taking into account several variables.

[0039] Complementarity is when all of the nucleotide bases in two nucleic acid strands have recognized base-pairing groups. Even if the "complete" or "total" rule is followed, the start Only a portion of the nucleotide bases of the primer and the DNA target are may be "partial", corresponding according to recognized base-pairing rules, and are the nucleotide bases of two nucleic acid strands that pair according to recognized base-pairing rules. In some cases, there is no corresponding "non-existent" sequence. The degree of complementarity between the mer and the DNA template is determined by the initiating capped oligonucleotide and the DNA template. A: It has a significant effect on the hybridization strength between the two templates and, accordingly, on the efficiency of the reaction. The term complementarity can also be used in reference to individual nucleotides. For example, a particular nucleotide in an oligonucleotide may be an initiating capped oligonucleotide. A complementary sequence between the remainder of the primer and the DNA strand, in contrast to or in comparison with the complementary sequence between the remainder of the primer and the DNA strand. It states its complementarity, or lack thereof, to the nucleotides in the strand. It may also be possible.

[0040] In this specification, the terms "complete", "completely" or "perfectly" are used. The complement is each of the nucleotide bases of the initial capped oligonucleotide primer and This means that the DNA targets correspond exactly according to recognized base-pairing rules. .

[0041] As used herein, the term "substantially complementary" refers to stringent hybridization. Substantially complementary refers to two sequences that hybridize under hybridization conditions. It will be understood that the target sequences need not hybridize along their entire length. In particular, a substantially complementary sequence contains a contiguous sequence of bases that does not hybridize with the target sequence. In some cases, the target sequence may hybridize under stringent hybridization conditions. The nucleic acid sequence may be located 3' or 5' of the contiguous sequence of bases that encodes the nucleic acid.

[0042] As used herein, the term "specific" refers to an initiating capped oligonucleotide primer. Initiation primers, when used in reference to a mer sequence and its ability to hybridize with a DNA template, When the capped oligonucleotide primer and the DNA strand are aligned, the DNA A sequence that has at least 50% sequence identity with a portion of the template. As a higher level of sequence identity, at least 75%, at least 80%, at least At least 85%, at least 90%, at least 95%, at least 99% and most preferably indicates 100% sequence identity.

[0043] As used herein, the term "nucleoside" refers to any nucleoside base found in nature and Includes all naturally occurring nucleosides, including all furanoside forms. The most common base rings found in existing nucleosides are the purine and pyrimidine rings. Naturally occurring purine rings include, for example, adenine, guanine and N 6 -Methyladenosine Naturally occurring pyrimidine rings include, for example, cytosine, thymine, 5-amino-2-phenylindole, -methylcytosine, pseudouracil, etc. Naturally occurring nucleosides For example, but not limited to, adenosine, guanosine, cytidine, thymidine, ribo, 2'-O-methyl of lysine, inosine, 7-methylguanosine or pseudouridine or 2'-deoxyribo derivatives.

[0044] As used herein, the terms "nucleoside analog," "modified nucleoside," or "nucleoside analog" are used interchangeably. "Nucleoside derivatives" include synthetic nucleosides as described herein. The nucleoside derivatives may also contain modified bases or / and bases with or without protecting groups. and nucleosides having a sugar moiety, e.g., 2'-deoxy-2'-fluoro- lysine, 5-fluorouridine, and the like. Methods and methods include the synthesis of such base rings and their synthetic analogs, as well as non-natural heterocyclic substituted base rings. Other nucleosides that can be used with the present invention include cyclic substituted sugars and acyclic substituted bases. Examples of nucleoside derivatives include LNA nucleosides, halogen-substituted purines (e.g., 6-fluoro oropurine), halogen-substituted pyrimidine, N6 -Ethyladenine, N 4 -(alkyl)- Examples include cytosine and 5-ethylcytosine (U.S. Pat. No. 6,762,298).

[0045] As used herein, the terms "universal base," "degenerate base," and "universal bases" are used interchangeably. As used herein, "degenerate base analogs" and "degenerate base analogs" include, for example, in certain embodiments, natural NTPs (e.g. For example, ATP, UTP, CTP, and GTP) or one of the other specific NTPs It has an artificial base that can be recognized by RNA polymerase as a substitute for Nucleoside analogues are also included. Universal or degenerate bases are described in Loakes, D., Nucleic Acids Res., Volume 29: 2437-2447 (20 01); Crey-Desbiolles, C. et al., Nucleic Acids R es., vol. 33: 1532-1543 (2005); Kincaid, K. et al., N Ucleic Acids Res., 33:2620-2628 (2005) ;Preparata,FP, Oliver,JS, J.Comput.Biol.75 3-765 (2004); and Hill, F. et al., Proc Natl Acad Sci. USA, vol. 95: 4258-4263 (1998) .

[0046] As used herein, the term "modified NTP" refers to any modified NTP that includes a sugar, a base, and a triphosphate chain. or any combination of these three positions. Examples of such NTPs are, for example, "Nucleoside 5'-triphosphates" ide Triphosphates and Their Analogs:Chem istry, Biotechnology and Biological Appl ications”, Vaghefi, M. et al., Taylor and Francis , Boca Raton (2005).

[0047] As used herein, the term "modified oligonucleotide" refers to, for example, modified nucleosides, Containing modified internucleotide linkages or modified nucleosides and internucleotide linkages The oligonucleotides include oligonucleotides having any combination of the following: Examples of linkage modifications include phosphorothioates, phosphotriesters, and methylphosphonates. (Stec, WJ et al., Chem. Int. Ed. Engl. 33:709-722 (1994); Lebedev, A. V. et al., E., P erspect. Drug Discov. Des., vol. 4: 17-40 (1996 and Zon et al., U.S. Patent Application No. 20070281308). Further examples of linker modifications are given in Waldner et al., Bioorg. Med. Chem. Lett. ers 6:2363-2366 (1996).

[0048] The term "promoter" is used herein to refer to a specific DNA sequence (e.g., a gene). A promoter is a region of a dsDNA template that directs and controls the initiation of transcription of a gene. Located on the same strand and upstream of the DNA (towards the 5' region of the sense strand). It is usually located immediately adjacent to (or partially overlapping) the DNA sequence to be transcribed. The nucleotide position in the target is relative to the transcription start site (position +1) where transcription of DNA begins. The initiation oligonucleotide primer is aligned to the start site of the promoter sequence (specific In this embodiment, the tetramer is located at positions +1 and +2, and in the case of the starting tetramer, the tetramer is located at positions +1, +2 and and +3).

[0049] As used herein, the term "transcription" or "transcription reaction" refers to a reaction that is complementary to a DNA template. To enzymatically produce RNA, thereby generating several RNA copies of a DNA sequence The term "transcriptional reaction" refers to a method known in the art for synthesizing RNA molecules. The term "RNA transcript," "primary transcript," or "transcript" is used herein. The transcription reaction involving the compositions and methods described herein is carried out using an "initiation capped oligonucleotide primer." Transcription of the DNA template can be exponential, non-linear or linear. The DNA template can be double-stranded linear DNA, partially double-stranded linear DNA, or circular double-stranded DNA. , DNA plasmids, PCR amplicons, modified nucleic acid templates compatible with RNA polymerase It could be.

[0050] As used herein, the term "acyl" refers to the group -C(O)R a (In the formula, R a Hydrogen, low alkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, etc.) Shows.

[0051] As used herein, the term "substituted acyl" refers to a group -C(O)R a’ (In the formula, R a’ teeth, Substituted lower alkyl, substituted cycloalkyl, substituted heterocyclyl, substituted aryl, substituted heterephthalic acid (e.g., aryl).

[0052] As used herein, the term "acyloxy" refers to the group -OC(O)R b (In the formula, R b teeth, Hydrogen, lower alkyl, substituted lower alkyl, cycloalkyl, substituted cycloalkyl, hetero Cyclyl, substituted heterocyclyl, aryl, substituted aryl, heteroaryl, substituted hetero aryl, etc.).

[0053] As used herein, the term "alkyl" refers, in some embodiments, to an alkyl group having 1 to 20 carbon atoms. In some embodiments, the hydrocarbon single bonds range from 1 to 8 carbon atoms. Examples of this include methyl, ethyl, propyl, isopropyl, n-butyl, and sec -Butyl, isobutyl, tert-butyl, pentyl, isoamyl, hexyl, octyl , dodecanyl, and the like.

[0054] As used herein, the term "lower alkyl" refers in some embodiments to a lower alkyl group having 1 to 6 aryl groups. Hydrocarbon straight chains in the range of carbon atoms, in some embodiments, from 2 to 5 carbon atoms. Refers to a chain or branched chain. Examples include ethyl, propyl, isopropyl, etc.

[0055] As used herein, the term "alkenyl" refers to an alkyl group having one or more double bonds, particularly Unless otherwise specified, from about 2 to about 20, and in some embodiments, from about 2 to about 10 carbon atoms. In some embodiments, the range is from about 2 to about 8 carbon atoms. In the present invention, straight or branched chain hydrocarbons containing carbon atoms in the range of about 2 to about 6 carbon atoms are used. Examples of alkenyl radicals are vinyl, allyl, 1,4-butadienyl, isopropenyl, etc.

[0056] As used herein, the term "alkenylaryl" refers to an alkenyl-substituted aryl group. "Substituted alkenylaryl" refers to one or more of the aryl groups as defined herein. It refers to an alkenylaryl group further bearing a substituent of the formula:

[0057] As used herein, the term "alkenylene" refers to an alkyl group having at least one carbon-carbon double bond. Typically, the alkyl group has 2 to 20 carbon atoms, and in some embodiments, has 2 to 12 carbon atoms. A divalent straight-chain or refers to a branched chain hydrocarbyl group, and "substituted alkenylene" refers to any of the groups shown herein. The term "alkenylene" refers to an alkenylene group further bearing one or more substituents such as the aryl, ... or aryl groups.

[0058] As used herein, the term "alkylene" refers to an alkylene group having 1 to 20 carbon atoms, in some embodiments. In the form of a cyclic alkyl group, in the range of 1 to 15 carbon atoms, two hydrogen atoms are from the same carbon atom, or Refers to a divalent hydrocarbyl group containing straight or branched chains taken from different carbon atoms. Examples of alkylene include, but are not limited to, methylene (-CH2-), ethylene ( -CH2CH2-) etc.

[0059] As used herein, the term "alkynyl" refers to an alkynyl group having one or more triple bonds and having about 2 up to 20 carbon atoms, in some embodiments in the range of about 2 to 10 carbon atoms, In some embodiments, in the range of about 2 to 8 carbon atoms, and in some embodiments, about 2 to 6 carbon atoms. Alkynyl radicals refer to straight or branched chain hydrocarbyls containing carbon atoms in the range of Examples of aryl include ethynyl, propynyl (propargyl), and butynyl.

[0060] As used herein, the term "alkynylaryl" refers to an alkynyl-substituted aryl group. "Substituted alkynylaryl" refers to one or more aryl groups as defined herein. It refers to an alkynylaryl group further bearing a substituent of the formula:

[0061] As used herein, the term "alkoxy" refers to the group -OR c (In the formula, R c is defined as Lower alkyl, substituted lower alkyl, aryl, substituted aryl, aralkyl, substituted aryl, etc. Raralkyl, heteroalkyl, heteroarylalkyl, cycloalkyl, substituted cycloalkyl cycloheteroalkyl, cycloheteroalkyl or substituted cycloheteroalkyl.

[0062] As used herein, the term "lower alkoxy" refers to the group -OR d (In the formula, R d is a low-level (It is a rukill).

[0063] As used herein, the term "alkylaryl" refers to an alkyl substituted aryl group. "Substituted alkylaryl" refers to an alkyl group having one or more substituents as set forth herein. It refers to an alkylaryl group which further carries a group.

[0064] As used herein, the term "alkylthio" refers to the group -SR h (In the formula, R h Is, Alki This refers to the

[0065] As used herein, the term "substituted alkylthio" refers to the group -SR i (In the formula, R i Is, place It refers to a substituted alkyl group.

[0066] As used herein, the term "alkynylene" refers to an alkynylene having at least one carbon-carbon triple bond. and typically has in the range of about 2 to 12 carbon atoms, and in some embodiments, about 2 to 8 "Substituted alkynyl" refers to a divalent linear or branched hydrocarbyl group having carbon atoms in the range of "Arylene" refers to an alkylene further bearing one or more substituents as set forth herein. Refers to an alkynylene group.

[0067] As used herein, the term "amide" refers to the group -C(O)NR j R j’ (In the formula, R j Oh BiR j’ are independently hydrogen, lower alkyl, substituted lower alkyl, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl or substituted heteroaryl) .

[0068] As used herein, the term "substituted amide" refers to the group -C(O)NR k R k’ (In the formula, R k and R k’ are independently hydrogen, lower alkyl, substituted lower alkyl, aryl, substituted aryl aryl, heteroaryl, or substituted heteroaryl, where R k and R k’ Of At least one of them is not hydrogen. R in combination with nitrogen k R k’ is optional may form a substituted heterocyclic or heteroaryl ring.

[0069] As used herein, the term "amino" or "amine" refers to the group -NR n R n’ (In the formula, R n and R n’ are independently hydrogen, lower alkyl, Substituted lower alkyl, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl or substituted heteroaryl). A "divalent amine" refers to the group -NH-. A "substituted divalent amine" refers to the group -NR-, where R is lower alkyl, substituted lower alkyl, Alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl or substituted heteroaryl Indicates that the reel is in the slot.

[0070] As used herein, the term "substituted amino" or "substituted amine" refers to a group -NR p R p’ (In the formula, R p and R p’ are independently hydrogen, lower alkyl, substituted lower alkyl, alkyl , substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl; , where R p and R p’ At least one of these is not hydrogen. Combined R p R p’ optionally forms a substituted heterocyclic or heteroaryl ring; obtain.

[0071] As used herein, the term "aroyl" refers to an aryl-carbonyl species such as benzoyl. "Substituted aroyl" refers to one or more substituted aroyl groups as defined herein. The term "aroyl group" refers to an aroyl group bearing an additional group.

[0072] As used herein, the term "aryl", alone or in combination, includes phenyl, naphthyl, or optionally 5-10 ring members, and in some embodiments 5-6 ring members. and / or optionally halo, hydroxy, alkoxy, aryl, Kilthio, alkylsulfinyl, alkylsulfonyl, acyloxy, aryloxy , heteroaryloxy, optionally an alkyl, aryl or heteroaryl group; optionally mono- or di-substituted amino, amidino, optionally alkyl, aryl urea optionally substituted with a heteroaryl, heteroaryl or heterocyclyl group; and N-mono- or N,N-disubstituted by alkyl, aryl or heteroaryl groups. Optionally aminosulfonyl, alkylsulfonylamino, arylsulfonyl amino, heteroarylsulfonylamino, alkylcarbonylamino, arylcarbonylamino arylamino, heteroarylcarbonylamino, etc. It refers to a fused aromatic heterocycle which may optionally be fused to a ring.

[0073] As used herein, the term "aryloxy" refers to the group -OAr, where Ar is an aryl aryl or substituted aryl group).

[0074] As used herein, the term "carbocycle" refers to a single ring or rings composed of linked carbon atoms. refers to a saturated, unsaturated or aromatic group having multiple fused rings. The ring or rings are Optionally unsubstituted or substituted with, for example, halogen, lower alkyl, alkoxy, Alkylthio, acetylene (-C=CH), amino, amide, azido, carboxyl, hydroxy hydroxyl, aryl, aryloxy, heterocycle, heteroaryl, substituted heteroaryl , nitro (-NO2), cyano (-CN), thiol (-SH), sulfamide (-S( O)2NH2 and the like.

[0075] As used herein, the term "guanidinyl" refers to the group -N=C(NH2)2 and is used interchangeably with " "Related guanidinyl" refers to the group -N=C(NR2)2, where each R is independently as defined herein. H, alkyl, substituted alkyl, aryl or substituted aryl as shown show.

[0076] As used herein, the term "halo" or "halogen" refers to all halogens, i.e. These include chloro (Cl), fluoro (F), bromo (Br) and iodo (I).

[0077] As used herein, the term "heteroaryl" refers to any heteroaryl independently selected from the groups O, S, and N. In some embodiments, one or more, in some embodiments, one to four, In some embodiments, the optional heteroatoms include 1 to 3, and in some embodiments, 1 to 2, heteroatoms. Optionally, halo, hydroxy, alkoxy, alkylthio, alkylsulfinyl, alkyl Sulfonyl, acyloxy, aryloxy, heteroaryloxy, optionally aryloxy, Amino groups which may be mono- or di-substituted by alkyl, aryl or heteroaryl groups; amidino, optionally alkyl, aryl, heteroaryl or heterocyclyl urea optionally substituted with an alkyl, aryl or heteroaryl group; Aminosulfonyl, alkylsulfonyl, arylsulfonylamino, arylsulfonylamino, heteroarylsulfonylamino, alkylcarbonylamino, arylcarbonylamino, heteroarylcarbonylamino A monocyclic ring containing 5 or 6 ring atoms, substituted with 1 to 3 groups or substituents such as Heteroaryl refers to an aromatic ring structure or a bicyclic aromatic group having 8 to 10 carbon atoms. Also, oxidized S or N-oxides such as sulfinyl, sulfonyl, and tertiary ring nitrogens. The carbon or nitrogen atom is not heteroaryl such that a stable aromatic ring is maintained. It is the attachment point of the aryl ring structure. Examples of heteroaryl groups are phthalimido, pyridinyl, , pyridazinyl, pyrazinyl, quinazolinyl, purinyl, indolyl, quinolinyl, pyridyl midinyl, pyrrolyl, oxazolyl, thiazolyl, thienyl, isoxazolyl, oxathiyl Diazolyl, isothiazolyl, tetrazolyl, imidazolyl, triazinyl, furanyl , benzofuryl, indolyl, etc. Substituted heteroaryls are preferred because they produce stable compounds. The substituents contain substituents attached at available carbon or nitrogen for binding to a ring.

[0078] As used herein, the term "substituted heteroaryl" refers to an optionally substituted heteroaryl group, Functional groups, such as halogen, lower alkyl, lower alkoxy, alkylthio, acetylene , amino, amide, carboxyl, hydroxyl, aryl, aryloxy, heterocycle, Substituted heterocycles, hetaryl, substituted hetaryl, nitro, cyano, thiol, sulfamide, etc. It refers to a heterocycle which is mono- or polysubstituted with, for example, aryl, aryls, or aryls.

[0079] As used herein, the term "heterocycle" refers to a ring that is optionally unsubstituted or For example, halogen, lower alkyl, lower alkoxy, alkylthio, acetylene, amino No, amide, carboxyl, hydroxyl, aryl, aryloxy, heterocycle, hetari aryl, substituted hetaryl, nitro, cyano, thiol, sulfamide, etc. may be monocyclic (e.g., morpholino, pyridyl, or furyl) or polycyclic condensed rings (e.g., , naphthopyridyl, quinoxalyl, quinolinyl, indolizinyl or benzo[b]thienoyl nyl) and has carbon atoms and at least one heteroatom such as N, O or S in the ring It refers to a saturated, unsaturated or aromatic group having the formula:

[0080] As used herein, the term "substituted heterocycle" refers to any heterocyclic ring that can be substituted in any manner to produce a stable compound. Optionally substituted alkyl, optionally substituted optionally substituted alkenyl, optionally substituted alkynyl, halo, hydro oxy, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl, acyl Oxy, aryl, substituted aryl, aryloxy, heteroaryloxy, amino, a amido, amidino, optionally alkyl, aryl, heteroaryl or heterocyclo urea optionally substituted with an alkyl, aryl or heteroaryl group; Aminosulfonyl, optionally N-monosubstituted or N,N-disubstituted by aryl groups; alkylsulfonylamino, arylsulfonylamino, heteroarylsulfonylamino, Alkylcarbonylamino, arylcarbonylamino, heteroarylcarbonylamino No, acyl, carboxyl, heterocycle, substituted heterocycle, hetaryl, substituted hetaryl, nitro , cyano, thiol, sulfonamide and oxo; or It refers to a heterocycle that is substituted with multiple, e.g., 1, 2 or 3, substituents.

[0081] As used herein, the term "hydrocarbyl" refers to a group whose backbone consists solely of carbon and hydrogen. Hydrocarbyl refers to any organic radical containing an alkyl, cycloalkyl, aryl ... alkyl, alkenyl, cycloalkenyl, alkynyl, aryl, alkylaryl, aryl aryl alkyl, aryl alkenyl, alkenyl aryl, aryl alkynyl, alkynyl aryl and the like.

[0082] As used herein, the term "substituted hydrocarbyl" refers to -O-, -S-, -NR- (formula wherein R is hydrogen, alkyl or substituted alkyl), -C(O)-, -C(S)-, -C(=NR')-, -C(=CR'2)- (wherein R' is an alkyl or substituted alkyl group) -OC(O)-, -OC(O)-O-, -OC(O)-NR- (also -NR-C(O)-O-), -NR-C(O)-, -NR-C(O)-NR-, -S- C(O)-, -SC(O)-O-, -SC(O)-NR-, -OS(O)2-, - OS(O)2-O-, -OS(O)2-NR-, -OS(O)-, -OS(O) -O-, -OS(O)-NR-, -O-NR-C(O)-, -O-NR-C(O)-O -, -O-NR-C(O)-NR-, -NR-OC(O)-, -NR-OC(O)- O-, -NR-OC(O)-NR-, -O-NR-C(S)-, -O-NR-C(S) -O-, -O-NR-C(S)-NR-, -NR-OC(S)-, -NR-OC(S )-O-, -NR-OC(S)-NR-, -OC(S)-, -OC(S)-O-, -OC(S)-NR-(or -NR-C(S)-O-), -NR-C(S)-, -N RC(S)-NR-, -SS(O)2-, -SS(O)2-O-, -SS(O) 2-NR-, -NR-OS(O)-, -NR-OS(O)-O-, -NR-OS( O)-NR-, -NR-OS(O)2-, -NR-OS(O)2-O-, -NR-O -S(O)2-NR-, -O-NR-S(O)-, -O-NR-S(O)-O-, -O- NR-S(O)-NR-, -O-NR-S(O)2-O-, -O-NR-S(O)2-N R-, -O-NR-S(O)2-, -OP(O)R2-, -SP(O)R2- or -NR-P(O)R2-, where each R is independently hydrogen, alkyl, or substituted alkyl. linked to the hydrocarbyl moiety by a linker / spacer moiety such as Hydroxy, hydrocarbyloxy, substituted hydrocarbyloxy, alkylthio, substituted a Arylthio, arylthio, substituted arylthio, amino, alkylamino, substituted alkyl Amino, carboxy, -C(S)SR, -C(O)SR, -C(S)NR2 (wherein each R are independently hydrogen, alkyl or substituted alkyl), nitro, cyano, halo, -SO -3M or -OSO3M, where M is H, Na, K, Zn, Ca or meglumine. ), guanidinyl, substituted guanidinyl, hydrocarbyl, substituted hydrocarbyl, hydro Carbyl carbonyl, substituted hydrocarbyl carbonyl, hydrocarbyloxy carbonyl , substituted hydrocarbyloxycarbonyl, hydrocarbylcarbonyloxy, substituted hydro Carbylcarbonyloxy, acyl, acyloxy, heterocycle, substituted heterocycle, heteroaryl aryl, substituted heteroaryl, heteroarylcarbonyl, substituted heteroarylcarbonyl, Carbamoyl, monoalkylcarbamoyl, dialkylcarbamoyl, arylcarbamoyl yl, carbamate group, dithiocarbamate group, aroyl, substituted aroyl, organic sulfonyl , substituted organic sulfonyl, organic sulfinyl, substituted alkylsulfinyl, alkylsulf phenylamino, substituted alkylsulfonylamino, arylsulfonylamino, substituted arylsulfonylamino one or more selected from the group consisting of arylsulfonylamino, sulfonamide, sulfuryl, etc. "R" refers to any of the above mentioned hydrocarbyl groups further bearing a substituent of the formula:

[0083] As used herein, the term "hydroxyl" or "hydroxy" refers to the group --OH. .

[0084] As used herein, the term "oxo" refers to an oxygen substituent that is double bonded to the carbon to which it is attached. Refers to the base.

[0085] As used herein the term "sulfinyl" refers to the group -S(O)-.

[0086] As used herein, the term "substituted sulfinyl" refers to the group -S(O)R t (In the formula, R t teeth , lower alkyl, substituted lower alkyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl alkylalkyl, substituted cycloalkylalkyl, heterocyclyl, substituted heterocyclyl, Hetereocyclyl alkyl, substituted heterocyclyl alkyl, aryl, Substituted aryl, heteroaryl, substituted heteroaryl, heteroaralkyl, substituted heteroaralkyl The aryl group is an alkyl, aralkyl or substituted aralkyl.

[0087] As used herein, the term "sulfonyl" refers to the group -S(O)2-.

[0088] As used herein, the term "substituted sulfonyl" refers to the group -S(O)R t (In the formula, R t teeth , lower alkyl, substituted lower alkyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl alkylalkyl, substituted cycloalkylalkyl, heterocyclyl, substituted heterocyclyl, Hetereocyclyl alkyl, substituted heterocyclyl alkyl, aryl, Substituted aryl, heteroaryl, substituted heteroaryl, heteroaralkyl, substituted heteroaralkyl The aryl group is an alkyl, aralkyl or substituted aralkyl.

[0089] As used herein, the term "sulfuryl" refers to the group -S(O)2-.

[0090] The present invention relates to an initiating capped oligonucleotide primer having the general form m7 Gppp[ N 2’Ome ] n[ N] m (In the formula, m7 G is an N7-methylated guanosine or any guanosine an anosine analog, N is any natural, modified or unnatural nucleoside, " can be any integer from 1 to 4, and "m" can be an integer from 1 to 9), 'Methods and compositions for synthesizing capped RNA are provided. In one aspect of the invention, , the initial capped oligonucleotide primer has Formula I: [ka] [In the formula, B1 to B 10 each is independently a natural, modified or non-natural nucleoside base; M is 0 or 1; L is 0 or 1; q1 is 1, Each of q2 to q9 is independently 0 or 1; R1 is H or methyl; R2 and R3 are independently H, OH, alkyl, O-alkyl, amine, azide, halo a gene, linker or detectable marker; X1 to X 13 each is independently O or S; Y1 to Y 13 each independently represents OH, SH, BH3, aryl, alkyl, O-aryl alkyl or O-aryl; Z1 to Z 22 each independently represents O, S, NH, CH2, C(halogen)2 or CH (halogen), R4 to R 12 each is independently H, OH, OMe, or a detectable marker. ] It has the structure shown below.

[0091] In certain embodiments, the initial capped oligonucleotide primer is a trimer (q 2 -q 9 =0), tetramer (q 3 -q 9 =0), pentamer (q 4 -q 9 =0), hexamer (q 5 - q 9 =0), heptamer (q 6 -q 9 =0), octamer (q 7 -q 9 =0), nonamer (q 8 -q 9 =0), decamer (q 9 =0) or 11-mer. Some examples of dotrimer primers are provided in Table I below:

[0092] [Table 1] JPEG2024150459000005.jpg70122

[0093] Other initiating capped oligonucleotide primers encompassed by the present invention include: Primers with known or novel base analogs are included. A method for synthesizing the primers is illustrated in the Examples below.

[0094] Transcription In eukaryotes, transcription of messenger RNA (mRNA) is initiated by RNA polymerase II. This is a complex multi-subunit enzyme with intricate regulation. To perform large-scale transcription in vitro, researchers generally use T7, T3, SP 6, single subunits derived from K1-5, K1E, K1F or K11 bacteriophages This family of polymerases uses accessory phage polymerases. It does not require a protein and has minimal constraints on the starting nucleotide sequence, approximately 17 nucleotides. The present application relates to a simple minimal promoter sequence for T7 RNA polymerase (T7 Although the present invention focuses on RNA polymerase P), those skilled in the art will appreciate that the present invention can be applied to other RNA polymerase Ps. It will be appreciated that the present invention may also be practiced using a laser.

[0095] T7 RNAP exists in at least two protein states. The first is the "A The second, called the "abortive complex," is involved in transcription initiation. This conformation is called the "elongation complex" and is highly processive. can be divided into six steps: 1) RNA polymerase promoter sequence 2) initiation of transcription; and 3) abortive transcription, during which the polymerase 4) non-processive elongation, which frequently releases the DNA template and short abortive transcripts, and 4) the open complex 5) processive elongation and 6) transcription termination. A significant amount of RNA consists of short abortive fragments of approximately 2–8 nucleotides in length (Bioc hemistry vol. 19: 3245-3253 (1980); Nucleic A cids Res. Volume 9: 31-45 (1981); Nucleic Acids Res. 15:8783-8798 (1987); Biochemistry 27:3966-3974 (1988). After synthesis of about 10-14 bases, RNA The polymerase escapes from abortive cycling and simultaneously aligns with the promoter DNA. They lose sequence-specific contacts and form a processive elongation complex, in which the RNA strand advances in a sequence-independent manner. (J. Mol. Biol. vol. 183: 165-177 (1985); P roc. Natl. Acad. Sci. USA Vol. 83: 3614-3618 ( 1986); Mol. Cell Biol. Vol. 7: pp. 3371-3379 (1987) year)).

[0096] The consensus sequence for the most active class III T7 promoters is It includes 17 bp of upstream sequence and 6 bp downstream (Cell 16: 815-25 (1979)). The position of the first transcribed nucleotide is generally the +1 transcript of the RNA. The second transcribed nucleotide is called the +2 transcribed nucleotide. During transcription, the two strands melt together, forming a transcription bubble, which separates the double-stranded The bottom strand (designated 3' to 5' in Table 2) is the template for transcription. For nucleotides +3 and above, the template strand is primarily Watson-Crick Base pairing interactions define the identity of the transcribed nucleotide. The nucleotide that codes for the RNA transcript nucleotide is defined as the +1 nucleotide of the template. In the example shown in Table 2, the +1 transcript nucleotide is G and the +1 template nucleotide is G. Similarly, the +4 transcript nucleotide is A and the +4 template nucleotide is C. The punch line is T.

[0097] [Table 2]

[0098] Unlike DNA polymerases, T7 RNAP synthesizes RNA in the absence of a primer. The first step in initiation is called de novo RNA synthesis, In this case, RNA polymerase recognizes a specific sequence on the DNA template and binds to positions +1 and + a first pair of nucleotide triphosphates that are complementary to the template residues of 2 and a dinucleotide triphosphate that is complementary to the template residues of 2; The initiating nucleotide is reacted with the polymerase The Kd value is calculated based on the first starting N The Kd for the first TP is 2 mM and for the second it is 80 μM, whereas the Kd for the second TP is 2 mM and for the third TP it is 80 μM. The actual NTP concentration is approximately 5 μM (J. Mol. Biol. (2007) Vol. 3, No. 1). 70, pp. 256-268). De novo synthesis is the rate-limiting step in transcription. It has been shown that T7 RNAP has a high affinity for GTP as the initiating nucleotide. (J. Biol. Chem. 248:2235-2244 (1999) Of the 17 T7 promoters in the genome, 15 are GTP-terminated (and 13 are pp pGpG) whereas there is no apparent NTP preference during transcription elongation (J. Mo Biol. 370:256-268 (2007). T7 RNA polymerase The enzyme initiates poorly on promoters that code for an A at position +1 and instead Transcription primarily initiates at the coded G at position +2 (J. Biol. Chem. vol. 278). Vol.:2819-2823 (2003).

[0099] During de novo RNA synthesis, the binding of the initiating nucleotide occurs via base stacking. , the specific interactions between the polymerase residues, the guanine portion of the initiating nucleotide, and the base This is mainly achieved by the free energy generated by complementary interactions (J. Mol. B iol. 370:256-268 (2007)).

[0100] T7 RNAP can also be initiated using short oligonucleotide primers. For example, 13 promoters in the T7 genome are expressed using pppGpG. It has been found that the β-amyloid ... Several groups have demonstrated that T7 RNAP binds dinucleotide primers (Biochemistry Vol. 24: 5716-5723) Axelrod et al. (1985) reported that the uncapped GpA dinucleotide The +1 and +2 constructs are 2'-deoxycytidine and 2'-deoxythymidine, respectively. They showed that it is possible to start with a 20-nucleotide template (the "CT" template). 0 micromolar (μM) dimer and 100 μM ATP, CTP, GTP and U Their reaction mixture also contained 100 μM 3' dATP, 3' dCTP They contained 50 μM 3' dUTP or 50 μM 3' dGTP. We observed only RNAs primed with a mixture of GpA and 5′-GTP primed RNAs. 'We did not observe any triphosphate RNA. This may be due to the reaction conditions used. 100 μM GTP inhibits the 2′ reaction of T7 polymerase to the first initiating guanosine. This is significantly lower than the mM Kd (J. Mol. Biol. (2007) Vol. 370, 25 (pp. 6-268). GTP competes for initiation with the initiator oligonucleotide. Using low GTP concentrations favors GpA initiation but results in low transcription yields. (maximum calculated yield estimated to be <150 μg / mL reaction). When transcription is initiated on a "CT" template using CpG, UpG, or GpG, they The 5′ nucleotide sequence is a transcription factor that mediates the formation of RNA transcripts and further untemplated 5′ nucleotides. The three observed morphologies were A, C, U, or G, respectively.

[0101] Axelrod et al. also used an uncapped GpG dinucleotide to The primers +1 and +2 are 2'-deoxycytidine ("CC" template) promoter. They observed low fidelity of initiation and produced three distinct transcripts. They observed that "examination of the autoradiographs shows that one member of each triplet Due to the initiation of a GpG at the normal (+1) position, the second member of each triplet is The third member of each triplet is the normal GpG at position (-1). This indicates that the GpG gene is due to the initiation of the guanosine triphosphate at position φ1. 0 promoter ("CC" template) and φ1.1A promoter ("CT" template) were used. If so, it is a relatively weak initiator and does not react with guanosine triphosphate at the concentration used. "The φ10 promoter is a key regulator of transcription and regulation of transcription in normal cells," they said. No initiation with the GpA dinucleotide was observed using the "CC" template. , ApC and ApA are initiators with either the above "TC" or "CC" templates. These did not act as targets for the template nucleic acid at positions +1 and +2, presumably because they This is because the method described by Axelrod et al. , which was designed for the generation of extremely small amounts of radioactive transcripts for sequencing. and is not suitable for large-scale production of useful pharmaceutical amounts of RNA. To increase the yield of RNA, NTPs containing an initiating dimer of 1000 and GTP are used. The expected result is that GTP reacts with the +1 nucleotide at an NTP concentration close to its Kd (2 mM). Since it competes effectively with the dimer for initiation of RNA, with a large proportion of RNA being generated starting with pppG.

[0102] Pitulle et al. showed that transcription using T7 RNAP results in the synthesis of uncapped oligonucleotides. We have shown that the ribozyme can be initiated using 2-mer to 6-mer ribozymes (Gene, Vol. 112:1 01-105 (1992). These oligonucleotides have a 5'-OH or 5 They also contained oligonucleotides of the structure biotin-ApG. All of the oligonucleotides used in this study were , which contained a 3'-terminal G. Pitulle et al. also Generates RNA transcripts with 2'-O-methylated or 2'-deoxy residues near the termini To achieve this, it is advisable to include 2'-O-methyl and deoxy residues in the primer sequence. In this publication, we have demonstrated that it is possible to ligate the 3' end of any starting oligonucleotide. It is clear that the anosine residue is paired with the +1 template nucleotide. This results in non-templated nucleotides being added to the 5' end of the RNA. The authors write, "Apart from the Y-terminal G, base pairing with the template DNA is not required, so this sequence "Sequence variations in the starting oligonucleotide fragment are easily possible." The nucleotide primer is perfectly compatible only with the template nucleotide "C" at position +1. is complementary to any nucleotide at subsequent positions (+2, +3, etc.) This is confirmed in a subsequent methodological paper by this group (M ethos Mol Biol. 74:99-110 (1997), Meth ods Mol. Biol. vol. 252: 9-17, (2004). indicates that all nucleotides of the initiating capped oligonucleotide primer are at position +1 and a nucleotide sequence as described herein that is completely complementary to the template nucleotide at the subsequent position. The method is different. Kleineidam et al. 5'-modified tRNA transcripts were generated by initiation with dimers or trimers modified with (Nucleic Acids Research, Vol. 21, pp. 1097-1101) 1993). Again, the authors concluded that the 3'-terminal guanosine of the primer is the +1 template nucleotide. It states that the treatment will start with leutidine "C".

[0103] Another study by Ishikawa et al. m7 GpppApG, m7 Gppp m6 A p.G. m7 GpppA 2’Ome pG or m7 Gppp m6 A 2’Ome pG cap The primer oligonucleotide trimer is a 2'-deoxycytosine trinucleotide at template positions +1 and +2. The template had a nucleotide residue ("CC" template; Nucleic Acids Symposium um Series No. 53:129 (2009)) demonstrated that it can initiate transcription. The authors write: m7 Different results were obtained from the case using G5'pppG at the -1 position. So, m7 G5'pppN1pG with an additional adenosine (N1) in the T7 promoter "This may result from base pairing between the 2'-deoxythymidines in the nucleoside analogues." The method of claim 1, wherein the +1 and +2 nucleotides of the initial capped oligonucleotide trimer are This is distinct from the method described in the present invention, which pairs with the +1 and +2 nucleotides of the template. Ishikawa et al. used 6 mM of starting oligonucleotide trimer and 0.9 mM GTP and 7.5 mM each of ATP, CTP, and UTP were used. Competing to direct the transcription reaction toward capped RNA over pppRNA A greater than six-fold excess of capped initiating oligonucleotide primer over GTP was used. This is the most expensive nucleotide component of the transcription reaction and accounts for the total amount of synthesized RNA. On the other hand, low concentrations of GTP (0.9 mM) increase the RNA synthesis rate in the transcription reaction. Limiting the total yield (theoretically to less than 1.4 mg / mL). The method described in the paper provides efficient RNA capping and higher yields of RNA (2 ~6mg / mL), thus achieving high-quality mRNA production at a commercially viable cost. To enable production, it is not necessary to limit the concentration of any NTP.

[0104] None of the publications discussed above directly measured RNA capping efficiency. Therefore, the extent of capping in those studies is unknown.

[0105] Importantly, in all of the studies described above, the +1 template nucleotide was 2 '-deoxycytidine (Biochemistry Vol. 24: 5716-5723 (1985), Gene, vol. 112:101-105 (1992), Meth ods Mol.Biol. 74:99-110 (1997), Methods Mol.Biol. 252:9-17, (2004), Nucleic Ac ids Research Vol. 21: 1097-1101 (1993) Nucle ic Acids Symposium Series No. 53:129 (2009) ).

[0106] In the more than 20 years since the publication of transcription initiation studies using oligonucleotide primers, Ucleic Acids Symposium Series No. 53: 129(20 Until the publication of a short report in 2009, the initiation oligonucleotides containing 5'-to-5' inverted cap structures were There have been no published examples of transcription initiation using mononucleotide primers.

[0107] Methods and compositions provided herein for preparing 5'-capped RNA These include, but are not limited to, mRNA, small nuclear RNA (snRNA), and nucleolus Small RNAs (snoRNAs) and small Cajal body-specific RNAs (scaRNAs) are These methods involve the synthesis of RNA using DNA as a template and under the control of a promoter. For the cap-containing oligonucleotide primer, nucleoside 5'-triphosphate ( In certain embodiments, the method includes the use of RNA synthesis In particular, the initiating capped oligonucleotide provides utility in the synthesis of capped RNA. The initiation oligonucleotide primer is the same as that of the natural RNA molecule. Cap0, Cap1, Cap2 or TMG-cap-like structures, MG-cap is at the 5' position of the RNA, cap 1 at the 2nd position, and cap 2 at the 3rd position. The natural Cap 0 structure contains a 2'-O-methylated nucleoside unit adjacent to the It does not have any 2'-O-methylated nucleoside units.

[0108] Methods and compositions for preparing RNA include, but are not limited to, mRNA, snRNA, snoRNA, carrying modifications at or near the 5' end scaRNA, transfer RNA (tRNA), ribosomal RNA (rRNA) and These methods include DNA synthesis, DNA synthesis, and transfer-messenger RNA (tmRNA). A capped RNA template for promoter-controlled synthesis of RNA with or without an initiating oligonucleotide primer, nucleoside 5'-triphosphate ( In certain embodiments, the method includes the use of RNA synthesis In particular, structural modifications that provide utility in the synthesis of 5'-modified RNAs are provided. A modified starting oligonucleotide primer that retains the

[0109] The initiating capped oligonucleotide primer has a nucleotide at the 3' end of the primer. RNA polymerase-mediated synthesis of RNA on a DNA template by adding a dopamine unit Initiating Capped Oligonucleotides that have an open 3'-OH group to allow for the initiation of synthesis The primer is aligned to the transcription initiation site (i.e., the initiation site is close to the 3' end of the promoter sequence). (which may be adjacent to and overlap with the promoter sequence) and is substantially complementary to the template DNA sequence. In certain embodiments, the initial capped oligonucleotide primer is a primer It directs RNA synthesis primarily in one direction ("forward"), starting from the 3' end of the In some aspects and embodiments, the initiating capped oligonucleotide primer is outcompete any nucleoside 5'-triphosphate for initiation of synthesis, thereby Maximize the production of RNA starting with a capped oligonucleotide primer and 5'- It minimizes the production of RNA that starts with a triphosphate-nucleoside (usually GTP).

[0110] Production of mRNA by in vitro transcription is a promising strategy to produce highly active phage RNA polymerases. RNA polymerases use enzymes (T3, T7, SP6 and others) to amplify the template nucleic acid. The control of specific promoters incorporated into DNA plasmid constructs prior to the primer sequence. The transcription process usually begins with the synthesis of purine nucleoside 5'-triphosphates (usually GTP ) and RNA polymerase encounters a termination sequence or completes the DNA template. Continue until.

[0111] As discussed above, the mCAP dinucleotide analog contains Cap 0 7m G( 5')ppp(5')N is used for initiation of in vitro transcription (See, e.g., RNA vol. 1:957-967 (1995)). These dinucleotides Capped RNA molecules produced using the analogues contain Cap 0. However, Only about 50% of the synthesized capped RNA molecules were in the correct "forward" orientation with cap 0. To convert RNA with cap 0 to RNA with cap 1, , a further enzymatic reaction using (nucleoside-2'-O) methyltransferase However, this conversion may not be quantitative and may be difficult to control. It is not easy to separate the remaining cap 0 RNA from the cap 1 RNA. In addition, competition from NTPs (specifically, GTP) to initiate transcription is This further reduces the amount of active capped RNA molecules produced.

[0112] Additionally, modifications having blocked 3' and / or 2' positions on the ribose 7m G Retaining residues, 7m G 3’Ome (5')ppp(5')N and other related ARCs Modified dinucleotide analogs, such as the A analog, are used to initiate transcription in vitro. (For example, RNA Vol. 7: pp. 1486-1495 (2001)). ARCA cap analogs direct RNA synthesis only in the "forward" direction, Therefore, the 5' end ( 7m (with 2' and / or 3' modifications of G residues) This produces an RNA molecule with the standard dinucleotide analogs , 7m In comparison with RNA prepared using G(5')ppp(5')N, RNA with ARCA cap 0 is more active than RNA with ARCA cap 1. The dinucleotide analogs discussed so far are required to convert the nucleotide sequence into RNA that can be synthesized by the Further enzymatic reactions using similar (nucleoside-2'-O) methyltransferases This method has been described in detail for mCAP dinucleotide analogs. The ARCA cap of RNA with cap 0 has the same disadvantages as those described above. The conversion of 1 to RNA may not be quantitative, and the reaction is not easy to control. It is difficult to separate the remaining cap 0 RNA from the cap 1 RNA. Competition from NTPs (specifically, GTP) for the initiation of transcription increases the amount of active capsid produced. This further reduces the amount of loop-forming RNA molecules.

[0113] A short oligonucleotide primer (2-6 mer) with a 3'-terminal guanosine residue was used. , which has been used to initiate in vitro transcription (Pitulle, C. et al., Ge ne, vol. 112:101-105 (1992). The primers contained modified and unmodified ribonucleoside residues (e.g., modified ribonucleoside residues). The nucleoside residues are 2'-O-methylated nucleoside residues and 2'-deoxyribonucleic acid residues. (containing nucleotide residues). Shorter oligonucleotide primers (dimers to tetramers) The pentamer essentially outcompetes GTP for the initiation of transcription, allowing the longer primer (the ~hexamer) is significantly less efficient at initiating transcription than GTP. These longer primers (as they are designed) bind to the DNA template at the initiation site. In contrast, the dimer, AG (designed The nucleotide sequence (such as the nucleotide sequence of ... RNA molecules generated using oligonucleotide primers have internal 2'-O-methyl groups 5'-cap0, cap1, cap2 or TMG- The RNA that does not have a cap structure is designated as Cap 1, Cap 2, or Cap 3. or using a capping enzyme to convert RNA to a TMG-capped structure However, such a conversion can be carried out in the same manner as described above. It has the same disadvantages as those described above.

[0114] Other short nucleotides containing cap structures and internal 2'-O-methylated nucleoside residues RNA oligonucleotides have been prepared chemically (Ohkubo et al., Org. Le tters vol. 15: 4386-4389 (2013). These short cap shapes The synthetic oligonucleotides were ligated with T4 DNA ligase and complementary DNA sprinter oligos. The 5'-capped nucleotides are used to "decap" long RNAs ( The final RNA fragment synthesized using this chemical-enzymatic method was A has both an internal 2'-O-methylated nucleoside residue and a 5'-TMG-cap structure. However, using this ligation approach, Only synthetic RNA (<200mer) was prepared. Furthermore, the yields were low (15–30%). T4 DNA ligation reactions are not easy to control and optimize, and polyacrylamide gel electrophoresis is difficult. The laborious separation process using arylamide gel electrophoresis and the removal of residual caps The remaining capped RNA must be isolated from the uncapped RNA by PAGE. Long (500-10,000 bases) capped mRNA from uncapped mRNA Separation of the two is not feasible.

[0115] Finally, to initiate in vitro transcription of RNA, 5'-modified nucleosides or is a 5'-modified mononucleotide or dinucleotide, usually a derivative of guanosine has been used (Gene, vol. 112:101-105 (1992) and Bi Chem., 10371-378 (1999). The ether nucleosides and nucleotides can be coupled to a label or affinity group (e.g., biotin). When incorporated into the 5' end of the RNA, This 5'-labeled or tagged antibody will allow for easy detection, isolation and purification. Tagged RNA may be necessary for some applications. However, this strategy requires the use of capsids. To prepare mRNA with cap 0, cap 1, cap 2 or TMG-cap structures, Not used.

[0116] In certain embodiments of the present invention, the composition of the initial capped oligonucleotide primer of formula I In a related embodiment, the RNA comprises an initial capped oligonucleotide of formula I. This method uses a primer to synthesize the DNA.

[0117] Initiating Capped Oligonucleotide Primer The initiation capped oligonucleotide primer of the present invention is a capped oligonucleotide primer that is capable of binding to a DNA template at the initiation site. The hybridization sequence may be complementary to the sequence provided herein. Hybridization sequences of primers for use in the methods and compositions described herein The length of the primer depends on the identity of the template nucleotide sequence and whether the primer is capable of in vitro transcription. During transcription, the DNA template hybridizes to the nuclease, depending on several factors, including the temperature used. The initiating capped oligonucleotide primer for use in transcription Determination of the desired length of a specific nucleotide sequence can be accomplished by one of ordinary skill in the art or through routine experimentation. For example, the length of the nucleic acid or oligonucleotide can be readily determined by the desired hybridization. The determination can be based on redization specificity or selectivity.

[0118] In some embodiments, the initiating capped oligonucleotide primer (inverted 5'- The nucleotide length of the 5' cap nucleotide (including the 5' cap nucleotide) is between 3 and about 9, with some In this embodiment, the nucleic acid of the initiating capped oligonucleotide primer (including the cap) The nucleotide length is between 3 and about 7, and in some embodiments, the initial capped oligo the nucleotide primer (including the cap) is between 3 and about 5 nucleotides in length; In some embodiments, the initial capped oligonucleotide primer (including the cap) The length of the initial capped oligonucleotide primer is approximately 3 nucleotides. The length of the hybridization sequence is determined by the length of the initiating capped oligonucleotide primer. The length of the slit may be less than or equal to the total length of the slit.

[0119] The presence of the hybridization sequence indicates that the initiating capped oligonucleotide primer , complementary to the DNA template at the initiation site only in the desired orientation (i.e., the "forward" orientation) In the forward orientation, the RNA transcripts are aligned primarily with inverted guanosine residues. (i.e. 7m G(5')ppp(5')N) over the incorrect "reverse" direction The predominance of forward primer alignment on the DNA template (Figure 1) is a key factor in the hybridization of The latter is maintained by the thermodynamics of the initial capped oligonucleotide. The length of the hybridization sequence of the nucleotide primer and the hybridization with the DNA template The desired forward orientation is determined by the identity of the bases involved in the redization. Hybridization occurs during in vitro transcription, when the DNA template and the initiation capsid are hybridized. The temperature at which the oligonucleotide primers are hybridized or used and It may also vary depending on the reaction conditions.

[0120] The initiating capped oligonucleotide primer of the present invention has the standard GTP, ATP, CT It enhances the efficiency of transcription initiation compared to the efficiency of initiation with P or UTP. In this embodiment, initiation of transcription occurs when synthesis of RNA does not occur from any NTP in the transcription mixture. It is not enhanced primarily when starting from an initial capped oligonucleotide primer. It is believed that the increased efficiency of transcription initiation leads to higher yields of RNA transcripts. The enhanced efficiency of transcription initiation was observed using conventional methods without an initiating capped primer. The synthesis of RNA was increased by approximately 10%, 20%, 40%, 60%, 80%, 90%, and 1 The amount of the ion exchange reaction may be increased by more than about 100%, about 150%, about 200%, or about 500%. In this embodiment, the "initiation capped oligonucleotide primer" is an arbitrary primer for initiation of transcription. The initiating capped oligonucleotide is then introduced into the ribosome to outcompete the NTP (including GTP). The level of substrate activity and efficacy of the tide primers can be readily determined. An example of a method for this is illustrated in Example 13. In certain embodiments, initiation is by NTP. This occurs from capped oligonucleotide primers rather than from siRNA, which results in higher levels of It results in the capping of the transcribed mRNA.

[0121] In some embodiments, the RNA comprises an initiating capped oligonucleotide having a substitution or modification. In some embodiments, a method is provided in which the primer is synthesized using an initiating capped primer. Substitutions and modifications of oligonucleotide primers do not substantially impair RNA synthesis A routine test synthesis was carried out to synthesize the modified initiating capped oligonucleotide primer. It can be examined whether the desired synthesis result can be obtained using the above method. Conducting such routine experiments to see whether the desired results can be obtained Substitutions or modifications of the initial capped oligonucleotide primer can be, for example: one or more modified nucleoside bases, one or more modified sugars, one or more or more modified internucleotide linkages and / or one or more modified triplet Contains phosphate bridges.

[0122] The methods and compositions provided herein may include one or more modifying groups. The modified initiating capped oligonucleotide primer is By incorporating into the initiation site, it can be extended by RNA polymerase on the DNA template. Capped oligonucleotide primers are made up of natural RNA and DNA nucleosides, modified The initial capped oligonucleotide may include a nucleoside or nucleoside analog. The nucleotide primer may be a naturally occurring internucleotide phosphodiester linkage or a modification thereof. It may contain combinations of these.

[0123] In one embodiment, the modifying group is modified at an increasing rate as the temperature of the enzyme reaction medium is increased. A thermally labile group that dissociates from the initiating capped oligonucleotide primer. Examples of thermally labile groups on oligonucleotides and NTPs are Nuclei c Acids Res., Vol. 36: e131 (2008), Collect.Sy mp. Ser., vol. 10: 259-263 (2008) and Analytica Chemistry, Vol. 81: 4955-4962 (2009) There are.

[0124] In some embodiments, at least one of the nucleotides may have a modification as disclosed herein. The method proposed here is that RNA is synthesized when one or more NTPs are added to the transcription reaction. In some embodiments, the modification of at least one NTP is RNA polymerase-mediated The modification of NTPs may, for example, include one or more modifications. modified nucleoside base, one or more modified sugars, one or more modified The modified NTP may include a 5'-triphosphate. It can be incorporated into the 3' end of the primer, does not block transcription, and supports further extension of the primer. Hold.

[0125] In another embodiment, the modification group of the initial capped oligonucleotide primer is a detectable Thus, after transcription, a detectable label or a detectable marker may be added. Target RNA containing the marker or markers is identified by size, mass, color and / or affinity capture. In some embodiments, the detectable label or marker is a fluorescent dye. and the affinity capture label is biotin. In certain embodiments, one or more of the transcription reactions contains multiple components (initial capped oligonucleotide primer and / or NTPs) Thus, after transcription, the RNA molecule can be identified, for example, by size, mass, affinity capture, or color. In one embodiment, the detectable label is a fluorescent dye and the affinity capture label is biotin.

[0126] Standard chemistry is used to synthesize the "initial capped oligonucleotide primer" of the present invention. Chemical and enzymatic synthesis methods are available and are disclosed herein in the Examples section.

[0127] kit A kit containing an "initiation capped oligonucleotide primer" for carrying out transcription For example, the kit may be used to synthesize a common RNA (e.g., FLuc mRNA). The kit may contain all the transfer reagents for synthesis. More specifically, the kit may include a "starter capped Oligonucleotide primers" mark vessels for transcription and carry out RNA synthesis and one or more modified or unmodified initiation caps. attached oligonucleotide primer, one or more unmodified NTPs, one or more modified NTPs (e.g., pseudouridine 5'-triphosphate), RNA polymerase , other enzymes, a reaction buffer, magnesium and a DNA template. The composition may contain one or more reagents that are

[0128] The initiating capped oligonucleotide primers of the present invention can be prepared by a variety of initiating nucleosides, Use of nucleotides and oligonucleotides or capsids such as mCAP and ARCA Current methods include the use of polyphosphate dinucleotide derivatives containing a .O . structure. and compositions having considerable advantages over the initiating capped oligonucleotide primer. The system is compatible with existing transcription systems and reagents and does not require additional enzymes or reagents. In addition, the use of an initiating capped oligonucleotide primer is a promising strategy for the detection of some non-enzymatic Eliminates chemical and enzymatic steps (e.g., capping and 2'-O-methylation) This in turn reduces the complexity and cost of the process of RNA synthesis.

[0129] Exemplary methods described herein include T7 RNA polymerase-mediated transcription reactions. Several other RNA fragments known in the art for use in transcription reactions include Any of a variety of natural enzymes can be utilized with the compositions and methods of the present invention. Other enzymes, including natural or mutated variants, include, for example, SP6 and T3R. RNA polymerases and RNA from other sources containing thermostable RNA polymerases Polymerases are included.

[0130] Some nucleic acid replication and amplification methods may involve transcription as part of the process. Among the methods are transcription-mediated amplification (TMA) known in the art, as well as nucleic acid sequence-based amplification. (NASBA), DNA and RNA sequencing and other nucleic acid extension reactions. Those skilled in the art will recognize that other methods, including variations on transcription reactions, that may be developed in the future may be used in place of the transcription method. Moreover, it will be appreciated that the method may be used in conjunction with a transfer method.

[0131] therapeutic use The present invention also provides an initiating capped oligonucleotide for use as a therapeutic agent in a pharmaceutical composition. The method comprises introducing RNA containing a nucleotide primer into a cell to treat a medical condition of the cell. or an initiating capped oligonucleotide primer is added to the RNA. These RNAs can be introduced into cells to produce proteins that can have a therapeutic effect on the host cell. An mRNA containing an initiating capped oligonucleotide primer for generating a protein Consider the generation of NA.

[0132] Treating conditions using RNA containing an initiating capped oligonucleotide primer One method for this purpose is to use a capped oligonucleotide primer containing an initial capped oligonucleotide primer of formula I. The RNA or a composition comprising such RNA is Administered to a subject having or suspected of having a condition that can be reduced or eliminated. The method includes the step of:

[0133] The RNA containing the initiating capped oligonucleotide primer of the "compound" of formula I is , at a concentration of 4 mg / ml or less, in a pharma- ceutically acceptable carrier and / or a pharma- ceutically acceptable carrier. When formulated into a salt form, the therapeutic efficacy of the compound is higher in untreated individuals than in untreated individuals using a pharma- ceutical acceptable carrier alone. At least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or is furthermore effective in resulting in a reduction in symptoms and / or symptomatology.

[0134] To treat a particular condition, the compound may be administered by injection or other suitable route known to those of skill in the art. For parenteral administration, pharmaceutical compositions can be formulated. Injectable compositions for parenteral administration are usually The active compound is contained in a suitable solution, such as sterile saline, and / or a pharmaceutical carrier. The compositions may also be present in lipid or phospholipid form, in liposomal suspensions, or in aqueous emulsions. It may be formulated as a suspension in water.

[0135] Methods for preparing various compositions and / or formulations are known to those skilled in the art and are described in Re. W.Mington's Pharmaceutical Sciences (19th ed.) (see Williams & Wilkins, 1995). , a pharma- ceutical , safe, effective, and effective method for increasing expression of a desired protein in a target cell or tissue. The composition contains an effective amount of a selected compound.

[0136] In some embodiments, the pharmaceutical composition comprises at least 0.1% (w / v) of the compound. As described above, in some embodiments, the pharmaceutical composition contains more than 0.1% In some embodiments, the pharmaceutical composition contains up to about 10%. In some embodiments, the pharmaceutical composition contains up to about 5%. The selection of an appropriate concentration depends on the desired dosage, frequency and and the method of delivery of the active agent.

[0137] For the treatment of a subject, such as a mammal or human, the subject's weight and overall health, treatment The dosage will be determined based on factors such as the condition being treated, the severity of the symptoms, and other factors. The dosage and concentration are determined to provide the desired benefit while avoiding any undesirable side effects. The typical dosage of the subject compounds is about 0.0005 to 500 mg / kg for human patients. Daily ranges, in some embodiments, range between about 1 and 100 mg / day. For example, Higher doses include, for example, 50-100, 75-100, or 50-75 mg / day. , lower dosages include, for example, 1 to 50, 25 to 50 or 1 to 25 mg per day.

[0138] Various aspects of the present invention will now be illustrated by the following non-limiting examples, which are provided for illustrative purposes only. However, it is not intended to be limiting of any implementation of the present invention. It will be understood that variations and modifications can be made without departing from the spirit and scope of the invention. If desired, the reagents and components described herein can be synthesized or commercially obtained. It is easy to understand how to do this. EXAMPLES

[0139] 7-Methylguanosine 5-diphosphate from guanosine 5'-diphosphate (pp 7m Key of G (Figure 1) A stirred solution of guanosine 5'-diphosphate (2.5 mmol) in 40.0 mL of water was diluted with vinegar. Acid is added to adjust the pH of the solution to 4.0. Dimethyl sulfate (4.0 mM) is added to this mixture. L) was added dropwise over a period of 30 minutes and the reaction mixture was cooled using 0.1 M NaOH solution. The reaction mixture is stirred at room temperature for 4 hours while maintaining the pH of the reaction mixture at about 4.0. The mixture was extracted with CH2Cl2 (3 x 50 mL) to remove unreacted dimethyl sulfate. The aqueous layer was diluted to 500 mL with water and adjusted to pH 6.5 with 1M TEAB. The mixture was then loaded onto a DEAE Sephadex column (3 x 50 cm). The product is eluted using a linear gradient (3 L) of AB, pH 7.5. 7m G( The fractions containing the triethylammonium salt are pooled, evaporated and dried under high vacuum. A fine white powder is obtained (yield: 80%). A similar procedure was also performed in Bioorgan.M. ed. Chem. Letters, vol. 17: 5295-5299 (2007) It has been done. EXAMPLES

[0140] p.p. 7m 7-Methylguanosine 5'-diphosphate imidazolide (Im-pp) from G 7m G Preparation of (Figure 2) In anhydrous DMF (20 mL), 7m Triethylammonium salt of G (0.4mmol l) in a solution of imidazole (4 mmol), triphenylphosphine (2 mmol) and 2 The reaction mixture was then reacted with 2'-dipyridyl disulfide (2 mmol) for 8 hours. The crude Im-pp was extracted by pouring into 50 mL of 0.2 M sodium perchlorate solution. 7m G precipitates. The mixture is cooled to -20°C and the resulting precipitate is collected by centrifugation. The Im-pp is washed with acetone (3 x 50 mL) and dried under high vacuum. 7m G The isolated yield of is approximately 100%. A similar procedure can be used to prepare Nucleosides, Nucleoside, Leotides, and Nucleic Acids Vol. 24: 1131-1134 pp. (2005) and J. Org. Chem. 64:5836-5840 (19 This was disclosed in 1999. EXAMPLES

[0141] 3'-O-methylguanosine 5'-phosphate (pG(3 ’Ome Preparation of (Figure 3) 3'-O-methylguanosine (10 mmol) was dissolved in triethyl phosphate ( The mixture is cooled to 0° C. in an ice-water bath and phosphorus oxychloride (30 mL) is added. mol) is added and the mixture is stirred at room temperature under argon for 3 hours. The reaction was quenched by slowly adding 0.05 mL of ethyl acetate (pH 8.5) with stirring. The mixture is stirred for 8 hours and diluted with 1 L of water. The resulting solution is dissolved in DEAE Se Load onto a phadex column (3 × 40 cm) and run a linear gradient of 0 to 100 mL of TEAB (pH 7.5). Elute the product using 0.05-1.0M (3 L). Combine fractions containing pure product. The mixture was evaporated to a solid residue and coevaporated with methanol (4 × 50 mL) to give pG( 3’Ome ) (triethylammonium salt) is obtained as a white solid (60% yield) A similar procedure is disclosed in U.S. Patent Application No. 2012 / 0156751. EXAMPLES

[0142] pG( 3’Ome ) to prepare 3'-O-methylguanosine 5'-phosphorylimidazolide (I m-pG( 3’Ome Preparation of (Figure 4) In anhydrous DMF (25 mL), pG( 3’Ome ) triethylammonium salt (0. 5mmol) in imidazole (5mmol), triphenylphosphine (2.5mmol) l) and 2,2'-dipyridyl disulfide (2.5 mmol) for 5 hours. The reaction mixture was poured into 400 mL of a 0.2 M sodium perchlorate in acetone solution. By this, crude Im-pG( 3’Ome ) precipitates. The mixture is cooled to -20°C. The resulting precipitate was collected by centrifugation and washed with acetone (3×60 mL); Dry under high vacuum (yield: 100%). A similar procedure is described in U.S. Patent Application No. 2012 This is disclosed in US Pat. No. 5,399,315. EXAMPLES

[0143] Im-pG( 3’Ome 3'-O-methylguanosine 5'-diphosphate (ppG( 3’Ome Preparation of (Figure 5) Im-pG( 3’Ome ) (7.0 mmol), Solid zinc chloride (14.0 mmol) is added in small portions. The mixture is stirred until the solid dissolves. Stir under argon for 15 min. 1 M tributylammonium phosphate in DMF (40 mL) A solution of 1,2-dichlorophenyl ether is added and the mixture is stirred at room temperature. After 5 hours, the mixture is diluted with 200 mL of water. The aqueous layer is diluted with water (1 L) and extracted with dichloromethane (2 x 200 mL). , loaded onto a DEAE Sephadex column (5 × 40 cm), and diluted with 0.05–1.0 M Elute with a linear gradient (6 L) of TEAB (pH 7.5). The fractions were combined, evaporated, and coevaporated with methanol (4 × 50 mL) to give ppG( 3’ Ome ) (triethylammonium salt) is obtained as a white solid (yield: 60%). A similar procedure is disclosed in U.S. Patent Application No. 2012 / 0156751. EXAMPLES

[0144] ppG( 3’Ome ) from 7-methyl-3'-O-methylguanosine 5-diphosphate (p p 7m G( 3’Ome Preparation of (Figure 6) ppG( 3’Ome ) (triethylammonium salt; 3.0 mmol A solution of 1,000 ml of sulphuric acid is prepared and the pH of the solution is adjusted to 4.0 by adding glacial acetic acid. Dimethyl acetate (10.0 mL) was added dropwise over 30 min, and 0.1 M NaOH solution was used. The reaction mixture is stirred at room temperature for 4 hours while maintaining the pH at 4.0±0.5 using ethanol. After a period, the reaction mixture was extracted with CH2Cl2 (3 × 150 mL) to remove unreacted di-sulfate. The aqueous layer was adjusted to pH 5.5, diluted with water (500 mL), and purified with DEAE. Load the product onto a Sephadex column (3 x 50 cm). Dilute the product with 0 to 1.0 M TEA. B, pH 7.5 linear gradient (3 L). 7m G 3’Ome ( The fractions containing the triethylammonium salt are pooled, evaporated and dried under high vacuum. A fine white powder is obtained (yield: 80%). A similar procedure was performed to prepare RNA Vol. 9:110. 8~1122 pages (2003); Nucleosides, Nucleotides&N Ucleic Acids vol. 25:337-340 (2006); and U.S. Pat. It is disclosed in application number 2012 / 0156751. EXAMPLES

[0145] p.p. 7m G( 3’Ome ) from 7-methyl-3'-O-methylguanosine 5-diphosphate Imidazolide (Im-pp 7m G( 3’Ome Preparation of (Figure 7) Im-pp 7m G( 3’Ome For the preparation of A similar procedure was described in RNA 14:1119-1131 (2008). As shown. EXAMPLES

[0146] pN( 2’-OR1 ) General procedure for the preparation of pN dinucleotides (R1 = H or Me) Procedure (Figure 8) 10 mL of acetonite containing 2.5 molar equivalents of activator (tetrazole) In a 1.0 mmol solution, phosphoramidite monomer (i) (1.0 mmol) and 2',3',N -Protected nucleoside (ii) (1.0 mmol) was reacted. After stirring at room temperature for 60 minutes, The intermediate product is then oxidized from P(III) to P(V) state with iodine to give dichloromethane. The organic layer was extracted with sodium sulfate (200 mL) and brine (200 mL). Dry over methanol and evaporate to a solid foam (intermediate (iii)).

[0147] To remove the DMT-protecting group, intermediate (iii) was dissolved in 10 mL of 80% acetic acid. After the reaction is complete (approximately 1-2 h), the mixture is evaporated and washed with methanol (5 × 30 mL). The acetic acid is removed by coevaporation with 5% methanol in dichloromethane as the eluent. The crude 5'-OH dimer (iv) was isolated by silica gel chromatography using Refine.

[0148] The 5'-OH dimer (iv) (1.0 mmol) was dissolved in 2 equivalents of acetonitrile in 10 mL of acetonitrile. amount of bis-cyanoethyl-N,N-diisopropyl-phosphoramidite and 2 equivalents It is phosphitylated using an activator (tetrazole). After stirring for 0 min, the 5'-phosphorylated dimer was converted from P(III) to P(V) using iodine. The mixture was oxidized to the HCl state and extracted with dichloromethane (150 mL) and brine (150 mL). The organic layer was evaporated to an oily residue and coevaporated with methanol (2 × 30 mL). The mixture was dissolved in 12 mL of methanol and concentrated ammonia (12 mL) was added. , pN 2’OR1 At room temperature for 48 hours until deprotection of the pN dimer (v) is complete. The mixture is evaporated and coevaporated with methanol (2 x 30 mL).

[0149] When R=methyl, the crude dimer (v) was purified by anion exchange and reverse phase chromatography. Purify directly by filtration (step 5). Evaporate the fractions to obtain the final pN 2’OR1 The pN dimer (v) (v; R1 = methyl; triethylammonium salt) was obtained as a white solid. (35% overall yield).

[0150] When R=TBDMS, the crude dimer (v) is treated with a HF-3TEA mixture. (Step 4B) to remove the 2'-OTBDMS protecting group (Org. Biomol. Chem. 3:3851-3868 (2005) and Nucl. Acids Res. 22:2430-2431 (1994). When the reaction is complete, The mixture was diluted with 0.05M TEAB and subjected to anion exchange and reverse phase chromatography. The fractions are evaporated to give a final pN 2’OR1 pN dimer ( v) (R1 = H; triethylammonium salt) is obtained as a white solid (30% overall yield). EXAMPLES

[0151] Synthesis of an initial oligonucleotide having a cap0, cap1 or cap2 structure General Procedure for (Examples Use 5'-Phosphorylated Dinucleotides) (Fig. 9) A. Approach 1: Im-pp in DMF (50 mL) 7m G( 3’Ome ) or Im-pp 7m G( 2 mmol; in the form of sodium salts) and 5′-phosphorylated dinucleotides (1 mmol; triethylammonium salt) at 35°C while stirring the mixture. 2 (1 g) is slowly added. After 24 h, a 25 mM solution of EDTA in water (500 The reaction is quenched by the addition of 1 mL of 1M sodium bicarbonate and neutralized. The mixture was diluted to 1 L with water and loaded onto a DEAE Sephadex column (3 x 50 cm). The product was isolated using a linear gradient (2 L) from 0 to 1 M ammonium bicarbonate, pH 7.2. The fractions containing the pure product were pooled, evaporated and dried under high vacuum. A fine white powder is obtained (yield: 60%). A similar approach is described in U.S. Pat. No. 2012 / 0156751; Bioorg. Med. Chem. Lett. Vol. 17 :5295-5299 (2007) and RNA Vol. 14:1119-1131 ( This was disclosed in the 2008 issue of the IEEE.

[0152] B. Approach 2: Im-pp 7m G( 3’Ome ) or Im-pp 7m G (2 mmol) was dissolved in MnCl N-methylmorpholine buffer (0.2 M, pH 7.0, 10 mL) and dissolve solid 5'-phosphorylated dinucleotide (1 mmol; triethylamine) in 10 mL of 100 mM NaCl. The reaction is stirred at room temperature. After 24-40 hours, the reaction is cooled to 10 The mixture is quenched with 1 mL of a 0.25 M solution of EDTA. The product was then loaded onto an ex column (3 × 50 cm). Elute using a linear gradient (2 L) of pH 7.2. Fractions containing pure product were pooled. Solvent, evaporate and dry under high vacuum to give a fine white powder (50% yield). A similar approach has been reported in Bioorganic & Medicinal Chemistry. Volume 21: 7921-7928 (2013), Nucleic Acids Res earch Vol. 37: pp. 1925-1935 (2009); J.Org.Chem. No. 64:5836-5840 (1999). (Note: 1. Example 9 Instead of the 5'-phosphorylated dinucleotide as exemplified in Monomer, tetramer, pentamer, hexamer, heptamer, octamer, nonamer or decamer oligonucleotide 2. Prepare an initial capped oligonucleotide with Cap 0 To achieve this, the 5'-phosphorylated oligonucleotide has a 2' 3. Starting capped with Cap1, e.g., pApG, which does not have an -O-methyl group To prepare the oligonucleotide, a 5'-phosphorylated oligonucleotide is prepared by first '-nucleoside residue, e.g., pA( 2’O me 4. To prepare the initial capped oligonucleotide having cap 2 The 5'-phosphorylated oligonucleotide has a phosphoryl group on the first and second 5'-nucleoside residues. pA( 2’Ome )pG( 2’Ome )pG). EXAMPLES

[0153] Structure of an initial capped oligonucleotide primer according to formula I FIG. 10 shows an initial capped oligonucleotide used in the examples according to structure I. The structure of the primer is shown below. A) m7 G 3’Ome pppG, B) m7 GpppG 2’Om e pG, C) m7 G 3’Ome pppG 2’Ome pG, D) m7 GpppA 2’Ome pG, E) m7 G 3’Ome pppA 2’Ome pG, F) m7 GpppC 2’Ome p G, G) m7 G 3’Ome pppC 2’Ome pG, H) m7 GpppA 2’Ome p.G. 2’Ome p.G. EXAMPLES

[0154] In vitro transcription using ARCA primers Double-stranded DNA transcription template encoding firefly luciferase by polymerase chain reaction The +1 template nucleotide was 2'-deoxycytidine. The mixture consisted of 25 μg / mL transcription template, 40 mM Tris-HCl (pH 8.0), 27 mM MgCl2, 2mM spermidine, 10mM DTT, 0.002% Triton X-100, 1000 units / mL mouse RNase inhibitor (New England Biolabs Catalog No. M0314), 2 units / mL inorganic pyrophosphatase (New England Biolabs Catalog No. M2403), 4000 units 1000 / mL T7 RNA polymerase (New England Biolabs catalog) No. M0251), 6mM ARCA( m7 G 3’Ome pppGG), 1.5 mM Constructed with 7.5 mM GTP, 7.5 mM ATP, 7.5 mM CTP and 7.5 mM UTP. The reaction was incubated for 2 hours at 37°C. HCl (pH 7.6), 2.5 mM MgCl2, 0.5 mM CaCl2, and 100 Units / mL DNase I (New England Biolabs catalog no. The mixture was supplemented with 100 mM NaCl (No. M0303) and incubated at 37°C for 1 hour. Neasy Maxi kit (Qiagen catalog no. 75162) was used according to the manufacturer's instructions. The mRNA was eluted with water and the solution was diluted with 50 mM Bis- Tris-propane HCl (pH 6.0), 1 mM MgCl2, 0.1 mM ZnCl 2 and 250 units / mg Antarctic phosphatase (New England nd Biolabs catalog number M0289) The reaction was incubated at 37°C for 1 hour. The resulting mRNA was purified using RNeasy M axi kit (Qiagen catalog no. 75162) according to the manufacturer's instructions. The mRNA was purified using 100% glycerol. EXAMPLES

[0155] In vitro transcription using trimer initiation capped oligonucleotide primers Some initiating capped oligonucleotide primers utilized in the transcription are listed in Table 1. The double-stranded firefly luciferase specific for each trimer was synthesized by polymerase chain reaction. These templates are generated by splitting DNA transcription templates that differ at template nucleotides +1 and +2. For a given trimer, template nucleotide +1 is B1 (transcript nucleotide +2). The nucleotide that was destined to become nucleotide +1 was complementary to the template nucleotide. Leotide +2 is B 10 (Nucleotide destined to be transcript nucleotide +2 The transcription reaction was performed at 25 μg / mL transcription factor (TFA) and 100 μg / mL transcription factor (TFA). Template: 40 mM Tris-HCl (pH 8.0), 27 mM MgCl2, 2 mM S Permidine, 10 mM DTT, 0.002% Triton X-100, 1000 units Units / mL mouse RNase inhibitor (New England Biolabs catalog) No. M0314), 2 units / mL inorganic pyrophosphatase (New England d Biolabs Catalog No. M2403), 4000 units / mL of T7 RNA Polymerase (New England Biolabs Catalog No. M0251) and and 6 mM initiating capped oligonucleotide primer, 1.5 mM GTP and 7 Assemble with 0.5 mM each of ATP, CTP, and UTP. This primer / NTP formulation is referred to as primer / NTP formulation 1. Other polymerases, such as T7, T3 or SP6 RNA polymerase, can be used By using NA polymerase instead of the respective promoters It is clear that the transcription reaction mixture can perform the same function as the GFP gene. The reaction mixture was incubated with 10 mM Tris-HCl (pH 7.6), 2.5 mM M MgCl2, 0.5 mM CaCl2 and 100 units / mL DNase I (New England Biolabs Cat. No. M0303) and incubated at 37°C. The resulting mRNA was purified using the RNeasy Maxi Kit (Q Purify using 100% glycerol (Cat. No. 75162) according to the manufacturer's instructions. The mRNA was eluted with water and the solution was diluted with 50 mM Bis-Tris-propane HCl (pH 6.0), 1mM MgCl2, 0.1mM ZnCl2 and 250 units / mg a Intactic phosphatase (New England Biolabs catalog no. The reaction mixture was incubated at 37°C for 1 hour. The obtained mRNA was incubated with RNeasy Maxi kit (Qiagen Catalog). Purify the mRNA using the PCR product (log number 75162) according to the manufacturer's instructions. Elute with water. EXAMPLES

[0156] Tetramer initiation capped oligonucleotide primer 7m G 3’Ome pppA 2’Om e p.G. 2’Ome In vitro transcription using pG In transcription, an initiating capped oligonucleotide primer 7m G 3’OMe ppp A 2’OMe p.G. 2’OMe pG was used. Transcription was carried out as in Example 11 as follows: The polymerase chain reaction was used to generate double-stranded DNA fragments specific for the tetramers. A transcription template was prepared. The template nucleotide +1 (2'-deoxythymidine) was adenosine The first nucleotide of the primer destined to be transcript nucleotide +1 Similarly, the template nucleotide +2 (2'-deoxycytidine) was complementary to The second nucleotide of the primer destined to become guanosine (transcript nucleotide +2) Similarly, the template nucleotide +3 (2'-deoxycytidine) was complementary to the template nucleotide +3 (2'-deoxycytidine). The nucleotide sequence of the primer destined to become guanosine (nucleotide +3 of the transcript) The transcription reaction consisted of 25 μg / mL transcription template, 40 mM Tris-HCl (pH 8.0), 27 mM MgCl2, 2 mM spermidine, 10mM DTT, 0.002% Triton X-100, 1000 units / mL Mouse RNase inhibitor (New England Biolabs Catalog No. M03 14), 2 units / mL inorganic pyrophosphatase (New England Biol abs Catalog No. M2403), 4000 units / mL T7 RNA polymerase (New England Biolabs Catalog No. M0251), 6 mM starting calcium Capped Oligonucleotide Primers 7m G 3’OMe pppA 2’OMe p.G. 2’O Me pG, 1.5 mM GTP, 7.5 mM ATP, 7.5 mM UTP and 7.5 The constructs were assembled using 1 mM CTP. Those skilled in the art will recognize that T7, T3 or SP6 RNA polymerases Other polymerases, such as T7 RNA polymerase, can be used in place of T7 RNA polymerase. It is clear that the same function can be performed by using each of the promoters. The transcription reaction mixture was incubated at 37°C for 2 hours. M Tris-HCl (pH7.6), 2.5mM MgCl2, 0.5mM CaCl 2 and 100 units / mL of DNase I (New England Biol. The mixture was supplemented with PBS (catalog number M0303) and incubated at 37°C for 1 hour. mRNA was purified using the RNeasy Maxi kit (Qiagen catalog no. 75162). The mRNA was purified using 100% PBS according to the manufacturer's instructions. 1 mM Bis-Tris-propane HCl (pH 6.0), 1 mM MgCl2, 0.1 mM ZnCl2 and 250 units / mg antarctic phosphatase (Ne by adjusting the The reaction was incubated at 37°C for 1 hour. The resulting mRNA was Neasy Maxi kit (Qiagen catalog no. 75162) was used according to the manufacturer's instructions. The purification was performed using the kit according to the manufacturer's instructions. The mRNA was eluted with water. EXAMPLES

[0157] 2'-deoxythymidine and 2'-deoxycytidine at template positions +1 and +2, respectively. A transcription template having a nucleotide residue is used. m7 GpppA 2’Ome pG initiation capped oligonucleotide In vitro transcription using nucleotide primers 2'-deoxythymidine and 2'-deoxysuccinimidine at template positions +1 and +2, respectively. A double-stranded firefly luciferase DNA transcription template with cysteine ​​residues was used. Two types of transcription The reaction mixture consisted of 25 μg / mL transcription template, 40 mM Tris-HCl (pH 8.0), 27 2 mM MgCl2, 2 mM spermidine, 10 mM DTT, 0.002% Trito n X-100, 1000 units / mL mouse RNase inhibitor (New Engl and Biolabs Catalog No. M0314), 2 units / mL inorganic pyrophosphatase Tase (New England Biolabs Catalog No. M2403), 4000 Units / mL of T7 RNA polymerase (New England Biolabs) The two transcripts were assembled using the same primer set (catalog number M0251). The amount of nucleotide, the amount of NTP and the identity of the NTP were different. (Nucleic Acids Symposium Series No. 5) 3:129(2009)). This transcription reaction was 6 mM m7 GpppA 2’Ome pG-initiated capped oligonucleotide primer, It contained 0.9 mM GTP and 7.5 mM ATP, CTP and UTP. In subsequent examples, this primer / NTP formulation is referred to as primer / NTP formulation 2. In this formulation, the initiating oligonucleotide primer is in >6-fold excess over GTP. The second transfer was m7 GpppA 2’Ome pG start capped oligo Nucleotide primers, 5 mM GTP, ATP, CTP and pseudouridine triphosphate In the subsequent examples, this primer / NTP formulation was used as a primer. This is called Primer / NTP Formulation 3. In Primer / NTP Formulation 3, commercially useful amounts of The GTP concentration was increased to produce RNA. Other polymerases, such as RNA polymerase, can be used instead of T7 RNA polymerase. can be used to perform the same functions as by using their respective promoters. The transcription reaction mixture is incubated at 37° C. for 2 hours. 10mM Tris-HCl (pH 7.6), 2.5mM MgCl2, 0.5mM CaCl2 and 100 units / mL DNase I (New England Biolabs Catalog No. M0303) and incubated at 37°C for 1 hour. The resulting mRNA was purified using the RNeasy Maxi kit (Qiagen Cat. No. 75 162) according to the manufacturer's instructions. The mRNA was eluted with water and purified. The solution was adjusted to 50 mM Bis-Tris-propane HCl (pH 6.0), 1 mM MgCl 2, 0.1 mM ZnCl2 and 250 units / mg Antarctic Phosphata The enzyme (New England Biolabs Catalog No. M0289) was used to The reactions were incubated at 37°C for 1 hour for primer / NTP formulation 2. , and primer / NTP formulation 3 was incubated for 3 hours. The resulting mRNA was RNeasy Maxi kit (Qiagen catalog no. 75162) was purchased from the manufacturer. The primers were purified according to the manufacturer's instructions. The mRNA was eluted with water. The purified transcript yields using methods 1 and 2 were 0.7 mg / ml, respectively. The primers / N were 3.9 mg / mL and 3.9 g / 1 mL transcription reaction. The yield using NTP formulation 2 was significantly higher than that obtained using primer / NTP formulation 1. was excellent. EXAMPLES

[0158] With a transcription template having cytidine residues at template positions +1 and +2, m7 GpppA 2’Om e In vitro transcription using pG-initiated capped oligonucleotide primers The +1 and +2 template nucleotides are cytidines, and therefore m7 GpppA 2’ Ome Duplexes that are not completely complementary to the pG initiation capped oligonucleotide primer A firefly luciferase DNA transcription template was used. Two transcription reactions were run at 25 μg / mL Transcription template, 40 mM Tris-HCl (pH 8.0), 27 mM MgCl2, 2 mM Spermidine, 10 mM DTT, 0.002% Triton X-100, 1000 Units / mL of mouse RNase inhibitor (New England Biolabs) Catalog number M0314), 2 units / mL inorganic pyrophosphatase (New England and Biolabs Catalog No. M2403), 4000 units / mL of T7 R NA polymerase (New England Biolabs Catalog No. M0251) The two transcripts were assembled using the initiating capped oligonucleotide and NTPs. The first transcript was identified by the method described by Ishikawa et al. (Nucleic Acids Symposium Series No. 53:129 (2009)) This transcription reaction contained primer / NTP formulation 2. The transcription of the nucleotide sequence was carried out using primer / NTP format 3. Those skilled in the art will recognize that the nucleotide sequence of ... Other polymerases, such as RNA polymerase, can be used in place of T7 RNA polymerase. can be used together to perform the same functions as by using their respective promoters. The transcription reaction mixture was incubated at 37°C for 2 hours. The material was 10 mM Tris-HCl (pH 7.6), 2.5 mM MgCl2, 0.5 mM M CaCl2 and 100 units / mL DNase I (New England Supplement with 100 mM NaCl (Biolabs Catalog No. M0303) and incubate at 37°C for 1 hour. The resulting mRNA was purified using the RNeasy Maxi kit (Qiagen Catalog No. 7 5162) according to the manufacturer's instructions or by reversed-phase high performance liquid chromatography. The mRNA was purified by chromatography. HCl (pH 6.0), 1 mM MgCl2, 0.1 mM ZnCl2, and 250 mM Nit / mg Antarctic phosphatase (New England Biola The reaction was dephosphorylated by adjusting the pH to 5.0 (BS catalog number M0289). 1 hour for primer / NTP formula 2 and 3 hours for primer / NTP formula 3 The resulting mRNA was purified using the RNeasy Maxi kit (Qiagen) and purified using the RNeasy Maxi kit (Qiagen). The product was purified using ribosomal RNA (catalog number 75162) according to the manufacturer's instructions. The NA was eluted with water. The transcription yields purified using primer / NTP formulations 1 and 2 were , 0.6 milligrams per milliliter transcription reaction (mg / mL) and 3.9 g, respectively. The yield using primer / NTP formulation 2 was 1 / 1 mL of primer / NTP. This was significantly superior to that obtained with TP formulation 1. EXAMPLES

[0159] Translation of mRNA in Huh-7 cells (Fig. 11) Luciferase mRNA generated using an initiating capped oligonucleotide primer The translation activity of A was assessed in triplicate in cultured hepatocytes. Huh-7 cells were cultured in 10% FB Supplemented with S, L-glutamine, non-essential amino acids and penicillin / streptomycin The cells were cultured in DMEM at 37°C in a 5% CO2 atmosphere. For comparison, cells were also transfected with ARCA. The cells were transfected with the Cap0 luciferase mRNA prepared according to the method described above. At each time point, cells were harvested and luciferase activity was assayed using the ONE-Glo luciferase assay. System kit (Promega catalog number E6120) was used according to the manufacturer's recommendations. Luminescence was measured using a GloMax-Multi+ detection system instrument as recommended by the manufacturer. Luciferase activity was measured for all mRNAs tested. The initiating capped oligonucleotide primer or the 3'-O-mer was used to detect the nucleotide sequence (Figure 11). mRNA generated using chill-initiated capped oligonucleotide primers was analyzed using ARC The fact that A-capped RNAs are translated with similar efficiency suggests that they are cotranscriptionally efficient. This shows that capping occurs efficiently. EXAMPLES

[0160] ARCA or initiating capped oligonucleotide using primer / NTP formulation 1 Capping efficiency of mRNA produced by co-transcriptional capping with a primer Assay to examine the rate (Figures 12A-12H) For each of the initiating capped oligonucleotide primers tested, liquid chromatography was performed. A sufficient amount of mRNA to be detected by liquid chromatography-mass spectrometry (LC-MS) was The small fragments were subjected to a capping assay, in which the small fragments were 5' The mRNA was cleaved from the termini and analyzed by LC-MS. To facilitate this, antarctic phosphatase (New England Biosciences) was used. olabs Catalog No. M0289) to obtain uncapped monophosphate, diphosphate, and The phosphate and triphosphate were converted to 5'OH. The phosphatase-treated mRNA was then The purified RNA was subjected to LC-MS analysis. The traces are shown for uncapped (5'OH after phosphatase treatment) and capped 1. The corresponding LC peaks are shown with their measured masses. The inset shows a schematic of the cleavage on the transcription template. The alignment of the initial oligonucleotide primers is shown. Note that the capping initiator nucleotide is shown base-paired with the template nucleotide. The subscript "m" indicates the 2'-O-methyl group, and the superscript "m7" indicates the base methyl group. For comparison, mRNAs cotranscriptionally capped with ARCA were The capping efficiency was estimated using the following formula (the number of capped (intensity of capped peak) / [(intensity of capped peak)+(intensity of 5'OH peak)] The actual cap formation percentages in FIG. m7 G 3’Ome pppG =79%, B) m7 GpppG 2’Ome pG=89%, C) m7 G 3’Ome pppG 2’Ome pG=87%, D) m7 GpppA 2’Ome pG=99%, E) m7 G 3’ Ome pppA 2’Ome pG=99%, F) m7 GpppC 2’Ome pG=98%, G) m7 G 3’Ome pppC 2’Ome pG=97%, H) m7 GpppA 2’Ome p.G. 2’Ome pG=50%. In each case, the starting trimer capping oligo Capping of co-transcriptionally capped transcripts using a nucleotide primer Efficiency is m7 G 3’Ome Higher than that observed with pppG(ARCA). It was. EXAMPLES

[0161] Insertion of 2'-deoxythymidine and 2'-deoxycytidine residues at template positions +1 and +2 with cytidine residues at template positions +1 and +2 versus a transcription template with m7 G pppA 2’Ome Comparison of capping using pG-initiated capped oligonucleotides (Figures 13A to 13D) The mRNA produced in Examples 14 and 15 was subjected to a capping assay. , 2'-deoxythymidine and 2'-deoxycytidine residues at template positions +1 and +2 Capped transcripts with cytidine residues at template positions +1 and +2 versus capped transcripts with cytidine residues at template positions +1 and +2 The relative efficiency and specificity of the synthesis were investigated by liquid chromatography-mass spectrometry (LC-MS). A sufficient amount of mRNA to be detected by the capping assay was subjected to the capping assay. In the assay, small fragments are cleaved from the 5' end of full-length mRNA and resolved by LC-MS. Prior to the cleavage of the mRNA, the Antarctic Hormone was added to facilitate the analysis. sphatase (New England Biolabs Catalog No. M0289) The uncapped monophosphates, diphosphates, and triphosphates were converted to 5'OH by treatment with The phosphatase-treated mRNA was then cleaved and purified. The uncapped and capped esters were subjected to LC-MS analysis. Figure 13 shows the LC traces. The LC peak corresponding to group 1 is shown with the observed mass. The subscript "m" indicates the alignment of the starting oligonucleotide primer at 2 13A and 13B show the -O-methyl group, and the superscript "m7" indicates base methylation. 3B transcripts were prepared using primer / NTP recipes 2 and 3, respectively. 2'-deoxythymidine and 2'-deoxycytidine at nucleotides +1 and +2 13C and 13D show the primer / NTP Using recipes 2 and 3, 2'-deoxycytidine was added to template nucleotides +1 and +2. The transcript was transcribed from a template having the following structure: m7 GpppA 2’Ome pG start oligo If the nucleotide is perfectly complementary to the +1 and +2 nucleotides, then The main products were: m7 GpppA 2’Ome The desired template starting with pG was used The transcript was the ChAP1 transcript (Figures 12A and 12B). The capping efficiency was estimated using the formula (intensity of the capped peak) / [(capped The primer was used to measure the intensity of the 5'OH peak (intensity of the 5'OH peak formed) + (intensity of the 5'OH peak). The capping efficiencies using primer / NTP formulation 2 and primer / NTP formulation 3 were 99% and 96%. Only a few abnormal initiation products were detected. In contrast, the template When nucleotides +1 and +2 are cytidines, m7 GpppA 2’Ome p.G. There is no perfect complementarity between the starting oligonucleotide and these template nucleotides. C and 12D are initiating capped oligonucleotides that start in two registers. In the first register, a 3' guanosine-initiated capped oligonucleotide is The +1 cytidine residue pairs with the +1 template cytidine to produce a 5' adenosine ( The transcripts were generated with caps (cap 1+A). "|" indicates a capped open Note that this indicates the base pairing of the initial nucleotide with the template nucleotide. In the second register, the 3' guanosine-initiated template nucleotide positions are shown. The capped oligonucleotide residue pairs with the +2 template cytidine and the +1 initiator cap The adenosine-tagged oligonucleotide forms a perfect hybrid with the +1 template nucleotide This is because the template 5' guanosine is replaced with a non-template adenosine. The capping efficiency was estimated using the following formula: Intensity of capped peak (cap 1+A) + (cap 1G to A) / [(cap Intensity of capped peaks (cap1+A) + (cap1G to A) + (5' The calculated capacitance in Figures 13C and 13D was calculated using the intensity of the OH peak. The capping efficiency was 97 and 77%. The fidelity of transcription initiation measured was determined by the initiation capped oligonucleotide primers. The surprising result was that the DNA fragments were quite highly complementary to the +1 and +2 nucleotides of the template. Furthermore, our method allows us to identify the NTP that drives cap formation. Efficient capping can be achieved without reducing the concentration of ribozyme, allowing for much higher transcription yields. Therefore, the capping method described by the present inventors is similar to that of Ishikawa et al. It is different from and superior to the EXAMPLES

[0162] (Figures 14A to 14B) Insertion of 2'-deoxythymidine and 2'-deoxycytidine residues at template positions +1 and +2 A transcription template with cytidine residues at template positions +1 and +2 versus a transcription template with cytidine residues at template positions +1 and +2 m7 Gpp pA 2’Ome Fractionation of mRNA generated using pG-initiated capped oligonucleotides Comparison of translation in transformed THP-1 cells To evaluate the expression of luciferase mRNA prepared in Examples 14 and 15, To do this, mRNA was transfected into THP-1 cells (ATCC, Cat. No. TIB-202). THP-1 cells were transfected with 10% FBS, sodium pyruvate, ATCC formulated RPMI-164 supplemented with ethanol and penicillin / streptomycin 0 (ATCC, Cat. No. 30-2001) at 37°C in a 5% CO2 atmosphere. The cells were cultured with the phorbol ester 12-O-tetradecanoylphorbol-13-amine. Acetate (TPA; Cell Signaling Technologies, Catalog In the presence of 2E+05 mAb / well in a 24-well plate, After 72 hours of seeding, cells were seeded at 100 n / well. For comparison, cells were also transfected with ARCA mRNA. Transfect with Cap 0 luciferase mRNA produced by Initiate Twenty hours after transfection, cells were harvested and luciferase activity was measured using ONE -Glo Luciferase Assay System Kit (Promega Catalog No. E612 0) according to the manufacturer's recommendations. Luminescence was measured using a GloMax-Multi+ Measurements were performed using a detection system instrument according to the manufacturer's recommendations. Data are presented for six replicates. Graphed as mean + / - standard deviation from the mean. Data analyzed using unpaired t-test. Pairwise p-values ​​are shown. m7 GpppA 2’Ome pG initiating capped oligonucleotide and 2' to template positions +1 and +2 - A template containing 2'-deoxythymidine and 2'-deoxycytidine residues ("TC" template) versus The clones were generated using a transcription template that has cytidine residues at positions +1 and +2 ("CC" template). The translation of the transcripts was compared in THP-1 cells. A) Transcription was performed using primer / N A) primer / NTP formulation 2 or B) primer / NTP formulation 3. Both formulations were When used, translation in cultured THP-1 cells, as described in the present invention, m7 Gp pP A 2’Ome The pG-initiated capped oligonucleotide primer is The results were significantly superior when the primers were perfectly complementary to templates +1 and +2 ("TC" templates). In FIG. 14B, we also show that the TC template was used to generate the ARCA-based The activity of capped (cap0) mRNA was assessed, which is cotranscriptionally ARCA Cap is the current industry standard for generating capped mRNA. The RNA formed is m7 GpppA 2’Ome pG initiating capped oligonucleotide If the primer is perfectly complementary to template nucleotides +1 and +2 ("TC" template), The transcripts produced in the 14-kDa gene had significantly lower activity than those produced in the 14-kDa gene (type 1).

[0163] In summary, the examples presented herein show that the methods described herein: Compared to previously published methods, the following features were achieved: 1) higher yield, 2) higher degree of capping, and 3) higher template yield. Produce RNA with a combination of high fidelity of transcription and 4) excellent activity in cells This proves that.

[0164] The invention illustratively described herein is not limited to any compound not specifically disclosed herein. may be performed appropriately in the absence of any element(s), limitation(s) not specified. Thus, for example, the terms "a" and "an" and "the "He)" and similar references are incorporated herein by reference in the context of describing the present invention, particularly in the following patent claims: Unless otherwise indicated herein or clearly indicated by context, Unless otherwise stated, the terms "a," "the," and "the" should be construed to cover both the singular and the plural. The words "comprising," "having," and "including" g), "containing," etc. must be read broadly and without restriction. (e.g., meaning "including but not limited to"). The recitation of ranges of values ​​simply includes each separate value falling within the range, unless otherwise stated in the specification. Each of the separate values ​​is intended to serve as a shorthand way of referring to the individual values ​​of The present invention is incorporated herein as if each individual were recited in the above-mentioned publication. All methods described herein are intended to be illustrative unless otherwise indicated by the context. Unless expressly contradicted by the principles of the present invention, the methods and apparatus provided herein may be performed in any suitable order. The use of any and all examples or exemplary language (eg, "such as") is merely to illustrate, but not to limit, the invention. The present invention is not intended to be limited in any way by the appended claims, and unless otherwise stated, no limitations are placed on the scope of the present invention. Any non-claimed element in the specification is not intended to be limiting to the practice of the invention. Furthermore, nothing in this specification should be construed as necessarily indicating The terms and expressions used are used as terms of description and not of limitation. In the use of such terms and expressions, any equivalent or equivalents of the features shown and described are intended to be understood as meaningless. It is not intended to be exhaustive, but various modifications are possible within the scope of the invention as claimed. It is recognized that the present invention is therefore directed to specific embodiments and optional Although specifically disclosed with reference to features, the disclosed specification is not embodied therein. It is understood that modifications and variations of the present invention may be made by those skilled in the art and that such modifications and variations may be made without departing from the spirit and scope of the present invention. It should be understood that variations and modifications are contemplated within the scope of the present invention. The present invention is therefore described specifically with reference to certain embodiments and optional features. Although disclosed herein, modifications, improvements and equivalents of the invention embodied therein are within the scope of the present invention. It is understood that modifications, improvements and variations may be made by those skilled in the art and that such modifications, improvements and variations are within the scope of the present invention. It should be understood that any method that uses the method is contemplated as being within the scope of the present invention. The materials, methods and examples provided herein are representative of specific embodiments. They are illustrative and not intended as limitations on the scope of the invention.

[0165] The invention has been described broadly and generically herein. Each of the species and subgeneric groups also form part of the present invention. Conditions or conditions that remove any subject from the genus, whether or not specifically recited herein, includes the generic description of the invention, with negative restrictions.

[0166] Furthermore, when features or aspects of the invention are described in terms of Markush groups, The present invention also relates to any individual member or members of the Markush group. It will be appreciated that this is described in terms of a subgroup of bars.

[0167] All publications, patent applications, patents and other references mentioned herein are expressly incorporated by reference in their entirety to the same extent as if each were individually incorporated by reference. In case of conflict, the present specification, including definitions, will control.

[0168] Applicant hereby reserves the right to modify, distribute, and / or transfer any such articles, patents, patent applications, or other physical and electronic The right to physically incorporate into this application any and all materials and information derived from the technical documentation Reserved.

[0169] Other embodiments are within the scope of the following claims.

Claims

1. A modified initiating capped oligonucleotide primer comprising the following structure: 【Chemistry 1】 [In the formula, each of B 1 through B 10 is independently a natural, modified or non-natural nucleoside base; M is 0 or 1; L is 0 or 1; q 1 is 1, and each of q 2 to q 9 is independently 0 or 1; R 1 is H or methyl; R 2 and R 3 are independently H, OH, alkyl, O-alkyl, halogen, a linker or a detectable marker, and at least one of R 2 and R 3 is H, alkyl, O-alkyl, halogen, a linker or a detectable marker; Each of X 1 to X 13 is independently O or S; each of Y 1 to Y 12 is independently OH, SH, BH 3 , aryl, alkyl, O-alkyl, or O-aryl; each of Z 1 to Z 22 independently is O, S, NH, CH 2 , C(halogen) 2 or CH(halogen); Each of R 4 through R 12 is independently H, OH, OMe, or a detectable marker. Where: B 1 is perfectly complementary to the nucleoside base on the nucleic acid template at transcription template position +1; B 2 through B 9 , if present, are perfectly complementary to their respective nucleoside bases on the nucleic acid template at the transcription initiation site from position +2 onwards; B 10 , the last nucleoside base of the initiating capped oligonucleotide primer is perfectly complementary to the last hybridized transcription template nucleotide.].

2. q 1 = 1 and 2. The modified initial capped oligonucleotide primer of claim 1, wherein each of q 2 to q 9 =0 or 1.

3. q 2 to q 9 = 0, and 2. The modified initial capped oligonucleotide primer of claim 1, wherein B 10 hybridizes to template nucleotide 2.

4. q 1 to q 2 = 1, q 3 to q 9 =0, and 2. The modified initial capped oligonucleotide primer of claim 1, wherein B2 and B10 hybridize to template nucleotides 2 and 3, respectively.

5. q 1 to q 3 = 1, q 4 to q 9 =0, and 2. The modified initial capped oligonucleotide primer of claim 1, wherein B2, B3 and B10 hybridize to template nucleotides 2, 3 and 4, respectively.

6. q 1 to q 4 =1, q 5 to q 9 =0, and 2. The modified initial capped oligonucleotide primer of claim 1, wherein B2, B3, B4 and B10 hybridize to template nucleotides 2, 3, 4 and 5, respectively.

7. q 1 to q 5 =1, q 6 to q 9 =0, and 2. The modified initial capped oligonucleotide primer of claim 1, wherein B2, B3, B4, B5 and B10 hybridize to template nucleotides 2, 3, 4, 5 and 6, respectively.

8. q 1 to q 6 =1, q 7 to q 9 =0, and 2. The modified initial capped oligonucleotide primer of claim 1, wherein B2, B3, B4, B5, B6 and B10 hybridize to template nucleotides 2, 3, 4, 5, 6 and 7, respectively.

9. q 1 to q 7 =1, q 8 to q 9 =0, and 2. The modified initial capped oligonucleotide primer of claim 1, wherein B2, B3, B4, B5, B6, B7 and B10 hybridize to template nucleotides 2, 3, 4, 5, 6, 7 and 8, respectively.

10. q 1 to q 8 =1, q 9 =0, and 2. The modified initial capped oligonucleotide primer of claim 1, wherein B2, B3, B4, B5, B6, B7, B8 and B10 hybridize to template nucleotides 2, 3, 4, 5, 6, 7, 8 and 9, respectively.

11. q 1 to q 9 = 1, and 2. The modified initial capped oligonucleotide primer of claim 1, wherein B2, B3, B4, B5, B6, B7, B8, B9 and B10 hybridize to template nucleotides 2, 3, 4, 5, 6, 7, 8, 9 and 10, respectively.

12. q 1 to q 2 = 1, q 3 to q 9 =0, and 2. The modified initial capped oligonucleotide primer of claim 1, wherein B2 and B10 hybridize to template nucleotides 2 and 3, respectively.

13. An RNA molecule comprising a modified initial capped oligonucleotide primer according to any one of claims 1 to 12.

14. A composition comprising the modified RNA molecule of claim 13, wherein the composition is free of a capping enzyme.

15. A composition comprising the modified RNA molecule of claim 13, wherein the modified initial capped oligonucleotide primer is a synthetic primer.

16. A cell containing a modified RNA molecule comprising a modified initial capped oligonucleotide primer according to any one of claims 1 to 12.

17. A pharmaceutical composition comprising a modified RNA molecule comprising the modified initial capped oligonucleotide primer of any one of claims 1 to 12 and a pharma- ceutically acceptable carrier.

18. A composition comprising an RNA molecule comprising an initial capped oligonucleotide primer comprising the following structure: 【Chemistry 2】 [In the formula, each of B 1 through B 10 is independently a natural, modified or non-natural nucleoside base; M is 0 or 1; L is 0 or 1; q 1 is 1, and each of q 2 to q 9 is independently 0 or 1; R 1 is H or methyl; R 2 and R 3 are independently H, OH, alkyl, O-alkyl, halogen, a linker, or a detectable marker; Each of X 1 to X 13 is independently O or S; each of Y 1 to Y 12 is independently OH, SH, BH 3 , aryl, alkyl, O-alkyl, or O-aryl; each of Z 1 to Z 22 independently is O, S, NH, CH 2 , C(halogen) 2 or CH(halogen); Each of R 4 through R 12 is independently H, OH, OMe, or a detectable marker. Where: B 1 is perfectly complementary to the nucleoside base on the nucleic acid template at transcription template position +1; B 2 through B 9 , if present, are perfectly complementary to their respective nucleoside bases on the nucleic acid template at the transcription initiation site from position +2 onwards; B 10 , the last nucleoside base of the initiating capped oligonucleotide primer is perfectly complementary to the last hybridized transcription template nucleotide. A composition in which a capping enzyme is absent.

19. A composition comprising an RNA molecule comprising an initial capped oligonucleotide primer comprising the following structure: 【Chemistry 3】 [In the formula, each of B 1 through B 10 is independently a natural, modified or non-natural nucleoside base; M is 0 or 1; L is 0 or 1; q 1 is 1, and each of q 2 to q 9 is independently 0 or 1; R 1 is H or methyl; R 2 and R 3 are independently H, OH, alkyl, O-alkyl, halogen, a linker, or a detectable marker; Each of X 1 to X 13 is independently O or S; each of Y 1 to Y 12 is independently OH, SH, BH 3 , aryl, alkyl, O-alkyl, or O-aryl; each of Z 1 to Z 22 independently is O, S, NH, CH 2 , C(halogen) 2 or CH(halogen); Each of R 4 through R 12 is independently H, OH, OMe, or a detectable marker. Where: B 1 is perfectly complementary to the nucleoside base on the nucleic acid template at transcription template position +1; B 2 through B 9 , if present, are perfectly complementary to their respective nucleoside bases on the nucleic acid template at the transcription initiation site from position +2 onwards; B 10 , the last nucleoside base of the initiating capped oligonucleotide primer is perfectly complementary to the last hybridized transcription template nucleotide. The composition, wherein the initial capped oligonucleotide primer is a compound primer.

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