Compositions and methods for synthesizing 5'-capped rnas

Abstract

Provided herein are methods and compositions for synthesizing 5' capped RNA, wherein the initiating capping oligonucleotide primer has the general form m7 Gppp[N 2’Ome ] n [N] m wherein m7 G is N7-methylated guanosine or any guanosine analog, N is any natural, modified or non-natural nucleoside, "n" can be any integer from 0 to 4 and "m" can be an integer from 1 to 9.

Description

[0001] This application is a divisional application. The original application was filed on September 20, 2016, with application number 2016800674586 (PCT / US2016 / 052670), and the invention title was "Composition and method for synthesizing 5'-capped RNA".

[0002] Cross-reference to related applications

[0003] This application claims priority to U.S. Provisional Application No. 62 / 221,248, filed September 21, 2015, the contents of which are incorporated herein by reference in their entirety.

[0004] sequence list

[0005] This application contains a sequence list that has been electronically submitted in ASCII format, and the entire sequence is incorporated herein by reference. The ASCII copy was created on November 16, 2016, and is named 095109-000500PC-1022543_SL.txt with a size of 785 bytes. Invention Field

[0006] This invention relates to methods and compositions for synthesizing 5'-capped RNA. In a specific aspect, the invention relates to novel cap-containing initiating oligonucleotide primers having a natural or modified 5'-cap 0, cap 1, cap 2, or trimethylguanosine-cap (TMG-cap) structure. In other aspects, the invention relates to providing methods for efficiently generating and using these methods to prepare 5'-capped RNA. Background Technology

[0007] The following description is provided to aid the reader's understanding. The information provided and any references cited are not to be acknowledged as prior art.

[0008] Messenger RNA (mRNA) encoding physiologically important proteins for therapeutic applications has shown significant advantages over DNA-based plasmids and viral vectors for delivering genetic material. Among these advantages, the most important is:

[0009] (i) High level of safety (reduced potential genomic damage from viral or plasmid integration),

[0010] (ii) mRNA delivery leads to immediate protein expression (unlike the delayed response that typically accompanies plasmids).

[0011] (iii) mRNA allows for robust dose-dependent control of protein expression, and

[0012] (iv) The simplicity of large-scale mRNA synthesis compared to the manufacture of plasmids and viral vectors.

[0013] Messenger RNAs can encode virtually any known protein and can be delivered to specific tissues and organs using a variety of methods known to those skilled in the art. Once delivered, these mRNAs direct the expression of ribosomal proteins within the target tissue, resulting in the production of hundreds of proteins according to the mRNA molecule.

[0014] Several structural elements present in each active mRNA molecule are utilized for the efficient translation of the encoded protein. One of these elements is the cap structure at the 5' end of the mRNA, which is present in all eukaryotes (and some viruses). The naturally occurring cap structure contains a riboguanosine residue methylated at the N7 position of the guanine base. This 7-methylguanosine ( 7m G) Linked to the 5′ end of the mRNA molecule via a 5′ to 5′ triphosphate chain, extending throughout this application. 7m and m7 are interchangeable and have equivalent meanings. (5′ end) 7m The presence of the Gppp fragment is crucial for mRNA maturation, and its:

[0015] Protecting mRNA from degradation by exonucleases,

[0016] Promotes the transport of mRNA from the nucleus to the cytoplasm, and

[0017] It plays a key role in the assembly of the translation initiation complex (Cell 9:645-653,(1976); Nature 266:235,(1977); Federation of Experimental Biologists Society Letter 96:1-11,(1978); Cell 40:223-24,(1985); Prog.Nuc.Acid Res.35:173-207,(1988); Ann.Rev.Biochem.68:913-963,(1999); J.Biol.Chem.274:30337-3040,(1999)).

[0018] Only those mRNAs carrying a cap structure are active in cap-dependent translation; the "decapitation" of mRNA results in the almost complete loss of its template activity for protein synthesis (Nature, 255:33-37, (1975); J. Biol. Chem., vol. 253:5228-5231, (1978); and Proc. Natl. Acad. Sci. USA, 72:1189-1193, (1975)).

[0019] Another element of eukaryotic mRNA is the presence of a 2'-O-methyl nucleoside residue at transcript position 1 (cap 1), and in some cases, at both transcript positions 1 and 2 (cap 2). 2'-O-methylation of mRNA is necessary for greater efficiency in in vivo mRNA translation (Proc. Natl. Acad. Sci. USA, 77:3952-3956 (1980)) and further enhances the nuclease stability of 5'-capped mRNA. mRNAs with cap 1 (and cap 2) serve as unique markers, allowing cells to recognize the true 5' end of mRNA and, in some cases, distinguishing it from transcripts derived from infectious genetic elements (Nucleic Acid Research 43:482–492 (2015)).

[0020] Primary mRNA transcripts carry a 5'-triphosphate group (5'-pppmRNA) resulting from RNA synthesis initiation at NTPs (generally GTPs) in vivo. The conversion of the 5'-triphosphated end of the mRNA transcript to a cap structure (cap 0) occurs through several enzymatic steps (J. Biol. Chem. 250:9322, (1975); J. Biol. Chem. 271:11936, (1996); J. Biol. Chem. 267:16430, (1992)). These enzymatic steps include:

[0021] Step 1: RNA triphosphatase converts the 5′-triphosphate of mRNA to a 5′-diphosphate, pppN1 (pN). x →ppN1(pN) x +Inorganic phosphate;

[0022] Step 2: RNA guanylate transferase uses GTP to transfer GMP residues to the 5′-bisphosphate group of mRNA, ppN1 (pN). x +GTP→G(5′)ppp(5′)N1(pN) x +Inorganic pyrophosphate; and

[0023] Step 3: Guanine-7-methyltransferase uses S-adenosylmethionine (AdoMet) as a cofactor and transfers the methyl group from AdoMet to the 7-nitro group of the guanine base, G(5′)ppp(5′)N1(pN). x +AdoMet→ 7m G(5′)ppp(5′)N1(pN) x +AdoHyc.

[0024] The RNA resulting from these enzymatic activities is called “5′ capped RNA” or “capped RNA”, and the combination of enzymes involved in the formation of “capped RNA” is called “capping enzyme”. Capping enzymes (including clonal forms of these enzymes) have been identified and purified from many sources and are well known in the art (Prog. Nucleic Acid Res. Mol. Biol. 66:1-40, (2001); Prog. Nucleic Acid Res. Mol. Biol. 50:101-129, (1995); and Microbiol. Rev. 44:175, (1980)). Capped RNA, which is produced by adding a cap nucleotide to the 5′ end of primary RNA via a capping enzyme, has been called capped RNA with a “cap 0 structure” (J. Biol. Chem. 269:14974-14981, (1994); J. Biol. Chem. 271:11936-11944, (1996)). Capping enzymes have been used to synthesize capped RNAs with a cap 0 structure in vitro (J. Biol. Chem. 255:11588, (1980); Proc. Natl. Acad. Sci. USA 94:9573, (1997); J. Biol. Chem. 267:16430, (1992); J. Biol. Chem. 269:14974, (1994); and J. Biol. Chem. 271:11936, (1996)).

[0025] Capped RNAs with a 5'-cap O structure can be further converted into a "Cap1" structure in vivo by (nucleoside-2′-O-)methyltransferases (J. Biol. Chem. 269:14974-14981, (1994); J. Biol. Chem. 271:11936-11944, (1996); and EMBO 21:2757-2768, (2002)). For example, vaccinia mRNA (nucleoside-2′-O)methyltransferases can catalyze the methylation of the 2′-hydroxyl group of the penultimate nucleotide of 5′-capped RNA with a cap O structure via the following reaction:

[0026] 7m G(5′)ppp(5′)N1pN2(pN) x +AdoMet→ 7m G(5′)ppp(5′)N1 2′-OMe pN2(pN) x +AdoHyc.

[0027] It has been reported that capped RNA with a cap 1 structure is translated more efficiently than capped RNA with a cap 0 structure (Nucleic Acids Res. 26:3208, (1998)). Eukaryotic cells utilize another (nucleoside-2′-O) methyltransferase (e.g., hMTR2 in human cells (Nucleic Acids Res. 39:4756 (2011)) to catalyze the methylation of the 2′-hydroxyl group of the second transcribed nucleotide of 5′-capped RNA to convert the cap 1 structure to the cap 2 structure via the following reaction:

[0028] 7m G(5′)ppp(5′)N1 2′-OMe pN2(pN) x +AdoMet→ 7m G(5′)ppp(5′)N1 2′-OMe pN2 2′-OMe (pN) x +AdoHyc.

[0029] Approximately 50% of eukaryotic mRNAs have a cap 2 structure.

[0030] To produce long functional RNA for various biological research purposes, methods for the in vitro enzymatic synthesis of primary RNA were developed in the mid-1980s (Methods Enzymol. 180: 51–62, (1989); Nucl. Acids Res., 10: 6353-6362, (1982); Meth. Enzymol., 180: 42-50 (1989); Nucl. Acids Res., 12: 7035-7056, (1984) and Nucleic Acid Research 15: 8783-8798, (1987)).

[0031] Following in vitro transcription, the original mRNA transcript carrying the 5'-triphosphate group can be further capped using a capping enzyme. However, in vitro enzymatic 5'-capping is expensive, laborious, inefficient, and difficult to control.

[0032] Considering these drawbacks, another method for synthesizing capped mRNA in vitro was developed, in which chemically synthesized dinucleotides... 7m G(5′)ppp(5′)G (also known as mCAP) is used to initiate transcription (RNA 1: 957-967, (1995)). The mCAP dinucleotide contains the 5′-cap O structure of mature mRNA, but does not have the 2′-O-methyl nucleotide characteristic of cap 1 and cap 2 structures.

[0033] However, two main drawbacks are attributed to the use of synthetic mCAP dinucleotides to initiate in vitro transcription. The first is the strong competition between mCAP and pppG for initiating mRNA synthesis. When mRNA is initiated with pppG, the resulting ppp-mRNA is non-translational and immunogenic due to the presence of the 5'-triphosphate. Conversely, when mRNA is initiated with mCAP, the resulting 5'-capped-mRNA is translationally active and less immunogenic.

[0034] To increase the ratio of 5'-capped to 5'-uncapped (or 5'-triphosphorylated; pppmRNA) mRNA, an excess of pppG relative to pppG must be used. 7m GpppG (4:1 to 10:1) is preferred for producing 5′-capped mRNA transcripts (up to 80-90%). The downside of this approach is that the rapid depletion of GTP supply during transcription and the need for large quantities of potentially expensive synthetic mCAP dimers lead to a significant decrease in overall mRNA yield. Post-transcriptionally, to reduce the immunogenicity of the synthesized mRNA, a crude mixture containing both 5′-capped mRNA and 5′-pppmRNA needs to be further treated with alkaline phosphatase to remove the capless 5′-triphosphate group from the pppmRNA. The capless 5′-OH form of mRNA obtained after phosphatase treatment is inactive and does not participate in the translation process.

[0035] Another drawback arises when using asymmetric mCAP dinucleotides: bidirectional initiation. 7m GpppG's G or 7m The 3′-hydroxyl group in the G moiety tends to act as the initiation site for transcriptional elongation with almost equal probability. Depending on the conditions of the transcriptional response, this generally leads to... 7m G(5′)pppG(pN) n and G(5′)ppp 7m G(pN) n The two isoform RNAs were synthesized in approximately equal proportions (RNA 1: 957-967, (1995)).

[0036] To eliminate the bidirectional initiation of mRNA synthesis using mCAP dinucleotides, novel modified mCAP analogs were developed, among which... 7m The 3′-OH group of the G residue is replaced by OCH3 (“OMe”): 7mG(3'-O-Me)pppG (also known as the Anti-Reverse Cap Analog (ARCA)). ARCA initiates mRNA synthesis only in the proper forward direction (RNA 7:1486-1495 (2001)). Several types of ARCA analogs are known in the art (see, for example, U.S. Patent No. 7,074,596). However, a large molar excess of ARCA relative to pppG is still required to ensure that most mRNA transcript molecules have a 5'-cap structure. A further disadvantage is that mRNAs with a cap 1 structure cannot be used... 7m GpppG 2′-OMe Synthesis of cap dimer (RNA 1:957, (1995)) or its ARCA analogue.

[0037] Currently, known pathways for producing active long mRNAs containing a cap 1 structure consist of the following: enzymatic capping of 5'-triphosphorylated mRNA transcripts and enzymatic 2'-O-methylation of mCAP-capped or ARCA-capped mRNA precursors (Nucleosides, Nucleotides, and Nucleic Acids, 25:337-340, (2006) and Nucleosides, Nucleotides, and Nucleic Acids 25(3):307-14, (2006)). Both methods are quite laborious and difficult to control, and even with substantial optimization, neither method can guarantee high yields of capped and methylated mRNA precursors (J. Gen. Virol., 91:112-121, (2010)). Moreover, methods for preparing mRNAs with a cap 2 structure are even more difficult and the results are unpredictable. The enzyme used to convert cap 1 to cap 2 is not currently commercially available.

[0038] Another significant complexity in the in vitro synthesis of mRNA, especially in large-scale manufacturing, is the need to isolate and purify capped active mRNA molecules from all inactive and, in some cases, immunogenic capless mRNA forms. Unfortunately, these methods are not trivial and often require the synthesis of modified mCAP analogs with a conjugated affinity tag moiety, which allows for easier isolation and purification of capped RNA transcripts. Methods for synthesizing mCAP analogs with affinity tags as reporter / affinity moieties and novel protocols for isolating capped RNA from transcription reaction mixtures are known in the art (see, for example, U.S. Patent 8,344,118). While these approaches are efficient, they require the use of more expensive mCAP analogs and they only allow for the preparation and isolation of mRNAs containing a cap 0 structure.

[0039] Natural and modified RNA synthesized in vitro is used in a variety of applications, including ribozymes, antisense, biophysical, and biochemical research. Additionally, capped mRNA transcripts are used in applications requiring protein synthesis, such as in vivo expression experiments (using microinjection, transfection, and infection), in vitro translation experiments and assays, and various applications in therapeutics, diagnostics, vaccine development, labeling, and detection.

[0040] Therefore, there is an industrial need for compositions and methods that allow for the large-scale synthesis of mRNA in the following ways: (a) less labor-intensive than conventional methods, (b) eliminating or reducing bidirectional initiation during transcription, (c) resulting in higher mRNA yields, (d) reduced costs compared to current methods, (e) reduced generation of heterologous products with different 5' sequences, and (f) eliminating the need for additional enzymatic reactions to incorporate cap 1 and cap 2 structures into the synthesized mRNA. There is also a need to synthesize various mRNAs containing modified and / or non-natural nucleosides, carrying specific modifications and / or affinity tags such as fluorescent dyes, radioisotopes, mass tags, and / or a partner of a molecular binding pair, such as biotin, at or near the 5' end of the molecule. Summary of the Invention

[0041] This article provides methods and compositions for synthesizing 5'-capped RNA. In one aspect of the invention, the initiating capped oligonucleotide primer comprises the general formula structure of formula I:

[0042]

[0043] in

[0044] B1 to B 10 Each of them is independently a natural, modified, or non-natural nucleoside base;

[0045] M is 0 or 1;

[0046] L is either 0 or 1;

[0047] q1 is 1;

[0048] Each of q2 to q9 is independently 0 or 1;

[0049] R1 is H or methyl;

[0050] R2 and R3 are independently H, OH, alkyl, O-alkyl, halogen, amine, azide, linker, or detectable marker;

[0051] X1 to X 13 Each of them is independently O or S;

[0052] Y1 to Y13 Each of these is independently OH, SH, BH3, aryl, alkyl, O-alkyl, or O-aryl;

[0053] Z0 is O;

[0054] Z1 to Z 22 Each of these is independently O, S, NH, CH2, C (halogen)2, or CH (halogen); and

[0055] R4 to R 12 Each of these can be independently H, OH, OMe, a linker, or a detectable marker.

[0056] In another aspect of the invention, an RNA molecule comprising a capped initiating oligonucleotide primer of Formula I is provided, a pharmaceutical composition comprising such RNA, a cell containing such RNA, and a cell containing a protein or peptide translated from such RNA are provided.

[0057] In another aspect of the invention, a method is provided in which an RNA molecule containing a capped initiating oligonucleotide primer of formula I is synthesized into a mixture containing a polynucleotide template and an RNA polymerase under conditions favorable for transcription by RNA polymerase via a polynucleotide template, and the mixture is subsequently incubated for a time sufficient to allow transcription of the template. Attached Figure Description

[0058] Figure 1 The preparation of 7-methylguanosine 5-diphosphate (pp) from guanosine 5'-bisphosphate is shown. 7m An exemplary method of G);

[0059] Figure 2 It shows the output from pp 7m G Preparation of 7-methylguanosine 5'-diphosphate imidazolide (Im-pp) 7m An exemplary method of G);

[0060] Figure 3 The preparation of 3'-O-methylguanosine 5'-phosphate (pG) from 3'-O-methylguanosine is shown. 3’Ome Exemplary methods;

[0061] Figure 4 An exemplary method for preparing 3'-O-methylguanosine 5'-phosphorimidazolide (Im-pG3'Ome) from pG3'Ome is shown;

[0062] Figure 5 It shows the results from Im-pG 3’Ome Preparation of 3'-O-methylguanosine 5'-bisphosphate (ppG) 3’Ome Exemplary methods;

[0063] Figure 6 It shows the method for using ppG 3’Ome Preparation of 7-methyl-3'-O-methylguanosine 5-bisphosphate (pp 7m G 3’Ome Exemplary methods;

[0064] Figure 7 It shows the method for using pp 7m G 3’Ome Preparation of 7-methyl-3'-O-methylguanosine 5-bisphosphate imidazole (Im-pp) 7m G 3’Ome Exemplary methods;

[0065] Figure 8 The preparation of pN is shown 2’-OR1 General procedure for pN oligonucleotides (R1 = H or Me);

[0066] Figure 9 A general procedure for synthesizing starting oligonucleotides with a cap 0, cap 1, or cap 2 structure is shown;

[0067] Figure 10A The structure of the starting capped oligonucleotide primer used in the example according to Formula I is shown, wherein: 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; X 13 For O; Y 13 Z0 is OH; Z1 is O; Z2 is O; Z 22 It is O;

[0068] Figure 10B The structure of the initiating capped oligonucleotide primer used in the example according to Formula I is shown, wherein: B1 is guanine; B 10 For guanine; M = 0; L = 1; q1 = 1; q2 to q9 = 0; R1 = H; R2 = H; R3 = H; X1 = O; X2 = O; X4 = O; X 13 Y1 is O; Y2 is OH; Y4 is OH; Y 13 Z0 is OH; Z1 is O; Z2 is O; Z4 is O; Z5 is O; Z 22 O; R4 is O-methyl;

[0069] Figure 10C The structure of the initiating capped oligonucleotide primer used in the example according to Formula I is shown, wherein: B1 is guanine; B 10 For guanine; M = 0; L = 1; q1 = 1; q2 to q9 = 0; R1 = H; R2 = H; R3 = O-methyl; X1 = O; X2 = O; X4 = O; X13 Y1 is O; Y2 is OH; Y4 is OH; Y 13 Z0 is OH; Z1 is O; Z2 is O; Z4 is O; Z5 is O; Z 22 O; R4 is O-methyl;

[0070] Figure 10D The structure of the initiating capped oligonucleotide primer used in the example according to Formula I is shown, wherein: B1 is adenine; B 10 For guanine; M = 0; L = 1; q1 = 1; q2 to q9 = 0; R1 = H; R2 = H; R3 = H; X1 = O; X2 = O; X4 = O; X 13 Y1 is O; Y2 is OH; Y4 is OH; Y 13 Z0 is OH; Z1 is O; Z2 is O; Z4 is O; Z5 is O; Z 22 O; R4 is O-methyl;

[0071] Figure 10E The structure of the initiating capped oligonucleotide primer used in the example according to Formula I is shown, wherein: B1 is adenine; B 10 For guanine; M = 0; L = 1; q1 = 1; q2 to q9 = 0; R1 = H; R2 = H; R3 = O-methyl; X1 = O; X2 = O; X4 = O; X 13 Y1 is O; Y2 is OH; Y4 is OH; Y 13 Z0 is OH; Z1 is O; Z2 is O; Z4 is O; Z5 is O; Z 22 O; R4 is O-methyl;

[0072] Figure 10F The structure of the initiating capped oligonucleotide primer used in the example according to Formula I is shown, wherein: B1 is cytosine; B 10 For guanine; M = 0; L = 1; q1 = 1; q2 to q9 = 0; R1 = H; R2 = H; R3 = H; X1 = O; X2 = O; X4 = O; X 13 Y1 is O; Y2 is OH; Y4 is OH; Y 13 Z0 is OH; Z1 is O; Z2 is O; Z4 is O; Z5 is O; Z 22 O; R4 is O-methyl;

[0073] Figure 10G The structure of the initiating capped oligonucleotide primer used in the example according to Formula I is shown, wherein: B1 is cytosine; B 10For guanine; M = 0; L = 1; q1 = 1; q2 to q9 = 0; R1 = H; R2 = H; R3 = O-methyl; X1 = O; X2 = O; X4 = O; X 13 Y1 is O; Y2 is OH; Y4 is OH; Y 13 Z0 is OH; Z1 is O; Z2 is O; Z4 is O; Z5 is O; Z 22 O; R4 is O-methyl;

[0074] Figure 10H The structure of the initiating capped oligonucleotide primer used in the example according to Formula I is shown, wherein: B1 is adenine; B2 is guanine; B 10 For guanine; M = 0; L = 1; q1 = 1; q2 = 1; q3 to q9 = 0; R1 = H; R2 = H; R3 = O-methyl; X1 = O; X2 = O; X4 = O; X5 = O; X 13 Y1 is O; Y2 is OH; Y4 is OH; Y5 is OH; Y 13 Z0 is OH; Z1 is O; Z2 is O; Z4 is O; Z5 is O;

[0075] Z6 is O; Z 22 R4 is O-methyl; R5 is O-methyl;

[0076] Figure 11 The luciferase activity of mRNA co-transcribed and capped was demonstrated;

[0077] Figures 12A-12H Together, they demonstrate the use of 12A)ARCA and 12B) m7 GpppG 2’Ome pG, 12C) m7 G 3’Ome pppG 2’Ome pG, 12D) m7 GpppA 2’Ome pG, 12E) m7 G 3’Ome pppA 2’-OMe pG, 12F) m7 GpppC 2’Ome pG, 12G) m7 G 3’Ome pppC 2’Ome pG, 12H) m7 G 3’Ome pppA 2’Ome pG 2’Ome Capping efficiency of pG co-transcribed capped mRNA. Figure 12B-12G The efficiency of adding a cap in the middle is equal to or significantly exceeds that in the middle. Figure 12A The efficiency of capping observed in the process; and

[0078] Figures 13A-13D Together they show the use of transcription templates m7 GpppA 2’Ome Capping efficiency and initiation fidelity of pG co-transcribed capped mRNA, among which Figure 13A An example is given of the application of primer / NTP formulation 2 with 2'-deoxythymidine and 2'-deoxycytidine residues at template positions +1 and +2; Figure 13B The application of primer / NTP formulation 3 with 2'-deoxythymidine and 2'-deoxycytidine residues at template positions +1 and +2 is illustrated. Figure 13C Examples of applications using primer / NTP formulation 2, with 2'-deoxycytidine residues at template positions +1 and +2, and... Figure 13D An example is shown of the application of primer / NTP formulation 3 at template positions +1 and +2 for 2'-deoxycytidine residues;

[0079] Figure 14A and 14B Together, they illustrate a comparison of the translation of mRNAs produced by initiating capped oligonucleotides with m7GpppA2'OmepG on transcription templates with 2'-deoxythymidine and 2'-deoxycytidine residues at template positions +1 and +2 versus those with cytidine residues at template positions +1 and +2 in differentiated THP-1 cells. Detailed Implementation

[0080] Unless otherwise defined, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. All patents, patent applications, and publications cited throughout this disclosure are incorporated herein by reference in their entirety. Where multiple definitions exist for terms herein, those definitions in this section shall prevail.

[0081] As used herein in conjunction with numerical values, the terms “approximately” or “about” mean an indication value plus or minus 30%, including all values ​​within the defined range, including the value stated herein.

[0082] As used herein, the terms “nucleic acid,” “nucleotide sequence,” or “nucleic acid sequence” refer to oligonucleotides, polynucleotides, or any fragments thereof, any ribose or deoxyribose derivatives, and to naturally occurring or synthetic molecules containing native and / or modified nucleotide residues and nucleotide linkers. These phrases also refer to DNA or RNA of natural (e.g., genomic) or synthetic origin, which may be single-stranded, double-stranded, triple-stranded, or quadruple-stranded and may represent sense or antisense strands, or refer to any DNA-like or RNA-like substance. Referring to a DNA sequence, the “RNA equivalent” consists of the same linear nucleotide sequence as the reference DNA sequence, except that all or most of the nitrogenous bases thymine are replaced by uracil, and the sugar backbone is composed of ribose instead of 2'-deoxyribose. Additional alternative nucleic acid backbones suitable for the methods and compositions provided herein include, but are not limited to, thiophosphates, selenophosphates, alkyl phosphates, aryl phosphates, alkyl phosphates, aryl phosphonates, phosphoboronates, morpholinonucleotides (MNA), locked nucleic acids (LNA), and peptide nucleic acids (PNA).

[0083] As used herein, the term "primer" or "oligonucleotide primer" refers to a ribose- or deoxyribose- or chimeric ribose / deoxyribose-oligonucleotide, single-stranded, which may be naturally occurring or synthetic, and often comprises a sequence of about 2 to about 10 nucleotides, about 3 to about 8 nucleotides, or about 3 to about 5 nucleotides. Oligonucleotide primers may contain one or more modifying groups. Oligonucleotide primers may include RNA, DNA, and / or other modified nucleosides. Those skilled in the art can design and prepare oligonucleotide primers suitable for transcribing DNA template sequences.

[0084] As used herein, the term "starting capped oligonucleotide analog" or "starting capped oligonucleotide primer" refers to a starting oligonucleotide primer containing a cap 0, cap 1, cap 2, or TMG-cap structure at the 5' end of the primer. The capped primer has an unmodified or open 3'-OH group and can be extended by RNA polymerase by incorporating an NTP to the 3' end of the primer. It enables in vitro transcription to be initiated under promoter control in a transcription system containing the necessary components (DNA template (e.g., DNA plasmid), RNA polymerase, nucleoside 5'-triphosphate, and suitable buffer). As also used herein, "starting primer" or "starting oligonucleotide primer" refers to an oligonucleotide carrying a terminal 3'-OH group that is an effective substrate for RNA polymerase. In some embodiments, the starting oligonucleotide primer is a substrate of RNA polymerase and can be extended by incorporating an NTP to the 3' end of the primer. The starting oligonucleotide primer is complementary to the DNA template at the starting site.

[0085] As used herein, in the context of initiating capped oligonucleotide primers and NTPs, the terms “unsubstituted” or “unmodified” refer to initiating capped oligonucleotide primers and NTPs that have not yet been modified.

[0086] As used herein, the term "modified initiating capped oligonucleotide primer" refers to an initiating capped oligonucleotide primer containing one or more additional modifying groups.

[0087] As used herein, the term "modifying group" refers to any chemical part that can be attached to the start primer at some position, including but not limited to sugars, nucleoside bases, triphosphate bridges, and / or internucleotide phosphates (e.g., U.S. Patent Application No. 20070281308). The modifying group of the start capped oligonucleotide primer can be a group of any nature compatible with the transcription process.

[0088] As used herein, the term "nucleotide linker" refers to one or more bonds between two nucleosides that link an oligonucleotide primer or nucleic acid, and can be a natural phosphodiester linker or a modified linker.

[0089] As used herein, the term "marker" or "detectable mark" refers to any compound or combination of compounds that can be attached to or otherwise associated with a molecule so that the molecule can be detected directly or indirectly by detecting the mark. Detectable marks can be radioactive isotopes (e.g., carbon, phosphorus, iodine, indium, sulfur, tritium, etc.) or mass isotopes (e.g., H₂). 2 C 13 or N 15 (e.g., dyes or fluorophores (e.g., cyanin, fluorescein, or coumarin), haptens (e.g., biotin), or any other reagent that can be detected directly or indirectly.

[0090] As used herein, the term “hybridization” or “specific hybridization” refers to the process of annealing a starter capped oligonucleotide primer to a DNA template under appropriately stringent conditions during a transcription reaction. Hybridization with DNA is performed via a starter capped oligonucleotide primer, which in some embodiments is 3–10 nucleotides in length—including a 5’–5’ inverted cap structure. Nucleic acid hybridization techniques are well known in the art (e.g., Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Press, Plainview, NY (1989); Ausubel, FM, et al., Current Protocols in Molecular Biology, John Wiley & Sons, Secaucus, NJ (1994)).

[0091] As used herein, in the context of the complex of an initiating capped oligonucleotide primer and a DNA template, the terms “complementary,” “complementary,” or “complementarity” refer to the standard Watson / Crick base pairing rule. For example, the sequence “5'-AGTC-3'” is complementary to the sequence “3'-TCAG-5'.” Certain non-natural or synthetic nucleotides may be included in the nucleic acids described herein; these include, but are not limited to, base- and sugar-modified nucleosides, nucleotides, and nucleic acids such as inosine, 7-deoxyguanosine, 2'-O-methylguanosine, 2'-fluoro-2'-deoxycytidine, pseudouridine, locked nucleic acids (LNA), and peptide nucleic acids (PNA). Complementarity does not need to be perfect; duplexes may contain mismatched base pairs, degenerate, or unmatched nucleotides. Those skilled in the art can determine duplex stability empirically by taking into account a number of variables, including, for example, the length of the oligonucleotide, its base composition and sequence, the incidence of mismatched base pairs, ionic strength, the composition of the hybridization buffer, and reaction conditions.

[0092] Complementarity can be “complete” or “full,” meaning all nucleotide bases of the two nucleic acid strands match according to accepted base pairing rules; it can be “partial,” meaning only some nucleotide bases of the initiating capped oligonucleotide primer match the DNA target according to accepted base pairing rules; or it can be “absent,” meaning none of the nucleotide bases of either nucleic acid strand match according to accepted base pairing rules. The degree of complementarity between the initiating capped oligonucleotide primer and the DNA template can have a significant impact on the hybridization strength and corresponding reaction efficiency between the initiating capped oligonucleotide and the DNA template. The term complementarity can also refer to the use of individual nucleotides. For example, it can indicate the complementarity or lack of a specific nucleotide within the oligonucleotide with a nucleotide within the other strand (relative to or compared to the complementarity between the rest of the initiating capped oligonucleotide primer and the DNA strand).

[0093] As used herein, the terms “complete,” “full,” or “perfect” complement each other, meaning that every nucleotide base of the initiating capped oligonucleotide primer is precisely matched to the DNA target according to accepted base pairing rules.

[0094] As used herein, the term “substantially complementary” refers to two sequences that hybridize under strict hybridization conditions. Those skilled in the art will understand that substantially complementary sequences do not need to hybridize along their entire length. Specifically, substantially complementary sequences may comprise consecutive base sequences that do not hybridize with the target sequence and may be located on the 3' or 5' side of consecutive base sequences that hybridize with the target sequence under strict hybridization conditions.

[0095] As used herein, when referring to the initiating capped oligonucleotide primer sequence and its ability to hybridize with a DNA template, the term "specific" means a sequence that has at least 50% sequence identity with a portion of the DNA template when the initiating capped oligonucleotide primer is aligned with the DNA strand. Preferred higher levels of sequence identity include at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, and most preferably 100% sequence identity.

[0096] As used herein, the term "nucleoside" includes all naturally occurring nucleosides, including all forms of nucleoside bases and furanyl glycosides found in nature. The most commonly found base rings in naturally occurring nucleosides are purine and pyrimidine rings. Naturally occurring purine rings include, for example, adenine, guanine, and N-acetylene. 6-Methyladenine. Naturally occurring pyrimidine rings include, for example, cytosine, thymine, 5-methylcytosine, and pseudouridine. Naturally occurring nucleosides include, but are not limited to, the ribose, 2'-O-methyl, or 2'-deoxyribose derivatives of adenosine, guanosine, cytidine, thymidine, uridine, inosine, 7-methylguanosine, or pseudouridine.

[0097] As used herein, the terms “nucleoside analog,” “modified nucleoside,” or “nucleoside derivative” include synthetic nucleosides as described herein. Nucleoside derivatives also include nucleosides with modified bases and / or sugar moieties, with or without protecting groups, and include, for example, 2’-deoxy-2’-fluorouridine, 5-fluorouridine, and analogs. The compounds and methods provided herein include such base rings and their synthetic analogs, as well as non-natural heterocyclic-substituted basic sugars and acyclic-substituted basic sugars. Other nucleoside derivatives that can be used in this invention include, for example, LNA nucleosides, halogenated purines (e.g., 6-fluoropurine), halogenated pyrimidines, N… 6 -Ethyl adenine, N 4 -(alkyl)-cytosine, 5-ethylcytosine and the like (US Patent No. 6,762,298).

[0098] As used herein, the terms “universal base,” “degenerate base,” “universal base analog,” and “degenerate base analog” include, for example, nucleoside analogs having an artificial base that, in some embodiments, can be recognized by RNA polymerase as one of the natural NTPs (e.g., ATP, UTP, CTP, and GTP) or a substitute for another specific NTP. Universal or degenerate bases are disclosed in Loakes, D., Nucleic Acids Res., 29:2437-2447 (2001); Crey-Desbiolles, C., et al., Nucleic Acids Res., 33:1532–1543 (2005); Kincaid, K., et al., Nucleic Acids Res., 33:2620-2628 (2005); Preparata, FP, Oliver, JS, J. Comput. Biol. 753-765 (2004); and Hill, F., et al., Proc Natl Acad. Sci. USA, 95:4258-4263 (1998)).

[0099] As used herein, the term “modified NTP” refers to a nucleoside 5'-triphosphate having a chemical moiety bonded at any position, including a sugar, a base, a triphosphate chain, or any combination of these three positions. Examples of such NTPs can be found, for example, in “Nucleoside Triphosphates and Their Analogs: Chemistry, Biotechnology and Biological Applications”, Vaghefi, M., ed., Taylor and Francis, Boca Raton (2005).

[0100] As used herein, the term "modified oligonucleotide" includes, for example, an oligonucleotide containing a modified nucleoside, a modified internucleotide linker, or any combination of a modified nucleoside and an internucleotide linker. Examples of internucleotide linker modifications of oligonucleotides include phosphate thioesters, phosphate triesters, and methylphosphonate derivatives (Stec, WJ, et al., Chem. Int. Ed. Engl., 33:709-722 (1994); Lebedev, AV, et al., E., Perspect. Drug Discov. Des., 4:17-40 (1996); and Zon, et al., U.S. Patent Application No. 20070281308). Other examples of internucleotide linker modifications can be found in Waldner, et al., Bioorg. Med. Chem. Letters 6:2363-2366 (1996).

[0101] As used herein, the term "promoter" refers to a region of the dsDNA template that directs and controls the initiation of transcription of a specific DNA sequence (e.g., a gene). The promoter is located on the same strand of the DNA and upstream (near the 5' region of the sense strand). The promoter is generally adjacent to (or partially overlaps with) the DNA sequence to be transcribed. The nucleotide positions in the promoter are designated relative to the transcription start site (position +1). The initiation oligonucleotide primer is complementary to the initiation site of the promoter sequence (which in some embodiments is at positions +1 and +2, and in the case of an initiation tetramer, at positions +1, +2, and +3).

[0102] As used herein, the terms “transcription” or “transcriptional reaction” refer to methods known in the art for the enzymatic preparation of RNA complementary to a DNA template, thereby producing multiple RNA copies of a DNA sequence. The RNA molecule synthesized in a transcriptional reaction is called an “RNA transcript,” “original transcript,” or simply “transcription.” Transcriptional reactions involving the compositions and methods provided herein employ “initiation capped oligonucleotide primers.” Transcription of a DNA template can be exponential, non-linear, or linear. The DNA template can be double-stranded linear DNA, partially double-stranded linear DNA, circular double-stranded DNA, DNA plasmids, PCR amplicons, or modified nucleic acid templates compatible with RNA polymerase.

[0103] As used herein, the term "acyl" refers to the group -C(O)R. a , where R a It can be hydrogen, lower alkyl, cycloalkyl, heterocyclic, aryl, heteroaryl and similar groups.

[0104] As used herein, the term "substituted acyl" refers to the group -C(O)R a’ , where R a’ It can be a substituted lower alkyl group, a substituted cycloalkyl group, a substituted heterocyclic group, a substituted aryl group, a substituted heteroaryl group, or a similar group.

[0105] As used herein, the term "acyloxy group" signifies the group -OC(O)R. b , where R b It can be hydrogen, lower alkyl, substituted lower alkyl, cycloalkyl, substituted cycloalkyl, heterocyclic, substituted heterocyclic, aryl, substituted aryl, heteroaryl, substituted heteroaryl and similar groups.

[0106] As used herein, the term "alkyl" refers to a single-bonded chain of hydrocarbons in some embodiments ranging from 1 to 20 carbon atoms, and in some embodiments from 1 to 8 carbon atoms; examples include methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, pentyl, isopentyl, hexyl, octyl, dodecyl, and similar groups.

[0107] As used herein, the term "lower alkyl" refers to a straight-chain or branched hydrocarbon, in some embodiments of 1-6 carbon atoms, and in some embodiments of 2-5 carbon atoms. Examples include ethyl, propyl, isopropyl, and similar groups.

[0108] As used herein, the term "alkenyl" refers to a straight-chain or branched hydrocarbon group having one or more double bonds and, unless otherwise specified, containing about 2 to about 20 carbon atoms, and in some embodiments about 2 to about 10 carbon atoms, and in some embodiments about 2 to about 8 carbon atoms, and in some embodiments about 2 to about 6 carbon atoms. Examples of alkenyl groups include vinyl, allyl, 1,4-butadienyl, isopropenyl, and similar groups.

[0109] As used herein, the term "alkenylaryl" refers to an alkenyl-substituted aryl group, and "substituted alkenylaryl" refers to an alkenylaryl group further having one or more substituents as described herein.

[0110] As used herein, the term "alkenylene" refers to a divalent straight-chain or branched hydrocarbon group having at least one carbon-carbon double bond and generally containing 2-20 carbon atoms, and in some embodiments, 2-12 carbon atoms, and in some embodiments, 2-8 carbon atoms, and "substituted alkenylene" refers to an alkenylene group further having one or more substituents as described herein.

[0111] As used herein, the term "alkylene" refers to a divalent hydrocarbon group containing 1-20 carbon atoms, and in some embodiments 1-15 carbon atoms, either straight-chain or branched, wherein two hydrogen atoms are removed from the same carbon atom or from different carbon atoms. Examples of alkylene groups include, but are not limited to, methylene (-CH2-), ethylene (-CH2CH2-), and similar groups.

[0112] As used herein, the term "alkynyl" refers to a straight-chain or branched hydrocarbon group having one or more triple bonds and containing about 2 to 20 carbon atoms, and in some embodiments about 2 to 10 carbon atoms, and in some embodiments about 2 to 8 carbon atoms, and in some embodiments about 2 to 6 carbon atoms. Examples of alkynyl groups include ethynyl, propynyl (propynyl), butynyl, and similar groups.

[0113] As used herein, the term "alkynylaryl" refers to an alkynyl-substituted aryl group, and "substituted alkynylaryl" refers to an alkynylaryl group further having one or more substituents as described herein.

[0114] As used herein, the term "alkoxy" refers to the -OR group. c , where R cIt is a lower alkyl group, a substituted lower alkyl group, an aryl group, a substituted aryl group, an aralkyl group, a substituted aralkyl group, a heteroalkyl group, a heteroarylalkyl group, a cycloalkyl group, a substituted cycloalkyl group, a cyclohexaalkyl group, or a substituted cyclohexaalkyl group, as defined.

[0115] As used herein, the term "lower alkoxy" refers to the group -OR d , where R d It is a lower alkyl group.

[0116] As used herein, the term "alkylaryl" refers to an alkyl-substituted aryl group, and "substituted alkylaryl" refers to an alkylaryl group further having one or more substituents as described herein.

[0117] As used herein, the term "alkylthio" refers to the -SR group. h , where R h It is an alkyl group.

[0118] As used herein, the term "substituted alkylthio" refers to the -SR group. i , where R i It is a substituted alkyl group.

[0119] As used herein, the term "ethynyl" refers to a divalent straight-chain or branched hydrocarbon group having at least one carbon-carbon triple bond and generally having a range of about 2 to 12 carbon atoms, and in some embodiments about 2 to 8 carbon atoms, and "substituted ethynyl" refers to an ethynyl group further having one or more substituents as described herein.

[0120] As used herein, the term "acylamino" refers to the group -C(O)NR. j R j’ , where R j and R j’ It can be hydrogen, lower alkyl, substituted lower alkyl, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl or substituted heteroaryl independently.

[0121] As used herein, the term "substituted amide" signifies the group -C(O)NR. k R k’ , where R k and R k’ Independently, it is hydrogen, a lower alkyl group, a substituted lower alkyl group, an aryl group, a substituted aryl group, a heteroaryl group, or a substituted heteroaryl group, provided that R is... k and R k’ At least one of them is not hydrogen. R k R k’ Combining with nitrogen can form optionally substituted heterocyclic or heteroaryl rings.

[0122] As used herein, the term "amino" or "amine" indicates the group –NR n R n’ , where R n and R n’ It can be independently hydrogen, lower alkyl, substituted lower alkyl, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl or substituted heteroaryl as defined herein. “Divalent amine” indicates the group -NH-. “Substituted divalent amine” indicates the group -NR-, where R is lower alkyl, substituted lower alkyl, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl or substituted heteroaryl.

[0123] As used herein, the term "substituted amino" or "substituted amine" indicates the group -NR p R p’ , where R p and R p’ Independently, it can be hydrogen, lower alkyl, substituted lower alkyl, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, or substituted heteroaryl, however, provided that R... p and R p’ At least one of them is not hydrogen. R p R p’ Combining with nitrogen can form optionally substituted heterocyclic or heteroaryl rings.

[0124] As used herein, the term "aromatic acyl" refers to an aryl carbonyl group, such as benzoyl, and "substituted aromatic acyl" refers to an aromatic acyl group further having one or more substituents as described herein.

[0125] As used herein, the term "aryl" alone or in combination refers to a phenyl, naphthyl, or fused aromatic heterocycle that optionally has 5-10 ring members and, in some embodiments, 5-6 ring members, a cycloalkyl group, and / or optionally substituted with 1 to 3 of the following groups or substituents: halogen, hydroxyl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl, acyloxy, aryloxy, heteroaryloxy, amino, amidoyl, optionally monosubstituted or disubstituted with alkyl, aryl, or heteroaryl, optionally substituted with alkyl, aryl, heteroaryl, or heterocyclic groups, optionally N-monosubstituted or N,N-disubstituted with alkyl, aryl, or heteroaryl groups, alkylsulfonyl, arylsulfonyl, heteroarylsulfonyl, alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, or similar groups.

[0126] As used herein, the term "aryloxy group" signifies the group -OAr, where Ar is an aryl or substituted aryl group.

[0127] As used herein, the term "carbocyclic ring" refers to a saturated, unsaturated, or aromatic group having a monocyclic or multiple fused rings consisting of linked carbon atoms. The ring (one or more) may optionally be unsubstituted or substituted with groups such as halogens, lower alkyl groups, alkoxy groups, alkylthio groups, acetylene (-C=CH), amino groups, amide groups, azide groups, carboxyl groups, hydroxyl groups, aryl groups, aryloxy groups, heterocyclic groups, heteroaryl groups, substituted heteroaryl groups, nitro groups (-NO2), cyano groups (-CN), mercapto groups (-SH), sulfonamide groups (-S(O)2NH2), and similar groups.

[0128] As used herein, the term “guanidinyl” means the group -N=C(NH2)2, and “substituted guanidinyl” means the group –N=C(NR2)2, wherein each R is independently H, alkyl, substituted alkyl, aryl, or substituted aryl as described herein.

[0129] As used herein, the term “halogen” or “halogen” refers to all halogens, namely chlorine (Cl), fluorine (F), bromine (Br), and iodine (I).

[0130] As used herein, the term "heteroaryl" refers to a monocyclic aromatic ring structure containing 5 or 6 ring atoms or a bicyclic aromatic group having 8-10 atoms: containing one or more independently selected from groups O, S, and N, and in some embodiments 1-4, in some embodiments 1-3, and in some embodiments 1-2 heteroatoms, and optionally substituted by 1-3 of the following groups or substituents: halogen, hydroxyl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl, acyloxy, aryloxy, heteroaryloxy, amino, amidoyl, optionally monosubstituted or disubstituted by alkyl, aryl, or heteroaryl, optionally substituted by alkyl, aryl, heteroaryl, or heterocyclic urea, optionally N-monosubstituted or N,N-disubstituted aminosulfonyl, alkylsulfonylamino, arylsulfonylamino, heteroarylsulfonylamino, alkylcarbonylamino, arylcarbonylamino, heteroarylcarbonylamino, or similar groups. Heteroaryl groups also aim to include oxidized S or N, such as sulfinyl, sulfonyl, and N-oxides of tertiary ring nitrogen. The carbon or nitrogen atom is the attachment point for the heteroaryl ring structure, thus maintaining a stable aromatic ring. Examples of heteroaryl groups are phthalimide, pyridinyl, pyrazinyl, quinazolinyl, purine, indole, quinolinyl, pyrimidinyl, pyrroleyl, etc. azole group, thiazolyl group, thiophene group, iso Azolyl, oxathiadiazolyl, isothiazolyl, tetrazolyl, imidazolyl, triazine, furanyl, benzofuranyl, indoleyl, and similar groups. Substituted heteroaryl groups contain substituents attached to an available carbon or nitrogen to produce a stable compound.

[0131] As used herein, the term "substituted heteroaryl" refers to a heterocycle optionally monosubstituted or polysubstituted with one or more functional groups, such as halogens, lower alkyl groups, lower alkoxy groups, alkylthio groups, acetylene groups, amino groups, amide groups, carboxyl groups, hydroxyl groups, aryl groups, aryloxy groups, heterocycles, substituted heterocycles, heteroaryl groups, substituted heteroaryl groups, nitro groups, cyano groups, mercapto groups, sulfonamide groups, and similar groups.

[0132] As used herein, the term "heterocyclic" refers to a saturated, unsaturated, or aromatic group having a monocyclic (e.g., morpholino, pyridinyl, or furanyl) or multiple fused rings (e.g., naphthyridinyl, quinoxalinyl, quinolinyl, indolizinyl, or benzo[b]thiophene) and having a carbon atom and at least one heteroatom (such as N, O, or S) within the ring, which may optionally be unsubstituted or substituted with: for example, halogens, lower alkyl groups, lower alkoxy groups, alkylthio groups, acetylene groups, amino groups, amide groups, carboxyl groups, hydroxyl groups, aryl groups, aryloxy groups, heterocyclic groups, heteroaryl groups, substituted heteroaryl groups, nitro groups, cyano groups, mercapto groups, sulfonamide groups, and similar groups.

[0133] As used herein, the term "substituted heterocycle" refers to a heterocycle substituted with one or more (e.g., one, two, or three) substituents selected from the following groups: optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, halogen, hydroxyl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl, acyloxy, aryl, substituted aryl, aryloxy, heteroaryloxy, amino, amide, amidine, optionally substituted with alkyl, aryl, heteroaryl, or heterocycle. Urea substituted with alkyl, optionally N-monosubstituted or N,N-disubstituted aminosulfonyl, alkylsulfonylamino, arylsulfonylamino, heteroarylsulfonylamino, alkylcarbonylamino, arylcarbonylamino, heteroarylcarbonylamino, acyl, carboxyl, heterocyclic, substituted heterocyclic, heteroaryl, substituted heteroaryl, nitro, cyano, mercapto, sulfonylamino, and oxo, wherein the substituent is attached at any available point to produce a stable compound.

[0134] As used herein, the term "hydrocarbon group" refers to any organic group whose skeleton contains only carbon and hydrogen. Thus, hydrocarbon groups include alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, alkylaryl, arylalkyl, arylalkenyl, alkenylaryl, arylalkynyl, alkynylaryl, and similar groups.

[0135] As used herein, the term "substituted hydrocarbon group" refers to any of the above-mentioned hydrocarbon groups further comprising one or more substituents selected from the following: hydroxyl, alkyloxy, substituted alkyloxy, alkathio, substituted alkathio, arylthio, substituted arylthio, amino, alkylamino, substituted alkylamino, carboxyl, -C(S)SR, -C(O)SR, -C(S)NR2, wherein each R is independently hydrogen, alkyl or substituted alkyl, nitro, cyano, halogen, -SO3M or -OSO3M, wherein M is H, Na, K, Zn, Ca, or meglumine, guanidinyl, substituted guanidinyl, hydrocarbon, substituted hydrocarbon, hydrocarbon carbonyl, substituted hydrocarbon Carbonyl, alkyloxycarbonyl, substituted alkyloxycarbonyl, alkylcarbonyloxy, substituted alkylcarbonyloxy, acyl, acyloxy, heterocycle, substituted heterocycle, heteroaryl, substituted heteroaryl, heteroarylcarbonyl, substituted heteroarylcarbonyl, carbamoyl, monoalkylcarbamoyl, dialkylcarbamoyl, arylcarbamoyl, carbamic acid, dithiocarbamic acid, aromatic acyl, substituted aromatic acyl, organic sulfonyl, substituted organic sulfonyl, organic sulfinyl, substituted alkyl sulfinyl, alkyl sulfonyl amino, substituted alkyl sulfonyl amino, aryl sulfonyl amino, substituted aryl sulfonyl amino, sulfonyl amino, sulfonyl amino, sulfonyl group and similar groups,Two or more of the groups mentioned above are attached to the hydrocarbon moiety via linker / spacer portions such as: -O-, -S-, -NR- — where R is hydrogen, alkyl, or a substituted alkyl; -C(O)-, -C(S)-, -C(=NR')-, -C(=CR'2)- — where R' is alkyl or a substituted alkyl; -OC(O)-, -OC(O)-O-, -OC(O)-NR- (or -NR-C(O)-O-), -NR-C(O)-, -NR-C(O)-NR -, -SC(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)-, -NR-C(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-OS(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-NR-, -O-NR-S(O)2-, -OP(O)R2-, -SP(O)R2- or -NR-P(O)R2-, wherein each R is independently hydrogen, alkyl or substituted alkyl, and similar groups.

[0136] As used herein, the term "hydroxyl (hydroxyl or hydroxy)" refers to the -OH group.

[0137] As used herein, the term “oxo” refers to an oxygen substituent bonded to an attached carbon double bond.

[0138] As used herein, the term "sulfinyl" signifies the group -S(O)-.

[0139] As used herein, the term "substituted sulfinyl group" signifies the group -S(O)R t , where R t It is a lower alkyl group, a substituted lower alkyl group, a cycloalkyl group, a substituted cycloalkyl group, a cycloalkyl alkyl group, a substituted cycloalkyl alkyl group, a heterocyclic group, a substituted heterocyclic group, a heterocyclic alkyl group, a substituted heterocyclic alkyl group, an aryl group, a substituted aryl group, a heteroaryl group, a substituted heteroaryl group, a heteroaryl alkyl group, a substituted heteroaryl alkyl group, an aryl group, or a substituted aryl alkyl group.

[0140] As used herein, the term "sulfonyl" signifies the group -S(O)2-.

[0141] As used herein, the term "substituted sulfonyl" refers to the group -S(O)2R. t , where R t It is a lower alkyl group, a substituted lower alkyl group, a cycloalkyl group, a substituted cycloalkyl group, a cycloalkyl alkyl group, a substituted cycloalkyl alkyl group, a heterocyclic group, a substituted heterocyclic group, a heterocyclic alkyl group, a substituted heterocyclic alkyl group, an aryl group, a substituted aryl group, a heteroaryl group, a substituted heteroaryl group, a heteroaryl alkyl group, a substituted heteroaryl alkyl group, an aryl group, or a substituted aryl alkyl group.

[0142] As used herein, the term “thioyl” signifies the group -S(O)2-.

[0143] This invention provides methods and compositions for synthesizing 5' capped RNA, wherein the initiating capped oligonucleotide primer has a general formula. m7 Gppp[N 2’Ome ] n[ N] m ,in m7 G is an N7-methylated guanosine or any guanosine analogue, N is any natural, modified, or non-natural nucleoside, "n" can be any integer from 1 to 4, and "m" can be an integer from 1 to 9. In one aspect of the invention, the initiating capped oligonucleotide primer has the structure of Formula I:

[0144]

[0145] in:

[0146] B1 to B 10 Each of them is independently a natural, modified, or non-natural nucleoside base;

[0147] M is 0 or 1;

[0148] L is 0 or 1;

[0149] q1 is 1;

[0150] Each of q2 to q9 is independently 0 or 1;

[0151] R1 is H or methyl;

[0152] R2 and R3 are independently H, OH, alkyl, O-alkyl, amine, azide, halogen, linker, or detectable marker;

[0153] X1 to X 13 Each of them is independently O or S;

[0154] Y1 to Y 13 Each of them is independently OH, SH, BH3, aryl, alkyl, O-alkyl, or O-aryl;

[0155] Z0 is 0; Z1 to Z 22 Each of these is independently O, S, NH, CH2, C (halogen)2 or CH (halogen), and

[0156] R4 to R 12 Each of them is independently H, OH, OMe, or a detectable marker.

[0157] In some implementations, the initiating 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), heptameric (q) 6 -q 9 =0), Octamer (q) 7 -q 9 =0), nonamer (q) 8 -q 9 =0), decamer (q) 9 =0) or undecimer. Several examples of primers for initiating capped oligonucleotide trimers are listed in Table I below:

[0158] Table I

[0159]

[0160]

[0161] Other initiating capped oligonucleotide primers covered by this invention include those having known or novel base analogues. Methods for synthesizing initiating capped oligonucleotide primers are illustrated in the examples below.

[0162] Transcription

[0163] In eukaryotic cells, transcription of messenger RNA (mRNA) is accomplished by RNA polymerase II. This is a complex multi-subunit enzyme with intricate regulation. For large-scale transcription in vitro, studies typically use single-subunit phage polymerases derived from T7, T3, SP6, K1-5, K1E, K1F, or K11 phages. This family of polymerases has a simple, minimal promoter sequence of ~17 nucleotides, requires no accessory proteins, and has minimal restrictions on the starting nucleotide sequence. While this application focuses on T7 RNA polymerase (T7 RNAP), those skilled in the art will understand that the invention can be practiced with other RNA polymerases.

[0164] T7 RNAP exists in at least two protein states. The first is called the “abortive complex” and is associated with transcription initiation. The second is a very progressive conformation called the “elongation complex.” In vitro transcription can be divided into six steps: 1) RNA polymerase binds to the promoter sequence, 2) transcription initiation, 3) non-progressive elongation, known as abortive transcription, during which the polymerase frequently releases the DNA template and short abortive transcripts, 4) the open complex converts to the closed complex, 5) progressive elongation, and 6) transcription termination. The large amount of RNA produced during transcription consists of short abortive fragments of 2–8 nucleotides in length (Biochemistry 19:3245–3253 (1980); Nucleic Acids Res. 9:31–45 (1981); Nucleic Acids Res. 15:8783–8798 (1987); Biochemistry 27:3966–3974 (1988)). After synthesizing approximately 10–14 bases, the RNA polymerase escapes from the ineffective cycle, loses its sequence-specific contact with the promoter DNA, and forms a progressive elongation complex in which the RNA chain elongates in a sequence-independent manner (J.Mol.Biol.183:165-177(1985); Proc.Natl.Acad.Sci.USA83:3614-3618(1986); Mol.Cell Biol.7:3371-3379(1987)).

[0165] The common sequences of the most active class III T7 promoters include a 17 bp sequence upstream of the transcription start site and a 6 bp sequence downstream of it (Cell 16:815-25.(1979)). The position of the first transcribed nucleotide is generally referred to as the +1 transcript nucleotide of RNA, the second transcribed nucleotide as the +2 transcript nucleotide, and so on (Table 2). During transcription, the two strands unwind (melt) to form a transcription bubble, and the lower strand of the double helix (shown as 3' to 5' in Table 2) serves as the template for transcription. For transcript nucleotides +3 and beyond, the template strand primarily defines the identity of the transcribed nucleotide through Watson-Crick base pairing interactions. Here, the nucleotide encoding the first RNA transcript nucleotide is defined as the +1 template nucleotide. In the examples shown in Table 2, the +1 transcript nucleotide is G, and the +1 template nucleotide is C. Similarly, the +4 transcript nucleotide is A, and the +4 template nucleotide is T.

[0166] Table 2

[0167]

[0168] Unlike DNA polymerase, T7 RNAP initiates RNA synthesis in the absence of a primer. The first step in initiation is called de novo RNA synthesis, in which RNA polymerase recognizes a specific sequence on the DNA template, selects the first pair of nucleotide triphosphates complementary to the template residues at positions +1 and +2, and catalyzes the formation of a phosphodiester bond to form a dinucleotide. The starting nucleotide has a lower affinity for the polymerase than those used during elongation. For the first initiating NTP, the Kd value is 2 mM, and for the second initiating NTP, the Kd value is 80 μM, while during elongation, the Kd is approximately 5 μM for NTPs (J. Mol. Biol. (2007) 370, 256–268). De novo synthesis has been found to be the rate-limiting step during transcription. T7 RNAP exhibits a strong bias towards GTP as the starting nucleotide (J. Biol. Chem. 248: 2235-2244 (1973)). Of the 17 T7 promoters in the genome, 15 initiate with GTP (and 13 with pppGpG), while no obvious NTP preference is observed during transcriptional elongation (J.Mol.Biol.370:256-268(2007)). T7 RNA polymerase rarely initiates at promoters encoding A at position +1; instead, transcription primarily initiates with promoters encoding G at position +2 (J.Biol.Chem.278:2819-2823(2003)).

[0169] During de novo RNA synthesis, the binding of the initiating nucleotide is primarily achieved through the free energy created by the base stack, specific interactions between polymerase residues, the guanine moiety of the initiating nucleotide, and base complementarity interactions (J.Mol.Biol.370:256-268(2007)).

[0170] T7 RNAP is known to be initiated using short oligonucleotide primers. For example, 13 promoters in the T7 genome are known to be initiated using pppGpG (J.Mol.Biol.370:256-268(2007)). Several groups have shown that T7 RNAP can be initiated using dinucleotide primers (Biochemistry 24:5716-5723(1985)). Axelrod et al. demonstrated that capless GpA dinucleotides can be initiated from +1 and +2 template nucleotides ("CT" templates) of 2'-deoxycytidine and 2'-deoxythymidine, respectively. The reaction conditions are 200 μM dimer and 100 μM ATP, CTP, GTP, and UTP. The reaction mixture also contains 100 μM 3'dATP, 3'dCTP, 3'dUTP, or 50 μM 3'dGTP. They observed only GpA-initiated RNA, and not a mixture of GpA-initiated RNA and 5' triphosphate RNA from GTP-initiated RNA. This is likely due to the reaction conditions used. For the first initiating guanosine, 100 μM GTP was much lower than 2 mM Kd T7 polymerase (J. Mol. Biol. (2007) 370, 256–268). Since GTP competes with the initiating oligonucleotide for initiation, using a low GTP concentration favors GpA initiation but results in low transcription yields (estimated maximum calculated yield of <150 μg / mL). When transcription was initiated on a "CT" template with ApG, CpG, UpG, or GpG, they observed the formation of RNA transcripts with additional non-templated 5' nucleotides (A, C, U, or G, respectively).

[0171] Axelrod et al. also used capless GpG dinucleotides to initiate RNA synthesis at promoters where template nucleotides +1 and +2 were 2'-deoxycytidine ("CC" template). They observed low fidelity initiation and three distinct transcripts. They stated, "Examination of autoradiography indicated that one member of each triplet was initiated with GpG at the normal (+1) position, the second member of each triplet was initiated with GpG at the abnormal (-1) position, and the third member of each triplet was initiated with guanosine triphosphate at the normal position. Therefore, GpG is effective for..." The promoter (“CC” template), and for The promoter (“CT” template) is a relatively weak initiator and does not prevent normal initiation via guanosine triphosphate at the concentration used. They did not observe any initiation via guanosine triphosphate. The promoter (“CC” template) is initiated with a GpA dinucleotide. CpA, ApC, and ApA do not act as initiators on either of the aforementioned “TC” and “CC” templates, presumably because they cannot hybridize with the template nucleotides at positions +1 and +2. The method described by Axelrod et al. is designed to produce very small amounts of radioactive transcripts for sequencing and is not suitable for large-scale production of useful drug-grade RNA. If higher concentrations (~5 mM) of initiator dimers and NTPs (including GTP) are used to increase RNA yield, the expected result is a low proportion of RNA starting with the initiator dimer because at NTP concentrations closer to Kd (2 mM), GTP effectively competes with the dimer for initiation starting from nucleotide +1, resulting in a high proportion of RNA starting with pppG.

[0172] Pitulle et al. demonstrated that transcription with T7 RNAP can be initiated with capless oligonucleotides (dimers to hexamers) (Gene, 112:101-105 (1992)). These oligonucleotides have a 5'-OH or a 5' monophosphate. They also initiated transcription with oligonucleotides with a biotin-ApG structure. All oligonucleotides used in this study contained a 3' G. Pitulle et al. also demonstrated that 2'-O-methyl residues and deoxy residues can be included in the primer sequence to produce RNA transcripts with 2'-O-methylation or 2'-deoxy residues at or near the 5' end of the RNA. It is clear in this publication that the 3' guanosine residue of any initiating oligonucleotide pairs with a +1 template nucleotide. This results in the attachment of a non-templated nucleotide to the 5' end of the transcribed RNA. Specifically, the authors state, "Since pairing with template DNA bases is not required except for the G at the Y-terminus, sequence variations in this segment are also possible." Therefore, all starting oligonucleotide primers are complementary only to the template nucleotide "C" at position +1, and not to any nucleotides at subsequent positions (+2, +3, etc.). This is confirmed in the group's subsequent method papers (Methods Mol Biol. 74:99-110 (1997), Methods Mol. Biol. 252:9-17, (2004)). Their method differs from the method described in this paper—in which all nucleotides of the starting capped oligonucleotide primer are fully complementary to the template nucleotides at positions +1 and beyond. Kleineidam et al. created 5'-modified tRNA transcripts by initiating with dimers or trimers modified with 2'-deoxy or 2'-O-methyl sugars (Nucleic Acids Research 21:1097-1101 (1993)). Again, the authors state that the 3'-terminal guanosine of the primer is initiated at the +1 template nucleotide "C".

[0173] Another study by Ishikawa et al. shows that... m7 GpppApG, m7 Gppp m6 ApG, m7 GpppA 2’Ome pG or m7 Gppp m6 A 2’Ome The capped initiation oligonucleotide trimer of the pG structure can initiate transcription on templates with 2'-deoxycytidine residues at positions +1 and +2 (“CC” template; Nucleic Acids Symposium Series No. 53:129 (2009)). The authors state, “derived from using…” m7 The different outcomes of the G5'pppG case can be due to... m7This is caused by the base pairing between the extra adenosine (N1) in G5'pppN1pG and the 2'-deoxythymidine at the -1 position in the T7 promoter. "This method is clearly distinguished from the method described in this invention—where the +1 and +2 nucleotides of the initiating capped oligonucleotide trimer pair with the +1 and +2 nucleotides of the template nucleotide. Ishikawa et al. used 6 mM of initiating oligonucleotide trimer, 0.9 mM of GTP, and 7.5 mM each of ATP, CTP, and UTP. The authors used a capped initiating oligonucleotide primer (the most expensive nucleotide component in the transcription reaction) with an excess of more than 6 times relative to the competitive GTP to drive the transcription reaction toward capped RNA relative to pppRNA, which increases the overall cost of RNA synthesis. On the other hand, the low concentration of GTP (0.9 mM) limits the total RNA yield in the transcription reaction (theoretically to less than 1.4 mg / mL). In contrast, the method described herein does not require limiting the concentration of any NTP to obtain both efficient RNA capping and higher RNA yields (2 to 6 mg / mL), and therefore allows for the production of high-quality mRNA at a commercially useful cost."

[0174] None of the publications discussed above directly measured RNA capping efficiency, so the degree of capping in those studies is unknown.

[0175] Importantly, in all the studies described above, the +1 template nucleotide was 2'-deoxycytidine (Biochemistry 24:5716-5723 (1985), Gene, 112:101-105 (1992), Methods Mol.Biol. 74:99-110 (1997), Methods Mol.Biol. 252:9-17 (2004), Nucleic Acids Research 21:1097-1101 (1993), Nucleic Acids Symposium Series No. 53:129 (2009)).

[0176] For over 20 years since the publication of studies on transcription initiation using oligonucleotide primers, no instances of transcription initiation using initiation oligonucleotide primers containing 5'- to 5' reverse cap structures had been published until the short report in Nucleic Acids Symposium Series No. 53:129 (2009).

[0177] The methods and compositions provided herein for the preparation of 5'-capped RNA include, but are not limited to, mRNA, small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), and small cajal body-specific RNA (scaRNA). These methods involve DNA-templated and promoter-controlled RNA synthesis using capped oligonucleotide primers, nucleoside 5'-triphosphates (NTPs), and RNA polymerase. In some aspects, the method uses an initiating capping oligonucleotide primer, which provides utility in RNA synthesis, particularly in the synthesis of capped mRNA. The initiating oligonucleotide primer has a structure similar to the cap 0, cap 1, cap 2, or TMG-cap of the native RNA molecule, comprising a 2'-O-methylated nucleoside unit at the 5'-position adjacent to the penultimate cap 1 and the penultimate cap 2. The native cap 0 structure does not have a 2'-O-methylated nucleoside unit.

[0178] Methods and compositions for preparing RNA, including but not limited to mRNA, snRNA, snoRNA, scaRNA, transfer RNA (tRNA), ribosomal RNA (rRNA), and transfer-messenger RNA (tmRNA)—which carries modifications at or near the 5' end of the molecule. These methods involve DNA-templated and promoter-controlled RNA synthesis using capped or non-capped initiating oligonucleotide primers, nucleoside 5'-triphosphates (NTPs), and RNA polymerase. In some aspects, the method uses modified initiating oligonucleotide primers carried in RNA synthesis; particularly in the synthesis of 5'-modified RNA, structural modifications that provide utility.

[0179] The start-capped oligonucleotide primer has an open 3'-OH group, which allows for the initiation of RNA polymerase-mediated RNA synthesis on the DNA template by adding a nucleotide unit to the 3' end of the primer. The start-capped oligonucleotide primer is substantially complementary to the template DNA sequence at the transcription start site (i.e., the start site is closer to the 3' end of the promoter sequence and can overlap with the promoter sequence). In some embodiments, the start-capped oligonucleotide primer primarily directs RNA synthesis in one direction (“forward”) starting from the 3' end of the primer. In some aspects and embodiments, the start-capped oligonucleotide primer is superior to any nucleoside 5'-triphosphate for initiating RNA synthesis, thereby maximizing RNA production initiated with the start-capped oligonucleotide primer and minimizing RNA production initiated with nucleoside 5'-triphosphate (generally GTP).

[0180] mRNA production via in vitro transcription utilizes highly active bacteriophage RNA polymerases (T3, T7, SP6, and others). RNA polymerases operate under the control of specific promoters that are incorporated into the template nucleotide sequence in the DNA plasmid construct. Transcription typically begins with purine nucleoside 5'-triphosphate (generally GTP) and continues until the RNA polymerase encounters a termination sequence or completes the DNA template.

[0181] As discussed above, mCAP dinucleotide analogs containing a 0 cap ( 7m G(5′)ppp(5′)N) has been used to initiate in vitro transcription (e.g., RNA 1:957-967 (1995)). Capped RNA molecules produced using these dinucleotide analogs contain a cap 0. However, only about 50% of the synthesized capped RNA molecules have the correct "positive" orientation of the cap 0. To convert RNA with a cap 0 to RNA with a cap 1, an additional enzymatic reaction must be performed using (nucleoside-2′-O) methyltransferase. However, this conversion may not be quantitative; it is not easy to control and it is difficult to separate the remaining cap 0 RNA from the cap 1 RNA. In addition, competition from NTPs (specifically GTPs) for transcription initiation further reduces the yield of active capped RNA molecules.

[0182] In addition, such as 7m G 3’Ome (5′)ppp(5′)N modified dinucleotide analogs and modifications that block the 3′ and / or 2′ positions on the ribose. 7m Other related ARCA analogs of the G residue have been used for the initiation of in vitro transcription (e.g., RNA 7:1486-1495 (2001)). These ARCA cap analogs direct RNA synthesis only in the "positive" direction and thus produce RNA molecules with a (natural) cap O at the 5' end (having the opposite of the G residue). 7m 2' and / or 3' modification of G residues). Compared to the use of standard dinucleotide analogs ( 7m RNA prepared using G(5′)ppp(5′)N is more active in the translation system. To convert RNA with an ARCA cap of 0 to RNA with an ARCA cap of 1, an additional enzymatic reaction must be performed using a (nucleoside-2′-O) methyltransferase, similar to that required for the previously discussed dinucleotide analogs. This method shares the same drawbacks as the method detailed regarding mCAP dinucleotide analogs; the conversion of RNA with a cap of 0 to RNA with an ARCA cap of 1 may not be quantitative; the reaction is difficult to control, making it difficult to separate the remaining cap 0 RNA from the cap 1 RNA; and competition from NTPs (specifically GTPs) for transcription initiation further reduces the yield of active capped RNA molecules.

[0183] Short oligonucleotide primers (dimers to hexamers) with 3'-terminal guanosine residues have been used for the initiation of in vitro transcription (Pitulle, C. et al., Gene, 112:101-105 (1992)). These oligonucleotide primers contain modified and unmodified ribonucleoside residues (e.g., modified ribonucleoside residues include 2'-O-methylated ribonucleoside residues and 2'-deoxyribonucleoside residues). Shorter oligonucleotide primers (dimers to tetramers) are generally superior to GTP for transcription initiation, while longer primers (pentamers to hexamers) are much less efficient at initiating transcription compared to GTP. This is likely because these longer primers (as they are designed) have a low percentage of complementarity with the DNA template at the initiation site. In contrast, the dimer AG (as designed) is complementary to the DNA template at the initiation site. The RNA molecules generated using oligonucleotide primers discussed in this section have internally 2'-O-methylated nucleosides but lack 5'-caps (0, 1, 2, or TMG-caps). To convert RNA without cap structures into RNA with cap 1, cap 2, or TMG-cap structures, an additional enzymatic reaction using a capping enzyme is necessary. However, this conversion shares the same drawbacks as those described above.

[0184] Other short RNA oligonucleotides containing a cap structure and internal 2'-O-methylated nucleoside residues have been chemically prepared (Ohkuboet.al., Org. Letters 15:4386-4389 (2013)). These short capped oligonucleotides were ligated to "decapitated" (without a 5'-cap structure) fragments of long RNA using T4 DNA ligase and complementary DNA fragment (splinter) oligonucleotides. The final RNA synthesized using this chemical-enzymatic method has both internal 2'-O-methylated nucleoside residues and a 5'-TMG-cap structure. However, this ligation method only produces short capped RNA (<200 polymers). Moreover, the yield is low (15-30%). The T4 DNA ligation reaction is not easily controlled and optimized, and requires a laborious separation process using polyacrylamide gel electrophoresis and separation of capped RNA from the remaining uncapped RNA. Separating long (500-10000 bases) capped mRNA from the remaining uncapped mRNA by PAGE is not feasible.

[0185] Finally, 5'-modified nucleosides or 5'-modified mononucleotides or 5'-modified dinucleotides (generally derivatives of guanosine) have been used to initiate in vitro RNA transcription (Gene, 112:101–105 (1992) and Bioconjug. Chem., 10371–378 (1999)). These initiating nucleosides and nucleotides can carry a label or affinity group (e.g., biotin) and, when incorporated at the 5' end of the RNA, will allow for easy detection, isolation, and purification of the synthesized RNA. This 5'-labeled or tagged RNA may be necessary for some applications. However, this strategy is not used to prepare mRNA with cap 0, cap 1, cap 2, or TMG-cap structures.

[0186] In some aspects of the invention, compositions of starter capped oligonucleotide primers of Formula I are provided. In related aspects, methods for synthesizing RNA using starter capped oligonucleotide primers of Formula I are provided.

[0187] Initiation capped oligonucleotide primer

[0188] The initiating capped oligonucleotide primers of this invention have hybridization sequences that are complementary to a sequence on a DNA template at the initiation site. The length of the hybridization sequence of the primers used in the methods and compositions provided herein depends on several factors, including the characteristics of the template nucleotide sequence and the temperature used when the primer hybridizes with the DNA template or during in vitro transcription. The desired length of a specific nucleotide sequence of the initiating capped oligonucleotide primer used for transcription can be readily determined by those skilled in the art or by conventional experimental methods. For example, the length of the nucleic acid or oligonucleotide can be determined based on the desired hybridization specificity or selectivity.

[0189] In some embodiments, the length of the initiating capped oligonucleotide primer (including the inverted 5'-5' cap nucleotide) is between 3 and 9 nucleotides; in some embodiments, the length of the initiating capped oligonucleotide primer (including the cap) is between 3 and 7 nucleotides; in some embodiments, the length of the initiating capped oligonucleotide primer (including the cap) is between 3 and 5 nucleotides; and in some embodiments, the length of the initiating capped oligonucleotide primer (including the cap) is about 3 nucleotides. The length of the hybridization sequence within the initiating capped oligonucleotide primer may be equal to or shorter than the total length of the initiating capped oligonucleotide primer.

[0190] The presence of hybridization sequences prompts the initiating capped oligonucleotide primer to align primarily with the complementary sequence of the DNA template at the start site in the desired direction (i.e., the "forward" direction). In the forward direction, the RNA transcript begins with the reverse guanosine residue (i.e., 7m G(5′)ppp(5′)N…). DNA template ( Figure 1The dominance of the forward alignment of primers relative to an incorrect "reverse" alignment is maintained by the thermodynamics of the hybridization complex. The latter is determined by the length of the hybridization sequence of the initiating capped oligonucleotide primer and the characteristics of the bases involved in hybridization with the DNA template. Hybridization in the desired forward orientation can also depend on the temperature and reaction conditions during hybridization of the DNA template and the initiating capped oligonucleotide primer, or during in vitro transcription.

[0191] Compared to initiation using standard GTP, ATP, CTP, or UTP, the initiation-capped oligonucleotide primers of the present invention enhance the efficiency of transcription initiation. In some embodiments, enhanced transcription initiation is considered to occur when RNA synthesis begins primarily with the initiation-capped oligonucleotide primer rather than with any NTP in the transcription mixture. This enhanced efficiency of transcription initiation results in a higher yield of RNA transcripts. The efficiency of enhanced transcription initiation can be about 10%, about 20%, about 40%, about 60%, about 80%, about 90%, about 100%, about 150%, about 200%, or about 500% higher relative to conventional methods using initiation-capped primers. In some embodiments, the “initiation-capped oligonucleotide primer” outperforms any NTP (including GTP) for transcription initiation. Those skilled in the art can readily determine the level of substrate activity and the efficiency of the initiation-capped oligonucleotide primer. An example of a method for determining substrate efficiency is illustrated in Example 13. In some implementations, initiation occurs from capped oligonucleotide primers instead of NTPs, which results in higher capping levels of transcribed mRNA.

[0192] In some aspects, methods are provided for synthesizing RNA using start-capped oligonucleotide primers with substitutions or modifications. In some aspects, the substitution and modification of the start-capped oligonucleotide primers do not significantly impair RNA synthesis. Routine assays can be performed to determine whether the desired synthetic result can be obtained with the modified start-capped oligonucleotide primers. Those skilled in the art can perform such routine experimental methods to determine whether the desired result can be obtained. Substitutions or modifications to the start-capped oligonucleotide primers include, for example, one or more modified nucleoside bases, one or more modified sugars, one or more modified internucleotide linkers, and / or one or more modified triphosphate bridges.

[0193] Modified initiating capped oligonucleotide primers, which may include one or more modifying groups of the methods and compositions provided herein, can be extended on a DNA template by RNA polymerase by incorporating an NTP into an open 3'-OH group. Initiating capped oligonucleotide primers may include natural RNA and DNA nucleosides, modified nucleosides, or nucleoside analogs. Initiating capped oligonucleotide primers may contain natural internucleotide phosphodiester linkers or modified forms thereof, or combinations thereof.

[0194] In one embodiment, the modifying group may be a thermally unstable group that dissociates from the modified initiating capped oligonucleotide primer at a rate that increases with increasing temperature of the enzyme reaction medium. Examples of thermally unstable oligonucleotides and NTPs are described in Nucleic Acids Res., 36:e131 (2008), Collect. Symp. Ser., 10:259-263 (2008) and Analytical Chemistry, 81:4955-4962 (2009).

[0195] In some respects, methods for synthesizing RNA are provided, wherein at least one or more NTPs are added to a transcription reaction, which may have modifications as disclosed herein. In some respects, the modification of the at least one NTP does not significantly impair RNA polymerase-mediated RNA synthesis. NTP modifications may include, for example, one or more modified nucleoside bases, one or more modified sugars, or one or more modified 5'-triphosphates. The modified NTP may be incorporated into the 3' end of an initiating capped oligonucleotide primer and does not block transcription and supports further primer elongation.

[0196] In another embodiment, the modifying group of the initiating capped oligonucleotide primer can be a detectable label or a detectable marker. Therefore, after transcription, the target RNA containing the detectable label or marker can be 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 marker is biotin. In some embodiments, one or more components of the transcription reaction (the initiating capped oligonucleotide primer and / or NTP) can be labeled with a detectable label or marker. Therefore, after transcription, the RNA molecule can be identified, for example, by size, mass, affinity capture, or color. In some embodiments, the detectable label is a fluorescent dye; and the affinity capture marker is biotin.

[0197] Standard chemical and enzymatic synthesis methods can be used to synthesize the “starting capped oligonucleotide primers” of this invention, and are disclosed in the Examples section of this document.

[0198] Reagent test kit

[0199] Kits that include “start-capped oligonucleotide primers” for performing transcription are also considered. For example, a kit may contain all the transcription reagents for synthesizing ordinary RNA (e.g., FLuc mRNA). More specifically, a kit may contain: “start-capped oligonucleotide primers”; a container labeled for transcription; instructions for performing RNA synthesis; and one or more reagents selected from one or more modified or unmodified start-capped oligonucleotide primers, one or more unmodified NTPs, one or more modified NTPs (e.g., pseudouridine 5'-triphosphate), RNA polymerase, other enzymes, reaction buffer, magnesium, and DNA template.

[0200] The initiating capped oligonucleotide primers of this invention offer significant advantages over current methods and compositions, involving the use of various initiating nucleosides, nucleotides, and oligonucleotides, or polyphosphate dinucleotide derivatives containing a cap O structure (such as mCAP and ARCA). The initiating capped oligonucleotide primers are compatible with existing transcription systems and reagents and do not require additional enzymes or reagents. Furthermore, the use of initiating capped oligonucleotide primers eliminates the need for several non-enzymatic and enzymatic steps (such as capping and 2'-O-methylation), thus reducing the complexity and cost of RNA synthesis.

[0201] While the exemplary methods described herein relate to T7 RNA polymerase-mediated transcription reactions, a variety of other RNA polymerases known in the art for transcription reactions can be used in conjunction with the compositions and methods of the present invention. Other enzymes, including naturally occurring or mutant variants that can be utilized, include, for example, SP6 and T3 RNA polymerases and RNA polymerases from other sources (including thermostable RNA polymerases).

[0202] Some nucleic acid replication and amplification methods may include transcription as part of the process. These methods include: transcription-mediated amplification (TMA) and nucleic acid sequence-based amplification (NASBA), DNA and RNA sequencing, and other nucleic acid extension reactions known in the art. Those skilled in the art will understand that other methods may be used in place of or in conjunction with transcription methods, including variations of transcription reactions developed in the future.

[0203] Therapeutic uses

[0204] The present invention also considers producing mRNA containing a start-capped oligonucleotide primer for use as a therapeutic agent in pharmaceutical compositions, introducing RNA containing a start-capped oligonucleotide primer into cells to treat a medical condition of the cells, or introducing RNA containing a start-capped oligonucleotide primer into cells that utilize those RNAs to produce proteins that can have therapeutic effects on the host cells.

[0205] A method for treating a condition using RNA containing a start-capped oligonucleotide primer includes the step of administering RNA containing a start-capped oligonucleotide primer of Formula I or a composition containing such RNA to a subject suffering from or suspected of suffering from the condition, wherein the symptoms / symptomology of the condition may be reduced or eliminated in severity.

[0206] When formulated at a concentration of 4 mg / ml or lower in a pharmaceutically acceptable carrier and / or a pharmaceutically acceptable salt, RNA containing a starter capped oligonucleotide primer of Formula I "compound" effectively produces at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more reduction in symptoms and / or symptom severity compared to untreated individuals using the pharmaceutically acceptable carrier alone.

[0207] Pharmaceutical compositions can be formulated for administration by injection or other suitable routes known to those skilled in the art for treating a particular condition. Injectable compositions for parenteral administration generally contain the active compound in a suitable solution and / or drug carrier (such as sterile saline). The composition can also be formulated as a suspension in lipids or phospholipids, in a liposome suspension, or in an aqueous emulsion.

[0208] Methods for preparing various compositions and / or formulations are known to those skilled in the art. See Remington's Pharmaceutical Sciences (19 th Ed., Williams & Wilkins, 1995. The composition to be given will contain a selected compound in a pharmaceutically safe and effective amount for increasing the expression of the desired protein in target cells or tissues.

[0209] In some embodiments, the pharmaceutical composition contains at least 0.1% (w / v) of the compound described above; in some embodiments, the pharmaceutical composition contains more than 0.1%; in some embodiments, the pharmaceutical composition contains up to about 10% of the compound; in some embodiments, the pharmaceutical composition contains up to about 5% of the compound; and in some embodiments, the pharmaceutical composition contains up to about 1% (w / v) of the compound. The selection of an appropriate concentration depends on factors such as the desired dose, frequency, and method of active agent delivery.

[0210] For treatment of subjects (such as mammals or humans), the dosage is determined based on factors such as the subject's weight and overall health, the condition being treated, and the severity of symptoms. The dosage and concentration are determined to produce the desired benefit while avoiding any undesirable side effects. For human patients, the typical dosage of the subject compound is in the range of about 0.0005 to 500 mg / day, and in some embodiments, in the range of about 1-100 mg / day. For example, higher dosage regimens include, for instance, 50-100 mg / day, 75-100 mg / day, or 50-75 mg / day, and lower dosage regimens include, for instance, 1-50 mg / day, 25-50 mg / day, or 1-25 mg / day.

[0211] Various aspects of the invention are illustrated by the following non-limiting examples. These examples are for illustrative purposes and are not intended to limit any practice of the invention. It should be understood that variations and modifications can be made without departing from the spirit and scope of the invention. Those skilled in the art will readily know how to synthesize or commercially obtain the reagents and components described herein.

[0212] This document also includes the following implementation methods:

[0213] Implementation Method 1. A starting capped oligonucleotide primer, comprising the following structure:

[0214]

[0215] in:

[0216] B1 to B 10 Each of them is independently a natural, modified, or non-natural nucleoside base;

[0217] M is 0 or 1;

[0218] L is 0 or 1;

[0219] q1 is 1 and each of q2 to q9 is independently 0 or 1;

[0220] R1 is H or methyl;

[0221] R2 and R3 are independently H, OH, alkyl, O-alkyl, halogen, linker, or detectable marker;

[0222] X1 to X 13 Each of them is independently O or S;

[0223] Y1 to Y 13 Each of these is independently OH, SH, BH3, aryl, alkyl, O-alkyl, or O-aryl;

[0224] Z0 is O;

[0225] Z1 to Z 22 Each of these is independently O, S, NH, CH2, C (halogen)2, or CH (halogen); and

[0226] R4 to R 12 Each of these can be independently H, OH, OMe, or a detectable marker.

[0227] Implementation Method 2. The initiating capped oligonucleotide primer according to Implementation Method 1, wherein:

[0228] q1 = 1, and

[0229] Each of q2-q9 is either 0 or 1.

[0230] Implementation Method 3. The initiating capped oligonucleotide primer according to Implementation Method 1 or 2, wherein:

[0231] B1 is completely complementary to the nucleoside base at position +1 on the transcription template of the nucleic acid template.

[0232] B2 to B9, if present, are completely complementary to the corresponding nucleoside bases at the transcription start site on the nucleic acid template, starting from position +2.

[0233] B 10 The last nucleoside base of the initiating capped oligonucleotide primer is completely complementary to the last hybridization transcription template nucleotide.

[0234] Implementation Method 4. The initiating capped oligonucleotide primer according to Implementation Method 1, wherein:

[0235] q2-q9=0, and

[0236] B10 hybridizes with template nucleotide 2.

[0237] Implementation Method 5. The initiating capped oligonucleotide primer according to Implementation Method 1, wherein:

[0238] q1-q2=1

[0239] q3-9=0, and

[0240] B2 and B10 hybridize with template nucleotides 2 and 3, respectively.

[0241] Implementation Method 6. The initiating capped oligonucleotide primer according to Implementation Method 1, wherein:

[0242] q1-q3=1,

[0243] q4-9=0, and

[0244] B2, B3, and B10 hybridize with template nucleotides 2, 3, and 4, respectively.

[0245] Implementation Method 7. The initiating capped oligonucleotide primer according to Implementation Method 1, wherein:

[0246] q1-q4=1,

[0247] q5-9=0, and

[0248] B2, B3, B4, and B10 hybridize with template nucleotides 2, 3, 4, and 5, respectively.

[0249] Implementation Method 8. The initiating capped oligonucleotide primer according to Implementation Method 1, wherein:

[0250] q1-q5=1,

[0251] q6-9=0, and

[0252] B2, B3, B4, B5, and B10 hybridize with template nucleotides 2, 3, 4, 5, and 6, respectively.

[0253] Implementation Method 9. The initiating capped oligonucleotide primer according to Implementation Method 1, wherein:

[0254] q1-q6=1,

[0255] q7-9=0, and

[0256] B2, B3, B4, B5, B6, and B10 hybridize with template nucleotides 2, 3, 4, 5, 6, and 7, respectively.

[0257] Implementation Method 10. The initiating capped oligonucleotide primer according to Implementation Method 1, wherein:

[0258] q1-q7=1,

[0259] q8-9=0, and

[0260] B2, B3, B4, B5, B6, B7, and B10 hybridize with template nucleotides 2, 3, 4, 5, 6, 7, and 8, respectively.

[0261] Implementation Method 11. The initiating capped oligonucleotide primer according to Implementation Method 1, wherein:

[0262] q1-q8=1,

[0263] q9 = 0, and

[0264] B2, B3, B4, B5, B6, B7, B8, and B10 hybridize with template nucleotides 2, 3, 4, 5, 6, 7, 8, and 9, respectively.

[0265] Implementation Method 12. The initiating capped oligonucleotide primer according to Implementation Method 1, wherein:

[0266] q1-q9=1, and

[0267] B2, B3, B4, B5, B6, B7, B8, B9, and B10 hybridize with template nucleotides 2, 3, 4, 5, 6, 7, 8, 9, and 10, respectively.

[0268] Implementation Method 13. The initiating capped oligonucleotide primer according to Implementation Method 1, wherein:

[0269] q1-q2=1,

[0270] q3-9=0, and

[0271] B2 and B10 hybridize with template nucleotides 2 and 3, respectively.

[0272] Implementation Method 14. An RNA molecule comprising the initiation capped oligonucleotide primer as described in Implementation Method 1.

[0273] Implementation Method 15. A cell containing an RNA molecule comprising an initiating capped oligonucleotide primer as described in Implementation Method 1.

[0274] Implementation Method 16. A cell containing a protein or peptide translated from an RNA molecule comprising an initiating capped oligonucleotide primer as described in Implementation Method 1.

[0275] Embodiment 17. A pharmaceutical composition comprising an RNA molecule and a pharmaceutically acceptable carrier, said RNA molecule comprising the initiating capped oligonucleotide primer according to Embodiment 1.

[0276] Implementation Method 18. A method for synthesizing a fully templated RNA molecule, comprising the following steps:

[0277] Introducing a starter capped oligonucleotide primer having the following structure into a mixture containing the RNA polymerase under conditions favorable to RNA polymerase transcription via a multinucleotide template, and

[0278] Incubate the mixture for a period of time sufficient to allow the template to be transcribed.

[0279]

[0280] in:

[0281] B1 to B 10 Each of them is independently a natural, modified, or non-natural nucleoside base;

[0282] M is 0 or 1;

[0283] L is 0 or 1;

[0284] R1 is H or methyl;

[0285] R2 and R3 are independently H, OH, alkyl, O-alkyl, halogen, linker, or detectable marker;

[0286] X1 to X 13 Each of them is independently O or S;

[0287] Y1 to Y 13 Each of these is independently OH, SH, BH3, aryl, alkyl, O-alkyl, or O-aryl;

[0288] Z0 is O;

[0289] Z1 to Z 22 Each of these is independently O, S, NH, CH2, C (halogen)2, or CH (halogen); and

[0290] R4 to R 12 Each of these can be independently H, OH, OMe, or a detectable marker.

[0291] Example 1

[0292] Preparation of 7-methylguanosine 5-diphosphate (pp) from guanosine 5'-diphosphate 7m G)( Figure 1 )

[0293] Acetic acid was added to a solution of guanosine 5'-bisphosphate (2.5 mmol) in 40.0 mL of water under stirring to adjust the pH of the solution to 4.0. Dimethyl sulfate (4.0 mL) was added dropwise to the mixture over a period of 30 minutes, and the reaction mixture was stirred at room temperature for 4 hours while maintaining the pH of the reaction mixture at approximately 4.0 using 0.1 M NaOH solution. After 4 hours, the reaction mixture was extracted with CH2Cl2 (3 × 50 mL) to remove unreacted dimethyl sulfate. The aqueous layer was diluted with water to 500 mL, adjusted to pH 6.5 with 1 M TEAB, and loaded onto a DEAE Sephadex column (3 × 50 cm). The product was eluted using a linear gradient of 0–1 M TEAB at pH 7.5 (3 L). The solution containing pure PP was then... 7m The G (triethylammonium salt) was partially combined, evaporated, and dried under high vacuum to obtain a fine white powder (yield: 80%). A similar procedure is disclosed in Bioorgan. Med. Chem. Letters 17:5295-5299 (2007).

[0294] Example 2

[0295] By pp7m G Preparation of 7-methylguanosine-5'-diphosphate imidazole (Im-pp) 7m G)( Figure 2 )

[0296] pp 7m G's triethylammonium salt (0.4 mmol) was reacted with imidazole (4 mmol), triphenylphosphine (2 mmol), and 2,2'-dipyridyl disulfide (2 mmol) in dry DMF (20 mL) for 8 hours. Crude Im-pp was precipitated by pouring the reaction mixture into 250 mL of 0.2 M sodium perchlorate solution. 7m G. The mixture was cooled to -20°C, and the precipitate was collected by centrifugation, washed with acetone (3 × 50 mL), and dried under high vacuum. Im-pp 7m The separation yield of G is approximately 100%. Similar procedures are disclosed in Nucleosides, Nucleotides, and Nucleic Acids 24:1131-1134 (2005) and J.Org.Chem.64:5836-5840 (1999).

[0297] Example 3

[0298] Preparation of 3'-O-methylguanosine 5'-phosphate (pG( 3’Ome ))( Figure 3 )

[0299] 3'-O-methylguanosine (10 mmol) was dissolved in triethyl phosphate (40 mL) at 60-70 °C. The mixture was cooled to 0 °C in an ice-water bath, phosphorus oxychloride (30 mmol) was added, and the mixture was stirred at room temperature under argon for 3 hours. The reaction was quenched by slowly adding 1 M TEAB (100 mL; pH 8.5) with stirring. The mixture was stirred for 8 hours and diluted with 1 L of water. The resulting solution was loaded onto a DEAE Sephadex column (3 × 40 cm) and eluted with TEAB (pH 7.5) in a linear gradient of 0.05 to 1.0 M (3 L). The fraction containing the purified product was combined, evaporated to a solid residue, and co-evaporated with methanol (4 × 50 mL) to give pG( 3’Ome (Triethylammonium salt) (60% yield). A similar procedure is disclosed in U.S. Patent Application Serial No. 2012 / 0156751.

[0300] Example 4

[0301] By pG( 3’Ome Preparation of 3'-O-methylguanosine 5'-phosphorylimidazole (Im-pG( 3’Ome ))( Figure 4)

[0302] pG( 3’Ome The triethylammonium salt (0.5 mmol) of α-triethylammonium salt was reacted with imidazole (5 mmol), triphenylphosphine (2.5 mmol), and 2,2'-dipyridyl disulfide (2.5 mmol) in dry DMF (25 mL) for 5 hours. Crude Im-pG was precipitated by pouring the reaction mixture into 400 mL of 0.2 M sodium perchlorate in acetone solution. 3’Ome The mixture was cooled to -20°C and the precipitate was collected by centrifugation, washed with acetone (3 × 60 mL), and dried under high vacuum (yield: 100%). A similar procedure is disclosed in U.S. Patent Application Serial No. 2012 / 0156751.

[0303] Example 5

[0304] By Im-pG( 3’Ome Preparation of 3'-O-methylguanosine 5'-bisphosphate (ppG( 3’Ome ))( Figure 5 )

[0305] Solid zinc chloride (14.0 mmol) was added in small portions to Im-pG ( 3’Ome A 40 mL solution of dried DMF containing 7.0 mmol was added. The mixture was stirred under argon for 15 minutes until all solids dissolved. A 40 mL solution of 1 M tributylammonium phosphate in DMF was added and the mixture was stirred at room temperature. After 5 hours, the mixture was diluted with 200 mL of water and extracted with dichloromethane (2 × 200 mL). The aqueous layer was diluted with water (1 L), loaded onto a DEAE Sephadex column (5 × 40 cm), and eluted with a linear gradient of 0.05 to 1.0 M (6 L) TEAB (pH 7.5). The fractions containing the pure product were combined, evaporated, and co-evaporated with methanol (4 × 50 mL) to give ppG as a white solid. 3’Ome (Triethylammonium salt) (Yield: 60%). A similar procedure is disclosed in U.S. Patent Application Serial No. 2012 / 0156751.

[0306] Example 6

[0307] By ppG ( 3’Ome Preparation of 7-methyl-3'-O-methylguanosine 5-bisphosphate (pp 7m G( 3’Ome ()( Figure 6 )

[0308] Preparation of ppG ( 3’Ome(Triethylammonium salt; 3.0 mmol) was dissolved in 50 mL of water and glacial acetic acid was added to adjust the pH of the solution to 4.0. Dimethyl sulfate (10.0 mL) was added dropwise to the mixture over a period of 30 minutes, and the reaction mixture was stirred at room temperature for 4 hours while maintaining the pH at 4.0 ± 0.5 using 0.1 M NaOH solution. After 4 hours, the reaction mixture was extracted with CH2Cl2 (3 × 150 mL) to remove unreacted dimethyl sulfate. The aqueous layer was adjusted to pH 5.5, diluted with water (500 mL), and loaded onto a DEAE Sephadex column (3 × 50 cm). The product was eluted using a linear gradient of 0–1.0 M TEAB, pH 7.5 (3 L). The product containing pure PP was then eluted. 7m G 3’Ome The triethylammonium salt was partially combined, evaporated, and dried under high vacuum to obtain a fine white powder (yield: 80%). Similar procedures are disclosed in RNA 9:1108-1122 (2003); Nucleoside Nucleotides & Nucleic acids 25:337-340 (2006); and U.S. Patent Application Serial No. 2012 / 0156751.

[0309] Example 7

[0310] By pp 7m G( 3’Ome Preparation of 7-methyl-3'-O-methylguanosine 5-bisphosphate imidazole (Im-pp) 7m G( 3’Ome ))( Figure 7 )

[0311] Im-pp was prepared using the method described in Example 1. 7m G( 3’Ome A similar procedure is disclosed in RNA14:1119–1131 (2008).

[0312] Example 8

[0313] Used to prepare pN ( 2’-OR1 General procedure for pN dinucleotides (R1 = H or Me) (Figure) Figure 8 )

[0314] The phosphoramidite monomer (i) (1.0 mmol) and the 2',3',N-protected nucleoside (ii) (1.0 mmol) were reacted in 10 mL of acetonitrile containing 2.5 molar equivalents of activator (tetrazole). After stirring at room temperature for 60 min, the intermediate was oxidized from P(III) to P(V) with iodine and extracted with dichloromethane (200 mL) and brine (200 mL). The organic layer was dried over sodium sulfate and evaporated to a solid foam (intermediate (iii)).

[0315] To remove the DMT-protecting group, intermediate (iii) was dissolved in 10 mL of 80% acetic acid, and after the reaction was complete (approximately 1–2 hours), the mixture was evaporated and co-evaporated with methanol (5 × 30 mL) to remove the acetic acid. The crude 5'-OH dimer (iv) was separated and purified by silica gel chromatography using 5% methanol in dichloromethane as the eluent.

[0316] The 5'-OH dimer (iv) (1.0 mmol) was phosphorylated in 10 mL of acetonitrile with 2 equivalents of bis-cyanoethyl-N,N-diisopropylphosphite and 2 equivalents of activator (tetrazole). After stirring at room temperature for 30 min, the 5'-phospholated dimer was oxidized from P(III) to P(V) with iodine and extracted with dichloromethane (150 mL) and brine (150 mL). The organic layer was evaporated to an oily residue, co-evaporated with methanol (2 × 30 mL), dissolved in 12 mL of methanol, and concentrated ammonia (12 mL) was added. The mixture was kept at room temperature for more than 48 hours until pN was completed. 2’OR1 Deprotection of pN dimer (v). The mixture was evaporated and co-evaporated with methanol (2 × 30 mL).

[0317] When R = methyl, the crude dimer (v) is purified directly by anion exchange and reversed-phase chromatography (step 5). The fractions are evaporated to obtain the final pN as a white solid. 2’OR1 pN dimer (v) (v; R1 = methyl; triethylammonium salt) (35% gross yield).

[0318] When R = TBDMS, the crude dimer (v) was treated with an 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 was complete, the mixture was diluted with 0.05 M TEAB and purified by anion exchange and reverse chromatography (step 5). The fractions were evaporated to give the final pN as a white solid. 2’OR1 pN dimer (v) (R1 = H; triethylammonium salt) (30% gross yield).

[0319] Example 9

[0320] General procedure for synthesizing starting oligonucleotides with cap 0, cap 1, or cap 2 structures (5'-phosphorylated dinucleotides are used in the examples) Figure 9 )

[0321] Method A 1:

[0322] To Im-pp 7m G( 3’Ome ) or Im-pp 7m A suspension of G (2 mmol; sodium salt form) and 5'-phosphorylated dinucleotide (1 mmol; triethylammonium salt) in DMF (50 mL) was slowly added to anhydrous ZnCl2 (1 g) while the mixture was stirred at 35 °C. After 24 hours, the reaction was terminated by adding 25 mM EDTA aqueous solution (500 mL) and neutralized with 1 M sodium bicarbonate solution. The mixture was diluted with water to 1 L and loaded onto a DEAE Sephadex column (3 × 50 cm). The product was eluted using a linear gradient of 0–1 M ammonium bicarbonate, pH 7.2 (2 L). The fractions containing pure product were combined, evaporated, and dried under high vacuum to give a fine white powder (yield: 60%). A similar method is disclosed in U.S. Patent Application Serial No. 2012 / 0156751; Bioorg.Med.Chem.Lett.17:5295-5299 (2007) and RNA14:1119-1131 (2008).

[0323] Method B:

[0324] Im-pp 7m G( 3’Ome ) or Im-pp 7mG (2 mmol) was dissolved in N-methylmorpholine buffer (0.2 M, pH 7.0, 10 mL) containing MnCl2 (2 mmol) and added to solid 5'-phosphorylated dinucleotide (1 mmol; triethylammonium salt). The reaction was stirred at room temperature. After 24–40 hours, the reaction was terminated with 10 mL of 0.25 M EDTA solution. The mixture was loaded onto a DEAE Sephadex column (3 × 50 cm). The product was eluted using a linear gradient of 0–1.0 M ammonium bicarbonate, pH 7.2 (2 L). The fractions containing pure product were combined, evaporated, and dried under high vacuum to give a fine white powder (50% yield). Similar methods are disclosed in Bioorganic & Medicinal Chemistry 21:7921-7928 (2013), Nucleic Acids Research 37:1925-1935 (2009); J. Org. Chem. 64:5836-5840 (1999). (Note: 1. 5'-phosphorylated trimers, tetramers, pentamers, hexamers, heptamers, octamers, nonamers, or decamers can be used instead of the 5'-phosphorylated dinucleotides described in Example 9. 2. To prepare a starting capped oligonucleotide with a cap of 0, the 5'-phosphorylated oligonucleotide does not have a 2'-O-methyl group on the first 5'-nucleoside residue, for example, pApG. 3. To prepare a starting capped oligonucleotide with a cap of 1, the 5'-phosphorylated oligonucleotide carries a 2'-O-methyl group on the first 5'-nucleoside residue, for example, pA( 2’Ome )pG. 4. To prepare an initiating capped oligonucleotide with a 2-cap, the 5'-phosphorylated oligonucleotide carries two 2'-O-methyl groups on the first and second 5'-nucleoside residues, for example, pA( 2’Ome )pG( 2’Ome )pG).

[0325] Example 10

[0326] Based on the structure of the initiating capped oligonucleotide primer according to Formula I

[0327] Figure 10 shows the structure of the initiating capped oligonucleotide primer used in the example according to structure I. A) m7 G 3’ Ome pppG, B) m7 GpppG 2’Ome pG, C) m7 G 3’Ome pppG 2’Ome pG, D) m7 GpppA 2’Ome pG, E) m7 G 3’Ome pppA2’Ome pG, F) m7 GpppC 2’Ome pG, G) m7 G 3’Ome pppC 2’Ome pG, H) m7 GpppA 2’Ome pG 2’Ome pG.

[0328] Example 11

[0329] Transcription was performed in vitro using ARCA primers.

[0330] A double-stranded DNA transcription template encoding firefly luciferase was generated via polymerase chain reaction. The +1 template nucleotide was 2'-deoxycytidine. The transcription reaction was performed with 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 mouse RNase inhibitor (New England Biolabs catalog #M0314), 2 units / mL inorganic pyrophosphatase (New England Biolabs catalog #M2403), 4000 units / mL T7 RNA polymerase (New England Biolabs catalog #M0251), and 6 mM ARCA (…). m7 G 3’Ome The mRNA was prepared using a combination of 1.5 mM MgCl2, 7.5 mM ATP, 7.5 mM CTP, and 7.5 mM UTP. Transcription was incubated at 37°C for 2 hours. The reaction was supplemented with 10 mM Tris-HCl (pH 7.6), 2.5 mM MgCl2, 0.5 mM CaCl2, and 100 units / mL DNase I (New England Biolabs catalog #M0303) and incubated at 37°C for 1 hour. The resulting mRNA was purified using the RNeasy Maxi kit (Qiagen catalog #75162) according to the manufacturer's instructions. The mRNA was eluted in water and dephosphorylated by adjusting the solution to 50 mM Bis-Tris-Propane HCl (pH 6.0), 1 mM MgCl2, 0.1 mM ZnCl2, and 250 units / mg Antarctic phosphatase (New England Biolabs catalog #M0289). The reaction was incubated at 37°C for 1 hour. The obtained mRNA was purified using the RNeasy Maxi kit (Qiagen catalog #75162) according to the manufacturer's instructions. The mRNA was then eluted in water.

[0331] Example 12

[0332] Transcription was performed in vitro using a trimer-initiated capped oligonucleotide primer.

[0333] Table 1 indicates some of the initiation capped oligonucleotide primers used for transcription. A specific double-stranded firefly luciferase DNA transcription template is generated for each trimer via polymerase chain reaction. These templates differ in template nucleotides +1 and +2. For a given trimer, template nucleotide +1 is complementary to B1 (which is destined to become transcript nucleotide +1). Similarly, template nucleotide +2 is complementary to B... 10 (This nucleotide is destined to become transcript nucleotide +2; see Example 10)) Complementary. The transcription reaction was performed with 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 mouse RNase inhibitor (New England Biolabs catalog #M0314), 2 units / mL inorganic pyrophosphatase (New England Biolabs catalog #M2403), 4000 units / mL T7 RNA polymerase (New England Biolabs catalog #M0251), and 6 mM initiating capped oligonucleotide primer, 1.5 mM GTP, and 7.5 mM each of ATP, CTP, and UTP. In subsequent examples, this primer / NTP formulation will be referred to as primer / NTP formulation 1. Those skilled in the art will understand that other polymerases, such as T7, T3, or SP6 RNA polymerases, can be used in place of T7 RNA polymerase to perform the same function by using their corresponding promoters. The transcription reaction mixture was incubated at 37°C for 2 hours. The reaction was supplemented with 10 mM Tris-HCl (pH 7.6), 2.5 mM MgCl2, 0.5 mM CaCl2, and 100 units / mL DNase I (New England Biolabs catalog #M0303), and incubated at 37°C for 1 hour. The resulting mRNA was purified using the RNeasy Maxi kit (Qiagen catalog #75162) according to the manufacturer's instructions. The mRNA was eluted in water and dephosphorylated by adjusting the solution to 50 mM Bis-Tris-Propane HCl (pH 6.0), 1 mM MgCl2, 0.1 mM ZnCl2, and 250 units / mg Antarctic phosphatase (New England Biolabs catalog #M0289). The reaction was incubated at 37°C for 1 hour. The resulting mRNA was purified using the RNeasy Maxi kit (Qiagen catalog #75162) according to the manufacturer's instructions. The mRNA was then eluted in water.

[0334] Example 13

[0335] Tetramer-initiated capped oligonucleotide primers 7m G 3’Ome pppA 2’Ome pG 2’Ome pG in vitro transcription

[0336] Initiation capped oligonucleotide primer 7m G 3’OMe pppA 2’OMe pG 2’OMe pG is used for transcription. Transcription is performed according to Example 11 with the following modifications. A specific double-stranded DNA transcription template is generated as a tetramer via polymerase chain reaction. Template nucleotide +1 (2'-deoxythymidine) is complementary to adenosine (the first nucleotide of the primer destined to become transcript nucleotide +1). Similarly, template nucleotide +2 (2'-deoxycytidine) is complementary to guanosine (the second nucleotide of the primer destined to become transcript nucleotide +2). Similarly, template nucleotide +3 (2'-deoxycytidine) is complementary to guanosine (the third nucleotide of the primer destined to become transcript nucleotide +3). Transcription reaction was performed using 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 mouse RNase inhibitor (New England Biolabs catalog #M0314), 2 units / mL inorganic pyrophosphatase (New England Biolabs catalog #M2403), 4000 units / mL LT7 RNA polymerase (New England Biolabs catalog #M0251), and 6 mM initiating capped oligonucleotide primers. 7m G 3’OMe pppA 2’ OMe pG 2’OMeThe reaction mixture consisted of pG, 1.5 mM GTP, 7.5 mM ATP, 7.5 mM UTP, and 7.5 mM CTP. Those skilled in the art will recognize that other polymerases, such as T7, T3, or SP6 RNA polymerases, can be used in place of T7 RNA polymerase to perform the same function using their corresponding promoters. The transcription reaction mixture was incubated at 37°C for 2 hours. The reaction was supplemented with 10 mM Tris-HCl (pH 7.6), 2.5 mM MgCl2, 0.5 mM CaCl2, and 100 units / mL DNase I (New England Biolabs catalog #M0303), and incubated at 37°C for 1 hour. The resulting mRNA was purified using the RNeasy Maxi kit (Qiagen catalog #75162) according to the manufacturer's instructions. The mRNA was eluted in water and dephosphorylated by adjusting the solution to 50 mM Bis-Tris-Propane HCl (pH 6.0), 1 mM MgCl2, 0.1 mM ZnCl2, and 250 units / mg Antarctic phosphatase (New England Biolabs catalog #M0289). The reaction was incubated at 37°C for 1 hour. The resulting mRNA was purified using the RNeasy Maxi kit (Qiagen catalog #75162) according to the manufacturer's instructions. The mRNA was then eluted in water.

[0337] Example 14

[0338] On transcription templates containing 2'-deoxythymidine and 2'-deoxycytidine residues at template positions +1 and +2, respectively, using m7 GpppA 2’Ome pG-initiated capped oligonucleotide primers were used for in vitro transcription.

[0339] Double-stranded firefly luciferase DNA was used as the transcription template, containing 2'-deoxythymidine and 2'-deoxycytidine residues at positions +1 and +2, respectively. Two transcription reactions were performed using 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 mouse RNase inhibitor (New England Biolabs catalog #M0314), 2 units / mL inorganic pyrophosphatase (New England Biolabs catalog #M2403), and 4000 units / mL T7 RNA polymerase (New England Biolabs catalog #M0251). The two transcriptions differed in the amount of initiating capped oligonucleotides, the amount of NTPs, and the characteristics of the NTPs. The first transcription was designed to mimic the transcription conditions described by Ishikawa et al. (Nucleic Acids Symposium Series No. 53:129 (2009)). This transcriptional reaction contained 6 mM of [unclear text - likely a specific ingredient or ingredient]. m7 GpppA 2’Ome pG-initiated capped oligonucleotide primer, 0.9 mM GTP, and 7.5 mM ATP, CTP, and UTP. In subsequent examples, this primer / NTP formulation will be referred to as primer / NTP formulation 2. In this formulation, the initiating oligonucleotide primer is in greater than 6-fold excess relative to GTP. The second transcription uses 5 mM ATP. m7 GpppA 2’OmepG-initiated capped oligonucleotide primers, 5 mM GTP, ATP, CTP, and pseudouridine triphosphate (ΨTP). In subsequent examples, this primer / NTP formulation will be referred to as primer / NTP formulation 3. In primer / NTP formulation 3, the GTP concentration is increased to produce a commercially useful amount of RNA. Those skilled in the art will recognize that other polymerases, such as T7, T3, or SP6 RNA polymerases, can be used in place of T7 RNA polymerase to perform the same function using their corresponding promoters. The transcription reaction mixture was incubated at 37°C for 2 hours. The reaction was supplemented with 10 mM Tris-HCl (pH 7.6), 2.5 mM MgCl2, 0.5 mM CaCl2, and 100 units / mL DNase I (NewEngland Biolabs catalog #M0303) and incubated at 37°C for 1 hour. The resulting mRNA was purified using the RNeasy Maxi kit (Qiagen catalog #75162) according to the manufacturer's instructions. mRNA was eluted in water and dephosphorylated by adjusting the solution to 50 mM Bis-Tris-Propane HCl (pH 6.0), 1 mM MgCl2, 0.1 mM ZnCl2, and 250 units / mg Antarctic phosphatase (New England Biolabs catalog #M0289). For primer / NTP formulation 2, the reaction was incubated at 37°C for 1 hour, and for primer / NTP formulation 3, the reaction was incubated at 37°C for 3 hours. The resulting mRNA was purified using the RNeasyMaxi kit (Qiagen catalog #75162) according to the manufacturer's instructions. The mRNA was eluted in water. The purification transcription yields for primer / NTP formulations 2 and 3 were 0.7 mg / mL and 3.9 mg / mL, respectively. The yield for primer / NTP formulation 3 was significantly better than that obtained for primer / NTP formulation 2.

[0340] Example 15

[0341] On transcription templates containing cytidine residues at template positions +1 and +2, using m7 GpppA 2’Ome pG-initiated capped oligonucleotide primers for in vitro transcription

[0342] The template was transcribed using double-stranded firefly luciferase DNA, where +1 and +2 template nucleotides were cytidine, and therefore not completely transcribed. m7 GpppA 2’OmeThe pG initiation capped oligonucleotide primers were complementary. Two transcription reactions were combined with 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 mouse RNase inhibitor (New England Biolabs catalog #M0314), 2 units / mL inorganic pyrophosphatase (New England Biolabs catalog #M2403), and 4000 units / mL T7 RNA polymerase (New England Biolabs catalog #M0251). The two transcriptions differed only in the amount of initiation capped oligonucleotides and NTPs. The first transcription was designed to mimic... Ishikawa Transcription conditions (Nucleic Acids Symposium Series No. 53:129 (2009)) were followed. This transcription reaction contained primer / NTP formulation 2. A second transcription was performed using primer / NTP formulation 3. Those skilled in the art will understand that other polymerases, such as T7, T3, or SP6 RNA polymerases, can be used in place of T7 RNA polymerase to perform the same function using their corresponding promoters. The transcription reaction mixture was incubated at 37°C for 2 hours. The reaction was supplemented with 10 mM Tris-HCl (pH 7.6), 2.5 mM MgCl2, 0.5 mM CaCl2, and 100 units / mL DNase I (New England Biolabs catalog #M0303) and incubated at 37°C for 1 hour. The resulting mRNA was purified according to the manufacturer's instructions using the RNeasy Maxi kit (Qiagen catalog #75162) or by reversed-phase high-performance liquid chromatography. mRNA was dephosphorylated by adjusting the solution to 50 mM Bis-Tris-Propane HCl (pH 6.0), 1 mM MgCl2, 0.1 mM ZnCl2, and 250 units / mg Antarctic phosphatase (NewEngland Biolabs catalog #M0289). For primer / NTP formulation 2, the reaction was incubated at 37°C for 1 hour, and for primer / NTP formulation 3, the reaction was incubated at 37°C for 3 hours. The resulting mRNA was purified using the RNeasy Maxi kit (Qiagen catalog #75162) according to the manufacturer's instructions. The mRNA was eluted in water. The purification transcription yields for primer / NTP formulations 2 and 3 were 0.6 mg / mL and 3.9 mg / mL, respectively. The yield for primer / NTP formulation 3 was significantly better than that obtained for primer / NTP formulation 2.

[0343] Example 16

[0344] Translate mRNA in Huh-7 cells ( Figure 11 )

[0345] Translational activity of luciferase mRNA generated using initiating capped oligonucleotide primers was evaluated in triplicate in cultured hepatocytes. Huh-7 cells were cultured at 37°C in DMEM supplemented with 10% FBS, L-glutamine, non-essential amino acids, and penicillin / streptomycin at 5% CO2. Cells were transfected with 400 ng of mRNA. For comparison, cells were also transfected with Cap0 luciferase mRNA, generated by initiation with ARCA. At 20 h, cells were harvested and luciferase activity was measured using the ONE-Glo luciferase assay kit (Promega catalog #E6120) according to the manufacturer's recommendations. Luminescence was measured using the GloMax-Multi+ detection system instrument according to the manufacturer's recommendations. Luciferase activity of all tested mRNAs was detected. Figure 11 The fact that mRNAs generated using initiating capped oligonucleotide primers or 3'-O-methyl initiating capped oligonucleotide primers have translation efficiency similar to that of ARCA-capped RNAs indicates that they are effectively co-transcribed and capped.

[0346] Example 17

[0347] An assay to determine the capping efficiency of mRNA generated by co-transcriptional capping using ARCA or primers employing primer / NTP formulation 1 ( Figures 12A-12H )

[0348] For each initiating capping oligonucleotide primer tested, a sufficient amount of mRNA for detection by liquid chromatography-mass spectrometry (LC-MS) was used for a capping assay. In this assay, a small fragment was cut from the 5' end of the full-length mRNA and analyzed by LC-MS. Prior to cutting, the mRNA was treated with Antarctic phosphatase (New England Biolabs catalog #M0289) to convert uncapped monophosphates, diphosphates, and triphosphates to 5'OH for analysis. The phosphatase-treated mRNA was then cut and purified. The purified RNA was analyzed by LC-MS. Figure 12 shows the LC traces. The LC peaks corresponding to uncapped (5'OH after phosphatase treatment) and capped 1 are indicated by the observed quality. The inset schematic shows the alignment of the initiating oligonucleotide primer on the transcription template. Note that in the schematic, "|" indicates a base pair between the capped initiating nucleotide and the template nucleotide. The subscript "m" indicates 2'-O-methyl and the superscript "m7" indicates base methylation. For comparison, a capping assay was performed using ARCA-co-transcribed capped mRNA. The capping efficiency is estimated using the following formula: (intensity of the capped peak) / [(intensity of the capped peak) + (intensity of the 5'OH peak)]. The % capping observed in Figure 12 is represented by A). 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 pG 2’Ome pG = 50%. In each case, the capping efficiency of transcripts co-transcribed with initiating trimer-capped oligonucleotide primers is greater than that of transcripts with pG = 50%. m7 G 3’Ome Capping efficiency observed by pppG(ARCA).

[0349] Example 18

[0350] On transcription templates with 2'-deoxythymidine and 2'-deoxycytidine residues at template positions +1 and +2 vs. on transcription templates with cytidine residues at template positions +1 and +2 m7 GpppA 2’Ome Comparison of pG-initiated capped oligonucleotide capping ( Figures 13A-13D )

[0351] Capping assays were performed on the mRNAs prepared in Examples 14 and 15 to determine the relative efficiency and specificity of capping transcripts with 2'-deoxythymidine and 2'-deoxycytidine residues at template positions +1 and +2 versus transcripts with cytidine residues at template positions +1 and +2. Sufficient amounts of mRNA, detectable by liquid chromatography-mass spectrometry (LC-MS), were used for the capping assays. In this assay, a small fragment was cut from the 5' end of the full-length mRNA and analyzed by LC-MS. Prior to cutting, the mRNA was treated with Antarctic phosphatase (New England Biolabs catalog #M0289) to convert uncapped monophosphates, diphosphates, and triphosphates to 5'OH to facilitate analysis. The phosphatase-treated mRNA was then cut and purified. The purified RNA was analyzed by LC-MS. Figure 13 shows the LC traces. The LC peaks corresponding to uncapped and capped 1 are indicated by the observed quality. The inset schematically illustrates the alignment of the starting oligonucleotide primers on the transcription template. The subscript "m" indicates 2'-O-methyl and the superscript "m7" indicates base methylation. Figure 13A and 13B Transcripts were transcribed from templates containing 2'-deoxythymidine and 2'-deoxycytidine at positions +1 and +2, respectively, using primer / NTP formulation 2 and primer / NTP formulation 3, respectively. Figure 13C and 13D These are transcripts transcribed from a template containing 2'-deoxycytidine at nucleotides +1 and +2, respectively, using primer / NTP formulation 2 and primer / NTP formulation 3. m7 GpppA 2’Ome When the pG-initiated oligonucleotide is fully complementary to both +1 and +2 nucleotides, the major product observed is... m7 GpppA 2’Ome pG...initiated desired templated cap 1 transcript ( Figure 13A and 13B ).for Figure 13A and 13B The capping efficiency was estimated using the following formula: (intensity of the capping peak) / [(intensity of the capping peak) + (intensity of the 5' OH peak)], and the capping efficiencies with primer / NTP formulation 2 and primer / NTP formulation 3 were 99% and 96%, respectively. Only a small amount of anomalous starting products were detected. Conversely, when template nucleotides +1 and +2 were cytidine, inm7 GpppA 2’Ome There is no perfect complementarity between the pG initiating oligonucleotide and these template nucleotides. Figure 13C and 13D The diagram illustrates the initiating capping oligonucleotides starting with two registers. In the first register, the 3' guanosine initiating capping oligonucleotide residue pairs with +1 template cytidine to produce a transcript with an additional non-templated 5' adenosine (cap1+A). Note that in the diagram, "|" indicates a base pair between the capping initiating nucleotide and the template nucleotide. The specified template nucleotide position is indicated. In the second register, the 3' guanosine initiating capping oligonucleotide residue pairs with +2 template cytidine and the +1 initiating capping oligonucleotide adenosine does not form a complete heterozygote with +1 template nucleotide. This produces a transcript where the templated 5' guanosine has been replaced by a non-templated adenosine (cap1, G to A). The capping efficiency is estimated using the following formula: (intensity of capping peak "Cap1+A" + "Cap1 G to A") / [(intensity of capping peak "Cap1+A" + "Cap1 G to A") + (intensity of 5' OH peak)]. Figure 13C and 13D The calculated capping efficiencies were 97% and 77%. Note the following surprising finding: when the initiating capping oligonucleotide primer is perfectly complementary to the corresponding template +1 and +2 nucleotides, the capping efficiency and templated transcription initiation fidelity are much higher. Furthermore, with our method, efficient capping can be achieved without reducing the concentration of the NTPs driving capping, allowing for significantly higher transcription yields. Therefore, our described capping method differs from and is superior to the capping method of Ishikawa et al.

[0352] Example 19 ( Figures 14A-14B )

[0353] On transcription templates with 2'-deoxythymidine and 2'-deoxycytidine residues at template positions +1 and +2 vs. on transcription templates with cytidine residues at template positions +1 and +2 m7 GpppA 2’Ome Comparison of translation of mRNAs derived from pG-initiated capped oligonucleotides in differentiated THP-1 cells

[0354] To assess the expression of the luciferase mRNA generated in Examples 14 and 15, the mRNA was transfected into THP-1 cells (ATCC, catalog #TIB-202) in six duplicates. THP-1 cells were cultured in ATCC-prepared RPMI-1640 (ATCC, catalog #30-2001) supplemented with 10% FBS, sodium pyruvate, and penicillin / streptomycin at 37°C and 5% CO2. Cells were seeded in 24-well plates (2E+05 cells per well) in the presence of phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA; Cell Signaling Technologies, catalog #4174) to induce differentiation. 72 hours post-seeding, cells were transfected with 100 ng of mRNA per well. For comparison, cells were also transfected with Cap0 luciferase mRNA, which is generated by initiation with ARCA. Twenty hours after transfection, cells were harvested and luciferase activity was measured using the ONE-Glo Luciferase Assay System Kit (Promega catalog #E6120) as recommended by the manufacturer. Luminescence was measured using the GloMax-Multi+ Detection System instrument as recommended by the manufacturer. Data were plotted as the mean + / - standard deviation of the mean from six replicates. Data were analyzed using an unpaired t-test to generate p-values ​​as a measure of significance. The p-values ​​indicate the difference between pairs. Comparisons were made using Luciferase in THP-1 cells. m7 GpppA 2’Ome Translation of transcripts generated by pG-initiated capped oligonucleotides and using a template containing 2'-deoxythymidine and 2'-deoxycytidine residues at template positions +1 and +2 (“TC” template) vs. a transcription template with cytidine residues at template positions +1 and +2 (“CC” template) was performed. Transcription was carried out using either primer / NTP formulation 2 (A) or primer / NTP formulation 3 (B). With both formulations, when... m7 GpppA 2’Ome When the pG-initiated capped oligonucleotide primer, as described in this invention, is fully complementary to the template nucleotides +1 and +2 (“TC” template), translation in cultured THP-1 cells is significantly superior. Figure 14B In this study, we also evaluated the activity of mRNA prepared using a "TC" template and capped with ARCA (0-cap). This is the current industry standard for producing co-transcribed capped mRNA. ARCA-capped RNA exhibits higher activity than mRNA prepared using a TC template. m7 GpppA 2’Ome Transcripts produced when the pG-initiated capped oligonucleotide primer is fully complementary to the template nucleotides +1 and +2 (“TC” template) have significantly lower activity.

[0355] In summary, the embodiments shown herein demonstrate that, compared to previously disclosed methods, the methods described herein generate RNA with the following combination: 1) high yield, 2) high degree of capping, 3) high fidelity of templated transcription, and 4) superior activity in cells.

[0356] The invention described herein can be suitably practiced in the absence of any one or more elements or limitations not specifically disclosed herein. Therefore, for example, in the context of describing the invention (especially in the context of the appended claims), the terms “a,” “an,” and “the,” and similar language will be interpreted to cover both singular and plural forms unless otherwise indicated herein or clearly contradicted by the context. The terms “comprising,” “having,” “including,” “containing,” etc., should be interpreted broadly without limitation (e.g., meaning “including but not limited to”). The enumeration of numerical ranges herein is merely intended to serve as shorthand for each individual value falling within that range, unless otherwise indicated herein, and each individual value is incorporated into the specification as if it were individually enumerated herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or clearly contradicted by the context. The use of any and all embodiments or exemplary language (e.g., “such as”) provided herein is merely intended to better elucidate the invention and not to limit the scope of the invention, unless otherwise stated. No language in the specification should be construed as indicating that any unstated element is necessary for the practice of the invention. Furthermore, the terminology and expressions used herein are descriptive rather than limiting, and their use is not intended to exclude any equivalents or portions thereof of the features shown and described, but rather to indicate that various modifications are possible within the scope of the invention as claimed. Therefore, it should be understood that while the invention has been specifically disclosed with reference to certain embodiments and optional features, modifications and variations of the invention as disclosed herein can be made by those skilled in the art, and such modifications and variations are considered to be within the scope of the invention. The materials, methods, and examples provided herein are representative of certain embodiments and are exemplary, not intended to limit the scope of the invention.

[0357] This invention has been described broadly and generally. Each narrow class and subgroup falling into the higher-level disclosure also constitutes part of this invention. This includes general descriptions with incidental conditions or negative limitations that exclude any subject matter from the broader class—whether or not such exclusions are specifically enumerated herein.

[0358] Furthermore, when describing features or aspects of the invention with respect to the Markush group, those skilled in the art will recognize that the invention is also described with respect to any individual member or subgroup of members of the Markush group.

[0359] All publications mentioned herein. Patent applications, patents and other references are expressly incorporated by reference in their entirety, as if each were individually incorporated by reference. In case of conflict, this specification (including definitions) shall prevail.

[0360] The applicant reserves the right to physically incorporate any and all materials and information from any such article, patent, patent application or other physical or electronic document into this application.

[0361] Other embodiments are proposed in the appended claims.

Claims

1. A compound having the following formula: or its salt, wherein: B1 and B2 are each independently selected from adenine, N... 6 -Methyladenine, guanine, cytosine, or uracil. R1 is CH3, and R2 is H or methyl.

2. The compound of claim 1, wherein B1 is selected from the group consisting of: adenine, guanine, and N. 6 -Methyladenine, wherein B2 is selected from the group consisting of: adenine, guanine, and N. 6 -Methyladenine.

3. The compound of claim 1, wherein B1 is selected from the group consisting of: adenine, guanine, and N. 6 -Methyladenine.

4. The compound of claim 1, wherein B2 is selected from the group consisting of: adenine, guanine, and N. 6 -Methyladenine.

5. The compound of claim 1, wherein B1 is selected from adenine or guanine.

6. The compound of claim 1, wherein B2 is selected from adenine or guanine.

7. The compound of claim 1, wherein B1 is adenine and B 10 It's guanine.

8. The compound of claim 1, wherein B1 is adenine and B 10 It's uracil.

9. The compound of claim 1, wherein B1 is guanine and B... 10 It's guanine.

10. The compound of claim 1, wherein B1 is cytosine and B 10 It's guanine.

11. The compound of any one of claims 1-10, wherein R2 is CH3.

12. The compound of claim 1, wherein the compound is selected from the group consisting of: m7 G 3’Ome pppApG; m7 G 3’Ome pppGpG; m7 G 3’Ome pppCpG; m7 G 3’Ome pppA 2’Ome pG; m7 G 3’Ome pppA 2’Ome pU; and m7 G 3’Ome pppG 2’Ome pC。 13. The compound of claim 1, wherein the compound is m7 G 3’Ome pppApG.

14. The compound of claim 1, wherein the compound is m7 G 3’Ome pppGpG.

15. The compound of claim 1, wherein the compound is m7 G 3’Ome pppCpG.

16. An RNA molecule comprising the compound of claim 1.

17. Isolated cells containing the RNA molecule according to claim 16.

18. A pharmaceutical composition comprising the RNA molecule according to claim 16 and a pharmaceutically acceptable carrier.

19. A pharmaceutical composition comprising a cell containing an RNA molecule according to claim 16.

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