A method for synthesizing capped RNA and a capped RNA transcription reaction solution

Abstract

The present application relates to the field of genetic engineering technology, and more specifically, to a method for synthesizing capped RNA and a capped RNA transcription reaction solution. The method for synthesizing capped RNA comprises mixing and incubating raw materials; the raw materials include a DNA template, a capping polynucleotide, NTPs, an RNA polymerase, and a solution containing magnesium ions; the DNA template includes an RNA promoter, and the base at position ‑1 of the template chain of the RNA promoter is cytosine; the bases at positions +1 to +n of the template chain of the RNA promoter are respectively connected to the bases B1 to B2 in the capping polynucleotide; n The present invention regulates the base at position ‑1 of the template strand of the RNA promoter to be cytosine, and at the same time regulates the bases at positions +1 to +n of the template strand of the RNA promoter to be respectively aligned with the bases at positions B1 to B2 in the capping polynucleotide. n Complementary pairing significantly improves the efficiency of RNA capping and is beneficial to improving the stability of RNA after transcription and synthesis.

Description

Technical Field

[0001] The present application relates to the field of genetic engineering technology, and in particular to a method for synthesizing capped RNA and a capped RNA transcription reaction solution. Background Art

[0002] Currently, the capping efficiency of RNA synthesized by enzymatic capping and co-transcriptional capping is low, resulting in low stability of the transcribed RNA, which is unable to resist enzyme degradation, increase the affinity for binding to eIF4E, and improve the efficiency of protein translation. Summary of the invention

[0003] The purpose of the embodiments of the present application is to provide a method for synthesizing capped RNA and a capped RNA transcription reaction solution, which aims to improve the problem of low efficiency of existing RNA capping.

[0004] The first aspect of the present application provides a method for synthesizing capped RNA, comprising mixing and incubating raw materials.

[0005] The raw materials include DNA template, capping polynucleotide, NTPs, RNA polymerase and a solution containing magnesium ions.

[0006] The structural formula of the capping polynucleotide is as follows:

[0007]

[0008] B1 to B n Each is independently a natural or modified nucleobase.

[0009] E is 0 or 1.

[0010] F is 0 or 1.

[0011] H1 is 1; H2 to H n Each independently is 0 or 1.

[0012] M1 to M n-1 Each is independently oxygen, hydroxyl, sulfur, mercapto, imino, amino, alkyl and alkoxy.

[0013] M n It is hydroxyl.

[0014] R1 and R2 are each independently hydrogen, alkyl or alkoxy.

[0015] R3 to R n+4 Each is independently hydrogen, hydroxy, alkyl, alkoxy, S-alkyl, NH-alkyl, O-aryl, S-aryl, NH-aryl or halo.

[0016] X1 to X n+4Each is independently oxygen, sulfur, imino and methylene.

[0017] Y1 to Y n+3 Each is independently oxygen, sulfur, selenium or borane.

[0018] Z1 to Z n+3 Each is independently a hydroxyl group, a mercapto group, a boryl group, an aryl group, an alkyl group, an alkoxy group or an O-aryl group.

[0019] Here, n is a natural number greater than or equal to 1.

[0020] The DNA template includes an RNA promoter, wherein the base at position -1 of the template strand of the RNA promoter is cytosine; the bases at positions +1 to +n of the template strand of the RNA promoter are respectively aligned with the bases B1 to B2 in the capping polynucleotide. n Complementary pairing.

[0021] Optionally, n is a natural number ranging from 8 to 12.

[0022] The present invention regulates the base at position -1 of the template chain of the RNA promoter to be cytosine, and at the same time regulates the bases at positions +1 to +n of the template chain of the RNA promoter to be respectively aligned with the bases B1 to B2 in the capping polynucleotide. n Complementary pairing significantly improves the efficiency of RNA capping and is beneficial to improving the stability of RNA after transcription and synthesis.

[0023] The second aspect of the present application provides a capped RNA transcription reaction solution, comprising a DNA template, a capped polynucleotide, NTPs, an RNA polymerase, and a solution containing magnesium ions.

[0024] The structural formula of the capping polynucleotide is as follows:

[0025]

[0026] B1 to B n Each is independently a natural or modified nucleobase.

[0027] E is 0 or 1.

[0028] F is 0 or 1.

[0029] H1 is 1; H2 to H n Each independently is 0 or 1.

[0030] M1 to M n-1 Each is independently oxygen, hydroxyl, sulfur, mercapto, imino, amino, alkyl and alkoxy.

[0031] M n It is hydroxyl.

[0032] R1 and R2 are each independently hydrogen, alkyl or alkoxy.

[0033] R3 to R n+4 Each is independently hydrogen, hydroxy, alkyl, alkoxy, S-alkyl, NH-alkyl, O-aryl, S-aryl, NH-aryl or halo.

[0034] X1 to X n+4 Each is independently oxygen, sulfur, imino and methylene.

[0035] Y1 to Y n+3 Each is independently oxygen, sulfur, selenium or borane.

[0036] Z1 to Z n+3 Each is independently a hydroxyl group, a mercapto group, a boryl group, an aryl group, an alkyl group, an alkoxy group or an O-aryl group.

[0037] Here, n is a natural number greater than or equal to 1.

[0038] The DNA template includes an RNA promoter, wherein the base at position -1 of the template strand of the RNA promoter is cytosine; the bases at positions +1 to +n of the template strand of the RNA promoter are respectively aligned with the bases B1 to B2 in the capping polynucleotide. n Complementary pairing.

[0039] Optionally, n is a natural number ranging from 8 to 12.

[0040] Optionally, the NTPs include at least one of ATP, CTP, GTP, UTP, 1-N-Me-Pseudo UTP, 5'-Me-UTP, 5'-OMe-UTP and PseudoUTP.

[0041] Optionally, NTPs include ATP, CTP, GTP and UTP; the concentration ratio of ATP, CTP, GTP and UTP to the capping polynucleotide is (2-10):(2-10):(2-10):(2-10):(2-40).

[0042] Optionally, the final molar concentration of magnesium ions in the solution containing magnesium ions in the capped RNA transcription reaction solution is 20-60 mM.

[0043] The present invention regulates the base at position -1 of the template chain of the RNA promoter in the capped RNA transcription reaction solution to be cytosine, and at the same time regulates the bases at positions +1 to +n of the template chain of the RNA promoter to be respectively aligned with the bases B1 to B2 in the capped polynucleotide. n Complementary pairing, combined with the concentration of magnesium ions and the concentration ratio of NTPs and capping polynucleotides, significantly improves the RNA capping efficiency after the transcription reaction solution is reacted, which is beneficial to improving the stability of the RNA after transcription synthesis. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present application and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.

[0045] Figure 1 The HPLC analysis chart of the RNA product of Example 1 of the present application is shown.

[0046] Figure 2 The HPLC analysis chart of the RNA product of Example 2 of the present application is shown.

[0047] Figure 3 The HPLC analysis chart of the RNA product of Example 3 of the present application is shown.

[0048] Figure 4 The HPLC analysis chart of the RNA product of Example 4 of the present application is shown.

[0049] Figure 5 The HPLC analysis chart of the RNA product of Example 5 of the present application is shown.

[0050] Figure 6 The HPLC analysis chart of the RNA product of Example 6 of the present application is shown.

[0051] Figure 7 The HPLC analysis chart of the RNA product of Example 7 of the present application is shown.

[0052] Figure 8 The HPLC analysis chart of the RNA product of Example 8 of the present application is shown.

[0053] Fig. 9 The HPLC analysis chart of the RNA product of Example 9 of the present application is shown.

[0054] Fig.10 The HPLC analysis chart of the RNA product of Example 10 of the present application is shown.

[0055] Fig.11 The HPLC analysis chart of the RNA product of Example 11 of the present application is shown.

[0056] Fig.12 The HPLC analysis chart of the RNA product of Example 12 of the present application is shown.

[0057] Fig.13The HPLC analysis chart of the RNA product of Comparative Example 1 of the present application is shown.

[0058] Fig.14 The HPLC analysis chart of the RNA product of Comparative Example 2 of the present application is shown.

[0059] Fig.15 The HPLC analysis chart of the RNA product of Comparative Example 3 of the present application is shown.

[0060] Fig.16 The HPLC analysis chart of the RNA product of Comparative Example 4 of the present application is shown.

[0061] Fig.17 The HPLC analysis chart of the RNA product of Comparative Example 5 of the present application is shown.

[0062] Fig.18 The HPLC analysis chart of the RNA product of Comparative Example 6 of the present application is shown.

[0063] Fig.19 The HPLC analysis chart of the RNA product of Comparative Example 7 of the present application is shown.

[0064] Fig. 20 The HPLC analysis chart of the RNA product of Comparative Example 8 of the present application is shown.

[0065] Fig.21 The HPLC analysis chart of the RNA product of Comparative Example 9 of the present application is shown.

[0066] Fig. 22 The HPLC analysis chart of the RNA product of Comparative Example 10 of the present application is shown.

[0067] Fig.23 The HPLC analysis chart of the RNA product of Comparative Example 11 of the present application is shown.

[0068] Fig.24 The HPLC analysis chart of the RNA product of Comparative Example 12 of the present application is shown.

[0069] Fig.25 The HPLC analysis chart of the RNA product of Comparative Example 13 of the present application is shown.

[0070] Fig.26 The HPLC analysis chart of the RNA product of Comparative Example 14 of the present application is shown.

[0071] Fig. 27 The HPLC analysis chart of the RNA product of Comparative Example 15 of the present application is shown. DETAILED DESCRIPTION

[0072] In order to make the purpose, technical scheme and advantages of the embodiments of the present application clearer, the technical scheme in the embodiments of the present application will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0073] The following is a detailed description of the capped RNA synthesis method and the capped RNA transcription reaction solution of the examples of the present application.

[0074] In this application, the unit "M" refers to mol / L; "mM" refers to mmol / L.

[0075] In the present application, the +1 position of the template chain of the RNA promoter refers to the position of the first nucleotide transcribed from the 3' to the 5' end of the DNA template by RNA synthesis (or capped RNA synthesis); the +2 position of the template chain of the RNA promoter refers to the position of the second nucleotide transcribed from the 3' to the 5' end of the DNA template by RNA synthesis (or capped RNA synthesis); the +n position of the template chain of the RNA promoter refers to the position of the nth nucleotide transcribed from the 3' to the 5' end of the DNA template by RNA synthesis (or capped RNA synthesis); the -1 position of the template chain of the RNA promoter refers to the position adjacent to the 3' end of the +1 position of the template chain of the RNA promoter; accordingly, the RNA promoter The base at position +1 of the template chain of the RNA promoter refers to the base on the first nucleotide transcribed from the 3' to the 5' end of the DNA template by RNA synthesis (or capped RNA synthesis); the base at position +2 of the template chain of the RNA promoter refers to the base on the second nucleotide transcribed from the 3' to the 5' end of the DNA template by RNA synthesis (or capped RNA synthesis); the base at position +n of the template chain of the RNA promoter refers to the base on the nth nucleotide transcribed from the 3' to the 5' end of the DNA template by RNA synthesis (or capped RNA synthesis); the base at position -1 of the template chain of the RNA promoter refers to the base at the nucleotide adjacent to the 3' end of the +1 position of the template chain of the RNA promoter.

[0076] In the present application, 5'-Me-UTP refers to uracil with a methyl group modified at the 5' end; 5'-OMe-UTP refers to uracil with a methoxy group modified at the 5' end.

[0077] The present application provides a method for synthesizing capped RNA, comprising mixing and incubating raw materials.

[0078] The raw materials include DNA template, capping polynucleotide, NTPs, RNA polymerase and a solution containing magnesium ions.

[0079] Among them, the structural formula of the capping polynucleotide is as follows:

[0080]

[0081] B1 to B n Each is independently a natural or modified nucleobase.

[0082] E is 0 or 1.

[0083] F is 0 or 1.

[0084] H1 is 1; H2 to H n Each independently is 0 or 1.

[0085] M1 to M n-1 Each is independently oxygen, hydroxyl, sulfur, mercapto, imino, amino, alkyl and alkoxy.

[0086] M n It is hydroxyl.

[0087] R1 and R2 are each independently hydrogen, alkyl or alkoxy.

[0088] R3 to R n+4 Each is independently hydrogen, hydroxy, alkyl, alkoxy, S-alkyl, NH-alkyl, O-aryl, S-aryl, NH-aryl or halo.

[0089] X1 to X n+4 Each is independently oxygen, sulfur, imino and methylene.

[0090] Y1 to Y n+3 Each is independently oxygen, sulfur, selenium or borane.

[0091] Z1 to Z n+3 Each is independently a hydroxyl group, a mercapto group, a boryl group, an aryl group, an alkyl group, an alkoxy group or an O-aryl group.

[0092] Here, n is a natural number greater than or equal to 1.

[0093] The DNA template includes an RNA promoter, wherein the base at position -1 of the template strand of the RNA promoter is cytosine; the bases at positions +1 to +n of the template strand of the RNA promoter are respectively aligned with the bases B1 to B2 in the capping polynucleotide. n Complementary pairing.

[0094] By way of example, n may be a natural number such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15.

[0095] It should be noted that the capped polynucleotide in the present application refers to a polynucleotide having a cap structure at the 5' end of the nucleotide chain, and the cap structure refers to a 7-methylguanosine inverted at the 5' end of the mRNA. As an example, the capped polynucleotide can be a capped dinucleotide or a capped trinucleotide or a capped tetranucleotide or other capped polynucleotide.

[0096] It should be noted that, in other embodiments of the present application, the structure of the capped polynucleotide is not limited to the above structure, as long as the capped polynucleotide has a cap structure.

[0097] In some embodiments of the present application, n is a natural number between 8 and 12. It should be noted that in other embodiments of the present application, n may not be limited to the above natural numbers.

[0098] NTPs are nucleoside triphosphates (containing ribose) and are the raw materials for synthesizing RNA; capped polynucleotides are the raw materials for synthesizing capped RNA. The magnesium ions in the raw materials form a complex with NTPs or capped polynucleotides and DNA templates to be recognized by RNA polymerase, thereby allowing the DNA template to be stably transcribed to synthesize RNA.

[0099] Since the cap structure is composed of a reverse 7-methylguanosine at the 5' end of the mRNA, the cap structure is conducive to the stability, nuclear export and translation of the mRNA. In order to obtain capped RNA (i.e., RNA with a cap structure) by co-transcription in vitro, the base at the -1 position of the template chain of the RNA promoter in the DNA template is regulated to be cytosine, and the bases at the +1 to +n positions (i.e., the 3' to 5' ends) of the template chain of the RNA promoter are regulated to be respectively aligned with the bases at B1 to B2 in the capping polynucleotide. n (i.e., the bases from the 5' to the 3' end) are complementary and paired, thereby significantly improving the efficiency of RNA capping after in vitro co-transcription (up to about 20%), which is beneficial to improving the stability of RNA after transcription and synthesis.

[0100] In some embodiments of the present application, the base at position -1 of the template strand of the RNA promoter is cytosine, the bases at positions +1 and +2 of the template strand of the RNA promoter are both cytosine, B1 and B2 in the capping polynucleotide are both guanine, and the bases at positions +3 to +n of the template strand of the RNA promoter are respectively the same as the bases at positions B3 to B4 in the capping polynucleotide. n Complementary pairing. By regulating the bases at positions -1, +1, and +2 of the template chain of the RNA promoter to be cytosine, and regulating B1 and B2 in the capping polynucleotide to be guanine, the bases at positions +3 to +n of the template chain of the RNA promoter are matched with the bases at positions B3 to B4 in the capping polynucleotide. n Complementary pairing can further improve RNA capping efficiency.

[0101] In some embodiments of the present application, the capped polynucleotide is a capped dimeric nucleotide, and the structural formula of the capped dimeric nucleotide is as follows:

[0102]

[0103] B1 is a natural or modified nucleobase.

[0104] E is 0 or 1.

[0105] F is 0 or 1.

[0106] M1 is a hydroxyl group.

[0107] R1 and R2 are each independently hydrogen, alkyl or alkoxy.

[0108] R3 to R5 are each independently hydrogen, hydroxy, alkyl, alkoxy, S-alkyl, NH-alkyl, O-aryl, S-aryl, NH-aryl or halo.

[0109] X1 to X5 are each independently oxygen, sulfur, imino and methylene.

[0110] Y1 to Y4 are each independently oxygen, sulfur, selenium or borane.

[0111] Z1 to Z4 are each independently hydroxyl, mercapto, boryl, aryl, alkyl, alkoxy or O-aryl.

[0112] The base at position -1 of the template strand of the RNA promoter is cytosine; the base at position +1 of the template strand of the RNA promoter complementarily pairs with B1 in the capping duplex.

[0113] As an example, ARCA (Chinese name: Anti-Reverse Cap Analog, English name: Anti Reverse Cap Analog) is used as the capping dinucleotide, and the structural formula of ARCA is as follows:

[0114]

[0115] When ARCA is selected as the capping dinucleotide, the base at the +1 position of the template strand of the RNA promoter is cytosine.

[0116] It should be noted that, in other embodiments of the present application, the structure of the capped dinucleotide is not limited to the above structure, as long as the capped dinucleotide has a cap structure.

[0117] By regulating the base at the -1 position of the template chain of the RNA promoter in the DNA template to be cytosine, and at the same time regulating the base at the +1 position (from the 3' to the 5' end) of the template chain of the RNA promoter to complementarily pair with B1 (i.e., the base from the 5' to the 3' end) in the capping dinucleotide, the RNA capping efficiency after in vitro co-transcription is significantly improved, which is beneficial to improving the stability of the RNA after transcription synthesis.

[0118] In some embodiments of the present application, the capped polynucleotide is a capped trimer nucleotide, and the structural formula of the capped trimer nucleotide is as follows:

[0119]

[0120] B1 and B2 are each independently a natural or modified nucleobase.

[0121] E is 0 or 1.

[0122] F is 0 or 1.

[0123] M1 is oxygen, sulfur, imino, alkyl and alkoxy.

[0124] M2 is a hydroxyl group.

[0125] R1 and R2 are each independently hydrogen, alkyl or alkoxy.

[0126] R3 to R6 are each independently hydrogen, hydroxy, alkyl, alkoxy, S-alkyl, NH-alkyl, O-aryl, S-aryl, NH-aryl or halo.

[0127] X1 to X6 are each independently oxygen, sulfur, imino and methylene.

[0128] Y1 to Y5 are each independently oxygen, sulfur, selenium or borane.

[0129] Z1 to Z5 are each independently hydroxyl, mercapto, boryl, aryl, alkyl, alkoxy or O-aryl.

[0130] The base at position -1 of the template strand of the RNA promoter is cytosine; the bases at positions +1 to +2 of the template strand of the RNA promoter are complementary paired with B1 to B2 in the capping trinucleotide, respectively.

[0131] As an example, the molecular formula of the capping trinucleotide can be m 7 G(5')ppp(5')(2'OMeA)pU、m 7 Gppp(A2'O-MOE)pG、m 7 G(5')ppp(5')(2'OMeA)pGorm 7 G(5')ppp(5')(2'OMeG)pG.

[0132] m 7 The structural formula of G(5')ppp(5')(2'OMeA)pU is as follows:

[0133]

[0134] When the capping trinucleotide is selected 7 When G(5')ppp(5')(2'OMeA)pU, the bases at positions +1 to +2 of the template strand of the RNA promoter are thymine and adenine, respectively.

[0135] m 7 The structural formula of Gppp(A2'O-MOE)pG is as follows:

[0136]

[0137] When the capping trinucleotide is selected 7 When Gppp(A2'O-MOE)pG, the bases at positions +1 to +2 of the template strand of the RNA promoter are thymine and cytosine, respectively.

[0138] m 7 The structural formula of G(5')ppp(5')(2'OMeA)pG is as follows:

[0139]

[0140] When the capping trinucleotide is selected 7 When G(5')ppp(5')(2'OMeA)pG, the bases at positions +1 to +2 of the template strand of the RNA promoter are thymine and cytosine, respectively.

[0141] m 7 The structural formula of G(5')ppp(5')(2'OMeG)pG is as follows:

[0142]

[0143] When the capping trinucleotide is selected 7 When G(5')ppp(5')(2'OMeG)pG, the bases at positions +1 to +2 of the template strand of the RNA promoter are all cytosine.

[0144] It should be noted that, in other embodiments of the present application, the structure of the capped trinucleotide is not limited to the above structure, as long as the capped trinucleotide has a cap structure.

[0145] By regulating the base at position -1 of the template chain of the RNA promoter in the DNA template to be cytosine, and at the same time regulating the bases at positions +1 to +2 (i.e., 3' to 5' ends) of the template chain of the RNA promoter to complementarily pair with B1 to B2 (i.e., 5' to 3' ends) in the capping polynucleotide, the RNA capping efficiency after in vitro co-transcription is significantly improved, which is beneficial to improving the stability of the RNA after transcription synthesis.

[0146] The DNA template refers to a double-stranded DNA sequence containing an RNA promoter, and its sources include but are not limited to PCR and plasmid DNA.

[0147] In the present application, the RNA polymerase may be selected from natural RNA polymerases and mutant RNA polymerases. As an example, the natural RNA polymerase may be selected from T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase, etc.; the mutant RNA polymerase may be selected from Hi-T7 RNA polymerase (NEB) and thermostable T7 RNA polymerase (TOYOBO), etc. It should be noted that in other embodiments of the present application, the RNA polymerase may not be selected from the above RNA polymerases.

[0148] In some embodiments of the present application, when the RNA polymerase is a thermostable RNA polymerase, the RNA capping efficiency after in vitro co-transcription can be further improved; for example, Hi-T7 RNA polymerase (brand: NEB, catalog number: M0251) and thermostable T7 RNA polymerase (brand: TOYOBO, catalog number: TRL-2017), etc.

[0149] NTPs include natural or modified nucleotides. In the present embodiment, NTPs include at least one of ATP (adenosine triphosphate), CTP (cytidine triphosphate), GTP (guanosine triphosphate), UTP (uridine triphosphate), 1-N-Me-Pseudo UTP, 5'-Me-UTP, 5'-OMe-UTP and PseudoUTP. It should be noted that in other embodiments of the present application, NTPs may not include the above-mentioned nucleotides, and NTPs may include analogs corresponding to each nucleotide, including but not limited to base or sugar-modified nucleotides.

[0150] In some embodiments of the present application, the solution containing magnesium ions is MgCl 2. It should be noted that in other embodiments of the present application, the solution containing magnesium ions may not be limited to the above solution, and the solution containing magnesium ions may also be other solutions.

[0151] In some embodiments of the present application, NTPs include at least one of ATP, CTP, GTP, UTP, 1-N-Me-Pseudo UTP, 5'-Me-UTP, 5'-OMe-UTP and PseudoUTP.

[0152] In this embodiment, the final molar concentration of magnesium ions in the solution containing magnesium ions in the raw material is 20 to 60 mM. As an example, the final molar concentration of magnesium ions in the solution containing magnesium ions in the raw material can be 20 mM, 25 mM, 30 mM, 40 mM, 46 mM, 50 mM, 60 mM, etc.

[0153] In this embodiment, NTPs include ATP, CTP, GTP and UTP; the concentration ratio of ATP, CTP, GTP and UTP to the capping polynucleotide is (2-10):(2-10):(2-10):(2-10):(2-40). As an example, the concentration ratio of ATP, CTP, GTP and UTP to the capping polynucleotide can be 2:2:2:2:2, 3.5:7.5:7.5:7.5:14.0, 7.5:7.5:7.5:3.5:14.0, 7.5:7.5:7.5:7.5:14.0, 3:3:3:3:9 and 3:3:3:9:25, etc.

[0154] Further, the concentration ratio of ATP, CTP, GTP and UTP to the capping polynucleotide is (3-9):(3-9):(3-9):(3-9):(3-25); further, the concentration ratio of ATP, CTP, GTP and UTP to the capping polynucleotide is (3.5-8):(3.5-8):(3.5-8):(3.5-8):(3.5-14).

[0155] The above molar ratio enables magnesium ions to better form a complex with NTPs or capping polynucleotides and DNA templates so as to be recognized by RNA polymerase, thereby enabling stable transcription to synthesize capped RNA.

[0156] In this embodiment, the raw materials for the synthesis of capped RNA also include at least one of inorganic pyrophosphatase and nuclease inhibitor. In some embodiments of the present application, the final concentration of inorganic pyrophosphatase is 0.0002-0.1U / μL; as an example, the final concentration of inorganic pyrophosphatase can be 0.0002U / μL, 0.001U / μL, 0.01U / μL, 0.02U / μL, 0.04U / μL, 0.08U / μL and 0.1U / μL, etc. In some embodiments of the present application, the final concentration of nuclease inhibitor is 0.2-2.0U / μL; as an example, the final concentration of nuclease inhibitor is 0.2U / μL, 0.4U / μL, 0.5U / μL, 1.0U / μL and 2.0U / μL, etc.

[0157] In this embodiment, the raw materials for synthesizing capped RNA further include at least one of Tris-HCl (tris(hydroxymethyl)aminomethane hydrochloride), spermidine, DTT (dithiothreitol) and Triton X-100 (polyethylene glycol octylphenyl ether).

[0158] In some embodiments of the present application, the pH of Tris-HCl is 7.2-8.3; as an example, the pH of Tris-HCl can be 7.2, 7.5, 7.9, 8.0 and 8.3, etc. In some embodiments of the present application, the final concentration of Tris-HCl is 10-100mM; as an example, the final concentration of Tris-HCl can be 10mM, 20mM, 40.0mM, 80mM and 100mM, etc. In some embodiments of the present application, the final concentration of spermidine is 0.1-10mM; as an example, the final concentration of spermidine can be 0.1mM, 0.5mM, 1mM, 2mM, 4mM and 10mM, etc. In some embodiments of the present application, the final concentration of DTT is 1-20mM; as an example, the final concentration of DTT can be 1mM, 5mM, 10mM, 15mM and 20mM, etc. In some embodiments of the present application, the percentage of Triton X-100 in the total volume of the raw material is 0.005% to 0.05%; as an example, the percentage of Triton X-100 in the total volume of the raw material can be 0.005%, 0.01%, 0.02%, 0.05%, etc.

[0159] The DNA template includes an RNA promoter, and the base at position -1 of the template strand of the RNA promoter is cytosine. As an example, the sequence of the template strand of the RNA promoter is shown in SEQ ID NO.1, or the sequence of the template strand of the RNA promoter is shown in SEQ ID NO.2, or the sequence of the template strand of the RNA promoter is shown in SEQ ID NO.3, as shown in Table 1.

[0160] Table 1

[0161]

[0162] In the table: the direction from sequence position -17 to +1 is the direction from 3' to 5' end of the template strand of the RNA promoter.

[0163] It should be noted that, in other embodiments of the present application, the template chain of the RNA promoter may not be limited to the above sequence, as long as the base at the -1 position of the template chain of the RNA promoter is cytosine, the bases starting from the +1 position from the 3' to 5' end of the template chain of the RNA promoter are complementary to the bases starting from B1 from the 5' to 3' end of the capping dinucleotide.

[0164] Further, the incubation temperature is 10 to 70°C and the incubation time is 1 to 6 hours. As an example, the incubation temperature can be 10°C, 20°C, 37°C, 50°C, 55°C, 60°C or 70°C. As an example, the incubation time can be 1h, 1.5h, 2.0h, 3.0h, 4.0h or 6.0h, etc. At the above incubation temperature and incubation time, the capped RNA can be stably synthesized, which is conducive to improving the RNA capping efficiency.

[0165] In some implementations of the present application, the incubation temperature is 37-65° C.; optionally, the incubation temperature is 50-60° C. When the incubation temperature is 50-60° C., the RNA capping efficiency can be further improved.

[0166] After the raw materials are mixed and incubated, an enzyme for digesting the DNA template is added to perform a digestion reaction. In this embodiment, after the raw materials are mixed and incubated, DNase I enzyme (Chinese name: deoxyribonuclease I, a nuclease that can digest single-stranded or double-stranded DNA to produce single deoxynucleotides or single-stranded or double-stranded oligodeoxynucleotides) is added and digested at 20 to 60°C for 10 to 40 minutes. As an example, the temperature of the digestion reaction can be 20°C, 25°C, 37°C, 40°C, 50°C, and 60°C, etc.; the time of the digestion reaction can be 10min, 15min, 20min, 30min, and 40min, etc. After incubation, an enzyme for digesting the DNA template is added to perform a digestion reaction on the DNA template, which can cause the DNA template to produce single deoxynucleotides or single-stranded or double-stranded oligodeoxynucleotides, which is convenient for subsequent purification.

[0167] After adding the enzyme that digests the DNA template for digestion reaction, purification treatment is performed. In this embodiment, after adding the enzyme that digests the DNA template for digestion reaction, LiCl is added and allowed to stand at -15 to -25°C for 20 to 40 minutes for purification treatment. As an example, the temperature of the purification treatment can be -15°C, -18°C, -20°C, -22°C, and -25°C, etc.; the standing time can be 20min, 25min, 28min, 30min, and 40min, etc. After purification, it is reasonable to improve the purity of the capped RNA.

[0168] The present application also provides a capped RNA transcription reaction solution, comprising a DNA template, a capped polynucleotide, NTPs, an RNA polymerase, and a solution containing magnesium ions.

[0169] Among them, the structural formula of the capping polynucleotide is as follows:

[0170]

[0171] B1 to B n Each is independently a natural or modified nucleobase.

[0172] E is 0 or 1.

[0173] F is 0 or 1.

[0174] H1 is 1; H2 to H n Each independently is 0 or 1.

[0175] M1 to M n-1 Each is independently oxygen, hydroxyl, sulfur, mercapto, imino, amino, alkyl and alkoxy.

[0176] M n It is hydroxyl.

[0177] R1 and R2 are each independently hydrogen, alkyl or alkoxy.

[0178] R3 to R n+4 Each is independently hydrogen, hydroxy, alkyl, alkoxy, S-alkyl, NH-alkyl, O-aryl, S-aryl, NH-aryl or halo.

[0179] X1 to X n+4 Each is independently oxygen, sulfur, imino and methylene.

[0180] Y1 to Y n+3 Each is independently oxygen, sulfur, selenium or borane.

[0181] Z1 to Z n+3 Each is independently a hydroxyl group, a mercapto group, a boryl group, an aryl group, an alkyl group, an alkoxy group or an O-aryl group.

[0182] Wherein, n is a natural number greater than or equal to 1. As an example, n can be a natural number such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 and 15.

[0183] The DNA template includes an RNA promoter, wherein the base at position -1 of the template strand of the RNA promoter is cytosine; the bases at positions +1 to +n of the template strand of the RNA promoter are respectively aligned with the bases B1 to B2 in the capping polynucleotide. n Complementary pairing.

[0184] In some embodiments of the present application, n is a natural number between 8 and 12. It should be noted that in other embodiments of the present application, n may not be limited to the above natural numbers.

[0185] In some embodiments of the present application, NTPs include at least one of ATP, CTP, GTP, UTP, 1-N-Me-Pseudo UTP, 5'-Me-UTP, 5'-OMe-UTP and PseudoUTP.

[0186] In some embodiments of the present application, NTPs include ATP, CTP, GTP and UTP; the concentration ratio of ATP, CTP, GTP and UTP to the capping polynucleotide is (2-10):(2-10):(2-10):(2-10):(2-40).

[0187] In some embodiments of the present application, the final molar concentration of magnesium ions in the solution containing magnesium ions in the raw material is 20-60 mM.

[0188] In some embodiments of the present application, the capped RNA transcription reaction solution further includes at least one of an inorganic pyrophosphatase and a nuclease inhibitor.

[0189] In some embodiments of the present application, the raw material further comprises at least one of Tris-HCl, spermidine, DTT and Triton X-100.

[0190] The capped RNA transcription reaction solution provided in this application has at least the following advantages:

[0191] By regulating the base at position -1 of the template chain of the RNA promoter in the capped RNA transcription reaction solution to be cytosine, and at the same time regulating the bases at positions +1 to +n of the template chain of the RNA promoter to be respectively aligned with the bases B1 to B n Complementary pairing, combined with the concentration of magnesium ions and the concentration ratio of NTPs and capping polynucleotides, significantly improves the RNA capping efficiency after the transcription reaction solution reacts, which is beneficial to improving the stability of the RNA synthesized after transcription.

[0192] The features and performance of the present application are further described in detail below in conjunction with the embodiments.

[0193] Example 1

[0194] This embodiment provides a capped RNA transcription reaction solution and a method for synthesizing capped RNA.

[0195] 1 μL 0.8M Tris-HCl (pH 7.9), 0.92 μL 1M MgCl2, 1 μL 40mM spermidine, 0.2 μL 1M DTT, 1 μL 0.2% Triton X-100 by volume, 1.5 μL 100mM ATP, 1.5 μL 100mM CTP, 1.5 μL 1.5mM UTP, 0.7 μL 100mM GTP, 2.8 μL 100mM ARCA, 1 μL 200U / μL T7 RNA polymerase, 0.8 μL 1U / μL inorganic pyrophosphatase, 1 μL 20U / μL nuclease inhibitor, 1 μL 1ug / μL DNA template and 4.08 μL nuclease-free water were mixed into a 20uL mixed system to obtain a capped RNA transcription reaction solution. Incubate the capped RNA transcription reaction solution at 37°C for 2 hours. Add 1uL 1U / μL DNase I enzyme and digest for 30 minutes at 37°C. Add 10.5μL 7.5M LiCl solution and let stand at -20°C for 20 minutes, then centrifuge at 7500rpm at 4°C for 15 minutes to obtain RNA precipitate, rinse twice with 75% ethanol to obtain RNA product precipitate.

[0196] Wherein, the sequence of the template strand of the RNA promoter in the DNA template is as follows:

[0197] Sequence position: -17 -1+1…

[0198] Template strand 3': -ATTATGCTGAGTGATACC...

[0199] Examples 2-12, Comparative Examples 1-15

[0200] Examples 2-12 and Comparative Examples 1-15 provide a capped RNA transcription reaction solution and a method for synthesizing capped RNA, respectively. Please refer to Example 1. The capped RNA transcription reaction solutions of Examples 2-12 and Comparative Examples 1-15 are different from those of the capped RNA transcription reaction solution of Example 1 in terms of the final concentration of ATP, the final concentration of CTP, the final concentration of UTP, the final concentration of GTP, the selection of capped polynucleotides, and the final concentration of capped polynucleotides, as shown in Table 1. The capped RNA transcription reaction solutions of Examples 2-12 and Comparative Examples 1-15 are different from those of the capped RNA transcription reaction solution of Example 1 in terms of the selection of RNA polymerase, the concentration of RNA polymerase, and the DNA template, as shown in Table 2; the methods for synthesizing capped RNA of Examples 2-12 and Comparative Examples 1-15 are different from those of the method for synthesizing capped RNA of Example 1 in terms of the incubation temperature and incubation time, as shown in Table 3.

[0201] Table 1

[0202]

[0203]

[0204] Table 2

[0205]

[0206]

[0207]

[0208] Note: The sequence position from -17 to +1 in the template chain sequence of the promoter in the DNA template is the 3' to 5' end direction of the template chain of the RNA promoter; the " / " is located between the sequence position -1 and +1, which is used to indicate the correspondence between the bases on the template chain of the RNA promoter and the sequence position.

[0209] Table 3

[0210]

[0211]

[0212] Test Example 1

[0213] The RNA capping efficiency test was performed on the capped RNA transcription reaction solutions provided in Examples 1-12 and Comparative Examples 1-15 and the RNA products obtained by the capped RNA synthesis methods.

[0214] Among them, the test method for RNA capping efficiency is: dissolve the RNA product precipitate with water, use alkaline phosphatase to remove the 5'-phosphate group that may exist in the RNA obtained by the capped RNA synthesis method (i.e., in vitro transcription reaction), and reduce the influence of the presence of the 5'-phosphate group on the results of HPLC (high performance liquid chromatography) and LC-MS (liquid chromatography-mass spectrometry). Then, the RNA treated with alkaline phosphatase is specifically cut and purified by RNaseH (ribonuclease H) to obtain a small fragment of RNA with uniform 3'-end. Finally, the molecular weight of the 5'-end small fragment of RNA is analyzed by liquid chromatography and LC-MS to determine the RNA capping situation, that is, the capping situation of the RNA product obtained by the capped RNA synthesis method. Figure 1-Figure 27 In the example 1, "|" indicates the complementary pairing of bases in the template strand of the RNA promoter in the DNA template with the capping polynucleotide; "5'OH-G", "5'OH-GG", "5'-OH", "5'OH-AGG" and "5'OH-GGG" indicate uncapped RNA products, and the peak indicated by the capping polynucleotide is the capped RNA product. In Experimental Example 1, the RNA capping efficiency of Examples 1-12 and Comparative Examples 1-15 is determined by the following method: capped RNA product peak area / (capped RNA product peak area+uncapped RNA product peak area), and the test results are shown in Table 4 and Figure 1-Figure 27 shown.

[0215] Table 4 RNA capping efficiency test results

[0216]

[0217]

[0218] From Table 4 we can see that:

[0219] In the present application, the RNA capping efficiency of Example-2 using ARCA as the capping polynucleotide is significantly higher than the RNA capping efficiency of Comparative Examples 1-4 using ARCA as the capping polynucleotide; 7 The RNA capping efficiency of Example 3-5 using Gppp(A2'O-MOE)pG as the capping polynucleotide is significantly higher than that using m 7 Gppp(A2'O-MOE)pG is the capping efficiency of comparative examples 5-8; and from comparative examples 5-6, it can be seen that when the base at the -1 position of the template strand of the RNA promoter in the DNA template is adenine, even if the capping polynucleotide m 7The A and G in Gppp(A2'O-MOE)pG are complementary to the +1 T and +2 C bases of the template strand of the RNA promoter, and the RNA capping efficiency of Comparative Examples 5-6 is also not high. Example 5 shows that when the concentration ratio of the capping polynucleotide to ATP, UTP, CTP, and GTP is 1:1:1:1:1, an RNA product with high capping efficiency can also be obtained; 7 The RNA capping efficiency of Examples 6-10 in which the base at position -1 of the RNA promoter in the capping polynucleotide DNA template G(5')ppp(5')(2'OMeG)pG is cytosine and the bases at positions +1 and +2 of the promoter template chain are both cytosine is significantly higher than that of Comparative Examples 9-13.

[0220] The RNA capping efficiency of Examples 1-12 of the present application is significantly higher than that of Comparative Examples 1-15. By regulating the base at position -1 of the template strand of the RNA promoter in the DNA template to be cytosine, and regulating the bases at positions +1 to +n (i.e., 3' to 5' ends) of the template strand of the RNA promoter to be respectively aligned with the bases at positions B1 to B2 in the capping polynucleotide, the capping polynucleotide is further enhanced. n (i.e., the bases from the 5' to the 3' end) are complementary paired, thereby significantly improving the RNA capping efficiency after in vitro co-transcription; further, the RNA capping efficiency is improved when the incubation temperature is 50°C compared to when the incubation temperature is 37°C, and the final RNA capping efficiency can be increased by up to about 20%.

[0221] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application. SEQUENCE LISTING <110> Shanghai Zhaowei Technology Development Co., Ltd. Shanghai Zhaowei Bioengineering Co., Ltd. <120> A method for synthesizing capped RNA and a capped RNA transcription reaction solution <130> 2021.09.09 <160> 3 <170> PatentIn version 3.5 <210> 1 <211> 18 <212> DNA <213> Synthesis <400> 1 attatgctga gtgatacc 18 <210> 2 <211> 18 <212> DNA <213> Synthesis <400> 2 taaatccact gtgatacc 18 <210> 3 <211> 18 <212> DNA <213> Synthesis <400> 3 ttaattgggt gtgattcc 18

Claims

1. A method for synthesizing capped RNA, characterized in that: include: Mixing and incubating the raw materials; The raw materials include a DNA template, a capping polynucleotide, NTPs, an RNA polymerase, and a solution containing magnesium ions; The capped polynucleotide is a capped dimeric nucleotide, and the structural formula of the capped dimeric nucleotide is as follows: B1 is a natural or modified nucleoside base; E is 1; F is 0; M1 is a hydroxyl group; R1 and R2 are hydrogen; R3 to R5 are each independently hydroxy or alkoxy; X1 to X5 are oxygen; Y1 to Y4 are oxygen; Z1 to Z4 are hydroxyl groups; The base at position -1 of the template strand of the RNA promoter is cytosine; the base at position +1 of the template strand of the RNA promoter is complementary to B1 in the capping dinucleotide; Alternatively, the capped polynucleotide is a capped trimer nucleotide, and the capped trimer nucleotide has the following structural formula: B1 and B2 are each independently a natural or modified nucleoside base; E is 1; F is 0; M1 is oxygen; M2 is a hydroxyl group; R1 and R2 are hydrogen; R3 to R6 are each independently hydroxyl or alkoxy; X1 to X6 are oxygen; Y1 to Y5 are oxygen; Z1 to Z5 are hydroxyl groups; The base at position -1 of the template strand of the RNA promoter is cytosine; the bases at positions +1 to +2 of the template strand of the RNA promoter are complementary paired with B1 to B2 in the capping trinucleotide, respectively.

2. The method for synthesizing capped RNA according to claim 1, characterized in that: The capping dinucleotide is ARCA, and the base at the +1 position of the template strand of the RNA promoter is cytosine.

3. The method for synthesizing capped RNA according to claim 1, characterized in that: The capping trinucleotide is selected from m 7 G(5')ppp(5')(2'OMeA)pU、m 7 Gppp(A2'O-MOE)pG、m 7 G(5')ppp(5')(2'OMeA)pG or m 7 G(5')ppp(5')(2'OMeG)pG.

4. The method for synthesizing capped RNA according to claim 1, characterized in that: The incubation temperature is 10-70°C.

5. The method for synthesizing capped RNA according to claim 4, characterized in that: The incubation temperature is 37-65°C.

6. The method for synthesizing capped RNA according to claim 4, characterized in that The incubation temperature is 50-60°C.

7. The method for synthesizing capped RNA according to claim 1, characterized in that: After the incubation, an enzyme for digesting the DNA template is added to perform a digestion reaction.

8. The method for synthesizing capped RNA according to claim 7, characterized in that: The enzyme for digesting the DNA template is DNase I enzyme.

9. The method for synthesizing capped RNA according to claim 7, characterized in that: The temperature of the digestion reaction is 20-60°C.

10. The method for synthesizing capped RNA according to claim 7, characterized in that: The digestion reaction time is 10 to 40 minutes.

11. The method for synthesizing capped RNA according to claim 7, characterized in that: The digestion reaction also includes purification treatment.

12. The method for synthesizing capped RNA according to claim 11, characterized in that: The purification process includes adding LiCl and standing at -15 to -25°C for 20 to 40 minutes.

13. The method for synthesizing capped RNA according to any one of claims 1 to 12, characterized in that: NTPs include ATP, CTP and GTP; the NTPs also include at least one of UTP, 1-N-Me-Pseudo UTP, 5'-Me-UTP, 5'-OMe-UTP and PseudoUTP.

14. The method for synthesizing capped RNA according to claim 13, characterized in that: NTPs include ATP, CTP, GTP and UTP; the concentration ratio of the ATP, CTP, GTP and UTP to the capping polynucleotide is (2-10): (2-10): (2-10): (2-10): (2-40).

15. The method for synthesizing capped RNA according to claim 13, characterized in that: The final molar concentration of magnesium ions in the solution containing magnesium ions in the raw material is 20-60 mM.

16. The method for synthesizing capped RNA according to any one of claims 1 to 12, characterized in that: The raw material also includes at least one of an inorganic pyrophosphatase and a nuclease inhibitor.

17. The method for synthesizing capped RNA according to claim 16, characterized in that: The raw materials also include Tris-HCl, spermidine, DTT and Triton X-100.

18. The method for synthesizing capped RNA according to any one of claims 1 to 12, characterized in that: The sequence of the template strand of the RNA promoter is shown in SEQ ID NO.1; Or, the sequence of the template strand of the RNA promoter is shown in SEQ ID NO.2; Alternatively, the sequence of the template strand of the RNA promoter is as shown in SEQ ID NO.

3.

19. A capped RNA transcription reaction solution, characterized in that: including a DNA template, a capping polynucleotide, NTPs, an RNA polymerase, and a solution containing magnesium ions; The capped polynucleotide is a capped dimeric nucleotide, and the structural formula of the capped dimeric nucleotide is as follows: B1 is a natural or modified nucleoside base; E is 1; F is 0; M1 is a hydroxyl group; R1 and R2 are hydrogen; R3 to R5 are each independently hydroxy or alkoxy; X1 to X5 are oxygen; Y1 to Y4 are oxygen; Z1 to Z4 are hydroxyl groups; The base at position -1 of the template strand of the RNA promoter is cytosine; the base at position +1 of the template strand of the RNA promoter is complementary to B1 in the capping dinucleotide; Alternatively, the capped polynucleotide is a capped trimer nucleotide, and the capped trimer nucleotide has the following structural formula: B1 and B2 are each independently a natural or modified nucleoside base; E is 1; F is 0; M1 is oxygen; M2 is a hydroxyl group; R1 and R2 are hydrogen; R3 to R6 are each independently hydroxyl or alkoxy; X1 to X6 are oxygen; Y1 to Y5 are oxygen; Z1 to Z5 are hydroxyl groups; The base at position -1 of the template strand of the RNA promoter is cytosine; the bases at positions +1 to +2 of the template strand of the RNA promoter are complementary paired with B1 to B2 in the capping trinucleotide, respectively.

20. The capped RNA transcription reaction solution according to claim 19, characterized in that NTPs include ATP, CTP and GTP; the NTPs also include at least one of UTP, 1-N-Me-Pseudo UTP, 5'-Me-UTP, 5'-OMe-UTP and PseudoUTP.

21. The capped RNA transcription reaction solution according to claim 19, characterized in that: NTPs include ATP, CTP, GTP and UTP; the concentration ratio of the ATP, CTP, GTP and UTP to the capping polynucleotide is (2-10): (2-10): (2-10): (2-10): (2-40).

22. The capped RNA transcription reaction solution according to claim 19, characterized in that: The final molar concentration of magnesium ions in the solution containing magnesium ions in the capped RNA transcription reaction solution is 20-60 mM.

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